Life history tradeoffs influence mortality associated with the amphibian pathogen Batrachochytrium dendrobatidis

Fatal amphibian chytridiomycosis has typically been associated with the direct costs of infection. However the relationship between exposure to the pathogen, infection and mortality may not be so straightforward. Using results from both field work and experiments we report how exposure of common toads to Batrachochytrium dendrobatidis influences development and survival and how developmental stage influences host responses. Our results show that costs are accrued in a dose dependent manner during the larval stage and are expressed at or soon after metamorphosis. Exposure to B. dendrobatidis always incurs a growth cost for tadpoles and can lead to larval mortality before or soon after metamorphosis even when individuals do not exhibit infection at time of death. In contrast, exposure after metamorphosis almost always results in infection, but body size dictates survival to a greater extent than does dose. These data show that amphibian survival in the face of challenge by an infectious agent is dependent on host condition as well as life history stage. Under current models of climate change, many species of amphibia are predicted to increasingly occur outside their environmental optima. In this case, condition-dependent traits such as we have demonstrated may weigh heavily on species survival.     

Effects of an infectious fungus, Batrachochytrium dendrobatidis, on amphibian predator-prey interactions

The effects of parasites and pathogens on host behaviors may be particularly important in predator-prey contexts, since few animal behaviors are more crucial for ensuring immediate survival than the avoidance of lethal predators in nature. We examined the effects of an emerging fungal pathogen of amphibians, Batrachochytrium dendrobatidis, on anti-predator behaviors of tadpoles of four frog species. We also investigated whether amphibian predators consumed infected prey, and whether B. dendrobatidis caused differences in predation rates among prey in laboratory feeding trials. We found differences in anti-predator behaviors among larvae of four amphibian species, and show that infected tadpoles of one species (Anaxyrus boreas) were more active and sought refuge more frequently when exposed to predator chemical cues. Salamander predators consumed infected and uninfected tadpoles of three other prey species at similar rates in feeding trials, and predation risk among prey was unaffected by B. dendrobatidis. Collectively, our results show that even sub-lethal exposure to B. dendrobatidis can alter fundamental anti-predator behaviors in some amphibian prey species, and suggest the unexplored possibility that indiscriminate predation between infected and uninfected prey (i.e., non-selective predation) could increase the prevalence of this widely distributed pathogen in amphibian populations. Because one of the most prominent types of predators in many amphibian systems is salamanders, and because salamanders are susceptible to B. dendrobatidis, our work suggests the importance of considering host susceptibility and behavioral changes that could arise from infection in both predators and prey.     

Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses

Innate immune mechanisms of defense are especially important to ectothermic vertebrates in which adaptive immune responses may be slow to develop. One innate defense in amphibian skin is the release of abundant quantities of antimicrobial peptides. Chytridiomycosis is an emerging infectious disease of amphibians caused by the skin fungus, Batrachochytrium dendrobatidis . Susceptibility to chytridiomycosis varies among species, and mechanisms of disease resistance are not well understood. Previously, we have shown that Australian and Panamanian amphibian species that possess skin peptides that effectively inhibit the growth of B. dendrobatidis in vitro tend to survive better in the wild or are predicted to survive the first encounter with this lethal pathogen. For most species, it has been difficult to experimentally infect individuals with B. dendrobatidis and directly evaluate both survival and antimicrobial peptide defenses. Here, we demonstrate differences in susceptibility to chytridiomycosis among four Australian species ( Litoria caerulea, Litoria chloris, Mixophyes fasciolatus and Limnodynastes tasmaniensis ) after experimental infection with B. dendrobatidis , and show that the survival rate increases with the in vitro effectiveness of the skin peptides. We also observed that circulating granulocyte, but not lymphocyte, counts differed between infected and uninfected Lit. chloris . This suggests that innate granulocyte defenses may be activated by pathogen exposure. Taken together, our data suggest that multiple innate defense mechanisms are involved in resistance to chytridiomycosis, and the efficacy of these defenses varies by amphibian species.     

Prevalence of Batrachochytrium dendrobatidis in American bullfrog and southern leopard frog larvae from wetlands on the Savannah River Site, South Carolina

Batrachochytrium dendrobatidis, an aquatic fungus, has been linked to recent amphibian population declines. Few surveys have assessed B. dendrobatidis infections in areas where the disease is suggested to be less virulent and population declines have not been observed, such as southeastern North America. Although adult Rana catesbeiana and Rana sphenocephala from the Savannah River Site, South Carolina collected in 1979 and 1982 were identified as having B. dendrobatidis, it is unknown whether the fungus is currently present at the site or if susceptibility to infection varies among species or wetlands with different histories of environmental contamination. From 15 May through 15 August 2004, we collected R. catesbeiana and R. sphenocephala tadpoles from three wetlands with differing contamination histories on the Savannah River Site, South Carolina. We found B. dendrobatidis in only one of the wetlands we surveyed. Batrachochytrium dendrobatidis infection was identified in 64% of the R. catesbeiana tadpoles sampled and histologically assessed (n=50) from a wetland contaminated with mercury, copper, and zinc. No R. sphenocephala tadpoles from this site (n=50) were infected. In combination with a recently published report, our data suggest that B. dendrobatidis has been present at the Savannah River Site for over 25 yr but has not caused any apparent population declines. This time period is similar to the known presence of 30 yr of B. dendrobatidis in northeastern North America. Our data suggest that R. sphenocephala larvae might be resistant to infection, even when occupying the same wetland as the infected R. catesbeiana. Our survey did not clarify the effects of environmental contamination on infection severity, but our study stresses the importance of additional field surveys to document how this pathogen is affecting amphibians globally.     

Evaluation of tadpole mouthpart depigmentation as a diagnostic test for infection by Batrachochytrium dendrobatidis for four California anurans

The objective of this study was to evaluate the utility of gross morphologic examination of larval mouthpart defects as a diagnostic screening test to detect Batrachochytrium dendrobatidis infection in four California, USA, anuran species. We examined mouthparts of 2,034 tadpoles of Bufo boreas, Pseudacris regilla, and Rana catesbeiana collected in 2003 and 2004 and Bufo canorus collected in 2004. Data were recorded for three morphologic features: upper toothrows, lower toothrows, and combined jaw sheaths. Mouthpart defects were observed in all four species (n=757), but only two species were infected with B. dendrobatidis (n=84). Sensitivity and specificity of the mouthparts test were 76% and 58%, respectively. Forty-two percent of B. dendrobatidis-negative animals would have been designated positive based on mouthpart defects. Observed prevalence was 43%, and true prevalence was 3.0%. Tests of the null hypothesis using logistic regression analysis showed that anuran larval mouthpart defects were not associated with B. dendrobatidis infection whether mouthparts scores were tested by individual morphologic feature or in combination (P=0.37). We conclude that B. dendrobatidis infection and anuran larval mouthpart defects are two separate processes that may occur concurrently and that evaluation of tadpole oral morphology is neither an accurate nor a reliable diagnostic test for B. dendrobatidis infection for the four species tested.     

Behaviour of Australian rainforest stream frogs may affect the transmission of chytridiomycosis

The amphibian disease chytridiomycosis, caused by the pathogen Batrachochytrium dendrobatidis, has been implicated in mass mortalities, population declines and extinctions of amphibians around the world. In almost all cases, amphibian species that have disappeared or declined due to chytridiomycosis coexist with non-declining species. One reason why some species decline from chytridiomycosis and others do not may be interspecific differences in behaviour. Host behaviour could either facilitate or hinder pathogen transmission, and transmission rates in the field are likely to vary among species according the frequency of factors such as physical contact between frogs, contact with infected water and contact with environmental substrates containing B. dendrobatidis. We tracked 117 frogs (28 Litoria nannotis, 27 L. genimaculata and 62 L. lesueuri) at 5 sites where B. dendrobatidis is endemic in the rainforest of tropical northern Queensland and recorded the frequency of frog-to-frog contact and the frequency of contact with stream water and environmental substrates. Frequency of contact with other frogs and with water were highest in L. nannotis, intermediate in L. genimaculata and lowest in L. lesueueri. Environmental substrate use also differed among species. These species-specific opportunities for disease transmission were correlated with conservation status: L. nannotis is the species most susceptible to chytridiomycosis-related declines and L. lesueuri is the least susceptible. Interspecific variation in transmission probability may, therefore, play a large role in determining why chytridiomycosis drives some populations to extinction and not others.     

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.     

Surveillance for batrachochytrium dendrobatidis using Mixophyes (Anura: Myobatrachidae) larvae

Fourteen populations of anuran larvae (tadpoles), including three populations of the endangered Fleay's Barred Frog (Mixophyes fleayi) and 11 populations of the common Great Barred Frog (Mixophyes fasciolatus), in creek sites in the southeast region of Queensland were selected. Site selection was based on a history (within the district) of adult frog population declines and/or disappearances or records of infection of adult frogs or larvae by Batrachochytrium dendrobatidis. Larvae were collected once from each creek site between October 2002 and October 2004, and were between Gosner developmental stages 25 and 40. Total body length ranged from 18 mm to 100 mm. Mouthparts were examined under a dissecting microscope for grossly visible abnormalities, and then examined for histologic evidence of B. dendrobatidis. The most consistent mouthpart abnormalities found were multifocal depigmentation of the jaw sheaths and loss or shortening of the tooth rows. At the individual larva level, presence of mouthpart abnormalities was strongly associated with histologic diagnosis of B. dendrobatidis (93%). At least one larva with abnormal mouthparts was detected at 12 of the 14 sites and histologic evidence of B. dendrobatidis was detected at 13 of the 14 sites. These findings suggest that larvae of Mixophyes species are suitable for surveillance for B. dendrobatidis. We conclude that surveillance of B. dendrobatidis where individual larva prevalences of mouthpart abnormalities and histologic evidence of B. dendrobatidis are as high as those observed in this study (66% and 78%, respectively), relatively small numbers of larvae are required to detect these infections. Medium to large larvae (body length >30 mm) were much more likely to be affected than small larvae (body length < or =30 mm), suggesting that larger individuals should be targeted for surveillance.     

Unlikely Remedy: Fungicide Clears Infection from Pathogenic Fungus in Larval Southern Leopard Frogs (Lithobates sphenocephalus)

Amphibians are often exposed to a wide variety of perturbations. Two of these, pesticides and pathogens, are linked to declines in both amphibian health and population viability. Many studies have examined the separate effects of such perturbations; however, few have examined the effects of simultaneous exposure of both to amphibians. In this study, we exposed larval southern leopard frog tadpoles (Lithobates sphenocephalus) to the chytrid fungus Batrachochytrium dendrobatidis and the fungicide thiophanate-methyl (TM) at 0.6 mg/L under laboratory conditions. The experiment was continued until all larvae completed metamorphosis or died. Overall, TM facilitated increases in tadpole mass and length. Additionally, individuals exposed to both TM and Bd were heavier and larger, compared to all other treatments. TM also cleared Bd in infected larvae. We conclude that TM affects larval anurans to facilitate growth and development while clearing Bd infection. Our findings highlight the need for more research into multiple perturbations, specifically pesticides and disease, to further promote amphibian heath.     

Transmission of Batrachochytrium dendrobatidis within and between amphibian life stages

Chytridiomycosis is an emerging infectious disease caused by the chytrid fungus Batrachochytrium dendrobatidis, which has been implicated in amphibian declines worldwide. The mountain yellow-legged frog Rana muscosa is a declining amphibian species that can be infected by B. dendrobatidis; however, transmission between conspecifics has not been documented. Here, we present experimental evidence that R. muscosa tadpoles can be infected by fungal zoospores and that they can transmit infection to each other and to postmetamorphic animals. We compared several techniques for detecting B. dendrobatidis transmission and found that histology with serial sectioning was able to detect infection before cytology or visual inspections. We also show that R. muscosa tadpoles appear healthy with B. dendrobatidis infection, while postmetamorphic animals experience mortality. In addition, we provide guidelines for visually detecting B. dendrobatidis in R. muscosa tadpoles, which may be useful in other affected species. Field surveys of infected and uninfected populations verify this identification technique.     

Disease associated with integumentary and cloacal parasites in tadpoles of northern red-legged frog Rana aurora aurora

A total of 6830 northern red-legged frog Rana aurora aurora tadpoles were examined under a dissecting microscope for oral disc, integumentary, and cloacal abnormalities in 13 ponds in and near Redwood National Park in northern California. Of these, 163 tadpoles were collected for histopathological investigation, including 115 randomly collected individuals, 38 collected with oral disc abnormalities, and 10 collected due to severe morbidity of unknown etiology. The tadpoles were infected with 8 parasites, including Batrachochytrium dendrobatidis (the amphibian chytrid), trematodes, leeches, and protozoa. Chytridiomycosis was detected at an overall prevalence of 6.4%, but prevalence was higher in tadpoles with oral disc lesions than in those with normal oral discs (43.5% versus 6.1%). Interestingly, infection was associated with some environmental and co-infection risk factors. Individual tadpoles possessed 0 to 5 species of parasites in varying intensities. Apiosoma sp. was the most prevalent (66%) and widespread. Tadpoles infected with B. dendrobatidis had a lower diversity of oral parasites than those uninfected. During the field portion of the study, a large number (approximately 500) of moribund and dead tadpoles was seen occurring at multiple locations within and surrounding Redwood National Park. Ten animals were collected for histological examination and a diverse protozoal infection was discovered, including some known pathogens of fish. This study is the first reporting parasitism and disease in natural populations of northern red-legged frogs.     

Effects of induced variation in anuran larval development on postmetamorphic energy reserves and locomotion

Anuran larvae exhibit high levels of phenotypic plasticity in growth and developmental rates in response to variation in temperature and food availability. We tested the hypothesis that alteration of developmental pathways during the aquatic larval stage should affect the postmetamorphic performance of the Iberian painted frog (Discoglossus galganoi). We exposed tadpoles to different temperatures and food types (animal- vs. plant-based diets) to induce variation in the length of the larval period and body size at metamorphosis. In this species, larval period varied with temperature but was unaffected by diet composition. In contrast, size at metamorphosis was shaped by the interaction between food quality and temperature; tadpoles fed on an animal-based diet became bulkier metamorphs than those fed on plant-based food at high (22°C) but not at low (12°C) temperature. Body condition of newly metamorphosed frogs was unrelated to the temperature or food type experienced during the premetamorphic stage. Frogs maintained at high temperature during the larval period showed reduced jumping ability, especially when fed on the plant-based diet. However, when considering size-independent jumping ability, cold-reared individuals exhibited the lowest performance, and herbivores reared at 17°C the highest. Cold-reared (12°C) frogs accumulated larger amounts of energy reserves than individuals raised at 17°C or 22°C. This was still the case after correction for differences in body mass, thus indicating some size-independent effect of developmental temperature. Despite the higher lipid content of the carnivorous diet, the differences in energy reserves between herbivores and carnivores were relatively weak and associated with differences in body size. These results suggest that the consequences of environmental variation in the larval habitat can extend to the terrestrial phase and influence juvenile growth and survival.     

Early-spring survey for Batrachochytrium dendrobatidis in wild Rana dybowskii in Heilongjiang Province, China

Batrachochytrium dendrobatidis has been investigated worldwide because of its importance in population declines in multiple species of amphibians; however, little is known regarding the disease status of all native amphibian species in China. The present study is the first survey of chytridiomycosis in free-ranging amphibian populations in China, and it examined the possible presence of B. dendrobatidis in Rana dybowskii in northeastern China (Heilongjiang Province). R. dybowskii is mainly distributed in the northeast part of China and is intensively hunted for human consumption, making populations vulnerable to extirpation in the event of additional stresses from disease epidemics. The survey was performed in early spring of 2008, using a PCR assay, histological examination of skin samples, and zoospore culture. In total, 191 frogs were examined; thus, a 95% confidence limit for prevalence of 1.57% was selected. Our results demonstrate that R. dybowskii is currently free from chytridiomycosis in Heilongjiang, even though the natural conditions of the sampling sites are suitable for the occurrence of B. dendrobatidis. Central and local governments should implement strict management measures to prevent the escape of non-native commercial amphibian species into this area, which might endanger local populations of native species.     

Batrachochytrium dendrobatidis infection of amphibians in the Doñana National Park, Spain

Amphibian chytridiomycosis, caused by infection with the non-hyphal, zoosporic chytrid fungus Batrachochytrium dendrobatidis (Bd), is an emerging infectious disease recognised as a cause of recent amphibian population declines and extinctions worldwide. The Do?ana National Park (DNP) is located in southwestern Spain, a country with widespread Bd infection. This protected area has a great diversity of aquatic habitats that constitute important breeding habitats for 11 native amphibian species. We sampled 625 amphibians in December 2007 and February to March 2008, months that correspond to the early and intermediate breeding seasons for amphibians, respectively. We found 7 of 9 sampled species to be infected with Bd and found differences in prevalence between sampling periods. Although some amphibians tested had higher intensities of infection than others, all animals sampled were apparently healthy and, so far, there has been no evidence of either unusually high rates of mortality or amphibian population declines in the DNP.     

Effect of temperature on host response to Batrachochytrium dendrobatidis infection in the mountain yellow-legged frog (Rana muscosa)

The pathogenic chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis, has been implicated in declines of amphibian populations throughout the world, including declines and extinctions of local populations of mountain yellow-legged frogs, Rana muscosa, in the California Sierra Nevada. Previous studies have shown B. dendrobatidis achieves its maximum growth rate in culture in the temperature range of 17-25 C, and exposure to very high temperatures can clear frogs of B. dendrobatidis infection. Here we present the results of a laboratory experiment in which experimentally infected R. muscosa tadpoles were followed through metamorphosis at temperatures of 17 and 22 C. All infected animals developed clinical disease within a similar time frame. However, frogs housed at 22 C exhibited a significantly lower mortality than those housed at 17 C. Within 35 days after metamorphosis, 50% of the frogs housed at 22 C died, while 95% of the frogs housed at 17 C died. Clinical signs subsided in the surviving frogs at 22 C, despite persistent infection. Because both temperatures are within the optimal thermal range for growth of B. dendrobatidis, we propose that the difference in outcome indicates the effect of temperature on the host's resistance to chytridiomycosis, rather than an effect on the fungus alone.     

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.     

Detection of chytridiomycosis caused by Batrachochytrium dendrobatidis in the endangered Sardinian newt (Euproctus platycephalus) in southern Sardinia, Italy

The chytridiomycete fungus Batrachochytrium dendrobatidis is known to be focally distributed across Europe, but has only been linked to "chytridiomycosis at a few locations in Spain. Here we report the second occurrence of chytridiomycosis in European amphibians. We found a population of endangered Sardinian newts (Euproctus platycephalus) exhibiting clinical signs of disease including loss of digits and patchy, discolored skin. Molecular examination of skin samples tested positive for B. dendrobatidis. The population of E. platycephalus has been in decline on a timescale consistent with the global emergence of chytridiomycosis, and the ecology of this salamander suggests that the disease in this species warrants concern.     

Production of polyclonal antibodies to Batrachochytrium dendrobatidis and their use in an immunoperoxidase test for chytridiomycosis in amphibians

Polyclonal antibodies were produced for diagnosing chytridiomycosis in amphibians. Two sheep and 4 rabbits were inoculated with homogenized whole culture of Batrachochytrium dendrobatidis in Freund's complete adjuvant or triple adjuvant. Antisera from all animals reacted strongly with all stages of B. dendrobatidis and stained the walls, cytoplasm, rhizoids and zoospores in an indirect immunoperoxidase test. Significant cross-reactivity occurred only with some fungi in the Chytridiomycota, and there are no members of this phylum besides B. dendrobatidis that infect frogs. The immunoperoxidase stain is a useful screening test when combined with recognition of the morphology and infection site of B. dendrobatidis.     

Co-localisation of Batrachochytrium dendrobatidis and keratin for enhanced diagnosis of chytridiomycosis in frogs

Chytridiomycosis is a disease of post-metamorphic frogs caused by the fungus Batrachochytrium dendrobatidis and is associated with large declines in frog populations on a global scale. B. dendrobatidis is found only in the keratinised tissues, which include the mouthparts of healthy tadpoles. The epidermis of infected post-metamorphic frogs is thickened (hyperkeratosis) and the superficial layer can sometimes slough. Diagnosis is most commonly performed on stained sections of toe clips or ventral skin. Accurate interpretation can be difficult and requires a high level of expertise, particularly in infected animals exhibiting hyperkeratosis with sloughing. Misdiagnosis can occur when zoosporangia of B. dendrobatidis are shed with the superficial keratin layers. We have developed a staining protocol based on previously described methods to detect both B. dendrobatidis and keratin, to improve the sensitivity and specificity of diagnosis of chytridiomycosis by inexperienced diagnosticians.     

Amphibian immune defenses against chytridiomycosis: impacts of changing environments

Eco-immunology is the field of study that attempts to understand the functions of the immune system in the context of the host's environment. Amphibians are currently suffering devastating declines and extinctions in nearly all parts of the world due to the emerging infectious disease chytridiomycosis caused by the chytrid fungus, Batrachochytrium dendrobatidis. Because chytridiomycosis is a skin infection and remains confined to the skin, immune defenses of the skin are critical for survival. Skin defenses include secreted antimicrobial peptides and immunoglobulins as well as antifungal metabolites produced by symbiotic skin bacteria. Low temperatures, toxic chemicals, and stress inhibit the immune system and may impair natural defenses against B. dendrobatidis. Tadpoles' mouth parts can be infected by B. dendrobatidis. Damage to the mouth parts can impair growth, and the affected tadpoles maintain the pathogen in the environment even when adults have dispersed. Newly metamorphosing frogs appear to be especially vulnerable to infection and to the lethal effects of this pathogen because the immune system undergoes a dramatic reorganization at metamorphosis, and postmetamorphic defenses are not yet mature. Here we review our current understanding of amphibian immune defenses against B. dendrobatidis and the ability of the pathogen to resist those defenses. We also briefly review what is known about the impacts of temperature, environmental chemicals, and stress on the host-pathogen interactions and suggest future directions for research.