Aquatic Salamander Research Articles

Long‐term injury records reveal the role of biting in male–male combat in the Japanese giant salamander

Abstract Biting is a common male–male combat tactic across jawed vertebrates, in which teeth or bony beaks serve as weapons. Because biting can cause severe injuries in some species, bite scars provide critical information about the degrees and patterns of fights. Amphibians have a remarkable diversity in tooth morphology and arrangement. However, the functional ecology of amphibian teeth is poorly studied. For example, unlike aquatic suction‐feeding frogs that lost teeth, aquatic salamanders that suction‐feed prey retain numerous sharp teeth, suggesting that teeth as weapons may partially explain their retention. Here, we analysed long‐term injury records of one of the fully aquatic, suction‐feeding salamanders (Japanese Giant Salamander, Andrias japonicus) to examine the previously unexplored potential of amphibian teeth as weapons for male–male combat. The majority of the injuries were missing toes and limbs, which most likely occurred during intra‐specific combat. We explored associations between injuries and independent variables such as sex, body size, body condition and stream sections. We also investigated possible injury‐pattern biases along the lateral (i.e. right vs. left) and longitudinal (i.e. anterior vs. posterior) axes, which could reveal how these animals fight. We found that males had more injuries, larger individuals had more injuries, and males with poorer conditions had more injuries. There was no association between injuries and stream sections. In addition, we found that salamanders had more injuries on the right and posterior sides of the body. The lateral asymmetry in injuries is likely associated with turning asymmetry during combat. Our results revealed the intense male–male combats of giant salamanders, providing evidence of the use of teeth as weapons. Combats via biting in amphibians may be much more common and severe than currently known. Our research suggests the function of amphibian teeth as weapons, which is likely to be a vital selective agent shaping its remarkable diversity.

Spatiotemporal variation in the ingestion of microplastics in aquatic and terrestrial salamanders endemic to Türkiye

Microplastics (MPs), small plastic particles measuring less than 5 mm in size, have pervaded whole ecosystems globally, posing a significant threat to both aquatic and terrestrial organisms due to their ingestion and subsequent bioaccumulation, which can result in various health effects. Do salamanders living in terrestrial habitats ingest more microplastics compared to those living in aquatic habitats? To answer this question, two endemic model organisms—one aquatic: Anatolia newt, Neurergus strauchii sensu lato, and the other terrestrial, Atıf’s Lycian salamander, Lyciasalamandra atifi —were chosen to examine microplastic accumulation in both aquatic and terrestrial species in southern and eastern Türkiye. Our hypothesis is that salamanders living in terrestrial habitats may ingest more microplastics due to the fact that plastics are primarily produced and used on land before entering aquatic ecosystems. Using FTIR stereoscopy and stereomicroscopy, a total of 31 MPs were found in 14 (78 %) of the 18 locations studied for the aquatic salamander species. In contrast, 12 (86 %) of the 14 locations examined for the terrestrial salamander species contained 48 MPs. Although the number of MPs detected per individual in the terrestrial species showed an increasing trend over the 10 years, such a trend was not observed in the aquatic species. In terrestrial species, MPs abundance and salamander body size were significantly correlated in a positive way. On the other hand, in aquatic species, a significant and positive relationship was observed between MPs abundance and specimen weight. In the age/sex-based evaluation, more MPs were found in the gastrointestinal tracts of the juveniles (0.39 MPs/indiv.) compared to females (0.34 items/indiv.) and males (0.32 MPs/indiv.). The levels of microplastics found in terrestrial and aquatic salamanders highlight the potential threat posed by human-induced plastic pollution to terrestrial and aquatic ecosystems. To the best of our knowledge, the present study was a pioneer in reporting the variations in microplastic accumulation in aquatic and terrestrial salamanders.

Bigfoot Swims in the Garden of Eden

Encounters with hellbenders, an aquatic salamander found throughout the Appalachian Mountain range, has inspired Americans to reimagine their relationship with nature since the colonial period. These salamanders spend the majority of their time curled up in the nests they build under rocks on the bottom of cool, fast flowing streams. There, hellbenders blend into their surroundings and are commonly out of human sight. Only rarely do humans see hellbenders. According to written and oral accounts, hellbenders are generally only seen by humans when they bite angler’s bait. This essay examines human accounts of encounters with hellbenders from the eighteenth century through the twenty-first century. It argues that since the colonial period, Americans utilized encounters with hellbenders to reexamine their relationship with nature. This essay draws from the literary and historical analysis of scholars who have studied Bigfoot myths to argue that hellbenders have functioned rhetorically in the same way as Bigfoot myths, serving as a totem that helps Americans connect to an imagined wild environment. As these accounts demonstrate, human encounters with seemingly exotic, and commonly unseen, non-human animals like hellbenders have played a key role in the epistemological process that gave birth to the American wildlife conversation movement. This examination of human encounters with hellbenders shows American’s have reimagined their relationship to the environment through encounters with hellbenders over three centuries. In the eighteenth century, encounters with hellbenders influenced the creation of the American national identity after the United States formally severed their ties to Europe and founded a new nation. From the nineteenth century onward, American men developed a conversation movement around the idea of protecting so-called wild spaces and seemingly exotic animals like hellbenders to reenforce the American identity and their masculinity.

Batrachochytrium salamandrivorans is necronotic: carcasses could play a role in Bsal transmission

IntroductionBatrachochytrium salamandrivorans (Bsal) poses a major threat to global amphibian biodiversity. It is essential we understand Bsal transmission to develop better-informed management strategies. Infected carcasses are an important source of transmission for several human and wildlife disease systems; however, they have not been examined as sources for Bsal exposure. Here, we evaluated whether infected newt carcasses could contribute to Bsal transmission dynamics.MethodsWe cohoused infected carcasses with susceptible newts in two cohousing chamber types (partitioned or non-partitioned) at three timepoints post-mortem ([0,24[, [24,48, [48,72] hrs). The partitioned chamber prevented newt-to-newt contact hence only allowed indirect, waterborne transmission of zoospores. We measured shedding rates of infected carcasses at each post-mortem timepoint and monitored infection status and mortality of susceptible newts which were exposed during cohousing events.ResultsOur results indicate carcasses are capable of transmitting Bsal to susceptible newts up to at least 72 hrs post-mortem, even without live newts directly contacting carcasses. All susceptible newts in each chamber type and post-mortem period became infected and >90% experienced disease-induced mortality. Bsal genomic copies/uL in skin swabs taken from infected carcasses were high, averaging 7.4x105, 8.6x105, and 2.0x106 at 24, 48, and 72 hrs post-mortem, respectively. Water samples collected from cohousing chambers averaged 2743 Bsal genomic copies/uL (approximately 1357 zoospores) and did not decline over 72 hrs. DiscussionOur results indicate Bsal infection can occur rapidly between infected carcasses and susceptible aquatic salamanders via indirect and direct transmission pathways, and carcasses may prolong outbreaks by increasing the duration that infected individuals remain infectious. Carcass removal may be a strategy to reduce Bsal transmission and the impacts of outbreaks.

Concurrent threats and extinction risk in a long-lived, highly fecund vertebrate with parental care.

Detecting declines and quantifying extinction risk of long-lived, highly fecund vertebrates, including fishes, reptiles, and amphibians, can be challenging. In addition to the false notion that large clutches always buffer against population declines, the imperiled status of long-lived species can often be masked by extinction debt, wherein adults persist on the landscape for several years after populations cease to be viable. Here we develop a demographic model for the eastern hellbender (Cryptobranchus alleganiensis), an imperiled aquatic salamander with paternal care. We examined the individual and interactive effects of three of the leading threats hypothesized to contribute to the species' demise: habitat loss due to siltation, high rates of nest failure, and excess adult mortality caused by fishing and harvest. We parameterized the model using data on their life history and reproductive ecology to model the fates of individual nests and address multiple sources of density-dependent mortality under both deterministic and stochastic environmental conditions. Our model suggests that high rates of nest failure observed in the field are sufficient to drive hellbender populations toward a geriatric age distribution and eventually to localized extinction but that this process takes decades. Moreover, the combination of limited nest site availability due to siltation, nest failure, and stochastic adult mortality can interact to increase the likelihood and pace of extinction, which was particularly evident under stochastic scenarios. Density dependence in larval survival and recruitment can severely hamper a population's ability to recover from declines. Our model helps to identify tipping points beyond which extinction becomes certain and management interventions become necessary. Our approach can be generalized to understand the interactive effects of various threats to the extinction risk of other long-lived vertebrates. As we face unprecedented rates of environmental change, holistic approaches incorporating multiple concurrent threats and their impacts on different aspects of life history will be necessary to proactively conserve long-lived species.

A Late Miocene occurrence of the extinct salamander Batrachosauroides (Caudata, Batrachosauroididae) and other new caudate fossils from Florida and Georgia, USA

Two partial vertebrae of the rare, large-bodied, aquatic salamander Batrachosauroides are reported from the Upper Miocene Love Bone Bed (late Clarendonian, ~10–9 Ma) Alachua County, Florida. They represent the latest occurrence of Batrachosauroides by 2.8–5.8 million years from previous records and are the latest account of the family Batrachosauroididae in the eastern United States, being either younger than or approximately coeval with fossils of Peratosauroides problematica from the Clarendonian San Pablo Formation of central California. While most Neogene Batrachosauroides in North America are from the warm interval spanning the Late Oligocene Warming (LOW) to the Middle Miocene Climatic Optimum (MMCO), this is the first unequivocal account well after the conclusion of the MMCO suggesting Florida was a post-MMCO refugium during global cooling in the Late Miocene. Batrachosauroides vertebrae from the late Hemingfordian Suwannee Springs site (Florida) and the late Barstovian Gragg Mine (southwestern Georgia) are also described. Two other caudate taxa are present at the Love Bone Bed, Ambystoma and a mid-sized Siren that is the most common amphibian in the Love Bone Bed Local Fauna. The presence and rarity of Ambystoma further corroborates the existence of peripheral or seasonal lentic aquatic habitats adjacent to the main lotic body of the Love Bone Bed deposit. Other salamanders from the paleocoastal Gragg Mine Local Fauna include Notophthalmus and Amphiuma n. sp., aff. Amphiuma pholeter. The latter represents the oldest record of the A. pholeter lineage and documents its presence in the Gulf Coastal Plain since the Middle Miocene. The Gragg Mine represents a unique interval in the southeast at the conclusion of the MMCO.

Fish and aquatic salamander responses to the creation of riparian canopy gaps along forested headwater streams

Many headwaters across temperate North America have uniform mid‐succession riparian forests recovering from historic land clearing. These young riparian stands contrast with late‐succession forests, which have complex structural characteristics including canopy gaps. Canopy gaps provide structural diversity that can be important for terrestrial species, and they are also hypothesized to be important features for aquatic environments. The light patches below gaps create productivity hotspots in streams and therefore create potential for increased stream apex predator abundances through bottom‐up food web drivers. However, increasing light may also affect stream temperature, a consideration for coldwater fish (salmonids). We established an experimental before‐after control‐impact study to explicitly assess how creating canopy gaps in the riparian forest affects the abundance and biomass of coastal cutthroat trout (Oncorhynchus clarkii clarkii) and Pacific giant salamanders (Dicamptodon tenebrosus) in paired reference and treatment reaches at five replicate streams. Gaps were designed to resemble those in old‐growth forests in the treatment reach of each system although wood was explicitly left out of the stream. At four of five sites, we found small and generally consistent positive responses in adult cutthroat trout and total vertebrate biomass to localized increases in light but only 2 years after treatment. Results suggest that opening riparian canopies adjacent to streams via gaps is a viable tool to enhance structural complexity of riparian forests without negatively impacting stream vertebrates; however, a single gap alone had small effects on aquatic vertebrates. More or larger gaps would likely be needed to notably enhance aquatic apex predators.

Disease state associated with chronic toe lesions in hellbenders may alter anti-chytrid skin defenses

Hellbenders (Cryptobranchus alleganiensis) are large, aquatic salamanders from the eastern United States. Both subspecies, eastern and Ozark hellbenders, have experienced declines resulting in federal listing of Ozark hellbenders. The globally distributed chytrid fungus, Batrachochytrium dendrobatidis (Bd) has been detected in both subspecies, and Batrachochytrium salamandrivorans (Bsal) poses a new threat if introduced into North America. Ozark hellbenders also suffer a high prevalence of toe lesions of unknown etiology, with changes in host immunocompetence hypothesized to contribute. Antimicrobial peptides (AMPs) secreted from dermal granular glands may play a role in hellbender health. We collected skin secretions from free-ranging hellbenders and enriched them for small cationic peptides used for growth inhibition assays against Bd and Bsal. Generalized linear mixed models revealed the presence of active toe lesions as the strongest and only significant predictor of decreased Bd inhibition by skin peptides. We also found skin secretions were more inhibitory of Bsal than Bd. MALDI-TOF mass spectrometry revealed candidate peptides responsible for anti-chytrid activity. Results support the hypothesis that hellbender skin secretions are important for innate immunity against chytrid pathogens, and decreased production or release of skin peptides may be linked to other sub-lethal effects of disease associated with toe lesions.

Characterization of the developing axolotl nasal cavity supports multiple evolution of the vertebrate choana.

All tetrapods (mammals, birds, reptiles, and amphibians) share the ability to breathe with their mouths closed due to the formation of choanae, which are openings that allow communication between the nasal and oral cavities. In most fishes, the nasal cavities serve a strictly olfactory function, possessing incurrent and excurrent nares that lie outside of the mouth and therefore, never communicate with the respiratory system. It is not until the evolution of tetrapods, in which the nasal cavities consistently open into the mouth, that they are used both for olfaction and for respiration. However, this developmental transition is poorly understood, with no consensus on the evolutionary origin of the choana in various groups despite decades of debate. Here, we use high-contrast 3D imaging in conjunction with histology and apoptotic cell analysis in non-mineralized embryonic tissues to study the formation of the choana in the axolotl (Ambystoma mexicanum), an aquatic salamander species. We show that the axolotl choana forms from an extension of the embryonic nasal sac, which pushes through intervening mesenchyme and connects with the palate epithelium of the oral cavity, eventually breaking through. This mechanism differs from caecilians, mammals and reptiles, where fusion across a bucconasal groove plays an active role in choana formation. Nevertheless, caecilians, mammals and axolotls converge on the development of a transient epithelial tissue that has to break down in order to develop a patent choana, adding another twist to the intriguing arguments on the evolutionary history of the choana.

Droughts Reduce Growth Rates and Increase Vulnerability to Increasingly Frequent and Severe Drying Events in an Aquatic Ectotherm

Many aquatic organisms are experiencing increasingly severe and frequent droughts and drying events. Simultaneously, drought effects are carrying over to nondrought years as ecosystems remain in incomplete states of recovery. Aquatic organisms are thus faced with fewer sequential years under degraded environmental conditions to prepare for increasingly severe droughts and potential drying events. We assessed the effect of droughts and sex on the growth, mass, and mass-dependent estivation potential of long-lived aquatic salamanders (Greater Sirens, Siren lacertina) that estivate during drying events brought on by severe droughts. We calculated growth rates of S. lacertina based on mark–recapture data spanning 11 yr of a severe drought local minimum (of past 50 yr) in the southeastern United States. Sirens showed a distinct seasonal gain in body length and mass from March through September and little growth for the rest of the year. Gains during the growth season were strongly reduced by drought conditions. Although male and female sirens were predicted to reach a similar maximum body size, females grew much slower. Recruitment into drying event “size refugia” is constrained by drying event severity (determines minimum size required), frequency (determines available time between events to grow), and environmental conditions between drying events (determines the rate of growth). Thus, increases in drying event severity and frequency will require faster growth to a larger body size for successful recruitment into a size class that is resistant to drying events. The slower growth of females and reduction of growth during suboptimal years (mild to moderate droughts) suggest that the life history strategy of Greater Sirens for persisting through drying events potentially increases their demographic susceptibility to the predicted effects of climate change.

Evaluation of Tagging Methods for Unique Identification of Individuals in Three Aquatic Eurycea Salamander Species

Marking or tagging salamanders increases efficiencies in the operations and management of captive populations and facilitates techniques for monitoring demographics of wild populations. Tags facilitate monitoring of longitudinal data for individual animals such as age or estimated age, growth, reproduction, and health. We evaluated three different tagging methods on Texas Blind, San Marcos, and Comal Springs Salamanders to determine success of each as a long-term marker of individual animals. For this study, we selected visible implant elastomer (VIE), visible implant alphanumeric tags (VIA), and passive integrated transponders (PIT). Of these, only VIE tagging has been evaluated in San Marcos Salamanders; none have been evaluated for use in the other two salamander species studied; and comparison studies among these tags for fully aquatic salamander species have not been performed. These tag types were selected for their effectiveness with other salamander species, ability to be quickly and easily identified tank-side, minimal negative effects on organisms, and perceived ease of implanting the tags. The VIE tags were retained by all species and had high readability scores. However, the issues of tag breakage and deterioration should be considered when using VIE tags. VIA tags were rejected in all but one San Marcos Salamander and all Comal Springs Salamanders, but 90% of Texas Blind Salamanders retained them. PIT tags were not tested on San Marcos, Comal Springs, and smaller Texas Blind Salamanders due to size of tag and low retention rate in larger salamanders. Of the tags evaluated in this study, VIE was the most effective in terms of retention, readability, and ease of insertion.

Development and assessment of an environmental DNA (eDNA) assay for a cryptic Siren (Amphibia: Sirenidae)

Environmental DNA (eDNA) assays have become a major aspect of surveys for aquatic organisms in the past decade. These methods are highly sensitive, making them well-suited for monitoring rare and cryptic species. Current efforts to study the Rio Grande Siren in southern Texas have been hampered due to the cryptic nature of these aquatic salamanders. Arid conditions further add to the difficulty in studying this species, as many water bodies they inhabit are ephemeral, sometimes constraining sampling efforts to a short window after heavy rain. Additionally, sirens are known to cease activity and reside underground when ponds begin to dry or as water temperatures increase. Conventional sampling efforts require extensive trap-hours to be effective, which is not always possible within the required sampling window. This study presents the development of a novel eDNA assay technique for this elusive species using conventional PCR and Sanger sequencing and compares eDNA sampling results with simultaneous trapping at multiple sites to assess the relative effectiveness of the procedure. Rio Grande Siren detection via eDNA sampling was significantly higher at all sites compared to trapping, confirming the utility of this assay for species detection. This methodology gives promise for future work assessing the distribution and status of the Rio Grande Siren and has potential for use on other southern Texas amphibians.

Metabolite compositions on skins of eastern hellbenders Cryptobranchus alleganiensis alleganiensis differ with location and captivity.

Eastern hellbenders Cryptobranchus alleganiensis alleganiensis, large aquatic salamanders, are declining over most of their range. The amphibian-killing fungus Batrachochytrium dendrobatidis (Bd) has contributed to global amphibian declines and has been detected on eastern hellbenders, but infection intensities were lower than those of species that are more susceptible to Bd. The factors limiting Bd on hellbenders may include antifungal metabolites produced by their skin microbiota. We used a metabolite fingerprinting technique to noninvasively identify the presence, but not identity, of metabolites associated with eastern hellbenders. We surveyed the skin of wild eastern hellbenders to test whether the composition and richness (i.e. number of metabolites) of their metabolites are explained by Bd status or location. Furthermore, we surveyed for metabolites on captive eastern hellbenders to test whether metabolite compositions were different between captive and wild eastern hellbenders. Bd detection was not associated with either metabolite richness or composition. Both metabolite composition and richness differed significantly on hellbenders from different locations (i.e. states). For metabolite composition, there was a statistical interaction between location and Bd status. Metabolite richness was greater on captive eastern hellbenders compared to wild hellbenders, and metabolite compositions differed between wild and captive eastern hellbenders. The methods we employed to detect metabolite profiles effectively grouped individuals by location even though metabolite composition and richness have high levels of intraspecific variation. Understanding the drivers and functional consequences of assemblages of skin metabolites on amphibian health will be an important step toward understanding the mechanisms that result in disease vulnerability.

Regeneration in the phylogenetic empire: an interview with Peter Currie.

Regeneration in the phylogenetic empire: an interview with Peter Currie.

Variation in Articular Cartilage Thickness Among Extant Salamanders and Implications for Limb Function in Stem Tetrapods

The size and shape of articular cartilage in the limbs of extant vertebrates are highly variable, yet they are critical for understanding joint and limb function in an evolutionary context. For example, inferences about unpreserved articular cartilage in early tetrapods have implications for how limb length, joint range of motion, and muscle leverage changed over the tetrapod water-land transition. Extant salamanders, which are often used as functional models for early limbed vertebrates, have much thicker articular cartilage than most vertebrate groups, but the exact proportion of cartilage and how it varies across salamander species is unknown. I aimed to quantify this variation in a sample of 13 salamanders representing a broad range of sizes, modes of life, and genera. Using contrast-enhanced micro-CT, cartilage dimensions and bone length were measured non-destructively in the humerus, radius, ulna, femur, tibia, and fibula of each specimen. Cartilage correction factors were calculated as the combined thickness of the proximal and distal cartilages divided by the length of the bony shaft. Articular cartilage added about 30% to the length of the long bones on average. Cartilage was significantly thicker in aquatic salamanders (42 ± 14% in the humerus and 35 ± 8 in the femur) than in terrestrial salamanders (21 ± 7% in both humerus and femur). There was no consistent relationship between relative cartilage thickness and body size or phylogenetic relatedness. In addition to contributing to limb length, cartilage caps increased the width and breadth of the epiphyses by amounts that varied widely across taxa. To predict the effect of salamander-like cartilage correction factors on muscle leverage, a simplified model of the hindlimb of the Devonian stem tetrapodAcanthostegawas built. In this model, the lever arms of muscles that cross the hip at an oblique angle to the femur was increased by up to six centimeters. Future reconstructions of osteological range of motion and muscle leverage in stem tetrapods and stem amphibians can be made more rigorous by explicitly considering the possible effects of unpreserved cartilage and justifying assumptions based on available data from extant taxa, including aquatic and terrestrial salamanders.

Macroinvertebrate Communities and Body Condition of Larval Eastern Hellbender Salamanders (Cryptobranchus alleganiensis) in North Carolina.

Salamanders are important biological indicators of freshwater aquatic ecosystems. The Eastern Hellbender, Cryptobranchus alleganiensis, is found primarily in streams across the southeastern, midwestern, and eastern portions of the United States. However, this unique large aquatic salamander is facing numerous threats and declines across its geographic range, including in Appalachia. Moreover, little is known regarding the early life history stages (gilled larvae), particularly regarding food availability (aquatic insects present in streams) and body condition. In this study, we assessed the macroinvertebrate communities of streams sampled for larval C. alleganiensis and report on the body condition index within western North Carolina streams. We found varying levels of diversity across sample locations for macroinvertebrates (total sampled = 3,619, representing over 30 genera), with the most prevalent insects from Trichoptera order, with an overall high percent of Ephemeroptera, Plecoptera, and Trichoptera (EPT) in all streams, ranging from 68.5% to 90.7%. Functional Feeding Groups included 40.5% filterers, 24.3% predators, 17.6% gatherers, 10.7% shredders, and 6.9% scrapers. Shannon Diversity Index in sample streams ranged from 2.101 to 2.698. Body condition or SMI (scaled mass index) ranged from 1.5 to 3.3 2 across sites with a largely consistent and strong linear relationship between log mass and total length (r = 0.910). Our results add to the body of knowledge on the larval ecology of this North American salamander and may aid in future management of hellbender stream habitats.

Translocation does not influence amphibian chytrid fungus prevalence among wild eastern hellbenders Cryptobranchus alleganiensis.

Disease monitoring is an essential step in translocation projects, specifically in amphibians where emerging pathogens such as the chytrid fungus Batrachochytrium dendrobatidis (Bd) are linked to population declines. The eastern hellbender Cryptobranchus alleganiensis is a large, fully aquatic salamander experiencing precipitous range-wide population declines; however, the role Bd plays in these declines is unclear. To augment declining hellbender populations and determine effects of translocation on Bd prevalence, we conducted a translocation study of wild adult hellbenders from 2 source streams with abundant hellbender populations to 2 streams with declining populations in east Tennessee, USA. In 2018, we implanted radio transmitters into 30 hellbenders and sampled them periodically for Bd until 17 of the 30 hellbenders were translocated in 2019. We attempted to recapture translocated hellbenders approximately every 45 d for 3 mo to determine Bd prevalence post-release. We used qPCR to detect Bd and quantify zoospore loads on positive samples. Hellbenders had a pre-translocation Bd prevalence of 50% (15 of 30), which decreased to 10% (1 of 10) post-translocation. The average zoospore load for positive samples was 73.63 ± 30.82, and no hellbenders showed signs of chytridiomycosis throughout the study. Although we detected no significant effect of translocation on Bd prevalence, we observed a reduction in Bd prevalence post-release. Our results indicate that translocation did not lead to an increase in pathogen prevalence in translocated wild adult hellbenders, suggesting that chytrid did not impact the success of short-term translocations of eastern hellbenders in the Blue Ridge ecoregion.

Environmental DNA Assessment Reveals Restoration Success for Mudpuppies (Necturus maculosus)

Mudpuppies (Necturus maculosus) are secretive, fully aquatic salamanders with a range that spans much of the eastern United States and Canada including the Great Lakes region. Although this species was once abundant, there have been widespread declines due to habitat loss and modification, pollution, lampricide use, and overcollection. We compared environmental DNA (eDNA) and trapping surveys conducted from 2014 to 2016 to determine Mudpuppy occupancy along the St. Clair-Detroit River System, where this indicator species could be a gauge for success of ongoing restoration. Mudpuppy eDNA was detected at all sites with positive trapping records, as well as one site where individuals have not been trapped previously. Sites with shoreline restoration had the highest occupancy estimates, whereas deep-water restoration did not affect Mudpuppy occupancy. Additionally, eDNA surveys resulted in higher detection probability than setline and minnow trap survey methods, illustrating the benefit of using eDNA to detect secretive species. This study demonstrates the success of restoration efforts in increasing the occupancy of an indicator species and can be used as a template for other restoration initiatives.

Shelter Guarding Behavior of the Eastern Hellbender (Cryptobranchus alleganiensis alleganiensis) in North Carolina Streams

Cryptobranchus alleganiensis alleganiensis (Eastern Hellbender) is a fully aquatic salamander of conservation concern across the southeastern US. Characterization of shelter guarding by males and interspecific behavior during the breeding season in wild populations within the Blue Ridge ecoregion of North Carolina is lacking. To this end, we characterized diurnal video sequences of natural shelter-rock and nest-guarding behavior by male Eastern Hellbenders. We documented several intraspecific behaviors between resident males and nonresident conspecifics (males and females) during the breeding season (late summer to early fall) of 2018–2019 using modified action cameras with 4–6 hour battery life deployed in the French Broad River Basin of western North Carolina. Breeding behavior was documented (gravid females entering shelter) in 5 shelters, with 4 nests later confirmed by researchers. Across 21 unique shelters, we documented several examples of aggressive behaviors by resident Eastern Hellbender males toward conspecifics, including defensive posturing, territorial behavior, fights, biting, and bite-holds. All males occupying shelters (residents) spent the majority of time actively guarding the shelter entrance (81.7% of video) followed by maintenance behavior (e.g., modifying the shelter entrance; 5.3% of video), and short bouts (mean = 3.2 minutes) away from the shelter rock (2.5% of video). This study provides the first quantitative report of male breeding-season behavior across multiple natural shelters using non-invasive, affordable, waterproof cameras in North Carolina. We report on the utility of this method for observing behavior in stream systems and its potential application for monitoring of nests and behavior in other diurnal aquatic species.

The first report on circulating microRNAs at Pre- and Post-metamorphic stages of axolotls.

MicroRNAs (miRNAs) are endogenously coded small RNAs, implicated in post-transcriptional gene regulation by targeting messenger RNAs (mRNAs). Circulating miRNAs are cell-free molecules, found in body fluids, such as blood and saliva, and emerged recently as potential diagnostic biomarkers. Functions of circulating miRNAs and their roles in target tissues have been extensively investigated in mammals, and the reports on circulating miRNAs in non-mammalian clades are largely missing. Salamanders display remarkable regenerative potential, and the Mexican axolotl (Ambystoma mexicanum), a critically endangered aquatic salamander, has emerged as a powerful model organism in regeneration and developmental studies. This study aimed to explore the circulating miRNA signature in axolotl blood plasma. Small RNA sequencing on plasma samples revealed 16 differentially expressed (DE) circulating miRNAs between neotenic and metamorphic stages out of identified 164 conserved miRNAs. Bioinformatics predictions provided functional annotation of detected miRNAs for both stages and enrichment of DE miRNAs in cancer-related and developmental pathways was notable. Comparison with previous reports on axolotl miRNAs unraveled common and unique members of the axolotl circulating miRNome. Overall, this work provides novel insights into non-mammalian aspects of circulating miRNA biology and expands the multi-omics toolkit for this versatile model organism.