Further evidence from common garden rearing experiments of heritable traits separating lean and siscowet lake charr (Salvelinus namaycush) ecotypes.

Genetic evidence of selection for complex and polygenically regulated phenotypes can easily become masked by neutral population genetic structure and phenotypic plasticity. Without direct evidence of genotype-phenotype associations it can be difficult to conclude to what degree a phenotype is heritable or a product of environment. Common garden laboratory studies control for environmental stochasticity and help to determine the mechanism that regulate traits. Here we assess lipid content, growth, weight, and length variation in full and hybrid F1 crosses of deep and shallow water sympatric lake charr ecotypes reared for nine years in a common garden experiment. Redundancy analysis (RDA) and quantitative-trait-loci (QTL) genomic scans are used to identify associations between genotypes at 19,714 single nucleotide polymorphisms (SNPs) aligned to the lake charr genome and individual phenotypes to determine the role that genetic inheritance plays in ecotype phenotypic diversity. Lipid content, growth, length, and weight differed significantly among lake charr crosses throughout the experiment suggesting that pedigree plays a large roll in lake charr development. Polygenic scores of 15 SNPs putatively associated with lipid content and/or condition factor indicated that ecotype distinguishing traits are polygenically regulated and additive. A QTL identified on chromosome 38 contained >200 genes, some of which were associated with lipid metabolism and growth, demonstrating the complex nature of ecotype diversity. The results of our common garden study further indicate that lake charr ecotypes observed in nature are predetermined at birth and that ecotypes differ fundamentally in lipid metabolism and growth.

Seasonal variation of pituitary gonadotropin subunit, brain-type aromatase and sex steroid receptor mRNAs, and plasma steroids during gametogenesis in wild sablefish.

Pituitary-hormone signaling plays critical roles in the onset and progression of gametogenesis in vertebrates. This study characterized expression patterns of pituitary gonadotropin beta-subunits (fshb and lhb), brain-type aromatase (cyp19a1b), androgen (ar1, ar2) and estrogen receptors (esr1, esr2a, esr2b), and changes in plasma steroid levels by liquid chromatography/tandem mass spectrometry in wild sablefish (Anoplopoma fimbria, order Scorpaeniformes) during a complete reproductive cycle. Transcripts for fshb increased during early gametogenesis and peaked in late vitellogenic females and late recrudescent males, while expression of lhb reached maximum levels in periovulatory and spermiating fish. Pituitary levels of cyp19a1b and ar1 were strongly correlated with those of lhb in females and males, increasing during gametogenesis and reaching maximum levels prior to spawning. By contrast, expression of ar2, and the three estrogen receptors differed between female and male sablefish. 17ß-estradiol (E2) was the dominant steroid in females during vitellogenesis, while a range of at least 6 steroids (11ß-hydroxyandrostenedione, testosterone [T], E2, 11-ketotestosterone [11KT], 11-deoxycortisol, and 17α,20ß,21-trihydroxyprogesterone) were detected at similar levels in males during testicular development. Prior to spawning, a marked increase in 4-androstenedione, T, 11KT and E2 was found in both periovulatory females and spermiating males. In conclusion, the concomitant changes in plasma androgen levels and pituitary ar1 expression during gametogenesis suggest a specific role for androgens in pituitary hormone regulation of reproduction in sablefish. Further, our data highlight the importance of E2 during final stages of maturation in this species, which may regulate the transcription of pituitary lhb in a paracrine fashion.

Investigating the extent of parallelism in morphological and genomic divergence among lake trout ecotypes in Lake Superior.

Understanding the emergence of species through the process of ecological speciation is a central question in evolutionary biology which also has implications for conservation and management. Lake trout (Salvelinus namaycush) is renowned for the occurrence of different ecotypes linked to resource and habitat use throughout North America. We aimed to unravel the fine genetic structure of the four lake trout ecotypes in Lake Superior. A total of 486 individuals from four sites were genotyped at 6822 filtered SNPs using RADseq technology. Our results revealed different extent of morphological and genetic differentiation within the different sites. Overall, genetic differentiation was weak but significant and was on average three times higher between sites (mean FST  = 0.016) than between ecotypes within sites (mean FST  = 0.005) indicating higher level of gene flow or a more recent shared ancestor between ecotypes within each site than between populations of the same ecotype. Evidence of divergent selection was also found between ecotypes and/or in association with morphological variation. Outlier loci found in genes related to lipid metabolism and visual acuity were of particular interest in this context of ecotypic divergence. However, we did not find clear indication of parallelism at the genomic level, despite the presence of phenotypic parallelism among some ecotypes from different sampling sites. Overall, the occurrence of different levels of both genomic and phenotypic differentiation between ecotypes within each site with several differentiated loci linked to relevant biological functions supports the presence of a continuum of divergence in lake trout.

Extending Immunological Profiling in the Gilthead Sea Bream, Sparus aurata, by Enriched cDNA Library Analysis, Microarray Design and Initial Studies upon the Inflammatory Response to PAMPs.

This study describes the development and validation of an enriched oligonucleotide-microarray platform for Sparus aurata (SAQ) to provide a platform for transcriptomic studies in this species. A transcriptome database was constructed by assembly of gilthead sea bream sequences derived from public repositories of mRNA together with reads from a large collection of expressed sequence tags (EST) from two extensive targeted cDNA libraries characterizing mRNA transcripts regulated by both bacterial and viral challenge. The developed microarray was further validated by analysing monocyte/macrophage activation profiles after challenge with two Gram-negative bacterial pathogen-associated molecular patterns (PAMPs; lipopolysaccharide (LPS) and peptidoglycan (PGN)). Of the approximately 10,000 EST sequenced, we obtained a total of 6837 EST longer than 100 nt, with 3778 and 3059 EST obtained from the bacterial-primed and from the viral-primed cDNA libraries, respectively. Functional classification of contigs from the bacterial- and viral-primed cDNA libraries by Gene Ontology (GO) showed that the top five represented categories were equally represented in the two libraries: metabolism (approximately 24% of the total number of contigs), carrier proteins/membrane transport (approximately 15%), effectors/modulators and cell communication (approximately 11%), nucleoside, nucleotide and nucleic acid metabolism (approximately 7.5%) and intracellular transducers/signal transduction (approximately 5%). Transcriptome analyses using this enriched oligonucleotide platform identified differential shifts in the response to PGN and LPS in macrophage-like cells, highlighting responsive gene-cassettes tightly related to PAMP host recognition. As observed in other fish species, PGN is a powerful activator of the inflammatory response in S. aurata macrophage-like cells. We have developed and validated an oligonucleotide microarray (SAQ) that provides a platform enriched for the study of gene expression in S. aurata with an emphasis upon immunity and the immune response.

Sea lampreys elicit strong transcriptomic responses in the lake trout liver during parasitism.

BACKGROUND: The sea lamprey (Petromyzon marinus) is a jawless vertebrate that parasitizes fish as an adult and, with overfishing, was responsible for the decline in lake trout (Salvelinus namaycush) populations in the Great Lakes. While laboratory studies have looked at the rates of wounding on various fish hosts, there have been few investigations on the physiological effects of lamprey wounding on the host. In the current study, two morphotypes of lake trout, leans and siscowets, were parasitized in the laboratory by sea lampreys and the liver transcriptomes of parasitized and nonparasitized fish were analyzed by RNA-seq (DESeq2 and edgeR) to determine which genes and gene pathways (Ingenuity Pathway Analysis) were altered by lamprey parasitism. RESULTS: Overall, genes encoding molecules involved in catalytic (e.g., enzymatic) and binding activities (factors and regulators) predominated the regulated gene lists. In siscowets, the top upregulated gene was growth arrest and DNA-damage-inducible protein and for leans it was interleukin-18-binding protein. In leans, the most significantly downregulated gene was UDP-glucuronosyltransferase 2A2 - DESeq2 or phosphotriesterase related - edgeR. For siscowets, the top downregulated gene was C-C motif chemokine 19 - DESeq2 or GTP-binding protein Rhes - edgeR. Gene pathways associated with inflammatory-related responses or factors (cytokines, chemokines, oxidative stress, apoptosis) were regulated following parasitism in both morphotypes. However, pathways related to energy metabolism (glycolysis, gluconeogenesis, lipolysis, lipogenesis) were also regulated. These pathways or the intensity or direction (up/downregulation) of regulation were different between leans and siscowets. Finally, one of the most significantly downregulated pathways in both leans and siscowets was the kynurenine (tryptophan degradation) pathway. CONCLUSIONS: The results indicate a strong transcriptional response in the lake trout to lamprey parasitism that entails genes involved in the regulation of inflammation and cellular damage. Responses to energy utilization as well as hydromineral balance also occurred indicating an adjustment in the host to energy demands and osmotic imbalances during parasitism. Given the role of the kynurenine pathway in promoting immunotolerance in mammals, the downregulation observed in this pathway during parasitism may signify an attempt by the host to inhibit any feedback suppression of the immune response to the lamprey.

Dimethylsulfoniopropionate (DMSP) Increases Survival of Larval Sablefish, Anoplopoma fimbria.

High concentrations of dimethylsulfoniopropionate (DMSP), a chemical compound released by lysed phytoplankton, may indicate high rates of grazing by zooplankton and may thus be a foraging cue for planktivorous fishes. Previous studies have shown that some planktivorous fishes and birds aggregate or alter locomotory behavior in response to this chemical cue, which is likely adaptive because it helps them locate prey. These behavioral responses have been demonstrated in juveniles and adults, but no studies have tested for effects on larval fish. Larvae suffer from high mortality rates and are vulnerable to starvation. While larvae are generally thought to be visual predators, they actually have poor vision and cryptic prey. Thus, larval fish should benefit from a chemical cue that provides information on prey abundance. We reared larval sablefish, Anoplopoma fimbria, for one week and supplemented feedings with varying concentrations of DMSP to test the hypothesis that DMSP affects larval survival. Ecologically relevant DMSP concentrations increased larval survival by up to 70 %, which has implications for production in aquaculture and recruitment in nature. These results provide a new tool for increasing larval production in aquaculture and also suggest that larvae may use DMSP as an olfactory cue. The release of DMSP may be a previously unappreciated mechanism through which phytoplankton affect larval survival and recruitment.

Extracellular DNA traps in a ctenophore demonstrate immune cell behaviors in a non-bilaterian.

The formation of extracellular DNA traps (ETosis) is a first response mechanism by specific immune cells following exposure to microbes. Initially characterized in vertebrate neutrophils, cells capable of ETosis have been discovered recently in diverse non-vertebrate taxa. To assess the conservation of ETosis between evolutionarily distant non-vertebrate phyla, we observed and quantified ETosis using the model ctenophore Mnemiopsis leidyi and the oyster Crassostrea gigas. Here we report that ctenophores - thought to have diverged very early from the metazoan stem lineage - possess immune-like cells capable of phagocytosis and ETosis. We demonstrate that both Mnemiopsis and Crassostrea immune cells undergo ETosis after exposure to diverse microbes and chemical agents that stimulate ion flux. We thus propose that ETosis is an evolutionarily conserved metazoan defense against pathogens.

Climate change can impair bacterial pathogen defences in sablefish via hypoxia-mediated effects on adaptive immunity.

Low-oxygen levels (hypoxia) in aquatic habitats are becoming more common because of global warming and eutrophication. However, the effects on the health/disease status of fishes, the world's largest group of vertebrates, are unclear. Therefore, we assessed how long-term hypoxia affected the immune function of sablefish, an ecologically and economically important North Pacific species, including the response to a formalin-killed Aeromonas salmonicida bacterin. Sablefish were held at normoxia or hypoxia (100% or 40% air saturated seawater, respectively) for 6-16 weeks, while we measured a diverse array of immunological traits. Given that the sablefish is a non-model organism, this involved the development of a species-specific methodological toolbox comprised of qPCR primers for 16 key immune genes, assays for blood antibacterial defences, the assessment of blood immunoglobulin (IgM) levels with ELISA, and flow cytometry and confocal microscopy techniques. We show that innate immune parameters were typically elevated in response to the bacterial antigens, but were not substantially affected by hypoxia. In contrast, hypoxia completely prevented the ∼1.5-fold increase in blood IgM level that was observed under normoxic conditions following bacterin exposure, implying a serious impairment of adaptive immunity. Since the sablefish is naturally hypoxia tolerant, our results demonstrate that climate change-related deoxygenation may be a serious threat to the immune competency of fishes.

Behavioural fever is a synergic signal amplifying the innate immune response.

Behavioural fever, defined as an acute change in thermal preference driven by pathogen recognition, has been reported in a variety of invertebrates and ectothermic vertebrates. It has been suggested, but so far not confirmed, that such changes in thermal regime favour the immune response and thus promote survival. Here, we show that zebrafish display behavioural fever that acts to promote extensive and highly specific temperature-dependent changes in the brain transcriptome. The observed coupling of the immune response to fever acts at the gene-environment level to promote a robust, highly specific time-dependent anti-viral response that, under viral infection, increases survival. Fish that are not offered a choice of temperatures and that therefore cannot express behavioural fever show decreased survival under viral challenge. This phenomenon provides an underlying explanation for the varied functional responses observed during systemic fever. Given the effects of behavioural fever on survival and the fact that it exists across considerable phylogenetic space, such immunity-environment interactions are likely to be under strong positive selection.

Luteinizing hormone stimulation of in vitro ovulation in brook trout (Salvelinus fontinalis) involves follicle contraction and activation of proteolytic genes.

Luteinizing hormone (LH) is an essential hormone for the stimulation of the ovulatory process in vertebrates. However, little is known in fish regarding the different mechanisms induced by LH during ovulation that facilitate the rupture of the follicle wall and the subsequent expulsion of the mature oocyte. In this study, the effects of salmon LH (sLH) on in vitro ovulation were investigated in brook trout (Salvelinus fontinalis) isolated follicles. sLH significantly stimulated in vitro ovulation and contraction of brook trout preovulatory follicles. In order to investigate the possible involvement of proteolytic events in the ovulatory action of LH, the expression of genes known to have a crucial role in the degradation of follicle wall structure was examined. Our results show that sLH clearly stimulated the mRNA expression levels of matrix metalloproteinases (MMPs; including mmp2 and mmp19) and other enzymes with proteolytic action during ovulation, such as a disintegrin and metalloproteinase with thrombospondin-like motifs 1 (adamts1) and plasminogen (plg), in brook trout preovulatory follicles. In addition, the expression of mmp2, adamts1 and plg increased in brook trout follicles during the progression of LH-induced ovulation. Interestingly, the expression of tissue inhibitor of matrix metalloproteinase 2 (timp2), a known regulator of MMP2 activity, paralleled that of mmp2, suggesting the existence of a controlled mechanism of MMP2 action. Therefore, the known increase in proteolytic activity during ovulation in fish could be the result of the stimulation of the expression of proteolytic enzymes by LH in preovulatory follicles. We propose that LH may stimulate ovulation in brook trout follicles by stimulating proteolysis of the follicle wall and by stimulating follicle contraction.

Characterization and evaluation of sex-specific expression of suppressors of cytokine signaling (SOCS)-1 and -3 in juvenile yellow perch (Perca flavescens) treated with lipopolysaccharide.

The suppressor of cytokine signaling (SOCS) proteins are a family of intracellular proteins that are centrally involved with vertebrate growth, development and immunity via their effects as negative feed-back regulators of cytokine (and hormone) signaling. The genes for SOCS-1 & -3 were cloned, sequences analyzed and expression patterns examined in the commercially-important teleost, yellow perch (Perca flavescens). The deduced (mature) proteins for yellow perch (yp)SOCS-1 and (yp)SOCS-3 consist of 211 and 205 amino acids, respectively. Functional domains such as the Src homology-2 (SH2) and SOCS-box were present in ypSOCS-1 and ypSOCS-3 and these domains were well conserved between teleost species. Sequence analysis showed that ypSOCS-1 & -3 share highest homology (among similar teleost sequences), to the stickleback (Gasterosteus aculatus) SOCS-1 & -3 protein homologs. To investigate sex-specific expression of the ypSOCS-1 and ypSOCS-3 mRNAs, juvenile male and female yellow perch were immunologically challenged with a single injection (10 µg/g bw) of lipopolysaccharide (LPS) and tissues (gill, head kidney, kidney, liver and spleen) were sampled over a 48-h time-course. Quantitative real-time PCR analysis showed that ypSOCS-1 & -3 were expressed in all tissues examined and at all sampling time-points. LPS injection significantly induced ypSOCS-1 & -3 mRNA levels in gill, head kidney, liver, kidney and spleen, with maximal induction occurring at 6 h post-injection in each tissue. By 48-h post-injection, expression levels for ypSOCS-1 & -3 mRNAs approached, or reached, control levels in all tissues examined. While there were statistical interactions among variables (treatment, time and sex) for ypSOCS-1, we only found a main effect of sex on SOCS-3 mRNA expression in head kidney with higher copy numbers occurring in males than in females treated with LPS. Sexually-dimorphic expression of SOCS-1 or -3 mRNA has not been examined, or described, in a teleost. Our findings suggest the involvement of the SOCS genes in the yellow perch immune response and that differences among the sexes are evident and should be explored further.

Identification of ovarian gene expression patterns during vitellogenesis in Atlantic cod (Gadus morhua).

Follicular maturational competence and ovulatory competence in teleost fish refer to the ability of the ovarian follicle to undergo final oocyte maturation and ovulation, respectively, in response to gonadotropin stimulation and other external cues. Some gene products related to competence acquisition are likely synthesized during vitellogenic growth, as these follicles gain in vivo responsiveness to exogenous gonadotropin stimulation and can be induced to undergo maturation and ovulation. In Atlantic cod (Gadus morhua), gonadotropin responsiveness has been shown to be oocyte size-dependent, and only ovaries containing late-stage vitellogenic follicles can be induced to ovulate. The purpose of the present study was to compare gene expression patterns between mid (unresponsive) and late (responsive) vitellogenic ovaries to identify genes involved in gonadotropin responsiveness and the acquisition of maturational and ovulatory competencies. Representational difference analysis was conducted in two reciprocal comparisons using intact ovarian fragments and follicle wall-enriched tissues, and genes of interest were used in real time quantitative PCR to confirm differential expression. Few differences were detected in intact ovarian fragments, but type IV ice-structuring protein and gephyrin were upregulated later in development and may be involved in lipid and sulfur metabolism, respectively. Candidate gene assays for luteinizing hormone receptor and aromatase also exhibited significant upregulation during vitellogenesis. Many genes were differentially expressed in follicle wall-enriched tissues, including endocrine maturational regulators and smooth muscle genes. Overall, maturational and ovulatory competencies during vitellogenesis in Atlantic cod are associated with up- and downregulation of many genes involved in lipid metabolism, endocrine regulation, and ovulatory preparation.

The Influence of Life History on the Response to Parasitism: Differential Response to Non-Lethal Sea Lamprey Parasitism by Two Lake Charr Ecomorphs.

The energetic demands of stressors like parasitism require hosts to reallocate energy away from normal physiological processes to survive. Life history theory provides predictions about how hosts will reallocate energy following parasitism, but few studies provide empirical evidence to test these predictions. We examined the sub-lethal effects of sea lamprey parasitism on lean and siscowet lake charr, two ecomorphs with different life history strategies. Leans are shorter lived, faster growing, and reach reproductive maturity earlier than siscowets. Following a parasitism event of 4 days, we assessed changes to energy allocation by monitoring endpoints related to reproduction, energy storage, and growth. Results indicate that lean and siscowet lake charr differ considerably in their response to parasitism. Severely parasitized leans slightly increased their reproductive effort and maintained growth and energy storage, consistent with expectations based on life history that leans are less likely to survive parasitism and have shorter lifespans than siscowets making investing in immediate reproduction more adaptive. Siscowets nearly ceased reproduction following severe parasitism and showed evidence of altered energy storage, consistent with a strategy that favors maximizing long-term reproductive success. These findings suggest that life history can be used to generalize stressor response between populations and can aid management efforts.

Divergent responses to peptidoglycans derived from different E. coli serotypes influence inflammatory outcome in trout, Oncorhynchus mykiss, macrophages.

BACKGROUND: Pathogen-associated molecular patterns (PAMPs) are structural components of pathogens such as lipopolysaccharide (LPS) and peptidoglycan (PGN) from bacterial cell walls. PAMP-recognition by the host results in an induction of defence-related genes and often the generation of an inflammatory response. We evaluated both the transcriptomic and inflammatory response in trout (O. mykiss) macrophages in primary cell culture stimulated with DAP-PGN (DAP; meso-diaminopimelic acid, PGN; peptidoglycan) from two strains of Escherichia coli (PGN-K12 and PGN-O111:B4) over time. RESULTS: Transcript profiling was assessed using function-targeted cDNA microarray hybridisation (n = 36) and results show differential responses to both PGNs that are both time and treatment dependent. Wild type E. coli (K12) generated an increase in transcript number/diversity over time whereas PGN-O111:B4 stimulation resulted in a more specific and intense response. In line with this, Gene Ontology analysis (GO) highlights a specific transcriptomic remodelling for PGN-O111:B4 whereas results obtained for PGN-K12 show a high similarity to a generalised inflammatory priming response where multiple functional classes are related to ribosome biogenesis or cellular metabolism. Prostaglandin release was induced by both PGNs and macrophages were significantly more sensitive to PGN-O111:B4 as suggested from microarray data. CONCLUSION: Responses at the level of the transcriptome and the inflammatory outcome (prostaglandin synthesis) highlight the different sensitivity of the macrophage to slight differences (serotype) in peptidoglycan structure. Such divergent responses are likely to involve differential receptor sensitivity to ligands or indeed different receptor types. Such changes in biological response will likely reflect upon pathogenicity of certain serotypes and the development of disease.

Disruption of the salmon reproductive endocrine axis through prolonged nutritional stress: changes in circulating hormone levels and transcripts for ovarian genes involved in steroidogenesis and apoptosis.

Mechanisms regulating the normal progression of ovarian follicular growth versus onset of atresia in fishes are poorly understood. To gain a better understanding of these processes, we exposed immature female coho salmon (Oncorhynchus kisutch) to prolonged fasting to induce follicular atresia and monitored body growth, development of the ovarian follicles, changes in reproductive hormones, and transcripts for ovarian genes. Prolonged fasting reduced body and ovary weight and increased the appearance of atretic follicles relative to normally fed controls. Endocrine analyses showed that fasting reduced plasma insulin-like growth factor 1 (IGF1), estradiol-17ß (E2), and pituitary, but not plasma, levels of follicle-stimulating hormone (FSH). Transcripts for ovarian fsh receptor (fshr) and steroidogenesis-related genes, such as steroidogenic acute regulatory protein (star), 3ß-hydroxysteroid dehydrogenase (hsd3b), and P450 aromatase (cyp19a1a) were significantly lower in fasted fish. Ovarian expression of apoptosis-related genes, such as Fas-associated death domain (fadd), caspase 8 (casp8), caspase 3 (casp3), and caspase 9 (casp9) were significantly elevated in fasted fish compared to fed fish, indicating that apoptosis is involved in the process of atresia in this species. Interestingly, some genes such as fadd, casp8, casp3, and hsd3b, were differentially expressed prior to increases in the number of atretic follicles and reductions in hormone levels induced by fasting, and may therefore have potential as early indicators of atresia. Together these results suggest that prolonged nutritional stress may disrupt the reproductive system and induce follicular atresia in part via reductions in ovarian IGF and FSH signaling, and downstream effects on steroidogenesis-related genes and E2 production.

The rise and fall of the ancient northern pike master sex-determining gene.

The understanding of the evolution of variable sex determination mechanisms across taxa requires comparative studies among closely related species. Following the fate of a known master sex-determining gene, we traced the evolution of sex determination in an entire teleost order (Esociformes). We discovered that the northern pike (Esox lucius) master sex-determining gene originated from a 65 to 90 million-year-old gene duplication event and that it remained sex linked on undifferentiated sex chromosomes for at least 56 million years in multiple species. We identified several independent species- or population-specific sex determination transitions, including a recent loss of a Y chromosome. These findings highlight the diversity of evolutionary fates of master sex-determining genes and the importance of population demographic history in sex determination studies. We hypothesize that occasional sex reversals and genetic bottlenecks provide a non-adaptive explanation for sex determination transitions.

RADSex: A computational workflow to study sex determination using restriction site-associated DNA sequencing data.

The study of sex determination and sex chromosome organization in nonmodel species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, restriction site-associated DNA sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analysed a published data set of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyse new RAD-Seq data sets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in nonmodel species thanks to its speed of analyses, low resource usage, ease of application and visualization options. Furthermore, our analysis of new data sets from 15 species provides new insights on sex determination in fish.

A genetic basis for the phenotypic differentiation between siscowet and lean lake trout (Salvelinus namaycush).

In Lake Superior there are three principal forms of lake trout (Salvelinus namaycush): lean, siscowet and humper. Wild lean and siscowet differ in the shape and relative size of the head, size of the fins, location and size of the eyes, caudal peduncle shape and lipid content of the musculature. To investigate the basis for these phenotypic differences, lean and siscowet lake trout, derived from gametes of wild populations in Lake Superior, were reared communally under identical environmental conditions for 2.5 years. Fish were analysed for growth, morphometry and lipid content, and differences in liver transcriptomics were investigated using Roche 454 GS-FLX pyrosequencing. The results demonstrate that key phenotypic differences between wild lean and siscowet lake trout such as condition factor, morphometry and lipid levels, persist in these two forms when reared in the laboratory under identical environmental conditions. This strongly suggests that these differences are genetic and not a result of environmental plasticity. Transcriptomic analysis involving the comparison of hepatic gene frequencies (RNA-seq) and expression (quantitative reverse transcription-polymerase chain reaction (qPCR)) between the two lake trout forms, indicated two primary gene groups that were differentially expressed; those involving lipid synthesis, metabolism and transport (acyl-CoA desaturase, acyl-CoA binding protein, peroxisome proliferator-activated receptor gamma, and apolipoproteins), and those involved with immunity (complement component C3, proteasome, FK506 binding protein 5 and C1q proteins). The results demonstrate that RNA-seq can be used to identify differentially expressed genes; however, some discrepancies between RNA-seq analysis and qPCR indicate that methods for deep sequencing may need to be refined and/or different RNA-seq platforms utilized.

Cellular and molecular evidence for a role of tumor necrosis factor alpha in the ovulatory mechanism of trout.

BACKGROUND: The relevance of immune-endocrine interactions to the regulation of ovarian function in teleosts is virtually unexplored. As part of the innate immune response during infection, a number of cytokines such as tumor necrosis factor alpha (TNF alpha) and other immune factors, are produced and act on the reproductive system. However, TNF alpha is also an important physiological player in the ovulatory process in mammals. In the present study, we have examined for the first time the effects of TNF alpha in vitro in preovulatory ovarian follicles of a teleost fish, the brown trout (Salmo trutta). METHODS: To determine the in vivo regulation of TNF alpha expression in the ovary, preovulatory brook trout (Salvelinus fontinalis) were injected intraperitoneally with either saline or bacterial lipopolysaccharide (LPS). In control and recombinant trout TNF alpha (rtTNF alpha)-treated brown trout granulosa cells, we examined the percentage of apoptosis by flow cytometry analysis and cell viability by propidium iodide (PI) staining. Furthermore, we determined the in vitro effects of rtTNF alpha on follicle contraction and testosterone production in preovulatory brown trout ovarian follicles. In addition, we analyzed the gene expression profiles of control and rtTNF alpha-treated ovarian tissue by microarray and real-time PCR (qPCR) analyses. RESULTS: LPS administration in vivo causes a significant induction of the ovarian expression of TNF alpha. Treatment with rtTNF alpha induces granulosa cell apoptosis, decreases granulosa cell viability and stimulates the expression of genes known to be involved in the normal ovulatory process in trout. In addition, rtTNF alpha causes a significant increase in follicle contraction and testosterone production. Also, using a salmonid-specific microarray platform (SFA2.0 immunochip) we observed that rtTNF alpha induces the expression of genes known to be involved in inflammation, proteolysis and tissue remodeling. Furthermore, the expression of kallikrein, TOP-2, serine protease 23 and ADAM 22, genes that have been postulated to be involved in proteolytic and tissue remodeling processes during ovulation in trout, increases in follicles incubated in the presence of rtTNF alpha. CONCLUSIONS: In view of these results, we propose that TNF alpha could have an important role in the biomechanics of follicle weakening, ovarian rupture and oocyte expulsion during ovulation in trout, primarily through its stimulation of follicular cell apoptosis and the expression of genes involved in follicle wall proteolysis and contraction.

Stimulation of growth and changes in the hepatic transcriptome by 17beta-estradiol in the yellow perch (Perca flavescens).

The effects of dietary 17beta-estradiol (E(2)) on growth and liver transcriptomics were investigated in the yellow perch (Perca flavescens). After a 3-mo treatment, E(2) significantly stimulated an increase in length and weight of juvenile male and female perch relative to control animals. The increase was significantly greater in females compared with males. Separate, unnormalized cDNA libraries were constructed from equal quantities of RNA from 6 male and 6 female livers of E(2)-treated and control perch, and 3,546 and 3,719 expressed sequence tags (ESTs) were obtained, respectively. To characterize E(2)-regulated transcripts, EST frequencies between libraries were calculated within contiguous sequences that were assembled from the combined ESTs of both libraries. Frequencies were also determined in EST transcript groupings produced by aligning all of the ESTs from both libraries at the nucleotide level. From these analyses, there were 28 annotated transcripts that were regulated by 75% between libraries and for which there were at least 5 ESTs of the same transcript between libraries. Regulation of a subset (14) of these transcripts was confirmed by quantitative reverse transcription-polymerase chain reaction (QPCR). Transcripts that were upregulated by E(2) included reproduction-related proteins, binding proteins, and proteases and protease inhibitors. While not part of the transcript frequency analysis, QPCR showed significant upregulation of estrogen receptor esr1 and of insulin-like growth factor I (IGF-I) in E(2) livers. E(2)-downregulated transcripts represented a variety of functional categories including components of the respiratory chain, lipid transport and metabolism, glycolysis, amino acid and nitrogen metabolism, binding proteins, a hydrolytic enzyme, and a transcriptional regulator. In perch it appears that exogenous estrogen drastically shifts liver metabolism toward the production of lipoproteins and carbohydrate binding proteins, and that the growth-promoting action may involve an increase in hepatic IGF-I production.