Isolation, characterization and molecular identification of a novel aquareovirus that infects endangered fountain darter Etheostoma fonticola.

The fountain darter Etheostoma fonticola (FOD) is a federally endangered fish listed under the US Endangered Species Act. Here, we identified and characterized a novel aquareovirus isolated from wild fountain darters inhabiting the San Marcos River. This virus was propagated in Chinook salmon embryo (CHSE)-214, rainbow trout gonad-2 and fathead minnow cells at 15°C. The epithelioma papulosum cyprini cell line was refractory at all temperatures evaluated. High throughput sequencing technologies facilitated the complete genome sequencing of this virus utilizing ribosomal RNA-depleted RNA extracted from infected CHSE-214 cells. Conventional PCR primer sets were developed for the detection and confirmation of this virus to assist diagnostic screening methods. Phylogenetic analysis suggests this virus belongs to the Aquareovirus A genus. This research provides requisite initial data critical to support hatchery and refugia biosecurity measures for this endangered species.

Environmental signatures and effects of an oil and gas wastewater spill in the Williston Basin, North Dakota.

Wastewaters from oil and gas development pose largely unknown risks to environmental resources. In January 2015, 11.4ML (million liters) of wastewater (300g/L TDS) from oil production in the Williston Basin was reported to have leaked from a pipeline, spilling into Blacktail Creek, North Dakota. Geochemical and biological samples were collected in February and June 2015 to identify geochemical signatures of spilled wastewaters as well as biological responses along a 44-km river reach. February water samples had elevated chloride (1030mg/L) and bromide (7.8mg/L) downstream from the spill, compared to upstream levels (11mg/L and <0.4mg/L, respectively). Lithium (0.25mg/L), boron (1.75mg/L) and strontium (7.1mg/L) were present downstream at 5-10 times upstream concentrations. Light hydrocarbon measurements indicated a persistent thermogenic source of methane in the stream. Semi-volatile hydrocarbons indicative of oil were not detected in filtered samples but low levels, including tetramethylbenzenes and di-methylnaphthalenes, were detected in unfiltered water samples downstream from the spill. Labile sediment-bound barium and strontium concentrations (June 2015) were higher downstream from the Spill Site. Radium activities in sediment downstream from the Spill Site were up to 15 times the upstream activities and, combined with Sr isotope ratios, suggest contributions from the pipeline fluid and support the conclusion that elevated concentrations in Blacktail Creek water are from the leaking pipeline. Results from June 2015 demonstrate the persistence of wastewater effects in Blacktail Creek several months after remediation efforts started. Aquatic health effects were observed in June 2015; fish bioassays showed only 2.5% survival at 7.1km downstream from the spill compared to 89% at the upstream reference site. Additional potential biological impacts were indicated by estrogenic inhibition in downstream waters. Our findings demonstrate that environmental signatures from wastewater spills are persistent and create the potential for long-term environmental health effects.

Evidence of estrogenic endocrine disruption in smallmouth and largemouth bass inhabiting Northeast U.S. national wildlife refuge waters: A reconnaissance study.

Intersex as the manifestation of testicular oocytes (TO) in male gonochoristic fishes has been used as an indicator of estrogenic exposure. Here we evaluated largemouth bass (Micropterus salmoides) or smallmouth bass (Micropterus dolomieu) form 19 National Wildlife Refuges (NWRs) in the Northeast U.S. inhabiting waters on or near NWR lands for evidence of estrogenic endocrine disruption. Waterbodies sampled included rivers, lakes, impoundments, ponds, and reservoirs. Here we focus on evidence of endocrine disruption in male bass evidenced by gonad histopathology including intersex or abnormal plasma vitellogenin (Vtg) concentrations. During the fall seasons of 2008-2010, we collected male smallmouth bass (n=118) from 12 sites and largemouth bass (n=173) from 27 sites. Intersex in male smallmouth bass was observed at all sites and ranged from 60% to 100%; in male largemouth bass the range was 0-100%. Estrogenicity, as measured using a bioluminescent yeast reporter, was detected above the probable no effects concentration (0.73ng/L) in ambient water samples from 79% of the NWR sites. Additionally, the presence of androgen receptor and glucocorticoid receptor ligands were noted as measured via novel nuclear receptor translocation assays. Mean plasma Vtg was elevated (>0.2mg/ml) in male smallmouth bass at four sites and in male largemouth bass at one site. This is the first reconnaissance survey of this scope conducted on US National Wildlife Refuges. The baseline data collected here provide a necessary benchmark for future monitoring and justify more comprehensive NWR-specific studies.

Comparative responses to endocrine disrupting compounds in early life stages of Atlantic salmon, Salmo salar.

Atlantic salmon (Salmo salar) are endangered anadromous fish that may be exposed to feminizing endocrine disrupting compounds (EDCs) during early development, potentially altering physiological capacities, survival and fitness. To assess differential life stage sensitivity to common EDCs, we carried out short-term (4 day) exposures using three doses each of 17 α-ethinylestradiol (EE2), 17 ß-estradiol (E2), and nonylphenol (NP) on four early life stages; embryos, yolk-sac larvae, feeding fry and 1 year old smolts. Differential response was compared using vitellogenin (Vtg, a precursor egg protein) gene transcription. Smolts were also examined for impacts on plasma Vtg, cortisol, thyroid hormones (T4/T3) and hepatosomatic index (HSI). Compound-related mortality was not observed in any life stage, but Vtg mRNA was elevated in a dose-dependent manner in yolk-sac larvae, fry and smolts but not in embryos. The estrogens EE2 and E2 were consistently stronger inducers of Vtg than NP. Embryos responded significantly to the highest concentration of EE2 only, while older life stages responded to the highest doses of all three compounds, as well as intermediate doses of EE2 and E2. Maximal transcription was greater for fry among the three earliest life stages, suggesting fry may be the most responsive life stage in early development. Smolt plasma Vtg was also significantly increased, and this response was observed at lower doses of each compound than was detected by gene transcription suggesting plasma Vtg is a more sensitive indicator at this life stage. HSI was increased at the highest doses of EE2 and E2, and plasma T3 was decreased at the highest dose of EE2. Our results indicate that all life stages are potentially sensitive to endocrine disruption by estrogenic compounds and that physiological responses were altered over a short window of exposure, indicating the potential for these compounds to impact fish in the wild.

A new bacilliform fathead minnow rhabdovirus that produces syncytia in tissue culture.

A pathogenic bacilliform virus 130-180 nm in length and 31-47 nm in diameter was isolated from moribund fathead minnows (Pimephales promelas) exhibiting hemorrhages in their eyes and skin. A cytopathic effect of multifocal syncytia was observed in the epithelioma papulosum cyprini cell line after a 48 h incubation at 20 degrees C. A similar cytopathic effect was also observed in other cell lines tested, but not in bluegill fry, koi fin, or Chinook salmon embryo cells. The filterable agent was inactivated by exposure to 50 degrees C for 10 min, 20% ether, 2 and 50% chloroform, pH 3, and pH 10, was unaffected by 5'-iodo-2 deoxyuridine, and appeared bacilliform and occasionally bullet-shaped by electron microscopy. These results are consistent with those of rhabdoviruses. Immunodot blots performed with antisera against selected fish rhabdoviruses, an aquareovirus, and a birnavirus were all negative. River's postulates were fulfilled in fathead minnows, but the agent did not replicate or cause disease in other cyprinids or salmonids during challenge experiments. Hepatic, splenic, and renal lesions were observed during histological analysis of diseased fish from viral challenges and from the original case. Structural proteins resolved via SDS-PAGE had molecular weights similar to those reported in lyssaviruses of the family Rhabdoviridae; however, syncytia formation is not a typical cytopathic effect of rhabdoviruses. This virus, has tentatively been named the fathead minnow rhabdovirus (FHMRV) and is most similar to the members of the family Rhabdoviridae, but atypical properties like syncytia formation may justify the assignment to a novel taxon.