Microbiome and epigenetic variation in wild fish with low genetic diversity.

Non-genetic sources of phenotypic variation, such as the epigenome and the microbiome, could be important contributors to adaptive variation for species with low genetic diversity. However, little is known about the complex interaction between these factors and the genetic diversity of the host, particularly in wild populations. Here, we examine the skin microbiome composition of two closely-related mangrove killifish species with different mating systems (self-fertilising and outcrossing) under sympatric and allopatric conditions. This allows us to partition the influence of the genotype and the environment on their microbiome and (previously described) epigenetic profiles. We find the diversity and community composition of the skin microbiome are strongly shaped by the environment and, to a lesser extent, by species-specific influences. Heterozygosity and microbiome alpha diversity, but not epigenetic variation, are associated with the fluctuating asymmetry of traits related to performance (vision) and behaviour (aggression). Our study identifies that a proportion of the epigenetic diversity and microbiome differentiation is unrelated to genetic variation, and we find evidence for an associative relationship between microbiome and epigenetic diversity in these wild populations. This suggests that both mechanisms could potentially contribute to variation in species with low genetic diversity.

Microbiomes in the context of developing sustainable intensified aquaculture.

With an ever-growing human population, the need for sustainable production of nutritional food sources has never been greater. Aquaculture is a key industry engaged in active development to increase production in line with this need while remaining sustainable in terms of environmental impact and promoting good welfare and health in farmed species. Microbiomes fundamentally underpin animal health, being a key part of their digestive, metabolic and defense systems, in the latter case protecting against opportunistic pathogens in the environment. The potential to manipulate the microbiome to the advantage of enhancing health, welfare and production is an intriguing prospect that has gained considerable traction in recent years. In this review we first set out what is known about the role of the microbiome in aquaculture production systems across the phylogenetic spectrum of cultured animals, from invertebrates to finfish. With a view to reducing environmental footprint and tightening biological and physical control, investment in "closed" aquaculture systems is on the rise, but little is known about how the microbial systems of these closed systems affect the health of cultured organisms. Through comparisons of the microbiomes and their dynamics across phylogenetically distinct animals and different aquaculture systems, we focus on microbial communities in terms of their functionality in order to identify what features within these microbiomes need to be harnessed for optimizing healthy intensified production in support of a sustainable future for aquaculture.

Feminizing effects of ethinylestradiol in roach (Rutilus rutilus) populations with different estrogenic pollution exposure histories.

Experimental exposures aimed at assessing the risks posed by estrogens in waste-water treatment work (WwTW) effluents to fish populations have rarely considered whether populations differ in their sensitivity to estrogenic compounds. This is despite evidence that selection at genes involved in the estrogen response has occurred in wild populations, and evidence that genotype can influence estrogen-response. In this study we compare the effects of a two-year exposure to a low measured concentration (1.3 ng/L) of ethinylestradiol (EE2) on the sexual development of roach (Rutilus rutilus) whose parental generation was sampled from two river stretches heavily contaminated with WwTW effluent and from two without any known WwTW effluent contamination. Exposure to EE2 significantly reduced the proportion of genetic males and induced a range of feminized phenotypes in males. Significantly, exposure also increased the proportion of genetic females with vitellogenic oocytes from 51 to 96%, raising the possibility that estrogen pollution could impact populations of annually spawning fish species through advancing female reproduction by at least a year. However, there was no evidence that river origin affected sensitivity to estrogens in either sex. Thus, we conclude that chronic exposure to low level EE2 has reproductive health outcomes for both male and female roach, but we find no evidence that the nature or magnitude of the response is affected by the population origin.

Early life stress causes persistent impacts on the microbiome of Atlantic salmon.

Farmed fish are commonly exposed to stress in intensive aquaculture systems, often leading to immune impairment and increased susceptibility to disease. As microbial communities associated with the gut and skin are vital to host health and disease resilience, disruption of microbiome integrity could contribute to the adverse consequences of stress exposure. Little is known about how stress affects the fish microbiome, especially during sensitive early life stages when initial colonisation and proliferation of host-associated microbial communities take place. Therefore, we compared the effects of two aquaculture-relevant early-life stressors on the gut and skin microbiome of Atlantic salmon fry (four months post hatching) using 16S rRNA amplicon sequencing. Acute cold stress applied during late embryogenesis had a pronounced, lasting effect on the structure of the skin microbiome, as well as a less consistent effect on the gut microbiome. Follow-up targeted qPCR assays suggested that this is likely due to disruption of the egg shell microbial communities at the initial stages of microbiome colonisation, with persistent effects on community structure. In contrast, chronic post hatching stress altered the structure of the gut microbiome, but not that of the skin. Both types of stress promoted similar Gammaproteobacteria ASVs, particularly within the genera Acinetobacter and Aeromonas, which include several important opportunistic fish pathogens. Our results demonstrate the sensitivity of the salmon microbiome to environmental stressors during early life, with potential associated health impacts on the host. We also identified common signatures of stress in the salmon microbiome, which may represent useful microbial stress biomarkers.

Quantitative assessment of fish larvae community composition in spawning areas using metabarcoding of bulk samples.

Accurate assessment of larval community composition in spawning areas is essential for fisheries management and conservation but is often hampered by the cryptic nature of many larvae, which renders them difficult to identify morphologically. Metabarcoding is a rapid and cost-effective method to monitor early life stages for management and environmental impact assessment purposes but its quantitative capability is under discussion. We compared metabarcoding with traditional morphological identification to evaluate taxonomic precision and reliability of abundance estimates, using 332 fish larvae from multinet hauls (0-50 m depth) collected at 14 offshore sampling sites in the Irish and Celtic seas. To improve quantification accuracy (relative abundance estimates), the amount of tissue for each specimen was standardized and mitochondrial primers (12S gene) with conserved binding sites were used. Relative family abundance estimated from metabarcoding reads and morphological assessment were positively correlated, as well as taxon richness (RS  = 0.81, P = 0.007) and diversity (RS  = 0.90, P = 0.002). Spatial patterns of community composition did not differ significantly between metabarcoding and morphological assessments. Our results show that DNA metabarcoding of bulk tissue samples can be used to monitor changes in fish larvae abundance and community composition. This represents a feasible, efficient, and faster alternative to morphological methods that can be applied to terrestrial and aquatic habitats.

Investigation into Adaptation in Genes Associated with Response to Estrogenic Pollution in Populations of Roach (Rutilus rutilus) Living in English Rivers.

Exposure of male fish to estrogenic substances from wastewater treatment works (WwTWs) results in feminization and reduced reproductive fitness. Nevertheless, self-sustaining populations of roach (Rutilus rutilus) inhabit river stretches polluted with estrogenic WwTW effluents. In this study, we examine whether such roach populations have evolved adaptations to tolerate estrogenic pollution by comparing frequency differences in single-nucleotide polymorphisms (SNPs) between populations sampled from rivers receiving either high- or low-level WwTW discharges. SNPs within 36 "candidate" genes, selected for their involvement in estrogenic responses, and 120 SNPs in reference genes were genotyped in 465 roaches. There was no evidence for selection in highly estrogen-dependent candidate genes, including those for the estrogen receptors, aromatases, and vitellogenins. The androgen receptor (ar) and cytochrome P450 1A genes were associated with large shifts in allele frequencies between catchments and in individual populations, but there is no clear link to estrogen pollution. Selection at ar in the effluent-dominated River Lee may have resulted from historical contamination with endocrine-disrupting pesticides. Critically, although our results suggest population-specific selection including at genes related to endocrine disruption, there was no strong evidence that the selection resulted from exposure to estrogen pollution.

A laboratory investigation into features of morphology and physiology for their potential to predict reproductive success in male frogs.

Amphibian populations are declining globally, however, the contribution of reduced reproduction to declines is unknown. We investigated associations between morphological (weight/snout-vent length, nuptial pad colour/size, forelimb width/size) and physiological (nuptial pad/testis histomorphology, plasma hormones, gene expression) features with reproductive success in males as measured by amplexus success and fertility rate (% eggs fertilised) in laboratory maintained Silurana/Xenopus tropicalis. We explored the robustness of these features to predict amplexus success/fertility rate by investigating these associations within a sub-set of frogs exposed to anti-androgens (flutamide (50 µg/L)/linuron (9 or 45 µg/L)). In unexposed males, nuptial pad features (size/colour/number of hooks/androgen receptor mRNA) were positively associated with amplexus success, but not with fertility rate. In exposed males, many of the associations with amplexus success differed from untreated animals (they were either reversed or absent). In the exposed males forelimb width/nuptial pad morphology were also associated with fertility rate. However, a more darkly coloured nuptial pad was positively associated with amplexus success across all groups and was indicative of androgen status. Our findings demonstrate the central role for nuptial pad morphology in reproductive success in S. tropicalis, however, the lack of concordance between unexposed/exposed frogs complicates understanding of the utility of features of nuptial pad morphology as biomarkers in wild populations. In conclusion, our work has indicated that nuptial pad and forelimb morphology have potential for development as biomarkers of reproductive health in wild anurans, however, further research is needed to establish this.

Cortisol-Related Signatures of Stress in the Fish Microbiome.

Exposure to environmental stressors can compromise fish health and fitness. Little is known about how stress-induced microbiome disruption may contribute to these adverse health effects, including how cortisol influences fish microbial communities. We exposed juvenile Atlantic salmon to a mild confinement stressor for two weeks. We then measured cortisol in the plasma, skin-mucus, and feces, and characterized the skin and fecal microbiome. Fecal and skin cortisol concentrations increased in fish exposed to confinement stress, and were positively correlated with plasma cortisol. Elevated fecal cortisol was associated with pronounced changes in the diversity and structure of the fecal microbiome. In particular, we identified a marked decline in the lactic acid bacteria Carnobacterium sp. and an increase in the abundance of operational taxonomic units within the classes Clostridia and Gammaproteobacteria. In contrast, cortisol concentrations in skin-mucus were lower than in the feces, and were not related to any detectable changes in the skin microbiome. Our results demonstrate that stressor-induced cortisol production is associated with disruption of the gut microbiome, which may, in turn, contribute to the adverse effects of stress on fish health. They also highlight the value of using non-invasive fecal samples to monitor stress, including simultaneous determination of cortisol and stress-responsive bacteria.

Comparative transcriptomics reveal conserved impacts of rearing density on immune response of two important aquaculture species.

Infectious diseases represent an important barrier to sustainable aquaculture development. Rearing density can substantially impact fish productivity, health and welfare in aquaculture, including growth rates, behaviour and, crucially, immune activity. Given the current emphasis on aquaculture diversification, stress-related indicators broadly applicable across species are needed. Utilising an interspecific comparative transcriptomic (RNAseq) approach, we compared gill gene expression responses of Atlantic salmon (Salmo salar) and Nile tilapia (Oreochromis niloticus) to rearing density and Saprolegnia parasitica infection. Salmon reared at high-density showed increased expression of stress-related markers (e.g. c-fos and hsp70), and downregulation of innate immune genes. Upon pathogen challenge, only salmon reared at low density exhibited increased expression of inflammatory interleukins and lymphocyte-related genes. Tilapia immunity, in contrast, was impaired at low-density. Using overlapping gene ontology enrichment and gene ortholog analyses, we found that density-related stress similarly impacted salmon and tilapia in key immune pathways, altering the expression of genes vital to inflammatory and Th17 responses to pathogen challenge. Given the challenges posed by ectoparasites and gill diseases in fish farms, this study underscores the importance of optimal rearing densities for immunocompetence, particularly for mucosal immunity. Our comparative transcriptomics analyses identified density stress impacted immune markers common across different fish taxa, providing key molecular targets with potential for monitoring and enhancing aquaculture resilience in a wide range of farmed species.

Environmental plasticity and colonisation history in the Atlantic salmon microbiome: A translocation experiment.

Microbial communities associated with the gut and the skin are strongly influenced by environmental factors, and can rapidly adapt to change. Historical processes may also affect the microbiome. In particular, variation in microbial colonisation in early life has the potential to induce lasting effects on microbial assemblages. However, little is known about the relative extent of microbiome plasticity or the importance of historical colonisation effects following environmental change, especially for nonmammalian species. To investigate this we performed a reciprocal translocation of Atlantic salmon between artificial and semi-natural conditions. Wild and hatchery-reared fry were transferred to three common garden experimental environments for 6 weeks: standard hatchery conditions, hatchery conditions with an enriched diet, and simulated wild conditions. We characterized the faecal and skin microbiome of individual fish before and after the environmental translocation, using a BACI (before-after-control-impact) design. We found evidence of extensive microbiome plasticity for both the gut and skin, with the greatest changes in alpha and beta diversity associated with the largest changes in environment and diet. Microbiome richness and diversity were entirely determined by environment, with no detectable effects of fish origin, and there was also a near-complete turnover in microbiome structure. However, we also identified, for the first time in fish, evidence of historical colonisation effects reflecting early-life experience, including ASVs characteristic of captive rearing. These results have important implications for host adaptation to local selective pressures, and highlight how conditions experienced during early life can have a long-term influence on the microbiome and, potentially, host health.

Transcriptomic response to aquaculture intensification in Nile tilapia.

To meet future global demand for fish protein, more fish will need to be farmed using fewer resources, and this will require the selection of nonaggressive individuals that perform well at high densities. Yet, the genetic changes underlying loss of aggression and adaptation to crowding during aquaculture intensification are largely unknown. We examined the transcriptomic response to aggression and crowding in Nile tilapia, one of the oldest and most widespread farmed fish, whose social structure shifts from social hierarchies to shoaling with increasing density. A mirror test was used to quantify aggression and skin darkening (a proxy for stress) of fish reared at low and high densities, and gene expression in the hypothalamus was analysed among the most and least aggressive fish at each density. Fish reared at high density were darker, had larger brains, were less active and less aggressive than those reared at low density and had differentially expressed genes consistent with a reactive stress-coping style and activation of the hypothalamus-pituitary-interrenal (HPI) axis. Differences in gene expression among aggressive fish were accounted for by density and the interaction between density and aggression levels, whereas for nonaggressive fish differences in gene expression were associated with individual variation in skin brightness and social stress. Thus, the response to crowding in Nile tilapia is context dependent and involves different neuroendocrine pathways, depending on social status. Knowledge of genes associated with the response to crowding may pave the way for more efficient fish domestication, based on the selection of nonaggressive individuals with increasing tolerance to chronic stress necessary for aquaculture intensification.

Contrasting effects of acute and chronic stress on the transcriptome, epigenome, and immune response of Atlantic salmon.

Stress experienced during early life may have lasting effects on the immune system, with impacts on health and disease dependent on the nature and duration of the stressor. The epigenome is especially sensitive to environmental stimuli during early life and represents a potential mechanism through which stress may cause long-lasting health effects. However, the extent to which the epigenome responds differently to chronic vs acute stressors is unclear, especially for non-mammalian species. We examined the effects of acute stress (cold-shock during embryogenesis) and chronic stress (absence of tank enrichment during larval-stage) on global gene expression (using RNA-seq) and DNA methylation (using RRBS) in the gills of Atlantic salmon (Salmo salar) four months after hatching. Chronic stress induced pronounced transcriptional differences, while acute stress caused few lasting transcriptional effects. However, both acute and chronic stress caused lasting and contrasting changes in the methylome. Crucially, we found that acute stress enhanced transcriptional immune response to a pathogenic challenge (bacterial lipopolysaccharide, LPS), while chronic stress suppressed it. We identified stress-induced changes in promoter and gene-body methylation that were associated with altered expression for a small proportion of immune-related genes, and evidence of wider epigenetic regulation within signalling pathways involved in immune response. Our results suggest that stress can affect immuno-competence through epigenetic mechanisms, and highlight the markedly different effects of chronic larval and acute embryonic stress. This knowledge could be used to harness the stimulatory effects of acute stress on immunity, paving the way for improved stress and disease management through epigenetic conditioning.

Transcriptomic response to parasite infection in Nile tilapia (Oreochromis niloticus) depends on rearing density.

BACKGROUND: Captive animal populations, be it for food production or conservation programmes, are often maintained at densities far beyond those in natural environments, which can have profound effects on behaviour, immune and stress levels, and ultimately welfare. How such alterations impact transcriptional responses to pathogen infection is a 'different kettle of fish' and remains poorly understood. Here, we assessed survival and gene expression profiles of infected fish reared at two different densities to elucidate potential functional genomic mechanisms for density-related differences in disease susceptibility. RESULTS: Utilising a whole-transcriptome sequencing (RNAseq) approach, we demonstrate that rearing density in tilapia (Oreochromis niloticus) significantly impacts susceptibility to the oomycete Saprolegnia parasitica, via altered transcriptional infection responses. Tilapia held at low densities have increased expression of genes related to stress, likely due to increased aggressive interactions. When challenged with Saprolegnia, low-density fish exhibit altered expression of inflammatory gene responses and enhanced levels of adaptive immune gene suppression compared to fish reared at higher density, resulting in significantly increased mortality rates. In addition, Saprolegnia infection substantially perturbs expression of circadian clock genes, with fish reared at low-density having higher levels of molecular clock dysregulation. CONCLUSIONS: Our results reveal the wide-scale impact of stocking density on transcriptional responses to infection and highlight the need to incorporate circadian biology into our understanding of disease dynamics in managed animals.

Data on optimisation of a multiplex HRM-qPCR assay for native and invasive crayfish as well as the crayfish plague in four river catchments.

The data presented here corresponds to the research paper "Simultaneous detection of invasive signal crayfish, endangered white-clawed crayfish and the crayfish plague using environmental DNA". A crayfish-specific assay was designed and optimised using three real-time PCR supermixes (SYBR™ Green, SsoFast™ EvaGreen® and HOT FIREPol® EvaGreen®). Diagnostic high resolution melt (HRM) data from direct application of assay on both ex-situ eDNA water samples and field samples from four catchments (two in Wales, two in England) is presented in this article, displaying positive HRM profiles for invasive signal crayfish (Pacifastacus leniusculus), native white-clawed crayfish (Austropotamobius pallipes) and crayfish plague causal agent (Aphanomyces astaci).

Interpopulation Variation in the Atlantic Salmon Microbiome Reflects Environmental and Genetic Diversity.

The microbiome has a crucial influence on host phenotype and is of broad interest to ecological and evolutionary research. Yet, the extent of variation that occurs in the microbiome within and between populations is unclear. We characterized the skin and gut microbiomes of seven populations of juvenile Atlantic salmon (Salmo salar) inhabiting a diverse range of environments, including hatchery-reared and wild populations. We found shared skin operational taxonomic units (OTUs) across all populations and core gut microbiota for all wild fish, but the diversity and structure of both skin and gut microbial communities were distinct between populations. There was a marked difference between the gut microbiomes of wild and captive fish. Hatchery-reared fish had lower intestinal microbial diversity, lacked core microbiota found in wild fish, and showed altered community structure and function. Skin and gut microbiomes were also less varied within captive populations, reflecting more uniform artificial rearing conditions. The surrounding water influenced the microbiome of the gut and, especially, the skin, but could not explain the degree of variation observed between populations. For both gut and skin, we found that there was greater difference in microbiome structures between more genetically distinct fish populations, and that population genetic diversity was positively correlated with microbiome diversity. However, diet is likely to be the major factor contributing to the large differences in gut microbiota between wild and captive fish. Our results highlight the scope of interpopulation variation in the Atlantic salmon microbiome and offer insights into the deterministic factors contributing to microbiome diversity and structure.IMPORTANCE Variation in the microbiome has a fundamental influence on host health, ecology, and evolution, but the scope and basis of this variation are not fully understood. We identified considerable variation in skin and gut microbial communities between seven wild and captive populations of Atlantic salmon, reflecting divergent environmental conditions and fish genetic diversity. In particular, we found very pronounced differences in the intestinal microbiomes of wild and hatchery-reared fish, likely reflecting differences in diet. Our results offer an insight into how the microbiome potentially contributes to the generation of local adaptations in this species and how domestication alters intestinal microbial communities, highlighting future research directions in these areas.

Immune-Related Transcriptional Responses to Parasitic Infection in a Naturally Inbred Fish: Roles of Genotype and Individual Variation.

Parasites are strong drivers of evolutionary change and the genetic variation of both host and parasite populations can co-evolve as a function of parasite virulence and host resistance. The role of transcriptome variation in specific interactions between host and parasite genotypes has been less studied and can be confounded by differences in genetic variation. We employed two naturally inbred lines of a self-fertilizing fish to estimate the role of host genotype in the transcriptome response to parasite infection using RNA-seq. In addition, we targeted several differentially expressed immune-related genes to further investigate the relative role of individual variation in the immune response using RT-qPCR, taking advantage of the genomic uniformity of the self-fertilizing lines. We found significant differences in gene expression between lines in response to infection both in the transcriptome and in individual gene RT-qPCR analyses. Individual RT-qPCR analyses of gene expression identified significant variance differences between lines for six genes but only for three genes between infected and control fish. Our results indicate that although the genetic background plays an important role in the transcriptome response to parasites, it cannot fully explain individual differences within genetically homogeneous lines, which can be important for determining the response to parasites.

Hepatic transcriptional responses to copper in the three-spined stickleback are affected by their pollution exposure history.

Some fish populations inhabiting contaminated environments show evidence of increased chemical tolerance, however the mechanisms contributing to this tolerance, and whether this is heritable, are poorly understood. We investigated the responses of two populations of wild three-spined stickleback (Gasterosteus aculeatus) with different histories of contaminant exposure to an oestrogen and copper, two widespread aquatic pollutants. Male stickleback originating from two sites, the River Aire, with a history of complex pollution discharges, and Siblyback Lake, with a history of metal contamination, were depurated and then exposed to copper (46µg/L) and the synthetic oestrogen ethinyloestradiol (22ng/L). The hepatic transcriptomic response was compared between the two populations and to a reference population with no known history of exposure (Houghton Springs, Dorset). Gene responses included those typical for both copper and oestrogen, with no discernable difference in response to oestrogen between populations. There was, however, some difference in the magnitude of response to copper between populations. Siblyback fish showed an elevated baseline transcription of genes encoding metallothioneins and a lower level of metallothionein induction following copper exposure, compared to those from the River Aire. Similarly, a further experiment with an F1 generation of Siblyback fish bred in the laboratory found evidence for elevated transcription of genes encoding metallothioneins in unexposed fish, together with an altered transcriptional response to 125µg/L copper, compared with F1 fish originating from the clean reference population exposed to the same copper concentration. These data suggest that the stickleback from Siblyback Lake have a differential response to copper, which is inherited by the F1 generation in laboratory conditions, and for which the underlying mechanism may include an elevation of baseline transcription of genes encoding metallothioneins. The genetic and/or epigenetic mechanisms contributing to this inherited alteration of metallothionein transcription have yet to be established.

Adaptive capabilities and fitness consequences associated with pollution exposure in fish.

Many fish populations are exposed to harmful levels of chemical pollution and selection pressures associated with these exposures have led to the evolution of tolerance. Our understanding of the physiological basis for these adaptations is limited, but they are likely to include processes involved with the absorption, distribution, metabolism and/or excretion of the target chemical. Other potential adaptive mechanisms include enhancements in antioxidant responses, an increased capacity for DNA and/or tissue repair and alterations to the life cycle of fish that enable earlier reproduction. Analysis of single-nucleotide polymorphism frequencies has shown that tolerance to hydrocarbon pollutants in both marine and estuarine fish species involves alteration in the expression of the xenobiotic metabolism enzyme CYP1A. In this review, we present novel data showing also that variants of the CYP1A gene have been under selection in guppies living in Trinidadian rivers heavily polluted with crude oil. Potential costs associated with these adaptations could reduce fitness in unpolluted water conditions. Integrating knowledge of local adaptation to pollution is an important future consideration in conservation practices such as for successful restocking, and improving connectivity within river systems.This article is part of the themed issue 'Human influences on evolution, and the ecological and societal consequences'.

Identification of conserved hepatic transcriptomic responses to 17ß-estradiol using high-throughput sequencing in brown trout.

Estrogenic chemicals are major contaminants of surface waters and can threaten the sustainability of natural fish populations. Characterization of the global molecular mechanisms of toxicity of environmental contaminants has been conducted primarily in model species rather than species with limited existing transcriptomic or genomic sequence information. We aimed to investigate the global mechanisms of toxicity of an endocrine disrupting chemical of environmental concern [17ß-estradiol (E2)] using high-throughput RNA sequencing (RNA-Seq) in an environmentally relevant species, brown trout (Salmo trutta). We exposed mature males to measured concentrations of 1.94, 18.06, and 34.38 ng E2/l for 4 days and sequenced three individual liver samples per treatment using an Illumina HiSeq 2500 platform. Exposure to 34.4 ng E2/L resulted in 2,113 differentially regulated transcripts (FDR < 0.05). Functional analysis revealed upregulation of processes associated with vitellogenesis, including lipid metabolism, cellular proliferation, and ribosome biogenesis, together with a downregulation of carbohydrate metabolism. Using real-time quantitative PCR, we validated the expression of eight target genes and identified significant differences in the regulation of several known estrogen-responsive transcripts in fish exposed to the lower treatment concentrations (including esr1 and zp2.5). We successfully used RNA-Seq to identify highly conserved responses to estrogen and also identified some estrogen-responsive transcripts that have been less well characterized, including nots and tgm2l. These results demonstrate the potential application of RNA-Seq as a valuable tool for assessing mechanistic effects of pollutants in ecologically relevant species for which little genomic information is available.

Global transcriptomic profiling demonstrates induction of oxidative stress and of compensatory cellular stress responses in brown trout exposed to glyphosate and Roundup.

BACKGROUND: Glyphosate, the active ingredient in Roundup formulations, is the most widely used herbicide worldwide, and as a result contaminates surface waters and has been detected in food residues, drinking water and human urine, raising concerns for potential environmental and human health impacts. Research has shown that glyphosate and Roundup can induce a broad range of biological effects in exposed organisms, particularly via generation of oxidative stress. However, there has been no comprehensive investigation of the global molecular mechanisms of toxicity of glyphosate and Roundup for any species. We aimed to characterise and compare the global mechanisms of toxicity of glyphosate and Roundup in the liver of brown trout (Salmo trutta), an ecologically and economically important vertebrate species, using RNA-seq on an Illumina HiSeq 2500 platform. To do this, we exposed juvenile female brown trout to 0, 0.01, 0.5 and 10 mg/L of glyphosate and Roundup (glyphosate acid equivalent) for 14 days, and sequenced 6 replicate liver samples from each treatment. RESULTS: We assembled the brown trout transcriptome using an optimised de novo approach, and subsequent differential expression analysis identified a total of 1020 differentially-regulated transcripts across all treatments. These included transcripts encoding components of the antioxidant system, a number of stress-response proteins and pro-apoptotic signalling molecules. Functional analysis also revealed over-representation of pathways involved in regulating of cell-proliferation and turnover, and up-regulation of energy metabolism and other metabolic processes. CONCLUSIONS: These transcriptional changes are consistent with generation of oxidative stress and the widespread induction of compensatory cellular stress response pathways. The mechanisms of toxicity identified were similar across both glyphosate and Roundup treatments, including for environmentally relevant concentrations. The significant alterations in transcript expression observed at the lowest concentrations tested raises concerns for the potential toxicity of this herbicide to fish populations inhabiting contaminated rivers.