title: Sex differentiation and determination in the invasive fish, Gambusia holbrooki
Creators: name: Mousavi, SE
Publisher: name: University of Tasmania
Year: 2020

description: The eastern mosquitofish, Gambusia holbrooki, is a freshwater viviparous fish that originates from North America. This along with its sister species G. affinis, were introduced throughout the world (i.e., over 110 countries), including Australia as mosquito control agents. Due to their high adaptability and tolerance, they have rapidly established populations in new ecosystems and caused decline in native aquatic fauna through competition and predation. For example, in Australia, G. holbrooki has been implicated in the decline of nine native fish species (genera Galaxias, Chlamydogobius, Melanotaenia, Craterocephalus, Mogurnda, Pseudomugil, Ambassis, Scaturiginichthys and Retropinna) and 10 species of frog. Therefore, G. holbrooki is considered as one of the highly invasive species. Their impacts have become apparent, particularly in freshwater ecosystems, where invasive species constitute the single greatest threat to biodiversity. Hence, development of an effective ‘threat abatement strategy’ is vital. One of the most promising solutions for eradication, is the Trojan Y chromosome (TYC) approach. The strategy works by manipulating the sex ratio of the target population through the introduction of sex-reversed females carrying two Y chromosomes (Trojan Y) that produce only male offspring. However, for this to be successful, the chromosomal sex mechanism must be known. In addition, developing a phenotypic sex marker could facilitate the process of genetic screening of the embryos prior to and post-hormonal treatments (i.e., production of sex-reversed embryos) for rapid identification of sex reversed individuals. To investigate the relationship between genetic sex and phenotypic traits amongst various embryonic stages, a detailed developmental study is needed. However, little information was available on embryonic development in this species, with the exception of an earlier detailed staging system for Gambusia sp. Although less complete than the present study, the earlier study accurately describes stages of embryonic development, and includes useful sets of hand-drawn illustrations. More recently, another study established time-saving (i.e., without sacrificing the brood) approach to predict the developmental progress, by applying the gravid spot index of pregnant females as surrogates for five broad stages. Generally, proposed classifications are a guide but not precise for G. holbrooki developmental staging due to divergent traits and morphology. Regardless, several basic keys specific to different organ systems (e.g., circulation), detailed morphology and precise indices for each stage were virtually non-existent. The developmental studies are also essential to investigate the genotype-phenotype relationships as several sex-related events occur during the embryogenesis. However, these remain uninvestigated, particularly in G. holbrooki development. Low mitotic index is a common problem in cytogenetic studies when using direct (i.e., harvesting cells from tissue of a living animal instead of in vitro tissue culture) chromosome preparation methods. Moreover, to improve cytogenetic studies, physical stretching/relaxation of metaphase chromosomes is essential, primarily to enhance the resolution of the fine chromosomal region (i.e., precise detection). Intercalated agents such ethidium bromide (EtBr) is known as effective in altering the conformational and physical properties of DNA helix of the chromosomes. Although there are several reports confirming the key roles of EtBr that intercalates between DNA bases and prevents DNA folding and condensing in mammalian models like mice, there is no report of using these agents in any fish species including G. holbrooki. Several studies have suggested a XX/XY sex-determining system for G. holbrooki based on the melanic color pattern inheritance from father to son and its allelic linkage group associated to males. However, a sex determination mechanism has not been cytogenetically confirmed in this or any poecilids, with the exception of P. reticulata. Therefore, due to inconclusive evidence in the literature, multiple evidence, including cytogenetic are required to verify the chromosomal sex determining mechanism in G. holbrooki. In this context, the overarching objectives of this study were to discover early phenotypic markers of sex differentiation and ascertain the sex determining mechanism in G. holbrooki, specifically: 1) standardise developmental staging of G. holbrooki embryos for determining the timing and onset of sex determination and differentiation; 2) develop a phenotypic sex marker to assist distinguishing the sex of live embryos at very early developmental stages (e.g., prior to sex-reversal of embryos), and; 3) identify and confirm a sex determination mechanism (i.e. male or female heterogamety) using classical karyomorphology and comparative genomic hybridisation (CGH). Objectives were addressed by morphological studies of embryos during development from zygote to parturition (chapter 2), with emphasis on heart development (chapter 3) and cytogenetics (chapters 4 and 5). First (chapter 2), a comprehensive developmental staging of embryos that document sequential events of development from zygote to parturition (30 days post fertilization) at 25 ± 0.5 °C was defined. To overcome limitations associated with superfetation and obtaining an adequate number of embryos, two approaches were simultaneously employed; first, embryos were harvested from wild caught gravid females, with varying intensity of gravid spot, during peak reproductive season to ensure availability of sufficient embryos at multiple developmental stages. Second, to calibrate developmental timing, genetically sexed females (i.e., at parturition) were grown up to maturity (2–3 months) and then individually mated with males to record time of mating (i.e., first copulation behaviour) as proxy for fertilisation. Post-copulation females (n = 1 or 2) were sampled, and embryos harvested every 6 h for ten days (n = 35), then every 12 h for the remaining 20 days (n = 55). Live embryos were photographed. Morphological diagnostics were used for preliminary staging of embryos according to indices described for developmental staging in Gambusia sp. and zebrafish. For stage identification, a combination of key morphometric measurements (egg size, egg diameter and embryo total length), indices (i.e., otic vesicle closure, heart rates); and meristic counts (e.g., the number of caudal fin rays, and number of caudal fin ray elements) were also employed, as necessary. The development of nervous, circulation, musculoskeletal, visual, and digestive systems along with craniofacial and external body features, were also employed for staging. This was complimented by quantitative pixel analysis of embryonic photographs (i.e., three-dimensional pixel distribution analysis of images). The data were collectively used to describe and define each embryonic stage. The developmental process was defined into seven broad developmental phases (i.e., zygote, cleavage, blastula, gastrula, segmentation, pharyngula and parturition) and 40 stages numerically (stages 0–39; zygote to parturition, respectively). The superfetation, inadequacy of embryos, time/age-controlled during embryogenesis, non-transparency of the eggs and very limited developmental information amongst livebearers for detailed comparison posed some challenges. However, these were overcome by employing multiple strategies including the pixel intensity analysis to assist with identification of stages during early development. Traits found during development could be sex-related, for example, both pigmentation and skeletal traits (i.e., teeth) are sometimes linked to sex chromosome in teleost (e.g., Malawi cichlids). Therefore, the outputs and knowledge from the developmental staging allowed investigating the relationship between developmental phenotypic traits and their genetic sex. This was further validated using a genetic sex marker. Second (chapter 3), the heart rates (HRs) during embryogenesis of G. holbrooki, from onset of heartbeat to just prior to parturition was investigated. Genetic sex of the embryos was postverified using a sex-specific genetic marker. Heartbeat of embryos (n = 10 each sex/stage) at early organogenesis, mid-organogenesis, late organogenesis, early pharyngula, late pharyngula, just prior to parturition and adults (n = 10 each sex) were videographed for 60 s at 25 ± 0.5 °C. The HR and its frequencies were determined using a non-invasive light-cardiography (LCG) method previously described for adult zebrafish. LCG profiles for embryos corresponded to contraction of the ventricle (increased average brightness) conversely, relaxation (decreased average brightness) within the prescribed region of interests (ROI). LCG profiles for adults constitute an opposite signal intensity compared to embryos. The peaks for each LCG oscillation were found using Gauss model and peak analyser function (Origin Pro 2020). The time duration (s) between ventricular systolic (contraction) and atrial diastolic (dilation) phases, constituted a complete cardiac cycle. The time delay between the atrioventricular (A-V delay) peak values of the extracted synchronous chronologies within the same cardiac cycles were measured using time history values for ventricle and atrium peaks. A-V delay was defined as the time duration (s) between the onset of atrium peak and the onset of subsequent ventricular peak, which represents the resting phase of the heart. The average resting time per minute for all developmental stages was calculated by multiplying the total A-V delay/sec by 60. For quantification of ventricle size in adults, both ventricular surface area (mm2 ) and volume of the ventricle (mm3 ) were calculated using ImageJ. Ventricle volume measurements were normalised by condition factor (a) derived separately for each sex (male = 0.01; female = 0.02) using allometric growth equation, W = aL3 . Results reveal that heart rates and resting time significantly increase (p &lt; 0.05) with progressive embryonic developmental stage. The total cardiac resting time per minute was approximately three times greater in the advanced embryos (just prior to parturition) compared to those at early organogenesis, when the heart first begins to beat. Initially, up to mid-organogenesis stage, the heart rates of both male and female embryos were comparable (p &gt; 0.05). However, at late organogenesis, both ventricular and arterial frequencies of female embryos were significantly higher (p &lt; 0.05) than those of their male sibs at the corresponding developmental stages and remained so at all later developmental stages evaluated. Consistent with the sex-specific size dimorphism in adults of this species, the size of the normalised heart (i.e., ventricle) of females were significantly larger than those of the males (p &lt; 0.05). In addition, a clear difference in average HR of the two sexes in adults was immediately evident with females having higher HRs (p &lt; 0.05). In addition, significant (p &lt; 0.05) differences in ventricular diastolic state was evident between adult females and males. Collectively the results suggest that the cardiac sex-dimorphism manifests as early as mid-organogenesis and persists through adulthood in this species. This appears to be the first study to demonstrate early onset of cardiac sex-dimorphism in any teleost species. These findings also suggest that the cardiac measurements can be employed to non-invasively and rapidly sex the developing embryos, well in advance of their phenotypic sex is discernible. Although the mechanism(s) underpinning the observed sex-specific cardiac functions are as yet unknown, the results highlight the amenability of G. holbrooki as a powerful vertebrate model to study these and sex-specific consequences of human cardiovascular diseases as well as to monitor anthropogenic and climatic impacts on heart physiology. Third (chapter 4), to facilitate cytogenetic studies in this species the use of baker’s yeast to optimise frequency of mitotic chromosomes and ethidium bromide to overcome the problems associated with chromosomal condensation were experimentally determined in four different tissues (cephalic kidney, liver, spleen, and gill). Mitotic stimulation with activated baker’s yeast, Saccharomyces cerevisiae (SC) was determined over 1–4 days. For synchronisation and elongation of mitotic chromosomes, individuals (both sexes) were injected (IP) with ethidium bromide (2 or 4 µg/ml) and colcemid 1 (µg/ml), and sacrificed at three timepoints (1, 4 and 8 h) post-injections. Results showed that, cells obtained from gill had the highest number of metaphases at 3 days post-SC exposure, with no significant (p &gt; 0.05) differences between sexes. Nonetheless, sex specific differences in the mitotic index were evidenced in spleen, kidney, and liver, suggesting sex specific difference in immune-response to yeast injections. Elongated metaphase chromosomes of female and male were obtained following injection of 4 µg/ml and 2 µg/ml of ethidium bromide, respectively, for an hour. Moreover, the average mitotic chromosome length of females was significantly (p &lt; 0.05) longer than males at 4 µg/ml ethidium bromide treatment at all the three timepoints (1, 4 and 8 h). Such difference in chromosomal elongation between sexes, could be attributed to the sex-specific episomal (e.g., DNA methylation) and histone modifications or variations in length of linker DNA sequences between the consecutive nucleosomes. This substantially facilitated downstream cytogenetic experiments in this study by making available (1) adequate number of arrested cells at metaphase for intensive cytogenetic studies e.g., comparative genomic hybridisation (CGH) and (2) elongated metaphase chromosome allowed investigating very fine heterochromatin structure (i.e., sex-specific fluorescent signals) differences between sexes. To identify and verify the sex determination mechanism (Chapter 5), the metaphase spreads of both males and females were prepared using a conventional air-drying method. Chromosomes were counterstained using 1 mg/ml 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI, 0.2 µg/ml final concentration) and mounted with Vectashield medium (without DAPI). Mitotic chromosomes were imaged at 100× magnification using a Leica epifluorescence microscope equipped with single band pass emission filters. Metaphase chromosome (n = 25/sex) arms were measured and ideograms for male and female fish generated using IdeoKar. Total genomic DNA was nick translated incorporating SpectrumGreen dUTP (Vysis) and Spectrum-Red dUTP (Vysis). Briefly, slides were denatured for 3 min at 70 °C in 70 % formamide, 2 X SSC, dehydrated through an ethanol series, air-dried, and kept at 37 °C until probe hybridisation. For each slide (made using one drop of fixed-cell solution), 250–500 ng of SpectrumGreen-labelled female and SpectrumRed-labelled male DNA was co-precipitated with (or without) 5–10 µg of boiled genomic DNA from the homogametic sex (as competitor), and 5–10 µg Salmon sperm (as carrier). Since the homogametic sex was not known, reciprocal experiments were performed using alternatively male and female DNA as competitor. As various fluorescent dyes could exhibit differential signal intensities, reciprocal experiments were done incorporating either of dyes (red/green) into total genomic DNA (male or female) as necessary. The co-precipitated probe DNA was resuspended in 35–50 µl hybridisation buffer, first denatured and then hybridised on chromosomes for 3 days at 37 °C. Post washings, slides were mounted with antifade medium Vectashield (with DAPI) and imaged. Phylogenetic analysis was done by multiple sequence alignments of mitochondrial cytochrome b (COI) gene segment (1140 bp) derived from eight Poeciliid species including G. holbrooki (Tasmania and Florida populations) using neighborjoining method (Timura-nei model) with 1000 replicates. Results from karyotyping and ideograms analysis showed no morphological (i.e., size) differences in any of the chromosome pairs in both sexes. However, fluorescent in situ hybridisation (FISH) results showed heterochromatinisation of all the centromeres (CG-positive and AT-negative) and few pericentric and distal regions of the metaphase chromosomes in both sexes. Two small size autosomes in both sexes were intensively heterochromatinised, indicative of DNA repeats accumulation. These two smallest autosomes had hybridisation patterns resembling those of ancestral sex chromosome pair, perhaps originating from whole genome duplication. In addition, a pair of DAPI-positive (i.e., AT-rich) microchromosomes were observed in 20% of the male metaphases and not in females. However, CGH results showed that a single microchromosome also exists in the female metaphases, showing high CG-rich heterochromatinisation (similar to male) but DAPI-negative. Interestingly, CGH in the male metaphase revealed a large male-specific signal (i.e., indicating preferential binding of the male-derived probe) on an interstitial arm of a large chromosome and also two conspicuous male-specific signals superimposed on two weak female-specific signals in two different similar sized chromosomes of the female mitotic complements. These patterns are consistent with the hypothesis of an XX/XY sex determining mechanism but require further verification. Notably, the male specific signal was in the proximal location of the chromosome implying that the accumulation of these repeats may have initiated the Y chromosome differentiation, as well as played a critical role towards the evolution and differentiation of sex chromosomes in this species. Direct estimation of the genetic divergence timing of the Y chromosome in the G. holbrooki was showed to range between 6– 10 million years ago. Therefore, G. holbrooki represents characteristics of nascent XY sex chromosomes, where the Y appears at an early stage of differentiation. In conclusion, this study for the first time described a detailed developmental staging guide in this species, identified sex-specific heart rate during the development which can be employed as a precise and non-invasive sexing marker and provided cytogenetic evidence suggesting the species is likely male heterogametic (XX/XY). Further characterisation of the putative sex chromosomes through isolation of sex-specific sequences and their mapping may be necessary to validate the identity and role of these and the microchromosomes, in this species.