Female frogs communicate with males through blinking.

Blinking is a behavior unique to animal taxa with motile eyelids, such as most amphibians and reptiles as well as all birds and mammals1. Eyelid movement has physiological functions, such as lubricating the cornea and washing away dust, but its potential signaling functions are not well understood1,2. 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 taxa1,3,4. 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.

How do different concentrations of aluminum and zinc affect the survival, body size, morphology and immune system of Physalaemus cuvieri (Fitzinger, 1826) tadpole?

The assessment of amphibian responses as bioindicators of exposure to chemical pollutants is an important tool for conservation of native species. This study aimed to investigate the effects of chronic aluminum (Al) and zinc (Zn) exposure on survival, body size, morphology (malformations), and immune system (leukocyte profile) in P. cuvieri tadpoles. Ecotoxicological analyses were performed utilizing chronic toxicity tests in which 210 tadpoles at the 25th Gosner developmental stage were exposed to Al and Zn. Individuals of P. cuvieri were maintained in glass containers containing various concentrations of aluminum sulfate (0.1, 0.2, or 0.3 mg/L) and zinc sulfate (0.18, 0.27 or 0.35 mg/L), and tests were performed in triplicate. After 14 days, amphibians were weighed, measured and survival rate, malformations in the oral and intestine apparatus, leukocyte profile, and ratio between neutrophils and lymphocytes determined. The differing concentrations of Al and Zn did not produce lethality in P. cuvieri where 95% of the animals survived 326 hr following metal exposure. Individuals exposed to Zn achieved greater body growth and weight gain compared to controls. Aluminum increased weight gain compared controls. These metals also produced malformations of the oral and intestine apparatus and enhanced occurrence of hemorrhages, especially at the highest doses. Lymphocytes were the predominant cells among leukocytes, with lymphopenia and neutrophilia observed following Al and Zn treatment, as evidenced by elevated neutrophil/lymphocyte ratio, an important indicator of stress in animals. Data suggest that further studies need to be carried out, even with metal concentrations higher than those prescribed by CONAMA, to ensure the conservation of this species.

Is thermal sensitivity affected by predation risk? A case study in tadpoles from ephemeral environments.

Changes in environmental temperature may induce variations in thermal tolerance and sensitivity in ectotherm organisms. These variations generate plastic responses that can be analyzed by examining their Thermal Performance Curves (TPCs). Additionally, some performance traits, like locomotion, could be affected by other factors such as biological interactions (e.g., predator-prey interaction). Here, we evaluate if the risk of predation modifies TPCs in Mendoza four-eyed frog (Pleurodema nebulosum, Burmeister, 1861) and Guayapa's four-eyed frog (Pleurodema guayapae, Barrio, 1964), two amphibian species that occur in ephemeral ponds in arid environments. We measured thermal tolerances and maximum swimming velocity at six different temperatures in tadpoles under three situations: control, exposure to predator chemical cues, and exposure to conspecific alarm cues. TPCs were fitted using General Additive Mixed Models. We found that curves of tadpoles at risk of predation differed from those of control mainly in thermal sensitivity parameters. Our work confirms the importance of biotic interactions have in thermal physiology.

Cold-induced skin darkening does not protect amphibian larvae from UV-associated DNA damage.

Amphibian declines are sometimes correlated with increasing levels of ultraviolet radiation (UVR). While disease is often implicated in declines, environmental factors such as temperature and UVR play an important role in disease epidemiology. The mutagenic effects of UVR exposure on amphibians are worse at low temperatures. Amphibians from cold environments may be more susceptible to increasing UVR. However, larvae of some species demonstrate cold acclimation, reducing UV-induced DNA damage at low temperatures. Understanding of the mechanisms underpinning this response is lacking. We reared Limnodynastes peronii larvae in cool (15°C) or warm (25°C) waters before acutely exposing them to 1.5 h of high intensity (80 µW cm-2 ) UVBR. We measured the color of larvae and mRNA levels of a DNA repair enzyme. We reared larvae at 25°C in black or white containers to elicit a skin color response, and then measured DNA damage levels in the skin and remaining carcass following UVBR exposure. Cold-acclimated larvae were darker and displayed lower levels of DNA damage than warm-acclimated larvae. There was no difference in CPD-photolyase mRNA levels between cold- and warm-acclimated larvae. Skin darkening in larvae did not reduce their accumulation of DNA damage following UVR exposure. Our results showed that skin darkening does not explain cold-induced reductions in UV-associated DNA damage in L. peronii larvae. Beneficial cold-acclimation is more likely underpinned by increased CPD-photolyase abundance and/or increased photolyase activity at low temperatures.

The effects of preformed vitamin A and provitamin A carotenoid supplementation on tadpoles of the poison frog Phyllobates vittatus.

Understanding the nutritional requirements of captive animals is necessary for proper animal husbandry, however, the specific dietary requirements for many amphibian species commonly kept in captivity are unknown. Like most vertebrates, frogs cannot synthesize carotenoids and must therefore obtain these essential nutrients through diet. It is unclear if amphibians can cleave provitamin A carotenoids to form vitamin A metabolically within the body, so common practice is to supplement their captive diets with both preformed vitamin A and provitamin A carotenoids. We carried out a feeding experiment in tadpoles of Phyllobates vittatus, a commonly kept poison frog species, to test the effects of supplementing a fish flake diet with a provitamin A carotenoid (2.5 mg/g ß-carotene) and vitamin A (0.033-0.066 µg/mL retinyl acetate), both individually and in combination. Contrary to our expectations, supplementation had either no effect or adverse effects on tadpole growth and survivorship. Tadpoles reared under supplemented diets with vitamin A showed higher mortality rates, coupled with symptoms of hypervitaminosis A. Survivors had a smaller body size and mass at metamorphosis. ß-carotene supplementation alone had no detectable effect. The vitamin A and ß-carotene levels in our supplemented diet have been shown to be harmless or benefit tadpoles of other species, yet our results indicate that adding these amounts to what is found in a generalist fish flake mix can have detrimental effects on P. vittatus tadpoles. More broadly, this study highlights the importance of creating husbandry guidelines based on the specific physiological needs of the species (or species groups) being kept in captivity, rather than general ones for all amphibians, as is often done.

Female Preferences for More Elaborate Signals Are an Emergent Outcome of Male Chorusing Interactions in Túngara Frogs.

AbstractIn chorusing species, conspecific interference exerts strong selection on signal form and timing to maximize conspicuousness and attractiveness within the signaling milieu. We investigated how túngara frog calling strategies were influenced by varied social environments and male phenotypes and how calling interactions influenced female preferences. When chorusing, túngara frog calls consist of a whine typically followed by one to three chucks. In experimental choruses we saw that as chorus size increased, calls increasingly had their chucks overlapped by the high-amplitude beginning section of other callers' whines. Playback experiments revealed that such overlap reduced the attractiveness of calls to females but that appending additional chucks mitigated this effect. Thus, more elaborate calls were preferred when calls suffered overlap, although they were not preferred when overlap was absent. In response to increasing risk of overlap in larger choruses, males increased call elaboration. However, males overwhelmingly produced two-chuck calls in even the largest choruses, despite our results suggesting that additional chucks would more effectively safeguard calls. Furthermore, aspects of male phenotypes predicted to limit call elaboration had negligible or uncertain effects, suggesting that other constraints are operating. These results highlight how complex interrelations among signal form, signaling interactions, and the social environment shape the evolution of communication in social species.

Evidence for individual vocal recognition in a pair-bonding poison frog, Ranitomeya imitator.

Individually distinctive vocalizations are widespread in nature, although the ability of receivers to discriminate these signals has only been explored through limited taxonomic and social lenses. Here, we asked whether anuran advertisement calls, typically studied for their role in territory defense and mate attraction, facilitate recognition and preferential association with partners in a pair-bonding poison frog (Ranitomeya imitator). Combining no- and two-stimulus choice playback experiments, we evaluated behavioral responses of females to male acoustic stimuli. Virgin females oriented to and approached speakers broadcasting male calls independent of caller identity, implying that females are generally attracted to male acoustic stimuli outside the context of a pair bond. When pair-bonded females were presented with calls of a mate and a stranger, they showed significant preference for calls of their mate. Moreover, behavioral responses varied with breeding status: females with eggs were faster to approach stimuli than females that were pair bonded but did not currently have eggs. Our study suggests a potential role for individual vocal recognition in the formation and maintenance of pair bonds in a poison frog and raises new questions about how acoustic signals are perceived in the context of monogamy and biparental care.

Invading nonnative frogs use different microhabitats and change physiology along an elevation gradient.

The coqui frog (Eleutherodactylus coqui) was introduced to the island of Hawai'i in the 1980s, and has spread across much of the island. There is concern they will invade higher elevation areas where negative impacts on native species are expected. It is not known if coqui change behavior and baseline physiology in ways that allow them to invade higher elevations. We investigated where coqui are found across the island and whether that includes recent invasion into higher elevations. We also investigated whether elevation is related to coqui's microhabitat use, including substrate use and height off the forest floor, and physiological metrics, including plasma osmolality, oxidative status, glucose, free glycerol, and triglycerides, that might be associated with invading higher elevations. We found coqui have increased the area they occupy along roads from 31% to 50% and have moved into more high-elevation locations (16% vs. 1%) compared to where they were found 14 years ago. We also found frogs at high elevation on different substrates and closer to the forest floor than frogs at lower elevations-perhaps in response to air temperatures which tended to be warmer close to the forest floor. We observed that blood glucose and triglycerides increase in frogs with elevation. An increase in glucose is likely an acclimation response to cold temperatures while triglycerides may also help frogs cope with the energetic demands of suboptimal temperatures. Finally, we found that female coqui have higher plasma osmolality, reactive oxygen metabolites (dROMs), free glycerol, and triglycerides than males. Our study suggests coqui behavior and physiology in Hawai'i may be influenced by elevation in ways that allow them to cope with lower temperatures and invade higher elevations.

Exposure to microcrystallized cellulose affects the health of tadpoles and sheds light on the threat these materials pose to amphibians.

The increasing use of cellulose-based materials (CBMs) has provided beneficial applications in different sectors. However, its release into environments may represent an ecological risk, therefore demanding that ecotoxicological studies be conducted to understand the risks (current and future) of CBM pollution. Thus, we evaluated the possible effects of microcrystalline cellulose (CMs) in Physalaemus cuvieri tadpoles. After seven days of exposure to CMs (at 58.29 and 100 mg/L), the animals were subjected to behavioral evaluation, and different biomarkers (biometric and biochemical) were evaluated. Although our data do not point to a neurotoxic effect of CMs (inferred by the absence of behavioral changes and changes in AChE and BChE activity), animals exposed to CMs showed differences in body condition. Furthermore, we noticed an increase in the frequency of erythrocyte nuclear abnormalities and DNA damage, which were correlated with the ingestion of CMs. We noticed that the antioxidant activity of tadpoles exposed to CMs (inferred by SOD, CAT, and DPPH radical scavenging activity) was insufficient to control the increase in ROS and MDA production. Furthermore, exposure to CMs induced a predominant Th2-specific immune response, marked by suppressed IFN-γ and increased IL-10 levels, with a consequent reduction in NO levels. Principal component analysis and IBRv-2 indicate, in general, a primarily more toxic response to animals exposed to the highest CM concentration. Therefore, our study evidence that CMs affect the health of P. cuvieri tadpoles and sheds light on the threat these materials pose to amphibians.

How hibernation in frogs drives brain and reproductive evolution in opposite directions.

Environmental seasonality can promote the evolution of larger brains through cognitive and behavioral flexibility but can also hamper it when temporary food shortage is buffered by stored energy. Multiple hypotheses linking brain evolution with resource acquisition and allocation have been proposed for warm-blooded organisms, but it remains unclear how these extend to cold-blooded taxa whose metabolism is tightly linked to ambient temperature. Here, we integrated these hypotheses across frogs and toads in the context of varying brumation (hibernation) durations and their environmental correlates. We showed that protracted brumation covaried negatively with brain size but positively with reproductive investment, likely in response to brumation-dependent changes in the socio-ecological context and associated selection on different tissues. Our results provide novel insights into resource allocation strategies and possible constraints in trait diversification, which may have important implications for the adaptability of species under sustained environmental change.

The global importance and interplay of colour-based protective and thermoregulatory functions in frogs.

Small-scale studies have shown that colour lightness variation can have important physiological implications in ectotherms, with darker species having greater heating rates, as well as protection against pathogens and photooxidative damage. Using data for 41% (3059) of all known frog and toad species (Anura) from across the world, we reveal ubiquitous and strong clines of decreasing colour lightness towards colder regions and regions with higher pathogen pressure and UVB radiation. The relative importance of pathogen resistance is higher in the tropics and that of thermoregulation is higher in temperate regions. The results suggest that these functions influence colour lightness evolution in anurans and filtered for more similarly coloured species under climatic extremes, while their concurrent importance resulted in high within-assemblage variation in productive regions. Our findings indicate three important functions of colour lightness in anurans - thermoregulation, pathogen and UVB protection - and broaden support for colour lightness-environment relationships in ectotherms.

Tiny spies: mosquito antennae are sensitive sensors for eavesdropping on frog calls.

Most mosquito and midge species use hearing during acoustic mating behaviors. For frog-biting species, however, hearing plays an important role beyond mating as females rely on anuran calls to obtain blood meals. Despite the extensive work examining hearing in mosquito species that use sound in mating contexts, our understanding of how mosquitoes hear frog calls is limited. Here, we directly investigated the mechanisms underlying detection of frog calls by a mosquito species specialized on eavesdropping on anuran mating signals: Uranotaenia lowii. Behavioral, biomechanical and neurophysiological analyses revealed that the antenna of this frog-biting species can detect frog calls by relying on neural and mechanical responses comparable to those of non-frog-biting species. Our findings show that in Ur. lowii, contrary to most species, males do not use sound for mating, but females use hearing to locate their anuran host. We also show that the response of the antennae of this frog-biting species resembles that of the antenna of species that use hearing for mating. Finally, we discuss our data considering how mosquitoes may have evolved the ability to tap into the communication system of frogs.

What makes a great invader? Anatomical traits as predictors of locomotor performance and metabolic rate in an invasive frog.

Invasive species are characterized by their ability to establish and spread in a new environment. In alien populations of anurans, dispersal and fitness-related traits such as endurance, burst performance and metabolism are key to their success. However, few studies have investigated inter-individual variation in these traits and more specifically have attempted to understand the drivers of variation in these traits. Associations of anatomical features may be excellent predictors of variation in performance and could be targets for selection or subject to trade-offs during invasions. In this study, we used marsh frogs (Pelophylax ridibundus), a species that has been introduced in many places outside its native range and which is now colonizing large areas of Western Europe. We first measured the inter-individual variation in resting metabolism, the time and distance they were able to jump until exhaustion, and their peak jump force, and then measured the mass of specific organs and lengths of body parts suspected to play a role in locomotion and metabolism. Among the 5000 bootstrap replicates on body size-corrected variables, our statistical models most often selected the stomach (75.42%), gonads (71.46%) and the kidneys (67.26%) as predictors of inter-individual variation in metabolism, and the gluteus maximus muscle (97.24%) mass was the most frequently selected predictor of jump force. However, endurance was poorly associated with the anatomical traits (R2distance=0.42, R2time=0.37). These findings suggest that selection on these predictors may lead to physiological changes that may affect the colonization, establishment and dispersal of these frogs.

Larval plastic responses to warming and desiccation delay gonadal maturation in postmetamorphic spadefoot toads.

Developmental plasticity allows organisms to adjust life-history traits to varying environmental conditions, which can have concomitant effects across life stages. Many amphibians are suitable model systems to study plasticity because their larvae can adjust growth and differentiation under fluctuating environments. It is unknown, however, whether somatic and gonadal differentiation are equally affected by environmentally induced plasticity or whether their decoupling alters gonadal maturation postmetamorphosis, which may affect fitness. We tested if developmental acceleration in response to warming and desiccation risk results in shifts in gonadal maturation during metamorphosis and postmetamorphic growth in western spadefoot toads (Pelobates cultripes). We found additive effects of increased temperature and desiccation risk on development and growth at metamorphosis, which largely constrained gonadal maturation in metamorphic and postmetamorphic individuals of both sexes. Furthermore, the conditions experienced by larvae incurred sex-specific carryover effects on the gonadal maturation of juveniles 5 months after metamorphosis. In females, high temperature delayed ovarian maturation regardless of the water level. In males, exposure to high temperature and high water levels slightly delayed the testes' maturation. These results highlight the relevance of larval plasticity in the gonadal maturation of species undergoing metamorphosis, which may have implications for population demographics and the evolution of life histories.

Response to comment on 'Parasite defensive limb movements enhance acoustic signal attraction in male little torrent frogs'.

Recently we showed that limb movements associated with anti-parasite defenses can enhance acoustic signal attraction in male little torrent frogs (Amolops torrentis), which suggests a potential pathway for physical movements to become co-opted into mating displays (Zhao et al., 2022). Anderson et al. argue for alternative explanations of our results and provide a reanalysis of part of our data (Anderson et al., 2023). We acknowledge some of the points raised and provide an additional analysis in support of our hypothesis.

The effects of temperature on elastic energy storage and release in a system with a dynamic mechanical advantage latch.

Changes in temperature alter muscle kinetics and in turn affect whole-organism performance. Some organisms use the elastic recoil of biological springs, structures which are far less temperature sensitive, to power thermally robust movements. For jumping frogs, the use of elastic energy in tendons is facilitated through a geometric latching mechanism that operates through dynamic changes in the mechanical advantage (MA) of the hindlimb. Despite the well-documented use of elastic energy storage, frog jumping is a locomotor behavior that is significantly affected by changes in temperature. Here, we used an in vitro muscle preparation interacting in real time with an in silico model of a legged jumper to understand how changes in temperature affect the flow of energy in a system using a MA latch. We used the plantaris longus muscle-tendon unit (MTU) to power a virtual limb with changing MA and a mass being accelerated through a real-time feedback controller. We quantified the amount of energy stored in and recovered from elastic structures and the additional contribution of direct muscle work after unlatching. We found that temperature altered the duration of the energy loading and recovery phase of the in vitro/in silico experiments. We found that the early phase of loading was insensitive to changes in temperature. However, an increase in temperature did increase the rate of force development, which in turn allowed for increased energy storage in the second phase of loading. We also found that the contribution of direct muscle work after unlatching was substantial and increased significantly with temperature. Our results show that the thermal robustness achieved by an elastic mechanism depends strongly on the nature of the latch that mediates energy flow, and that the relative contribution of elastic and direct muscle energy likely shapes the thermal sensitivity of locomotor systems.

Widespread learned predator recognition to an alien predator across populations in an amphibian species.

Alien predators are a major cause of decline and extinction of species worldwide, since native organisms are rarely equipped with specific antipredatory strategies to cope with them. However, phenotypic plasticity and learned predator recognition may help prey populations to survive novel predators. Here we examine geographical variation in the learning ability of larval spadefoot toads (Pelobates cultripes) to recognize invasive predatory crayfish (Procambarus clarkii). We compare the learning-mediated behavioural responses of tadpoles from six populations across two regions in Spain (central and southern), with different histories of exposure to the presence of the invasive species. Two of the populations showed innate recognition of chemical cues from the invasive crayfish, whereas three of them learned to recognize such cues as a threat after conditioning with conspecific alarm cues. Learning abilities did not differ among southern populations, but they did among central populations. We assessed patterns of genetic variation within and among these two regions through microsatellite markers and found low genetic divergence among the southern populations but greater differentiation among the central ones. We hypothesize that similar responses to the invasive crayfish in southern populations may have arisen from a combination of extended historical exposure to this introduced predator (~ 50 y) and higher levels of gene flow, as they inhabit a highly interconnected pond network. In contrast, populations from central Spain show lower connectivity, have been exposed to the invasive crayfish for a shorter period of time, and are more divergent in their plastic responses.

Female treefrog preference for breeding sites matches offspring performance in the presence of two anuran competitors.

Preference-performance theory predicts that females should select breeding sites that maximize offspring performance. Amphibians have been a model system for investigating habitat selection, yet most studies have focused on habitat selection in response to predators and conspecifics. We investigated female oviposition site selection and larval performance in eastern gray treefrogs (Hyla versicolor) among pools with and without one of two ecologically distinct heterospecific larvae, the green frog (Rana clamitans) and the American bullfrog (Rana catesbeiana). Pools without heterospecifics were used on more nights and had more eggs deposited compared to pools with heterospecifics. In a competition experiment, treefrog larval performance matched this female preference: tadpoles developed faster and grew larger in the absence of either heterospecific. Although we hypothesized that bullfrogs would more strongly affect female treefrog preference and offspring performance because of previous work demonstrating that bullfrogs had stronger negative effects on other tadpoles, both heterospecifics elicited similar responses. The effects of heterospecifics on anuran breeding site selection are understudied and not well understood, and our results demonstrate that female selection of breeding sites is an adaptive behavior for offspring in the presence of heterospecific competitors.

Novel observations of "freeze resistance" and dynamic blue and green dorsal coloration in frozen and thawing Dryophytes chrysoscelis.

Freeze tolerant animals survive the winter by tolerating the freezing and thawing of up to 70% of body water and the respective cessation and resumption of essential functions including circulation and respiration during each freeze-thaw cycle. Cope's gray treefrog Dryophytes chrysoscelis is a freeze tolerant anuran that uses a system of cryoprotectants to prevent intracellular freezing and mitigate osmotic stress during freezing and thawing episodes. Morphological features were documented in D. chrysoscelis using a repeated freeze-thaw protocol. Dorsal skin in frozen frogs was distinctly blue and green before reverting to brown during thawing. The dorsal color change in frozen frogs does not function similarly to other known color change events in amphibians. The return to brown skin color in thawing animals coincides with recovery of vital functions in freeze tolerant frogs, suggesting that dorsal color change is an indicator of postfreeze recovery in D. chrysoscelis. We also provide evidence of "freeze resistance" in D. chrysoscelis. Two individuals did not freeze following three successive bouts of ice inoculation at -2.5°C and maintained brown dorsal color despite ice crystallization on the dorsum and contact with frozen substrate. Both frogs had similar plasma osmolality, circulating cryoprotectants, and incidence of cryoinjury compared to frogs that were frozen and thawed once or three times. Freeze resistance may be explained by physical changes in the skin including lipid accumulation and dehydration. This integrative study presents novel attributes of organismal freeze tolerance in D. chrysoscelis.

Social regulation of androgenic hormones and gestural display behavior in a tropical frog.

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.