Flow cytometric assessments of metabolic activity in bacterial assemblages provide insight into ecosystem condition along the Buffalo National River, Arkansas.

The Buffalo National River (BNR), on karst terrain in Arkansas, is considered an extraordinary water resource. Water collected in Spring 2017 along BNR was metagenomically analyzed using 16S rDNA, and for 17 months (5/2017-11/2018), bacterial responses were measured in relation to nutrients sampled along a stretch of BNR near a concentrated animal feed operation (CAFO) on Big Creek. Because cell count and esterase activity can increase proportionally with organic enrichment, they were hypothesized to be elevated near the CAFO. Counts (colony forming units; CFUs) were different among sites for 73 % of the months; Big Creek generated highest CFUs 27 % of the time, with the closest downstream site at 13.3 %. Esterase activity was different among sites 94 % of the time, with Big Creek exhibiting lowest activity 71 % of the time. Over the months, activity was similar across sites at ~70 % active, except at Big Creek (56 %). The α-diversity of BNR microbial consortia near a wastewater treatment plant (WWTP) and the CAFO was related to distance from the WWTP and CAFO. The inverse relationship between high CFUs and low esterase activity at Big Creek (r = -0.71) actuated in vitro exposures of bacteria to organic wastewater contaminants (OWC) previously identified in the watershed. Exponential-phase Escherichia coli (stock strain), Streptococcus suis (avirulent, from swine), and S. dysgalactiae (virulent, from silver carp, Hypophthalmichthys molitrix) were incubated with atrazine, pharmaceuticals (17 α-ethynylestradiol and trenbolone), and antimicrobials (tylosin and butylparaben). Bacteria were differentially responsive. Activity varied with exposure time and OWC type, but not concentration; atrazine decreased it most. Taken together - the metagenomic taxonomic similarities along BNR, slightly higher bacterial growth and lower bacterial esterase at the CAFO, and the lab exposures of bacterial strains showing that OWC altered metabolism - the results indicated that bioactive OWC entering the watershed can strongly influence microbial processes in the aquatic ecosystem.

Juvenile African Clawed Frogs (Xenopus laevis) Express Growth, Metamorphosis, Mortality, Gene Expression, and Metabolic Changes When Exposed to Thiamethoxam and Clothianidin.

Neonicotinoids (NEO) represent the main class of insecticides currently in use, with thiamethoxam (THX) and clothianidin (CLO) primarily applied agriculturally. With few comprehensive studies having been performed with non-target amphibians, the aim was to investigate potential biomarker responses along an adverse outcome pathway of NEO exposure, whereby data were collected on multiple biological hierarchies. Juvenile African clawed frogs, Xenopus laevis, were exposed to commercial formulations of THX and CLO at high (100 ppm) and low (20 ppm) concentrations of the active ingredient. Mortality, growth, development, liver metabolic enzyme activity, and gene expression endpoints were quantified. Tadpoles (n > 1000) from NF 47 through tail resorption stage (NF 66) were exposed to NEO or to NEO-free media treatments. Liver cell reductase activity and cytotoxicity were quantified by flow cytometry. Compared to control reference gene expressions, levels of expression for NEO receptor subunits, cell structure, function, and decontamination processes were measured by RT-qPCR by using liver and brain. Mortality in THX high was 21.5% compared to the control (9.1%); the metabolic conversion of THX to CLO may explain these results. The NF 57 control tadpoles were heavier, longer, and more developed than the others. The progression of development from NF 57-66 was reduced by THX low, and weight gain was impaired. Liver reductases were highest in the control (84.1%), with low NEO exhibiting the greatest reductions; the greatest cytotoxicity was seen with THX high. More transcriptional activity was noted in brains than in livers. Results affirm the utility of a study approach that considers multiple complexities in ecotoxicological studies with non-target amphibians, underscoring the need for simultaneously considering NEO concentration-response relationships with both whole-organism and biomarker endpoints.

Sperm quality biomarkers complement reproductive and endocrine parameters in investigating environmental contaminants in common carp (Cyprinus carpio) from the Lake Mead National Recreation Area.

Lake Mead National Recreational Area (LMNRA) serves as critical habitat for several federally listed species and supplies water for municipal, domestic, and agricultural use in the Southwestern U.S. Contaminant sources and concentrations vary among the sub-basins within LMNRA. To investigate whether exposure to environmental contaminants is associated with alterations in male common carp (Cyprinus carpio) gamete quality and endocrine- and reproductive parameters, data were collected among sub-basins over 7 years (1999-2006). Endpoints included sperm quality parameters of motility, viability, mitochondrial membrane potential, count, morphology, and DNA fragmentation; plasma components were vitellogenin (VTG), 17ß-estradiol, 11-keto-testosterone, triiodothyronine, and thyroxine. Fish condition factor, gonadosomatic index, and gonadal histology parameters were also measured. Diminished biomarker effects were noted in 2006, and sub-basin differences were indicated by the irregular occurrences of contaminants and by several associations between chemicals (e.g., polychlorinated biphenyls, hexachlorobenzene, galaxolide, and methyl triclosan) and biomarkers (e.g., plasma thyroxine, sperm motility and DNA fragmentation). By 2006, sex steroid hormone and VTG levels decreased with subsequent reduced endocrine disrupting effects. The sperm quality bioassays developed and applied with carp complemented endocrine and reproductive data, and can be adapted for use with other species.

Reproductive health of yellow perch Perca flavescens in selected tributaries of the Chesapeake Bay.

Reduced recruitment of yellow perch has been noted for a number of years in certain urbanized watersheds (South and Severn Rivers) of the Chesapeake Bay. Other rapidly developing watersheds such as Mattawoman Creek are more recently showing evidence of reduced recruitment of anadromous fishes. In this study, we used a battery of biomarkers to better document the reproductive health of adult yellow perch collected during spring spawning in 2007-2009. Perch were collected in the South and Severn Rivers, Mattawoman Creek and the less developed Choptank and Allen's Fresh watersheds for comparison. Gonadosomatic indices, plasma reproductive hormone concentrations, plasma vitellogenin concentrations and gonad histology were evaluated in mature perch of both sexes. In addition, sperm quantity (cell counts) and quality (total and progressive motility, spermatogenic stage and DNA integrity), were measured in male perch. Many of these biomarkers varied annually and spatially, with some interesting statistical results and trends. Male perch from the Choptank and Allen's Fresh had generally higher sperm counts. In 2008 counts were significantly lower in the perch from the Severn when compared to other sites. The major microscopic gonadal abnormality in males was the proliferation of putative Leydig cells, observed in testes from Severn and less commonly, Mattawoman Creek perch. Observations that could significantly impact egg viability were an apparent lack of final maturation, abnormal yolk and thin, irregular zona pellucida. These were observed primarily in ovaries from Severn, South and less commonly Mattawoman Creek perch. The potential association of these observations with urbanization, impervious surface and chemical contaminants is discussed.

Sperm quality assessments for endangered razorback suckers Xyrauchen texanus.

Flow cytometry (FCM) and computer-assisted sperm motion analysis (CASA) methods were developed and validated for use with endangered razorback suckers Xyrauchen texanus collected (n=64) during the 2006 spawning season. Sperm motility could be activated within osmolality ranges noted during milt collections (here 167-343 mOsm/kg). We hypothesized that sperm quality of milt collected into isoosmotic (302 mOsm/kg) or hyperosmotic (500 mOsm/kg) Hanks' balanced salt solution would not differ. Pre-freeze viabilities were similar between osmolalities (79%±6 (s.e.m.) and 76%±7); however, post-thaw values were greater in hyperosmotic buffer (27%±3 and 12%±2; P=0.0065), as was mitochondrial membrane potential (33%±4 and 13%±2; P=0.0048). Visual estimates of pre-freeze motility correlated with total (r=0.7589; range 23-82%) and progressive motility (r=0.7449) by CASA and were associated with greater viability (r=0.5985; P<0.0001). Count (FCM) was negatively correlated with post-thaw viability (r=-0.83; P=0.0116) and mitochondrial function (r=-0.91; P=0.0016). By FCM-based assessments of DNA integrity, whereby increased fluorochrome binding indicated more fragmentation, higher levels were negatively correlated with count (r=-0.77; P<0.0001) and pre-freeze viabilities (r=-0.66; P=0.0004). Fragmentation was higher in isotonic buffer (P=0.0234). To increase reproductive capacity of natural populations, the strategy and protocols developed can serve as a template for use with other imperiled fish species, biomonitoring, and genome banking.