An evaluation of germline mutations and reproductive impacts in fathead minnow (Pimephales promelas) exposed to contaminated sediment.

Polycyclic aromatic hydrocarbons (PAHs) have become ubiquitous in the aquatic environment. Some PAHs are mutagenic, potentially causing germline mutations in fish that inhabit PAH contaminated waters. We evaluated the effect of exposure to sediment-borne PAHs on reproduction and germline mutation rates in fathead minnows (Pimephales promelas). Exposure to the contaminated sediment had no significant impact on the reproductive endpoints measured in this study. Germline mutations rates at three microsatellite DNA loci were 1.69 × 10-3 in fish exposed to PAH-contaminated sediment and 0.55 × 10-3 in control fish, with zero mutations being observed in fish exposed to sediment from a reference site. While the difference in mutation rates between treatments was not statistically significant for the sample size used (15-19 families per treatment), the observed mutations rates enabled us to estimate the sample size required to detect a significant effect. To our knowledge, this is the first report of germline mutation rates in fathead minnow exposed to an environmental contaminant, providing baseline data for use in the design of future experiments.

A subchronic in situ exposure method for evaluating effects in small-bodied fish at contaminated sites.

In situ fish-caging studies at contaminated sites can provide information that is more realistic compared to traditional laboratory-based studies. However, few methods have been developed for exposing sentinel fish species for subchronic durations, and fewer still are optimized for exposing small-bodied fish while maintaining fish health and growth throughout the caging trial. Those methods typically lack a feeding regimen during the fish caging trial. While that may be acceptable or even appropriate for typical short-term toxicity testing, it does limit the duration of the exposure, and may not be suitable when post-caging trials or observations are necessary. Returning healthy fish to the lab following the in situ exposure would be important, for example, in studies designed to examine long-term or multigenerational effects following an in situ exposure. In this article we describe a subchronic method for caging small fish at contaminated sites while maintaining growth and reproductive development. Fathead minnow (Pimephales promelas) were caged in situ for 6 weeks, after which time they were returned to the lab where they were evaluated for health and reproductive performance. Growth and reproductive endpoints revealed no adverse effect on fish due to fish caging and related handling, demonstrating the suitability of our caging and feeding method for long-term caging studies.

Transcriptome analyses in juvenile yellow perch (Perca flavescens) exposed in vivo to clothianidin and chlorantraniliprole: Possible sampling bias.

The St. Lawrence River is an important North American waterway that is subject to anthropogenic pressures including intensive urbanization, and agricultural development. Pesticides are widely used for agricultural activities in fields surrounding the yellow perch (Perca flavescens) habitat in Lake St. Pierre (Quebec, Canada), a fluvial lake of the river where the perch population has collapsed. Clothianidin and chlorantraniliprole were two of the most detected insecticides in surface waters near perch spawning areas. The objectives of the present study were to evaluate the transcriptional and biochemical effects of these two pesticides on juvenile yellow perch exposed for 28d to environmental doses of each compound alone and in a mixture under laboratory/aquaria conditions. Hepatic mRNA-sequencing revealed an effect of chlorantraniliprole alone (37 genes) and combined with clothianidin (251 genes), but no effects of clothianidin alone were observed in perch. Dysregulated genes were mostly related to circadian rhythms and to Ca2+ signaling, the latter effect has been previously associated with chlorantraniliprole mode of action in insects. Moreover, chronic exposure to clothianidin increased the activity of acetylcholinesterase in the brain of exposed fish, suggesting a potential non-target effect of this insecticide. Further analyses of three clock genes by qRT-PCR suggested that part of the observed effects of chlorantraniliprole on the circadian gene regulation of juvenile perch could be the result of time-of-day of sacrifice. These results provide insight into biological effects of insecticides in juvenile perch and highlight the importance of considering the circadian rhythm in experimental design and results analyses.

Phylogeographic Genetic Diversity in the White Sucker Hepatitis B Virus across the Great Lakes Region and Alberta, Canada.

Hepatitis B viruses belong to a family of circular, double-stranded DNA viruses that infect a range of organisms, with host responses that vary from mild infection to chronic infection and cancer. The white sucker hepatitis B virus (WSHBV) was first described in the white sucker (Catostomus commersonii), a freshwater teleost, and belongs to the genus Parahepadnavirus. At present, the host range of WSHBV and its impact on fish health are unknown, and neither genetic diversity nor association with fish health have been studied in any parahepadnavirus. Given the relevance of genomic diversity to disease outcome for the orthohepadnaviruses, we sought to characterize genomic variation in WSHBV and determine how it is structured among watersheds. We identified WSHBV-positive white sucker inhabiting tributaries of Lake Michigan, Lake Superior, Lake Erie (USA), and Lake Athabasca (Canada). Copy number in plasma and in liver tissue was estimated via qPCR. Templates from 27 virus-positive fish were amplified and sequenced using a primer-specific, circular long-range amplification method coupled with amplicon sequencing on the Illumina MiSeq. Phylogenetic analysis of the WSHBV genome identified phylogeographical clustering reminiscent of that observed with human hepatitis B virus genotypes. Notably, most non-synonymous substitutions were found to cluster in the pre-S/spacer overlap region, which is relevant for both viral entry and replication. The observed predominance of p1/s3 mutations in this region is indicative of adaptive change in the polymerase open reading frame (ORF), while, at the same time, the surface ORF is under purifying selection. Although the levels of variation we observed do not meet the criteria used to define sub/genotypes of human and avian hepadnaviruses, we identified geographically associated genome variation in the pre-S and spacer domain sufficient to define five WSHBV haplotypes. This study of WSHBV genetic diversity should facilitate the development of molecular markers for future identification of genotypes and provide evidence in future investigations of possible differential disease outcomes.

Genome Sequences of 26 White Sucker Hepatitis B Virus Isolates from White Sucker, Catostomus commersonii, Inhabiting Transboundary Waters from Alberta, Canada, to the Great Lakes, USA.

We report 26 genome sequences of the white sucker hepatitis B virus (WSHBV) from the white sucker, Catostomus commersonii The genome length ranged from 3,541 to 3,543 bp, and nucleotide identity was 96.7% or greater across genomes. This work suggests a geographical range of this virus that minimally extends from the Athabasca River, Alberta, Canada, to the Great Lakes, USA.

Sex- and tissue-specific effects of waterborne estrogen on estrogen receptor subtypes and E2-mediated gene expression in the reproductive axis of goldfish.

This research examined the gene expression profile of three goldfish estrogen receptor (ER) subtypes in multiple tissues in relation to mRNA levels of aromatase B and vitellogenin (VTG) following waterborne estrogen exposures. The protocol consisted of: i) adult male goldfish in late gonadal recrudescence exposed to 1 nM 17beta-estradiol (E2); ii) adult male and female goldfish in early sexual regression exposed to 1 nM E2 for 3, 6, 12 and 24h; and, iii) sexually mature, adult male goldfish exposed to 0.3 nM 17alpha-ethynylestradiol (EE2) for 24h. Liver produced the most consistent response with up-regulation of ERalpha in sexually regressed, mature and recrudescing males and in sexually regressed females. The dose and length of exposure, reproductive state and sex affected the auto-regulation of ERbeta1 by E2. ERbeta2 was not affected in any experiments suggesting it may not be auto-regulated by E2. Aromatase B and VTG gene expression were affected by E2, but also by other experimental conditions. EE2 induced liver ERalpha and VTG mRNA levels indicating that high environmental EE2 levels induce E2-mediated gene expression in a model teleost. These studies reveal a more complicated action of estrogenic compounds that has important implications on estrogenic endocrine disruptors in teleosts.

A synthesis of implementation science frameworks and application to global health gaps.

BACKGROUND: Implementation science has been growing as discipline in the past decades, producing an increasing number of models in the area. On the other hand, most frameworks are intended to guide the implementation of programs, focusing on identifying elements and stages that increase their success. This article aims to structure this discussion, proposing a simplified tool that synthesizes common elements of other frameworks, and highlight the usefulness to use implementation science not only in identifying successful implementation strategies but as a tool to assess gaps in global health initiatives. METHODS: The study was carried out through a combined methodology that included an initial search of implementation science frameworks, experts' opinions, and the use of references in frameworks to elaborate a list of articles to be reviewed. A total of 52 articles were analyzed, identifying their definitions of implementation science and the elements of different frameworks. RESULTS: The analysis of articles allowed identifying the main goals and definitions of implementation science. In a second stage, frameworks were classified into "time-based", "component-based" and "mixed", and common elements of each type of model were used to propose a synthetic framework with six elements: Diagnosis, Intervention provider/ system, Intervention, Recipient, Environment, and Evaluation. Finally, this simplified framework was used to identify gaps in global health was using The Lancet Global Health Series. Potential areas of intervention arise for five different global health issues: malaria, non-communicable diseases, maternal and child health, HIV/AIDS, and tuberculosis. Prioritization strategies differ for the different health issues, and the proposed framework can help identify and classify all these different proposals. CONCLUSIONS: There is a huge variety of definitions and models in implementation science. The analysis showed the usefulness of applying an implementation science approach to identify and prioritize gaps in implementation strategies in global health.

Potential endocrine disruption of sexual development in free ranging male northern leopard frogs (Rana pipiens) and green frogs (Rana clamitans) from areas of intensive row crop agriculture.

Intensive row crop agriculture (IRCA) for corn and soybean production is predominant in eastern and central North America. IRCA relies heavily on pesticide and nutrient inputs to maximize production under conventional systems. In 2003-2005, we assessed the occurrence of a suite of potential endocrine effects in amphibians inhabiting farm ponds and agricultural drains in IRCA areas of southwestern Ontario. Effects were compared to amphibians from two agricultural reference sites as well as four non-agricultural reference sites. Pesticide and nutrient concentrations were also determined in water samples from those sites. Atrazine and metolachlor were detected in most samples, exceeding 1 microg L(-1) at some sites. Blood samples were taken from northern leopard frogs (Rana pipiens) and green frogs (Rana clamitans) for analysis of circulating sex steroids and vitellogenin-like protein (Vtg-lp), a biomarker of exposure to environmental estrogens. Gonads were histologically examined for evidence of abnormalities. Some evidence of exposure to endocrine disrupting compounds was apparent from the data. The occurrence of testicular ovarian follicles (TOFS) in male R. pipiens was significantly higher (42%; p<0.05) at agricultural sites, particularly those in Chatham county compared to frogs from reference sites (7%). There was no difference in circulating sex steroid levels between frogs from agricultural and reference sites and sex steroid levels did not correlate with pesticide concentrations in the environment. No differences were detected in the gonadosomatic indices or stage of spermatogenesis between frogs from agricultural and non-agricultural regions (p>0.05). Plasma Vtg-lp was detected in only one male R. pipiens from an agricultural site. Neither gonad size, gonad maturity nor sex steroid levels differed between normal males and those with testicular oocytes. Although the proportion of testicular oocytes did not correlate directly with atrazine concentrations, it did correlate with a mixture of pesticides and nutrients, particularly atrazine and nitrate, while the number of pesticides detected at each site was also important.

Bioaccumulation of pharmaceuticals and personal care product chemicals in fish exposed to wastewater effluent in an urban wetland.

The bioaccumulation of a broad range of pharmaceuticals and personal care product chemicals (PPCPs) was studied in Cootes Paradise Marsh (CPM), an urban wetland that receives tertiary treated municipal waste waters as well as urban storm runoff. We measured PPCPs in caged and wild goldfish, as well as wild carp, and compared observed bioaccumulation factors (BAFP) using concentrations in surface waters and fish blood plasma, with modeled BAFs. Thirty-two PPCPs were detected in water from the central CPM site (CPM3) while 64 PPCPs were found at higher concentrations at a site immediately downstream of the effluent outflow (CPM1). Following a 3-week deployment, 15 PPCPs were detected in the plasma of caged goldfish at CPM1, and 14 at CPM3, compared to only 3 in goldfish caged at a reference site. The highest BAFP in goldfish were for the antidepressant Σfluoxetine averaging 386 L/kg in caged and 906 L/kg in wild goldfish, respectively. In carp, ΣDiazepam (diazepam and oxazepam) had the highest BAFP (927 L/kg). This study identified a broader range of PPCPs in fish and surface waters than previously reported. However, modeled BAFs did not show good agreement with observed whole body or plasma BAFs, demonstrating that more work is needed to better explain bioaccumulation of PPCPs.

Endocrine disruption and altered gonadal development in white perch (Morone americana) from the lower Great Lakes region.

High prevalences of gonadal intersex have been observed in wild fish populations in areas affected by domestic and industrial effluents. For this study, fish were collected in 1998 from the Cootes Paradise region of Hamilton Harbour in western Lake Ontario, Canada, to determine whether gonadal abnormalities, including intersex, were present in young of the year (YOY) fish. No gonadal abnormalities were observed in goldfish (Carassius auratus), common carp (Cyprinus carpio), gizzard shad (Dorosoma cepedianum), brown bullhead (Ictalurus ameiurus), pumpkinseed (Lepomis gibbosus), and bluegill (Lepomis macrochirus). However, intersex gonads were observed in 8 of 16 male white perch (Morone americana) examined in this survey. Subsequently, in 1999 and 2000 white perch estimated to be YOY to approximately 2 years of age were collected from Cootes Paradise and from two other sites in the lower Great Lakes region. Gonadal intersex was observed in male white perch collected from the Bay of Quinte (22-44%) and Lake St. Clair (45%), although the prevalence and the extent of the intersex condition were lower relative to the 83% prevalence in white perch collected in Cootes Paradise. Intersex was not observed in hatchery-reared white perch or in white perch collected from an uncontaminated reference site (i.e., Deal Lake) in the United States. An analysis of plasma collected in the spring of 2002 from male adult white perch in Cootes Paradise revealed high concentrations of vitellogenin, ranging from 49 to 1,711 microg/mL. These observations indicate that male white perch are exposed to estrogenic endocrine-disrupting substances that may be responsible for the induction of gonadal intersex.

Bioaccumulation of the pharmaceutical 17alpha-ethinylestradiol in shorthead redhorse suckers (Moxostoma macrolepidotum) from the St. Clair River, Canada.

17alpha-ethynylestradiol (EE2), a synthetic estrogen prescribed as a contraceptive, was measured in Shorthead Redhorse Suckers (ShRHSs) (Moxostoma macrolepidotum) collected near a wastewater treatment plant (WWTP) in the St. Clair River (Ontario, Canada). We detected EE2 in 50% of the fish samples caught near the WWTP (Stag Island), which averaged 1.6+/-0.6ng/g (wet weight) in males and 1.43+/-0.96ng/g in females. No EE2 was detected in the samples from the reference site (Port Lambton) which was 26km further downstream of the Stag Island site. Only males from Stag Island had VTG induction, suggesting the Corunna WWTP effluent as a likely source of environmental estrogen. EE2 concentrations were correlated with total body lipid content (R(2)=0.512, p<0.01, n=10). Lipid normalized EE2 concentrations were correlated with delta(15)N (R(2)=0.436, p<0.05, n=10), suggesting higher EE2 exposures in carnivores. Our data support the hypothesis of EE2 bioaccumulation in wild fish.