Leopard Frog Research Articles

Broad-scale predictions of herpetofauna occupancy and colonization in an agriculturally dominated landscape.

Predictions of species occurrence allow land managers to focus conservation efforts on locations where species are most likely to occur. Such analyses are rare for herpetofauna compared to other taxa, despite increasing evidence that herptile populations are declining because of landcover change and habitat fragmentation. Our objective was to create predictions of occupancy and colonization probabilities for 15 herptiles of greatest conservation need in Iowa. From 2006-2014, we surveyed 295 properties throughout Iowa for herptile presence using timed visual-encounter surveys, coverboards, and aquatic traps. Data were analyzed using robust design occupancy modeling with landscape-level covariates. Occupancy ranged from 0.01 (95% CI = -0.01, 0.03) for prairie ringneck snake (Diadophis punctatus arnyi) to 0.90 (95% CI = 0.898, 0.904) for northern leopard frog (Lithobates pipiens). Occupancy for most species correlated to landscape features at the 1-km scale. General patterns of species' occupancy included negative effects of agricultural features and positive effects of water features on turtles and frogs. Colonization probabilities ranged from 0.007 (95% CI = 0.006, 0.008) for spiny softshell turtle (Apalone spinifera) to 0.82 (95% CI = 0.62, 1.0) for western fox snake (Pantherophis ramspotti). Colonization probabilities for most species were best explained by effects of water and grassland landscape features. Predictive models had strong support (AUC > 0.70) for six out of 15 species (40%), including all three turtles studied. Our results provide estimates of occupancy and colonization probabilities and spatial predictions of occurrence for herptiles of greatest conservation need across the state of Iowa.

Correction: Genetic assessment and monitoring of wild, captive, and reintroduced northern leopard frog populations

Correction: Genetic assessment and monitoring of wild, captive, and reintroduced northern leopard frog populations

Diverse Relationships between Batrachochytrium Infections and Antimicrobial Peptide Defenses Across Leopard Frog Populations.

Antimicrobial peptides (AMPs) play a fundamental role in the innate defense against microbial pathogens, as well as other immune and non-immune functions. Their role in amphibian skin defense against the pathogenic fungus Batrachochytrium dendrobatidis (Bd) is exemplified by experiments in which depletion of host's stored AMPs increases mortality from infection. Yet, the question remains whether there are generalizable patterns of negative or positive correlations between stored AMP defenses and the probability of infection or infection intensity across populations and species. This study aims to expand on prior field studies of AMP quantities and compositions by correlating stored defenses with an estimated risk of Bd exposure (prevalence and mean infection intensity in each survey) in five locations across the United States and a total of three species. In all locations, known AMPs correlated with the ability of recovered secretions to inhibit Bd in vitro. We found that stored AMP defenses were generally unrelated to Bd infection except in one location where the relative intensity of known AMPs was lower in secretions from infected frogs. In all other locations, known AMP relative intensities were higher in infected frogs. Stored peptide quantity was either positively or negatively correlated with Bd exposure risk. Thus, future experiments coupled with organismal modeling can elucidate whether Bd infection affects secretion/synthesis and will provide insight into how to interpret amphibian ecoimmunology studies of AMPs. We also demonstrate that future AMP isolating and sequencing studies can focus efforts by correlating mass spectrometry peaks to inhibitory capacity using linear decomposition modeling.

Anuran occupancy varies with stream characteristics and flow across Arizona wilderness areas

Abstract Riparian ecosystems comprise less than 2% of the landscape in the arid western United States, yet they provide habitat and resources to over half of arid‐land wildlife species, including a broad diversity of anurans (frogs and toads). Despite this, relatively few studies describe riparian herpetofaunal communities in the Southwest, and remote wilderness areas are particularly undersurveyed. We employed passive acoustic monitoring to capture anuran advertisement calls in wilderness area tributaries of the Verde River, Arizona, USA. In the spring and summer of 2021 and 2022, 13–29 autonomous recording units (ARUs) were deployed along perennial, intermittent and ephemeral tributary stream reaches. We characterized sites based on the per cent of pool, riffle, run and side channel habitat within 100 m of each ARU and quantified substrate, wetted channel width and canopy cover. To relate anuran occupancy to environmental and hydrological variables, we evaluated acoustic detection data using single‐species, single‐season occupancy models. Four species were detected in this study: canyon treefrog (Hyla arenicolor), red‐spotted toad (Anaxyrus punctatus), Woodhouse's toad (Anaxyrus woodhousii) and non‐native American bullfrog (Lithobates catesbeianus), with canyon treefrog being the most ubiquitous species observed. Vocal activity of canyon treefrog was higher at perennial and intermittent sites compared to ephemeral sites, and the presence of pools was most strongly associated with canyon treefrog occupancy. Persistent, slow‐moving water best predicted the presence of breeding canyon treefrogs. Notably, this study did not detect several species with historical records in the middle Verde River catchment, including Arizona toad (Anaxyrus microscaphus) and northern leopard frog (Lithobates pipiens). Given climate change‐related flow declines and intensifying demands for water in arid lands globally, maintaining stream flows that provide consistent and suitable hydroregimes for anuran breeding and larval development is of increasing importance. Determining habitat use and flow regimes necessary to support anuran populations can aid in prioritization of conservation actions related to water management and predict how changes in water availability may impact stream‐breeding anurans.

Effects of perfluoroalkyl sulfonic acids on developmental, physiological, and immunological measures in northern leopard frog tadpoles

The chronic toxicity of short chain perfluoroalkyl sulfonic acids (PFSAs), such as perfluorobutanesulfonic acid (PFBS) and perfluorohexanesulfonic acid (PFHxS), are relatively understudied despite the increasing detection of these compounds in the environment. We investigated the chronic toxicity and bioconcentration of PFBS and PFHxS using northern leopard frog (Rana [Lithobates] pipiens) tadpoles. We exposed Gosner stage (GS) 25 tadpoles to either PFBS or PFHxS at nominal concentrations of 0.1, 1, 10, 100, and 1000 μg/L until metamorphosis (GS42). We then assessed tadpole growth, development, stress, and immune metrics, and measured fatty acid (FA) composition and PFSA concentrations in liver and whole-body tissues. Tadpole growth and development measures were relatively unaffected by PFSA exposure. However, tadpoles exposed to 1000 μg/L PFBS or PFHxS had significantly increased hepatosomatic indexes (HSI) relative to controls. Further, tadpoles from the 1000 μg/L PFHxS treatment had altered FA profiles relative to controls, with increased total FAs, saturated FAs, monounsaturated FAs, and omega-6 polyunsaturated FAs. In addition, tadpoles from the 1000 μg/L PFHxS treatment had a higher probability of waterborne corticosterone detection. These results suggest that PFBS and PFHxS influence the hepatic health of tadpoles, and that PFHxS may alter lipid metabolism in tadpoles. We also observed a higher probability of tadpoles being phenotypically female after exposure to an environmentally relevant concentration (0.1 μg/L) of PFHxS, suggesting that PFHxS may exert endocrine disrupting effects on tadpoles during early development. The measured bioconcentration factors (BCFs) for both compounds were ≤10 L kg−1 wet weight, suggesting low bioconcentration potential for PFBS and PFHxS in tadpoles. Many of the significant effects observed in this study occurred at concentrations several orders of magnitude above those measured in the environment; however, our work shows effects of PFSAs exposure on amphibians and provides essential information for ecological risk assessments of these compounds.

Responses of Showy Leopard Frog Larvae (Lithobates spectabilis) to the Presence of Introduced Rainbow Trout in Mexico

Responses of Showy Leopard Frog Larvae (<scp><i>Lithobates spectabilis</i></scp>) to the Presence of Introduced Rainbow Trout in Mexico

Discovery, development and optimisation of a novel frog antimicrobial peptide with combined mode of action against drug-resistant bacteria

Antimicrobial peptides (AMP) have emerged as promising candidates for addressing the clinical challenges posed by the rapid evolution of antibiotic-resistant microorganisms. Brevinins, a representative frog-derived AMP family, exhibited broad-spectrum antimicrobial activities, attacking great attentions in previous studies. However, their strong haemolytic activity and cytotoxicity, greatly limit their further development. In this work, we identified and characterised a novel brevinin-1 peptide, brevinin-1pl, from the skin secretions of the northern leopard frog, Rana pipiens. Like many brevinins, brevinin-1pl also displayed strong haemolytic activity, resulting in a lower therapeutic index. We employed several bioinformatics tools to analyse the structure and potential membrane interactions of brevinin-1pl, leading to a series of modifications. Among these analogues, des-Ala16-[Lys4]brevinin-1pl exhibited great enhanced therapeutic efficacy in both in vitro and in vivo tests, particularly against some antibiotics-resistant Escherichia coli strains. Mechanistic studies suggest that des-Ala16-[Lys4]brevinin-1pl may exert bactericidal effects through multiple mechanisms, including membrane disruption and DNA binding. Consequently, des-Ala16-[Lys4]brevinin-1pl holds promise as a candidate for the treatment of drug-resistant Escherichia coli infections.

Effects of amphibian genetic diversity on ecological communities.

The amount of genetic diversity within a population can affect ecological processes at population, community, and ecosystem levels. However, the magnitude, consistency, and scope of these effects are largely unknown. To investigate these issues, we conducted two experiments manipulating the amount of genetic diversity and environmental factors in larval amphibians. The first experiment manipulated wood frog genetic diversity, the presence or absence of caged predators, and competition from leopard frogs to test whether these factors affected survival, growth, and morphology of wood frogs and leopard frogs. The second experiment manipulated wood frog genetic diversity, the presence or absence of uncaged predators, and resource abundance to test whether these factors affected wood frog traits (survival, morphology, growth, development, and behavior) and other components of the ecological community (zooplankton abundance, phytoplankton, periphyton, and bacterial community structure). Genetic diversity did not affect wood frog survival, growth, and development in either experiment. However, genetic diversity did affect the mean morphology of wood frog tadpoles in the first experiment and the abundance and distribution of zooplankton in the second experiment. It did not affect phytoplankton abundance, periphyton abundance, or bacterial community structure. While effect sizes (Cohen's d) of genetic diversity were approximately half those of environment treatments, the greatest effect sizes were for interaction effects between genetic diversity and environment. Our results indicate that genetic diversity can have a large effect on ecological processes, but the direction of those effects is highly dependent upon environmental conditions, and not easily predicted from simple measures of traits.

Genetic assessment and monitoring of wild, captive, and reintroduced northern leopard frog populations

The northern leopard frog (Lithobates pipiens) has undergone dramatic declines in population size and range over recent decades in western Canada and the United States. In British Columbia, only a single population remains at the Creston Valley Wildlife Management Area. Yet, the continuing viability of this population is uncertain. In this paper, the current genetic structure of northern leopard frog populations in western Canada was assessed using microsatellite markers. Historical samples from the extinct population of Fort Steele in British Columbia were compared with the Creston Valley population to understand changes in population genetic parameters over time. Genotypic data from four populations (Creston Valley, Drain K, Prince Spring, and Cypress Hill) sampled in 2004 and 2019 were compared. To evaluate changes in the genetic diversity of the Creston Valley population over time, allelic richness and expected heterozygosity of the population were compared at three time points using genotypes from 2000, 2004, and 2019. Northern leopard frog populations in western Canada showed high genetic differentiation, with genetic diversity decreasing from east to west. Although there weren’t notable changes in genetic parameters between 2004 and 2019, there was evidence of a decline in diversity between 2000 and 2019. The extinct population of Fort Steele had private alleles, while the current Creston Valley population did not, suggesting a genetic bottleneck in the Creston Valley population. Therefore, genetic rescue, specifically for the endangered Creston Valley population, can be considered as an action to support recovery. Additionally, continued genetic monitoring will help in the effective management of the species by providing information on the success of conservation actions.

Compatibility of eugenol anesthesia with classroom physiology experiments on nerve and muscle of frogs

Frogs serve as valuable model organisms for studying physiological responses of nerve, skeletal muscle and the heart in undergraduate biology labs. Heretofore, induced hypothermia has been the preferred technique for pain and stress reduction prior to euthanasia by pithing. However, AVMA guidelines discourage hypothermia for restraint or euthanasia of amphibians and advocate anesthetic overdose as preferable to pithing alone. Unfortunately, the primary anesthetic used in fish and other aquatic invertebrates, MS-222, blocks normal nerve and muscle function in leopard frogs and renders them useless for classroom physiology experiments, as demonstrated by Medler (2019). Eugenol is a safe and affordable anesthetic that is effective in amphibians. We sought to test the hypothesis that eugenol anesthesia is compatible with classroom nerve and muscle experiments on frogs. Bullfrogs and leopard frogs were immersed in a bath of eugenol suspended in tap water at 275 to 350 mg/L. At various time points, a four step reflex test was performed including tests of righting response, superficial pain reflex, deep pain reflex, and corneal blink reflex. Once all reflexes were absent, the frog was euthanized by pithing and the gastrocnemius muscle was prepared for force recordings. The sciatic nerve was stimulated with a bipolar stimulating electrode while gastrocnemius force was measured using an isometric force transducer. The threshold voltage eliciting a muscle contraction and the peak isometric twitch force were recorded at 15 minute intervals for 2 hours. Control frogs were sedated by immersion in ice water for 20 to 30 minutes, pithed when reflexes were substantially weakened, and then subjected to the same stimulation protocol and force measurements. At these doses, eugenol reliably induces loss of reflexes in 20 to 35 minutes and neuromuscular preparations maintain the ability to respond to electrical stimulation. Preliminary results suggest that eugenol may produce a modest elevation of threshold voltage and a modest reduction in maximum twitch force compared to control frogs euthanized without chemical anesthesia. Eugenol may be an effective anesthetic to prepare bullfrogs and leopard frogs for classroom experiments on nerve and muscle. Funding was provided by the Bill and Linda Frost Fund. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Assessing the Combined Effects of Host and Parasite Exposure to Forever Chemicals in an Amphibian-Echinostome System.

Per- and polyfluoroalkyl substances (PFAS) are environmental contaminants of growing concern due to their potential negative effects on wildlife and human health. Per- and polyfluoroalkyl substances have been shown to alter immune function in various taxa, which could influence the outcomes of host-parasite interactions. To date, studies have focused on the effects of PFAS on host susceptibility to parasites, but no studies have addressed the effects of PFAS on parasites. To address this knowledge gap, we independently manipulated exposure of larval northern leopard frogs (Rana pipiens) and parasites (flatworms) via their snail intermediate host to environmentally relevant PFAS concentrations and then conducted trials to assess host susceptibility to infection, parasite infectivity, and parasite longevity after emergence from the host. We found that PFAS exposure to only the host led to no significant change in parasite load, whereas exposure of parasites to a 10-µg/L mixture of PFAS led to a significant reduction in parasite load in hosts that were not exposed to PFAS. We found that when both host and parasite were exposed to PFAS there was no difference in parasite load. In addition, we found significant differences in parasite longevity post emergence following exposure to PFAS. Although some PFAS-exposed parasites had greater longevity, this did not necessarily translate into increased infection success, possibly because of impaired movement of the parasite. Our results indicate that exposure to PFAS can potentially impact host-parasite interactions. Environ Toxicol Chem 2024;43:1537-1546. © 2024 SETAC.

Something in the water: aquatic microbial communities influence the larval amphibian gut microbiota, neurodevelopment and behaviour.

Microorganisms colonize the gastrointestinal tract of animals and establish symbiotic host-associated microbial communities that influence vertebrate physiology. More specifically, these gut microbial communities influence neurodevelopment through the microbiota-gut-brain (MGB) axis. We tested the hypothesis that larval amphibian neurodevelopment is affected by the aquatic microbial community present in their housing water. Newly hatched Northern Leopard Frog (Lithobates pipiens) tadpoles were raised in pond water that was unmanipulated (natural) or autoclaved. Tadpoles raised in autoclaved pond water had a gut microbiota with reduced bacterial diversity and altered community composition, had decreased behavioural responses to sensory stimuli, were larger in overall body mass, had relatively heavier brains and had altered brain shape when compared with tadpoles raised in natural pond water. Further, the diversity and composition of the gut microbiota were associated with tadpole behavioural responses and brain measurements. Our results suggest that aquatic microbial communities shape tadpole behaviour and brain development, providing strong support for the occurrence of the MGB axis in amphibians. Lastly, the dramatic role played by aquatic microbial communities on vertebrate neurodevelopment and behaviour should be considered in future wildlife conservation efforts.

The effects of two short-chain perfluoroalkyl carboxylic acids (PFCAs) on northern leopard frog (Rana pipiens) tadpole development

Short-chain perfluoroalkyl carboxylic acids (PFCAs) have been detected in the environment globally. The presence and persistence of these compounds in the environment may lead to chronic wildlife exposure. We used northern leopard frog (Rana pipiens) tadpoles to investigate the chronic toxicity and the bioconcentration of two short-chain PFCAs, perfluorobutanoic acid (PFBA) and perfluorohexanoic acid (PFHxA). We exposed Gosner stage 25 tadpoles to PFBA and PFHxA (as individual chemicals) at nominal concentrations of 0.1, 1, 10, 100, and 1000 µg/L for 43–46 days. Tadpoles exposed to 0.1 to 100 µg/L of PFBA and PFHxA had significantly higher mean snout-to-vent lengths, mean masses, and scaled mass indexes than control tadpoles. These results indicate that exposure to short-chain PFCAs influences tadpole growth. Further investigation into the mechanism(s) causing the observed changes in tadpole growth is warranted. We observed a significantly higher proportion of males in the PFBA 1 µg/L treatment group, however further histological analyses are required to confirm visual sex identification before making concrete conclusions on the effects of PFCAs on amphibian sex ratios. PFBA concentrations in tissues were higher than PFHxA concentrations; a pattern that contrasts with previously published studies using fish, suggesting potential differences between taxa in PFBA and PFHxA bioconcentration. Bioconcentration factors were <10 L/kg wet weight, indicating low bioconcentration potential in tadpoles. Our results suggest that PFBA and PFHxA may have effects at environmentally-relevant concentrations (0.1–10 µg/L) and further investigation is required before these compounds can be deemed a “safe” alternative to their long-chain counterparts.

Comparing Transcriptomic Responses to Chemicals Across Six Species Using the EcoToxChip RNASeq Database.

The EcoToxChip project includes RNA-sequencing data from experiments involving model (Japanese quail, fathead minnow, African clawed frog) and ecological (double-crested cormorant, rainbow trout, northern leopard frog) species at multiple life stages (whole embryo and adult) exposed to eight chemicals of environmental concern known to perturb a wide range of biological systems (ethinyl estradiol, hexabromocyclododecane, lead, selenomethionine, 17β trenbolone, chlorpyrifos, fluoxetine, and benzo[a]pyrene). The objectives of this short communication were to (1) present and make available this RNA-sequencing database (i.e., 724 samples from 49 experiments) under the FAIR principles (FAIR data are data which meet principles of findability, accessibility, interoperability, and reusability), while also summarizing key meta-data attributes and (2) use ExpressAnalyst (including the Seq2Fun algorithm and EcoOmicsDB) to perform a comparative transcriptomics analysis of this database focusing on baseline and differential transcriptomic changes across species-life stage-chemical combinations. The database is available in NCBI GEO under accession number GSE239776. Across all species, the number of raw reads per sample ranged between 13 and 58 million, with 30% to 79% of clean reads mapped to the "vertebrate" subgroup database in EcoOmicsDB. Principal component analyses of the reads illustrated separation across the three taxonomic groups as well as some between tissue types. The most common differentially expressed gene was CYP1A1 followed by CTSE, FAM20CL, MYC, ST1S3, RIPK4, VTG1, and VIT2. The most common enriched pathways were metabolic pathways, biosynthesis of cofactors and biosynthesis of secondary metabolites, and chemical carcinogenesis, drug metabolism, and metabolism of xenobiotics by cytochrome P450. The RNA-sequencing database in the present study may be used by the research community for multiple purposes, including, for example, cross-species investigations, in-depth analyses of a particular test compound, and transcriptomic meta-analyses. Environ Toxicol Chem 2024;00:1-6. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Sequence capture identifies fastidious chytrid fungi directly from host tissue

The chytrid fungus Batrachochytrium dendrobatidis (Bd) was discovered in 1998 as the cause of chytridiomycosis, an emerging infectious disease causing mass declines in amphibian populations worldwide. The rapid population declines of the 1970s-1990s were likely caused by the spread of a highly virulent lineage belonging to the Bd-GPL clade that was introduced to naïve susceptible populations. Multiple genetically distinct and regional lineages of Bd have since been isolated and sequenced, greatly expanding the known biological diversity within this fungal pathogen. To date, most Bd research has been restricted to the limited number of samples that could be isolated using culturing techniques, potentially causing a selection bias for strains that can grow on media and missing other unculturable or fastidious strains that are also present on amphibians. We thus attempted to characterize potentially non-culturable genetic lineages of Bd from distinct amphibian taxa using sequence capture technology on DNA extracted from host tissue and swabs. We focused our efforts on host taxa from two different regions that likely harbored distinct Bd clades: (1) wild-caught leopard frogs (Rana) from North America, and (2) a Japanese Giant Salamander (Andrias japonicus) at the Smithsonian Institution’s National Zoological Park that exhibited signs of disease and tested positive for Bd using qPCR, but multiple attempts failed to isolate and culture the strain for physiological and genetic characterization. We successfully enriched for and sequenced thousands of fungal genes from both host clades, and Bd load was positively associated with number of recovered Bd sequences. Phylogenetic reconstruction placed all the Rana-derived strains in the Bd-GPL clade. In contrast, the A. japonicus strain fell within the Bd-Asia3 clade, expanding the range of this clade and generating additional genomic data to confirm its placement. The retrieved ITS locus matched public barcoding data from wild A. japonicus and Bd infections found on other amphibians in India and China, suggesting that this uncultured clade is widespread across Asia. Our study underscores the importance of recognizing and characterizing the hidden diversity of fastidious strains in order to reconstruct the spatiotemporal and evolutionary history of Bd. The success of the sequence capture approach highlights the utility of directly sequencing pathogen DNA from host tissue to characterize cryptic diversity that is missed by culture-reliant approaches.

Chronic Exposure to a PFAS Mixture Resembling AFFF-Impacted Surface Water Decreases Body Size in Northern Leopard Frogs (Rana pipiens).

Per- and polyfluoroalkyl substances (PFAS) occur in the environment as mixtures, yet mixture toxicity remains poorly understood. Aqueous film-forming foams (AFFFs) are a common source of PFAS. Our objective was to examine chronic effects of a complex PFAS mixture on amphibian growth and development. We tested toxicity of a five-chemical PFAS mixture summing to 10 μg/L and that accounts for >90% of the PFAS in AFFF-affected surface waters: perfluorooctane sulfonate (PFOS, 40%), perfluorohexane sulfonic acid (PFHxS, 30%), perflurooctanoic acid (PFOA, 12.5%), perfluorohexanoic acid (PFHxA, 12.5%), and perfluoropentanoic acid (PFPeA, 5%). We also included treatments to determine whether PFOS drove mixture toxicity and whether PFOS and mixture components act additively. We exposed Northern leopard frog (Rana pipiens) larvae through metamorphosis (∼130 d) in outdoor mesocosms. After 21 days of exposure, the larval body condition fell ∼5% relative to controls in the 4 μg/L PFOS treatment and mixtures lacking PFOS. At metamorphosis, the full 5-component 10 μg/L PFAS mixture reduced mass by 16% relative to controls. We did not observe effects on development. Our results indicate that toxicity of PFOS and other PFAS mixtures typical of AFFF sites act additively and that PFOS is not more inherently toxic than other mixture components.

Successful eradication of invasive American bullfrogs leads to coextirpation of emerging pathogens

AbstractInterventions of the 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 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 two 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 coextirpated 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.

Environmental DNA surveys can underestimate amphibian occupancy and overestimate detection probability: implications for practice

AbstractEnvironmental DNA analysis of water samples has recently emerged as a powerful tool for studying the distribution, ecology, and conservation of many amphibian species. Validating efficacy through comparison with established methods of detection is important for any new method. We used multi‐method and single‐method occupancy models to explore the effectiveness of 3 techniques for detecting northern leopard frogs (Lithobates pipiens) and boreal chorus frogs (Pseudacris maculata). We conducted surveys using automated recording units, environmental DNA sampling, and visual encounter surveys in spring (30 Apr–3 Jun) and summer (23 Jul–27 Aug) of 2014 at 68 sites in southern Alberta, Canada. The multi‐method analysis incorporated data from all 3 survey methods while accounting for the lack of independence of detections within a sampling occasion, and we compared this to single‐method analyses to explore occupancy biases associated with each method and differences in method‐specific detection probability. Occupancy and detection probabilities estimated from environmental DNA analyses were biased for northern leopard frogs, overestimating probability of detection and underestimating occupancy. We could not assess bias for boreal chorus frogs because of overdispersion present in in the models. Although no single covariate explained this bias, it was ameliorated (albeit by reducing precision) by including a raw visual count of individuals as a proxy for abundance during the breeding but not the post‐metamorphic season. These results emphasize the need for careful consideration of temporal and spatial aspects of sampling design, conducting pilot studies, and external validation of eDNA‐only occupancy monitoring schemes prior to widespread implementation.

A molecular and ecological study of Macracanthorhynchus ingens (von Linstow, 1879) (Acanthocephala: Archiacanthocephala), in its paratenic and definitive hosts in southeastern Mexico and the Eastern USA.

The acanthocephalan Macracanthorhynchus ingens (von Linstow 1879) (Acanthocephala: Archiacanthocephala) is a parasite that infects the gut of carnivores (racoons, coyotes, wolves, foxes, badgers, skunks, opossum, mink and bears) as an adult and the body cavity of lizards, snakes, and frogs as a cystacanth in the Americas. In this study, adults and cystacanths of M. ingens from southeastern Mexico and southern Florida, USA, were identified morphologically by having a cylindrical proboscis armed with 6 rows of hooks each with 6 hooks. Hologenophores were used to sequence the small (SSU) and large (LSU) subunits of ribosomal DNA and cytochrome c oxidase subunit 1 (cox 1) from mitochondrial DNA.Phylogenetic analysis of the new SSU and LSU sequences of M. ingens placed them in a clade with other sequences available in GenBank identified as M. ingens. The cox 1 tree showed that the nine new sequences and six previously published sequences of M. ingens from the USA form a clade with other sequences previously identified as M. ingens from GenBank. The intraspecific genetic divergence among isolates from the Americas ranged from 0 to 2%, and in combination with the phylogenetic trees confirmed that the isolates belonged to the same species. The cox 1 haplotype network inferred with 15 sequences revealed 10 haplotypes separated from each other by a few substitutions. Rio Grande Leopard Frogs and Vaillant´s Frogs harbored cystacanths with low prevalence, 28% and 37% respectively, in Mexico. Brown Basilisks, an invasive lizard in Florida, USA, had high values of prevalence, 92% and 93% in males and females, respectively. Females harbored more cystacanths than males (0-39 vs 0-21) for unknown reasons that may, however, be related to ecological differences.

Developing in drying ponds could leave frogs unprepared for climate change

Dependable wet seasons are essential for the survival of many frogs, which rely on full pools in spring for their aquatic young to develop and mature. But recently, some regions have suffered unusually dry winters, threatening vital watercourses, and spring conditions are predicted to become even drier. ‘Development is a particularly sensitive period for many animals’, says Michel Ohmer, at University of Mississippi, USA, explaining that growing as a tadpole in a dwindling warm pool could affect the animal later in life. But no one knew how. As the conditions that southern leopard frog (Rana sphenocephala) tadpoles grow up in are predicted to change, Ohmer, Jeff Bednark and Trina Wantman (while at University of Pittsburgh, USA) headed down to Fort Polk, Louisiana, USA, to collect eggs freshly laid by the frogs to find out how the animals fared as young adults after a dry or warm start.Recreating 18 tadpole ponds in plastic pools in Louisiana, the team covered each with cloth providing 50% shade for shelter before introducing 40 tadpoles to their new homes and warming some to simulate future climate conditions. Then, the team recreated a drought by lowering the water rapidly in six tanks over 9 weeks until almost dry, while draining the water more slowly from six other tanks and keeping the water level unchanged in the remaining ponds. By doing so, the team created a range of conditions from future droughts to the current climate. But how did the different pond environments affect the developing youngsters?The team measured the tadpoles’ growth and development, and it was clear that the loss of water from their tank homes caused the tadpoles to develop faster, while growing in the warmer pools drove them to develop even faster, emerging as fully formed froglets in about 10 weeks. And when the team compared the tadpoles’ size when they finally lost their tails, the youngsters in the drying tanks were always smaller than the froglets from the deeper pools. But how did the changing and warming environments impact the frogs later in life?After transporting the froglets to Pittsburgh, Ohmer spooked the 1- to 2-month-old amphibians into jumping at different temperatures by gently prodding them, and found that the froglets that had developed in a cool, deep pond outleapt the froglets from the warmer, drier ponds, bounding 12% further at 32°C. The team also checked how well the froglets could withstand the heat, discovering that the fastest developing youngsters were unable to cope with high temperatures, regardless of the water conditions in their pools. And up to the age of 7 months, the froglets that had developed in the warmest and most rapidly drying pools were more timid and less keen to explore than the other frogs, while preferring to hang out in warmer conditions.‘The environment tadpoles develop in can impact frogs both in the short- and long-term, leading to changes not only in how they can handle the heat, but also the temperatures they choose in their habitat’, says Ohmer. So, growing up in drying ponds could be detrimental for adult frogs as the climate continues warming. And Ohmer warns that the impacts of climate change ‘may be amplified beyond what has been predicted’, as more timid frogs that develop in dwindling pools may avoid picking up microbes that offer protection from other pathogens. However, she points out that the frogs’ preference for warmer conditions may also make them less vulnerable to fungal infections that thrive in the cold.