One Health Approach to Globalizing, Accelerating, and Focusing Amphibian and Reptile Disease Research-Reflections and Opinions from the First Global Amphibian and Reptile Disease Conference.

The world's reptiles and amphibians are experiencing dramatic and ongoing losses in biodiversity, changes that can have substantial effects on ecosystems and human health. In 2022, the first Global Amphibian and Reptile Disease Conference was held, using One Health as a guiding principle. The conference showcased knowledge on numerous reptile and amphibian pathogens from several standpoints, including epidemiology, host immune defenses, wild population effects, and mitigation. The conference also provided field experts the opportunity to discuss and identify the most urgent herpetofaunal disease research directions necessary to address current and future threats to reptile and amphibian biodiversity.

Encouraging news for in situ conservation: Translocation of salamander larvae has limited impacts on their skin microbiota.

The key role of symbiotic skin bacteria communities in amphibian resistance to emerging pathogens is well recognized, but factors leading to their dysbiosis are not fully understood. In particular, the potential effects of population translocations on the composition and diversity of hosts' skin microbiota have received little attention, although such transfers are widely carried out as a strategy for amphibian conservation. To characterize the potential reorganization of the microbiota over such a sudden environmental change, we conducted a common-garden experiment simulating reciprocal translocations of yellow-spotted salamander larvae across three lakes. We sequenced skin microbiota samples collected before and 15 days after the transfer. Using a database of antifungal isolates, we identified symbionts with known function against the pathogen Batrachochytrium dendrobatidis, a major driver of amphibian declines. Our results indicate an important reorganization of bacterial assemblages throughout ontogeny, with strong changes in composition, diversity and structure of the skin microbiota in both control and translocated individuals over the 15 days of monitoring. Unexpectedly, the diversity and community structure of the microbiota were not significantly affected by the translocation event, thus suggesting a strong resilience of skin bacterial communities to environmental change-at least across the time-window studied here. A few phylotypes were more abundant in the microbiota of translocated larvae, but no differences were found among pathogen-inhibiting symbionts. Taken together, our results support amphibian translocations as a promising strategy for this endangered animal class, with limited impact on their skin microbiota.

Lost reproductive value reveals a high burden of juvenile road mortality in a long-lived species.

Adult mortality is often the most sensitive vital rate affecting at-risk wildlife populations. Therefore, road ecology studies often focus on adult mortality despite the possibility for roads to be hazardous to juvenile individuals during natal dispersal. Failure to quantify concurrent variation in mortality risk and population sensitivity across demographic states can mislead the efforts to understand and mitigate the effects of population threats. To compare relative population impacts from road mortality among demographic classes, we weighted mortality observations by applying reproductive value analysis to quantify expected stage-specific contributions to population growth. We demonstrate this approach for snapping turtles (Chelydra serpentina) observed on roads at two focal sites in Ontario, Canada, where we collected data for both live and dead individuals observed on roads. We estimated reproductive values using stage-classified matrix models to compare relative population-level impacts of adult and juvenile mortality. Reproductive value analysis is a tractable approach to assessing demographically variable effects for applications covering large spatial scales, nondiscrete populations, or where abundance data are lacking. For one site with long-term life-history data, we compared demographic frequency on roads to expected general population frequencies predicted by the matrix model. Our application of reproductive value is sex specific but, as juvenile snapping turtles lack external secondary sex characters, we estimated the sex ratio of road-crossing juveniles after dissecting and sexing carcasses collected on roads at five sites across central Ontario, Canada. Juveniles were more abundant on roads than expected, suggesting a substantial dispersal contribution, and the road-killed juvenile sex ratio approached 1:1. A higher proportion of juveniles were also found dead compared with adults, and cumulative juvenile mortality had similar population-level importance as adult mortality. This suggests that the impact of roads needs to be considered across all life stages, even in wildlife species with slow life histories, such as snapping turtles, that are particularly sensitive to adult mortality.

Frog Virus 3 Genomes Reveal Prevalent Recombination between Ranavirus Lineages and Their Origins in Canada.

Ranaviruses are pathogens associated with the decline of amphibian populations across much of their distribution. In North America, frog virus 3 (FV3) is a widely distributed pathogen with wild populations of amphibians harboring different lineages and putative recombinants between FV3 and common midwife toad virus (CMTV). These recombinants have higher pathogenicity, and CMTV-derived genes associated with virulence are reported in wild strains in Canada. However, while FV3 is linked to amphibian die-offs in North America, CMTVs have been reported only in commercial frog farms in North America. We sequenced complete genomes of 18 FV3 isolates from three amphibian species to characterize genetic diversity of the lineages in Canada and infer possible recombinant regions. The 18 FV3 isolates displayed different signals of recombination, varying from none to interspersed recombination with previously isolated CMTV-like viruses. In general, most recombination breakpoints were located within open reading frames (ORFs), generating new ORFs and proteins that were a mixture between FV3 and CMTV. A combined spatial and temporal phylogeny suggests the presence of the FV3 lineage in Canada is relatively contemporary (<100 years), corroborating the hypothesis that both CMTV- and FV3-like viruses spread to North America when the international commercial amphibian trade started. Our results highlight the importance of pathogen surveillance and viral dynamics using full genomes to more clearly understand the mechanisms of disease origin and spread.IMPORTANCE Amphibian populations are declining worldwide, and these declines have been linked to a number of anthropogenic factors, including disease. Among the pathogens associated with amphibian mortality, ranaviruses have caused massive die-offs across continents. In North America, frog virus 3 (FV3) is a widespread ranavirus that can infect wild and captive amphibians. In this study, we sequenced full FV3 genomes isolated from frogs in Canada. We report widespread recombination between FV3 and common midwife toad virus (CMTV). Phylogenies indicate a recent origin for FV3 in Canada, possibly as a result of international amphibian trade.

Spatiotemporal heterogeneity decouples infection parameters of amphibian chytridiomycosis.

Emerging infectious diseases are responsible for declines in wildlife populations around the globe. Mass mortality events associated with emerging infectious diseases are often associated with high number of infected individuals (prevalence) and high pathogen loads within individuals (intensity). At the landscape scale, spatial and temporal variation in environmental conditions can alter the relationship between these infection parameters and blur the overall picture of disease dynamics. Quantitative estimates of how infection parameters covary with environmental heterogeneity at the landscape scale are scarce. If we are to identify wild populations at risk of disease epidemics, we must elucidate the factors that shape, and potentially decouple, the link between pathogen prevalence and intensity of infection over complex ecological landscapes. Using a network of 41 populations of the amphibian host Rana pipiens in Ontario, Canada, we present the spatial and temporal heterogeneity in pathogen prevalence and intensity of infection of the chytrid fungus Batrachochytrium dendrobatidis (Bd), across a 3-year period. We then quantify how covariation between both infection parameters measured during late summer is modified by previously experienced spatiotemporal environmental heterogeneity across 14 repeat sampled populations. Late summer Bd infection parameters are governed, at least in part, by different environmental factors operating during separate host life-history events. Our results provide evidence for a relationship between Bd prevalence and thermal regimes prior to host breeding at the site level, and a relationship between intensity of infection and aquatic conditions (precipitation, hydroshed size and river density) throughout host breeding period at the site level. This demonstrates that microclimatic variation within temporal windows can drive divergent patterns of pathogen dynamics within and across years, by effecting changes in host behaviour which interfere with the pathogen's ability to infect and re-infect hosts. A clearer understanding of the role that spatiotemporal heterogeneity has upon infection parameters will provide valuable insights into host-pathogen epidemiology, as well as more fundamental aspects of the ecology and evolution of interspecific interactions.

Low intraspecific variation of Frog virus 3 with evidence for novel FV3-like isolates in central and northwestern Canada.

Frog virus 3 (FV3) and FV3-like ranaviruses can infect a variety of cold-blooded aquatic species and present a primary threat to amphibians across the globe. Previous studies of FV3-like viruses have largely investigated higher-level phylogenetic distinctions of these pathogens via portions of the conserved major capsid protein (MCP), and the putative virulence gene vIF-2α. Few studies, however, have investigated the spatial distribution of FV3 variants at the population level3-data that can be used to further understand the spatial epidemiology of this disease. In this study, we sequenced the MCP and vIF-2α of 127 FV3-positive amphibians sampled from Canadian water bodies in Ontario, northeastern Alberta, and southern Northwest Territories to explore whether intraspecific genetic variation exists within FV3. There was a lack of variation at the 2 markers across these regions, suggesting that there is a lack of FV3 sequence diversity in Canada, which may hint at a single source of infection that has spread. However, an undocumented variant termed Wood Buffalo ranavirus (WBRV) was detected in samples from 3 sites in Alberta and Northwest Territories that clustered within the FV3-like lineage with 99.3% sequence homology for MCP. For vIF-2α, all sequences were the expected truncated variant except for 6 samples in Ontario. These latter sequences were suggestive of recombination with common midwife toad virus (CMTV). The lack of variation suggests that higher-resolution genome analyses will be required to further explore the spatial spread and intraspecific variation of the disease.

Mining-caused changes to habitat structure affect amphibian and reptile population ecology more than metal pollution.

Emissions from smelting not only contaminate water and soil with metals, but also induce extensive forest dieback and changes in resource availability and microclimate. The relative effects of such co-occurring stressors are often unknown, but this information is imperative in developing targeted restoration strategies. We assessed the role and relative effects of structural alterations of terrestrial habitat and metal pollution caused by century-long smelting operations on amphibian and reptile communities by collecting environmental and time- and area-standardized multivariate abundance data along three spatially replicated impact gradients. Overall, species richness, diversity, and abundance declined progressively with increasing levels of metals (As, Cu, and Ni) and soil temperature (T(s)) and decreasing canopy cover, amount of coarse woody debris (CWD), and relative humidity (RH). The composite habitat variable (which included canopy cover, CWD, T(s), and RH) was more strongly associated with most response metrics than the composite metal variable (As, Cu, and Ni), and canopy cover alone explained 19-74% of the variance. Moreover, species that use terrestrial habitat for specific behaviors (e.g., hibernation, dispersal), especially forest-dependent species, were more severely affected than largely aquatic species. These results suggest that structural alterations of terrestrial habitat and concomitant changes in the resource availability and microclimate have stronger effects than metal pollution per se. Furthermore, much of the variation in response metrics was explained by the joint action of several environmental variables, implying synergistic effects (e.g., exacerbation of metal toxicity by elevated temperatures in sites with reduced canopy cover). We thus argue that the restoration of terrestrial habitat conditions is a key to successful recovery of herpetofauna communities in smelting-altered landscapes.

Amphibian pathogens at northern latitudes: presence of chytrid fungus and ranavirus in northeastern Canada.

Infections by the fungal pathogen Batrachochytrium dendrobatidis (Bd) and members of the genus Ranavirus (Rv) are increasingly reported as significant determinants of amphibian population die-offs. The complexity associated with their transmission and spatial distribution leads to an increase in demand for comprehensive reporting systems and global mapping of their distribution. Here, we document the distribution of these 2 pathogens in a remote northern temperate lowland where environmental sensitivity is high, providing important insight into the pathogens' natural history and infection patterns. Wood frog Lithobates sylvaticus tissues were collected from the James Bay area in northeastern Canada and were screened for the presence of Bd and Rv using conventional and real-time PCR. Both pathogens were present in the study area, which is the northernmost record in eastern North America. Interestingly, different patterns of distribution were observed between the eastern and western coasts of James Bay, suggesting differences in the spatial and transmission dynamics for each pathogen. Anthropogenic introduction may still influence the distribution patterns observed, even at these latitudes. The presence of infections in this remote area also raises further questions on the risk these pathogens pose to northern amphibian communities. We encourage further research in remote locations for a better understanding of these pathogens, their transmission dynamics, and especially their respective impacts on amphibian populations worldwide.

Complete genome analysis of a frog virus 3 (FV3) isolate and sequence comparison with isolates of differing levels of virulence.

BACKGROUND: Frog virus 3 (FV3) is the type species of the genus Ranavirus, and in the past few decades, FV3 infections have resulted in considerable morbidity and mortality in a range of wild and cultivated amphibian species in the Americas, Europe, and Asia. The reasons for the pathogenicity of FV3 are not well understood. FINDINGS: We investigated three FV3 isolates designated SSME, wt-FV3, and aza-Cr, and reported that our wt-FV3 and aza-Cr strains showed similar levels of virulence, while SSME was the least virulent in an in vivo study with Lithiobates pipiens tadpoles. Using 454 GS-FLX sequencing technology, we sequenced SSME and compared it to the published wt-FV3 genome. SSME had multiple amino acid deletions in ORFs 49/50L, 65L, 66L, and 87L, which may explain its reduced virulence. We also investigated repeat regions and found that repeat copy number differed between isolates, with only one group of 3 isolates and 1 pair of isolates being identical at all 3 locations. CONCLUSIONS: In this study we have shown that genetic variability is present between closely related FV3 isolates, both in terms of deletions/insertions, and even more so at select repeat locations. These genomic areas with deletions/insertions may represent regions that affect virulence, and therefore require investigation. Furthermore, we have identified repeat regions that may prove useful in future phylogeographical tracking and identification of ranaviral strains across different environmental regions.

Strong restructuration of skin microbiota during captivity challenges ex-situ conservation of amphibians.

In response to the current worldwide amphibian extinction crisis, conservation instances have encouraged the establishment of ex-situ collections for endangered species. The resulting assurance populations are managed under strict biosecure protocols, often involving artificial cycles of temperature and humidity to induce active and overwintering phases, which likely affect the bacterial symbionts living on the amphibian skin. However, the skin microbiota is an important first line of defense against pathogens that can cause amphibian declines, such as the chytrid Batrachochytrium dendrobatidis (Bd). Determining whether current husbandry practices for assurance populations might deplete amphibians from their symbionts is therefore essential to conservation success. Here, we characterize the effect of the transitions from the wild to captivity, and between aquatic and overwintering phases, on the skin microbiota of two newt species. While our results confirm differential selectivity of skin microbiota between species, they underscore that captivity and phase-shifts similarly affect their community structure. More specifically, the translocation ex-situ is associated with rapid impoverishment, decrease in alpha diversity and strong species turnover of bacterial communities. Shifts between active and overwintering phases also cause changes in the diversity and composition of the microbiota, and on the prevalence of Bd-inhibitory phylotypes. Altogether, our results suggest that current husbandry practices strongly restructure the amphibian skin microbiota. Although it remains to be determined whether these changes are reversible or have deleterious effects on their hosts, we discuss methods to limit microbial diversity loss ex-situ and emphasize the importance of integrating bacterial communities to applied amphibian conservation.

Ranavirus Amplification in Low-Diversity Amphibian Communities.

In an era where emerging infectious diseases are a serious threat to biodiversity, epidemiological patterns need to be identified, particularly the complex mechanisms driving the dynamics of multi-host pathogens in natural communities. Many amphibian species have faced unprecedented population declines associated with diseases. Yet, specific processes shaping host-pathogen relationships within and among communities for amphibian pathogens such as ranaviruses (RV) remain poorly understood. To address this gap, we conducted a comprehensive study of RV in low-diversity amphibian communities in north-western Canada to assess the effects of biotic factors (species identity, species richness, abundance) and abiotic factors (conductivity, pH) on the pathogen prevalence and viral loads. Across 2 years and 18 sites, with communities of up to three hosts (wood frog, Rana sylvatica; boreal chorus frog, Pseudacris maculata; Canadian toad, Anaxyrus hemiophrys), we observed that RV prevalence nearly doubled with each additional species in a community, suggesting an amplification effect in aquatic, as well as terrestrial life-history stages. Infection intensity among infected wood frogs and boreal chorus frogs also significantly increased with an increase in species richness. Interestingly, we did not observe any effects of host abundance or abiotic factors, highlighting the importance of including host identity and species richness when investigating multi-host pathogens. Ultimately, only such a comprehensive approach can improve our understanding of complex and often highly context-dependent host-pathogen interactions.

Effects of two amphibian pathogens on the developmental stability of green frogs.

Developmental instability, measured as fluctuating asymmetry (FA), is often used as a tool to measure stress and the overall quality of organisms. Under FA, it is assumed that control of symmetry during development is costly and that under stress the trajectory of development is disturbed, resulting in asymmetric morphologies. Amphibian emergent infectious diseases (EIDs), such as Ranavirus and chytrid fungus, have been involved in several mortality events, which makes them stressors and allows for the study of FA. We analyzed nine populations of green frogs (Rana clamitans) for the presence or absence of Ranavirus and chytrid fungus. Individuals were measured to determine levels of FA in seven traits under the hypothesis that FA is more likely to be observed in individuals infected by the pathogens. Significantly higher levels of FA were found in individuals with Ranavirus compared with uninfected individuals among all populations and all traits. We did not observe FA in individuals infected with chytrid fungus for any of the traits measured. Additionally, we observed a significant association between Ranavirus infection and levels of FA in both males and females, which may indicate this viral disease is likely to affect both sexes during development. Altogether, our results indicate that some EIDs may have far-reaching and nonlethal effects on individual development and populations harboring such diseases and that FA can be used as a conservation tool to identify populations subject to such a stress.

Environmental and Host Effects on Skin Bacterial Community Composition in Panamanian Frogs.

Research on the amphibian skin microbiota has focused on identifying bacterial taxa that deter a pathogenic chytrid fungus, and on describing patterns of microbiota variation. However, it remains unclear how environmental variation affects amphibian skin bacterial communities, and whether the overall functional diversity of the amphibian skin microbiota is associated to such variation. We sampled skin microbial communities from one dendrobatoid frog species across an environmental gradient along the Panama Canal, and from three dendrobatoid frog species before and after the onset of the wet season in one site. We found frog skin microbial alpha diversity to be highest in frogs from sites with low soil pH, but no clear effect of the onset of the wet season. However, we found frog skin microbial community structure to be affected by soil pH and the onset of the wet season, which also resulted in a decrease in between-sample variation. Across the sampled frog species, bacterial functional groups changed with the onset of the wet season, with certain bacterial functional groups entirely disappearing and others differing in their relative abundances. In particular, we found the proportion of Bd-inhibitory bacteria to correlate with mean soil pH, and to increase in two of the frog species with the onset of the wet season. Taken together, our results suggest that structure and predicted function of amphibian bacterial skin communities may be influenced by environmental variables such as pH and precipitation, site effects, and host effects.

Copper and nickel effects on survival and growth of northern leopard frog (Lithobates pipiens) tadpoles in field-collected smelting effluent water.

Trace metals can have subtle yet chronic impacts on organisms by inducing physiological stress that reduces their survival or impedes their ability to tolerate additional environmental stressors. The toxicity literature indicates, however, that aquatic organisms react differently to trace metals depending on the environments in which they reside. The objective of the present study was to understand the response of northern leopard frog (Lithobates pipiens) larvae to ionic copper (Cu), nickel (Ni), and their combination within an effluent water collected downstream of a tailings wetland area. Tadpoles were assigned randomly to 1 of 8 Cu concentrations (8-200 µg/L), 7 Ni concentrations (160-1200 µg/L), or 8 Cu and Ni combined concentrations (8:160-200:1200 µg/L) and showed significant differences in survival and life history traits among treatments. In the Cu and Cu and Ni combined treatments, tadpole survival decreased with increased Cu exposure starting at Cu = 160 µg/L and in the Ni treatment, tadpole survival decreased with increased Ni exposure starting at Ni = 650 µg/L. All Cu-exposed treatments induced a growth increase as the concentration increased, whereas the tadpoles showed a significant decrease in growth rate in Ni treatments. These contrasting outcomes suggest a plastic response to trace metals whereby tadpoles allocate energy reserves toward either escaping or coping with stress. Finally, the authors' argue that future studies will benefit from examining the impacts of multiple stressors in aquatic ecosystems to provide better environmental mitigation.

Mitigating reptile road mortality: fence failures compromise ecopassage effectiveness.

Roadways pose serious threats to animal populations. The installation of roadway mitigation measures is becoming increasingly common, yet studies that rigorously evaluate the effectiveness of these conservation tools remain rare. A highway expansion project in Ontario, Canada included exclusion fencing and ecopassages as mitigation measures designed to offset detrimental effects to one of the most imperial groups of vertebrates, reptiles. Taking a multispecies approach, we used a Before-After-Control-Impact study design to compare reptile abundance on the highway before and after mitigation at an Impact site and a Control site from 1 May to 31 August in 2012 and 2013. During this time, radio telemetry, wildlife cameras, and an automated PIT-tag reading system were used to monitor reptile movements and use of ecopassages. Additionally, a willingness to utilize experiment was conducted to quantify turtle behavioral responses to ecopassages. We found no difference in abundance of turtles on the road between the un-mitigated and mitigated highways, and an increase in the percentage of both snakes and turtles detected dead on the road post-mitigation, suggesting that the fencing was not effective. Although ecopassages were used by reptiles, the number of crossings through ecopassages was lower than road-surface crossings. Furthermore, turtle willingness to use ecopassages was lower than that reported in previous arena studies, suggesting that effectiveness of ecopassages may be compromised when alternative crossing options are available (e.g., through holes in exclusion structures). Our rigorous evaluation of reptile roadway mitigation demonstrated that when exclusion structures fail, the effectiveness of population connectivity structures is compromised. Our project emphasizes the need to design mitigation measures with the biology and behavior of the target species in mind, to implement mitigation designs in a rigorous fashion, and quantitatively evaluate road mitigation to ensure allow for adaptive management and optimization of these increasingly important conservation tools.

Sex-biased predation by polecats influences the mating system of frogs.

In agile frogs, Rana dalmatina, an increase in male-biased operational sex ratio and in male abundance results in the emergence of alternative male mating behaviour in the form of searching. As a consequence, females are coerced into mating with multiple males, which in turn increases the level of conflict between the sexes. Selective predation on males by the European polecat, Mustela putorius, decreases the occurrence of polyandry. In ponds visited by polecats, the sex ratio is less male biased than in ponds where polecats are absent. As a result most males call to attract females and fewer males actively search for females. Females are able to choose between calling males and mate with a single male. Thus, predation by polecats is found to influence sex ratio, male abundance and sexual conflict in a frog mating system, restricting the opportunity for multiple mating.

Inbreeding and road effect zone in a Ranidae: the case of Agile frog, Rana dalmatina Bonaparte, 1840.

Inbreeding has often been invoked in the extinction of local populations. In eleven western France populations of Agile frog studied, observed heterozygosity was significantly lower than expected in all cases, giving new evidence of such a depression in small populations. It especially occurred in ponds located near an highway rather than in undisturbed populations (FIS = 0.544 and 0.315, respectively). Thus, our results argue for a "road effect zone". Discussing about road distance and conservation policies, we propose that roads are directly involved in inbreeding and in local extinction. Thus, road construction ought to consider conservation management.

Frog alien species: a way for genetic invasion?

European water frogs are characterized by anthropic introductions and Rana ridibunda may be considered as an invasive species. As such translocations may result in introgression of exotic genes in native populations, i.e. genetic pollution, we studied genetic characteristics (on 11 allozymic loci) of natural versus introduced water frogs. Our study contributed to (1) disclose 3 genetic markers allowing the identification of exotic frogs; (2) quantify the proportion of exotic frogs found in natural populations; and (3) suggest how genetic pollution may arise in these frogs.

A novel technique to measure chronic levels of corticosterone in turtles living around a major roadway.

Conservation biology integrates multiple disciplines to expand the ability to identify threats to populations and develop mitigation for these threats. Road ecology is a branch of conservation biology that examines interactions between wildlife and roadways. Although the direct threats of road mortality and habitat fragmentation posed by roads have received much attention, a clear understanding of the indirect physiological effects of roads on wildlife is lacking. Chronic physiological stress can lower immune function, affect reproductive rates and reduce life expectancy; thus, it has the potential to induce long-lasting effects on populations. Reptiles are globally in decline, and roads are known to have negative effects on reptile populations; however, it is unknown whether individual responses to roads and traffic result in chronic stress that creates an additional threat to population viability. We successfully extracted reliable measures of corticosterone (CORT), a known, commonly used biomarker for physiological stress, from claw trimmings from painted turtles (Chrysemys picta) captured at three study sites (road-impacted site, control site and validation site). Corticosterone levels in claws were evaluated as a measure of chronic stress in turtles because CORT is deposited during growth of the claw and could provide an opportunity to examine past long-term stress levels. While male turtles had higher CORT levels on average than females, there was no difference in the level of CORT between the road-impacted and control site, nor was there a relationship between CORT and turtle body condition. In validating a novel approach for non-invasive measurement of long-term CORT levels in a keratinized tissue in wild reptiles, our study provides a new avenue for research in the field of stress physiology.