Infected Frogs Research Articles

Neutralization of bacterial endotoxins by frog antimicrobial peptides

The ability of skin antimicrobial peptides of the southern bell frog, Litoria raniformis, to neutralize in vitro the endotoxin, proinflammatory lipopolysaccharide (LPS) complex, from two different gram-negative bacterial pathogens, human pathogen Escherichia coli (0111:B4) and frog pathogen Klebsiella pneumoniae, was investigated. The LPS neutralization activity of the natural mixture of skin antimicrobial peptides was measured using chromogenic Limulus amebocyte lysate assays. These skin antimicrobial peptides neutralized the LPSs from both pathogens at physiologically relevant concentrations (IC(50) < 100 µg/mL) showing their potential for non-specific LPS neutralization in vivo in the skin of infected frogs and for development of anti-endotoxin agents.

Influence of Hepatozoon parasites on host-seeking and host-choice behaviour of the mosquitoes Culex territans and Culex pipiens

Hepatozoon species are heteroxenous parasites that commonly infect the blood of vertebrates and various organs of arthropods. Despite their ubiquity, little is known about how these parasites affect host phenotype, including whether or not these parasites induce changes in hosts to increase transmission success. The objectives of this research were to investigate influences of the frog blood parasite Hepatozoon clamatae and the snake blood parasite Hepatozoon sipedon on host-seeking and host-choice behaviour of the mosquitoes Culex territans and Culex pipiens, respectively. During development of H. sipedon in C. pipiens, significantly fewer infected mosquitoes fed on uninfected snakes compared to uninfected mosquitoes. When H. sipedon was mature in C. pipiens, the number of infected and uninfected C. pipiens that fed on snakes was not significantly different. Higher numbers of mosquitoes fed on naturally infected snakes and frogs compared to laboratory-reared, uninfected control animals. However, experiments using only laboratory-raised frogs revealed that infection did not significantly affect host choice by C. territans. Behaviour of C. pipiens in the presence of H. sipedon may increase transmission success of the parasite and provide the first evidence of phenotypic changes in the invertebrate host of Hepatozoon parasites.

New Host and Distribution Records forAplectana hamatospicula(Ascaridida: Cosmocercidae) inGastrophryne olivacea(Anura: Microhylidae) from the Great Plains U.S.A.

Aplectana hamatospicula is a nematode that parasitizes the large intestine of anurans, and has been reported from bufonids, eleutherodactylids, hylids, microhylids, and ranids from North and Central America. Aplectana hamatospicula was first described, over 70 yr ago, from Hyla eximia and Bufo peltocephalus from Mexico and Cuba, respectively, and reported from Gastrophryne carolinensis from Florida. Since then there have been no reports of this nematode in North America north of Mexico. The life cycle of A. hamatospicula is not known, and there is limited information on Aplectana spp. from North America. During 2010-2011, we collected 351 anurans of 8 species from 4 locations in Stillwater, Payne County, Oklahoma, U.S.A., and examined them for the presence of Aplectana spp. Of the 8 species of anurans surveyed, A. hamatospicula infected only the Great Plains narrowmouth toad (Gastrophryne olivacea). The prevalence of A. hamatospicula was 85.7% (12/14) with a mean abundance of 28.4 + 24.0 and a mean intensity of 33.1 + 22.8 worms per infected toad. We provide new morphological measurements for male and female A. hamatospicula along with new locality records for this nematode in North America.

Benefits and costs of using a lungworm parasite to control invasive toads in Australia

Pizzatto & Shine (2012) noted the serious impacts that the introduced cane toad Rhinella marina has caused to its predators in Australia. The toad’s range is expanding at an increasing rate and additional negative consequences are predicted. Control measures have been ineffective or in some cases have not been tried. Female toads have a very high fecundity, and the species has a high potential growth rate. High removal rates of the toad population are needed to eliminate the species, suggesting that physical removal is not practical (Shine & Doody, 2010). One approach to controlling the toad species is biological control, which has been effective in some cases in Australia and elsewhere (Saunders et al., 2010). Biological control usually involves introduction of a species that in its native range controls the population of the pest species. However, introducing an exotic species as a biocontrol agent can have negative effects on nontarget species and potentially on ecosystem function (Simberloff & Stiling, 1996). Use of a native species as a biological control agent eases these concerns, but may involve a protocol to increase the abundance and distribution of a native control agent. In the case of the cane toad, one goal of biocontrol can be slowing the rate of expansion. Individuals at the leading edge of expansion have greater locomotor endurance, and the authors suggest that this may be due to escape from a lungworm parasite that infects toads behind the leading edge. Therefore, the authors test aspects of a general hypothesis that posits that lungworm parasites can be used as a biocontrol agent to slow the toads’ advance. This may not be an entirely satisfactory solution, but slowing the toads’ advance might buy time to develop other ways to control the species’ numbers. As the lungworm is already present in the toads and likely was present in the toads when they were introduced to Australia in 1935, it is not considered an exotic species. The authors suggest that one way to increase the prevalence of lungworm infection is to infect a native tree frog species, Litoria caerulea, which does not seem to be negatively affected by the parasite. They also propose three primary assumptions necessary for biocontrol to work in this case: the parasite does not harm the tree frogs, the tree frogs can maintain an infection over the long term, and infective larvae of the parasite can be transmitted to cane toads and reduce viability of the toads. They test aspects of these assumptions in their paper. Pizzatto and Shine showed that the tree frogs were infected at a 50% rate at the end of the experiment, and this was after placing the lungworm in the mouths of the frogs. All parasite-exposed frogs survived, and their growth rate was not different from that of uninfected controls. It would be important to compare the growth of infected frogs and uninfected frogs as the ‘parasite-exposed’ treatment apparently contained frogs that were not infected or had cleared the infection. Of those frogs that were infected, they had on average a low intensity (1.67 parasites per frog). It is unclear that this intensity would have the desired effect of transmission of larvae to toads. To test for transmission to the toads, lungworm larvae were put in boxes housing five toads. About half of the toads were infected under these laboratory conditions. In terms of fitness effects on the toads, there was no survival difference between infected and uninfected, no difference in body mass and no difference in sprint speeds. The authors did find that toads exposed to lungworms had lower stamina than did controls. Laboratory conditions are likely to maximize transmission of parasites as toads were under high density conditions, and their movements were constrained. However, under these conditions, parasite prevalence in frogs and toads averaged 50%. Importantly, only minor fitness effects were seen in the toads. Based on these results, it seems doubtful that control would be effective in nature. There are other reasons to be skeptical that biocontrol would be effective in this case. Is the lungworm a factor in population control in other parts of the toads’ range where prevalence is high? If not, why would it be expected to work bs_bs_banner

Is Typhoid Mary a good role model?

Cane toads, Bufo marinus, are among the iconic poster children of invasive species, in part thanks to Mark Lewis’s 1988 Australian documentary, ‘Cane Toads: An Unnatural History’, and subsequent follow-up, ‘Cane Toads: The Conquest’. Unfortunately, the conquest in his latest video refers to what the cane toads have achieved, not the Australians’ efforts to control them, despite the impressive research program of Rick Shine and his colleagues at the University of Sidney, among others. In this issue’s featured paper, Pizzatto & Shine (2012) show that a lungworm parasite of the invasive cane toad, apparently brought to Australia along with the toad, can infect native green tree frogs, Litoria caerulea, with no evident detrimental effect on the frogs. These frogs can maintain the infection for up to 120 days postexposure, and infective lungworm larvae in the native frogs’ feces can then infect cane toads, substantially reducing their stamina. This leads to the intriguing suggestion that biological control of the cane toad could be undertaken by deliberately infecting these native frogs, which could then disseminate the lungworm to the cane toads, as a sort of ‘Typhoid Mary’ – an asymptomatic carrier of the pathogen that, while remaining healthy, spreads disease to others. The authors are not ready to advocate this approach in this system, ‘except in areas of high conservation value where toads are likely to have major impacts’, primarily on the grounds of possible undetected effects on green tree frog viability and secondarily on animal welfare grounds – although if, in fact, the parasite has no effect on green tree frogs, it is not clear how infecting them is imposing suffering. A more important reason to avoid the ‘Typhoid Mary’ approach in this case would seem to be because a close relative of the green tree frog, the splendid tree frog (Litoria splendida), has been found to be very sensitive to this lungworm parasite, at least in laboratory trials (Pizzatto & Shine, 2011a). I wonder if the likelihood of spread to L. splendida by deliberately infected L. caerulea would actually be greater than spread to cane toads, considering the two native frogs are congeners. The concept is certainly novel, and we need novel ways to control invasive pests. However, this approach, while using a native species to spread the parasite, ultimately depends on the use of an exotic organism. Although it is true that many parasites and parasitoid insects are quite host specific, unfortunately, this is not always the case. A parasitoid fly introduced as a biological control agent for gypsy moth and other pest Lepidoptera was subsequently found to impact populations of several native giant silk moth species in North America (Boettner, Elkinton & Boettner, 2000). Other examples of negative effects of biological control agents are cited by Howarth (1991), Simberloff & Stiling (1996) and Barratt et al. (2010). Current regulations emphasize the need for host specificity of any introduction, and concern for non-target impacts is more and more a concern in the regulatory environment (Hajek, McManus & Junior, 2007; Barratt et al., 2010). The cane toad is also an iconic poster child for biological control gone wrong, and in our efforts to control it, we must ensure that things are not made worse – if that is possible. Typhoid Mary unknowingly spread the bacteria that cause typhoid fever to other members of her own species, as a rather unsanitary cook to a whole series of human families. A possible approach in the cane toad system might be to take heavily infected cane toads and release them at the invasion front (where the lungworm is often absent), or otherwise enhance the level of lungworm parasites, if this can be done in a way that would not significantly increase the toad population – perhaps by releasing small infected metamorphs that would likely be cannibalized by larger cane toads (Pizzatto & Shine, 2011b). So far, efforts to identify a truly cane toad-specific pathogen for use as a biological control agent have been unsuccessful, as have been efforts to induce autoimmunity and disrupt metamorphosis (Pallister et al., 2011). Alarm pheromones and translocation of parasitic lungworms to the invasion front may provide new tools in the management of this damaging pest species (Saunders et al., 2010). Unfortunately, at present, a truly effective and safe ‘integrated toad management’ program still seems elusive.

Immune evasion or avoidance: Fungal skin infection linked to reduced defence peptides in Australian green-eyed treefrogs, Litoria serrata

Many parasites and pathogens suppress host immunity to maintain infection or initiate disease. On the skin of many amphibians, defensive peptides are active against the fungus Batrachochytrium dendrobatidis (Bd), the causative agent of the emerging infectious disease chytridiomycosis. We tested the hypothesis that infection with the fungus may be linked to lower levels of defensive peptides. We sampled both ambient (or constitutive) skin peptides on the ventral surface immediately upon capture, and stored skin peptides induced from granular glands by norepinephrine administration of Australian green-eyed treefrogs, Litoria serrata. Upon capture, uninfected frogs expressed an array of antimicrobial peptides on their ventral surface, whereas infected frogs had reduced skin peptide expression. Expression of ambient skin peptides differed with infection status, and antimicrobial peptides maculatin 1.1 and 2.1 were on average three times lower on infected frogs. However, the repertoire of skin peptides stored in granular glands did not differ with infection status; on average equal quantities were recovered from infected and from uninfected frogs. Our results could have at least two causes: (1) frogs with reduced peptide expression are more likely to become infected; (2) Bd infection interferes with defence peptides by inhibiting release or causing selective degradation of peptides on the skin surface. Immune evasion therefore may contribute to the pathogenesis of chytridiomycosis and a mechanistic understanding of this fungal strategy may lead to improved methods of disease control.

Hematologic and plasma biochemical reference intervals for health monitoring of wild Australian tree frogs

Few hematologic and biochemical reference intervals for wild amphibians have been established. Reference values would aid in early detection of emerging infectious diseases, which are a significant problem for amphibian conservation efforts. We aimed to establish reference intervals for a wide range of hematologic and plasma biochemistry variables for 2 species of Australian tree frogs, describe morphologic features of leukocytes, and analyze the effects of season, year, and parasite status on blood values. Blood specimens were collected from reference populations of wild adult Australian tree frogs, Litoria caerulea and L infrafrenata, for analysis of hematologic (manual) variables, plasma biochemical (automated) analytes, and plasma and serum proteins using automated methods, refractometry, and electrophoresis. Inter- and intraspecies differences were found in L caerulea (n = 80) and L infrafrenata (n = 66) frogs for hematologic and biochemical variables. Intraspecies differences were largely associated with seasonal variations. In the dry season, both species had higher WBC counts, with higher lymphocyte counts in L caerulea and higher neutrophil counts in L infrafrenata, and uric acid concentrations. In the wet season, both species had higher glucose and potassium concentrations, L caerulea frogs had higher neutrophil counts, and L infrafrenata frogs had higher total protein, phosphorus, and sodium concentrations, AST activity, PCV, hemoglobin concentration, and RBC, thrombocyte, and basophil counts. Hemogregarines were identified in 19% of blood samples from L infrafrenata frogs; multiple hematologic and biochemical variables were altered in infected frogs. Wide interspecies and seasonal variations highlight the need to establish species- and season-specific reference intervals for amphibians. Hematologic and plasma biochemical reference values should be useful in assessing the health status and in detecting emerging diseases in wild amphibians.

Prevalence of Batrachochytrium dendrobatidis infection is extremely low in direct-developing Australian microhylids

The emerging infectious disease chytridiomycosis has been implicated in declines and disappearances of amphibian populations around the world. However, susceptibility to infection and the extent of pathological effects of infection vary among hosts, and species with life histories that include parental care of direct-developing terrestrial eggs may tend to be less susceptible. We examined samples from a total of 595 individuals of 9 species of direct-developing Australian frogs in the family Microhylidae for the presence of infection by Batrachochytrium dendrobatidis (Bd). Between 1995 and 2004, 336 samples were collected; 102 of these were analysed histologically and 234 were tissues stored in alcohol, which were examined using diagnostic quantitative PCR (qPCR). Swab samples were collected from 259 frogs from 2005 to 2008 and were examined using qPCR. None of the 595 samples showed evidence of infection by Bd. If these data are regarded as a single sample representative of Australian microhylids, the upper 95% binomial confidence limit for the prevalence of infection in frogs of this family is 0.0062 (<1%). Even if only the data from the more powerful diagnostic qPCR tests are used, the upper 95% confidence limit for prevalence is 0.0075 (<1%). Our data suggest that Australian microhylids have a very low prevalence of infection by Bd in nature, and thus are either not susceptible, or are only slightly susceptible, to chytridiomycosis. This could be due solely to, or in combination with, low rates of transmission and to factors that promote resistance to infection, including ecological or behavioural characteristics, innate immune functions such as antimicrobial skin peptides, or antimicrobial symbionts in skin flora.

Molecular Biology Techniques Q&amp;A

Molecular Biology Techniques Q&amp;A

Size and sex matter: infection dynamics of an invading parasite (the pentastome Raillietiella frenatus) in an invading host (the cane toad Rhinella marina)

Correlations between host phenotype and vulnerability to parasites can clarify the processes that enhance rates of parasitism, and the effects of parasites on their hosts. We studied an invasive parasite (the pentastome Raillietiella frenatus, subclass Pentastomida, order Cephalobaenida) infecting a new host (the invasive cane toad Rhinella marina), in tropical Australia. We dissected toads over a 27-month period to investigate seasonal changes in pentastome population dynamics and establish which aspects of host phenotype are related to infection. Pentastome prevalence and intensity varied seasonally; male toads were 4 times more likely to be infected than were females; and prevalence was highest in hosts of intermediate body size. The strong sex effect may reflect habitat or dietary divergence between the sexes, resulting in males encountering parasites more often. The relationship between pentastome prevalence and host size likely reflects a role for acquired immunity in preventing re-infection. Infection did not influence host body condition (fatbody size), suggesting that R. frenatus does not impose high energy costs in cane toads. Infected toads had heavier spleens (likely an immune response to infection) and larger testes (perhaps since reproductively active hosts have altered microhabitat use and/or immunocompetence) than did uninfected conspecifics. Although experimental studies are required to identify the causal bases of such patterns, our data confirm that infection status within a population can be strongly linked to host phenotypic traits.

Germ Tube Mediated Invasion of Batrachochytrium dendrobatidis in Amphibian Skin Is Host Dependent

Batrachochytrium dendrobatidis (Bd) is the causative agent of chytridiomycosis, a fungal skin disease in amphibians and driver of worldwide amphibian declines.We focussed on the early stages of infection by Bd in 3 amphibian species with a differential susceptibility to chytridiomycosis. Skin explants of Alytes muletensis, Litoria caerulea and Xenopus leavis were exposed to Bd in an Ussing chamber for 3 to 5 days. Early interactions of Bd with amphibian skin were observed using light microscopy and transmission electron microscopy. To validate the observations in vitro, comparison was made with skin from experimentally infected frogs. Additional in vitro experiments were performed to elucidate the process of intracellular colonization in L. caerulea.Early interactions of Bd with amphibian skin are: attachment of zoospores to host skin, zoospore germination, germ tube development, penetration into skin cells, invasive growth in the host skin, resulting in the loss of host cell cytoplasm. Inoculation of A. muletensis and L. caerulea skin was followed within 24 h by endobiotic development, with sporangia located intracellularly in the skin. Evidence is provided of how intracellular colonization is established and how colonization by Bd proceeds to deeper skin layers. Older thalli develop rhizoid-like structures that spread to deeper skin layers, form a swelling inside the host cell to finally give rise to a new thallus.In X. laevis, interaction of Bd with skin was limited to an epibiotic state, with sporangia developing upon the skin. Only the superficial epidermis was affected. Epidermal cells seemed to be used as a nutrient source without development of intracellular thalli. The in vitro data agreed with the results obtained after experimental infection of the studied frog species. These data suggest that the colonization strategy of B. dendrobatidis is host dependent, with the extent of colonization most likely determined by inherent characteristics of the host epidermis.

Getting under—and through—the skin: ecological genomics of chytridiomycosis infection in frogs

Amphibian species around the world are currently becoming endangered or lost at a rate that outstrips other vertebrates—victims of a combination of habitat loss, climate change and susceptibility to emerging infectious disease (Stuart et al. 2004). One of the most devastating such diseases is caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), which infects hundreds of amphibian species on multiple continents. While Bd itself has been characterized for some time, we still know little about the mechanisms that make it so deadly. In this issue of Molecular Ecology, Rosenblum et al. describe a genomic approach to this question, reporting the results of a genome-wide analysis of the transcriptional response to Bd in the liver, skin and spleen of mountain yellow-legged frogs (Rana mucosa and R. sierrae: Fig. 1) (Rosenblum et al. 2012). Their results indicate that the skin is not only the first, but likely the most important, line of defence in these animals. Strikingly, they describe a surprisingly modest immune response to infection in Rana, a result that may help explain variable Bd susceptibility across populations and species.

Sodium Chloride Inhibits the Growth and Infective Capacity of the Amphibian Chytrid Fungus and Increases Host Survival Rates

The amphibian chytrid fungus Batrachochytrium dendrobatidis is a recently emerged pathogen that causes the infectious disease chytridiomycosis and has been implicated as a contributing factor in the global amphibian decline. Since its discovery, research has been focused on developing various methods of mitigating the impact of chytridiomycosis on amphibian hosts but little attention has been given to the role of antifungal agents that could be added to the host's environment. Sodium chloride is a known antifungal agent used routinely in the aquaculture industry and this study investigates its potential for use as a disease management tool in amphibian conservation. The effect of 0–5 ppt NaCl on the growth, motility and survival of the chytrid fungus when grown in culture media and its effect on the growth, infection load and survivorship of infected Peron's tree frogs (Litoria peronii) in captivity, was investigated. The results reveal that these concentrations do not negatively affect the survival of the host or the pathogen. However, concentrations greater than 3 ppt significantly reduced the growth and motility of the chytrid fungus compared to 0 ppt. Concentrations of 1–4 ppt NaCl were also associated with significantly lower host infection loads while infected hosts exposed to 3 and 4 ppt NaCl were found to have significantly higher survival rates. These results support the potential for NaCl to be used as an environmentally distributed antifungal agent for the prevention of chytridiomycosis in susceptible amphibian hosts. However, further research is required to identify any negative effects of salt exposure on both target and non-target organisms prior to implementation.

Lungworm infection modifies cardiac response to exercise in cane toads

AbstractParasites can affect the locomotor performance of their hosts via a range of mechanisms. Cane toads (Rhinella marina) in Australia often contain native‐range lung nematodes (Rhabdias pseudosphaerocephala), and the parasite reduces endurance (and thus, dispersal rate) of this invasive anuran. The mechanism of impact plausibly involves reduced oxygen supply from infected lungs; if so, we expect to see that exercise will increase heartbeat rates more in infected toads than in uninfected conspecifics. Our data on 103 field‐collected toads (53 of which contained lungworms) support this prediction. Exercise induced a greater increase in heartbeat rate in infected toads than in uninfected conspecifics, but no shift in oxygen saturation of the haemoglobin.

Bovine colostrum: A veterinary nutraceutical

A total of 116 adult frogs were captured from Moniya area of Ibadan and University of Ibadan Fish Farm, Oyo State of Nigeria between 31st March and 8th June 2009 and investigated for blood parasites by microscopic examination of stained blood films. Some haematological parameters were estimated. The mean packed cell volume, haemoglobin, red blood cells, white blood cells, platelet count, mean cell volume, mean cell haemoglobin concentration and mean cell haemoglobin of the samples from the low rain forest area of the south Western Nigeria were 26.90 and 29.67%, 8.85 and 9.83 g/dl, 2.71 and 2.73 × 106 µl-1, 15618.97 and 16704.31 × 103 µl-1, 173344.83 and 163124.14 ×109 µl-1, 102.83 and 117.67 fl, 32.95 and 33.80 g/dl, and 33.87 and 38.77 pg. Only haemoflagellate from the genus Trypanosoma was identified with different morphological forms which are attributed to Trypanosoma rotatorium complex. Prevalence of infection in the two study sites were estimated as 32.8 and 29.3%, respectively. The infected frogs had double nucleated red blood cells. Key words: Haematology,common frog, Rana temporaria, blood parasites, Ibadan.

Xenopus laevis oocytes infected with multi-drug–resistant bacteria: implications for electrical recordings

The Xenopus laevis oocyte has been the workhorse for the investigation of ion transport proteins. These large cells have spawned a multitude of novel techniques that are unfathomable in mammalian cells, yet the fickleness of the oocyte has driven many researchers to use other membrane protein expression systems. Here, we show that some colonies of Xenopus laevis are infected with three multi-drug–resistant bacteria: Pseudomonas fluorescens, Pseudomonas putida, and Stenotrophomonas maltophilia. Oocytes extracted from infected frogs quickly (3–4 d) develop multiple black foci on the animal pole, similar to microinjection scars, which render the extracted eggs useless for electrical recordings. Although multi-drug resistant, the bacteria were susceptible to amikacin and ciprofloxacin in growth assays. Supplementing the oocyte storage media with these two antibiotics prevented the appearance of the black foci and afforded oocytes suitable for whole-cell recordings. Given that P. fluorescens associated with X. laevis has become rapidly drug resistant, it is imperative that researchers store the extracted oocytes in the antibiotic cocktail and not treat the animals harboring the multi-drug–resistant bacteria.

Helminth Parasites of the Wood Frog, Lithobates sylvaticus, in Prairie Pothole Wetlands of the Northern Great Plains

Parasites are ubiquitous members of biotic communities. Because their persistence and abundance is closely tied to other taxa and numerous environmental factors, information about parasite diversity may provide unique insights into ecosystem health. Parasites may also impact host health and population dynamics. Because they are relatively inconspicuous, however, little is known about parasite diversity and prevalence in specific host species in most geographic regions. We sampled parasites from wood frogs in the ecologically unique Prairie Pothole Region of North Dakota, USA. Most frogs were infected with helminths. We found a minimum of 7 species of trematodes, 3 of nematodes, and 1 cestode. Two species had prevalence >50%: the lung nematode Rhabdias bakeri and the trematode Echinoparyphium rubrum. Helminth species richness ranged from 0 to 6 taxa per host, with a median of 2. Total helminth abundance within infected frogs ranged from 1 to 503 worms. Males caught during the spring breeding season were infected with more taxa of helminths than either recent metamorphs or frogs captured later in the summer. The total abundance of helminths was also greater in breeding frogs. These data provide the foundation for further analyses of the ecology of amphibian-parasite interactions on the Northern Plains.

Temperature, hydric environment, and prior pathogen exposure alter the experimental severity of chytridiomycosis in boreal toads

Prevalence of the pathogen Batrachochytrium dendrobatidis (Bd), implicated in amphibian population declines worldwide, is associated with habitat moisture and temperature, but few studies have varied these factors and measured the response to infection in amphibian hosts. We evaluated how varying humidity, contact with water, and temperature affected the manifestation of chytridiomycosis in boreal toads Anaxyrus (Bufo) boreas boreas and how prior exposure to Bd affects the likelihood of survival after re-exposure, such as may occur seasonally in long-lived species. Humidity did not affect survival or the degree of Bd infection, but a longer time in contact with water increased the likelihood of mortality. After exposure to approximately 10(6) Bd zoospores, all toads in continuous contact with water died within 30 d. Moreover, Bd-exposed toads that were disease-free after 64 d under dry conditions, developed lethal chytridiomycosis within 70 d of transfer to wet conditions. Toads in unheated aquaria (mean = 15 degrees C) survived less than 48 d, while those in moderately heated aquaria (mean = 18 degrees C) survived 115 d post-exposure and exhibited behavioral fever, selecting warmer sites across a temperature gradient. We also found benefits of prior Bd infection: previously exposed toads survived 3 times longer than Bd-naïve toads after re-exposure to 106 zoospores (89 vs. 30 d), but only when dry microenvironments were available. This study illustrates how the outcome of Bd infection in boreal toads is environmentally dependent: when continuously wet, high reinfection rates may overwhelm defenses, but periodic drying, moderate warming, and previous infection may allow infected toads to extend their survival.

Helminth community structure of Scinax nasicus (Anura: Hylidae) from a South American subtropical area

The main goal of this study was to evaluate the relative influence of season, year of study, host body size, and host sex on abundance of helminth species parasitic in the frog Scinax nasicus. A total of 273 frogs was collected between December 2004 and November 2006 over all seasons in Corrientes City, Province of Corrientes, Argentina. Helminth community included 21 taxa, and was dominated particularly by larval trematodes. Infected frogs harboured a maximum of 7 species. Host sex and season played no significant effect in determining infracommunity parasite species abundance. Similarly, species richness was similar for both host sexes, and across time (year and season). However, occurrence of the parasites Opisthogonimus sp. 2 and Travtrema aff. stenocotyle greatly varied over time. Host body size was the main factor for determining infrapopulation structure of Centrorhynchus sp. Species richness was significantly and positively correlated with host body size. Strong associations were observed mainly between metacercariae of some species. The transmission strategies of parasites suggest that this hylid acquires infections by ingestion of infective larvae and through direct contact with larval parasites from aquatic and terrestrial habitats.

Batrachochytrium dendrobatidis and the Collapse of Anuran Species Richness and Abundance in the Upper Manu National Park, Southeastern Peru

Amphibians are declining worldwide, but these declines have been particularly dramatic in tropical mountains, where high endemism and vulnerability to an introduced fungal pathogen, Batrachochytrium dendrobatidis (Bd), is associated with amphibian extinctions. We surveyed frogs in the Peruvian Andes in montane forests along a steep elevational gradient (1200-3700 m). We used visual encounter surveys to sample stream-dwelling and arboreal species and leaf-litter plots to sample terrestrial-breeding species. We compared species richness and abundance among the wet seasons of 1999, 2008, and 2009. Despite similar sampling effort among years, the number of species (46 in 1999) declined by 47% between 1999 and 2008 and by 38% between 1999 and 2009. When we combined the number of species we found in 2008 and 2009, the decline from 1999 was 36%. Declines of stream-dwelling and arboreal species (a reduction in species richness of 55%) were much greater than declines of terrestrial-breeding species (reduction of 20% in 2008 and 24% in 2009). Similarly, abundances of stream-dwelling and arboreal frogs were lower in the combined 2008-2009 period than in 1999, whereas densities of frogs in leaf-litter plots did not differ among survey years. These declines may be associated with the infection of frogs with Bd. B. dendrobatidis prevalence correlated significantly with the proportion of species that were absent from the 2008 and 2009 surveys along the elevational gradient. Our results suggest Bd may have arrived at the site between 1999 and 2007, which is consistent with the hypothesis that this pathogen is spreading in epidemic waves along the Andean cordilleras. Our results also indicate a rapid decline of frog species richness and abundance in our study area, a national park that contains many endemic amphibian species and is high in amphibian species richness.