Genetic Variants Underlying Plasticity in Natural Populations of Spadefoot Toads: Environmental Assessment versus Phenotypic Response.

Many organisms facultatively produce different phenotypes depending on their environment, yet relatively little is known about the genetic bases of such plasticity in natural populations. In this study, we describe the genetic variation underlying an extreme form of plasticity--resource polyphenism--in Mexican spadefoot toad tadpoles, Spea multiplicata. Depending on their environment, these tadpoles develop into one of two drastically different forms: a carnivore morph or an omnivore morph. We collected both morphs from two ponds that differed in which morph had an adaptive advantage and performed genome-wide association studies of phenotype (carnivore vs. omnivore) and adaptive plasticity (adaptive vs. maladaptive environmental assessment). We identified four quantitative trait loci associated with phenotype and nine with adaptive plasticity, two of which exhibited signatures of minor allele dominance and two of which (one phenotype locus and one adaptive plasticity locus) did not occur as minor allele homozygotes. Investigations into the genetics of plastic traits in natural populations promise to provide novel insights into how such complex, adaptive traits arise and evolve.

Systematic assessment of the brown tree frog (Anura: Pelodryadidae: Litoria ewingii) reveals two endemic species in South Australia.

The brown tree frog (Litoria ewingii) is a relatively widespread, commonly encountered pelodryadid frog from south-eastern Australia, known for its characteristic whistling call. The distribution of Litoria ewingii spans over more than 350,000 km2, encompassing a range of moist temperate habitats, and is fragmented by well-known biogeographic barriers. A preliminary analysis of mitochondrial DNA sequences revealed evidence for deep phylogenetic structure between some of these fragmented populations. In this study, we sought to re-evaluate the systematics and taxonomy of Litoria ewingii sensu lato by analysing variation in nuclear and mitochondrial DNA, adult morphology and male advertisement calls throughout the species range. Our analyses reveal two additional, deeply divergent and allopatric lineages in South Australia. We herein re-describe Litoria ewingii from Tasmania, southern New South Wales, Victoria and south-eastern South Australia, resurrect the name Litoria calliscelis for a species occurring in the Mount Lofty Ranges and Fleurieu Peninsula in South Australia, and describe a new species, Litoria sibilus sp. nov., endemic to Kangaroo Island.

Reclamation history and land use types across multiple spatial scales shape anuran communities in the coastal land reclamation region.

Land reclamation is a widely adopted method for managing land shortage and promoting coastal economic development globally. However, its impacts on biodiversity vary based on distinct reclamation histories and land use management strategies in different regions. This study aims to examine the effects of reclamation history and land use types at different spatial scales on anuran communities in coastal reclaimed land, which are an important taxon in the coastal ecosystem. We used visual and acoustic encounter methods to survey anurans in 2016 and 2017 across 20 1-km radius coastal land reclamation landscapes with different reclamation histories (10, 20, and 60 y after reclamation) in Nanhui Dongtan of Shanghai, an important coastal land reclamation region along the Yangtze River Estuary. Landscape variables (farmlands, woodlands, and impermeable surface covers, and the landscape Shannon diversity index) at four different spatial scales (250 m, 500 m, 750 m and 1000 m) and water salinity in each landscape were measured. Our findings reveal differences in anuran communities between study sites with 10, 20, and 60 years of reclamation history. Abundances of the ornamented pygmy frog (Microhyla fissipes) and Beijing gold-striped pond frog (Pelophylax plancyi) in landscapes with a 10-year reclamation history were significantly lower compared to those with histories of 20 and 60 years. Zhoushan toad (Bufo gargarizans) abundance was significantly negatively related to farmland cover at the 1000 m scale and impermeable surface cover at the 250 m scale; Hong Kong rice-paddy frog (Fejervarya multistriata) abundance was significantly positively related to farmland cover at the 1000 m scale; ornamented pygmy frog abundance was positively related to farmland cover at the 1000 m scale; and Beijing gold-striped pond frog abundance was significantly positively and negatively related to the landscape Shannon diversity index at the 1000 m scale and to water salinity, respectively. Amphibians quickly migrated and colonized coastal reclaimed land from older natural lands. However, two anuran species with specific habitat requirements tended to avoid areas with shorter reclamation histories. The single-species models revealed different responses to various land uses at the various scales, which indicated that land use management was important to amphibian conservation in coastal reclamation regions.

Assessment of toxicity, genotoxicity and oxidative stress in Fejervarya limnocharis exposed to tributyltin.

Tributyltin (TBT) is widely used in various commercial applications due to its biocidal properties. Toxicological and genotoxicological data on TBT exposure to amphibians is insufficient. Our study aimed to determine the acute toxicity and genotoxic potential of TBT in Fejervarya limnocharis tadpoles. Furthermore, oxidative stress was also investigated in TBT-treated tadpoles. Tadpoles of Gosner stage (26-30) were screened and subjected to increasing concentrations of TBT (0, 3, 7, 11, 15, 19, 23 µg/L) for determining the LC50 values for 24 h, 48 h, 72 h, and 96 h. LC50 values of TBT for 24 h, 48 h, 72 h, and 96 h were found to be 19.45, 15.07, 13.12, and 11.84 µg/L respectively. Based on the 96 h LC50 value (11.84 µg/L), tadpoles were exposed to different sub-lethal concentrations of TBT for the evaluation of its genotoxic potential and effects on oxidative balance. The role of TBT on survivability, growth, and time to metamorphosis was also assessed. TBT exposure significantly altered the life history traits measured, increased mortality, and delayed the time taken to metamorphosis. Results indicated significant induction of micronucleus (MN, p < 0.001) and other erythrocytic nuclear aberrations (ENA, p < 0.01) in the TBT-treated groups. Significant alterations in comet parameters and oxidative balance were also observed in the treated groups. The present study findings might add to the cause of the gradual population decline seen in the amphibians. This study also demonstrates the alteration of the life-history traits, oxidative balance, and DNA damage upon TBT exposure which can have long-term consequences for the anuran amphibian F. limnocharis.

Benefits and costs of female and male care in amphibians: a meta-analytical approach.

The origin of parental care is a central question in evolutionary biology, and understating the evolution of this behaviour requires quantifying benefits and costs. To address this subject, we conducted a meta-analysis on amphibians, a group in which parental care has evolved multiple times. We found that both male and female parents increase egg survival, regardless of whether the breeding site is concealed or exposed. Parental care also increases survival and growth of tadpoles and juveniles, independent of the caring sex. However, parental care reduces parental body condition, particularly when parents remain stationary near the offspring. Females tend to experience higher reproductive costs, but sample size is restricted to few species. In some frog species, paternal care increases male reproductive success because females prefer caring males. The benefits of parental care in amphibians resembles those reported for arthropods but differ from fish, in which parental care does not improve offspring survival. Moreover, the decrease in body condition, which is not found in fish, is influenced by the form of parental care, suggesting a trade-off between caring and foraging, as already reported for certain arthropods. Finally, the reproductive costs of parental care for both sexes remain unexplored and deserve further research.

Structural, Functional, and Mutational Studies of a Potent Subtilisin Inhibitor from Budgett's Frog, Lepidobatrachus laevis.

Subtilases play a significant role in microbial pathogen infections by degrading the host proteins. Subtilisin inhibitors are crucial in fighting against these harmful microorganisms. LL-TIL, from skin secretions of Lepidobatrachus laevis, is a cysteine-rich peptide belonging to the I8 family of inhibitors. Protease inhibitory assays demonstrated that LL-TIL acts as a slow-tight binding inhibitor of subtilisin Carlsberg and proteinase K with inhibition constants of 91 pM and 2.4 nM, respectively. The solution structures of LL-TIL and a mutant peptide reveal that they adopt a typical TIL-type fold with a canonical conformation of a reactive site loop (RSL). The structure of the LL-TIL-subtilisin complex and molecular dynamics (MD) simulations provided an in-depth view of the structural basis of inhibition. NMR relaxation data and molecular dynamics simulations indicated a rigid conformation of RSL, which does not alter significantly upon subtilisin binding. The energy calculation for subtilisin inhibition predicted Ile31 as the highest contributor to the binding energy, which was confirmed experimentally by site-directed mutagenesis. A chimeric mutant of LL-TIL broadened the inhibitory profile and attenuated subtilisin inhibition by 2 orders of magnitude. These results provide a template to engineer more specific and potent TIL-type subtilisin inhibitors.

Assessment of predation risk through conspecific cues by anuran larvae.

Accurate assessment of predation risk is critical for prey survival during predator-prey interactions. Prey can assess predation risk by the presence of cues dropped by predators themselves, but they can also gather information about risk level through cues released by other prey, avoiding the hazard of being in close proximity to predators. In this study, we examine the ability of anuran larvae (Pelobates cultripes) to detect predation risk indirectly when they are in contact with conspecifics that have been recently exposed to chemical stimuli from natural predators (larvae of aquatic beetles). In a first experiment, we confirmed that larvae exposed to predator cues exhibited innate defensive behavior, indicating that they perceived the risk of predation and, thus, could potentially act as risk indicators for naïve conspecifics. In a second experiment, we observed that unexposed larvae paired with a startled conspecific adjusted their antipredator behavior, presumably by mirroring conspecifics' behavior and/or using chemical cues from their partners as a risk information source. This cognitive ability of tadpoles to assess predation risk through conspecific cues might play an important role in their interaction with predators, facilitating the early detection of potential threats to elicit appropriate antipredator responses and increase the chances of survival.

Toxicity assessment of polyethylene microplastics in combination with a mix of emerging pollutants on Physalaemus cuvieri tadpoles.

Studies in recent years have shown that aquatic pollution by microplastics (MPs) can be considered to pose additional stress to amphibian populations. However, our knowledge of how MPs affect amphibians is very rudimentary, and even more limited is our understanding of their effects in combination with other emerging pollutants. Thus, we aimed to evaluate the possible toxicity of polyethylene MPs (PE-MPs) (alone or in combination with a mix of pollutants) on the health of Physalaemus cuvieri tadpoles. After 30 days of exposure, multiple biomarkers were measured, including morphological, biometric, and developmental indices, behavioral parameters, mutagenicity, cytotoxicity, antioxidant and cholinesterase responses, as well as the uptake and accumulation of PE-MPs in animals. Based on the results, there was no significant change in any of the parameters measured in tadpoles exposed to treatments, but induced stress was observed in tadpoles exposed to PE-MPs combined with the mixture of pollutants, reflecting significant changes in physiological and biochemical responses. Through principal component analysis (PCA) and integrated biomarker response (IBR) assessment, effects induced by pollutants in each test group were distinguished, confirming that the exposure of P. cuvieri tadpoles to the PE-MPs in combination with a mix of emerging pollutants induces an enhanced stress response, although the uptake and accumulation of PE-MPs in these animals was reduced. Thus, our study provides new insight into the danger to amphibians of MPs coexisting with other pollutants in aquatic environments.

Ontogeny of risk assessment and escape-hatching performance by red-eyed treefrog embryos in two threat contexts.

Arboreal embryos of red-eyed treefrogs, Agalychnis callidryas, hatch prematurely in response to hypoxia when flooded and to mechanosensory cues in snake attacks, but hatching later improves tadpole survival. We studied ontogenetic changes in risk assessment and hatching performance of embryos in response to flooding and physical disturbance. We hypothesized that risk assessment decreases as hatchling survival improves and hatching performance increases as embryos develop. Because snakes eat faster than embryos asphyxiate, we hypothesized that embryos decide to hatch sooner and hatch faster in response to mechanosensory cues. We video-recorded individual embryos hatching in response to each cue type, then compared the incidence and timing of a series of events and behaviors from cue onset to complete hatching across ages and stimuli. Latency from cue to hatching decreased developmentally in both contexts and was shorter with mechanosensory cues, but the elements contributing to those changes differed. Hypoxia assessment involved position changes, which decreased developmentally along with assessment time. Mechanosensory cue assessment occurred more rapidly, without movement, and decreased with age. The first stages of hatching, membrane rupture and head emergence, were surprisingly age independent but faster with mechanosensory cues, congruent with greater effort under more immediate risk. In contrast, body emergence and compression showed ontogenetic improvement consistent with morphological constraints but no cue effect. Both appropriate timing and effective performance of hatching are necessary for continued development. Different stages of the process vary with development and environmental context, suggesting combinations of adaptive context- and stage-dependent behavior, cue-related constraints on information acquisition, and ontogenetic constraints on elements of performance.

First molecular assessment on Cosmocerca spp. from Brazilian anurans and description of a new species of Cosmocerca (Ascaridomorpha: Cosmocercoidea) from the white-spotted humming frog Chiasmocleis albopunctata (Boettger, 1885) (Anura: Microhylidae).

Cosmocerca spp. are common nematode parasites of amphibians. We provide herein molecular data for two species of Cosmocerca and describe a new species, Cosmocerca albopunctata n. sp., using light microscopy and molecular data (cytochrome c oxidase subunit 1 - COI mtDNA). Cosmocerca albopunctata n. sp. can be easily distinguished from other congeneric species by the combination of characteristics such as body size, length of spicules and gubernaculum, and the arrangements and number of caudal papillae (7 + 1:1 + 1:6). The phylogenetic results based on the partial COI mtDNA sequences clustered the new species in a monophyletic clade along with the other sequences of Cosmocerca spp. Therefore, our results contribute to the knowledge about the species diversity and genetic data for Cosmocerca spp. in the Neotropical region.

Effects of waterborne Pb/Cu mixture on Chinese toad, Bufo gargarizans tadpoles: morphological, histological, and intestinal microbiota assessment.

Coexistence of heavy metals in aquatic environments exert complex effects on amphibians. Here, the adverse effects of Pb (0.14 µM) combined with Cu at concentrations of 0, 0.25, and 1.0 µM were investigated in Bufo gargarizans tadpoles. Tadpoles were chronically exposed from Gosner stage (Gs) 26 to Gs 38, and morphology of tadpoles as well as intestinal histology and bacterial community were assessed. Our results indicated that Pb+Cu1.0 exposure induced significant retardation of somatic mass, total length, intestine mass, and intestine length as well as intestinal histological alterations. Pb+Cu0.25 and Pb+Cu1.0 exposure were associated with the loss of gut bacterial diversity. Proteobacteria and Bacteroidetes were two dominant phyla in tadpoles independently of heavy metal exposure, but the abundance of Proteobacteria increased significantly in Pb+Cu1.0 group and Bacteroidetes decreased significantly in all treatment groups. Furthermore, functional prediction indicated that metabolic disorders were associated with Pb+Cu0.25 and Pb+Cu1.0 exposure. Overall, relative limited shifts in intestinal bacterial diversity, composition, and functionality caused by Pb+Cu0 exposure, while coexistence of Pb and Cu induced gut dysbiosis and might further cause disturbance of metabolic homeostasis. The findings of this study provide insights into the effects of Pb and Cu coexistence on the health of amphibians.

Risk assessment for the native anurans from an alien invasive species, American bullfrogs (Lithobates catesbeianus), in South Korea.

The invasive species are of global concern, and the Invasive American Bullfrog (IAB; Lithobates catesbeianus) is one of the worst invasive amphibian species worldwide. Like other countries, South Korea is also facing challenges from IAB. Although many studies indicated impacts of IAB on native anurans in Korea, the actual risk at the specific level is yet to evaluate. Considering the putative invasiveness of IAB, it is hypothesized that any species with the possibility of physical contact or habitat sharing with them, will have a potential risk. Thus, we estimated and observed their home range, preferred habitats, morphology, behavior, and ecology. Then, comparing with existing knowledge, we assessed risks to the native anurans. We found a home range of 3474.2 ± 5872.5 m2 and identified three types of habitats for IAB. The analyses showed at least 84% of native anurans (frogs and toads) were at moderate to extreme risks, which included all frogs but only 33% of toads. Finally, we recommended immediate actions to conserve the native anurans based on our results. As this study is the first initiative to assess the specific risk level from the invasiveness of L. catesbeianus, it will help the managers to set conservation priorities and strategies.

Frog embryos use multiple levels of temporal pattern in risk assessment for vibration-cued escape hatching.

Stereotyped signals can be a fast, effective means of communicating danger, but animals assessing predation risk must often use more variable incidental cues. Red eyed-treefrog, Agalychnis callidryas, embryos hatch prematurely to escape from egg predators, cued by vibrations in attacks, but benign rain generates vibrations with overlapping properties. Facing high false-alarm costs, embryos use multiple vibration properties to inform hatching, including temporal pattern elements such as pulse durations and inter-pulse intervals. However, measures of snake and rain vibration as simple pulse-interval patterns are a poor match to embryo behavior. We used vibration playbacks to assess if embryos use a second level of temporal pattern, long gaps within a rhythmic pattern, as indicators of risks. Long vibration-free periods are common during snake attacks but absent from hard rain. Long gaps after a few initial vibrations increase the hatching response to a subsequent vibration series. Moreover, vibration patterns as short as three pulses, separated by long periods of silence, can induce as much hatching as rhythmic pulse series with five times more vibration. Embryos can retain information that increases hatching over at least 45 s of silence. This work highlights that embryo behavior is contextually modulated in complex ways. Identical vibration pulses, pulse groups, and periods of silence can be treated as risk cues in some contexts and not in others. Embryos employ a multi-faceted decision-making process to effectively distinguish between risk cues and benign stimuli.

An in vivo study of the toxic effects of triclosan on Xenopus laevis (Daudin, 1802) frog: Assessment of viability, tissue damage and mitochondrial dysfunction.

The present study describes the in vivo effect of triclosan on the frog Xenopus laevis (Daudin, 1802). We have found a dose-dependence of the effect of triclosan on the survival of frogs. At a dose of 2 mg/L, the death of frogs was observed already on the 4th day of the experiment, while at a concentration of 0.5 mg/L, the frogs remained viable for 11 days. Triclosan caused damage to the liver tissue, which was expressed in an increase in the area of hemorrhage and the number of melanomacrophage centers. 0.5 mg/L of this agent did not affect the number of frog red blood cells, but reduced their osmotic resistance. Keeping animals in water containing triclosan (0.5 mg/L for 96 h) led to the suppression of the state 3 respiration rate of frog liver mitochondria. This effect was accompanied by suppression of the combined activity of complexes II and III of the mitochondrial respiratory chain. In parallel with this, we observed a reduction in the Ca2+ retention capacity of frog liver mitochondria, indicating a decrease in the resistance of organelles to mitochondrial permeability transition pore opening. The paper discusses the effects of triclosan on aquatic organisms.

Ecotoxicity assessment of triphenyl phosphate (TPhP) exposure in Hoplobatrachus rugulosus tadpoles.

Triphenyl phosphate (TPhP), a widely used aromatic organophosphate flame retardant, is known to accumulate in organisms through water, air, and soil, consequently, causing toxicity. This study is the first to evaluate the acute and sub-chronic toxicities of TPhP to amphibians. In the acute toxicity analysis, the 96-h median lethal concentration (LC50) for GS35 Hoplobatrachus rugulosus tadpoles was 2.893 mg/L, and the 10% effect concentration (EC10) was 289 µg/L. After two weeks of exposure to low TPhP concentrations, the survival and metamorphosis rates of H. rugulosus tadpoles decreased, and the metamorphosis time was prolonged as the TPhP concentration increased. The threshold concentration that affected tadpole survival and metamorphosis time was 50 µg/L and 100 µg/L, respectively. No significant differences were observed in the condition factor and hepatic somatic index of the tadpole after metamorphosis; however, tadpole body mass and TPhP concentration were negatively correlated. Further, TPhP inhibited the expressions of Cu-Zn sod and cat, thereby reducing the activities of superoxide dismutase and catalase in the tadpole liver. The threshold for affecting gene expression and enzymatic activity was 100 µg/L. These findings provide significant insights on the stress ecology of aquatic organisms.

An assessment of the taxonomic validity of three species of marsupial frogs (Anura: Hemiphractidae: Gastrotheca) from the Yungas of Bolivia based on external morphology and cranial osteology.

The genus Gastrotheca (Anura: Hemiphractidae) is a group of marsupial frogs particularly diverse in Andean regions. Several taxonomic studies of this genus have been conducted in the humid cloud forestsor Yungasof the Andean eastern slopes of central Bolivia (departments of Cochabamba and Santa Cruz). Yet, the distinction among three species that occur sympatrically in these forests, G. lauzuricae (proposed as a junior synonym of G. coeruleomaculatus in 2015), G. piperata, and G. splendens, remains unclear since the morphological characters that purportedly support their differentiation are variable and partly shared among them. We have carried out external morphological studies, including multivariate morphometric analyses, to assess how they support the taxonomic status of these three species. We also evaluated characters of the cranial osteology of a sample of six individuals using micro CT-scanning. Principal component and linear discriminant analyses resulted in a great overlap among the putative species. Cranial osteological comparisons did not reveal highly significant differences among them, but suggested that different degrees of hyperossification could be related to the developmental state of individuals. Our results indicate that most morphological and osteological reported differences between the three species likely represent intraspecific variation. Thus, we propose that the three nominal species belong to a single biological entity, for which the name Gastrotheca splendens (Schmidt, 1857) has priority. We also restrict the name Gastrotheca coeruleomaculatus (Werner, 1899) to externally similar congeneric populations from the Yungas forests of department of La Paz, but highlighting the need of a detailed evaluation of their taxonomic identity.

Assessment of exposure to ionizing radiation in Chernobyl tree frogs (Hyla orientalis).

Ionizing radiation can damage organic molecules, causing detrimental effects on human and wildlife health. The accident at the Chernobyl nuclear power plant (1986) represents the largest release of radioactive material to the environment. An accurate estimation of the current exposure to radiation in wildlife, often reduced to ambient dose rate assessments, is crucial to understand the long-term impact of radiation on living organisms. Here, we present an evaluation of the sources and variation of current exposure to radiation in breeding Eastern tree frogs (Hyla orientalis) males living in the Chernobyl Exclusion Zone. Total absorbed dose rates in H. orientalis were highly variable, although generally below widely used thresholds considered harmful for animal health. Internal exposure was the main source of absorbed dose rate (81% on average), with 90Sr being the main contributor (78% of total dose rate, on average). These results highlight the importance of assessing both internal and external exposure levels in order to perform a robust evaluation of the exposure to radiation in wildlife. Further studies incorporating life-history, ecological, and evolutionary traits are needed to fully evaluate the effects that these exposure levels can have in amphibians and other taxa inhabiting radio-contaminated environments.

Red List assessment of amphibian species of Ecuador: A multidimensional approach for their conservation.

Ecuador is one of the most biodiverse countries in the world, but faces severe pressures and threats to its natural ecosystems. Numerous species have declined and require to be objectively evaluated and quantified, as a step towards the development of conservation strategies. Herein, we present an updated National Red List Assessment for amphibian species of Ecuador, with one of the most detailed and complete coverages for any Ecuadorian taxonomic group to date. Based on standardized methodologies that integrate taxonomic work, spatial analyses, and ecological niche modeling, we assessed the extinction risk and identified the main threats for all Ecuadorian native amphibians (635 species), using the IUCN Red List Categories and Criteria. Our evaluation reveals that 57% (363 species) are categorized as Threatened, 12% (78 species) as Near Threatened, 4% (26 species) as Data Deficient, and 27% (168 species) as Least Concern. Our assessment almost doubles the number of threatened species in comparison with previous evaluations. In addition to habitat loss, the expansion of the agricultural/cattle raising frontier and other anthropogenic threats (roads, human settlements, and mining/oil activities) amplify the incidence of other pressures as relevant predictors of ecological integrity. Potential synergic effects with climate change and emergent diseases (apparently responsible for the sudden declines), had particular importance amongst the threats sustained by Ecuadorian amphibians. Most threatened species are distributed in montane forests and paramo habitats of the Andes, with nearly 10% of them occurring outside the National System of Protected Areas of the Ecuadorian government. Based on our results, we recommend the following actions: (i) An increase of the National System of Protected Areas to include threatened species. (ii) Supporting the ex/in-situ conservation programs to protect species considered like Critically Endangered and Endangered. (iii) Focalizing research efforts towards the description of new species, as well as species currently categorized as Data Deficient (DD) that may turn out to be threatened. The implementation of the described actions is challenging, but urgent, given the current conservation crisis faced by amphibians.

Pathologic Lesions of the Budgett Frog (Lepidobatrachus laevis), an Emerging Laboratory Animal Model.

Lepidobatrachus laevis, commonly called the Budgett frog, is a member of the horned frog family (Ceratophryidae), which has become increasingly popular among amphibian hobbyists. L. laevis is also used in biologic research on embryonic development, providing a novel model species for the study of organogenesis, regeneration, evolution, and biologic scaling. However, little scientific literature details disease processes or histologic lesions in this species. Our objective was to describe spontaneous pathologic lesions in L. laevis to identify disease phenotypes. We performed a retrospective analysis of 14 captive L. laevis frogs (wild-caught and captive-bred), necropsied at the NC State University College of Veterinary Medicine between 2008 and 2018. The majority of frogs exhibited renal changes, including varying combinations of tubular epithelial binucleation, karyomegaly, and cytoplasmic vacuolation; polycystic kidney disease; and renal carcinoma. Many of the renal changes are reminiscent of a condition described in Japanese (Bufo japonicus) and Chinese (Bufo raddei) toad hybrids that progresses from tubular epithelial atypia and tubular dilation to polycystic kidney disease to renal carcinoma. A second common finding was variably sized, randomly distributed bile duct clusters (biliary proliferation). Other noteworthy findings included regional or generalized edema, intestinal adenocarcinoma, aspiration pneumonia, and parasitism. This retrospective analysis is the first description of histologic lesions identified in captive L. laevis populations, providing new insight into spontaneous disease processes occurring in this species for use in disease diagnosis and clinical management.

Acute toxicity assessment of arsenic, chromium and almix 20WP in Euphlyctis cyanophlyctis tadpoles.

Heavy metals and herbicide are gaining serious environmental concern in aquatic toxicology due to its adverse effects on aquatic organisms especially amphibians. Accordingly, present study first time evaluated the acute toxicity of two heavy metals [arsenic (As3+) and chromium (Cr6+)] and a herbicide (Almix) to Indian skittering frog tadpole, Euphlyctis cyanophlyctis. The LC50 values of As, Cr and Almix for 24, 48, 72 and 96 h were 73.58, 56.31, 43.58 and 32.58 mg L-1; 326.68, 224.31, 171.92 and 141.99 mg L-1; and 1297.85, 1148.22, 1033.62 and 955.17 mg L-1, respectively. It also revealed the concentration- and time-dependent increased mortality rate under these toxicants. The safety concentrations (SC) of As, Cr and Almix to tadpoles were 3.26, 14.20 and 95.52 mg L-1, respectively. The findings disclosed that As is highly toxic to E. cyanophlyctis than Cr and Almix. Alkaline phosphatase (ALP) activity showed varied responses to exposed chemicals. In particularly, ALP activity reduced significantly for Cr treatment. Glutathione S-transferase (GST) activity in E. cyanophlyctis was significantly inhibited by As treatment (p < 0.05); however, GST activity was remain unchanged for Cr and Almix (p > 0.05). The As toxicity correlates positively with GST inhibition (r = 0.779, p < 0.01); contrarily, Cr and Almix revealed negative correlation with GST induction (r = -0.461 and -0.19, respectively; p > 0.05). This result indicated that GST play a crucial role for regulating the tadpole mortality and intoxication by As, Cr and Almix. Overall, our findings demonstrate the different levels of toxic sensitivity (adverse effects) under different toxicants on E. cyanophlyctis tadpoles. Finally, the present findings could be used as baseline information of toxicosis for metalloid, heavy metal and herbicide exposures in wild frog populations.