Intersex, or the presence of characteristics of both sexes, in fishes that are normally gonochoristic has been used as an indicator of exposure to estrogenic compounds. In 2003, during health assessments conducted in response to kills and a high prevalence of skin lesions observed in smallmouth bass Micropterus dolomieu in the South Branch of the Potomac River, the presence of immature oocytes within testes was noted. To evaluate this condition, a severity index (0-4) was developed based on the distribution of oocytes within the testes. Using gonad samples collected from 2003 to 2005, the number of histologic sections needed to accurately detect the condition in mature smallmouth bass was statistically evaluated. The reliability of detection depended on the severity index and the number of sections examined. Examining five transverse sections taken along the length of the gonad resulted in a greater than 90% probability of detecting testicular oocytes when the severity index exceeded 0.5. Using the severity index we compared smallmouth bass collected at selected sites within the South Branch during three seasons in 2004. Seasonal differences in severity and prevalence were observed. The highest prevalence and severity were consistently noted during the prespawn-spawning season, when compared with the postspawn season. In 2005, smallmouth bass were collected at selected out-of-basin sites in West Virginia where fish kills and external skin lesions have not been reported, as well as at sites in the Shenandoah River, Virginia (part of the Potomac drainage), where kills and lesions occurred in 2004-2005. The prevalence of testicular oocytes is discussed in terms of human population and agricultural intensity.

Taxonomic identification of pollen has historically been accomplished via light microscopy but requires specialized knowledge and reference collections, particularly when identification to lower taxonomic levels is necessary. Recently, next-generation sequencing technology has been used as a cost-effective alternative for identifying bee-collected pollen; however, this novel approach has not been tested on a spatially or temporally robust number of pollen samples. Here, we compare pollen identification results derived from light microscopy and DNA sequencing techniques with samples collected from honey bee colonies embedded within a gradient of intensive agricultural landscapes in the Northern Great Plains throughout the 2010-2011 growing seasons. We demonstrate that at all taxonomic levels, DNA sequencing was able to discern a greater number of taxa, and was particularly useful for the identification of infrequently detected species. Importantly, substantial phenological overlap did occur for commonly detected taxa using either technique, suggesting that DNA sequencing is an appropriate, and enhancing, substitutive technique for accurately capturing the breadth of bee-collected species of pollen present across agricultural landscapes. We also show that honey bees located in high and low intensity agricultural settings forage on dissimilar plants, though with overlap of the most abundantly collected pollen taxa. We highlight practical applications of utilizing sequencing technology, including addressing ecological issues surrounding land use, climate change, importance of taxa relative to abundance, and evaluating the impact of conservation program habitat enhancement efforts.

Fishes were collected at 16 sites within the three major river drainages (Delaware, Susquehanna, and Ohio) of Pennsylvania. Three species were evaluated for biomarkers of estrogenic/antiandrogenic exposure, including plasma vitellogenin and testicular oocytes in male fishes. Smallmouth bass Micropterus dolomieu, white sucker Catostomus commersonii, and redhorse sucker Moxostoma species were collected in the summer, a period of low flow and low reproductive activity. Smallmouth bass were the only species in which testicular oocytes were observed; however, measurable concentrations of plasma vitellogenin were found in male bass and white sucker. The percentage of male bass with testicular oocytes ranged from 10 to 100 %, with the highest prevalence and severity in bass collected in the Susquehanna drainage. The percentage of males with plasma vitellogenin ranged from 0 to 100 % in both bass and sucker. Biological findings were compared with chemical analyses of discrete water samples collected at the time of fish collections. Estrone concentrations correlated with testicular oocytes prevalence and severity and with the percentage of male bass with vitellogenin. No correlations were noted with the percentage of male sucker with vitellogenin and water chemical concentrations. The prevalence and severity of testicular oocytes in bass also correlated with the percent of agricultural land use in the watershed above a site. Two sites within the Susquehanna drainage and one in the Delaware were immediately downstream of wastewater treatment plants to compare results with upstream fish. The percentage of male bass with testicular oocytes was not consistently higher downstream; however, severity did tend to increase downstream.Electronic supplementary materialThe online version of this article (doi:10.1007/s10661-014-3868-5) contains supplementary material, which is available to authorized users.

Skin lesions and spring mortality events of smallmouth bass Micropterus dolomieu and selected other species were first noted in the South Branch of the Potomac River in 2002. Since that year morbidity and mortality have also been observed in the Shenandoah and Monocacy rivers. Despite much research, no single pathogen, parasite, or chemical cause for the lesions and mortality has been identified. Numerous parasites, most commonly trematode metacercariae and myxozoans; the bacterial pathogens Aeromonas hydrophila, Aeromonas salmonicida, and Flavobacterium columnare; and largemouth bass virus have all been observed. None have been consistently isolated or observed at all sites, however, nor has any consistent microscopic pathology of the lesions been observed. A variety of histological changes associated with exposure to environmental contaminants or stressors, including intersex (testicular oocytes), high numbers of macrophage aggregates, oxidative damage, gill lesions, and epidermal papillomas, were observed. The findings indicate that selected sensitive species may be stressed by multiple factors and constantly close to the threshold between a sustainable (healthy) and nonsustainable (unhealthy) condition. Fish health is often used as an indicator of aquatic ecosystem health, and these findings raise concerns about environmental degradation within the Potomac River drainage. Unfortunately, while much information has been gained from the studies conducted to date, due to the multiple state jurisdictions involved, competing interests, and other issues, there has been no coordinated approach to identifying and mitigating the stressors. This synthesis emphasizes the need for multiyear, interdisciplinary, integrative research to identify the underlying stressors and possible management actions to enhance ecosystem health.

Identifying plant taxa that honey bees (Apis mellifera) forage upon is of great apicultural interest, but traditional methods are labor intensive and may lack resolution. Here we evaluate a high-throughput genetic barcoding approach to characterize trap-collected pollen from multiple North Dakota apiaries across multiple years. We used the Illumina MiSeq platform to generate sequence scaffolds from non-overlapping 300-bp paired-end sequencing reads of the ribosomal internal transcribed spacers (ITS). Full-length sequence scaffolds represented ~530 bp of ITS sequence after adapter trimming, drawn from the 5’ of ITS1 and the 3’ of ITS2, while skipping the uninformative 5.8S region. Operational taxonomic units (OTUs) were picked from scaffolds clustered at 97% identity, searched by BLAST against the nt database, and given taxonomic assignments using the paired-read lowest common ancestor approach. Taxonomic assignments and quantitative patterns were consistent with known plant distributions, phenology, and observational reports of pollen foraging, but revealed an unexpected contribution from non-crop graminoids and wetland plants. The mean number of plant species assignments per sample was 23.0 (+/- 5.5) and the mean species diversity (effective number of equally abundant species) was 3.3 (+/- 1.2). Bray-Curtis similarities showed good agreement among samples from the same apiary and sampling date. Rarefaction plots indicated that fewer than 50,000 reads are typically needed to characterize pollen samples of this complexity. Our results show that a pre-compiled, curated reference database is not essential for genus-level assignments, but species-level assignments are hindered by database gaps, reference length variation, and probable errors in the taxonomic assignment, requiring post-hoc evaluation. Although the effective per-sample yield achieved using custom MiSeq amplicon primers was less than the machine maximum, primarily due to lower “read2” quality, further protocol optimization and/or a modest reduction in multiplex scale should offset this difficulty. As small quantities of pollen are sufficient for amplification, our approach might be extendable to other questions or species for which large pollen samples are not available.

Understanding the diet of an endangered species illuminates the animal’s ecology, habitat requirements, and conservation needs. However, direct observation of diet can be difficult, particularly for small, nocturnal animals such as the Pacific pocket mouse (Heteromyidae: Perognathus longimembris pacificus). Very little is known of the dietary habits of this federally endangered rodent, hindering management and restoration efforts. We used a metabarcoding approach to identify source plants in fecal samples (N = 52) from the three remaining populations known. The internal transcribed spacers (ITS) of the nuclear ribosomal loci were sequenced following the Illumina MiSeq amplicon strategy and processed reads were mapped to reference databases. We evaluated a range of threshold mapping criteria and found the best-performing setting generally recovered two distinct mock communities in proportions similar to expectation. We tested our method on captive animals fed a known diet and recovered almost all plant sources, but found substantial heterogeneity among fecal pellets collected from the same individual at the same time. Observed richness did not increase with pooling of pellets from the same individual. In field-collected samples, we identified 4–14 plant genera in individual samples and 74 genera overall, but over 50 percent of reads mapped to just six species in five genera. We simulated the effects of sequencing error, variable read length, and chimera formation to infer taxon-specific rates of misassignment for the local flora, which were generally low with some exceptions. Richness at the species and genus levels did not reach a clear asymptote, suggesting that diet breadth remained underestimated in the current pool of samples. Large numbers of scat samples are therefore needed to make inferences about diet and resource selection in future studies of the Pacific pocket mouse. We conclude that our minimally invasive method is promising for determining herbivore diets given a library of sequences from local plants.

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.

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