Distribution of Batrachochytrium dendrobatidis and pathology in the skin of green tree frogs Litoria caerulea with severe chytridiomycosis

Although histopathology is used routinely for diagnosis of chytridiomycosis in live and dead amphibians, there are no quantitative data on the distribution of the causative fungus, Batrachochytrium dendrobatidis, in the skin. We performed quantitative histological examinations on 6 sites on the body and 4 toes of 10 free-ranging adult green tree frogs Litoria caerulea found recently dead or dying from chytridiomycosis. Large numbers of sporangia occurred in all areas of ventral skin and toes; on average there were 94.3 sporangia mm(-1) of superficial epidermis. The number of sporangia was highly variable and this appeared to be related to the stage in the cycle of sloughing. The stratum corneum tends to build up with high intensities of infection and then sheds entirely rather than being shed continuously. Very few or no sporangia occurred on dorsal skin. This distribution could be explained by the dryness of the dorsal skin or possibly by the greater number of serous glands, which produce antifungal peptides, on the dorsum. In some frogs, ulceration and erosions occurred on skin on the back in the absence of sporangia. Other pathological changes such as hyperkeratosis and congestion occurred much more frequently on ventral surfaces.     

Pathophysiology in mountain yellow-legged frogs (Rana muscosa) during a chytridiomycosis outbreak

The disease chytridiomycosis is responsible for declines and extirpations of amphibians worldwide. Chytridiomycosis is caused by a fungal pathogen (Batrachochytrium dendrobatidis) that infects amphibian skin. Although we have a basic understanding of the pathophysiology from laboratory experiments, many mechanistic details remain unresolved and it is unknown if disease development is similar in wild amphibian populations. To gain a better understanding of chytridiomycosis pathophysiology in wild amphibian populations, we collected blood biochemistry measurements during an outbreak in mountain yellow-legged frogs (Rana muscosa) in the Sierra Nevada Mountains of California. We found that pathogen load is associated with disruptions in fluid and electrolyte balance, yet is not associated with fluctuations acid-base balance. These findings enhance our knowledge of the pathophysiology of this disease and indicate that disease development is consistent across multiple species and in both laboratory and natural conditions. We recommend integrating an understanding of chytridiomycosis pathophysiology with mitigation practices to improve amphibian conservation.     

Clinical diagnosis and treatment of epidermal chytridiomycosis in African clawed frogs (Xenopus tropicalis)

An investigation was conducted to determine the cause of morbidity and mortality in a collection of 55 adult male Xenopus (Silurana) tropicalis at the University of California, Berkeley. More than 80% of affected frogs died during the epizootic. All frogs were anorectic and lethargic, had dark pigmentation and excess skin sloughing, and lacked a slime layer. Histologic examination revealed severe hyperplastic and spongiotic dermatitis associated with colonization of the stratum corneum by large numbers of zoosporangia diagnostic of Batrachochytrium dendrobatidis. Treatment with a commercial formalin/malachite green solution at a dilution of 0.007 ml/L of tank water for 24 h, repeated every other day for four treatments, eliminated the organism and was curative. These findings are indicative of epidermal chytridiomycosis as a primary cause of death in this collection of X. tropicalis.     

Use of immunohistochemistry to diagnose chytridiomycosis in dyeing poison dart frogs (Dendrobates tinctorius)

Chytridiomycosis, caused by Batrachochytrium dendrobatidis, is an emerging disease of both wild and captive amphibians, posing a threat to their survival in many parts of the world. As the disease can be difficult to diagnose on routine pathologic sections, the purpose of this study was to develop an additional method for visualization. To accomplish this, immunohistochemical staining was applied to histologic skin sections from four experimentally infected Dyeing poison dart frogs (Dendrobates tinctorius). Staining of the positive tissue sections was distinct and readily visualized, making this technique a valuable ancillary diagnostic test for this important disease.     

Explosive jumping: extreme morphological and physiological specializations of Australian rocket frogs (Litoria nasuta)

Abstract Anuran jumping is an ideal system for examining the relationships between key morphological, physiological, and kinematic parameters. We used the Australian rocket frog (Litoria nasuta) as a model species to investigate extreme specialization of the vertebrate locomotor system for jumping. We measured the ground reaction forces applied during maximal jumps using a custom-designed force platform, which allowed us to calculate instantaneous measures of acceleration, velocity, power output, and total jump distance. We quantified the mechanical properties of the plantaris longus muscle using the work loop technique. We found that L. nasuta achieved the second-longest relative jumping distance for any anuran (55.2 body lengths for one individual) and the highest published anuran values for isolated net mean muscle power output measured using work loops (93.5 W kg(-1) muscle mass), hindlimb length to snout-vent length ratio (2.02), and relative hindlimb muscle mass (33% of body mass). Litoria nasuta also had a higher ratio of tibia length to snout-vent length than 19 related species. We found that the mean power output expended during the takeoff phase of jumping in the individual that jumped the farthest was about three times greater than our estimate of available muscle power output.     

The ebb and flow of antimicrobial skin peptides defends northern leopard frogs (Rana pipiens) against chytridiomycosis

Many amphibian species are threatened with extinction by the emerging infectious disease, chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis. This unprecedented global crisis threatens to reduce the biodiversity of the entire amphibian class. The fungus invades the skin and impairs the uptake and retention of essential ions leading to cardiac arrest. Antimicrobial peptides (AMPs) secreted into the mucus of some amphibians are thought to be an important defense against chytridiomycosis. However, little is known about the quantities of AMPs secreted under natural conditions, whether they are sufficient to protect against this pathogen, and how they interact with commensal microbes. To understand how defensive peptides and skin microbes may interact, it is essential to know the precise quantities of AMPs present under natural conditions. Using matrix‐assisted laser desorption time‐of‐flight mass spectrometry and growth inhibition assays, we show that northern leopard frogs (Rana pipiens) at rest constitutively release low amounts of AMPs that inhibit B. dendrobatidis in vitro, and AMP defenses are elevated following a simulated predator attack. Using a synthetic peptide analogue of brevinin‐1Pb as an external control, we quantified the amounts of four previously described AMPs (brevinin 1Pa, brevinin‐1Pb, brevinin‐1Pd, and ranatuerin‐2P) at several time points after secretion. Once secreted onto the skin, the peptides are most active for 15 min, and small quantities persist for at least 2 h. Taken together, our data suggest that small amounts of AMPs are rapidly available and quite stable on the skin of R. pipiens. They are effective inhibitors of B. dendrobatidis at these low constitutive concentrations but degrade within 2 h, protecting the integrity of the skin and commensal bacteria.     

Contrasting fecal corticosterone metabolite levels in captive and free-living colonial tuco-tucos (Ctenomys sociabilis)

The environment in which an animal lives can profoundly influence its biology, including physiological responses to external stressors. To examine the effects of environmental conditions on physiological stress reactions in colonial tuco-tucos (Ctenomys sociabilis), we measured glucocorticoid (GC) levels in captive and free-living members of this species of social, subterranean rodent. Analyses of plasma and fecal samples revealed immunoreactive corticosterone (metabolites) to be the most prevalent GC in this species. An adrenocorticotropic hormone challenge confirmed that fecal corticosterone metabolites are responsive to exogenous stressors and provided validation of the commercial enzyme immunoassay kit used to detect these metabolites. Comparisons of adult female C. sociabilis from natural and captive environments revealed significantly higher baseline concentrations of corticosterone metabolites and significantly greater individual variation in metabolite concentrations among free-living animals. These findings suggest that the natural environment in which these animals occur is more challenging and more variable than the captive housing conditions employed. In addition to providing the first evaluation of GC levels in captive and wild colonial tuco-tucos, our findings indicate that the influence of environmental conditions on stress physiology may have important implications for understanding the social behavior of this species in the laboratory and the field.     

Mechanisms of competition between tadpoles of Australian frogs (Litoria spp.) and invasive cane toads (Rhinella marina)

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Large-scale seasonal variation in the prevalence and severity of chytridiomycosis

The chytrid fungus Batrachochytrium dendrobatidis has been implicated as the causative agent of mass mortalities, population declines and the extinctions of amphibian species worldwide. Although several studies have shown that the prevalence of chytridiomycosis (the disease caused by the fungus) increases in cooler months, the magnitude and timing of these seasonal fluctuations have yet to be accurately quantified. We conducted disease sampling in a single population of stony creek frogs Litoria wilcoxii on 13 occasions over a 21-month period and used quantitative real-time polymerase chain reaction to detect and quantify the number of B. dendrobatidis zoospores present on samples. Disease prevalence varied significantly across sampling sessions, peaking at 58.3% (in early spring) and dropping to as low as 0% on two occasions (late summer and early autumn). There was a significant negative relationship between disease prevalence and mean air temperature in the 30 days prior to sampling. These large-scale seasonal fluctuations in chytridiomycosis levels will strongly influence conservation programs and amphibian disease research.     

Thermoregulatory behaviour affects prevalence of chytrid fungal infection in a wild population of Panamanian golden frogs

Predicting how climate change will affect disease dynamics requires an understanding of how the environment affects host–pathogen interactions. For amphibians, global declines and extinctions have been linked to a pathogenic chytrid fungus, Batrachochytrium dendrobatidis. Using a combination of body temperature measurements and disease assays conducted before and after the arrival of B. dendrobatidis, this study tested the hypothesis that body temperature affects the prevalence of infection in a wild population of Panamanian golden frogs (Atelopus zeteki). The timing of first detection of the fungus was consistent with that of a wave of epidemic infections spreading south and eastward through Central America. During the epidemic, many golden frogs modified their thermoregulatory behaviour, raising body temperatures above their normal set point. Odds of infection decreased with increasing body temperature, demonstrating that even slight environmental or behavioural changes have the potential to affect an individual''s vulnerability to infection. The thermal dependency of the relationship between B. dendrobatidis and its amphibian hosts demonstrates how the progression of an epidemic can be influenced by complex interactions between host and pathogen phenotypes and the environments in which they are found.     

Effects of corticosterone treatment on male aggressive behavior in a lizard (Anolis sagrei)

Social dominance in several species of lizards appears related to adrenal gland activity. The purpose of this study was to test whether the adrenal hormone corticosterone (CS) would inhibit the aggressive behavior of males of the lizard Anolis sagrei. Reproductively active males were randomly assigned to either CS or placebo (P) treatment groups or to an untreated stimulus male group. Both CS and P were administered in pellet form. Corticosterone pellets were designed to release approximately 4.8 micrograms of CS/day. Initially (Day 1), treatment males were implanted subcutaneously with a single pellet of either CS or P. After 1 week (Day 8), behavior tests were staged between treatment males and untreated stimulus males. The behavior of treatment males in response to stimulus males was videotaped. Significantly fewer CS-treated males than P-treated males approached the stimulus males, erected a crest, or showed biting behavior. Corticosterone-treated males also displayed significantly less frequently than did P-treated males and gave fewer displays of a type associated with dominance. Mean combined testis weight at the end of the experiment was significantly less in CS-treated males than in P-treated males and spermatogenic activity in CS-treated males was reduced. Plasma testosterone in CS-treated males was nondetectable, whereas P-treated males had normal levels of this steroid. These results indicate that CS can inhibit male aggressive behavior and testicular function in a lizard and support the hypothesis that the behavior changes observed in subordinate animals following defeat in agonistic encounters may in part be due to increased plasma CS levels.     

Hindlimb muscle fiber types in two frogs (Rana catesbeiana and Litoria caerulea) with different locomotor behaviors: histochemical and enzymatic comparison

To test how differences in locomotor behaviors may be reflected in muscle fiber-type diversity within anurans, a comparison of hindlimb muscles between the powerful terrestrial hopper, Rana catesbeiana, and the tree frog, Litoria caerulea, was done. One postural muscle (tibialis posticus, TP) and one primary hopping muscle (plantaris longus, PL), were characterized to identify muscle fiber types using standard histochemical methods. In addition, spectophotometric analysis of activity levels of the oxidative enzyme citrate synthase (CS) and the glycolytic enzyme lactate dehydrogenase (LDH) were done in each muscle. In spite of presumed differences in behavior between the species, we found no significant differences in the proportions of the identified fiber types when the muscles were compared across species. In addition, there were no significant differences in the proportions of the different fiber types between the postural versus phasic muscles within species. Within Rana, the postural muscle (TP) had greater oxidative capacity (as measured by CS activity) than did the phasic muscle (PL). Both muscles had equivalent LDH activities. Within Litoria, PL and TP did not differ in either LDH or CS activities. Both PL and TP of Litoria had less LDH activity and greater CS activity than their homologs in Rana. Thus, in spite of the uniform populations of fiber types between muscles and species, the metabolic diversity based on enzyme activity is consistent with behavioral differences between the species. These results suggest that the range of functional diversity within fiber types may be very broad in anurans, and histochemical fiber typing alone is not a clear indicator of their metabolic or functional properties.     

Intraspecific variation in laryngeal and ear morphology in male cricket frogs (Acris crepitans)

In a previous report, the authors found significant population variation in the calls of cricket frogs ( Acris crepitans ) that could not be explained by geographic variation in body size alone. Here we extend that work by investigating intraspecific population variation in the morphological characteristics underlying acoustic communication in male cricket frogs from several sites in Texas. We measured the volumes of laryngeal and auditory components responsible for the generation or reception of species-specific vocalizations in male frogs from eight populations. We found significant differences among populations in body size, as well as all the laryngeal and ear components we measured. With the exception of vocal cord and extracolumella volumes, the volumes of these anatomical structures differ among populations independently of body size as determined by a covariate analysis with snout-vent length as the covariate. Call dominant frequency differs among populations in a clinal pattern and head width, arytenoid cartilage, vocal cord and dilator muscle volume show a similar pattern when the residuals of the regression of morphological component on SVL are assessed for this trend. The results show that both larynx and ear structures can change in size independently of body size, yielding significant geographic variation in the behavioral and physiological expressions of the acoustic communication system underlying mate choice.     

Host mating system and the prevalence of disease in a plant population

A modified susceptible-infected-recovered (SIR) host-pathogen model is used to determine the influence of plant mating system on the outcome of a host-pathogen interaction. Unlike previous models describing how interactions between mating system and pathogen infection affect individual fitness, this model considers the potential consequences of varying mating systems on the prevalence of resistance alleles and disease within the population. If a single allele for disease resistance is sufficient to confer complete resistance in an individual and if both homozygote and heterozygote resistant individuals have the same mean birth and death rates, then, for any parameter set, the selfing rate does not affect the proportions of resistant, susceptible or infected individuals at equilibrium. If homozygote and heterozygote individual birth rates differ, however, the mating system can make a difference in these proportions. In that case, depending on other parameters, increased selfing can either increase or decrease the rate of infection in the population. Results from this model also predict higher frequencies of resistance alleles in predominantly selfing compared to predominantly outcrossing populations for most model conditions. In populations that have higher selfing rates, the resistance alleles are concentrated in homozygotes, whereas in more outcrossing populations, there are more resistant heterozygotes.     

Differential sensitivity to androgens within a sexually dimorphic muscle of male frogs (Xenopus laevis)

Male frogs use their forelimb flexor muscles to clasp females during the mating behavior known as amplexus. We investigated the effects of testosterone on a principal forelimb flexor, the flexor carpi radialis muscle (FCR), using morphological and histochemical techniques. Male Xenopus laevis were surgically manipulated to produce high or low levels of circulating testosterone for an 8-week period. After this treatment, measurement of fibers in muscle cross-sections revealed that average fiber size was positively correlated with testosterone level. This effect was not the same for all muscle fibers, however. Fibers in the shoulder region were more sensitive to testosterone than fibers in other regions of the muscle. Histochemical staining of cross-sections showed that the patterns of staining for myosin ATPase or succinic dehydrogenase (SDH) were not influenced by testosterone levels, but total SDH activity was increased by testosterone treatment. When sensitivity to testosterone was correlated with ATPase activity, fibers with high ATPase activity were found to be more sensitive to testosterone than fibers with low activity, regardless of position within the muscle. Most fibers with high ATPase activity were located in the shoulder region of the muscle. These fibers are innervated by different motor axons than are fibers in the elbow region of the muscle, and contractions of shoulder (but not elbow) region fibers, elicited by stimulation of motor axons, are slowed by testosterone treatment (Regnier and Herrera, 1993, J. Physiol. 461:565–581). © 1993 John Wiley & Sons, Inc.     

Long-distance movements of individuals in a free-living bank vole population: an important element of male breeding strategy

The experiment involved two stages: the first one consisted of studying the dynamics of long-distance movements and the characteristics of moving individuals in a free-living population of bank voles and the second one — of an analysis of paternity of young individuals born to females representing specific breeding colonies (basing on an analysis of six microsatellite fragments). The study has shown that both male and female bank voles are capable of covering long distances of up to almost 1 km. Males moved significantly more frequently than females. The proportion of travelling males was the highest in spring and the lowest in autumn. Almost all moving males were adult and they were sexually active. Almost all moving females were adult, however, the majority of them were sexually inactive. This indicates that the character and causes of long-distance movements of females and males differ. Female movements are supposed to be related to the search for living and breeding grounds by young, already adult individuals, driven out from their mothers’ territory. Multiple paternity occurred in 25% of all litters analysed. Young whose fathers were males from outside of their mother’s breeding colonies occurred in litters throughout the whole breeding season. They made up 59% of all young analysed and in the spring (June) all the young animals were born to fathers originating outside of the female’s breeding colony. The results obtained may confirm the hypothesis that long-distance movements of male bank voles constitute a part of their breeding strategy, involving them in searching for breeding partners over an extensive area.     

Seasonal changes in angiotensin converting enzyme activity in male and female frogs (Rana esculenta)

Gonad, lung, kidney and serum angiotensin converting enzyme (ACE) activities were determined by specific substrate hydrolysis in male and female Rana esculenta over 1 year. Ovary ACE activity showed the highest values among the different tissues, with a significant peak (223+/-52 nmol min(-1) mg protein(-1)) in late winter-early spring. Testis ACE activity followed a significant seasonal cycle, increasing from September to peak in April (2.5+/-0.8 nmol min(-1) mg protein(-1)) and then decreased in the post-reproductive period. Lung and kidney ACE activities were not correlated with the annual reproductive cycle phases. In serum a peak of activity was present in the post-reproductive period both in male and female frogs. The present data show a correlation between ACE and the annual reproductive cycle of R. esculenta.