Evaluating translocation success of wild eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) in Blue Ridge Ecoregion streams using pre- and post-translocation home range sizes and movement metrics.

Translocations of freshwater species have become a widespread conservation strategy to mitigate the impacts of habitat fragmentation, yet they are not often rigorously monitored using animal movement data to determine their success. We demonstrate the value of monitoring pre- and post-translocation movements and home-range sizes of a fully-aquatic, benthic stream salamander, the eastern hellbender (Cryptobranchus a. alleganiensis) to determine translocation success. We studied the home range sizes, movements, and habitat use of individuals (n = 27) in two self-sustaining populations (S1 & S2) for one year, and then subsequently collected similar data from a subset of these individuals (n = 17) that were translocated into two nearby streams (T1 & T2) with dam-isolated, declining populations in the Blue Ridge Ecoregion of Tennessee. We collected 1,571 location data points (869 pre-translocation and 715 post-translocation) from four study sites, and evaluated effects of mass, sex, and pre-translocation home range size/sedentariness, as well as habitat covariates on home range size and movements. Hellbender home range sizes increased from pre-translocation estimates at both sites, but response depended primarily on physical characteristics of release sites. Home range and fine-scale movement metrics indicated that hellbenders translocated from S1 to T1 settled in more quickly, had greater site fidelity, and smaller home ranges than hellbenders translocated from S2 to T2. Hellbender movements were influenced by cover rock size and density rather than individual characteristics. Study-long survival rates of translocated hellbenders increased from S1 to T1 (80% to 100%) and decreased from S2 to T2 (76% to 33%). Monitoring pre- and post-translocation movements was a valuable method for evaluating short-term translocation success in a freshwater environment. For future hellbender translocations, managers should prioritize selecting suitable release sites with contiguous boulder-dense areas (1-2 per m2), adequate prey (crayfish) densities (>1/m2), and habitats with low risk of predation.

Prevalence and Molecular Analysis of Hellbender (Cryptobranchus alleganiensis) Trypanosomes in Tennessee.

Populations of eastern hellbenders (Cryptobranchus alleganiensis alleganiensis) have been declining for multiple decades because of a variety of stressors associated with anthropogenic habitat disturbance followed by riparian sedimentation. The influence of parasite-associated morbidity and mortality on wild hellbender populations is poorly understood. Research has detected widespread trypanosome infection in hellbenders in Virginia, US, with no other reported detections within the eastern hellbender's extensive range. In our study, trypanosomes mostly closely resembling Trypanosoma cryptobranchi were observed in a blood smear of a hellbender from Tennessee during a population survey. Banked whole blood from this hellbender along with 51 other hellbenders was molecularly tested for the 18s rRNA gene of amphibian trypanosomes using newly designed PCR primers. In total, 3/52 (5.8%) hellbenders were PCR- and sequence-positive for trypanosomes. This is the first report of the partial 18s rRNA sequence from hellbender trypanosomes from North America. Further research into trypanosome epidemiology and hellbender health implications is warranted.

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.

Subterranean movement inferred by temporary emigration in Barton Springs salamanders (Eurycea sosorum).

Movement behavior is an important aspect of animal ecology but is challenging to study in species that are unobservable for some portion of their lives, such as those inhabiting subterranean environments. Using four years of robust-design capture-recapture data, we examined the probability of movement into subterranean habitat by a population of endangered Barton Springs salamanders (Eurycea sosorum), a species that inhabits both surface and subterranean groundwater habitats. We tested the effects of environmental variables and body size on survival and temporary emigration, using the latter as a measure of subterranean habitat use. Based on 2,046 observations of 1,578 individuals, we found that temporary emigration was higher for larger salamanders, 79% of which temporarily emigrated into subterranean habitat between primary sampling intervals, on average. Body size was a better predictor of temporary emigration and survival compared to environmental covariates, although coefficients from lower ranked models suggested turbidity and dissolved oxygen may influence salamander movement between the surface and subsurface. Surface population dynamics are partly driven by movement below ground and therefore surface abundance estimates represent a fraction of the superpopulation. As such, while surface habitat management remains an important conservation strategy for this species, periodic declines in apparent surface abundance do not necessarily indicate declines of the superpopulation associated with the spring habitat.

Physiological impacts of temperature variability and climate warming in hellbenders (Cryptobranchus alleganiensis).

Cold-adapted hellbender salamanders that inhabit cool mountain streams are expected to fare poorly under warmer projected climate scenarios. This study investigated the physiological consequences of long-term, naturalistic temperature variation on juvenile hellbenders under simulated current and warmer (+1.6 C) climates vs. controlled steady temperatures. Mean temperature and temperature variability were both important predictors of growth as indicated by monthly body mass change (%), stress as indicated by neutrophil:lymphocyte (N:L) ratio and bacteria-killing ability of blood. Cold exposure in hellbenders was associated with weight loss, increased N:L ratios and reduced Escherichia coli killing ability of blood, and these effects were less pronounced under a warmer climate scenario. These observations suggest that cold periods may be more stressful for hellbenders than previously understood. Growth rates peaked in late spring and late fall around 14-17°C. Hellbenders experiencing warmer simulated climates retained body condition better in winter, but this was counter-balanced by a prolonged lack of growth in the 3-month summer period leading up to the fall breeding season where warmer simulated conditions resulted in an average loss of -0.6% body mass/month, compared to a gain +1.5% body mass/month under current climate scenario. Hellbenders can physiologically tolerate projected warmer temperatures and temperature fluctuations, but warmer summers may cause animals to enter the fall breeding season with a caloric deficit that may have population-level consequences.

The relationship between spindly leg syndrome incidence and water composition, overfeeding, and diet in newly metamorphosed harlequin frogs (Atelopus spp.).

Spindly Leg Syndrome (SLS) is a persistent animal welfare issue associated with the rearing of amphibians in captivity. We conducted two experiments to investigate the effects of diet, water composition and overfeeding on prevalence of SLS in newly metamorphosed harlequin frogs (Atelopus spp.). In our first experiment, we offered 400 full-sibling tadpoles of Atelopus certus isocaloric diets in treatments of 31%, 37%, 42% and 48% crude protein respectively. Tadpoles fed higher protein diets metamorphosed faster, but the incidence of SLS exceeded 80% in all treatments leading to the conclusion that variation in dietary protein was not responsible for causing SLS. We used 720 full-sibling Atelopus glyphus tadpoles in a second experiment to examine the effects of diet type, water composition and diet ration on SLS. We found that an overall incidence of 58% spindly leg in tadpoles reared in tap water, but reduced to about 10% in water treated by reverse osmosis and then reconstituted. It is possible that the reverse osmosis treatment removed some factor that caused the SLS, or that the reconstitution may have added a mineral lacking in the original tap water. Within tap water treatments, overfeeding tadpoles in tanks increased the incidence of SLS. We recommend further experimental research into this condition to identify the causative factors in the water. Additional research into the nutritional composition of food available to wild tadpoles would be useful in formulating captive diets, that have to date been solely based on surrogate species.

Cryptic impacts of temperature variability on amphibian immune function.

Ectothermic species living in temperate regions can experience rapid and potentially stressful changes in body temperature driven by abrupt weather changes. Yet, among amphibians, the physiological impacts of short-term temperature variation are largely unknown. Using an ex situ population of Cryptobranchus alleganiensis, an aquatic North American salamander, we tested the hypothesis that naturally occurring periods of temperature variation negatively impact amphibian health, either through direct effects on immune function or by increasing physiological stress. We exposed captive salamanders to repeated cycles of temperature fluctuations recorded in the population's natal stream and evaluated behavioral and physiological responses, including plasma complement activity (i.e. bacteria killing) against Pseudomonas aeruginosa, Escherichia coli and Aeromonas hydrophila. The best-fit model (ΔAICc=0, wi=0.9992) revealed 70% greater P. aeruginosa killing after exposure to variable temperatures and no evidence of thermal acclimation. The same model predicted 50% increased E. coli killing, but had weaker support (ΔAICc=1.8, wi=0.2882). In contrast, plasma defenses were ineffective against A. hydrophila, and other health indicators (leukocyte ratios, growth rates and behavioral patterns) were maintained at baseline values. Our data suggest that amphibians can tolerate, and even benefit from, natural patterns of rapid warming/cooling. Specifically, temperature variation can elicit increased activity of the innate immune system. This immune response may be adaptive in an unpredictable environment, and is undetectable by conventional health indicators (and hence considered cryptic). Our findings highlight the need to consider naturalistic patterns of temperature variation when predicting species' susceptibility to climate change.