Aquatic Salamander Research Articles

Scavenging Behavior of the Aquatic Eastern Hellbender Salamander (Cryptobranchus Alleganiensis) in North Carolina

Abstract: Vertebrates may scavenge, or utilize carrion resources more than has been widely assumed (Devault et al. 2003). While documentation of scavengers in terrestrial and marine systems has received increased attention (Beasley et al. 2015; Davenport et al. 2016), little is known regarding scavengers in freshwater stream systems. Dominant fully aquatic salamanders are important foragers in these systems and may rival the biomass of predatory fish species (Davic and Welsh 2004). One fully aquatic salamander in Appalachian streams, the eastern hellbender (Cryptobranchus alleganiensis), occurs in NC streams which receive potentially large seasonal influxes of hatchery reared trout (NCWRC 2017). Moreover, few reports exist in the published literature on the food preferences of eastern hellbender salamanders. While hellbender diet is comprised of 90% crayfish based on stomach content analysis (Peterson et al. 1989), a small portion of their diet is thought to include fish or fish bait (Nickerson and Mays 1973). Barring a few anecdotal reports from fisherman, little is known regarding scavenging by hellbenders in NC.

Skin glands of an aquatic salamander vary in size and distribution and release antimicrobial secretions effective against chytrid fungal pathogens.

Amphibian skin is unique among vertebrate classes, containing a large number of multicellular exocrine glands that vary among species and have diverse functions. The secretions of skin glands contain a rich array of bioactive compounds including antimicrobial peptides (AMPs). Such compounds are important for amphibian innate immune responses and may protect some species from chytridiomycosis, a lethal skin disease caused by the fungal pathogens Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal). While the bioactivity of skin secretions against Bd has been assessed for many amphibian taxa, similar studies are lacking for Bsal, a chytrid fungus that is especially pathogenic for salamanders. We studied the skin glands and their potential functions in an aquatic salamander, the three-toed amphiuma (Amphiuma tridactylum). Skin secretions of captive adult salamanders were analyzed by RP-HPLC and tested against the growth of Bd and Bsal using in vitro assays. We found that compounds within collected skin secretions were similar between male and female salamanders and inhibited the growth of Bd and Bsal. Thus, skin secretions that protect against Bd may also provide protection against Bsal. Histological examination of the skin glands of preserved salamanders revealed the presence of enlarged granular glands concentrated within caudal body regions. A site of potential gland specialization was identified at the tail base and may indicate specialized granular glands related to courtship and communication.

Anthropogenic Associated Mortality in the Eastern Hellbender (Cryptobranchus alleganiensis alleganiensis)

Anthropogenic disturbance to natural habitats has been identified as a major threat to freshwater biodiversity. Human activities can destroy critical habitat of aquatic species by altering hydrodynamic stream characteristics. Movement and removal of shelter rocks within and along streams has been postulated to be detrimental to survival of aquatic salamanders; however, empirical data are lacking. Cryptobranchus alleganiensis alleganiensis (Eastern Hellbender) is a fully aquatic salamander species that shelters under cobble and boulder as both a juvenile and as an adult. Herein, we document mortality of both adult and larval Eastern Hellbender salamanders associated with anthropogenic habitat disturbance (i.e., moving and stacking of rocks to build small dams). To our knowledge, these observations are the first of their kind in the scientific literature. Given the high number of visitors to and increasing recreational use of rivers on public lands in the southeastern US, managers should consider implementing outreach education programs and monitoring popular recreation areas to deter in-stream habitat alteration.

Using Environmental DNA to Assess Hellbender Populations in the West Fork Greenbrier River, West Virginia

The eastern hellbender (Cryptobranchus alleganiensis alleganiensis) is a fully aquatic giant salamander whose habitats are commonly defined as swift running, fairly shallow, highly oxygenated waters. Hellbenders have been previously established as a sentinel species for water quality and aquatic ecosystem health. Unfortunately, populations have undergone a century long decline due to various anthropogenic influences. Measuring hellbender populations can be challenging and extremely invasive, which can be injurious for the individuals being sampled. Recent advances in non-invasive genetic sampling allows for the processing and analysis of environmental DNA (eDNA) from water samples; which has proven effective for detecting low population density aquatic macroorganisms. In this study, we sought to identify hellbender populations along the West Fork Greenbrier River in West Virginia. We utilized an aqueous filtering approach in conjunction with chloroform-isoamyl extractions to isolate eDNA. To positively identify sites with hellbender presence we utilized PCR to test for two hellbender specific regions of the mitochondrial gene, cytochrome-b; final results were compared to a traditional hellbender survey which had been conducted simultaneously with the eDNA sample collection.

Cutaneous microbiota of the Japanese giant salamander (Andrias japonicus), a representative of an ancient amphibian clade

Andrias japonicus, the Japanese giant salamander, is the second largest amphibian species in the world. The biology of this long-lived, fully aquatic salamander is still incompletely known, and studying the threats it experiences is important for conservation management. We used 16S amplicon sequencing to provide the first data on the composition and diversity of the cutaneous microbiome of this species. Skin bacterial communities of adult and larval giant salamanders were composed primarily of taxa belonging to the phyla Proteobacteria and Bacteroidetes, and, their community structure differed significantly from that of two other syntopic amphibians (Cynops pyrrhogaster and Glandirana rugosa). We also found differences between wild A. japonicus and captive individuals, with the latter having an increased bacterial diversity. The fungal pathogen Batrachochytrium dendrobatidis (Bd) was detected only in captive individuals (40% prevalence), and did not correlate with a particular bacterial community structure. We identified eight bacteria that were significantly more abundant on A. japonicus compared to syntopic amphibians, one of which was Janthinobacterium lividum, a bacterial species known to exert Bd-inhibiting effects. Our study provides baseline data for future in-depth studies on the microbial ecology of cutaneous bacteria and the contribution of cutaneous bacteria to Bd resistance in giant salamanders.

Antipredator behavior of the Barton Springs salamander (Eurycea sosorum) in response to aquatic invertebrates: potential consequences of habitat restoration

The Barton Springs Salamander, Eurycea sosorum, is a fully aquatic salamander found in Barton Springs in Texas, USA, and has benefited from habitat restoration efforts. While important to improve overall habitat quality for this imperiled species, current management and restoration practices may also inadvertently increase the abundance of non-target organisms such as predatory invertebrates. Fish represent major predators of this species, but little is known about the role of invertebrates as potential predators. It is important to understand the role of these aquatic invertebrates as predators of E. sosorum, especially if habitat restoration also increases predator abundance. Using adult, predator-naive salamanders, we examined the antipredator response of E. sosorum to chemical cues from the following treatments: crayfish, dragonfly larvae, snails, and water. Salamanders decreased activity (antipredator behavior) only in response to the crayfish treatment. The responses to dragonfly larvae, snails, and water did not differ, suggesting that dragonfly larvae are not perceived as predators by these salamanders. Our study provides preliminary evidence suggesting that habitat restoration has unexpectedly increased crayfish abundance, which in turn may negatively affect E. sosorum, and that future management strategies should consider crayfish removal if salamander abundances decline with increasing crayfish abundance.

Natural gas drilling in the Marcellus Shale region: well pad densities and aquatic communities

Few studies have examined the relationship between natural gas development and surface water quality using well pad density (the number of well pads/km2) as an indicator of potential impacts to aquatic life, specifically fish, salamander, and crayfish assemblages. We tested the hypothesis that animal assemblages, assessed by densities of ecologically important species, will differ among groups of watersheds with different well pad densities. In 2011, sites were sampled in Pennsylvania between July 13 and September 14 (N = 28; 10 = no well pads, 8 = low density, 10 = moderate density). We did not detect evidence of impact on fish, salamander, and crayfish assemblages. It should be noted that this study primarily assessed infrastructure-related impacts. Watersheds under investigations in this study area included no stream withdrawals for drilling and relatively low well pad density (≤0.541 well pads/km2), which reduces the amount of roads and pipeline as well as sediment runoff reaching the stream from this and other infrastructure during development. In addition, setback and landscape limitations also kept well pads relatively far from surface waters, and these watersheds remained highly forested after development because most were in state forests. Therefore, these findings represent the least intrusive scenario for impacts to aquatic communities while extracting natural gas.

Augmentation programme for the endangered Ozark hellbender Cryptobranchus alleganiensis bishopi in Missouri

The Ozark hellbender Cryptobranchus alleganiensis bishopi is an endangered aquatic salamander that inhabits cold‐water streams in the Ozark Highlands of Missouri and Arkansas in the USA. As a result of the drastic population declines over the past 40+ years, with a prominent shift in age structure (large, mature individuals predominate; young classes virtually absent), the Ozark Hellbender Working Group (OHWG) was established in 2001 to develop a conservation strategy for the species. The OHWG comprises representatives from state and federal agencies, Saint Louis Zoo (MO, USA), universities and interested individuals. Based on data from the Hellbender Population and Viability Assessment it was determined that without intervention, including a captive‐propagation, head‐start and augmentation programme, it was highly likely (>96% probability) that the species would go extinct within the next 75 years. Inter‐agency collaboration has facilitated significant progress since the inception of the captive‐propagation programme in 2002. The joint strategy of the world's first captive breeding of Ozark hellbenders in 2011 and subsequent captive breeding in the 2012–2015 period, together with the hatching and rearing of wild‐collected eggs, means 6737 larvae/juveniles have been produced and/or reared at the Saint Louis Zoo. Since 2008, 3138 juvenile Ozark hellbenders reared at Saint Louis Zoo have been released into the wild in Missouri to augment remaining populations in four different streams (Bryant Creek, North Fork of the White River, Current River and Eleven Point River). Based on the success of the captive‐propagation programme, post‐release survival and dedicated individuals of various organizations the future of the Ozark hellbender is looking optimistic.

Hearing and Sound Production in the Aquatic Salamander, Amphiuma means

Abstract: In contrast to anurans, acoustic communication and hearing within Order Urodela is poorly understood. Several studies have documented sound production in a few salamander species; however, these studies did not examine auditory ranges of these salamanders simultaneously. Two-Toed Amphiumas (Amphiuma means) were used as a model to examine the ecological significance of sound production in salamanders. We conducted a series of behavioral trials to validate sound production in this species, and auditory-evoked potentials (AEPs) were measured to assess hearing abilities. Amphiuma means produced three unique broadband acoustic signals: low-, mid-, and high-frequency clicks. All acoustic signals were produced when A. means were placed in groups, but not when housed individually. The audiogram was relatively flat at 100–700 Hz; above this range, sensitivity declined with increasing frequency. No auditory responses were detected between 1.5 and 10 kHz. The dominant frequencies of the acoustic signals (...

Vertebral shape and body elongation in Triturus newts

Body elongation in vertebrates is often related to a lengthening of the vertebrae and an increase in their number. Changes in the number and shape of vertebrae are not necessarily linked. In tailed amphibians, a change in body shape is mostly associated with an increase in the number of trunk and tail vertebrae. Body elongation without a numerical change of vertebrae is rare. In Triturus aquatic salamanders body elongation is achieved by trunk elongation through an increase in the number of trunk vertebrae. We used computed microtomography and three-dimensional geometric morphometrics to document the size, shape and number of trunk vertebrae in seven Triturus species. The data suggest that body elongation has occurred more frequently than body shortening, possibly related to a more aquatic versus a more terrestrial locomotor style. Our results show that body elongation is achieved through an increase in the number of trunk vertebrae, and that interspecific differences in vertebral shape are correlated with this pattern of elongation. More gracile trunk vertebrae were found in the more elongated species. The shape differences are such that single trunk vertebrae can be used for the identification of species with a possible application in the identification of subfossil and fossil material.

Ancient Lake Maxima and Substrate-dependent Riverine Migration Have Defined the Range of the Mudpuppy (<i>Necturus maculosus</i>) in Southern Ontario Following the Wisconsinan Glaciation

The Mudpuppy (Necturus maculosus) is an entirely aquatic salamander whose geographic range is thus defined by immigration routes in watersheds that permit feasible travel. Significant barriers, such as large waterfalls, effectively bar this species from further colonization upstream. We compared the contemporary distribution of Mudpuppies in southern Ontario with varying post-glacial ancient lake maxima and riverine outlet-flow conditions. Topography does not appear to be a range-limiting factor, but the type of river grade (waterfalls versus riffles) does. The distribution of modern records of this species in Ontario aligns closely with maxima from the Nipissing phase occurring 4000–5000 years ago, leading us to suggest that this is when Mudpuppies invaded and proliferated in the Great Lakes Basin.

A Non-Invasive Water-Borne Assay of Stress Hormones in Aquatic Salamanders

Declines in amphibian populations are due to an array of anthropogenic and natural factors requiring a need to detect and monitor populations that are either in decline or at risk of decline. Physiological biomarkers, such as glucocorticoid stress hormones, present a viable option to monitor stress levels and, thus, the physiological health of vertebrate populations. We performed preliminary experiments necessary for the use of water-borne hormone assays to measure corticosterone (CORT) as an assessment of aspects of chronic stress for the management and conservation of three species of aquatic salamanders in central Texas (Eurycea, Plethodontidae). We examined the time for CORT levels to peak in response to capture and handling in populations of E. nana, E. sosorum, and E. tonkawae. Peak CORT release rates differed among species, with peak releases generally occurring in under 3 h. This highlights the need to obtain a timeline for CORT to mount so that peak rates are captured during the leaching phase. We also examined the responsiveness of the hypothalamic-pituitary-interrenal (HPI) axis using an adrenocorticotropic hormone (ACTH) challenge or exposure to an external stressor (agitation). Eurycea sosorum (one population) and E. tonkawae showed HPI responsiveness. To examine the relationship between field and laboratory CORT release rates, we compared baseline CORT release rates between captive and wild-caught populations of all three species. Once the preliminary studies have been performed, water-borne CORT release rates can be an effective biomarker to monitor whether a population is experiencing chronic stress. Water-borne hormone collection also allows for repeated sampling of individuals over time. Our experiments provide a foundation for future studies relating stress to HPI responsiveness in aquatic Eurycea, which, in turn, will help inform management of both wild and captive populations.

Spatial - Temporal Variation in Algal Community in Freshwater Springs Inhabited by Aquatic Salamander Neurergus Crocatus

Spatial - Temporal Variation in Algal Community in Freshwater Springs Inhabited by Aquatic Salamander Neurergus Crocatus

Trap characteristics and species morphology explain size-biased sampling of two salamander species

Demographic studies often depend on sampling techniques providing representative samples from populations. However, the sequence of events leading up to a successful capture or detection is susceptible to biases introduced through individual-level behaviour or physiology. Passive sampling techniques may be especially prone to sampling bias caused by size-related phenomena (e.g., physical limitations on trap entrance). We tested for size-biased sampling among five types of passive traps using a 9-year data set for two species of aquatic salamanders that have a 20 and 61 fold change in length over their ontogeny (Amphiuma means, Siren lacertina). Size-biased trapping was evident for both species, with body size distributions (body length mean and SD) of captured individuals differing among sampling techniques. Because our two species differed in girth at similar lengths, we were able to show that size biases (in length) were most likely caused by girth limitations on trap entry rates, and potentially by differences in retention rates. Accounting for the biases of sampling techniques may be critical when assessing current population status and demographic change.

Polydactyly and other limb abnormalities in the Jurassic salamander Chunerpeton from China

Polydactyly and other malformations such as supernumerary limbs are relatively common in extant salamander populations. They generally result from biotic or abiotic disruption of normal regeneration following non-lethal predator or conspecific bite injury. Here, we record, for the first time, similar malformations in a fossil salamander. Fourteen specimens of the perennibranchiate Jurassic salamander Chunerpeton tianyiensis (Urodela, Cryptobranchidae), recovered from Jianping County, Liaoning Province, China, display polydactyly or abnormal phalangeal counts in one or more limbs, and two also possess supernumerary hind limbs. Polydactyly in Chunerpeton is mainly post-axial, occurring on the feet more often than the hands, and on the right side of the body more often than on the left. The extra legs also occurred on the right side of the body. Similar conditions can be observed in living salamanders, so these specimens suggest that the challenges faced by aquatic salamanders, and the developmental processes triggered in response, have changed relatively little in 160 million years.

Side-by-side secretion of Late Palaeozoic diverged courtship pheromones in an aquatic salamander.

Males of the advanced salamanders (Salamandroidea) attain internal fertilization without a copulatory organ by depositing a spermatophore on the substrate in the environment, which females subsequently take up with their cloaca. The aquatically reproducing modern Eurasian newts (Salamandridae) have taken this to extremes, because most species do not display close physical contact during courtship, but instead largely rely on females following the male track at spermatophore deposition. Although pheromones have been widely assumed to represent an important aspect of male courtship, molecules able to induce the female following behaviour that is the prelude for successful insemination have not yet been identified. Here, we show that uncleaved sodefrin precursor-like factor (SPF) protein pheromones are sufficient to elicit such behaviour in female palmate newts (Lissotriton helveticus). Combined transcriptomic and proteomic evidence shows that males simultaneously tail-fan multiple ca 20 kDa glycosylated SPF proteins during courtship. Notably, molecular dating estimates show that the diversification of these proteins already started in the late Palaeozoic, about 300 million years ago. Our study thus not only extends the use of uncleaved SPF proteins outside terrestrially reproducing plethodontid salamanders, but also reveals one of the oldest vertebrate pheromone systems.

Trade-offs between growth and maturation: the cost of reproduction for surviving environmental extremes.

Life-history trade-offs and the costs of reproduction are central concepts in evolution and ecology. Episodic climatic events such as drought and extreme temperatures provide strong selective pressures that can change the balance of these costs and trade-offs. We used size-structured matrix models parameterized from field and laboratory studies to examine the effect of periodic drought on two species of aquatic salamanders (greater siren, Siren lacertina; lesser siren, Siren intermedia) that differ in size at reproduction and maximum body size. Post-drought body size distributions of the larger species (S. lacertina) are consistent with size-dependent mortality. Smaller individuals were extirpated from the population during each drought while large animals persisted, a pattern that contrasted with that seen in several ectotherms. This appears to be largely explained by estivation proficiency and a positive relationship between body size and estivation potential. Increased body size, however, may come at the cost of fecundity and maturation rate compared to a closely related congener. The cost of somatic allocation in this case may manifest itself via reduced per-capita competitive ability, which (at least in simulation studies) allows the smaller, fast-maturing species to outcompete the larger, slow-maturing species when drought is minimal or nonexistent.

Pathogenic chytrid fungus Batrachochytrium dendrobatidis, but not B. salamandrivorans, detected on eastern hellbenders.

Recent worldwide declines and extinctions of amphibian populations have been attributed to chytridiomycosis, a disease caused by the pathogenic fungus Batrachochytrium dendrobatidis (Bd). Until recently, Bd was thought to be the only Batrachochytrium species that infects amphibians; however a newly described species, Batrachochytrium salamandrivorans (Bs), is linked to die-offs in European fire salamanders (Salamandra salamandra). Little is known about the distribution, host range, or origin of Bs. In this study, we surveyed populations of an aquatic salamander that is declining in the United States, the eastern hellbender (Cryptobranchus alleganiensis alleganiensis), for the presence of Bs and Bd. Skin swabs were collected from a total of 91 individuals in New York, Pennsylvania, Ohio, and Virginia, and tested for both pathogens using duplex qPCR. Bs was not detected in any samples, suggesting it was not present in these hellbender populations (0% prevalence, 95% confidence intervals of 0.0–0.04). Bd was found on 22 hellbenders (24% prevalence, 95% confidence intervals of 0.16 ≤ 0.24 ≤ 0.34), representing all four states. All positive samples had low loads of Bd zoospores (12.7 ± 4.9 S.E.M. genome equivalents) compared to other Bd susceptible species. More research is needed to determine the impact of Batrachochytrium infection on hellbender fitness and population viability. In particular, understanding how hellbenders limit Bd infection intensity in an aquatic environment may yield important insights for amphibian conservation. This study is among the first to evaluate the distribution of Bs in the United States, and is consistent with another, which failed to detect Bs in the U.S. Knowledge about the distribution, host-range, and origin of Bs may help control the spread of this pathogen, especially to regions of high salamander diversity, such as the eastern United States.

Using environmental DNA methods to improve detectability in a hellbender (Cryptobranchus alleganiensis) monitoring program

Isolation of environmental DNA (eDNA) is becoming a valuable tool for detecting presence of rare or secretive aquatic species. The recent use of quantitative PCR (qPCR) with eDNA sampling presents the possibility of using this method to infer population abundance and status. This approach would be especially useful for species such as the Eastern hellbender (Cryptobranchus alleganiensis alleganiensis), a declining, secretive, aquatic salamander that requires intense field survey effort to study. In 2012, we conducted eDNA sampling at sites across the range of the species in North Carolina. Our objectives were to assess presence across 61 sites, test for a correlation of abundance and biomass with eDNA estimates at a subset of 23 sites, and sample at multiple spatial and temporal scales in three river systems. Overall, we detected hellbender eDNA at 33 sites, including all sites with 2012 hellbender records, 71% of all recent or historic sites with hellbender presence, and at nine sites that lack species occurrence records. We did not find a correlation between eDNA estimates and field survey counts of individuals or biomass. We detected a strong temporal increase in eDNA during the September breeding period, but no consistent evidence of a spatial relationship with eDNA. Overall, our results demonstrate the efficacy of eDNA for detecting hellbender populations. Furthermore, the potential utility of qPCR to assess population status in hellbenders requires further study and testing, although it may be promising for determining population reproductive status.

A Test of Local Enhancement in Amphibians

AbstractLocal enhancement is an underexplored social learning mechanism that is often observed in organisms that live in groups. This mechanism occurs when individuals are attracted to areas where conspecifics have previously been, but which are not present when the animal actually moves into the area. We tested for local enhancement in wood frog tadpoles (Lithobates sylvatica) and spotted salamander larvae (Ambystoma maculatum) in three experiments that exposed individuals to one side of a test chamber which was empty and another that contained a group of three conspecifics. Side preference of the focal individual was recorded once the conspecifics were removed. Tadpoles showed a clear preference for moving to areas where a group of tadpoles had previously been located. Conversely, this preference was not observed in salamander larvae. In addition, salamander larvae took significantly more time to initially choose a side. These results indicate that tadpoles exhibit local enhancement, whereas aquatic salamander larvae do not. This difference in social learning could be largely due to differences in aquatic ecology between tadpoles and salamander larvae.