Naegleria fowleri Detected in Grand Teton National Park Hot Springs.

The free-living thermophilic amoeba Naegleria fowleri (N. fowleri) causes the highly fatal disease primary amoebic meningoencephalitis. The environmental conditions that are favorable to the growth and proliferation of N. fowleri are not well-defined, especially in northern regions of the United States. In this study, we used culture-based methods and multiple molecular approaches to detect and analyzeN. fowleri and other Naegleria spp. in water, sediment, and biofilm samples from five hot spring sites in Grand Teton National Park, Wyoming, U.S.A. These results provide the first detections of N. fowleri in Grand Teton National Park and provide new insights into the distribution of pathogenic N. fowleri and other nonpathogenic Naegleria spp. in natural thermal water systems in northern latitudes.

Fecal metabarcoding of the endangered Pacific pocket mouse (Perognathus longimembris pacificus) reveals a diverse and forb rich diet that reflects local habitat availability.

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.

Freshwater unionid mussels threatened by predation of Round Goby (Neogobius melanostomus).

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.

Novel microbiome dominated by Arcobacter during anoxic excurrent flow from an ocean blue hole in Andros Island, The Bahamas.

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.

An updated genetic marker for detection of Lake Sinai Virus and metagenetic applications.

BACKGROUND: Lake Sinai Viruses (LSV) are common RNA viruses of honey bees (Apis mellifera) 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 A. mellifera 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. METHODS: 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). RESULTS: The first sample set in our pilot assessment compared cDNA pools from managed A. mellifera hives in California (n = 8) and Maryland (n = 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, n = 24 paired samples), collected from managed A. mellifera 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 Halictus ligatus (four of 63 specimens tested, 6.3%) at a similar rate as A. mellifera (nine of 115 specimens, 7.8%), but only one H. ligatus 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.

Influenza A Virus Detected in Native Bivalves in Waterfowl Habitat of the Delmarva Peninsula, USA.

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.

Exposure to synthetic hydraulic fracturing waste influences the mucosal bacterial community structure of the brook trout (Salvelinus fontinalis) epidermis.

Production of natural gas using unconventional technologies has risen as demand for alternative fuels has increased. Impacts on the environment from waste generated from these processes are largely unexplored. In particular, the outcomes of organismal exposure to hydraulic fracturing waste have not been rigorously evaluated. We evaluated the effects of exposure to surrogate hydraulic fracturing waste (HF waste) on mucosal bacterial community structure of the brook trout (Salvelinus fontinalis) epidermis. Brook trout are fish native to streams at risk to HF waste exposure. Here, fish were exposed to four treatments (control, 0.00%; low, 0.01%; medium, 0.10%; and high, 1.0% concentrations) of surrogate HF waste synthesized to mimic concentrations documented in the field. Epidermal mucus samples were collected and assessed 15 days post-exposure to determine if the associated bacterial community varied among treatments. We observed differences in epidermal mucosal bacterial community composition at multiple taxonomic scales among treatments. These community changes reflected compositional differences in taxa dominance and community similarity rather than losses or gains in taxonomic richness. The dominant bacterial genus that explained the greatest variation in community structure between exposed and unexposed fish was Flavobacterium. Two genera associated with salmonid diseases, Flavobacterium and Pseudomonas, were statistically more abundant in high treatments than controls. These results suggest that exposure to low levels of HF waste influences bacterial colonization and may lead to a disruption that favors bacterial populations associated with fish disease.

Draft Genome Sequence of a Picorna-Like Virus Associated with Gill Tissue in Clinically Normal Brook Trout, Salvelinus fontinalis.

Here, we report a draft genome sequence of a picorna-like virus associated with brook trout, Salvelinus fontinalis, gill tissue. The draft genome comprises 8,681 nucleotides, excluding the poly(A) tract, and contains two open reading frames. It is most similar to picorna-like viruses that infect invertebrates.

Low Pathogenic Avian Influenza Viruses in Wild Migratory Waterfowl in a Region of High Poultry Production, Delmarva, Maryland.

Migratory waterfowl are natural reservoirs for low pathogenic avian influenza viruses (AIVs) and may contribute to the long-distance dispersal of these pathogens as well as spillover into domestic bird populations. Surveillance for AIVs is critical to assessing risks for potential spread of these viruses among wild and domestic bird populations. The Delmarva Peninsula on the east coast of the United States is both a key convergence point for migratory Atlantic waterfowl populations and a region with high poultry production (>4,700 poultry meat facilities). Sampling of key migratory waterfowl species occurred at 20 locations throughout the Delmarva Peninsula in fall and winter of 2013-14. Samples were collected from 400 hunter-harvested or live-caught birds via cloacal and oropharyngeal swabs. Fourteen of the 400 (3.5%) birds sampled tested positive for the AIV matrix gene using real-time reverse transcriptase PCR, all from five dabbling duck species. Further characterization of the 14 viral isolates identified two hemagglutinin (H3 and H4) and four neuraminidase (N2, N6, N8, and N9) subtypes, which were consistent with isolates reported in the Influenza Research Database for this region. Three of 14 isolates contained multiple HA or NA subtypes. This study adds to the limited baseline information available for AIVs in migratory waterfowl populations on the Delmarva Peninsula, particularly prior to the highly pathogenic AIV A(H5N8) and A(H5N2) introductions to the United States in late 2014.

RNA sequencing analysis of transcriptional change in the freshwater mussel Elliptio complanata after environmentally relevant sodium chloride exposure.

To identify potential biomarkers of salt stress in a freshwater sentinel species, we examined transcriptional responses of the common mussel Elliptio complanata to controlled sodium chloride (NaCl) exposures. Ribonucleic acid sequencing (RNA-Seq) of mantle tissue identified 481 transcripts differentially expressed in adult mussels exposed to 2 ppt NaCl (1.2 ppt chloride) for 7 d, of which 290 had nonoverlapping intervals. Differentially expressed gene categories included ion and transmembrane transport, oxidoreductase activity, maintenance of protein folding, and amino acid metabolism. The rate-limiting enzyme for synthesis of taurine, an amino acid frequently linked to osmotic stress in aquatic species, was upregulated, as was the transmembrane ion pump sodium/potassium adenosine 5'-triphosphatase. These patterns confirm a primary transcriptional response to the experimental dose, albeit likely overlapping with nonspecific secondary stress responses. Substantial involvement of the heat shock protein 70 chaperone family and the water-transporting aquaporin family was not detected, however, in contrast to some studies in other bivalves. A subset of the most significantly regulated genes was confirmed by quantitative polymerase chain reaction in an independent sample. Cluster analysis showed separation of mussels exposed to 2 ppt NaCl from control mussels in multivariate space, but mussels exposed to 1 ppt NaCl were largely indistinguishable from controls. Transcriptome-scale analysis of salt exposure under laboratory conditions efficiently identified candidate biomarkers for further functional analysis and field validation. Environ Toxicol Chem 2017;36:2352-2366. © Published 2017 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Complete Genome Sequence of a Novel Aquareovirus That Infects the Endangered Fountain Darter, Etheostoma fonticola.

Here, we report the complete genome of a novel aquareovirus isolated from clinically normal fountain darters, Etheostoma fonticola, inhabiting the San Marcos River, Texas, USA. The complete genome consists of 23,958 bp consisting of 11 segments that range from 783 bp (S11) to 3,866 bp (S1).

Metabarcoding of Fecal Samples to Determine Herbivore Diets: A Case Study of the Endangered Pacific Pocket Mouse.

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.

Transforming growth factor-ß1 expression in endangered age-0 shortnose suckers (Chasmistes brevirostris) from Upper Klamath Lake, OR relative to histopathology, meristic, spatial, and temporal data.

During July-September of 2008, 2009, and 2010 endangered age-0 juvenile shortnose suckers were sampled from Upper Klamath Lake, OR in a health evaluation that included the measurement of transforming growth factor - beta (TGF-ß) expression in spleen in combination with a histopathology assessment. This analysis was performed to determine if the expression of this immuno-regulator could be used as a component of a larger health evaluation intended to identify potential risk-factors that may help to explain why very few of these fish survive to age-1. Potential associations between TGF-ß1 expression, histopathological findings, meristic data as well as temporal and spatial data were evaluated using analysis-of-variance. In this analysis, the absence or presence of opercula deformity and hepatic cell necrosis were identified as significant factors in accounting for the variance in TGF-ß1 expression observed in age-0 shortnose suckers (n = 122, squared multiple R = 0.989). Location of sample collection and the absence or presence of anchor worms (Lernaea spp.) were identified as significant cofactors. The actual mechanisms involved with these relationships have yet to be determined. The strength, however, of our findings support the concept of using TGF-ß1 expression as part of a broader fish health assessment and suggests the potential for using additional immunologic measures in future studies. Specifically, our results indicate that the measure of TGF-ß1 expression in age-0 shortnose sucker health assessments can facilitate the process of identifying disease risks that are associated with the documented lack of recruitment into the adult population.

Taxonomic Characterization of Honey Bee (Apis mellifera) Pollen Foraging Based on Non-Overlapping Paired-End Sequencing of Nuclear Ribosomal Loci.

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.

Efficacy of pH elevation as a bactericidal strategy for treating ballast water of freight carriers.

Treatment of ship ballast water with sodium hydroxide (NaOH) is one method currently being developed to minimize the risk to introduce aquatic invasive species. The bactericidal capability of sodium hydroxide was determined for 148 bacterial strains from ballast water collected in 2009 and 2010 from the M/V Indiana Harbor, a bulk-freight carrier plying the Laurentian Great Lakes, USA. Primary culture of bacteria was done using brain heart infusion agar and a developmental medium. Strains were characterized based on PCR amplification and sequencing of a portion of the 16S rRNA gene. Sequence similarities (99+ %) were determined by comparison with the National Center for Biotechnology Information (NCBI) GenBank catalog. Flavobacterium spp. were the most prevalent bacteria characterized in 2009, comprising 51.1% (24/47) of the total, and Pseudomonas spp. (62/101; 61.4%) and Brevundimonas spp. (22/101; 21.8%) were the predominate bacteria recovered in 2010; together, comprising 83.2% (84/101) of the total. Testing was done in tryptic soy broth (TSB) medium adjusted with 5 N NaOH. Growth of each strain was evaluated at pH 10.0, pH 11.0 and pH 12.0, and 4 h up to 72 h. The median cell count at 0 h for 148 cultures was 5.20 × 10(6) cfu/mL with a range 1.02 × 10(5)-1.60 × 10(8) cfu/mL. The TSB adjusted to pH 10.0 and incubation for less than 24 h was bactericidal to 52 (35.1%) strains. Growth in pH 11.0 TSB for less than 4 h was bactericidal to 131 (88.5%) strains and pH 11.0 within 12 h was bactericidal to 141 (95.3%). One strain, Bacillus horikoshii, survived the harshest treatment, pH 12.0 for 72 h.

Spread of the Rat Lungworm (Angiostrongylus cantonensis) in Giant African Land Snails (Lissachatina fulica) in Florida, USA.

The rat lungworm (Angiostrongylus cantonensis) is a parasitic nematode that causes rat lungworm disease. It is the leading cause of eosinophilic meningitis and is a zoonotic health risk. We confirmed the presence of A. cantonensis using species-specific, quantitative PCR in 18 of 50 (36%) giant African land snails (Lissachatina fulica) collected from Miami, Florida, US in May 2013. These snails were collected from seven of 21 core areas that the Florida Department of Agriculture and Consumer Services monitor weekly. Rat lungworms have not previously been identified in these areas. Duplicate DNA extractions of foot muscle tissue from each snail were tested. Of the seven core areas we examined, six were positive for A. cantonensis and prevalence of infection ranged from 27% to 100%. Of the 18 positive snails, only five were positive in both extractions. Our results confirm an increase in the range and prevalence of rat lungworm infection in Miami. We also emphasize the importance of extracting sufficient host tissue to minimize false negatives.

Description of two new gill myxozoans from smallmouth (Micropterus dolomieu) and largemouth (Micropterus salmoides) bass.

Two previously undescribed species of myxozoan parasites were observed in the gills of bass inhabiting the Potomac and James River basins. They are described using morphological characteristics and small-subunit (SSU) rDNA gene sequences. Both were taxonomically identified as new species of Myxobolus; Myxobolus branchiarum n. sp. was found exclusively in smallmouth bass, and Myxobolus micropterii n. sp. was found in largemouth and smallmouth bass. Small, spherical, white plasmodia of M. branchiarum from smallmouth bass were observed grossly in the gills; these plasmodia had an average length of 320.3 µm and width of 246.1 µm. The development of the plasmodia is intralamellar in the secondary lamellae of the gills. Mature spores were pyriform in shape with a length of 12.8 ± 1.4 (8.1-15.1) µm and width of 6.9 ± 1.1 (4.0-9.0) µm. Analysis of SSU rDNA identified M. branchiarum in a sister-group to 3 species of Henneguya , although morphologically caudal appendages were absent. Myxobolus micropterii observed in the gills of largemouth and smallmouth bass had larger, ovoid, cream-colored plasmodia with an average length of 568.1 µm and width of 148.1 µm. The cysts developed at the distal end of the gill filament within the primary lamellae. The mature spores were ovoid in shape with a length of 10.8 ± 0.7 (9.2-12.2) µm and width of 10.6 ± 0.6 (9.0-11.8) µm. SSU rDNA analysis placed M. micropterii in a sister group with Henneguya lobosa and Myxobolus oliveirai . The highest prevalence of M. branchiarum was observed in the gills of bass collected from the Cowpasture River (50.9%). Prevalence was 44.6% in bass from the Potomac River and only 4.3% in bass collected from the Shenandoah River. A seasonal study of M. branchiarum , which included both infected and uninfected smallmouth bass, determined that a significantly higher intensity was observed in the spring than in the summer (P < 0.001) or fall (P  =  0.004). In an analysis excluding uninfected bass, a higher intensity was observed in the spring than in the summer (P  =  0.001) or fall (P  =  0.008). Prevalence and seasonal differences were not determined for M. micropterii .

Morphology and 18S rDNA of Henneguya gurlei (Myxosporea) from Ameiurus nebulosus (Siluriformes) in North Carolina.

Henneguya gurlei was isolated from Ameiurus nebulosus captured in North Carolina and redescribed using critical morphological features and 18S small-subunit ribosomal RNA (SSU rDNA) gene sequence. Plasmodia are white, spherical, or subspherical, occur in clusters, measure up to 1.8 mm in length, and are located on the dorsal, pectoral, and anal fins. Histologically, plasmodia are located in the dermis and subdermally, and the larger cysts disrupt the melanocyte pigment layer. The spore body is lanceolate, 18.2 +/- 0.3 microm (range 15.7-20.3) in length, and 5.4 +/- 0.1 microm (range 3.8-6.1) in width in valvular view. The caudal appendages are 41.1 +/- 1.1 microm (range 34.0-49.7) in length. Polar capsules are pyriform and of unequal size. The longer polar capsule measures 6.2 +/- 0.1 microm (range 5.48-7.06), while the shorter is 5.7 +/- 0.1 microm (range 4.8-6.4) in length. Polar capsule width is 1.2 +/- 0.03 microm (range 1.0-1.54). The total length of the spore is 60.9 +/- 1.2 microm (range 48.7-68.5). Morphologically, this species is similar to other species of Henneguya that are known to infect ictalurids. Based on SSU rDNA sequences, this species is most closely related to H. exilis and H. ictaluri, which infect Ictalurus punctatus.