Connecting the Pipes: Agricultural Tile Drains and Elevated Imidacloprid Brain Concentrations in Juvenile Northern Leopard Frogs (Rana pipiens).

Neonicotinoids are neurotoxic insecticides and are often released into nearby wetlands via subsurface tile drains and can negatively impact nontarget organisms, such as amphibians. Previous studies have indicated that imidacloprid, a commonly used neonicotinoid, can cross the amphibian blood-brain barrier under laboratory conditions; however, little is known about the impact of low concentrations in a field-based setting. Here, we report aqueous pesticide concentrations at wetland production areas that were either connected or not connected to agricultural tile drains, quantified imidacloprid and its break down products in juvenile amphibian brains and livers, and investigated the relationship between imidacloprid brain concentration and brain size. Imidacloprid concentrations in brain and water samples were nearly 2.5 and 5 times higher at tile wetlands (brain = 4.12 ± 1.92 pg/mg protein; water = 0.032 ± 0.045 µg/L) compared to reference wetlands, respectively. Tile wetland amphibians also had shorter cerebellums (0.013 ± 0.001 mm), depicting a negative relationship between imidacloprid brain concentration and cerebellum length. The metabolite, desnitro-imidacloprid, had liver concentrations that were 2 times higher at tile wetlands (2 ± 0.3 µg/g). Our results demonstrate that imidacloprid can cross the amphibian blood-brain barrier under ecological conditions and may alter brain dimensions and provide insight into the metabolism of imidacloprid in amphibians.

PREVALENCE OF RANAVIRUS IN SPOTTED SALAMANDER (AMBYSTOMA MACULATUM) LARVAE FROM CREATED VERNAL POOLS IN WEST VIRGINIA, USA.

Ranavirosis is a disease of high concern for amphibians due to widespread documentation of its lethal and sublethal impacts and its high transmission potential across populations and species. We investigated whether spotted salamander (Ambystoma maculatum) ranavirus prevalence and viral load were associated with habitat characteristics, genetic diversity, corticosterone levels, and body size. In 2015 and 2016, we sampled 34 recently created vernal pools in the Monongahela National Forest, West Virginia, USA. We collected tail clippings from 1,128 spotted salamander larvae and waterborne hormone samples from 436 of those larvae, along with eight environmental characteristics of the pools. Over the 2-yr period, we detected ranavirus in 62% of pools, with prevalence ranging from 0% to 63% (mean, 7.68%). Spotted salamander size was positively correlated with ranavirus presence and viral load; however, we did not find associations between ranavirus prevalence or viral load and habitat characteristics, spotted salamander genetic diversity, relatedness, effective number of breeders, or corticosterone levels. The widespread occurrence of ranavirus in the vernal pools illustrates the potential for rapid natural introduction of the pathogen to created wetlands. Managers could consider monitoring local distributions of ranavirus before creation of new vernal pools to guide strategic placement of the wetlands to minimize occurrence and prevalence of this pathogen.

Community development, implementation, and assessment of a NIBLSE bioinformatics sequence similarity learning resource.

As powerful computational tools and 'big data' transform the biological sciences, bioinformatics training is becoming necessary to prepare the next generation of life scientists. Furthermore, because the tools and resources employed in bioinformatics are constantly evolving, bioinformatics learning materials must be continuously improved. In addition, these learning materials need to move beyond today's typical step-by-step guides to promote deeper conceptual understanding by students. One of the goals of the Network for Integrating Bioinformatics into Life Sciences Education (NIBSLE) is to create, curate, disseminate, and assess appropriate open-access bioinformatics learning resources. Here we describe the evolution, integration, and assessment of a learning resource that explores essential concepts of biological sequence similarity. Pre/post student assessment data from diverse life science courses show significant learning gains. These results indicate that the learning resource is a beneficial educational product for the integration of bioinformatics across curricula.

Ranavirus is widespread in Costa Rica and co-occurs with threatened amphibians.

Amphibians are globally threatened by emerging infectious diseases, and ranaviruses are among the most concerning pathogens to threaten species in the wild. We sampled for ranaviruses in wild amphibians at 8 sites in Costa Rica, spanning broad climatic zones and taxonomic associations. Seven of these sites are inhabited by highly threatened amphibian species that persist at low global population sizes after population declines due to amphibian chytridiomycosis. One of the surveyed sites is occupied by an introduced amphibian species, which is relatively rare in Central America but may be an important pathway for long-distance transport of ranaviruses. We detected ranavirus using quantitative polymerase chain reaction in 16.3% of the 243 individuals and among 5 of our 8 sites, but not at the site with the introduced species. Infection prevalence varied among species and sites, but not with mean annual temperature or mean annual precipitation. Infection intensity did not vary with species, site, temperature, or precipitation. Our results show that ranavirus infection is spatially widespread in Costa Rica, affecting a broad range of host species, and occurs across climatic zones-though we encountered no mortality or morbidity in our sampled species. Ranaviruses are known to cause intermittent mass mortality in amphibian populations, and the threatened species sampled here are likely vulnerable to population impacts from emerging ranaviruses. Therefore, we believe the potential impacts of ranaviruses on amphibian populations in tropical regions have likely been underestimated, and that they should be viewed as a potential major stressor to threatened amphibians in tropical regions.

Genotyping and differential bacterial inhibition of Batrachochytrium dendrobatidis in threatened amphibians in Costa Rica.

Amphibians have declined around the world in recent years, in parallel with the emergence of an epidermal disease called chytridiomycosis, caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd). This disease has been associated with mass mortality in amphibians worldwide, including in Costa Rica, and Bd is considered an important contributor to the disappearance of this group of vertebrates. While many species are susceptible to the disease, others show tolerance and manage to survive infection with the pathogen. We evaluated the pathogen Bd circulating in Costa Rica and the capacity of amphibian skin bacteria to inhibit the growth of the pathogen in vitro. We isolated and characterized - genetically and morphologically - several Bd isolates from areas with declining populations of amphibians. We determined that the circulating chytrid fungus in Costa Rica belongs to the virulent strain Bd-GPL-2, which has been related to massive amphibian deaths worldwide; however, the isolates obtained showed genetic and morphological variation. Furthermore, we isolated epidermal bacteria from 12 amphibian species of surviving populations, some in danger of extinction, and evaluated their inhibitory activity against the collection of chytrid isolates. Through bioassays we confirmed the presence of chytrid-inhibitory bacterial genera in Costa Rican amphibians. However, we observed that the inhibition varied between different isolates of the same bacterial genus, and each bacterial isolation inhibited fungal isolation differently. In total, 14 bacterial isolates belonging to the genera Stenotrophomonas, Streptomyces, Enterobacter, Pseudomonas and Klebsiella showed inhibitory activity against all Bd isolates. Given the observed variation both in the pathogen and in the bacterial inhibition capacity, it is highly relevant to include local isolates and to consider the origin of the microorganisms when performing in vivo infection tests aimed at developing and implementing mitigation strategies for chytridiomycosis.

Serratia marcescens shapes cutaneous bacterial communities and influences survival of an amphibian host.

Ongoing investigations into the interactions between microbial communities and their associated hosts are changing how emerging diseases are perceived and ameliorated. Of the numerous host-microbiome-disease systems of study, the emergence of chytridiomycosis (caused by Batrachochytrium dendrobatidis, hereafter Bd) has been implicated in ongoing declines and extinction events of amphibians worldwide. Interestingly, there has been differential survival among amphibians in resisting Bd infection and subsequent disease. One factor thought to contribute to this resistance is the host-associated cutaneous microbiota. This has raised the possibility of using genetically modified probiotics to restructure the host-associated microbiota for desired anti-fungal outcomes. Here, we use a previously described strain of Serratia marcescens (Sm) for the manipulation of amphibian cutaneous microbiota. Sm was genetically altered to have a dysfunctional pathway for the production of the extracellular metabolite prodigiosin. This genetically altered strain (Δpig) and the functional prodigiosin producing strain (wild-type, WT) were compared for their microbial community and anti-Bd effects both in vitro and in vivo. In vitro, Bd growth was significantly repressed in the presence of prodigiosin. In vivo, the inoculation of both Sm strains was shown to significantly influence amphibian microbiota diversity with the Δpig-Sm treatment showing increasing alpha diversity, and the WT-Sm having no temporal effect on diversity. Differences were also seen in host mortality with Δpig-Sm treatments exhibiting significantly decreased survival probability when compared with WT-Sm in the presence of Bd. These results are an important proof-of-concept for linking the use of genetically modified probiotic bacteria to host microbial community structure and disease outcomes, which in the future may provide a way to ameliorate disease and address critical frontiers in disease and microbial ecology.

SEASONALITY IN BATRACHOCHYTRIUM DENDROBATIDIS DETECTION IN AMPHIBIANS IN CENTRAL OKLAHOMA, USA.

Chytridiomycosis, an infectious disease caused by the fungus Batrachochytrium dendrobatidis (chytrid or Bd), has not been well studied in Oklahoma. This is of particular concern regarding the connection between seasonality and chytrid infection. To further investigate this connection, chytrid prevalence and infection load were quantified within amphibians in central Oklahoma from March to October, across two sites in Oklahoma Co. and two sites in Cleveland Co. The results show a trend between seasonality and chytrid, with spring and fall showing higher prevalence and summer showing lower prevalence, which coincides closely with the preferred chytrid growth temperatures. Additionally, periods of high rainfall in May 2015 are linked to increased chytrid prevalence, as has been suggested by other research. Additionally, species exhibiting high chytrid prevalence follow the results of previous studies: Blanchard's cricket frog (Acris blanchardi), American bullfrog (Rana catesbeiana), and southern leopard frog (Rana sphenocephala).

Community richness of amphibian skin bacteria correlates with bioclimate at the global scale.

Animal-associated microbiomes are integral to host health, yet key biotic and abiotic factors that shape host-associated microbial communities at the global scale remain poorly understood. We investigated global patterns in amphibian skin bacterial communities, incorporating samples from 2,349 individuals representing 205 amphibian species across a broad biogeographic range. We analysed how biotic and abiotic factors correlate with skin microbial communities using multiple statistical approaches. Global amphibian skin bacterial richness was consistently correlated with temperature-associated factors. We found more diverse skin microbiomes in environments with colder winters and less stable thermal conditions compared with environments with warm winters and less annual temperature variation. We used bioinformatically predicted bacterial growth rates, dormancy genes and antibiotic synthesis genes, as well as inferred bacterial thermal growth optima to propose mechanistic hypotheses that may explain the observed patterns. We conclude that temporal and spatial characteristics of the host's macro-environment mediate microbial diversity.

Assessment of Bacterial Communities Associated With the Skin of Costa Rican Amphibians at La Selva Biological Station.

Amphibian skin is a suitable environment for rich communities of microorganisms, both beneficial and detrimental to the host. The amphibian cutaneous microbiota has been hypothesized to play an important role as symbionts, protecting their hosts against disease. Costa Rica has one of the most diverse assemblages of amphibians in the world and we know very little about the microbiota of these tropical animals. For comparison with other studies, we explore the diversity of the skin bacterial communities employing16S rRNA amplicon sequencing of swab samples from twelve species of frogs at La Selva Biological Station in Sarapiquí, Heredia province. The predominant phylum detected in our studies was Proteobacteria, followed by Bacteroidetes and Actinobacteria, with these three phyla representing 89.9% of the total bacterial taxa. At the family level, Sphingobacteriaceae and Comamonadaceae were highly represented among samples. Our results suggest that host species and host family are significant predictors of the variation in microbiota composition. This study helps set the foundation for future research about microbiota composition and resilience to unfavorable conditions, leading to improvement in managing strategies for endangered amphibian species.

Optimizing, validating, and field testing a multiplex qPCR for the detection of amphibian pathogens.

Amphibian populations worldwide are facing numerous threats, including the emergence and spread of infectious diseases. In the past 2 decades, Batrachochytrium dendrobatidis (Bd), a parasitic fungus, and a group of viruses comprising the genus Ranavirus have become widespread and resulted in mass mortality events and extirpations worldwide. In 2013, another novel fungus, B. salamandrivorans (Bsal), was attributed to dramatic declines in populations of fire salamander Salamandra salamandra in the Netherlands. Experimental infections demonstrated that Bsal is highly pathogenic to numerous salamander genera. In an effort to prevent the introduction of Bsal to North America, the US Fish and Wildlife Service (USFWS) listed 201 salamander species as injurious wildlife under the Lacey Act. To determine infection status and accurately assess amphibian health, the development of a sensitive and specific diagnostic assay was needed. We describe the optimization and validation of a multiplex quantitative polymerase chain reaction (qPCR) protocol for the simultaneous detection of Bd, Bsal, and frog virus 3-like ranaviruses. A synthetic genome template (gBlock®) containing the target genes from all 3 pathogens served as the positive control and allowed accurate quantification of pathogen genes. The assay was validated in the field using an established non-lethal swabbing technique to survey local amphibian populations throughout a range of habitats. This multiplex qPCR demonstrates high reproducibility, sensitivity, and was capable of detecting both Bd and ranavirus in numerous locations, species, and life stages. Bsal was not detected at any point during these sampling efforts.

Differential patterns of Batrachochytrium dendrobatidis infection in relict amphibian populations following severe disease-associated declines.

Global amphibian biodiversity has declined dramatically in the past 4 decades, and many amphibian species have declined to near extinction as a result of emergence of the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). However, persistent or recovering populations of several amphibian species have recently been rediscovered, and such populations may illustrate how amphibian species that are highly susceptible to chytridiomycosis may survive in the presence of Bd. We conducted field surveys for Bd infection in 7 species of Costa Rican amphibians (all species that have declined to near extinction but for which isolated populations persist) to characterize infection profiles in highly Bd-susceptible amphibians post-decline. We found highly variable patterns in infection, with some species showing low prevalence (~10%) and low infection intensity and others showing high infection prevalence (>80%) and either low or high infection intensity. Across sites, infection rates were negatively associated with mean annual precipitation, and infection intensity across sites was negatively associated with mean average temperatures. Our results illustrate that even the most Bd-susceptible amphibians can persist in Bd-enzootic ecosystems, and that multiple ecological or evolutionary mechanisms likely exist for host-pathogen co-existence between Bd and the most Bd-susceptible amphibian species. Continued monitoring of these populations is necessary to evaluate population trends (continuing decline, stability, or population growth). These results should inform efforts to mitigate impacts of Bd on amphibians in the field.

Detection of amphibian chytrid fungus on waterfowl integument in natural settings.

The chytrid fungus Batrachochytrium dendrobatidis (Bd), the causal agent of the amphibian disease chytridiomycosis, has spread at an alarming rate since its discovery. Bd was initially thought to only infect keratinizing epithelial cells in amphibians, a core component of amphibian skin. However, recent studies have detected Bd on the integument of non-amphibian hosts. We conducted a survey of 3 duck species (gadwalls, green-winged teals, and mallards) to determine whether Bd DNA could be found on their feet. Bd was found on the feet, by quantitative PCR, of individuals from all 3 species (5/11 gadwalls, 4/8 green-winged teals, and 13/21 mallards), though there were no significant differences in zoospore presence or load between species. We conclude that these waterfowl species may act as vector hosts for Bd, adding to the growing list of potential waterfowl vectors. Future studies are needed to determine whether Bd on waterfowl feet is viable and infectious to amphibian hosts.

Characterization of Batrachochytrium dendrobatidis Inhibiting Bacteria from Amphibian Populations in Costa Rica.

Global amphibian declines and extinction events are occurring at an unprecedented rate. While several factors are responsible for declines and extinction, the fungal pathogen Batrachochytrium dendrobatidis (Bd) has been cited as a major constituent in these events. While the effects of this chytrid fungus have been shown to cause broad scale population declines and extinctions, certain individuals and relict populations have shown resistance. This resistance has been attributed in part to the cutaneous bacterial microbiome. Here, we present the first study characterizing anti-Bd bacterial isolates from amphibian populations in Costa Rica, including the characterization of two strains of Serratia marcescens presenting strong anti-Bd activity. Transcriptome sequencing was utilized for delineation of shifts in gene expression of the two previously uncharacterized strains of S. marcescens grown in three different treatments comprising Bd, heat-killed Bd, and a no Bd control. These results revealed up- and down-regulation of key genes associated with different metabolic and regulatory pathways. This information will be valuable in continued efforts to develop a bacterial-based approach for amphibian protection as well as providing direction for continued mechanistic inquiries of the bacterial anti-Bd response.

Integrative behavioral ecotoxicology: bringing together fields to establish new insight to behavioral ecology, toxicology, and conservation.

The fields of behavioral ecology, conservation science, and environmental toxicology individually aim to protect and manage the conservation of wildlife in response to anthropogenic stressors, including widespread anthropogenic pollution. Although great emphasis in the field of toxicology has been placed on understanding how single pollutants affect survival, a comprehensive, interdisciplinary approach that includes behavioral ecology is essential to address how anthropogenic compounds are a risk for the survival of species and populations in an increasingly polluted world. We provide an integrative framework for behavioral ecotoxicology using Tinbergen's four postulates (causation and mechanism, development and ontogeny, function and fitness, and evolutionary history and phylogenetic patterns). The aims of this review are: 1) to promote an integrative view and re-define the field of integrative behavioral ecotoxicology; 2) to demonstrate how studying ecotoxicology can promote behavior research; and 3) to identify areas of behavioral ecotoxicology that require further attention to promote the integration and growth of the field.

Design- and model-based recommendations for detecting and quantifying an amphibian pathogen in environmental samples.

Accurate pathogen detection is essential for developing management strategies to address emerging infectious diseases, an increasingly prominent threat to wildlife. Sampling for free-living pathogens outside of their hosts has benefits for inference and study efficiency, but is still uncommon. We used a laboratory experiment to evaluate the influences of pathogen concentration, water type, and qPCR inhibitors on the detection and quantification of Batrachochytrium dendrobatidis (Bd) using water filtration. We compared results pre- and post-inhibitor removal, and assessed inferential differences when single versus multiple samples were collected across space or time. We found that qPCR inhibition influenced both Bd detection and quantification in natural water samples, resulting in biased inferences about Bd occurrence and abundance. Biases in occurrence could be mitigated by collecting multiple samples in space or time, but biases in Bd quantification were persistent. Differences in Bd concentration resulted in variation in detection probability, indicating that occupancy modeling could be used to explore factors influencing heterogeneity in Bd abundance among samples, sites, or over time. Our work will influence the design of studies involving amphibian disease dynamics and studies utilizing environmental DNA (eDNA) to understand species distributions.

Infection risk decreases with increasing mismatch in host and pathogen environmental tolerances.

The fungal pathogen Batrachochytrium dendrobatidis (Bd) has caused the greatest known wildlife pandemic, infecting over 500 amphibian species. It remains unclear why some host species decline from disease-related mortality whereas others persist. We introduce a conceptual model that predicts that infection risk in ectotherms will decrease as the difference between host and pathogen environmental tolerances (i.e. tolerance mismatch) increases. We test this prediction using both local-scale data from Costa Rica and global analyses of over 11 000 Bd infection assays. We find that infection prevalence decreases with increasing thermal tolerance mismatch and with increasing host tolerance of habitat modification. The relationship between environmental tolerance mismatches and Bd infection prevalence is generalisable across multiple amphibian families and spatial scales, and the magnitude of the tolerance mismatch effect depends on environmental context. These findings may help explain patterns of amphibian declines driven by a global wildlife pandemic.

Host species composition influences infection severity among amphibians in the absence of spillover transmission.

Wildlife epidemiological outcomes can depend strongly on the composition of an ecological community, particularly when multiple host species are affected by the same pathogen. However, the relationship between host species richness and disease risk can vary with community context and with the degree of spillover transmission that occurs among co-occurring host species. We examined the degree to which host species composition influences infection by Batrachochytrium dendrobatidis (Bd), a widespread fungal pathogen associated with amphibian population declines around the world, and whether transmission occurs from one highly susceptible host species to other co-occurring host species. By manipulating larval assemblages of three sympatric amphibian species in the laboratory, we characterized the relationship between host species richness and infection severity, whether infection mediates growth and survivorship differently across various combinations of host species, and whether Bd is transmitted from experimentally inoculated tadpoles to uninfected tadpoles. We found evidence of a dilution effect where Bd infection severity was dramatically reduced in the most susceptible of the three host species (Anaxyrus boreas). Infection also mediated survival and growth of all three host species such that the presence of multiple host species had both positive (e.g., infection reduction) and negative (e.g., mortality) effects on focal species. However, we found no evidence that Bd infection is transmitted by this species. While these results demonstrate that host species richness as well as species identity underpin infection dynamics in this system, dilution is not the product of reduced transmission via fewer infectious individuals of a susceptible host species. We discuss various mechanisms, including encounter reduction and antagonistic interactions such as competition and opportunistic cannibalism that may act in concert to mediate patterns of infection severity, growth, and mortality observed in multihost communities.

The effects of a fungicide and chytrid fungus on anuran larvae in aquatic mesocosms.

The amphibian disease chytridiomycosis, caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd), has been linked to significant amphibian declines over the past three decades. The most severe effects of the pathogen have been primarily observed in relatively pristine areas that are not affected by many anthropogenic factors.One hypothesis concerning improved amphibian persistence with Bd in disturbed landscapes is that contaminants may abate the effects of Bd on amphibians. Recent laboratory studies have shown that pesticides, specifically the fungicide thiophanate-methyl (TM), can kill Bd outside of hosts and clear Bd infections within hosts. Using aquatic mesocosms, we tested the hypothesis that TM (0.43 mg/L) would alter growth and development of Lithobates sphenocephalus (southern leopard frog) tadpoles and Bd-infection loads in infected individuals. We hypothesized that the scope of such alterations and infection clearing would be affected by aquatic community variables, specifically zooplankton. TM altered zooplankton diversity (reduced cladoceran and increased copepod and ostracod abundances) and caused mortality to all tadpoles in TM-exposed tanks. In TM-free tanks, Bd-exposed tadpoles in high-density treatments metamorphosed smaller than Bd-unexposed, effects that were reversed in low-density treatments. Our study demonstrates the potential adverse effects of a fungicide and Bd on tadpoles and aquatic systems.

Acute toxicity tests and meta-analysis identify gaps in tropical ecotoxicology for amphibians.

Amphibian populations are declining worldwide, particularly in tropical regions where amphibian diversity is highest. Pollutants, including agricultural pesticides, have been identified as a potential contributor to decline, yet toxicological studies of tropical amphibians are very rare. The present study assesses toxic effects on amphibians of 10 commonly used commercial pesticides in tropical agriculture using 2 approaches. First, the authors conducted 8-d toxicity assays with formulations of each pesticide using individually reared red-eyed tree frog (Agalychnis callidryas) tadpoles. Second, they conducted a review of available data for the lethal concentration to kill 50% of test animals from the US Environmental Protection Agency's ECOTOX database to allow comparison with their findings. Lethal concentration estimates from the assays ranged over several orders of magnitude. The nematicides terbufos and ethoprophos and the fungicide chlorothalonil were very highly toxic, with evident effects within an order of magnitude of environmental concentrations. Acute toxicity assays and meta-analysis show that nematicides and fungicides are generally more toxic than herbicides yet receive far less research attention than less toxic herbicides. Given that the tropics have a high diversity of amphibians, the findings emphasize the need for research into the effects of commonly used pesticides in tropical countries and should help guide future ecotoxicological research in tropical regions.