A hundred-year-old mystery-the reproductive mode and larval morphology of the enigmatic frog genus Allophryne (Amphibia; Anura; Allophrynidae).

Frogs of the Allophrynidae are an enigmatic family from South America. To date, published information is lacking regarding this group's reproductive biology and larval morphology. Here, we provide the first detailed description of the reproductive mode, developmental mode, and tadpole morphology for Allophryne ruthveni. We developed a captive breeding and rearing protocol for this species and then conducted a series of observations to describe aspects of its reproductive biology. In captivity, this species exhibits aquatic oviposition, where single eggs are laid ungrouped within a simple jelly capsule and are scattered free in the water column before sinking to develop on benthic substrates. We did not observe parental care nor any parental interactions with eggs post-fertilization. Tadpoles are characterized by an oval body, anteroventral oral disc, a labial tooth row formula of 2(2)/3, and a dextral vent tube. The buccopharyngeal cavity is marked by the presence of two pairs of infralabial papilla and four lingual papillae. Cranial morphology is characterized by the presence of the commissura quadratoorbital. This species possesses an additional slip of the m. rectus cervicis and of the m. levator arcuum branchialium III. We discuss our results in comparison with glassfrogs (Centrolenidae).

Evaluating potential developmental toxicity of perfluoroalkyl and polyfluoroalkyl substances in Xenopus laevis embryos and larvae.

As part of the US Environmental Protection Agency's perfluoroalkyl and polyfluoroalkyl substances (PFAS) Action Plan, the agency is committed to increasing our understanding of the potential ecological effects of PFAS. The objective of these studies was to examine the developmental toxicity of PFAS using the laboratory model amphibian species Xenopus laevis. We had two primary aims: (1) to understand the developmental toxicity of a structurally diverse set of PFAS compounds in developing embryos and (2) to characterize the potential impacts of perfluorooctanesulfonic acid (PFOS), perfluorohexanesulfonic acid (PFHxS), perfluorooctanoic acid (PFOA), and hexafluoropropylene oxide-dimer acid (HFPO-DA a.k.a. GenX), on growth and thyroid hormone-controlled metamorphosis. We employed a combination of static renewal and flow-through exposure designs. Embryos were exposed to 17 structurally diverse PFAS starting at the midblastula stage through the completion of organogenesis (96 h). To investigate impacts on PFOS, PFOA, PFHxS, and HFPO-DA on development and metamorphosis, larvae were exposed from premetamorphosis (Nieuwkoop Faber stage 51 or 54) through pro metamorphosis. Of the PFAS tested in embryos, only 1H,1H,10H,10H-perfluorodecane-1,10-diol (FC10-diol) and perfluorohexanesulfonamide (FHxSA) exposure resulted in clear concentration-dependent developmental toxicity. For both of these PFAS, a significant increase in mortality was observed at 2.5 and 5 mg/L. For FC10-diol, 100% of the surviving embryos were malformed at 1.25 and 2.5 mg/L, while for FHxSA, a significant increase in malformations (100%) was observed at 2.5 and 5 mg/L. Developmental stage achieved was the most sensitive endpoint with significant effects observed at 1.25 and 0.625 mg/L for FC10-diol and FHxSA, respectively. In larval studies, we observed impacts on growth following exposure to PFHxS and PFOS at concentrations of 100 and 2.5 mg/L, respectively, while no impacts were observed in larvae when exposed to PFOA and HFPO-DA at concentration of 100 mg/L. Further, we did not observe impacts on thyroid endpoints in exposed larvae. These experiments have broadened our understanding of the impact of PFAS on anuran development.

Vital role of oxidative stress in tadpole liver damage caused by polystyrene nanoparticles.

Polystyrene nanoparticles are emerging as contaminants in freshwater environments, posing potential risks to amphibians exposed to extended periods of water contamination. Using tadpoles as a model, this study aimed to evaluate the toxicity of PS NPs. Pyrolysis-gas chromatography-tandem mass spectrometry (Py-GCMS) analysis revealed a concentration-dependent increase in polystyrene nanoparticles (PS NPs) levels in tadpoles with escalating exposure concentrations. Following exposure to 100 nm fluorescent microspheres, fluorescence was observed in the intestines and gills, peaking at 48 hours. Histopathological analysis identified degenerative necrosis and inflammation in the liver, along with atrophic necrosis of glomeruli and tubules in the kidneys. These results indicate a discernible impact of PS NPs on antioxidant levels, including reduced superoxide dismutase and catalase activities, elevated glutathione content, and increased malondialdehyde levels. Electron microscopy observations revealed the infiltration of PS NPs into Kupffer's cells and hepatocytes, leading to visible lesions such as nuclear condensation and mitochondrial disruption. The primary objective of this research was to elucidate the adverse effects of prolonged PS NPs exposure on amphibians.

Invading new climates at what cost? Ontogenetic differences in the thermal dependence of metabolic rate in an invasive amphibian.

Global warming can either promote or constrain the invasive potential of alien species. In ectotherm invaders that exhibit a complex life cycle, success is inherently dependent on the capacity of each developmental stage to cope with environmental change. This is particularly relevant for invasive anurans, which disperse on land while requiring water for reproduction. However, it remains unknown how the different life stages respond in terms of energy expenditure under different climate change scenarios. We here quantified the oxygen uptake of frogs at rest (a proxy of the standard metabolic rate) in the aquatic phase (at the tadpole and climax, i.e. during metamorphosis, stages) and in the terrestrial phase (metamorphosed stage) at three environmental temperatures. To do so, we used marsh frogs (Pelophylax ridibundus), an amphibian with the largest invasive range within the palearctic realm and for which their adaptation to global warming might be key to their invasion success. Beyond an increase of metabolic rate with temperature, our data show variation in thermal adaptation across life stages and a higher metabolic cost during metamorphosis. These results suggest that the cost to shift habitat and face changes in temperature may be a constraint on the invasive potential of species with a complex life cycle which may be particularly vulnerable during metamorphosis.

Does the accounting of the local symmetry fragments in quasi-SMILES improve the predictive potential of the QSAR models of toxicity toward tadpoles?

Models of toxicity to tadpoles have been developed as single parameters based on special descriptors which are sums of correlation weights, molecular features, and experimental conditions. This information is presented by quasi-SMILES. Fragments of local symmetry (FLS) are involved in the development of the model and the use of FLS correlation weights improves their predictive potential. In addition, the index of ideality correlation (IIC) and correlation intensity index (CII) are compared. These two potential predictive criteria were tested in models built through Monte Carlo optimization. The CII was more effective than IIC for the models considered here.

Increasingly cautious sampling, not the black colouration of unpalatable prey, is used by fish in avoidance learning.

The efficiency of aposematic colouration of prey is based on the innate bias or facilitation of avoidance learning of predators. In many toxic bufonids, larvae are uniformly black, which is considered a warning signal. We compared fish predation on normal (black) and 'transient albino' (greyish) common toad Bufo bufo tadpoles that did not differ in toxicity or activity. In a two-stage experiment, each fish was presented with tadpoles of one colour in the first trial and the other colour in a subsequent trial. While tadpoles sampled by fish were typically not ingested, some died from injuries. The attack rate did not differ between tadpole phenotypes nor trials, irrespective of which phenotype was the first exposed to the fish. However, during the second trial, the sampled tadpoles, independent of colouration, were mouthed by fish for shorter periods and tadpole mortality decreased. The duration of mouthing also declined with an increasing number of attacks during subsequent trials. We conclude that in single-species prey populations, black tadpole colouration is not a warning signal as it does not accelerate predator learning about prey unprofitability. Our results indicate that with growing experience, predators sample potentially toxic prey more cautiously. This may explain why natural selection does not eliminate aposematic morphs even if predators continuously sample conspicuous prey.

Effects of predation risk on the sensory asymmetries and defensive strategies of Bufotes balearicus tadpoles.

Lateralization consists of the differential use of bilateral organs or limbs and is well described in many taxa and in several contexts. Common ecological frameworks where it can be observed are foraging and predatory ones, with benefits related to both visual and auditory lateralization such as faster response or increasing neural processing ability. Anuran amphibians are considered relevant models for investigating lateralization, due to their great ecological variety and the possibility of easily being raised under laboratory conditions. By adopting the "rotational preference test", we used Balearic green toad tadpoles to test the effects of behavioural defensive responses triggered by different predator types (native vs alien, i.e. dragonfly larvae Aeshna cyanea and adult red swamp crayfish Procambarus clarkii) and diets (fasted vs. tadpole-fed predators) on their lateralization. We recorded tadpoles' responses to five different chemical cues: clean water (control treatment), fasted dragonfly larvae and crayfish, and tadpole-fed dragonfly larvae and crayfish. Green toad tadpoles did not show a bias in a predominant direction, although lateralization occurred at the individual level, as shown by the intensity index (LA). Perceived predation risk was the highest in tadpoles exposed to the combined chemical cues of conspecific prey and native predators, which elicited both changes in the intensity of lateralization and a marked reduction in tadpoles' activity level. Our results suggest that contextual predation threat may induce very rapid changes in the expression of asymmetries at the individual level, and might play a role as part of the complex defensive strategies adopted by prey in the attempt to escape predators.

Combined effects of landscape composition and agrochemicals on frog communities amid sugarcane-dominated agroecosystems.

Global demand for crops will continue increasing over the next few decades to cover both food and biofuel needs. This demand will put further pressure to expand arable land and replace natural habitats. However, we are only beginning to understand the combined effects of agrochemicals and land-use change on tropical freshwater biodiversity. In this study, we analyzed how pond-dwelling anuran larvae responded to pond characteristics, landscape composition, and agrochemical contamination in a sugarcane-dominated agroecosystem in Brazil. Then we used an information theoretical approach with generalized linear models to relate species richness and abundance to predictor variables. The variation in tadpole abundance was associated with both agrochemical concentration (e.g., ametryn, diuron, and malathion) and landscape variables (e.g., percentage of forest, percentage of agriculture, and distance to closest forest). The relationship between species abundance and agrochemicals was species-specific. For example, the abundances of Scinax fuscovarius and Physalaemus nattereri were negatively associated with ametryn, and Dendropsophus nanus was negatively associated with tebuthiuron, whereas that of Leptodactylus fuscus was positively associated with malathion. Conversely, species richness was associated with distance to forest fragments and aquatic vegetation heterogeneity, but not agrochemicals. Although we were unable to assign a specific mechanism to the variation in tadpole abundance based on field observations, the lower abundance of three species in ponds with high concentrations of agrochemicals suggest they negatively impact some frog species inhabiting agroecosystems. We recommend conserving ponds near forest fragments, with abundant stratified vegetation, and far from agrochemical runoffs to safeguard more sensitive pond-breeding species.

Ontogeny of the anuran urostyle and the developmental context of evolutionary novelty.

Developmental novelties often underlie the evolutionary origins of key metazoan features. The anuran urostyle, which evolved nearly 200 MYA, is one such structure. It forms as the tail regresses during metamorphosis, when locomotion changes from an axial-driven mode in larvae to a limb-driven one in adult frogs. The urostyle comprises of a coccyx and a hypochord. The coccyx forms by fusion of caudal vertebrae and has evolved repeatedly across vertebrates. However, the contribution of an ossifying hypochord to the coccyx in anurans is unique among vertebrates and remains a developmental enigma. Here, we focus on the developmental changes that lead to the anuran urostyle, with an emphasis on understanding the ossifying hypochord. We find that the coccyx and hypochord have two different developmental histories: First, the development of the coccyx initiates before metamorphic climax whereas the ossifying hypochord undergoes rapid ossification and hypertrophy; second, thyroid hormone directly affects hypochord formation and appears to have a secondary effect on the coccygeal portion of the urostyle. The embryonic hypochord is known to play a significant role in the positioning of the dorsal aorta (DA), but the reason for hypochordal ossification remains obscure. Our results suggest that the ossifying hypochord plays a role in remodeling the DA in the newly forming adult body by partially occluding the DA in the tail. We propose that the ossifying hypochord-induced loss of the tail during metamorphosis has enabled the evolution of the unique anuran bauplan.

Recovery of Ceratophrys ornata tadpoles exposed to environmental concentrations of chlorpyrifos: evaluation of biomarkers of exposure.

Chlorpyrifos (CPF) is one of the most widely used insecticides worldwide despite the fact that many authors have warned about its effects in non-target biota. The effects of CPF on anurans are well known, but the process of recovery from these effects after exposure is less explored. The aim of this study was to evaluate the duration of sublethal effects induced by environmental concentrations of CPF on Ceratophrys ornata tadpoles after exposure. The experimental design consisted of an exposure phase (96 h) in which tadpoles were individually exposed to three concentrations of CPF (0, 0.01 and 0.02 mg CPF/L) and a post-exposure phase (72 h) in which exposed tadpoles were transferred to CPF-free media. Individuals that survived the exposure phase to CPF showed neither long-term lethal effects nor long-term swimming alterations and altered prey consumption after being transferred to CPF-free media. No morphological abnormalities were observed either. However, at the end of both phases, tadpoles emitted shorter sounds with a higher dominant frequency than the tadpoles in the control group, i.e., the tadpoles did not recover their normal sounds. Thus, for the first time in this species, we have shown that effects on sounds should be prioritized as biomarkers of exposure, as they not only provide longer detection times after cessation of exposure, but also involve non-destructive methods. The following order of priority could be established for the selection of biomarkers that diagnose the health status of individuals and precede irreversible responses such as mortality: alterations in sounds > swimming alterations > prey consumption.

Comparative efficacy of natural aquatic predators for biological control of mosquito larvae: A neglected tool for vector control.

Mosquitoes are a dominant fraction of dipteran fauna, occupying a variety of niches. The most common method deployed for their control is the use of insecticides. Throughout their life cycle they are exposed to a wide range of predators in different habitats, thus biological control of mosquitoes by using aquatic predators has been suggested. Therefore, the present study was carried out to explore the type of natural predators coexisting with the mosquito larvae in still water bodies and to determine their efficacy as predators for mosquito larvae. A coexistence of different predators with mosquito larvae was observed in 27 standing water bodies of Chandigarh, India. The predation efficiency of tadpoles of frog was comparable to Gambusia fish, as 97% of the mosquito larvae of all instars of the medically important mosquito genera Anopheles, Aedes, Culex and Armigeres were preyed. The toad tadpoles were found to be least effective and their predation rate was found to be negligible. Further studies on larval source management by frog tadpoles in combination with insecticides or stand-alone would be worthwhile.

Habituation in anuran tadpoles and the role of risk uncertainty.

The ability to learn in the context of predation allows prey to respond to threats by adjusting their behavior based on specific information acquired from their current environment. Habituation is a process that allows animals to adapt to environmental changes. Very little is known about habituation in wild animals in general and there are no studies on habituation in anuran tadpoles in particular. Here, we performed three experiments to investigate the behavioral response of predator naïve Pleurodema thaul tadpoles to repeated stimulation with two predation risk cues (injured conspecific and predator fed cues) which a priori provide different information regarding risk. Experiment 1 showed that P. thaul tadpoles habituate the antipredator response when undergo predation risk chemical cues from injured conspecific and that response is long term. Experiment 2 showed that P. thaul tadpoles did not habituate their antipredator response when exposed to cues derived from an event of nymph odonate preying on P. thaul tadpoles (predator fed cues). Experiment 3 specifically evaluated the risk imposed by each of the risk cues used in Experiment 1 and Experiment 2 and showed that the degree of perceived risk in tadpoles appear to be similar in a single experience with any risk stimuli. We suggest that the behavioral habituation of tadpoles in the context of predation could be modulated by the level of uncertainty associated with risk stimuli.

Amphibian loss alters periphyton structure and invertebrate growth in montane streams.

Amphibians are declining worldwide due to a combination of stressors such as climate change, invasive species, habitat loss, pollution and emergent diseases. Although their losses are likely to have important ecological consequences on the structure and functioning of freshwater ecosystems, this issue has been scarcely explored. We conducted an experiment in three montane streams-where primary production is the main source of energy and carbon-to assess the effects of amphibian disappearance (i.e. presence or absence of the common midwife toad Alytes obstetricans, a common species found in pools of these streams) on several aspects of ecosystem functioning and structure: periphyton biomass and chlorophyll a concentration, algal assemblage structure, and growth of macroinvertebrate grazers. We compared four types of experimental enclosures: (i) without macroinvertebrates or amphibians; (ii) with larvae of the caddisfly Allogamus laureatus; (iii) with A. obstetricans tadpoles; and (iv) with both A. laureatus larvae and A. obstetricans tadpoles. The absence of tadpoles increased periphyton biomass, but did not cause differences on inorganic sediment accrual. The algal assemblage had a higher diversity in the absence of tadpoles, and their characteristic taxa differed from the assemblages in presence of tadpoles. A. laureatus presented higher mass in presence of tadpoles; however, tadpole length was not affected by presence of macroinvertebrates. Our results suggest that presence of tadpoles is a driver of periphyton accrual and assemblage structure, acting as top-down control and with key potential consequences on the functioning of montane stream ecosystems.

La población de anfibios está reduciéndose globalmente debido a una combinación de factores como el cambio climático, las especies invasoras, la pérdida de hábitat, la contaminación y las enfermedades emergentes. Aunque su desaparición probablemente causará consecuencias ecológicas importantes en la estructura y el funcionamiento de los ecosistemas de agua dulce, es un aspecto que ha sido poco explorado. Se realizó un experimento en tres arroyos de montaña, donde la producción primaria es la principal fuente de energía y carbono, para determiner los efectos de la desaparición de los anfibios (presencia o ausencia del sapo partero común Alytes obstetricans, una especie común en pozas de estos arroyos) en varios aspectos del funcionamiento y la estructure del ecosistema: la biomasa de perifiton y la concentración de clorofila, la estructura de la comunidad algal y el crecimiento de los macroinvertebrados raspadores. Comparamos cuatro tipos de recintos: (i) sin macroinvertebrados ni anfibios; (ii) con larvas del tricóptero Allogamus laureatus; (iii) con renacuajos de A. obstetricans; y (iv) con tanto larvas de A. laureatus como renacuajos de A. obstetricans. La ausencia de renacuajos aumentaba la biomasa de perifiton, aunque no causaba diferencias en la acumulación de sedimento inorgánico. La comunidad de algas presentaba una mayor diversidad en ausencia de renacuajos, y sus taxones característicos eran distintos de los de las comunidades con presencia de renacuajos. A. laureatus tenía una mayor masa en presencia de renacuajos, pero la longitud de los renacuajos no se veía afectada por la presencia de macroinvertebrados. Nuestros resultados sugieren que la presencia de renacuajos controla la acumulación de perifiton y la estructura de las comunidades, ejerciendo un control de arriba hacia abajo, con consecuencias clave en el funcionamiento de los ecosistemas de arroyos de montaña.

Influence of nano and bulk copper on agile frog development.

Nanotechnology, as one of the fastest-growing industries, offers many benefits in various fields. However, properties that contribute to its positive effects, in other context, can cause adverse effects to various organisms, such as amphibians. Identifying possible negative effects on its survival is crucial since amphibians are the most threatened group of vertebrates. In that context, we investigated the influence of both nano and bulk copper on embryonic development of agile frog, Rana dalmatina. The embryos were exposed to various concentrations (0.01 mg/L, 0.075 mg/L, 0.15 mg/L or 0.3 mg/L) of either nano (CuO, declared size 40-80 nm) or bulk form (CuSO4·5H2O) for 16 days. Upon the experiment, tadpoles were measured and weighted, then homogenized and their protein, lipid, and carbohydrates content determined, as well as the activity of LDH. Our results suggest stronger negative influence of nano copper to size and weight of tadpoles, and bulk copper on lipid content, while both had strong negative effect on carbohydrates content, and LDH activity. In addition, our results suggest agile frog to be more susceptible to negative influence of both, nano and bulk copper, than commonly used Xenopus laevis.

Olfactory subsystems in the peripheral olfactory organ of anuran amphibians.

Anuran amphibians (frogs and toads) typically have a complex life cycle, involving aquatic larvae that metamorphose to semi-terrestrial juveniles and adults. However, the anuran olfactory system is best known in Xenopus laevis, an animal with secondarily aquatic adults. The larval olfactory organ contains two distinct sensory epithelia: the olfactory epithelium (OE) and vomeronasal organ (VNO). The adult organ contains three: the OE, the VNO, and a "middle cavity" epithelium (MCE), each in its own chamber. The sensory epithelia of Xenopus larvae have overlapping sensory neuron morphology (ciliated or microvillus) and olfactory receptor gene expression. The MCE of adults closely resembles the OE of larvae, and senses waterborne odorants; the adult OE is distinct and senses airborne odorants. Olfactory subsystems in other (non-pipid) anurans are diverse. Many anuran larvae show a patch of olfactory epithelium exposed in the buccal cavity (bOE), associated with a grazing feeding mode. And other anuran adults do not have a sensory MCE, but many have a distinct patch of epithelium adjacent to the OE, the recessus olfactorius (RO), which senses waterborne odorants. Olfaction plays a wide variety of roles in the life of larval and adult anurans, and some progress has been made in identifying relevant odorants, including pheromones and feeding cues. Increased knowledge of the diversity of olfactory structure, of odorant receptor expression patterns, and of factors that affect the access of odorants to sensory epithelia will enable us to better understand the adaptation of the anuran olfactory system to aquatic and terrestrial environments.

Osteological development of a small and fast metamorphic frog, Microhyla fissipes (Anura, Neobatrachia, Microhylidae).

Describing osteological development is of great importance for understanding vertebrate phenotypic variations, form-functional transitions and ecological adaptations. Anurans exhibit dramatic changes in their morphology, habitat preferences, diet and behaviour between the tadpole and frog stages. However, the anatomical details of their cranial and postcranial development have not been extensively studied, especially in Microhylidae. In this work, we studied the microhylid Microhyla fissipes, commonly known as the ornamented pygmy frog, a small-sized frog with fast metamorphosis. Its osteological development was comprehensively described based on 120 cleared and stained specimens, including six tadpoles for each stage between 28 and 45, six juveniles and six adults. Additionally, 22 osteological traits of these specimens involved in food acquisition, respiration, audition and locomotion were selected and measured to reflect the changes in tadpole ecological functions during metamorphosis. Our study provides the first detailed qualitative and quantitative developmental information about these structures. Our results have confirmed that skeletal elements (viz., neopalatines, omosternum, clavicles and procoracoids) absent in adults are not detected during development. Our data reveal that morphologically, radical transformations of the cranial structures related to feeding and breathing are completed within stages 42-45 (72 h), but the relative length and width of these skeletons have changed in earlier stages. The postcranial skeletons correlated with locomotion are well developed before stage 42 and approach the adult morphology at stage 45. Indeed, the relative length of the pectoral girdle and forelimb reaches the adult level at stage 42 and stage 45, respectively, whereas that of the vertebral column, pelvic girdle and hind limbs increases from their appearance until reaching adulthood. Based on published accounts of 19 species from Neobatrachia, Mesobatrachia and Archaeobatrachia, cranial elements are among the first ossified skeletons in most studied species, whereas sphenethmoids, neopalatines, quadratojugals, mentomeckelians, carpals and tarsals tend to ossify after metamorphosis. These results will help us to better understand the ecomorphological transformations of anurans from aquatic to terrestrial life. Meanwhile, detailed morphological and quantitative accounts of the osteological development of Microhyla fissipes will provide a foundation for further study.

Contextual behavioural plasticity in Italian agile frog (Rana latastei) tadpoles exposed to native and alien predator cues.

Predation is a strong driver for the evolution of prey behaviour. To properly assess the actual risk of predation, anuran tadpoles mostly rely on water-borne chemical cues, and their ability to evaluate environmental information is even more crucial when potential predators consist of unknown alien species. Behavioural plasticity - that is, the capacity to express changes in behaviour in response to different environmental stimuli - is crucial to cope with predation risk. We explored the defensive behaviour of Italian agile frog (Rana latastei) tadpoles when exposed to the chemical cues of two predator species, one native (dragonfly larvae) and one alien (red swamp crayfish). Firstly, we observed whether a plastic life history trait (i.e. hatching time) might be affected by native predatory cues. Secondly, we recorded a suite of behavioural responses (activity level, lateralization and sinuosity) to each cue. For assessing lateralization and sinuosity, we developed a C++ code for the automatic analysis of digitally recorded tadpole tracks. Hatching time seemed not to be affected by the potential risk of predation, while both predator species and diet affected tadpoles' defensive behaviour. Tadpoles responded to a predator threat by two main defensive strategies: freezing and 'zig-zagging'. While the first behaviour had previously been reported, the analysis of individual trajectories indicated that tadpoles can also increase path complexity, probably to prevent predators from anticipating their location. We also recorded a decrease in lateralization intensity, which suggests that under predation risk, tadpoles tend to scrutinize the surrounding environment equally on both sides.

Xenopus laevis tadpoles exposed to metamifop: Changes in growth, behavioral endpoints, neurotransmitters, antioxidant system and thyroid development.

Pesticides are a major cause of the reduction in the global amphibian population. In this study, the acute toxicity and chronic effects of metamifop on Xenopus laevis (X. laevis) tadpoles were investigated. The 96 h-LC50 value of metamifop on X. laevis tadpoles was 0.634 mg/L, which indicated that metamifop was highly toxic to tadpoles. In the chronic toxicity study, tadpoles were exposed to 0.063 mg/L of metamifop. After 14, 21 and 35 d of exposure, metamifop significantly inhibited the body weight and neurotransmitter synthesis of tadpoles, caused abnormal behavior and interfered with fat metabolism. According to the results of antioxidant enzymes and malondialdehyde (MDA), tadpoles exposed to 0.063 mg/L metamifop suffered severe lipid oxidative damage. Compared with the control group, the thyroid hormone (TH) levels and related gene expression in tadpoles in the treatment group were affected, reflecting the endocrine interference effect of metamifop. The data of this study can enrich our knowledge of the effects of aryloxyphenoxy propionate pesticides on amphibians and highlight the role of metamifop and other pesticides play in global decline of amphibians.

Copper-induced sublethal effects in Bufo gargarizans tadpoles: growth, intestinal histology and microbial alternations.

Copper (Cu) is one of the environmental contaminations which can pose significant risks for organisms. The current study explores the effects of Cu exposure on the growth, intestinal histology and microbial ecology in Bufo gargarizans. The results revealed that 0.5-1 µM Cu exposure induced growth retardation (including reduction of total body length and wet weight) and intestinal histological injury (including disordered enterocyte, changes in the villi and vacuoles) of tadpoles. Also, high-throughput sequencing analysis showed that Cu exposure caused changes in richness, diversity and structure of intestinal microbiota. Moreover, the composition of intestinal microbiota was altered in tadpoles exposed to different concentrations of Cu. At the phylum level, we observed the abundance of proteobacteria was increased, while the abundance of fusobacteria was decreased in the intestinal microbiota of tadpoles exposed to 1 µM Cu. At the genus level, a reduced abundance of kluyvera and aeromonas was observed in the intestinal microbiota of tadpoles under the exposure of 0-0.5 µM Cu. Finally, functional predictions revealed that tadpoles exposed to copper may be at a higher risk of developing metabolic disorders or diseases. Above all, our results will develop a comprehensive view of the Cu exposure in amphibians and will yield a new consideration for sublethal effects of Cu on aquatic organisms.

Gut microbiota of invasive bullfrog tadpoles responds more rapidly to temperature than a noninvasive congener.

Environmental temperature can alter the composition, diversity, and function of ectothermic vertebrate gut microbial communities, which may result in negative consequences for host physiology, or conversely, increase phenotypic plasticity and persistence in harsh conditions. The magnitude of either of these effects will depend on the length of time animals are exposed to extreme temperatures, and how quickly the composition and function of the gut microbiota can respond to temperature change. However, the temporal effects of temperature on gut microbiota are currently unknown. Here, we investigated the length of time required for increased temperature to alter the composition of gut bacterial communities in tadpoles of two frog species, the green frog, Lithobates clamitans, and its congener, the globally invasive American bullfrog, L. catesbeianus. We also explored the potential functional consequences of these changes by comparing predicted metagenomic profiles across temperature treatments at the last experimental time point. Bullfrog-associated microbial communities were more plastic than those of the green frog. Specifically, bullfrog communities were altered by increased temperature within hours, while green frog communities took multiple days to exhibit significant changes. Further, over ten times more bullfrog bacterial functional pathways were temperature-dependent compared to the green frog. These results support our hypothesis that bullfrog gut microbial communities would respond more rapidly to temperature change, potentially bolstering their ability to exploit novel environments. More broadly, we have revealed that even short-term increases in environmental temperature, expected to occur frequently under global climate change, can alter the gut microbiota of ectothermic vertebrates.