Batrachochytrium dendrobatidis Infection in the Recently Rediscovered Atelopus mucubajiensis (Anura, Bufonidae), a Critically Endangered Frog from the Venezuelan Andes

The harlequin frog, Atelopus mucubajiensis, is a critically endangered species that was believed to be extinct until 2004, when a single adult female was found in the Venezuelan Andes. Its disappearance after 1994 has been attributed to an increase in the prevalence of the fungal pathogen, Batrachochytrium dendrobatidis, in populations of these frogs. Using histology and Real-Time PCR assays, we tested this adult female positive for B. dendrobatidis. We report for the first time, to our knowledge, infection in a live specimen of A. mucubajiensis. The presence of this pathogen in remaining individuals poses a problem for the long-term persistence of this critically endangered species, as new epidemic episodes of chytridiomycosis could jeopardize the survival of remnant populations.     

Transmission of Batrachochytrium dendrobatidis to wood frogs (Lithobates sylvaticus) via a bullfrog (L. catesbeianus) vector

Chytridiomycosis, an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis, threatens anuran populations worldwide. Effects of B. dendrobatidis on frog species are variable. Some species typically develop nonlethal infections and may function as carriers; others typically develop lethal infections that can lead to population declines. Nonlethal infections in the bullfrog (Lithobates catesbeianus) are well-documented. In contrast, recently metamorphosed wood frogs (L. sylvaticus) can die from chytridiomycosis. We conducted an ex-situ experiment between May and July 2010 to determine whether B. dendrobatidis-infected bullfrogs could transmit the fungus to wood frog tadpoles when the two species shared a body of water. We tested for B. dendrobatidis infections with quantitative polymerase chain reactions (qPCR) in a subsample of the wood frog tadpoles and in all metamorphosed wood frogs and compared risk of death of froglets exposed and unexposed to infected bullfrogs. We detected B. dendrobatidis sporadically in subsampled treatment tadpoles (nine of 90, 10%) and frequently in treatment froglets (112 of 113, 99.1%). Pooled risk of froglet death was higher (P<0.001) in treatment enclosures than in control enclosures. Our results indicate that, at the low infection loads bullfrogs tend to carry, swabbing for PCR analyses may underestimate prevalence of B. dendrobatidis in this species. We highlight bullfrog disease screening as a management challenge, especially in light of exotic bullfrog colonies on multiple continents and large-scale global trade in this species. We document the importance of quantifying lethal and sublethal effects of bullfrog vectors on B. dendrobatidis-susceptible species.     

Presence of the amphibian chytrid fungus Batrachochytrium dendrobatidis in threatened corroboree frog populations in the Australian Alps

Since the early 1980s, the southern corroboree frog Pseudophryne corroboree and northern corroboree frog P. pengilleyi have been in a state of decline from their sub-alpine and high montane bog environments on the southern tablelands of New South Wales, Australia. To date, there has been no adequate explanation as to what is causing the decline of these species. We investigated the possibility that a pathogen associated with other recent frog declines in Australia, the amphibian chytrid fungus Batrachochytrium dendrobatidis, may have been implicated in the decline of the corroboree frogs. We used histology of toe material and real-time PCR of skin swabs to investigate the presence and infection rates with B. dendrobatidis in historic and extant populations of both corroboree frog species. Using histology, we did not detect any B. dendrobatidis infections in corroboree frog populations prior to their decline. However, using the same technique, high rates of infection were observed in populations of both species after the onset of substantial population declines. The real-time PCR screening of skin swabs identified high overall infection rates in extant populations of P. corroboree (between 44 and 59%), while significantly lower rates of infection were observed in low-altitude P. pengilleyi populations (14%). These results suggest that the initial and continued decline of the corroboree frogs may well be attributed to the emergence of B. dendrobatidis in populations of these species.     

The role of habitat in structuring Halipegus occidualis metapopulations in the green frog

The transmission dynamics of the trematode Halipegus occidualis in its definitive host, Rana clamitans, have been examined over a 5-yr period in a North Carolina pond. The breeding season of green frogs coincides with the period of worm recruitment, during which time male frogs are territorial and females show strong site fidelity. This site fidelity allows inferences to be made regarding the suitability of a particular habitat for worm transmission based on frog infection intensities within that habitat. Four foci of infection were identified in the pond by plotting worm infrapopulation size against site of host capture. Sites within infection foci are characterized by shallow water and emergent vegetation, factors favorable for overlapping distributions of the 4 hosts in the life cycle of H. occidualis. Consistent year-to-year worm prevalences and intensities, despite fluctuations in frog population size, are thought to be the result of a relatively constant proportion of the frog population being present in infection foci each year. Removal of worms from heavily infected frogs in the fifth year resulted in further heavy worm recruitment by treated frogs suggesting that site selection can predispose a frog to heavy infection. Further, the sum of removed parasites and those recruited after parasite removal by treated frog hosts was higher than worm infrapopulations observed in previous years, indicating that worm density regulates parasite infrapopulation size in heavily infected frogs.     

High Prevalence of Batrachochytrium dendrobatidis in Wild Populations of Lowland Leopard Frogs Rana yavapaiensis in Arizona

Batrachochytrium dendrobatidis (Bd) is a fungus that can potentially lead to chytridiomycosis, an amphibian disease implicated in die-offs and population declines in many regions of the world. Winter field surveys in the last decade have documented die-offs in populations of the lowland leopard frog Rana yavapaiensis with chytridiomycosis. To test whether the fungus persists in host populations between episodes of observed host mortality, we quantified field-based Bd infection rates during nonwinter months. We used PCR to sample for the presence of Bd in live individuals from nine seemingly healthy populations of the lowland leopard frog as well as four of the American bullfrog R. catesbeiana (a putative vector for Bd) from Arizona. We found Bd in 10 of 13 sampled populations. The overall prevalence of Bd was 43% in lowland leopard frogs and 18% in American bullfrogs. Our results suggest that Bd is widespread in Arizona during nonwinter months and may become virulent only in winter in conjunction with other cofactors, or is now benign in these species. The absence of Bd from two populations associated with thermal springs (water >30°C), despite its presence in nearby ambient waters, suggests that these microhabitats represent refugia from Bd and chytridiomycosis.     

Prevalence of Batrachochytrium dendrobatidis in three species of wild frogs on Prince Edward Island, Canada

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has resulted in the decline or extinction of approximately 200 frog species worldwide. It has been reported throughout much of North America, but its presence on Prince Edward Island (PEI), on the eastern coast of Canada, was unknown. To determine the presence and prevalence of Bd on PEI, skin swabs were collected from 115 frogs from 18 separate sites across the province during the summer of 2009. The swabs were tested through single round end-point PCR for the presence of Bd DNA. Thirty-one frogs were positive, including 25/93 (27%) green frogs Lithobates (Rana) clamitans, 5/20 (25%) northern leopard frogs L. (R.) pipiens, and 1/2 (50%) wood frogs L. sylvaticus (formerly R. sylvatica); 12 of the 18 (67%) sites had at least 1 positive frog. The overall prevalence of Bd infection was estimated at 26.9% (7.2-46.7%, 95% CI). Prevalence amongst green frogs and leopard frogs was similar, but green frogs had a stronger PCR signal when compared to leopard frogs, regardless of age (p < 0.001) and body length (p = 0.476). Amongst green frogs, juveniles were more frequently positive than adults (p = 0.001). Green frogs may be the most reliable species to sample when looking for Bd in eastern North America. The 1 wood frog positive for Bd was found dead from chytridiomycosis; none of the other frogs that were positive for Bd by PCR showed any obvious signs of illness. Further monitoring will be required to determine what effect Bd infection has on amphibian population health on PEI.     

Altitudinal distribution of chytrid (Batrachochytrium dendrobatidis) infection in subtropical Australian frogs

The disappearance of amphibian populations from seemingly pristine upland areas worldwide has become a major focus of conservation efforts in the last two decades, and a parasitic chytrid fungus, Batrachochytrium dendrobatidis, is thought to be the causative agent of the population declines. We examined the altitudinal distribution of chytrid infections in three stream‐dwelling frog species (Litoria wilcoxii, L. pearsoniana and L. chloris) in southeast Queensland, Australia, and hypothesized that if B. dendrobatidis were responsible for the disappearance of high‐altitude frog populations, infection prevalence and intensity would be greatest at higher altitudes. Overall, 37.7% of the 798 adult frogs we sampled were infected with B. dendrobatidis, and infections were found in all the populations we examined. Contrary to our initial hypothesis, we found no consistent evidence that high‐altitude frogs were more likely to be infected than were lowland frogs. Further, the intensity of fungal infections (number of zoospores) on high‐altitude frogs did not differ significantly from that of lowland frogs. Batrachochytrium dendrobatidis appears to be capable of infecting frogs at all altitudes in the subtropics, suggesting that all populations are at risk of decline when conditions favour disease outbreaks. We did find evidence, however, that chytrid infections persist longer into summer in upland as compared with lowland areas, suggesting that montane amphibian populations remain susceptible to disease outbreaks for longer periods than do lowland populations. Further, we found that at high altitudes, temperatures optimal for chytrid growth and reproduction coincide with frog metamorphosis, the life‐stage at which frogs are most susceptible to chytrid infections. While these altitudinal differences may account for the differential population‐level responses to the presence of B. dendrobatidis, the reason why many of southeast Queensland's montane frog populations declined precipitously while lowland populations remained stable has yet to be resolved.     

Susceptibility of frogs to chytridiomycosis correlates with increased levels of immunomodulatory serotonin in the skin

Chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), is a skin disease responsible for the global decline of amphibians. Frog species and populations can vary in susceptibility, but this phenomenon remains poorly understood. Here, we investigated serotonin in the skin of infected and uninfected frogs. In more susceptible frog populations, skin serotonin rose with increasing infection intensity, but decreased in later stages of the disease. The more resistant population maintained a basal level of skin serotonin. Serotonin inhibited both Bd sporangial growth and Jurkat lymphocyte proliferation in vitro. However, serotonin accumulates in skin granular glands, and this compartmentalisation may prevent inhibition of Bd growth in vivo. We suggest that skin serotonin increases in susceptible frogs due to pathogen excretion of precursor tryptophan, but that resistant frogs are able to control the levels of serotonin. Overall, the immunosuppressive effects of serotonin may contribute to the susceptibility of frogs to chytridiomycosis.     

Condition-dependent reproductive effort in frogs infected by a widespread pathogen

To minimize the negative effects of an infection on fitness, hosts can respond adaptively by altering their reproductive effort or by adjusting their timing of reproduction. We studied effects of the pathogenic fungus Batrachochytrium dendrobatidis on the probability of calling in a stream-breeding rainforest frog (Litoria rheocola). In uninfected frogs, calling probability was relatively constant across seasons and body conditions, but in infected frogs, calling probability differed among seasons (lowest in winter, highest in summer) and was strongly and positively related to body condition. Infected frogs in poor condition were up to 40% less likely to call than uninfected frogs, whereas infected frogs in good condition were up to 30% more likely to call than uninfected frogs. Our results suggest that frogs employed a pre-existing, plastic, life-history strategy in response to infection, which may have complex evolutionary implications. If infected males in good condition reproduce at rates equal to or greater than those of uninfected males, selection on factors affecting disease susceptibility may be minimal. However, because reproductive effort in infected males is positively related to body condition, there may be selection on mechanisms that limit the negative effects of infections on hosts.     

Human-mediated Escalation of a Formerly Eradicable Problem: The Invasion of Caribbean Frogs in the Hawaiian Islands

Two species of neotropical frog, Eleutherodactylus coqui and E. planirostris, have been introduced into the State of Hawaii via the horticulture trade. E. coqui was introduced prior to 1988 and E. planirostris was first reported in 1994. Since these dates frog colonies have rapidly spread accidentally and intentionally and frog abundance within colonies has grown rapidly. Although these frogs were originally restricted to horticulture sites, they are now found in residential areas, resorts and hotels, and public lands. Due to the high potential biomass of introduced frogs there are realistic anthropogenic and ecological concerns associated with the spread of these frogs. Though there currently is a tool that can be used for localized control of frogs in limited circumstances, overall efforts by Federal, State, and County agencies to control the frog in Hawaii have been hampered by limited authorities and funds, disbelief in the threat, and the reluctance to act.     

Experimental transmission of cutaneous chytridiomycosis in dendrobatid frogs

In a series of three experiments during March-October, 1998, two species of captive-bred poison dart frogs (Dendrobates tinctorius and D. auratus) were exposed to Batrachochytrium dendrobatidis, a recently-described chytridiomycete fungus (chytrid) that was originally isolated from a blue poison dart frog (D. azureus). All frogs exposed to the chytrids developed a fatal skin disease, whereas none of the control frogs developed skin lesions. The most consistent clinical sign in chytrid-exposed frogs was excessive shedding of skin. Gross lesions were subtle, usually affected the legs and ventrum, and consisted of mild skin thickening and discoloration. Microscopic examination of shed skin pieces and/or skin imprints demonstrated the presence of chytrids and was used for ante mortem and post mortem confirmation of chytrid infection. Histologically, there was epidermal hyperkeratosis, hyperplasia, and hypertrophy associated with low to moderate numbers of chytrids in the keratinized layers. These experiments demonstrated that Batrachochytrium dendrobatidis can be a fatal pathogen in poison dart frogs. The experimentally-induced disease in these frogs resembled cases of cutaneous chytridiomycosis that have recently been described in several other species of captive and wild amphibians.     

Chytridiomycosis in native Arizona frogs

Twenty-seven adult/sub-adult lowland leopard frogs (Rana yavapaiensis), two larval lowland leopard frogs, two adult Chirichahua leopard frogs (Rana chiricahuensis), and two adult canyon tree frogs (Hyla arenicolor) collected from populations experiencing mortality events at eight sites were found to have characteristic lesions of chytrid fungus infection (Batrachochytrium dendrobatidis). The mortalities occurred during December 1992 and between October and February in 1997-98 and December and February in 1998-99. Gross lesions varied from none to diffuse reddening of the skin of the abdomen, pelvic area, and legs. Microscopic lesions were characteristic of those previously reported for the disease and included diffuse epidermal hyperplasia, hyperkeratosis, and colonization of the keratinized layers of the epidermis by sporangia of the chytrid. Bacterial cultures did not yield a primary pathogenic agent. Virus isolation from frog tissues was negative. Batrachochytrium dendrobatidis was isolated from the skin of two of 10 R. yavapaiensis and one of two H. arenicolor cultured following necropsy. An additional nine of 11 clinically affected or dead R. yavapaiensis from the same locations, but not necropsied, were culture positive for B. dendrobatidis.     

Stable isotope analyses—A method to distinguish intensively farmed from wild frogs

Consumption of frog legs is increasing worldwide, with potentially dramatic effects for ecosystems. More and more functioning frog farms are reported to exist. However, due to the lack of reliable methods to distinguish farmed from wild‐caught individuals, the origin of frogs in the international trade is often uncertain. Here, we present a new methodological approach to this problem. We investigated the isotopic composition of legally traded frog legs from suppliers in Vietnam and Indonesia. Muscle and bone tissue samples were examined for δ¹⁵N, δ¹³C, and δ¹⁸O stable isotope compositions, to elucidate the conditions under which the frogs grew up. We used DNA barcoding (16S rRNA) to verify species identities. We identified three traded species (Hoplobatrachus rugulosus, Fejervarya cancrivora and Limnonectes macrodon); species identities were partly deviating from package labeling. Isotopic values of δ¹⁵N and δ¹⁸O showed significant differences between species and country of origin. Based on low δ¹⁵N composition and generally little variation in stable isotope values, our results imply that frogs from Vietnam were indeed farmed. In contrast, the frogs from the Indonesian supplier likely grew up under natural conditions, indicated by higher δ¹⁵N values and stronger variability in the stable isotope composition. Our results indicate that stable isotope analyses seem to be a useful tool to distinguish between naturally growing and intensively farmed frogs. We believe that this method can be used to improve the control in the international trade of frog legs, as well as for other biological products, thus supporting farming activities and decreasing pressure on wild populations. However, we examined different species from different countries and had no access to samples of individuals with confirmed origin and living conditions. Therefore, we suggest improving this method further with individuals of known origin and history, preferably including samples of the respective nutritive bases.     

Strain differences in the amphibian chytrid Batrachochytrium dendrobatidis and non-permanent, sub-lethal effects of infection

The chytrid fungus Batrachochytrium dendrobatidis (Bd) is likely the cause of numerous recent amphibian population declines worldwide. While the fungus is generally highly pathogenic to amphibians, hosts express a wide range of responses to infection, probably due to variation among hosts and environmental conditions, but possibly also due to variation in Bd. We investigated variation in Bd by exposing standardized host groups to 2 Bd strains in a uniform environment. All exposed frogs became infected, but subsequent lethal and sub-lethal (weight loss) responses differed among groups. These results demonstrate variation in Bd and suggest variation occurs even at small geographical scales, likely explaining some of the variation in host responses. With lower than expected mortality among infected frogs, we continued our study opportunistically to determine whether or not frogs could recover from chytridiomycosis. Using heat, we cleared infection from half of the surviving frogs, leaving the other half infected, then continued to monitor mortality and weight. Mortality ceased among disinfected frogs but continued among infected frogs. Disinfected frogs gained weight significantly more than infected frogs, to the point of becoming indistinguishable from controls, demonstrating that at least some of the effects of sub-lethal chytridiomycosis on hosts can be non-permanent and reversible.     

Batrachochytrium dendrobatidis in amphibians confiscated from illegal wildlife trade and used in an ex situ breeding program in Brazil

This paper describes an outbreak of chytridiomycosis affecting a group of Dendrobates tinctorius, a Neotropical anuran species, confiscated from the illegal wildlife trade and housed in a private zoo in Brazil as part of an ex situ breeding program. We examined histological sections of the skin of 30 D. tinctorius and 20 Adelphobates galactonotus individuals. Twenty D. tinctorius (66.7%) and none of the A. galactonotus were positive for Batrachochytrium dendrobatidis (Bd). Multiple development stages of Bd infection were observed. The reasons for the inter-specific difference in the rate of infection could not be determined, and further studies are advised. Because the examined population consisted of confiscated frogs, detailed epidemiological aspects could not be investigated, and the source of the fungus remains uncertain. The existence of ex situ amphibian populations is important for protecting species at higher risk in the wild, and ex situ amphibian conservation and breeding programs in Brazil may be established using confiscated frogs as founders. However, this paper alerts these programs to the urgency of strict quarantine procedures to prevent the introduction of potential pathogens, particularly Bd, into ex situ conservation programs.     

Seasonality of Batrachochytrium dendrobatidis infection in direct-developing frogs suggests a mechanism for persistence

Batrachochytrium dendrobatidis (Bd), a disease-causing amphibian-specific fungus, is widely distributed in Puerto Rico, but is restricted to elevations above 600 m. The effect of this pathogen in the wild was studied by monitoring Eleutherodactylus coqui and E. portoricensis in 2 upland forests at El Yunque, a site characterized by historic population declines in the presence of chytridiomycosis. We tested a potential synergistic interaction between climate and Bd by measuring prevalence of infection and level of infection per individual sampled (number of zoospores), across the dry and wet seasons for 2 yr (between 2005 and 2007). Infection levels in adult frogs were significantly higher during the dry season in both species studied, suggesting a cyclic pattern of dry/ cool-wet/warm climate-driven synergistic interaction. These results are consistent with ex situ experiments in which E. coqui infected with Bd were more susceptible to chytridiomycosis when subjected to limited water treatments resembling drought. Long-term data on the prevalence of Bd in the populations studied versus intensity of infection in individual frogs provided contradictory information. However, the conflicting nature of these data was essential to understand the status of Bd in the species and geographical area studied. We conclude that in Puerto Rico, Bd is enzootic, and vulnerability of eleutherodactylid frogs to this pathogen is related to seasonal climatic variables. Our data suggest a mechanism by which this disease can persist in tropical frog communities without decimation of its hosts, but that complex interactions during severe droughts may lead to population crashes.     

Genetic detection of multiple exotic water frog species in Belgium illustrates the need for monitoring and immediate action

The recent genetic screening of water frogs (genus Pelophylax) in Belgium has shown that the invasion of two water frog species from Eastern Europe and the Mediterranean region, P. ridibundus and P. cf. bedriagae is widespread. Possibly other exotic water frogs are invading and establishing themselves through commercial trade. We used a genetic identification approach to rapidly detect and identify morphologically cryptic exotic water frog species in a large number of populations throughout the northern part of Belgium. Among a total of 944 individuals, we found 506 non-indigenous specimens, seven of which belonged to species not recorded before with certainty in Belgium or neighbouring regions in the wild. One of them was identified genetically as the Iberian green frog (P. perezi), but was most likely a P. perezi × P. esculentus hybrid. Six individuals of the Levantine frog (P. bedriagae) were found in a pond in the vicinity of a pet shop where the species is sold. All other exotic frogs belonged to four different haplotypes of P. ridibundus, established in Belgium since c. 1970, and two haplotypes of P. cf. bedriagae, a poorly-known eastern Mediterranean sister species of the latter. Overall, our study underscores the extent of exotic water frog invasions associated with the pet trade. Although two of the exotic species were recorded in small numbers, their early detection is essential with regards to adequate control and eradication of invasive species.     

Survey protocol for detecting chytridiomycosis in all Australian frog populations

Spread of the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) has caused the decline and extinction of frogs, but the distribution of Bd is not completely known. This information is crucial to implementing appropriate quarantine strategies, preparing for outbreaks of chytridiomycosis due to introduction of Bd, and for directing conservation actions towards affected species. This survey protocol provides a simple and standard method for sampling all frog populations in Australia to maximise the chances of detecting Bd. In order to structure and prioritise the protocol, areas are divided by bioregion and frog species are allocated depending on the water bodies they utilize into 3 groups representing different levels of risk of exposure to Bd. Sixty individuals per population need to be tested to achieve 95% certainty of detecting 1 positive frog, based on the minimum apparent prevalence of > or =5% in infected Australian frog populations and using a quantitative real-time TaqMan PCR test. The appropriate season to sample varies among bioregions and will ideally incorporate temperatures favourable for chytridiomycosis (e.g. maximum air temperatures generally <27 degrees C). Opportunistic collection and testing of sick frogs and tadpoles with abnormal mouth-parts should also be done to increase the probability of detecting Bd. The survey priorities in order are (1) threatened species that may have been exposed to Bd, (2) bioregions surrounding infected bioregions/ecological groups, and (3) species of frogs of unknown infection status in infected bioregions. Within these priority groups, sampling should first target ecological groups and species likely to be exposed to Bd, such as those associated with permanent water, and areas within bioregions that have high risk for Bd as indicated by climatic modelling. This protocol can be adapted for use in other countries and a standard protocol will enable comparison among amphibian populations globally.     

Host invasion by Batrachochytrium dendrobatidis: fungal and epidermal ultrastructure in model anurans

The chytridiomycete fungus Batrachochytrium dendrobatidis (Bd) colonizes mouthparts of amphibian larvae and superficial epidermis of post-metamorphic amphibians, causing the disease chytridiomycosis. Fungal growth within host cells has been documented by light and transmission electron microscopy; however, entry of the fungus into host cells has not. Our objective was to document how Bd enters host cells in the wood frog Lithobates sylvaticus, a species at high mortality risk for chytridiomycosis, and the bullfrog L. catesbeianus, a species at low mortality risk for chytridiomycosis. We inoculated frogs and documented infection with transmission electron microscopy. Zoospores encysted on the skin surface and produced morphologically similar germination tubes in both host species that penetrated host cell membranes and enabled transfer of zoospore contents into host cells. Documenting fungal and epidermal ultrastructure during host invasion furthers our understanding of Bd development and the pathogenesis of chytridiomycosis.