Potential biomarkers of endocrine and habitat disruption identified via RNA-Seq in Salvelinus fontinalis with proximity to fracking operations in Pennsylvania headwater stream ecosystems.

Unconventional natural gas development (fracking) has been a rapidly expanding technique used for the extraction of natural gas from the Marcellus Shale formation in Pennsylvania. There remains a knowledge gap regarding the ecological impacts of fracking, especially regarding the long-term health of native Brook trout (Salvelinus fontinalis) populations. During the summer of 2015, Brook trout were sampled from twelve streams located in forested, northwestern Pennsylvania in order to evaluate the impacts of fracking on Brook trout. Four stream sites were undisturbed (no fracking activity), three had a developed well pad without fracking activity, and five had active fracking with natural gas production. Liver tissue was isolated from two to five fish per stream and underwent RNA-Seq analysis to identify differentially expressed genes between ecosystems with differing fracking status. Data were analyzed individually and with samples pooled within-stream to account for hierarchical data structure and variation in sample coverage within streams. Differentially expressed and differentially alternatively spliced genes had functions related to lipid and steroid metabolism, mRNA processing, RNA polymerase and protein regulation. Unique to our study, genes related to xenobiotic and stress responses were found as well as potential markers for endocrine disruption and saline adaptation that were identified in watersheds with active fracking activity. These results support the utility of RNA-Seq to assess trout health and suggest detrimental impacts of fracking on sensitive trout populations.

Temporal Transcriptomics of Gut Escherichia coli in Caenorhabditis elegans Models of Aging.

Host-bacterial interactions over the course of aging are understudied due to complexities of the human microbiome and challenges of collecting samples that span a lifetime. To investigate the role of host-microbial interactions in aging, we performed transcriptomics using wild-type Caenorhabditis elegans (N2) and three long-lived mutants (daf-2, eat-2, and asm-3) fed Escherichia coli OP50 and sampled at days 5, 7.5, and 10 of adulthood. We found host age is a better predictor of the E. coli expression profiles than host genotype. Specifically, host age was associated with clustering (permutational multivariate analysis of variance [PERMANOVA], P = 0.001) and variation (Adonis, P = 0.001, R2 = 11.5%) among E. coli expression profiles, whereas host genotype was not (PERMANOVA, P > 0.05; Adonis, P > 0.05, R2 = 5.9%). Differential analysis of the E. coli transcriptome yielded 22 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways and 100 KEGG genes enriched when samples were grouped by time point [LDA, linear discriminant analysis; log(LDA), ≥2; P ≤ 0.05], including several involved in biofilm formation. Coexpression analysis of host and bacterial genes yielded six modules of C. elegans genes that were coexpressed with one bacterial regulator gene over time. The three most significant bacterial regulators included genes relating to biofilm formation, lipopolysaccharide production, and thiamine biosynthesis. Age was significantly associated with clustering and variation among transcriptomic samples, supporting the idea that microbes are active and plastic within C. elegans throughout life. Coexpression analysis further revealed interactions between E. coli and C. elegans that occurred over time, building on a growing literature of host-microbial interactions. IMPORTANCE Previous research has reported effects of the microbiome on health span and life span of Caenorhabditis elegans, including interactions with evolutionarily conserved pathways in humans. We build on this literature by reporting the gene expression of Escherichia coli OP50 in wild-type (N2) and three long-lived mutants of C. elegans. The manuscript represents the first study, to our knowledge, to perform temporal host-microbial transcriptomics in the model organism C. elegans. Understanding changes to the microbial transcriptome over time is an important step toward elucidating host-microbial interactions and their potential relationship to aging. We found that age was significantly associated with clustering and variation among transcriptomic samples, supporting the idea that microbes are active and plastic within C. elegans throughout life. Coexpression analysis further revealed interactions between E. coli and C. elegans that occurred over time, which contributes to our growing knowledge about host-microbial interactions.

Regions of genetic divergence in depth-separated Sebastes rockfish species pairs: Depth as a potential driver of speciation.

Depth separation is a proposed driver of speciation in marine fishes, with marine rockfish (genus Sebastes) providing a potentially informative study system. Sebastes rockfishes are commercially and ecologically important. This genus encompasses more than one hundred species and the ecological and morphological variance between these species provides opportunity for identifying speciation-driving adaptations, particularly along a depth gradient. A reduced-representation sequencing method (ddRADseq) was used to compare 95 individuals encompassing six Sebastes species. In this study, we sought to identify regions of divergence between species that were indicative of divergent adaptation and reproductive barriers leading to speciation. A pairwise comparison of S. chrysomelas (black-and-yellow rockfish) and S. carnatus (gopher rockfish) FST values revealed three major regions of elevated genomic divergence, two of which were also present in the S. miniatus (vermilion rockfish) and S. crocotulus (sunset rockfish) comparison. These corresponded with regions of both elevated DXY values and reduced nucleotide diversity in two cases, suggesting a speciation-with-gene-flow evolutionary model followed by post-speciation selective sweeps within each species. Limited whole-genome resequencing was also performed to identify mutations with predicted effects between S. chrysomelas and S. carnatus. Within these islands, we identified important SNPs in genes involved in immune function and vision. This supports their potential role in speciation, as these are adaptive vectors noted in other organisms. Additionally, changes to genes involved in pigment expression and mate recognition shed light on how S. chrysomelas and S. carnatus may have become reproductively isolated.

Genomic characterization of sex-identification markers in Sebastes carnatus and Sebastes chrysomelas rockfishes.

Fish have evolved a variety of sex-determining (SD) systems including male heterogamy (XY), female heterogamy (ZW) and environmental SD. Little is known about SD mechanisms of Sebastes rockfishes, a highly speciose genus of importance to evolutionary and conservation biology. Here, we characterize the sex determination system in the sympatrically distributed sister species Sebastes chrysomelas and Sebastes carnatus. To identify sex-specific genotypic markers, double digest restriction site - associated DNA sequencing (ddRAD-seq) of genomic DNA from 40 sexed individuals of both species was performed. Loci were filtered for presence in all of the individuals of one sex, absence in the other sex and no heterozygosity. Of the 74 965 loci present in all males, 33 male-specific loci met the criteria in at least one species and 17 in both. Conversely, no female-specific loci were detected, together providing evidence of an XY sex determination system in both species. When aligned to a draft reference genome from Sebastes aleutianus, 26 sex-specific loci were interspersed among 1168 loci that were identical between sexes. The nascent Y chromosome averaged 5% divergence from the X chromosome and mapped to reference Sebastes genome scaffolds totalling 6.9Mbp in length. These scaffolds aligned to a single chromosome in three model fish genomes. Read coverage differences were also detected between sex-specific and autosomal loci. A PCR-RFLP assay validated the bioinformatic results and correctly identified sex of five additional individuals of known sex. A sex-determining gene in other teleosts gonadal soma-derived factor (gsdf) was present in the model fish chromosomes that spanned our sex-specific markers.

Comparative population genetic analysis of bocaccio rockfish Sebastes paucispinis using anonymous and gene-associated simple sequence repeat loci.

Comparative population genetic analyses of traditional and emergent molecular markers aid in determining appropriate use of new technologies. The bocaccio rockfish Sebastes paucispinis is a high gene-flow marine species off the west coast of North America that experienced strong population decline over the past 3 decades. We used 18 anonymous and 13 gene-associated simple sequence repeat (SSR) loci (expressed sequence tag [EST]-SSRs) to characterize range-wide population structure with temporal replicates. No F(ST)-outliers were detected using the LOSITAN program, suggesting that neither balancing nor divergent selection affected the loci surveyed. Consistent hierarchical structuring of populations by geography or year class was not detected regardless of marker class. The EST-SSRs were less variable than the anonymous SSRs, but no correlation between F(ST) and variation or marker class was observed. General linear model analysis showed that low EST-SSR variation was attributable to low mean repeat number. Comparative genomic analysis with Gasterosteus aculeatus, Takifugu rubripes, and Oryzias latipes showed consistently lower repeat number in EST-SSRs than SSR loci that were not in ESTs. Purifying selection likely imposed functional constraints on EST-SSRs resulting in low repeat numbers that affected diversity estimates but did not affect the observed pattern of population structure.

GCAT-SEEKquence: genome consortium for active teaching of undergraduates through increased faculty access to next-generation sequencing data.

To transform undergraduate biology education, faculty need to provide opportunities for students to engage in the process of science. The rise of research approaches using next-generation (NextGen) sequencing has been impressive, but incorporation of such approaches into the undergraduate curriculum remains a major challenge. In this paper, we report proceedings of a National Science Foundation-funded workshop held July 11-14, 2011, at Juniata College. The purpose of the workshop was to develop a regional research coordination network for undergraduate biology education (RCN/UBE). The network is collaborating with a genome-sequencing core facility located at Pennsylvania State University (University Park) to enable undergraduate students and faculty at small colleges to access state-of-the-art sequencing technology. We aim to create a database of references, protocols, and raw data related to NextGen sequencing, and to find innovative ways to reduce costs related to sequencing and bioinformatics analysis. It was agreed that our regional network for NextGen sequencing could operate more effectively if it were partnered with the Genome Consortium for Active Teaching (GCAT) as a new arm of that consortium, entitled GCAT-SEEK(quence). This step would also permit the approach to be replicated elsewhere.

Characterization of a genomic divergence island between black-and-yellow and gopher Sebastes rockfishes.

Islands of high genomic divergence that contain genes of evolutionary significance may form between diverging species. The gopher rockfish, Sebastes carnatus, and black-and-yellow rockfish, S. chrysomelas, are sympatrically distributed temperate marine species inhabiting rocky reefs and kelp forests on the west coast of the United States. Prior studies documented low levels of genetic divergence between the two species, except at a single microsatellite locus that displayed high divergence, Sra.7-2. To better characterize genome wide divergence, we scored 25 additional microsatellite loci. Mean neutral divergence between species (F(ST) = 0.01) changed little from prior estimates. Sra.7-2 continued to be an extreme divergence outlier. Five novel microsatellites within ± 15 kb of Sra.7-2 were characterized. High divergence, consistently low diversity in S. chrysomelas, and linkage disequilibrium were detected at these loci, suggesting the influence of recent selection. However, coalescent modelling of divergence at neutral and Sra.7-2 regions showed that initial divergence at Sra.7-2 was ancient, likely predating divergence at neutral regions. It is therefore unlikely that Sra.7-2 divergence represents solely recent ecological divergence within one species and may represent the action of recurrent selection. Introgressive gene flow (2N(E) m) was much higher (>>1) at neutral than Sra.7-2 regions (<<1) despite evidence that two S. carnatus individuals have recently mixed ancestry at the Sra.7-2 region. The Sra.7-2 genomic region is likely one of several regions containing genes involved in initiating and maintaining species integrity. Completion of the final stages of speciation appears to be a slow and ongoing process for these species.

GEOGRAPHIC DISTRIBUTION OF MOLECULAR VARIANCE WITHIN THE BLUE MARLIN (MAKAIRA NIGRICANS): A HIERARCHICAL ANALYSIS OF ALLOZYME, SINGLE-COPY NUCLEAR DNA, AND MITOCHONDRIAL DNA MARKERS.

This study presents a comparative hierarchical analysis of variance applied to three classes of molecular markers within the blue marlin (Makaira nigricans). Results are reported from analyses of four polymorphic allozyme loci, four polymorphic anonymously chosen single-copy nuclear DNA (scnDNA) loci, and previously reported restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA). Samples were collected within and among the Atlantic and Pacific Oceans over a period of several years. Although moderate levels of genetic variation were detected at both polymorphic allozyme (H = 0.30) and scnDNA loci (H = 0.37), mtDNA markers were much more diverse (h = 0.85). Allele frequencies were significantly different between Atlantic and Pacific Ocean samples at three of four allozyme loci and three of four scnDNA loci. Estimates of allozyme genetic differentiation (θO ) ranged from 0.00 to 0.15, with a mean of 0.08. The θO values for scnDNA loci were similar to those of allozymes, ranging from 0.00 to 0.12 with a mean of 0.09. MtDNA RFLP divergence between oceans (θO = 0.39) was significantly greater than divergence detected at nuclear loci (95% nuclear confidence interval = 0.04-0.11). The fourfold smaller effective population size of mtDNA and male-mediated gene flow may account for the difference observed between nuclear and mitochondrial divergence estimates.