First Evidence of <Emphasis Type="Italic">Batrachochytrium dendrobatidis</Emphasis> in China: Discovery of Chytridiomycosis in Introduced American Bullfrogs and Native Amphibians in the Yunnan Province,China

Although the chytrid fungus Batrachochytrium dendrobatidis (Bd), the etiological agent of amphibian chytridiomycosis, has been implicated in mass mortality and population declines on several continents around the world, there have been no reports on the presence of Bd infections in amphibians in China. We employed quantitative PCR and histological techniques to investigate the presence of Bd in introduced North American bullfrogs (Rana catesbeiana) (referred to hereafter as bullfrog) and native amphibians in bullfrog-invaded areas of the Yunnan Province, China. A total of 259 samples at five wild sites were collected between June and September in 2007 and 2008, including bullfrogs and four native amphibian species (Rana pleuraden, Rana chaochiaoensis, Odorrana andersonii, and Bombina maxima). In addition, 37 samples of adult bullfrogs were obtained from a food market. Bd infections were discovered in bullfrogs and three native amphibian species from all of the surveyed sites. Of the 39 Bd-positive samples, 35 were from wild-caught bullfrog tadpoles, postmetamorphic bullfrogs, R. pleuraden, R. chaochiaoensis, and O. andersonii, and four were from adult bullfrogs from the market. Our results provide the first evidence of the presence of Bd in Chinese amphibians, suggesting that native amphibian diversity in China is at risk from Bd. There is an urgent need to monitor the distribution of Bd in amphibians in China and understand the susceptibility of native amphibian species to chytridiomycosis. Strict regulations on the transportation of bullfrogs and the breeding of bullfrogs in markets and farms should be drafted in order to stop the spread of Bd by bullfrogs.     

Diet composition of post-metamorphic bullfrogs (Rana catesbeiana) in the Zhoushan archipelago, Zhejiang Province, China

Bullfrogs (Rana catesbeiana) are listed as one of the 100 worst invasive alien species in the world. They are generalist predators and thus may affect native species through predation. In previous studies, the food contents of bullfrogs were mostly examined at a single site. In the present study, the diet composition of post-metamorphic bullfrogs on eight islands (Daishan, Liuheng, Xiushan, Fodu, Taohua, Xiashi, Cezi, and Putuoshan) in the Zhoushan Archipelago, Zhejiang Province of China, were examined by using the stomach flushing method from June 30 to August 11 in 2005. A total of 391 individual frogs were measured, including 113 adults and 278 juveniles. The analysis of the stomach contents shows that, for adult bullfrogs, the most important prey (by diet volume) overall were Decapoda, Coleoptera, Odonata, Mesogastropoda, Raniformes, and Cypriniformes. For juvenile bullfrogs, these were Decapoda, Coleoptera, Cypriniformes, Odonata, Orthoptera, Hymenoptera, Lepidoptera larvae, Mesogastropoda, and Raniformes. Moreover, the prey size and diet volume increased with the body size of both adult and juvenile bullfrogs. The diet composition of primary preys of bullfrogs was significantly different among the islands. The results indicate that bullfrogs exert different predatory influences on native fauna at different sites and that bullfrogs are generalist predators with extensive ecological impacts on native fauna. __________ Translated from Biodiversity Science, 2006, 14 (5): 363–371 [译自: 生 物多样性]     

舟山群岛变态后牛蛙的食性研究

牛蛙(Ranacatesbeiana)是极其危险的外来入侵种,对当地生物类群的捕食是其主要的危害机制,但目前有关其食性的研究均只局限于一个研究地点。为了全面评估牛蛙对当地生物类群的捕食影响,2005年6月30日至8月11日期间,作者采用洗胃法对舟山群岛8个岛屿(岱山、六横、秀山、佛渡、桃花、虾峙、册子和普陀山)变态后牛蛙的食性进行了研究。共采集到变态后的牛蛙391只,包括牛蛙成体113只和亚成体278只。食性分析结果表明:(1)按食物体积计算,成年牛蛙以十足目、鞘翅目、蜻蜓目、中腹足目、蛙形目和鲤形目为主要食物,亚成体牛蛙以十足目、鞘翅目、鲤形目、蜻蜓目、直翅目、膜翅目、鳞翅目幼虫、中腹足目和蛙形目为主要食物;成体和亚成体牛蛙胃中食物的平均大小均随其体长增加而增大;(2)不同岛屿上的牛蛙在主要食物类型构成上存在差异。这表明牛蛙在不同地方对不同生物类群造成的捕食危害是不一样的,充分反映了其生态危害的广泛性。     

Diet composition of post-metamorphic bullfrogs (Rana catesbeiana) in the Zhoushan archipelago, Zhejiang Province, China

Bullfrogs (Runa catesbeiana) are listed as one of the 100 worst invasive alien species in the world.They are generalist predators and thus may affect native species through predation.In previous studies,the food contents of bullfrogs were mostly examined at a single site.In the present study,the diet composition of post-metamorphic bullfrogs on eight islands (Daishan,Liuheng,Xiushan,Fodu,Taohua,Xiashi,Cezi,and Putuoshan) in the Zhoushan Archipelago,Zhejiang Province of China,were examined by using the stomach flushing method from June 30 to August 11 in 2005.A total 0f391 individual frogs were measured,including 113 adults and 278 juveniles.The analysis of the stomach contents shows that,for adult bullfrogs,the most important prey (by diet volume) overall were Decapoda,Coleoptera,Odonata,Mesogastropoda,Raniformes,and Cypriniformes.For juvenile bullfrogs,these were Decapoda,Coleoptera,Cypriniformes,Odonata,Orthoptera,Hymenoptera,Lepidoptera larvae,Mesogastropoda,and Raniformes.Moreover,the prey size and diet volume increased with the body size of both adult and juvenile bullfrogs.The diet composition of primary preys of bullfrogs was significantly different among the islands.The results indicate that bullfrogs exert different predatory influences on native fauna at different sites and that bullfrogs are generalist predators with extensive ecological impacts on native fauna.     

Population genetics of introduced bullfrogs, Rana (Lithobates) catesbeianus, in the Willamette Valley, Oregon, USA

The American bullfrog, Rana (Lithobates) catesbeianus, is endemic to eastern North America, but has been introduced to approximately 40 countries on four continents and is considered one of the hundred worst invasive alien species in the world. Here, we investigated the genetics of invasive bullfrogs in the Willamette Valley, Oregon, USA, where bullfrogs are widespread and abundant to determine: (1) the minimum number of bullfrog introductions; (2) the native source population(s); and (3) whether genetic variation is reduced compared to source populations. To answer these questions, we analyzed partial sequences of the mitochondrial cytochrome b gene for 251 bullfrogs from the Willamette Valley and the native range. We found that bullfrogs from the Mississippi River basin and Great Lakes region were introduced at least once to the Willamette Valley. Genetic variation measured as haplotype diversity (h) and nucleotide diversity (?? _n ) was not significantly different between Willamette Valley and source populations. Our results were in contrast to a recent genetic analysis of invasive bullfrog populations in Europe, which found that genetic variation in European bullfrog populations was much lower than in source populations. European bullfrogs also originated from different source populations than Willamette Valley bullfrogs. The difference in genetic composition between Willamette Valley and European bullfrogs is likely due to differences in their invasion histories and may have implications for the potential of bullfrogs in these different regions to adapt and expand.     

Development of vasotocin pathways in the bullfrog brain

The brain of adult bullfrogs (Rana catesbeiana) contains six populations of cells which are immunoreactive for the neurohypophysial peptide arginine vasotocin (AVT). It is unknown when some of these cell populations first appear during development and when the sexual differences in AVT distribution first become apparent. We therefore used immunocytochemistry to examine development of AVT pathways in developing bullfrog tadpoles and in newly metamorphosed froglets of both sexes. AVT-immunoreactive (AVT-ir) cells were already present in the three diencephalic areas (magnocellular preoptic nucleus, suprachiasmatic nucleus and hypothalamus) at stage III (Taylor and Kollros stages), the earliest stage examined. Cell size in the magnocellular nucleus was not bimodally distributed in either tadpoles or froglets. AVT-ir cells in the telencephalic septal nucleus and amygdala did not appear until stage VI. There was no sexual difference in the density of AVT-ir cells or fibers in the amygdala of tadpoles or froglets. Finally, cells in the hindbrain pretrigeminal nucleus appeared much later-after stage XX. Thus, different populations of neurons begin to express AVT at unique times during development. The sexual dimorphism in AVT content observed in the amygdala of adult bullfrogs must appear during juvenile development or at adulthood.     

The invasive bullfrog <Emphasis Type="Italic">Lithobates catesbeianus</Emphasis> in oases of Baja California Sur,Mexico: potential effects in a fragile ecosystem

The American bullfrog (Lithobates catesbeianus), considered as one of the world’s worst invasive species because of its negative effects on native fauna, has been introduced into over 40 countries in four continents. The invasion status in Mexico is poorly known. The first known record for this species in the Peninsula of Baja California, northwestern Mexico is an individual collected 35 years ago. A great portion of the peninsula is an arid desert where the sources of freshwater are the spring-fed oasis and mountain arroyos that support rare and endemic species closely associated with wetland conditions. Humans, who have introduced alien species, inhabit many of those oases. The aim of this work was to document the presence of the exotic bullfrog in 40 oases and mountain arroyos in Baja California Sur state during 2006–2009. Bullfrogs were found in 25% of the oases sampled; 15% have confirmed records of breeding populations and five oases are new location records. Our results show a considerable increase (50%) in the number of oasis invaded by bullfrogs in only 8 years. The animal populations of rare or endemic species associated with oases would be threatened by the arrival of bullfrogs. Actions for their control and eradication are urgently needed.     

Development of arginine vasotocin innervation in two species of anuran amphibian:Rana catesbeiana andRana sylvatica

Arginine vasotocin (AVT) is a neurotransmitter in the amphibian central nervous system and is released from the neurohypophysis in the regulation of hydromineral balance and other homeostatic functions. Many amphibians experience drastic changes in habitat with respect to water availability during their transformation from aquatic larvae to terrestrial adults. To examine whether metamorphosis is accompanied by a reorganization of central vasotocinergic neurons, the developmental organization of vasotocin neurons and nerve fibers was studied with immunocytochemistry in the brains of bullfrogs (Rana catesbeiana) and woodfrogs (R. sylvatica). In bullfrogs, early limb-bud-stage tadpoles had AVT-immunoreactive neurons and nerve fibers in the lateral septal nucleus, amygdala, preoptic hypothalamus, suprachiasmatic nucleus, and posterodorsal tegmentum. Woodfrog larvae showed similar patterns of hypothalamic AVT immunoreactivity, although neuronal staining in the amygdala did not appear until metamorphic climax, and never appeared in septal neurons or in the posterodorsal tegmentum. Whereas the highly terrestrialR. sylvatica adults must adapt to an adult habitat with prolonged periods of dehydration,R. catesbeiana adults remain semiaquatic and, as such, need not develop extreme mechanisms for water retention. Nonetheless, vasotocinergic pathways showed developmental similarities in the two species. The early appearance of AVT innervation in bothRana suggests that AVT has neuroregulatory functions well before metamorphosis.     

Influence of temperature on the optimal hematocrit of the bullfrog (Rana catesbeiana)

Summary The influence of temperature on blood viscosity and consequently on the potential for oxygen transport by blood was determined using a controlled flow, variable pressure tube viscometer, and blood from adult bullfrogs. Blood viscosity was determined as a function of hematocrit and temperature, and oxygen capacity was determined as a function of hematocrit. These data were used to describe 1) the potential for oxygen transport in the tube viscometer, and 2) the relation between the optimal hematocrit, the hematocrit which provided the greates oxygen transport, and temperature. The optimal hematocrit increased at a rate of 0.237% per °C increase in temperature. This value is close to the rate of change inin vivo hematocrit of 0.246 and 0.240% per °C increase in body temperature (T_b) observed in winter bullforgs acclimated to 5 and 20°C, respectively. During the summer the hematocrit ratio showed no consistent relation to T_b. These results suggest that in bullfrogs the cardiovascular adjustments to change in T_b involve the optimal hematocrit in winter, but not in summer.     

American bullfrog <Emphasis Type="Italic">Lithobates catesbeianus</Emphasis> (Shaw, 1802) invasion in Argentina

The American bullfrog (Lithobates catesbeianus) has been introduced throughout the world over the past two centuries. This taxa is a potentially devastating invader because of its large size, prolific reproductive output, and broad ecological niche. Consequently, the extent of this ongoing biological invasion is an increasing conservation concern. In Argentina, several introduced populations have been reported. In most cases, these introductions have been intentional or incidental releases from breeding facilities, yet the consequences and effective controls for captive-breeding programs have not been assessed by government environmental agencies. Further studies are needed regarding the trophic ecology, reproductive biology, ecological niche, and chytrid fungus infection prevalence to predict the ultimate impacts of this species on native ecosystems. The aim of this work is to report a new alien population of L. catesbeianus at La Candelaria, Salta province, Argentina. This record represents the first population of American bullfrogs detected in northwestern Argentina.     

Structural changes in ribosomes during development

Basic ribosomal proteins, extracted from immature erythrocytes of both pre-metamorphic and adult bullfrogs (Rana catesbeiana), were characterized on urea-polyacrylamide gels. Ribosomes from larval and adult organisms display quantitative and qualitative differences in the banding patterns of their basic ribosomal proteins. Such alterations in ribosomal structure may be important in the control of gene expression during development.     

The emerging amphibian pathogen Batrachochytrium dendrobatidis globally infects introduced populations of the North American bullfrog, Rana catesbeiana

Batrachochytrium dendrobatidis is the chytridiomycete fungus which has been implicated in global amphibian declines and numerous species extinctions. Here, we show that introduced North American bullfrogs (Rana catesbeiana) consistently carry this emerging pathogenic fungus. We detected infections by this fungus on introduced bullfrogs from seven of eight countries using both PCR and microscopic techniques. Only native bullfrogs from eastern Canada and introduced bullfrogs from Japan showed no sign of infection. The bullfrog is the most commonly farmed amphibian, and escapes and subsequent establishment of feral populations regularly occur. These factors taken together with our study suggest that the global threat of B. dendrobatidis disease transmission posed by bullfrogs is significant.     

拉萨国家级自然保护区拉鲁湿地发现牛蛙

原产于美国落基山脉以东地区的牛蛙(Rana Catesbeiana )是一种非常危险的外来物种。 2014年6月14-24日在拉萨市国家级自然保护区拉鲁湿地内发现成 、幼体 的牛蛙。建议借助政府、知名宗教人士、媒介等力量,以制度、呼吁、宣传等有效渠道,尽快解决湿地放生牛蛙的问题,以降低湿地现有牛蛙的种群增长及栖息地的进一步扩大。     

Role of Antimicrobial Peptides in Amphibian Defense Against Trematode Infection

Antimicrobial peptides (AMPs) contribute to the immune defenses of many vertebrates, including amphibians. As larvae, amphibians are often exposed to the infectious stages of trematode parasites, many of which must penetrate the host’s skin, potentially interacting with host AMPs. We tested the effects of the natural AMPs repertoires on both the survival of trematode infectious stages as well as their ability to infect larval amphibians. All five trematode species exhibited decreased survival of cercariae in response to higher concentrations of adult bullfrog AMPs, but no effect when exposed to AMPs from larval bullfrogs. Similarly, the use of norepinephrine to remove AMPs from larval bullfrogs, Pacific chorus frogs, and gray treefrogs had only weak (gray treefrogs) or non-significant (other tested species) effects on infection success by Ribeiroia ondatrae. We nonetheless observed strong differences in parasite infection as a function of both host stage (first- versus second-year bullfrogs) and host species (Pacific chorus frogs versus gray treefrogs) that were apparently unrelated to AMPs. Taken together, our results suggest that AMPs do not play a significant role in defending larval amphibians against trematode cercariae, but that they could be one mechanism helping to prevent infection of post-metamorphic amphibians, particularly for highly aquatic species.     

Pattern of distribution of the American bullfrog <Emphasis Type="Italic">Rana catesbeiana</Emphasis> in Europe

Despite early reports of its presence, no recent data exist on the distribution of the American bullfrog in Europe, the causes of introduction, or the trends of populations. We monitored the European situation at two spatial scales. In SW France, we performed call surveys over 2,500 wetlands. We found bullfrogs over about 2,000 km², apparently the European area in which the strongest expansion of bullfrogs is taking place. In addition, we used questionnaires to investigate the situation at the continental scale. At least 25 independent introductions occurred in Europe; eradication attempts were successful three times, and bullfrog populations are present in five countries. Education programs and monitoring are necessary to reduce the rate of introduction and to start management action as soon as possible. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Attenuated cardiovascular effects of prostaglandin I2 and prostaglandin F2 alpha in cold acclimated American bullfrogs, Rana catesbeiana

American bullfrogs, Rana catesbeiana respond to prostaglandins with changes in heart rate and blood pressure. These studies compare responses of warm (22 degrees C) and cold acclimated (5 degrees C) bullfrogs to prostaglandins. Gas chromatographic analysis determined equivalent fatty acid profiles in total lipids of heart and artery tissue from warm and cold acclimated animals. Arachidonic acid was the fatty acid precursor found in greatest abundance in both groups. For cardiovascular experiments, bullfrogs were cannulated by using a T-cannula implanted in the right sciatic artery. In warm acclimated bullfrogs, preinfusion systemic arterial pressure (SAP) was 14.7 +/- 0.5 mm Hg, and heart rate was 33.0 +/- 1.7 beats/min. Cold acclimated bullfrogs had SAP values of 8.0 +/- 0.8 mm Hg, and heart rate was 6.9 +/- 0.3 beats/min. Arachidonic and eicosapentaenoic acid infusions (2,000 micrograms/kg body weight [bw]) were hypertensive in cold acclimated and hypotensive in warm acclimated animals. These effects were blocked by indomethacin (4 mg/kg bw). In both warm and cold acclimated bullfrogs, prostaglandin F2 alpha (3-100 micrograms/kg bw) was hypertensive, while prostaglandin I2 (0.03-3 micrograms/kg bw) was hypotensive, with both prostaglandins stimulating a greater absolute response in warm acclimated animals. In addition, both prostaglandins increased heart rate in warm but not in cold acclimated bullfrogs. The results suggest diminished cardiovascular sensitivity to prostaglandins at low environmental temperatures.     

Novel,panzootic and hybrid genotypes of amphibian chytridiomycosis associated with the bullfrog trade

Global amphibian declines are linked with the presence of specific, highly virulent genotypes of the emerging fungal disease chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) known as the global panzootic lineage (Bd‐GPL). The global trade in amphibians for human consumption is suspected to have facilitated emergence of the disease, but evidence to support this is largely lacking. Here, we investigated the role the Lithobates catesbeianus (North American bullfrog) trade in spreading Bd genotypes by comparing strains associated with L. catesbeianus to a global panel using 36 sequenced loci from multiple chromosomal regions. Most bullfrogs were infected with Bd‐GPL genotypes, but we also detected novel, highly divergent Bd genotypes (Bd‐Brazil) from a live bullfrog in a US market and from native Brazilian anurans in the Atlantic Forest where bullfrogs are widely farmed. Sexual reproduction was also detected for the first time in Bd in the form of a hybrid genotype between the Bd‐GPL and Bd‐Brazil lineages in the Atlantic Forest. Despite the demonstration that ribosomal RNA types in Bd fail to undergo concerted evolution (over 20 sequence types may be found in a single strain), the Bd‐GPL and Bd‐Brazil lineages form largely separate clusters of related internal transcribed spacer (ITS) RNA sequences. Using ITS sequences, we then demonstrate the presence of Bd‐Brazil in Japan, primarily on invasive L. catesbeianus. The finding that Bd is capable of sexual reproduction between panzootic and endemic genotypes emphasizes the risk of international wildlife trade as a source of additional Bd epizootics owing to hybridization.     

Why islands are easier to invade: human influences on bullfrog invasion in the Zhoushan archipelago and neighboring mainland China

Islands are often considered easier to invade than mainland locations because of lower biotic resistance, but this hypothesis is difficult to test. We compared invasion success (the probability of establishing a wild reproducing population) for bullfrogs (Rana catesbeiana) introduced to enclosures on 26 farms on islands in the Zhoushan archipelago and 15 farms in neighboring mainland China. Bullfrogs were more likely to invade farms located on islands with lower native frog species richness than mainland farms, consistent with the biotic resistance hypothesis. However, human frog hunting pressure also differed between islands and the mainland and, along with the number of bullfrogs raised in enclosures, was a stronger predictor of invasion success than native frog richness in multiple regression. Variation in hunting pressure was also able to account for the difference in invasion success between islands and mainlands: islands had lower hunting pressure and thus higher invasion probability. We conclude that the ease with which bullfrogs have invaded islands of the Zhoushan archipelago relative to the mainland has little to do with biotic resistance but results from variation in factors under human control.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

An exotic species is the favorite prey of a native enemy

Although native enemies in an exotic species'' new range are considered to affect its ability to invade, few studies have evaluated predation pressures from native enemies on exotic species in their new range. The exotic prey naiveté hypothesis (EPNH) states that exotic species may be at a disadvantage because of its naïveté towards native enemies and, therefore, may suffer higher predation pressures from the enemy than native prey species. Corollaries of this hypothesis include the native enemy preferring exotic species over native species and the diet of the enemy being influenced by the abundance of the exotic species. We comprehensively tested this hypothesis using introduced North American bullfrogs (Lithobates catesbeianus, referred to as bullfrog), a native red-banded snake (Dinodon rufozonatum, the enemy) and four native anuran species in permanent still water bodies as a model system in Daishan, China. We investigated reciprocal recognition between snakes and anuran species (bullfrogs and three common native species) and the diet preference of the snakes for bullfrogs and the three species in laboratory experiments, and the diet preference and bullfrog density in the wild. Bullfrogs are naive to the snakes, but the native anurans are not. However, the snakes can identify bullfrogs as prey, and in fact, prefer bullfrogs over the native anurans in manipulative experiments with and without a control for body size and in the wild, indicating that bullfrogs are subjected to higher predation pressures from the snakes than the native species. The proportion of bullfrogs in the snakes'' diet is positively correlated with the abundance of bullfrogs in the wild. Our results provide strong evidence for the EPNH. The results highlight the biological resistance of native enemies to naïve exotic species.