Effects of perfluoroalkyl sulfonic acids on developmental, physiological, and immunological measures in northern leopard frog tadpoles.

The chronic toxicity of short chain perfluoroalkyl sulfonic acids (PFSAs), such as perfluorobutanesulfonic acid (PFBS) and perfluorohexanesulfonic acid (PFHxS), are relatively understudied despite the increasing detection of these compounds in the environment. We investigated the chronic toxicity and bioconcentration of PFBS and PFHxS using northern leopard frog (Rana [Lithobates] pipiens) tadpoles. We exposed Gosner stage (GS) 25 tadpoles to either PFBS or PFHxS at nominal concentrations of 0.1, 1, 10, 100, and 1000 µg/L until metamorphosis (GS42). We then assessed tadpole growth, development, stress, and immune metrics, and measured fatty acid (FA) composition and PFSA concentrations in liver and whole-body tissues. Tadpole growth and development measures were relatively unaffected by PFSA exposure. However, tadpoles exposed to 1000 µg/L PFBS or PFHxS had significantly increased hepatosomatic indexes (HSI) relative to controls. Further, tadpoles from the 1000 µg/L PFHxS treatment had altered FA profiles relative to controls, with increased total FAs, saturated FAs, monounsaturated FAs, and omega-6 polyunsaturated FAs. In addition, tadpoles from the 1000 µg/L PFHxS treatment had a higher probability of waterborne corticosterone detection. These results suggest that PFBS and PFHxS influence the hepatic health of tadpoles, and that PFHxS may alter lipid metabolism in tadpoles. We also observed a higher probability of tadpoles being phenotypically female after exposure to an environmentally relevant concentration (0.1 µg/L) of PFHxS, suggesting that PFHxS may exert endocrine disrupting effects on tadpoles during early development. The measured bioconcentration factors (BCFs) for both compounds were ≤10 L kg-1 wet weight, suggesting low bioconcentration potential for PFBS and PFHxS in tadpoles. Many of the significant effects observed in this study occurred at concentrations several orders of magnitude above those measured in the environment; however, our work shows effects of PFSAs exposure on amphibians and provides essential information for ecological risk assessments of these compounds.

Milkweed in agricultural field margins - A neonicotinoid exposure route for pollinators at multiple life stages.

Neonicotinoid insecticides move from targeted crops to wildflowers located in adjacent field margins, acting as a potential exposure source for wild pollinators and insect species of conservation concern, including monarch butterflies. Monarchs rely on milkweed over multiple life stages, including as a host plant for eggs and a food source for both larvae (leaves) and adults (flowers). Milkweeds, which are closely associated with field margins, can contain neonicotinoid residues, but previous assessments are constrained to a single plant tissue type. In 2017 and 2018, we sampled milkweeds from 95 field margins adjacent to crop fields (corn, soybean, hay, wheat, and barley) in agricultural landscapes of eastern Ontario, Canada. Milkweeds were sampled during the flower blooming period and leaves and flower tissues were analysed. The neonicotinoids acetamiprid, clothianidin, thiamethoxam, and thiacloprid were detected. Maximum concentrations in leaf samples included 10.30 ng/g of clothianidin in 2017, and 24.4 ng/g of thiamethoxam in 2018. Clothianidin and thiamethoxam percent detections in flowers (72 % and 61 %, respectively) were significantly higher than detections in leaves (24 % and 31 %, respectively). Thiamethoxam concentrations were significantly higher in paired flower samples than leaf samples (median 0.33 ng/g vs <0.07 ng/g) while clothianidin concentrations also trended higher in flowers (median 0.18-0.55 ng/g vs <0.18 ng/g). Only thiamethoxam showed significant differences between years, and we found no effect of crop type, with hay, soybean and corn fields all yielding 50-56 % detections in leaves. We found significantly higher concentrations in older milkweed flowers than young flowers or leaves (medians 0.87 ng/g vs <0.18 ng/g and 0.45 ng/g vs <0.07 ng/g for clothianidin and thiamethoxam, respectively). Our results highlight the importance of considering variation in milkweed tissue type and age of flowers in neonicotinoid exposure risk assessments. Efforts to increase milkweed availability in agricultural landscapes should consider how exposure to neonicotinoids can be mitigated.

Contrasting trophic transfer patterns of cadmium and mercury in the Arctic marine food web of east Hudson Bay, Canada.

We investigated trophic transfer of cadmium (Cd) through an Arctic marine food web in Hudson Bay and compared it with mercury (Hg), a metal known to strongly biomagnify. We evaluated blue mussel, sea urchin, common eider, sculpin, Arctic cod, and ringed seal for the influence of dietary and biological variables on variation in Cd and Hg concentrations. Age and size influenced metal concentrations among individuals within a vertebrate species. Consumer carbon and sulfur isotope values were correlated with their Cd and Hg concentrations, indicating habitat-specific feeding influenced metal bioaccumulation. Trophic transfer patterns for Cd depended on the vertebrate tissue, with food web biodilution observed for the muscle but not the liver. Liver Cd concentrations were higher in ringed seal and some common eider relative to prey. In contrast, we observed mercury biomagnification for both tissues. Tissue- and species-specific physiology can explain discrepancies of Cd trophic transfer in this Arctic marine food web.

The effects of two short-chain perfluoroalkyl carboxylic acids (PFCAs) on northern leopard frog (Rana pipiens) tadpole development.

Short-chain perfluoroalkyl carboxylic acids (PFCAs) have been detected in the environment globally. The presence and persistence of these compounds in the environment may lead to chronic wildlife exposure. We used northern leopard frog (Rana pipiens) tadpoles to investigate the chronic toxicity and the bioconcentration of two short-chain PFCAs, perfluorobutanoic acid (PFBA) and perfluorohexanoic acid (PFHxA). We exposed Gosner stage 25 tadpoles to PFBA and PFHxA (as individual chemicals) at nominal concentrations of 0.1, 1, 10, 100, and 1000 µg/L for 43-46 days. Tadpoles exposed to 0.1 to 100 µg/L of PFBA and PFHxA had significantly higher mean snout-to-vent lengths, mean masses, and scaled mass indexes than control tadpoles. These results indicate that exposure to short-chain PFCAs influences tadpole growth. Further investigation into the mechanism(s) causing the observed changes in tadpole growth is warranted. We observed a significantly higher proportion of males in the PFBA 1 µg/L treatment group, however further histological analyses are required to confirm visual sex identification before making concrete conclusions on the effects of PFCAs on amphibian sex ratios. PFBA concentrations in tissues were higher than PFHxA concentrations; a pattern that contrasts with previously published studies using fish, suggesting potential differences between taxa in PFBA and PFHxA bioconcentration. Bioconcentration factors were <10 L/kg wet weight, indicating low bioconcentration potential in tadpoles. Our results suggest that PFBA and PFHxA may have effects at environmentally-relevant concentrations (0.1-10 µg/L) and further investigation is required before these compounds can be deemed a "safe" alternative to their long-chain counterparts.

Basolateral amygdala neuropeptide Y system modulates binge ethanol consumption.

Neuropeptide Y (NPY) signaling regulation of corticolimbic communication is known to modulate binge-like ethanol consumption in rodents. In this work we sought to assess the impact of intra-BLA NPY system modulation on binge-like ethanol intake and to assess the role of the NPY1R+ projection from the BLA to the mPFC in this behavior. We used "drinking-in-the-dark" (DID) procedures in C57BL6J mice to address these questions. First, the impact of intra-BLA administration of NPY on binge-like ethanol intake was assessed. Next, the impact of repeated cycles of DID intake on NPY1R expression in the BLA was assessed with use of immunohistochemistry (IHC). Finally, chemogenetic inhibition of BLA→mPFC NPY1R+ projections was assessed to determine if limbic communication with the mPFC was specifically involved in binge-like ethanol intake. Importantly, as both the BLA and NPY system are sexually dimorphic, both sexes were assessed in these studies. Intra-BLA NPY dose-dependently decreased binge-like ethanol intake in males only. Repeated DID reduced NPY1R expression in the BLA of both sexes. Silencing of BLA→mPFC NPY1R+ neurons significantly reduced binge-like ethanol intake in both sexes in a dose-dependent manner. We provide novel evidence that (1) intra-BLA NPY reduces binge-like ethanol intake in males; (2) binge-like ethanol intake reduces NPY1R levels in the BLA; and (3) chemogenetic inhibition of BLA→mPFC NPY1R+ neurons blunts binge-like drinking in male and female mice. These observations provide the first direct evidence that NPY signaling in the BLA, and specifically BLA communication with the mPFC, modulates binge-like ethanol consumption.

Somatostatin signaling modulates binge drinking behavior via the central nucleus of the amygdala.

Somatostatin (SST) is a neuropeptide widely expressed in the central nervous system with dense expression in limbic regions such as the extended amygdala. It has recently gained attention for playing a role in modulating alcohol use disorders and co-morbid neuropsychiatric disorders. However, the role of SST in the central nucleus of the amygdala (CeA), a key region for neuropeptide regulation of alcohol and anxiety related behaviors, in alcohol consumption has not been assessed. In this work we perform an initial examination of the interaction between the CeA SST system and binge ethanol intake. Binge intake is a dangerous pattern of excessive ethanol consumption associated with health complications and the transition into alcohol dependence. We use the Drinking in the Dark (DID) model of binge intake in C57BL/6J male and female mice to examine: 1) the impact of 3 DID cycles on CeA SST expression; 2) the effect of intra-CeA SST injection on binge-like ethanol consumption; and 3) if the SST receptor 2 or 4 (SST2R or SST4R) mediate any effect on consumption. Our results show binge-like ethanol intake decreases SST expression in the CeA, but not neighboring basolateral amygdala. We further found intra-SST CeA administration reduces binge ethanol intake. This decrease was replicated by the administration of an SST4R agonist. These effects were not sex-dependent. Overall, this work lends further support for SST playing a role in alcohol related behaviors and as a potential therapeutic target.

Astrocyte expression in the extended amygdala of C57BL/6J mice is sex-dependently affected by chronic intermittent and binge-like ethanol exposure.

Excessive ethanol drinking is a major problem within the United States, causing alterations in brain plasticity and neurocognitive function. Astrocytes are glial cells that regulate neurosynaptic plasticity, modulate neurochemicals, and monitor other homeostatic roles. Astrocytes have been found to play a part in modulating excessive ethanol consumption. The basolateral amygdala (BLA), central amygdala (CeA), and bed nucleus of the stria terminalis (BNST) are brain regions that process stress, anxiety, and reward; they are also implicated in modulating ethanol intake. Little is understood, however, about how astrocyte expression in each region is modulated by chronic and binge-like ethanol drinking patterns. In the present report, we utilized two separate animal models of excessive drinking: chronic intermittent ethanol (CIE) and "Drinking-in-the-dark" (DID). Following these paradigms, animal brains were processed through immunohistochemistry (IHC) and stained for glial fibrillary acidic protein (GFAP). Collected data illustrated a sex-dependent relationship between ethanol intake and GFAP immunoreactivity (IR) in the BLA and BNST, but not in the CeA. Specifically, CIE and DID ethanol drinking resulted in blunted GFAP-IR (specifically via GFAP-positive cell count) in the BLA and BNST, particularly in males. These findings may implicate sex-dependent ethanol-induced changes in BLA and BNST astrocytes, providing a potential therapeutic target for anxiety and stress disorders.

Chronic ethanol consumption exacerbates future stress-enhanced fear learning, an effect mediated by dorsal hippocampal astrocytes.

BACKGROUND: Alcohol use disorder (AUD) and post-traumatic stress disorder (PTSD) are highly comorbid, yet there is a lack of preclinical research investigating how prior ethanol (EtOH) dependence influences the development of a PTSD-like phenotype. Furthermore, the neuroimmune system has been implicated in the development of both AUD and PTSD, but the extent of glial involvement in this context remains unclear. A rodent model was developed to address this gap in the literature. METHODS: We used a 15-day exposure to the 5% w/v EtOH low-fat Lieber-DeCarli liquid diet in combination with the stress-enhanced fear learning (SEFL) paradigm to investigate the effects of chronic EtOH consumption on the development of a PTSD-like phenotype. Next, we used a reverse transcription quantitative real-time polymerase chain reaction to quantify mRNA expression of glial cell markers GFAP (astrocytes) and CD68 (microglia) following severe footshock stress in EtOH-withdrawn rats. Finally, we tested the functional contribution of dorsal hippocampal (DH) astrocytes in the development of SEFL in EtOH-dependent rats using astrocyte-specific Gi designer receptors exclusively activated by designer drugs (Gi -DREADD). RESULTS: Results demonstrate that chronic EtOH consumption and withdrawal exacerbate future SEFL. Additionally, we found significantly increased GFAP mRNA expression in the dorsal and ventral hippocampus and amygdalar complex following the severe stressor in EtOH-withdrawn animals. Finally, the stimulation of the astroglial Gi -DREADD during EtOH withdrawal prevented the EtOH-induced enhancement of SEFL. CONCLUSIONS: Collectively, results indicate that prior EtOH dependence and withdrawal combined with a severe stressor potentiate future enhanced fear learning. Furthermore, DH astrocytes significantly contribute to this change in behavior. Overall, these studies provide insight into the comorbidity of AUD and PTSD and the potential neurobiological mechanisms behind increased susceptibility to a PTSD-like phenotype in individuals with AUD.

Lateral habenula-projecting central amygdala circuits expressing GABA and NPY Y1 receptor modulate binge-like ethanol intake in mice.

The central nucleus of the amygdala (CeA) is a critical brain region in the integration of emotional behaviors and is one of the major output areas of the amygdaloid complex. The CeA is composed of GABAergic interneurons and projection neurons which co-express a range of peptides including neuropeptide Y (NPY). Importantly, GABA and NPY signaling, via the NPY Y1 receptor (Y1R), in the CeA modulate binge-like ethanol intake in rodents and these systems undergo neuroplastic alterations following a history of ethanol consumption. Here we assessed the roles of GABAergic and Y1R+ circuits arising from the CeA and innervating the lateral habenula (LHb), a brain region that modulates the aversive properties of ethanol, in modulating binge-like ethanol intake in mice using "drinking in the dark" (DID) procedures. Using an anterograde cre-inducible reporter virus we established the CeA → LHb circuit in male and female vgat-ires-cre and NPY1r-cre mice. Next, we found that chemogenetic silencing of both the GABAergic or Y1R+ CeA → LHb circuit significantly blunted binge-like intake of a 20% ethanol solution but this same procedure failed to alter the consumption of a 3% sucrose solution. Finally, one, 4-day cycle of DID failed to alter basal or effects of ethanol or NPY on inhibitory transmission in Y1R+ CeA → LHb neurons. The present results suggest that blunting GABAergic tone in LHb-projecting CeA neurons may represent a new approach to preventing the development of AUDs. Drugs that target NPY Y1Rs are potential attractive targets.

Chemogenetic inhibition of corticotropin-releasing factor neurons in the central amygdala alters binge-like ethanol consumption in male mice.

Repetitive bouts of binge drinking can lead to neuroplastic events that alter ethanol's pharmacologic effects and perpetuate excessive consumption. The corticotropin-releasing factor (CRF) system is an example of ethanol-induced neuroadaptations that drive excessive ethanol consumption. Our laboratory has previously shown that CRF antagonist, when infused into the central amygdala (CeA), reduces binge-like ethanol consumption. The present study extends this research by assessing the effects of silencing CRF-producing neurons in CeA on binge-like ethanol drinking stemming from "Drinking in the Dark" (DID) procedures. CRF-ires-Cre mice underwent surgery to infuse Gi/o-coupled Designer Receptors Exclusively Activated by Designer Drugs (DREADD) virus or a control virus into either the CeA or basolateral amygdala (BLA). Gi/o-DREADD-induced CRF-neuronal inhibition in the CeA resulted in a 33% decrease in binge-like ethanol consumption. However, no effect on ethanol consumption was seen after DREADD manipulation in the BLA. Moreover, CeA CRF-neuronal inhibition had no effect on sucrose consumption. The effects of silencing CRF neurons in the CeA on ethanol consumption are not secondary to changes in motor function or anxiety-like behaviors as assessed in the open-field test (OFT). Finally, the DREADD construct's functional ability to inhibit CRF-neuronal activity was demonstrated by reduced ethanol-induced c-Fos following DREADD activation. Together, these data suggest that the CRF neurons in the CeA play an important role in binge ethanol consumption and that inhibition of the CRF-signaling pathway remains a viable target for manipulating binge-like ethanol consumption. (PsycInfo Database Record (c) 2022 APA, all rights reserved).

Exposure to the synthetic phenolic antioxidant 4,4'-thiobis(6-t-butyl-m-cresol) disrupts early development in the frog Silurana tropicalis.

Many chemicals in commonly used household and industrial products are being released into the environment, yet their toxicity is poorly understood. The synthetic phenolic antioxidant, 4,4'-thiobis(6-t-butyl-m-cresol) (CAS 96-69-5; TBBC) is present in many common products made of rubber and plastic. Yet, this phenolic antioxidant has not been tested for potential toxicity and developmental disruption in amphibians, a sensitive and susceptible class. We investigated whether acute and chronic exposure to TBBC would interfere with thyroid hormone-dependent developmental processes in the frog Silurana tropicalis and thus affect its early life-stage development. We exposed S. tropicalis embryos at the Nieuwkoop-Faber (NF) 9-10 stage to TBBC at nominal concentrations (0, 25, 50, 75, 100, 200 and 400 µg/L) to determine the 96h lethal concentrations and sublethal effects. We conducted a chronic exposure starting at stage NF47-48 to three sublethal TBBC nominal concentrations (0, 0.002, 0.1 and 5 µg/L) for 48-52 days to evaluate effects on growth and metamorphosis. The 96h lethal and effective (malformations) TBBC concentrations (LC50 and EC50) were 70.5 and 76.5 µg/L, respectively. Acute exposure to all TBBC concentrations affected S. tropicalis growth and was lethal at 200 and 400 µg/L. Chronic exposure to sublethal TBBC concentrations reduced body size by 8% at 5 µg/L and body mass by 17% at 0.002 µg/L when metamorphosis was completed. This study demonstrates that TBBC is toxic, induces malformations and inhibits tadpole growth after acute and chronic exposures. These findings call for further investigations on the mode of actions of TBBC and related antioxidants for developmental disruption in amphibians.

Meta-analysis shows environmental contaminants elevate cortisol levels in teleost fish - Effect sizes depend on contaminant class and duration of experimental exposure.

Glucocorticoid hormones (GCs) help vertebrates maintain homeostasis during and following challenging events. Short-term elevations in GC levels are necessary for survival, whereas longer-term changes can lead to reduced reproductive output and immunosuppression. Persistent environmental contaminants (ECs) are widespread globally. Experimental exposure of individuals to ECs is associated with varying GC responses, within, and across, species and contaminants. Individuals exposed to ECs over long durations are expected to have prolonged GC elevations, which likely affect their health. We conducted a meta-analysis to test for a relationship between fish GC levels and experimental exposure to ECs, and to explore potential moderators, including duration of exposure, that could help explain the variation in effect sizes within and between studies. We report almost exclusively on cortisol responses of teleost fish to ECs. Although there was much variation in effect sizes, captive-bred fish exposed to ECs had baseline GC levels 1.5× higher than unexposed fish, and fish exposed to pharmaceuticals (estradiols and stimulants being mainly considered) had baseline GC levels approximately 2.5× higher than unexposed fish. We found that captive-bred and wild-caught fish did not differ in GC levels after exposure to the same classes of ECs - studies on captive bred fish may thus enable inferences about GC responses to ECs for wild species. Furthermore, effect sizes did not differ between baseline and challenge-induced GC measures. In different analyses, duration of exposure was negatively correlated to effect size, suggesting that the GC response may acclimate after chronic exposure to some ECs which could potentially alter the GC response of EC-exposed fish to novel stressors. Future studies should explore the effect of multiple stressors on the fish GC response and perform tests on a broader array of contaminant types and vertebrate classes.

Trematodes coupled with neonicotinoids: effects on blood cell profiles of a model amphibian.

Habitat loss, climate change, environmental contaminants, and parasites and pathogens are among the main factors thought to act singly or together in causing amphibian declines. We tested for combined effects of neonicotinoid pesticides and parasites (versus parasites-only) on mortality, growth, and white blood cell profiles of a model amphibian: the northern leopard frog (Rana pipiens). We first exposed infectious stages of frog trematodes (cercariae of Echinostoma spp.) to low and high concentrations of thiamethoxam or clothianidin versus water-only controls. There were no differences in survival of trematode cercariae between treatments. For the main experiment, we exposed tadpoles to clean water versus high concentrations of clothianidin or thiamethoxam for 2 weeks and added trematode cercariae to all tanks after 1 week. Exposure of tadpoles and parasites to high concentrations of thiamethoxam or clothianidin did not affect parasite infection success. Tadpole survival was not different between treatments before or after parasite addition and there were no significant differences in tadpole snout-to-vent lengths or developmental stages between treatments. Tadpoles exposed to thiamethoxam + parasites had smaller widths than parasite-only tadpoles, whereas tadpoles exposed to clothianidin + parasites had higher eosinophil to leukocyte ratios compared to parasite-only tadpoles. Tadpoles of both neonicotinoid + parasite treatments had significantly lower monocyte to leukocyte ratios relative to parasite-only tadpoles. High concentrations of neonicotinoid combined with parasites appear to influence tadpole immune function important for further defense against parasites and pathogens. This work highlights the need for more holistic approaches to ecotoxicity studies, using multiple stressors.

The Effects of Ditch Management in Agroecosystems on Embryonic and Tadpole Survival, Growth, and Development of Northern Leopard Frogs (Lithobates pipiens).

Agricultural drainage ditches help remove excess water from fields and provide habitat for wildlife. Drainage ditch management, which includes various forms of vegetation clearing and sediment dredging, can variably affect the ecological function of these systems. To determine whether ditch conditions following dredging/vegetation clearing management affected the survival, growth, and development of embryos and tadpoles of northern leopard frogs (Lithobates pipiens), we conducted three field studies using in situ cages over 2 years. We measured nutrients, pesticides, and other water quality properties in vegetated/unmanaged (i.e., no clearing or dredging) and newly cleared/dredged (i.e., treeless, then dredged), clay-bottomed drainage ditches in a river basin in Eastern Ontario, Canada. Nutrients, atrazine, and total neonicotinoid concentrations were generally lower at the cleared/dredged sites, whereas glyphosate was at higher concentrations. In contrast, water-quality variables measured in situ, particularly temperature, dissolved oxygen, and turbidity, tended to be higher in the cleared/dredged sites. Total phosphorous and total organic carbon concentrations at all sites were above the recommended limits for amphibian assays. No significant differences were detected in the survival, hatching success, or development of embryos among the ditch management treatments, but premature hatching was observed at one vegetated/unmanaged site where high specific conductivity may have been formative. We found the cleared/dredged sites supported earlier tadpole growth and development, likely as a result of the higher water temperatures. Increased temperature may have offset other growth/development stressors, such as those related to water chemistry. However, the long-term consequences of these differences on amphibian populations requires further study.

Effects of two pesticides on northern leopard frog (Lithobates pipiens) stress metrics: Blood cell profiles and corticosterone concentrations.

Amphibians are declining globally. Exposure to pesticides has been implicated in decreasing amphibian immune function, thus increasing their susceptibility to parasites and disease and thereby negatively affecting individuals and populations. Amphibians are likely exposed to neonicotinoids because these widely used insecticides are highly soluble in water and because amphibian freshwater habitats are often embedded in agroecosystems. Herein, we investigate the effects of long-term exposure to two individual neonicotinoids (clothianidin or thiamethoxam) at either low or high concentrations (2.5 or 250 µg/L) on northern leopard frog (Lithobates pipiens) blood cell profiles and concentrations of corticosterone, an energy-mediating hormone associated with stress. Larval frogs from Gosner stage 25 to 46 were exposed to pesticide and control treatments in outdoor mesocosms. Corticosterone concentrations were measured after 6 d of exposure, and blood cell profiles were assessed once frogs reached Gosner stage 46 (following 8 w of exposure). No significant changes were found in erythrocyte counts, leukocyte counts, monocyte to leukocyte ratios or corticosterone concentrations between treatments. However, exposure to either 2.5 or 250 µg/L of clothianidin, or 250 µg/L of thiamethoxam decreased neutrophil to lymphocyte ratios and neutrophil to leukocyte ratios, and exposure to 2.5 µg/L of clothianidin or 250 µg/L of thiamethoxam decreased eosinophil to leukocyte ratios. Our results indicate that long-term exposure to neonicotinoids can alter leukocyte profiles, indicative of a stress response. Future studies should investigate whether chronic exposure to neonicotinoids affect multiple measures of stress differently or influences the susceptibility of amphibians to parasites and pathogens. Our work underscores the importance of continued use of multiple measures of stress for different amphibian species when undertaking ecotoxicological assessments.

Clothianidin alters leukocyte profiles and elevates measures of oxidative stress in tadpoles of the amphibian, Rana pipiens.

Neonicotinoid pesticide use is widespread and highly debated, as evidenced by recent attention received from the public, academics and pesticide regulatory agencies. However, relatively little is known about the physiological effects of neonicotinoid insecticides on aquatic vertebrates. Amphibians (larval stages in particular) are excellent vertebrate bioindicators in aquatic systems due to their risk of exposure and sensitivity to environmental stressors. Previous work with wood frog (Rana sylvatica) tadpoles exposed to formulated products containing thiamethoxam or clothianidin in outdoor mesocosms found significant shifts in leukocyte profiles, suggesting the tadpoles were physiologically stressed. The main objective of the present study was to characterize this stress response further using complementary measures of stress after exposure to clothianidin on northern leopard frogs (Rana pipiens) during their aquatic larval stages. Laboratory static-renewal exposures were conducted over eight weeks with the technical product clothianidin at 0, 0.23, 1, 10 and 100 µg/L, and diquat dibromide at 532 µg/L was used as a positive control. We assessed tadpole leukocyte profiles and measures of oxidative stress as these sub-lethal alterations could affect amphibian fitness. We found changes in several types of leukocytes at 1 and 10 µg/L, suggesting that these tadpoles exhibited signs of mild physiological stress. Clothianidin also induced an oxidative stress response at 0.23, 1 and 100 µg/L. However, we found no differences in survival, growth, development time or hepatosomatic index in frogs exposed to clothianidin. Our study indicates that tadpoles chronically exposed to clothianidin have increased stress responses, but in the absence of concentration-response relationships and effects on whole-organism endpoints, the implications on the overall health and fitness of these changes are unclear.

Distinct and Overlapping Patterns of Acute Ethanol-Induced C-Fos Activation in Two Inbred Replicate Lines of Mice Selected for Drinking to High Blood Ethanol Concentrations.

The inbred high drinking in the dark (iHDID1 and iHDID2) strains are two replicate lines bred from the parent HS/Npt (HS) line for achieving binge levels of blood ethanol concentration (≥80 mg/dL BEC) in a four-hour period. In this work, we sought to evaluate differences in baseline and ethanol-induced c-Fos activation between the HS, iHDID1, and iHDID2 genetic lines in brain regions known to process the aversive properties of ethanol. METHODS: Male and female HS, iHDID1, and iHDID2 mice underwent an IP saline 2 3 g/kg ethanol injection. Brain sections were then stained for c-Fos expression in the basolateral/central amygdala (BLA/CeA), bed nucleus of the stria terminals (BNST), A2, locus coeruleus (LC), parabrachial nucleus (PBN), lateral/medial habenula (LHb/MHb), paraventricular nucleus of the thalamus (PVT), periaqueductal gray (PAG), Edinger-Westphal nuclei (EW), and rostromedial tegmental nucleus (RMTg). RESULTS: The iHDID1 and iHDID2 lines showed similar and distinct patterns of regional c-Fos; however, in no region did the two both significantly differ from the HS line together. CONCLUSIONS: These data lend further support to altered baseline or ethanol-induced activation in brain regions associated with processing the aversive properties of ethanol in the iHDID1 and iHDID2 genetic lines.

Size and survival of two freshwater snail species in relation to shedding of cercariae of castrating Echinostoma spp.

Trematode-induced castration of snails is widespread and can lead to other life history changes of snails such as changes in trajectories of size and growth or survival. The changes produced likely depend on whether the parasite or host controls allocation of host resources remaining after partial or complete cessation of host current reproduction by castrating trematodes. Documenting host life history changes, like changes in host size in response to castration, is a first step in assessing whether these changes are beneficial to the parasite (increasing transmission success) or to the host (outliving the infection) or to neither. Herein, we test for differences in size and survival among individuals of two snail species in relation to infection by Echinostoma spp. trematodes. Active shedding of Echinostoma spp. was associated with castration of all Stagnicola elodes snails from a site in Eastern Ontario. Snails actively shedding cercariae were not different in size from non-shedding, egg-laying snails but had a higher mortality than egg-laying snails. Active shedding of Echinostoma spp. cercariae was also associated with castration of nearly all Helisoma trivolvis monitored, from a site in Southwestern Ontario. Actively shedding, non-laying H. trivolvis hosts were smaller on average than non-shedding egg-laying hosts, but both non-laying and egg-laying snails survived equally well. We discuss these results in light of what is known about effects of castration on snail hosts in terms of growth and survival for these and other trematode species and speculate on whether changes in size or survival benefits parasite or host.

Ecotoxicity assessment and bioconcentration of a highly brominated organophosphate ester flame retardant in two amphibian species.

Restrictions on the production and use of some highly toxic and persistent flame retardants has resulted in the increased use of alternative phosphate flame retardants that are less-well characterized. The brominated organophosphate ester flame retardant, tris(tribromoneopentyl) phosphate (CAS 19186-97-1, molecular formula C15H24Br9O4P, molecular weight 1018.47 g/mol, acronym TTBrNP) is a compound with potential to bioaccumulate and disrupt endocrine functions. To determine the toxicity of TTBrNP, two Canadian native amphibian species, Lithobates sylvaticus and L. pipiens, were acutely (embryos and Gosner stage 25 (GS25) tadpoles) or sub-chronically (GS25-41 tadpoles) exposed to the following nominal concentrations of TTBrNP: 0 (water and solvent controls), 30.6, 61.3, 122.5 and 245.0 µg/L. Note, measured concentrations declined with time (i.e., 118%-30% of nominal). There was high survival for both species after acute and sub-chronic exposures, where 75%-100% survived the exposures, respectively. There were no differences in the occurrence of abnormalities or hatchling size between controls and TTBrNP treatments for either species exposed acutely as embryos or tadpoles. Furthermore, after 30 d of sub-chronic exposure of L. pipiens tadpoles to TTBrNP there were no effects on size, developmental stage, liver somatic index or sex ratio. Bioconcentration factors were low at 26 ± 3.1 L/kg ww in tadpoles from all treatments, suggesting biotransformation or limited bioavailability via aquatic exposures. Thus, using two species of anurans at different early larval stages, we found TTBrNP up to 245 µg/L to have no overt detrimental effects on survival or morphological responses that would suggest fitness-relevant consequences.

A role for the neuropeptide somatostatin in the neurobiology of behaviors associated with substances abuse and affective disorders.

In recent years, neuropeptides which display potent regulatory control of stress-related behaviors have been extensively demonstrated to play a critical role in regulating behaviors associated with substance abuse and affective disorders. Somatostatin (SST) is one neuropeptide known to significantly contribute to emotionality and stress behaviors. However, the role of SST in regulating behavior has received relatively little attention relative to other stress-involved peptides, such as neuropeptide Y or corticotrophin releasing factor. This review characterizes our current understanding of the role of SST and SST-expressing cells in general in modulating several behaviors intrinsically linked to substance abuse and affective disorders, specifically: anxiety and fear; stress and depression; feeding and drinking; and circadian rhythms. We further summarize evidence of a direct role for the SST system, and specifically somatostatin receptors 2 and 4, in substance abuse disorders. This article is part of the special issue on 'Neuropeptides'.