Allelic diversification after transposable element exaptation promoted gsdf as the master sex determining gene of sablefish.

Concepts of evolutionary biology suggest that morphological change may occur by rare punctual but rather large changes, or by more steady and gradual transformations. It can therefore be asked whether genetic changes underlying morphological, physiological, and/or behavioral innovations during evolution occur in a punctual manner, whereby a single mutational event has prominent phenotypic consequences, or if many consecutive alterations in the DNA over longer time periods lead to phenotypic divergence. In the marine teleost, sablefish (Anoplopoma fimbria), complementary genomic and genetic studies led to the identification of a sex locus on the Y Chromosome. Further characterization of this locus resulted in identification of the transforming growth factor, beta receptor 1a (tgfbr1a) gene, gonadal somatic cell derived factor (gsdf), as the main candidate for fulfilling the master sex determining (MSD) function. The presence of different X and Y Chromosome copies of this gene indicated that the male heterogametic (XY) system of sex determination in sablefish arose by allelic diversification. The gsdfY gene has a spatio-temporal expression profile characteristic of a male MSD gene. We provide experimental evidence demonstrating a pivotal role of a transposable element (TE) for the divergent function of gsdfY By insertion within the gsdfY promoter region, this TE generated allelic diversification by bringing cis-regulatory modules that led to transcriptional rewiring and thus creation of a new MSD gene. This points out, for the first time in the scenario of MSD gene evolution by allelic diversification, a single, punctual molecular event in the appearance of a new trigger for male development.

Cardiac remodeling in response to embryonic crude oil exposure involves unconventional NKX family members and innate immunity genes.

Cardiac remodeling results from both physiological and pathological stimuli. Compared with mammalian hearts, fish hearts show a broader array of remodeling changes in response to environmental influences, providing exceptional models for dissecting the molecular and cellular bases of cardiac remodeling. We recently characterized a form of pathological remodeling in juvenile pink salmon (Oncorhynchus gorbuscha) in response to crude oil exposure during embryonic cardiogenesis. In the absence of overt pathology (cardiomyocyte death or inflammatory infiltrate), cardiac ventricles in exposed fish showed altered shape, reduced thickness of compact myocardium and hypertrophic changes in spongy, trabeculated myocardium. Here, we used RNA sequencing to characterize molecular pathways underlying these defects. In juvenile ventricular cardiomyocytes, antecedent embryonic oil exposure led to dose-dependent upregulation of genes involved in innate immunity and two NKX homeobox transcription factors not previously associated with cardiomyocytes, nkx2.3 and nkx3.3 Absent from mammalian genomes, the latter is largely uncharacterized. In zebrafish embryos, nkx3.3 demonstrated a potent effect on cardiac morphogenesis, equivalent to that of nkx2.5, the primary transcription factor associated with ventricular cardiomyocyte identity. The role of nkx3.3 in heart growth is potentially linked to the unique regenerative capacity of fish and amphibians. Moreover, these findings support a cardiomyocyte-intrinsic role for innate immune response genes in pathological hypertrophy. This study demonstrates how an expanding mechanistic understanding of environmental pollution impacts - i.e. the chemical perturbation of biological systems - can ultimately yield new insights into fundamental biological processes.

Temporal Dynamics of DNA Methylation Patterns in Response to Rearing Juvenile Steelhead (Oncorhynchus mykiss) in a Hatchery versus Simulated Stream Environment.

Genetic selection is often implicated as the underlying cause of heritable phenotypic differences between hatchery and wild populations of steelhead trout (Oncorhynchus mykiss) that also differ in lifetime fitness. Developmental plasticity, which can also affect fitness, may be mediated by epigenetic mechanisms such as DNA methylation. Our previous study identified significant differences in DNA methylation between adult hatchery- and natural-origin steelhead from the same population that could not be distinguished by DNA sequence variation. In the current study, we tested whether hatchery-rearing conditions can influence patterns of DNA methylation in steelhead with known genetic backgrounds, and assessed the stability of these changes over time. Eyed-embryos from 22 families of Methow River steelhead were split across traditional hatchery tanks or a simulated stream-rearing environment for 8 months, followed by a second year in a common hatchery tank environment. Family assignments were made using a genetic parentage analysis to account for relatedness among individuals. DNA methylation patterns were examined in the liver, a relatively homogeneous organ that regulates metabolic processes and somatic growth, of juveniles at two time points: after eight months of rearing in either a tank or stream environment and after a subsequent year of rearing in a common tank environment. Further, we analyzed DNA methylation in the sperm of mature 2-year-old males from the earlier described treatments to assess the potential of environmentally-induced changes to be passed to offspring. Hepatic DNA methylation changes in response to hatchery versus stream-rearing in yearling fish were substantial, but few persisted after a second year in the tank environment. However, the early rearing environment appeared to affect how fish responded to developmental and environmental signals during the second year since novel DNA methylation differences were identified in the livers of hatchery versus stream-reared fish after a year of common tank rearing. Furthermore, we found profound differences in DNA methylation due to age, irrespective of rearing treatment. This could be due to smoltification associated changes in liver physiology after the second year of rearing. Although few rearing-treatment effects were observed in the sperm methylome, strong family effects were observed. These data suggest limited potential for intergenerational changes, but highlight the importance of understanding the effects of kinship among studied individuals in order to properly analyze and interpret DNA methylation data in natural populations. Our work is the first to study family effects and temporal dynamics of DNA methylation patterns in response to hatchery-rearing.

Initiation of sex change and gonadal gene expression in black sea bass (Centropristis striata) exposed to exemestane, an aromatase inhibitor.

Many teleost fishes exhibit sequential hermaphroditism, where male or female gonads develop first and later undergo sex change. Model sex change species are characterized by social hierarchies and coloration changes, which enable experimental manipulations to better understand these processes. However, other species such as the protogynous black sea bass (Centropristis striata) do not exhibit these characteristics and instead receive research attention due to their importance in fisheries or aquaculture. Black sea bass social structure is unknown, which makes sex change sampling difficult, and few molecular resources are available. The purpose of the present study was to induce sex change using exemestane, an aromatase inhibitor, and assess gonadal gene expression using sex markers (amh, zpc2) and genes involved in steroidogenesis (cyp19a1a, cyp11b), estrogen signaling (esr1, esr2b), and apoptosis or atresia (aen, casp9, fabp11, parg, pdcd4, rif1). Overall, dietary exemestane treatment was effective, and most exposed females exhibited early histological signs of sex change and significantly higher rates of ovarian atresia relative to control females. Genes associated with atresia did not reflect this, however, as expression patterns in sex changing gonads were overall similar to those of ovaries, likely due to a whole ovary dilution effect of the RNA. Still, small but insignificant expression decreases during early sex change were detected for ovary-related genes (aen, casp9, fabp11, zpc2) and anti-apoptotic factors (parg, rif1). Exemestane treatment did not impact spermatogenesis or testicular gene expression, but testes were generally characterized by elevated steroidogenic enzyme and estrogen receptor mRNAs. Further research will be needed to understand these processes in black sea bass, using isolated ovarian follicles and multiple stages of sex change.

Characterization of Genetic and Epigenetic Variation in Sperm and Red Blood Cells from Adult Hatchery and Natural-Origin Steelhead, Oncorhynchus mykiss.

While the goal of most conservation hatchery programs is to produce fish that are genetically and phenotypically indistinguishable from the wild stocks they aim to restore, there is considerable evidence that salmon and steelhead reared in hatcheries differ from wild fish in phenotypic traits related to fitness. Some evidence suggests that these phenotypic differences have a genetic basis (e.g., domestication selection) but another likely mechanism that remains largely unexplored is that differences between hatchery and wild populations arise as a result of environmentally-induced heritable epigenetic change. As a first step toward understanding the potential contribution of these two possible mechanisms, we describe genetic and epigenetic variation in hatchery and natural-origin adult steelhead, Oncorhynchus mykiss, from the Methow River, WA. Our main objectives were to determine if hatchery and natural-origin fish could be distinguished genetically and whether differences in epigenetic programming (DNA methylation) in somatic and germ cells could be detected between the two groups. Genetic analysis of 72 fish using 936 SNPs generated by Restriction Site Associated DNA Sequencing (RAD-Seq) did not reveal differentiation between hatchery and natural-origin fish at a population level. We performed Reduced Representation Bisulfite Sequencing (RRBS) on a subset of 10 hatchery and 10 natural-origin fish and report the first genome-wide characterization of somatic (red blood cells (RBCs)) and germ line (sperm) derived DNA methylomes in a salmonid, from which we identified considerable tissue-specific methylation. We identified 85 differentially methylated regions (DMRs) in RBCs and 108 DMRs in sperm of steelhead reared for their first year in a hatchery environment compared to those reared in the wild. This work provides support that epigenetic mechanisms may serve as a link between hatchery rearing and adult phenotype in steelhead; furthermore, DMRs identified in germ cells (sperm) highlight the potential for these changes to be passed on to future generations.

Novel adverse outcome pathways revealed by chemical genetics in a developing marine fish.

Crude oil spills are a worldwide ocean conservation threat. Fish are particularly vulnerable to the oiling of spawning habitats, and crude oil causes severe abnormalities in embryos and larvae. However, the underlying mechanisms for these developmental defects are not well understood. Here, we explore the transcriptional basis for four discrete crude oil injury phenotypes in the early life stages of the commercially important Atlantic haddock (Melanogrammus aeglefinus). These include defects in (1) cardiac form and function, (2) craniofacial development, (3) ionoregulation and fluid balance, and (4) cholesterol synthesis and homeostasis. Our findings suggest a key role for intracellular calcium cycling and excitation-transcription coupling in the dysregulation of heart and jaw morphogenesis. Moreover, the disruption of ionoregulatory pathways sheds new light on buoyancy control in marine fish embryos. Overall, our chemical-genetic approach identifies initiating events for distinct adverse outcome pathways and novel roles for individual genes in fundamental developmental processes.

High-throughput sequencing and pathway analysis reveal alteration of the pituitary transcriptome by 17α-ethynylestradiol (EE2) in female coho salmon, Oncorhynchus kisutch.

Considerable research has been done on the effects of endocrine disrupting chemicals (EDCs) on reproduction and gene expression in the brain, liver and gonads of teleost fish, but information on impacts to the pituitary gland are still limited despite its central role in regulating reproduction. The aim of this study was to further our understanding of the potential effects of natural and synthetic estrogens on the brain-pituitary-gonad axis in fish by determining the effects of 17α-ethynylestradiol (EE2) on the pituitary transcriptome. We exposed sub-adult coho salmon (Oncorhynchus kisutch) to 0 or 12 ng EE2/L for up to 6 weeks and effects on the pituitary transcriptome of females were assessed using high-throughput Illumina(®) sequencing, RNA-Seq and pathway analysis. After 1 or 6 weeks, 218 and 670 contiguous sequences (contigs) respectively, were differentially expressed in pituitaries of EE2-exposed fish relative to control. Two of the most highly up- and down-regulated contigs were luteinizing hormone ß subunit (241-fold and 395-fold at 1 and 6 weeks, respectively) and follicle-stimulating hormone ß subunit (-3.4-fold at 6 weeks). Additional contigs related to gonadotropin synthesis and release were differentially expressed in EE2-exposed fish relative to controls. These included contigs involved in gonadotropin releasing hormone (GNRH) and transforming growth factor-ß signaling. There was an over-representation of significantly affected contigs in 33 and 18 canonical pathways at 1 and 6 weeks, respectively, including circadian rhythm signaling, calcium signaling, peroxisome proliferator-activated receptor (PPAR) signaling, PPARα/retinoid x receptor α activation, and netrin signaling. Network analysis identified potential interactions between genes involved in circadian rhythm and GNRH signaling, suggesting possible effects of EE2 on timing of reproductive events.

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.

Temporal and spatial variability in nearshore bacterioplankton communities of Lake Michigan.

The spatial and temporal variability of bacterial communities were determined for the nearshore waters of Lake Michigan, an oligotrophic freshwater inland sea. A freshwater estuary and nearshore sites were compared six times during 2006 using denaturing gradient gel electrophoresis (DGGE). Bacterial composition clustered by individual site and date rather than by depth. Seven 16S rRNA gene clone libraries were constructed, yielding 2717 bacterial sequences. Spatial variability was detected among the DGGE banding patterns and supported by clone library composition. The clone libraries from deep waters and the estuary environment revealed highest overall bacterial diversity. Betaproteobacteria sequence types were the most dominant taxa, comprising 40.2-67.7% of the clone libraries. BAL 47 was the most abundant freshwater cluster of Betaproteobacteria, indicating widespread distribution of this cluster in the nearshore waters of Lake Michigan. Incertae sedis 5 and Oxalobacteraceae sequence types were prevalent in each clone library, displaying more diversity than previously described in other freshwater environments. Among the Oxalobacteraceae sequences, a globally distributed freshwater cluster was determined. The nearshore waters of Lake Michigan are a dynamic environment that experience forces similar to the coastal ocean environment and share common bacterial diversity with other freshwater habitats.

Bacteroidales diversity in ring-billed gulls (Laurus delawarensis) residing at Lake Michigan beaches.

This study investigated the occurrence and diversity of Bacteroidales fecal bacteria in gulls residing in the Great Lakes region. Members of this bacterial order have been widely employed as human and bovine host-specific markers of fecal pollution; however, few studies have focused on gulls, which can be a major source of fecal indicator bacteria and pathogens at beaches. We found a low but consistent occurrence of Bacteroidales in gulls at five beaches in three different counties spanning the Wisconsin shoreline of Lake Michigan. The percentages of gulls positive for Bacteroidales were 4 to 8% at beaches in the southern part of the state and 8 to 50% at beaches in the north. Sequencing of 931 clones from seven gull Bacteroidales 16S rRNA gene libraries revealed a large amount of diversity in both individual and pooled gull fecal samples. Two libraries constructed from pooled gull fecal samples (n = 5 and n = 6) did not have a greater richness of sequences than individual samples, suggesting that even within a single gull diversity is high and an extensive sequencing effort is needed to characterize the populations. Estimates of the numbers of operational taxonomic units (OTUs) for the libraries obtained using different similarity levels revealed a large amount of microdiveristy with a limited number of OTUs at the 95% similarity level. Gull sequences were clustered by the beach from which they were collected, suggesting that there were geographic effects on the distribution of Bacteriodales. More than 53% of the 16S rRNA gene sequences from gulls at the southern beaches were associated with the family Porphyromonadaceae, primarily the genus Parabacteroides, whereas sequences from gulls at the northern beaches were comprised of Bacteroidaceae and Prevotellaceae sequences. Comparison of gull sequences with sequences from goose, canine, raccoon, and sewage sources revealed distinct clusters of closely related gull sequences; however, these sequences were widely dispersed across a dendrogram that included all other sources, including previously characterized gull Bacteroidales from other studies, suggesting that geographic influence or simply sample representation plays a greater role in the observed population structure than strictly the host gut environment.

Pathogen-associated gene expression profiles in rainbow trout macrophages.

Pathogens can be distinctively recognized by the cells of the immune system through interactions between the Pathogen-Associated Molecular Patterns (PAMPs) that they produce and the innate immune receptors of leukocytes. The present paper reports on the PAMP-modulated expression of a group of genes expressed in trout macrophages. The genes were identified in subtracted libraries from lipopolysaccharide (LPS)-stimulated macrophages and their expression was analyzed using quantitative real time PCR following stimulation of the cells with E. coli LPS, poly (I:C) and zymosan; representing Gram-negative bacteria, viruses and fungi, respectively. Genes (SPINT1L, DDIT4L, STEAP4, and TNFAIP3), the expression of which was induced by LPS and zymosan, were not significantly up-regulated by poly(I:C) and the opposite was found for transcripts (HMGB1 and PSMB9) up-regulated by poly(I:C). Overall, the differences in gene expression were greater at a later stage of macrophage activation (24 h) at a time when stimulation with poly(I:C) resulted in substantially different responses as compared to LPS and, to a lesser extent, zymosan.