BACKGROUND
identifying environmental cues and their perception and transduction mechanisms is a central focus of research in developmental biology within an ecological context  <cit> .
changes in environmental variables can have profound influences on differentiation, growth and reproduction in many organisms  <cit> .
temperature is the main abiotic factor that affects many biological functions at different levels of organization by changing the rates of chemical reactions and physiological processes, or by changing the three-dimensional shapes of biomolecules  <cit> .
fish exhibit enormous diversity in their morphology, in habitat occupancy, and in their biology  <cit> .
this diversity is also remarkable as regards to their reproductive strategies including sex determination and differentiation  <cit> , two processes that contribute to the establishment of the sex ratio, a crucial parameter for population viability and for the continuation of all species with sexual reproduction.
the sex ratio can be affected by environmental factors, mainly by temperature  <cit> .
among vertebrates with genetic sex determination  master sex determining genes are not conserved and eight genes with such a function have been identified so far: sry in mammals, dmrt- <dig> in birds, dm-w in xenopus laevis, and dmy, amhr <dig>  amhy, sdy and gsdf in fish  <cit> .
on the other hand, genes involved in the sex differentiation process  are fairly conserved in structure and dimorphic expression from fish to mammals  <cit> .
genes involved in testis differentiation include dmrt <dig>  dax <dig>  sox <dig>  arb, amh, cyp11b , sox9a <dig>  tbx1a and tbx1b  <cit> ; whereas genes involved in ovarian differentiation include cyp19a1a, foxl <dig>  er, fst , hsd3b and star  <cit> .
however, the order of expression and interactions among these genes may change between groups  <cit>  or depending on environmental conditions  <cit> .
in any case, estrogens are essential for proper ovarian differentiation in all non-mammalian vertebrates  <cit> .
thus, cyp19a1a, the gene that codes for aromatase, the enzyme that catalyzes the irreversible conversion of androgens into estrogens, is a major player in vertebrate sd and crucial for the establishment of the sex ratio.
aromatase gene expression is susceptible to environmental temperature influences.
therefore, and regardless of the sex ratio response pattern to temperature  <cit> , in reptiles and fish, the two types of vertebrates with temperature-dependent sex determination , the effects of environmental temperature on sex ratios are mediated by changes in cyp19a1a expression.
in all fish species analyzed so far  <cit>  more males are produced with increasing temperatures and cyp19a1a is always inhibited at male-producing temperatures and stimulated at female-producing temperatures  <cit> , in general, elevated temperatures increase the expression of male-related genes such as amh, dmrt <dig> or arb after the sd period, while decreasing the expression of female-related genes in addition to cyp19a1a, such as esr <dig>  esr <dig>  erb <dig>  fshr or foxl <dig> .
effects of temperature on fish sex ratios are more pronounced if animals are exposed to elevated temperatures during early development.
however, the number of genes known to be affected is limited and the metabolic and signaling pathways affected are essentially unknown.
in this regard, several studies have explored the effects of cold and cold acclimation  <cit>  but focusing on tissues other than the gonads, such as liver, skeletal white muscle and gills ; brain  <cit>  or heart  <cit> .
unfortunately, comparative transcriptomic studies on the effect of heat on the gonads are essentially limited to just one study with the pejerrey, odontesthes bonariensis, a fish with tsd , and carried out on juvenile or adult mature gonads, not with differentiating gonads.
thus, it is difficult to ascertain whether observed altered patterns of gene expression are the cause or the consequence of a given gonadal phenotype resulting from exposure to elevated temperature.
the european sea bass is a eurythermal marine teleost able to live between  <dig> and 27 °c and a gonochoristic species with a polygenic system of sex determination  <cit> , where genetics and temperature contribute essentially equally to sex ratios  <cit> .
recent studies in the sea bass have shown that cyp19a1a and cyp11b are good markers of female and male sex differentiation, respectively  <cit> .
in all fish species studied so far, high temperatures cause masculinization.
this is also the case of the european sea bass, where elevated temperatures during the thermosensitive period , which is located 0– <dig> dph, result in masculinization of about 50 % of the fish that otherwise, under more natural temperatures, would have developed into females, as assessed by sex ratio analysis .
recently, we discovered that the effects of early temperature include hypermethylation of the cyp19a1a promoter in one-year-old juvenile females, with concomitant suppression of cyp19a1a expression and resulting in male instead of female development, the first evidence of an epigenetic link between environmental temperature and sex ratios in vertebrates  <cit> .
we were interested in understanding the underlying mechanisms responsible for this masculinization at the time when the differentiating gonad is being affected.
therefore, instead of sampling one-year-old juveniles, as done before by us and others, in which gonads have already completed the process of sex differentiation, and where it is difficult to ascertain whether observed transcriptomic profiles and epigenetic changes are the cause or the consequence of that process, we sampled fish at  <dig> dph, i.e., 110 days after the end of temperature treatment but shortly after the start of sex differentiation .
thus, the objective was to gain a better understanding of the genes and the pathways involved in sex differentiation that are directly affected by temperature at the time of sex differentiation.
RESULTS
growth, body indices and sex ratios
because of the differences in rearing temperature between  <dig> and  <dig> dph, fish from the ht group were significantly  larger than those of the lt group at  <dig> dph in both sl and bw .
growth differences between groups had disappeared by  <dig> dph.
sexual growth dimorphism, in favor of females , was present only in the lt group .
as observed in our previous studies survival was unrelated to temperature treatment .
survival was ~60 % to the end of the larval stage and ~90 % thereafter.table  <dig> growth of european sea bass juveniles at 170 days post hatch, classified according to treatment and cyp19a1a expression levels, as shown in fig.
1a
data as mean ± sem
lt low temperature, ht high temperature, n sample size
different letters indicate significant  differences between treatments
data as mean ± sem
lt low temperature, ht high temperature, n sample size
the same superscript a indicates lack of significant  differences between treatments.
asterisks indicate statistical differences  between females and males within the same treatment group, i.e., sexual growth dimorphism
at  <dig> dph, from each temperature group we randomly sampled  <dig> fish .
however, since, as stated above, elevated temperature results in masculinization of some females, within the randomly selected fish we choose for microarray analysis, in each group,  <dig> fish with high cyp19a1a levels, which are typically associated with female development, and for this we used a previously validated clustering method based on cyp19a1a qrt-pcr expression levels .
one-way anova showed statistical differences due to the expression levels, temperature treatment and their interaction .
thus, in the lt or control group, fish with high cyp19a1a levels undoubtedly represented developing females, whereas in the ht group fish with high cyp19a1a levels  also represented females that had been subjected to elevated temperature.
it is important to state that despite overall differences in the sampled fish  there were no differences in sl or bw between the  <dig> fish selected from the two groups for microarray analysis.
thus, the observed transcriptomic differences between ht and lt fish  were due to temperature and not to size-related differences in gonadal development.fig.
<dig>
a individual gonadal aromatase  expression levels  as assessed by qrt-pcr in the low  and high  temperature groups at  <dig> dph.
blue squares and red circles correspond to individual fish with low  and high  cyp19a1a levels, respectively.
the horizontal line marks mean expression for each experimental group.
the cyp19a1a expression of one of the putative females in the lt group  was much higher than the rest and it has been deliberately omitted from the graph for clarity purposes.
different letters indicate significant  differences between the four groups, i.e., high and low cyp19a1a expressors of the lt and ht treatments.
b sex ratios of juvenile european sea bass sampled at 332 days post hatch.
stacked bars showing male  and female  percent in the low  and high  temperature groups.
statistical differences  between groups are marked with three asterisks.
sample size: lt,  <dig> fish; ht,  <dig> fish
visual assessment of the sex ratio of  <dig> dph juveniles combined with histological verification showed that the lt group had  <dig>  % males while the ht group had  <dig>  % males  , showing a masculinizing effect of the elevated temperature.
however, within each sex no differences were observed in the presence and in the abundance of the different cellular types between the two temperature groups.
females had immature ovaries containing oocytes at the cortical alveolar  stage while males had testis containing all germ cell types, including spermatozoa .
the gsi percent values at  <dig> dph for lt males and females were  <dig>  ±  <dig>  and  <dig>  ±  <dig> , respectively, and for ht males and females were  <dig>  ±  <dig>  and  <dig>  ±  <dig> , respectively.
a two-way anova analysis showed statistical differences due to sex  but no effect due to the thermal treatment or to the interaction between sex and temperature.
microarray analysis
microarray analysis of sexually differentiating gonads at  <dig> dph obtained from fish with a high cyp19a1a expression, i.e., putative females, revealed the presence of  <dig> significantly and differentially expressed  genes when comparing the ht vs. the lt group , of which  <dig> genes were upregulated  and  <dig> were downregulated .
a heatmap representation of the de genes grouped fish according to their thermal history, with the exception of one lt fish, which had an intermediate position, and one ht fish, which was classified as an outlier by a principal component analysis and was not further considered in the analysis .
some of the upregulated genes were related to reproduction, i.e., cryptochrome dash  and troponin i , or to epigenetic gene expression regulation, i.e., histone deacetylase  <dig> .
some of the downregulated genes also showed reproduction-related functions such as cdc <dig> effector protein  <dig> , insulin-like growth factor  or smoothelin  .fig.
<dig> heatmap of the microarray expression data for the  <dig> up- and  <dig> downregulated genes, where each row represents a gene and each column represents an individual fish .
key color representing the level of expression .
the dendograms provide information of the similarity between genes and between the different samples.
notice that all ht samples and all but one lt samples cluster together.
lt, low temperature group; ht, high temperature group.
see additional file 1: table s <dig> for a complete list of gene names and abbreviations
the amigo web-based tool was used to recover the sequence of these de genes and those sequences were then uploaded to blast2go in order to enrich results with go terms and extract more information about these de genes.
a fisher’s exact test with multiple testing corrections for false discovery rate  showed that five go term categories were overrepresented when compared to a reference test containing all the annotated sequences from our custom-made array .
further analysis of the go terms provided their distribution among the three main categories: biological process, molecular function and cell component for the up- and downregulated genes separately .
the kyoto encyclopedia of genes and genomes  database provided more information on the pathways containing these de genes .
several pathways involved in protein synthesis as well as in immunological processes were found.
microarray validation
out of the  <dig> de genes, we selected eight , i.e., about one third of the de genes, for validation by qrt-pcr.
these genes were selected because they are known to be involved in reproduction in other species or because they are involved in epigenetic regulatory mechanisms.
in addition, together they exhibited a wide dynamic range of changes.
qrt-pcr analysis showed significant differences  for cg <dig> and hdac <dig> among the upregulated genes and for the cdc42ep <dig> and smtn <dig> among the downregulated ones .
importantly, all but one  of these selected genes had the same direction of change  when analyzed by qrt-pcr as compared to microarray results.
we consider this sufficient for the validation of the microarray.fig.
<dig> validation of microarray results by analyzing ten fish by qrt-pcr according to treatment .
a-d four upregulated genes in the ht vs. the lt group comparison: dmei_cg <dig> ; histone deacetylase  <dig> ; cryptochrome dash  and troponin i .
e-h four downregulated genes for the same comparison: cell division cycle  <dig> effector protein  <dig> ; extracellular matrix protein  <dig> ; carbonic anhydrase  <dig>  and smoothelin .
data as mean ± sem.
letters mark statistical significance  between groups.
ten fish  were analyzed by a qrt-pcr
enrichment analysis
a go enrichment analysis of the de genes showed up- and downregulation of the same bp categories, albeit containing different de genes .
these altered categories contained more go terms that were upregulated and mainly related to metabolic and cellular processes , while other processes such as reproduction , growth , immune processes  or signaling  were downregulated at elevated temperatures.
regarding mf go categories , the catalytic and binding activities were the most represented subcategories for both up- and downregulated go terms.
analysis of the cc categories showed that upregulated processes were taking place mainly in the organelle , macromolecular complex , membrane-enclosed lumen  or membrane  , while the downregulated processes were taking place in the membrane, organelle, macromolecular complex  or extracellular region  .
further analysis of the go enriched terms of the de genes in comparison to the microarray reference set , showed that terms related to the negative regulation of the nerve impulse and synaptic transmission were overrepresented, as well as the adenylate cyclase inhibiting g-protein coupled receptor signaling pathway .fig.
<dig> biological process-related go terms for the ht versus lt group comparison.
a biological process go terms for the upregulated genes, and  for the downregulated genes
blast2go analysis of the de genes showed that eleven of the  <dig> differentially regulated pathways  had higher expression in ht fish than in lt fish and that these pathways were related to catabolism , biosynthesis  or signal transduction .
however, the downregulated pathways were related, in agreement with the results of the go term analysis, to immunology  and nitrogen metabolism.
david analysis of the de genes showed that there were  <dig> upregulated categories, of which four were highly significant: 1) progesterone-mediated oocyte maturation , 2) tight junction , 3) chemokine signaling , and 4) hormone-mediated signaling  pathways.
in all of them, adcy <dig> is involved as a signaling initiation factor.
after running an annotation clustering with david, seven clusters were downregulated and related to cell component, dna binding, transcription and signaling processes.
reproduction and stress-related genes
fifteen known reproduction-related genes were analyzed by qrt-pcr .
results showed that genes involved in testicular differentiation, such as doublesex-mab-3-related transcription factor  <dig> , were significantly  upregulated in ht fish, as also was the steroidogenic acute regulatory protein .
in contrast, some genes involved in ovarian differentiation, such as aromatase , were significantly  downregulated in ht fish, as also was aquaporin  <dig> .
the expression of eight of these genes is shown in fig.
<dig>  since activation of the stress response has been associated with temperature effects on sex ratios in fish , we tested the effects of ht in our model in two stress response-related genes; the 11ß-hydroxysteroid dehydrogenase  and the glucocorticoid receptor , involved in cortisol synthesis and response.
while nr3c <dig> was significantly  upregulated in ht fish, hsd11b was not significantly different .fig.
<dig> quantitative rt-pcr results for eight known sex differentiation-related genes per temperature treatment groups.
a-d male pathway: anti-müllerian hormone , doublesex- and mab-3-related transcription factor  <dig> ; female pathway: cytochrome p <dig>  family <dig>  subfamily a, polypeptide 1a  and sry-related hmg-box transcription factor sox <dig> , respectively.
e steroidogenic acute regulatory protein ,  vasa protein ,  insulin-like growth factor  <dig>  and  cytochrome p <dig>  family <dig>  subfamily a, polypeptide 1b .
data as mean ± sem.
asterisk marks statistical differences between groups .
ten fish  were analyzed by a qrt-pcr
fig.
<dig> quantitative rt-pcr results for  11β-hydroxysteroid dehydrogenase  and  glucocorticoid receptor .
data as mean ± sem.
asterisk marks statistical differences between groups .
ten fish  were analyzed by a qrt-pcr
epigenetic mechanisms-related genes
according to a growing body of evidence, several genes involved in epigenetic regulatory mechanisms have been implicated in sex determination/differentiation .
based on this, we analyzed the genes related to epigenetic mechanisms and implicated in sex determination/differentiation  <cit>  that were present in our microarray  even if they were found to be not de.
seven genes, representative of different categories of epigenetic regulatory mechanisms, including dicer  <dig>  a helicase needed to produce an active small rna component that represses gene expression; ehmt <dig>  a histone methyltransferase; jarid2a, a dna-binding protein that acts as a transcriptional repressor; pcgf <dig>  which contains a ring finger motif and forms protein-protein interactions to maintain transcriptional repression; hdac <dig>  a histone deacetylase; mettl <dig>  a methyltransferase-like protein; and suz <dig>  a suppressor of trithorax zeste  <dig> homolog gene, were selected and analyzed by qrt-pcr.
four of these genes were upregulated  in the ht group: dicer <dig>  jarid2a, pcgf <dig> and hdac11) .fig.
<dig> quantitative rt-pcr results for the epigenetic regulatory mechanisms-related genes.
endoribonuclease dicer , euchromatic histone-lysine n-methyltransferase  <dig> , protein jumonji  and polycomb group ring finger  <dig> .
data as mean ± sem.
asterisks mark statistical differences between groups .
ten fish  were analyzed by a qrt-pcr
discussion
to the best of our knowledge, this is the first study on the analysis of the gonadal transcriptome at the time of sex differentiation, using a species-specific microarray, of fish previously exposed to elevated temperature during the thermosensitive period.
since we took care that the  <dig> fish selected from each group for microarray analysis at  <dig> dph  had the same size, it is safe to assume that differences in gene expression observed are solely due to temperature, not to differences in growth.
consistent with previous studies  <cit> , temperature induced a male-biased sex ratio .
the higher gsi values for ht-treated females observed at one year of age may be due to a persistent effect on gonadal growth relative to somatic growth with no obvious effect at the histological level.
the number of de genes was low as could be anticipated because in the ht vs. lt comparison we actually compared fish of the same age, at a similar developmental stage and similar size, as just stated above, and most likely of the same sex, females, as assessed by high cyp19a1a expression levels, a good marker of phenotypic sex.
this is actually what we intended since females, not males, are the ones mainly affected by temperature.
moreover, at  <dig> dph gonads were cleanly isolated and there was no contamination of the surrounding tissues.
what we could gain by looking at earlier age, e.g., at the end of the tsp at about  <dig> dph, we would lose by having to include non-gonadal tissues .
among the genes analyzed by qrt-pcr, there were three patterns of response to heat .
many genes had increased levels of expression in the ht group.
among them, there were genes upregulated during normal testis differentiation such as dmrt <dig>  in accordance with the masculinizing effect of elevated temperatures  <cit> .. the vasa helicase is a germ cell marker known to be more expressed in ovaries than in testis and previously found to be upregulated by elevated temperatures  <cit> .
in our study the difference in vasa expression levels did not reach statistical significance.
cholesterol is important in maintaining membrane integrity and sterol synthesis and its levels have previously been found also to be increased by temperature  <cit> .
however, genes ultimately related to steroid hormone production were not significantly affected in the present study, including the gene encoding gonadotropin-releasing hormone   <cit> , and star, which is involved in cholesterol import  <cit> .
in our study, genes such as wisp <dig>  known to be involved in cellular growth, were downregulated by heat, whereas genes such as nr3c <dig>  related to immune system regulation responses, were upregulated, suggesting growth and immunological stress response adjustments in the gonads.
there were other downregulated genes.
among these there was cyp19a1a, confirming results of an independent experiment aimed to find molecular signatures of male and female differentiation  <cit> .
aquaporin  <dig> , a water channel protein which plays a major role in oocyte hydration in fish  <cit> , was also downregulated by heat.
the steroidogenic enzyme 11ß-hydroxysteroid-dehydrogenase  <dig> , which converts the stress hormone cortisol into the inactive metabolite cortisone, and also converts 11ß-hydroxy androgens such as 11ß-hydroxyadrostenedione into 11-ketotestosterone, a potent piscine androgen  <cit> , was not significantly affected by heat.
in contrast, the glucocorticoid receptor  was upregulated in the ht group with significant differences with respect to the lt group.
these results are interesting because in the pejerrey , a fish with tsd, fernandino et al.
<cit>  observed upregulation of both hsd <dig> and nr3c <dig> in masculinized gonads by ht and attributed this to the fact that ht elicits both masculinizing and stress responses.
in our study, nr3c <dig> was upregulated 110 days after the end of the temperature treatment, suggesting a persistent stress response probably maintained by an epigenetic regulatory mechanism.
in fact, studies in rodents have shown that nr3c <dig> is able to exhibit sustained expression, even a long time after the stimulus ended, through changes in methylation of its regulatory region  <cit> .
the differences between our results and those of fernandino et al.
<cit>  with the pejerrey suggests that gender or developmental stage may be important in explaining these differences, since while fernandino et al.
<cit>  sampled juvenile, sexually differentiated males, in our case we selected not only sexually differentiating fish but also those ones that exhibited cyp9a1a expression levels at  <dig> dph compatible with ovarian differentiation.
furthermore, when comparing our results with those obtained with microarray analysis of dimorphic gene expression in a turtle with tsd  <cit> , genes that are normally highly expressed in the testis such as dmrt <dig> were also upregulated in our study while genes such as cyp19a1a were downregulated due to temperature, highlighting the conserved masculinizing effect of heat across different vertebrate groups.
the third pattern of response was represented by genes whose expression was not affected by heat, including sox <dig>  a gene that has been related with ovarian development in the european sea bass and other fish  <cit> .
the brain aromatase gene  belonged to this group, thus corroborating earlier observations of our group  <cit> , and opposite to the increase observed in tilapia when applying ht during early development  <cit> .
other genes also in this group were tesc, important during male gonadal development, and col18a <dig>  implicated in organ morphogenesis.
since the hypermethylation of the cyp19a1a promoter in both females and masculinized females at one year of age was not so evident at  <dig> dph  <cit> , it may be that other epigenetic regulatory mechanisms are responsible for the “memory” of early ht exposure.
to gain further evidence, we examined the expression of seven epigenetic regulatory mechanisms-related genes that have been directly or indirectly connected with sex determination and gonadogenesis  <cit>  and were present in our microarray .
the selected genes for qrt-pcr analysis were hdac <dig>  jarid2a, ehmt <dig>  dicer <dig>  suz <dig>  pcgf <dig> and mettl <dig> to have representatives of different epigenetic mechanisms.
interestingly, four of them were upregulated in the ht group, displaying significant differences : hdac <dig>  jarid2a, dicer <dig> and pcgf <dig>  although further studies are clearly needed, it is interesting to note that, although in different ways, these genes are involved in transcriptional repression functions, which here may be connected with the long-lasting effects of early heat exposure.
whole gonad transcriptomic analysis showed  <dig> de genes .
some of these de genes were related to metabolic processes, and some to epigenetic regulatory mechanism including cg <dig> , hdac <dig>  or tep <dig> .
other genes are involved in reproductive processes in other species such as cdc42ep <dig>  where the mrna interacts with the human fertility protein pumilio <dig> in the testis  <cit> .
cry-dash, has an ancestral circadian role in light perception and related to massive spawning in corals during full moon  <cit>  while smtn <dig> is a regulator of the progesterone receptor during mice pregnancy  <cit>  and tnn <dig> controls ovulatory contraction of non-striated actomyosin networks in caenorhabditis elegans  <cit> .
enrichment analysis showed an upregulation of the overall catalytic activity, a process known to be affected by heat since elevated temperature produces changes in chemical reaction rates and increases protein denaturalization  <cit> .
this corresponded to an overrepresentation of the catalytic pathways and of the signal transduction due to heat.
the observation that most of the altered pathways were related to catabolism and signal reception-transmission corroborates the idea that the effects of heat were still persistent 110 days after the thermal treatment had finished.
in order to further understand the biological meaning of the set of de genes, david clustering analysis showed that dna binding and transcription were enriched, suggesting that despite protein and amino acid catabolism, protein synthesis and replacement was also occurring in ht gonads, most likely to compensate for the destabilizing temperature effects on protein structure.
likewise, downregulation of the immunology-related pathway may be because our samples were obtained from differentiating females .
cossins et al.
<cit>  investigated the transcriptomal response of seven carp tissues to cold.
from that study, we selected the  <dig> genes that showed the greatest increase in expression in response to cold: 92 kda type iv collagenase precursor , adp/atp translocase  <dig>  atp-binding cassette, subfamily f member  <dig>  apolipoproteins, rna-binding protein, nadp-dependent malic enzyme, mitochondrial uncoupling protein  <dig>  calmodulin, cofilin, granulin alpha, tubulins alpha, beta and gamma chains and high mobility group  <dig>  all are involved in a variety of functions including protein turnover, unsaturated fatty acid synthesis, homeostasis or stress protein production.
most of these  <dig> genes were present in our microarray and, interestingly, when we examined their behavior, we found that, in general, expression levels tended to be lower in our ht group, although without significant differences .
thus, many of the genes that cossins et al.
<cit>  found upregulated by cold in the carp intestine with transport and regulatory functions  had, when present, a downward tendency in the ht gonads.
we realize that this may be an anecdotal coincidence, but the observation that the above-mentioned genes seem to behave depending on whether fish are exposed to heat or cold and regardless of tissue or species, warrants their further study in other species exposed to temperature changes.
it could be possible that these genes could serve as markers of previous thermal history.
in contrast to what has been observed in killifish livers  <cit> , heat shock proteins were downregulated in our study, suggesting that the short heat exposure took place enough time ago to allow the return to their normal expression levels and become downregulated.
in addition, cholesterol and genes involved in the lipid metabolism were affected by heat.
thus, for example, cholesterol synthase and hmg-coa reductase were downregulated as a result of chronic temperature elevation, as previously reported by podrabsky and somero  <cit> .
the presence of translation elongation factors or proteasomes with a high level of expression due to heat is also in agreement with previous studies in other tissues  <cit> .
apart from common transcriptional responses to heat, each tissue seems to have different strategies to cope with temperature changes: brain modulates glycolytic activity, liver turns on lipid metabolism and muscle remodels its contractile apparatus  <cit> .
from the present study, we can add that gonads increase catabolism and signal transduction, but reproductive and immune related functions decrease.
this is in agreement with the documented deleterious effects of high temperature on gonadal function  <cit> .
CONCLUSIONS
this paper provides information on the effect of temperature at the whole gonadal transcriptomic level and at the time of sex differentiation in a fish with mixed genetic and environmental sex determination.
at about 3– <dig> months after the end of exposure, downregulation of the expression of female-related genes such as cyp19a1a and an increase in male-related ones such as dmrt <dig> was readily observed.
furthermore, some signaling, catabolic, biosynthetic, growth and reproduction pathways were still affected.
our study shows that the response to a change in the early environment, in this case of temperature, is evident months after the environmental perturbation has finished.
these transcriptomic changes determine the course of sex differentiation and ultimately alter the population sex ratio.
furthermore, this study, in addition to reporting changes in the expression of cognate genes and signaling pathways related to vertebrate sex differentiation, identifies genes previously not implicated in thermal-induced gonadal development, which seem conserved across species, as well as genes related to epigenetic regulatory mechanisms responsible for acquiring and maintaining cell identity, of which some were upregulated by high temperatures.
this data provide an advance to our understanding of the underlying mechanisms responsible for the environmentally driven transcriptomic changes, leading, in turn, to changes in an important phenotype such as the sexual phenotype.

 identifying environmental cues and their perception and transduction mechanisms is a central focus of research in developmental biology within an ecological context. changes in environmental variables can have profound influences on differentiation, growth and reproduction in many organisms. temperature is the main abiotic factor that affects many biological functions at different levels of organization by changing the rates of chemical reactions and physiological processes, or by changing the three-dimensional shapes of biomolecules. fish exhibit enormous diversity in their morphology, in habitat occupancy, and in their biology. this diversity is also remarkable as regards to their reproductive strategies including sex determination and differentiation, two processes that contribute to the establishment of the sex ratio, a crucial parameter for population viability and for the continuation of all species with sexual reproduction. the sex ratio can be affected by environmental factors, mainly by temperature. among vertebrates with genetic sex determination master sex determining genes are not conserved and eight genes with such a function have been identified so far: sry in mammals, dmrt- in birds, dm-w in xenopus laevis, and dmy, amhr amhy, sdy and gsdf in fish. on the other hand, genes involved in the sex differentiation process are fairly conserved in structure and dimorphic expression from fish to mammals. genes involved in testis differentiation include dmrt dax sox arb, amh, cyp11b, sox9a tbx1a and tbx1b; whereas genes involved in ovarian differentiation include cyp19a1a, foxl er, fst, hsd3b and star. however, the order of expression and interactions among these genes may change between groups or depending on environmental conditions. in any case, estrogens are essential for proper ovarian differentiation in all non-mammalian vertebrates. thus, cyp19a1a, the gene that codes for aromatase, the enzyme that catalyzes the irreversible conversion of androgens into estrogens, is a major player in vertebrate sd and crucial for the establishment of the sex ratio. aromatase gene expression is susceptible to environmental temperature influences. therefore, and regardless of the sex ratio response pattern to temperature, in reptiles and fish, the two types of vertebrates with temperature-dependent sex determination, the effects of environmental temperature on sex ratios are mediated by changes in cyp19a1a expression. in all fish species analyzed so far more males are produced with increasing temperatures and cyp19a1a is always inhibited at male-producing temperatures and stimulated at female-producing temperatures, in general, elevated temperatures increase the expression of male-related genes such as amh, dmrt or arb after the sd period, while decreasing the expression of female-related genes in addition to cyp19a1a, such as esr esr erb fshr or foxl. effects of temperature on fish sex ratios are more pronounced if animals are exposed to elevated temperatures during early development. however, the number of genes known to be affected is limited and the metabolic and signaling pathways affected are essentially unknown. in this regard, several studies have explored the effects of cold and cold acclimation but focusing on tissues other than the gonads, such as liver, skeletal white muscle and gills; brain or heart. unfortunately, comparative transcriptomic studies on the effect of heat on the gonads are essentially limited to just one study with the pejerrey, odontesthes bonariensis, a fish with tsd, and carried out on juvenile or adult mature gonads, not with differentiating gonads. thus, it is difficult to ascertain whether observed altered patterns of gene expression are the cause or the consequence of a given gonadal phenotype resulting from exposure to elevated temperature. the european sea bass is a eurythermal marine teleost able to live between and 27 °c and a gonochoristic species with a polygenic system of sex determination, where genetics and temperature contribute essentially equally to sex ratios. recent studies in the sea bass have shown that cyp19a1a and cyp11b are good markers of female and male sex differentiation, respectively. in all fish species studied so far, high temperatures cause masculinization. this is also the case of the european sea bass, where elevated temperatures during the thermosensitive period, which is located 0– dph, result in masculinization of about 50 % of the fish that otherwise, under more natural temperatures, would have developed into females, as assessed by sex ratio analysis. recently, we discovered that the effects of early temperature include hypermethylation of the cyp19a1a promoter in one-year-old juvenile females, with concomitant suppression of cyp19a1a expression and resulting in male instead of female development, the first evidence of an epigenetic link between environmental temperature and sex ratios in vertebrates. we were interested in understanding the underlying mechanisms responsible for this masculinization at the time when the differentiating gonad is being affected. therefore, instead of sampling one-year-old juveniles, as done before by us and others, in which gonads have already completed the process of sex differentiation, and where it is difficult to ascertain whether observed transcriptomic profiles and epigenetic changes are the cause or the consequence of that process, we sampled fish at dph, i.e., 110 days after the end of temperature treatment but shortly after the start of sex differentiation. thus, the objective was to gain a better understanding of the genes and the pathways involved in sex differentiation that are directly affected by temperature at the time of sex differentiation.  growth, body indices and sex ratios because of the differences in rearing temperature between and dph, fish from the ht group were significantly larger than those of the lt group at dph in both sl and bw. growth differences between groups had disappeared by dph. sexual growth dimorphism, in favor of females, was present only in the lt group. as observed in our previous studies survival was unrelated to temperature treatment. survival was ~60 % to the end of the larval stage and ~90 % thereafter.table growth of european sea bass juveniles at 170 days post hatch, classified according to treatment and cyp19a1a expression levels, as shown in fig. 1a data as mean ± sem lt low temperature, ht high temperature, n sample size different letters indicate significant differences between treatments data as mean ± sem lt low temperature, ht high temperature, n sample size the same superscript a indicates lack of significant differences between treatments. asterisks indicate statistical differences between females and males within the same treatment group, i.e., sexual growth dimorphism at dph, from each temperature group we randomly sampled fish. however, since, as stated above, elevated temperature results in masculinization of some females, within the randomly selected fish we choose for microarray analysis, in each group, fish with high cyp19a1a levels, which are typically associated with female development, and for this we used a previously validated clustering method based on cyp19a1a qrt-pcr expression levels. one-way anova showed statistical differences due to the expression levels, temperature treatment and their interaction. thus, in the lt or control group, fish with high cyp19a1a levels undoubtedly represented developing females, whereas in the ht group fish with high cyp19a1a levels also represented females that had been subjected to elevated temperature. it is important to state that despite overall differences in the sampled fish there were no differences in sl or bw between the fish selected from the two groups for microarray analysis. thus, the observed transcriptomic differences between ht and lt fish were due to temperature and not to size-related differences in gonadal development.fig. a individual gonadal aromatase expression levels as assessed by qrt-pcr in the low and high temperature groups at dph. blue squares and red circles correspond to individual fish with low and high cyp19a1a levels, respectively. the horizontal line marks mean expression for each experimental group. the cyp19a1a expression of one of the putative females in the lt group was much higher than the rest and it has been deliberately omitted from the graph for clarity purposes. different letters indicate significant differences between the four groups, i.e., high and low cyp19a1a expressors of the lt and ht treatments. b sex ratios of juvenile european sea bass sampled at 332 days post hatch. stacked bars showing male and female percent in the low and high temperature groups. statistical differences between groups are marked with three asterisks. sample size: lt, fish; ht, fish visual assessment of the sex ratio of dph juveniles combined with histological verification showed that the lt group had  % males while the ht group had  % males, showing a masculinizing effect of the elevated temperature. however, within each sex no differences were observed in the presence and in the abundance of the different cellular types between the two temperature groups. females had immature ovaries containing oocytes at the cortical alveolar stage while males had testis containing all germ cell types, including spermatozoa. the gsi percent values at dph for lt males and females were  ±  and  ± , respectively, and for ht males and females were  ±  and  ± , respectively. a two-way anova analysis showed statistical differences due to sex but no effect due to the thermal treatment or to the interaction between sex and temperature. microarray analysis microarray analysis of sexually differentiating gonads at dph obtained from fish with a high cyp19a1a expression, i.e., putative females, revealed the presence of significantly and differentially expressed genes when comparing the ht vs. the lt group, of which genes were upregulated and were downregulated. a heatmap representation of the de genes grouped fish according to their thermal history, with the exception of one lt fish, which had an intermediate position, and one ht fish, which was classified as an outlier by a principal component analysis and was not further considered in the analysis. some of the upregulated genes were related to reproduction, i.e., cryptochrome dash and troponin i, or to epigenetic gene expression regulation, i.e., histone deacetylase. some of the downregulated genes also showed reproduction-related functions such as cdc effector protein, insulin-like growth factor or smoothelin.fig. heatmap of the microarray expression data for the up- and downregulated genes, where each row represents a gene and each column represents an individual fish. key color representing the level of expression. the dendograms provide information of the similarity between genes and between the different samples. notice that all ht samples and all but one lt samples cluster together. lt, low temperature group; ht, high temperature group. see additional file 1: table s for a complete list of gene names and abbreviations the amigo web-based tool was used to recover the sequence of these de genes and those sequences were then uploaded to blast2go in order to enrich results with go terms and extract more information about these de genes. a fisher’s exact test with multiple testing corrections for false discovery rate showed that five go term categories were overrepresented when compared to a reference test containing all the annotated sequences from our custom-made array. further analysis of the go terms provided their distribution among the three main categories: biological process, molecular function and cell component for the up- and downregulated genes separately. the kyoto encyclopedia of genes and genomes database provided more information on the pathways containing these de genes. several pathways involved in protein synthesis as well as in immunological processes were found. microarray validation out of the de genes, we selected eight, i.e., about one third of the de genes, for validation by qrt-pcr. these genes were selected because they are known to be involved in reproduction in other species or because they are involved in epigenetic regulatory mechanisms. in addition, together they exhibited a wide dynamic range of changes. qrt-pcr analysis showed significant differences for cg and hdac among the upregulated genes and for the cdc42ep and smtn among the downregulated ones. importantly, all but one of these selected genes had the same direction of change when analyzed by qrt-pcr as compared to microarray results. we consider this sufficient for the validation of the microarray.fig. validation of microarray results by analyzing ten fish by qrt-pcr according to treatment. a-d four upregulated genes in the ht vs. the lt group comparison: dmei_cg; histone deacetylase; cryptochrome dash and troponin i. e-h four downregulated genes for the same comparison: cell division cycle effector protein; extracellular matrix protein; carbonic anhydrase and smoothelin. data as mean ± sem. letters mark statistical significance between groups. ten fish were analyzed by a qrt-pcr enrichment analysis a go enrichment analysis of the de genes showed up- and downregulation of the same bp categories, albeit containing different de genes. these altered categories contained more go terms that were upregulated and mainly related to metabolic and cellular processes, while other processes such as reproduction, growth, immune processes or signaling were downregulated at elevated temperatures. regarding mf go categories, the catalytic and binding activities were the most represented subcategories for both up- and downregulated go terms. analysis of the cc categories showed that upregulated processes were taking place mainly in the organelle, macromolecular complex, membrane-enclosed lumen or membrane, while the downregulated processes were taking place in the membrane, organelle, macromolecular complex or extracellular region. further analysis of the go enriched terms of the de genes in comparison to the microarray reference set, showed that terms related to the negative regulation of the nerve impulse and synaptic transmission were overrepresented, as well as the adenylate cyclase inhibiting g-protein coupled receptor signaling pathway.fig. biological process-related go terms for the ht versus lt group comparison. a biological process go terms for the upregulated genes, and for the downregulated genes blast2go analysis of the de genes showed that eleven of the differentially regulated pathways had higher expression in ht fish than in lt fish and that these pathways were related to catabolism, biosynthesis or signal transduction. however, the downregulated pathways were related, in agreement with the results of the go term analysis, to immunology and nitrogen metabolism. david analysis of the de genes showed that there were upregulated categories, of which four were highly significant: 1) progesterone-mediated oocyte maturation, 2) tight junction, 3) chemokine signaling, and 4) hormone-mediated signaling pathways. in all of them, adcy is involved as a signaling initiation factor. after running an annotation clustering with david, seven clusters were downregulated and related to cell component, dna binding, transcription and signaling processes. reproduction and stress-related genes fifteen known reproduction-related genes were analyzed by qrt-pcr. results showed that genes involved in testicular differentiation, such as doublesex-mab-3-related transcription factor, were significantly upregulated in ht fish, as also was the steroidogenic acute regulatory protein. in contrast, some genes involved in ovarian differentiation, such as aromatase, were significantly downregulated in ht fish, as also was aquaporin. the expression of eight of these genes is shown in fig.  since activation of the stress response has been associated with temperature effects on sex ratios in fish, we tested the effects of ht in our model in two stress response-related genes; the 11ß-hydroxysteroid dehydrogenase and the glucocorticoid receptor, involved in cortisol synthesis and response. while nr3c was significantly upregulated in ht fish, hsd11b was not significantly different.fig. quantitative rt-pcr results for eight known sex differentiation-related genes per temperature treatment groups. a-d male pathway: anti-müllerian hormone, doublesex- and mab-3-related transcription factor; female pathway: cytochrome p family subfamily a, polypeptide 1a and sry-related hmg-box transcription factor sox, respectively. e steroidogenic acute regulatory protein, vasa protein, insulin-like growth factor and cytochrome p family subfamily a, polypeptide 1b. data as mean ± sem. asterisk marks statistical differences between groups. ten fish were analyzed by a qrt-pcr fig. quantitative rt-pcr results for 11β-hydroxysteroid dehydrogenase and glucocorticoid receptor. data as mean ± sem. asterisk marks statistical differences between groups. ten fish were analyzed by a qrt-pcr epigenetic mechanisms-related genes according to a growing body of evidence, several genes involved in epigenetic regulatory mechanisms have been implicated in sex determination/differentiation. based on this, we analyzed the genes related to epigenetic mechanisms and implicated in sex determination/differentiation that were present in our microarray even if they were found to be not de. seven genes, representative of different categories of epigenetic regulatory mechanisms, including dicer a helicase needed to produce an active small rna component that represses gene expression; ehmt a histone methyltransferase; jarid2a, a dna-binding protein that acts as a transcriptional repressor; pcgf which contains a ring finger motif and forms protein-protein interactions to maintain transcriptional repression; hdac a histone deacetylase; mettl a methyltransferase-like protein; and suz a suppressor of trithorax zeste homolog gene, were selected and analyzed by qrt-pcr. four of these genes were upregulated in the ht group: dicer jarid2a, pcgf and hdac11).fig. quantitative rt-pcr results for the epigenetic regulatory mechanisms-related genes. endoribonuclease dicer, euchromatic histone-lysine n-methyltransferase, protein jumonji and polycomb group ring finger. data as mean ± sem. asterisks mark statistical differences between groups. ten fish were analyzed by a qrt-pcr discussion to the best of our knowledge, this is the first study on the analysis of the gonadal transcriptome at the time of sex differentiation, using a species-specific microarray, of fish previously exposed to elevated temperature during the thermosensitive period. since we took care that the fish selected from each group for microarray analysis at dph had the same size, it is safe to assume that differences in gene expression observed are solely due to temperature, not to differences in growth. consistent with previous studies, temperature induced a male-biased sex ratio. the higher gsi values for ht-treated females observed at one year of age may be due to a persistent effect on gonadal growth relative to somatic growth with no obvious effect at the histological level. the number of de genes was low as could be anticipated because in the ht vs. lt comparison we actually compared fish of the same age, at a similar developmental stage and similar size, as just stated above, and most likely of the same sex, females, as assessed by high cyp19a1a expression levels, a good marker of phenotypic sex. this is actually what we intended since females, not males, are the ones mainly affected by temperature. moreover, at dph gonads were cleanly isolated and there was no contamination of the surrounding tissues. what we could gain by looking at earlier age, e.g., at the end of the tsp at about dph, we would lose by having to include non-gonadal tissues. among the genes analyzed by qrt-pcr, there were three patterns of response to heat. many genes had increased levels of expression in the ht group. among them, there were genes upregulated during normal testis differentiation such as dmrt in accordance with the masculinizing effect of elevated temperatures.. the vasa helicase is a germ cell marker known to be more expressed in ovaries than in testis and previously found to be upregulated by elevated temperatures. in our study the difference in vasa expression levels did not reach statistical significance. cholesterol is important in maintaining membrane integrity and sterol synthesis and its levels have previously been found also to be increased by temperature. however, genes ultimately related to steroid hormone production were not significantly affected in the present study, including the gene encoding gonadotropin-releasing hormone, and star, which is involved in cholesterol import. in our study, genes such as wisp known to be involved in cellular growth, were downregulated by heat, whereas genes such as nr3c related to immune system regulation responses, were upregulated, suggesting growth and immunological stress response adjustments in the gonads. there were other downregulated genes. among these there was cyp19a1a, confirming results of an independent experiment aimed to find molecular signatures of male and female differentiation. aquaporin, a water channel protein which plays a major role in oocyte hydration in fish, was also downregulated by heat. the steroidogenic enzyme 11ß-hydroxysteroid-dehydrogenase, which converts the stress hormone cortisol into the inactive metabolite cortisone, and also converts 11ß-hydroxy androgens such as 11ß-hydroxyadrostenedione into 11-ketotestosterone, a potent piscine androgen, was not significantly affected by heat. in contrast, the glucocorticoid receptor was upregulated in the ht group with significant differences with respect to the lt group. these results are interesting because in the pejerrey, a fish with tsd, fernandino et al. observed upregulation of both hsd and nr3c in masculinized gonads by ht and attributed this to the fact that ht elicits both masculinizing and stress responses. in our study, nr3c was upregulated 110 days after the end of the temperature treatment, suggesting a persistent stress response probably maintained by an epigenetic regulatory mechanism. in fact, studies in rodents have shown that nr3c is able to exhibit sustained expression, even a long time after the stimulus ended, through changes in methylation of its regulatory region. the differences between our results and those of fernandino et al. with the pejerrey suggests that gender or developmental stage may be important in explaining these differences, since while fernandino et al. sampled juvenile, sexually differentiated males, in our case we selected not only sexually differentiating fish but also those ones that exhibited cyp9a1a expression levels at dph compatible with ovarian differentiation. furthermore, when comparing our results with those obtained with microarray analysis of dimorphic gene expression in a turtle with tsd, genes that are normally highly expressed in the testis such as dmrt were also upregulated in our study while genes such as cyp19a1a were downregulated due to temperature, highlighting the conserved masculinizing effect of heat across different vertebrate groups. the third pattern of response was represented by genes whose expression was not affected by heat, including sox a gene that has been related with ovarian development in the european sea bass and other fish. the brain aromatase gene belonged to this group, thus corroborating earlier observations of our group, and opposite to the increase observed in tilapia when applying ht during early development. other genes also in this group were tesc, important during male gonadal development, and col18a implicated in organ morphogenesis. since the hypermethylation of the cyp19a1a promoter in both females and masculinized females at one year of age was not so evident at dph, it may be that other epigenetic regulatory mechanisms are responsible for the “memory” of early ht exposure. to gain further evidence, we examined the expression of seven epigenetic regulatory mechanisms-related genes that have been directly or indirectly connected with sex determination and gonadogenesis and were present in our microarray. the selected genes for qrt-pcr analysis were hdac jarid2a, ehmt dicer suz pcgf and mettl to have representatives of different epigenetic mechanisms. interestingly, four of them were upregulated in the ht group, displaying significant differences: hdac jarid2a, dicer and pcgf although further studies are clearly needed, it is interesting to note that, although in different ways, these genes are involved in transcriptional repression functions, which here may be connected with the long-lasting effects of early heat exposure. whole gonad transcriptomic analysis showed de genes. some of these de genes were related to metabolic processes, and some to epigenetic regulatory mechanism including cg, hdac or tep. other genes are involved in reproductive processes in other species such as cdc42ep where the mrna interacts with the human fertility protein pumilio in the testis. cry-dash, has an ancestral circadian role in light perception and related to massive spawning in corals during full moon while smtn is a regulator of the progesterone receptor during mice pregnancy and tnn controls ovulatory contraction of non-striated actomyosin networks in caenorhabditis elegans. enrichment analysis showed an upregulation of the overall catalytic activity, a process known to be affected by heat since elevated temperature produces changes in chemical reaction rates and increases protein denaturalization. this corresponded to an overrepresentation of the catalytic pathways and of the signal transduction due to heat. the observation that most of the altered pathways were related to catabolism and signal reception-transmission corroborates the idea that the effects of heat were still persistent 110 days after the thermal treatment had finished. in order to further understand the biological meaning of the set of de genes, david clustering analysis showed that dna binding and transcription were enriched, suggesting that despite protein and amino acid catabolism, protein synthesis and replacement was also occurring in ht gonads, most likely to compensate for the destabilizing temperature effects on protein structure. likewise, downregulation of the immunology-related pathway may be because our samples were obtained from differentiating females. cossins et al. investigated the transcriptomal response of seven carp tissues to cold. from that study, we selected the genes that showed the greatest increase in expression in response to cold: 92 kda type iv collagenase precursor, adp/atp translocase atp-binding cassette, subfamily f member apolipoproteins, rna-binding protein, nadp-dependent malic enzyme, mitochondrial uncoupling protein calmodulin, cofilin, granulin alpha, tubulins alpha, beta and gamma chains and high mobility group all are involved in a variety of functions including protein turnover, unsaturated fatty acid synthesis, homeostasis or stress protein production. most of these genes were present in our microarray and, interestingly, when we examined their behavior, we found that, in general, expression levels tended to be lower in our ht group, although without significant differences. thus, many of the genes that cossins et al. found upregulated by cold in the carp intestine with transport and regulatory functions had, when present, a downward tendency in the ht gonads. we realize that this may be an anecdotal coincidence, but the observation that the above-mentioned genes seem to behave depending on whether fish are exposed to heat or cold and regardless of tissue or species, warrants their further study in other species exposed to temperature changes. it could be possible that these genes could serve as markers of previous thermal history. in contrast to what has been observed in killifish livers, heat shock proteins were downregulated in our study, suggesting that the short heat exposure took place enough time ago to allow the return to their normal expression levels and become downregulated. in addition, cholesterol and genes involved in the lipid metabolism were affected by heat. thus, for example, cholesterol synthase and hmg-coa reductase were downregulated as a result of chronic temperature elevation, as previously reported by podrabsky and somero. the presence of translation elongation factors or proteasomes with a high level of expression due to heat is also in agreement with previous studies in other tissues. apart from common transcriptional responses to heat, each tissue seems to have different strategies to cope with temperature changes: brain modulates glycolytic activity, liver turns on lipid metabolism and muscle remodels its contractile apparatus. from the present study, we can add that gonads increase catabolism and signal transduction, but reproductive and immune related functions decrease. this is in agreement with the documented deleterious effects of high temperature on gonadal function.  this paper provides information on the effect of temperature at the whole gonadal transcriptomic level and at the time of sex differentiation in a fish with mixed genetic and environmental sex determination. at about 3– months after the end of exposure, downregulation of the expression of female-related genes such as cyp19a1a and an increase in male-related ones such as dmrt was readily observed. furthermore, some signaling, catabolic, biosynthetic, growth and reproduction pathways were still affected. our study shows that the response to a change in the early environment, in this case of temperature, is evident months after the environmental perturbation has finished. these transcriptomic changes determine the course of sex differentiation and ultimately alter the population sex ratio. furthermore, this study, in addition to reporting changes in the expression of cognate genes and signaling pathways related to vertebrate sex differentiation, identifies genes previously not implicated in thermal-induced gonadal development, which seem conserved across species, as well as genes related to epigenetic regulatory mechanisms responsible for acquiring and maintaining cell identity, of which some were upregulated by high temperatures. this data provide an advance to our understanding of the underlying mechanisms responsible for the environmentally driven transcriptomic changes, leading, in turn, to changes in an important phenotype such as the sexual phenotype. 
previous studies on the effects of temperature on gene expression have been restricted to a few cognate genes, mostly related to testis or ovarian development, and analyzed in gonads once they had completed the process of sex differentiation. however, studies on the effect of temperature at the whole gonadal transcriptomic level are scarce in fish and, in addition, temperature effects at the time of sex differentiation at the transcriptomic level are also unknown. transcriptomic analysis of the gonads was carried out about three months after the end of temperature exposure, shortly after the beginning of the process of sex differentiation. results also show the usefulness of comparing the effects of heat on the behavior of cognate genes related to sex differentiation as well as that of genes involved in establishing and maintaining cell identity through epigenetic mechanisms. sex in fish is plastic and in several species can be influenced by environmental factors. taken together, the results of this study contribute to the understanding of how the early environment sets permanent changes that result in long-lasting consequences, in this case in the sexual phenotype. transcriptomic analysis of early differentiating female gonads showed how heat caused: 1) an up-regulation of genes related to cholesterol transport, the stress response and testis differentiation, 2) a decrease in the expression of genes related to ovarian differentiation such as cyp19a1a, and 3) an increase in the expression of several genes related to epigenetic regulatory mechanisms. here, we used the european sea bass, a gonochoristic teleost with a polygenic sex determination system influenced by temperature, and exposed larvae to elevated temperature during the period of early gonad formation.
 sex in fish is plastic and in several species can be influenced by environmental factors. in sensitive species, elevated temperatures have a masculinizing effect. previous studies on the effects of temperature on gene expression have been restricted to a few cognate genes, mostly related to testis or ovarian development, and analyzed in gonads once they had completed the process of sex differentiation. however, studies on the effect of temperature at the whole gonadal transcriptomic level are scarce in fish and, in addition, temperature effects at the time of sex differentiation at the transcriptomic level are also unknown. here, we used the european sea bass, a gonochoristic teleost with a polygenic sex determination system influenced by temperature, and exposed larvae to elevated temperature during the period of early gonad formation. transcriptomic analysis of the gonads was carried out about three months after the end of temperature exposure, shortly after the beginning of the process of sex differentiation.  elevated temperature doubled the number of males with respect to untreated controls. transcriptomic analysis of early differentiating female gonads showed how heat caused: 1) an up-regulation of genes related to cholesterol transport, the stress response and testis differentiation, 2) a decrease in the expression of genes related to ovarian differentiation such as cyp19a1a, and 3) an increase in the expression of several genes related to epigenetic regulatory mechanisms.  taken together, the results of this study contribute to the understanding of how the early environment sets permanent changes that result in long-lasting consequences, in this case in the sexual phenotype. results also show the usefulness of comparing the effects of heat on the behavior of cognate genes related to sex differentiation as well as that of genes involved in establishing and maintaining cell identity through epigenetic mechanisms. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords gonadal developmenttemperatureepigeneticssex ratioissue-copyright-statement© the author 2015 