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Detecting pathogens in the live animal trade is critical for tracking and preventing their movement, introduction and spillover into susceptible fauna. However, the scale of the live animal trade makes individually testing animals infeasible for all but the most economically important taxa. For instance, while the fungal pathogen,Batrachochytrium salamandrivorans(Bsal), threatens amphibian, particularly caudate diversity, in Europe and the Americas, screening even a fraction of the millions of live amphibians imported into the United States, alone, is impractically laborious and expensive. A promising alternative to individual‐level sampling (e.g. swabbing the skin of salamanders) is to instead collect DNA from the animals' environment (e.g. housing container or water) which allows us to screen a whole group of animals at a time.

We used a series of experiments withBsal‐spiked water and substrates and experimentally infected rough‐skinned newts (Taricha granulosa) to determine which methods yield the mostBsalenvironmental DNA (eDNA) and evaluate the capacity of these methods to detectBsal‐infected animals in conditions found in captive settings and trade.

We found that filtering water housing infected animals for even an hour can consistently recover detectable levels ofBsaleDNA, that there is little evidence ofBsaleDNA being clumped in housing containers or swamped or inhibited by dirty housing containers, and that eDNA‐based methods achieves an equivalent or higher chance of detectingBsalinfections in a (virtual) population of co‐housed newts with fewer samples than individual swabs.

By sampling the genetic materials accumulated from a whole group of animals, eDNA‐based methods are a powerful means of detecting pathogens, such asBsal, in shipments and captive populations. These methods bring routine pathogen surveillance into reach in many more contexts and can thus be an important tool in conservation and disease control.

 

Salinity (sodium chloride, NaCl) from anthropogenic sources is a persistent contaminant that negatively affects freshwater taxa. Amphibians can be susceptible to salinity, but some species are innately or adaptively tolerant. Physiological mechanisms mediating tolerance to salinity are still unclear, but changes in osmoregulatory hormones such as corticosterone (CORT) and aldosterone (ALDO) are prime candidates. We exposed larval barred tiger salamanders (Ambystoma mavortium) to environmentally relevant NaCl treatments (<32–4000 mg·L⁻¹) for 24 days to test effects on growth, survival, and waterborne CORT responses. Of those sampled, we also quantified waterborne ALDO from a subset. Using a glucocorticoid antagonist (RU486), we also experimentally suppressed CORT signaling of some larvae to determine if CORT mediates effects of salinity. There were no strong differences in survival among salinity treatments, but salinity reduced dry mass, snout–vent length, and body condition while increasing water content of larvae. High survival and sublethal effects demonstrated that salamanders were physiologically challenged but could tolerate the experimental concentrations. CORT signaling did not attenuate sublethal effects of salinity. Baseline and stress‐induced (after an acute stressor, shaking) CORT were not influenced by salinity. ALDO was correlated with baseline CORT, suggesting it could be difficult to decouple the roles of CORT and ALDO. Future studies comparing ALDO and CORT responses of adaptively tolerant and previously unexposed populations could be beneficial to understand the roles of these hormones in tolerance to salinity. Nevertheless, our study enhances our understanding of the roles of corticosteroid hormones in mediating effects of a prominent anthropogenic stressor.

 

Greater knowledge of how host–microbiome interactions vary with anthropogenic environmental change and influence pathogenic infections is needed to better understand stress-mediated disease outcomes. We investigated how increasing salinization in freshwaters (e.g. due to road de-icing salt runoff) and associated increases in growth of nutritional algae influenced gut bacterial assembly, host physiology and responses to ranavirus exposure in larval wood frogs (Rana sylvatica). Elevating salinity and supplementing a basic larval diet with algae increased larval growth and also increased ranavirus loads. However, larvae given algae did not exhibit elevated kidney corticosterone levels, accelerated development or weight loss post-infection, whereas larvae fed a basic diet did. Thus, algal supplementation reversed a potentially maladaptive stress response to infection observed in prior studies in this system. Algae supplementation also reduced gut bacterial diversity. Notably, we observed higher relative abundances of Firmicutes in treatments with algae—a pattern consistent with increased growth and fat deposition in mammals—that may contribute to the diminished stress responses to infection via regulation of host metabolism and endocrine function. Our study informs mechanistic hypotheses about the role of microbiome mediation of host responses to infection that can be tested in future experiments in this host–pathogen system 

In mammals, the cytokine hormone leptin promotes wound healing by increasing inflammation, cellular recruitment, angiogenic regrowth, and re-epithelialization; however, it is not known whether leptin has conserved actions on wound healing in other vertebrates. Here, we tested the hypothesis that leptin promotes both the quality and speed of wound healing in the South African clawed frog, Xenopus laevis . First, fluorescent immunohistochemistry using a polyclonal antibody specific to Xenopus leptin showed that in juvenile dorsal skin, leptin protein is expressed in the dorsal epidermal layer, as well in blood vessel endothelial cells and sensory nerves that run along the base of the dermis. Injection of recombinant Xenopus leptin (rXleptin) stimulates phosphorylated STAT3 (pSTAT3), indicative of leptin-activated JAK/STAT signaling in the epidermis. Similar to mammals, leptin protein expression increases at the wound site after injury of the epidermis. We then cultured “punch-in-a-punch” full-thickness dorsal skin explants in three doses of rXleptin (0, 10, and 100 ng/ml) and showed that leptin treatment doubled the rate of wound closure after 48 h relative to skin punches cultured without leptin. Food restriction prior to wound explant culture reduced the amount of wound closure, but leptin injection prior to euthanasia rescued closure to similar control levels. Leptin treatment also significantly reduced bacterial infection of these epidermal punches by 48 h in culture. This study shows that leptin is likely an endogenous promoter of wound healing in amphibians. Leptin-based therapies have the potential to expedite healing and reduce the incidence of secondary infections without toxicity issues, the threat of antibiotic resistance, or environmental antibiotic contamination. The conservation of leptin’s actions on wound healing also suggests that it may have similar veterinary applications for other exotic species. 

Ranaviruses are emerging pathogens that have caused mortality events in amphibians worldwide. Despite the negative effects of ranaviruses on amphibian populations, monitoring efforts are still lacking in many areas, including in the Prairie Pothole Region (PPR) of North America. Some PPR wetlands in Montana and North Dakota (USA) have been contaminated by energy-related saline wastewaters, and increased salinity has been linked to greater severity of ranavirus infections. In 2017, we tested tissues from larvae collected at 7 wetlands that ranged in salinity from 26 to 4103 mg Cl l^-1. In 2019, we used environmental DNA (eDNA) to test for ranaviruses in 30 wetlands that ranged in salinity from 26 to 11754 mg Cl l^-1. A previous study (2013-2014) found that ranavirus-infected amphibians were common across North Dakota, including in some wetlands near our study area. Overall, only 1 larva tested positive for ranavirus infection, and we did not detect ranavirus in any eDNA samples. There are several potential reasons why we found so little evidence of ranaviruses, including low larval sample sizes, mismatch between sampling and disease occurrence, larger pore size of our eDNA filters, temporal variation in outbreaks, low host abundance, or low occurrence or prevalence of ranaviruses in the wetlands we sampled. We suggest future monitoring efforts be conducted to better understand the occurrence and prevalence of ranaviruses within the PPR.

 

Abstract Why do some biological systems and communities persist while others fail? Robustness, a system's stability, and resilience, the ability to return to a stable state, are key concepts that span multiple disciplines within and outside the biological sciences. Discovering and applying common rules that govern the robustness and resilience of biological systems is a critical step toward creating solutions for species survival in the face of climate change, as well as the for the ever-increasing need for food, health, and energy for human populations. We propose that network theory provides a framework for universal scalable mathematical models to describe robustness and resilience and the relationship between them, and hypothesize that resilience at lower organization levels contribute to robust systems. Insightful models of biological systems can be generated by quantifying the mechanisms of redundancy, diversity, and connectivity of networks, from biochemical processes to ecosystems. These models provide pathways towards understanding how evolvability can both contribute to and result from robustness and resilience under dynamic conditions. We now have an abundance of data from model and non-model systems and the technological and computational advances for studying complex systems. Several conceptual and policy advances will allow the research community to elucidate the rules of robustness and resilience. Conceptually, a common language and data structure that can be applied across levels of biological organization needs to be developed. Policy advances such as cross-disciplinary funding mechanisms, access to affordable computational capacity, and the integration of network theory and computer science within the standard biological science curriculum will provide the needed research environments. This new understanding of biological systems will allow us to derive ever more useful forecasts of biological behaviors and revolutionize the engineering of biological systems that can survive changing environments or disease, navigate the deepest oceans, or sustain life throughout the solar system. 

Ranaviruses are emerging pathogens of poikilothermic vertebrates. In 2015 the Global Ranavirus Reporting System (GRRS) was established as a centralized, open access, online database for reports of the presence (and absence) of ranavirus around the globe. The GRRS has multiple data layers (e.g., location, date, host(s) species, and methods of detection) of use to those studying the epidemiology, ecology, and evolution of this group of viruses. Here we summarize the temporal, spatial, diagnostic, and host-taxonomic patterns of ranavirus reports in the GRRS. The number, distribution, and host diversity of ranavirus reports have increased dramatically since the mid 1990s, presumably in response to increased interest in ranaviruses and the conservation of their hosts, and also the availability of molecular diagnostics. Yet there are clear geographic and taxonomic biases among the reports. We encourage ranavirus researchers to add their studies to the portal because such collation can provide collaborative opportunities and unique insights to our developing knowledge of this pathogen and the emerging infectious disease that it causes.

 

The stress-induced susceptibility hypothesis, which predicts chronic stress weakens immune defences, was proposed to explain increasing infectious disease-related mass mortality and population declines. Previous work characterized wetland salinization as a chronic stressor to larval amphibian populations. Thus, we combined field observations with experimental exposures quantifying epidemiological parameters to test the role of salinity stress in the occurrence of ranavirus-associated mass mortality events. Despite ubiquitous pathogen presence (94%), populations exposed to salt runoff had slightly more frequent ranavirus related mass mortality events, more lethal infections, and 117-times greater pathogen environmental DNA. Experimental exposure to chronic elevated salinity (0.8–1.6 g l −1 Cl − ) reduced tolerance to infection, causing greater mortality at lower doses. We found a strong negative relationship between splenocyte proliferation and corticosterone in ranavirus-infected larvae at a moderate elevation of salinity, supporting glucocorticoid-medicated immunosuppression, but not at high salinity. Salinity alone reduced proliferation further at similar corticosterone levels and infection intensities. Finally, larvae raised in elevated salinity had 10 times more intense infections and shed five times as much virus with similar viral decay rates, suggesting increased transmission. Our findings illustrate how a small change in habitat quality leads to more lethal infections and potentially greater transmission efficiency, increasing the severity of ranavirus epidemics. 

Mechanistic models are critical for our understanding of both within-host dynamics (i.e., pathogen replication and immune system processes) and among-host dynamics (i.e., transmission). Within-host models, however, are not often fit to experimental data, which can serve as a robust method of hypothesis testing and hypothesis generation. In this study, we use mechanistic models and empirical, time-series data of viral titer to better understand the replication of ranaviruses within their amphibian hosts and the immune dynamics that limit viral replication. Specifically, we fit a suite of potential models to our data, where each model represents a hypothesis about the interactions between viral replication and immune defense. Through formal model comparison, we find a parsimonious model that captures key features of our time-series data: The viral titer rises and falls through time, likely due to an immune system response, and that the initial viral dosage affects both the peak viral titer and the timing of the peak. Importantly, our model makes several predictions, including the existence of long-term viral infections, which can be validated in future studies.