Effects of in situ experimental selenium exposure on finescale dace (Phoxinus neogaeus) gut microbiome.

Selenium (Se) is an environmental contaminant of global concern that can cause adverse effects in fish at elevated levels. Fish gut microbiome play essential roles in gastrointestinal function and host health and can be perturbed by environmental contaminants, including metals and metalloids. Here, an in-situ Se exposure of female finescale dace (Phoxinus neogaeus) using mesocosms was conducted to determine the impacts of Se accumulation on the gut microbiome and morphometric endpoints. Prior to this study, the gut microbiome of finescale dace, a widespread Cyprinid throughout North America, had not been characterized. Exposure to Se caused a hormetic response of alpha diversity of the gut microbiome, with greater diversity at the lesser concentration of 1.6 µg Se/L, relative to that of fish exposed to the greater concentration of 5.6 µg Se/L. Select gut microbiome taxa of fish were differentially abundant between aqueous exposure concentrations and significantly correlated with liver-somatic index (LSI). The potential effects of gut microbiome dysbiosis on condition of wild fish might be a consideration when assessing adverse effects of Se in aquatic environments. More research regarding effects of Se on field-collected fish gut microbiome and the potential adverse effects or benefits on the host is warranted.

16S rRNA metabarcoding unearths responses of rare gut microbiome of fathead minnows exposed to benzo[a]pyrene.

Activities of gut microbiomes are often overlooked in assessments of ecotoxicological effects of environmental contaminants. Effects of the polycyclic aromatic hydrocarbon, benzo[a]pyrene (BaP) on active gut microbiomes of juvenile fathead minnows (Pimephales promelas) were investigated. Fish were exposed for two weeks, to concentrations of 0, 1, 10, 100, or 1000 µg BaP g-1 in the diet. The active gut microbiome was characterized using 16S rRNA metabarcoding to determine its response to dietary exposure of BaP. BaP reduced alpha-diversity at the greatest exposure concentrations. Additionally, exposure to BaP altered community composition of active microbiome and resulted in differential proportion of taxa associated with hydrocarbon degradation and fish health. Neighborhood selection networks of active microbiomes were not reduced with greater concentrations of BaP, which suggests ecological resistance and/or resilience of gut microbiota. The active gut microbiome had a similar overall biodiversity as that of the genomic gut microbiota, but had a distinct composition from that of the 16S rDNA profile. Responses of alpha- and beta-diversities of the active microbiome to BaP exposure were consistent with that of genomic microbiomes. Normalized activity of microbiome via the ratio of rRNA to rDNA abundance revealed rare taxa that became active or dormant due to exposure to BaP. These differences highlight the need to assess both 16S rDNA and rRNA metabarcoding to fully derive bacterial compositional changes resulting from exposure to contaminants.

Effects of aqueous fluoxetine exposure on gut microbiome of adult Pimephales promelas.

The microbiome of the gut is vital for homeostasis of hosts with its ability to detoxify and activate toxicants, as well as signal to the immune and nervous systems. However, in the field of environmental toxicology, the gut microbiome has only recently been identified as a measurable indicator for exposure to environmental pollutants. Antidepressants found in effluents of wastewater treatment plants and surface waters have been shown to exhibit antibacterial-like properties in vitro, where some bacteria are known to express homologous proteins that bind antidepressants in vertebrates. Therefore, it has been hypothesized that exposure to antidepressant drugs might affect gut microbiota of aquatic organisms. In this study, the common antidepressant, fluoxetine, was investigated to determine whether it can modulate the gut microbiome of adult fathead minnows. A 28-day, sub-chronic, static renewal exposure was performed with nominal fluoxetine concentrations of 0.01, 10 or 100 µg/L. Using 16S rRNA amplicon sequencing, shifts among the gut-associated microbiota were observed in individuals exposed to the greatest concentration, with greater effects observed in females. These changes were associated with a decrease in relative proportions of commensal bacteria, which can be important for health of fish including bacteria essential for fatty acid oxidation, and an increase in relative proportions of pathogenic bacteria associated with inflammation. Results demonstrate, for the first time, how antidepressants found in some aquatic environments can influence gut microbiota of fishes.

Using zooplankton metabarcoding to assess the efficacy of different techniques to clean-up an oil-spill in a boreal lake.

Regulators require adequate information to select best practices with less ecosystem impacts for remediation of freshwater ecosystems after oil spills. Zooplankton are valuable indicators of aquatic ecosystem health as they play pivotal roles in biochemical cycles while stabilizing food webs. Compared with morphological identification, metabarcoding holds promise for cost-effective, high-throughput, and benchmarkable biomonitoring of zooplankton communities. The objective of this study was to apply DNA and RNA metabarcoding of zooplankton for ecotoxicological assessment and compare it with traditional morphological identification in experimental shoreline enclosures in a boreal lake. These identification methods were also applied in context of assessing response of the zooplankton community exposed to simulated spills of diluted bitumen (dilbit), with experimental remediation practices (enhanced monitored natural recovery and shoreline cleaner application). Metabarcoding detected boreal zooplankton taxa up to the genus level, with a total of 24 shared genera, and while metabarcoding-based relative abundance served as an acceptable proxy for biomass inferred by morphological identification (ρ ≥ 0.52). Morphological identification determined zooplankton community composition changes due to treatments at 11 days post-spill (PERMANOVA, p = 0.0143) while metabarcoding methods indicated changes in zooplankton richness and communities at 38 days post-spill (T-test, p < 0.05; PERMANOVA, p ≤ 0.0429). Shoreline cleaner application overall seemed to have the largest impact on zooplankton communities relative to enhanced monitored natural recovery, regardless of zooplankton identification method. Both metabarcoding and morphological identification were able to discern the differences between the two experimental remediation practices. Metabarcoding of zooplankton could provide informative results for ecotoxicological assessment of the remediation practices of dilbit, advancing our knowledge of best practices for remediating oil-impacted aquatic ecosystems while serving to accelerate the assessment of at-risk freshwater ecosystems.

Responses of juvenile fathead minnow (Pimephales promelas) gut microbiome to a chronic dietary exposure of benzo[a]pyrene.

The microbiome has been described as an additional host "organ" with well-established beneficial roles. However, the effects of exposures to chemicals on both structure and function of the gut microbiome of fishes are understudied. To determine effects of benzo[a]pyrene (BaP), a model persistent organic pollutant, on structural shifts of gut microbiome in juvenile fathead minnows (Pimephales promelas), fish were exposed ad libitum in the diet to concentrations of 1, 10, 100, or 1000 µg BaP g-1 food, in addition to a vehicle control, for two weeks. To determine the link between exposure to BaP and changes in the microbial community, concentrations of metabolites of BaP were measured in fish bile and 16S rRNA amplicon sequencing was used to evaluate the microbiome. Exposure to BaP only reduced alpha-diversity at the greatest exposure concentrations. However, it did alter community composition assessed as differential abundance of taxa and reduced network complexity of the microbial community in all exposure groups. Results presented here illustrate that environmentally-relevant concentrations of BaP can alter the diversity of the gut microbiome and community network connectivity.

Effects of the husky oil spill on gut microbiota of native fishes in the North Saskatchewan River, Canada.

In July 2016, a Husky Energy pipeline spilled 225,000 L of diluted heavy crude oil, with a portion of the oil entering the North Saskatchewan River near Maidstone, SK, Canada. This event provided a unique opportunity to assess potential effects of a crude oil constituent (namely polycyclic aromatic hydrocarbons, PAHs) on a possible sensitive indicator of freshwater ecosystem health, the gut microbiota of native fishes. In summer 2017, goldeye (Hiodon alosoides), walleye (Sander vitreus), northern pike (Esox lucius), and shorthead redhorse (Moxostoma macrolepidotum) were collected at six locations upstream and downstream of the spill. Muscle and bile were collected from individual fish for quantification of PAHs and intestinal contents were collected for characterization of the microbial community of the gut. Results suggested that host species is a significant determinant of gut microbiota, with significant differences among the species across sites. Concentrations of PAHs in dorsal muscle were significantly correlated with gut community compositions of walleye, but not of the other fishes. Concentrations of PAHs in muscle were also correlated with abundances of several families of bacteria among fishes. This study represents one of the first to investigate the response of the gut microbiome of wild fishes to chemical stressors.

Differential responses of gut microbiota of male and female fathead minnow (Pimephales promelas) to a short-term environmentally-relevant, aqueous exposure to benzo[a]pyrene.

In addition to aiding in digestion of food and uptake of nutrients, microbiota in guts of vertebrates are responsible for regulating several beneficial functions, including development of an organism and maintaining homeostasis. However, little is known about effects of exposures to chemicals on structure and function of gut microbiota of fishes. To assess effects of exposure to polycyclic aromatic hydrocarbons (PAHs) on gut microbiota, male and female fathead minnows (Pimephales promelas) were exposed to environmentally-relevant concentrations of the legacy PAH benzo[a]pyrene (BaP) in water. Measured concentrations of BaP ranged from 2.3 × 10-3 to 1.3 µg L-1. The community of microbiota in the gut were assessed by use of 16S rRNA metagenetics. Exposure to environmentally-relevant aqueous concentrations of BaP did not alter expression levels of mRNA for cyp1a1, a "classic" biomarker of exposure to BaP, but resulted in shifts in relative compositions of gut microbiota in females rather than males. Results presented here illustrate that in addition to effects on more well-studied molecular endpoints, relative compositions of the microbiota in guts of fish can also quickly respond to exposure to chemicals, which can provide additional mechanisms for adverse effects on individuals.

Female reproductive impacts of dietary methylmercury in yellow perch (Perca flavescens) and zebrafish (Danio rerio).

The purpose of this study was to evaluate the effects of environmentally relevant dietary MeHg exposures on adult female yellow perch (Perca flavescens) and female zebrafish (Danio rerio) ovarian development and reproduction. Yellow perch were used in the study for their socioeconomic and ecological importance within the Great Lakes basin, and the use of zebrafish allowed for a detailed analysis of the molecular effects of MeHg following a whole life-cycle exposure. Chronic whole life dietary exposure of F1 zebrafish to MeHg mimics realistic wildlife exposure scenarios, and the twenty-week adult yellow perch exposure (where whole life-cycle exposures are difficult) captures early seasonal ovarian development. For both species, target dietary accumulation values were achieved prior to analyses. In zebrafish, several genes involved in reproductive processes were shown to be dysregulated by RNA-sequencing and quantitative real-time polymerase chain reaction (QPCR), but no significant phenotypic changes were observed regarding ovarian staging, fecundity, or embryo mortality. Yellow perch were exposed to dietary MeHg for 12, 16, or 20 weeks. In this species, a set of eight genes were assessed by QPCR in the pituitary, liver, and ovary, and no exposure-related changes were observed. The lack of genomic resources in yellow perch hinders the characterization of subtle molecular impacts. The ovarian somatic index, circulating estradiol and testosterone, and ovarian staging were not significantly altered by MeHg exposure in yellow perch. These results suggest that environmentally relevant MeHg exposures do not drastically reduce the reproductively important endpoints in these fish, but to capture realistic exposure scenarios, whole life-cycle yellow perch exposures are needed.

Retrospective analysis of mercury content in feathers of birds collected from the state of Michigan (1895-2007).

Museum specimens were used to analyze temporal trends in feather mercury (Hg) concentrations in birds collected from the state of Michigan between the years 1895 and 2007. Hg was measured in flank and secondary feathers from three species of birds that breed in the Great Lakes region; common terns (n = 32), great blue herons (n = 35), and herring gulls (n = 35). More than 90% of the Hg in feathers should be organic, but some of the heron and gull feathers collected prior to 1936 showed evidence of contamination with inorganic Hg, likely from museum preservatives. The data presented here therefore consist of organic Hg in pre-1936 samples and total Hg in post-1936 samples. Insufficient tissue was available from terns to assess organic Hg content. Mean Hg concentrations ranged from 2.9 ± 2.5 µg/g Hg in tern flank feathers to 12.4 ± 10.6 µg/g Hg in gull flank feathers. No linear trend of Hg contamination over time was detected in herons and gulls. Though a significant decrease was noted for terns, these data are presented with caution given the strong likelihood that earlier samples were preserved with inorganic mercury. When data were separated into 30-year intervals, Hg content in heron and gull feathers collected from birds sampled between 1920 and 1949 were consistently highest but not to a level of statistical significance. For example, Hg concentrations in gull secondary feathers collected in the second time interval (1920-1949) were 11.5 ± 7.8. This value was 67% higher than the first time interval (1890-1919), 44% higher than the third interval (1950-1979), and 187% higher than the fourth interval (1980-2009). Studies on Great Lakes sediments also showed greatest Hg accumulations in the mid-twentieth century. Through the use of museum specimens, these results present a unique snapshot of Hg concentrations in Great Lakes biota in the early part of the twentieth century.