Fantastically plastic: fish larvae equipped for a new world

Teleost fish are more diverse than any other vertebrate group, and yet only a limited number of species are fished and farmed globally. Efforts to expand the quantity and diversity of fish produced are hampered by the extreme diversity of ontogenetic responses of fish, especially during larval development. This review looks at advances in molecular phylogeny, endocrine and nutrient influences and long-term studies of the phenotypes of commercially important fish to put the sources and consequences of this plasticity into context. This nested context of evolutionary forces of the fish-specific genome duplication, epigenetic influences, ontogenetically conserved processes like metamorphosis and cell determination is further presented in relation to how fish larvae translate the environment into somatic signals, the teleostian diversity of internal processes like sex differentiation and somatogenesis, and the long-term practical consequences of changes in timing or anthropogenic influences. This review aims to present a new baseline of knowledge of marine fish larvae which is useful to scientists, managers and producers.

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title: Phenotypic Plasticity and the Origins of Diversity
abstract: Phenotypic plasticity is the ability of a single genotype to produce more than one alternative form of morphology, physiological state, and/or behavior in response to environmental conditions. "Plasticity" and "development" are related terms that are becoming increasingly common in evolutionary biology and ecology. Both phenomena have passed through a period of neglect. In the 1960s Wigglesworth (228, p. 107) described some geneticists as being "apologetic" about environmentally cued polymorphisms, which they considered examples of unfortunate defects in the delicate genetic apparatus: "As R. A. Fisher once said to me, it is not surprising that such elaborate machinery should sometimes go wrong." And Bradshaw (19, p. 148) noted that botanists were carefully avoiding any mention of plasticity; environmental effects in experiments were considered "only an embarrassment." Until recently, genetic considerations have predominated in discussions of evolution and selection. Compared to the enormous progress made in genetics, there has been relatively little systematic effort to analyze environmental effects on the phenotype, and their evolutionary consequences. The plastic phenotype, stigmatized by poorly understood environmental influences and the ghost of Lamarck, has sometimes been lost from view as the focus of selection (e.g. 46; but see 48, 49). Much recent progress has been made toward integrating developmental and evolutionary biology, especially in vertebrate morphology (2, 12, 16, 216), developmental genetics (16, 163, 164), and molecular biology (103; also see 10, 111). "Developmental constraints" is a term symptomatic of this progress, though an unfortunate one because it seems to imply that the main effect of

title: Evolutionary Analyses of Morphological and Physiological Plasticity in Thermally Variable Environments
abstract: SYNOPSIS. Morphological and physiological plasticity is often thought to represent an adaptive response to variable environments. However, determining whether a given pattern of plasticity is in fact adaptive is analytically challenging, as is evaluating the degree of and limits to adaptive plasticity. Here we describe a general methodological framework for studying the evolution of plastic responses. This framework synthesizes recent analytical advances from both evolutionary ecology and functional biology, and it does so by integrating field experiments, functional and physiological analyses, environmental data, and genetic studies of plasticity. We argue that studies of plasticity in response to the thermal environment may be particularly valuable in understanding the role of environmental variation in the evolution of plasticity: not only can thermally-relevant traits often be mechanistically and physiologically linked to the thermal environment, but also the variability and predictability of the thermal environment itself can be quantified on ecologically relevant time scales. We illustrate this approach by reviewing a case study of seasonal plasticity in the extent of wing melanization in Western White Butterflies ( Pontia occidentalis ). This review demonstrates that 1) wing melanin plasticity is heritable, 2) plasticity does increase fitness in nature, but the effect varies between seasons and between years, 3) selection on existing variation in the magnitude of plasticity favors increased plasticity in one melanin trait that affects thermoregulation, but 4) the marked unpredictability of short-term (within-season) weather patterns substantially limits the capacity of plasticity to match optimal wing phenotypes to the weather conditions actually experienced. We complement the above case study with a casual review of selected aspects of thermal acclimation responses. The magnitude of thermal acclimation (“flexibility”) is demonstrably modest rather than fully compensatory. The magnitude of genetic variation (crucial to evolutionary responses to selection) in thermal acclimation responses has been investigated in only a few species to date. In conclusion, we suggest that an understanding of selection and evolution of thermal acclimation will be enhanced by experimental examinations of mechanistic links between traits and environments, of the physiological bases and functional consequences of acclimation, of patterns of environmental variability and predictability, of the fitness consequences of acclimation in nature, and of potential genetic constraints.

title: Gene duplication as a mechanism of genomic adaptation to a changing environment
abstract: A subject of extensive study in evolutionary theory has been the issue of how neutral, redundant copies can be maintained in the genome for long periods of time. Concurrently, examples of adaptive gene duplications to various environmental conditions in different species have been described. At this point, it is too early to tell whether or not a substantial fraction of gene copies have initially achieved fixation by positive selection for increased dosage. Nevertheless, enough examples have accumulated in the literature that such a possibility should be considered. Here, I review the recent examples of adaptive gene duplications and make an attempt to draw generalizations on what types of genes may be particularly prone to be selected for under certain environmental conditions. The identification of copy-number variation in ecological field studies of species adapting to stressful or novel environmental conditions may improve our understanding of gene duplications as a mechanism of adaptation and its relevance to the long-term persistence of gene duplications.

title: The Sequence of the Human Genome
abstract: A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies—a whole-genome assembly and a regional chromosome assembly—were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional ∼12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

title: Epigenetics and phenotypic variation in mammals
abstract: What causes phenotypic variation? By now it is clear that phenotype is a result of the interaction between genotype and environment, in addition to variation not readily attributable to either. Epigenetic phenomena associated with phenotypic variation at the biochemical, cellular, tissue, and organism level are now well recognized and are likely to contribute to the “intangible variation” alluded to. While it is clear that epigenetic modifications are mitotically heritable, the fidelity of this process is not well understood. Inheritance through more than one generation of meioses is even less well studied. So it remains unclear to what extent epigenetic changes contribute to phenotypic variation in natural populations. How might such evidence be obtained? What are the features of phenotypes that might suggest an epigenetic component? How much of the epigenetic component is truly independent of genetic changes? The answers to such questions must come from studies designed specifically to detect subtle, stochastically determined phenotypic variation in suitable animal models.

title: Epigenetic transgenerational actions of environmental factors in disease etiology
abstract: The ability of environmental factors to promote a phenotype or disease state not only in the individual exposed but also in subsequent progeny for successive generations is termed transgenerational inheritance. The majority of environmental factors such as nutrition or toxicants such as endocrine disruptors do not promote genetic mutations or alterations in DNA sequence. However, these factors do have the capacity to alter the epigenome. Epimutations in the germline that become permanently programmed can allow transmission of epigenetic transgenerational phenotypes. This review provides an overview of the epigenetics and biology of how environmental factors can promote transgenerational phenotypes and disease.

title: Chromatin Higher Order Folding--Wrapping up Transcription
abstract: Eukaryotic genomes are organized into condensed, heterogeneous chromatin fibers throughout much of the cell cycle. Here we describe recent studies indicating that even transcriptionally active loci may be encompassed within 80- to 100-nanometer-thick chromonema fibers. These studies suggest that chromatin higher order folding may be a key feature of eukaryotic transcriptional control. We also discuss evidence suggesting that adenosine-5′-triphosphate–dependent chromatin-remodeling enzymes and histone-modifying enzymes may regulate transcription by controlling the extent and dynamics of chromatin higher order folding.

title: Scale formation in selected fundulid and cyprinodontid fishes
abstract: Patterns of scale formation (onset, points of origin, completion, and spatial pattern) were examined for six species of killifishes in two families (Cyprinodontidae: Cyprinodon variegatus, and Fundulidae: Fundulus confluentus, F. heteroclitus, F. luciae, F. majalis, and Lucania parva) to determine if the patterns are another useful indicator for the transition from the larval to juvenile periods. In some species, the patterns were very similar, with scale formation originating on/near the caudal peduncle, then the dorsal surface of the head (in fundulids only), and later on the lateral surfaces of the head, and on the ventral surface of the trunk at the level of the pectoral fin. The timing of scale formation, relative to fish size, was later than or overlapped with other morphological characters (e.g., fin ray formation, juvenile/adult body shape) often used to mark the larval/juvenile transition. The onset of scale formation, across all species, occurred between 8 and 13 mm TL. Completion of scale formation occurred between 18 and 23 mm TL. At completion, scales covered 86–99% of the trunk and head. Completion of scale formation in these fishes is one of the last external morphological changes to occur during the larval to juvenile transition. For these species, and other flatfishes we have examined in detail, it appears that scale formation may be useful in helping to define the end of the larval period and the beginning of the juvenile period. Further studies, of divergent groups of teleosts, are necessary to determine if this length-based approach has broad validity.

title: Establishment of long term cultures of neural stem cells from adult sea bass, Dicentrarchus labrax.
abstract: Long term cell cultures could be obtained from brains of adult sea bass (Dicentrarchus labrax) up to 5 days post mortem. On three different occasions, sea bass brain tissues were dissected, dispersed and cultured in Leibovitz's L-15 media supplemented with 10% fetal bovine serum. The resulting cellular preparations could be passaged within 2 or 3 weeks of growth. The neural cells derived from the first trial (SBB-W1) have now been passaged over 24 times within two years. These cells have been cryopreserved and thawed successfully. SBB-W1 cells are slow growing with doubling times requiring at least 7 days at 22 degrees C. These long term cell cultures could be grown in suspension as neurospheres that were immunopositive for nestin, a marker for neural stem cells, or grown as adherent monolayers displaying both glial and neural morphologies. Immunostaining with anti-glial fibrillary acidic protein (a glial marker) and anti-neurofilament (a neuronal marker), yielded positive staining in most cells, suggesting their possible identity as neural stem cells. Furthermore, Sox 2, a marker for neural stem cells, could be detected from these cell extracts as well as proliferating cell nuclear antigen, a marker for proliferating cells. SBB-W1 could be transfected using pEGFP-N1 indicating their viability and suitability as convenient models for neurophysiological or neurotoxicological studies.

title: Spatial, temporal and interindividual epigenetic variation of functionally important DNA methylation patterns
abstract: DNA methylation is an epigenetic modification that plays an important role in gene regulation. It can be influenced by stochastic events, environmental factors and developmental programs. However, little is known about the natural variation of gene-specific methylation patterns. In this study, we performed quantitative methylation analyses of six differentially methylated imprinted genes (H19, MEG3, LIT1, NESP55, PEG3 and SNRPN), one hypermethylated pluripotency gene (OCT4) and one hypomethylated tumor suppressor gene (APC) in chorionic villus, fetal and adult cortex, and adult blood samples. Both average methylation level and range of methylation variation depended on the gene locus, tissue type and/or developmental stage. We found considerable variability of functionally important methylation patterns among unrelated healthy individuals and a trend toward more similar methylation levels in monozygotic twins than in dizygotic twins. Imprinted genes showed relatively little methylation changes associated with aging in individuals who are >25 years. The relative differences in methylation among neighboring CpGs in the generally hypomethylated APC promoter may not only reflect stochastic fluctuations but also depend on the tissue type. Our results are consistent with the view that most methylation variation may arise after fertilization, leading to epigenetic mosaicism.

title: Comparative organ differentiation during early life stages of marine fish.
abstract: The basic developmental mechanisms of teleosts are similar, but there are differences with respect to the timing of developmental events. These events are controlled by genetic and environmental factors. Direct comparisons of organogenesis are complicated due to large variations in egg sizes and incubation temperatures between species. But in general, cultivated small marine pelagic fish larvae originating from rather small eggs (like gadoids, flatfishes, sparids) hatch with a relatively large yolk sac, a larval finfold and subdermal space and under-developed organs. Developmental status at hatch differs between species and the duration of the yolk sac period varies. Main organs and organ systems become functional by first feeding and differentiate during the larval stage and metamorphosis. Species developing directly via large yolk-rich eggs and a long incubation period have a juvenile like morphology and organ functionality at first feeding, sometimes immediately after hatch (like wolffishes). Histomorphological and cell- or organ functional studies of developing embryos and larvae of cultivated species constitute basic information for understanding species-specific events, of utmost importance for improving production protocols. Information is still lacking on early functionality of endocrine and immunocompetent tissues and organs, areas that deserve future focus.

title: Epigenetics and transgenerational transfer: a physiological perspective
abstract: Epigenetics, the transgenerational transfer of phenotypic characters without modification of gene sequence, is a burgeoning area of study in many disciplines of biology. However, the potential impact of this phenomenon on the physiology of animals is not yet broadly appreciated, in part because the phenomenon of epigenetics is not typically part of the design of physiological investigations. Still enigmatic and somewhat ill defined is the relationship between the overarching concept of epigenetics and interesting transgenerational phenomena (e.g. ‘maternal/parental effects’) that alter the physiological phenotype of subsequent generations. The lingering effect on subsequent generations of an initial environmental disturbance in parent animals can be profound, with genes continuing to be variously silenced or expressed without an associated change in gene sequence for many generations. Known epigenetic mechanisms involved in this phenomenon include chromatin remodeling (DNA methylation and histone modification), RNA-mediated modifications (non-coding RNA and microRNA), as well as other less well studied mechanisms such as self-sustaining loops and structural inheritance. In this review we: (1) discuss how the concepts of epigenetics and maternal effects both overlap with, and are distinct from, each other; (2) analyze examples of existing animal physiological studies based on these concepts; and (3) offer a construct by which to integrate these concepts into the design of future investigations in animal physiology.

title: Variability of our somatic (epi)genomes.
abstract: A motto of the human genome project could have been “everything that happens in the world depends on [DNA] sequences” (); the Project's quasi-completion was solemnly celebrated in 2000. Still, in 2010, chief-genomicists Francis Collins and Craig Venter reportedly admit that its impact on

title: The implications for aquaculture practice of epigenomic programming of components of the endocrine system of teleostean embryos: lessons learned from mammalian studies
abstract: Epigenetics is the study of changes in gene expression patterns that occur without any modification of the underlying nucleotide sequence of the DNA. Modifications of the so-called epigenome include complex transient or permanent chemical changes of the DNA or histone proteins resulting in the suppression or enhancement of gene expression, together with an array of post-translational events that modify the translational products. Epigenomic programming (EP) of the genome is an essential component of embryonic development in animals from the totipotent fertilized egg to the pluripotent stem cells, stem cell differentiation and final tissue and organ formation. Many of these EP processes are influenced transiently and some permanently by environmental influences. In eutherian mammals, environmentally related EP of embryos is linked to permanent changes in the phenotype of the progeny, some of which have been associated with adult onset metabolic disorders. Moreover, because some of the epigenetic remodelling occurs in both the soma and germ line, the resultant phenotypic characteristics (some of which are linked to disease states) may be heritable. Although far less is known about the effects of environmentally linked EP on the ontogeny of fishes, the available information suggests that the EP processes are similar amongst all vertebrates, and there are clear parallels between fish and mammals that are discussed in this paper. Our perspective takes the well-established findings in mammals and uses them to proactively extrapolate to the as yet under-recognized implications of EP for fish biology and for fish production in intensive aquaculture.

title: The history of a developmental stage: Metamorphosis in chordates
abstract: Metamorphosis displays a striking diversity in chordates, a deuterostome phylum that comprises vertebrates, urochordates (tunicates), and cephalochordates (amphioxus). In anuran amphibians, the tadpole loses its tail, develops limbs, and undergoes profound changes at the behavioral, physiological, biochemical, and ecological levels. In ascidian tunicates, the tail is lost and the head tissues are drastically remodeled into the adult animal, whereas in amphioxus, the highly asymmetric larva transforms into a relatively symmetric adult. This wide diversity has led to the proposal that metamorphosis evolved several times independently in the different chordate lineages during evolution. However, the molecular mechanisms involved in metamorphosis are largely unknown outside amphibians and teleost fishes, in which metamorphosis is regulated by the thyroid hormones (TH) T3 and T4 binding to their receptors (thyroid hormone receptors). In this review, we compare metamorphosis in chordates and then propose a unifying definition of the larva‐to‐adult transition, based on the conservation of the role of THs and some of their derivatives as the main regulators of metamorphosis. According to this definition, all chordates (if not, all deuterostomes) have a homologous metamorphosis stage during their postembryonic development. The intensity and the nature of the morphological remodeling varies extensively among taxa, from drastic remodeling like in some ascidians or amphibians to more subtle events, as in mammals.  2008.

title: The Molecular and Endocrine Basis of Flatfish Metamorphosis
abstract: A significant component of aquaculture is the production of good quality larvae, and, in the case of flatfish, this is tied up with the change from a symmetric larva to an asymmetric juvenile. Despite the pioneering work carried out on the metamorphosis of the Japanese flounder (Paralichthys olivaceus) and summer flounder (Paralichthys dentatus), the underlying molecular basis of flatfish metamorphosis is still relatively poorly characterized. It is a thyroid hormone (TH) driven process, and the role of other hormones in the regulation of the process along with the interplay of abiotic factors are still relatively poorly characterized as is the extent of tissue and organ remodeling, which underlie the profound structural and functional modifications that accompany the larval/juvenile transition. The isolation of genes for hormones, receptors, binding proteins, and other accessory factors has provided powerful tools with which to pursue this question. The application of molecular methodologies such as candidate gene approaches and microarray analysis coupled to functional genomics has started to contribute to understanding the complexity of tissue and organ modifications that accompany flatfish metamorphosis. A better understanding of the biology of normal metamorphosis is essential to identify factors contributing to abnormal metamorphosis.

title: Photoreceptor development in the pineal organ and the eye of Plecoglossus altivelis and Paralichthys olivaceus (Teleostei)
abstract: SummaryThe ontogenetic apperance of pineal photo-receptors was compared with that of retinal photoreceptors in the ayu Plecoglossus altivelis and the lefteye flounder Paralichthys olivaceus, which hatched 10 days and 3 days after fertilization, respectively. Despite the disparity in incubation time, the outer segments (containing membranous lamellae) of the pineal photoreceptors first appeared from 3 to 4 days after fertilization in both species. In contrast, the outer segments of the retinal photoreceptors first became visible 5 to 6 days after fertilization, although a characteristic retinal stratification and the optic tract leaving the ganglion cell layer were already found 4 days after fertilization in both species. The functional significance of these temporal disparities and/or similarities in photoreceptor development are discussed with special reference to the timing of daily rhythmic activities during the early developmental period of the teleosts.

title: Molecular identification and developmental expression of UV and green opsin mRNAs in the pineal organ of the Atlantic halibut.
abstract: The pineal organ is the only differentiated photoreceptor organ present in embryos and early larvae of the Atlantic halibut (Hippoglossus hippoglossus). We investigated the molecular identity of opsins in the pineal organ, and their expression during different life stages. Using RT-PCR we identified two 681-bp gene sequences, named HPO1 and HPO4, in cDNA from adult pineal and whole embryos. The predicted amino acid sequences showed highest identity to the transmembrane regions of teleostean RH2 green cone opsins (HPO1, 72-91%) and SWS-1 UV cone opsins (HPO4, 71-83%). In situ hybridization revealed expression of HPO1 and HPO4 mRNA transcripts in photoreceptors in the pineal organ of embryos, larvae and adults. HPO1 and HPO4 mRNA transcripts were also expressed in the larval retina. Our study provides molecular evidence for short and middle wavelength light sensitive photoreceptors in the pineal organ of Atlantic halibut throughout life, and suggests that pineal photoreception may play an important role during embryonic and larval life stages, especially at the time when the retina does not possesses corresponding photoreceptor capacity.

title: Early development of the pineal photoreceptors prior to the retinal differentiation in the embryonic rainbow trout, Oncorhynchus mykiss (Teleostei).
abstract: The development of the pineal and retinal photoreceptors in the embryonic rainbow trout, Oncorhynchus mykiss was investigated by means of light and electron microscopy. The pineal photoreceptors endowed with photoreceptive outer segments and signal-transmitting synapses appeared first 15 days after fertilization, and well developed by 21 days. In contrast, the retinal photoreceptors appeared as late as 27 days after fertilization (1 day before hatching). This temporal disparity in photoreceptor development has been compared with the photoperiodic formation of otolith increments, which is initiated during an early developmental stage lacking retinal photoreception. It has been suggested that the early development of pineal photoreceptors may be involved in light-dark recognition, establishment of the diel and circadian rhythms, and entrainment to the environmental photoperiod in the embryonic rainbow trout.

title: Effect of daily thermo- and photo-cycles of different light spectrum on the development of Senegal sole (Solea senegalensis) larvae
abstract: This paper investigates the impact of different thermo- and photo-cycles of distinct wavelengths on Solea senegalensis larvae from day 1 to 30 post-hatching. In experiment 1, larvae were exposed to 12 h light:12 h dark (12 L:12D) cycle and (A) constant temperature (20.7 °C), (B) thermocycle of 12 h thermophase: 12 h cryophase, 22.1 °C day: 19.0 °C night (referred to as TC) or (C) 12 h cryophase: 12 h thermophase, 19.2 °C day: 22.0 °C night (referred to as CT). In experiment 2, larvae were kept under constant temperature (20.8 °C) and exposed to (A) continuous light (LL), (B) continuous darkness (DD), and LD 12 L:12D cycles of (C) white light (LD W ), (D) blue light (LD B ) or (E) red light (LD R ). The sole larvae achieved the best performance, and showed fastest development and lowest degree of deformity under natural thermocycle conditions (TC) with a deformity percentage of 31.1% and LD B cycles with 27.7% of malformation, conditions which were nearest their natural aquatic environment. Larvae reared under TC started eye migration at 9 day post-hatching (DPH), while larvae exposed to CT started eye migration at 11 DPH. In larvae under the LD B treatment the migration of the eye started earlier than in the other treatments (9 DPH), while larvae reared under LL and DD photoperiods died before metamorphosis. These findings highlight the importance of light and temperature cycles during the early development of S. senegalensis larvae, which should be taken into consideration in experimental or rearing protocols.

title: The pineal organ of teleost fishes
abstract: The pineal organ of teleost fish is a directly photosensory organ that contains photoreceptor cells similar to those of the retina. It conveys photoperiod information to the brain via neural pathways and by release of indoleamines, primarily melatonin, into the circulation. The photoreceptor cells respond to changes in ambient illumination with a gradual modulation of neurotransmission to second-order neurons that innervate various brain centres, and by modulation of indoleamine synthesis. Melatonin is produced rhythmically, and melatonin synthesis may be regulated either directly by ambient photoperiod, or by an endogenous circadian oscillator that is entrained by the photoperiod. During natural conditions, melatonin is produced at highest levels during the night. Although the pineal organ undoubtedly influences a variety of physiological parameters, as assessed by experimental removal of the pineal organ and/or administration of exogenous indoleamines, its role in any physiological situation is not clear cut. The effects of any interference with pineal functions appear to vary with the time of year and experimental photothermal regimes. There are strong indications that the pineal organ is one component in a central neural system that constitutes the photoperiod-responding system of the animal, i.e. the system that is responsible for correct timing of daily and seasonal physiological rhythms. It is important to envisage the pineal organ as a part of this system; it interacts with other photosensory structures (the retina, possibly extraretinal non-pineal photoreceptors) and circadian rhythm generators

title: Expression of pineal ultraviolet- and green-like opsins in the pineal organ and retina of teleosts.
abstract: In teleostean bony fishes, studies on the adults of various species have shown that pineal photoreceptors are maximally sensitive to short- and middle-wavelength light, possibly utilising both rod-like and pineal-specific opsins. Until recently, however, very little was known about the pineal opsins present in embryonic and larval teleosts and their relationships to opsins expressed by retinal photoreceptors. Our immunocytochemical studies have revealed that, in Atlantic halibut, herring and cod, pineal photoreceptors express principal phototransduction molecules during embryonic life before they appear in retinal photoreceptors. In cDNA from embryonic and adult halibut, we identified two partial opsin gene sequences, HPO1 and HPO4, with highest homology to teleost green and ultraviolet cone opsins (72-83% and 71-83% amino acid identity, respectively). In halibut, these opsins are expressed in the pineal organ of embryos and appear in the retina of larvae. Our recent in situ hybridisation studies with RNA probes for HPO1 and HPO4 demonstrate the presence of green-like opsin mRNAs in the pineal organ and the retina of herring, cod, turbot, haddock, Atlantic salmon, zebrafish and three species of cichlid, and of ultraviolet opsins in the retinas of zebrafish, Atlantic salmon, turbot and the three cichlid species. We conclude that the halibut pineal organ appears to have the potential for both ultraviolet and green photosensitivity from the embryonic stage and that the retina may acquire the same potential during the larval stages. In the other teleosts studied, although both pineal and retinal photoreceptors seem to utilise a green-like opsin from the larval stage, ultraviolet photoreception appears to be restricted to the retina.

title: Daily and circadian melatonin release in vitro by the pineal organ of two nocturnal teleost species: Senegal sole (Solea senegalensis) and tench (Tinca tinca).
abstract: This research aimed to investigate melatonin rhythms in the pineal organ of two nocturnal fish species, sole and tench, which show high sensitivity to light. Pineal organs were cultured in vitro under an LD (12 h light:12 h dark) cycle to study the daily rhythmicity of melatonin release. In addition, the in vitro culture was performed under conditions of constant darkness (DD) to study the endogenous control of the rhythm. In the pineal organs cultured under an LD cycle, rhythmic melatonin release was evident in both species, with low values observed during the photophase (15.6+/-7.2 and 22.6+/-2.6 pg/mL for sole and tench, respectively) and high values coinciding with the scotophase (74.0+/-8.2 and 82.1+/-9.1 pg/mL, for sole and tench, respectively). Under LD, the rhythm had a period of 24 h (p<0.001) and presented similar acrophases for both species, located around 9-10 h after lights off (2 and 3 h before the end of the dark phase). When the pineal organs were cultured under DD, the results differed between the species studied. A marked circadian rhythm in melatonin release by the pineal was registered in tench, with lower values during the subjective day, i.e. the period that was previously day (6.2+/-1.6 pg/mL) and higher values during the subjective night, i.e. the period that was previously night (20.4+/-5.5 pg/mL). The rhythm had a mean tau of 24.1 h (p<0.01) and the acrophase was located around 12 h after lights off (the beginning of the subjective day), slightly later than that registered under LD conditions. In contrast, melatonin values in sole remained high during darkness (around 92.0+/-6.9 pg/mL) for four consecutive days, including subjective day periods. In short, these findings revealed that the rhythm of melatonin release in tench is under endogenous control by a circadian oscillator within the pineal organ, while no such pacemaker was evident in sole, which melatonin rhythm appeared to be exclusively light-driven.

title: Evolution of photosensory pineal organs in new light: the fate of neuroendocrine photoreceptors.
abstract: Pineal evolution is envisaged as a gradual transformation of pinealocytes (a gradual regression of pinealocyte sensory capacity within a particular cell line), the so-called sensory cell line of the pineal organ. In most non-mammals the pineal organ is a directly photosensory organ, while the pineal organ of mammals (epiphysis cerebri) is a non-sensory neuroendocrine organ under photoperiod control. The phylogenetic transformation of the pineal organ is reflected in the morphology and physiology of the main parenchymal cell type, the pinealocyte. In anamniotes, pinealocytes with retinal cone photoreceptor-like characteristics predominate, whereas in sauropsids so-called rudimentary photoreceptors predominate. These have well-developed secretory characteristics, and have been interpreted as intermediaries between the anamniote pineal photoreceptors and the mammalian non-sensory pinealocytes. We have re-examined the original studies on which the gradual transformation hypothesis of pineal evolution is based, and found that the evidence for this model of pineal evolution is ambiguous. In the light of recent advances in the understanding of neural development mechanisms, we propose a new hypothesis of pineal evolution, in which the old notion 'gradual regression within the sensory cell line' should be replaced with 'changes in fate restriction within the neural lineage of the pineal field'.

title: Does light have an influence on fish growth
abstract: Light compares a complex of external and ecological factors, including colour spectrum, intensity and photoperiod. Light characteristics are very specific in an aquatic environment and light is extremely variable in nature. `Receptivity' of fish to light profoundly changes according to the species and the developmental status. Specific photoreceptor cells are present in both eye and pineal. If it is easy to change the light in experimentation and to observe the effects on fish growth, it is much more difficult in nature to make such determinations. In larvae, many studies have been dedicated to the influence of intensity and photoperiod on growth: generally, species need a minimal threshold intensity to be able to develop normally and grow. This is probably related to the aptitude to localize, catch and ingest prey. Light is also indispensable for body pigmentation, an important phenomenon involved in early development and growth. Too intense light can be stressful or even lethal. A few species are able to develop and grow at very low intensities or, sometimes, in the absence of light. Generally, long daylength improves larval rearing quality. The synergistic effect of `food availability-daylength' appears to be determining at this stage. In older fish, there is very little information about the influence of light `quality' but more about intensity and much more about photoperiod. Light intensity effects are not so clear and depend on the species and the experimental procedures: it is probably not an important factor for growth stimulation. Daylength appears much more important. Many species, including both marine species and salmonids, react to photoperiod treatments and long daylength stimulates growth. The most studied species is the Atlantic salmon, which is very sensitive, both during the freshwater stage, with the parr–smolt transformation very dependent on the photoperiod, and also in sea water. In this last condition, lighting also influences early maturation. An important point is to be certain that light affects fish growth through a better food conversion efficiency and not just through stimulated food intake. Also included in this review is a discussion about the endolymph–otolith system, which is very sensitive to daylight and seasonal cycles and a review of the present knowledge on the involvement of light influence on hormone levels (melatonin, somatotropin, thyroid hormones and other hormones).

title: Ontogeny of the GnRH systems in zebrafish brain: in situ hybridization and promoter-reporter expression analyses in intact animals
abstract: The ontogeny of two gonadotropin-releasing-hormone (GnRH) systems, salmon GnRH (sGnRH) and chicken GnRH-II (cGnRH-II), was investigated in zebrafish (Danio rerio). In situ hybridization (ISH) first detected sGnRH mRNA-expressing cells at 1 day post-fertilization (pf) anterior to the developing olfactory organs. Subsequently, cells were seen along the ventral olfactory organs and the olfactory bulbs, reaching the terminal nerve (TN) ganglion at 5–6 days pf. Some cells were detected passing posteriorly through the ventral telencephalon (10–25 days pf), and by 25–30 days pf, sGnRH cells were found in the hypothalamic/preoptic area. Continuous documentation in live zebrafish was achieved by a promoter-reporter expression system. The expression of enhanced green fluorescent protein (EGFP) driven by the sGnRH promoter allowed the earlier detection of cells and projections and the migration of sGnRH neurons. This expression system revealed that long leading processes, presumably axons, preceded the migration of the sGnRH neuron somata. cGnRH-II mRNA expressing cells were initially detected (1 day pf) by ISH analysis at lateral aspects of the midbrain and later on (starting at 5 days pf) at the midline of the midbrain tegmentum. Detection of red fluorescent protein (DsRed) driven by the cGnRH-II promoter confirmed the midbrain expression domain and identified specific hindbrain and forebrain cGnRH-II-cells that were not identified by ISH. The forebrain DsRed-expressing cells seemed to emerge from the same site as the sGnRH-EGFP-expressing cells, as revealed by co-injection of both constructs. These studies indicate that zebrafish TN and hypothalamic sGnRH cell populations share a common embryonic origin and migratory path, and that midbrain cGnRH-II cells originate within the midbrain.

title: Ontogeny and distribution of gonadotropin-releasing hormone (GnRH) neuronal systems in the brain of the cichlid fish Cichlasoma dimerus
abstract: Using immunocytochemistry we have described the distribution and ontogeny of three distinct gonadotropin-releasing hormone (GnRH) neural systems, emphasizing the analysis during the period of sex differentiation in the South American cichlid fish Cichlasoma dimerus. In the forebrain a group of neurones immunoreactive to salmon GnRH that formed clusters in the nucleus olfacto retinalis (NOR), was located at the junction of the olfactory bulb and the telencephalon. These neurones differentiated 3 days after fertilization from the olfactory placodes. GnRH immunoreactive neurones along the olfactory nerves through the rostrobasal olfactory bulb were observed on day 4 and at the NOR on day 10. A group of neurones immunoreactive to chicken GnRH II was seen in the dorsal midbrain tegmentum. They originate from the ventricular ependyma between days 5 and 6. These neurones remained close to blood vessels throughout development. Between days 22 and 30 a group of neurones immunoreactive to seabream GnRH was detected in the anterior basal preoptic area. GnRH innervation of the pituitary was detected after the differentiation of preoptic neurones and in coincidence with gonadal differentiation. We hypothesize that the GnRH neural systems have three distinct embryonic origins. Furthermore, we show that the NOR and the midbrain GnRH neurones might have functions other than gonadal development, whereas the preoptic GnRH neurones in C. dimerus might be associated with gonadal sex differentiation.

title: Developmental expression of the G protein-coupled receptor 54 and three GnRH mRNAs in the teleost fish cobia.
abstract: The cDNAs of the G protein-coupled receptor 54 (GPR54) and three prepro-gonadotropin-releasing hormones, GnRH-I (seabream GnRH), GnRH-II (chicken GnRH-II), and GnRH-III (salmon GnRH) were isolated and cloned from the brain of the teleost fish cobia, Rachycentron canadum. The cobia GPR54 cDNA was 95 and 51-56% identical to those of tilapia and mammalian models respectively. The GnRH cDNA sequences of cobia showed strong identities to those of tilapia, Atlantic croaker, red drum, and the seabass and seabream species. The real-time quantitative RT-PCR methods allowed detection of all three GnRH mRNAs on the first day after hatching (DAH). The GnRH-I mRNA levels, which were the lowest among the three GnRHs, increased gradually with two distinct peaks in larvae at 3 and 4 DAH. On the other hand, GnRH-II and GnRH-III mRNAs were significantly higher in larvae at 2 and 6 DAH compared with those on the preceding days. In addition, significant peaks of all the three GnRH mRNAs were observed in the brains of 26-day-old fish. The finding of higher GnRH-I and GnRH-II mRNAs in males than females at 153 DAH may be related to early puberty observed during the first year in laboratory-reared male cobia. Moreover, this study demonstrates for the first time the expression of GPR54 mRNA during larval development in a vertebrate species. The concomitant expression patterns of GPR54 and GnRH mRNAs during different stages of larval and juvenile developments, and during early puberty in male cobia suggest a potential relationship between GPR54 and multiple GnRHs during these stages of development consistent with the role of GPR54 in controlling GnRH release in mammals. The increase in GPR54 and GnRH mRNAs observed during early puberty in cobia is consistent with a similar change reported in pubertal rats. This finding together with the localization of GPR54 mRNAs on GnRH neurons in fish and mammals suggests that the GPR54-GnRH interactions may be conserved in different vertebrate groups.

title: Seabream GnRH immunoreactivity in brain and pituitary of XX and XY Nile tilapia, Oreochromis niloticus during early development.
abstract: Seabream gonadotropin-releasing hormone (sbGnRH)-the chief preoptic area-hypothalamus (POA-H) form of GnRH in tilapia is involved in sexual maturation. In this study, we investigated the qualitative changes in ontogeny of sbGnRH immunoreactivity (ir-), between sexes to understand its impending role during sex differentiation. For this, the differences in immunocytochemical localization of sbGnRH in genetically male (XY) and female (XX) fish were studied from 1 day after hatching (dah), through the critical period of sex differentiation (7-21 dah) to 40 dah and mature Nile tilapia. Specific antisera against sbGnRH were used for immunolocalization. SbGnRH ir- neurons were observed in POA-H as early as 5 and 15 dah in XY fish and XX fish, respectively. Higher ir- was detected in the POA-H of XY tilapia compared with XX population till 10 dah. There was a qualitative drop in sbGnRH ir- neurons/cell bodies in POA-H around 20 dah till 30 dah in XY population compared with other durations. SbGnRH ir- cells were detected in pituitary of XX fish by 15 dah and in XY fish around 10 dah but seemed to drop down by 20 dah in XY whereas it continued to remain steady in XX fish. The sbGnRH ir- in XY fish showed a rise from 35 dah and thence till 40 dah. This study revealed subtle differences in POA-H and pituitary sbGnRH ir- during early development between genetic male and female fish with possible implications in sex differentiation.

title: Developmental Expression of Three Forms of Gonadotropin-Releasing Hormone and Ontogeny of the Hypothalamic-Pituitary-Gonadal Axis in Gilthead Seabream (Sparus aurata)1
abstract: To address the complexity of the origin of the GnRH system in perciforms, we investigated the ontogenic expression of three GnRHs in gilthead seabream. Using in situ hybridization, chicken (c) GnRH-II mRNA-expressing cells were detected in the hindbrain at 1.5 days postfertilization (DPF) and in the midbrain at 2 DPF and thereafter; the hindbrain signals became undetectable after 10 DPF. Salmon (s) GnRH mRNA-expressing cells were first seen in the olfactory placode at 3 DPF, started caudal migration at 14 DPF, and reached the preoptic areas at 59 DPF. Seabream (sb) GnRH mRNA-expressing cells were first detected in the terminal nerve ganglion cells (TNgc), ventral part of the ventral telencephalon, nucleus preopticus parvocellularis, and thalamus at 39 DPF, and extended to the nucleus preopticus magnocellularis at 43 DPF, ventrolateral hypothalamus at 51 DPF, and nucleus lateralis tuberis and posterior tuberculum at 59 DPF. Coexpression of sbGnRH and sGnRH transcripts was found in the TNgc. Using real-time fluorescence-based quantitative polymerase chain reaction, transcript levels of cGnRH-II and sGnRH were first detected at 1 and 1.5 DPF, respectively, and increased and remained high thereafter. Transcript levels of sbGnRH remained low after first detection at 1 DPF. Furthermore, these GnRH expression profiles were correlated with the expression profiles of reproduction-related genes in which at least four concomitant increases of GnRH, GnRH receptor, gonadotropin, gonadotropin receptor, and Vasa transcripts were found at 5, 8, 14, and 28 DPF. Our data provide an expanded view of the ontogeny of the GnRH system and reproductive axis in perciforms.

title: Transcriptional regulation of pituitary POMC is conserved at the vertebrate extremes despite great promoter sequence divergence.
abstract: The stress response involves complex physiological mechanisms that maximize behavioral efficacy during attack or defense and is highly conserved in all vertebrates. Key mediators of the stress response are pituitary hormones encoded by the proopiomelanocortin gene (POMC). Despite conservation of physiological function and expression pattern of POMC in all vertebrates, phylogenetic footprinting analyses at the POMC locus across vertebrates failed to detect conserved noncoding sequences with potential regulatory function. To investigate whether ortholog POMC promoters from extremely distant vertebrates are functionally conserved, we used 5'-flanking sequences of the teleost fish Tetraodon nigroviridis POMCalpha gene to produce transgenic mice. Tetraodon POMCalpha promoter targeted reporter gene expression exclusively to mouse pituitary cells that normally express Pomc. Importantly, transgenic expression in mouse corticotrophs was increased after adrenalectomy. To understand how conservation of precise gene expression mechanisms coexists with great sequence divergence, we investigated whether very short elements are still conserved in all vertebrate POMC promoters. Multiple local sequence alignments that consider phylogenetic relationships of ortholog regions identified a unique 10-bp motif GTGCTAA(T/G)CC that is usually present in two copies in POMC 5'-flanking sequences of all vertebrates. Underlined nucleotides represent totally conserved sequences. Deletion of these paired motifs from Tetraodon POMCalpha promoter markedly reduced its transcriptional activity in a mouse corticotropic cell line and in pituitary POMC cells of transgenic mice. In mammals, the conserved motifs correspond to reported binding sites for pituitary-specific nuclear proteins that participate in POMC transcriptional regulation. Together, these results demonstrate that mechanisms that participate in pituitary-specific and hormonally regulated expression of POMC have been preserved since mammals and teleosts diverged from a common ancestor 450 million years ago despite great promoter sequence divergence.

title: Stress responsiveness of the pituitary-interrenal axis during early life stages of common carp (Cyprinus carpio).
abstract: Whole-body levels of ACTH, alpha-MSH and cortisol in eggs and larvae of the common carp (Cyprinus carpio) were determined periodically up until 168 h after fertilisation. ACTH, alpha-MSH and cortisol immunoreactivity was detected in unfertilised eggs, and endogenous production of ACTH and alpha-MSH was observed 24 h after fertilisation and that of cortisol 36 h after fertilisation. ACTH immunoreactivity reached peak levels before hatching (56-72 h after fertilisation) and remained relatively stable thereafter, while alpha-MSH immunoreactivity started to increase after hatching. At 36 h after fertilisation, whole-body cortisol levels increased rapidly reaching peak levels at the end of hatching (72 h after fertilisation), remaining stable until the end of the experiment. From 50 h after fertilisation onwards, embryos and larvae increased their whole-body cortisol levels when subjected to handling (mechanical pressure during egg stage or netting during the larval stage). It is concluded that the pituitary-interrenal axis in carp is fully functional at the time of hatching. No indications of a stress non-responsive period after hatching were observed. To characterise ACTH and alpha-MSH immunoreactivities in carp larvae, whole-body homogenates were analysed by HPLC, with pituitary homogenates of adult carp serving as a reference. ACTH and alpha-MSH immunoreactivity in carp larvae homogenates consisted of three and two products respectively. HPLC of adult carp pituitaries revealed the presence of two ACTH immunoreactive products, which may represent a phosphorylated and a non-phosphorylated ACTH variant, while the three alpha-MSH peaks most likely represent des-acetylated, mono-acetylated and di-acetylated alpha-MSH, the latter being the predominant form. In carp larvae, however, one of the ACTH immunoreactive products co-eluted with the non-phosphorylated ACTH, while the two alpha-MSH products identified co-eluted with des-acetylated and mono-acetylated alpha-MSH, indicating that POMC processing at this stage of development is different from prohormone processing in adult fish.

title: Development of the corticosteroid stress axis and receptor expression in zebrafish.
abstract: Using zebrafish embryos and larvae, we examined the temporal patterns of cortisol and expression of genes involved in corticosteroid synthesis and signaling. Embryonic cortisol levels decreased approximately 70% from 1.5 h postfertilization (hpf) to hatch (approximately 42 hpf) and then increased 27-fold by 146 hpf. The mRNA abundances of steroidogenic acute regulatory protein, 11beta-hydroxylase and 11beta-hydroxysteroid dehydrogenase type 2, increased severalfold after hatch and preceded the rise of cortisol levels. In contrast to other teleosts that possess two glucocorticoid receptors (GRs) and one mineralocorticoid receptor (MR), only one GR and MR were identified in zebrafish, which were cloned and sequenced. GR mRNA abundance decreased from 1.5 to 25 hpf, rebounded, and then was stable from 49 to 146 hpf. MR transcripts increased continuously from 1.5 hpf and were 52-fold higher by 97 hpf. An acute cortisol response to a stressor was not detected until 97 hpf, whereas melanocortin type 2 receptor mRNA increased between 25 and 49 hpf. Collectively, the patterns of cortisol and the expression of cortisol biosynthetic genes and melanocortin type 2 receptor suggest that the corticoid stress axis in zebrafish is fully developed only after hatch. The temporal differences in GR, MR, and 11beta-hydroxysteroid dehydrogenase type 2 gene expression lead us to propose a key role for MR signaling by maternal cortisol during embryogenesis, whereas cortisol secretion after hatch may be regulating GR expression and signaling in zebrafish.

title: Ontogeny, tissue distribution, and hormonal regulation of insulin-like growth factor binding protein-2 (IGFBP-2) in a marine fish, Sparus aurata.
abstract: In this study, we have cloned insulin-like growth factor binding protein (IGFBP)-2 from a marine hermaphroditic fish species, the gilthead sea bream (Sparus aurata), and determined its structure, ontogeny, tissue distribution, and hormonal regulation. The sea bream IGFBP-2 precursor consists of 286 amino acids, including a putative signal peptide of 22 residues and a mature protein of 264 residues. The overall sequence of sea bream preIGFBP-2 is 52-39% identical to that of zebrafish, chick, mouse, rat, pig, sheep, bovine, and human preIGFBP-2. The cysteine-rich N- and C-terminal domains, which are believed to be important for IGF binding, show a greater degree of conservation with a sequence identity of 73-56% and 53-50%, respectively. Sea bream IGFBP-2 contains 18 cysteine residues and their alignment is identical to those of other vertebrate IGFBP-2s. The Arg-Gly-Asp (RGD) sequence, present in the C-terminal domain of all known IGFBP-2, is also present in the sea bream. Northern blot analysis of RNA samples extracted from adult liver and developing larvae, revealed two transcripts of about 1.3 and 2.5 kb. RT-PCR analysis showed that IGFBP-2 was expressed in all adult tissues studied, with the highest levels found in liver and skin. IGFBP-2 transcripts were detected in gonad during reproductive cycle of S. aurata. Highest levels of IGFBP-2 mRNA were found in bisexual young gonads, and the levels decreased with gonad development and relatively high levels of IGFBP-2 mRNA were found in the ovary during spawning. By contrast, testicular IGFBP-2 mRNA levels were very low. RT-PCR detected IGFBP-2 mRNA throughout development in unfertilized eggs, embryos, and larvae, with highest levels observed after day 3 post-hatching, suggesting that this mRNA is the product of both the maternal and embryonic genomes. High variability in steady-state levels of hepatic IGFBP-2 mRNA was noted in adult fish, resulting in a statistically insignificant response to growth hormone treatment. These results suggest that the structure of IGFBP-2 is conserved in sea bream and that IGFBP-2 mRNA is expressed during early development and in gonad during the reproductive cycle, suggesting that it may play a role in gilthead sea bream development and reproduction.

title: Expression and cellular localization of insulin-like growth factor-II protein and mRNA in Sparus aurata during development.
abstract: The spatial localization of IGF-II protein and mRNA was investigated during larval and postlarval developmental stages of the gilthead sea bream (Sparus aurata) by immunohistochemistry and in situ hybridization, using specific antisera and riboprobes. Steady-state levels of IGF-II mRNA in larvae were determined by Northern blot analysis and were found to be increased. Immunoreactivity towards IGF-II was found in larval skin, muscle, gills, gut, olfactory epithelium and kidney. After metamorphosis, the strongest immunoreactivity was found in red skeletal muscle. Positive reaction with IGF-II antibodies was also found in the olfactory epithelium and in the epithelia of pharynx, oesophagus, stomach and kidney. In the adult, the most intense signal was observed in the red and pink musculature and in heart musculature. Immunostaining was also found in saccus vasculosus, thymus, spleen and ovary. IGF-II mRNA was detected by in situ hybridization in the brain, olfactory epithelium, eye, pharynx, skeletal musculature and liver. The spatial distribution of IGF-II shown in this study is consistent with previous findings on the cellular localization of IGF type 1 receptor in the sea bream and supports a role for IGF-II during development and growth of sea bream. Furthermore, these results suggest that IGF-II acts in an autocrine/paracrine manner.

title: Comparative thyroidology: thyroid gland location and iodothyronine dynamics in Mozambique tilapia (Oreochromis mossambicus Peters) and common carp (Cyprinus carpio L.)
abstract: In teleosts, the thyroid gland is mostly found in the subpharyngeal region. However, in several species thyroid follicles are found in, for example, heart, head kidney and kidney. Such heterotopic thyroid follicles are active, and considered to work in concert with the subpharyngeal thyroid. In Mozambique tilapia (Oreochromis mossambicus) thyroid activity is, indeed, restricted to the subpharyngeal region; in common carp (Cyprinus carpio) the functional endocrine thyroid is associated with renal tissues. The subpharyngeal follicles of carp comprise only 10% of the total thyroid tissue, and these follicles neither accumulate iodide nor synthesize or secrete thyroid hormones to a significant degree. Although the shape and size of carp subpharyngeal and renal follicles vary, the epithelial cell height of the thyrocytes and thyroxine immunoreactivity do not differ, which suggests that the activity of the carp subpharyngeal thyroid follicles is dormant. Differences in thyroid physiology between the two fish species were further assessed at the level of peripheral thyroid hormone metabolism. Carp clears plasma of thyroid hormones faster than tilapia does. Furthermore, a significant amount of conjugated thyroid hormones was observed in the plasma of tilapia, which was preceded by the occurrence of thyroid hormone conjugates in the subpharyngeal region and coincides with the appearance of conjugates in the surrounding water. Apparently, plasma thyroid hormone conjugates in tilapia originate from the thyroid gland and function in the excretion of thyroid hormones. Our data illustrate the variability in teleostean thyroidology, an important notion for those studying thyroid physiology.

title: The thyroid gland and thyroid hormones in Senegalese sole (Solea senegalensis) during early development and metamorphosis.
abstract: We here describe the ontogeny and morphology of the thyroid gland in Senegalese sole (Solea senegalensis), and correlate these with whole body concentrations of thyroid hormones during early development and metamorphosis. Under our rearing conditions at 19.5 degrees C, most larvae entered metamorphosis in stage 1 at 15 days post-hatching (dph), and completed metamorphosis in stage 4 at 25dph. The onset of metamorphosis coincided with surges in whole body T4 and T3 concentrations. Crossmon's trichrome stain colored the lumen of follicular structures brightly red, and this co-localized with a T4-immunoreactivity. Thyroid follicles were first observed in stage 0 pre-metamorphic larvae at 5dph of age, and were detected exclusively in the subpharyngeal region, surrounding the ventral aorta. Increases in whole body thyroid hormone levels coincided with a 2(1/2)-fold increase in the total thyroidal colloid area in stage 1 larvae (aged 15dph) compared to stage 0 larvae (12dph). This was preceded by an approximately 40%-increase in the follicles' epithelial cell height in stage 0 larvae at 12dph compared to larvae at 5dph, and by an increase in the whole body T3/T4 ratio, indicative of an increase in outer ring deiodination. We conclude that in S. senegalensis there is a clear chronology in the activation of the thyroid gland that starts in early pre-metamorphic larvae.

title: Central and peripheral integration of interrenal and thyroid axes signals in common carp (Cyprinus carpio L.).
abstract: In teleostean fishes the hypothalamic-pituitary-thyroid axis (HPT axis) and the hypothalamic-pituitary-interrenal axis (HPI axis) regulate the release of thyroid hormones (THs) and cortisol respectively. Since many actions of both hormones are involved in the regulation of metabolic processes, communication between both signal pathways can be anticipated. In this study, we describe central and peripheral sites for direct interaction between mediators of both neuroendocrine axes in the common carp (Cyprinus carpio). Despite suggestions in the literature that CRH is thyrotropic in some fish; we were not able to establish stimulatory effects of CRH on the expression of the pituitary TSHbeta subunit gene. In preoptic area tissue incubated with 10(-7) M thyroxine (T(4)) a 2 x 9-fold increase in the expression of CRH-binding protein (CRHBP) was observed. Thus, T(4) could reduce the bioavailable hypothalamic crh via the up regulation of crhbp expression and hence down regulate the HPI axis. At the peripheral level, cortisol (10(-6) M), ACTH (10(-7) M), and alpha-MSH (10(-7) M) stimulate the release of T(4) from kidney and head kidney fragments, which contain all functional thyroid follicles in carp, by two- to fourfold. The substantiation of three pituitary thyrotropic factors, viz. TSH, ACTH, and alpha-MSH, in common carp, allows for an integration of central thyrotropic signals. Clearly, two sites for interaction between the HPT axis, the HPI axis, and alpha-MSH are present in common carp. These interactions may be key to the proper regulation of general metabolism in this fish.

title: Ontogeny of the thyroid hormones and cortisol in the gilthead sea bream, Sparus aurata.
abstract: The aim of the study was to elucidate the pattern of changes in the whole body concentrations of thyroid hormones (TH) and cortisol in eggs, pre-larvae, and larvae in the gilthead sea bream, Sparus aurata. The ontogeny of these hormones was related to specific morphological characteristics that characterize early development. TH and cortisol were detected in substantial amounts in the eggs of sea bream. T3 concentration in eggs was found to be higher than that of T4 (T3, 4.02+/-0.38 ng g-1; T4, 1.63+/-0.28 ng g-1), as it is often observed in marine teleosts. T4 decreased until hatching (T4, 1.01+/-0.58 ng g-1), while T3 reached a minimum at the time of first feeding (T3, 0.19+/-0.02 ng g-1). As development proceeded, both hormones displayed a similar pattern of changes with increasing concentrations until the completion of metamorphosis (T3, 15.84+/-2.92 ng g-1; T4, 9.78+/-2.39 ng g-1), indicating that the hypothalamo-adenohypophysial-thyroid axis begins to function soon after first feeding. The pattern of changes of cortisol (F) concentration was almost parallel to that of T4, starting from 0.83 ng g-1 in eggs. Minimum F concentration was observed at hatching (0.39+/-0.03 ng g-1) and maximum at melanophores and scale formation (14.82+/-2.71 and 14.82+/-3.12 ng g-1, respectively). Results provide data for the first time on the ontogeny of thyroid hormones and cortisol during the early development in sea bream and are in agreement with results in other fish species for an important action of TH during early development.

title: Ontogeny of thyroid hormones, cortisol, hsp70 and hsp90 during silver sea bream larval development.
abstract: We studied the profiles of silver sea bream (Sparus sarba) thyroxine (T(4)), triiodothyronine (T(3)), cortisol and the heat shock protein (hsp) families hsp70 and hsp90 during larval development. Eggs from sexually mature female sea bream were fertilized and larvae were collected at incremental time intervals between 1-46 days post hatch (dph). Both T(4) and T(3) were detected in 1 dph larvae and it was found that both increased as development progressed with a distinct surge in amounts between 21-35 dph, a time associated with direct development of larvae to juveniles. Cortisol increased from 1 dph reaching a maximum and constant level from 35 dph onwards. Using RT-PCR coupled with radioisotope hybridization of immobilized cDNA we assessed the transcript levels of hsp70 and it was found that transcript remained unaltered between 1-14 dph before progressively increasing. Immunoblotting was used to study the larval concentrations of hsp70 and hsp90 and it was found that hsp70 was not significantly changed between 1-14 dph whereas hsp90 increased from 1 dph onwards. These findings suggest an important role for hsp90 in the corticosteroid receptor complex during silver sea bream larval development.

title: Thyroxine surge in metamorphosing flounder larvae.
abstract: The tissue concentration of thyroxine (T4) in larval flounder (Paralichthys olivaceus) was studied at various stages of their metamorphosis using a specific radioimmunoassay developed for samples obtained by quantitative extraction of the hormone from fish eggs and larvae. T4 concentrations were below the limit of detection in extracts of fertilized eggs and larvae until the end of prometamorphosis. Several dorsal fin rays continued growing from premetamorphosis until the end of prometamorphosis. The hormone became detectable at the onset of metamorphic climax when the elongated dorsal fin rays stopped growing. A sharp increase in tissue T4 concentration was observed in midclimax of metamorphosis, and the high level of T4 (11-13 ng/g body weight) was maintained until the end of the climax. The period of elevated T4 levels coincided with resorption of the elongated dorsal fin rays. The right eye completed translocation from the right to the left side of the body during metamorphic climax. The tissue T4 level declined to about a half of the peak value during postclimax, and then increased moderately in later adult-type juveniles. The results of an additional experiment, in which fertilized eggs and newly hatched larvae were treated with T4 and/or thiourea, suggested the presence of negative feedback regulation of pituitary-thyroid axis, implying an involvement of thyroid hormones even in early premetamorphic larvae. These results suggest that metamorphic climax is induced by a surge of thyroid hormone, and that thyroid hormone may also regulate development before and after the metamorphosis.

title: The role of thyroid hormone in tissue development in metamorphosing flounder
abstract: Japanese flounder exhibit extensive morphological and functional changes in various organs during metamorphosis. The larval-type muscle characterized by abundant basophilic sarcoplasm and a small number of myofibrils, changes into the well developed adult-type muscle with abundant myofibrils. Concomitant with these morphological alterations, there are changes in the protein constituents of muscle, including troponin T and myosin light chains. Premetamorphic larvae have large, round erythrocytes with small, round nuclei. These larval erythrocytes are replaced by smaller and elliptical adult-type erythrocytes during metamorphosis. The composition of hemoglobin also changes during this period. Additionally, the gastric glands develop, and pepsinogen is first produced during metamorphosis. Four cDNAs encoding thyroid hormone receptors (THRs) were cloned from a cDNA library constructed from whole bodies of metamorphosing flounder. Deduced amino acid sequences of these four genes show high homology of two with those of α-type THRs and the other two with β-type THRs of higher vertebrates.

title: Thyroxine as a Mediator of Metamorphosis of Atlantic Halibut, Hippoglossus Hippoglossus
abstract: The response of morphological, histological and endocrinological development to exogenous 1-thyroxine (T4) and to water depth during metamorphosis in Atlantic halibut, Hippoglossus hippoglossus, was investigated. Exogenous T4 was given in daily doses of 0.1, 0.05 ppm or a control treatment to halibut larvae at 550 daydegrees (posthatch, premetamorphic) for 14 days. Water depths of 40 cm, 10 cm or 1.5 cm were used to rear halibut larvae from 590 daydegrees for 21 days. Halibut larvae given exogenous T4 at 0.1 ppm had accelerated eye migration relative to MH in fish given 0.05 ppm and in control fish. Pigmentation was correlated with dosage after 14 days. The volume of thyroid tissue was expressed in a dose-dependent manner and exhibited a size-dependency within each treatment. However, the follicles were atypical with reduced colloid, increased lumen and low epithelial cells even in the control group. The results indicate that T4 is a mediator in halibut metamorphosis. In the water depth experiment, only cortisol levels of larvae reared in 1.5 cm water were significantly affected after 21 days, but this was not correlated with metamorphic rate. Hormone profiles, morphological changes and size suggest the existence of a ‘window of opportunity’ for metamorphosis in halibut extending from about 16 mm and tapering off about 21 mm SL. The pooled hormone profiles indicate the commencement of a hormonal cascade similar to that of other flatfishes during metamorphosis. The results indicate that growth, neural and skeletal transformation, and pigmentation are biochemically separate processes in the metamorphosis of Atlantic halibut.

title: The Hypothalamic-Pituitary-Thyroid (HPT) Axis in Fish and Its Role in Fish Development and Reproduction
abstract: Bony fishes represent the largest vertebrate class and are a very diverse animal group. This chapter provides a thorough review of the available scientific literature on the thyroid system in these important vertebrate animals. The molecular components of the hypothalamic-pituitary-thyroid (HPT) axis in this group correspond closely to those of mammals. The thyroid tissue in the fishes is organized as diffuse follicles, with a few exceptions, rather than as an encapsulated gland as is found in most other vertebrate species. The features of this diffuse tissue in fishes are reviewed with an emphasis on feedback relationships within the HPT axis, the molecular biology of the thyroid system in fishes, and comparisons versus the thyroid systems of other vertebrate taxa. A review of the role of thyroid hormone in fish development and reproduction is included. Available information about the HPT axis in fishes is quite detailed for some species and rather limited or absent in others. This review focuses on species that have been intensively studied for their value as laboratory models in assays to investigate disruption in normal function of the thyroid system. In addition, in vitro and in vivo assay methods for screening chemicals for their potential to interfere with the thyroid system are reviewed. It is concluded that there are currently no in vitro or in vivo assays in fish species that are sufficiently developed to warrant recommendation for use to efficiently screen chemicals for thyroid disruption. Methods are available that can be used to measure thyroid hormones, although our ability to interpret the causes and implications of potential alterations in T4 or T3 levels in fishes is nonetheless limited without further research.

title: Thyroid hormones in eggs of various freshwater, marine and diadromous teleosts and their changes during egg development
abstract: Thyroid hormone concentrations in unfertilized eggs of 26 species of various freshwater, marine and diadromous teleosts were examined, together with changes in their concentrations during egg development in some species. Significant quantities of both thyroxine (T4) and triiodothyronine (T3) were found in eggs of all species examined. Mean T4 and T3 concentrations in eggs varied from 0.04 (marbled sole) to 15.00 ng/g (chum salmon), and from 0.07 (goldfish) to 9.95 ng/g (Pacific herring), respectively. T4 concentrations were significantly greater than T3 concentrations in eggs of most freshwater fishes, whereas T3 concentrations were greater in seawater fishes. During the course of development, thyroid hormones in eggs decreased markedly before hatching. These findings suggest that thyroid hormones are consistently present in teleost eggs, and thus may play an important role in the egg development.

title: Metamorphosis of Summer Flounder (Paralichthys dentatus): Thyroid Status and the Timing of Gastric Gland Formation
abstract: We investigated the timing of stomach formation in metamorphosing summer floun- der (Paralichthys dentatus) and the role of thyroid status in gastric gland appearance and pepsin- ogen production. In the first experiment, untreated summer flounder from successive developmental stages were analyzed using histochemistry and immunocytochemistry. During prometamorphosis, the epithelial folds in the region of the incipient stomach increased. Early in metamorphosis, a few gastric glands appeared, but pepsinogen was not identified until mid-metamorphosis. The stomach of late-metamorphic summer flounder had well-developed gastric glands comparable to those in the juvenile. In the second and third experiments, premetamorphic summer flounder were maintained in seawater containing the vehicle DMSO (control) or seawater containing either 100 ppb L-thyroxine sodium salt (T4) or 30 ppm thiourea (TU, an inhibitor of T4 synthesis). Thy- roxine induced metamorphosis within 1 week in the second experiment and within 3 days in the third experiment, whereas TU delayed or inhibited the transition into metamorphosis. The timing of gastric gland differentiation and pepsinogen production was accelerated by 2 weeks in T4-treated larvae and delayed or, in the case of pepsinogen production, inhibited in TU-treated larvae. Thus, T4 stimulates the formation of gastric glands and is necessary for the appearance of pepsinogen in metamorphosing summer flounder. In the third experiment, T4 induced gastric glands within 3 days and in larvae that had not entered metamorphosis, showing that differ- entiation of gastric glands could be dissociated from metamorphosis. This artificially induced heterochrony has potential application in aquaculture.  1998.  Differentiation of the gastrointestinal tract of summer flounder (Paralichthys dentatus) during metamorphosis is of interest for both basic and practical reasons. The digestive system of fishes transitioning between habitats and diets undergoes dynamic remodeling (Dabrowski, '86; Govoni et al., '86). Chemicals signals of the endocrine system co- ordinate much of the transformation both of the organism and of the gastrointestinal tract, and it has been proposed that thyroid hormones in par- ticular prepare the digestive system for shifts in habitat (Specker, '88). This topic has received re- cent review for vertebrates generally (Collie, '95) and for the period of early development in mam- malian neonates (Koldovsky, '94) and metamor- phosing amphibians (Shi and Ishizuya-Oka, '96). Summer flounder, like most marine fishes (Tanaka, '73), are altricial at hatch, and their gas- trointestinal tract is fairly undifferentiated (Bisbal and Bengtson, '95). In the growing practice of cul- turing flatfish, this simple gut has necessitated feeding larval flounder live organisms such as ro- tifers and brine shrimp. Not until metamorphosis— the period of transformation from the larvae to the juvenile (see Youson, '88)—can they be fed cheaper formulated diets and, even during metamorphosis, survival on formulated diets is lower than survival on live food (Bengtson et al., '98). As various flat-

title: Normal table of postembryonic zebrafish development: Staging by externally visible anatomy of the living fish
abstract: The zebrafish is a premier model organism yet lacks a system for assigning postembryonic fish to developmental stages. To provide such a staging series, we describe postembryonic changes in several traits that are visible under brightfield illumination or through vital staining and epiflourescent illumination. These include the swim bladder, median and pelvic fins, pigment pattern, scale formation, larval fin fold, and skeleton. We further identify milestones for placing postembryonic fish into discrete stages. We relate these milestones to changes in size and age and show that size is a better indicator of developmental progress than is age. We also examine how relationships between size and developmental progress vary with temperature and density, and we document the effects of histological processing on size. To facilitate postembryonic staging, we provide images of reference individuals that have attained specific developmental milestones and are of defined sizes. Finally, we provide guidelines for reporting stages that provide information on both discrete and continuous changes in growth and development. Developmental Dynamics 238:2975–3015, 2009.

title: Looking closer at the determining of a phenotype? Compare by stages or size, not age
abstract: During ontogeny, individual fish larvae and juveniles will develop at different paces despite apparently identical age and environment, and even genetic background. The urgent need to elucidate mechanisms critical to juvenile production, recruitment or even lifelong growth patterns implies that measures and results are compared by a meaningful standard such as developmental stage of the individual fish. Characterizations of developmental stages based on relatively invariable characteristics such as the sequence of cranial ossification have proven to be not only commercially and scientifically applicable in Atlantic halibut, but also point to a practical use for a character which is preserved in most fishes examined. Once described, useful proxies for stage determinants may be size parameters such as standard length, or preferably myotome height (particularly for flatfishes). The worst proxy for development of a batch of fish is age, exacerbated by the expense of analyzing irrelevant intervals.

title: Recent insights into organogenesis of the adrenal cortex
abstract: The primary endocrine organs responsible for steroid hormone biosynthesis--the adrenal cortex and gonads--are derived from the urogenital ridge. Several recent discoveries in human and mouse genetics have begun to unravel the complex genetic cascade that dictates adrenocortical cell lineage, proliferation and differentiation. The factors that regulate adrenocortical organogenesis and the maintenance of growth promote or block a cascade of transcription factors that differentially coordinate the proliferation and differentiation of the gland. Here, we outline the developmental milestones of the adrenal cortex with recent contributions to the field, focusing on factors that have been shown to play a role in vivo in humans and mice.

title: The ontogenesis of the steroidogenic tissues
abstract: Based on the common function of steroid hormone‐producing tissues and homologous regulation by the hypothalamo‐pituitary axis, the adrenal cortex and the gonads have been suggested to have an intimate ontogenic relationship. This assumption is also supported by the findings of common transcription factors implicated in the differentiation of both types of tissue and further supported by concomitant defects in such tissues due to the disruption of a single gene. Similarly, simultaneous anomalies in those tissues are also observed in some diseases caused by mutations of the genes encoding those transcription factors. A recent immunohistochemical study with one of the transcription factors, Ad4BP/SF‐1, definitely demonstrated the presence of a particular cell population designated the ‘adreno‐genital primordium (AGP)’ which gives rise to both the adrenal cortex and the gonads. In the process of differentiation from the AGP to the mature adrenal cortex and the gonads of the two sexes, several interesting issues can be raised as to the next targets for the study. To address these issues it is important to elucidate the upstream regulatory mechanisms and downstream target genes of such transcription factors as WT1, SRY, SOX9 and DAX1, in addition to Ad4BP/SF‐1, all of which are implicated in steroidogenic tissue differentiation.

title: The Chromaffin Cell and its Development
abstract: This article summarizes some of the recent progress in understanding the development of chromaffin cells. These cells are derivatives of the neural crest and are intimately associated with the sympathetic nervous system. Although a common sympathoadrenal (SA) progenitor cell for chromaffin cells and sympathetic neurons has been postulated, there is evidence to suggest that chromaffin progenitors are already distinct, at least in part, from neuronal SA progenitors prior to invading the adrenal gland. The concept of an essential role of glucocorticoid signalling for chromaffin cell development has been shaken by the observation that chromaffin cells in mice lacking the glucocorticoid receptor develop largely normal. Distinct developmental requirements of chromaffin cells and sympathetic neurons must also be assumed based on the analyses of mice carrying targeted mutations of the genes for two transcription factors, MASH1 and Phox2B. Both genes are expressed by SA progenitors, but are distinctly required for the development of chromaffin cells and sympathetic neurons. There is an ongoing search for molecules selectively operating at the sites, where chromaffin cells develop. Such molecules may be candidates for triggering the distinct developmental pathway of chromaffin cells, as opposed to sympathetic neurons.

title: DEVELOPMENT OF THE PITUITARY, THYROID AND INTERRENAL GLANDS AND APPLICATIONS OF ENDOCRINOLOGY TO THE IMPROVED REARING OF MARINE FISH LARVAE
abstract: A key to success in the culture of marine fish is the mass production of high quality fry, a process largely dependent on successful first feeding and normal development and growth of fish larvae. In this regard it is important to examine the structural and functional development of the endocrine system during early ontogeny of marine fish. To characterize early endocrine development a comparative study was conducted with various marine fish. The age when the pituitary gland differentiated, assessed by immunohistochemical detection of GH and PRL, varied among species, but generally coincided with the time of eye pigmentation, when yolk absorption is very rapid in most marine fish larvae. Thyroid follicles and pancreatic islets first appeared at about the same time, while interrenal cells appeared later. Tissue concentrations of thyroid hormones decreased during embryonic development to nearly undetectable levels at the completion of yolk absorption. During early larval life thyroid hormone levels increased gradually and were significantly elevated during the transformation to the juvenile stage, especially in flatfish. Tissue concentrations of cortisol showed nearly the same developmental pattern, but increased prior to thyroid hormone levels. GH and PRL exhibited different developmental patterns. GH increased continuously during larval growth while PRL exhibited nearly the same pattern as cortisol in Japanese flounder. Based on these findings it is concluded that the endocrine system becomes functional before the completion of yolk absorption in most marine fish, when the switch from a maternal (yolk) to an endogenous larval hormone source occurs. A preliminary experiment on T 4 supplementation indicated that intact protein ingestion by pinocytosis in the rectal epithelium and fat absorption in the intestinal epithelium were enhanced. Potential benefits of exogenous hormone treatment are discussed particularly in relation to digestive functions.

title: Parallel Early Development of Zebrafish Interrenal Glands and Pronephros: Differential Control by wt1 and ff1b
abstract: Adrenal deficiencies are often associated with problems related to its development, which is not fully understood. To better understand adrenocortical development, we studied zebrafish because of the ease of embryo manipulation. The adrenocortical equivalent in zebrafish is called the interrenal, because it is embedded in the kidney. We find that interrenal development parallels that of the embryonic kidney (pronephros). Primordial interrenal cells first appear as bilateral intermediate mesoderm expressing ff1b in a region ventral to the third somite. These cells then migrate toward the axial midline and fuse together. The pronephric primordia are wt1-expressing cells located next to the interrenal. They also migrate to the axial midline and fuse to become glomeruli at later developmental stages. Our gene knockdown experiments indicate that wt1 is required for its initial restricted expression in pronephric primordia, pronephric cell migration, and fusion. wt1 also appears to be involved in interrenal development and ff1b expression. Similarly, ff1b is required for interrenal differentiation and activation of the differentiated gene, cyp11a1. Our results show that the zebrafish interrenal and pronephros are situated close together and go through parallel developmental processes but are governed by different signaling events.

title: Sex determination and sex differentiation in fish: an overview of genetic, physiological, and environmental influences
abstract: A great deal of information is known regarding the process of sex differentiation in fish, and the mechanisms involved in primary sex determination are now beginning to be defined. A range of gonadal differentiation types have been described for fish, including gonochoristic species possessing purely ovarian or testicular tissues, as well as hermaphroditic species that can initially mature either as males (protandrous) or females (protogynous). Sex determination in fish is a very flexible process with respect to evolutionary patterns observed among genera and families, and within individuals is subject to modification by external factors. These influences can affect the fate of both somatic and germ cells within the primordial gonad, and include the action of genetic, environmental (e.g. temperature), behavioural, and physiological factors. Exogenous sex steroids administered at the time of sex determination can strongly influence the course of sex differentiation in fish, suggesting that they play a critical role in assignment of gonad determination as well as subsequent differentiation. Detailed information is available from fish systems describing the production of sex steroids, as well as the enzymes involved in steroid production. Both estradiol and the maturation hormone 17α, 20β-dihydroxy-4-pregnen-3-one (17α, 20β-DP) are produced by a two-step process involving different cell layers in the gonad, and have effects on the differentiation of gonadal and nongonadal tissues. Gonadal development and differentiation in some fish is also controlled by hormones from the pituitary gland (gonadotropins) that are regulated by release hormones (GnRH) and other neuroendocrine and gonadal factors. Genetic determination of sex in fish can involve monogenic or polygenic systems, with factors located on the autosomes or on sex chromosomes. In the latter case, both male (XY) and female (ZW) heterogametic systems have been described, as well as many subtle variations on these themes. Sex chromosomes are found in approximately 10% of fish species examined, and sex-linked phenotypic traits, and protein and molecular genetic markers have been identified in several fish systems. Some species of fish reproduce gynogenetically, producing all-female populations. Several gene families known to be involved in sex determination in other vertebrates have recently been shown to be similarly involved in fish, suggesting conservation of sex determination pathways. The lability of sex-determination systems in fish makes some species sensitive to environmental pollutants capable of mimicking or disrupting sex hormone actions. Such observations provide important insight into potential impacts from endocrine disruptors, and can provide useful monitoring tools for impacts on aquatic environments.

title: Sex differentiation in the hagfish Eptatretus stouti.
abstract: A detailed study was made of sex differentiation in three anteroposteriorly distributed body regions of the long gonadal fold in young Pacific hagfish (Eptatretus stouti) from 12.4 to 45 cm in length. In general, the posterior region, adjacent to the cloaca, remained undifferentiated, and only in specimens more than 28 cm long was there development of this region into a differentiated testis. On the other hand, the anterior gonadal region of all specimens under 20 cm developed continually and progressively through stages that included cystic nests of cells derived from the germinal epithelium, acquisition of follicular investments around these cells, growth of these young oocytes and break-up of their original follicular groupings, and, finally, in larger specimens, vitellogenesis. Although the earliest oocytes formed in cyst-like nests of cells, later oocytes were derived individually directly from the germinal epithelium. In larger specimens, in which testicular development occurred in the posterior part of the gonad, the anterior gonad involuted, leaving a thin structure that lacks germ cells. There were numerous exceptions to this general description of events, indicating a certain degree of lability of determination of a particular gonadal region to either male or female differentiation. Some earlier stages of female differentiation could occur in the posterior gonadal region of a few smaller animals. Some posteriorly located ovotestes (differentiated elements of both sexes) developed in a few specimens between 16 and 21 cm in length, but were not seen in larger animals. Some testicular follicles appeared in the anterior gonadal regions of three specimens between 20 and 25 cm in length, but not in specimens larger than that. Three hermaphroditic specimens between 29 and 33 cm in length were found in which there was a well-differentiated testis posteriorly and a well-differentiated ovary (vitellogenic eggs) anteriorly. Questions of hermaphroditism, lability of sex determination, and possible epigenetic influences on these phenomena are taken up in the discussion. Since this study provides the first correlation of stages of sex differentiation with animal length, it forms the basis for experimental approaches in E. stouti to analysis of mechanisms of sex determination and differentiation.

title: Guidance of Primordial Germ Cell Migration by the Chemokine SDF-1
abstract: The signals directing primordial germ cell (PGC) migration in vertebrates are largely unknown. We demonstrate that sdf-1 mRNA is expressed in locations where PGCs are found and toward which they migrate in wild-type as well as in mutant embryos in which PGC migration is abnormal. Knocking down SDF-1 or its receptor CXCR4 results in severe defects in PGC migration. Specifically, PGCs that do not receive the SDF-1 signal exhibit lack of directional movement toward their target and arrive at ectopic positions within the embryo. Finally, we show that the PGCs can be attracted toward an ectopic source of the chemokine, strongly suggesting that this molecule provides a key directional cue for the PGCs.

title: Migration of zebrafish primordial germ cells: a role for myosin contraction and cytoplasmic flow.
abstract: The molecular and cellular mechanisms governing cell motility and directed migration in response to the chemokine SDF-1 are largely unknown. Here, we demonstrate that zebrafish primordial germ cells whose migration is guided by SDF-1 generate bleb-like protrusions that are powered by cytoplasmic flow. Protrusions are formed at sites of higher levels of free calcium where activation of myosin contraction occurs. Separation of the acto-myosin cortex from the plasma membrane at these sites is followed by a flow of cytoplasm into the forming bleb. We propose that polarized activation of the receptor CXCR4 leads to a rise in free calcium that in turn activates myosin contraction in the part of the cell responding to higher levels of the ligand SDF-1. The biased formation of new protrusions in a particular region of the cell in response to SDF-1 defines the leading edge and the direction of cell migration.

title: Histological Studies on the Sex Differentiation in Rainbow Trout
abstract: The gonads of rainbow trout ramained in a sexually indifferent condition during the sac-fry period. In the swim-up fry approximately two months after fertilization, about one half of the individuals had gonads showing signs of meiosis. Primary growth of oocytes and lamella formations were subsequently observed in the gonads (ovaries) of about one half of the fingerlings. On the other hand, the gonads of the rest of the fingerlings did not show any signs of meiotic activity, but Sertoli-like cells and lobule and sperm duct formations were observed. Therefore, it may be concluded that rainbow trout belongs to the differentiated type of gonochoristic teleosts.

title: Changes in the number of germ cells in the gonads of the rainbow trout (Salmo gairdneri) during the first 10 post-hatching weeks.
abstract: The number of germ cells in fry gonads was determined from histological sections sampled periodically over a 10-week post-hatching period. Several successive types of germ cells were identified: primordial germ cells (PGC), two kinds of gonocytes (G1 and G2) and all the stages of female meiotic prophase. The mean diameters are shown in table 1. The number of germ cells increased regularly from 47 +/- 35 (SD) after hatching (PGC) to 166 +/- 25 at 2 post-hatching weeks (G1). At 5 post-hatching weeks, two groups of fish could be distinguished by the number of their germ cells (G2) - one group had less than 926 cells and the other more than 1 577 - and by their gonadal morphology (Filiform or with an enlarged anterior part due to germ cell concentration). At 6 post-hatching weeks, the ovary differentiated with organization of the ovarian lamellae and the appearance of oocytes in meiotic prophase (4 out of 10 trout). The gonads of the other fish stayed at the indifferent stage. The number of germ cells was significantly higher in female-type gonads (6 335 +/- 3 558) than in indifferent gonads (1 696 +/- 467). The situation was the same at 8 to 10 weeks.

title: Mass Isolation of Primordial Germ Cells from Transgenic Rainbow Trout Carrying the Green Fluorescent Protein Gene Driven by the vasa Gene Promoter
abstract: Mass isolation of live primordial germ cells (PGCs) was demonstrated for the first time in ectothermal vertebrates. To establish a stem cell-mediated gene transfer system in fish, a stem cell line that retains the ability to develop into gametes is necessary. PGCs are well suited for use as the initial material for such a stem cell line. We established transgenic rainbow trout (Oncorhynchus mykiss) strains carrying the green fluorescent protein (GFP) gene driven by a rainbow trout vasa-like gene (RtVLG) promoter/enhancer. Because GFP expression was specific to the PGCs, PGCs were successfully visualized in all developmental stages examined. Isolated genital ridges containing GFP-labeled PGCs were enzymatically dissociated. To isolate PGCs from the complex pools of dissociated genital ridges, GFP-labeled cells were sorted by flow cytometry. The sorted GFP-positive cells were large and round with a large nucleus, typical characters of PGC morphology. The expression of RtVLG was detected only in the GFP-positive cell population, confirming that these cells were PGCs. This simple and efficient technique to purify a large number of viable PGCs opens the way for establishing a stem cell line, which can differentiate into the germline. The purified PGCs would also be a novel tool for cellular and molecular study of vertebrate germline stem cells.

title: Sexual plasticity of rainbow trout germ cells
abstract: The sexual plasticity of fish gonads declines after the sex-differentiation period; however, the plasticity of the germ cells themselves after this stage remains poorly understood. We characterized the sexual plasticity of gonial germ cells by transplanting them into sexually undifferentiated embryonic gonads in rainbow trout (Oncorhynchus mykiss). Spermatogonia or oogonia isolated from the meiotic gonads of vasa-green fluorescent protein (Gfp) gene transgenic trout were transplanted into the peritoneal cavity of newly hatched embryos of both sexes, and the behavior of the GFPlabeled donor cells was observed. The transplanted spermatogonia and oogonia migrated towards the recipient gonadal anlagen, and were subsequently incorporated into them. We also confirmed that the donor-derived gonial germ cells resumed gametogenesis in the recipient somatic microenvironment synchronously with the endogenous germ cells. Surprisingly, the donor-derived spermatogonia started to proliferate and differentiate into oocytes in female recipients. At 2 years post-transplantation, the eggs from mature female recipients were artificially inseminated with sperm from intact male rainbow trout. Normal, live offspring with the donor-derived haplotype were obtained. In addition, oogonia-derived sperm were produced in the male recipients. These donor-derived sperm were shown to be fully functional, as live offspring carrying GFP-labeled germ cells with the donor haplotype were obtained in the first filial (F1) generation. These findings indicate that rainbow trout pre-meiotic germ cells, which are likely to be spermatogonial or oogonial stem cells, possess a high level of sexual plasticity, and that the sexual differentiation of germ cells is controlled solely by the somatic microenvironment, rather than being cell autonomous.

title: Visualization and motility of primordial germ cells using green fluorescent protein fused to 3'UTR of common carp nanos-related gene
abstract: Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. We previously visualized the PGCs of several teleostean embryos by injecting RNA synthesized from constructs encoding green fluorescent protein (GFP) fused to the 3'UTR of the zebrafish ( Danio rerio ) nanos 1 gene ( nos 1). However, this technique was not always suitable for visualizing PGCs in embryos from all teleost species. In this study, we compared the visualization of PGCs in common carp ( Cyprinus carpio ) embryos using two artificial constructs containing GFP fused to the 3'UTR of nanos from either common carp or zebrafish. Visualization was better using GFP fused to the 3'UTR of the nanos gene from common carp, compared with that from zebrafish. The visualized PGCs successfully migrated toward the gonadal ridge after transplantation into goldfish host embryos, suggesting that they maintained normal migratory motility. These techniques could be useful for the production of inter-specific germline chimeras using common carp donor PGCs.

title: A light- and electron-microscopic study on the migration of primordial germ cells in the teleost, Oryzias latipes
abstract: SummaryThe primordial germ cells (PGCs) of Oryzias latipes in migration to the gonadal anlage have been investigated by light and electron microscopy. The ultrastructure of the PGCs, which occur in the subendodermal space on the syncytial periblast, differ conspicuously from that of the surrounding endodermal cells. After the PGCs move to the cavity between lateral plate and ectoderm, they are taken into the somatomesodermal layer and transferred to the dorsal mesentery where they form gonadal anlage with mesodermal cells. During their translocation to the dorsal mesentery through the somatic mesoderm, apparently without formation of pseudopods, the PGCs are completely surrounded by mesodermal cells. Since these conditions seem unfavorable to the active translocation of the PGCs to the dorsal mesentery, it is more likely that the PGCs are transferred passively by the morphogenic activity of the lateral-plate mesoderm.Counts of the number of the PGCs revealed that they are mitotically dormant during the migratory period. After the completion of the migration, they regain their proliferative activity. The PGCs in the female proliferate more actively than those in the male, which provides the first morphological indication of sex differentiation in this species of fish.

title: Morphological development of the gonads in zebrafish
abstract: Gonadogenesis of zebrafish Danio rerio was investigated by means of light microscopy to test the suitability of gonad histology as an endpoint in hazard assessment of endocrine-active compounds. At age 2 weeks post-fertilization (pf), primordial germ cells were found in a dorsocaudal position in the body cavity. At 4 weeks pf, the majority of the fish (86%) possessed paired gonads with meiotic germ cells; these gonads represented presumptive ovaries. At week 5 pf, 87% of the fish examined had ovaries with perinucleolar oocytes. Further development of the gonads in female zebrafish up to week 11 pf was characterized by an increase in gonad size as well as in the number and size of perinucleolar oocytes. Starting with week 5, some fish showed alterations of gonad morphology, including a decrease in the number and size of the oocytes, an enhanced basophilia and irregular shape of the oocytes, and finally their degeneration into residual bodies. With the decline in oocyte number, stromal cells became more numerous and they infiltrated the gonadal matrix. In several 7 week-old zebrafish with altered gonadal morphology, enhanced numbers of gonial cells arranged in cyst-like groups appeared. These gonads were interpreted as presumptive testes. In one fish out of 32 individuals examined, spermatocytes were detected, in addition to the gonial cells. During the subsequent weeks, the percentage of fish showing early testes with spermatogonia, spermatocytes and spermatids increased and reached 40% at 11 weeks pf. The sequence of gonadal alterations taking place in some of the individuals from week 5 pf onwards was interpreted to reflect the transition of protogynic ovaries into testes. The developmental pattern described identifies zebrafish to be a juvenile hermaphrodite. The results of this study are of relevance for the use of gonadal histopathology as endpoint in endocrine disruption testing, particularly in order to avoid false diagnoses of ‘intersex gonads’ in zebrafish.

title: Gonadal differentiation in coho salmon, Oncorhynchus kisutch, after a single treatment with androgen or estrogen at different stages during ontogenesis
abstract: The labile period for hormonal treatment for purposes of controlling sexual differentiation in coho salmon was deternined by administration of estradiol-17β (E2) and 17α-methyltestosterone (MT) through immersion for 2 h at a concentration of 400 μg/l, to groups of coho salmon sampled weekly from the late eyed-egg stage until the third week of feeding. Histological samples of the gonadal region were prepared from a control group at each weekly treatment interval using a plastic embedding process. It was found that a single estrogen treatment significantly increased the proportion of phenotypic females when administered between 8 days pre-hatch and 13 days post-hatch, while a single androgen treatment significantly increased the proportion of phenotypic males when administered between 6 and 13 days post-hatch. The maximum response for E2-treated groups (84% females) occurred as a result of treatment 1 day before 50% hatch while the maximum response for MT-treated groups (73.1% males) occurred as a result of treatment 1 week later. Thus, the labile period during which the fish were able to respond to a single administration of exogenous steroid was determined to last 3 weeks, and coincided with the time of hatching and the first days of the alevin stage. Sexual differentiation was first observed at 27 days post-hatching, 21–28 days after the time of maximum steroid sensitivity and coinciding approximately with the time of first feeding.

title: Ultrastructural study on the differentiation and development of steroid-producing cells during ovarian differentiation in the amago salmon, Oncorhynchus rhodurus
abstract: In order to examine the relationship between steroid hormones and sex differentiation, the initial appearance and development of the steroid-producing thecal cells (SPCs) and steroid-converting granulosa cells in the ovary were observed ultrastructurally during the process of ovarian differentiation in the amago salmon, Oncorhynchus rhodurus. Initial ovarian differentiation was identified by the transformation of oogonia to meiotic oocytes in the gonads of fish at 20–25 days after hatching. SPCs having the ultrastructural features of SPCs of other fishes were first observed on the periphery of clusters containing meiotic oocytes at the onset of ovarian differentiation. At the same time, granulosa cells were also observed inside the basement lamina of the clusters. Some SPCs and granulosa cells developed at the periphery of auxocytes as the ovary developed further. However, extensive development of organelles in SPCs was not observed as the ovary developed further. These results suggest the possibility of steroid production at the time of ovarian differentiation in the amago salmon.

title: Vertebrates: Comparative Anatomy, Function, Evolution
abstract: 1 Introduction 2 Origin of Chordates 3 The Vertebrate Story 4 Biological Design 5 Life History 6 Integument 7 Skeletal System: The Skull 8 Skeletal System: The Axial Skeleton 9 Skeletal System: The Appendicular Skeleton 10 The Muscular System 11 The Respiratory System 12 The Circulatory System 13 The Digestive System 14 The Urogenital System 15 The Endocrine System 16 The Nervous System 17 Sensory Organs 18

title: Evolution of the bilaterian germ line: lineage origin and modulation of specification mechanisms.
abstract: A key focus of evolutionary developmental biology (evo-devo) in recent years has been to elucidate the evolution of developmental mechanisms as a means of reconstructing the hypothetical last common ancestors of various clades. Prominent among such reconstructions have been proposals as to the nature of the mysterious "Urbilateria," originally defined as the last common ancestor of the extant Bilateria (protostomes and deuterostomes). Indeed, drawings of this animal can now be found, as well as detailed information on the genetics and morphological processes that it used to construct its gut, heart, eyes, appendages, segments, and body regions. Perhaps surprisingly, however, no explanations have yet been offered as to how this animal might have achieved the successful reproduction that must have been necessary for it to give rise to those lineages that are ancestral to today's diverse clades. The present article examines the comparative data available to date on the specification of the only cells containing the genetic hereditary material, the germ cells, and speculates on the possible evolutionary and developmental origin of the Urbilaterian germ line.

title: Evolution of germ cell development in tetrapods: comparison of urodeles and amniotes
abstract: The embryonic development of germ cells in tetrapods is described, focusing on groups with the inductive mode of germ cell specification. In mammals PGCs are induced early in the gastrulation process, they are internalized with future extraembryonic mesoderm in the early posterior primitive streak, and specified soon thereafter. Strong evidence indicates that a similar process occurs in turtles and some other reptiles. In amniotes, the PGCs appear well before formation of the gonad in the posterior trunk, resulting in a period in which they are located outside the embryo before their migration to the gonad. In contrast, in urodeles the PGCs appear relatively late, and throughout development maintain a position close to precursors of the somatic cells of the gonad so that migration is not required. In lampreys early development of germ cells is strikingly similar to that in urodeles, suggesting this is the primitive process. As amniotes evolved large yolky eggs and better access to nutrition, development of the posterior half of the trunk became more dependent on cell proliferation; this was followed or accompanied by a shift of early germ cell development to the equivalent of the early primitive streak. A similar process may have occurred as some basal vertebrates developed large yolky eggs.

title: Growth and the regulation of myotomal muscle mass in teleost fish
abstract: Teleost muscle first arises in early embryonic life and its development is driven by molecules present in the egg yolk and modulated by environmental stimuli including temperature and oxygen. Several populations of myogenic precursor cells reside in the embryonic somite and external cell layer and contribute to muscle fibres in embryo, larval, juvenile and adult stages. Many signalling proteins and transcription factors essential for these events are known. In all cases, myogenesis involves myoblast proliferation, migration, fusion and terminal differentiation. Maturation of the embryonic muscle is associated with motor innervation and the development of a scaffold of connective tissue and complex myotomal architecture needed to generate swimming behaviour. Adult muscle is a heterogeneous tissue composed of several cell types that interact to affect growth patterns. The development of capillary and lymphatic circulations and extramuscular organs – notably the gastrointestinal, endocrine, neuroendocrine and immune systems – serves to increase information exchange between tissues and with the external environment, adding to the complexity of growth regulation. Teleosts often exhibit an indeterminate growth pattern, with body size and muscle mass increasing until mortality or senescence occurs. The dramatic increase in myotomal muscle mass between embryo and adult requires the continuous production of muscle fibres until 40–50% of the maximum body length is reached. Sarcomeric proteins can be mobilised as a source of amino acids for energy metabolism by other tissues and for gonad generation, requiring the dynamic regulation of muscle mass throughout the life cycle. The metabolic and contractile phenotypes of muscle fibres also show significant plasticity with respect to environmental conditions, migration and spawning. Many genes regulating muscle growth are found as multiple copies as a result of paralogue retention following whole-genome duplication events in teleost lineages. The extent to which indeterminate growth, ectothermy and paralogue preservation have resulted in modifications of the genetic pathways regulating muscle growth in teleosts compared to mammals largely remains unknown. This review describes the use of compensatory growth models, transgenesis and tissue culture to explore the mechanisms of muscle growth in teleosts and provides some perspectives on future research directions.

title: Distribution of ionocytes in the saccular epithelium of the inner ear of two teleosts (Oncorhynchus mykiss and Scophthalmus maximus)
abstract: The saccular membranes of trout (Oncorhynchus mykiss) and turbot (Scophthalmus maximus) were examined to characterize specialized epithelial cells that might be responsible for ion exchange. The approach for localizing cell types was new for this tissue, as observations were made with a stereomicroscope and a light microscope in order to have a general view of the epithelium. No important differences between the two species were seen. The saccular tissue is a monolayer epithelium (except for the macula neural zone) surrounded by a layer of connective tissue invaded by many blood vessels. The use of the fluorescent probe DAPSMI and zinc iodide/osmium fixation-coloration defined two areas in which ionocytes were present. In the first, large ionocytes were grouped into a nearly complete, crowned meshwork around, but separated from, the macula. In the second area, opposite the macula, the ionocytes were smaller, cubical, and grouped in patches. Cells rich in Na+, K+-ATPase and carbonic anhydrase II were present in both areas. Contrary to previous studies in mammals and fish, ionocytes were also found in the epithelium of the saccule.

title: Glycoconjugates in the otolithic membrane of herring larvae: a possible framework for encoding the life history recorder in fishes
abstract: Glycoconjugates in the inner ear of herring Clupea harengus larvae, investigated by laser confocal and electron microscopy, were located mainly in the gelatinous layer of the otolithic membranes, forming a collar around the proximal surfaces of the otoliths. The site of secretion was located on the surface of the sensory macula, from which a colonnade of glycoconjugate streamers projected through the subcupular region to connect with the gelatinous layer of each otolith. An electron dense component of the outer gelatinous layers, shown by TEM to be closely associated with the sensory kinocilia, suggested that they provided a basis for the streamers and offered a potential role in directing the path of secretion. It is hypothesized that this highly structured glycoconjugate framework could provide a mechanism for localizing and containing ionic and protein gradients previously detected in this vicinity and which are considered to have a key role in driving the differential growth and mineralization of the otoliths.

title: Otopetrin 1 is required for otolith formation in the zebrafish Danio rerio.
abstract: Orientation with respect to gravity is essential for the survival of complex organisms. The gravity receptor is one of the phylogenetically oldest sensory systems, and special adaptations that enhance sensitivity to gravity are highly conserved. The fish inner ear contains three large extracellular biomineral particles, otoliths, which have evolved to transduce the force of gravity into neuronal signals. Mammalian ears contain thousands of small particles called otoconia that serve a similar function. Loss or displacement of these structures can be lethal for fish and is responsible for benign paroxysmal positional vertigo (BPPV) in humans. The distinct morphologies of otoconial particles and otoliths suggest divergent developmental mechanisms. Mutations in a novel gene Otopetrin 1 (Otop1), encoding multi-transmembrane domain protein, result in nonsyndromic otoconial agenesis and a severe balance disorder in mice. Here we show that the zebrafish, Danio rerio, contains a highly conserved gene, otop1, that is essential for otolith formation. Morpholino-mediated knockdown of zebrafish Otop1 leads to otolith agenesis without affecting the sensory epithelium or other structures within the inner ear. Despite lack of otoliths in early development, otolith formation partially recovers in some fish after 2 days. However, the otoliths are malformed, misplaced, lack an organic matrix, and often consist of inorganic calcite crystals. These studies demonstrate that Otop1 has an essential and conserved role in the timing of formation and the size and shape of the developing otolith.

title: Control of Crystal Size and Lattice Formation by Starmaker in Otolith Biomineralization
abstract: The stone-like otoliths from the ears of teleost fishes are involved in balance and hearing and consist of calcium carbonate crystallites embedded in a protein framework. We report that a previously unknown gene, starmaker, is required in zebrafish for otolith morphogenesis. Reduction of starmaker activity by injection of modified antisense oligonucleotides causes a change in the crystal lattice structure and thus a change in otolith morphology. The expression pattern of starmaker, along with the presence of the protein on the growing otolith, suggest that the expression levels of starmaker control the shape of the otoliths.

title: Molecular structure and composition of fish otoliths
abstract: Recent and fossil otoliths from 25 different fishes have been studied for their amino acid content and for their C13/C12 and O18/O16 distribution in the carbonate fraction. The selection includes specimens from a wide phylogenetic range as well as from various freshwater and marine habitats. All otoliths are composed of aragonite, and their total organic matter ranges from 0.2 to 10%. The organic matter is a protein (MW>150,000), which is characterized by a high abundance of acidic amino acids. In comparison to molluscs that exhibit a wide variety of different mineralized tissues which are species specific, the proteinaceous matter of all otoliths is chemically rather uniform. The high abundance of oxygen-rich amino acids accounts for the ease of mineralization of the organic template. Namely, oxygen supplied by carboxyl grops is used for the coordination of Ca++ ions, resulting in the formation of metal ion coordination polyhedra. Carbonate groups linked via hydrogen bridges to the template will exchange their oxygen with that of the metal polyhedra to stabilize the structure; Ca++O9 polyhedra are the consequence. Subsequent nucleation and crystal growth will lead to aragonite. Oxygen and carbon isotope data indicate that the aragonite is formed close to isotopic equilibrium with the sea. This is surprising, because seawater has no direct access to the inner ear where the otolith orginates. Isotope data may serve a threefold purpose: (1) to determine the mean water temperature where the fish lived, (2) to distinguish between fresh water and marine fish in ancient deposits, and (3) to reveal information on migrtory tendencies of fish.

title: Biochemical Properties of Water-Soluble Otolith Proteins and the Immunobiochemical Detection of the Proteins in Serum and Various Tissues in the Tilapia Oreochromis niloticus
abstract: The water-soluble proteins (WSP) of otoliths were electrophoretically and immunochemically stud ied in the tilapia Oreochromis niloticus. WSP were separated into three or more undefined bands by gradient native PAGE and into 14-16 bands with a main band at 52 kDa by gradient SDS-PAGE. The main band was PAS-positive and had a moderate calcium-binding capacity. Proteins extracted from the sacculus showed an intense calcium-binding capacity in a single band in the low molecular region. Immunoelectrophoresis and immunoblotting revealed that anti-WSP rabbit serum was positive to the WSP, serum, and extracts from various tissues, including the sacculus, semicircular canal, heart, liver, gill lamella, and brain. These immunoreactions, except those of the WSP and the brain, disappeared af ter absorption with tilapia serum. Two-dimensional electrophoresis revealed that at least two serum pro teins at 20 kDa and 21 kDa immunoreacted with the antiserum. The amino acid composition of the 20 kDa protein was dominated by Glx but was not necessarily similar to that reported for tilapia otoliths.

title: Analysis of the soluble matrix of vaterite otoliths of juvenile herring (Clupea harengus): do crystalline otoliths have less protein?
abstract: Otoliths are calcium carbonate concretions laid down in the inner ear of fish and used in fish age estimation. Otoliths precipitate in the form of aragonite but aberrant precipitation may result in vaterite formation instead of aragonite. Vaterite otoliths are more translucent than aragonite. The quantity of HCl-soluble proteins (SP) was measured in the vaterite otoliths and their aragonite pairs of one year old reared herring Clupea harengus to assess the changes induced by the precipitation of vaterite in the amount of soluble proteins in the otolith. Results showed that vaterite otoliths had as much soluble proteins as their aragonite pairs (p>0.05). Due to the lower density of the vaterite, vaterite otoliths were lighter than their aragonite pairs (p<0.05) which explained that protein concentrations were significantly higher (p<0.05) than in aragonite otoliths. These results indicate that the precipitation of vaterite in otoliths did not affect the inclusion of soluble proteins. Furthermore, they suggest that soluble proteins do not affect the translucent or opaque appearance of otoliths. Differences in translucency may instead be caused by the amounts of insoluble proteins or by differences in the physical properties of proteins. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of the otolith proteins revealed two bands at 50 and 62 kDa in both aragonite and vaterite otoliths suggesting that the precipitation of vaterite in the otolith is not controlled by either of these two proteins present in the otolith.

title: Otolith matrix proteins OMP-1 and Otolin-1 are necessary for normal otolith growth and their correct anchoring onto the sensory maculae
abstract: Fish otoliths are highly calcified concretions deposited in the inner ear and serve as a part of the hearing and balance systems. They consist mainly of calcium carbonate and a small amount of organic matrix. The latter component is considered to play important roles in otolith formation. Previously, we identified two major otolith matrix proteins, OMP-1 (otolith matrix protein-1) and Otolin-1, from salmonid species. To assess the function of these proteins in otolith formation, we performed antisense morpholino oligonucleotide (MO)-mediated knockdown of omp-1 and otolin-1 in zebrafish embryos. We first identified zebrafish cDNA homologs of omp-1 (zomp-1) and otolin-1 (zotolin-1). Whole-mount in situ hybridization then revealed that the expression of both zomp-1 and zotolin-1 mRNAs is restricted to the otic vesicles. zomp-1 mRNA was expressed from the 14-somite stage in the otic placode, but the zOMP-1 protein was detected only from 26-somite stage onwards. In contrast, zotolin-1 mRNA expression became clear around 72 hpf. MOs designed to inhibit zomp-1 and zotolin-1 mRNA translation, respectively, were injected into 1-2 cell stage embryos. zomp-1 MO caused a reduction in otolith size and an absence of zOtolin-1 deposition, while zotolin-1 MO caused a fusion of the two otoliths, and an increased instability of otoliths after fixation. We conclude that zOMP-1 is required for normal otolith growth and deposition of zOtolin-1 in the otolith, while zOtolin-1, a collagenous protein, is involved in the correct arrangement of the otoliths onto the sensory epithelium of the inner ear and probably in stabilization of the otolith matrix.

title: Identification of a novel matrix protein contained in a protein aggregate associated with collagen in fish otoliths
abstract: In the biomineralization processes, proteins are thought to control the polymorphism and morphology of the crystals by forming complexes of structural and mineral‐associated proteins. To identify such proteins, we have searched for proteins that may form high‐molecular‐weight (HMW) aggregates in the matrix of fish otoliths that have aragonite and vaterite as their crystal polymorphs. By screening a cDNA library of the trout inner ear using an antiserum raised against whole otolith matrix, a novel protein, named otolith matrix macromolecule‐64 (OMM‐64), was identified. The protein was found to have a molecular mass of 64 kDa, and to contain two tandem repeats and a Glu‐rich region. The structure of the protein and that of its DNA are similar to those of starmaker, a protein involved in the polymorphism control in the zebrafish otoliths [Söllner C, Burghammer M, Busch‐Nentwich E, Berger J, Schwarz H, Riekel C & Nicolson T (2003) Science302, 282–286]. 45Ca overlay analysis revealed that the Glu‐rich region has calcium‐binding activity. Combined analysis by western blotting and deglycosylation suggested that OMM‐64 is present in an HMW aggregate with heparan sulfate chains. Histological observations revealed that OMM‐64 is expressed specifically in otolith matrix‐producing cells and deposited onto the otolith. Moreover, the HMW aggregate binds to the inner ear‐specific short‐chain collagen otolin‐1, and the resulting complex forms ring‐like structures in the otolith matrix. Overall, OMM‐64, by forming a calcium‐binding aggregate that binds to otolin‐1 and forming matrix protein architectures, may be involved in the control of crystal morphology during otolith biomineralization.

title: COST OF GROWTH IN CELLS AND ORGANISMS: GENERAL RULES AND COMPARATIVE ASPECTS
abstract: In a crude fashion it can be said that metabolizable energy (M) is partitioned into metabolic work, paid for by 'oxidations' (R), and 'assimilation', i.e. production (P), so that M = R+P. However, a fraction of R is required to meet the expenses of production and if these expenses represent, Joule for Joule, a constant proportion of the amount produced, then Rt = Rm+cP, where Rt = total metabolic expenditures, Rm = metabolic expenditures for maintaining the non-producing organism, and cP = Rp = metabolic expenditures connected with the processes of production. The partitioning of metabolizable energy into R and P as well as into Rm and Rp may vary depending on the phylogeny and life-history of the species concerned and on ecological circumstances. Thus selection is expected to act on both ratios, R/P and Rm/Rp. By comparing the ratios P/(P+Rp) (the apparent efficiency of production) and Rp/P (the apparent metabolic cost of production) in different types of organisms, one finds that a value of P/(P+Rp) = 0.75, equal to 75% efficiency, 10 mgdbm/mmol ATP, and 16 mumolO2/mg dbm (when I mg identical to 22 J), can be used as a 'consensus value' for the average efficiency, or cost, of the transformation of metabolizable energy into production in a wide range of organisms, from bacteria to mammals. This value corresponds to about three times the theoretical cost of synthesizing the same amount of tissue on the basis of known biochemical principles. The reasons why the empirical costs of production are higher than the theoretical costs of synthesis by what appears to be a common factor may be quite different in bacteria, small ectothermic and large endothermic organisms. Deviations from the consensus value may be due to differences in energy density of the nutrients assimilated and the tissues synthesized. Further complications arise because of interactions between P, Rp, and Rm. In microorganisms the existence of a constant and a variable component of maintenance metabolism has been postulated, the latter decreasing with increasing rate of production. In small ectothermic metazoans, on the other hand, the nonlinear relationship between growth metabolism and growth rate has led to the speculation that above a critical value of Pg certain energy consuming functions of maintenance are suppressed and the energy thus gained used for fuelling growth processes. There is some evidence that, at least in ectothermic metazoans, the apparent cost of growth decreases with the rate of growth, reaching a low plateau of about 10 mumolO2/mgdbm at growth rates exceeding about 8 mgdbm/g/h.(ABSTRACT TRUNCATED AT 400

title: Otolith growth in trout Oncorhynchus mykiss: supply of Ca2+ and Sr2+ to the saccular endolymph.
abstract: Kinetic and pharmacological characteristics of Ca2+ fluxes across the saccular epithelium of trout were studied using a perfused isolated inner ear. 45Ca2+influx from the Ringer solution to the endolymph was 3-4 nmoles h(-1)microl(-1) endolymph, which corresponds to a global turnover rate of the endolymph calcium of 200 % h(-1). Ca2+ entry into the proximal endolymph was faster than into the distal fluid. Net Ca2+ movement across the saccular epithelium depended on the direction and intensity of the chemical gradient of calcium between the Ringer solution and the endolymph. Increasing the calcium concentration in the Ringer solution up to 4.4 mmol l(-1) provoked an accumulation of Ca2+ in both proximal and distal endolymphs, and equilibrium was reached about 30 min after the beginning of perfusion. Perfusion with calcium-free Ringer partially emptied the proximal compartment of calcium, whereas the calcium levels in the distal endolymph did not vary during 70 min of perfusion. Verapamil (10(-5) mol l(-1)) and cyanide (CN, 10(-3) mol l(-1)) did not modify the accumulation of Ca2+ within the endolymph in the presence of a favourable calcium chemical gradient. Furthermore the relationship between Ca2+ net fluxes and the chemical calcium gradient across the saccular epithelium was linear, indicating a passive diffusional mechanism via a paracellular pathway. Similar relationships were found for Sr2+ fluxes across the saccular epithelium in the presence of positive chemical gradients (1, 2 and 4 mmol l(-1) Sr2+). In vivo experiments in which trout were intraperitoneously injected with CaCl2 solution confirmed the tight relationship between the calcium levels in plasma and endolymph (both proximal and distal). Sampling proximal and distal endolymphs in trout and turbot saccules revealed a decreasing proximo-distal calcium gradient in endolymph of both fish species. The present results strongly suggest that the endolymph is supplied with Ca2+ and Sr2+ via a paracellular pathway located in the proximal area of the saccular epithelium.

title: On the role of the central nervous system in regulating the mineralisation of inner-ear otoliths of fish
abstract: Stato- or otoliths are calcified structures in the organ of balance and equilibrium of vertebrates, the inner ear, where they enhance its sensitivity to gravity. The compact otoliths of fish are composed of the calcium carbonate polymorph aragonite and a small fraction of organic molecules. The latter form a protein skeleton which determines the morphology of an otolith as well as its crystal lattice structure. This short review addresses findings according to which the brain obviously plays a prominent role in regulating the mineralisation of fish otoliths and depends on the gravity vector. Overall, otolith mineralisation has thus been identified to be a unique, neuronally guided biomineralisation process. The following is a hypothetical model for regulation of calcification by efferent vestibular neurons: (1) release of calcium at tight junctions in the macular epithelia, (2) macular carbonic anhydrase activity (which in turn is responsible for carbonate deposition), (3) chemical composition of matrix proteins. The rationale and evidence that support this model are discussed.

title: Thyroid hormones are necessary for teleostean otolith growth
abstract: Experiments were conducted to understand the effects of thyroid hormones and thiourea (TU) on otolith growth during metamorphosis of leptocephali. TU is an anti-thyroid hormone drug that inhibits the production of thyroxine (T 4 ) and triiodothyronine (T 3 ) in the thyroid tissue. Fully grown leptocephali of tarpon Megalops cyprinoides collected at the mouth of the Gong-Shy-Tyan Brook, N Taiwan, were immediately treated with 10 ppb T 3 and 300 ppm TU for the experimental period of 13 d. The newly grown otolith lengths measured from the maximum radius were 77.0 ± 12.0, 91.9 ± 7.2 and 47.9 ± 6.3 pm in the control, T 3 and TU groups, respectively. The newly grown otolith length of the T 3 group was significantly longer than those of the control and TU groups, and the control group had significantly larger length values than the TU group. The T 3 treatment stimulated otolith growth, increasing daily increments from 7 to 13 cm (control) to 9 to 16 pm (T 3 ) during the growth peak of metamorphosis. Maximum daily increments were only 6 to 10 pm in TU-treated tarpons. The otoliths of the fish treated with T 3 and TU had increments deposited daily. Otoliths of T 3 -treated and control fish grew throughout the experimental period, but otoliths of TU-treated fish stopped growth beginning on Day 6 to Day 9. These results suggest that thyroid hormones are essential for otolith growth, probably by regulating the expressions and functions of the otolith protein matrix and ion transporters in the saccular epithelium.

title: The deposition of otolith rings in Atlantic salmon, Salmo salar L., embryos
abstract: Salmon embryos, age 48-8 1 days post-fertilization (dpf), were incubated under three different temperature and photoperiod regimes to test the effects of incubation conditions on otolith ring deposition. Otolith rings were first observed at 50 dpf and certain developmental events were marked by distinct, heavy rings. The rate of ring deposition increased with increasing temperature and exposure to light, ranging from 0.50 to 2.33 rings per day. The possible relation to physiological activity as measured by oxygen consumption is discussed.

title: Otolith micro-increment formation in herring Clupea harengus larvae in relation to growth rate
abstract: Estimating the age of individual fish larvae using otolith micro-increments has become a widely used tool in early life-history ecology. A basic assumption of the method is that the micro- increments are formed on a regular (usually daily) basis. Validation experiments are recommended to test this assumption, since otolith microstructure varies between species and interpretation of the structures can be problematic. Whilst many such experiments have demonstrated daily deposition rates, those examining slow-growing larvae have often not supported this assumption. In response, it has been suggested that increments are in fact being formed on a daily basis, but that they are too narrow to be resolved by optical microscopy. In this study, we raised herring larvae over a range of growth rates by manipulating prey levels. At 16 and 37 d old, the otoliths were marked by immersion of the larvae in a solution of alizarin complexone. The rearing experiment was terminated at 51 d post-hatch, and after suitable preparation, otoliths were examined using both light and scanning electron microscopy. Micro-increment counts based on light microscopy were lower than expected (assuming daily deposition) when larval growth rates were less than 0.42 mm d -1 . These results are in accord with previous reports in the literature. Examination of the otolith region between the alizarin marks by scanning electron microscopy (SEM) produced slightly higher increment counts compared with light microscopy (average + 2), but this was not sufficient to support the assumption of daily deposition of micro-increments between Days 16 and 37 in slow-growing larvae. Rather than leading to the regular deposition of narrow increments, slow growth in laboratory-reared herring larvae often appears to affect otolith structure. This results in fewer than the expected number of increments being deposited, and affects counts made both by light and scanning electron microscopy. Growth rates attained in this laboratory study are typical of those that may be experi- enced by cohorts of herring larvae in the wild, particularly from autumn spawning stocks. The use of otolith microstructure to estimate absolute ages of slow-growing herring larvae may therefore lead to significant underestimates of true age, even if SEM is used.

title: Sagittal otolith size and shape variability to identify geographical intraspecific differences in three species of the genus Merluccius
abstract: A study was carried out on the morphology (size and shape) of the saccular otolith (sagitta) by means of image analysis on three species of the genus Merluccius (M. gayi, M. hubbsi and M. merluccius). By digitization of the sagittae, morphometry and outline (Fourier harmonic) as form descriptor measurements were obtained, that were subsequently analysed by means of multivariant methods, allowing the intraspecific variability to be quantified. The differences in the intraspecific sagittal otolith variability confirmed spatial differences of predicted groups. The results were associated with environmental and population aspects. The use of combined morphometric measurements for the whole otolith and its corresponding sulcus acusticus were very appropriate for determining the origin of the analysed otoliths. Using both principal components analysis and discriminant analyses, a clear geographical differentiation was obtained for Merluccius gayi (from Chile and Peru) and M. merluccius (from the Atlantic and Mediterranean). In both species, the discriminant analysis predicted that all the sagittae could be correctly assigned within each of the groups studied. However, it was a little less effective at differentiating between the two groups of M. hubbsi (from the San Matias Gulf on the Patagonian Shelf and Argentinian–Uruguayan Common Fishing Zone). As a result, the morphometric analysis of the sagittae otoliths acted as a clear diagnostic tool to differentiate individuals from different geographical distribution areas.

title: Otolith size trends in marine fish communities from different depth strata
abstract: A comparison of 681 saccular otoliths (sagitta) from 134 species belonging to six demersal communities from different depth strata and the epipelagic community from the north-western Mediterranean Sea was made in order to study otolith relative size and function related morphologies. A relationship between otolith size composition, habitat and depth was found. The epipelagic community was characterized by species with very small and small otolith sizes (68% of the epipelagic species). In the demersal communities, the proportion of species with large sagitta increased with depth until 750 m (reached 50% of the species of the upper slope). The abyssal community (between 1000 and 2000 m), however, was characterized by a decrease in the mean otolith size and an increase in the proportion of species with very small otoliths. With exception of the abyssal community, endogenous causes (a mixture of geneaology, plesiomorph characters shared by the all species of the taxonomic group and recent adaptive ones) may be even more important than exogenous factors in determining the otolith relative size. Within the endogenous causes that condition sagitta size, the adaptive features associated with specialization in acoustic communication are relevant.

title: New insights into gill ionocyte and ion transporter function in euryhaline and diadromous fish
abstract: Teleost fishes are able to acclimatize to seawater by secreting excess NaCl by means of specialized "ionocytes" in the gill epithelium. Antibodies against Na(+)/K(+)-ATPase (NKA) have been used since 1996 as a marker for identifying branchial ionocytes. Immunohistochemistry of NKA by itself and in combination with Na(+)/K(+)/2Cl(-) cotransporter and CFTR Cl(-) channel provided convincing evidence that ionocytes are functional during seawater acclimation, and also revealed morphological variations in ionocytes among teleost species. Recent development of antibodies to freshwater- and seawater-specific isoforms of the NKA alpha-subunit has allowed functional distinction of ion absorptive and secretory ionocytes in Atlantic salmon. Cutaneous ionocytes of tilapia embryos serve as a model for branchial ionocytes, allowing identification of 4 types: two involved in ion uptake, one responsible for salt secretion and one with unknown function. Combining molecular genetics, advanced imaging techniques and immunohistochemistry will rapidly advance our understanding of both the unity and diversity of ionocyte function and regulation in fish osmoregulation.

title: 8 - Osmoregulation in Estuarine and Intertidal Fishes
abstract: Intertidal and estuarine fishes stand out among euryhaline fishes because of their physiological plasticity in response to frequent salinity changes and other environmental challenges, including polar ice and tropical heat. Examples are the northern killifish and tropical Nile tilapia. Estuarine fishes combine low water permeability of skin and gill epithelia with efficient NaCl secretion to live in seawater and hypersaline conditions, but have variable abilities to absorb NaCl from dilute environments, with some species requiring dietary salt intake for survival in freshwater. Changing salinity produces temporary shifts in plasma osmolality, and osmosensing ionocytes respond by respectively increasing or shutting off NaCl secretion if shrunken or swollen osmotically. Aerial stranding and semi-terrestrial living stress respiratory, osmoregulatory, and acid–base systems, requiring large coping capabilities. Some estuarine fishes go through a tolerance phase before launching true acclimating mechanisms, so the response to cycling salinities is slight. This delayed response calls for more investigations through genomics and proteomics.

title: Ontogeny of osmoregulation in postembryonic fish: a review.
abstract: Salinity and its variations are among the key factors that affect survival, metabolism and distribution during the fish development. The successful establishment of a fish species in a given habitat depends on the ability of each developmental stage to cope with salinity through osmoregulation. It is well established that adult teleosts maintain their blood osmolality close to 300 mosM kg(-1) due to ion and water regulation effected at several sites: tegument, gut, branchial chambers, urinary organs. But fewer data are available in developing fish. We propose a review on the ontogeny of osmoregulation based on studies conducted in different species. Most teleost prelarvae are able to osmoregulate at hatch, and their ability increases in later stages. Before the occurrence of gills, the prelarval tegument where a high density of ionocytes (displaying high contents of Na+/K+-ATPase) is located appears temporarily as the main osmoregulatory site. Gills develop gradually during the prelarval stage along with the numerous ionocytes they support. The tegument and gill Na+/K+-ATPase activity varies ontogenetically. During the larval phase, the osmoregulatory function shifts from the skin to the gills, which become the main osmoregulatory site. The drinking rate normalized to body weight tends to decrease throughout development. The kidney and urinary bladder develop progressively during ontogeny and the capacity to produce hypotonic urine at low salinity increases accordingly. The development of the osmoregulatory functions is hormonally controlled. These events are inter-related and are correlated with changes in salinity tolerance, which often increases markedly at the metamorphic transition from larva to juvenile. In summary, the ability of ontogenetical stages of fish to tolerate salinity through osmoregulation relies on integumental ionocytes, then digestive tract development and drinking rate, developing branchial chambers and urinary organs. The physiological changes leading to variations in salinity tolerance are one of the main basis of the ontogenetical migrations or movements between habitats of different salinity regimes.

title: Ontogeny of osmoregulation and salinity tolerance in the gilthead sea bream Sparus aurata.
abstract: The gilthead sea bream, Sparus aurata, is a euryhaline teleost that hatches in the open sea. The larvae drift to the coast and juveniles migrate into estuaries and lagoons where the salinity of the water may vary from brackish to hyper-saline. The ontogeny of osmoregulation in Sparus aurata was studied at successive stages, from day 1 (D1) post-hatch to the late juvenile stage (D300) after exposure to different salinities ranging from fresh water to 45.1 per thousand, at 18 degrees C. Survival ranged from between 5.1 and 39.1 per thousand at D3, and from 1.0 to 45.1 per thousand from D75. The fish were hyper-hypo-osmotic regulators at all studied stages. The acquisition of the full ability to hypo- and hyper-regulate occurred in four steps. The osmoregulatory capacity appeared age-dependent and reached its maximum level after D96, and the localization of ionocytes in the integument and gills occurred concurrently during development of the sea bream. However, the main site of osmoregulation shifted from the integument to the gills from D30 to D70, with a corresponding sharp increase in the osmoregulatory ability. Our results suggest that the early development of osmoregulatory ability, and thus of salinity tolerance in the sea bream may provide an advantageous flexibility for the timing of the migration between sea and estuaries and lagoons.

title: Alternative reproductive styles in seabreams (Pisces: Sparidae)
abstract: review of sexuality in the Sparidae shows that protandrous, protogynous, simultaneous and rudimentary hermaphroditism have all been reported in the family. Careful histological study shows that even in those species reputed to have separate sexes, intersexuality is found in the juvenile condition. We suggest that two reproductive styles, sex change and ‘late’ gonochorism are found in the family. The bi-potentiality of the sparid gonad is considered to be a pre-adaptation for the development of sequential hermaphroditism in species in which reproductive success is size related. In these species sex change is an alternative reproductive style that enables individuals to maximise their lifetime reproductive success by functioning as one sex when small and the other sex when large.

title: Sex-changing fish as a manipulable system for the study of the determination, differentiation, and stability of sex in vertebrates.
abstract: Two fundamental questions concerning vertebrate sexuality are what controls the stability of gender throughout the life of an individual and what are the genetic, immunological, and endocrine factors controlling sex determination and differentiation. Significant aspects of both issues cannot readily be examined experimentally in standard laboratory vertebrates partly because these species are gonochores and provide no opportunity to examine plasticity in sexual systems, and partly because few means have yet been found experimentally to manipulate immunogenetic factors, such as sex-specific DNA or cell-surface antigens, thought to be involved in the determination and differentiation of sex. Behaviorally induced adult sex change in hermaphroditic fishes constitutes a unique system of controlled sexual plasticity in which these issues can be addressed. Several species of tropical marine fishes satisfy the requirements for an appropriate laboratory experimental animal. Sex change in them is known to involve alterations in external coloration, behavior, gonadal structure, hormonal enzyme activity, levels of circulating steroid hormones, and concentrations of H-Y antigen. Genetic, immunological, endocrine, and behavioral factors interact with one another causally in ways permitting relatively simple manipulations simultaneously to analyze the network of sequential causes for sex change and to address the above-stated fundamental issues concerning sexuality generally in vertebrates. Since sex change can be started at will, these fish species become powerful model systems for the analysis of basic mechanisms of sex determination, differentiation, and long-term stability.

title: The Birds and the Bees and the Flowers and the Trees: Lessons from Genetic Mapping of Sex Determination in Plants and Animals
abstract: The ability to identify genetic markers in nonmodel systems has allowed geneticists to construct linkage maps for a diversity of species, and the sex-determining locus is often among the first to be mapped. Sex determination is an important area of study in developmental and evolutionary biology, as well as ecology. Its importance for organisms might suggest that sex determination is highly conserved. However, genetic studies have shown that sex determination mechanisms, and the genes involved, are surprisingly labile. We review studies using genetic mapping and phylogenetic inferences, which can help reveal evolutionary pattern within this lability and potentially identify the changes that have occurred among different sex determination systems. We define some of the terminology, particularly where confusion arises in writing about such a diverse range of organisms, and highlight some major differences between plants and animals, and some important similarities. We stress the importance of studying taxa suitable for testing hypotheses, and the need for phylogenetic studies directed to taxa where the patterns of changes can be most reliably inferred, if the ultimate goal of testing hypotheses regarding the selective forces that have led to changes in such an essential trait is to become feasible.

title: Sexual Dimorphic Expression of Genes in Gonads During Early Differentiation of a Teleost Fish, the Nile Tilapia Oreochromis niloticus1
abstract: The Nile tilapia, a gonochoristic teleost fish with an XX/XY sex-determining system, provides an excellent model for studying gonadal sex differentiation because genetic all-females and all-males are available. In this study, we used quantitative real-time RT-PCR to determine the precise timing of the gonadal expression of 17 genes thought to be associated with gonadal sex differentiation in vertebrates. Gonads were isolated from all-female and all-male tilapia before (5–15 days after hatching [dah]) and after (25–70 dah) morphological sex differentiation. The transcript of aromatase (cyp19a1a), an enzyme responsible for producing estradiol-17beta, was expressed only in XX gonads at 5 dah, with a marked elevation in expression thereafter. In contrast, mRNA expression of steroid 11beta-hydroxylase (cyp11b2), an enzyme responsible for the synthesis of 11-ketotestosterone (11-KT, a potent androgen in fish), was found in XY gonads from 35 dah only. These results, combined with the presence of transcripts for other steroidogenic enzymes and estrogen receptors in XX gonads at 5–7 dah, are consistent with our earlier suggestion that estradiol-17beta plays a critical role in ovarian differentiation in tilapia, whereas a role for 11-KT in testicular differentiation is questionable. A close relationship between the expression of foxl2, but not nr5a1 (Ad4BP/SF-1), and that of cyp19a1a in XX gonads suggests an important role for Foxl2 in the transcriptional regulation of cyp19a1a. Dmrt1 exhibited a male-specific expression in XY gonads from 6 dah onward, suggesting an important role for Dmrt1 in testicular differentiation. Sox9 and amh (anti-Mullerian hormone) showed a testis-specific expression, being evident only in the later stages of testicular differentiation. It is concluded that the sex-specific expression of foxl2 and cyp19a1a in XX gonads and dmrt1 in XY gonads during early gonadal differentiation (5–6 dah) is critical for undifferentiated gonads to differentiate into either the ovary or testis in the Nile tilapia.

title: [Sex determination in the teleost medaka, Oryzias latipes].
abstract: Although the sex of most animals is determined by genetic information, sex-determining genes had been identified only in mammals, several flies, and the worm Caenorhabditis elegans until the recent discovery of DMY (DM-domain gene on the Y chromosome) in the sex-determining region on the Y chromosome of the teleost fish medaka, Oryzias latipes. Functional and expression analyses of DMY have shown it to be the master gene for male sex determination in the medaka. The only sex-determining genes found so far in vertebrates are Sry and DMY. Therefore, the medaka is expected to become a good experimental animal for investigating the precise mechanisms involved in primary sex determination in nonmammalian vertebrates. This article reviews the origin of DMY and the sexual development of gonads in the medaka. The putative functions of DMY are also discussed.

title: Linkage and Physical Mapping of Sex Region on LG23 of Nile Tilapia (Oreochromis niloticus)
abstract: Evidence supports that sex determination (SD) in tilapia is controlled by major genetic factors that may interact with minor genetic as well as environmental factors, thus implying that SD should be analyzed as a quantitative trait. Quantitative trait loci (QTL) for SD in Oreochromis niloticus were previously detected on linkage groups (LG) 1 and 23. Twenty-one short single repeats (SSR) of >12 TGs and one single nucleotide polymorphism were identified using the unpublished tilapia genome sequence on LG23. All markers showed two segregating alleles in a mapping family that was obtained by a cross between O. niloticus male (XY) and sex-reversed female (ΔXY) yielding 29 females (XX) and 61 males (XY and YY). Interval mapping analysis mapped the QTL peak between SSR markers ARO172 and ARO177 with a maximum F value of 78.7 (P < 7.6 × 10−14). Twelve adjacent markers found in this region were homozygous in females and either homozygous for the alternative allele or heterozygous in males. This segment was defined as the sex region (SR). The SR encompasses 1.5 Mbp on a single tilapia scaffold (no. 101) harboring 51 annotated genes. Among 10 candidate genes for SD that were tested for gene expression, anti-Müllerian hormone (Amh), which is located in the center of the SR, showed the highest overexpression in male vs. female embryos at 3 to 7 days postfertilization.

title: Minireview: sex differentiation.
abstract: Mammalian sex differentiation is a hormone-dependent process in the male following the determination of a testis from the indifferent gonad through a cascade of genetic events. Female sex differentiation is not dependent on ovarian hormones, yet there is evidence that members of the Wnt family of developmental signaling molecules play a role in Müllerian duct development and in suppressing Leydig cell differentiation in the ovary. The testis induces male sex differentiation (including testis descent) through a time-dependent production of optimal concentrations of anti-Müllerian hormone, insulin-like factor(s) and androgens. Observations in several human syndromes of disordered fetal sex development corroborate findings in murine embryo studies, although there are exceptions in some gene knockout models. The ubiquitously expressed AR interacts in a ligand-dependent manner with coregulators to control the expression of androgen-responsive genes. Preliminary studies suggest the possibility of hormone resistance syndromes associated with coregulator dysfunction. Polymorphic variants in genes controlling androgen synthesis and action may modulate androgenic effects on sex differentiation.

title: A morphological and experimental study of gonadal sex differentiation in the rainbow trout, Salmo gairdneri
abstract: SummaryGonadal sex differentiation in rainbow trout takes place between day (D) 45 and D 55 after fertilization. Until D 400 the male GSI is maintained at about 0.4‰, and that of the female at about 1‰. Treatment with N,N-dimethylformamide (DMF) influences the sex ratio and GSI, and is therefore unsuitable as a solvent for steroids to be added to the aquarium water for examination of their effects on gonadal sex differentiation. Triton X-100 has no such effect, and is used as the steroid solvent in the present experiments. Progesterone (300 μg/l) administered for 4 weeks from hatching, or from D 43, significantly affects the sex ratio in favour of females. Methyltestosterone given from hatching results in sterilization of the gonads, especially when the treatment is carried out for 8 weeks, or when a relatively high dose (300 μg/l) is used for 4 weeks. When given from D 43 methyl testosterone has a masculinizing effect. It is concluded that progesterone and androgen(s) are important in inducing the onset of ovarian and testis development, respectively.

title: Oocyte apoptosis during the transition from ovary-like tissue to testes during sex differentiation of juvenile zebrafish.
abstract: Large numbers of apoptotic early diplotene oocytes were observed during the transition from ovary-like undifferentiated gonadal tissue to testes during sex differentiation in presumptive males of the zebrafish (Danio rerio). The percentage of terminal-deoxynucleotidyl-transferase-mediated dUTP nick-end labelling (TUNEL)-positive apoptotic oocytes in the gonads of presumptive males was approximately eight- to 12-fold higher than in genetic all-females. By 29 days post-hatching, all oocytes had disappeared from the gonads of presumptive males. In these males, we also observed apoptotic somatic cells in the ovarian cavity between 23 and 35 days post-hatching. Therefore, the disappearance of oocytes and the decomposition of the ovarian cavity caused by apoptosis during sex differentiation were male-specific events. In genetic all-females, apoptosis in a proportion of early diplotene oocytes was found in the undifferentiated gonads at 15-19 days post-hatching, probably as a result of programmed oocyte loss during ovarian development. These findings suggest that oocyte apoptosis is the mechanism of testicular and ovarian differentiation in zebrafish.

title: Genetic Sex Differentiation in Fish
abstract: Sex Determination Hermaphroditism Patterns of Sex Differentiation Impact of New Techniques Differentiation in Gonochores Sex Chromosomes Sex Determining Genes Sex Differentiation Genes Sex Steroid Receptor Genes Gonadal Differentiation Model Fshes Genetic Cascades Gonadal Ducts Puberty and Maturation Acrosomeless Sperm and Micropylar Eggs Secondary Sex Characters Morphotypes and Differentiation Reproductive Senescence Stem Cells and Germ Cells Differentiation in Hermaphrodites Patterns of Hermaphroditism Simultaneous Hermaphrodites Sex Changing Hermaphrodites Structural and Temporal Costs Sex Chromosomes and Genes Sex Differentiation Genes Gonadal Differentiation Morphotypes and Differentiation Strelity and Mutants Gonadectomy Abnormals and Steriles Mutants

title: Environmental sex determination: interaction of temperature and genotype in a fish.
abstract: Sex determination in an atherinid fish, the Atlantic silverside (Menidia menidia), is under the control of both genotype and temperature during a specific period of larval development. The sex ratios of the progeny of different females are variable and differ in their responsiveness to temperature. This demonstrates that sex ratio in fishes that normally have separate sexes can be influenced by the environment.

title: Evidence of temperature-dependent sex determination in the European sea bass (Dicentrarchus labrax L.).
abstract: To test the hypothesis that sex determination in the European sea bass (Dicentrarchus labrax L.) Can be affected by the incubating temperature during the very early developmental stages, eggs from the same batch of spontaneously spawned broodstock were divided at the stage of half-epiboly into three groups according to rearing temperature: G13 = 13 degrees C, G15 = 15 degrees C, and G20 = 20 degrees C. Temperature treatment lasted until the middle of metamorphosis (17-18 mm total length, [TL]), and, with the exclusion of water temperature, all biotic and abiotic conditions were identical for the three experimental groups. The on-growing phase was performed under ambient photoperiod and temperature conditions for all groups. Sex proportions were determined by histological examination of the gonads of fish at 308, 467, and 568 days posthatch (DPH). At 308 DPH (TL: 135-201 mm), 100% of the specimens had differentiated into males and females. A significantly higher (P < 0.01) proportion of females was found in groups G13 (72-74%) and G15 (67-73%) than in group G20 (24-28%). At the final sampling there was no statistically significant difference in body weight between the experimental groups. However, in all groups, female fish were larger than males (P < 0.001). Results provide for the first time clear evidence that temperature during the very early developmental stages is the crucial factor affecting the process of sex differentiation of the sea bass, with low rearing temperatures (13 or 15 degrees C) resulting in sex proportions consistently skewed in favor of females.

title: The influence of temperature on sex determination in sockeye salmon Oncorhynchus nerka
abstract: Thermolability in phenotypic expression of sex was examined in hime salmon, a land-locked type of sockeye salmon Oncorhynchus nerka, which is a commercially valuable species in cold-water lakes of Japan and is expected to have good potential for aquaculture. Eyed-eggs or alevins of genetically all-female fish were subjected to high temperature. Water temperature was raised from 9 to 18 °C (1) in the middle of the eyed-egg stage, 42 days post-fertilization (dpf); (2) just before starting of hatch, 57 dpf; (3) just after completion of hatch, 78 dpf; or (4) 20 days thereafter, 98 dpf, and kept constant at 18 °C until 63 days after emergence of alevins. Fewer survivals were observed when the temperature was raised early in development. The survival rate in the earliest treatment decreased below 5% at 105 dpf. Observation of gonads at 335 to 456 dpf and 733 dpf revealed that 12 of 14 fish (85.7%) exposed to high temperature on and after 57 dpf exhibited a sex change to phenotypic males. Proportions of sex reversal from the genetical female to the phenotypic male decreased contrary to the survival rates as the temperature treatment started later. Sockeye salmon proved to exhibit thermolability in phenotypic expression of sex, and the temperature-sensitive window was demonstrated to exist in the early life stages on and after 57 dpf, which appears to conform with the exogenous androgen-sensitive window. The new technique through temperature control shown in the present study should contribute to producing all-female populations of this species in a safe, simple, environmental friendly, and acceptable manner.

title: Sex determination and temperature-induced sex differentiation in three natural populations of Nile tilapia (Oreochromis niloticus) adapted to extreme temperature conditions
abstract: As a species of major interest for aquaculture, the sex determination system (SDS) of Nile tilapia, Oreochromis niloticus, has been widely investigated. In this species, sex determination is considered to be governed by the interactions between a complex system of genetic sex determination factors (GSD) and the influence of temperature (TSD) during a critical period. Previous studies were exclusively carried out on domestic stocks with the genetic and maintenance limitations associated. Given the wide distribution and adaptation potential of the Nile tilapia, we investigated under controlled conditions the sex determination system of natural populations adapted to three extreme thermal regimes: stable extreme environments in Ethiopia, either cold temperatures in a highland lake (Lake Koka), or warm temperatures in hydrothermal springs (Lake Metahara), and an environment with large seasonal variations in Ghana (Kpandu, Lake Volta). The sex ratio analysis was conducted on progenies reared under constant basal (27 °C) or high (36 °C) temperatures during the 30 days following yolk-sac resorption. Sex ratios of the progenies reared at standard temperature suggest that the three populations share a similar complex GSD system based on a predominant male heterogametic factor with additional influences of polymorphism at this locus and/or action of minor factors. The three populations presented a clear thermosensitivity of sex differentiation, with large variations in the intensity of response depending on the parents. This confirms the presence of genotype-environment interactions in TSD of Nile tilapia. Furthermore the existence of naturally sex-reversed individuals is strongly suggested in two populations (Kpandu and Koka). However, it was not possible here to infer if the sex-inversion resulted from minor genetic factors and/or environmental influences. The present study demonstrated for the first time the conservation of a complex SDS combining polymorphic GSD and TSD components in natural populations of Nile tilapia. We discuss the evolutionary implications of our findings and highlight the importance of field investigations of sex determination.

title: Genetic, endocrine, and environmental components of sex determination and differentiation in the European sea bass (Dicentrarchus labrax L.).
abstract: The European sea bass (Dicentrarchus labrax L.) is a differentiated gonochoristic marine teleost of the family Moronidae (closely related to the hermaphrodites of the family Serranidae), where many juvenile males exhibit intratesticular oocytes, suggesting a certain sexual lability. Like most fish, the sea bass does not have recognizable heterochromosomes or sex-linked markers but there are clear parental effects on the sex ratios. The data available so far indicate that the proportion of females resulting from individual crossings may range from as little as 1 to about 70%. Sex differentiation proceeds in a caudo-cranial fashion and starts when fish reach 8-9 cm standard length (usually about 200 days post-hatching, dph, under typical rearing conditions), with females differentiating first. Both forms of aromatase have been cloned in this species and their temporal expression has been studied. Brain aromatase is detectable already in the larval stages but its involvement in sex differentiation is not yet clear. The ovarian form increases after 100 dph before ovarian differentiation, with high levels in females and basal levels in males. Thus, ovarian aromatase seems to be involved in female differentiation. On the other hand, androgen receptor (AR) gene expression levels show the opposite pattern, with higher levels in males than in females. It is not yet known whether androgens are necessary for testicular differentiation or rather they are the result of it. Of the several environmental factors tested, temperature is the only one that has been shown to be able to clearly influence sex ratios. Larval and juvenile sea bass reared in captivity at high temperature usually develop as males. Recent research suggests that the high incidence of males under aquaculture conditions is due to the high water temperature used, and that the effects of temperature would be mediated by an inhibition of aromatase mRNA expression and activity in genotypic females. However, other effects of temperature mediated through alterations in developmental rates cannot be discarded. This paper reviews the current knowledge on sex determination and differentiation in the sea bass and suggests some directions for future research.

title: [Gonadal ontogenesis and sex differentiation in the sea bass, Dicentrarchus labrax, under fish-farming conditions].
abstract: The histology of the different stages of gonadal development (appearance of PGC, edification of gonad primordium, organization of an undifferentiated gonad, testicular or ovarian development) has been studied in fingerlings and juveniles of sea-bass in fish-culture conditions. Sex differentiation with a caudo-cranial gradient was direct and more in accordance with length than with age. Ovarian and testicular differentiation occurred in fish 11 to 23 months old and from 90 to 187 mm SL. Testis ova were frequently observed.

title: Temperature sex determination in the European sea bass, Dicentrarchus labrax (L., 1758) (Teleostei, Perciformes, Moronidae): critical sensitive ontogenetic phase.
abstract: The temperature sex determination (TSD) mechanism in the European sea bass (Dicentrarchus labrax L.) was studied in respect to: a) the TSD sensitivity during the different developmental stages; and b) the intrapopulation correlation of sex determination with the growth rate up to the end of the TSD-sensitive period. At the stage of half-epiboly, eggs from the same batch were divided into four groups and subjected to different thermal treatments: a) 15 degrees C (G15 group) and b) 20 degrees C (G20 group) up to the middle of metamorphosis stage; c) 15 degrees C up to the end of yolk-sac larval stage and subsequently to 20 degrees C (G15-5 group); and d) 15 degrees C up to the end of the preflexion stage and then to 20 degrees C (G15-10 group). At the end of the treatments, size grading was applied and four additional populations were established from the upper (L) and lower (S) size portions of the G15 and G20 populations: G15L, G15S, G20L, and G20S. During the following growing phase, all populations were subjected to common rearing conditions. The sex ratios of each population were macroscopically determined at 190-210 mm mean total length. Female incidence was significantly affected (P < 0.05) by the different thermal treatments: 66.1% in the G15, 47.1% in the G15-10, 37.6% in the G15-5, and 18.1% in the G20 group. In addition, sex ratio was correlated with the growth rate of the fish up to the end of the TSD-sensitive period, with the larger fish presenting a significantly higher (P < 0.01) female incidence than the smaller fish in both thermal regimes tested: 73.1% in G15L vs. 57% in G15S, and 36.6% in G20L vs. 22.5% in G20S group. Results provide, for the first time, clear evidence that the sea bass is sensitive to TSD during all different ontogenetic stages up to metamorphosis, and that sex ratio is correlated with the growth rate of the fish well before the differentiation and maturation of the gonads.

title: Balancing the effects of rearing at low temperature during early development on sex ratios, growth and maturation in the European sea bass (Dicentrarchus labrax). Limitations and opportunities for the production of highly female-biased stocks
abstract: In many cultured fish species, including the European sea bass, undesirable highly male-biased sex ratios are nevertheless frequent. High temperatures (∼ 21 °C), typically used during the larval and early juvenile stages are thought to cause sex-reversal of genotypic females. Rearing at lower temperatures (

title: Thyroxine enhances larval development and survival in Sarotherodon (Tilapia) mossambicus Ruppell
abstract: Yolk-sac larvae were obtained from the mouth of brooding female tilapia (Sarotherodon mossambicus Ruppell). Treatment of these larvae with l -thyroxine-sodium (Eltroxin, Glaxo) by immersion in 0.1 ppm solution (changed daily) markedly accelerated their development and enhanced their survival. This effect of thyroxine, if confirmed in other species, may find applications in the improvement of larval rearing.

title: Thyroid hormones promote early metamorphosis in grouper (Epinephelus coioides) larvae.
abstract: The response of grouper larvae to the thyroid hormones, thyroxine (T4) and triiodothyronine (T3), was examined. Two-, 3-, and 4-week-old grouper larvae were reared in seawater containing either T4 or T3 at 0.01, 0.1, and 1 ppm. T4 or T3 induced metamorphosis in all age groups in a dose-dependent manner. Regardless of the size of the larvae, metamorphosis was completed in 2 days in larvae treated with 1 ppm of either T4 or T3; 3-4 days in larvae exposed to 0.1 ppm; and 5-6 days in larvae immersed in 0.01 ppm. None of the fish in the control group completed metamorphosis during this period. Compared with the control fish, survival rates were higher in groups exposed to 0.01 ppm and lower in those exposed to 1 ppm of T3. In 4-week-old larvae, T4 treatment (0.01 to 1.0 ppm) resulted in higher survival compared to the control. These results suggest that a dose of 0.01 ppm is appropriate for acceleration of metamorphosis and improvement of survival in 3- and 4-week-old grouper larvae. A lower dose may be apropriate for earlier stages.

title: Thyroid hormone induces metamorphosis of flounder larvae.
abstract: The role of thyroid hormone in flounder metamorphosis was studied by use of thyroxine and the antithyroidal agent, thiourea (TU). T4 stimulated metamorphosis of pelagic larvae, producing miniatures of naturally metamorphosed benthic juveniles. In contrast, TU induced metamorphic stasis, resulting in giant pelagic larvae. These results indicate that the thyroid plays a role in flounder metamorphosis comparable to its well-known developmental role in amphibian metamorphosis.

title: Effects of various doses of thyroxine and triiodothyronine on the metamorphosis of flounder (Paralichthys olivaceus).
abstract: Two experiments were carried out to clarify the effects of thyroxine (T4) and triiodothyronine (T3) on flounder metamorphosis. In the first experiment, three doses of T4 were administered to premetamorphic flounder larvae together with thiourea (TU). Thiourea treatment (30 ppm in ambient seawater) alone caused metamorphic stasis in the fish, although the beginning of the right eye migration was not completely inhibited. T4 at 100 and 10 ppb in ambient seawater induced metamorphosis in the thiourea-treated larvae. The fish of these groups completed translocation of the right eye to the left side and resorption of elongated dorsal fin rays. However, 1 ppb T4 failed to induce metamorphic climax. The elongated dorsal fin rays continued to grow until the end of the experiment in the group treated with TU alone and in combination with 1 ppb T4. In the second experiment, premetamorphic flounder larvae were treated with two doses of T4 and three doses of T3. The T3 treatment was several times more potent than T4 treatment in the induction of metamorphosis. The results of the present study suggest that the metamorphic events of flounder larvae are dependent on the thyroid hormone concentration, and the relative biological potency of T4 and T3 on flounder metamorphosis is similar to that observed in mammals and amphibia.

title: Effects of thyroxine and thiourea on the development of larval red sea bream Pagrus major
abstract: The role Of thyroid hormone on the development of larval and early juvenile red sea bream was studied by administrating thyroxine (T4) and thiourea (TU) as an anti-thyroidal agent. T4 treatment accelerated the appearance of black stripes on the body and the change in the habitat from the surface to the bottom of the aquarium. In contrast, TU treatment caused stasis of black stripes formation and delayaed the change in the habitat. These results indicate that thyroid hormone plays the role of an inducer in the morphological and behavioral transformation from post-larva to juvenile in red sea bream.

title: Thyroid hormone stimulates gastric development in flounder larvae during metamorphosis
abstract: To elucidate the hormonal regulatory mechanisms of gastric development of the flounder, Paralichthys olivaceus, premetamorphic flounder larvae were treated with either 100 ppb of L-thyroxine sodium salt (T4), or 30 ppm of thiourea (TU, a goitrogen) by dissolving drugs in ambient seawater. Control fish were reared in untreated seawater. Measurement of gastric gland volume with an image analyzing system revealed that the T4 treatment markedly increased the volume of the glands. Immunocytochemistry using an antiserum raised against purified quail pepsinogen as the primary antibody showed precocious appearance of pepsinogen-like immunoreactivity in the gastric glands of the larvae treated with T4. In contrast, the gastric glands of the larvae treated with TU remained undifferentiated throughout the experiment. The volume of the glands of TU-treated larvae was very small and immunoreactivity for antiquail pepsinogen was very weak even at the end of the experiment (4 weeks after the start of the experiment) when control fish had well-developed gastric glands. These results strongly suggest that thyroid hormone stimulates differentiation of gastric glands during metamorphosis of the flounder.

title: Combined application of cortisol and triiodothyronine in the culture of larval marine finfish
abstract: Newly-hatched larvae of Pacific threadfin (or ‘moi’; Polydactylus sexfilis) exposed to triiodthyronine (T3; 2.6 p.p.m.) and cortisol (0.1 p.p.m.) by immersion for 1 h exhibited earlier development than untreated controls and larvae exposed to the individual hormones. The onset of initial gut formation and initial somatic pigmentation were accelerated by this treatment. Survival to 29 days of age was also improved (P < 0.05, Experiment 1; P < 0.005, Experiment 2) in hormone-treatment groups as compared with untreated controls. Our ongoing experiments address two working hypotheses: (1) advanced digestive function induced by T3 and cortisol imparts improvements in food utilization during the critical time of first-feeding and (2) uniform growth following hormone treatment leads to reduced rates of cannibalism. 
 
These results appear to be consistent with established development-promoting interactions of T3 and cortisol in Japanese flounder, amphibians, and other vertebrates. These observations suggest that hormone therapy may have practical utility in the culture of marine larvae.

title: Changes in whole body concentrations of thyroid hormones and cortisol in metamorphosing conger eel
abstract: SummaryTo clarify the hormonal regulation of metamorphosis of the conger eel (Conger myriaster), changes in whole body concentrations of thyroid hormones, thyroxine (T4) and triiodothyronine (T3), and cortisol during metamorphosis were examined, as well as the changes in the histological activity of the thyroid gland. In larvae before metamorphosis, T4 and T3 levels were less than 5 and 0.15 ng·g-1 respectively. Levels of T4 increased to about 30 ng·g-1 during early metamorphosis, and decreased subsequently. Levels of T3 increased gradually in early metamorphosis, and then increased abruptly to about 2.0 ng·g-1 in late metamorphosis. Before metamorphosis, cortisol levels of the leptocephali less than 11 cm in total length were greater than 200 ng·g-1. Cortisol levels decreased rapidly in larger premetamorphic leptocephali, and low levels were maintained throughout the metamorphic period. Histological observation revealed an activation of the thyroid gland in early metamorphosis; thyroid follicle epithelial cells became columnar and their nuclei larger. Active uptake of colloid by these cells and intensive vascularization of the gland were also observed. By the end of metamorphosis, follicle epithelial cells became squamous, indicating a low level of glandular activity. These results suggest that thyroid hormone plays an important role in regulation of conger eel metamorphosis.

title: Cortisol enhances the stimulating action of thyroid hormones on dorsal fin-ray resorption of flounder larvae in vitro.
abstract: Effects of thyroid hormones and cortisol on fin-ray resorption of the larval flounder were studied in vitro. Dorsal fin rays were dissected from the prometamorphic larvae of the Japanese flounder (Paralichthys olivaceus) and were cultured for 7-8 days in a medium supplemented with different concentrations of thyroxine (T4) or triiodothyronine (T3) or combinations of either thyroid hormone or cortisol (0.1 micrograms/ml). Both T4 and T3 induced shortening of the second fin ray. T3 was more potent than T4; doses of 0.1 and 1.0 micrograms/ml T4 significantly accelerated shortening of the fin rays, whereas T3 was effective at concentrations ranging from 0.001 to 1.0 micrograms/ml. Cortisol further enhanced the effects of both T4 and T3 but was ineffective in the absence of thyroid hormones. When the prometamorphic larvae were reared in water containing T4 (0.01 micrograms/ml), T4 + cortisol (0.10 micrograms/ml), or cortisol alone for 15 days, both the T4- and T4 + cortisol-treated groups showed an advancement of the morphological changes associated with metamorphosis relative to the control group without hormone treatment. However, no clear synergistic effect of cortisol was observed in the in vivo experiment; a sufficient quantity of cortisol may be produced by the larval interrenal.

title: Hormones and Egg/Larval Quality in Fish
abstract: Recent evidence suggests that hormones are passed on to eggs by broodfish. This store of maternal hormones may fill the regulatory needs of fish larvae for growth, development, osmoregulation, stress response and other physiological functions prior to the functional development of their own endocrine glands. Thus the hormonal levels in eggs/larvae may be an important determinant of egg/larval quality. 
 
 
 
Thyroid hormones have been best studied in this regard. Both thyroxine (T4) and triiodothyronine (T3) are present in fish eggs and in most cases, the levels decrease as development proceeds until the onset of endogenous thyroid hormone production, which usually occurs before or at around yolk-sac resorption. Enhancement of T4/T3 levels in newly-hatched larvae through immersion or maternal injection has been shown to promote larval growth, development and/or survival in several fish species, although overdose causes thyrotoxicosis. 
 
 
 
Other hormones such as corticosteroids, sex steroids, growth hormone and prolactin are also being studied, but to a much lesser extent. 
 
 
 
The paper reviews the evidence and discusses the practical implications of the various findings for larval rearing in aquaculture.

title: Physiological changes in fish from stress in aquaculture with emphasis on the response and effects of corticosteroids
abstract: Stress in fish caused by physical disturbances encountered in aquaculture, such as handling and transport, evokes a variety of responses that may be adaptive or maladaptive. The overall effect of stress may be considered as a change in biological condition beyond the normal resting state that challenges homeostasis and, thus, presents a threat to the fish's health. These stress-induced changes are grouped as primary; secondary, which includes metabolic, hematological, hydromineral, and structural; and tertiary or ‘whole animal’ responses. Many of these responses can be used as quantitative indicators of stress although investigators need to be aware of the various ‘nonstress’ factors that can also influence these conditions. A major focus of current research is on the response of the hypothalamic-pituitary-interrenal axis and the resultant elevation of circulating corticosteroids. Stress, through the action of corticosteroids, may (a) reduce immunocompetence by influencing lymphocyte numbers and antibody-production capacity, and (b) affect reproduction by altering levels and patterns of reproductive hormones that influence maturation. Stress may also alter metabolic scope in fish and affect growth, partly as a result of the catabolic or gluconeogenic effect of corticosteroids. Although certain stressors encountered during normal aquacultural procedures may be unavoidable, a number of practical approaches are suggested that would help to alleviate the detrimental effects of stress in fish.

title: Metabolic Responses Associated with Confinement Stress in Tilapia: The Role of Cortisol
abstract: We examined the effect of 2- or 24-hr confinement stress and cortisol treatment on plasma cortisol, glucose, lactate and free amino acids concentration and hepatic glycogen content and activities of certain enzymes involved in the intermediary metabolism in tilapia (Oreochromis mossambicus). Confinement of tilapia for 2 or 24 hr resulted in significantly higher plasma cortisol, glucose, lactate (2 hr), total and some of the free amino acids concentration (especially at 24 hr) and hepatic pyruvate kinase (PK), phosphoenolpyruvate carboxykinase (PEPCK; 24 hr) and lactate dehydrogenase activities (24 hr). Hepatic glycogen content was lower at 2 and 24 hr in the confined fish compared with the unstressed fish. Tilapia given cortisol implants (50 mg·kg−1 body wt) had significantly higher plasma cortisol, glucose and some of the free amino acids concentration and hepatic PEPCK and aspartate aminotransferase activities, whereas PK activity ratio was significantly lower compared with the sham group. The results suggest that glucose production 2 hr after confinement may be due to glycogenolysis, whereas the maintenance of higher glucose at 24 hr after confinement is essentially due to gluconeogenesis. Furthermore, the changes in plasma metabolites and hepatic enzyme activities with cortisol implantation suggest that cortisol plays a role in the metabolic adjustment to 24-hr confinement stress in tilapia.

title: The stress response in fish
abstract: The stress response in teleost fish shows many similarities to that of the terrestrial vertebrates. These concern the principal messengers of the brain-sympathetic-chromaffin cell axis (equivalent of the brain-sympathetic-adrenal medulla axis) and the brain-pituitary-interrenal axis (equivalent of the brain-pituitary-adrenal axis), as well as their functions, involving stimulation of oxygen uptake and transfer, mobilization of energy substrates, reallocation of energy away from growth and reproduction, and mainly suppressive effects on immune functions. There is also growing evidence for intensive interaction between the neuroendocrine system and the immune system in fish. Conspicuous differences, however, are present, and these are primarily related to the aquatic environment of fishes. For example, stressors increase the permeability of the surface epithelia, including the gills, to water and ions, and thus induce systemic hydromineral disturbances. High circulating catecholamine levels as well as structural damage to the gills and perhaps the skin are prime causal factors. This is associated with increased cellular turnover in these organs. In fish, cortisol combines glucocorticoid and mineralocorticoid actions, with the latter being essential for the restoration of hydromineral homeostasis, in concert with hormones such as prolactin (in freshwater) and growth hormone (in seawater). Toxic stressors are part of the stress literature in fish more so than in mammals. This is mainly related to the fact that fish are exposed to aquatic pollutants via the extensive and delicate respiratory surface of the gills and, in seawater, also via drinking. The high bioavailability of many chemicals in water is an additional factor. Together with the variety of highly sensitive perceptive mechanisms in the integument, this may explain why so many pollutants evoke an integrated stress response in fish in addition to their toxic effects at the cell and tissue levels. Exposure to chemicals may also directly compromise the stress response by interfering with specific neuroendocrine control mechanisms. Because hydromineral disturbance is inherent to stress in fish, external factors such as water pH, mineral composition, and ionic calcium levels have a significant impact on stressor intensity. Although the species studied comprise a small and nonrepresentative sample of the almost 20,000 known teleost species, there are many indications that the stress response is variable and flexible in fish, in line with the great diversity of adaptations that enable these animals to live in a large variety of aquatic habitats.

title: Thyroid hormone regulation of apoptotic tissue remodeling: implications from molecular analysis of amphibian metamorphosis.
abstract: Organogenesis and tissue remodeling are critical processes during postembryonic animal development. Anuran metamorphosis has for nearly a century served as an excellent model to study these processes in vertebrates. Frogs not only have essentially the same organs with the same functions as higher vertebrates such as humans, but also employ similar organogenic processes involving highly conserved genes. Development of frog organs takes place during metamorphosis, which is free of any maternal influences but absolutely dependent on the presence of thyroid hormone. Furthermore, this process can be easily manipulated both in intact tadpoles and in organ cultures by controlling the availability of thyroid hormone. These interesting properties have led to extensive morphological, cellular, and biochemical studies on amphibian metamorphosis. More recently, the cloning of thyroid hormone receptors and the demonstration that they are transcription factors have encouraged enormous interest in the molecular pathways controlling tissue remodeling induced by thyroid hormone during metamorphosis. This article summarizes some of the recent studies on the mechanisms of gene regulation by thyroid hormone receptors and isolation and functional characterization of genes induced by thyroid hormone during Xenopus metamorphosis. Particular focus is placed on the remodeling of the animal intestine, which involves both apoptosis (programmed cell death) of larval cells and de novo development of adult tissues, and the roles of thyroid hormone-induced genes that encode matrix metalloproteinases during this process.

title: Thyroid hormones are important for embryonic to larval transitory phase in zebrafish.
abstract: In zebrafish, like many other teleost species, the development and differentiation of many major organs continue unabated into the yolk-sac larval stage before culminating in a free-swimming larva capable of exogenous feeding. We investigated the role of thyroid hormone (TH) in this important embryonic to larval transitory phase. Thyroid hormone receptor (TR) alpha and beta mRNAs are expressed during the early stages of zebrafish embryonic development. Beginning from the midblastula stage, the level of TR beta mRNAs increases dramatically and is maintained until the end of the transitory phase. Excessive exogenous thyroxine (T4; 30 nM) is toxic and causes severe developmental defects. Cotreatment of embryos with amiodarone, an antagonist of TR, and goitrogen methimazole (MMI) lead to severe retardation in the maturation of the gastrointestinal system, swim bladder, and the lower jaw cartilages and the resorption of the yolk sac. The developmental arrest is lethal, and treated larvae do not survive beyond 7 day postfertilization (dpf), but can be completely rescued by the presence of 10 nM T4. We propose that the embryonic to larval transitory phase in many teleost species is characterized by its dependency on the timely synthesis of TH and the concomitant autoinductive increase in TR beta mRNA levels.

title: Feeding behaviour and digestive physiology in larval fish: current knowledge, and gaps and bottlenecks in research
abstract: Food uptake follows rules defined by feeding behaviour that determines the kind and quantity of food ingested by fish larvae as well as how live prey and food particles are detected, captured and ingested. Feeding success depends on the progressive development of anatomical characteristics and physiological functions and on the availability of suitable food items throughout larval development. The fish larval stages present eco-morpho-physiological features very different from adults and differ from one species to another. The organoleptic properties, dimensions, detectability, movements characteristics and buoyancy of food items are all crucial features that should be considered, but is often ignored, in feeding regimes. Ontogenetic changes in digestive function lead to limitations in the ability to process certain feedstuffs. There is still a lack of knowledge about the digestion and absorption of various nutrients and about the ontogeny of basic physiological mechanisms in fish larvae, including how they are affected by genetic, dietary and environmental factors. The neural and hormonal regulation of the digestive process and of appetite is critical for optimizing digestion. These processes are still poorly described in fish larvae and attempts to develop optimal feeding regimes are often still on a ‘trial and error’ basis. A holistic understanding of feeding ecology and digestive functions is important for designing diets for fish larvae and the adaptation of rearing conditions to meet requirements for the best presentation of prey and microdiets, and their optimal ingestion, digestion and absorption. More research that targets gaps in our knowledge should advance larval rearing.

title: Amphibian intestinal villin: isolation and expression during embryonic and larval development.
abstract: An actin-binding protein of M(r) 105,000 has been isolated from anuran amphibian intestinal mucosa. Polyclonal antibodies directed against chicken and pig intestinal villins and anti-porcine villin headpiece monoclonal antibody crossreact with the amphibian M(r) 105,000 protein. Furthermore, the latter possesses an NH2-terminal sequence that is very homologous to those of avian and mammalian villins. In addition, polyclonal antibodies directed against amphibian intestinal M(r) 105,000 protein crossreact with chicken and mouse intestinal epithelial cell villins. These data indicate that the amphibian intestinal M(r) 105,000 protein is immunologically and structurally related to villin, an actin-binding protein expressed in specific epithelial tissues in vertebrates. Morphological, immunocytochemical and immunoblotting techniques were then used to investigate the expression of villin during embryonic and larval intestinal development of Xenopus laevis. Villin is not found in the egg or the endoderm of the early embryo. It is first detected just before hatching in the apical domain of endodermal cells at a time when few surface microvilli are visible by transmission electron microscopy. In the newly hatched larva, villin accumulates as these cells differentiate. These results provide a detailed developmental profile of Xenopus intestinal villin expression and demonstrate that this protein is a useful marker for the presumptive intestinal endoderm.

title: The earliest changes in gene expression in tadpole intestine induced by thyroid hormone.
abstract: Genes induced by thyroid hormone (TH) to change their expression in the Xenopus laevis gastrointestinal (GI) tract have been isolated using a subtractive hybridization method. An exhaustive search for down-regulated genes identified a single gene. Thirty-two different cDNA fragments derived from the up-regulated mRNA of tadpole intestine 18 h after addition of TH were cloned. They map to no more than 22 distinct genes. The isolation of multiple cDNA fragments derived from a single mRNA indicates that the complexity of up-regulated genes is limited. Both ubiquitous and intestine-specific up-regulated genes were found in this screen. The majority of these genes respond directly to TH induction as judged by the resistance of up-regulation to inhibitors of protein synthesis. The biological significance of these genes is supported by their dramatic regulation in the GI tract during spontaneous metamorphosis.

title: Cell-cell interactions during remodeling of the intestine at metamorphosis in Xenopus laevis.
abstract: Amphibian metamorphosis is accompanied by extensive intestinal remodeling. This process, mediated by thyroid hormone (TH) and its nuclear receptors, affects every cell type. Gut remodeling in Xenopus laevis involves epithelial and mesenchymal proliferation, smooth muscle thickening, neuronal aggregation, formation of intestinal folds, and shortening of its length by 75%. Transgenic tadpoles expressing a dominant negative TH receptor (TRDN) controlled by epithelial-, fibroblast-, and muscle-specific gene promoters were studied. TRDN expression in the epithelium caused abnormal development of virtually all cell types, with froglet guts displaying reduced intestinal folds, thin muscle and mesenchyme, absence of neurons, and reduced cell proliferation. TRDN expression in fibroblasts caused abnormal epithelia and mesenchyme development, and expression in muscle produced fewer enteric neurons and a reduced inter-muscular space. Gut shortening was inhibited only when TRDN was expressed in fibroblasts. Gut remodeling results from both cell-autonomous and cell-cell interactions.

title: Studies on Xenopus laevis intestine reveal biological pathways underlying vertebrate gut adaptation from embryo to adult
abstract: BackgroundTo adapt to its changing dietary environment, the digestive tract is extensively remodeled from the embryo to the adult during vertebrate development. Xenopus laevis metamorphosis is an excellent model system for studying mammalian gastrointestinal development and is used to determine the genes and signaling programs essential for intestinal development and maturation.ResultsThe metamorphosing intestine can be divided into four distinct developmental time points and these were analyzed with X. laevis microarrays. Due to the high level of conservation in developmental signaling programs and homology to mammalian genes, annotations and bioinformatics analysis were based on human orthologs. Clustering of the expression patterns revealed co-expressed genes involved in essential cell processes such as apoptosis and proliferation. The two largest clusters of genes have expression peaks and troughs at the climax of metamorphosis, respectively. Novel conserved gene ontology categories regulated during this period include transcriptional activity, signal transduction, and metabolic processes. Additionally, we identified larval/embryo- and adult-specific genes. Detailed analysis revealed 17 larval specific genes that may represent molecular markers for human colonic cancers, while many adult specific genes are associated with dietary enzymes.ConclusionsThis global developmental expression study provides the first detailed molecular description of intestinal remodeling and maturation during postembryonic development, which should help improve our understanding of intestinal organogenesis and human diseases. This study significantly contributes towards our understanding of the dynamics of molecular regulation during development and tissue renewal, which is important for future basic and clinical research and for medicinal applications.

title: FACS-assisted microarray profiling implicates novel genes and pathways in zebrafish gastrointestinal tract development.
abstract: The zebrafish Danio rerio is an excellent model system for mammalian gastrointestinal development. To identify differentially regulated genes important in gastrointestinal organogenesis, we profiled the transcriptome of the zebrafish developing gastrointestinal tract.



Embryos from a transgenic zebrafish line expressing green fluorescent protein (GFP) in the developing intestine, liver, and pancreas were dissociated at 4 developmental time points, their cells sorted based on GFP expression with fluorescence-activated cell sorting (FACS), and analyzed with microarrays. To improve our analysis, we annotated the Affymetrix Zebrafish GeneChip with human orthologs.



Transcriptional profiling showed significant differences between GFP(+) and GFP(-) cells. Up-regulated genes and pathways were consistent with mammalian gastrointestinal development, such as hepatic nuclear factor gene networks and cancer. We implicate the phosphatidylinositol 3 kinase (PI3K) pathway and show that inhibition with LY294002 causes gastrointestinal defects in zebrafish. We identified novel genes, such as the microRNAs miR-217 and miR-122, the tight junction protein claudin c, the gene fam136a, and a zebrafish tetraspanin. Novel pathways include genes containing a putative transcription factor binding sequence, GGAANCGGAANY, and a nucleolar gene network. The zebrafish microarrays also identify a set of 32 genes that may mediate the effects of gain of chromosome arm 8q in human colon, liver, and pancreatic cancers.



We successfully combine FACS and microarray profiling to follow organogenesis throughout development. These experiments identify novel genes and pathways that probably play a role in mammalian gastrointestinal development and are potential targets for therapeutic intervention in the management of gastrointestinal disease and cancer.

title: Sensitive determination of iodine species, including organo-iodine, for freshwater and seawater samples using high performance liquid chromatography and spectrophotometric detection
abstract: In order to more effectively use iodine isotope ratios, 129 I / 127 I , as hydrological and geochemical tracers in aquatic systems, a new high performance liquid chromatography (HPLC) method was developed for the determination of iodine speciation. The dissolved iodine species that dominate natural water systems are iodide, iodate, and organic iodine. Using this new method, iodide was determined directly by combining anion exchange chromatography and spectrophotometry. Iodate and the total of organic iodine species are determined as iodide, with minimal sample preparation, compared to existing methods. The method has been applied to quantitatively determine iodide, iodate as the difference of total inorganic iodide and iodide after reduction of the sample by NaHSO 3 , and organic iodine as the difference of total iodide (after organic decomposition by dehydrohalogenation and reduction by NaHSO 3 ) and total inorganic iodide. Analytical accuracy was tested: (1) against certified reference material, SRM 1549, powdered milk (NIST); (2) through the method of standard additions; and (3) by comparison to values of environmental waters measured independently by inductively coupled plasma mass spectrometry (ICP-MS). The method has been successfully applied to measure the concentrations of iodide species in rain, surface and ground water, estuarine and seawater samples. The detection limit was ∼1 nM (0.2 ppb), with less than 3% relative standard deviation (R.S.D.) for samples determined by standard additions to an iodide solution of 20 nM in 0.1 M NaCl. This technique is one of the few methods sensitive enough to accurately quantify stable iodine species at nanomolar concentrations in aquatic systems across a range of matrices, and to quantitatively measure organic iodine. Additionally, this method makes use of a very dilute mobile phase, and may be applied to small sample volumes without pre-column concentration or post-column reactions.

title: Uptake of iodide from water in Atlantic halibut larvae (Hippoglossus hippoglossus L.)
abstract: The natural diet of marine fish larvae, copepods, contain 60–350 mg I kg− 1, while live feed used in commercial hatcheries have iodine concentrations in the range of 1 mg kg− 1. Seawater is also considered to be an important source of iodine for marine fish. The question asked in this study is whether Atlantic halibut larvae are capable of absorbing iodide from the water and if so, can the seawater sustain the iodine requirement during larval development and metamorphosis. Levels of iodide and iodate in seawater samples from four different rearing facilities were analysed. All samples contained relative low levels of iodide (0–22 nM) and except for samples from one site; the levels of iodide and iodate were in agreement with previously published data. The uptake of iodide from seawater was measured by incubating Atlantic halibut larvae in water with a constant level of radioactive iodide (125I−) and increasing levels of cold iodide (127I−). To evaluate whether the uptake of iodide would change during metamorphosis, three different developmental stages (pre metamorphic, metamorphic and post metamorphic) were examined. The uptake was similar for all three stages, increasing with increasing concentration of iodide in the water. The highest level of iodide used was 2000 nM, 100 times higher than what was measured in the seawater samples. The uptake curves did not seem to reach equilibrium. This may be due to a constant non-specific uptake or that the equilibrium level is higher than 2000 nM. The uptake was partly blocked by perchlorate (ClO3−) which is a known inhibitor of the sodium iodide symporter. This indicates that the Atlantic halibut larvae accumulate iodide through both specific and non-specific uptake pathways.

title: Correlation between environmental iodide concentrations and larval growth, survival, and whole body concentrations of thyroid hormones and cortisol in Pacific threadfin (Polydactylus sexfilis)
abstract: Pacific threadfin ( Polydactylus sexfilis ), known locally by its Polynesian name “moi”, is rapidly becoming a premier aquaculture species in Hawaii and throughout the Indo-Pacific. Nevertheless, threadfin culture is suffering from extraordinary loss of seed stock during the pre-metamorphic and metamorphic periods, and from dramatic size variation between animals after metamorphosis that leads to cannibalism. The objective of the present study was to examine effects of iodide concentration of rearing tanks on larval growth, survival, metamorphosis, and whole body concentrations of thyroid hormones and cortisol. Threadfin larvae raised in ocean water grew significantly larger, and showed increased survival compared with larvae raised in water from a seawater-injected well that had lower iodide concentrations. Larvae raised in ocean water developed more rapidly than in well seawater. At 14 days post-hatch, 50% of the larvae raised in ocean water reached flexion-stage, compared to 15% in well seawater. In ocean water-raised larvae, whole body thyroxine (T 4 ) concentrations increased sharply from 0.4–0.5 ng/g at hatching through day 13 post-hatch to 1.9 ng/g on day 15, and decreased gradually to 0.5 ng/g by day 23. Larvae raised in well seawater did not show a peak in T 4 , as levels increased gradually from 0.2 ng/g on day 1 to 2.4 ng/g by day 25. Profiles of triiodothyronine (T 3 ) were similar between the two groups, decreasing from 0.17 ng/g at 8 h before hatching to 0.02–0.04 ng/g by day 7 post-hatch, and then gradually increasing to 0.45 ng/g by day 23. Whole body concentration of cortisol was 0.5 ng/g prior to hatching for both groups, increasing to 27–30 ng/g by day 5. Cortisol levels fluctuated during days 7 to 25 post-hatch between 12 and 26 ng/g for ocean water-raised larvae, and between 14 and 32 ng/g for larvae raised in well seawater. Absence of a peak in the T 4 profile in well seawater-raised larvae may indicate incomplete synchronization of development or metamorphosis. These results suggest the importance of environmental iodide in larval metamorphosis, and subsequent survival and growth, possibly through the synthesis of thyroid hormones.

title: EFFECTS OF OZONATION ON THE SPECIATION OF DISSOLVED IODINE IN ARTIFICIAL SEAWATER
abstract: Iodine in the form of iodide is required for synthesis of tri-iodothyronine and thyroxine in fish. Iodine chemical speciation in aliquots of raw artificial seawater mix was measured before, during, and after exposure for fixed time periods to air only and to concentrations of ozone required to achieve oxidation–reduction potentials typical of a protein skimmer (400 mV) and an ozone contact chamber (800 mV). Chemical species of iodine were also measured in tank water from a large, recirculating, ozonated aquarium system that has a low-grade incidence of thyroid lesions (e.g., thyroiditis, hyperplasia, adenoma, and adenocarcinoma) in its fish. With increasing exposure to ozone, concentrations of iodide and dissolved organic iodine (DOI) decreased, whereas iodate levels increased. As a result of exposure to 400 mV, iodide concentration dropped to less than half the amount found in raw artificial seawater mix. After exposure to 800 mV, initial iodide levels decreased by 67%, and DOI became undetectable, whereas iodate concentration increased by 155%, with no remarkable change in total iodine concentration. These results indicate ozone-induced conversions from iodide to iodate, and DOI to iodide or iodate (or both). Iodide and DOI were not detectable in the aquarium system's water samples. Ozonation of artificial seawater may alter the relative concentrations of iodine species in a closed tank system, so that iodide supplementation of the diet or tank water of captive teleosts and elasmobranchs living in ozonated seawater is advisable.

title: Determination of iodide and iodate by ion chromatography with postcolumn reaction and UV/visible detection
abstract: Iodide and iodate can be determined by two new methods using anion-exchange chromatography with postcolumn reaction and UV/visible detection. Iodide is determined as IBr2- at 249 nm. Iodate is determined as I3- at 288 nm. The analyses can be run completely automatically and do not require any sample pretreatment. The detection limits for iodide and iodate are 0.1 μg/L. The methods have been successfully applied to determine iodide and iodate in several mineral waters and in drinking water as well as for the determination of iodide in table salt.

title: Different iodine and thyroid hormone levels between Atlantic halibut larvae fed wild zooplankton or Artemia from first exogenous feeding until post metamorphosis
abstract: The iodine concentration in wild zooplankton was 700 times higher than in Artemia and threefold higher in Atlantic halibut Hippoglossus hippoglossus larvae fed wild zooplankton than in those fed Artemia. In larvae fed wild zooplankton thyroxine (T4) and triiodothyronine (T3) concentrations were significantly higher than in those fed Artemia at the commencement of metamorphosis, 68–77 days post hatching. There was no difference in the amino acids phenylalanine and tyrosine levels in larvae fed Artemia in comparison with those fed wild zooplankton. The selenium level was significantly higher in larvae fed Artemia than in those fed wild zooplankton, but concentrations in the prey were not different. The results indicate sufficient amounts of phenylalanine, tyrosine and selenium in Artemia, but indicated a lower thyroid status due to an insufficient iodine supply at commencement of metamorphosis in larvae fed Artemia. This may partly explain the higher frequency of juveniles with complete eye migration and asymmetric pigmentation in the group fed wild zooplankton.

title: Measurement and regulation of thyroidal status in teleost fish
abstract: SummaryWe have reviewed the stages in teleost thyroid function and its regulation, from the initial biosynthesis of the TH to their eventual interaction with putative receptors.TH biosynthesis depends on an adequate plasma iodide level, determined partly by dietary iodide and partly by active branchial iodide uptake from the water, Pulse-injected radioiodide can be used to evaluate thyroidal iodide uptake, aspects of TH biosynthesis and TH thyroidal secretion. However, owing to variable plasma iodide levels, care is required in interpretating these parameters. TH biosynthesis, thyroglobulin properties and intrathyroidal secretion mechanisms have received limited recent attention. Histological indices of thyroid tissue changes, while useful in many situations, do not always correlate with more direct estimates of thyroidal secretion and can be misleading.Thyroid function is regulated by the hypothalamo-pituitary-thyroid axis, but neither the identities of the hypothalamic factors nor a reliable immunoassay for TSH have been established. Currently, activity of the hypothalamic-pituitary axis is usually determined by pituitary thyrotrope histological appearance or bioassay of pituitary TSH. Plasma free T4 feeds back at both the pituitary and hypothalamic levels and inhibits TSH release. Thyroidal T4 secretory activity is presumably adjusted to maintain a constant plasma T4level according to physiologic state.Plasma T4 is probably the most commonly used index of thyroidal status. However, (1) T4 is probably not the active form of TH, (2) the T4 plasma level may be influenced by the binding properties of plasma proteins, and (3) the T4 concentration alone makes no provision for the rate of T4 turnover in plasma. The most practical way to measure thyroidal T4SR is to determine plasma T4DR, and assuming steady-state conditions, equate it to T4SR. The T4DR is determined from kinetic studies employing*T4, which also enable estimates of sizes of vascular and extravascular T4 pools and their rates of exchange. Excretion of T4 or its derivatives in urine or bile can be determined also. A high proportion of T4 is enzymatically monodeiodinated in liver and other tissues, generating T3 for local (intracellular) and vascular systemic compartments.Bothin vivo andin vitro methods have been used to quantify T4 deiodinase activity, which is highly responsive to physiologic state and environmental variables. T3 production is inhibited by a moderate T3 excess indicating an autoregulatory system, whereby tissue T3 levels are maintained at a set-point appropriate for a particular physiologic state. The rate of T3 production provides an informative measure of thyroidal status in a given tissue. However, other pathways also contribute to the maintenance of T3 homeostasis at a particular set-point. These include the rate of T3 degradation to 3,3′-T2, the rate of T4 substrate diversion to rT3 (an inactive isomer) and by the excretion of parent compounds or conjugates in bile and urine. Potential losses across branchial or integumentary surfaces have yet to be evaluated.The most fundamental measure of thyroidal status is represented by the amount of T3 saturably bound to receptors/nucleus for the cell type of interest. This is estimated most accurately in double isotope studies in which T3 contributions from both vascular and intracellular compartments are evaluated. Less satisfactory but meaningful indices of T3 availability to receptor sites may be obtained from the plasma T3 (or free T3) level and from the tissue T3 level. The former is appropriate if the cell type in question obtains its T3 primarily from plasma; the latter should be measured if the cell type derives its T3 mainly through intracellular deiodinase activity. If the proportion of vascular T3/intracellular T3 bound to receptors is known, it may indicate the degree of receptor activation. However, even cytosolic T3 levels may not vary in proportion to nuclear T3 levels.Differences in thyroidal function between teleosts and homeotherms can be attributed to distinctive strategies in iodide economy and to fundamental differences in control of thyroidal status. Owing to more certain iodide availability (branchial iodide pump and plasma iodide-binding proteins), teleosts are probably more liberal in their iodide use and have less efficient mechanisms for recovery and retention of hormonal iodide than homeotherms. Also, primary control of teleost thyroidal function appears peripheral. It is the finely regulated conversion of T4 to T3 in tissues which may largely determine the T4 secretion rate. Thus, T4, as a prohormone, may be produced more to satisfy the substrate needs for T4 conversion rather than to drive T3 production. Because TH are mainly implicated in tissue- or cell-specific processes involved in development, growth and reproduction in teleosts, it may be advantageous for their thyroidal status to be determined locally through T4-to-T3 deiodination. In homeotherms, primary control is mainly central through the hypothalamic-pituitary axis, which regulates thyroidal secretion of T4 and significant amounts of T3. The level of T4 (free T4) is believed to drive the production of T3 in most peripheral tissues. Because TH are extensively involved in the systemically integrated adjustment of basal metabolic rate in homeotherms, it may have been advantageous to evolve a system leaning towards central control by the hypothalamus, the brain centre associated with thermoregulation.

title: Thyroid hormones in growth and development of fish.
abstract: The thyroid hormones (THs), thyroxine (T(4)) and triiodothyronine (T(3)) are products of the thyroid gland in all vertebrates. Their role in early development and metamorphosis is well established in mammals and amphibians, respectively, and recently several studies in fish have highlighted the importance of THs during flatfish metamorphosis. THs are present in high quantities in fish eggs and are presumably of maternal origin. During embryogenesis the concentration of T(4) and T(3) in the eggs decrease until endogenous production starts. Thyroid hormone receptors (TR) have been isolated from several teleosts and in common with tetrapods two receptor isoforms have been identified, TR alpha and TR beta. Both the receptors are expressed in early embryos and larvae of the Japanese flounder (Paralichthys olivaceus), zebrafish (Danio rerio) and seabream (Sparus aurata) although a different temporal pattern is apparent. The role of THs and TRs in fish embryogenesis, larval development and during metamorphosis will be discussed.

title: Characterization of an iodothyronine 5'-deiodinase in gilthead seabream (Sparus auratus) that is inhibited by dithiothreitol.
abstract: Iodothyronine deiodinases catalyze the conversion of the thyroid prohormone T(4) to T(3) by outer ring deiodination (ORD) of the iodothyronine molecule. The catalytic cycle of deiodinases is considered to be critically dependent on a reducing thiol cosubstrate that regenerates the selenoenzyme to its native state. The endogenous cosubstrate has still not been firmly identified; in studies in vitro the sulfhydryl reagent dithiothreitol (DTT) is commonly used to activate ORD. We now have characterized an ORD activity in the teleost gilthead seabream (Sparus auratus) that is inhibited by DTT. DTT inhibited reverse T(3) (rT(3)) ORD by 70 and 100% in kidney homogenates (IC(50) 0.4 mmol/liter) and microsomes (IC(50) 0.1 mmol/liter), respectively. The omission of DTT from the incubation medium restored renal ORD Michaelis-Menten kinetics with a Michaelis constant value of 5 mumol/liter rT(3) and unmasked the inhibition by 6-n-propyl-2-thiouracil. A putative seabream deiodinase type 1 (saD1), derived from kidney mRNA, showed high homology (> or = 41% amino acid identity) with vertebrate deiodinases type 1. Features of this putative saD1 include a selenocysteine encoded by an in-frame UGA codon, consensus sequences, and a predicted secondary structure for a selenocysteine insertion sequence and an amino acid composition of the catalytic center that is identical with reported consensus sequences for deiodinase type 1. Remarkably, three of six cysteines that are present in the deduced saD1 protein occur in the predicted amino terminal hydrophobic region. We suggest that the effects of DTT on rT(3) ORD can be explained by interactions with the cysteines unique to the putative saD1 protein.

title: Dietary low-glucosinolate rapeseed meal affects thyroid status and nutrient utilization in rainbow trout (Oncorhynchus mykiss).
abstract: Two rapeseed (Brassica napus) meals, RM1 and RM2, with two levels of glucosinolates (GLS; 5 and 41 mumol/g DM respectively) were incorporated at the levels of 300 and 500 g/kg of the diets of juvenile rainbow trout (Oncorhynchus mykiss) in replacement of fish meal, and compared with a fish-meal-based diet. A decrease in the digestibility of the DM, protein, gross energy and P was observed with high-rapeseed meal (RM) incorporation. In trout fed on RM-based diets, growth performance was reduced even after only 3 weeks of feeding. Feed efficiency was adversely affected by RM and GLS intake. Protein and energy retention coefficients were significantly lower in fish fed on the diet containing the higher level of GLS. P retention was significantly lower with all the RM-based diets than with the fish-meal diet. Irrespective of the degree of growth inhibition, fish fed on RM-based diets exhibited similar typical features of hypothyroid condition due to GLS intake, expressed by lower plasma levels of triiodothyronine and especially thyroxine and a hyperactivity of the thyroid follicles. This hypothyroidal condition led to a strong adjustment of the deiodinase activities in the liver, the kidney and the brain. A significant increase of the outer ring deiodinase activities (deiodinases type I and II respectively) and a decrease of the inner ring deiodinase activity (deiodinase type III) were observed. It is concluded that the observed growth depression could be attributed to the concomitant presence of GLS, depressing the thyroid function, and of other antinutritional factors affecting digestibility and the metabolic utilization of dietary nutrients and energy.

title: Hypothyroidism induces type I iodothyronine deiodinase expression in tilapia liver.
abstract: In the current study, the authors examined the effects of experimentally induced hypothyroidism on peripheral thyroid hormone metabolism and growth in two closely related tilapia species: the Nile tilapia (Oreochromis niloticus) and the slower growing black tilapia (Sarotherodon melanotheron). Hypothyroidism, induced by administration of 0.2% methimazole through the food, significantly decreased plasma T(3) and T(4) in both species. This decrease in circulating thyroid hormones was accompanied by an increase in hepatic type II deiodinase (D2) and a decrease in hepatic type III deiodinase (D3). Hepatic type I deiodinase (D1), which is barely expressed in euthyroid tilapia, was significantly upregulated during hypothyroidism. The changes in hepatic D1 and D2 enzyme activity were paralleled by changes in D1 and D2 mRNA levels, indicating pretranslational regulation. Hypothyroidism also resulted in severe growth retardation that was accompanied by an increase in condition factor. Because hyperthyroidism has been shown to decrease the condition factor, these results suggest that thyroid hormones play an essential role in the control of proportional body growth in fish. The authors conclude that (1) hepatic D1 expression is induced by hypothyroidism in tilapia, (2) the changes in hepatic iodothyronine deiodinases during hypothyroidism in tilapia are predominantly regulated at a pretranslational level, and (3) thyroid hormones are involved in the control of proportional body growth in fish.

title: Iodine enrichment of Artemia and enhanced levels of iodine in Atlantic halibut larvae (Hippoglossus hippoglossus L.) fed the enriched Artemia
abstract: Flatfish metamorphosis is initiated by the actions of thyroid hormones (TH) and iodine is an essential part of these hormones. Hence, an iodine deficiency may lead to insufficient levels of TH and incomplete metamorphosis. In this study, different iodine sources for enrichment of Artemia were evaluated and the levels of iodine obtained in Artemia were within the range of 60-350 [mu]g g[superscript [-]1] found in copepods. Larval Atlantic halibut was fed Artemia enriched with either normal DC-DHA Selco or DC-DHA Selco (commercial enrichments) supplemented with iodine from days 9 to 60 postfirst feeding. There was no significant difference in growth, mortality or metamorphic development between the groups. The analyses showed that we were able to enrich Artemia with iodine. Further, the larvae-fed iodine-enriched Artemia had higher whole body iodine concentration compared to larvae-fed Artemia without iodine enrichment.

title: Reduction in occurrence frequency of albinism in juvenile flounder Paralichthys olivaceus hatchery-reared on wild zooplankton.
abstract: The effect of a wild zooplankton supply on the appearance of albinism in juvenile flounder P. olivaceus was studied by two rearing experiments. Flounder larvae were initially reared with only rotifer Brachionus plicatilis and then given wild zooplankton and rotifer or only wild zooplankton, from 5.50-5.57mm TL to complete metamorphosis. These were compared with those given Brazilian Artemia sauna nauplii and rotifer during the same stage. Larvae given wild zooplankton showed poorer growth and lower survival rate than those given Artemia. However the frequency occurrence of albinism in the former was very much lower (0.1-1.8%) than in the latter (97.2-100.0%). When flounder larvae were given wild zooplankton and rotifer, they took 40.0-76.3% rotifer in number during pelagic stage, These results suggested the possibility of preventing albinism through the improvement of cultured foods during early life stage.

title: Lipid and fatty acid composition of normal and malpigmented Atlantic halibut (Hippoglossus hippoglossus) fed enriched Artemia : a comparison with fry fed wild copepods
abstract: Atlantic halibut larvae originating from a single spawn of one female and one male were fed either Super Selco™ enriched, enrichment-grade Artemia nauplii or extensively grown zooplankton. The feeding experiment was conducted in 1.5-m3 outdoor tanks to evaluate if diet affected pigmentation. Pigmentation rates were found to be higher in the zooplankton-fed fish: 99.2% compared to 66.4% in the Artemia-fed halibut. Lipid and fatty acid analyses were performed on five malpigmented Artemia-fed fry, five normally pigmented Artemia-fed fry and 10 copepod-fed fry after 43 days feeding. Lipid class analysis was carried out on the eyes and carcasses of the halibut fry by HPTLC and densitometry. Fatty acid analyses of the same tissues were performed by gas liquid chromatography. Copepod-fed fry showed significantly higher levels of docosahexaenoic acid (22:6n−3; DHA) than their Artemia-fed counterparts (54.9% and 15.8% DHA in eye phosphatidylethanolamine (PE), respectively). Ratios of DHA: eicosapentaenoic acid (20:5n−3; EPA) were also higher in the copepod-fed fish: 11.1, 4.1 and 2.3 in, respectively, PE, phosphatidylcholine (PC) and triglyceride (TAG) in the eye lipid fractions of copepod-fed fry compared to 0.7, 0.3 and 0.1 in the same lipid of eyes of Artemia-fed fry. Within the Artemia-fed population, DHA and EPA levels were slightly, but significantly, raised in the eye PC fractions of normally pigmented fish compared to malpigmented ones. However, there was no significant difference in DHA:EPA ratios between normal and malpigmented fish fed Artemia. EPA: arachidonic acid (ARA) ratios were higher in zooplankton-fed fish but there was no significant difference in these ratios between normal and malpigmented Artemia-fed halibut.

title: Natural copepods are superior to enriched artemia nauplii as feed for halibut larvae (Hippoglossus hippoglossus) in terms of survival, pigmentation and retinal morphology: relation to dietary essential fatty acids.
abstract: Replicate groups of halibut larvae were fed to d 71 post-first feeding (PFF) either the marine copepod, Eurytemora velox, or Artemia nauplii doubly enriched with the marine chromist or golden algae, Schizochytrium sp., (Algamac 2000) and a commercial oil emulsion (SuperSelco). The fatty acid compositions of eyes, brains and livers from larvae fed the two diets were measured, and indices of growth, eye migration and skin pigmentation were recorded along with histological examinations of eye and liver. The docosahexaenoic acid [22:6(n-3); DHA]/eicosapentaenoic acid [20:5(n-3); EPA] ratios in Artemia nauplii enriched with the SuperSelco and Algamac 2000 were 0.4 and 1.0, respectively. The E. velox copepods were divided into two size ranges (125-250 and 250-400 microm) with the smaller size range containing the highest level of (n-3) highly unsaturated fatty acids (HUFA). The DHA/EPA ratios for the two size ranges of copepods were 2.0 and 0.9, respectively. The total lipids of eyes, brains and livers of larvae fed copepods had higher levels of DHA and lower levels of EPA than those of larvae fed enriched Artemia. The percentage of survival of the halibut larvae was significantly higher when copepods rather than enriched Artemia nauplii were fed, but larval specific growth rates did not differ. The indices of eye migration were high and not significantly different in larvae fed the two diets, but the percentage of larvae undergoing successful metamorphosis (complete eye migration and dorsal pigmentation) was higher in larvae fed copepods (40%) than in larvae fed enriched Artemia (4%). The rod/cone ratios in histological sections of the retina were 2.5 +/- 0.7 in larvae fed copepods and 1.3 +/- 0.6 in larvae fed enriched Artemia (P < 0.01). Histological examination of the livers and intestines of the larvae were consistent with better assimilation of lipid from copepods than lipid from Artemia nauplii up to 46 d post-first feeding. Thus, marine copepods are superior to enriched Artemia as food for halibut larvae in terms of survival, eye development and pigmentation, and this superiority can be related to the level of DHA in the feed.

title: First feeding of Atlantic halibut (Hippoglossus hippoglossus) using different combinations of Artemia and wild zooplankton
abstract: Larvae of Atlantic halibut were offered different combinations of Artemia and wild Zooplankton during first feeding to examine the effects on feeding incidence, growth, survival and pigmentation. Free amino acid and fatty acid composition in feeds and fish larvae were also analysed. The free amino acid concentration in unenriched Artemia averaged 45 μmol/g w.w., compared to 116 μmol/ g w.w. in the wild Zooplankton. Super Selco™ (Artemia Systems, Belgium)-enriched Artemia increased the concentration to 57 μmol/g w.w. on average, and also increased the lipid content in Artemia to approximately the levels found in wild Zooplankton (3.0% of wet weight), though with a much lower n − 3 PUFA content. Larvae fed 19 days on enriched Artemia (EA) were significantly larger than larvae fed wild Zooplankton (Z), wild Zooplankton for 7 days then unenriched Artemia (Z7) or unenriched Artemia (UA). The EA group also showed the best survival at day 19, 28.7% (19.9–34.4%) compared to 14.0% (13.2–14.7%) in the Z group and 13.6% (10.5–18.3%) in the Z7 group. Also the UA group had significantly higher survival 22.8% (18.8–25.5%) than the Z group, but showed slower growth. The fatty acid composition of larvae fed Artemia showed a development towards the composition of their feeds, while the fatty acid composition in the larvae fed wild Zooplankton remained relatively similar to that of prefed larvae. After 57 days of feeding, high frequencies of malpigmented juveniles were observed in the UA and EA groups (95.7 and 70.6%, respectively) as well as in the Z7 group (80.8%). All larvae fed wild Zooplankton or larvae transferred from an unenriched Artemia diet to a diet of wild Zooplankton from day 19 onwards, became normally pigmented. The present experiment showed that use of Artemia could be successful in providing growth and survival in the early larval stage of exogenous feeding in Atlantic halibut, but had negative effects on pigmentation later on. However, by introduction of wild Zooplankton prior to a critical stage (found to be beyond 19 days of feeding) these effects could be eliminated.

title: Estimation of larval stage determining the appearance of albinism in Hatchery-reared juvenile flounder Paralichthys olivaceus
abstract: The larval stage of the flounder P. olivaceus, when their body color (normal coloration or albinism of juveniles) was determined, was estimated by two ways of feeding. One was to feed Brazilian Artemia nauplii and rotifers which induce albinism easily in a high percentage. The other was to feed wild zooplankton and rotifers which usually induce normal coloration in a high percentage. Larvae of 10 days old were divided into 8 lots of 1, 000 larvae each and were fed for 29-30 days on different feeding schedules at different growth stages by a combination of the 2 feeding methods. Based upon the percentage of normal coloration and albinism at the end of experiment, 8 experimental lots were divided into 3 groups. In the first group (Tanks 1-3) in which Artemia nauplii were changed to wild zooplankton before the larvae grew up to D stage, the highest percentage of normal coloration was observed (60.2-75.4%). In the third group (Tanks 5-8) fish began to accept wild zooplankton from E stage, the highest percentage of albinism was ob-served (90.9-100.0%). In the second group (Tank 4 only), wild zooplankton were given to larvae in place of Artemia nauplii when the larval developmental stages consisted of 25% D and 75% E, and this composition agreed well with the percentage occurrences of normal coloration (27%) and albinism (73%). From these results, D stage was estimated to be the critical stage deter-mining the juvenile's coloration in terms of kinds of live foods.

title: Effects of docosahexaenoic, eicosapentaenoic, and arachidonic acids on the early growth, survival, lipid composition and pigmentation of yellowtail flounder (Limanda ferruginea): a live food enrichment experiment
abstract: The role of dietary ratios of docosahexaenoic acid (DHA, 22:6n−3), eicosapentaenoic acid (EPA, 20:5n−3) and arachidonic acid (AA, 20:4n−6) on early growth, survival, lipid composition, and pigmentation of yellowtail flounder was studied. Rotifers were enriched with lipid emulsions containing high DHA (43.3% of total fatty acids), DHA+EPA (37.4% and 14.2%, respectively), DHA+AA (36.0% and 8.9%), or a control emulsion containing only olive oil (no DHA, EPA, or AA). Larvae were fed differently enriched rotifers for 4 weeks post-hatch. At week 4, yellowtail larvae fed the high DHA diet were significantly larger (9.7±0.2 mm, P<0.05) and had higher survival (22.1±0.4%), while larvae fed the control diet were significantly smaller (7.3±0.2 mm, P<0.05) and showed lower survival (5.2±1.9%). Larval lipid class and fatty acid profiles differed significantly among treatments with larvae fed high polyunsaturated fatty acid (PUFA) diets having higher relative amounts of triacylglycerols (18–21% of total lipid) than larvae in the control diet (11%). Larval fatty acids reflected dietary levels of DHA, EPA and AA while larvae fed the control diet had reduced amounts of monounsaturated fatty acids (MUFA) and increased levels of PUFA relative to dietary levels. A strong relationship was observed between the DHA/EPA ratio in the diet and larval size (r2=0.75, P=0.005) and survival (r2=0.86, P=0.001). Following metamorphosis, the incidence of malpigmentation was higher in the DHA+AA diet (92%) than in all other treatments (∼50%). Results suggest that yellowtail larvae require a high level of dietary DHA for maximal growth and survival while diets containing elevated AA exert negative effects on larval pigmentation.

title: Quantitative interlake comparison of thyroid pathology in Great Lakes coho (Oncorhynchus kisutch) and chinook (Oncorhynchus tschawytscha) salmon.
abstract: Coho salmon (Oncorhynchus kisutch) and chinook salmon (Oncorhynchus tshawytscha) from Lakes Ontario, Michigan, Erie, or Huron were found to suffer epizootics of thyroid hyperplasia and goiters which appeared to have an environmental etiology. There were 13-fold differences in goiter prevalence within the Great Lakes, and the differences in goiter frequency were correlated with the degree of thyroid hyperplasia. A means of assessing the degree of thyroid hyperplasia (thyroid index) is described, and the derived index was used to facilitate statistical interlake and interspecies comparisons. Despite the hyperplastic (or goitered) condition in all prespawning or spawning Great Lakes salmon, serum thyroid hormone levels were generally higher than in prespawning coho salmon from the Fraser River, British Columbia, indicating that the Great Lakes fish were not necessarily hypothyroid. The hyperplastic lesions appear to undergo progressive changes: (a) large follicles, partly colloid depleted, surrounded by cuboidal epithelial cells; (b) small follicles, largely colloid depleted, surrounded by columnar epithelial cells (in this form, the follicles commonly assume a trabeculate arrangement); (c) "microfollicles" with greatly enlarged columnar epithelial cells encompassing very small follicles; (d) apparently afollicular lesions with little or no colloid in evidence. There was some evidence of benign invasiveness, although the lesions generally resembled simple hyperplastic parenchymatous goiters seen in humans.

title: Iodine‐enriched rotifers and Artemia prevent goitre in Senegalese sole (Solea senegalensis) larvae reared in a recirculation system
abstract: To study the effect of dietary supplementation of iodine in Solea senegalensis, larvae were randomly distributed in six tanks. Larvae in three tanks were given rotifers and Artemia enriched with iodine in addition to Rich Advance or Super Selco from 2 days after hatch (DAH) until 31 DAH. Larvae in a second set of three tanks were fed control rotifers and Artemia, enriched only with Rich Advance or Super Selco. Samples were collected at 2, 5, 10, 15 and 31 DAH to determine dry weight, total length, myotome height and thyroid status. Larvae fed the iodine-enriched diet had significantly higher weight at 31 DAH and higher levels of whole body iodine concentration, compared to control larvae. At 31 DAH, larvae from the control treatment showed typical goitrous thyroid follicles. Thyroid cells of larvae from this treatment appeared columnar or afollicular, with the colloid partly or completely depleted, representative of hyperplasia (goitre). The lower growth rate in fish larvae from the control treatment was possibly a consequence of the hyperplasia, and the iodine enrichment prevented Senegalese sole larvae from developing goitre. This study demonstrates the importance of iodine enrichment of live feed for fish reared in a recirculation system.

title: Thyroid gland development in Senegalese sole (Solea senegalensis Kaup 1858) during early life stages: A histochemical and immunohistochemical approach
abstract: A key to success in the culture of marine fish species is the mass production of high quality fry, a process largely dependent on successful first feeding and normal development and growth of fish larvae. In this regard it is important to examine the structural and functional development of the endocrine system (pituitary, thyroid, interrenal glands) during early ontogeny of marine fish. Particularly, the thyroid hormones, thyroxine (T4) and triiodothyronine (T3), influence numerous metabolic processes, such as growth, differentiation, metamorphosis, reproduction, respiration, migratory behaviour, central nervous system activity, seasonal adaptation, etc. Therefore the aim of this study was to describe the development of the thyroid gland and the ontogeny appearance of the thyroid hormones in Solea senegalensis larvae by means of histological and immunohistochemical techniques. The first thyroid follicle was present at 4 days-post-hatch (dph) coinciding with first feeding. During metamorphosis (12–20 dph) the follicles increased in both number and size, and by 30 dph presented the same characteristics as that seen in adult fish. Tissue immunostaining of both thyroid hormones decreased during the endogenous larvae development to nearly undetectable levels at the completion of yolk-sac absorption. During larvae exogenous phase, T3 and T4 immunostaining was first detected by 6 dph and an increase of specific staining for both hormones was detected between 12 and 20 dph, during metamorphosis phase.

title: Growth and physiological changes during metamorphosis of Senegal sole reared in the laboratory
abstract: The metamorphosis of Solea senegalensis was studied in larvae reared at 20° C and fed four different feeding regimes. A, Artemia (4 nauplii ml 1 ); B, Artemia (2 nauplii ml -1 ); C, mixed diet (2 nauplii ml -1 and 3 mg ml -1 microencapsulated diet); and D, microencapsulated diet (3.7 mg ml -1 ) Rotifers were also supplied in all cases during the first days of feeding. These feeding regimes supported different growth rates during the pre-metamorphosis period (regime A, G=0.376 day -1 ; regime B, G=0.253 day -1 ; regime C, G=0.254 day -1 ; regime D, G=0.162 day -1 ). Larvae started metamorphosis 9 days after hatching (DAH) when fed the regime A, 13 DAH with regime B, 11 DAH with regime C and 15 DAH with regime D. A minimum 5.6-5.9 mm L T was required under all feeding regimes to initiate the metamorphosis. Eye translocation was completed when the larvae reached 8.6-8.7 mm L T (regimes A, B and C), but only 7.3 mm L T with regime D, 4.4-6.2 days were required to complete eye migration under the regimes A, B and C, and 18.3 days under the regime D. This transformation is concomitant with changes in body reserves, and with the pattern of some digestive enzymes.

title: Developmental expression, differential hormonal regulation and evolution of thyroid and glucocorticoid receptor variants in a marine acanthomorph teleost (Sciaenops ocellatus).
abstract: Interactions between the thyroid hormone (TH) and corticosteroid (CS) hormone axes are suggested to regulate developmental processes in vertebrates with a larval phase. To investigate this hypothesis, we isolated three nuclear receptors from a larval acanthomorph teleost, the red drum (Sciaenops ocellatus), and established their orthologies as thraa, thrb-L and gra-L using phylogenomic and functional analyses. Functional characterization of the TH receptors in COS-1 cells revealed that Thraa and Thrb-L exhibit dose-dependent transactivation of a luciferase reporter in response to T3, while SoThraa is constitutively active at a low level in the absence of ligand. To test whether interactions between the TH and CS systems occur during development, we initially quantified the in vivo receptor transcript expression levels, and then examined their response to treatment with triiodothyronine (T3) or cortisol. We find that sothraa and sothrb-L are autoregulated in response to exogenous T3 only during early larval development. T3 did not affect sogra-L expression levels, nor did cortisol alter levels of sothraa or sothrb-L at any stage. While differential expression of the receptors in response to non-canonical ligand hormone was not observed under the conditions in this study, the correlation between sothraa and sogra-L transcript abundance during development suggests a coordinated function of the TH and CS systems. By comparing the findings in the present study to earlier investigations, we suggest that the up-regulation of thraa may be a specific feature of metamorphosis in acanthomorph teleosts.

title: Onset of the primary stress in European sea bass Dicentrarhus labrax, as indicated by whole body cortisol in relation to glucocorticoid receptor during early development
abstract: The temporal patterns of whole body cortisol, the onset of the primary stress response and mRNA abundance of the two glucocorticoid receptors isoforms during early ontogeny was investigated for the first time in the European sea bass, Dicentrarchus labrax . The ontogenetic profile of basal cortisol content was similar to that observed in other pelagic marine species, with minimum values at hatching (0.12 ± 0.01 ng g −1 ), a slight increase at first feeding (2.44 ± 0.71 ng g −1 ) and a first peak at flexion (47.91 ± 6.72 ng g −1 ). However, basal cortisol content at flexion and at the more advanced developmental stages was one of the highest reported for fish. Exposure to a physical stressor (high currents) increased whole body cortisol above control levels at all developmental stages examined (first feeding, pre-larvae, flexion, post flexion, end of metamorphosis and juveniles). However, the magnitude of the cortisol response was low at first feeding and high in juvenile fish. There was a greater number of GR-1 than GR-2 transcripts levels in embryos, pre-larvae and larvae of European sea bass. GR-1 transcripts showed minimum mRNA abundance in embryos and at post flexion and maximum at the end of metamorphosis and in juveniles. The mRNA transcript levels of GR-2 showed a slight increase at hatch to reach a peak in post flexion larvae and in juveniles but these differences were not statistically significant. These results indicate that the onset of cortisol production in European sea bass is near the transition from endogenous to exogenous feeding and show that fish are capable of a stress-induced stimulation of cortisol production even at first feeding.

title: The onset of cortisol synthesis and the stress response is independent of changes in CYP11B or CYP21 mRNA levels in larval red drum (Sciaenops ocellatus).
abstract: Although cortisol plays an important role in teleost development, the onset of cortisol production and the cortisol stress response in teleosts remain poorly understood. Here we have reported basal cortisol levels and the development of the cortisol stress response in larval red drum (Sciaenops ocellatus). We isolated partial nucleic acid sequences encoding two key corticosteroidogenic enzymes, CYP11B and CYP21 and assessed ontogenetic patterns of their mRNA levels relative to basal and stress-induced cortisol production. Basal cortisol was first detected 3 days post-hatch (DPH) and reached a maximum at 9 DPH. Cortisol did not increase in response to an acute stressor prior to 6 DPH. From 6 DPH forward, stress caused significant increases in larval cortisol content. Stress-induced cortisol levels in 6-9 DPH larvae were highest 1h post-stress. In larvae 11 DPH and older, the highest cortisol measurements occurred 0.5h post-stress. Elevated cortisol was still evident after 3h in 6 DPH larvae. From 11 DPH onward, basal cortisol levels were reestablished in larvae by 1h post-stress. CYP11B and CYP21 transcripts were detected in red drum 12h prior to hatching and in all post-hatch larvae examined. Changes in CYP11B and CYP21 mRNA levels did not occur in association with the ontogenetic appearance of cortisol, or the onset of the stress response. As larvae developed, the dynamics of the cortisol stress response matured from a low magnitude, slow recovery response, to a response similar to that observed in juvenile and adult fish.

title: Ontogeny of the cortisol stress response in yellow perch (Perca flavescens)
abstract: The ontogeny of the cortisol stress response in yellow perch (Perca flavescens) was documented by measuring resting and stress-induced whole-body cortisol levels in fish at several early development stages. In newly fertilized eggs resting cortisol levels averaged 41.0 ± 3.0 pg/embryo, and decreased to 8.0 ± 3.0 pg/embryo by day 6 where they remained constant until hatch on day 10. Acute stress challenge tests (30 sec cold shock) conducted on embryonic (days 6 and 8 post-fertilization) and larval (hatch and 2 days post-hatch) yellow perch revealed no differences in whole-body cortisol levels between stressed and unstressed individuals. In acute stress challenge tests (30 sec net confinement) conducted on post-hatch yellow perch, differences in cortisol levels between stressed (30 min post-stress) and unstressed fish were detected one week after hatching (12.3 ± 1.6 ng/g and 7.0 ± 1.3 ng/g, respectively). The magnitude of the stress response was greater in 2- to 5-week-old fish than in one-week-old fish. Changes in post-stress cortisol levels were closely associated with changes in interrenal morphology. Cumulatively, these results indicate that cortisol biosynthesis, integration and maturation of the hypothalamic-pituitary-interrenal (HPI) axis can be observed in yellow perch as early as one week after hatching.

title: Cortisol content of eggs and larvae of teleosts.
abstract: The whole-animal content of the cortisol was measured in embryos and larvae of tilapia (Oreochromis mossambicus), rainbow trout (Oncorhynchus mykiss), ayu (Plecoglossus altivelis), milkfish (Chanos chanos), and yellowfin bream (Acanthropagrus latus) by radioimmunoassay following the validation of an extraction method. The total cortisol content in tilapia was 50.3 +/- 19.1 pg immediately following fertilization, then decreased abruptly and maintained a lower level of 10-17 pg until larval hatching; after hatching the cortisol content increased to 47.2 +/- 11.9 pg by the seventh day. Newly hatched rainbow trout had 60.3 +/- 6.4 pg cortisol and then increased their cortisol level slowly to 83.0 +/- 7.2 pg by the fifth day after hatching. Ayu larvae contained 5.2 pg cortisol immediately following hatching. On the other hand, pelagic milkfish revealed a much lower cortisol level, being undetectable from hatching until the second day and ranging from 0.4 to 3.7 pg from the third to seventh day after hatching. Yellowfin bream, demonstrating a similarity to milkfish, were not found to have any detectable cortisol from hatching until the third day, but presented 1.6-7.7 pg from the fifth to seventh day after hatching. The presence and clearance of cortisol during early development of fertilized eggs of tilapia suggest a maternal origin of the hormone. The amount of cortisol deposited in the larval body of tilapia increased after hatching from 25% to nearly 100% of the total cortisol in whole larvae, while that in the larval yolk sac decreased to an undetectable level, implying that the increased cortisol may be produced or secreted by the larva. The possible role of cortisol in larval development is discussed.

title: Whole-body corticosteroid and plasma cortisol concentrations in larval and juvenile Atlantic cod Gadus morhua L. following acute stress
abstract: Methods were developed to assess whole-body immunoreactive corticosteroid concentrations (IRC) and plasma levels of cortisol in Atlantic cod subjected to several common, acute stressors. A measurable corticosteroid stress response was observed at the first sampling in whole bodies of 8-day post-hatch (dph) larvae. Two groups of juveniles (∼5 and 30 g) were subjected to a 30 s net stressor and whole-body IRC and plasma cortisol levels were determined. Post-stressor IRC in smaller fish rose approximately 14-fold, peaked at 1 h, were sustained for 3–6 h and returned to pre-stressor levels within 24 h. Post-stressor plasma cortisol levels in larger fish rose approximately 18-fold, peaked at 0.5–1 h, were sustained for 1–3 h and then returned to near pre-stressor levels after 24 h. Immunoreactive corticosteroid concentrations appeared to remain elevated longer than plasma cortisol levels suggesting that steroids other than cortisol were contributing to total immunoreactivity in homogenates. Juveniles exposed to either a grading procedure or high density transport had maximal IRC and plasma cortisol levels within 90 min which returned to pre-stressor levels within 24–72 h. A reduction in water salinity (20 g L−1) did not moderate the corticosteroid response in juveniles. The results show that Atlantic cod respond to common, acute stressors in a manner similar to other teleosts. Whole-body homogenates can be used to identify changes in IRC in response to acute stress in cod with the caveat that recovery IRC may differ from plasma cortisol concentrations.

title: Ontogeny of the corticotropin-releasing factor system in zebrafish.
abstract: The corticotropin-releasing factor (CRF) system in fish functions to maintain homeostasis during stress in part by regulating cortisol production via the hypothalamus-pituitary-interrenal (HPI) axis. Towards understanding the role of the CRF system in vertebrate development, we describe the ontogeny of the CRF system, cortisol, and the stress response in the zebrafish, Danio rerio. Early embryonic expression of mRNA encoding CRF, urotensin I (UI), CRF-binding protein (CRF-BP), and two CRF receptors (CRF-R1 and CRF-R2) suggest a function in the early organization of the developing embryo. The expression patterns of CRF, UI, and CRF-BP in the larval brain are consistent with the adult distribution patterns for these genes and support HPI-axis independent functions. The relative amounts of CRF and UI mRNA in the heads and tails of developing and adult zebrafish suggest that CRF functions primarily in the brain while UI also plays an important role in the caudal neurosecretory system. The amount of cortisol in developing zebrafish is low and relatively constant through the first 6 days of development. The commencement of feeding after 4 dpf, however, significantly increases basal cortisol production. Finally, we show that zebrafish larvae are able to respond to an osmotic stressor as early as 3 dpf. Overall, results from this study establish the zebrafish as a model species for research on stress during ontogeny and offer new insights into an HPI-axis independent function for the CRF system during embryogenesis.

title: Molecular programming of the corticosteroid stress axis during zebrafish development.
abstract: The functions of the hypothalamus-pituitary-interrenal (HPI) axis in teleosts have been studied primarily in juvenile and adult fish, whereas little is known about the molecular events leading to the onset of the stressor-induced cortisol response during development. Here we summarize a number of studies that have examined changes in the expression of genes encoding proteins critical for the functioning of the HPI axis, and the associated cortisol response in developing zebrafish embryos and larvae. The mRNA transcripts for some of these genes, including corticotropin releasing factor (CRF), proopiomelanocortin (POMC), melanocortin 2 receptor (MC2R), steroidogenic acute regulatory protein (StAR) and cytochrome P450 side chain cleavage (P450scc) have been detected during embryogenesis prior to hatch. The mRNA levels of MC2R, StAR and P450scc are up-regulated immediately prior to the dramatic rise in basal larval cortisol levels after hatch. Although all the components of the HPI axis are expressed and cortisol is synthesized at hatch, a stressor-induced cortisol response was not evident until 97 hpf. We hypothesize that this disconnect in the timing of the basal cortisol synthesis and stressor-induced cortisol synthesis is due to the delayed development of peripheral and central neural inputs relaying stressor stimuli to the hypothalamus. Overall, zebrafish appear to be an excellent model for elucidating the molecular mechanisms leading to the development of the corticoid stress axis in vertebrates.

title: Ontogeny of the cortisol stress response in larval rainbow trout.
abstract: The ontogeny of the interrenal stress response in rainbow trout was characterized by measuring resting and acute-stress-induced changes in whole-body cortisol levels in embryos and larvae at different early developmental stages. In Experiment 1, resting cortisol levels averaged 6.0 ng/g in newly fertilized eggs, fell to less than 0.3 ng/g by the time of hatching at Week 4 (incubation at 10 degrees), and increased to 1.4 ng/g by Week 5. Cortisol levels did not change in response to acute stress in 3-, 4-, or 5-week-old fish. In Experiment 2, resting cortisol averaged 1.4 ng/g in newly fertilized eggs, fell to less than 0.03 ng/g by Week 2, and then steadily increased between Weeks 3 and 6 to a peak of 4.8 ng/g before falling to 1.2 ng/g by Week 7. Cortisol levels did not change in response to acute stress in 3-, 4-, or 5-week-old fish. Six-week-old fish showed a 2.3-fold increase in cortisol levels at 1 hr poststress, indicating that the hypothalamic-pituitary-interrenal axis first develops responsiveness to stress 2 weeks after hatching and 1 week before the onset of exogenous feeding. The stress hyporesponsive period after hatching in rainbow trout may be homologous to the 2-week stress hyporesponsive period after birth in rodents, the function of which may be to maintain low, constant corticosteroid levels during a critical developmental period when these steroids can have permanent effects on neural organization. As suggested for mammals, this period may be a time when rainbow trout are particularly vulnerable to environmental effects on their subsequent development.

title: Plasma cortisol response to stress in juvenile rainbow trout is influenced by their life history during early development and by egg cortisol content.
abstract: In this study, the consequences of the exposure of rainbow trout (Oncorhynchus mykiss) to a brief stress during early development were explored on the later response of fingerlings to stress. Firstly, we analyzed the ontogeny of cortisol production and that of the initial cortisol response to stress in developing fish. It is only at the eyed stage that the embryos started to produce some basal cortisol. The HPI (hypothalamic-pituitary-interrenal axis) was however not functional before hatching, as exposure of the embryos to a stress did not trigger any cortisol response. A cortisol response to an acute stress was detected 9 days after hatching. In a second set of experiments, we showed that a very brief stress applied at 3 different early stages (eyed, hatching, and yolk resorption) reduced the later cortisol response to stress of 5-month-old fingerlings. This reduction is not likely to be due to alterations in the fish interrenal sensitivity because the 5-month-old fingerlings responded to ACTH treatment (only one dose tested) within the same magnitude as the fish that were not stressed during early development. An experimentally induced increase in egg cortisol just after fertilization also induced a reduction in stress sensitivity of 5 month old fingerlings, which was dose-dependent This study shows for the first time that the responsiveness of the corticotrope axis in 5-6 months old rainbow trout was influenced both by early stress exposure and by initial egg cortisol levels. Whether the HPI was functional or not at the time the initial stress was applied only had a small influence on the later unambiguous effect of early stress exposure.

title: Maturation of the pancreatic and intestinal digestive functions in sea bass (Dicentrarchus labrax): effect of weaning with different protein sources
abstract: The maturation of the digestive functions in sea bass (Dicentrarchus labrax) larvae was evaluated by the enzymatic profile of pancreas and intestine brush border membranes. Sea bass larvae were weaned at day 25 with three simplified diets different by their protein nature: 100% fish meal (FP), 100% casein mixture (CP) and 50% fish meal-50% casein mixture (CFP). The casein mixture contained 35% of hydrolysate. The control group was fed live preys. The specific activity of amylase decreased with age irrespectively of the diets whereas the specific activity of trypsin was enhanced. The casein mixture reduced pancreatic secretion in amylase and trypsin. The CFP group differed from the other groups fed on compound diets, exhibiting as soon as day 32 high activities of brush border enzymes, similar to controls. This sharp increase between day 25 and 32 appeared to be crucial for larval survival. The addition of a protein hydrolysate in a weaning diet seems to facilitate this maturation process.

title: Unique and conserved aspects of gut development in zebrafish.
abstract: Although the development of the digestive system of humans and vertebrate model organisms has been well characterized, relatively little is known about how the zebrafish digestive system forms. We define developmental milestones during organogenesis of the zebrafish digestive tract, liver, and pancreas and identify important differences in the way the digestive endoderm of zebrafish and amniotes is organized. Such differences account for the finding that the zebrafish digestive system is assembled from individual organ anlagen, whereas the digestive anlagen of amniotes arise from a primitive gut tube. Despite differences of organ morphogenesis, conserved molecular programs regulate pharynx, esophagus, liver, and pancreas development in teleosts and mammals. Specifically, we show that zebrafish faust/gata-5 is a functional ortholog of gata-4, a gene that is essential for the formation of the mammalian and avian foregut. Further, extraembryonic gata activity is required for this function in zebrafish as has been shown in other vertebrates. We also show that a loss-of-function mutation that perturbs sonic hedgehog causes defects in the development of the esophagus that parallel those associated with targeted disruption of this gene in mammals. Perturbation of sonic hedgehog also affects zebrafish liver and pancreas development, and these effects occur in a reciprocal fashion, as has been described during mammalian liver and ventral pancreas development. Together, these data define aspects of digestive system development necessary for the characterization of zebrafish mutants. Given the similarities of teleost and mammalian digestive physiology and anatomy, these findings have implications for developmental and evolutionary studies as well as research of human diseases, such as diabetes, liver cirrhosis, and cancer.

title: Ontogeny of the gut motility control system in zebrafish Danio rerio embryos and larvae
abstract: Using digital motion analysis, the ontogeny of the cholinergic, tachykinin and pituitary adenylate cyclase-activating polypeptide (PACAP) control systems was studied in zebrafish Danio rerio larvae, in vivo. For the first time we show that the regular propagating anterograde waves that occur in the zebrafish larval gut before and around the onset [at 5–6 days post fertilization (d.p.f.)] of feeding are modulated by acetylcholine or atropine, PACAP and NKA (neurokinin A). At 3 d.p.f., when no spontaneous motility has developed, application of acetylcholine did not affect the gut. However, at 4 d.p.f., acetylcholine increased and atropine reduced the frequency of propagating anterograde waves. At 5 d.p.f., NKA increased and PACAP reduced the wave frequency. This suggests that both excitatory and inhibitory pathways develop at an early stage in the gut, independent of exogenous feeding. Immunohistochemistry established the presence of gut neurons expressing PACAP and NKA in the proximal part of the developing gut from the first stage investigated (2 d.p.f.) and before regular motility was observed. 1 d.p.f. (PACAP) or 2 d.p.f. (NKA) stages later the whole gut was innervated. This supports physiological results that gut motility is under neuronal control during the period when regular motility patterns develop.

title: Formation of the digestive system in zebrafish: III. Intestinal epithelium morphogenesis.
abstract: Recent analysis of a novel strain of transgenic zebrafish (gutGFP) has provided a detailed description of the early morphological events that occur during the development of the liver and pancreas. In this paper, we aim to complement these studies by providing an analysis of the morphological events that shape the zebrafish intestinal epithelium. One of our goals is to provide a framework for the future characterization of zebrafish mutant phenotypes in which intestinal epithelial morphogenesis has been disrupted. Our analysis encompasses the period between 26 and 126 h post-fertilization (hpf) and follows the growth, lumen formation and differentiation of a continuous layer of endoderm into a functional intestinal epithelium with three morphologically distinct segments: the intestinal bulb, mid-intestine and posterior intestine. Between 26 hpf and 76 hpf, the entire intestinal endoderm is a highly proliferative organ. To make a lumen, the zebrafish endoderm cells undergo apical membrane biogenesis, adopt a bilayer configuration and form small cavities that coalesce without cell death. Thereafter, the endoderm cells polarize and differentiate into distinct cell lineages. Enteroendocrine cells are distinguished first at 52 hpf in the caudal region of the intestine in a new stable transgenic line, Tg[nkx2.2a:mEGFP]. The differentiation of mucin-containing goblet cells is first evident at 100 hpf and is tightly restricted to a middle segment of the intestine, designated the mid-intestine, that is also demarcated by the presence of enterocytes with large supranuclear vacuoles. Meanwhile, striking expansion of the lumen in the rostral intestine forms the intestinal bulb. Here the epithelium elaborates folds and proliferating cells become progressively restricted to a basal compartment analogous to the crypts of Lieberkühn in mammals. At 126 hpf, the posterior intestine remains an unfolded monolayer of simple columnar epithelium.

title: Intestinal growth and differentiation in zebrafish
abstract: Intestinal development in amniotes is driven by interactions between progenitor cells derived from the three primary germ layers. Genetic analyses and gene targeting experiments in zebrafish offer a novel approach to dissect such interactions at a molecular level. Here we show that intestinal anatomy and architecture in zebrafish closely resembles the anatomy and architecture of the mammalian small intestine. The zebrafish intestine is regionalized and the various segments can be identified by epithelial markers whose expression is already segregated at the onset of intestinal differentiation. Differentiation of cells derived from the three primary germ layers begins more or less contemporaneously, and is preceded by a stage in which there is rapid cell proliferation and maturation of epithelial cell polarization. Analysis of zebrafish mutants with altered epithelial survival reveals that seemingly related single gene defects have different effects on epithelial differentiation and smooth muscle and enteric nervous system development.

title: Ontogeny and physiology of the digestive system of marine fish larvae
abstract: The ontogeny of the digestive tract of marine fish larvae has been the subject of many studies these last twenty years. Beyond the scientific interest for such animal species, particularly for developmental aspects, most of these studies aimed to come up with the expectations of commercial hatcheries by reducing the bottlenecks in larvae culture and weaning processes (switch from live preys to compound diet feeding sequence). Consequently, the profile and dietary adaptation of digestive enzymes, the description and histology of the main organs have been extensively studied, when others aspects of the physiology of the larvae digestive tract (gut hormones, intestinal transport…) were poorly investigated and this imbalanced knowledge is showed through this review. For a long time, it has been hypothesized that marine fish larvae had insufficient digestive enzyme capacity to handle compound diets compared to juvenile fish, and several weaning strategies were built on the supply of exogenous enzymes (Kolkovski, 2001) but were never very conclusive. More recently, it was considered that ontogenetic differences between the larval type and adult type organ function do not mean that larvae face physiological or digestive deficiencies during early life stages. Fish larvae should not be considered as primitive organisms but represent a transitional period in which both ontogeny and growth cause substantial changes in structure, physiology, size and body shape. The basic mechanisms of organ development are similar in all teleosts, even though there are considerable differences regarding the relative timing in the ontogeny. The time in organ development and its associated physiological functions is affected by the general life history of each species and a number of abiotic and biotic factors, such as water temperature, food availability and composition during early life stages...

title: Biochemistry of intestinal development.
abstract: In biochemical terms, the rat small intestine is relatively immature at birth and for the first two postnatal weeks. Then during the third week a dramatic array of enzymic changes begins, and by the end of the fourth week the intestine has the digestive and absorptive properties of the adult. Selective examples of these changes are discussed with emphasis on their implications for toxicological studies. The review also includes a detailed consideration of the roles of the dietary change of weaning and of glucocorticoid and thyroid hormones in the regulation of intestinal development.

title: Control of development of gastrointestinal system in neonates.
abstract: Our recent studies of structure and function of gastrointestinal tract mucosa revealed that the domestification of Sus scrofa corresponds with the significant slowing of the organ development. On top of genetic potential, the nutritional factors (or more precisely - lack of certain biologically active substances in the feed of pregnant sows) are responsible. Moreover, feeding neonates with milk replacers instead of mother's milk further slows down the development. This is manifested by reduced mitotic activity in the crypts and enhanced apoptosis of enterocytes. The negative effects consist of slower replacement of fetal type, vacuolated enterocytes to adult type enterocytes, modified profile of brush border enzymes, alterations in intestinal mucosa barrier, higher susceptibility to infectious agents, and many others. On the other hand, farmers in order to intensify the production, shorten the suckling period imposing the neonatal piglets to be weaned at 3-4 weeks of life and even earlier. Altogether, it makes the weaning disorders one of the most important problems in pig husbandry, and the mortality of piglets in the leading pig-producing countries still reaches 10%. A number of strategies have been developed to counteract the post-weaning problems. One of them is to stimulate the development of the gastrointestinal tract of the neonate by supplementation of the sow diet with certain biologically active substances and plants. The other idea is to speed up the postnatal development of the gut mucosa for example by plant lectins. Lessons from pig studies can be also useful in human nutrition and medicine since the development of porcine gastrointestinal tract shows a great similarity to that of humans.

title: Blue Chromatophores in Two Species of Callionymid Fish
abstract: Dendritic chromatophores that contained blue pigmentary organelles were found in the bluish parts of the skin of two callionymid species, the mandarin fish, Synchiropus splendidus, and the psychedelic fish, S. picturatus. We named these novel cells “cyanophores” and the organelles “cyanosomes”. In response to various stimulatory cues, the cyanophores responded by the aggregation or dispersion of cyanosomes. In addition to their role in the revelation of bluish hues, these cyanophores may participate in the changes in shades of blue of these gorgeous fish.

title: Dual appearance of xanthophores, and ontogenetic changes in other pigment cells during early development of Japanese flounder Paralichthys olivaceus
abstract: Flatfishes display a left–right asymmetry that is unique in the animal kingdom. In order to clarify the mechanisms of the asymmetrical development of pigment cells, changes in pigment cell densities were examined in Japanese flounder Paralichthys olivaceus. During development from symmetrical larvae to asymmetrical juveniles, pigment cell densities were monitored on the skin on both the left side (ocular side in juvenile; eventually has two eyes) and the right side (blind side in juvenile; eventually has no eyes). A symmetrical and constant decrease was observed in leucophores and larval type melanophores. A mostly symmetrical (slightly delayed on the blind side) and constant increase in iridophores from metamorphosis was observed. Adult-type melanophores appeared and then increased only after metamorphosis on the ocular side. However, the pattern of xanthophores was complicated: they first existed symmetrically and decreased symmetrically until metamorphosis, and they later increased only on the ocular side. The dual appearance of the xanthophores, as well as the differences between their depths and sizes on the ocular and blind sides, may suggest the presence of two types of xanthophores—just as melanophores are well known to exhibit two types. The ontogenetic study of pigment cells described here is likely to help to elucidate the process of abnormal pigmentation in flatfishes.

title: Structural and functional diversity of proopiomelanocortin in fish with special reference to barfin flounder
abstract: Proopiomelanocortin (POMC) is a precursor of adrenocorticotropic hormone (ACTH), melanocyte-stimulating hormone (MSH), and endorphin (END). We have characterized POMC systems in barfin flounder. The results revealed unique aspects of POMC systems. Notable features in terms of pituitary functions are the occurrence of three functional POMC genes, the mutation of an essential sequence in the beta-END in one of the genes, occurrence of alpha-MSH in addition to ACTH in the pars distalis of the pituitary, and expression of the three genes in a single cell. While MSHs stimulate pigment dispersion, expression of the POMC gene and plasma levels of MSH do not always respond to background color changes between black and white. The functions of MSHs in skin pigmentation are very unique, because acetylation at the N-terminal of alpha-MSH inhibits its pigment dispersing activity. This is in contrast to results from other teleosts and amphibians, in which acetylation increases the activity. In the skin, the POMC gene is expressed in the non-chromatophoric dermal cells, indicating that MSH produced in the skin de novo has a paracrine function. The detection of MSH peptides in skin extracts seems to show that the control of skin pigmentation by MSHs is twofold-endocrine control by the pituitary, and paracrine control by the skin itself. Thus, fish provide an interesting model to help understand the structural and functional diversity of POMC systems. In this review, we provide an overview of our recent studies on the characterization of molecules and biological significance of POMC systems in barfin flounder.

title: Functions of melanin-concentrating hormone in fish.
abstract: Melanin-concentrating hormone (MCH) was originally discovered in fish, in which it causes aggregation or concentration of melanin granules in melanophores, thus regulating body color. MCH is a cyclic neuropeptide synthesized as a preprohormone in the hypothalamus of all vertebrates. Mammalian MCH plays an important role as a neurotransmitter or neuromodulator in regulating food intake and energy homeostasis. MCH signaling system may involve in regulating food intake also in fish. This neuropeptide binds to G-protein-coupled seven transmembrane receptor[s] to mediate its functions. This article reviews MCH and MCH receptor signaling systems in body color change and food intake in fish.

title: The development of the MCH system
abstract: Although a great deal is published on the MCH neurons, very few works were devoted to the study of their development. However, existing literature points out two important traits: first, these neurons differentiate a MCH phenotype very early in all species studied so far, which might suggest a role for the MCH peptide during development; second, in the rat, birth date greatly influence the phenotype of MCH neurons. At least two sub-populations were described on the basis of their chemical phenotype, projection pattern and birth date. The understanding of processes involved in the differentiation of these sub-populations may help understand the medio-lateral differentiation of the tuberal hypothalamus.

title: Central administration of melanin-concentrating hormone (MCH) suppresses food intake, but not locomotor activity, in the goldfish, Carassius auratus

abstract: Melanin-concentrating hormone (MCH) is a hypothalamo-pituitary peptide, which was first identified in the salmon pituitary as a hormone affecting body color. Recently, MCH has been implicated in the regulation of feeding behavior and energy homeostasis in mammals. Despite a growing body of knowledge concerning MCH in mammals, however, there is little information about the effect of MCH on appetite and behavior in fish. The aim of the present study was to investigate the action of MCH on feeding behavior and spontaneous locomotor activity in the goldfish. We administered synthetic MCH by intracerebroventricular (ICV) injection and examined its effect on food intake and locomotor activity using an automatic monitoring system. Both types of synthetic MCH we employed, which are of fish and human origin, were effective in stimulating aggregation of melanin granules in the melanophores of goldfish scales. Cumulative food intake was significantly decreased by ICV injection of both MCHs in a dose-dependent manner. ICV injection of fish MCH at the same doses as those used for examination of food intake induced no marked changes in locomotor activity during the observation period. These results suggest that MCH influences feeding behavior, but not spontaneous locomotor activity, in the goldfish, and may exert an anorexigenic action in the goldfish brain, unlike its orexigenic action in mammals.

title: Development of melanin-concentrating hormone-immunoreactive elements in the brain of gilthead seabream (Sparus auratus)
abstract: The development of the hypothalamic melanin-concentrating hormone (MCH) system of the teleost Sparus auratus has been studied by immunocytochemistry using an anti-salmon MCH serum. Immunoreactive perikarya and fibers are found in embryos, larvae, and juvenile specimens. In juveniles, most labeled neurons are present in the nucleus lateralis tuberis; some are dispersed in the nucleus recessus lateralis and nucleus periventricularis posterior. From the nucleus lateralis tuberis, MCH neurons project a conspicuous tract of fibers to the ventral hypothalamus; this penetrates the pituitary stalk and reaches the neurohypophysis. Most fibers end close to the cells of the pars intermedia, and some reach the adenohypophysial rostral pars distalis. Immunoreactive fibers can also be seen in extrahypophysial localizations, such as the preoptic region and the nucleus sacci vasculosi. In embryos, MCH-immunoreactive neurons first appear at 36 h post-fertilization in the ventrolateral margin of the developing hypothalamus. In larvae, at 4 days post-hatching, perikarya can be observed in the ventrolateral border of the hypothalamus and in the mid-hypothalamus, near the ventricle. At 26 days post-hatching, MCH perikarya are restricted to the nucleus lateralis tuberis. The neurohypophysis possesses MCH-immunoreactive fibers from the second day post-hatching. The results indicate that MCH plays a role in larval development with respect to skin melanophores and cells that secrete melanocyte-stimulating hormone.

title: Chronological study of the appearance of adenohypophysial cells in the ayu (Plecoglossus altivelis)
abstract: We previously reported the chronological appearance of adenohypophysial cells in freshwater teleosts using an immunocytochemical technique. The present study investigated the chronological appearance of adenohypophysial cells in the ayu, which is spawned and has its early development in brackish water, and the results were compared with those obtained in freshwater and seawater teleosts, as well as in other vertebrates. In the adult teleostean adenohypophysis, seven or eight types of secretory cells have been distinguished, each of which produce different hormones: prolactin (PRL), growth hormone (GH), thyroid stimulating hormone (TSH), gonadotropic hormones (GTH I and GTH II), adrenocorticotropic hormone (ACTH), melanophore stimulating hormone (MSH) and somatolactin (SL). In the pituitary of adult ayu, seven distinct types of glandular cells (PRL, GH, TSH, GTH, ACTH, MSH and SL cells) were identified. Chronologically, a few immunoreactive (ir)-PRL and ir-GH cells appeared in the ventral side of the pituitary one day before hatching. Then, just after hatching, ir-GTH cells were observed in the central to dorsal portion; ir-ACTH cells were found distributed in the anterior portion and some ir-MSH and a few ir-SL cells were seen in the posterior portion of the pituitary. Finally, a small number of ir-TSH cells were identified 50 days after hatching. These results differed from those obtained in other fishes previously reported with regard to the times of appearance of the PRL and GH cells. PRL cells appeared first, followed by GH cells in the freshwater teleosts, PRL and GH cells appeared at the same time in the brackishwater teleosts, while GH cells appeared first and PRL cells appeared last in the seawater teleosts. These results reflect the fact that PRL plays a major role in osmoregulation among freshwater teleosts, as compared with GH, which plays a similar role in seawater teleosts. It seems that both PRL and GH may play important roles in osmoregulation in brackishwater fish.

title: Effects of arachidonic acid on pigmentation and fatty acid composition of Japanese flounder, Paralichthys olivaceus (Temminck and Schlegel)
abstract: The dietary effect of arachidonic acid (20:4n-6, AA) on the composition of the head and the body, and on the pigmentation of Japanese flounder, Paralichthys olivaceus (T. & S.), was studied by means of microbound diets. Increasing levels of this essential fatty acid produced higher growth and pigmentation success in juveniles after 45 days of feeding. The role played by AA in the composition of phosphatidylinositol and eicosanoid synthesis is discussed in relation to the normal function of the neural system and the production of hormones involved in the metamorphosis and pigmentation of flatfish.

title: Arachidonic acid requirements in larval summer flounder, Paralichthys dentatus
abstract: The arachidonic acid (20:4n-6,AA) requirements of larval summer flounder weredetermined for the rotifer- and Artemia-feeding stages. Experimental emulsionscontained adequate n-3 highly unsaturated fattyacid (HUFA) ratios and emulsion levels of AAwere set at 0, 3, 6, 9, and 12% (AA0, AA3,AA6, AA9, and AA12). Examination of fatty acidlevels in live feeds and larval tissuesconfirmed the physiological incorporation offatty acids relative to dietary levels. In thefirst experiment, survival, growth, andsalinity tolerance (2-h in 70‰) were measuredat 18 days after hatch (dah) after feeding thelarvae the various levels of AA. Larvae fedAA6-enriched rotifers were better able tosurvive the salinity tolerance test. AAenrichment up to 12% had no effect on growthand survival. In the second experiment, larvaewere fed AA0- or AA6-enriched rotifers until 23dah, followed by unenriched 24- and 48-h Artemia nauplii until 32 dah. These larvaethen were subdivided and fed AA-enriched Artemia from 33-45 dah. At the end of thisexperiment, larvae fed AA6-enriched rotifershad higher survival, increased growth, andsurvived better in the salinity tolerance test(2-h in 80‰). The enrichment of Artemiadid not have any effect on these variables.Thus, the provision of AA6-enriched rotifers(10 mg AA g−1 DW) early in larvaldevelopment may serve to enhance larval stresstolerance at the rotifer stage while alsoincreasing larval survival, growth, and stresstolerance later in the Artemia stage.

title: Arachidonic acid, arachidonic/eicosapentaenoic acid ratio, stearidonic acid and eicosanoids are involved in dietary‐induced albinism in Senegal sole (Solea senegalensis)
abstract: Senegal sole larvae were fed live prey enriched with different amounts of arachidonic acid (ARA, 20:4n-6) and eicosapentaenoic acid (EPA, 20:5n-3) to re-evaluate the effect of these two fatty acids on flatfish pigmentation. Echium oil, a plant-derived oil rich in gamma-linolenic acid (18:3n-6) and stearidonic acid (18:4n-3) was also used as a component of one of the enrichment emulsions. Although ARA content did not have any effect on growth there was a clear influence on pigmentation that correlated clearly with prostaglandin production. Inclusion of Echium oil, on the contrary, exerted a positive effect on pigmentation rate even though dietary ARA levels were as high as in the other emulsions. The relationships between dietary ARA levels and dietary ARA/EPA ratio, prostaglandin production and pigmentation are discussed.

title: Growth, survival, lipid composition and pigmentation of turbot (Scophthalmus maximus) larvae fed live-prey enriched in Arachidonic and Eicosapentaenoic acids
abstract: Turbot larvae were fed live-prey enriched with different levels of arachidonic (ARA) and eicosapentaenoic (EPA) acids to study the effects of these fatty acids on body composition and pigmentation success. Significantly reduced pigmentation was obtained in those fish fed medium and high ARA diets for 43 days. Growth and survival were the same for all groups. The incorporation of ARA and EPA in fish eyes, brains, livers and carcasses reflected the percentage of these fatty acids in the diets. ARA accumulation was similar in all tissues, but brain accumulated EPA was less efficient than the other tissues examined. A highly significant, negative correlation was found between the %ARA in turbot juvenile brain total lipids and pigmentation success. A weaker, positive correlation was found between brain EPA and pigmentation. Increasing dietary ARA affected the fatty acid composition of turbot brain phosphoglycerides more than increasing dietary EPA, especially in phosphatidylinositol (PI) and phosphatidylethanolamine (PE). A negative relationship was found between percentage normal pigmentation and ARA levels in brain phosphatidylcholine (PC), PE and phosphatidylserine (PS). Elevated levels of ARA in PI also resulted in malpigmented juveniles, but EPA:ARA ratios ≥1 in PI were associated with normal pigmentation. We conclude that, given a sufficiency of dietary docosahexaenoic acid (DHA), the optimum dietary level of EPA is not a function of DHA, but of dietary ARA.

title: Ontogeny of ACTH and MSH cells in Japanese flounder (Paralichthys olivaceus) in relation to albinism
abstract: Japanese flounder larvae were reared for 43 days in glass aquariums with and without sand in the bottom to study the influence of the substrate on the pigmentation pattern, pituitary growth and ACTH and MSH production. Similar results were obtained in terms of survival, growth and ocular side pigmentation in both groups. However, a clear difference was observed in blind side pigmentation and only 28% of the fish cultured with sand developed this abnormality, contrasting with 58% of those reared without sand. A clear increase in the pituitary volume was observed during larval development, being maximal at post metamorphosis. No differences in the pituitary volume could be found among pigmented, albino and ambicoloured juveniles, although those reared with sand had a larger pituitary than those reared without sand. ACTH and MSH producing cells were detected by immunohistochemistry. The proportion of the pituitary occupied by ACTH or MSH cells (ratio) was highest at early larval stages, and decreased with development to a minimum value at the climax of metamorphosis. No significant difference in the ACTH ratio was found between the two groups, although juveniles reared without sand showed a slightly higher ratio, probably reflecting the stress experienced by these fish. The MSH ratio was significantly higher in fish reared with sand. No significant differences were found in the ACTH and MSH ratios among pigmented, albino and ambicoloured fish. Fish produced with a sandy bottom showed a lower degree of hyperpigmentation and possessed larger pituitaries that had higher MSH ratios than those reared without sand.

title: Arachidonic acid enriched live prey induces albinism in Senegal sole (Solea senegalensis) larvae
abstract: We examined the effect of dietary arachidonic acid (20:4n-6, ARA) on growth, survival, pigmentation and fatty acid composition of Senegal sole larvae using a dose–response design. From 3 to 37 days post-hatch (dph), larvae were fed live food (rotifers from 3 to 9 dph, Artemia nauplii from −37 dph) that had been enriched using one of three experimental emulsions containing 3 graduated concentrations (1.3, 68 and 120.1 mg ARA g −1 dry weight) of ARA and constant docosahexaenoic acid (22:6n-3, DHA). A commercial enrichment product (DHA-Protein Selco, ARA content 7.8 mg g −1 dry weight) was used as a reference diet. Final concentration of ARA in Artemia nauplii ranged from 0.2 to 17.5 mg g −1 lipids. Growth and survival were independent of dietary levels of ARA tested. However, there was a correlation between dietary ARA and a significant reduction in pigmentation leading to increased albinism. Tissue fatty acid concentrations reflected the corresponding dietary composition. Eicosapentaenoic acid (20:5n-3, EPA) levels in all the tissues examined were inversely related to dietary ARA. There was almost a 100-fold increase in the proportion of docosapentaenoic acid (22:5n-3, DPA) in the tissues relative to the diet, which might indicate chain elongation from EPA as a result of inadequate dietary DHA. A negative, linear correlation was found between the pigmentation rate and the ARA content in the head, as well as with dietary ARA/EPA ratio.

title: Recent advances in lipid nutrition in fish larvae
abstract: Due to the importance of dietary lipid utilization for larval rearing success, increasing attention has been paid during the last years to different aspects of larval lipid nutrition such as digestion, absorption, transport and metabolism, which are frequently studied by different research groups. The present study reviews the published information on these aspects, including some recent results obtained in our laboratory, that contribute to a better understanding of larval lipid nutrition.Neutral lipase activity was found in the digesta of larval gilthead seabream as early as first feeding, followed by a significant increase which reached up 8 times the initial levels at day 15 and was clearly influenced by the fatty acid composition of dietary lipids. Accordingly, the capacity for lipid absorption by the intestinal epithelium has been also observed at the onset of exogenous feeding, although the specific location in the different digestive tract segments differ with species. Whereas the capacity to absorb lipid increases with development in live prey-fed larvae, this improvemment is delayed in larvae fed formulated diet. Increasing dietary phosphatidyl cholines levels enhanced lipid absorption regardless of whether it is of soybean or marine origin, but the latter improved hepatic lipid utilization. Enzymatic, histological and biochemical evidences suggest that marine fish larvae are able to effectively digest and absorb n-3 HUFA-rich triacylglycerols, but feeding with phosphoacylglycerols, particularly if they are rich in n-3 HUFA, would enhance phosphoacylglycerols digestion and specially lipid transport alowing a better n-3 HUFA incorporation into larval membrane lipids and promoting fish growth. Although the essentiality of n-3 HUFA for larval marine fish has been studied extensively, only recently has the importance of dietary arachidonic acid in the larvae of few species been recognised. Evidences for competitive interactions among these essential fatty acids suggest that besides a minimum dietary requirement for each essential fatty acid, their relative ratios must also be considered.

title: Effect of arachidonic acid levels in broodstock diet on larval and egg quality of Japanese flounder Paralichthys olivaceus.
abstract: This study investigated the effect of dietary arachidonic acid (AA) in broodstock of Japanese flounder on subsequent egg and larval quality. Diets with similar proximate composition and n-3 HUFA level, but with different AA levels (0.1%, 0.6% and 1.2% of diet), were fed to the broodstock from 3 months before and during the spawning season. Spawning was observed from March to May. Total egg production over the spawning season was highest in fish fed the 0.6% AA diet and lowest in fish fed the 1.2% AA diet. All parameters measured as egg quality (percentage of buoyant eggs, hatching rate, larval survival and normality of larvae.) were highest in fish fed the 0.6% AA diet. AA content in eggs proportionally increased with the dietary AA level. EPA content of polar lipids of eggs had a negative correlation with the AA level in diets whereas the DHA content was independent of dietary AA. The results of this study indicate that a supplement of AA at 0.6 g/100 g diet improved the reproductive performance of Japanese flounder, but a higher level of AA (1.2 g/100 g diet) negatively affected both egg and larval quality due to a potential inhibitory effect on EPA bioconversion.

title: The effect of tissue docosahexaenoic and arachidonic acids levels on hypersaline tolerance and leucocyte composition in striped bass (Morone saxatilis) larvae
abstract: Larval striped bass (M. saxatilis), tissue docosahexaenoic (DHA) and arachidonic (AA) acids levels were modulated through dietary enrichments and the effect on growth and survivorship examined. Mean growth was significantly greater in larvae enriched with AA than in larvae enriched with DHA (F-value for AA treatment was 20.5 versus only 5.1 for the DHA treatment). Dietary treatment did not have a significant effect on larval survivorship (56.0±2.4%, p > 0.05). When challenged with hypersaline (25 psu) immersion, DHA enriched larvae survived better than AA enriched larvae, but larvae with body tissue levels of 15.4 mg AA g−1 and 7.2–15.4 mg DHA g−1 dry weight provided maximal survivorship to the challenge. Elevated levels of body tissue AA was generally associated with elevated levels of whole body cortisol. On the other hand, increasing levels of DHA mainly affected the kinetics of cortisol increase to hypersaline exposure. Larvae injected intraperitoneally with formalin fixed Staphylococcus aureus responded by altering the proportion of lymphocytes, monocytes and neutrophils in perpherial blood. Lymphocytes, which accounted for the largest percentage of white blood cells (over 70%), decreased in all challenged larvae during the first 6 hours post injection then returned to pre-challenge levels after 44 hours. Conversely, the relative proportion of monocytes and neutrophils rose from 14% and 2% up to 28% and 6% of the total circulating leucocytes, respectively. The largest increase occurred in larvae fed a moderate level of both DHA and AA.

title: Critical levels of essential fatty acids for normal pigmentation in Atlantic halibut (Hippoglossus hippoglossus L.) larvae
abstract: High levels of n-3 polyunsaturated fatty acids and a low level of arachidonic acid (ARA, 20:4n-6) in larval diets seem to be necessary for normal pigmentation of Atlantic halibut juveniles, whereas energy status and fatty acid composition seem to modulate eye migration in flatfish in general. However, we do not know the limits or the critical combinations of essential fatty acids that will give normal development of the larvae. In the present study we fed Atlantic halibut larvae enriched Artemia with small differences in fatty acid composition. Artemia enriched with the high TAG (triacylglycerol) emulsion contained average eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) of 13.9 and 12.5% of total fatty acids, respectively, while in Artemia enriched with the high MAG (monoacylglycerol) emulsion, the levels were 12.9 and 9.4%. The fatty acid composition of the larvae reflected the composition of Artemia , in addition ARA was slightly lower in the high TAG than in the high MAG enriched Artemia , i.e. 3.7–4.0 and 4.2–4.3% of total fatty acids, respectively. There were no differences in lipid level, measured as fatty acid methyl esters, neither between the Artemia types, nor between the larval groups. The percentages of normal pigmentation in Atlantic halibut juveniles that had been fed the high TAG and high MAG enriched Artemia were 77 ± 2 and 46 ± 16%, respectively, the eye migration was also slightly better in the high TAG group, but the final weight was lowered from 1.98 ± 0.17 g in the high MAG group to 1.56 ± 0.13 g in the high TAG group. There was a correlation between pigmentation and eye migration, since fish where the left eye had passed the mid-dorsal ridge did not get pigmentation on the blind side. It is concluded that DHA in larval whole body should be higher than 13% of total fatty acids to obtain normal pigmentation in Atlantic halibut larvae when the EPA:ARA ratio is approximately 3.5. The differences in fatty acid composition in the present study had only minor effects on eye migration.

title: Effect of dietary arachidonic acid levels on growth and survival of gilthead sea bream /Sparus aurata L. larvae
abstract: In order to determine the effect of different levels of arachidonic acid (AA) in microdiets on growth and survival of gilthead seabream larvae, two experiments were carried out. In the first experiment, 17-day old larvae were fed microdiets for 14 days. In this trial, we tested four diets with a similar docosahexaenoic acid (DHA)/eicosapentaenoic acid (EPA) ratio and AA in the range of 0.1%–1.8% dry weight (d.w.) and a fifth diet with 1.0% d.w. of AA and a different DHA/EPA ratio. In the second experiment, 16-day old larvae were fed for 21 days on two microdiets with levels of 0.1% and 1.0% d.w. AA. All diets in the two experiments had the same total n−3 HUFA level. Growth was measured by total length and dry body weight of larvae. Larval lipids were extracted and fatty acid compositions of total neutral lipid (NL) and total polar lipid (PL) fractions were determined by gas chromatography. In the first experiment, the diet containing 1.8% d.w. of AA showed the best survival (P<0.05) but did not improve growth rate. In the second experiment, an increase in the AA dietary level from 0.1% to 1.0% d.w. significantly improved (P<0.05) larval growth. EPA accumulation in the PLs of the larvae was negatively affected by the inclusion of dietary AA. When working with a fixed dietary n−3 HUFA level (2.2% d.w.), the effect of dietary AA on larval growth was masked by that of the dietary DHA/EPA ratio. However, when DHA/EPA ratio in diet was unchanged (∼1.8), it was possible to improve larval growth by supplementing the diet with 1.0 d.w. of AA. The negative effect produced by the increment of dietary AA on the EPA incorporation into the larval PLs may be related to a competition interaction.

title: Recent developments in the essential fatty acid nutrition of fish
abstract: Because of competitive interactions in the metabolism of polyunsaturated fatty acids, tissue and bodily requirements for each of the three dietary essential fatty acids in marine fish, 22:6 n −3, 20:5 n −3 and 20:4 n −6, cannot be meaningfully considered in isolation. Rather, it is necessary to consider requirements in relative as well as absolute amounts, i.e., in terms of the ratio of 22:6 n −3:20:5 n −3:20:4 n −6. This is illustrated by recent research in our laboratories which has suggested that the optimal dietary ratio of 22:6 n −3:20:5 n −3 in sea bass larvae is circa 2:1 with the optimal dietary ratio of 20:5 n −3:20:4 n −6 being circa 1:1. The optimal dietary ratio of 22:6 n −3:20:5 n −3 in turbot and halibut larvae is similarly circa 2:1 but the optimal dietary ratio of 20:5 n −3:20:4 n −6 in these species is 10:1 or greater. In addition, studies with salmon parr point to dietary 18:3 n −3 and 18:2 n −6 being important in determining the optimal tissue ratio of 20:5 n −3:20:4 n −6 for successful parr–smolt transition. We deduce that differences in essential fatty acid requirements for different species of fish reflect different dietary and metabolic adaptations to different habitats and consider how such knowledge can be exploited to develop improved diets for fish, especially in their early stages of development.

title: Effects of PGF2alpha on human melanocytes and regulation of the FP receptor by ultraviolet radiation.
abstract: Prostaglandins are potent lipid hormones that activate multiple signaling pathways resulting in regulation of cellular growth, differentiation, and apoptosis. In the skin, prostaglandins are rapidly released by keratinocytes following ultraviolet radiation and are chronically present in inflammatory skin lesions. We have shown previously that melanocytes, which provide photoprotection to keratinocytes through the production of melanin, express several receptors for prostaglandins, including the PGE2 receptors EP1 and EP3 and the PGF2alpha receptor FP, and that PGF2alpha stimulates melanocyte dendricity. We now show that PGF2alpha stimulates the activity and expression of tyrosinase, the rate-limiting enzyme in melanin synthesis. Analysis of FP receptor regulation showed that the FP receptor is regulated by ultraviolet radiation in melanocytes in vitro and in human skin in vivo. We also show that ultraviolet irradiation stimulates production of PGF2alpha by melanocytes. These results show that PGF2alpha binding to the FP receptor activates signals that stimulate a differentiated phenotype (dendricity and pigmentation) in melanocytes. The regulation of the FP receptor and the stimulation of production of PGF2alpha in melanocytes in response to ultraviolet radiation suggest that PGF2alpha could act as an autocrine factor for melanocyte differentiation.

title: Molecular bases of tyrosinase-negative oculocutaneous albinism: a single base insertion or a missense point mutation in the tyrosinase gene.
abstract: We have identified two different mutations in the tyrosinase genes of Japanese patients with tyrosinase-negative oculocutaneous albinism (OCA). One is a single base insertion in the exon 2 of the tyrosinase gene that shifts the reading frame and introduces a premature termination codon (TGA) after the amino acid residue 298 (codon 316). The other is a G to A transition at residue 312, leading to a single amino acid substitution, arginine at position 59 (codon 77) to glutamine. The promoter activity of the patients' tyrosinase genes was evaluated in the cell-free transcription system prepared from pigmented melanoma cells, indicating that the patients' genes were accurately transcribed in vitro. It is therefore conceivable that the tyrosinase gene is expressed in their melanocytes. Furthermore, transient expression of the mutated genes indicates that the truncated tyrosinase or the tyrosinase containing glutamine 59 is unable to form melanin in melanocytes. We therefore propose that these mutations in the tyrosinase genes lead to a phenotype of tyrosinase-negative OCA.

title: Melanocyte‐stimulating Properties of Secretory Phospholipase A2
abstract: Phospholipase A2 (PLA2) catalyzes the release of free fatty acids from membrane phospholipids, and its products derived from these fatty acids, such as prostaglandins and leukotrienes, significantly up‐regulate the key mela‐nogenic enzyme, tyrosinase, in melanocytes. This has led to suggestions that PLA2 itself triggers melanin synthesis in melanogenesis following UV irradiation or inflammation.

title: Pigmentation and eye migration in Atlantic halibut (Hippoglossus hippoglossus L.) larvae: new findings and hypotheses
abstract: Atlantic halibut juveniles, which have been fed Artemia during larval development, frequently demonstrate malpigmentation and impaired eye migration. This is in contrast to the high percentage of normally developed larvae fed copepods, reared under similar conditions. Nutrition is therefore an important component influencing larval development. Analyses of the nutrient composition of Artemia and copepods show that Atlantic halibut larvae fed Artemia probably receive sufficient amounts of vitamin A by converting canthaxanthin, while iodine may be deficient, possibly leading to interrupted thyroid hormone synthesis. An unbalanced fatty acid composition, such as high levels of arachidonic acid and low levels of docosahexaenoic acid, can be another limiting factor in Artemia. Vitamin A, fatty acids and thyroid hormones have all been shown to affect pigmentation in flatfish. They are ligands to nuclear receptors, thyroid hormone receptors, retinoic acid receptors, retinoic X receptors and peroxisomal proliferator-activated receptors, which are members of the superfamily of steroid hormone receptors. The receptors interact with each other to promote gene expression that modulates proliferation and differentiation of cells. Our hypothesis is that these interactions are important for development during flatfish metamorphosis. Very little data exist on the topic of impaired eye migration. However, energy limitation, iodine deficiency and an unbalanced fatty acid composition have been proposed as possible explanations. Here, we review the literature on development of pigment cells and the possible mechanisms behind the effects of vitamin A, fatty acids and thyroid hormone on pigmentation and eye migration during development of Atlantic halibut larvae. 2 : featfish larvae, Atlantic halibut, pigmentation,

title: Nutrition in pregnant or lactating rats programs lipid metabolism in the offspring.
abstract: Epidemiological studies in human show that size in early life is related to blood cholesterol concentrations in adult life, raising the hypothesis that early nutrition programs later lipid metabolism, affecting risk for later vascular disease. Here, we tested the hypothesis that nutrition during pregnancy or lactation in the rat programs lipid metabolism in the offspring, studied in adult life (mean 6 months). Rats (n 35) from normally-fed dams (controls) were compared with (1) rats (n 22) from dams protein-restricted in pregnancy and lactation; (2) rats (n 9) born to normally-fed mother crossed to protein-restricted lactating dams and (3) those (n 9) born of protein-restricted dams and crossed to normally-fed lactating animals. In these latter three groups the offspring showed long-term reduction in plasma cholesterol, HDL-cholesterol and triacylglycerol concentrations compared with controls. The effects were predominantly in males. These findings suggest that in the rat the sensitive period for nutritional programming of cholesterol and triacylglycerol metabolism is both pre- and postnatal (pre-weaning) and that rats may be 'indirectly' programmed by altering the maternal nutritional milieu during gestation or lactation. Whilst it has been hypothesized that early human undernutrition programs risk for vascular disease, one aspect of undernutrition, low maternal protein intake, in this rat model programmed lower plasma cholesterol and triacylglycerol concentrations.

title: Birth-date selection in early life stages of plaice Pleuronectes platessa in the eastern Irish Sea (British Isles)
abstract: For species with extended spawning seasons, short periods of beneficial conditions often lead to disproportionate survival of sub-sets of the offspring. This has been demonstrated for freshwater fish and for marine pelagic species, but has been less frequently tested for other teleost groups. Using offshore plankton surveys, we constructed egg production curves for plaice Pleuro- nectes platessa L. in the eastern Irish Sea. Data from 5 yr showed that spawning began sometime before January and was completed by the end of April. Over these years the timing of peak spawn- ing varied between mid-February and mid-March. In 2001, 2002 and 2003, we also sampled post- settlement plaice from inshore nursery grounds. Otolith micro-increment analyses and temperature- dependent egg development rates were used to back-calculate the birth dates of the settled fish. By June, immigration to the nursery grounds was largely completed and the shape of the reconstructed birth-date distributions corresponded closely to the relevant egg production curves. In contrast to many other studies, there was little evidence for significantly disproportionate survival of specific portions of the egg production curve between spawning and the recently post-settled stages. Such a survival pattern could be generated by a gradual switch from starvation to predation as the principal cause of larval mortality as the spawning season progressed.

title: Age estimation in common sole Solea solea larvae: validation of daily increments and evaluation of a pattern recognition technique
abstract: Close similarities have been found between the otoliths of sea-caught and laboratory- reared larvae of the common sole Solea solea (L.), given appropriate temperatures and nourishment of the latter. But from hatching to mouth formation, and during metamorphosis, sole otoliths have proven difficult to read because the increments may be less regular and low contrast. In this study, the growth increments in otoliths of larvae reared at 12'C were counted by light microscopy to test the hypothesis of daily deposition, with some results verified using scanning electron microscopy (SEM), and by image analysis in order to compare the reliability of the 2 methods in age estimation. Age was first estimated (in days posthatch) from light micrographs of whole mounted otoliths. Counts were initiated from the increment formed at the time of mouth opening (Day 4). The average incremental deposition rate was consistent with the daily hypothesis. However, the light-micrograph readings tended to underestimate the mean ages of the larvae. Errors were probably associated with the low-contrast increments: those deposited after the mouth formation during the transition to first feeding, and those deposited from the onset of eye migration (about 20 d posthatch) during metamorphosis. SEM failed to resolve these low- contrast areas accurately because of poor etching. A method using image analysis was applied to a sub- sample of micrograph-counted otoliths. The image analysis was supported by an algorithm of pattern recognition (Growth Demodulation Algorithm, GDA). On each otolith, the GDA method integrated the growth pattern of these larval otoliths to averaged data from different radial profiles, in order to demod- ulate the exponential trend of the signal before spectral analysis (Fast Fourier Transformation, FFT). This second method both allowed more precise designation of increments, particularly for low-contrast areas, and more accurate readings but increased error in mean age estimation. The variability is prob- ably due to a still rough perception of otolith increments by the GDA method, counting being achieved through a theoretical exponential pattern and mean estimates being given by FFT. Although this error variability was greater than expected, the method provides for improvement in both speed and accu- racy in otolith readings.

title: Otolith Microstructure of the Lesser Sandeel, Ammodytes Marinus
abstract: Daily increments were demonstrated in the sagittae of the lesser sandeel, Ammodytes marinus , maintained in outdoor enclosures. Daily increment formation was also inferred for wild sandeels from an analysis of changes in age composition within a series of field samples. A comparison between sagitta microstructure and morphological development of larval and juvenile sandeels indicated that the first sagitta increment was formed around the time of hatching. Yolk-sac absorption and larval metamorphosis were accompanied by changes in otolith microstructure. The lesser sandeel, Ammodytes marinus (Raitt), is important as a prey item for many species of fish, seabirds and marine mammals (Furness, 1990) and supports major fisheries in the North Sea (Anon., 1991). However, little is known about the ecological processes affecting year-class strength in this species. Otolith microstructure has been demonstrated to be a potentially useful tool for investigating early survivorship in fishes, since otoliths may provide a chronological record of early growth and life-history events (see Campana & Neilson, 1985). This paper examines the usefulness of otolith microstructure for studies of sandeel growth, from investigations of increment periodicity and a comparison between otolith microstructure and morphological development.

title: Changes in otolith microstructure at metamorphosis of plaice, Pleuronectes platessa L.
abstract: Individually reared plaice were monitored by morphologically defined stages until 10 d after metamorphosis. Otoliths (sagittae) were sampled prior to, at, and after metamorphosis. All but one individual had circular sagittae without secondary growth centres (accessory primordia) before metamorphosis. Four days after metamorphosis, all sagittae displayed one or more accessory primordia. The numbers of accessory primordia on the left and right sagittae were not significantly different. Thus, the use of accessory primordia in combination with ageing techniques to back-calculate time to and from metamorphosis of sampled juveniles is validated.

title: Vaterite Sagittal Otoliths in Hatchery-Reared Juvenile Red Drums
abstract: uncommon, have been reported in many fish species. We examined sagittae, lapilli, and asterisci from 1,140 wild and 1,723 hatchery-reared juvenile red drums (Sciaenops ocellatus). Aberrant sagittae were found in 20 hatchery-reared fish. These otoliths, which appeared to be formed from a conglomeration of small spheres, were composed of the vaterite polymorph of calcium carbonate instead of the aragonite polymorph normally found in sagittae. No internal ring structure was present; however, gross external morphological features were similar to normal sagittae. Affected red drums had one normal sagitta, and all lapilli and asterisci were normal. No abnormal sagittae were found in wild fish.

title: Morphometry and composition of aragonite and vaterite otoliths of deformed laboratory reared juvenile herring from two populations
abstract: Vaterite otoliths were sampled from two reared populations (Celtic and Clyde Seas) of juvenile herring Clupea harengus. The crystallography, elemental composition and morphometry were analysed and compared with those of normal aragonite otoliths. The incidence of vaterite otoliths in the juveniles sampled (n ¼ 601) ranged from 7� 8% in the Clyde population to 13� 9% in the Celtic Sea population, and was 5� 5% in the small sample (n ¼ 36) of wild adults examined. In all but one case fish had only one vaterite otolith; the corresponding otolith of the pair was completely aragonite. Although the majority of the juveniles sampled showed craniofacial deformities, there was no link between the skull or jaw malformation and the incidence of vaterite otoliths. All vaterite otoliths had an aragonite inner area, and vaterite deposition began sometime after the age of 90 days. The vaterite otoliths were larger and lighter than their corresponding aragonite partners, and were less dense as a consequence of the vaterite crystal structure. The vaterite areas of the otoliths were depleted in Sr, Na and K. Concentrations of Mn were higher in the vaterite areas. The transition between the aragonite inner areas and the vaterite areas was sharply delineated. Within a small spatial scale (20mm 3 ) in the vaterite areas, however, there was co-precipitation of both vaterite and aragonite. The composition of the aragonite cores in the vaterite otoliths was the same as in the cores of the normal aragonite otoliths indicating that the composition of the aragonite cores did not seed the shift to vaterite. Vaterite is less dense than aragonite, yet the concentrations of Ca analysed with wavelengthdispersive spectrometry (WDS) were the same between the two polymorphs, indicating that Ca concentrations measured with WDS are not a good indicator of hypermineralized zones with high mineral density. The asymmetry in density and size of the otoliths may cause disruptions of hearing and pressure sensitivity for individual fish with one vaterite otolith, however, the presence of vaterite otoliths did not seem to affect the growth of these laboratory reared juvenile herring.

title: Crystalline otoliths in teleosts: Comparisons between hatchery and wild coho salmon (Oncorhynchus kisutch) in the Strait of Georgia
abstract: Otoliths, or ‘ear stones’, are calcium carbonate structures found in all vertebrates. In teleosts, they have a number of sensory functions, including hearing. Daily growth increments of these structures have permitted advanced age and population studies of teleosts. Whereas ‘normal’ otoliths are composed of crystals imbedded within a protein matrix as aragonite, a ‘crystalline’ form of calcium carbonate termed vaterite is also found. A review of the otolith literature demonstrates a significant level of understanding of the structure and function of otoliths, but the cause for crystalline otolith structure remains speculative. Pairs of otoliths from hatchery and wild juvenile and adult coho salmon (Oncorhynchus kisutch) were examined visually for determination of otolith microstructure type. The vateritic or crystalline otoliths were found in much higher percentages in juvenile hatchery-reared coho salmon than in juvenile wild coho salmon, supporting previous studies. There did not seem to be any negative impact on size or survival. There was also no correlation between crystalline otoliths and premature maturation in coho males. A preliminary study of adult coho salmon returning to Big Qualicum and Chilliwack hatcheries showed even higher ratios of vateritic otoliths than observed in juveniles.

title: The effect of vaterite deposition on sound reception, otolith morphology, and inner ear sensory epithelia in hatchery-reared Chinook salmon (Oncorhynchus tshawytscha)
abstract: The inner ear of teleost fishes contains three calcareous structures (otoliths) that are part of the organs for hearing and balance. The largest of these structures, the sagitta, is usually composed of calcium carbonate crystals in the form of aragonite, but the calcium carbonate also occurs less frequently in a clear crystallized form called vaterite. We investigated the functional consequences of otolith crystal structure on hearing in juvenile Chinook salmon (Oncorhynchus tshawytscha) using the auditory brainstem response technique. A significant loss of sensitivity (2.5–6.5 dB) occurred within the primary hearing range (100–300 Hz) among salmon that had at least one vateritic sagitta. Auditory thresholds were not significantly different in fish with one vs. two vaterite sagittae. Crystallized sagittae were significantly larger and less dense than their aragonite counterparts. Saccular epithelium shape and hair bundle orientation patterns did not differ between saccules with different crystal types. Th...

title: Vateritic Sagitta in Wild and Stocked Lake Trout: Applicability to Stock Origin
abstract: Aragonite is the normal form of calcium carbonate found in teleost otoliths, but it is sometimes replaced by vaterite, an alternate crystalline structure. We investigated the assumption that sagittal otoliths with vaterite replacement were unique to stocked lake trout Salvelinus namaycush in the Laurentian Great Lakes. Earlier studies had attributed these abnormalities to stocking stress, and proposed that the presence of vaterite could separate individual unmarked stocked lake trout from their wild counterparts. We examined and described the frequency of vateritic sagittae in two wild and three stocked populations of lake trout from the Great Lakes and a wild population from a remote inland lake in northern Canada. Among lake trout caught 2–12 years after being stocked, prevalence of vateritic sagittae was 66% for Lake Superior fish, 75% for Lake Huron fish, and 86% for Lake Ontario fish. Among wild fish caught, vateritic sagittae were present in 37% of Lake Superior fish, 22% of Lake Huron fish...

title: Morphology and microchemistry of abnormal otoliths in the ayu, Plecoglossus altivelis
abstract: The sagittal otolith morphology and microchemistry of reared juvenile ayu, Plecoglossus altivelis, were examined to describe the occurrence and microchemical characteristics of the abnormal otoliths in this species. Juvenile ayu (N = 31) were collected in June 2004 at three different locations, Wakayama, Kumamoto, and Biwa Lake in Japan, where they were being reared in freshwater aquaculture ponds after having been collected in the wild as larvae. Otolith abnormality was found in the sagittae of 26% (N = 8) of the individuals examined, of which five fish had abnormal otoliths only on one side, while the otolith on the other side was normal. Abnormal otoliths were more transparent and crystalline in appearance with irregular shapes compared to normal ones that were more opaque and less irregular. Abnormal otoliths were divided into two types, semi-abnormal (Type 1) with a normal part in the center, and fully-abnormal (Type 2) that were completely crystalline in appearance. The line transects and whole otolith concentration maps showed that the contents of Sr, Na and K were lower in the abnormal otolith regions compared to the normal ones, while those of Ca and S were almost constant in both. The appearance and microchemical properties of the abnormal ayu otoliths were similar to the abnormal otoliths in other species in which vaterite replaces the aragonite. Abnormal formation of otoliths occurred in ayu from Biwa Lake (30%) and Kumamoto (45%), while the Wakayama samples had no abnormality. The microchemical analyses of the normal and abnormal otoliths indicated that some abnormal otoliths had formed before the fish were captured and transferred to the hatchery, so the possible causes of otolith abnormality in ayu are discussed.

title: Polymorphic crystalline structure of fish otoliths
abstract: Although most otoliths of teleost fishes contain aragonite, a detailed survey of the otoliths of serveral species confirms that other crystalline forms of calcium carbonate occur. Otoliths of Hoplostethus atlanticus, Pagrus major, Macruronus novaezelandiae, Merluccius australis, Congiopodus coriaceus, Kathetostoma giganteum, Argentina elongata, Rhomobosolea tapirina, Neophrynichthys latus, Coelorinchus aspercephalus, Paranothothenia microlepidota, and Gonorhynchus gonorhynchus contained the aragonite, calcite, and vaterite morphs of calcium carbonate in varying proportions. Aragonitic otoliths of Allocyttus niger, Hoplostethus altlanticus, and Pagrus major contained sequences of calcite‐like crystals. The surface of the vateritic otolith of Acipenser brevirostrum is shown in detail. Three classes of information are stored in the crystalline structure of the otolith: shape conservation, coexisting crystal morphs, and consecutive changes in crystal morph. Analysis of this crystalline information supports the hypothesis of control of growth of the otolith by proteins from the sensory epithelium or macula. Protein variation involved may be genetic in origin, or non‐genetic arising from “stuttering” of the translation process. Proteins extracted from vateric and aragonitic morphs of the otolith of Macruronus novaezelandiae showed differences in infared absorption spectra that were consistent with two different amino acid sequences.

title: The effect of salinity on growth rate, survival and swimbladder inflation in gilthead seabream, Sparus aurata, larvae
abstract: The effects of salinity (15–40 p.p.t. salinity; 450–1200 mOsm kg−1) on growth, survival and swim bladder (SB) inflation in gilthead seabream, Sparus aurata, were evaluated. Over the salinity range tested, 1–32 day old seabream larvae were found to be osmotic regulators. Within the range tested, 1 day old larvae maintained an osmotic pressure of 224 mOsm kg−1, significantly lower than 410 mOsm kg−1 in 13 and 24 day old larvae. In addition, water content in larvae was negatively correlated with age, being over 86% in 4 day old larvae and 83% in 32 day old larvae. A negative relationship was found between salinity and survival; as the rearing salinity decreased from 40 to 25 p.p.t., survival increased from 5.3 to 18.6%. Larval final dry weight (DW) and wet weight (WW) were over 16% higher at 25 than 40 p.p.t. salinity. Finally, reduced salinity induced a significant increase in the rate of SB inflation; 65 to 92.5% in 40 and 25 p.p.t. acclimated larvae, respectively.

title: Seawater acclimation of tilapia, Oreochromis spilurus spilurus Günter, fry and fingerlings
abstract: The effect of size and acclimation period to full-strength sea water was investigated using the mortality, growth, chloride cell proliferation and plasma Na concentration in four size groups (0.03-1.6 g mean initial weight) of mixed-parentage, sex-reversed, all-male fry and fingerlings of Oreochromis spilurus spilurus Gunter obtained from University of Stirling. The fish were transferred from fresh water to sea water (36.6%0) through a continuous salinity increase during a 48, 72 or 120 h acclimation period. Survival was high in all groups, although the smallest fish (0.03 g) showed significantly higher mortality than the larger fish. The acclimation regime had no effect on growth rate, but 120 h acclimation gave highest final weight in the newly released group. Regardless of acclimation period, size and plasma Na concentration during the acclimation period, the plasma Na level stabilized within 12 h after full salinity in the two largest size groups. Ovoid voluminous chloride cells proliferated in all groups during the acclimation period, and numbers stabilized with stable salinity. It is concluded in this study that O. spilurus spilurus can successfully be acclimated to sea water with a gradual continuous acclimation period of 48 h, even as newly released fry (0.03 g). However, an acclimation period of 120 h gave the highest final weight for this size group.

title: How should salinity influence fish growth?
abstract: Development and growth (continuous in fish) are controlled by 'internal factors' including CNS, endocrinological and neuroendocrinological systems. Among vertebrates, they also are highly dependent on environmental conditions. Among other factors, many studies have reported an influence of water salinity on fish development and growth. In most species, egg fertilization and incubation, yolk sac resorption, early embryogenesis, swimbladder inflation, larval growth are dependent on salinity. In larger fish, salinity is also a key factor in controlling growth. Do the changes in growth rate, that depend on salinity, result from an action on: (1) standard metabolic rate; (2) food intake; (3) food conversion; and/or (4) hormonal stimulation? Better growth at intermediate salinities (8-20 psu) is very often, but not systematically, correlated to a lower standard metabolic rate. Numerous studies have shown that 20 to >50% of the total fish energy budget are dedicated to osmoregulation. However, recent ones indicate that the osmotic cost is not as high (roughly 10%) as this. Data are also available in terms of food intake and stimulation of food conversion, which are both dependent on the environmental salinity. Temperature and salinity have complex interactions. Many hormones are known to be active in both osmoregulation and growth regulation, e.g. in the control of food intake. All of these factors are reviewed. As often, multiple causality is likely to be at work and the interactive effects of salinity on physiology and behaviour must also be taken into account.

title: A retrospective approach to fractionize variation in body mass of Atlantic cod Gadus morhua.
abstract: Eggs of a single spawning batch from wild-caught Norwegian Atlantic cod Gadus morhua were hatched and first fed on either natural zooplankton or enriched rotifers Brachionus plicatilis during the larval period. Juvenile G. morhua (initial mass 14·2 g) from the two first-feeding groups were then reared for 3 months under a variety of temperature (10 and 14° C) and salinity (15 and 32) combinations. All fish were individually tagged and microsatellite markers were used in a multiplex to trace the pedigree of all fish and body mass variation analysed according to different environmental and genetic sources. After the termination of the laboratory trial, the fish were transferred to land-based tanks and later to sea pens and reared at ambient conditions for 26 months until they were harvested in March 2009. Growth gain from the larval and juvenile periods was persistent during the 26 months of sea pen ongrowing. The final mass of the zooplankton group was 12% higher compared to the B. plicatilis group. Similarly, rearing under a temperature of 14° C and salinity of 15 during the initial 3 month period during the early juvenile stage resulted in 7-13% larger size at harvesting compared to the other three temperature and salinity combinations. The study indicates that the first-feeding method and temperature and salinity manipulation explain nearly 90% of the body mass variation explained by the model. The genetic effect (measured as body mass variation within the families studied) only accounted for c. 2% during the initial rearing period, whereas it has a large effect on growth variation (30%) during the long-term rearing at ambient conditions. Sex proportion and final maturation did not differ between family groups, and no interaction between sex and family group was seen.

title: The influence of temperature on the embryonic development of three cyprinid fishes, Abramis brama, Chalcalburnus chalcoides mento and Vimba vimba
abstract: The influence of temperature on the embryonic development of three European cyprinid fishes, Abramis brama, Chalcalburnus chalcoides mento and Vimba vimba, was studied with respect to hatching efficiency and the functional relationship with the duration of development. The functional dependance of the duration of development on temperature is well described by a power function with a correcting constant for temperature. Comparison with published data for other cyprinids shows that the response of embryos to temperature, together with the various temperature controlled responses of adult fishes, is a valuable aid for the ecological characterization of the different species.

title: Hatching time in spherical, pelagic, marine fish eggs in response to temperature and egg size
abstract: SynopsisThe relationship between egg diameter ((π, mm), temperature (T, °C) and egg development time to hatching (D, in days) was established for approximately spherical, pelagic marine fish eggs as log10D = 7.10 + 0.608 log10 π − 4.09 log10 (T + 26), which explains 82% of the variance of a data set of 140 cases, covering 84 species of teleost fishes, for temperatures from 2.8 to 29.5° C and eggs of 0.6 to 3.4 mm diameter.From this relationship it appears that temperature has 4.7 times as much effect on development time as egg diameter when all variables are expressed in standard deviation units. A discussion of these and related factors is given.

title: Egg Size and Viability and Seasonal Offspring Production of Young Atlantic Cod
abstract: Experiments monitoring the egg and larval production of captive northwest Atlantic cod Gadus morhua indicated that first-time spawners perform poorly compared to second-time spawners. They breed for a shorter period, produce fewer egg batches, exhibit lower fecundity, and produce smaller eggs with lower fertilization and hatching rates; moreover, their larvae are less likely to hatch in environmental conditions favorable for survival. Batch-average egg diameters of first-time spawners underwent a continuous rapid decline between initial and terminal batches, whereas second-time spawners commonly exhibited a parabolic curve (slight increase followed by a decrease). Seasonal composite hatching rates of all eggs spawned by first- and second-time spawners were 13 and 62%, respectively. Larval production per maternal gram was an order of magnitude greater for second-time than for first-time spawners (200 versus 20 larvae/g). Batch-average egg diameter declined 11% and larval dry weight declined 29% fr...

title: Influence of Temperature on Energy Utilization of Embryonic and Prolarval Tautog, Tautoga onitis
abstract: When daily energy budgets were calculated by two methods for tautog, Tautoga onitis, from fertilization to complete yolk absorption at three temperatures (16, 19, and 22 C), neither method indicated a deficiency of yolk energy for growth and metabolism prior to exogenous feeding capability. Deficits were indicated on the day of feeding capability at all temperatures by the oxygen consumption method, the more precise but less inclusive of the two. The dry weight method, which gave the more conservative estimates, indicated no deficit for the entire period of yolk absorption at 16 C, a deficit one day after feeding capability at 19 C, and a deficit on the day of feeding capability at 22 C.Regression analysis of oxygen consumption indicated that 22 C may be in the upper temperature limits for normal metabolism by embryonic and prolarval tautog. Yolk utilization efficiencies and postlarval size at complete yolk absorption tended to be inversely related to temperature. The ecological implications and the effec...

title: Effect of temperature on yolk utilization of Channa striatus
abstract: 1. 
1.|Embryonic development of Channa striatus lasted or 56, 48, 36 and 30 h at 20, 24, 28 and 32°C, respectively. 
 
2. 
2.|Larvae reared at 32°C utilized the adhering yold much faster (102 h) than those reared at 20°C (156 h). 
 
3. 
3.|No significant differences were found in total body length and water content. 
 
4. 
4.|However, the ash content and ash free dry weight increased with increasing temperature. 
 
5. 
5.|The yolk utilization efficiency increased with increasing temperature at hatching and decreased from hatching to yolk absorption.

title: Feeding, physiology and growth responses in first-feeding gilthead seabream (Sparus aurata L.) larvae in relation to prey density
abstract: The effects of prey density (0.1, 1 and 10 rotifer ml −1 ) on feeding, oxygen uptake, growth, survival and energetic efficiencies in Sparus aurata larvae were investigated. Dry weights and total lengths at day 15 after hatching were similar in larvae fed with 10 and 1 rotifer ml −1 but were significantly higher than in larvae fed with 0.1 rotifer ml −1 . These differences were a consequence of a delay in starting feeding and growth in larvae fed with the lowest rotifer concentrations tested. Growth rates were similar in larvae fed on 0.1 and 1 rotifer ml −1 , while larvae fed with 10 rotifers ml −1 showed the smallest growth rate. Survival decreased markedly (from 91 to 7%) when prey density decreased. Ingestion rates of the actively feeding larvae increased with age at the three prey densities but larvae fed with 0.1 rotifer ml −1 showed lower ingestion rates. The specific oxygen uptake increased from hatching up to 35–40 μg dry weight; above this size the specific oxygen consumption remained almost constant. Survival at the end of the experimental period was closely related to the percentage of larvae able to start feeding at day 6. In larvae fed with 0.1 rotifer ml −1 , the energy ingested during the first days of feeding roughly met maintenance requirements. Therefore, the survival of larvae able to start feeding is not guaranteed at such low prey density.

title: Temperature effects on cranial deformities in European sea bass, Dicentrarchus labrax (L.)
abstract: The present study investigates the effect of water temperature on the development of deformities during embryonic and larval stages of European sea bass, Dicentrarchus labrax (L.). Two temperature conditions were examined in duplicate by studying the presence of skeletal deformities in a sample of 45 to 51 fish taken from each population at the end of the rearing trials [20–23 mm total length (TL)]. The results indicated that water temperature during the embryonic and larval phase has significant effects on deformation of the branchiostegal rays (P   0.05). At 15°C, 27.2–33.4% of the examined fish had branchiostegal rays of abnormal shape and/or orientation, whereas at 20°C this deformity had a frequency of only 4.0–4.1%. The frequency distribution graph of branchiostegal counts demonstrated a significant deviation in the deformed fish from the normal (seven rays on each side of the body) phenotype at both temperatures tested. This deviation was mainly expressed as a lack of one to four rays (56.7% of deformed fish), or the formation of one extra ray (26.7% of deformed fish) (P < 0.001, G-test). The results are discussed in respect to the possible mechanisms of temperature effects on the development of skeletal deformities.

title: Environment and plasticity of myogenesis in teleost fish
abstract: Embryonic development in teleosts is profoundly affected by environmental conditions, particularly temperature and dissolved oxygen concentrations. The environment determines the rate of myogenesis, the composition of sub-cellular organelles, patterns of gene expression, and the number and size distribution of muscle fibres. During the embryonic and larval stages, muscle plasticity to the environment is usually irreversible due to the rapid pace of ontogenetic change. In the early life stages, muscle can affect locomotory performance and behaviour, with potential consequences for larval survival. Postembryonic growth involves myogenic progenitor cells (MPCs) that originate in the embryo. The embryonic temperature regime can have long-term consequences for the growth of skeletal muscle in some species, including the duration and intensity of myotube formation in adult stages. In juvenile and adult fish, abiotic (temperature, day-length, water flow characteristics, hypoxia) and biotic factors (food availability, parasitic infection) have complex effects on the signalling pathways regulating the proliferation and differentiation of MPCs, protein synthesis and degradation, and patterns of gene expression. The phenotypic responses observed to the environment frequently vary during ontogeny and are integrated with endogenous physiological rhythms, particularly sexual maturation. Studies with model teleosts provide opportunities for investigating the underlying genetic mechanisms of muscle plasticity that can subsequently be applied to non-model species of more ecological or commercial interest.

title: What determines growth potential and juvenile quality of farmed fish species
abstract: Enhanced production of high quality and healthy fry is a key target for a successful and competitive expansion of the aquaculture industry. Although large quantities of fish larvae are produced, survival rates are often low or highly variable and growth potential is in most cases not fully exploited, indicating significant gaps in our knowledge concerning optimal nutritional and culture conditions. Understanding the mechanisms that control early development and muscle growth are critical for the identification of time windows in development that introduce growth variation, and improve the viability and quality of juveniles. This literature review of the current state of knowledge aims to provide a framework for a better understanding of fish skeletal muscle ontogeny, and its impact on larval and juvenile quality as broadly defined. It focuses on fundamental biological knowledge relevant to larval phenotype and quality and, in particular, on the factors affecting the development of skeletal muscle. It also discusses the available methodologies to assess growth and larvae/juvenile quality, identifies gaps in knowledge and suggests future research directions. The focus is primarily on the major farmed non-salmonid fish species in Europe that include gilthead sea bream, European sea bass, turbot, Atlantic cod, Senegalese sole and Atlantic halibut.

title: New insights into temperature-induced white muscle growth plasticity during Dicentrarchus labrax early life: a developmental and allometric study
abstract: Dicentrarchus labrax is a major finfish of interest in Mediterranean aquaculture. As the development of its hatchery production had gone with an increase of its larval rearing temperature, we studied the effect of a constant high (20°C) and two lower (13 and 15°C) temperatures on its early white muscle growth, with developmental and allometric approaches. D. labrax, sampled at hatching and at three developmental stages corresponding to main events in fish early life (first exogenous feeding, notochord flexion and completion of fin ray counts), were histologically processed in order to follow changes in the white myotomal muscle size, cellularity and localisation of proliferative nuclei. Morphometric analyses showed that, for 13, 15 and 20°C incubated/reared D. labrax, the total cross-sectional area of white muscle increased slowly in eleutheroembryos shorter than 5.2 mm, and four times faster in longer fish (free-swimming larvae). White muscle growth occurred both by hypertrophy (increase in muscle fibre size) and hyperplasia (increase in total number of muscle fibres) in all sampled fish, but both processes were higher in free-swimming larvae than in eleutheroembryos. The morphometric establishment of a higher hyperplastic growth of white muscle in free-swimming larvae was confirmed at immunological level by a high occurrence of proliferative nuclei. Early thermal environment was demonstrated to affect the growth process of white muscle on a different way in eleutheroembryos and free-swimming larvae: in the former, white muscle hypertrophy was stimulated at 20°C and hyperplasia at lower temperature, whereas in the latter, both white muscle hypertrophy and hyperplasia were more stimulated at 20°C than at lower temperatures. This was verified at once when the fish length and the white muscle total cross-sectional area were used as explicative variables.

title: Larval programming of post-hatch muscle growth and activity in Atlantic salmon (Salmo salar)
abstract: Larval muscle development in Atlantic salmon is known to be affected by temperature; however, the long term effects and possible mechanisms involved are less well understood. Therefore, the aim of this study was to evaluate the influence of egg incubation temperature on post-hatch muscle growth and fish activity. Salmon eggs were incubated at either 10°C or 5°C from fertilization until hatching, then subsequently both groups were reared at 5°C. Fish from both groups were sampled at the eyed stage, 6 and 21 weeks after first feeding, for muscle cellularity analysis and immunocytochemistry. In addition, to try to establish a mechanism for altered growth, the activity of the fish was measured at 3, 6 and 21 weeks after first feeding. Our results demonstrate that whereas fish incubated at 10°C grow faster, the fish incubated at 5°C show a more sustained period of muscle growth and by 21 weeks are significantly longer, heavier and have more muscle fibres than those fish incubated at a higher temperature. We also demonstrate that fish raised at 5°C show increased food seeking activity throughout development and that this may explain their sustained growth and muscle development. These results taken together, demonstrate that egg incubation temperature up to hatching in salmon is critical for longer term muscle growth, twinned with increased activity. This is of interest to the aquaculture industry in term of the production of good quality fish protein.

title: Influence of swim training on cardiac activity, tissue capillarization, and mitochondrial density in muscle tissue of zebrafish larvae.
abstract: Larval zebrafish (Danio rerio) of two different age classes ("swim-up" larvae, 9 days old; "free-swimming" larvae, 21 days old) were exposed to either an endurance/continuous training or interval training. Control animals were kept in stagnant water. A comparison of cardiac activity of trained (either endurance or interval) and untrained animals at the end of the training regime revealed no differences in heart rate, end-diastolic and end-systolic ventricular volume, and cardiac output. Training also had no influence on the concentration of erythrocytes in the blood. Thus, at the level of total oxygen transport in the blood, training did not provoke any improvement during the first 32 days of development. Significant changes, however, were observed at the tissue level. In free-swimming larvae [i.e., between 21 and 32 days postfertilization (dpf)] endurance training increased the capillarization of both axial muscle caudal to the anus and the tail fin. In addition, mitochondrial density of red and intermediate muscle fibers increased significantly. In contrast to capillarization, even swim-up larvae, trained between 9 and 15 dpf, were affected. The observed increase in mitochondrial content indicates a high demand for oxygen and energy-rich metabolites for oxidative phosphorylation. In older larvae, this is met by the increase in capillarization that improves the blood supply and with it the required oxygen and metabolite supply of muscle tissue. Both of these adaptational changes result in a reduction of diffusion distances (between capillary and muscle fiber as well as mitochondria) and may contribute to a higher resistance toward oxygen deficiency. Furthermore, this study indicates that plasticity of muscle tissue is already established in early stages of development at both the tissue and cellular levels.

title: Muscle cellularity in relation to somatic growth in the European sea bass Dicentrarchus labrax (L.)
abstract: Sea bass, Dicentrarchus labrax (L.), juveniles (1.32 g) grew at a rate of 4.2% per day, when fish were fed ad libitum and reared at optimal thermal conditions (ambient sea water temperature, 20-26°C). At 13°C, feeding and spontaneous activity were severely restricted and somatic growth was reduced to 0.6% per day. Over a period of 6 weeks, both muscle fibre hyperplasia and nuclear division were higher in the ambient-temperature group compared with the fish reared at 13°C. Despite the differences in growth rate and spontaneous activity, muscle fibre hyperplasia was paralleled by nuclear division in the lateral axial muscle in both temperature groups and the number of nuclei per myofibre did not differ significantly between the two temperature groups. It is concluded that at optimal thermal and feeding conditions, somatic growth of sea bass juveniles is mediated through an increase in the number of nuclei and muscle fibres.

title: Embryonic temperature modulates muscle growth characteristics in larval and juvenile herring
abstract: The influence of embryonic and larval temperature regime on muscle growth was investigated in Atlantic herring (Clupea harengus L.). Eggs of spring-spawning Clyde herring were incubated at 5 degrees C, 8 degrees C or 12 degrees C until hatching and then reared until after metamorphosis at rising temperatures to simulate a seasonal warming. Metamorphosis to the juvenile stage was complete at 37 mm total length (TL), after an estimated 177 days as a larva at 5 degrees C, 117 days at 8 degrees C and 101 days at 12 degrees C. Growth rate and the development of median fins were retarded in relation to body length at 5 degrees C compared with 8 degrees C and 12 degrees C. Between hatching (at 8-9 mm TL) and 16 mm TL, there was a threefold increase in total muscle cross-sectional area, largely due to the hypertrophy of the embryonic red and white muscle fibres. The recruitment of additional white muscle fibres started at approximately 15 mm TL at all temperatures, and by 37 mm was estimated to be 66 fibres day-1 at 5 degrees C and 103 fibres day-1 at 8 degrees C and 12 degrees C. Peptide mapping studies revealed a change in myosin heavy chain composition in white muscle fibres between 20 and 25 mm TL. Embryonic red muscle fibres expressed fast myosin light chains until 24-28 mm TL at 5 degrees C and 22 mm TL at 12 degrees C, and new red fibres were added at the horizontal septum starting at the same body lengths. Following metamorphosis, the total cross-sectional area of muscle was similar at different temperatures, although the number of red and white fibres per myotome was significantly greater at the warmest than at the coldest regime. For example, the mean number of white muscle fibres per myotome in 50 mm TL juveniles was calculated to be 23.4 % higher at 12 degrees C (12 065) than at 5 degrees C (9775). In other experiments, spring-spawning (Clyde) and autumn-spawning (Manx) herring were reared at different temperatures until first feeding and then transferred to ambient seawater temperature and fed ad libitum for constant periods. These experiments showed that, for both stocks, the temperature of embryonic development influenced the subsequent rate of muscle fibre recruitment and hypertrophy as well as the density of muscle nuclei. Labelling experiments with 5'-bromo-2-deoxyuridine showed that both the hypertrophy and recruitment of muscle fibres involved a rapidly proliferating population of myogenic precursor cells. The cellular mechanisms underlying the environmental modulation of muscle growth phenotype are discussed.

title: Early temperature effects on muscle growth dynamics and histochemical profile of muscle fibres of sea bass Dicentrarchus labrax L., during larval and juvenile stages
abstract: Recently, it has been found that the thermal experience during the earliest phases of development could determine the larval and postlarval growth characteristics of teleosts. In order to investigate the effects of the early temperature regime on the advanced stages of growth of sea bass, Dicentrarchus labrax L., this species was reared during the vitelline phase at two temperatures: natural temperature (i15 jC) and 17.7F0.1 jC, and then larvae transferred to common temperature (natural temperature). Muscle growth was studied by morphometric and histochemical techniques (mATPase and NADH-TR). Body length and body mass were also measured. During the vitelline phase, muscle growth was similar in both experimental groups, but at 25 days, both hypertrophy and hyperplasia of white muscle fibres were greater in the prewarmed group (p<0.05). At the end of metamorphosis (80 days) and at 120 days, the average diameter of white muscle fibres, as well as the body length, were greater in the prewarmed group (p<0.05), but the number of white fibres did not differ significantly between groups. The morphological mosaic of white muscle fibres was observed at the end of metamorphosis, and the histochemical mosaic appeared gradually since the early postlarval stages. Thus, at 120 days, some specimens in both experimental groups showed three or four different mATPase staining white fibres: low (L), moderate (M), high (H) and/or very high (vH), whereas in other specimens, only L or M mATPase activity fibres were observed. Early T influenced the histochemical maturity of the

title: Functional consequences and population differences in the developmental plasticity of muscle to temperature in Atlantic herring Clupea harengus
abstract: The development of the axial muscles was investigated in Atlantic herring Clupea harengus from 2 spring-spawning (Clyde and Blackwater) and 1 autumn-spawning (Manx) popula- tions in 2 successive years. The results indicate significant developmental differences between stocks. Myofibril synthesis and the development of acetylcholinesterase activity at the neuromuscular junctions occurred later with respect to embryonic stage at 5°C than at 8 and 12°C in Clyde herring, with much less pronounced differences between temperatures for the other populations. In a second series of experiments, Clyde herring were incubated at 5 and 12°C until shortly after hatching, and then reared at ambient temperature in triplicate tanks. The 5°C group had a faster growth rate than the 12°C group with respect to effective day-degrees from hatch. However, flexion of the notochord and the development of the dorsal and anal fin ray muscles occurred at shorter body lengths in the 12 than 5°C groups. The maximum escape velocity of larvae during fast-starts was determined using a high-speed video. Over the size range 11.2 to 18.5 mm total length, the adjusted mean maximum velocity was 24% higher in larvae hatched from 12 than 5°C eggs. Cruising swimming behaviour also differed between groups with the 12°C larvae showing reduced yaw relative to the 5°C larvae, indicating a more developmentally advanced sub-carangiform style of locomotion.

title: Temperature sensitivity of skeletal ontogeny in Oreochromis mossambicus
abstract: Sensitivity of skeletal ontogenesis to temperature was assessed in Mozambican tilapia Oreochromis mossambicus and culture temperature manipulations (22, 27 and 32° C) were used to establish if age or length gave the most suitable metric for standardization. Oreochromis mossambicus larval growth was composed of two growth stanzas: an initial period of rapid growth, followed by a slower growth phase. Irrespective of culture temperature chondrogenesis occurred during the first rapid growth phase and ossification was initiated during the second slower growth phase. The sequence of events and rate at which ossification occurred was much more sensitive to temperature than chondrogenesis. Cumulative counts provide a useful developmental index for skeletal ontogenesis; overall, age (effective days-degrees) gave the best estimation of developmental status during chondrogenesis and the initiation of ossification, although standard length (as log10LS) was a better metric for completion of ossification. The timing of development of functionally important structures, such as Meckel's cartilage, the branchial arches, the centra and the cleithrum important for breathing, feeding and swimming, was well conserved at all temperatures and may be a good index of teleost developmental stages.

title: Osteological development of the vertebral column and of the fins in Pagellus erythrinus (L. 1758). Temperature effect on the developmental plasticity and morpho-anatomical abnormalities
abstract: The osteological ontogeny of Pagellus erythrinus (Linnaeus, 1758) and its temperature-induced plasticity is studied. The ontogeny of the skeleton follows the pattern described in all other Sparidae studied so far: the onset of the ontogeny of the vertebral column and the caudal fin is at 4.3 mm total length (TL) and the completion at 17.6 and 15.4 mm TL, respectively. Dorsal and anal fin development appears at about 5.2 mm TL and completes at 7.5 mm TL. The pelvic are the last fins to develop (7.5–11.5 mm TL). Although the pectorals are the only fins present before first feeding, their ontogeny is completed only after 12.0 mm TL. Temperature affects the developmental rate of the skeleton giving as much as 10.9% of differentiation in total length between 16 and 21 °C (caudal lepidotrichia), 9.2% between 16 and 18 °C (caudal lepidotrichia) and 6.7% between 18 and 21 °C (pelvic lepidotrichia). The role of temperature in developmental plasticity is enhanced by the induction of abnormalities mainly in the area of the caudal fin, which reached (in total) 75% occurrence in the higher temperature regime (23 °C) tested. The results of the skeletal ontogeny are compared with the pattern of development of the Sparidae, and are discussed in respect to the role of temperature in the developmental plasticity of fish.

title: Role of nutrients in skeletal metabolism and pathology in fish — An overview
abstract: The development of skeletal disorders in larval and juvenile fish is linked to a poorly understood relationship between nutrition, environment and genetic factors. These disorders are often a complex mixture, including vertebral and spinal malformations. In marine fish hatcheries, neck deformities, and vertebral and spinal disorders are most abundant. Fish bones are found in all shapes and sizes. They have a wide diversity of cartilages and bones with chondro- and osteogenic cells. Bone and cartilages may develop during embryonic, larval, juvenile or adult stages under normal ontogeny as well as during pathological states, wound repair and bone regeneration. Three types of cells play a significant role in the bone remodeling process and bone formation, resorption and mineralization; osteoblasts (bone forming cells), osteocytes (entrapped inside the bone matrix), and osteoclasts (multinucleated bone resorbing cells). Unfortunately, limited research effort has been directed to characterize the pathological changes associated with disorders linked to nutrient deficiencies in fish. Fish contain a significant amount of lipid and micronutrients in skeletal tissues including collagen, which are particularly susceptible to lipid peroxidation. A brief review of the current state of knowledge on the role of key nutrients in the pathogenesis of skeletal deformities with an emphasis on minerals (calcium, phosphorus and trace elements), vitamins (A, D, C, E and K), lipid and nutrient interactions is presented. Bone deformities associated with nutrient deficiencies and toxicities in various fish can provide an excellent model to study gene action, cell differentiation, morphogenesis, species differences in phenotypic expression of genetic abnormalities, deposition of calcium, phosphorus and other trace elements in cartilage and skeletal tissues in response to vitamins and hormones and nutrient interactions.

title: Intake of high levels of vitamin A and polyunsaturated fatty acids during different developmental periods modifies the expression of morphogenesis genes in European sea bass (Dicentrarchus labrax).
abstract: The effect of the feeding period on larval development was investigated in European sea bass larvae by considering the expression level of some genes involved in morphogenesis. Larvae were fed a control diet except during three different periods (period A: from 8 to 13 d post-hatching (dph); period B: from 13 to 18 dph; period C: from 18 to 23 dph) with two compound diets containing high levels of vitamin A or PUFA. European sea bass morphogenesis was affected by these two dietary nutrients during the early stages of development. The genes involved in morphogenesis could be modulated between 8 and 13 dph, and our results indicated that retinoids and fatty acids influenced two different molecular pathways that in turn implicated two different gene cascades, resulting in two different kinds of malformation.  A delayed development, reducing the number of vertebral segments and disturbing bone formation in the cephalic region. These malformations were correlated to an upregulation of retinoic acid receptor gamma, retinoid X receptor (RXR) alpha and bone morphogenetic protein (BMP)4. An excess of PUFA accelerated the osteoblast differentiation process through the upregulation of RXRalpha and BMP4, leading to a supernumerary vertebra. These results suggest that the composition of diets devoted to marine fish larvae has a particularly determining effect before 13 dph on the subsequent development of larvae and juvenile fish.

title: Optimal levels of dietary vitamin A for reduced deformity incidence during development of European sea bass larvae (Dicentrarchus labrax) depend on malformation type
abstract: The purpose of this study was to examine the impact of graded levels of dietary vitamin A on sea bass larval performances and to determine optimal retinol levels at different larval stages to avoid specific skeletal malformations. Retinol was incorporated into larval feeds at 0, 5, 10, 15, 25, 35 and 70 mg kg − 1 dry matter (giving rise to RET0, RET5, RET10, RET15, RET25, RET35, RET70 groups, respectively). Analysis of the several types of deformities affecting the skull, vertebral column or fins of the fish were observed depending on experimental groups. On one hand, the incidence of skull malformations affecting the maxillary and premaxillary bones, dentaries, operculum, branchiostegal rays and glossohyal was lower for the RET0 and RET5 groups. On the other hand, the frequency of vertebral (slight fusions and kyphosis of the anterior five vertebrae, over-mineralization and lordosis of the haemal vertebrae, the transformation of the last pre-haemal vertebra into haemal) and fin (deformations of the dorsal and anal pterygiophores, deviations of the related rays, modifications of the anatomy of the caudal supporting elements, partial to complete lack of the pelvic fins) deformities were lower for the RET5–70 groups. In the RET0 group, lower level of Hoxd-9 expression coincided with partial or complete lack of pelvic fin. Our results suggest that the optimal level of retinol for harmonious ontogenesis fluctuate along sea bass larvae development and that inadequate dietary retinol levels alters morphogenesis through the modulation of Hox gene expression, at least for the pelvic fin.

title: The effect of behavioural and morphological plasticity on foraging efficiency in the threespine stickleback (Gasterosteus sp.)
abstract: We conducted an experiment to assess the change in foraging efficiency resulting from diet-induced morphological and behavioural plasticity in a species of freshwater, threespine stickleback (Gasterosteus sp.). Different degrees of morphological and behavioural change were induced using two prey items commonly found in the diet of this species, allowing us to estimate the relative importance of each type of plasticity. The purpose of the experiment was twofold. First, earlier work had suggested that diet variability might be an important factor in the evolution of trophic morphological plasticity in sticklebacks. The present results extend this work by revealing the adaptive significance of morphological plasticity. The current experiment also qualitatively assessed the compatibility of the time scale of morphological change with that of the natural resource variability experienced by this species. The results indicate that diet-induced plasticity improves foraging efficiency continuously for up to 72 days of prey exposure. This is probably due in part to plasticity of the external trophic morphology but our results also suggest a complex interplay between morphology and behaviour. The time scale appears to be matched to that of natural diet variability although it is possible that some traits exhibit non-labile plasticity. Our discussion highlights the important distinction between conditions favouring the evolution of labile versus non-labile plasticity. The second objective of the experiment was to determine the relative importance of morphological and behavioural plasticity. Few studies have attempted to quantify the adaptive significance of morphological plasticity and no study to our knowledge has separated the effects of morphological and behavioural plasticity. Our experiment reveals that both behavioural and morphological plasticity are important and it also suggests a dichotomy between the two: behavioural plasticity predominately affects searching efficiency whereas morphological plasticity predominately affects handling efficiency.

title: Prolonged fasting and cortisol reduce myostatin mRNA levels in tilapia larvae; short-term fasting elevates.
abstract: Myostatin negatively regulates muscle growth and development and has recently been characterized in several fishes. We measured fasting myostatin mRNA levels in adult tilapia skeletal muscle and in whole larvae. Although fasting reduced some growth indexes in adults, skeletal muscle myostatin mRNA levels were unaffected. By contrast, larval myostatin mRNA levels were sometimes elevated after a short-term fast and were consistently reduced with prolonged fasting. These effects were specific for myostatin, as mRNA levels of glyceraldehyde-3-phosphate dehydrogenase and glucose-6-phosphatase were unchanged. Cortisol levels were elevated in fasted larvae with reduced myostatin mRNA, whereas in addition immersion of larvae in 1 ppm (2.8 microM) cortisol reduced myostatin mRNA in a time-dependent fashion. These results suggest that larval myostatin mRNA levels may initially rise but ultimately fall during a prolonged fast. The reduction is likely mediated by fasting-induced hypercortisolemia, indicating divergent evolutionary mechanisms of glucocorticoid regulation of myostatin mRNA, since these steroids upregulate myostatin gene expression in mammals.

title: Myostatin, a Negative Regulator of Muscle Growth, Functions by Inhibiting Myoblast Proliferation*
abstract: Myostatin, a member of the transforming growth factor-β (TGF-β) superfamily, has been shown to be a negative regulator of myogenesis. Here we show that myostatin functions by controlling the proliferation of muscle precursor cells. When C2C12 myoblasts were incubated with myostatin, proliferation of myoblasts decreased with increasing levels of myostatin. Fluorescence-activated cell sorting analysis revealed that myostatin prevented the progression of myoblasts from the G1- to S-phase of the cell cycle. Western analysis indicated that myostatin specifically up-regulated p21Waf1, Cip1, a cyclin-dependent kinase inhibitor, and decreased the levels and activity of Cdk2 protein in myoblasts. Furthermore, we also observed that in myoblasts treated with myostatin protein, Rb was predominately present in the hypophosphorylated form. These results suggests that, in response to myostatin signaling, there is an increase in p21 expression and a decrease in Cdk2 protein and activity thus resulting in an accumulation of hypophosphorylated Rb protein. This, in turn, leads to the arrest of myoblasts in G1-phase of cell cycle. Thus, we propose that the generalized muscular hyperplasia phenotype observed in animals that lack functional myostatin could be as a result of deregulated myoblast proliferation.

title: Carbohydrates in fish nutrition: effects on growth, glucose metabolism and hepatic enzymes
abstract: The utilisation of dietary carbohydrates and their effects on fish metabolism are reviewed. Details on how dietary carbohydrates affect growth, feed utilisation and deposition of nutrients are discussed. Variations in plasma glucose concentrations emphasizing results from glucose tolerance tests, and the impact of adaptation diets are interpreted in the context of secondary carbohydrate metabolism. Our focus then shifts to selected aspects of hormonal regulation of carbohydrate metabolism and dietary carbohydrates and their variable effects on glycogen and glucose turnover. We analyse the interaction of carbohydrates with other nutrients, especially protein and protein sparing, and de novo synthesis of lipids, and finish by discussing the correlation of dietary carbohydrates with fish health.

title: Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) digest and metabolize nutrients differently. Effects of water salinity and dietary starch level
abstract: Atlantic salmon (average weight: 199 g, group size: 55) and rainbow trout (average weight: 565 g, group size: 25) of the same age, kept in freshwater (FW) or full-strength seawater (SW), were fed diets containing 7% (LCH) or 23% (HCH) precooked maize starch, replacing dietary protein. The fish were fed ad libitum in triplicate groups. Feed intake was monitored. The species appeared quite similar regarding thermal growth coefficient (TGC); 3.09% and 3.14% for the salmon and the trout, respectively, and retention of digestible protein; 45.9% and 43.9%, and energy; 45.7% and 46.9%, respectively. Rainbow trout showed higher digestibilities than Atlantic salmon, most pronounced with the HCH diet. Digestibilities were negatively affected by starch level in Atlantic salmon, less severe in rainbow trout. Hepatosomatic index was higher in rainbow trout than Atlantic salmon when fed the HCH diet. Liver lipid, liver glycogen, plasma glucose and plasma insulin were higher in rainbow trout than in Atlantic salmon at both starch levels. The difference was greatest with the HCH diet. In Atlantic salmon, the HCH diet elevated only liver glycogen. Specific activities of maltase and iso-maltase were substantially higher in the rainbow trout than in the Atlantic salmon. In seawater, Atlantic salmon showed higher TGC than in FW. Rainbow trout seemed unaffected by salinity. Salinity did not affect body composition significantly, except for ash, which was higher in SW than in FW in both species. Digestibility of protein, starch, freeze dry matter and energy was higher in FW than in SW. For starch digestibility, this was true for Atlantic salmon only. Retention of protein, but not energy, was higher in SW than FW. No significant effects of water salinity were observed with regards to condition factor, dress-out percentage, HSI, liver lipid, liver glycogen, plasma glucose or plasma insulin. The high starch diet reduced TGC in both species with no significant effect on FCR or body composition. Negative effects of starch level were observed on digestibility of macronutrients and energy in Atlantic salmon, but not in rainbow trout. Retention of digestible energy was not significantly affected by starch level, indicating that starch energy can replace protein energy. The results clearly showed stimulating effects of dietary starch on mucosal maltase, sucrase and lactase activities in both Atlantic salmon and rainbow trout, contrary to results of previous research. The response in leucine aminopeptidase showed adaptation according to protein level.

title: Dietary regulation of activities and mRNA levels of trypsin and amylase in sea bass (Dicentrarchus labrax) larvae
abstract: Specific activities and mRNA levels of trypsin and amylase were studied in sea bass larvae. From day 20 to day 40, Dicentrarchus labrax were fed two rations of one day old Artemia: satiation (LP) and one-eighth of the satiation ration (LP/8) or two isoenergetic compound diets that varied in protein (30 and 60%) and carbohydrate (37 and 7%) content (FP30 and FP60 respectively). Trypsin mRNA levels and specific activities were mainly influenced by the nature of dietary protein and the Artemia ration. By using fish meal as protein source, dietary protein concentration did not affect either mRNA level nor specific activity of trypsin. These results suggested that the trypsin synthesis was not affected at a transcriptional level by the protein ration, i.e., Artemia ration. Decrease in amylase mRNA observed from day 29 in the four dietary groups suggested that this decrease in amylase expression is genetically programmed during sea bass larvae development. Nevertheless, the composition and the quantity of the diet influenced the amylase specific activities revealing primarily translational regulation of amylase. This study shows for the first time that the molecular mechanisms which control the dietary adaptation of trypsin and amylase are independently regulated, age-dependent and influenced by the composition and the quantity of the diet.

title: Activities of selected digestive enzymes during larval development of large yellow croaker (Pseudosciaena crocea)
abstract: Large yellow croaker is an important marine fish species for aquaculture in China. Larval rearing of this species is still based on live prey feeding. The aim of this study was to describe the onset and development of the main digestive enzymes during larval development of this species, in order to obtain essential data for the formulation of a compound diet adapted to larvae. Enzymatic assays were conducted from day 1 post hatching to day 40 in larvae fed the following live prey sequence: rotifers, Artemia nauplii, copepods, and then frozen copepods. Assays were done on whole larvae until day 19, then on dissected larvae and on purified brush border membranes of intestine. Pancreatic enzymes, amylase, and trypsin, showed high activities from early stages, even before mouth opening. Mechanisms of pancreatic secretion were achieved from day 19. Enzymes of brush border membranes, alkaline phosphatase, and aminopeptidase N, increased abruptly between day 23 and 25, showing maturation of the intestinal digestive process. These data, together with data describing mouth enlargement during development, suggest that large yellow croaker larvae could be fed successfully with a compound diet from early stages, at least from day 23.

title: Gluconeogenic enzyme gene expression is decreased by dietary carbohydrates in common carp (Cyprinus carpio) and gilthead seabream (Sparus aurata).
abstract: Our objective is to understand the low metabolic utilization of dietary carbohydrates in fish. We compared the regulation of gluconeogenic enzymes at a molecular level in two fish species, the common carp (Cyprinus carpio) and gilthead seabream (Sparus aurata), known to be relatively tolerant to dietary carbohydrates. After cloning of partial cDNA sequences for three key gluconeogenic enzymes (glucose-6-phosphatase (G6Pase), fructose biphosphatase (FBPase) and phosphoenolpyruvate carboxykinase (PEPCK) in the two species, we analyzed gene expressions of these enzymes 6 and 24 h after feeding with (20%) or without carbohydrates. Our data show that there is at least one gluconeogenic enzyme strongly regulated (decreased expression after feeding) in the two fish species, i.e. the PEPCK for common carp and G6Pase/FBPase for gilthead seabream. In these fish species, the regulation seems to be similar to the mammals at least at the molecular level.

title: Early feeding of carnivorous rainbow trout (Oncorhynchus mykiss) with a hyperglucidic diet during a short period: effect on dietary glucose utilization in juveniles.
abstract: Based on the concept of nutritional programming in higher vertebrates, we tested whether an acute hyperglucidic stimulus during early life could induce a long-lasting effect on carbohydrate utilization in carnivorous rainbow trout. The trout were fed a hyperglucidic diet (60% dextrin) at two early stages of development: either at first feeding (3 days, stimulus 1) or after yolk absorption (5 days, stimulus 2). Before and after the hyperglucidic stimulus, they received a commercial diet until juvenile stage (>10 g). Fish that did not experience the hyperglucidic stimuli served as controls. The short- and long-term effects of the stimuli were evaluated by measuring the expression of five key genes involved in carbohydrate utilization: alpha-amylase, maltase (digestion), sodium-dependent glucose cotransporter (SGLT1; intestinal glucose transport), and glucokinase and glucose-6-phosphatase, involved in the utilization and production of glucose, respectively. The hyperglucidic diet rapidly increased expressions of maltase, alpha-amylase, and glucokinase in stimulus 1 fish and only of maltase in stimulus 2 fish, probably because of a lower plasticity at this later stage of development. In the final challenge test with juveniles fed a 25% dextrin diet, both digestive enzymes were upregulated in fish that had experienced the hyperglucidic stimulus at first feeding, confirming the possibility of modification of some long-term physiological functions in rainbow trout. In contrast, no persistent molecular adaptations were found for the genes involved in glucose transport or metabolism. In addition, growth and postprandial glycemia were unaffected by the stimuli. In summary, our data show that a short hyperglucidic stimulus during early trout life may permanently influence carbohydrate digestion.

title: Polyunsaturated fatty acids, Part 1: Occurrence, biological activities and applications.
abstract: Polyunsaturated fatty acids form a unique class of food constituents that show a wide range of functions in biological systems. Investigations over the past two decades have uncovered their roles and those of their eicosanoid metabolites, and have highlighted their homeostatic functions in mammals. A growing number of common human medical conditions are thought to be traceable to dysfunctions in the eicosanoid system, which could in turn be due to imbalances in the intake and/or metabolism of polyunsaturated fatty acids. This, together with medical advances, has spurred the introduction of biomedical products, nutritionals, fortified foods and health supplements.

title: Regulation of FADS2 expression and activity in European sea bass (Dicentrarchus labrax, L.) fed a vegetable diet.
abstract: Supplies of marine fish oils are limited, and continued growth in aquaculture production dictates that lipid substitutes in fish diets must be used without compromising fish health and product quality. In this study, the total substitution of a fish meal and fish oil by a blend of vegetable meals (corn, soybean, wheat and lupin) and linseed oil in the diet of European sea bass (Dicentrachus labrax) was investigated. Two groups of European sea bass were fed with fish diet (FD) or vegetable diet (VD) for 9months. VD, totally deprived of eicosapentaenoate (EPA; 20:5n-3) and docosahexaenoate (DHA; 22:6n-3), revealed a nutritional deficiency and affected growth performance. Whilst VD induced a significant increase in fatty acid desaturase 2 (FADS2) and sterol binding regulatory element-binding protein 1 (SREBP-1) mRNA levels, the desaturation rate of [1-(14)C]18:3n-3 into [1-(14)C]18:4n-3, analysed in microsomal preparations using HPLC method, did not show an upregulation of FADS2 activities in liver and intestine of fish fed VD. Moreover Western-blot analysis did not revealed any significant difference of FADS2 protein amount between the two dietary groups. These data demonstrate that sea bass exhibits a desaturase (FADS2) activity whatever their diet, but a post-transcriptional regulation of fads2 RNA prevents an increase of enzyme in fish fed a HUFA-free diet. This led to a lower fish growth and poor muscle HUFA content.

title: A vertebrate fatty acid desaturase with Delta 5 and Delta 6 activities.
abstract: Delta5 and Delta6 fatty acid desaturases are critical enzymes in the pathways for the biosynthesis of the polyunsaturated fatty acids arachidonic, eicosapentaenoic, and docosahexaenoic acids. They are encoded by distinct genes in mammals and Caenorhabditis elegans. This paper describes a cDNA isolated from zebrafish (Danio rerio) with high similarity to mammalian Delta6 desaturase genes. The 1,590-bp sequence specifies a protein that, in common with other fatty acid desaturases, contains an N-terminal cytochrome b(5) domain and three histidine boxes, believed to be involved in catalysis. When the zebrafish cDNA was expressed in Saccharomyces cerevisiae it conferred the ability to convert linoleic acid (18:2n-6) and alpha-linolenic acid (18:3n-3) to their corresponding Delta6 desaturated products, 18:3n-6 and 18:4n-3. However, in addition it conferred on the yeast the ability to convert di-homo-gamma-linoleic acid (20:3n-6) and eicosatetraenoic acid (20:4n-3) to arachidonic acid (20:4n-6) and eicosapentaenoic acid (20:5n-3), respectively, indicating that the zebrafish gene encodes an enzyme having both Delta5 and Delta6 desaturase activity. The zebrafish Delta5/Delta6 desaturase may represent a component of a prototypic vertebrate polyunsaturated fatty acids biosynthesis pathway.

title: Ontogenic effects of early feeding of sea bass (Dicentrarchus labrax) larvae with a range of dietary n-3 highly unsaturated fatty acid levels on the functioning of polyunsaturated fatty acid desaturation pathways.
abstract: Four replicated groups of sea bass (Dicentrarchus labrax) larvae were fed diets containing an extra-high level of highly unsaturated fatty acids (HUFA) (XH; 3.7 % EPA+DHA), a high level of HUFA (HH; 1.7 %), a low level of HUFA (LH; 0.7 %) or an extra-low level of HUFA (XLH; 0.5 %) from day 6 to day 45 (experiment 1; XH1, HH1, LH1, XLH1). After a subsequent 1-month period feeding a commercial diet (2.7 % EPA+DHA), the capacity of the four initial groups to adapt to an n-3 HUFA-restricted diet (0.3 % EPA+DHA; R-groups: XH2R, HH2R, LH2R, XLH2R) was tested for 35 d. Larval dietary treatments had no effect on larval and juvenile survival rates. The wet weight of day 45 larvae was higher in XH1 and HH1 (P < 0.001), but the R-juvenile mass gains were similar in all treatments. Delta-6-desaturase (Delta6D) mRNA level was higher in LH1 and XLH1 at day 45 (P < 0.001), and higher in LH2R and XLH2R, with a significant increase at day 118.Concomitantly, PPARalpha and PPARbeta mRNA levels were higher in XLH1 at day 45, and PPARbeta and gamma mRNA levels were higher in XLH2R at day 118, suggesting possible involvement of PPAR in stimulation of Delta6D expression, when drastic dietary larval conditioning occurred. The low DHA content in the polar lipids (PL) of LH1 and XLH1 revealed an n-3-HUFA deficiency in these groups. Larval conditioning did not affect DHA content in the PL of R-juveniles. The present study showed (i) a persistent Delta6D mRNA enhancement in juveniles pre-conditioned with an n-3 HUFA-deficient larval diet, over the 1-month intermediate period, and (ii) brought new findings suggesting the involvement of PPAR in the Delta6D mRNA level stimulation. However, such nutritional conditioning had no significant effect on juvenile growth and lipid composition.

title: Epigenetic variations in early cardiovascular performance and hematopoiesis can be explained by maternal and clutch effects in developing zebrafish (Danio rerio).
abstract: This is the first study to show maternal and clutch effects on early developmental parameters like blood cell concentration and cardiac performance (heart rate, stroke volume and cardiac output) in developing zebrafish larvae (2-8 days post fertilization, dpf). Ten individuals per parental pair and developmental stage were analyzed. A pronounced interclutch variation of heart rate has been found in younger stages (2-4 dpf), while interclutch variation of heart rate was small in later stages (8 dpf). This effect was more pronounced in offspring from parental fish nourished with living food. The opposite effect was observed in interclutch variation of blood cell concentration. Here only older stages showed significant interclutch variations. Stroke volume and cardiac output had very small interclutch variations throughout all stages. Heart rate was strongly dependent with developmental stage in all groups. Nutritional maternal effects on heart performance and also in blood cell concentration could be detected in the offspring of parent animals either fed with flake food or with living food. Red blood cell count, calculated as a product from red blood cell concentration, was not significantly different in both feeding groups. The number of spawned eggs was not different. In summary, these data indicate that "clutch effects" caused by maternal and/or genetic influences can affect the developmental pattern of cardiac performance and blood cell concentration.

title: Effects of nutrition and environmental factors on the fetal programming of the reproductive axis.
abstract: Research from a wide range of scientific disciplines has shown that the reproductive performance of animals in adult life is determined, in part, by a variety of extraneous influences acting at different stages of development from before conception until after birth. These effects are probably mediated through changes in the hypothalamic-pituitary and gonadal axes but the physiological system that is affected depends on the stage of development at which the influence is applied. The physiological mechanisms through which environmental influences are transmitted to the target organs are, in many cases, complex and poorly understood. Gonadotrophins seem to play a pivotal role in the development of the fetal testis, although effects of environmental influences on GnRH secretion have yet to be demonstrated. Other studies have shown that, at earlier stages of fetal development, the normal ontogeny of gonadal development and function can be disrupted by undernutrition or the influence of endocrine-disrupting compounds. Specifically, in female fetuses, the onset of meiosis is delayed, whereas, in male fetuses, testosterone synthesis is increased as a result of enhanced testicular steroidogenic enzyme activity. Although reproductive performance is clearly influenced by prenatal factors, much further work is required to identify the relationships between developmental abnormalities and adult reproductive function. Work is also required to elucidate further the critical windows in development and the mechanisms by which environmental factors affect the reproductive organs of developing offspring.

title: Programming and reproductive functioning
abstract: Here, we explore the influence of fetal programming and early life exposures on lifelong reproductive health through modification of the hypothalamic-pituitary-gonadal axis. A range of programming issues are considered with examples from the literature demonstrating that environmental or nutritive exposures have a crucial role in reproductive performance, fetal growth, postnatal development and reproduction-related disease risk. We pay particular attention to recent research on associations between indicators of fetal and postnatal growth and the etiology of polycystic ovary syndrome in women. We conclude that the concept of programming can be applied to reproductive development and related health outcomes, and that the complex potential for interactions between parameters controlling fetal development and postnatal exposures invokes a need to adopt a perspective across the life course of an individual.

title: Photoperiodic modulation of metamorphosis in Atlantic halibut (Hippoglossus hippoglossus L.)
abstract: The effect of abrupt changes in photoperiod on induction of metamorphosis in Atlantic halibut was investigated. Larvae were reared in a 3.7-m 3 tank on enriched brine shrimp ( Artemia ) from first feeding at 265 day degrees (d°C) (44 days post hatch, DPH) until 66 days (DPH) under continuous illumination or to a standard length of 17 mm corresponding to the beginning of metamorphosis. Thereafter, the larvae were divided into two groups; one further exposed to continuous light (24L:0D), the other given a 12L:12D regime, with four replicates within each treatment. The experiment was terminated at 116 DPH. An abrupt decrease in photoperiod at commencement of metamorphosis briefly stimulated eye migration and slowed growth. Growth was not significantly different at the end of the experiment. The eye migration to standard length ratio was significantly higher under 12L:12D than in larvae under continuous light at 116 DPH. Continuous light accelerated the appearance of haemoglobin in the circulation, gave a higher frequency of normal pigmentation, and higher survival than did 12L:12D in metamorphosing halibut larvae. The sequence of measurable metamorphic events was generally neural change, growth and skeletal change, circulatory change and finally establishment of the pigmentation pattern. Heterochrony of metamorphic events was observed for eye migration and for the appearance of haemoglobin with respect to age but not size. The results indicate that a short period of decreased photoperiod may act as an initiator for some metamorphic events The underlying mechanism may be the action of photoperiod on melatonin and stage specific thyroid hormone interaction. The interaction between ontogeny, photoperiod and endocrinology should be further investigated.

title: Effect of Early Introduction of Microencapsulated Diet to Larval Atlantic Halibut, Hippoglossus hippoglossus L. Assessed by Microarray Analysis
abstract: An experimental microdiet prepared using an internal gelation method was used to partially replace the traditional live feed (Artemia) for larval Atlantic halibut, Hippoglossus hippoglossus L. Three trials were conducted with microdiet introduced at 20, 32, and 43 days post first feeding and larvae were sampled at approximately 2, 13, 23, and 33 days after microdiet introduction in each trial. The success of feeding was assessed by morphometrics and histological analysis of gut contents. Microdiet particles were readily consumed after a period of adaptation and provided an adequate source of nutrients with no significant increase in mortality in the microdiet-fed group compared to the control group. However, growth was limited and there was an increased incidence of malpigmentation of the eye and skin. Subtle changes in underlying digestive and developmental physiology were revealed by microarray analysis of RNA from control and experimental fish given microdiet from day 20 post first feeding. Fifty-eight genes were differentially expressed over the four sampling times in the course of the trial and the 28 genes with annotated functions fell into five major categories: metabolism and biosynthesis, cell division and proliferation, protein trafficking, cell structure, and stress. Interestingly, several of these genes were involved in pigmentation and eye development, in agreement with the phenotypic abnormalities seen in the larvae.

title: Quantifying Developmental Progress for Comparative Studies of Larval Fishes
abstract: Several convenient metrics for quantifying the ontogenetic state of fish larvae (based on age, size, and thermal history) were assessed in terms of intrinsic variability, the influence of temperature, and variability among species. Data for 12 ontogenetic events were collected from Atlantic menhaden (Clupeidae: Brevoortia tyrannus) and 15 events from red drum (Sciaenidae: Sciaenops ocellatus). Each species was reared at two constant temperatures 4-5 C apart. Precise timing of events and the variability in timing were calculated using a method analogous to a "dose-response" relationship. The logarithm of total length had the lowest intrinsic variability of all metrics examined; dry weight and age had the highest variability. Warmer temperatures accelerated ontogenetic events on all measurement scales, especially age-based metrics. Logarithmic transformations of day-degrees and effective day-degrees were insensitive to temperature, but these metrics require information that is often not available. An ontogenetic index, which compares species on the basis of their size or age at metamorphosis, performed best in interspecific comparisons.

title: Effects of temperature regime on growth and development of post-larval striped trumpeter (Latris lineata).
abstract: The striped trumpeter ( Latris lineata ) is a promising new candidate for diversification of aquaculture in temperate regions of Australasia. Striped trumpeter is also of scientific interest due to an unusually prolonged post-larval phase. The research aimed to identify the optimal temperature for rearing post-larval striped trumpeter approaching metamorphosis. Three-hundred-day-old post-hatch post-larvae (12.1 ± 0.2 g, 114.0 ± 0.5 mm, mean ± SE) were reared at 12, 14, 16 and 18 °C, over 84 days. Survival, growth and metamorphosis into juveniles were recorded every 21 days. Fish were fed to apparent satiation and reared in oxygen saturated water (95.9 ± 2.6%). At 14 °C, fish exhibited the best growth, had significantly higher lipid content and the majority (> 90%) of the population metamorphosed into juveniles. The performance of fish reared at 16 °C was similar to those at 14 °C but the carcass had a significantly higher protein content and a significantly smaller proportion of the population (66.2 ± 3.0%) metamorphosed into juveniles. A specific growth rate model showed that growth was highest at 14.4 °C. At 12 °C, fish showed the highest food conversion efficiency and all of the fish metamorphosed into juveniles. At 18 °C, fish showed the lowest growth, metamorphosis, and protein and energy retention. This is the first study on the effects of temperature on growth and development of striped trumpeter post-larvae. The results have important implications for aquaculture and fisheries management of striped trumpeter, in particular the rearing of post-larvae in hatcheries, timing of stocking into sea cages especially prior to metamorphosis, and for wild stock recruitment models.

title: Start the clock! Circadian rhythms and development
abstract: The contribution of timing cues from the environment to the coordination of early developmental processes is poorly understood. The day–night cycle represents one of the most important, regular environmental changes that animals are exposed to. A key adaptation that allows animals to anticipate daily environmental changes is the circadian clock. In this review, we aim to address when a light‐regulated circadian clock first emerges during development and what its functions are at this early stage. In particular, do circadian clocks regulate early developmental processes? We will focus on results obtained with Drosophila and vertebrates, where both circadian clock and developmental control mechanisms have been intensively studied. Developmental Dynamics 236:142–155, 2007.

title: Several micronutrients in the rotifer Brachionus sp. may not fulfil the nutritional requirements of marine fish larvae
abstract: The current best practice intensive culture of larval Atlantic cod includes feeding rotifers from onset of exogenous feeding until 25–30 days after hatching. These larvae grow considerably slower and develop higher frequencies of deformities than larvae reared in semi-extensive systems, using copepods as feed. The present study compares the micronutrient concentrations in rotifers with those of copepods, with the aim of identifying nutrients that may be limiting for normal growth and development of cod larvae. An additional criterion used is the nutrient requirements given for fish in general, by NRC (1993), as nutrient requirements of cod remains to be determined. Rotifers were fed on four different diets, consisting of baker’s yeast with cod liver oil (3.3 : 1 dry weight (DW)/v), baker’s yeast with Algamac 2000 TM (3.5 : 1 DW), baker’s yeast with live algae Chlorella (4.1 : 1 DW), and Culture Selco 3000 TM (CS). CS was a complete commercial diet for rotifers while the other diets are considered as based on raw ingredients. Compared with copepod nutrient levels, rotifers grown on yeast-based diets supplemented with either cod liver oil, Algamac 2000 or Chlorella were apparently sufficient for covering the requirements in cod larvae for all the B-vitamins, except thiamine. Rotifers cultured on the CS diet also had sufficient amounts of thiamine. Of the minerals, only calcium and magnesium were sufficient, using this criterion while iron was on the borderline. However, with reference to the requirements given for larger fish (NRC 1993), only thiamine, vitamin A, manganese, selenium and perhaps copper, appear too low in the rotifers cultured without extra micronutrient supplementation. The other nutrients were present at levels intermediate between copepod and fish requirement levels. This study suggests that it is necessary to develop enrichment techniques to produce rotifers with sufficient amounts of all micronutrients. Such techniques will also be important tools for determining which nutrients are present at levels below the actual requirements in cod larvae.

title: Live feeds for early stages of fish rearing.
abstract: 28 paginas, 2 tablas. Special Issue: Basic and Applied Aspects of Aquaculture Nutrition: Healthy Fish for Healthy Consumers. The definitive version is available at

title: Food microparticles for larval fish prepared by internal gelation
abstract: A novel experimental microdiet for larval fish was prepared using a modification of the internal gelation microencapsulation procedure. In these microspheres, a complete formulated diet is entrapped in a matrix of Ca-alginate. When compared with our previous methodology of polymerisation of dietary protein, this procedure has the advantage of using more environmentally friendly reagents at a lower cost: vegetable oil and acetic acid vs. cyclohexane and trimesic acid. In addition, it allows a more balanced formulation because no high proportion of pure protein is required. These features represent an advantage for a potential upscaling of this microdiet to pilot and industrial level. The present study describes the preparation procedure and the basic characteristics of these food particles. In addition, the microdiet was tested with fish larvae. Growth and developmental performance of Sparus aurata larvae obtained at 15 days post hatching (dph) fed this microdiet were similar to those previously obtained with microcapsules prepared by crosslinking of dietary protein. Solea senegalensis larvae were able to reach 400 μg dry weight when fed exclusively on alginate microdiet from 7 dph up to 30 dph.

title: Effects of climate change on fish reproduction and early life history stages
abstract: Seasonal change in temperature has a profound effect on reproduction in fish. Increasing temperatures cue reproductive development in spring-spawning species, and falling temperatures stimulate reproduction in autumn-spawners. Elevated temperatures truncate spring spawning, and delay autumn spawning. Temperature increases will affect reproduction, but the nature of these effects will depend on the period and amplitude of the increase and range from phase-shifting of spawning to complete inhibition of reproduction. This latter effect will be most marked in species that are constrained in their capacity to shift geographic range. Studies from a range of taxa, habitats and temperature ranges all show inhibitory effects of elevated temperature albeit about different environmental set points. The effects are generated through the endocrine system, particularly through the inhibition of ovarian oestrogen production. Larval fishes are usually more sensitive than adults to environmental fluctuations, and might be especially vulnerable to climate change. In addition to direct effects on embryonic duration and egg survival, temperature also influences size at hatching, developmental rate, pelagic larval duration and survival. A companion effect of marine climate change is ocean acidification, which may pose a significant threat through its capacity to alter larval behaviour and impair sensory capabilities. This in turn impacts on population replenishment and connectivity patterns of marine fishes.

title: Transcriptomics for understanding marine fish larval development
abstract: The larval phase is a crucial period in the life of marine fish. During this phase, the organism will acquire the phenotype of an adult fish through the development of tissues and organs and the maturation of some of the principal physiological functions. Many biological processes (differentiation, cellular proliferation, growth, etc.) are regulated during this period. These regulations take place at different biological levels and particularly concern the expression of genes involved in larval ontogenesis processes. The development of bioinformatic resources (DNA or cDNA sequences) and molecular tools enabling high throughput gene expression analysis (microarrays) have allowed the transcriptome of marine fish species to be studied. In the present review, we summarize the main findings from transcriptomic investigations of development of marine fish larvae. Special attention is paid to investigations of transcriptomic patterns during postembryonic development and to the impact of environmental or nutritional factors on the transcriptome of marine fish larvae. Transcriptomic approaches will be especially useful in the future for investigating the effect of temperature and water acidification (or pH) on the development of different fish species in the context of global climate change.

title: Expression of Developmental-Stage-Specific Genes in the Gilthead Sea Bream Sparus aurata L.
abstract: The mechanism of early fish development as well as the control of egg quality is of great importance for the ability of the oocyte to develop after fertilization. Embryonic development is initially regulated by maternally provided mRNAs and later by the zygotic genome. Maternal mRNAs have an important role in initiating processes crucial to patterning the developing fish embryo. Furthermore, it has been shown that maternal RNA plays an important role in egg quality. The identification and characterization of candidate maternal genes in non-model fish species with important aquaculture interest like the gilthead sea bream Sparus aurata L. is of importance for future studies related to egg quality. The broodstock of the gilthead sea bream produces large quantities of eggs with a high and non-controllable quality variation. In the present study, we have studied the gene expression of 16 genes (gapdh 1 and 2, cathepsin D, L, S and Z, erk1, jnk1, p38 alpha and p38 delta, ppar alpha, beta and gamma, tubulin beta, ferritin M, cyclinA2) of different functional categories in seven developmental stages. The 16 genes were chosen based on their putative involvement in egg quality and regulation of early development. In total, 11 showed a characteristic gene expression pattern pinpointing to the possible function as maternal genes and thus may function as molecular biomarker for egg quality.

title: Effects of global climate change on marine and estuarine fishes and fisheries
abstract: AbstractGlobal climate change is impacting and will continue to impact marine and estuarine fish and fisheries. Data trends show global climate change effects ranging from increased oxygen consumption rates in fishes, to changes in foraging and migrational patterns in polar seas, to fish community changes in bleached tropical coral reefs. Projections of future conditions portend further impacts on the distribution and abundance of fishes associated with relatively small temperature changes. Changing fish distributions and abundances will undoubtedly affect communities of humans who harvest these stocks. Coastal-based harvesters (subsistence, commercial, recreational) may be impacted (negatively or positively) by changes in fish stocks due to climate change. Furthermore, marine protected area boundaries, low-lying island countries dependent on coastal economies, and disease incidence (in aquatic organisms and humans) are also affected by a relatively small increase in temperature and sea level. Our interpretations of evidence include many uncertainties about the future of affected fish species and their harvesters. Therefore, there is a need to research the physiology and ecology of marine and estuarine fishes, particularly in the tropics where comparatively little research has been conducted. As a broader and deeper information base accumulates, researchers will be able to make more accurate predictions and forge relevant solutions.