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Multiple species of frogs in the Ranidae family have been observed to 'skitter' across the water surface, but little is understood about the biomechanical or physical mechanisms that underlie this behavior. All documented descriptions are anecdotal, asserting simply that the frogs can cross the water surface without sinking. To study this form of interfacial locomotion, we recorded high-speed video of the northern cricket frog Acris crepitans and quantified its kinematics. We also compared its semi-aquatic behavior with the frogs' terrestrial locomotion. Contrary to expectations based on anecdotal knowledge, we found that cricket frogs do not maintain an above-surface position throughout the locomotor cycle. Instead, the frogs are completely submerged during both the launching and landing phase of a jump cycle, similar to porpoising in other animals. It is possible that leg-retraction time constrains these frogs from performing true surface-only locomotion., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Infanticide is widespread across the animal kingdom, but the physiological drivers of infanticide versus care or neglect are relatively unexplored. Here, we identified salient environmental and physiological antecedents of infanticide in the mimic poison frog (Ranitomeya imitator), a biparental amphibian. We explored potential environmental cues influencing infant-directed behavior by evaluating changes in the frequency of food provisioning and tadpole mortality after either cross-fostering tadpoles between family units or displacing tadpoles within the terraria of their parents. We found that changes in offspring location reduce care and increase infanticide. Specifically, parents fed their displaced offspring less and, in some instances, tadpole mortality increased. We also investigated whether care and infanticide were related to changes in steroid hormone concentrations in an unfamiliar setting. Infanticide of fertilized eggs and hatchlings in the new territory included cannibalism and was associated with lower testosterone concentrations, but not with changes in corticosterone. Overall, our results support earlier findings that familiarity with offspring location drives parental investment in poison frogs, while indicating an association between low androgen levels and infanticidal behavior in an amphibian., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Polylactic acid microplastics (PLA-MPs), biobased plastics made from renewable resources, are considered to be a potential solution for alleviating the pollution pressure of plastics; however, the potential environmental risks of PLA-MPs must be further evaluated. In this study, the effects of PLA-MPs on the tadpoles of Pelophylax nigromaculatus were investigated by designing different PLA-MP exposure experiments. We found that PLA-MPs negatively affected the survival, growth and development of tadpoles. In addition, in open field tests, PLA-MPs also reduced tadpole locomotion while resulting in more repetitive searching behavior within a restricted area. This effect was more pronounced at higher concentrations of PLA-MPs (20 mg/mL) and in combination with the heavy metal Cd 2+ . In the tank tests, PLA-MPs increased tadpole aggregation, and their combined effect with Cd 2+ resulted in a tendency for tadpole aggregation to increase and then decrease, with the distribution tending to favor aggregation in edge regions. PLA-MPs also strongly inhibited the spatiotemporal exploratory activities of tadpoles in the tanks. This study provides a more detailed investigation of the behavioral effects of PLA-MPs on tadpoles and provides a theoretical basis for subsequent ecotoxicological studies of PLA-MPs., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Some snakes emit hissing calls which are imitated by birds to deter potential predators. However, the effect of these snake and bird hisses on anuran risk recognition is not yet explored. Here we hypothesize that these hisses may advertise dangers to frogs and evoke their anti-predator responses. We used little torrent frogs (Amolops torrentis) as subjects and conducted sound playbacks to test their anti-predator behaviors. We found that little torrent frogs changed their calling behaviors during sympatric snake hiss playbacks, but showed no response to white noise and allopatric snake hiss playbacks. They did not respond to sympatric avian hiss that has low acoustic similarity with snake sounds. However, they decreased calling activity in response to sympatric avian hiss that has high acoustic similarity with snakes. As compared to other treatments, more individuals ceased calling during the playbacks of the highly similar bird hiss. These results suggest that frogs may recognize risks from snake and snake-like hissing calls and perform anti-predator responses., (© 2024. The Author(s).)

The pervasive distribution of microplastics (MPs) in aquatic ecosystems presents a significant threat to wildlife, with amphibians being particularly vulnerable due to their complex life cycles and ecological roles. This study investigates physiological and ecological impacts of aquatic MP exposure on juvenile black-spotted pond frogs (Pelophylax nigromaculatus), focusing on juvenile frog stage, history of life after metamorphosis. MP examinations in the intestine and body revealed accumulation primarily in the gastrointestinal tracts without evidence of systemic distribution. Experimental exposure to different concentrations of MPs demonstrated adverse effects on growth, physiological stress, and immune function. Notably, higher MP concentrations led to significant reductions in growth and innate immunity, indicative of compromised health. High concentrations of MPs were associated with elevated levels of corticosterone and antioxidant enzymes, indicating physiological stress. However, there was no evidence of extreme hormonal surges or imbalances in antioxidant enzyme activity, suggesting that amphibians were able to effectively cope with the levels of MPs used in the study. Changes in gastrointestinal morphology and fecal microbiota composition were observed, reflecting response of metabolic adaptation to MP exposure. At low concentrations of MPs, adaptive changes in digestive tract morphology and the maintenance of gut microbiota balance were observed, indicating that the frogs were able to manage the exposure below a certain threshold. In contrast, high concentrations of MPs had clear negative effects on amphibians, which could impact biodiversity and ecosystem stability. These findings also suggest that MPs may trigger adaptive responses at lower concentrations, while still posing significant environmental risks at higher levels., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2024. Published by Elsevier Ltd.)

Pesticide exposure and its interaction with other natural stressors can play a role in amphibian population declines because disruptions in stress hormone regulatory mechanisms may inhibit immune responses during metamorphosis. Here, we determined the interactive effects of predation risk and sublethal concentration of two pesticides on immunological and physiological responses in tadpoles of the tropical frog Lithobates taylori. Using mesocosms, we used chronic exposure to three levels of chlorothalonil and β-endosulfan in the presence or absence of Odonate larvae. Our results show that β-endosulfan in high concentrations reduced the weight of the tadpoles and increased the neutrophil count and corticosterone (CORT) levels. Larval development was accelerated by high concentrations of chlorothalonil. Also, this pesticide in low and high concentrations increases the absolute values of lymphocytes. Tadpoles exposed to chlorothalonil increased the numbers of monocytes (in low concentrations), and lymphocytes (in high and low concentrations). The interactions of the low concentrations of both pesticides with and without the predator's presence also increased the number of lymphocytes. A combination of pesticides increases the number of lymphocytes in the blood due to synergistic cytotoxicity. This study proves that β- endosulfan elevates circulating CORT and thus generates physiological stress in tadpoles. Given that both pesticides are widely used within the distribution of L. taylori in Costa Rica, it is likely that tadpoles' development and immune function are altered by pesticide use. In combination with stressors such as emerging diseases and altered precipitation regimes, widespread agrochemical uses likely caused this species enigmatic decline in recent decades., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The ways in which animals sense the world changes throughout development. For example, young of many species have limited visual capabilities, but still make social decisions, likely based on information gathered through other sensory modalities. Poison frog tadpoles display complex social behaviors that have been suggested to rely on vision despite a century of research indicating tadpoles have poorly-developed visual systems relative to adults. Alternatively, other sensory modalities, such as the lateral line system, are functional at hatching in frogs and may guide social decisions while other sensory systems mature. Here, we examined development of the mechanosensory lateral line and visual systems in tadpoles of the mimic poison frog (Ranitomeya imitator) that use vibrational begging displays to stimulate egg feeding from their mothers. We found that tadpoles hatch with a fully developed lateral line system. While begging behavior increases with development, ablating the lateral line system inhibited begging in pre-metamorphic tadpoles, but not in metamorphic tadpoles. We also found that the increase in begging and decrease in reliance on the lateral line co-occurs with increased retinal neural activity and gene expression associated with eye development. Using the neural tracer neurobiotin, we found that axonal innervations from the eye to the brain proliferate during metamorphosis, with few retinotectal connections in recently-hatched tadpoles. We then tested visual function in a phototaxis assay and found tadpoles prefer darker environments. The strength of this preference increased with developmental stage, but eyes were not required for this behavior, possibly indicating a role for the pineal gland. Together, these data suggest that tadpoles rely on different sensory modalities for social interactions across development and that the development of sensory systems in socially complex poison frog tadpoles is similar to that of other frog species., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Gametogenesis produces gametes as a piece of genetic information transmitted to the offspring. While during sexual reproduction, progeny inherits a mix of genetic material from both parents, asexually reproducing organisms transfer a copy of maternal or paternal DNA to the progeny clonally. Parthenogenetic, gynogenetic and hybridogenetic animals have developed various mechanisms of gametogenesis, however, their inheritance is not fully understood. Here, we focused on the inheritance of asexual gametogenesis in hybrid Pelophylax esculentus (RL), emerging after crosses of P. lessonae (LL) and P. ridibundus (RR). To understand the mechanisms of gametogenesis in hybrids, we performed three-generation experiments of sexual P. ridibundus females and hybrids from all-male hybrid populations. Using fluorescent in situ hybridization, micronuclei analysis, flow cytometry and genotyping, we found that most adult hybrid males simultaneously produced two types of clonal sperm. Also, most male tadpole progeny in two successive backcrossed generations simultaneously eliminated L and R parental genomes, while some progeny produced only one type of sperm. We hypothesize that the reproductive variability of males producing two kinds of sperm is an adaptive mechanism to reproduce in mixed populations with P. ridibundus and may explain the extensive distribution of the all-male lineage across the European River Basin., (© 2024. The Author(s).)

Examples of resilience in nature give us hope amid a growing biodiversity crisis. While resilience has many definitions across disciplines, here I discuss resilience as the ability to continue to adapt and persist. Naturally, as biologists, we seek to uncover the underlying mechanisms that can help us explain the secrets of resilience across scales, from individuals to species to ecosystems and beyond. Perhaps we also ponder what the secrets to resilience are in our own lives, in our own research practices, and academic communities. In this paper, I highlight insights gained through studies of amphibian resilience following a global disease outbreak to uncover shared patterns and processes linked to resilience across amphibian communities. I also reflect on how classical resilience heuristics could be more broadly applied to these processes and to our own academic communities. Focusing on the amphibian systems that I have worked in-the Golden Frogs of Panama (Atelopus zeteki/varius) and the Mountain Yellow-Legged Frogs of California (Rana muscosa/sierrae)-I highlight shared and unique characteristics of resilience across scales and systems and discuss how these relate to adaptive renewal cycles. Reflecting on this work and previous resilience scholarship, I also offer my own thoughts about academia and consider what lessons we could take from mapping our own adaptive trajectories and addressing threats to our own community resilience., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Global warming may affect the early developmental stages of high-altitude amphibians, thereby influencing their later fitness. Yet, this has been largely unexplored. To investigate whether and how the temperatures experienced by embryonic and larval stages affect their fitness at later developmental stages, we designed two experiments in which the embryos and larvae were treated with three temperatures (24, 18 and 12 °C), respectively. Then, the life history traits of the tadpoles during the metamorphotic climax in all treatments were evaluated, including growth rate, survival rate, morphology, thermal physiology, swimming performance, standard metabolic rate (SMR), oxidative and antioxidative system, and metabolic enzyme activities. The results revealed that elevated temperature accelerated metamorphosis but decreased body size at metamorphosis. Additionally, warming during the embryonic and larval stages decreased the thermal tolerance range and induced increased oxidative stress. Furthermore, high embryonic temperature significantly decreased the hatching success, but had no significant effect on swimming performance and SMR. Warming during larval periods was harmful to the survival and swimming performance of tadpoles. The effect size analysis revealed that the negative impacts of embryonic temperature on certain physiological traits, such as growth and development, survival and swimming performance, were more pronounced than those of larval temperature. Our results highlight the necessity for particular attention to be paid to the early stages of amphibians, notably the embryonic stages when evaluating the impact of global warming on their survival., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Research has shown that leached plant toxins negatively impact the growth and development of larval amphibians. However, tadpoles may encounter these same toxins in food material, and differential exposure routes and distribution of toxic chemicals can yield variable downstream effects on animals. To date, most research understanding the interactions between dietary plant toxins and herbivores has been conducted in terrestrial systems. Despite the abundance of plant toxins in food and water sources, the effects of dietary plant toxins on larval amphibians have not been studied, and tannins could negatively affect these species. Here, green frog tadpoles (Lithobates clamitans) were fed diets with or without 2% tannic acid to test how their growth, development, behavior, and gut microbiome respond to dietary tannins. At the end of the trial, we conducted a behavioral assay to measure tadpole activity and boldness and inventoried the gut microbiome using 16S rRNA sequencing. Dietary tannins significantly decreased body mass by 66% and length by 28%, without influencing tadpole developmental stage. We found significant differences in exploratory behavior and boldness during the first minute of our behavioral assay, demonstrating that tannins have the potential to influence behavior during novel or stressful events. Finally, tannins significantly sculpted the gut microbiome, with an increase in the measurement of Shannon entropy. We observed 7 microbial phyla and 153 microbial genera that exhibited significantly differential abundances differences between control and tannic acid-fed tadpoles. Collectively, our results demonstrate that dietary tannins have the potential to alter amphibian growth, behavior, and microbiome., (© 2023 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.)

When amphibians thermoregulate, they face a fundamental trade-off between the ability to maintain activity and an increased rate of dehydration at higher temperatures. Canopy coverage affects both the thermal and hydric conditions of the environment and can therefore influence amphibian thermoregulation. Frogs require proper conditions to thermoregulate to successfully grow, survive, and reproduce. But while we know how canopy and environmental variables typically affect operative temperature, less is known about effects on amphibian water loss rates. In this study, we measure the effect of canopy coverage on the conditions available for thermoregulation at a breeding pond of the California red-legged frog, Rana draytonii. We use agar frog models to estimate the thermal and hydric capacities frogs would experience in locations with different canopy coverage and microhabitats. At each site, we deployed models under four microhabitat treatments: wet/sun, wet/shade, dry/sun, and dry/shade. We modeled how environmental variables affected operative temperature and evaporative water loss from agar frogs. We found positive effects of air temperature, the sun treatment, and reduced canopy cover on operative temperature, and negative direct or indirect effects of these variables on evaporative water loss, consistent with the hypothesized trade-off between thermoregulatory behavior to increase temperature and the increased desiccation risk due to higher water loss. Additionally, our results indicate that the availability of wet microhabitats can allow frogs to reduce water loss, potentially mitigating the risk of desiccation when thermoregulating to achieve higher operative temperatures. Our findings suggest, that with access to proper microhabitats, amphibians can mitigate the fundamental trade-off and receive benefits of thermoregulating at high temperatures., Competing Interests: Declaration of competing interest None, (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Amphibian diversity is most prominent in the warm and humid tropical and subtropical regions across the globe. Nonetheless, amphibians also inhabit high-altitude tropical mountains and regions at medium and high latitudes, exposing them to subzero temperatures and requiring behavioural or physiological adaptations to endure freezing events. While freeze tolerance has been predominantly reported in high-latitude zones where species endure prolonged freezing (several weeks or months), less is known about mid-latitudes amphibians exposed to occasional subzero temperatures. In this study, we employed a controlled ecological protocol, subjecting three frog species from the Iberian Peninsula (Rana parvipalmata, Epidalea calamita, and Pelobates cultripes) to a 2-h exposure to temperatures of -2 °C to investigate the accumulation of urea and glucose as physiological mechanisms associated with survival at freezing temperatures. Our results revealed a moderate response in the production of cryoprotectant metabolites under experimental freezing conditions, particularly urea, with notable findings in R. parvipalmata and E. calamita and no response in P. cultripes. However, no significant alterations in glucose concentrations were observed in any of the studied frog species. This relatively weak freezing tolerance response differs from the strong response exhibited by amphibians inhabiting high latitudes and enduring prolonged freezing conditions, suggesting potential reliance on behavioural adaptations to cope with occasional freezing episodes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the study reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Blinking is a behavior unique to animal taxa with motile eyelids, such as most amphibians and reptiles as well as all birds and mammals 1 . Eyelid movement has physiological functions, such as lubricating the cornea and washing away dust, but its potential signaling functions are not well understood 1 , 2 . The use of eyeblinks as a social signal is currently thought to be restricted to some primates, especially humans and their companion animals, but has not been verified in other taxa 1 , 3 , 4 . Here, through field observation and experiments, we demonstrate that female concave-eared torrent frogs (Odorrana tormota), which inhabit noisy streams, use eyeblinks to communicate with males to urge them to initiate amplexus for mating. Our findings reveal that eyeblinks may serve as a social signal in non-primate species., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Contamination of aquatic ecosystems is pervasive around the world and there has been a growing interest in understanding the ecological and evolutionary impacts. For contaminants such as pesticides, researchers are discovering widespread evolution of increased tolerance in target and non-target species and the role of phenotypic plasticity in facilitating this evolution. In contrast, we know much less about the evolution of tolerance in response to the increasing problem of freshwater salinization. In amphibians, recent studies have discovered that some populations from ponds with high salt pollution (from deicing road salts) have evolved higher tolerance. In this study, we examined whether populations of wood frog tadpoles (Rana sylvatica) possess rapid, inducible tolerance to salinity in a manner similar to their inducible tolerance to pesticides. Using newly hatched tadpoles from nine populations, we discovered that eight of the populations were able to alter their tolerance to salt. However, seven of the eight inducible populations experienced a higher sensitivity to salt while the eighth population experienced a higher tolerance to salt. Such inducible responses likely reflect the interplay of salt dynamics in the ponds, combined with the available genetic variation and selection intensity of each pond. This appears to be the first example of inducible salt tolerance in any animal and future studies should examine the generality of the response and how it may affect the evolution of tolerance to the global issue of freshwater salinization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Climate change can exacerbate the effects of environmental pollutants on aquatic organisms. Pollutants such as human antidepressants released from wastewater treatment plants have been shown to impact life-history traits of amphibians. We exposed tadpoles of the wood frog Lithobates sylvaticus to two temperatures (20 °C and 25 °C) and two antidepressants (fluoxetine and venlafaxine), and measured timing of metamorphosis, mass at metamorphosis, and two behaviors (startle response and percent motionless). Antidepressants significantly shortened time to metamorphosis at 20 °C, but not at 25 °C. At 25 °C, tadpoles metamorphosed significantly faster than those at 20 °C independent of antidepressant exposure. Venlafaxine reduced body mass at 25 °C, but not at 20 °C. Temperature and antidepressant exposure affected the percent of tadpoles showing a startle response. Tadpoles at 20 °C displayed significantly more responses than at 25 °C. Exposure to fluoxetine also increased the percent of tadpoles showing a startle response. Venlafaxine reduced the percent of motionless tadpoles at 25 °C but not at 20 °C. While our results showed that antidepressants can affect the timing of metamorphosis in tadpoles, warmer temperatures overrode these effects and caused a reduction in an important reaction behavior (startle response). Future studies should address how warmer global temperatures may exacerbate or negate the effects of environmental pollutants., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Many animals use forms of gesture and dance to communicate with conspecifics in the breeding season, though the mechanisms of this behavior are rarely studied. Here, we investigate the hormone basis of such visual signal behavior in Bornean rocks frogs (Staurois parvus). Our results show that males aggregating at breeding waterfalls have higher testosterone (T) levels, and we speculate that this hormone increase is caused by social cues associated with sexual competition. To this end, we find that T levels in frogs at the waterfall positively predict the number waving gestures-or "foot flags"-that males perform while competing with rivals. By contrast, T does not predict differences in male calling behavior. In these frogs, vocal displays are used largely as an alert signal to direct a rival's attention to the foot flag; thus, our results are consistent with the view that factors related to reproductive context drive up T levels to mediate displays most closely linked to male-male combat, which in this case is the frog's elaborate gestural routine., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Environmental challenges early in development can result in complex phenotypic trade-offs and long-term effects on individual physiology, performance and behavior, with implications for disease and predation risk. We examined the effects of simulated pond drying and elevated water temperatures on development, growth, thermal physiology and behavior in a North American amphibian, Rana sphenocephala. Tadpoles were raised in outdoor mesocosms under warming and drying regimes based on projected climatic conditions in 2070. We predicted that amphibians experiencing the rapid pond drying and elevated pond temperatures associated with climate change would accelerate development, be smaller at metamorphosis and demonstrate long-term differences in physiology and exploratory behavior post-metamorphosis. Although both drying and warming accelerated development and reduced survival to metamorphosis, only drying resulted in smaller animals at metamorphosis. Around 1 month post-metamorphosis, animals from the control treatment jumped relatively farther at high temperatures in jumping trials. In addition, across all treatments, frogs with shorter larval periods had lower critical thermal minima and maxima. We also found that developing under warming and drying resulted in a less exploratory behavioral phenotype, and that drying resulted in higher selected temperatures in a thermal gradient. Furthermore, behavior predicted thermal preference, with less exploratory animals selecting higher temperatures. Our results underscore the multi-faceted effects of early developmental environments on behavioral and physiological phenotypes later in life. Thermal preference can influence disease risk through behavioral thermoregulation, and exploratory behavior may increase risk of predation or pathogen encounter. Thus, climatic stressors during development may mediate amphibian exposure and susceptibility to predators and pathogens into later life stages., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

Acoustic communication plays a vital role in frog reproduction. In most anuran species, long-distance sound communication is one-way from males to females; during the reproductive season, males produce species-specific advertisement calls to attract gravid females, and females are generally silent but perform phonotactic movements that lead to amplexus. One exception is the concave-eared torrent frog (Odorrana tormota). In this species, females produce courtship calls that elicit antiphonal vocalizations by males, followed by precise phonotactic movements. The large odorous frog O. graminea (previously Odorrana livida) in southern China is subject to the same environmental constraints as O. tormota, with which it is sympatric; it is unclear whether their sound communication is one-way or bidirectional. Here, we provide the first data on female O. graminea vocalizations and their functions. Using playbacks of female calls, we conducted acoustic behavioral experiments in the laboratory in response to which males emitted single- or multi-note antiphonal calls with a varying fundamental frequency. Moreover, they were attracted to female call playbacks, exhibiting precise phonotaxis. The female courtship call-male response interaction thus forms a duet between partners of a receptive pair. These results demonstrate that this unique communication system likely reflects an adaptation to an environment in which short-distance communication is at a premium given the high levels of ambient noise., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

While most anuran species are highly vocal, few of them seem to be endowed with a complex call repertoire. Odorrana tormota, combines a remarkable vocalization complexity with auditory sensitivity over an extended spectral range spanning from audible to ultrasonic frequencies. This species is also exceptional for its ability to modify its middle ear tuning by closing the Eustachian tubes (ET). Using scanning laser Doppler vibrometry, the tympanal vibrations were measured to investigate if the tuning shift caused by the ET closure contributes to intraspecific acoustic communication. To gain insight into the inner ear frequency selectivity and sensitivity of this species, distortion product otoacoustic emissions were recorded at multiple frequency-level combinations. Our measurements of inner ear responses indicated that in O. tormota each sex is more sensitive to the frequencies of the other sex's vocalizations, female ears are more sensitive to 2-7 kHz, while male ears are more sensitive to 3-15 kHz. We also found that in both sexes the ET closure impacts the sensitivity of the middle and inner ear at frequencies used for communication with conspecifics. This study broadens our understanding of peripheral auditory mechanisms contributing to intraspecific acoustic communication in anurans., (© 2022. The Author(s).)

Amphibians have been facing global declines over the last decades from direct and indirect effects of anthropogenic activities. A contributor to declines is waterway contamination from agricultural runoffs of pesticides such as neonicotinoids. Beyond direct and indirect effects of the pesticide, few studies have investigated the possible interactions between neonicotinoids and natural environmental stressors across larval development, which could alter the strength and direction of observed neonicotinoid effects. The present study used a fully crossed design to investigate how a concentration of imidacloprid (a neonicotinoid; 10 µg/L) measured in surface waters interacted with low and high population densities (0.33 and 1 tadpole/L, respectively), an important environmental stressor, to influence behavior and development across metamorphosis in wood frogs (Rana sylvatica), known to breed in agricultural landscapes. Behaviors were measured in the absence and presence of predation cues using open-field tests at three distinct developmental stages, up to the metamorph stage. We found that imidacloprid did not interact with population density or independently affect behaviors in the absence of predation cues. However, individuals raised at high density compared with low density were more active at an early developmental stage but less active at metamorphic climax. Furthermore, both density and imidacloprid independently decreased the natural freezing response of tadpoles to predation cues. Finally, we found that distance traveled in the open-field test was weakly repeatable between aquatic stages but not repeatable across metamorphosis, a pattern that was not affected by treatments. The present study provides novel insights on the ecotoxicology of imidacloprid in the presence of a natural stressor, highlighting the importance of including behavioral assays and natural stressors in studies of amphibian ecotoxicology. Environ Toxicol Chem 2022;41:2968-2980. © 2022 SETAC., (© 2022 SETAC.)

Among others, the global change involves a worldwide increase in cropland area, with the concomitant rise in nitrogenous fertilizer supplementation and species range alterations, including parasites and pathogens. As most animals rely on their immune systems against these infectious agents, studying the potential effects of nitrogenous compounds on animal immune response is vital to understand their susceptibility to infections under these altered circumstances. Being subjected to an alarming process of global declines, amphibians are the object of particular attention, given their sensitivity to these compounds, especially to ammonium. Moreover, whereas adults can actively avoid polluted patches, larvae are confined within their waterbodies, thus exposed to contaminants in it. In this work, we test whether chronic exposure to a sublethal dose of ammonium during the larval stage of Pelophylax perezi frogs, released from all contamination after metamorphosis, leads to impaired inflammatory response to phytohemagglutinin in adults. We also test whether such a response differs between agrosystem individuals as compared with conspecifics from natural habitats. We found negative carryover effects of chronic exposure of larvae to ammonium on adult inflammatory response, which could imply a greater susceptibility to pathogens and parasites. However, this damage is only true for males, which, according to the immunocompetence handicap hypothesis, could be a consequence of a testosterone-triggered impairment of male immune function. In disagreement with our prediction, however, we detected no differences in the inflammatory response of agrosystem frogs to phytohemagglutinin as compared with natural habitat conspecifics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

We assessed the macrogeographic and neuroendocrine correlates of behavioral variation exhibited by juveniles, an important life stage for dispersal, across the expansive range of the wood frog. By rearing animals from eggs in a common garden then using a novel environment test, we uniquely demonstrated differential expression of juvenile behaviors among 16 populations spanning 8° latitude. On the individual level, cluster analysis indicated three major behavior profiles and principal component analysis resolved four unique axes of behavior, including escape, foraging, food intake, feeding efficiency. We found that increased escape behavior was associated with lower adrenocorticotropic hormone (ACTH)-induced circulating corticosterone (CORT) levels, however, foraging and food intake behaviors were not associated with either resting or ACTH-induced CORT. At the population level, the expression of foraging behaviors increased with latitude while food intake behaviors declined with latitude, which raised several hypotheses of eco-evolutionary processes likely driving this variation. Given that these behaviors covary along the same ecological gradient as locally adapted developmental traits, genomic studies in this species could provide deep insights into how HPA/I activity is associated with the eco-evolutionary processes that structure intraspecific variation in morphology and behavior., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Environmental stress is a major driver of ecological and evolutionary processes in nature. To cope with stress, organisms can adjust through phenotypic plasticity and/or adapt through genetic change. Here, we compared short-term behavioural (activity) and physiological (corticosterone levels, CORT) responses of Rana arvalis tadpoles from two divergent populations (acid origin, AOP, versus neutral origin, NOP) to acid and predator stress. Tadpoles were initially reared in benign conditions at pH 7 and then exposed to a combination of two pH (acid versus neutral) and two predator cue (predator cue versus no predator cue) treatments. We assessed behavioural activity within the first 15 min, and tissue CORT within 8 and 24 h of stress exposure. Both AOP and NOP tadpoles reduced their activity in acidic pH, but the response to the predator cue differed between the populations: AOP tadpoles increased whereas NOP tadpoles decreased their activity. The AOP and NOP tadpoles differed also in their CORT responses, with AOP being more responsive (CORT levels of NOP tadpoles did not differ statistically across treatments). After 8 h exposure, AOP tadpoles had elevated CORT levels in the acid-predator cue treatment and after 24 h exposure they had elevated CORT levels in all three stress treatments (relative to the benign neutral-no-cue treatment). These results suggest that adaptation to environmental acidification in R. arvalis is mediated, in part, via behavioural and hormonal plasticity., (© 2022. The Author(s).)

Artificial light at night (ALAN) is a pervasive pollutant that influences wildlife at both the individual and community level. In this study, we tested the individual-level effects of ALAN on three species of tadpole prey and their newt predators by measuring prey pigmentation and predator and prey mass. Then we evaluated whether the individual-level effects of ALAN on pigmentation and mass had cascading community-level effects by assessing the outcome of predator-prey interactions. We found that spring peepers exposed to ALAN were significantly darker than those reared under control conditions. Additionally, wood frogs reared in ALAN conditions were significantly smaller than those reared in control conditions. In contrast, Eastern newts collected earlier in the spring that were exposed to ALAN were significantly larger than controls while those collected later in the spring were not affected by ALAN, suggesting phenological differences in the effect of ALAN. To understand how changes in pigmentation and size due to ALAN influence predation rates, we ran predation assays in both ALAN-polluted and ALAN-free outdoor environments. After the predation assay, the size disparity in wood frogs reared in ALAN was eliminated such that there was no longer a treatment difference in wood frog size, likely due to size-selective predation. This demonstrates the beneficial nature of predators' selective pressure on prey populations. Lastly, despite individual-level effects of ALAN on pigmentation and mass, we did not detect cascading community-level effects on predation rates. Overall, this study highlights important species-level distinctions in the effects of ALAN. It also emphasizes the need to incorporate ecological complexity to understand the net impact of ALAN., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The freeze-tolerant wood frog, Rana sylvatica, is one of the very few vertebrate species known to endure full body freezing in winter and thaw in early spring without any significant sign of damage. Once frozen, wood frogs show no cardiac or lung activity, brain function, or physical movement yet resume full physiological and biochemical functions within hours after thawing. The miraculous ability to tolerate such extreme stresses makes wood frogs an attractive model for identifying the molecular mechanisms that can promote freeze/thaw endurance. Recapitulating these pro-survival strategies in transplantable human cells and organs could improve viability post-thaw leading to better post-transplant outcomes, in addition to providing more time for adequate distribution of these transplantable materials across larger geographical areas. Indeed, several laboratories are beginning to mimic the pro-survival responses observed in wood frogs to preservation of human cells, tissues and organs and, to date, a few trials have been successful in extending preservation time prior to transplantation. In this review, we discuss the biology of the freeze-tolerant wood frog, current advances in biobanking based on these animals, and extend our discussion to future prospects for cryopreservation as an aid to regenerative medicine., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Wood frogs, Rana sylvatica, endure the freezing of ∼65% of total body water while overwintering in cold climates, enduring not only internal ice formation but also long-term anoxia due to cessation of heartbeat and breathing. Thawing restores perfusion but rapid reoxygenation can increase vulnerability to reactive oxygen species and induce oxidative damage. This study provides a first assessment of antioxidant capacity, DNA damage, and DNA repair responses comparing freeze/thaw and anoxia/reoxygenation in liver and skeletal muscle of wood frogs. Oxidation of guanine resides in DNA did not change under either stress but total antioxidant capacity rose in both tissues under anoxia. Relative expression of eight proteins involved in double-stranded break repair (Mre11, Rad50, phospho-p95, XLF, DNA ligase IV, XRCC4, Ku70, Rad51) were assessed in both tissues. Freezing suppressed Ku70 and Rad51 in liver and Rad51 in muscle but levels rose again after thawing. Anoxia exposure suppressed XLF, Ku70 and Rad51 proteins in muscle. However, in liver, anoxia exposure led to elevated Mre11, Ku70 and DNA ligase IV, the former two belonging to the MRN complex that binds DNA and marks sites of double stranded breaks (DSBs). Large increases in Mre11 and Ku70 expression suggested DSB damage in liver under anoxia but not during freezing, whereas muscle was resistant to DSB damage under both stresses. These data indicate that DNA damage is minimal during whole body freezing due to tissue and stress specific regulation of antioxidant capacity and DNA damage repair to preserve genomic integrity., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Wildlife monitoring programs are instrumental for the assessment of species, habitat status, and for the management of factors affecting them. This is particularly important for species found in freshwater ecosystems, such as amphibians, as they have higher estimated extinction rates than terrestrial species. We developed and validated two species-specific environmental DNA (eDNA) protocols and applied them in the field to detect the Hazara Torrent Frog (Allopaa hazarensis) and Murree Hills Frog (Nanorana vicina). Additionally, we compared eDNA surveys with visual encounter surveys and estimated site occupancy. eDNA surveys resulted in higher occurrence probabilities for both A. hazarensis and N. vicina than for visual encounter surveys. Detection probability using eDNA was greater for both species, particularly for A. hazarensis. The top-ranked detection model for visual encounter surveys included effects of both year and temperature on both species, and the top-ranked occupancy model included effects of elevation and year. The top-ranked detection model for eDNA data was the null model, and the top-ranked occupancy model included effects of elevation, year, and wetland type. To our knowledge, this is the first time an eDNA survey has been used to monitor amphibian species in the Himalayan region., (© 2022. The Author(s).)

Competition within and among species can play a key role in structuring the assemblages of anuran tadpoles. Previous studies have reported that tadpoles of the invasive cane toad (Rhinella marina) are more strongly disadvantaged by the presence of native frog tadpoles than by the same number of conspecific toad tadpoles. That effect might arise from a lack of coevolution of the invasive toad with its competitors; and/or from a generalized superiority of frog tadpoles over toad tadpoles. To clarify those possibilities, we conducted experimental trials using the larvae of a native rather than invasive toad (Bufo japonicus formosus in Japan) exposed to larvae of native anurans (the sympatric frogs Rana japonica and Rana ornativentris and the parapatric toad Bufo japonicus japonicus). In intraspecific competition trials, higher densities of B. j. formosus prolonged the larval period and reduced size at metamorphosis, but did not affect survival. In interspecific competition trials, the effects of the other anuran species on B. j. formosus were similar to the effects of the same number of conspecific larvae. This similarity in impact of interspecific versus intraspecific competition argues against any overall competitive superiority of frog larvae over toad larvae. Instead, the vulnerability of larval cane toads to frog tadpoles may result from a lack of coevolutionary history., (© 2022. The Author(s).)

Predators are the key regulators of prey populations in different environments. However, they are not always present but often come and go. Moreover, the level of predation risk varies based on habitat complexity, resource availability and other ecological factors. Hence, it is adaptive for prey animals to match their antipredator responses with the level of predation risk as such responses are costly to produce and maintain, and there exists a trade-off between fitness-enhancing and fitness-reducing activities. To test whether larval Indosylvirana adjust their antipredator responses based on the level of predation risk, we designed an experiment in which tadpoles of Indosylvirana indica were exposed to nil, low, moderate or high levels of predation risk to assess the effect of risk on growth, behaviour, morphology and life-history traits. We also determined the whole-body corticosterone levels to assess the physiological changes associated with the level of predation risk. Our results show that the growth rate of tadpoles experiencing varying levels of predation risk was similar although there was a trend towards a higher growth rate at moderate and high risks. Surprisingly, tadpoles experiencing predation risk did not reduce their activity. However, the activity of tadpoles experiencing differential predation risk was comparable. Similarly, the use of shelter was comparable among tadpole groups, with an overall higher level of activity in the afternoon compared to other times. Although a few morphological traits were different among tadpole groups, there was no trend or pattern. Moreover, these morphological alterations did not contribute to overall tadpole shape changes. Physiologically, tadpoles experiencing predation risk had significantly lower levels of corticosterone compared to those without risk. Interestingly, corticosterone titres among tadpoles facing varying levels of predation risk were similar. Metamorphic traits of individuals in the various predation risk groups were significantly different. Tadpoles experiencing moderate and high risks emerged at a larger size, and those experiencing the highest level of risk metamorphosed at the earliest. The results of our study thus show that the antipredator responses of larval I. indica do not match the level of predation risk, indicating that physiological and ecological constraints could limit the extent to which prey species respond to predation risk and its intensity.

For animals to survive until reproduction, it is crucial that juveniles successfully detect potential predators and respond with appropriate behavior. The recognition of cues originating from predators can be innate or learned. Cues of various modalities might be used alone or in multi-modal combinations to detect and distinguish predators but studies investigating multi-modal integration in predator avoidance are scarce. Here, we used wild, naive tadpoles of the Neotropical poison frog Allobates femoralis ( Boulenger, 1884) to test their reaction to cues with two modalities from two different sympatrically occurring potential predators: heterospecific predatory Dendrobates tinctorius tadpoles and dragonfly larvae. We presented A. femoralis tadpoles with olfactory or visual cues, or a combination of the two, and compared their reaction to a water control in a between-individual design. In our trials, A. femoralis tadpoles reacted to multi-modal stimuli (a combination of visual and chemical information) originating from dragonfly larvae with avoidance but showed no reaction to uni-modal cues or cues from heterospecific tadpoles. In addition, visual cues from conspecifics increased swimming activity while cues from predators had no effect on tadpole activity. Our results show that A. femoralis tadpoles can innately recognize some predators and probably need both visual and chemical information to effectively avoid them. This is the first study looking at anti-predator behavior in poison frog tadpoles. We discuss how parental care might influence the expression of predator avoidance responses in tadpoles., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

The skins of frogs of the family Ranidae are particularly rich sources of biologically active peptides, among which antimicrobial peptides (AMPs) constitute the major portion. Some of these have attracted the interest of researchers because they possess both antimicrobial and anticancer activities. In this study, with 'shotgun' cloning and MS/MS fragmentation, three AMPs, homologues of family brevinin-1 (brevinin-1HL), and temporin (temporin-HLa and temporin-HLb), were discovered from the skin secretion of the broad-folded frog, Hylarana latouchii. They exhibited various degrees of antimicrobial and antibiofilm activities against test microorganisms and hemolysis on horse erythrocytes. It was found that they could induce bacteria death through disrupting cell membranes and binding to bacterial DNA. In addition, they also showed different potencies towards human cancer cell lines. The secondary structure and physicochemical properties of each peptide were investigated to preliminarily reveal their structure-activity relationships. Circular dichroism spectrometry showed that they all adopted a canonical α-helical conformation in membrane-mimetic solvents. Notably, the prepropeptide of brevinin-1HL from H. latouchii was highly identical to that of brevinin-1GHd from Hylarana guentheri, indicating a close relationship between these two species. Accordingly, this study provides candidates for the design of novel anti-infective and antineoplastic agents to fight multidrug-resistant bacteria and malignant tumors and also offers additional clues for the taxonomy of ranid frogs., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In the present study, responses of the Chinese brown frog (Rana chensinensis) to exposure to different doses and duration of Octyphenol (OP) which degraded from alkylphenol ethoxylates (APEs) were characterized during the adult periods. The effects of OP on growth, development and reproduction and the expression of genes in gonad were investigated. The expression levels of fshβ, lhβ, fshr and lhr had significant differences as the exposure time increased. The pathological and morphological changes were also observed in the OP treatments. Furthermore, the number of TUNEL positive cells and the TUNEL index was elevated after exposed to OP. Besides that, OP treatment could influence its mating behavior and reduce the fertilization rates. Taken together, these results indicated that OP disrupt sex steroid signaling, normal development of spermatogenesis, courtship behavior of male frogs and decline fertilization rate in R. chensinensis., (Copyright © 2021 Elsevier B.V. All rights reserved.)

For the first time, the results of a comparative analysis of free amino acids in the blood plasma of amphibians of the Ural fauna are presented: an endemic species (Siberian salamander (Salamandrella keyserlingii Dybovsky, 1870)) and an invasive species (marsh frog (Pelophylax ridibundus Pallas, 1771)). The species diversity of adaptive strategies of thermoresistant amphibionts both to negative and positive temperatures is shown., (© 2021. Pleiades Publishing, Ltd.)

Multimodal communication is common in the animal kingdom. It occurs when animals display by stimulating two or more receiver sensory systems, and often arises when selection favors multiple ways to send messages to conspecifics. Mechanisms of multimodal display behavior are poorly understood, particularly with respect to how animals coordinate the production of different signals. One important question is whether all components in a multimodal display share an underlying physiological basis, or whether different components are regulated independently. We investigated the influence of androgen receptors (ARs) on the production of both visual and vocal signal components in the multimodal display repertoire of the Bornean rock frog (Staurois parvus). To assess the role of AR in signal production, we treated reproductively active adult males with the antiandrogen flutamide (FLUT) and measured the performance of each component signal in the multimodal display. Our results show that blocking AR inhibited the production of multiple visual signals, including a conspicuous visual signal known as the "foot flag," which is produced by rotating the hind limb above the body. However, FLUT treatment caused no measurable change in vocal signaling behavior, or in the frequency or fine temporal properties of males' calls. Our study, therefore, suggests that activation of AR is not a physiological prerequisite to the coordination of multiple signals, in that it either does not regulate all signaling behaviors in a male's display repertoire or it does so only in a context-dependent manner., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

Wood frogs (Rana sylvatica) can survive extended periods of whole body freezing. Freezing imparts multiple stresses on cells that include anoxia and dehydration, but these can also be experienced as independent stresses. Under anoxia stress, energy metabolism is suppressed, and pro-survival pathways are prioritized to differentially regulate some transcription factors including OCT1 and OCT4. Jumonji C domain proteins (JMJD1A and JMJD2C) are hypoxia responsive demethylases whose expression is accelerated by OCT1 and OCT4 which act to demethylate genes related to the methionine cycle. The responses by these factors to 24 h anoxia exposure and 4 h aerobic recovery was analyzed in liver and skeletal muscle of wood frogs to assess their involvement in metabolic adaptation to oxygen limitation. Immunoblot results showed a decrease in JMJD1A levels under anoxia in liver and muscle, but an increase was observed in JMJD2C demethylase protein in anoxic skeletal muscle. Protein levels of adenosylhomocysteinase (AHCY) and methionine adenosyl transferase (MAT), enzymes of the methionine cycle, also showed an increase in the reoxygenated liver, whereas the levels decreased in muscle. A transcription factor ELISA showed a decrease in DNA binding by OCT1 in the reoxygenated liver and anoxic skeletal muscle, and transcript levels also showed tissue specific gene expression. The present study provides the first analysis of the role of the OCT1 transcription factor, associated proteins, and lysine demethylases in mediating responses to anoxia by wood frog tissues., (Copyright © 2021 Elsevier B.V. All rights reserved.)

A decline in sperm quality with age is a common prediction of senescence-based hypotheses and empirical studies. While widely studied across taxa, there is little known on the effect of ageing on sperm quality in amphibians, especially in captive populations used for controlled propagation and reintroduction efforts. Here, we investigated variation in sperm quality metrics (i.e., motility, concentration, and morphology) in the endangered Mississippi gopher frog (Lithobates sevosus) among males of three age categories using individuals from captive breeding populations housed at three different zoological institutions. Different aged males across the species expectant lifespan (1-9, 1-2, 3-4, and 8-9-year-old subcategories) were chosen in an attempt to identify an optimal breeding age relevant for captive breeding programs. Moreover, we explored and statistically controlled for potential differences in sperm quality which may be attributed to the type of induction hormones and source populations that differed among institutions. Results indicated that males of different ages did not differ in sperm motility or concentration. However, we did find that older males (8-9 years old) had significantly longer sperm than other age categories and younger males (1-2 years old) had significantly more atypical sperm than other age categories. Furthermore, we found no significant differences in any sperm quality metrics between the different induction hormones or source populations used at the different institutions. Within a captive breeding program, this information is especially valuable as our results indicate that males that have only recently sexually matured may not be ready to breed, while older males maintain sperm quality metrics presumably related to fertilization success., (© 2021 Wiley Periodicals LLC.)

Host's defense against external challenges activates an inflammatory response regulated by a set of chemical signals, including hormones. These immunomodulatory hormones, such as corticosterone, testosterone, and melatonin, trigger the systemic immune responses responsible for inflammatory assembly and resolution. This study aimed to investigate the effects of an immune challenge on endocrine and innate immune responses in the bullfrog (Lithobates catesbeianus). Adult males were intraperitoneally injected with lipopolysaccharide (LPS; 2 mg/kg) or saline, and blood samples were collected 6 and 24 h after injection for measurement of neutrophil/lymphocyte ratio, blood leukocyte phagocytosis, plasma bacterial killing ability, and plasma levels of corticosterone, melatonin, and testosterone. Our results showed LPS-induced increased neutrophil/lymphocyte ratio and leukocyte phagocytosis, and decreased melatonin and testosterone plasma levels, which were more pronounced 24 h after injection. Overall, we conclude that LPS intraperitoneal injection can activate the innate immune response and modulate the hormonal profile of the bullfrogs, with effects more pronounced 24 h than 6 h after treatment., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Complete functional descriptions of the induction sequences of phenotypically plastic traits (perception to physiological regulation to response to outcome) should help us to clarify how plastic responses develop and operate. Ranid tadpoles express several plastic antipredator traits mediated by the stress hormone corticosterone, but how they influence outcomes remains uncertain. We investigated how predator-induced changes in the tail morphology of wood frog (Rana sylvatica) tadpoles influenced their escape performance over a sequence of time points when attacked by larval dragonflies (Anax junius). Tadpoles were raised with no predator exposure, chemical cues of dragonflies added once per day, or constant exposure to caged dragonflies crossed with no exogenous hormone added (vehicle control only), exogenous corticosterone, or metyrapone (a corticosteroid synthesis inhibitor). During predation trials, we detected no differences after four days, but after eight days, tadpoles exposed to larval dragonflies and exogenous corticosterone had developed deeper tail muscles and exhibited improved escape performance compared to controls. Treatment with metyrapone blocked the development of a deeper tail muscle and resulted in no difference in escape success. Our findings further link the predator-induced physiological stress response of ranid tadpoles to the development of an antipredator tail morphology that confers performance benefits.

Phylogeography can reconstruct historical evolutionary processes by comparing historical patterns of gene flow, divergence among species and by using species distribution models (SDM) upon geographic distribution. We investigate the phylogeographic patterns of Anatolian brown frogs including R. macrocnemis and R. tavasensis as well as the Hyrcanian brown frog, R. pseudodalmatina, using a fragment of the mitochondrial 16S rRNA gene for 145 specimens across the entire range of these frogs. We calculate parameters of molecular diversity, such as the number of variable sites (S), the number of haplotypes (h), haplotype diversity (Hd) and nucleotide diversity (π). We generated a haplotype network and used three methods (Neutrality tests, mismatch distributions and Bayesian skyline plots) to reconstruct the demographic histories of R. macrocnemis and R. pseudodalmatina. Finally, we used SDMs to predict the habitat suitability for three periods: The Present Day, the Last Glacial Maximum (LGM) and the future until 2070 for R. macrocnemis and R. pseudodalmatina. Our phylogenetic analyses support a late Miocene origin of Anatolian and Hyrcanian lineages. Hyrcanian brown frogs were enclosed in lowlands of the southern coast of the Caspian Sea after the uplift of the Elburz range and the Armenian plateau. The formation of a salinity belt from the north Aegean corridor (the south western Turkey) to northward during the Late Tortonian led to the subdivision of ancestor of the Anatolian lineage into today isolated western and eastern populations. The salinity belt had a considerable impact on the divergence of R. tavasensis from R. macrocnemis. Combined historical demographic analyses and SDMs revealed a rapid expansion occurring during the Pleistocene in R. macrocnemis and R. pseudodalmatina. Currently, suitable habitat for R. macrocnemis has declined compared to the LGM, and the species is predicted to do even worse under future climatic conditions. In contrast, R. pseudodalmatina found suitable habitat from the LGM to present within its restricted distribution area; it is predicted to do fine even under future climate., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Restrictions on the production and use of some highly toxic and persistent flame retardants has resulted in the increased use of alternative phosphate flame retardants that are less-well characterized. The brominated organophosphate ester flame retardant, tris(tribromoneopentyl) phosphate (CAS 19186-97-1, molecular formula C15H24Br9O4P, molecular weight 1018.47 g/mol, acronym TTBrNP) is a compound with potential to bioaccumulate and disrupt endocrine functions. To determine the toxicity of TTBrNP, two Canadian native amphibian species, Lithobates sylvaticus and L. pipiens, were acutely (embryos and Gosner stage 25 (GS25) tadpoles) or sub-chronically (GS25-41 tadpoles) exposed to the following nominal concentrations of TTBrNP: 0 (water and solvent controls), 30.6, 61.3, 122.5 and 245.0 μg/L. Note, measured concentrations declined with time (i.e., 118%-30% of nominal). There was high survival for both species after acute and sub-chronic exposures, where 75%-100% survived the exposures, respectively. There were no differences in the occurrence of abnormalities or hatchling size between controls and TTBrNP treatments for either species exposed acutely as embryos or tadpoles. Furthermore, after 30 d of sub-chronic exposure of L. pipiens tadpoles to TTBrNP there were no effects on size, developmental stage, liver somatic index or sex ratio. Bioconcentration factors were low at 26 ± 3.1 L/kg ww in tadpoles from all treatments, suggesting biotransformation or limited bioavailability via aquatic exposures. Thus, using two species of anurans at different early larval stages, we found TTBrNP up to 245 μg/L to have no overt detrimental effects on survival or morphological responses that would suggest fitness-relevant consequences., Competing Interests: Declaration of competing interest None., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

Animals show a rich diversity of signals and displays. Among the many selective forces driving the evolution of communication signals, one widely recognized factor is the structure of the environment where animals communicate. In particular, animals communicating by sounds often emit acoustic signals from specific locations, such as high up in the air, from the ground or in the water. The properties of these different display sites may impose different constraints on sound production, and therefore drive signal evolution. Here, we used comparative phylogenetic analyses to assess the relationship between calling site (aquatic versus nonaquatic), body size and call dominant frequency of 160 frog species from the families Ranidae, Leptodactylidae and Hylidae. We found that the frequency of frogs calling from the water was lower than that of species calling outside of the water, a trend that was consistent across the three families studied. Furthermore, phylogenetic path analysis revealed that call site had both direct and indirect effects on call frequency. Indirect effects were mediated by call site influencing male body size, which in turn was negatively associated with call frequency. Our results suggest that properties of display sites can drive signal evolution, most likely not only through morphological constraints imposed on the sound production mechanism, but also through changes in body size, highlighting the relevance of the interplay between morphological adaptation and signal evolution. Changes in display site may therefore have important evolutionary consequences, as it may influence sexual selection processes and ultimately may even promote speciation., (© 2020 The Authors. Journal of Evolutionary Biology published by John Wiley & Sons Ltd on behalf of European Society for Evolutionary Biology.)

Amphibians are experiencing worldwide declines due to increasing anthropogenetic disturbances. However, the genetic variability and hence adaptability are still unknown for most frogs. We integrated the mitochondrial (ND2 gene), nuclear (TYR gene) and major histocompatibility complex (MHC) loci, to clarify the demographic patterns and immune-gene diversity of the Lolokou Sucker Frog (Amolops loloensis). Demographic analysis of the ND2 and TYR genes suggested that the Lolokou Sucker Frog experienced a population expansion within the last 10,000 years. High MHC diversity was detected, which has likely resulted from positive selection, indicating the current diversity bodes well for the species' adaptive potential to pathogenic challenges. These findings broaden our knowledge on the population history and evolution adaptation of the reclusive torrent frog, and conservation implications are provided.

Increasing evidence suggests that phenotypic plasticity can play a critical role in ecotoxicology. More specifically, induced pesticide tolerance, in which populations exposed to a contaminant show increased tolerance to the contaminants later, has been documented in multiple taxa. However, the physiological mechanisms of induced tolerance remain unclear. We hypothesized that induced pesticide tolerance is the result of a generalized stress response based on previous studies showing that both natural stressors and anthropogenic stressors can induce tolerance to pesticides. We tested this hypothesis by first exposing larval wood frogs (Rana sylvatica) to either an anthropogenic stressor (sublethal carbaryl concentration), a natural stressor (cues from a caged predator), or a simulated stressor via exogenous exposure to the stress hormone corticosterone (125 nM). We also included treatments that inhibited corticosterone synthesis with the compound metyrapone (MTP). We then exposed the larvae to a lethal carbaryl treatment to assess time to death. We found that prior exposure to 125 nM of exogenous CORT and predator cues induced tolerance to a lethal concentration of carbaryl through a slight delay in time to death. Pre-exposure to sublethal carbaryl, as well as MTP alone or in combination with predator cues, did not induce tolerance to the lethal carbaryl concentration relative to the ethanol vehicle control treatment. Our study provides evidence that pesticide tolerance can be induced by a generalized stress response both in the presence and absence (exogenous CORT) of specific cues and highlights the importance of considering physiological ecology and environmental context in ecotoxicology.

Natural populations often persist at the landscape scale as metapopulations, with breeding units (subpopulations) experiencing temporal extinction and recolonization events. Important parameters to forecast population viability in these systems include the ratio of the effective number of breeders (N b ) to the total number of adults (N a ) and migration rates among subpopulations. Here, we present the results of a 10-year integrative monitoring program of a metapopulation of the Iberian green frog (Pelophylax perezi) in central Spain. We characterized population dynamics at two main breeding ponds (Gravera and Laguna) using capture-mark-recapture data to estimate N a in each breeding season, and multilocus genotypes to estimate the effective population size (N e ), N b , individual breeding success, and migration rates. Both ponds experienced population decline after a dry season, with Gravera subsequently recovering and Laguna suffering a bottleneck associated with genetic impoverishment. In this subpopulation, average allelic richness and private alleles decreased from 2010 (10.87 and 1.67, respectively) to 2018 (8.0 and 0.20). The N b /N a ratio in Laguna in 2018 was twice as high (0.95) than in Gravera (0.41) or in pre-bottleneck Laguna (0.50), suggesting plasticity or genetic compensation through increased individual breeding success. Migration rates were asymmetric between ponds, with a stronger contribution from Gravera to Laguna (29.9% vs. 16.2% in the opposite direction) that may result in a rescue effect. This study emphasizes the importance of integrative demographic approaches for the monitoring of natural populations based on a better understanding of their spatio-temporal dynamics, which provides valuable information for conservation actions., (© 2020 International Society of Zoological Sciences, Institute of Zoology/Chinese Academy of Sciences and John Wiley & Sons Australia, Ltd.)

Invasive species pose a major threat to global biodiversity. The effects of invasive species can be strongly influenced and potentially mediated by their reproductive characteristics, such as fecundity, egg production, and duration and number of reproductive events. Selection for smaller body size at first reproduction can also play a role in their establishment, facilitating colonization and spread. The American bullfrog, native to the eastern U.S. (Lithobates catesbeianus), is a species that has invaded more than 40 countries across 4 continents. This species has become especially prevalent in the western United States since its introduction in the early 1900s. This study characterized reproductive characteristics of bullfrogs with emphasis on the minimum size at which males and females reach sexual maturity in the Willamette Valley, Oregon, USA invasion range. We collected and dissected 121 individuals in 2013 and 2017, quantifying characteristics of sexual maturity including snout-vent length, total length, sex, tympanum diameter, presence of distended oviducts or eggs for females, and testes length and sperm activity in males. Our results showed that the minimum reproductive size of both males and females was smaller relative to bullfrogs in their native range as well as in populations across their invasive range. Reduction in size at reproductive maturity is likely impacting the invasive success of American bullfrogs and this study gives us insight on management actions to control the invasion. Applying this insight, managers can adjust their definition of reproductively active adults, increasing the target population of culling and other control methods.

The lungs and skin are important respiratory organs in Anura, but the pulmonary structure of amphibians remains unclear due to the lack of a suitable procedure. This study improved the procedure used for fixing lungs tissues and used light microscopy, transmission electron microscopy and scanning electron microscopy to reveal the differences in the lung and skin morphologies between Pelophylax nigromaculatus (P. nigromaculatus) and Bufo gargarizans (B. gargarizans). In P. nigromaculatus and B. gargarizans, the cystic lungs comprise a continuous outer pulmonary wall on which primary, secondary, and tertiary septa attach, and a number of regular lattices form from raised capillaries and the pulmonary epithelium on the surfaces of the pulmonary wall and septa. Each lattice in P. nigromaculatus consists of several elliptical sheets and flat bottom, and the septa are distributed with denser sheets and have a larger stretching range than the pulmonary wall. The lattice in B. gargarizans consists of thick folds and an uneven bottom with several thin folds, and the septa have more developed thick and thin folds than the pulmonary wall. However, the density of the pulmonary microvilli, the area of a single capillary, the thicknesses of the blood-air barrier, pulmonary wall and septum, and the lung/body weight percentage obtained for B. gargarizans were higher than those found for P. nigromaculatus. In P. nigromaculatus, the dorsal skin has dense capillaries and a ring surface structure with mucus layer on the stratum corneum, and the ventral skin is slightly keratinized. In B. gargarizans, the stratum corneum in both the dorsal and ventral skins is completely keratinized. A fine ultrastructure analysis of P. nigromaculatus and B. gargarizans revealed that the pulmonary septa are more developed than the pulmonary walls, which means that the septa have a stronger respiratory function. The more developed lungs are helpful for the adaptation of B. gargarizans to drought environments, whereas P. nigromaculatus has to rely on more vigorous skin respiration to adapt to a humid environment.