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.

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.

Pre- and postnatal effects of experimentally manipulated maternal corticosterone on growth, stress reactivity and survival of nestling house wrens.

Corticosterone plays a central role in maintaining homeostasis, promoting energy acquisition, and regulating the stress response in birds. Exposure to elevated levels of corticosterone during development can profoundly alter offspring behaviour and physiology, but the effects of elevated maternal corticosterone on offspring development remain poorly understood.We tested two competing hypotheses concerning the effect of maternally derived corticosterone on growth and development of free-living house wrens: (i) elevated maternal corticosterone causes damaging effects on nestling phenotype and fitness (collateral damage hypothesis) and (ii) increased maternal corticosterone enhances offspring fitness by preparing nestlings for the environment experienced by their mother (environmental/maternal-matching hypothesis).We used a non-invasive means to increase maternal corticosterone by providing females with corticosterone-injected mealworms prior to and during egg production in the absence of any overt pre-natal maternal stress. To disentangle pre- and post-natal effects of this elevation in maternal corticosterone, we cross-fostered young in two experiments: (i) nestlings of control and experimental females were reared by unmanipulated, natural females in a uniform maternal environment; (ii) a split-brood design that enabled us to assess the interaction between the mother's corticosterone treatment and that of the nestlings.There were significant pre-natal effects of increased maternal corticosterone on nestling growth and survival. Offspring of females experiencing experimentally increased corticosterone were heavier and larger than offspring of control females. There also was a significant interaction between maternal corticosterone treatment and the corticosterone treatment to which young were exposed within the egg in their effect on nestling survival while in the nest; experimental young exhibited greater survival than control young, but only when reared by control mothers. There was also a significant effect of maternal corticosterone treatment on nestling stress reactivity and, in both experiments, on the eventual recruitment of offspring as breeding adults in the local population.These patterns are broadly consistent with the environmental/maternal-matching hypothesis, and highlight the importance of disentangling pre- and post-natal effects of manipulations of maternal hormone levels on offspring phenotype.

Group management influences reproductive function of the male cheetah (Acinonyx jubatus).

Although the free-ranging cheetah is generally socially solitary, as many as 60% of males live in same-sex (usually sibling) coalitions. Under ex situ conditions, the cheetah experiences low reproductive success with only ~18% of males having ever produced young. Most male cheetahs (85%) are managed in captivity in coalitions, but with no data on the influence of social grouping on reproductive parameters. We examined the influence of singleton versus coalition management on various male cheetah physiological traits, including ejaculate quality and gonadal and adrenal hormone metabolite concentrations. We also assessed behaviour within coalitions for evidence of social hierarchy through initiation of interactions with group mates and relatedness to physiological traits. Ejaculate quality (including total motile and structurally normal spermatozoa per ejaculate) and androgen concentration profiles were higher (P<0.05) in coalition compared with singleton males. These results support the conclusion that testis function in the cheetah, specifically related to the development of normal, motile spermatozoa and androgen production, is influenced by management with same-sex conspecifics. The findings have implications for ex situ conservation breeding programs by suggesting that reproductive quality can be enhanced through group maintenance of cheetah males.

Reduced immune function predicts disease susceptibility in frogs infected with a deadly fungal pathogen.

The relationship between amphibian immune function and disease susceptibility is of primary concern given current worldwide declines linked to the pathogenic fungus Batrachochytrium dendrobatidis (Bd). We experimentally infected lowland leopard frogs (Lithobates yavapaiensis) with Bd to test the hypothesis that infection causes physiological stress and stimulates humoral and cell-mediated immune function in the blood. We measured body mass, the ratio of circulating neutrophils to lymphocytes (a known indicator of physiological stress) and plasma bacterial killing ability (BKA; a measure of innate immune function). In early exposure (1-15 days post-infection), stress was elevated in Bd-positive vs. Bd-negative frogs, whereas other metrics were similar between the groups. At later stages (29-55 days post-infection), stress was increased in Bd-positive frogs with signs of chytridiomycosis compared with both Bd-positive frogs without disease signs and uninfected control frogs, which were similar to each other. Infection decreased growth during the same period, demonstrating that sustained resistance to Bd is energetically costly. Importantly, BKA was lower in Bd-positive frogs with disease than in those without signs of chytridiomycosis. However, neither group differed from Bd-negative control frogs. The low BKA values in dying frogs compared with infected individuals without disease signs suggests that complement activity might signify different immunogenetic backgrounds or gene-by-environment interactions between the host, Bd and abiotic factors. We conclude that protein complement activity might be a useful predictor of Bd susceptibility and might help to explain differential disease outcomes in natural amphibian populations.

Motile Sperm Output by Male Cheetahs (Acinonyx jubatus) Managed Ex Situ Is Influenced by Public Exposure and Number of Care-Givers.

The collective cheetah (Acinonyx jubatus) population in zoological institutions has never been self-sustaining because of challenges in natural reproduction. A retrospective analysis of North American zoo-breeding records has revealed that >90% of litters produced since 2003 occurred in facilities 'off-display' from the public. We examined seminal, endocrine, and behavioral traits of 29 adult male cheetahs that were: 1) managed in public exhibit or off-display facilities; 2) maintained by different numbers of cheetah-specific care-givers; and 3) living adjacent to varying numbers of adult conspecifics. Cheetahs housed off-display produced more total motile sperm/ejaculate (P = 0.04) than on-exhibit males. This finding was mirrored in our laboratory's historical records where two-fold more total motile sperm (P < 0.01) were measured in ejaculates from individuals with no public exposure (n = 43) compared to on-exhibit (n = 116) counterparts. Males at institutions with ≤3 care-givers also produced more total motile sperm/ejaculate (P < 0.03) and spent more time behaviorally active (P < 0.01) than at facilities using >3 care-givers. Exposure to high numbers of conspecifics within the same institution did not impact (P > 0.05) seminal traits, and presence of the public, care-giver number, or animals/facility had no influence (P > 0.05) on androgen or glucocorticoid excretion or other behavioral metrics. Findings indicate that male cheetahs are sensitive to general public exposure and too many care-givers, resulting in compromised motile sperm output/ejaculate with mechanism of action unrelated to altered androgen or glucocorticoid excretion.

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.

Cultivating creativity in conservation science.

Conservation practitioners and scientists are often faced with seemingly intractable problems in which traditional approaches fail. While other sectors (e.g., business) frequently emphasize creative thinking to overcome complex challenges, creativity is rarely identified as an essential skill for conservationists. Yet more creative approaches are urgently needed in the effort to sustain Earth's biodiversity. We identified 4 strategies to develop skills in creative thinking and discuss underlying research and examples supporting each strategy. First, by breaking down barriers between disciplines and surrounding oneself with unfamiliar people, concepts, and perspectives, one can expand base knowledge and experiences and increase the potential for new combinations of ideas. Second, by meeting people where they are (both literally and figuratively), one exposes oneself to new environments and perspectives, which again broadens experiences and increases ability to communicate effectively with stakeholders. Third, by embracing risk responsibly, one is more likely to develop new, nontraditional solutions and be open to high-impact outcomes. Finally, by following a cycle of learning, struggle, and reflection, one can trigger neurophysiological changes that allow the brain to become more creative. Creativity is a learned trait, rather than an innate skill. It can be actively developed at both the individual and institutional levels, and learning to navigate the relevant social and practical barriers is key to the process. To maximize the success of conservation in the face of escalating challenges, one must take advantage of what has been learned from other disciplines and foster creativity as both a professional skill and an essential component of career training and individual development.

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.

Different patterns of metabolic cryo-damage in domestic cat (Felis catus) and cheetah (Acinonyx jubatus) spermatozoa.

Felid spermatozoa are sensitive to cryopreservation-induced damage, but functional losses can be mitigated by post-thaw swim-up or density gradient processing methods that selectively recover motile or structurally-normal spermatozoa, respectively. Despite the importance of sperm energy production to achieving fertilization, there is little knowledge about the influence of cryopreservation or post-thaw processing on felid sperm metabolism. We conducted a comparative study of domestic cat and cheetah sperm metabolism after cryopreservation and post-thaw processing. We hypothesized that freezing/thawing impairs sperm metabolism and that swim-up, but not density gradient centrifugation, recovers metabolically-normal spermatozoa. Ejaculates were cryopreserved, thawed, and processed by swim-up, Accudenz gradient centrifugation, or conventional washing (representing the 'control'). Sperm glucose and pyruvate uptake, lactate production, motility, and acrosomal integrity were assessed. Mitochondrial membrane potential (MMP) was measured in cat spermatozoa. In both species, lactate production, motility, and acrosomal integrity were reduced in post-thaw, washed samples compared to freshly-collected ejaculates. Glucose uptake was minimal pre- and post-cryopreservation, whereas pyruvate uptake was similar between treatments due to high coefficients of variation. In the cat, swim-up, but not Accudenz processing, recovered spermatozoa with increased lactate production, pyruvate uptake, and motility compared to controls. Although confounded by differences in non-specific fluorescence among processing methods, MMP values within treatments were positively correlated to sperm motility and acrosomal integrity. Cheetah spermatozoa isolated by either selection method exhibited improved motility and/or acrosomal integrity, but remained metabolically compromised. Collectively, findings revealed a metabolically-robust subpopulation of cryopreserved cat, but not cheetah, spermatozoa, recovered by selecting for motility rather than morphology.

Oxidative phosphorylation is essential for felid sperm function, but is substantially lower in cheetah (Acinonyx jubatus) compared to domestic cat (Felis catus) ejaculate.

Compared with the normospermic domestic cat, sperm metabolic function is compromised in the teratospermic cat and cheetah, but the pathway(s) involved in this deficiency are unknown. Glycolysis is essential for sperm motility, yet it appears to function normally in spermatozoa of either species regardless of structural morphology. We conducted a comparative study to further understand the mechanisms of energy production in felid spermatozoa, with the hypothesis that oxidative phosphorylation is required for normal sperm function and is impaired in teratospermic ejaculates. Electroejaculates from both species were stained with MitoTracker to quantify mitochondrial membrane potential (MMP) or were incubated to assess changes in sperm function (motility, acrosomal integrity, and lactate production) after mitochondrial inhibition with myxothiazol. Sperm midpiece dimensions also were quantified. Sperm mitochondrial fluorescence (directly proportional to MMP) was ~95% lower in the cheetah compared with the normospermic and teratospermic cat, despite the cheetah having a 10% longer midpiece. In both species, MMP was increased 5-fold in spermatozoa with retained cytoplasm compared with structurally normal cells. Inhibition of oxidative phosphorylation impaired sperm function in both species, but a 100-fold higher inhibitor concentration was required in the cat compared with the cheetah. Collectively, findings revealed that oxidative phosphorylation was required for sperm function in the domestic cat and cheetah. This pathway of energy production appeared markedly less active in the cheetah, indicating a species-specific vulnerability to mitochondrial dysfunction. The unexpected, cross-species linkage between retained cytoplasmic droplets and elevated MMP may reflect increased concentrations of metabolic enzymes or substrates in these structures.

Glycolytic enzyme activity is essential for domestic cat (Felis catus) and cheetah (Acinonyx jubatus) sperm motility and viability in a sugar-free medium.

We have previously reported a lack of glucose uptake in domestic cat and cheetah spermatozoa, despite observing that these cells produce lactate at rates that correlate positively with sperm function. To elucidate the role of glycolysis in felid sperm energy production, we conducted a comparative study in the domestic cat and cheetah, with the hypothesis that sperm motility and viability are maintained in both species in the absence of glycolytic metabolism and are fueled by endogenous substrates. Washed ejaculates were incubated in chemically defined medium in the presence/absence of glucose and pyruvate. A second set of ejaculates was exposed to a chemical inhibitor of either lactate dehydrogenase (sodium oxamate) or glyceraldehyde-3-phosphate dehydrogenase (alpha-chlorohydrin). Sperm function (motility and acrosomal integrity) and lactate production were assessed, and a subset of spermatozoa was assayed for intracellular glycogen. In both the cat and cheetah, sperm function was maintained without exogenous substrates and following lactate dehydrogenase inhibition. Lactate production occurred in the absence of exogenous hexoses, but only if pyruvate was present. Intracellular glycogen was not detected in spermatozoa from either species. Unexpectedly, glycolytic inhibition by alpha-chlorohydrin resulted in an immediate decline in sperm motility, particularly in the domestic cat. Collectively, our findings reveal an essential role of the glycolytic pathway in felid spermatozoa that is unrelated to hexose metabolism or lactate formation. Instead, glycolytic enzyme activity could be required for the metabolism of endogenous lipid-derived glycerol, with fatty acid oxidation providing the primary energy source in felid spermatozoa.

Evidence for compromised metabolic function and limited glucose uptake in spermatozoa from the teratospermic domestic cat (Felis catus) and cheetah (Acinonyx jubatus).

Cheetahs and certain other felids consistently ejaculate high proportions (≥ 60%) of malformed spermatozoa, a condition known as teratospermia, which is prevalent in humans. Even seemingly normal spermatozoa from domestic cat teratospermic ejaculates have reduced fertilizing capacity. To understand the role of sperm metabolism in this phenomenon, we conducted a comparative study in the normospermic domestic cat versus the teratospermic cat and cheetah with the general hypothesis that sperm metabolic function is impaired in males producing predominantly pleiomorphic spermatozoa. Washed ejaculates were incubated in chemically defined medium containing glucose and pyruvate. Uptake of glucose and pyruvate and production of lactate were assessed using enzyme-linked fluorescence assays. Spermatozoa from domestic cats and cheetahs exhibited similar metabolic profiles, with minimal glucose metabolism and approximately equimolar rates of pyruvate uptake and lactate production. Compared to normospermic counterparts, pyruvate and lactate metabolism were reduced in teratospermic cat and cheetah ejaculates, even when controlling for sperm motility. Rates of pyruvate and lactate (but not glucose) metabolism were correlated positively with sperm motility, acrosomal integrity, and normal morphology. Collectively, our findings reveal that pyruvate uptake and lactate production are reliable, quantitative indicators of sperm quality in these two felid species and that metabolic function is impaired in teratospermic ejaculates. Furthermore, patterns of substrate utilization are conserved between these species, including the unexpected lack of exogenous glucose metabolism. Because glycolysis is required to support sperm motility and capacitation in certain other mammals (including dogs), the activity of this pathway in felid spermatozoa is a target for future investigation.

Improved quality of cryopreserved cheetah (Acinonyx jubatus) spermatozoa after centrifugation through Accudenz.

Sperm cryopreservation, in combination with assisted reproductive techniques, is a valuable tool for the genetic management of endangered felids. However, the acrosome of the cheetah spermatozoon is especially sensitive to cryopreservation, with approximately 40% of spermatozoa experiencing acrosomal damage immediately after thawing and then another approximately 15% loss during the next 4 hours in vitro. Additionally, thawing causes a reduction in sperm motility by approximately 20% with another decrease of approximately 12% during subsequent incubation in vitro. We hypothesized that slow removal of glycerol from cryopreserved cheetah spermatozoa using an Accudenz gradient would improve acrosomal integrity, sperm motility longevity, and structural morphology. Accudenz was compared with traditional cheetah sperm processing methods for glycerol removal that involves washing, multistep resuspension, and swim-up processing. Electroejaculates (n = 21 total from 8 males) were washed in Ham F10 medium, and sperm pellets were resuspended in TEST-yolk buffer with 0% glycerol. Samples were cryopreserved in straws in 4% final glycerol, thawed, and assessed for percent intact acrosomes (% IA), percent motility (% M), and forward progressive status (FPS; scale, 0-5). Sperm motility index (SMI) was calculated as (% M + [FPS x 20]) / 2. In study 1, glycerol removal by centrifugation through an Accudenz gradient (4%, 10%) was compared with traditional sperm washing (control) and multistep resuspension protocols. At each time after centrifugation (hourly for 4 hours), % IA was improved (P < .05) for Accudenz (range, 36%-39%) compared with control (30%-33%) and multistep (29%-33%) treatments. In study 2, a modified Accudenz protocol was compared with traditional washing and was found to improve (P < .05) SMI (range, 52-64) compared with controls (range, 41-52) at each time postthaw after centrifugation. In study 3, swim-up processed sperm were compared with those treated by centrifugation through Accudenz and traditional sperm washing for improving sperm morphology. The percentage of structurally-normal sperm recovered postthawing increased (P < .05) for both the Accudenz (38%) and swim-up (33%) treatments compared with controls (21%). Percent IA and SMI also were improved (P < .05) for Accudenz (range, 39%-47% and 46-59, respectively) compared with controls (range, 26%-33% and 40-53, respectively). Results indicate that using Accudenz for glycerol removal from cryopreserved cheetah sperm mitigates the significant loss in sperm quality that occurs after freeze-thawing. This alleviation of cellular damage resulting from cryopreservation contributes to a more than 10% improvement in overall sperm motility and, more importantly, allows retention of 40% or more of sperm with intact acrosomes.

Molecular beacon genotyping for globoid cell leukodystrophy from hair roots in the twitcher mouse and rhesus macaque.

Rapid and accurate genotype determination is ideal for the maintenance of breeding colonies of laboratory animal models of genetic disease. The rhesus macaque and murine (twitcher) models of globoid cell leukodystrophy have a dinucleotide deletion or single nucleotide substitution, respectively, which abolish ceramide beta-galactosidase activity and are authentic models of Krabbe disease. We report a molecular beacon PCR assay for each species which allows unambiguous determination of the genotype in under 4h. The assay works reliably with DNA extracted from hair roots using Chelex-100 in a 20 min, 100 degrees C incubation. We demonstrate that genotyping from hair roots is a preferred alternative to collecting blood or tissue for DNA extraction because it reduces animal distress, uses an inexpensive reagent, and is simpler and faster. Following amplification on a standard thermocycler with a 96-well plate format, these molecular beacon assays can be read on a standard laboratory fluorescent plate reader, eliminating the need to use a real-time thermocycler or to open the plate for subsequent restriction enzyme digestion and gel electrophoresis. The multiplexed ratio of fluorescence from wild-type- and mutant-specific beacons reporting at 560 nm and 535 nm wavelengths is distinct for each genotype.