ALBERT

Description: Chelonid alphaherpesvirus 5 (ChHV5) is a herpesvirus associated with fibropapillomatosis (FP) in sea turtles worldwide. Single-locus typing has previously shown differentiation between Atlantic and Pacific strains of this virus, with low variation within each geographic clade. However, a lack of multi-locus genomic sequence data hinders understanding of the rate and mechanisms of ChHV5 evolutionary divergence, as well as how these genomic changes may contribute to differences in disease manifestation. To assess genomic variation in ChHV5 among five Hawaii and three Florida green sea turtles, we used high-throughput short-read sequencing of long-range PCR products amplified from tumor tissue using primers designed from the single available ChHV5 reference genome from a Hawaii green sea turtle. This strategy recovered sequence data from both geographic regions for approximately 75% of the predicted ChHV5 coding sequences. The average nucleotide divergence between geographic populations was 1.5%; most of the substitutions were fixed differences between regions. Protein divergence was generally low (average 0.08%), and ranged between 0 and 5.3%. Several atypical genes originally identified and annotated in the reference genome were confirmed in ChHV5 genomes from both geographic locations. Unambiguous recombination events between geographic regions were identified, and clustering of private alleles suggests the prevalence of recombination in the evolutionary history of ChHV5. This study significantly increased the amount of sequence data available from ChHV5 strains, enabling informed selection of loci for future population genetic and natural history studies, and suggesting the (possibly latent) co-infection of individuals by well-differentiated geographic variants.

Description: Use of environmental DNA (eDNA) to assess distributions of aquatic and semi-aquatic macroorganisms is promising, but sampling schemes may need to be tailored to specific objectives. Given the potentially high variance in aquatic eDNA among replicate grab samples, compositing smaller water volumes collected over a period of time may be more effective for some applications. In this study, we compared eDNA profiles from composite water samples aggregated over three hours with grab water samples. Both sampling patterns were performed with identical autosamplers paired at two different sites in a headwater stream environment, augmented with exogenous fish eDNA from an upstream rearing facility. Samples were filtered through 0.8 μm cellulose nitrate filters and DNA was extracted with a cetyl trimethylammonium bromide procedure. Eukaryotic and bacterial community profiles were derived by amplicon sequencing of 12S ribosomal, 16S ribosomal, and cytochrome oxidase I loci. Operational taxa were assigned to genus with a lowest common ancestor approach for eukaryotes and to family with the RDP Classifier software for prokaryotes. Eukaryotic community profiles were more consistent with composite sampling than grab sampling. Downstream, rarefaction curves suggested faster taxon accumulation for composite samples, and estimated richness was higher for composite samples as a set than for grab samples. Upstream, composite sampling produced lower estimated richness than grab samples, but with overlapping standard errors. Furthermore, a bimodal pattern of richness as a function of sequence counts suggested the impact of clumped particles on upstream samples. Bacterial profiles were insensitive to sample method, consistent with the more even dispersion expected for bacteria compared with eukaryotic eDNA. Overall, samples composited over 3 h performed equal to or better than triplicate grab sampling for quantitative community metrics, despite the higher total sequencing effort provided to grab replicates. On the other hand, taxon-specific detection rates did not differ appreciably and the two methods gave similar estimates of the ratio of the common fish genera Salmo and Coregonus at each site. Unexpectedly, Salmo eDNA dropped out substantially faster than Coregonus eDNA between the two sites regardless of sampling method, suggesting that differential settling affects the estimation of relative abundance. We identified bacterial patterns that were associated with eukaryotic diversity, suggesting potential roles as biomarkers of sample representativeness.

Description: Lake Sinai Viruses (Sinaivirus) are commonly detected in honey bees (Apis mellifera) but no disease phenotypes or fitness consequences have yet been demonstrated. This viral group is genetically diverse, lacks obvious geographic structure, and multiple lineages can co-infect individual bees. While phylogenetic analyses have been performed, the molecular evolution of LSV has not been studied extensively. Here, I use LSV isolates from GenBank as well as contigs assembled from honey bee Sequence Read Archive (SRA) accessions to better understand the evolutionary history of these viruses. For each ORF, substitution rate variation, codon usage, and tests of positive selection were evaluated. Outlier regions of high or low diversity were sought with sliding window analysis and the role of recombination in creating LSV diversity was explored. Phylogenetic analysis consistently identified two large clusters of sequences that correspond to the current LSV1 and LSV2 nomenclature, however lineages sister to LSV1 were the most frequently detected in honey bee SRA accessions. Different expression levels among ORFs suggested the occurrence of subgenomic transcripts. ORF1 and RNA-dependent RNA polymerase had higher evolutionary rates than the capsid and ORF4. A hypervariable region of the ORF1 protein-coding sequence was identified that had reduced selective constraint, but a site-based model of positive selection was not significantly more likely than a neutral model for any ORF. The only significant recombination signals detected between LSV1 and LSV2 initiated within this hypervariable region, but assumptions of the test (single-frame coding and independence of substitution rate by site) were violated. LSV codon usage differed strikingly from that of honey bees and other common honey-bee viruses, suggesting LSV is not strongly co-evolved with that host. LSV codon usage was significantly correlated with that of Varroa destructor, however, despite the relatively weak codon bias exhibited by the latter. While codon usage between the LSV1 and LSV2 clusters was similar for three ORFs, ORF4 codon usage was uncorrelated between these clades, implying rapid divergence of codon use for this ORF only. Phylogenetic placement and relative abundance of LSV isolates reconstructed from SRA accessions suggest that detection biases may be over-representing LSV1 and LSV2 in public databases relative to their sister lineages.

Description: Endocrine disrupting contaminants are of continuing concern for potentially contributing to reproductive dysfunction in largemouth and smallmouth bass in the Chesapeake Bay watershed (CBW) and elsewhere. Exposures to atrazine (ATR) have been hypothesized to have estrogenic effects on vertebrate endocrine systems. The incidence of intersex in male smallmouth bass from some regions of CBW has been correlated with ATR concentrations in water. Fish early life stages may be particularly vulnerable to ATR exposure in agricultural areas, as a spring influx of pesticides coincides with spawning and early development. Our objectives were to investigate the effects of early life stage exposure to ATR or the model estrogen 17α-ethinylestradiol (EE2) on sexual differentiation and gene expression in gonad tissue. We exposed newly hatched largemouth bass (LMB, Micropterus salmoides) from 7 to 80 days post-spawn to nominal concentrations of 1, 10, or 100 µg ATR/L or 1 or 10 ng EE2/L and monitored histological development and transcriptomic changes in gonad tissue. We observed a nearly 100% female sex ratio in LMB exposed to EE2 at 10 ng/L, presumably due to sex reversal of males. Many gonad genes were differentially expressed between sexes. Multidimensional scaling revealed clustering by gene expression of the 1 ng EE2/L and 100 µg ATR/L-treated male fish. Some pathways responsive to EE2 exposure were not sex-specific. We observed differential expression in male gonad in LMB exposed to EE2 at 1 ng/L of several genes involved in reproductive development and function, including star, cyp11a2, ddx4 (previously vasa), wnt5b, cyp1a and samhd1. Expression of star, cyp11a2 and cyp1a in males was also responsive to ATR exposure. Overall, our results confirm that early development is a sensitive window for estrogenic endocrine disruption in LMB and are consistent with the hypothesis that ATR exposure induces some estrogenic responses in the developing gonad. However, ATR-specific and EE2-specific responses were also observed.

Description: Background Hoary bats (Lasiurus cinereus) are among the bat species most commonly killed by wind turbine strikes in the midwestern United States. The impact of this mortality on species census size is not understood, due in part to the difficulty of estimating population size for this highly migratory and elusive species. Genetic effective population size (Ne) could provide an index of changing census population size if other factors affecting Ne are stable. Methods We used the NeEstimator package to derive effective breeding population size (Nb) estimates for two temporally spaced cohorts: 93 hoary bats collected in 2009–2010 and an additional 93 collected in 2017–2018. We sequenced restriction-site associated polymorphisms and generated a de novo genome assembly to guide the removal of sex-linked and multi-copy loci, as well as identify physically linked markers. Results Analysis of the reference genome with psmc suggested at least a doubling of Ne in the last 100,000 years, likely exceeding Ne = 10,000 in the Holocene. Allele and genotype frequency analyses confirmed that the two cohorts were comparable, although some samples had unusually high or low observed heterozygosities. Additionally, the older cohort had lower mean coverage and greater variability in coverage, and batch effects of sampling locality were observed that were consistent with sample degradation. We therefore excluded samples with low coverage or outlier heterozygosity, as well as loci with sequence coverage far from the mode value, from the final data set. Prior to excluding these outliers, contemporary Nb estimates were significantly higher in the more recent cohort, but this finding was driven by high values for the 2018 sample year and low values for all other years. In the reduced data set, Nb did not differ significantly between cohorts. We found base substitutions to be strongly biased toward cytosine to thymine or the complement, and further partitioning loci by substitution type had a strong effect on Nb estimates. Minor allele frequency and base quality bias thresholds also had strong effects on Nb estimates. Instability of Nb with respect to common data filtering parameters and empirically identified factors prevented robust comparison of the two cohorts. Given that confidence intervals frequently included infinity as the stringency of data filtering increased, contemporary trends in Nb of North American hoary bats may not be tractable with the linkage disequilibrium method, at least using the protocol employed here.

Description: Background Environmental DNA (eDNA) surveys are appealing options for monitoring aquatic biodiversity. While factors affecting eDNA persistence, capture and amplification have been heavily studied, watershed-scale surveys of fish communities and our confidence in such need further exploration. Methods We characterized fish eDNA compositions using rapid, low-volume filtering with replicate and control samples scaled for a single Illumina MiSeq flow cell, using the mitochondrial 12S ribosomal RNA locus for taxonomic profiling. Our goals were to determine: (1) spatiotemporal variation in eDNA abundance, (2) the filtrate needed to achieve strong sequencing libraries, (3) the taxonomic resolution of 12S ribosomal sequences in the study environment, (4) the portion of the expected fish community detectable by 12S sequencing, (5) biases in species recovery, (6) correlations between eDNA compositions and catch per unit effort (CPUE) and (7) the extent that eDNA profiles reflect major watershed features. Our bioinformatic approach included (1) estimation of sequencing error from unambiguous mappings and simulation of taxonomic assignment error under various mapping criteria; (2) binning of species based on inferred assignment error rather than by taxonomic rank; and (3) visualization of mismatch distributions to facilitate discovery of distinct haplotypes attributed to the same reference. Our approach was implemented within the St. Regis River, NY, USA, which supports tribal and recreational fisheries and has been a target of restoration activities. We used a large record of St. Regis-specific observations to validate our assignments. Results We found that 300 mL drawn through 25-mm cellulose nitrate filters yielded greater than 5 ng/µL DNA at most sites in summer, which was an approximate threshold for generating strong sequencing libraries in our hands. Using inferred sequence error rates, we binned 12S references for 110 species on a state checklist into 85 single-species bins and seven multispecies bins. Of 48 bins observed by capture survey in the St. Regis, we detected eDNA consistent with 40, with an additional four detections flagged as potential contaminants. Sixteen unobserved species detected by eDNA ranged from plausible to implausible based on distributional data, whereas six observed species had no 12S reference sequence. Summed log-ratio compositions of eDNA-detected taxa correlated with log(CPUE) (Pearson’s R = 0.655, P 〈 0.001). Shifts in eDNA composition of several taxa and a genotypic shift in channel catfish (Ictalurus punctatus) coincided with the Hogansburg Dam, NY, USA. In summary, a simple filtering apparatus operated by field crews without prior expertise gave useful summaries of eDNA composition with minimal evidence of field contamination. 12S sequencing achieved useful taxonomic resolution despite the short marker length, and data exploration with standard bioinformatic tools clarified taxonomic uncertainty and sources of error.