Wild Frogs Research Articles

低温胁迫下虎纹蛙的生存力及免疫和抗氧化能力

全球气候变化是造成世界范围内两栖类种群衰退和灭绝的重要因素之一。随着极端天气出现变得日趋频繁,非季节性的、短期且剧烈的气温变化可能会严重干扰两栖类动物的生存与种群稳定。监测了浙江省金华市南山野生虎纹蛙(<em>Hoplobatrachus rugulosus</em>)分布区冬季的环境气温,并参考监测数据在实验室条件下研究了虎纹蛙在短期梯度降温(2 ℃/ 24 h)和急性冷暴露(即冷休克)(2 ℃)下的生存力及冷休克对机体免疫功能和抗氧化能力的影响。结果表明,虎纹蛙在冬季(2009-12-01-2010-03-31)经历的温度范围普遍在0-14 ℃之间,主要遭遇的低温区间在0-4 ℃,主要高温区间在10-14 ℃。通过梯度降温实验,发现温度降至12 ℃累积死亡率约为28.1%,10 ℃为87.5%,8 ℃为100%。在一定温度范围内,虎纹蛙死亡率与环境温度呈显著负相关(Pearson test, <em>r</em>=-0.952, <em>P</em><0.05)。经曲线拟合,回归方程计算可得半数致死温度为11.5 ℃。虎纹蛙在冷休克处理下,在第6 h累积死亡率为45%,12 h为80%,24 h达到100%。虎纹蛙死亡率与冷休克时间呈显著正相关(Pearson test, <em>r</em>=0.91, <em>P</em><0.05),半数致死时间为7.6 h。此外,冷休克(2 ℃,6 h)显著抑制了虎纹蛙脾脏巨噬细胞呼吸爆发强度(<em>t</em>= 3.827, d<em>f</em>=6, <em>P</em><0.05)、全血吞噬活性(<em>t</em>=5.388, d<em>f</em>=3.037, <em>P</em><0.05)及胃溶菌酶活力(<em>t</em>=6.37, d<em>f</em>=6, <em>P</em><0.05);肝脏(<em>t</em>=0.773, d<em>f</em>=8, <em>P</em> > 0.05)和肾脏(<em>t</em>=0.164, d<em>f</em>=4.542, <em>P</em> > 0.05)组织脂质过氧化物产物丙二醛(MDA)的含量虽无明显变化,但肝脏(<em>t</em>=-2.817, d<em>f</em>= 6, <em>P</em><0.05)和肾脏(<em>t</em>=-11.302, d<em>f</em>=6, <em>P</em><0.05)组织抗氧化物谷胱甘肽(GSH)含量及肝脏(<em>t</em>=-3.3, d<em>f</em>=6, <em>P</em><0.05)超氧化物歧化酶(SOD)活性均显著升高。上述结果表明虎纹蛙对低温较为敏感,冷休克能够诱导机体的免疫抑制,并导致机体对抗氧化物质需求的增加。可以推测,当遭遇极端低温天气时,低温胁迫可能会严重干扰虎纹蛙生理机能,加大种群的生存压力和疾病感染的风险。;Global climate change has been considered to be one of the key factors leading to declines and losses of amphibian populations. Though global warming is one of the acknowledged causes of fluctuations of amphibian population worldwide, unseasonal sharp fall in air temperature as result of global climate change may also affect the survival and stability of amphibian population severely. As amphibians are typical poikilothermic animals, environmental temperature can influence their physiological functions and enhance their susceptibility to infectious diseases. The immunological competence of amphibians may, therefore, make them vulnerable to parasites and disease particularly after discontinuous and extreme events (e.g. winter rainfall, late spring coldness). In this article, we have investigated how cold stress affected the physiology and viability of tiger frogs (<em>Hoplobatrachus rugulosus</em>). The winter temperatures experienced by wild tiger frogs in natural winter in the Southern Mountain of Jinhua in Zhejiang Province was monitored, and then replicated in subsequent experiments. The aim of the experiments was to determine how gradient cooling (2 ℃/24 h) and cold shock (2 ℃) affected the mortality rate of tiger frogs, and affected various aspects of their immune function and oxidation resistance. The results showed that the survival temperature range of wild overwintering was between 0-14 ℃ in 12/1/2009-3/31/2010, and minimum and maximum temperature ranged between 0-4 ℃ and 10-14 ℃ respectively. During the process of gradient cooling, we found that the accumulated mortality rate was 28.1% at 12 ℃, 87.5% at 10 ℃ and 100% at 8 ℃. A significant negative correction was found between environmental temperature and frog mortality within a certain temperature range (Pearson test, <em>r</em>=-0.952, <em>P</em><0.05). And lethal temperature of 50% occurred at 11.2 ℃ based on a fitted curve regression equation. Under cold shock,45% mortality was observed after being directly stimulated at 2 ℃ for 6 h,80% mortality appeared at 2 ℃ for 12 h, and increased to 100% after 24 h. There appeared to be positive correction between cold shock time and mortality (Pearson test, <em>r</em>=<em></em>0.91,<em> P</em><0.05), and lethal time of 50% is 7.6 h at 2 ℃ according to fitted curve regression equation. Moreover, cold shock significantly inhibited the respiratory burst of spleen macrophages (<em>t</em>=3.827, d<em>f</em>=6, <em>P</em><0.05), peripheral blood phagocytic activity (<em>t</em>=5.388, d<em>f</em>= 3.037,<em> P</em><0.05) and gastric lysozyme activity (<em>t</em>=6.37, d<em>f</em>=6, <em>P</em><0.05) at 2 ℃ for (6 h). Although there was no significant change in malonaldehyde (MDA) content, the glutathione (GSH) content of the liver (<em>t</em>=-2.817, d<em>f</em>=6, <em>P</em><0.05) and kidney (<em>t</em>=-11.302, d<em>f</em>=6, <em>P</em><0.05) increased. The activity of superoxide dismutase (SOD) also increased significantly in the liver (<em>t</em>=-3.3, d<em>f</em>=6, <em>P</em><0.05), while there was no apparent change within the kidney. The results indicate that tiger frogs are sensitive to low temperatures and that cold shock might lead to greater oxidative stress, more requirements for antioxidants and produce immunosuppression in tiger frogs. Consequently, low temperature stress will stimulate organism endocrine disorder and subject the frogs to a more intensive disturbance of physiological function, increase the survival pressure and susceptibility to infectious disease, which may affect development and dynamics of amphibian populations.

Urinary hormone metabolites identify sex and imply unexpected winter breeding in an endangered, subterranean-nesting frog

Urinary hormone analysis has proved accurate for identifying sex and breeding periods in dimorphic amphibians with known reproductive cycles. We examined whether these techniques could provide this much needed information for a monomorphic anuran with an unconfirmed mating season in the wild. We analysed urinary estrone conjugate, testosterone, and progesterone metabolites to infer the time of breeding and to identify sex in the endangered Maud Island frog, Leiopelma pakeka. Testosterone metabolites in males and estrone and progesterone metabolites in females were at their peak during winter for both wild and captive frogs. These urinary metabolite patterns were consistent with the high proportion of females exhibiting enlarged ovarian follicles in winter months. Sex identification based on urinary estrone metabolite levels was 94% correct in this monomorphic species, in which the sexes overlap in snout-to-vent length (SVL) for over half of their adult size range and in which no other sexually dimorphic trait is known. The seasonal profiles imply unexpected winter or early spring breeding in L. pakeka. Overall, these results demonstrate use of urinary hormone metabolites for reproductive monitoring and sex identification in one of the world’s most threatened and evolutionarily distinct amphibians.

120 THE DEVELOPMENTAL COMPETENCE OF TADPOLES PRODUCED IN VITRO FROM THE ENDANGERED DUSKY GOPHER FROG (RANA SEVOSA) USING EXOGENOUS HORMONE TREATMENT

Dusky gopher frogs once existed throughout the states of Mississippi, Alabama and Louisiana. Presently, the USA Fish and Wildlife Service estimates that there are less than 100 frogs left in the wild, with almost all of these residing in a single pond in Mississippi, making the dusky gopher frog America's most endangered frog species. Their habitat has been threatened by residential and forestry development, as well as from fire suppression and the decline of gopher tortoises, whose burrows the frogs use for shelter. The USA Fish and Wildlife Service brought the first dusky gopher frogs into captivity in 2001 and they have been kept at the Memphis Zoo since 2003 and at Omaha's Henry Doorly Zoo since 2004. Natural breeding attempts in captivity have been unsuccessful despite efforts to artificially mimic what is known to be the appropriate seasonal environmental (e.g. light, humidity, temperature) and social cues (e.g. calls from wild frogs during the breeding season) that stimulate reproduction in situ. Also, there was a concern about the incidences of abnormalities (e.g. spindly legs) from inadequate husbandry conditions (e.g. nutritional deficiencies) or diseases in tadpoles and froglets produced ex situ (e.g. dermomycoides). The objectives of this study were (1) to examine the developmental competence of tadpoles produced by exogenous hormone treatment for IVF to complete normal metamorphosis into viable froglets ex situ and (2) to determine the incidence of developmental abnormalities or dermomycoides in the froglets produced ex situ. Males and females were first separated by the identification of black male nuptial pads used during amplexus. Males and females were stimulated to spermiate and ovulate, respectively, using a variation of a standard amphibian hormonal stimulation protocol (Kouba et al. 2012 Reprod. Fertil. Dev. 24). As a result, more than 1460 viable tadpoles (61% of fertilized eggs) were produced from 18 segregated genetic pairings. To date, 602 (41%) of the tadpoles have completed normal metamorphosis into viable froglets (over 6 times the known existing population in situ), 51% were either culled to examine for dermomycoides or died for a variety of known (accidental) and unknown reasons (abnormal appendage development accounting for less than 7%) and 8% have yet to metamorphose after 9 months. The incidence of dermomycoides in the culled individuals was 100%; however, a study being conducted concurrently in their native habitat has confirmed that the organism does exist in situ with the dusky gopher frogs, which act as natural carriers. Efforts are currently in place to establish reintroduction programs for the dusky gopher froglets produced ex situ to increase the dwindling wild population now that tadpoles produced by IVF have been shown to be developmentally competent. In conclusion, IVF can be used to produce tadpoles of the dusky gopher frog with a low incidence of abnormalities, but the tadpoles were found to be carriers of the organism dermomycoides similar to their counterparts in the wild.

Ranavirus-associated mass mortality in wild amphibians, The Netherlands, 2010: A first report

In 2010, a mass die-off of over 1000 wild water frogs (Pelophylax spp.) and at least 10 common newts (Lissotriton vulgaris) occurred in a pond in The Netherlands. Haemorrhagic disease with hepatomegaly and splenomegaly was evident. Microscopically, multiple organs presented cells with multifocal intracytoplasmic inclusion bodies, in which ranavirus-like particles were demonstrated ultrastructurally. All specimens examined tested positive for ranavirus by PCR. The sequence obtained showed a 100% identity with the one deposited for common midwife toad virus (CMTV). This is the first report of ranavirus-associated mortality in wild amphibian populations in The Netherlands. It is also the first time CMTV or a CMTV-like virus has been reported in these two species in the adult stage and outside of Spain.

Anurans as paratenic hosts in the transmission of Hepatozoon caimani to caimans Caiman yacare and Caiman latirostris

Prevalence of Hepatozoon caimani has been reported in 76% of caimans Caiman yacare from the Pantanal region. Culex (Melanoconion) spp. mosquitoes were recently identified as natural vectors of this parasite. However, culicids are not typically eaten by crocodilians, suggesting that the main transmission route is through ingestion of insectivorous vertebrates, such as anurans. The susceptibility of wild frogs Leptodactylus chaquensis, Leptodactylus podicipinus and Scinax nasicus to infection by H. caimani was verified. Wild-caught anurans were force fed with sporulated oocysts from laboratory-bred Culex (Melanoconion) mosquitoes. Frogs were killed 30 days postinfection, and their internal organs were fed to caimans C. yacare and Caiman latirostris. Cystozoites were identified in fresh liver impression smears of L. chaquensis. C. yacare fed on anuran organ presented gametocytes in peripheral blood circulation between 74 and 80 days postinoculation (dpi). Gametocytes were also verified in C. latirostris fed on the internal organs of L. podicipinus and S. nasicus between 60-70 and 69-75 dpi, respectively. Since frogs used in experiment are sympatric with C. yacare and C. latirostris and may occur in the diet of these caimans, the results suggest these amphibians are paratenic hosts in the natural transmission cycle of H. caimani in Pantanal.

Urinary corticosterone metabolite responses to capture and captivity in the cane toad ( Rhinella marina)

Urinary corticosterone metabolite responses to capture have recently been shown for the first time in amphibians, and in the present study urinary corticosterone metabolite responses to capture and to confinement in captivity were measured in adult cane toads ( Rhinella marina) in Queensland, Australia. An adrenocorticotropic hormone (ACTH) challenge was used to provide a biological validation for urinary corticosterone metabolite concentrations measured by radioimmunoassay (RIA). Urinary corticosterone metabolite increased 1–2 days after ACTH but not saline injection and then returned to initial values, indicating that the RIA could detect changes in corticosterone secretion in toads. Urinary corticosterone metabolite responses to short-term capture and restraint in plastic bags were first apparent 2 h after capture of wild toads. Toads held communally in captivity for 5 days had elevated urinary corticosterone metabolite concentrations. Mean corticosterone concentrations declined significantly after a further 7 days in individual housing chambers. There was no sex difference in urinary corticosterone metabolite responses of toads to ACTH challenge, short-term capture or captivity. The relative amount of variation in the mean corticosterone responses was quantified by calculating coefficients of variation (CV) for each mean corticosterone response. Mean corticosterone at 0 min was more variable for captive toads than wild toads. Furthermore, initial corticosterone concentrations (0 min) were more variable than concentrations during the ACTH challenge, short-term capture and captivity. There was little change in the amount of variation of mean corticosterone levels between male and female toads with increasing time in captivity (12–29 days). This study has shown individual corticosterone responses of amphibians for the first-time, and has provided a novel method for quantifying the relative amount of variation in amphibian corticosterone responses.

Bioaccumulation, maternal transfer and elimination of polybrominated diphenyl ethers in wild frogs

To investigate bioaccumulation, maternal transfer and elimination of polybrominated diphenyl ethers (PBDEs) in amphibians, we collected adult frogs (Rana limnocharis) from a rice field in an e-waste recycling site in China. We found that ∑PBDEs in the whole frogs and various tissues (brain, liver, testis and egg) ranged from 17.10 to 141.11ngg−1 wet weight. Various tissues exhibited a similar PBDE congener profile, which was characterized by intermediate brominated congeners (BDE-99 and BDE-153) as the largest contributors, with less lower brominated congeners (BDE-28 and BDE-47) and higher brominated congeners (BDE-209). The maternal transfer capacity of PBDEs declined with the increase in bromine numbers of PBDE congeners. We suggest that the bromine atom number (the molecular size, to some degree) might be a determining factor for the maternal transport of a PBDE congener rather than Kow (Octanol–Water partition coefficient), which expresses a compound’s lipophilicity. ∑PBDEs concentrations in frogs decreased over time during a depuration period of 54days when these wild frogs were brought to the lab from the e-waste recycling site. The half-life of ∑PBDEs was 35days, with about 14days for BDE-47, and 36 and 81days for BDE-99 and BDE-153, respectively. The data shows that the elimination of PBDEs has no essential difference from aquatic and terrestrial species.

A randomized phase II study of gemcitabine (G) plus the cardiac glycoside huachansu (H) in the treatment of patients with locally advanced (LAPC) or metastatic pancreatic cancer (MPC).

4127 Background: Wild toad venom has been used for over 1,000 years as a traditional Chinese medicine; H, the extract of dried toad skin glands, is a potent cardiac glycoside without significant to...

Morphometric discrimination of wild from farmed Dybowski’s frog (Rana dybowskii) based on hindlimb length

Commercial farming of anuran species that are declining in the wild raises a need to discriminate wild from farmed frogs. We hypothesized wild frogs might have extended hindlimbs due to greater frequency or intensity of jumping relative to farmed frogs, highlighting a morphometric approach to discrimination of wild from farmed frogs using hindlimb length. In the present study, Dybowski’s frog (Rana dybowskii) was used to test this hypothesis. We measured body mass (Mb) and hindlimb length (Lh) of 2-year old farmed frogs and wild frogs aged 2 to 5 years. Dybowski’s frog demonstrated significant dimorphism in Mb and Lh. Mb was significantly greater among farmed 2-year old frogs in both sexes (p=0.000), while only among females was Lh significantly greater for wild frogs (p=0.000). Lh/Mb was used as an index for origin discrimination to eliminate the influence of Mb due to variation of husbandry conditions among farms. Mean Lh/Mb for farmed frogs was significantly lower than for wild frogs (p=0.000) in the 2-year old age class. Discrimination correctly classified 84.4% of farmed and 96.3% of wild male frogs. Among females, 92.9% of farmed frogs and 90.1% wild frogs were correctly classified. The overall correctness of classification was 92.1% and 90.8% for males and females, respectively. However, Lh/Mb revealed variation with age, resulting in reduced discriminative power for frogs ≥3 years old. We introduced a coefficient Ce to adjust the Lh/Mb of frogs ≥3 years to the level equivalent to 2-year frogs. The adjustment achieved 89.5% for overall correctness of origin for wild males and 92.4% for wild females ≥3 years old. These results show that Lh/Mb is an effective index to discriminate wild from farmed Dybowski’s frog. Since the physical demands of jumping are common among anurans, this index is also potentially applicable to other anuran species.

Early life progestin exposure causes arrested oocyte development, oviductal agenesis and sterility in adult Xenopus tropicalis frogs

Levonorgestrel (LNG) is a commonly used pharmaceutical progestin found in the environment. Information on the long-term toxicity of progestins following early life exposure is scant. We investigated the effects of developmental LNG exposure on sex differentiation, reproductive organ development and fertility in the model frog Xenopus tropicalis. Tadpoles were exposed to 0, 0.06 or 0.5 nM LNG via the water from hatching until metamorphosis. At metamorphosis effects on gonadal differentiation were evaluated using a subsample of frogs. Remaining animals were held unexposed for nine months, at which time reproductive organ structure, function and fertility were determined. LNG exposure severely impaired oviduct and ovary development and fertility. All adult females in the 0.5 nM group ( n = 10) completely lacked oviducts. They also displayed a significantly larger fraction of immature oocytes, arrested in meiotic prophase, than control females. Upon mating with unexposed males, only one of 11 LNG-exposed females laid eggs, whereas all control females did. No effects on testicular development, sperm count or male fertility were observed. At metamorphosis, no effects on sex ratio or gonadal histology were evident. The effects on ovarian and oviductal development were detected at adult age but not at metamorphosis, emphasising the importance of investigating the long-term consequences of developmental exposure. This is the first developmental reproductive toxicity study of a progestin in an aquatic vertebrate. Considering that several progestins are present in contaminated surface waters, further investigation into the sensitivity of frogs to progestins is warranted to understand the risk such compounds may pose to wild frog populations.

A randomized phase II study of gemcitabine (G) plus the cardiac glycoside huachansu (H) in the treatment of patients with locally advanced (LAPC) or metastatic pancreatic cancer (MPC).

284 Background: Wild toad venom has been used for over 1,000 years as a traditional Chinese medicine; H, the extract of dried toad skin glands, is a potent cardiac glycoside without significant toxicity. H has demonstrated preliminary evidence of antitumor activity in phase I clinical trial evaluation (Cancer 2009, 115:5309-18). Methods: We conducted a randomized, single-blind, phase II clinical trial of G + H versus G + placebo in patients with LAPC or MPC. Plasma bufalin levels were performed in H-treated subjects. 80 evaluable subjects accrued from October 2007 to May 2010 and were treated with G 1,000mg/m2 (iv on days 1, 8, 15, q 28d) plus either H (20 mL/m2 iv daily for 21 days followed by 7 days off, q 28d) or placebo. Demographic data is demonstrated in the table below. Results: Grade 3/4 adverse events, objective radiographic response rates (ORR), time to progression (TTP), quality of life (QOL), and overall survival (OS) were similar in the two groups (Table). There was not a statistically significant difference in the average plasma bufalin levels in patients with a radiographic partial response (PR) or stable disease versus those with progressive disease (PD) (0.098 ± 0.093 ng/mL vs. 0.071 ± 0.048 ng/mL, p=0.679). Conclusions: H when added to G was well tolerated but did not improve OS, TTP, QOL, or ORR. Further investigation of a more potent oral formulation of H is planned. H provided by Shenzhen 999 Traditional Chinese Medicine &amp; Development Co., Ltd. Supported by NIH U19CA121503-01. [Table: see text] No significant financial relationships to disclose.

Sparganosis, Henan Province, Central China

To the Editor: Sparganosis is a parasitic zoonosis caused by invasion of the spargana, the plerocercoid larvae of various diphyllobothroid tapeworms belonging to the genus Spirometra (1). Although human sparganosis is cosmopolitan, it is most frequently found in eastern and southeastern Asia (2). During 1927–2009 in the People’s Republic of China, >1,000 cases in humans in 27 provinces were reported; most cases were in southern China, where human infections were mainly acquired by eating raw or insufficiently cooked meat of frogs and snakes or by placing frog or snake flesh on open wounds for treatment of skin ulcers or on eyes to treat inflammation (3,4). Sparganosis is rarely seen in central and northern China. Before 2006, only 3 imported cases from southern China had been reported in Henan Province in central China (5). However, since 2006 in Henan Province, 20 autochthonous cases caused by ingestion of live tadpoles have emerged. To assess the risk for human infection with sparganosis in this province and to strengthen public safety awareness, we investigated spargana infection in the animal hosts of Spirometra tapeworms. During July 2007–July 2010, wild frogs and frog tadpoles were collected from the cities of Shangqiu, Zhoukou, and Luohe in Henan Province. Necropsies identified plerocercoids in 11.93% (163/1,366) of tadpoles and in 26.58% (172/647) of frogs. By frog species, plerocercoids were found in 31.09% (111/357) of Rana limmocharis and 26.29% (61/232) of R. nigromaculata frogs, each significantly (p<0.05) more numerous in these species than in R. temporaria frogs (0/58). In addition, 177 wild frogs sold at markets in Luohe were also examined; plerocercoids were detected in 30.39% (31/102) of R. limmocharis and 28% (21/75) of R. nigromaculata frogs. Thus, in Henan Province, R. limmocharis and R. nigromaculata frogs are the main intermediate hosts of Spirometra tapeworms. No pathologic changes associated with the tapeworms were found in dissected tadpoles and frogs. We found 250 plerocercoids in tadpoles and 1,387 in frogs. Tadpoles contained 1–14 (mean 1.53) and frogs 1–87 (mean 6.85) tapeworms. In frogs, most plerocercoids were located in the muscles of hind legs and back; some were in the muscles of the abdominal wall and forelegs. Plerocercoids dissected from tadpole and frog tissues were wrinkled, whitish, and ribbon-shaped and continuously moved while in normal saline. Plerocercoids from tadpoles were 1–8 mm long and 0.2–0.5 mm wide; those from frogs were 1–13 cm long and 1–2.5 mm wide. Cyclops were collected from ponds and ditches by using a 425-µm mesh (no. 40) sieve and species were identified by microscopic appearance as Mesocyclops leuckarti (6,7). Procercoids were microscopically found in the hemocele of 3.53% (3/85) of cyclops; 3–5 worms per cyclop were found. Fecal examination of dogs and cats found Spirometra mansoni tapeworm eggs in 19.35% (6/31) of dogs and 33.33% (1/3) cats. In addition, a 3-month-old specific pathogen–free cat was orally inoculated with 33 plerocercoids from tadpoles, fecal samples were microscopically examined by sedimentation during 10–25 days postinfection (dpi), and the cat was euthanized and examined for adult worms. S. mansoni tapeworm eggs were found in the feces during 12–25 dpi, and 17 adult worms, 26–45 cm long, were recovered from the small intestines at 25 dpi; however, no plerocercoids were seen in the tissues. The adult worms were morphologically identified as S. mansoni according to the following features: scolex with 2 longitudinal grooves, mature and gravid proglottids with conspicuous uterus at the center of segments, spiral-shaped uterus (unlike the rosette-shaped uterus of Diphyllobothrium latum), and cirrus and vaginal pore with separate openings (8,9). Although in recent years in Henan Province wild frogs have been sold clandestinely at markets, persons in this province do not routinely eat raw frog meat or use raw meat as poultices. However, some villagers in Henan do believe that eating live tadpoles has a medicinal role for skin diseases and, thus, they contract sparganosis. Accordingly, the route of plerocercoid infection for humans in Henan Province differs from that in southern China. Because this disease is rare in central and northern China, sparganosis is often neglected and misdiagnosed. Our survey showed that in Henan Province, 11.93% of tadpoles are infected with plerocercoids and 3.53% of cyclops are infected with procercoids. Therefore, eating live tadpoles poses a high risk for plerocercoid infection and must be discouraged. In addition, drinking raw water containing cyclops also poses a slight risk for sparganosis.

Inflammation in digits of unmarked and toe-tipped wild hylids

Context The use of toe-tipping (removal of the toe pad) in mark–recapture studies of anurans is controversial because of the unknown risk of infection and potential impact on animal survival. Aims We compared the inflammation rates of digits in unmarked and toe-tipped wild hylid frogs (Litoria genimaculata, L. nannotis, L. rheocola and Nyctimystes dayi) to determine if marked amphibians present with a greater proportion of affected digits than unmarked animals. Methods We examined marked and unmarked frogs captured on streams in northern Queensland over a 12-month period for phalangeal inflammation. Key results Of 777 unmarked frogs, 0.4% presented with digits showing inflammation because of natural causes. After toe-tipping, 59.1% of the frogs were recaptured within a mean of 97 days ± standard deviation of 102 days, with macroscopic inflammation observed in 1.3% of marked animals. The proportion of marked frogs with inflamed, toe-tipped digits did not differ significantly from that of inflamed digits in unmarked frogs (Fisher exact test P = 0.085) but the odds ratio (3.417, 0.851–13.730) indicated a greater likelihood of inflamed digits occurring in toe-tipped than unmarked frogs. Conclusions Although the risk of inflammation increased 3-fold in marked hylids, the low proportion of frogs affected (1.3%) indicated the potential pathological risk of toe-tipping did not outweigh the benefits of using the marking method. Implications This study suggests that toe-tipping may be an appropriate and relatively safe marking method in stream-dwelling hylid populations.

Use of femur bone density to segregate wild from farmed Dybowski's frog ( Rana dybowskii)

Wildlife has been utilized by humans throughout history and demand continues to grow today. Farming of wildlife can supplement the supply of wild-harvested wildlife products and, in theory, can reduce pressure on free-ranging populations. However, poached wildlife products frequently enter legal markets where they are fraudulently sold as farmed wildlife products. To effectively close this illegal trade in wild-captured wildlife, there is a need to discriminate wild products from farmed products. Because of the strong market demand for wild-captured frog meat and the resulting strong downward pressure on wild populations, we undertook research to develop a method to discriminate wild from farmed Dybowski's frog ( Rana dybowskii) based on femur bone density. We measured femur bone density ( D f ) as the ratio of bone mass to bone volume. D f of wild frogs revealed a slightly increasing linear trend with increasing age ( R 2 = 0.214 in males and R 2 = 0.111 in females, p = 0.000). Wild males and wild females of age classes from 2 to ≥5 years had similar D f values. In contrast, 2-year-old farmed frogs showed significantly higher D f values ( p = 0.000) among males (mean D f = 0.623 ± 0.011 g/ml, n = 32) than females (mean D f = 0.558 ± 0.011 g/ml, n = 27). For both sexes, D f of wild frogs was significantly higher than that of farmed frogs ( p = 0.000). Among males, 87.5% (28 of 32 individuals) of farmed frogs were correctly identified as farmed frogs and 86.3% (69 of 80 individuals) of wild frogs were correctly identified as wild frogs. These results suggest that femur bone density is one reliable tool for discriminating between wild and farmed Dybowski's frog. This study also highlights a novel strategy with explicit forensic potential to discriminate wild from captive bred wildlife species.

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.

Determination and occurrence of retinoic acids and their 4‐oxo metabolites in Liaodong Bay, China, and its adjacent rivers

Retinoic acids (RAs) and their metabolites play an important role in abnormal morphological development and are speculated to be a possible cause for the increased rates of deformities in wild frog populations. In the current study, a method using ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry was developed for simultaneously analyzing all-trans-RA (at-RA), 13-cis-RA (13c-RA), 9-cis-RA (9c-RA), and their 4-oxo metabolites, all-trans-4-oxo-RA (at-4-oxo-RA), 13-cis-4-oxo-RA (13c-4-oxo-RA), and 9-cis-4-oxo-RA (9c-4-oxo-RA) in wastewaters and surface waters. Method detection limits were matrix dependent, ranging from 0.02 to 0.37 ng/L. The method was used to investigate the occurrence of RAs and 4-oxo-RAs in Liaodong Bay and its adjacent rivers. Of these six retinoids, at-RA, 13c-RA, at-4-oxo-RA, and 13c-4-oxo-RA were detected in river waters at detection frequencies of 100%, 92%, 48.6%, and 21.6%, and concentrations of 0.05 to 1.23 ng/L, less than 0.03 to 0.41 ng/L, less than 0.02 to 1.00 ng/L, and less than 0.06 to 0.81 ng/L, respectively. Retinoic acids were detected for the first time in the aquatic environment and were found to be more persistent than 4-oxo-RAs. The hazard quotient for mortality of frog embryos caused by induction by retinoids detected in the current study was then estimated, and the value was calculated to be 0.09. No retinoid was detected in seawaters.

Pathology of cutaneous trombidiosis caused by larval trombiculid mites in a wild Lesueur's tree frog (Litoria wilcoxii)

Cutaneous trombidiosis caused by larvae of trombiculid mites (Vercammenia gloriosa and V. zweifelorum) in the skin of a wild tree frog, Litoria wilcoxii, in northern Queensland, Australia manifested as small, domed vesicular lesions on the dorsal and lateral surfaces posterior to the eyes. The lesions contained small, orange trombiculid mites, with a surrounding minimal inflammatory reaction. The general health and behaviour of the frog appeared unaffected. Provisional diagnosis of cutaneous trombidiosis can be made from its distinctive clinical appearance and confirmed by biopsy with direct microscopic examination of mites. This case report represents a new host record.

Assessing the long‐term impact of Ranavirus infection in wild common frog populations

AbstractAmphibians are declining worldwide, and one cause of this is infectious disease emergence. Mass mortalities caused by a virus or a group of viruses belonging to the genus Ranavirus have occurred in wild common frogs Rana temporaria in England since the 1980s, and ranaviral disease is widespread in amphibians in North America and Canada, where it can also cause mass die‐offs. Although there have been numerous reports of Ranavirus‐associated mass mortality events, no study has yet evaluated the long‐term impacts of this disease. This study follows up archived records of English common frog mortalities likely caused by Ranavirus. There is a preliminary indication that common frog populations can respond differently to the emergence of disease: emergence may be transient, catastrophic, or persistent with recurrent mortality events. We subsequently focused on populations that had recurring mortality events (n=18), and we report median declines of 81% in the number of adult frogs in these populations from 1996 to 2008. Comparable uninfected populations (n=16) showed no change in population size over the same time period. Regressions show that larger frog populations may be more likely to experience larger declines than smaller populations, and linear models show that percentage population size change is significantly correlated with disease status, but that habitat age (a possible proxy for environmental quality) has no significant effect on population size change. Our results provide the first evidence of long‐term localized population declines of an amphibian species which appear to be best explained by the presence of Ranavirus infection.

Minimising exposure of amphibians to pathogens during field studies

Many of the recent global amphibian mass mortalities, declines and extinctions have been attributed to the emerging infectious disease chytridiomycosis. There have been mass mortalities due to ranaviral disease but no major declines or extinctions. Controlling the transmission and spread of disease is of utmost importance, especially where there is the potential for human involvement. We have reviewed current hygiene guidelines for working with wild frogs, identified potential flaws and recommended those most suitable and effective for the field environment. Our within-site hygiene measures aim to reduce the risk of transmission among individuals. These measures encompass the capture, handling and holding of amphibians, skin disinfection before and after invasive procedures, marking frogs, sealing open wounds and treatment of accessory equipment. Our between-site hygiene measures aim to mitigate the risk of pathogen spread among populations. We have designed a risk calculator to help simplify and standardise the decision-making process for determining the level of risk and appropriate risk mitigation strategies to reduce the risk of increasing pathogen spread above background levels. Calculation of an overall risk score for pathogen spread takes into account the prior activity of field workers, the proposed activity, remoteness of the site, presence of known pathogens and the consequences of increased pathogen spread for amphibians in a given area.

Peering into molecular mechanisms of action with frogSCOPE

Exposure of critical life stages to harmful chemicals at low, environmentally-relevant concentrations can alter how hormones function, and change metabolic pathways or developmental processes that impact reproduction, behavior, or susceptibility to disease later in life. These alterations can be captured through evaluation of changes to transcriptomes, proteomes, and metabolomes occurring at those critical life stages thereby enabling more effective and earlier identification of mechanism of action, individual susceptibilities and adaptation, and prediction of detrimental sublethal effects. Amphibians are “wet canaries in the coalmine” as indicators for environmental health. There are more than 6000 species living in a variety of ecological niches worldwide yet limited ‘omics resources and approaches exist. To provide for a means of addressing this challenge, frogSCOPE (frog Sentinel species Comparative “ Omics” for the Environment) combines transcriptomics, proteomics, and metabolomics together to form the foundation for the identification of biological response indicators of harmful effects on a species of wild frog ( Rana catesbeiana) at a sensitive tadpole stage. Various exposure and sampling methodologies are possible including standard in vivo exposures, tail fin biopsies, and the C-fin assay. frogSCOPE establishes methodological and analytical approaches applicable to wildlife by using a uniquely-designed frog cDNA array developed to accommodate cross-species hybridization and quantitative real-time polymerase chain reaction (QPCR) assays on poorly genetically-characterized wildlife species. Combination with proteomics (isobaric tags for relative and absolute protein quantitation; iTRAQ) and metabolomics (mass spectrometry) enable the generation of molecular fingerprints to identify mechanisms of action in a more comprehensive fashion to better define suitable indicators of deleterious biological outcomes to wildlife.