Cross-species apical microinjected selenomethionine toxicity in embryo-larval fishes.

Selenium (Se) is an essential micronutrient that becomes toxic when exposures minimally exceed those that are physiologically required. Studies on Se contaminated aquatic environments have identified that embryo-larval fishes are at particular risk of Se toxicity, primarily due to maternal Se transfer to developing eggs during oogenesis. This study emulated these exposures in embryo-larval fathead minnow (FHM), rainbow trout (RBT), white sucker (WSu), and white sturgeon (WSt) using embryonic selenomethionine (SeMet) microinjections. Adverse Se-outcomes observed across these species included spinal and edematous deformities, total individuals deformed, and reduced survival. Spinal deformity was the most sensitive sublethal endpoint and developed at the lowest concentrations in WSt (10 % effects concentration (EC10) = 12.42 µg (total) Se/g dry weight (d.w.)) followed by WSu (EC10 = 14.49 µg Se/g d.w.) and FHM (EC10 = 18.10 µg Se/g d.w.). High mortality was observed in RBT, but SeMet influences were confounded by the species' innate sensitivity to the microinjections themselves. 5 % hazardous concentrations derived across exposure type data subsets were ∼49 % higher when derived from within-species maternal transfer exclusive data as opposed to all, or within-species microinjection exclusive, data. These results support the current exclusion of SeMet microinjections during regulatory guideline derivation and their inclusion when studying mechanistic Se toxicity across phylogenetically distant fishes.

Relationships among tissues, biofluids, and otolith selenium concentrations in wild female burbot (Lota lota).

In the Lake Koocanusa-Kootenai River system (Montana, USA and British Columbia, Canada), selenium (Se) contamination has become an international concern and is suspected to contribute to the observed burbot (Lota lota) population collapse. Due to our limited ability to sample burbot in Lake Koocanusa for monitoring studies, we used a reference population to develop tools to model tissue Se disposition for a focal species in systems with elevated Se. Total Se concentrations in otoliths, biofluids (blood and endolymph), and tissues (muscle, liver, and ovary) from burbot in reference lakes in northwestern Ontario, Canada, were measured to document tissue-to-tissue Se relationships and evaluate the potential for otoliths to retrace Se exposure in fish. Among burbot tissue, Se concentrations were the highest in the ovary (mean ± SD = 4.55 ± 2.23 µg g-1 dry mass [dm]), followed by the liver (2.69 ± 1.96 µg g-1 dm) and muscle (1.87 ± 1.14 µg g-1 dm), and decreased with body size (p < 0.05). In otoliths, Se was detected at low levels (<1 µg g-1 ). Selenium concentrations in burbot samples were positively correlated among muscle, ovary, liver, and endolymph tissues, but not for the most recent annually averaged or lifetime-averaged Se concentrations in otoliths. We hypothesize that Se concentrations were too low in this study to establish links between otoliths and other fish tissues and to detect significant lifetime variation in individuals, and that further validation using archived otoliths from burbot exposed to elevated Se levels in Lake Koocanusa-Kootenai River is needed to reconstruct exposure histories. However, intercompartmental models proved valuable for estimating Se concentrations in burbot tissues only available by means of lethal sampling (i.e., ovary), although additional work should confirm whether the established models are reliable to predict concentrations in Se-impaired systems as tissue distributions are likely to differ with increasing Se levels. Integr Environ Assess Manag 2023;00:1-11. © 2023 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

Hair Cortisol and Testosterone Concentrations in Relation to Maturity and Breeding Status of Male Feral Horses.

Steroid-hormone concentrations from non-invasively obtained biomarkers, like hair, can provide a representation of circulating hormones diffused over relatively long time periods (e.g., weeks or months). The hormone cortisol is often associated with physiological or even psychological stress, while testosterone is strongly associated with male development and reproductive success. Increasingly, studies are using hormone levels derived from hair to make inferences among both domestic animals and wildlife. For horses, all previous hair hormone analysis has been done on companion or working animals. We evaluated the levels of hair cortisol (n = 153) and testosterone (n = 48) from 136 feral horses living on Sable Island, Canada that have been part of a long-term individual-based study since 2008. This population has been undisturbed and unmanaged for over 50 years, and exhibits the natural social organization for horses, harem defense polygyny. Hair samples were collected in mid to late summer and the segment analyzed corresponds with hair grown during, and following, the peak of the reproductive season. Social position was determined based on the male's role as either a dominant breeding Stallion (Stallion), a non-breeding subordinate male (tag), adult Bachelor (5 years old or older), or Immature male (2-4 years of age). While there was no difference in hair-cortisol concentration among any class of adult males (i.e., Stallion, tag, or Bachelor), Immature males had significantly lower hair cortisol concentrations than the other groups (p = 0.001). Hair testosterone levels among the four social positions were significantly higher among Stallions (p = 0.04). Hair testosterone concentration was also significantly related to the probability of a male being either a Bachelor or Stallion and was the only variable remaining in AICc model selection (p = 0.016, AICc = 32.3, Null AICc = 38.8). While not a significant relationship, Stallions had a negative correlation between hair cortisol concentrations and testosterone (R2 = -0.20, p = 0.383), and Bachelors, conversely, had a positive association (R2 = 0.43, p = 0.246). Our observations of hormone concentrations in relation to physiological, social, or reproductive parameters in this population suggest trends that are similar to what has been established using blood or other matrices.

Hair Cortisol Concentrations in Feral Horses and the Influence of Physiological and Social Factors.

Cortisol is a glucocorticoid hormone produced during activation of the hypothalamic-pituitary-adrenal axis (HPA) in response to psychological or physiological demands. High amounts of circulating cortisol can be found in individuals experiencing energetically demanding physiological events, such as pregnancy, lactation, injury, or starvation, but, also, in individuals who may have less obvious HPA activation from social situations. The feral horse population on Sable Island (Nova Scotia, Canada) provides an opportunity to look at hair cortisol concentration (HCC) as a proxy for circulating cortisol concentration to better understand physiological correlates. The horse's complex social structure also allows us to look at how the population and group structure may influence HPA activation. Hair samples (n = 282) were analyzed from 113 females and 135 males. Females with dependent offspring (foals) had higher HCC than those females without dependent offspring (p = 0.005). Horses in poor body condition were also more likely to have higher HCC (females: p < 0.001, males: p = 0.028); females had greater variation in the body condition index (BCI), which also correlated with foal production. In general, the top-ranked models describing female cortisol levels included age, BCI, presence of a foal, as well as social measures such as harem size and the number of bachelors in the vicinity. The top model describing male cortisol levels included age, BCI, and year of collection only, and the number of bachelors in the home range appeared in subsequent, though still high-ranked, models. Among the variables not of direct interest, we found some significant results relating to hair color and hair texture. Differences in HCC patterns between feral and domestically kept horses (e.g., age and sex) are likely linked to periods of resource limitations, particularly for individuals experiencing energetically demanding processes such as reproduction, illness/parasitism, or related to experiencing the full range of social and reproductive behaviors.

Stress and reproductive hormones in hair associated with contaminant metal(loid)s of European brown bear (Ursus arctos).

Environmental contaminants like arsenic (As), cadmium (Cd), mercury (Hg) or lead (Pb) may disrupt hypothalamic-pituitary-adrenal (HPA) and hypothalamic-pituitary-gonadal (HPG) axes due to their endocrine toxicity potential. Resulting long-term physiological stress or adverse effects on wildlife reproduction and ontogeny may cause detrimental effects at the individual and population levels. However, data on environmental metal(loid)s' impact on reproductive and stress hormones in wildlife, especially large terrestrial carnivores, are scarce. Hair cortisol, progesterone and testosterone concentrations were quantified and modelled with hair As, Cd, total Hg, Pb, biological, environmental and sampling factors to test for potential effects in free-ranging brown bears (Ursus arctos) from Croatia (N = 46) and Poland (N = 27). Testosterone in males (N = 48) and females (N = 25) showed positive associations with Hg and an interaction between Cd and Pb, but a negative association with interaction between age and Pb. Higher testosterone was found in hair during its growth phase compared to quiescent phase. Body condition index was negatively associated with hair cortisol and positively associated with hair progesterone. Year and conditions of sampling were important for cortisol variation, while maturity stage for progesterone variation (lower concentrations in cubs and yearlings compared to subadult and adult bears). These findings suggest that environmental levels of Cd, Hg and Pb might influence the HPG axis in brown bears. Hair was shown to be a reliable non-invasive sample for investigating hormonal fluctuations in wildlife while addressing individual and sampling specificities.

Can scale cortisol concentration be quantified non-lethally in wild fish species?

Cortisol, the primary glucocorticoid in fishes, is secreted into the bloodstream in response to stress. Circulating cortisol accumulates in scales, a durable calcified structure that can be easily sampled from many fish species. As such, the use of scale cortisol concentration (SCC) is currently being explored as a means of chronic stress biomonitoring in wild fishes. Scales serve an important role in fish physiology and thus the number of scales required for reliable cortisol analysis is a limiting factor in the non-lethal collection of such samples. To date, scale cortisol quantification has also only been performed non-lethally in captive fishes and due to differences in stress responsiveness SCCs likely differ in wild species. As such, this study aimed to (1) apply our fish scale processing and analysis protocol to wild fish species and (2) apply it to five north temperate fish species to provide information useful to future non-lethal scale sampling regimes. Cortisol was successfully measured in scales collected from wild northern pike (Esox lucius), walleye (Sander vitreus), whitefish (Coregonus clupeaformis), white sucker (Catostomus commersonii) and captive rainbow trout (Oncorhynchus mykiss). SCCs were significantly different between species and thus the sample mass required for reliable cortisol analysis differed as well. In addition to the size of the fish and the mass of their scales this is an important consideration for future scale cortisol analyses as these factors could make SCC an attainable non-lethal sample matrix in some species of fish but impractical in others.

Chronic stress causes cortisol, cortisone and DHEA elevations in scales but not serum in rainbow trout.

Fish scales have been reported to incorporate cortisol over long periods of time and thus provide a promising means of assessing long-term stress in many species of teleost fish. However, the quantification of other stress related hormones has only been accomplished in our previous study conducted in goldfish (Carassius auratus). DHEA is a precursory androgen with anti-stress effects used alongside cortisol to diagnose chronic stress via the cortisol:DHEA ratio in mammals. Included in DHEA's anti-stress mechanisms are changes in the metabolism of cortisol to its inactive metabolite cortisone suggesting the relationships between cortisol, DHEA and cortisone may be additionally informative in the assessment of long-term stress. Therefore, to further explore these concepts in a native fish species and generate more comprehensive comparisons between scale and serum hormone concentrations than was possible in our previous study we implemented a 14-day stress protocol in adult rainbow trout (Oncorhynchus mykiss) and quantified resulting scale and serum cortisol, cortisone and DHEA concentrations. As predicted, elevations in scale concentrations of all hormones were observed in stressed trout compared to controls but were not reflected in serum samples. Significant differences in the cortisol:DHEA and cortisone:cortisol ratios were also found between control and stressed group scales but not serum. These results suggest not only that scales provide a superior medium for the assessment of long-term stress but also that the addition of scale cortisone and DHEA may provide additional relevant information for such assessments.

Perceived predation risk predicts glucocorticoid hormones, but not reproductive success in a colonial rodent.

The Cort-Adaptation hypothesis suggests that elevated glucocorticoids (GCs) can facilitate an adaptive response to environmental and physiological challenges. Most previous studies have focused on avian species, which may limit their generalizability to mammals, where lactation is known to be a major physiological challenge. Furthermore, the effect of predation risk on GC levels has not been tested in the Cort-Adaptation hypothesis. We sought to test this hypothesis in a colonial prey species, black-tailed prairie dogs (Cynomys ludovicianus). We predicted that individuals located near fewer neighboring conspecifics would perceive an increased risk of predation and, in turn, have increased GCs (measured through hair cortisol concentration (HCC)) and reduced annual reproductive success compared to more centrally located individuals. We also investigated other putative influences on HCC: age, lactation status, body condition, and season of hair growth. Levels of vigilance behavior were higher for those with fewer neighboring conspecifics, suggesting variation in perceived risk of predation. Further, the risk of predation appeared to represent a chronic, detrimental stressor as evidenced by a significant increase in HCC for prairie dogs with fewer neighbors. Lactation status and season also influenced HCC. We found support for the Cort-Adaptation hypothesis where increased HCC during the reproductive season correlated with whether a female produced a litter, but not litter size, suggesting a minimum threshold of GCs is required for successful reproduction in this species. Our work illustrates that HCC may operate as an indicator of perceived predation risk, but care should be taken to consider the variety of factors influencing GC homeostasis, in particular lactation, when drawing conclusions using HCC as a marker of long-term stress.

Differential selenium uptake by periphyton in boreal lake ecosystems.

The largest and most variable step of selenium (Se) assimilation into aquatic ecosystems is the rapid uptake of aqueous Se by primary producers. These organisms can transfer more harmful forms of Se to higher trophic levels via dietary pathways, although much uncertainty remains around this step of Se assimilation due to site-specific differences in water chemistry, hydrological and biogeochemical characteristics, and community composition. Thus, predictions of Se accumulation are difficult, and boreal lake systems are relatively understudied. To address these knowledge gaps, five static-renewal field experiments were performed to examine the bioaccumulation of low, environmentally relevant concentrations of Se, as selenite, by naturally grown periphyton from multiple boreal lakes. Periphyton rapidly accumulated Se at low aqueous Se concentrations, with tissue Se concentrations ranging from 8.0 to 24.9 µg/g dry mass (dm) in the 1-2 µg Se/L treatments. Enrichment functions ranged from 2870 to 12 536 L/kg dm in the 4 µg Se/L treatment, to 11 867-22 653 L/kg dm in the 0.5 µg Se/L treatment among lakes. Periphyton Se uptake differed among the five study lakes, with periphyton from mesotrophic lakes generally accumulating more Se than periphyton from oligotrophic lakes. Higher proportions of charophytes and greater dissolved inorganic carbon in more oligotrophic lakes corresponded to less periphyton Se uptake. Conversely, increased proportions of bacillariophytes and total dissolved phosphorus in more mesotrophic lakes corresponded to greater periphyton Se uptake. Periphyton community composition and water chemistry variables were correlated, limiting interpretation of differences in periphyton Se accumulation among lakes. The results of this research provide insight on the biodynamics of Se assimilation at the base of boreal lake food webs at environmentally relevant concentrations, which can potentially inform ecological risk assessments in boreal lake ecosystems in North America.

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.

Response of Crustacean Zooplankton and Benthic Macroinvertebrate Communities to Selenium Additions in a Boreal Lake.

Selenium (Se) is a contaminant of concern in Canada mainly due to its teratogenic effects on fish and birds. However, few studies have assessed the effects of Se on invertebrates in a field setting. The objective of this experiment was to assess potential community-level impacts of Se additions on zooplankton and benthic macroinvertebrates in a boreal lake ecosystem. From June to August 2018, Se (as selenite) was added to six limnocorrals in Lake 239 at the International Institute for Sustainable Development-Experimental Lakes Area, Northwestern Ontario, Canada, to achieve mean measured aqueous concentrations of 0.4, 0.8, 1.6, 3.4, 5.6 and 7.9 µg Se/L, with three untreated limnocorrals serving as controls (background Se = 0.08-0.09 µg/L). Periphyton, phytoplankton, and invertebrates (zooplankton and benthos) were monitored for 63 days. Zooplankton community composition shifted as a function of Se exposure, with Cladocera biomass and density decreasing with increasing Se concentrations. Similarly, cumulative abundance and biomass of Heptageniidae decreased with increasing Se treatment throughout the experimental period. The present study demonstrated that Se can have impacts on aquatic invertebrates at environmentally relevant exposure levels, and that future ecological risk assessments should consider the impacts of Se on both vertebrates and invertebrates. Environ Toxicol Chem 2022;41:95-107. © 2021 SETAC.

Protein biomarkers in serum as a conservation tool to assess reproduction: a case study on brown bears (Ursus arctos).

Monitoring the reproductive characteristics of a species can complement existing conservation strategies by understanding the mechanisms underlying demography. However, methodology to determine important aspects of female reproductive biology is often absent in monitoring programs for large mammals. Protein biomarkers may be a useful tool to detect physiological changes that are indicative of reproductive state. This study aimed to identify protein biomarkers of reproductive status in serum collected from free-ranging female brown bears (Ursus arctos) in Alberta, Canada, from 2001 to 2018. We hypothesized that the expression of proteins related to reproduction in addition to energetics and stress can be used to answer specific management-focused questions: (i) identify when a female is pregnant, (ii) detect if a female is lactating, (iii) determine age of sexual maturity (i.e. primiparity) and (iv) assess female fertility (i.e. reproduction rate). Furthermore, we investigated if silver spoon effects (favourable early life conditions provide fitness benefits through adulthood) could be determined using protein expression. A target panel of 19 proteins with established relationships to physiological function was measured by peptide-based analysis using liquid chromatography and multiple reaction monitoring mass spectrometry and their differential expression was evaluated using a Wilcoxon signed-rank test. We found biomarkers of pregnancy (apolipoprotein B-100 and afamin), lactation (apolipoprotein B-100 and alpha-2-macroglobulin) and sexual maturity (corticosteroid-binding globulin), but there were no statistically significant relationships with protein expression and fertility. The expression of proteins related to reproduction (afamin) and energetics (vitamin-D binding protein) was associated with the nutritional quality of the individual's present habitat rather than their early life habitat. This study highlights potential biomarkers of reproductive status and provides additional methods for monitoring physiological function in wildlife to inform conservation.

Effects of Wash Protocol and Contamination Level on Concentrations of Cortisol and Dehydroepiandrosterone (DHEA) in Swine Hair.

The effect of washing procedure and contamination level on the concentrations of cortisol and dehydroepiandrosterone (DHEA) in swine hair was explored over two studies. Hair shaved from finisher pigs (n = 8) and sows (n = 8, cortisol study 1 only) was split into two treatments (two hair samples/pig) to receive either three isopropanol or methanol washes, and two paired subsamples of hair were contaminated with feces and urine, mildly or severely. Samples were further subdivided and received one, three, or five methanol washes. Hormone concentrations were quantified from the hair and wash solvent, and the ratio of hormones in the solvent to that in the hair calculated. When grouping sow and grower hair together for analysis, hair cortisol concentrations were 13% greater after three isopropanol washes compared to methanol (22.84 ± 3.12 vs. 19.77 ± 2.64 pg/mg, respectively). When analyzing sow and grower hair separately, sow hair cortisol concentrations were 20% higher following three isopropanol washes compared to methanol washes (22.06 ± 5.21 vs. 27.72 ± 5.65 pg/mg), with no differences in grower pig hair concentrations. The solvent cortisol concentrations did not differ with wash solvent. No differences were seen for DHEA. Contamination level did not influence hormone concentrations. Hair cortisol concentrations were 24% higher after one wash compared to five washes (11.98 ± 1.47 vs. 9.05 ± 0.92 pg/mg), whereas the solvent cortisol concentrations were 80% and 84% higher after one wash compared to three and five washes, respectively (21.09 ± 4.04 vs. 4.21 ± 1.62 vs. 3.36 ± 1.32 pg/mg). The solvent-hair cortisol ratio was 65% and 73% higher following one wash compared to three and five washes (1.36 ± 0.80 vs. 0.47 ± 0.12 vs. 0.37 ± 0.14). Hair DHEA concentrations were 39% higher after one wash compared to five washes (42.39 ± 6.87 vs. 26.02 ± 5.69 pg/mg). The solvent DHEA concentrations, and the solvent-hair ratio for DHEA were 94% and 98% and 92% and 98% higher going from one wash to three and five washes, respectively (solvent: 5.07 ± 0.26 vs. 0.28 ± 0.12 vs. 0.12 ± 0.09 pg/mg and solvent-hair ratio: 0.13 ± 0.006 vs. 0.010 ± 0.004 vs. 0.003 ± 0.002). Following three methanol washes, the non-contaminated hair had 46% and 48% higher hair (17.47 ± 1.12 vs. 9.35 ± 0.80 vs. 9.05 ± 1.06 pg/mg) and a 76% and 72% higher solvent (16.31 ± 8.07 vs. 3.92 ± 0.50 vs. 4.50 ± 2.31 pg/mg) cortisol concentration compared to mild and severely contaminated hair, respectively. Wash solvent influences cortisol concentrations in swine hair, but not DHEA. Contaminated swine hair should be avoided in analyses when possible.

A Multi-Life Stage Comparison of Silver Nanoparticle Toxicity on the Early Development of Three Canadian Fish Species.

Information on the effects of silver nanoparticles (AgNPs) in fish has mostly been generated from standard laboratory species and short-term toxicity tests. However, there is significant uncertainty regarding AgNP toxicity to native species of concern in North America, particularly in northern freshwater ecosystems. We assessed the chronic toxicity of AgNPs in early life stages of three North American fish species: rainbow trout (Oncorhynchus mykiss), lake trout (Salvelinus namaycush), and northern pike (Esox lucius). Newly fertilized embryos were exposed to nominal aqueous concentrations of 0.1, 0.3, 1.0, 3.0, 10.0, or 30.0 nM AgNPs for 126 (rainbow trout), 210 (lake trout), and 25 (northern pike) days. Endpoints included cumulative developmental time (°C × day or degree-days to 50% life-stage transition), mortality, fork length, embryonic malformations, cumulative survival, and histopathology of gill and liver in larvae/alevins. The results showed life stage-specific differences in responses, with endpoints during the embryonic stage occurring more often and at lower concentrations compared to larval/alevin and juvenile stages. Sensitivities among species were highly dependent on the endpoints measured, although developmental time appeared to be the most consistent endpoint across species. At embryonic and larval/alevin stages, northern pike was the most sensitive species (lowest observable effect concentration of 0.1 nM using developmental time). Rainbow trout displayed similar responses to lake trout across multiple endpoints and therefore seems to be an adequate surrogate for trout species in ecotoxicology studies. Moreover, while mortality during individual life stages was not generally affected, the cumulative mortality across life stages was significantly affected, which highlights the importance of chronic, multi-life-stage studies. Environ Toxicol Chem 2021;40:3337-3350. © 2021 SETAC.

Correcting for enzyme immunoassay changes in long term monitoring studies.

Enzyme immunoassays (EIAs) are a common tool for measuring steroid hormones in wildlife due to their low cost, commercial availability, and rapid results. Testing technologies improve continuously, sometimes requiring changes in protocols or crucial assay components. Antibody replacement between EIA kits can cause differences in EIA sensitivity, which can hinder monitoring hormone concentration over time. The antibody in a common cortisol EIA kit used for long-term monitoring of stress in wildlife was replaced in 2014, causing differences in cross reactivity and standard curve concentrations. Therefore, the objective of this study was to develop a method to standardize results following changes in EIA sensitivity. We validated this method using cortisol concentrations measured in the hair of brown bears (Ursus arctos).•We used a simple linear regression to model the relationship between cortisol concentrations using kit 1 and kit 2.•We found a linear relationship between the two kits (R2 = 0.85) and used the regression equation (kit2 = (0.98 × kit1) + 1.65) to predict cortisol concentrations in re-measured samples.•Mean predicted percent error was 16% and 72% of samples had a predicted percent error <20%, suggesting that this method is well-suited for correcting changes in EIA sensitivity.

Landscape condition influences energetics, reproduction, and stress biomarkers in grizzly bears.

Environmental change has been shown to influence mammalian distribution, habitat use, and behavior; however, few studies have investigated the impact on physiological function. This study aimed to determine the influence of landscape condition on the expression of target proteins related to energetics, reproduction, and stress in grizzly bears. We hypothesized that changes in landscape condition explains protein expression. Skin biopsies were collected from free-ranging grizzly bears in Alberta, Canada from 2013-2019 (n = 86 individuals). We used an information theoretic approach to develop 11 a priori candidate generalized linear mixed models to explain protein expression. We compared models using Akaike Information Criteria (AICc) weights and averaged models with ΔAICc < 2 for each protein. Food resources, represented by increased distance to coal mines and decreased crown closure, positively influenced energetic proteins (adiponectin and alpha-1-acid glycoprotein). Proteins related to reproduction (ceruloplasmin and serpin B5) were positively associated with increased wetland and upland food resources in addition to movement, but negatively associated with increased distance to roads. One stress related protein, complement C3, was positively influenced by increased percent conifer. Given the need to detect emerging threats to wildlife, we suggest the assessment of physiological function will lead to improved monitoring of species in rapidly changing landscapes.

Cortisol levels in blood and hair of unanesthetized grizzly bears (Ursus arctos) following intravenous cosyntropin injection.

Hair cortisol concentration (HCC) is being used increasingly to evaluate long-term stress in many mammalian species. Most of the cortisol is assumed to passively diffuse from circulating blood into hair follicles and gradually accumulate in growing hair. However, our research with free-ranging grizzly bears (Ursus arctos) suggests HCC increases significantly within several hours following capture, a time too brief to be explained by this mechanism alone. In this study with captive grizzly bears, we sought to determine if a brief spike in blood cortisol concentration, thus mimicking a single stressful event, would cause an increase in HCC over a 7-day period. To do this, we administered a single intravenous dose (5 µg/kg) of cosyntropin to three captive unanaesthetised adult female grizzly bears on two occasions, during April when hair growth was arrested and during August when hair was growing. In both trials, the cosyntropin caused a two-fold or greater increase in serum cortisol levels within 1 hr but did not appear to influence HCC at 1, 48, and 168 hr following cosyntropin administration. We conclude the cosyntropin-induced cortisol spike was likely insignificant when compared to the adrenocortical response that occurs in free-ranging bears when captured. We suggest further study with a larger sample of captive bears to evaluate the combined effects of anaesthesia and multiple doses of cosyntropin administered over several hours would better simulate the adrenocortical response of free-ranging grizzly bears during capture.

Trophic dynamics of selenium in a boreal lake food web.

Selenium (Se) is both an essential micronutrient and a contaminant of concern that is of particular interest in mining-influenced waterbodies in Canada. The objective of this research was to characterize the trophic dynamics of selenium along a gradient of exposure concentrations in a Canadian boreal lake ecosystem. From June 20 to August 22, 2018, six limnocorrals (littoral, ∼3000 L enclosures) were spiked with mean measured concentrations of 0.4, 0.8, 1.6, 3.4, 5.6 and 7.9 µg Se/L as selenite, and three limnocorrals served as untreated controls (background aqueous Se = 0.08-0.09 µg/L). Total Se (TSe) concentrations in water, periphyton, phytoplankton, sediment, benthic macroinvertebrates, zooplankton and female finescale dace (Phoxinus neogaeus; added on day 21 of the experiment) were measured throughout and at the end of the experiment. Total Se bioaccumulation by organisms was generally non-linear. Greater uptake by phytoplankton than periphyton was observed. Taxonomic differences in accumulation of TSe by invertebrates (Heptageniidae = Chironomidae > zooplankton) were observed as well. Fish muscle and ovary tissue TSe bioaccumulation was more variable than that at lower trophic levels and uptake patterns indicated that fish did not reach steady state concentrations. This research provides field-derived models for the uptake of Se by algae and invertebrates, and contributes to a better understanding of the dynamics of TSe bioaccumulation over a gradient of exposure concentrations in cold-water lentic systems.

Development and validation of protein biomarkers of health in grizzly bears.

Large carnivores play critical roles in the maintenance and function of natural ecosystems; however, the populations of many of these species are in decline across the globe. Therefore, there is an urgent need to develop novel techniques that can be used as sensitive conservation tools to detect new threats to the health of individual animals well in advance of population-level effects. Our study aimed to determine the expression of proteins related to energetics, reproduction and stress in the skin of grizzly bears (Ursus arctos) using a liquid chromatography and multiple reaction monitoring mass spectrometry assay. We hypothesized that a suite of target proteins could be measured using this technique and that the expression of these proteins would be associated with biological (sex, age, sample location on body) and environmental (geographic area, season, sample year) variables. Small skin biopsies were collected from free-ranging grizzly bears in Alberta, Canada, from 2013 to 2019 (n = 136 samples from 111 individuals). Over 700 proteins were detected in the skin of grizzly bears, 19 of which were chosen as targets because of their established roles in physiological function. Generalized linear mixed model analysis was used for each target protein. Results indicate that sample year influenced the majority of proteins, suggesting that physiological changes may be driven in part by responses to changes in the environment. Season influenced the expression of proteins related to energetics, reproduction and stress, all of which were lower during fall compared to early spring. The expression of proteins related to energetics and stress varied by geographic area, while the majority of proteins that were affected by biological attributes (age class, sex and age class by sex interaction) were related to reproduction and stress. This study provides a novel method by which scientists and managers can further assess and monitor physiological function in wildlife.

Hair Cortisol Concentration and Body Mass in Moose (Alces alces) Infested with Deer Keds (Lipoptena cervi).

The deer ked (Lipoptena cervi), a hematophagous ectoparasite of cervids, is currently spreading in Scandinavia, and the moose (Alces alces) is its main host. However, little is known about the impact of deer keds on moose. We analyzed the hair cortisol concentration (HCC) from 262 moose harvested in the fall in relation to age class, sex, body mass (BM), and deer ked infestation intensity, and BM in relation to age class, sex, and infestation intensity. We found that HCC decreased with increasing deer ked intensity at low ked intensities, but for the higher levels of ked intensities, there was a positive relationship between HCC and ked intensity. The HCC was higher in males than in females and lower in yearlings than in calves and adults. Our failure to find any association between BM and deer ked intensity suggested a negligible impact of deer ked infestation on moose foraging and metabolism at the level of infestation observed early in the infestation, but did not exclude an effect later in winter. Our findings suggested that moose generally tolerated moderate parasitism by keds. However, the increase in HCC at higher ked intensities suggested that the tolerance strategy could be disrupted with further increases in intensities and consequently may negatively affect animal health and welfare.