Infected Frogs Research Articles

Composition of symbiotic bacteria predicts survival in Panamanian golden frogs infected with a lethal fungus

Symbiotic microbes can dramatically impact host health and fitness, and recent research in a diversity of systems suggests that different symbiont community structures may result in distinct outcomes for the host. In amphibians, some symbiotic skin bacteria produce metabolites that inhibit the growth of Batrachochytrium dendrobatidis (Bd), a cutaneous fungal pathogen that has caused many amphibian population declines and extinctions. Treatment with beneficial bacteria (probiotics) prevents Bd infection in some amphibian species and creates optimism for conservation of species that are highly susceptible to chytridiomycosis, the disease caused by Bd. In a laboratory experiment, we used Bd-inhibitory bacteria from Bd-tolerant Panamanian amphibians in a probiotic development trial with Panamanian golden frogs, Atelopus zeteki, a species currently surviving only in captive assurance colonies. Approximately 30% of infected golden frogs survived Bd exposure by either clearing infection or maintaining low Bd loads, but this was not associated with probiotic treatment. Survival was instead related to initial composition of the skin bacterial community and metabolites present on the skin. These results suggest a strong link between the structure of these symbiotic microbial communities and amphibian host health in the face of Bd exposure and also suggest a new approach for developing amphibian probiotics.

Genetic structure analysis of Spirometra erinaceieuropaei isolates from central and southern China.

BackgroundSparganosis caused by invasion of the plerocercoid larvae (spargana) of Spirometra erinaceieuropaei have increased in recent years in China. However, the population genetic structure regarding this parasite is still unclear. In this study, we used the sequences of two mitochondrial genes cytochrome b (cytb) and cytochrome c oxidase subunit I (cox1) to analyze genetic variation and phylogeographic structure of the S. erinaceieuropaei populations.Methodology/Principal FindingsA total of 88 S. erinaceieuropaei isolates were collected from naturally infected frogs in 14 geographical locations of China. The complete cytb and cox1 genes of each sample was amplified and sequenced. Total 61 haplotypes were found in these 88 concatenated sequences. Each sampled population and the total population have high haplotype diversity (Hd), accompanied by very low nucleotide diversity (Pi). Phylogenetic analyses of haplotypes revealed two distinct clades (HeN+HuN+GZ-AS clade and GX+HN+GZ-GY clade) corresponding two sub-networks yielded by the median-joining network. Pairwise F ST values supported great genetic differentiation between S. erinaceieuropaei populations. Both negative Fu’s F S value of neutrality tests and unimodal curve of mismatch distribution analyses supported demographic population expansion in the HeN+HuN+GZ-AS clade. The BEAST analysis showed that the divergence time between the two clades took place in the early Pleistocene (1.16 Myr), and by Bayesian skyline plot (BSP) an expansion occurred after about 0.3 Myr ago.Conclusions S. erinaceieuropaei from central and southern China has significant phylogeographic structure, and climatic oscillations during glacial periods in the Quaternary may affect the demography and diversification of this species.

Landscape context influences chytrid fungus distribution in an endangered European amphibian

AbstractWildlife disease is an emerging threat to biodiversity. The amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd), which causes the disease chytridiomycosis, has been documented in over 500 amphibian species globally. Understanding conditions under which amphibians are vulnerable to Bd is important for evaluating species risk and developing surveillance strategies. Here, we investigate the spatial distribution of Bd infection in the ephemeral pond‐breeding yellow‐bellied toad Bombina variegata, a species of high conservation concern in the European Union. We sampled 550 toads from 60 ponds in a traditional agricultural landscape in Southern Transylvania, Romania. Overall, Bd prevalence was low in B. variegata, but infected toads were widely dispersed through the landscape and were found in a quarter of all sampled ephemeral ponds. At the pond level, increased Bd occurrence was associated with short distances to perennial water sources and high forest cover. These findings suggest that perennial water sources may act as source habitat for Bd, with amphibian movements resulting in Bd spillover into ephemeral ponds. Increased Bd occurrence in ponds surrounded by high levels of forest cover is likely related to cooler and wetter conditions that are more favourable for Bd. Throughout the study landscape, patchy environmental suitability for Bd appears to restrict the pathogen to a subset of B. variegata habitat. Ephemeral ponds in open landscapes, without nearby perennial habitat, likely provide an environmental refuge from Bd, where the risk of infection is low. From a conservation perspective, these findings highlight the importance of maintaining ephemeral ponds in open landscapes, but these are currently threatened by land‐use change.

Helminth infestation of the moor frog (Rana arvalis Nilsson, 1842) and the Siberian tree frog (Rana amurensis Boulenger, 1886) in Western Siberia

The composition of helminths and infestation indices of two frog species of living in Western Siberia (Rana arvalis (n = 1082) and Rana amurensis (n = 33)) have been studied. Nine helminth species, including four nematodes and five trematodes, have been found. The following species were dominant and infected both frog species: nematodes Rhabdias bufonis, Oswaldocruzia filiformis, and Cosmocerca ornata and trematodes Haplometra cylindracea. The moor frog also hosted Dolichosaccus rastellus, Opisthioglyphe ranae, Pharingostomum cordatum (larvae), Parastrigea robusta (larvae), and Neyraplectana schneideri. The moor frog was characterized by higher species richness and diversity of helminths than the Siberian tree frog. The overall rates of helminth infestation have been the same in the moor frog and the Siberian tree frog. The invasion extensity was 85–100%; the index of abundance was 5.2–27.5. Differences in infestation rates of certain species by helminths were due to the biological peculiarities of the frog species.

The Effects of a Nematode Lungworm (Rhabdias hylae) on its Natural and Invasive Anuran Hosts

Biological invasions can bring both the invader and native taxa into contact with novel parasites. As cane toads ( Rhinella marina ) have spread through Australia, they have encountered lungworms (Rhabdias hylae) that occur in native frogs. Field surveys suggest that these lungworms have not host-switched to toads. In our laboratory studies, R. hylae infected cane toads as readily as it infected native frogs, but failed to reach the lungs of the novel host (i.e., were killed by the toads' immune response). Plausibly, then, R. hylae might reduce the viability both of their native hosts (frogs, that can exhibit high parasite burdens) and cane toads (that must deal with infective larvae traveling through the host body). Our laboratory trials suggest, however, that the impacts of the parasite on infected anuran hosts (both frogs and toads) were minimal, with no significant decrements to host survival, activity, growth, or locomotor performance. Ironically, the lack of impact of the parasite on its native hosts appears to be an outcome of co-evolution (frogs tolerate the lungworm), whereas the lack of impact on the novel host is due to a lack of co-evolution (toads can recognize and eliminate the lungworm).

Low humidity is a failed treatment option for chytridiomycosis in the critically endangered southern corroboree frog

Context One of the major drivers of the current worldwide amphibian decline and extinction crisis is the spread of the amphibian chytrid fungus, Batrachochytrium dendrobatidis (Bd). Captive assurance colonies may be the only lifeline for some species. Current antifungal chemotherapies can be effective for clearing infection but may have detrimental side effects. The only non-chemotherapeutic treatment that has been clinically tested is heat, which is impractical for cold-adapted species. The fungus does not survive desiccation, and therefore a plausible alternative non-chemotherapeutic treatment would be a low-humidity regimen. Aims We tested the efficacy of a low-humidity treatment regimen for treating Bd-infected animals of the critically endangered species Pseudophryne corroboree. This species has high tolerance of dry environments, and is thus an ideal candidate for this treatment trial. Methods Forty frogs were exposed to 1 × 106 zoospores. At three weeks after exposure access to water was reduced to once daily for 10 days for 20 animals. Ten of these animals were also rinsed daily with the aim of removing zoospores. Key results The drying regimen neither increased survival nor decreased infection load, and treatment may have hastened mortality. All infected frogs died between 21 and 80 days after exposure, except one untreated frog. Conclusions Our results suggest that a drying regimen is not a viable treatment for chytridiomycosis. Infection may decrease the host’s ability to cope with water stress. Implications The failed drying treatment in P. corroboree suggests that drying is not an appropriate treatment for chytridiomycosis and treatment efforts should be focussed on chemotherapies for cold-adapted species. P. corroboree is a critically endangered species for which chytridiomycosis is the primary cause of decline, yet few experiments have been carried out on this species with regards to chytridiomycosis. All experimental information about this species and disease will benefit the management and protection of the species.

Skin sloughing rate increases with chytrid fungus infection load in a susceptible amphibian

Summary Amphibian chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is responsible for the greatest disease‐driven loss of vertebrate biodiversity in recorded history. Understanding drivers of host susceptibility to this cutaneous disease is hindered by gaps in our knowledge of the host–pathogen relationship. One such overlooked aspect of susceptibility is variation in skin maintenance processes, particularly skin turnover via routine sloughing. It has been suggested that sloughing plays a role in immune defence, by removing skin‐associated microbes. Thus, skin sloughing may play an important role in the pathogenesis of chytridiomycosis. To determine the relationship between skin sloughing and disease progression, we exposed adult Australian green tree frogs (Litoria caerulea) to a local Bd strain and monitored sloughing rates and individual infection loads on a naturalistic cycling temperature regime (15–23 °C). We determined sloughing rates in real‐time by using an array of infrared video cameras to film frog behaviour and monitored infection load before and after sloughing by swabbing and analysis with quantitative PCR. We found that sloughing rate increased with Bd infection load in infected frogs, but sloughing itself did not affect Bd load on the ventral skin surface. Furthermore, Bd infection did not affect the duration of characteristic sloughing behaviour, and sloughing retained rhythmicity even at high infection loads. Although an increased sloughing rate might be considered advantageous for Bd‐infected animals, it does not appear to curb the progression of disease and may actually contribute to the loss of physiological homoeostasis seen in terminally ill frogs by further inhibiting water and electrolyte transport across the skin. By measuring sloughing rates directly for the first time, our results shed light on how Bd interacts with the physiological processes of the skin and indicate that variation in skin sloughing frequency may play a role in the observed inter‐ and intraspecific variation in susceptibility to disease.

Ultrastructural study of the egg wall surrounding the developing miracidia of the digenean Prosotocus confusus (Looss, 1894) (Plagiorchiida: Pleurogenidae), with the description of a unique cocoon-like envelope.

Helminth eggs play a critical role in movement of the parasite from definitive to intermediate host. Eggs of the pleurogenid digenean trematode Prosotocus confusus (Looss, 1894), a parasite of naturally infected frogs Pelophylax lessonae (Amphibia: Ranidae) in Europe, are described here for the first time. Particular emphasis is placed on the ultrastructure on the egg wall and on the detailed description of a unique cocoon-like envelope. Each embryonating egg is composed of an early embryo surrounded by a four-layered egg wall: (1) an outer, anucleate layer external to the eggshell, which forms a thick cocoon; (2) the operculate eggshell; (3) not fully formed, a differentiating outer embryonic envelope containing large nuclei of macromeres; and (4) situated below, an undifferentiated layer of the future inner embryonic envelope containing mesomere nuclei. Layers enveloping the egg apparently play an important role in the protection, metabolism, and storage of nutritive reserves for the developing miracidium. The outer anucleate layer, or cocoon, is situated externally to the eggshell and composed of an electron-lucent substance with numerous electron-dense islands attached to its peripheral membrane. A cocoon envelope such as this has never been seen in previous TEM studies of the eggs of parasitic platyhelminths, with the exception of another pleurogenid Brandesia turgida. The origin, formation, functional ultrastructure, and chemical composition of this peculiar layer remain enigmatic, although its function appears to be protective. The thick, electron-dense eggshell resembles that of other trematodes, exhibiting a characteristic fissure zone around the operculum. Numerous lysosome-like structures observed in some eggs may be involved in the autolysis of both the embryonic envelopes (particularly the early degeneration of macromere nuclei of the outer envelope, characteristic for this species) and in the disintegration of several early micromeres. The inner envelope, which forms later from mesomeres, persists longer during embryogenesis.

인공사육장에서 패혈증으로 집단폐사한 북방산개구리의 증례 보고

Frog culture industry is not yet familiar but has much potential. Generally, in farm, the population density is higher than that of in nature and frog farm is not the exception. But when population density is high, it can easily leads to stressful condition, poor sanitation. When a disease occur, it is a primary factor that makes the population more susceptible and the results more grave. Because of severe Rhabditoidea- helminth infection and subsequent bacterial septicemia, 50~70% of the total population had been died in a farm in Jeong-sun in Gangwon-do and Chungju in Chungcheongbuk-do from late June, 2012 to September, 2012. Diseased frogs showed ruptured lung, bloody ascites, liver discoloration, myocardium weakness, congested kidney, microcytic anemia and so on. Enterobacteriacea, Citrobacter.sp, Cupriavidus metallidurans, Acinetobacter.sp were isolated as major bacterium that had caused septicemia in frogs. Among isolated bacterium, Cupriavidus metallidurans, Ewingella americana, Shewanella aquimarina and Pseudoalteromonas sp. have not reported as potential pathogens in frogs before. It is a good example that severe helminth infection in frogs can lead to secondary infection of bacteria.

Quantifying Anuran Microhabitat Use to Infer the Potential for Parasite Transmission between Invasive Cane Toads and Two Species of Australian Native Frogs

Parasites that are carried by invasive species can infect native taxa, with devastating consequences. In Australia, invading cane toads (Rhinella marina) carry lungworm parasites (Rhabdias pseudosphaerocephala) that (based on previous laboratory studies) can infect native treefrogs (Litoria caerulea and L. splendida). To assess the potential of parasite transmission from the invader to the native species (and from one infected native frog to another), we used surveys and radiotelemetry to quantify anuran microhabitat use, and proximity to other anurans, in two sites in tropical Australia. Unsurprisingly, treefrogs spent much of their time off the ground (especially by day, and in undisturbed forests) but terrestrial activity was common at night (especially in anthropogenically modified habitats). Microhabitat overlap between cane toads and frogs was generally low, except at night in disturbed areas, whereas overlap between the two frog species was high. The situations of highest overlap, and hence with the greatest danger of parasite transmission, involve aggregations of frogs within crevices by day, and use of open ground by all three anuran species at night. Overall, microhabitat divergence between toads and frogs should reduce, but not eliminate, the transmission of lungworms from invasive toads to vulnerable native frogs.

Molecular characterization of a Spirometra mansoni antigenic polypeptide gene encoding a 28.7 kDa protein.

The Spirometra mansoni antigenic polypeptide (SmAP) gene was expressed in Escherichia coli, and its characteristics and value as an antigen for the serodiagnosis of sparganosis were investigated. The recombinant SmAP protein (rSmAP) has the molecular weight of 28.7 kDa. On Western blotting analysis, the rSmAP strongly reacted with the sera of mice infected with spargana, but not with normal sera; the anti-rSmAP serum obviously recognized the 28.7-kDa band in the crude antigens and excretory-secretory (ES) antigens of spargana. The immunofluorescence test (IFT) results showed that the positive staining was observed at different stages of spargana from the infected frogs and mice, but not adult worm of S. mansoni. An immunolocalization analysis identified SmAP in the teguments and parenchymal tissues of spargana. ELISA with rSmAP antigen or sparganum ES antigens were evaluated for the serodiagnosis of sparganosis. The results showed that the sensitivity of rSmAP-ELISA and ES-ELISA was 83.3% (25/30) and 100% (30/30), respectively, for the detection of anti-sparganum IgG antibodies in sera of the experimentally infected mice (P > 0.05), the specificities of both ELISA were 100% (67/67). It is suggested that the rSmAP might be a potential candidate antigen for serodiagnosis of sparganosis.

Helminth community structure in the Argentinean bufonid Melanophryniscus klappenbachi: importance of habitat use and season

The goal of this study was to evaluate the relative influence of terrestrial habits, season, and host body size on the species richness and abundance of helminth parasites in the toad Melanophryniscus klappenbachi, for which a greater abundance of nematode parasites was expected. A total of 90 toads were collected in the Chaco Province, Argentina. The helminth community found in infected toads included 17 taxa and was dominated particularly by larval parasites. Contrary to our expectations, nematode species showed lower values of infection parameters. Infected toads harbored a maximum of seven species, and the mean helminth richness was 3.16 ± 1.66 species per infected toads. Season played a significant effect on determining the species richness and abundance of the parasite infracommunity. Similarly, the prevalence of infection of several helminth species (8/47%) varied greatly over time. Host body size was the main factor in determining the infrapopulation structure of helminth parasites. Species richness was significantly and negatively correlated with host body size. Strong associations were observed mainly between larvae of some species. The transmission strategies of parasites suggest that this bufonid acquires infections through direct contact with larval parasites from aquatic and terrestrial habitats and by ingestion of infective larvae. The characteristic of the host tegument, such as the presence of alkaloids, could significantly contribute to the low occurrence of infection by skin-penetrating nematodes. Results also showed that diets and mobility of the host and the local microhabitat conditions play an important role in parasitic infections of toads.

Helminths of the American Toad,Anaxyrus americanus americanus, and Fowler's Toad,Anaxyrus fowlerifrom the Silver Creek Area and Lake Michigan Shoreline in Western Michigan, U.S.A

In total, 105 toads (71 American toads, Anaxyrus americanus americanus Holbrook, 1836; 31 Fowler's toads, Anaxyrus fowleri (Hinckley, 1882); and 3 A. a. americanus × A. fowleri hybrids) were collected in 2011 and 2012 from the lower Silver Creek area and Lake Michigan shoreline (LMS) in western lower Michigan and examined for helminths. Five helminth species (1 Digenea, Gorgoderina sp.; 1 Cestoda, Mesocestoides sp.; and 3 Nematoda,-Cosmocercoides variabilis, Oswaldocruzia pipiens, and Rhabdias americanus) were found in American toads. Mesocestoides sp. and R. americanus were the most common helminths of American toads. Four helminth species (1 Digenea, Haematoloechus sp.; 1 Cestoda, Mesocestoides sp.; and 2 Nematoda, C. variabilis and O. pipiens) infected Fowler's toad, with Mesocestoides sp. being most common. Anaxyrus a. americanus × A. fowleri hybrids were infected with Mesocestoides sp., R. americanus, and C. variabilis. The prevalences and intensities of helminth species found in both toad species and the mean numbers of helminth species in infected toads and in all examined toads were always higher in American toads than in Fowler's toads. Although C. variabilis and R. americanus infected American toads and C. variabilis and O. pipiens infected Fowler's toads on the LMS, it is believed that they were not infected there. The occurrence of these nematodes in toads on the LMS reflects infections that occurred in their breeding ponds and other moist areas. This is the first survey on the helminths of Fowler's toads from Michigan and from a shoreline dune area for both toad species.

Fighting a losing battle: vigorous immune response countered by pathogen suppression of host defenses in the chytridiomycosis-susceptible frog Atelopus zeteki.

The emergence of the disease chytridiomycosis caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd) has been implicated in dramatic global amphibian declines. Although many species have undergone catastrophic declines and/or extinctions, others appear to be unaffected or persist at reduced frequencies after Bd outbreaks. The reasons behind this variance in disease outcomes are poorly understood: differences in host immune responses have been proposed, yet previous studies suggest a lack of robust immune responses to Bd in susceptible species. Here, we sequenced transcriptomes from clutch-mates of a highly susceptible amphibian, Atelopus zeteki, with different infection histories. We found significant changes in expression of numerous genes involved in innate and inflammatory responses in infected frogs despite high susceptibility to chytridiomycosis. We show evidence of acquired immune responses generated against Bd, including increased expression of immunoglobulins and major histocompatibility complex genes. In addition, fungal-killing genes had significantly greater expression in frogs previously exposed to Bd compared with Bd-naïve frogs, including chitinase and serine-type proteases. However, our results appear to confirm recent in vitro evidence of immune suppression by Bd, demonstrated by decreased expression of lymphocyte genes in the spleen of infected compared with control frogs. We propose susceptibility to chytridiomycosis is not due to lack of Bd-specific immune responses but instead is caused by failure of those responses to be effective. Ineffective immune pathway activation and timing of antibody production are discussed as potential mechanisms. However, in light of our findings, suppression of key immune responses by Bd is likely an important factor in the lethality of this fungus.

Evaluation of amphotericin B and chloramphenicol as alternative drugs for treatment of chytridiomycosis and their impacts on innate skin defenses.

Chytridiomycosis, an amphibian skin disease caused by the emerging fungal pathogen Batrachochytrium dendrobatidis, has been implicated in catastrophic global amphibian declines. The result is an alarming decrease in amphibian diversity that is a great concern for the scientific community. Clinical trials testing potential antifungal drugs are needed to identify alternative treatments for amphibians infected with this pathogen. In this study, we quantified the MICs of chloramphenicol (800 μg/ml), amphotericin B (0.8 to 1.6 μg/ml), and itraconazole (Sporanox) (20 ng/ml) against B. dendrobatidis. Both chloramphenicol and amphotericin B significantly reduced B. dendrobatidis infection in naturally infected southern leopard frogs (Rana [Lithobates] sphenocephala), although neither drug was capable of complete fungal clearance. Long-term exposure of R. sphenocephala to these drugs did not inhibit antimicrobial peptide (AMP) synthesis, indicating that neither drug is detrimental to this important innate skin defense. However, we observed that chloramphenicol, but not amphotericin B or itraconazole, inhibited the growth of multiple R. sphenocephala skin bacterial isolates in vitro at concentrations below the MIC against B. dendrobatidis. These results indicate that treatment with chloramphenicol might dramatically alter the protective natural skin microbiome when used as an antifungal agent. This study represents the first examination of the effects of alternative antifungal drug treatments on amphibian innate skin defenses, a crucial step to validating these treatments for practical applications.

Analysis of skin and secretions of Dybowski’s frogs (Rana dybowskii) exposed to Staphylococcus aureus or Escherichia coli identifies immune response proteins

The aim of the present study was to investigate responses in Dybowski’s frogs (Rana dybowskii) exposed to bacteria, using proteomic and transcriptomic approaches. Staphylococcus aureus and Escherichia coli were used as representative Gram-positive and Gram-negative bacteria, respectively, in an infectious challenge model. Frog skin and skin secretions were collected and protein expression in infected frogs compared to control frogs by two-dimensional gel electrophoresis, silver staining, and image analysis. Proteins that demonstrated differential expression were analysed by mass spectrometry and identified by searching protein databases.More than 180 protein spots demonstrated differential expression in E. coli- or S. aureus-challenged groups and, of these, more than 55 spots were up- or down-regulated at least sixfold, post-infection. Proteins with a potential function in the immune response were identified, such as stathmin 1a, annexin A1, superoxide dismutase A, C-type lectin, lysozyme, antimicrobial peptides, cofilin-1-B, mannose receptor, histone H4, prohormone convertase 1, carbonyl reductase 1 and some components of the Toll-like receptor (TLR) signalling pathway. These molecules are potential candidates for further investigation of immune mechanisms in R. dybowskii; in particular, TLR-mediated responses, which might be activated in frogs exposed to pathogenic bacteria as part of innate immune defence, but which might also impact on adaptive immunity to infection.

Helminths of ten species of Litoria frogs (Anura: Hylidae) from Papua New Guinea

One hundred and ninety-one frogs collected in Papua New Guinea from 10 species of the genus Litoria, namely, L. angiana, L. arfakiana, L. caerulea, L. congenita, L. eurynastes, L. gasconi, L. infrafrenata, L. iris, L. kumae and L. modica, were examined for helminths. Eighteen species of helminths were found: one species of Monogenea, Parapolystoma bulliense; three species of Digenea, Diplodiscus amphichrus, Halipegus zweifeli and Mesocoelium monas; 13 species of Nematoda, gravid specimens of Aplectana macintoshii, Cosmocerca novaeguineae, Falcaustra papuensis, Maxvachonia adamsoni, Maxvachonia flindersi, Maxvachonia ingens, Meteterakis crombiei, Moaciria moraveci, Parathelandros mastigurus, Physalopteroides milnensis, Pseudorictularia dipsarilis, Seuratascaris numidica, and larvae (in cysts) of Abbreviata sp. and, one species of Acanthocephala, Pseudoacanthocephalus bufonis. Mean number of helminth species per host species was 3.0 ± 1.4 SD (range 1–5), mean number of helminth species per infected frog was 1.1 ± 0.4 SD (range 1–2), and mean number of helminths per infected host was 33.3 ± 5.8 SD (range 1–36). Twenty-eight new host records and two new locality records are reported. The biogeography of the recovered helminths is discussed.

The Role of Gamont Entry into Erythrocytes in the Specificity ofHepatozoonSpecies (Apicomplexa: Adeleida) for Their Frog Hosts

Hepatozoon species are apicomplexan parasites that infect blood cells and viscera of terrestrial vertebrates. One species, Hepatozoon clamatae, primarily infects green frogs, Rana clamitans , whereas another, Hepatozoon catesbianae, primarily infects bullfrogs, Rana catesbeiana , although both species of parasite are capable of infecting either species of frog. The aim of this study was to determine whether the basis for this partial host specificity is manifested at the gamont, or intraerythrocytic, stage of the parasite's life cycle. Blood was drawn from infected frogs and treated in vitro with a saline solution to induce intracellular gamonts to emerge from host erythrocytes. This treated blood was added to in vitro samples of uninfected blood of green frogs and bullfrogs. After 1 hr, samples were analyzed to determine the level of re-entry of the parasites into uninfected erythrocytes. Results obtained using multiple combinations of donor and recipient frogs indicate that extracellular gamonts of both parasite species do not exhibit preference for erythrocytes of 1 frog species over those of another. These results suggest that the basis for the observed host specificity is not determined at the gamont stage and is more likely dependent on another stage in the parasite life cycle.

Effect of Rhabdias pseudosphaerocephala on prey consumption of free-ranging cane toads (Rhinella marina) during Australian tropical wet seasons

The development of biological control measures to reduce the impact of invasive species is a desired goal. Rhabdias species have recently been advocated as biological control agents for invasive anurans. This study describes a field-based approach to support laboratory results on the potential impact of the lung nematode Rhabdias pseudosphaerocephala on the prey consumption of its host, the invasive cane toad (Rhinella marina, Bufonidae). Toads were sampled from various populations in the Wet Tropics of Australia during the wet seasons of 2010 and 2012. Consumed prey items were counted in 212 cane toads and identified to class and order levels and the number of lung nematodes was counted for each toad. The number of R. pseudosphaerocephala in free-ranging cane toads affected negatively the diversity of prey items consumed, but was not related to the number of prey items or the number of ants consumed. The results suggest that infection of free-ranging cane toads by the lung nematode reduces their range of prey items. Possible reasons could be a reduced locomotor activity resulting in changes of foraging modes of infected toads which was reported from some laboratory trials. Infection of cane toads by R. pseudosphaerocephala may therefore have the potential to alter the impact of cane toads on invertebrate communities and their competition for food resources with native Australian anurans.

Host Stress Response Is Important for the Pathogenesis of the Deadly Amphibian Disease, Chytridiomycosis, in Litoria caerulea

Chytridiomycosis, a disease caused by Batrachochytrium dendrobatidis, has contributed to worldwide amphibian population declines; however, the pathogenesis of this disease is still somewhat unclear. Previous studies suggest that infection disrupts cutaneous sodium transport, which leads to hyponatremia and cardiac failure. However, infection is also correlated with unexplained effects on appetite, skin shedding, and white blood cell profiles. Glucocorticoid hormones may be the biochemical connection between these disparate effects, because they regulate ion homeostasis and can also influence appetite, skin shedding, and white blood cells. During a laboratory outbreak of B. dendrobatidis in Australian Green Tree Frogs, Litoria caerulea, we compared frogs showing clinical signs of chytridiomycosis to infected frogs showing no signs of disease and determined that diseased frogs had elevated baseline corticosterone, decreased plasma sodium and potassium, and altered WBC profiles. Diseased frogs also showed evidence of poorer body condition and elevated metabolic rates compared with frogs showing no signs of disease. Prior to displaying signs of disease, we also observed changes in appetite, body mass, and the presence of shed skin associated with infected but not yet diseased frogs. Collectively, these results suggest that elevated baseline corticosterone is associated with chytridiomycosis and correlates with some of the deleterious effects observed during disease development.