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Infectious diseases are a major driver of the global amphibian decline. In addition, many factors, from genetics and stress to pollution and climate change, can influence the response to pathogens. Therefore, it is important to be able to evaluate amphibian immunity in the field and in the laboratory. The phytohemagglutinin (PHA) assay is an inexpensive and relatively non-invasive tool that has been used extensively to assess immunocompetence, especially in birds, and more recently in amphibians. However, there is substantial variation in experimental methodology among amphibian PHA studies in terms of species and life stages, PHA doses and injection sites, and use of experimental controls. Here, we compile and compare all known PHA studies in amphibians in order to identify knowledge gaps and develop best practices for future work. We found that research has only been conducted on a limited number of species, which may not reflect the diversity of amphibians as a whole. There is also a lack of validation studies in most species, so that doses and timing of PHA injection and subsequent swelling measurements may not effectively evaluate immunocompetence. Based on these and other findings, we put forward a set of recommendations to make future PHA studies more consistent and improve the ability to utilize this assay in wild populations, where immune surveillance is greatly needed.

Forest harvest is a primary landscape-scale management action affecting riparian forests. Although concerns about impacts of forest harvest on stream amphibians is generally limited to areas adjacent to harvest, there is a paucity of information regarding potential downstream effects of forest harvest on these species. We designed a before-after, control-impact (BACI) experiment to quantify potential impacts of clearcut logging that included 12-m buffers or smaller variable-width buffers on the distribution and abundance of headwater stream amphibians in adjacent and downstream areas. We sampled larval coastal tailed frogs (Ascaphus truei), coastal giant salamanders (Dicamptodon tenebrosus), and Columbia torrent salamanders (Rhyacotriton kezeri) across 3,915 sampling occasions that spanned 13 study reaches in 2008–2011 (pre-harvest) and 2013–2016 (post-harvest) as part of the Trask River Watershed Study in the Oregon Coast Range, U.S.A. We analyzed these data using occupancy models to estimate occupancy and (when possible) relative abundance, while accounting for various sources of imperfect detection. All species exhibited reduced occupancy adjacent to clearcuts with variable-width buffers (odds ratios [ORs] ranged = 0.24–0.48), and these negative impacts were not always diminished when increasing the buffer size to 12 m (ORs ranged = 0.20–3.56). Dicamptodon tenebrosus was the only species to have occupancy impacted in downstream areas, and this negative impact was related to clearcut logging with uniform 12-m buffers (OR = 0.60). This species was also the only species to have abundance negatively impacted by forest harvest in downstream areas (OR = https://0.41 with uniform 12-m buffers, OR = https://0.38 with variable-width buffers), albeit impacts to abundance were not evaluated for R. kezeri. Ascaphus truei abundance increased in areas downstream of clearcut logging with uniform 12-m buffers (OR = 2.92). Although we found the direction and magnitude of responses varied by species, our study confirms that clearcut logging can have negative impacts on amphibians that inhabit the adjacent stream areas. Perhaps more importantly, we also found that forest harvest can have negative effects on stream amphibians downstream of the harvested area and that increasing the buffer size to 12 m did not necessarily diminish these impacts in adjacent and downstream areas. Altogether, our study provides a nuanced picture of adjacent and downstream effects of forest harvest on three endemic headwater stream amphibians, and our findings demonstrate that forest management practices should consider downstream effects on aquatic taxa when assessing the impact of harvesting trees near headwater streams.

Mercury (Hg) is a toxic contaminant that has been mobilized and distributed worldwide and is a threat to many wildlife species. Amphibians are facing unprecedented global declines due to many threats, including contaminants. While the bi-phasic life history of many amphibians creates a potential nexus for methylmercury (MeHg) exposure in aquatic habitats and subsequent health effects, the broad-scale distribution of MeHg exposure in amphibians remains unknown. We used non-lethal sampling to assess MeHg bioaccumulation in 3,241 juvenile and adult amphibians during 2017–2021. We sampled 26 populations (14 species) across 11 states in the United States, including several imperiled species that could not have been sampled by traditional lethal methods. We examined whether life history traits of species and whether concentration of total mercury in sediment or dragonflies could be used as indicators of MeHg bioaccumulation in amphibians. Methylmercury contamination was widespread, with a 33-fold difference in concentrations across sites. Variation among years and clustered subsites was less than variation across sites. Life history characteristics such as size, sex, and whether the amphibian was a frog, toad, newt, or other salamander were the factors most strongly associated with bioaccumulation. Total Hg in dragonflies was a reliable indicator of bioaccumulation of MeHg in amphibians (R2 ? 0.67) whereas total Hg in sediment was not (R2 ? 0.04). Our study, the largest broadscale assessment of MeHg bioaccumulation in amphibians, highlights methodological advances that allow for non-lethal sampling of rare species and reveals immense variation among species, life histories, and sites. Our findings can help identify sensitive populations and provide environmentally relevant concentrations for future studies to better quantify potential threats of MeHg to amphibians.

Non-invasive methods are important to conservation physiology to reduce negative effects on the species being studied. Glucocorticoid (GC) hormones are often used to assess health of individuals, but collection methods can be invasive. Many amphibians are imperiled worldwide, and saliva is a non- or semi-invasive matrix to measure GCs that has been partially validated for only four amphibian species. Validation ensures that assays are reliable and can detect changes in saliva corticosterone (sCORT) following exposure to stressors, but it is also necessary to ensure sCORT concentrations are correlated with plasma concentrations. To help validate the use of saliva in assessing CORT responses in amphibians, we captured uniquely marked Columbia Spotted Frogs (Rana luteiventris) on sequential days and collected baseline and stress-induced (after handling) samples. For a subset of individuals, we collected and quantified CORT in both saliva and blood samples, which have not been compared for amphibians. We tested several aspects of CORT responses and, by collecting across separate days, measured repeatability of CORT responses across days. We also evaluated whether methods common to amphibian conservation, such as handling alone or handling, clipping a toe, and tagging elevated sCORT. Similar to previous studies, we show that sCORT is reliable concerning parallelism, recovery, precision, and sensitivity. Saliva CORT was weakly correlated with plasma CORT (R2 = 0.21), and we detected elevations in sCORT after handling, demonstrating biological validation. Toe-clipping and tagging did not increase sCORT over handling alone, but repeated handling elevated sCORT for ~72 hours. However, sCORT responses were highly variable and repeatability was low within individuals and among capture sessions, contrary to previous studies with urinary and waterborne CORT. Saliva CORT is a semi-invasive and rapid technique that could be useful to assess effects of anthropogenic change, and conservation efforts, but will require careful study design and future validation.

Interventions of host-pathogen dynamics provide strong tests of relationships, yet they are still rarely applied across multiple populations. After American Bullfrogs (Rana catesbeiana) invaded a wildlife refuge where federally threatened Chiricahua Leopard Frogs (R. chiricahuensis) were reintroduced 12 years prior, managers launched a landscape-scale eradication effort to help ensure continued recovery of the native species. We used a before-after-control-impact (BACI) design and environmental DNA sampling of 19 eradication sites and 18 control sites between fall 2016 and winter 2020–2021 to measure community-level responses to bullfrog eradication, including for 2 pathogens. Dynamic occupancy models revealed successful eradication from 94% of treatment sites. Native amphibians did not respond to bullfrog eradication, but the pathogens amphibian chytrid fungus (Batrachochytrium dendrobatidis) and ranaviruses were co-extirpated with bullfrogs. Our spatially replicated experimental approach provides strong evidence that management of invasive species can simultaneously reduce predation and disease risk for imperiled species.

Mercury (Hg) is a toxic contaminant that has been mobilized and distributed worldwide and is a threat to many wildlife species. Amphibians are facing unprecedented global declines due to many threats including contaminants. While the biphasic life history of many amphibians creates a potential nexus for methylmercury (MeHg) exposure in aquatic habitats and subsequent health effects, the broad-scale distribution of MeHg exposure in amphibians remains unknown. We used nonlethal sampling to assess MeHg bioaccumulation in 3,241 juvenile and adult amphibians during 2017–2021. We sampled 26 populations (14 species) across 11 states in the United States, including several imperiled species that could not have been sampled by traditional lethal methods. We examined whether life history traits of species and whether the concentration of total mercury in sediment or dragonflies could be used as indicators of MeHg bioaccumulation in amphibians. Methylmercury contamination was widespread, with a 33-fold difference in concentrations across sites. Variation among years and clustered subsites was less than variation across sites. Life history characteristics such as size, sex, and whether the amphibian was a frog, toad, newt, or other salamander were the factors most strongly associated with bioaccumulation. Total Hg in dragonflies was a reliable indicator of bioaccumulation of MeHg in amphibians (R2 ? 0.67), whereas total Hg in sediment was not (R2 ? 0.04). Our study, the largest broad-scale assessment of MeHg bioaccumulation in amphibians, highlights methodological advances that allow for nonlethal sampling of rare species and reveals immense variation among species, life histories, and sites. Our findings can help identify sensitive populations and provide environmentally relevant concentrations for future studies to better quantify the potential threats of MeHg to amphibians.

Amphibians are among the most sensitive taxa to climate change, and species inhabiting arid and semiarid landscapes at the extremes of their range are especially vulnerable to drought. The Jack Creek, Oregon, USA, population of Oregon spotted frogs (Rana pretiosa) faces unique challenges because it occupies the highest elevation site in the species' extant range and one that has been transformed by loss of American beavers (Castor canadensis[/]), which historically maintained open water. We evaluated the effects of drought mitigation (addition of excavated ponds) on relationships between local and regional water availability, inactive legacy beaver dams, and Oregon spotted frog population dynamics in the Jack Creek system. We conducted egg mass surveys and capture-mark-recapture sampling at a treatment reach with excavated ponds and 3 reference reaches over 13 years; surveys spanned a period before and after pond excavation at the treatment and 1 primary comparison reference reach. We analyzed data using a combination of robust design capture-mark-recapture estimators and generalized linear mixed models to characterize population dynamics. Adult Oregon spotted frog survival was approximately 19.5% higher at the treatment reach than the primary reference reach during the study period. Annual survival was most strongly associated with late summer vegetation greenness, a proxy for water availability, and males had higher survival than females. Among the 4 study reaches, the treatment reach consistently had higher late summer vegetation greenness, and the hydrology functioned more independently of regional precipitation patterns relative to the reference reaches; however, these dynamics were not linked to pond excavation. Breeding was concentrated in 2 legacy beaver ponds that were deepened by excavation during the study compared to an unexcavated beaver pond, 2 excavated ponds without legacy beaver dams, and 9 reference ponds. These results point to the benefit of enhancing existing beaver structures and indicate that management actions aimed at maintaining surface water for breeding in spring and saturated soils and ponded water for adults in late summer would benefit this unique population of Oregon spotted frogs in the face of drought.