BACKGROUND
microarray technology provides a powerful tool for investigating gene regulation and its significance for biological function.
however, our understanding of such relationships during environmental stress remains fragmentary, especially in vertebrates.
in particular, the commonality, or otherwise, of the responses of vertebrates to different environmental stresses remain poorly understood.
recently, some understanding of responses to individual stresses, in particular those related to thermal stress in teleost fishes, has been gained.
in these species, thermal stress can lead to changes in ventilation and circulation rates  <cit> , changes in mitochondrial densities and their properties  <cit> , and a reduction in cellular oxygen levels  <cit> .
reduced cellular oxygen levels can lead to increased levels of oxidative stress and hence, the cellular response to thermal stress often includes responses aimed at alleviating oxidative stress  <cit> .
for example, antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase are commonly activated during thermal stress  <cit> .
oxidative stress, and the cellular damage associated with it, can in turn induce a heat shock response primarily aimed at the molecular repair of protein damage  <cit> .
in addition, thermal stress can lead to extensive changes in gene expression  <cit> .
these transcriptional responses are likely the result of the stress-dependent activation of only a limited number of upstream regulators, namely the activation mitogen-activated protein kinases , in particular the jnk and p <dig> signalling pathways  <cit> , and immediate early genes  <cit> .
these complex transcriptional responses to stress necessarily precede adjustments at the protein level, and thus, are expected to form an important component of the cellular response to stress.
to advance our current limited understanding of the consistency of transcriptional stress responses across different environmental stress types, it is necessary to examine stress responses to a diversity of stressors simultaneously and a suitable model organism is required.
teleost fishes, and coral reef fishes in particular, are one such potential model.
being ectothermic and aquatic and because their physiology is tightly linked to the environment, teleost fishes are particularly amenable to experimental manipulation of environmental stresses and have hence become important models for environmental stress studies  <cit> .
coral reef fishes inhabit relatively stable environments compared to those experienced by many other teleost fishes, and are so likely to have increased sensitivity towards thermal and environmental anomalies.
by identifying the most sensitive biological processes in organisms with limited tolerance towards environmental anomalies, we gain understanding of the types of processes that must have been the targets of adaptation in organisms that exhibit broad environmental tolerances .
in this way, studying environmental stress responses in coral reef fishes may provide fundamental insights into mechanisms of environmental adaptation.
while there are currently no species-specific microarrays available for any species of coral reef fish, we have previously shown that the compugen  <dig> k oligonucleotide microarray developed for the zebrafish, danio rerio, can be successfully used to study gene responses in the coral reef fish pomacentrus moluccensis  <cit> .
in this previous study, comparative genomic hybridisation experiments showed good cross-hybridisation between the two fish species for most genes represented on the microarray, and differential expression of a set of candidate genes were confirmed by quantitative real-time pcr  <cit> .
here, we use this previously validated heterologous microarray approach to measure transcriptional responses of the coral reef fish pomacentrus moluccensis to different environmental stressors, including hypoxia, hyposmotic stress, cold, and heat.
we also compare heat stress responses of winter- and summer-acclimated fish to estimate seasonal effects on gene regulation.
the duration of environmental stress treatments was varied, permitting different aspects of the biological response to be examined.
short-term exposure experiments over three hours investigated early gene responses and upstream regulators of transcriptional stress responses, while prolonged exposure to heat over five days investigated medium-term effects of heat stress.
by grouping genes into functional categories and identifying those functional categories most commonly associated with gene responses to different types of environmental stress, we identify components of a common stress response in p. moluccensis as well as features unique to different types of environmental stress.
we use these gene function responses to infer stress-induced changes in biological and physiological function and discuss the types of biological functions affected by exposure to environmental stress.
RESULTS
early gene responses to different environmental stresses
to characterise environmental stress responses, we analysed the gene expression profiles of  <dig> individuals.
these individuals were from a natural population of p. moluccensis and were either subjected to one of the environmental stress treatments or kept at ambient conditions.
the microarray platform, data series, and raw data including the .tif image and .spot files are available from the gene expression omnibus website  <cit>  under accessions gpl <dig>  gse <dig>  and gsm181765-gsm <dig>  most genes that showed significant expression changes in response to short-term cold, heat, hypoxic or hyposmotic shock were down-regulated compared to the expression levels measured in fish kept at ambient conditions .
short-term heat shock at 34°c in summer led to significant expression changes at six gene loci, amongst which were pdip <dig>  rhoa, and zcchc <dig>  and three genes showed greater than two-fold expression changes .
in contrast to the summer response, the short-term 34°c heat shock response in winter involved many more genes, and the observed expression changes generally were of greater magnitude .
in total,  <dig> gene loci were differentially expressed in the winter-response to short-term heat shock at 34°c.
all but six of these genes were down-regulated in heat-stressed fish with fold changes up to  <dig> .
the early transcriptional response to hypoxia involved ten differentially expressed genes, amongst which were acin1a, β-actin, lbr, and ela <dig>  and a total of thirteen genes with greater than two-fold expression changes .
hyposmotic shock resulted in no significant expression changes, and none of the genes assayed showed greater than two-fold expression changes.
it is possible that other tissues, for example gills, might have shown a greater response to hyposmotic challenge, but measurement of expression changes in multiple tissues was outside the scope of this study.
due to this lack of response in liver tissue, hyposmotic stress is not discussed further.
gene regulation in response to prolonged heat
prolonged exposure to 31°c over five days resulted in  <dig> differentially expressed genes, most of which – in contrast to the short-term exposure experiments – were induced and  <dig> of which showed greater than two-fold expression changes .
genes encoding protein kinase c substrate 80k-h, rab escort protein  <dig>  semaphorin 3ab, distal-less homeobox  <dig>  and ttf-i interacting peptide  <dig> showed the greatest regulation .
differentially expressed genes belonged to a variety of gene functions, amongst which were protein processing, signal transduction, and transcription and translation.
only four percent had been previously associated with the response to stress, while  <dig> percent were of yet unknown gene function .
common effects of stress on biological function
the gene expression responses to different environmental stressors can be tested for the presence of common gene function responses.
such an approach can aid the interpretation of gene regulation in terms of the types of biological functions that are affected.
we thus grouped individual genes on the basis of their gene function.
we then tested whether genes of a certain function were more likely to be differentially expressed in response to environmental stress than expected if differential expression was independent of gene function.
such gene set analysis revealed significant association of a number of gene functional groups with the gene responses to different types of environmental stress .
in particular, gene functions related to cell growth and cytoskeleton, protein turnover, and metabolism were consistently associated with gene responses to environmental stress, regardless of the type of stressor applied .
for each gene ontology an aggregate score was computed based on the p-values for differential expression of the genes in that category.
the significance of the resulting gene set score was determined by random sampling of the data using the software erminej  <cit> .
p-values are fdr-corrected with values smaller than  <dig>  indicated in bold. "
<dig> h" indicates three hour exposure," <dig> d" five day exposure to stressor, 'sum."
refers to exposure experiments performed in summer, "hyposm."
refers to hyposmotic exposure experiments.
variable gene responses across stress treatments
having identified common gene function responses, we wanted to visualise the responses of individual genes across stress treatments and test for the presence of a set of commonly induced, or suppressed, genes.
for this purpose, we extracted the expression data from all differentially expressed genes across all treatments and organised the expression profiles using unsupervised hierarchical clustering.
hierarchical clustering identified four main clusters, with genes belonging to the same cluster showing coordinated responses across stressors .
while we identified groups of co-regulated genes, gene responses varied considerably across stressors.
individual genes that showed differential expression in one treatment did not necessarily show a significant response in another treatment, and some genes showed expression changes in opposite directions in response to different stressors .
cluster i was comprised of genes which were strongly induced in the 31°c five days treatment, but showed variable responses in the short-term treatments.
a subset of cluster i genes was generally down-regulated in the short-term treatments .
in order to determine whether there was a relationship between cluster identity and gene function, i.e.
whether co-regulated genes shared similar gene functions, we performed gene class testing on the clusters identified by hierarchical clustering using over-representation analysis.
the gene ontology 'mhc class i protein binding' was significantly over-represented amongst cluster i genes .
cluster ii contained a series of genes that were strongly down-regulated in the 34°c winter treatment and which showed variable expression responses in the other treatments.
a subset of cluster ii genes was down-regulated in most stress treatments examined .
cluster ii genes showed significant over-representation of gene functions related to cell growth and cytoskeleton .
cluster iii was comprised of genes that were strongly suppressed during prolonged heat at 31°c and most of which were induced in the short-term treatments, especially group c genes .
metabolic gene functions were significantly over-represented amongst cluster iii genes .
lastly, cluster iv included genes that were strongly induced in the 31°c five days treatment and which were generally also induced in the short-term treatments, especially genes in group d .
stress-related gene functions showed a trend for over-representation amongst cluster iv genes, but just exceeded the threshold for significance after fdr-correction .
we tested for significant over-representation of gene ontologies of genes within a cluster compared to the total complement of genes represented on the microarray.
gene clusters were identified by hierarchical clustering of significant gene responses across stressors
discussion
this is the first study to examine the effects of different types and durations of environmental stress on transcriptional regulation in an ectothermic vertebrate, in this case the coral reef fish p. moluccensis.
we identified a series of gene functions that were consistently associated with gene responses to environmental stress, suggesting common effects of different types of stress on biological function.
however, these common responses were achieved by the regulation of largely independent sets of genes.
thus, there was conservation of gene function responses, but variability in the responses of individual genes.
while the expression response of individual genes varied depending on the types and durations of stresses applied, we have identified groups of co-regulated genes, the genes within which shared similar gene functions.
common gene function responses elucidate how stress affects p. moluccensis
some gene functions were consistently associated with the stresses applied here, and thus are likely to reflect the common effects of environmental stress on biological function in this species.
consistent with the observation that stress negatively affects cell growth  <cit>  and that, therefore, actin cytoskeletal elements are commonly regulated in response to stress  <cit> , the gene ontologies 'actin cytoskeleton organization and biogenesis', 'anti-apoptosis', 'induction of apoptosis', and 'cell cycle' were associated with most stress responses measured here.
individual stress-responsive genes in this category included β-actin, plectin  <dig>  dynein, spectrin, tubulin beta- <dig>  filamin  <dig>  acin1a, grim <dig>  tp53inp <dig>  septin  <dig>  cyclin g <dig>  cdca <dig>  and bax .
three genes encoding for nucleobindin 2a, growth arrest-specific  <dig>  and cyclin g <dig> and associated with cell cycle and cell growth, showed the most consistent and significant induction across all stress treatments examined , making them good candidates for molecular stress biomarkers in p. moluccensis.
gene functions related to protein turnover, such as 'regulation of translation', 'protein folding', and 'ubiquitin-dependent protein catabolism', were also commonly associated with the gene responses of p. moluccensis to stress.
individual genes that contributed to this component of the common stress response included pdip <dig>  usp <dig>  psmc <dig>  fbxl <dig>  the translation initiation factors eif2b <dig>  eif2c <dig>  eif4b, and the ribosomal proteins rps <dig>  mrpl <dig>  mrpl <dig>  stress can increase rates of protein damage and ubiquitin-dependent protein catabolism  <cit> .
association of the gene functions 'protein folding' and 'ubiquitin-dependent protein catabolism' with gene responses observed in this study suggest that environmental stress leads to increased protein damage, an adjustment of the proteome, and de novo synthesis of proteins in this species.
protein synthesis in ectotherms accounts for about  <dig> percent of the cellular energy budget  <cit> .
de novo protein synthesis and protein repair in response to environmental stress are thus likely to incur substantial energetic costs in p. moluccensis and may reduce the energy available for other organismal functions such as growth and reproduction.
these results are concordant with studies of the yeast saccharomyces cerevisiae that have shown that yeast responds to a range of environmental stressors with a stereotypical change in gene expression and the suppression of around ten percent of assayed genes, many of which function in protein synthesis and cell growth  <cit> .
a third group of gene functions commonly associated with environmental stress responses in p. moluccensis included metabolic gene functions, in particular gene functions related to carbohydrate and lipid metabolism.
stress-responsive genes with metabolic functions included sc4mol, chst <dig>  amy2a, acad, dgat2l, dcxr, and fuca <dig>  metabolic adjustments and the re-allocation of energy resources may reflect mechanisms that allow for increased levels of protein and cellular repair during stress.
genes with stress-related functions, and functions in cellular repair, were also commonly associated with transcriptional stress responses in p. moluccensis, and included dnajb <dig>  peroxiredoxin  <dig>  ptges <dig>  apoea, hif1an, and a gene encoding elastase  <dig>  elastase  <dig> hydrolyses collagen-iv and elastin and its activity has been shown to increase in the presence of reactive oxygen species  <cit> .
regulation of elastase  <dig>  therefore, suggests increased levels of oxidative stress under conditions of environmental stress.
in addition, genes with immune functions and the gene ontology 'response to pest, pathogen or parasite' were commonly associated with environmental stress responses, suggesting a challenge to the immune system.
this functional category included the genes chst <dig>  t-cell receptor alpha, and haptoglobin.
finally, the gene function 'dna repair' was significantly associated with many of the stress responses measured here, suggesting increased levels of dna damage under conditions of environmental stress.
such responses are consistent with the observation that different types of stress can lead to immunosuppression  <cit>  and genotoxic effects  <cit> .
here we have shown that a multi-stressor approach, and the identification of gene functions most often associated with gene expression responses to stress, can enhance our understanding of the biological processes altered by environmental stress.
our results suggest that common effects of environmental stress in p. moluccensis include suppression of cell growth, increased protein damage and de novo synthesis of proteins, metabolic adjustments and a reallocation of energy resources possibly related to increased protein and cellular repair, induction of stress genes and cellular repair systems, and a challenge of immune functions.
different types of stressors have been previously associated with a 'minimal stress proteome', comprised in particular of proteins involved in dna, protein and molecular repair and energy metabolism  <cit> .
our results therefore provide experimental support for the presence of a stress proteome, and common stress responses, also in an ectothermic vertebrate, the coral reef fish pomacentrus moluccensis.
early gene responses to environmental stress
in addition to the common stress responses discussed above, there were also differences between the responses to individual stressors.
for example, there was a remarkable difference in the number of genes that were regulated in response to heat shock at 34°c in summer and winter.
mean winter and summer temperatures differ by approximately 4°c at the study site.
seasonal acclimation can lead to extensive transcriptional adjustments in teleosts  <cit>  and may thus account for some of the transcriptional differences observed here.
we cannot exclude, however, the possibility that some of the changes in gene expression were due to the male fish employed in the microarray analyses being at different stages of the reproductive cycle at the time of sampling.
the different experimental designs employed in the summer and winter analyses  may have further confounded these results as biological samples had been pooled in the winter analyses, but not in the summer analyses.
further studies will be required to more precisely define the sources of these differences in stress responses between summer and winter.
many responses to hypoxia compensate for reduced cellular oxygen levels by increasing capacities for oxygen delivery, and by enhancing oxygen-independent atp production by means of glycolysis  <cit> .
the hypoxia-inducible transcription factor hif- <dig> plays an important role in coordinating transcriptional responses to hypoxia  <cit> .
in the present study, hif-1a showed only limited gene regulation  which is consistent with hif regulation occurring largely at the protein level, not at the mrna expression level.
however, hypoxia-inducible factor  <dig> alpha subunit inhibitor , which modulates hif- <dig> transcriptional activity, was significantly down-regulated in response to prolonged heat exposure .
while further data are required to understand the role of hif-1a and hif1an in response to hypoxic and heat stress in p. moluccensis, association of the gene ontologies 'glycolysis' and 'gluconeogenesis' with the response to hypoxia is consistent with the need to increase oxygen-independent atp production.
furthermore, amongst the most significant expression responses to hypoxia was induction of amy2a, encoding the amylase- <dig> protein that endohydrolyses  <dig> -α-d-glucosidic linkages in polysaccharides such as starch and glycogen to make dextrin, which can then be reduced to α-d-glucose.
during hypoxia, this protein appears to free stored sugars for use in glycolysis and would, therefore, facilitate oxygen-independent atp production.
gene expression responses to prolonged heat
given current models of climatic change and the prediction of rising sea surface temperatures, we were particularly interested in gene responses to prolonged heat, and how early gene responses may change following prolonged exposure to heat.
we found that while the gene ontology 'protein folding' remained associated with the response to heat even after prolonged exposure 'ubiquitin-dependent protein catabolism' was not.
these results suggest that initial adjustments of the proteome during stress involve increased degradation of  proteins and increased levels of ubiquitin-dependent protein catabolism.
over time, rates of protein catabolism appear to return to baseline levels, while protein chaperone systems remain induced.
prolonged heat exposure was further associated with the gene ontology 'response to oxidative stress' and induction of the genes encoding peroxiredoxin  <dig> and apolipoprotein ea, two proteins with antioxidant function.
oxidative stress is caused by the formation of reactive oxygen species and has been associated with many forms of stress  <cit> .
it is likely that oxidative stress, and the associated cellular and molecular damage, incur substantial energetic costs during prolonged heat exposure.
the fact that the gene ontologies 'carbohydrate metabolism' and 'lipid metabolism' remain associated with gene responses even after prolonged exposure to heat may reflect long-term re-allocation of energy resources.
such energetic re-organisation may compensate for the energetic needs associated with protein and cellular repair arising from prolonged heat exposure and the associated oxidative stress.
for example, acad, dgat2l, dcxr, hadh <dig>  crabp <dig>  glb <dig>  elovl6l, fuca <dig>  and agpat <dig> all encode proteins that function in carbohydrate or lipid metabolism and were regulated in response to prolonged heat.
stress commonly suppresses immune function and chronic stress is typically associated with an increased risk of developing pathologies  <cit> .
the association of 'response to pest, pathogen or parasite' with gene responses to prolonged heat suggests a continuous challenge of the immune system, and potentially, an elevated risk of disease.
the ontology 'hemoglobin complex' was also associated with the response to prolonged heat suggesting that prolonged heat exposure may compromise oxygen supply.
during heat stress, oxygen demand can exceed oxygen supply, leading to hypoxia in the cells  <cit> .
genes encoding hemoglobin beta embryonic- <dig>  hemoglobin alpha embryonic- <dig>  and haptoglobin were induced in response to prolonged heat in this study, likely reflecting this need to enhance oxygen delivery systems during heat stress.
our data suggest that prolonged heat exposure in the coral reef fish p. moluccensis is associated with oxidative stress, proteomic and metabolic adjustments, an induction of oxygen delivery systems, and a challenge of immune functions.
CONCLUSIONS
understanding thermal and environmental stress responses in coral reef fishes is paramount as current models of climate change predict a significant rise in sea surface temperatures within the next decades  <cit> .
the predicted rise in sea surface temperatures adds to other anthropogenic threats, which have already significantly affected the functionality of coral reef ecosystems  <cit> , and may lead to increased levels of physiological stress and mortality in coral reef organisms.
for example, elevated sea surface temperatures a few degrees celsius above normal can lead to coral bleaching  <cit> .
while we have recently gained considerable knowledge about the relationship between sea surface temperatures and the incidence of coral bleaching, we presently have only limited data to estimate the potential effects of elevated temperatures on the physiology of coral reef fishes and, in particular, how heat alters biological function in these fishes.
our results provide the first evidence that the transcriptome and biological function of coral reef fishes can be significantly altered in response to a three-degree celsius increase in temperature.
such a temperature rise is well within the range of predicted temperature increases of current climate change models.
it is possible that the responses to heat observed here are altered following longer-term exposure.
further research is needed, therefore, to investigate the longer-term effects of increased temperatures on gene regulation as well as on growth, reproduction, and immune function.
since the functions of many heat-responsive genes are still unknown, experimental characterisation of gene functions during stress would also enhance our understanding of the effects of elevated temperatures on coral reef fishes and other ectothermic vertebrates.
future work may also establish closer links between the genome and phenome under conditions of environmental stress by relating liver gene responses to those observed in other tissues and by estimating the severity of stress using other indicators, such as blood cortisol levels or the expression of heat shock proteins.
in addition, future work may investigate the changes in transcriptional regulation over the full time course of the biological response, from a few minutes to several weeks and months of exposure.
irrespective of the complexity of environmental stress responses discussed above, this study has demonstrated that there are a set of gene functions commonly associated with environmental stress responses in a warm-adapted ectothermic vertebrate, that these common responses are effectuated by a largely independent set of genes, and that the multi-stressor approach applied here is useful for delineating common responses from those unique to different types of environmental stress.
a more complete understanding of environmental stress responses in ectothermic vertebrates will depend on the successful integration of environmental genomic data with data obtained from cellular, physiological and organismal studies, and our ability to unravel the complex interactions that occur between different levels of biological organisation.

 microarray technology provides a powerful tool for investigating gene regulation and its significance for biological function. however, our understanding of such relationships during environmental stress remains fragmentary, especially in vertebrates. in particular, the commonality, or otherwise, of the responses of vertebrates to different environmental stresses remain poorly understood. recently, some understanding of responses to individual stresses, in particular those related to thermal stress in teleost fishes, has been gained. in these species, thermal stress can lead to changes in ventilation and circulation rates, changes in mitochondrial densities and their properties, and a reduction in cellular oxygen levels. reduced cellular oxygen levels can lead to increased levels of oxidative stress and hence, the cellular response to thermal stress often includes responses aimed at alleviating oxidative stress. for example, antioxidant enzymes, such as superoxide dismutase, catalase, and glutathione peroxidase are commonly activated during thermal stress. oxidative stress, and the cellular damage associated with it, can in turn induce a heat shock response primarily aimed at the molecular repair of protein damage. in addition, thermal stress can lead to extensive changes in gene expression. these transcriptional responses are likely the result of the stress-dependent activation of only a limited number of upstream regulators, namely the activation mitogen-activated protein kinases, in particular the jnk and p signalling pathways, and immediate early genes. these complex transcriptional responses to stress necessarily precede adjustments at the protein level, and thus, are expected to form an important component of the cellular response to stress. to advance our current limited understanding of the consistency of transcriptional stress responses across different environmental stress types, it is necessary to examine stress responses to a diversity of stressors simultaneously and a suitable model organism is required. teleost fishes, and coral reef fishes in particular, are one such potential model. being ectothermic and aquatic and because their physiology is tightly linked to the environment, teleost fishes are particularly amenable to experimental manipulation of environmental stresses and have hence become important models for environmental stress studies. coral reef fishes inhabit relatively stable environments compared to those experienced by many other teleost fishes, and are so likely to have increased sensitivity towards thermal and environmental anomalies. by identifying the most sensitive biological processes in organisms with limited tolerance towards environmental anomalies, we gain understanding of the types of processes that must have been the targets of adaptation in organisms that exhibit broad environmental tolerances. in this way, studying environmental stress responses in coral reef fishes may provide fundamental insights into mechanisms of environmental adaptation. while there are currently no species-specific microarrays available for any species of coral reef fish, we have previously shown that the compugen k oligonucleotide microarray developed for the zebrafish, danio rerio, can be successfully used to study gene responses in the coral reef fish pomacentrus moluccensis. in this previous study, comparative genomic hybridisation experiments showed good cross-hybridisation between the two fish species for most genes represented on the microarray, and differential expression of a set of candidate genes were confirmed by quantitative real-time pcr. here, we use this previously validated heterologous microarray approach to measure transcriptional responses of the coral reef fish pomacentrus moluccensis to different environmental stressors, including hypoxia, hyposmotic stress, cold, and heat. we also compare heat stress responses of winter- and summer-acclimated fish to estimate seasonal effects on gene regulation. the duration of environmental stress treatments was varied, permitting different aspects of the biological response to be examined. short-term exposure experiments over three hours investigated early gene responses and upstream regulators of transcriptional stress responses, while prolonged exposure to heat over five days investigated medium-term effects of heat stress. by grouping genes into functional categories and identifying those functional categories most commonly associated with gene responses to different types of environmental stress, we identify components of a common stress response in p. moluccensis as well as features unique to different types of environmental stress. we use these gene function responses to infer stress-induced changes in biological and physiological function and discuss the types of biological functions affected by exposure to environmental stress.  early gene responses to different environmental stresses to characterise environmental stress responses, we analysed the gene expression profiles of individuals. these individuals were from a natural population of p. moluccensis and were either subjected to one of the environmental stress treatments or kept at ambient conditions. the microarray platform, data series, and raw data including the.tif image and.spot files are available from the gene expression omnibus website under accessions gpl gse and gsm181765-gsm most genes that showed significant expression changes in response to short-term cold, heat, hypoxic or hyposmotic shock were down-regulated compared to the expression levels measured in fish kept at ambient conditions. short-term heat shock at 34°c in summer led to significant expression changes at six gene loci, amongst which were pdip rhoa, and zcchc and three genes showed greater than two-fold expression changes. in contrast to the summer response, the short-term 34°c heat shock response in winter involved many more genes, and the observed expression changes generally were of greater magnitude. in total, gene loci were differentially expressed in the winter-response to short-term heat shock at 34°c. all but six of these genes were down-regulated in heat-stressed fish with fold changes up to. the early transcriptional response to hypoxia involved ten differentially expressed genes, amongst which were acin1a, β-actin, lbr, and ela and a total of thirteen genes with greater than two-fold expression changes. hyposmotic shock resulted in no significant expression changes, and none of the genes assayed showed greater than two-fold expression changes. it is possible that other tissues, for example gills, might have shown a greater response to hyposmotic challenge, but measurement of expression changes in multiple tissues was outside the scope of this study. due to this lack of response in liver tissue, hyposmotic stress is not discussed further. gene regulation in response to prolonged heat prolonged exposure to 31°c over five days resulted in differentially expressed genes, most of which – in contrast to the short-term exposure experiments – were induced and of which showed greater than two-fold expression changes. genes encoding protein kinase c substrate 80k-h, rab escort protein semaphorin 3ab, distal-less homeobox and ttf-i interacting peptide showed the greatest regulation. differentially expressed genes belonged to a variety of gene functions, amongst which were protein processing, signal transduction, and transcription and translation. only four percent had been previously associated with the response to stress, while percent were of yet unknown gene function. common effects of stress on biological function the gene expression responses to different environmental stressors can be tested for the presence of common gene function responses. such an approach can aid the interpretation of gene regulation in terms of the types of biological functions that are affected. we thus grouped individual genes on the basis of their gene function. we then tested whether genes of a certain function were more likely to be differentially expressed in response to environmental stress than expected if differential expression was independent of gene function. such gene set analysis revealed significant association of a number of gene functional groups with the gene responses to different types of environmental stress. in particular, gene functions related to cell growth and cytoskeleton, protein turnover, and metabolism were consistently associated with gene responses to environmental stress, regardless of the type of stressor applied. for each gene ontology an aggregate score was computed based on the p-values for differential expression of the genes in that category. the significance of the resulting gene set score was determined by random sampling of the data using the software erminej. p-values are fdr-corrected with values smaller than indicated in bold. " h" indicates three hour exposure," d" five day exposure to stressor,'sum." refers to exposure experiments performed in summer, "hyposm." refers to hyposmotic exposure experiments. variable gene responses across stress treatments having identified common gene function responses, we wanted to visualise the responses of individual genes across stress treatments and test for the presence of a set of commonly induced, or suppressed, genes. for this purpose, we extracted the expression data from all differentially expressed genes across all treatments and organised the expression profiles using unsupervised hierarchical clustering. hierarchical clustering identified four main clusters, with genes belonging to the same cluster showing coordinated responses across stressors. while we identified groups of co-regulated genes, gene responses varied considerably across stressors. individual genes that showed differential expression in one treatment did not necessarily show a significant response in another treatment, and some genes showed expression changes in opposite directions in response to different stressors. cluster i was comprised of genes which were strongly induced in the 31°c five days treatment, but showed variable responses in the short-term treatments. a subset of cluster i genes was generally down-regulated in the short-term treatments. in order to determine whether there was a relationship between cluster identity and gene function, i.e. whether co-regulated genes shared similar gene functions, we performed gene class testing on the clusters identified by hierarchical clustering using over-representation analysis. the gene ontology'mhc class i protein binding' was significantly over-represented amongst cluster i genes. cluster ii contained a series of genes that were strongly down-regulated in the 34°c winter treatment and which showed variable expression responses in the other treatments. a subset of cluster ii genes was down-regulated in most stress treatments examined. cluster ii genes showed significant over-representation of gene functions related to cell growth and cytoskeleton. cluster iii was comprised of genes that were strongly suppressed during prolonged heat at 31°c and most of which were induced in the short-term treatments, especially group c genes. metabolic gene functions were significantly over-represented amongst cluster iii genes. lastly, cluster iv included genes that were strongly induced in the 31°c five days treatment and which were generally also induced in the short-term treatments, especially genes in group d. stress-related gene functions showed a trend for over-representation amongst cluster iv genes, but just exceeded the threshold for significance after fdr-correction. we tested for significant over-representation of gene ontologies of genes within a cluster compared to the total complement of genes represented on the microarray. gene clusters were identified by hierarchical clustering of significant gene responses across stressors discussion this is the first study to examine the effects of different types and durations of environmental stress on transcriptional regulation in an ectothermic vertebrate, in this case the coral reef fish p. moluccensis. we identified a series of gene functions that were consistently associated with gene responses to environmental stress, suggesting common effects of different types of stress on biological function. however, these common responses were achieved by the regulation of largely independent sets of genes. thus, there was conservation of gene function responses, but variability in the responses of individual genes. while the expression response of individual genes varied depending on the types and durations of stresses applied, we have identified groups of co-regulated genes, the genes within which shared similar gene functions. common gene function responses elucidate how stress affects p. moluccensis some gene functions were consistently associated with the stresses applied here, and thus are likely to reflect the common effects of environmental stress on biological function in this species. consistent with the observation that stress negatively affects cell growth and that, therefore, actin cytoskeletal elements are commonly regulated in response to stress, the gene ontologies'actin cytoskeleton organization and biogenesis','anti-apoptosis','induction of apoptosis', and'cell cycle' were associated with most stress responses measured here. individual stress-responsive genes in this category included β-actin, plectin dynein, spectrin, tubulin beta- filamin acin1a, grim tp53inp septin cyclin g cdca and bax. three genes encoding for nucleobindin 2a, growth arrest-specific and cyclin g and associated with cell cycle and cell growth, showed the most consistent and significant induction across all stress treatments examined, making them good candidates for molecular stress biomarkers in p. moluccensis. gene functions related to protein turnover, such as'regulation of translation','protein folding', and'ubiquitin-dependent protein catabolism', were also commonly associated with the gene responses of p. moluccensis to stress. individual genes that contributed to this component of the common stress response included pdip usp psmc fbxl the translation initiation factors eif2b eif2c eif4b, and the ribosomal proteins rps mrpl mrpl stress can increase rates of protein damage and ubiquitin-dependent protein catabolism. association of the gene functions'protein folding' and'ubiquitin-dependent protein catabolism' with gene responses observed in this study suggest that environmental stress leads to increased protein damage, an adjustment of the proteome, and de novo synthesis of proteins in this species. protein synthesis in ectotherms accounts for about percent of the cellular energy budget. de novo protein synthesis and protein repair in response to environmental stress are thus likely to incur substantial energetic costs in p. moluccensis and may reduce the energy available for other organismal functions such as growth and reproduction. these results are concordant with studies of the yeast saccharomyces cerevisiae that have shown that yeast responds to a range of environmental stressors with a stereotypical change in gene expression and the suppression of around ten percent of assayed genes, many of which function in protein synthesis and cell growth. a third group of gene functions commonly associated with environmental stress responses in p. moluccensis included metabolic gene functions, in particular gene functions related to carbohydrate and lipid metabolism. stress-responsive genes with metabolic functions included sc4mol, chst amy2a, acad, dgat2l, dcxr, and fuca metabolic adjustments and the re-allocation of energy resources may reflect mechanisms that allow for increased levels of protein and cellular repair during stress. genes with stress-related functions, and functions in cellular repair, were also commonly associated with transcriptional stress responses in p. moluccensis, and included dnajb peroxiredoxin ptges apoea, hif1an, and a gene encoding elastase elastase hydrolyses collagen-iv and elastin and its activity has been shown to increase in the presence of reactive oxygen species. regulation of elastase therefore, suggests increased levels of oxidative stress under conditions of environmental stress. in addition, genes with immune functions and the gene ontology'response to pest, pathogen or parasite' were commonly associated with environmental stress responses, suggesting a challenge to the immune system. this functional category included the genes chst t-cell receptor alpha, and haptoglobin. finally, the gene function'dna repair' was significantly associated with many of the stress responses measured here, suggesting increased levels of dna damage under conditions of environmental stress. such responses are consistent with the observation that different types of stress can lead to immunosuppression and genotoxic effects. here we have shown that a multi-stressor approach, and the identification of gene functions most often associated with gene expression responses to stress, can enhance our understanding of the biological processes altered by environmental stress. our results suggest that common effects of environmental stress in p. moluccensis include suppression of cell growth, increased protein damage and de novo synthesis of proteins, metabolic adjustments and a reallocation of energy resources possibly related to increased protein and cellular repair, induction of stress genes and cellular repair systems, and a challenge of immune functions. different types of stressors have been previously associated with a'minimal stress proteome', comprised in particular of proteins involved in dna, protein and molecular repair and energy metabolism. our results therefore provide experimental support for the presence of a stress proteome, and common stress responses, also in an ectothermic vertebrate, the coral reef fish pomacentrus moluccensis. early gene responses to environmental stress in addition to the common stress responses discussed above, there were also differences between the responses to individual stressors. for example, there was a remarkable difference in the number of genes that were regulated in response to heat shock at 34°c in summer and winter. mean winter and summer temperatures differ by approximately 4°c at the study site. seasonal acclimation can lead to extensive transcriptional adjustments in teleosts and may thus account for some of the transcriptional differences observed here. we cannot exclude, however, the possibility that some of the changes in gene expression were due to the male fish employed in the microarray analyses being at different stages of the reproductive cycle at the time of sampling. the different experimental designs employed in the summer and winter analyses may have further confounded these results as biological samples had been pooled in the winter analyses, but not in the summer analyses. further studies will be required to more precisely define the sources of these differences in stress responses between summer and winter. many responses to hypoxia compensate for reduced cellular oxygen levels by increasing capacities for oxygen delivery, and by enhancing oxygen-independent atp production by means of glycolysis. the hypoxia-inducible transcription factor hif- plays an important role in coordinating transcriptional responses to hypoxia. in the present study, hif-1a showed only limited gene regulation which is consistent with hif regulation occurring largely at the protein level, not at the mrna expression level. however, hypoxia-inducible factor alpha subunit inhibitor, which modulates hif- transcriptional activity, was significantly down-regulated in response to prolonged heat exposure. while further data are required to understand the role of hif-1a and hif1an in response to hypoxic and heat stress in p. moluccensis, association of the gene ontologies'glycolysis' and'gluconeogenesis' with the response to hypoxia is consistent with the need to increase oxygen-independent atp production. furthermore, amongst the most significant expression responses to hypoxia was induction of amy2a, encoding the amylase- protein that endohydrolyses -α-d-glucosidic linkages in polysaccharides such as starch and glycogen to make dextrin, which can then be reduced to α-d-glucose. during hypoxia, this protein appears to free stored sugars for use in glycolysis and would, therefore, facilitate oxygen-independent atp production. gene expression responses to prolonged heat given current models of climatic change and the prediction of rising sea surface temperatures, we were particularly interested in gene responses to prolonged heat, and how early gene responses may change following prolonged exposure to heat. we found that while the gene ontology'protein folding' remained associated with the response to heat even after prolonged exposure'ubiquitin-dependent protein catabolism' was not. these results suggest that initial adjustments of the proteome during stress involve increased degradation of proteins and increased levels of ubiquitin-dependent protein catabolism. over time, rates of protein catabolism appear to return to baseline levels, while protein chaperone systems remain induced. prolonged heat exposure was further associated with the gene ontology'response to oxidative stress' and induction of the genes encoding peroxiredoxin and apolipoprotein ea, two proteins with antioxidant function. oxidative stress is caused by the formation of reactive oxygen species and has been associated with many forms of stress. it is likely that oxidative stress, and the associated cellular and molecular damage, incur substantial energetic costs during prolonged heat exposure. the fact that the gene ontologies'carbohydrate metabolism' and'lipid metabolism' remain associated with gene responses even after prolonged exposure to heat may reflect long-term re-allocation of energy resources. such energetic re-organisation may compensate for the energetic needs associated with protein and cellular repair arising from prolonged heat exposure and the associated oxidative stress. for example, acad, dgat2l, dcxr, hadh crabp glb elovl6l, fuca and agpat all encode proteins that function in carbohydrate or lipid metabolism and were regulated in response to prolonged heat. stress commonly suppresses immune function and chronic stress is typically associated with an increased risk of developing pathologies. the association of'response to pest, pathogen or parasite' with gene responses to prolonged heat suggests a continuous challenge of the immune system, and potentially, an elevated risk of disease. the ontology'hemoglobin complex' was also associated with the response to prolonged heat suggesting that prolonged heat exposure may compromise oxygen supply. during heat stress, oxygen demand can exceed oxygen supply, leading to hypoxia in the cells. genes encoding hemoglobin beta embryonic- hemoglobin alpha embryonic- and haptoglobin were induced in response to prolonged heat in this study, likely reflecting this need to enhance oxygen delivery systems during heat stress. our data suggest that prolonged heat exposure in the coral reef fish p. moluccensis is associated with oxidative stress, proteomic and metabolic adjustments, an induction of oxygen delivery systems, and a challenge of immune functions.  understanding thermal and environmental stress responses in coral reef fishes is paramount as current models of climate change predict a significant rise in sea surface temperatures within the next decades. the predicted rise in sea surface temperatures adds to other anthropogenic threats, which have already significantly affected the functionality of coral reef ecosystems, and may lead to increased levels of physiological stress and mortality in coral reef organisms. for example, elevated sea surface temperatures a few degrees celsius above normal can lead to coral bleaching. while we have recently gained considerable knowledge about the relationship between sea surface temperatures and the incidence of coral bleaching, we presently have only limited data to estimate the potential effects of elevated temperatures on the physiology of coral reef fishes and, in particular, how heat alters biological function in these fishes. our results provide the first evidence that the transcriptome and biological function of coral reef fishes can be significantly altered in response to a three-degree celsius increase in temperature. such a temperature rise is well within the range of predicted temperature increases of current climate change models. it is possible that the responses to heat observed here are altered following longer-term exposure. further research is needed, therefore, to investigate the longer-term effects of increased temperatures on gene regulation as well as on growth, reproduction, and immune function. since the functions of many heat-responsive genes are still unknown, experimental characterisation of gene functions during stress would also enhance our understanding of the effects of elevated temperatures on coral reef fishes and other ectothermic vertebrates. future work may also establish closer links between the genome and phenome under conditions of environmental stress by relating liver gene responses to those observed in other tissues and by estimating the severity of stress using other indicators, such as blood cortisol levels or the expression of heat shock proteins. in addition, future work may investigate the changes in transcriptional regulation over the full time course of the biological response, from a few minutes to several weeks and months of exposure. irrespective of the complexity of environmental stress responses discussed above, this study has demonstrated that there are a set of gene functions commonly associated with environmental stress responses in a warm-adapted ectothermic vertebrate, that these common responses are effectuated by a largely independent set of genes, and that the multi-stressor approach applied here is useful for delineating common responses from those unique to different types of environmental stress. a more complete understanding of environmental stress responses in ectothermic vertebrates will depend on the successful integration of environmental genomic data with data obtained from cellular, physiological and organismal studies, and our ability to unravel the complex interactions that occur between different levels of biological organisation. 
we have shown that different types of environmental stress induce expression changes in genes with similar gene functions, but that the responses of individual genes vary between stress types. in this study, we used a multi-stressor approach to identify components of a common stress response as well as components unique to different types of environmental stress. we still know, however, comparatively little about how environmentally induced stress affects gene expression in vertebrates, and the consistency of transcriptional stress responses to different types of environmental stress. many heat-responsive genes had functions associated with protein turnover, metabolism, and the response to oxidative stress. the functions of heat-responsive genes suggest that prolonged heat exposure leads to oxidative stress and protein damage, a challenge of the immune system, and the re-allocation of energy sources. this is the first environmental genomic study to measure gene regulation in response to different environmental stressors in a natural population of a warm-adapted ectothermic vertebrate. our understanding of the importance of transcriptional regulation for biological function is continuously improving. we identified a series of gene functions that were involved in all stress responses examined here, suggesting some common effects of stress on biological function. this study hence offers insight into the effects of environmental stress on biological function and sheds light on the expected sensitivity of coral reef fishes to elevated temperatures in the future. we also compared winter and summer responses to heat shock to examine the capacity for such responses to vary with acclimation to different ambient temperatures. we exposed individuals of the coral reef fish pomacentrus moluccensis to hypoxic, hyposmotic, cold and heat shock and measured the responses of approximately genes in liver. in response to heat exposure over five days, a total of gene loci were differentially expressed. these common responses were achieved by the regulation of largely independent sets of genes; the responses of individual genes varied greatly across different stress types.
 our understanding of the importance of transcriptional regulation for biological function is continuously improving. we still know, however, comparatively little about how environmentally induced stress affects gene expression in vertebrates, and the consistency of transcriptional stress responses to different types of environmental stress. in this study, we used a multi-stressor approach to identify components of a common stress response as well as components unique to different types of environmental stress. we exposed individuals of the coral reef fish pomacentrus moluccensis to hypoxic, hyposmotic, cold and heat shock and measured the responses of approximately genes in liver. we also compared winter and summer responses to heat shock to examine the capacity for such responses to vary with acclimation to different ambient temperatures.  we identified a series of gene functions that were involved in all stress responses examined here, suggesting some common effects of stress on biological function. these common responses were achieved by the regulation of largely independent sets of genes; the responses of individual genes varied greatly across different stress types. in response to heat exposure over five days, a total of gene loci were differentially expressed. many heat-responsive genes had functions associated with protein turnover, metabolism, and the response to oxidative stress. we were also able to identify groups of co-regulated genes, the genes within which shared similar functions.  this is the first environmental genomic study to measure gene regulation in response to different environmental stressors in a natural population of a warm-adapted ectothermic vertebrate. we have shown that different types of environmental stress induce expression changes in genes with similar gene functions, but that the responses of individual genes vary between stress types. the functions of heat-responsive genes suggest that prolonged heat exposure leads to oxidative stress and protein damage, a challenge of the immune system, and the re-allocation of energy sources. this study hence offers insight into the effects of environmental stress on biological function and sheds light on the expected sensitivity of coral reef fishes to elevated temperatures in the future. 