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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.

Andros Island, The Bahamas, composed of porous carbonate rock, has about 175 inland blue holes and over 50 known submerged ocean caves along its eastern barrier reef. These ocean blue holes can have both vertical and horizontal zones that penetrate under the island. Tidal forces drive water flow in and out of these caves. King Kong Cavern has a vertical collapse zone and a deep penetration under Andros Island that emits sulfidic, anoxic water and masses of thin, mucoid filaments ranging to meters in length and off-white turbid water during ebb flow. Our objective was to determine the microbial composition of this mucoid material and the unconsolidated water column turbidity based on the concept that they represent unique lithoautotrophic microbial material swept from the cave into the surrounding ocean. Bacterial DNA extracted from these filaments and surrounding turbid water was characterized using PCR that targeted a portion of the 16S rRNA gene. The genus Arcobacter dominated both the filaments and the water column above the cave entrance. Arcobacter nitrofigilis and Arcobacter sp. UDC415 in the mucoid filaments accounted for as much as 80% of mapped DNA reads. In the water column Arcobacter comprised from 65% to over 85% of the reads in the depth region from about 18 m to 34 m. Bacterial species diversity was much higher in surface water and in water deeper than 36 m than in the intermediate zone. Community composition indicates that ebb flow from the cavern influences the entire water column at least to within 6 m of the surface and perhaps the near surface as well.

Indigenous freshwater mussels (Unionidae) are integral to riverine ecosystems, playing a pivotal role in aquatic food webs and providing ecological services. With populations on the decline worldwide, freshwater mussels are of conservation concern. In this study, we explore the propensity of the invasive Round Goby (Neogobius melanostomus) fish to prey upon indigenous freshwater mussels. First, we conducted lab experiments where Round Gobies were given the opportunity to feed on juvenile unionid mussels and macroinvertebrates, revealing rates and preferences of consumption. Several Round Gobies consumed whole freshwater mussels during these experiments, as confirmed by mussel counts and x-ray images of the fishes. Next, we investigated Round Gobies collected from stream habitats of the French Creek watershed, which is renowned for its unique and rich aquatic biodiversity. We developed a novel DNA metabarcoding method to identify the specific species of mussels consumed by Round Goby and provide a new database of DNA gene sequences for 25 indigenous unionid mussel species. Several of the fishes sampled had consumed indigenous mussels, including the Elktoe (non-endangered), Creeper (non-endangered), Long Solid (state endangered), and Rayed Bean (federally endangered) species. The invasive Round Goby poses a growing threat to unionid mussels, including species of conservation concern. The introduction of the invasive Round Goby to freshwaters of North America is shaping ecosystem transitions within the aquatic critical zone having widespread implications for conservation and management.

In 2017 the rusty patched bumble bee (Bombus affinis) became the first bee listed under the Endangered Species Act in the continental United States due to population declines and an 87% reduction in the species’ distribution. Bombus affinis decline began in the 1990s, predating modern bee surveying initiatives, and obfuscating drivers of decline. While understood to be a highly generalist forager, little is known about the role that resource limitation or shifting floral community composition could have played in B. affinis decline. Determining which floral species support B. affinis could assist conservation efforts where B. affinis persists and identify floral species for restoration efforts. We constructed a historical foraging profile of B. affinis via DNA sequencing of pollen from museum specimens spanning seven states collected from 1913 to 2013. Molecular analysis revealed no temporal changes in the floral richness or composition of B. affinis pollen samples across our sampling period. Likewise, we found no temporal changes in the presence or proportion of native vs. introduced species in pollen samples, though we observed much greater use of introduced floral species than previously determined for B. affinis. Floral community composition was regionally dissimilar, inconsistent with patterns of B. affinis decline by state. Our results suggest B. affinis decline was unlikely to have been driven by spatial or temporal limitations of specific floral species. This work greatly expands the known forage of B. affinis and will provide managers with insight to aid the conservation of B. affinis.

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.

Colony monitoring devices used to track and assess the health status of honey bees are becoming more widely available and used by both beekeepers and researchers. These devices monitor parameters relevant to colony health at frequent intervals, often approximating real time. The fine-scale record of hive condition can be further related to static or dynamic features of the landscape, such as weather, climate, colony density, land use, pesticide use, vegetation class, and forage quality. In this study, we fit commercial honey bee colonies in two apiaries with pollen traps and digital scales to monitor floral resource use, pollen quality, and honey production. One apiary was situated in low-intensity agriculture; the other in high-intensity agriculture. Pollen traps were open for 72 h every two weeks while scales recorded weight every 15 min throughout the growing season. From collected pollen, we determined forage quantity per day, species identity using DNA sequencing, pesticide residues, amino acid content, and total protein content. From scales, we determined the accumulated hive weight change over the growing season, relating to honey production and final colony weight going into winter. Hive scales may also be used to identify the occurrence of environmental pollen and nectar dearth, and track phenological changes in plant communities. We provide comparisons of device-derived data between two apiaries over the growing season and discuss the potential for employing apiary monitoring devices to infer colony health in the context of divergent agricultural land use conditions.

Information on diet breadth and preference can assist in understanding links between food resources and population growth and inform habitat restoration for rare herbivores. We assessed the diet of the endangered Pacific pocket mouse using metabarcoding of fecal samples and compared it to plant community composition in long‐term study plots in two populations on Marine Corps Base Camp Pendleton, San Diego County, CA. Fecal samples (n = 221) were collected between spring 2016 and fall 2017 during monthly live‐trap surveys. Concurrently, percent cover and plant phenology were measured in plots centered on trap locations. Fecal samples were sequenced with paired‐end reads of the internal transcribed spacer 2 region of the nuclear ribosomal gene, and the resulting amplicons were matched to a regionally specific database. Seventy‐three plant taxa were detected, which were mostly forbs and perennial herbs (70–90%). Diet composition differed between populations, years, seasons, and plots. Overall, diet and local habitat composition in plots were significantly correlated. However, we detected some differences in above‐ground seed availability and proportion in fecal samples that indicate diet preferences for some forbs, perennial herbs, and native bunch grasses over perennial shrubs and non‐native grasses. This is the first study of PPM to pair plant phenology surveys with diet metabarcoding to estimate resource selection, and results suggest that managing habitat for diverse native forb communities and reducing non‐native grass cover may be beneficial for this critically endangered species. Fecal metabarcoding revealed high plant diversity, spatial and temporal differences in diet composition correlated with habitat availability, and a suggested preference for forbs and annual herbs in the Pacific pocket mouse in southern California. Results can help inform seed palettes for habitat restoration and reestablishment efforts for this critically endangered species. This work is highly relevant to resource managers and agencies tasked with protection and restoration of habitat for the Pacific pocket mouse. More broadly, it provides a methodological example applicable to herbivore conservation.

Current pollinator survey methods exhibit bias, require highly‐trained practitioners, and are difficult to scale to large sample sizes. High‐throughput sequencing of terrestrial eDNA could provide a complementary tool for studying pollinator communities, but eDNA methods have not been extensively evaluated. We conducted metagenetic analysis of whole arthropod community eDNA from 20 flower and seven honey bee‐collected pollen samples and compared eDNA‐derived data with traditional netting‐based surveys of the pollinator communities present during sampling. We focused our analysis on Anthophila (bees) and detected eight bee genera belonging to four families across COI, 16S, and 28S markers. Results varied considerably by marker and eDNA substrate. Detected bee genera were plausible for the study system and about 43 percent of total bee genera were detected with both eDNA and net‐based surveys, though netting resulted in more detections across a wider diversity of genera. Data from sequenced controls suggest that eDNA identifications were unlikely to have resulted from cross‐contamination. Our results demonstrate that bee communities can be documented with eDNA techniques and that the choice of marker and substrate substantially influences detection. Future improvements to our methods are required, but eDNA surveys appear well‐suited to characterize diverse pollinator communities and provide novel sampling perspectives within plant‐pollinator networks. Future efforts should focus on improving the selection of markers available for pollinator eDNA metagenetics, addressing taxonomic gaps within reference sequence databases and optimizing sampling and eDNA isolation protocols. We anticipate that such improvements are highly feasible and that eDNA will be a useful tool to those who study pollinators and plant‐pollinator interactions. Venn diagram showing bee genera detected by molecular eDNA analysis, netting surveys, and both methods. Genera are shaded according to their approximate abundance, as measured by total specimens netted across all surveys. Rank abundance of genera detected with both methods was significantly greater than rank abundance of those detected by netting alone.

Lake Sinai Viruses (LSV) are common RNA viruses of honey bees ( ) that frequently reach high abundance but are not linked to overt disease. LSVs are genetically heterogeneous and collectively widespread, but despite frequent detection in surveys, the ecological and geographic factors structuring their distribution in are not understood. Even less is known about their distribution in other species. Better understanding of LSV prevalence and ecology have been hampered by high sequence diversity within the LSV clade. Here we report a new polymerase chain reaction (PCR) assay that is compatible with currently known lineages with minimal primer degeneracy, producing an expected 365 bp amplicon suitable for end-point PCR and metagenetic sequencing. Using the Illumina MiSeq platform, we performed pilot metagenetic assessments of three sample sets, each representing a distinct variable that might structure LSV diversity (geography, tissue, and species). The first sample set in our pilot assessment compared cDNA pools from managed hives in California (  = 8) and Maryland (  = 6) that had previously been evaluated for LSV2, confirming that the primers co-amplify divergent lineages in real-world samples. The second sample set included cDNA pools derived from different tissues (thorax vs. abdomen,  = 24 paired samples), collected from managed hives in North Dakota. End-point detection of LSV frequently differed between the two tissue types; LSV metagenetic composition was similar in one pair of sequenced samples but divergent in a second pair. Overall, LSV1 and intermediate lineages were common in these samples whereas variants clustering with LSV2 were rare. The third sample set included cDNA from individual pollinator specimens collected from diverse landscapes in the vicinity of Lincoln, Nebraska. We detected LSV in the bee (four of 63 specimens tested, 6.3%) at a similar rate as (nine of 115 specimens, 7.8%), but only one sequencing library yielded sufficient data for compositional analysis. Sequenced samples often contained multiple divergent LSV lineages, including individual specimens. While these studies were exploratory rather than statistically powerful tests of hypotheses, they illustrate the utility of high-throughput sequencing for understanding LSV transmission within and among species.

We evaluated the prevalence of influenza A virus (IAV) in different species of bivalves inhabiting natural water bodies in waterfowl habitat along the Delmarva Peninsula and Chesapeake Bay in eastern Maryland. Bivalve tissue from clam and mussel specimens (Macoma balthica, Macoma phenax, Mulinia sp., Rangia cuneata, Mya arenaria, Guekensia demissa, and an undetermined mussel species) from five collection sites was analyzed for the presence of type A influenza virus by qPCR targeting the matrix gene. Of the 300 tissue samples analyzed, 13 samples (4.3%) tested positive for presence of influenza virus A matrix gene. To our knowledge, this is the first report of detection of IAV in the tissue of any bivalve mollusk from a natural water body.