First case of ranavirus-associated mass mortality in a natural population of the Huanren frog (Rana huanrenensis) tadpoles in South Korea

Globally, ranavirus is often responsible for the mass mortality of a variety of captive and wild amphibians. In Asia, several mass mortality cases of captive amphibians by ranavirus are known, but one mass mortality case in the wild has been reported in a non-endemic larval bullfrog population in Japan. In order to verify factors involved in mass mortality of Rana huanrenensis tadpoles (> 200 tadpoles) in a mountain stream in South Korea, we investigated possible infections by ranavirus, chytrid fungus, and lethal bacteria by conducting PCR assays of pathogens with specific primers. We found that all R. huanrenensis tadpoles collected (two alive and ten carcasses) showed positive PCR results for two different ranavirus primer sets targeting partial genes of a major capsid protein (MCP). The identified MCP sequence was more closely related to Rana catesbeiana virus JP MCP, isolated from invasive bullfrog tadpoles in Japan. We could not detect any lethal bacteria or chytrid fungus in the specimens. Our finding is the first report in Asia that ranavirus is involved in the mass mortality of endemic wild amphibians.     

Evaluating environmental DNA‐based quantification of ranavirus infection in wood frog populations

A variety of challenges arise when monitoring wildlife populations for disease. Sampling tissues can be invasive to hosts, and obtaining sufficient sample sizes can be expensive and time‐consuming, particularly for rare species and when pathogen prevalence is low. Environmental DNA (eDNA)‐based detection of pathogens is an alternative approach to surveillance for aquatic communities that circumvents many of these issues. Ranaviruses are emerging pathogens of ectothermic vertebrates linked to die‐offs of amphibian populations. Detecting ranavirus infections is critical, but nonlethal methods have the above issues and are prone to false negatives. We report on the feasibility and effectiveness of eDNA‐based ranavirus detection in the field. We compared ranavirus titres in eDNA samples collected from pond water to titres in wood frog (Lithobates sylvaticus; n = 5) tadpoles in sites dominated by this one species (n = 20 pond visits). We examined whether ranavirus DNA can be detected in eDNA from pond water when infections are present in the pond and if viral titres detected in eDNA samples correlate with the prevalence or intensity of ranavirus infections in tadpoles. With three 250 mL water samples, we were able to detect the virus in all visits with infected larvae (0.92 diagnostic sensitivity). Also, we found a strong relationship between the viral eDNA titres and titres in larval tissues. eDNA titres increased prior to observed die‐offs and declined afterwards, and were two orders of magnitude higher in ponds with a die‐off. Our results suggest that eDNA is useful for detecting ranavirus infections in wildlife and aquaculture.     

Ranavirus in wood frogs (Rana sylvatica): potential sources of transmission within and between ponds

Members of the genus Ranavirus (family Iridoviridae) can cause catastrophic mortality of pond-breeding amphibians and are associated with an emerging infectious disease that may be contributing to amphibian declines. We conducted three experiments to examine factors that may affect transmission both within and between local breeding populations of the wood frog (Rana sylvatica). In a laboratory study, when exposed to moribund tadpoles collected during a local ranaviral die-off, uninfected tadpoles died as soon as 4 days after exposure. The onset of death was accelerated when tadpoles were allowed to scavenge on carcasses of infected tadpoles. In a mesocosm experiment that was conducted in outdoor wading pools, die-offs of tadpoles began approximately 19 days after infected tadpoles were added to pools containing uninfected tadpoles. Mass die-offs with greater than 98% mortality occurred in all pools, regardless of the initial tadpole density. In a second mesocosm experiment, the addition of water and bottom sediments that were collected from a pond during a ranaviral die-off did not result in lower tadpole survival or growth relative to controls. Only a small percentage of tadpoles appeared to be sick, and most tadpoles survived until the first individuals began metamorphosing within a pool. However, tests for ranavirus using polymerase chain reaction were positive for most pools that received contaminated sediment, suggesting that some infections were sublethal. Our results indicate that transmission within ponds is enhanced by scavenging and that spread between local ponds could occur via the transport of contaminated sediment by animals or humans.     

The effect of habitat fragmentation on finescale population structure of wood frogs (Rana sylvatica)

We examined the impact of recent anthropogenic habitat fragmentation on the genetic structure of wood frog (Rana sylvatica) breeding sites in Wellington County of Ontario, Canada. In addition to geographic distance (average pairwise distance ~22 km, greatest distance ~50.22 km), four landscape features hypothesized to contribute to genetic differentiation between breeding sites were considered: road density, a major highway (highway 401), canopy cover, and watershed discontinuity. Analysis of data from 396 samples across nine breeding sites using eight microsatellite DNA loci, revealed a small degree of significant genetic structure between breeding sites. The presence of highway 401 and road density were correlated with small but statistically significant structure observed between several groups of sites. One outlier breeding site outside of Wellington County located within the city of Toronto, had significantly lower allelic richness and much larger population differentiation with the Wellington sites. Our data suggest that recent fragmentation has had an effect on wood frog population structure and also demonstrate the importance of dispersal for this species in maintaining levels of genetic diversity.     

Natural Stressors and Ranavirus Susceptibility in Larval Wood Frogs (Rana sylvatica)

Chronic exposure to stressors has been shown to suppress immune function in vertebrates, making them more susceptible to pathogens. It is less clear, however, whether many natural stressors are immunosuppressive. Moreover, whether stressors make disease more likely or more severe in populations is unclear because animals respond to stressors both behaviorally and physiologically. We tested whether chronic exposure to three natural stressors of wood frog tadpoles—high-densities, predator-cues, and low-food conditions—influence their susceptibility to a lethal ranavirus both individually in laboratory experiments, and collectively in outdoor mesocosms. Prior to virus exposure, we observed elevated corticosterone only in low-food treatments, although other treatments altered rates of growth and development as well as tadpole behavior. None of the treatments, however, increased susceptibility to ranavirus as measured by the proportion of tadpoles that became infected or died, or the time to death compared to controls. In fact, mortality in the mesocosms was actually lower in the high-density treatment even though most individuals became infected, largely because of increased rates of metamorphosis. Overall we find no support for the hypothesis that chronic exposure to common, ecologically relevant challenges necessarily elevates corticosterone levels in a population or leads to more severe ranaviral disease or epidemics. Conditions may, however, conspire to make ranavirus infection more common in metamorphosing amphibians.     

First case of ranavirus-associated morbidity and mortality in natural populations of the South American frog Atelognathus patagonicus

Atelognathus patagonicus is an endangered leptodactylid frog endemic to a small region in and around Laguna Blanca National Park in northern Patagonia, Argentina. All of the lakes and small ponds of the region (except Laguna Blanca itself) contain A. patagonicus and in all but one of these lakes the species shows clinical signs of a previously undiagnosed disease, the characteristics of which suggested a ranavirus. We collected symptomatic and asymptomatic A. patagonicus frogs and tadpoles from 4 small lakes and analyzed tissues for ranavirus and the chytrid fungus Batrachochytrium dendrobatidis using PCR amplification of pathogen DNA. Of the 32 specimens tested, 25 were positive for ranavirus major capsid protein (MCP). Sequence alignments of the ranavirus MCP from these specimens showed 100% similarity with published FV3 and FV3-like viruses from anurans, 98 to 99 % similarity with Bohle iridovirus, and 95 % similarity with Ambystoma tigrinum virus (ATV) and Regina ranavirus (RRV). A search of the NCBI Blast nucleotide database using the 500 base pair MCP sequence obtained from these samples did not suggest any homology to any other pathogen. In addition, 1 sample (3 pooled individuals) from 1 lake tested positive for B. dendrobatidis. The clinical signs observed primarily in late-stage tadpoles and recent metamorphs, which have reoccurred each year since at least 2001, are consistent with ranaviral disease, but until histopathology of diseased individuals is carried out, chytridiomycosis or other diseases cannot be ruled out.     

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

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

The influence of landscape and environmental factors on ranavirus epidemiology in a California amphibian assemblage

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Do rivers function as genetic barriers for the plateau wood frog at high elevations?

Using the plateau wood frog Rana kukunoris from the Hengduan Mountains as a model system, we tested whether rivers form significant genetic barriers (the riverine barrier hypothesis) to high elevation amphibians. Samples were collected from eight sites across three major river drainages, the Min, the Dadu and the Yalong Rivers, and the population genetic structure of these frogs was evaluated with data from eight microsatellite DNA loci. A large amount of genetic structure was found, and the pairwise F _ST ranged from 0.022 to 0.508 and a global F _ST was 0.215. Both analysis of molecular variance and isolation by distance analysis suggested that rivers, mountain ridges and geographic distances all contributed significantly to the population structure. However, no single landscape has prominent barrier effect to the plateau wood frog populations. An assignment analysis using the computer program Structure grouped the eight populations into four population clusters, and no single type of landscape can sufficiently explain the clustering. In conclusion, rivers do not appear to be the leading genetic barriers for the plateau wood frog. The strong population genetic structure is likely the consequence of attributes of the species, as opposed to environmental fragmentation, and the barrier effect of the landscapes is largely swamped by the large amount of intrinsic population structure.     

Localisation by immunofluorescence of thyrotropin-releasing hormone in the cutaneous glands of the frog, Rana ridibunda.

Using the technique of indirect immunofluorescence, thyrotropin-releasing hormone (TRH) has been localised in certain cells of the cutaneous glands of the frog, $\text{Rana ridibunda}$. No specific fluorescence was found in other cells in the dermis, nor were any positive cells found in the epidermis. Previous studies have shown by radioimmunoassay that large amounts of TRH are present in frog skin, particularly in the dorsal region. Since dorsal frog skin contains many more glands than its ventral counterpart, this non-homogeneous distribution of TRH can be explained by its presence in a population of cells found in one type of cutaneous gland.     

Microsatellite DNA markers for the Chinese wood frog (Rana chensinensis) and tests for their cross-utility in 15 ranid frog species

We developed 22 microsatellite markers for the Chinese wood frog (Rana chensinensis) to study the impact of landscape features on its population structure. Thirty‐four individuals from one breeding site were examined and 14 loci were polymorphic. The number of alleles, expected heterozygosity and observed heterozygosity varied from two to 14, from 0.0833 to 0.9118, and from 0.1376 to 0.8667, respectively. Cross‐species amplification was tested for 15 ranid frog species. The Plateau brown frog, Rana kukunoris (n = 23), was successfully amplified at 18 loci, and 15 were polymorphic with number of alleles varying from two to 18. Ten other species were also amplified at a limited number of loci.     

Microgeographical variation in thermal preference by an amphibian

Ectotherms use behaviour to buffer effects of temperature on growth, development and survival. While behavioural thermoregulation is widely reported, localized adaptation of thermal preference is poorly documented. Larval amphibians live in wetlands ranging from entirely open to heavily shaded by vegetation. We hypothesized that populations undergo localized selection leading to countergradient patterns of thermal preference behaviour. Specifically, we predicted that wood frog (Rana sylvatica) larvae from closed canopy ponds would be more strongly temperature selective and would prefer higher temperatures than conspecifics from populations found in open canopy ponds. In a study of six breeding ponds in north‐eastern Connecticut, USA, these predictions were upheld. The countergradient, microgeographical variation in thermal preference documented here implies that wood frog populations may have diverged rapidly in the face of contrasting selection pressures. Rapid, behaviourally mediated responses to changing thermal environments have important implications for understanding population responses to climate change.     

Water Temperature Affects Susceptibility to Ranavirus

The occurrence of emerging infectious diseases in wildlife populations is increasing, and changes in environmental conditions have been hypothesized as a potential driver. For example, warmer ambient temperatures might favor pathogens by providing more ideal conditions for propagation or by stressing hosts. Our objective was to determine if water temperature played a role in the pathogenicity of an emerging pathogen (ranavirus) that infects ectothermic vertebrate species. We exposed larvae of four amphibian species to a Frog Virus 3 (FV3)-like ranavirus at two temperatures (10 and 25°C). We found that FV3 copies in tissues and mortality due to ranaviral disease were greater at 25°C than at 10°C for all species. In a second experiment with wood frogs (Lithobates sylvaticus), we found that a 2°C change (10 vs. 12°C) affected ranaviral disease outcomes, with greater infection and mortality at 12°C. There was evidence that 10°C stressed Cope’s gray tree frog (Hyla chrysoscelis) larvae, which is a species that breeds during summer—all individuals died at this temperature, but only 10% tested positive for FV3 infection. The greater pathogenicity of FV3 at 25°C might be related to faster viral replication, which in vitro studies have reported previously. Colder temperatures also may decrease systemic infection by reducing blood circulation and the proportion of phagocytes, which are known to disseminate FV3 through the body. Collectively, our results indicate that water temperature during larval development may play a role in the emergence of ranaviruses.     

Observations on myiasis by the calliphorids, Bufolucilia silvarum and Bufolucilia elongata, in wood frogs, Rana sylvatica, from southeastern Wisconsin

Larvae of certain species of blowflies (Calliphoridae) can cause myiasis in frogs and toads, but there are few reports from North American amphibians. Of these, most are from toads (bufonids). In this study, we observe primary myiasis in a population of juvenile wood frogs, Rana sylvatica, collected on 22-23 August 2003, from southeastern Wisconsin and compare our observations with previous studies on myiasis from toads. Two (5%) of 39 frogs were infected by the blow fly Bufolucilia silvarum, with an intensity of 28 and 31, whereas 1 (2.5%) of 39 frogs was infected by the blow fly Bufolucilia elongata with an intensity of 14. We found that (1) B. silvarum lay eggs on healthy wood frogs, (2) eggs hatch, with first-instar maggots penetrating under the skin, (3) maggots develop to mature third instars within 13-16 hr of egg hatching, (4) maggots kill the host within 7-47 hr of egg hatching, and (5) maggots consume the entire frog carcass reducing it to bones within 42-59 hr of egg hatching. Our observations on the time of death and how quickly carcasses of wood frogs were consumed by these maggots compared with previous studies on toads suggest that finding infected juvenile wood frogs may be uncommon. Therefore, myiasis by these flies on wood frogs and other small terrestrial anurans may be a phenomenon that is much more common than is currently observed. This is the first report of B. silvarum and B. elongata causing myiasis in wood frogs.     

泽蛙、日本林蛙、饰纹姬蛙不同地理居群的核型多样性

本文研究了温州地区的泽蛙、日本林蛙、饰纹姬蛙的核型,并分析了9个地理居群泽蛙的核型、4个地理居群日本林蛙的核型和3个地理居群饰纹姬蛙的核型。结果表明,不同地理居群的同种蛙均有相同的染色体数和核型模式。泽蛙、日本林蛙都为 2n=26,NF=52,核模式5＋8;饰纹姬蛙 2n=24,NF=48,核模式6＋6。但同一种蛙的不同地理居群之间在SM数目和顺序、次缢痕或随体的位置等都有所不同,说明不同地理居群的同种蛙的染色体具有丰富的多样性。故保护蛙品种资源多样性,不仅要从整个群体上考虑,而且要针对每个品种（或类群）进行保护。 Abstract：The karyotypes of Rana limmocharis boie,Rana j.Japonica,and Microhyla ornata from Wenzhou were studied.The karyotypes of nine populations of Rana limmocharis boie,four populations of Rana j.Japonica and three populations of Microhyla ornata from different geographical regions were compared.The results demonstrated that the same species of different geographical populations have the same amount of chromosome and karyotypic formulae. Rana limmocharis boie and Rana j.Japonica have 2n=26,NF=52 and 5＋8 karyotypic formulae.Microhyla ornata has 2n=24,NF=48 and 6＋6 karyotypic formulae.But some dissimilarities were found among them.First,the number and sequence of submetacentric chromosome were different among them,and then the secondary contriction （SC）or satellite （Sat）were also different.It was showed that the chromosomes of same species of different geographical population have diversities.Conservation of frog genetic diversity must be considered of not only the genetic diversity conservation of the total frog population but also that of every frog breed.     

A new species of Rhabdias from lungs of the wood frog, Rana sylvatica, in North America: the last sibling of Rhabdias ranae?

Rhabdias bakeri n. sp. is described from specimens found in lungs of the wood frog, Rana sylvatica, from North Dakota. The new species has previously been mistakenly identified as Rhabdias ranae Walton, 1929, a common parasite of the leopard frog, Rana pipiens. The new species differs from R. ranae and Rhabdias joaquinensis Ingles, 1935 by the shape and size of pseudolabia, shape and size of buccal capsule, and wider esophageal bulb. Molecular analysis based on the partial sequences of nuclear 18S rDNA gene, complete sequences of internal transcribed spacer region, and partial sequences of 28S gene demonstrates clear differences between Rhabdias from Ra. sylvatica and Ra. pipiens, and supports the status of R. bakeri as a new species.     

Widespread occurrence of ranavirus in pond-breeding amphibian populations

Ranaviruses are an emerging threat for many amphibian populations, yet their distribution in amphibian communities and the association of infection with possible stressors and species is not fully understood due to historically sparse surveillance. Agricultural practices that reduce the water quality of amphibian breeding habitats (e.g., cattle access to wetlands) and environmental stressors (e.g., lower temperatures) may contribute to ranavirus emergence. We tested larval amphibians for ranavirus infection across four seasons in farm ponds (n?=?40) located in Tennessee, USA. Cattle at various densities were allowed access to half of the sampled ponds. Ranavirus infections were detected in nine species and in 33 of the sampled ponds (83%), illustrating widespread occurrence of the pathogen. Species within the family Ranidae were the most frequently infected. In 13 of the ponds containing infected individuals, prevalence exceeded 40% during at least one season. Infections were detected in multiple seasons in 20 of the sampled ponds containing infections, suggesting that ranaviruses are relatively persistent in these systems. Cattle had negative effects on water quality (turbidity and ammonia) and there was a positive association between cattle abundance and ranavirus prevalence in the summer. Counter to previous field studies in North America, we found a significant positive association between water temperature and ranavirus prevalence in the fall sampling events. Despite these findings, the influences of cattle and temperature on ranavirus prevalence were not consistent across seasons. As such, the mechanisms driving high ranavirus prevalence across the landscape and over time remain unclear. Given the widespread occurrence of ranaviruses in wild amphibians, we encourage the implementation of surveillance programs to help identify potential drivers of emergence. Sites with high ranavirus prevalence should be monitored annually for outbreaks, and the long-term effects on population size determined.     

Myiasis by Lucilia silvarum (Calliphoridae) in amphibian species in boreal Alberta, Canada

We report myiasis by Lucilia silvarum with an overall prevalence of 0.9% in amphibian populations in boreal Alberta. In the period 1998--1999, we documented L. silvarum infestations in wild populations of wood frog (Rana sylvatica), boreal chorus frog (Pseudacris maculata), boreal toad (Bufo boreas boreas), and Canadian toad (B. hemiophrys). We believe this is the first record of this parasite from boreal and Canadian toads. Almost all previous records of L. silvarum parasitism in North America indicate that myiasis is fatal to an anuran host. Here, we provide the first record of adult individuals from 2 species (wood frog and boreal toad) surviving infestations. Although we actively captured and examined amphibians in Alberta from 1996 to 2006, we only found parasitism in 1998 and 1999. This is the most northerly record of anuran infestations by this parasite.