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Abstract Chytridiomycosis, a lethal fungal disease caused by Batrachochytrium dendrobatidis (Bd), is responsible for population declines and extinctions of amphibians worldwide. However, not all amphibian species are equally susceptible to the disease; some species persist in Bd enzootic regions with no population reductions. Recently, it has been shown that the amphibian skin microbiome plays a crucial role in the defense against Bd. Numerous bacterial isolates with the capacity to inhibit the growth of Batrachochytrium fungi have been isolated from the skin of amphibians. Here, we characterized eight Acinetobacter bacteria isolated from the frogs Agalychnis callidryas and Craugastor fitzingeri at the genomic level. A total of five isolates belonged to Acinetobacter pittii,Acinetobacter radioresistens, or Acinetobactermodestus, and three were not identified as any of the known species, suggesting they are members of new species. We showed that seven isolates inhibited the growth of Bd and that all eight isolates inhibited the growth of the phytopathogen fungus Botrytis cinerea. Finally, we identified the biosynthetic gene clusters that could be involved in the antifungal activity of these isolates. Our results suggest that the frog skin microbiome includes Acinetobacter isolates that are new to science and have broad antifungal functions, perhaps driven by distinct genetic mechanisms. We characterized the genomes and the antifungal capacities of Acinetobacter bacteria isolated from the skin of two tropical frog species.

The vertebrate microbiome contributes to disease resistance, but few experiments have examined the link between microbiome community structure and disease resistance functions. Chytridiomycosis, a major cause of amphibian population declines, is a skin disease caused by the fungus, Batrachochytrium dendrobatidis (Bd). In a factorial experiment, bullfrog skin microbiota was reduced with antibiotics, augmented with an anti-Bd bacterial isolate (Janthinobacterium lividum), or unmanipulated, and individuals were then either exposed or not exposed to Bd. We found that the microbial community structure of individual frogs prior to Bd exposure influenced Bd infection intensity one week following exposure, which, in turn, was negatively correlated with proportional growth during the experiment. Microbial community structure and function differed among unmanipulated, antibiotic-treated, and augmented frogs only when frogs were exposed to Bd. Bd is a selective force on microbial community structure and function, and beneficial states of microbial community structure may serve to limit the impacts of infection.

Phyllomedusid treefrogs hatch prematurely to escape egg predation, but escape success varies among species. Snake attacks elicited 55% less escape hatching in spontaneously hatching Agalychnis spurrelli than in less developed A. callidryas. Agalychnis callidryas use their vestibular system and, secondarily, their lateral line to sense physical disturbances that cue hatching. Since A. spurrelli develop faster, we hypothesized that heterochronic shifts in the onset timing of vestibular mechanosensory function, relative to hatching ability, might explain their lower escape response to mechanosensory cues. To test this, we compared onset timings of hypoxia- and mechanosensory-cued hatching (MCH) and vestibular mechanosensory function in developmental series of both species. Across species, most sibships began responding to each cue at the same developmental stage and vestibular function development, measured by the vestibulo-ocular reflex (VOR), was similar. These results demonstrate that low escape-hatching success in A. spurrelli is not caused by a delay in the onset of vestibular mechanosensory function. MCH onset in A. spurrelli was associated with VOR, but with a higher threshold than in A. callidryas. The absence of MCH before strong vestibular function suggests multimodal mechanosensing may not contribute to antipredator responses of A. spurrelli embryos. Our observations of higher escape success of larger clutches in snake attacks and hatching complications in flooded A. spurrelli suggest that differences in clutch and egg capsule structure may contribute directly to species differences in escape-hatching success. Moreover, hatching complications in A. spurrelli may select against false alarms, increasing the stringency of hatching decision rules.Significance statementHatching is an essential life-stage transition during development. For some species, it also functions as an effective defense against egg-stage risks. However, the causes for variation in environmentally cued hatching responses are poorly understood. We assessed why two closely related treefrogs exhibit different escape-hatching responses to snake attacks. Comparing the onset of induced-hatching responses and vestibular function in Agalychnis callidryas and A. spurrelli revealed that the primary mechanism for mechanosensing and its developmental timing are conserved within this group, but the sensory function threshold for behavioral response differs. It also revealed that egg-clutch properties, including egg-clutch size and individual egg structure, affect the escape-hatching response. This motivates further research assessing the role of egg-clutch properties and their biomechanics in embryo escape-hatching responses.

Hatching timing is under strong selection and environmentally cued in many species. Embryos use multiple sensory modalities to inform hatching timing and many have spontaneous hatching patterns adaptively synchronized to natural cycles. Embryos can also adaptively shift their hatching timing in response to environmental cues indicating immediate threats or opportunities. Such cued shifts in hatching are widespread among amphibians; however, we know little about what, if anything, regulates their spontaneous hatching. Moreover, in addition to selection on hatching timing, embryos may experience benefits or suffer costs due to the spatial orientation of hatching. Amphibian eggs generally lack internal constraints on hatching direction but embryos might, nonetheless, use external cues to inform hatching orientation. The terrestrial embryos of red-eyed treefrogs, Agalychnis callidryas , hatch rapidly and prematurely in response to vibrational cues in egg-predator attacks and hypoxia if flooded. Here we examined A. callidryas’ use of light cues in hatching timing and orientation. To assess patterns of spontaneous hatching and the role of light cues in their diel timing, we recorded hatching times for siblings distributed across three light environments: continuous light, continuous dark, and a 12L:12D photoperiod. Under a natural photoperiod, embryos showed a clear diel pattern of synchronous hatching shortly after nightfall. Hatching was desynchronized in both continuous light and continuous darkness. It was also delayed by continuous light, but not accelerated by continuous dark, suggesting the onset of dark serves as a hatching cue. We examined hatching orientation and light as a potential directional cue for flooded embryos. Embryos flooded in their clutches almost always hatched toward open water, whereas individual eggs flooded in glass cups often failed to do so, suggesting the natural context provides a directional cue. To test if flooded embryos orient hatching toward light, we placed individual eggs in tubes with one end illuminated and the other dark, then flooded them and recorded hatching direction. Most embryos hatched toward the light, suggesting they use light as a directional cue. Our results support that A. callidryas embryos use light cues to inform both when and where to hatch. Both the spatial orientation of hatching and the timing of spontaneous hatching may affect fitness and be informed by cues in a broader range of species than is currently appreciated.

Some crepuscular and nocturnal animals are brightly marked yet the adaptive significance of their colorful patterns in low light, as found at twilight and night, is poorly understood. This phenomenon is particular prevalent in amphibians. Of the nearly 80% of nocturnal frogs, many exhibit color patterns with red, yellow, green and blue hues and/or contrasting spots and stripes. Despite the prevalence of these conspicuous visual signals in frogs, the function and adaptive significance of bright coloration for crepuscular/nocturnal frogs is still poorly understood. A critical first step in linking color pattern evolution with premating reproductive isolation and lineage divergence is determining whether color pattern plays a role in mate recognition in dim light. We studied the brightly colored Red-eyed Treefrog (Agalychnis callidryas), a crepuscular/nocturnal Neotropical treefrog that exhibits noteworthy geographic variation in color pattern and female choice for local male phenotypes. We measured retinal photoreceptor cell absorbance via microspectrophotometry and used visual modeling to assess whether distinct color pattern phenotypes were distinguishable as luminosity and chromaticity cues. We found that the Red-eyed Treefrog visual system is capable of discriminating differences in color patterns as brightness (luminosity) in their perception of nighttime visual cues. Differences in color (chromaticity) were also detectable in dim light, although less prominent than brightness. Combined, our data indicate that differences in these visual traits are discernable, can function for species and population recognition, and evolve through sexual selection. These social signals are thus analogous to the widespread visual displays exhibited by diurnal vertebrates, suggesting that the richness of similar sensory interactions among animals at twilight and after dark might be severely underappreciated. More generally, we demonstrate that combining studies of the visual system with population genetics, behavior, and natural history provides a framework for testing the evolution and adaptive function of color pattern.

The arboreal embryos of red-eyed treefrogs (Agalychnis callidryas) hatch prematurely to escape from egg predators, and escape success increases with age. We assessed developmental changes in the behavior and hatching performance of embryos attacked by wasps (Polybia rejecta) and their contributions to improved embryo survival. We recorded videos of 4- and 5-day-old embryos exposed to wasp attacks and determined each embryo’s fate. For a stratified random sample of embryos that escaped and died, we determined the occurrence, sequence, and timing of events during wasp-embryo interactions. We constructed path diagrams of event sequences, tested for age effects on transition probabilities, and measured the durations of periods between key events. Overall, escape success was 38% higher in older embryos. They were more likely to hatch pre-emptively than younger ones, thus less likely to experience direct attacks, suggesting that developmental gains in mechanosensory sensitivity may increase hatching responses to indirect cues. During direct attacks, embryos were equally likely to be captured by wasps at both ages, and hatching speed was similar, suggesting no relevant difference in escape-hatching performance. After a wasp ruptured their egg capsule, older embryos were more likely to exit, and they did so much sooner; younger embryos remained in ruptured capsules for longer and were more likely to be attacked again. This developmental change in embryo behavior indicates decreased tolerance for egg-stage risk as the chance of tadpole survival increases, suggesting that ontogenetic adaptation to changing risk trade-offs contributes strongly to the developmental increase in escape success.Significance statementAs animals develop, both their abilities and the optimal strategies for antipredator defense change, and some show ontogenetic adaptation of defensive behavior. This is documented in the larval and juvenile stages but rarely examined during the embryonic period, when development is fastest. We document substantial changes in embryo behavior across a 1-day period following the onset of predator-induced hatching in red-eyed treefrogs, with rapidly decreasing tolerance of egg-stage risk contributing to higher survival under wasp predation as the costs of hatching decrease. This provides evidence for ontogenetic adaptation of defensive behavior in a natural predation context and across an earlier developmental period than previously known, thereby strengthening results from prior work with vibration playbacks across later developmental stages. It suggests that even during periods of rapid development of morphology and abilities, understanding behavioral decisions may be crucial to explaining changes in survival.

Emerging diseases caused by fungi are a serious threat to wildlife biodiversity. The widespread fungal pathogen Batrachochytrium dendrobatidis (Bd) has caused dramatic amphibian population declines and species extinctions worldwide. While many amphibians have been negatively affected by Bd, some populations/species have persisted despite its presence. One factor contributing to amphibian protection against this fungus is the host-associated skin microbiome. In this study, we aimed to identify gene clusters associated with the antifungal activity of amphibian skin bacteria. Specifically, we explored skin bacteria isolated from species that have persisted in the wild despite the presence of Bd: the frogs Agalychnis callidryas , Craugastor fitzingeri , Dendropsophus ebraccatu s, and the axolotl Ambystoma altamirani . Bacterial isolates were tested in vitro for their capacity to inhibit the growth of two fungal pathogens: Bd and the phytopathogen Botrytis cinerea (Bc). Genome mining of these bacterial isolates revealed a diverse repertoire of Biosynthetic Gene Clusters (BGCs) and chitin-degrading gene families (ChDGFs) whose composition and abundance differed among bacterial families. We found specific BGCs and ChDGFs that were associated with the capacity of bacteria to inhibit the growth of either Bd or Bc, suggesting that distinct fungi could be inhibited by different molecular mechanisms. By using similarity networks and machine learning, we identified BGCs encoding known antifungal compounds such as viscosin, fengycin, zwittermicin, as well as siderophores and a novel family of beta-lactones. Finally, we propose that the diversity of BGCs found in amphibian skin bacteria comprise a substantial genetic reservoir that could collectively explain the antifungal properties of the amphibian skin microbiome.

The lemur tree frog (Agalychnis lemur), a critically endangered species, can benefit from ex situ conservation programs; however, managing amphibians under human care presents challenges, including the provision of appropriate nutrition. House crickets (Acheta domesticus), a common feeder insect, have an inverse calcium to phosphorus ratio (Ca:P; 0.15:1) and low calcium content (<0.3%). While gut-loading crickets with an 8% calcium diet can improve their calcium concentrations, no study has assessed the effects of dietary calcium on bone development in Agalychnis spp. Moreover, no study has examined how diet impacts the gut–skin axis and skin microbiome of these frogs. This study examined how crickets gut-loaded with either a 1.3% or 8% calcium diet affected lemur tree frog bone density and skin microbiome. We hypothesized that frogs consuming the 8% calcium diet would exhibit significantly higher Hounsfield units (HU; bone density) over time, as measured by micro-computed tomography (mCT), and that dietary calcium concentration would have no effect on skin bacterial and fungi microbiomes. Eleven juvenile lemur tree frogs underwent mCT scans at baseline and 90 and 180 days. Total body volume of interest analysis showed a significant increase in HU in the 8% calcium group compared to the 1.3% group (F = 9.9, p = 0.01). There was no significant difference noted in the alpha or beta diversities for the bacterial and fungal microbiomes between dietary groups. This study provides the first evidence of dietary calcium’s impact on bone density in lemur tree frogs, offering valuable insights for improving ex situ management of this species.

Environmentally cued plasticity in hatching timing is widespread in animals. As with later life-history switch points, plasticity in hatching timing may have carryover effects that affect subsequent interactions with predators and competitors. Moreover, the strength of such effects of hatching plasticity may be context dependent. We used red-eyed treefrogs, Agalychnis callidryas , to test for lasting effects of hatching timing (four or six days post-oviposition) under factorial combinations of resource levels (high or low) and predation risk (none, caged, or lethal Pantala flavescens dragonfly naiads). Tadpoles were raised in 400-L mesocosms in Gamboa, Panama, from hatching until all animals had metamorphosed or died, allowing assessment of effects across a nearly six-month period of metamorphosis. Hatching early reduced survival to metamorphosis, increased larval growth, and had context-dependent effects on metamorph phenotypes. Early during the period of metamorph emergence, early-hatched animals were larger than late-hatched ones, but this effect attenuated over time. Early-hatched animals also left the water with relatively longer tails. Lethal predators dramatically reduced survival to metamorphosis, with most mortality occurring early in the larval period. Predator effects on the timing of metamorphosis and metamorph size and tail length depended upon resources. For example, lethal predators reduced larval periods, and this effect was stronger with low resources. Predators affected metamorph size early in the period of metamorphosis, whereas resource levels were a stronger determinant of phenotype for animals that metamorphosed later. Effects of hatching timing were detectable on top of strong effects of larval predators and resources, across two subsequent life stages, and some were as strong as or stronger than effects of resources. Plasticity in hatching timing is ecologically important and currently underappreciated. Effects on metamorph numbers and phenotypes may impact subsequent interactions with predators, competitors, and mates, with potentially cascading effects on recruitment and fitness.

Adaptive plasticity at switch points in complex life cycles (e.g., hatching, metamorphosis) is well known, but the evolutionary history of such plasticity is not. Particularly unclear is how a single plastic response (e.g., shifts in hatching timing) evolves to respond to different threats and cues (e.g., abiotic and biotic). We conducted a comparative phylogenetic study of hatching plasticity in a group of frogs with arboreal embryos to determine when risk-accelerated hatching evolved in the clade, whether responses to two common egg-stage risks (snake predation and flooding) evolved independently, and whether the overall capacity for hatching plasticity was evolutionarily more conservative than responses to specific cues. Red-eyed treefrogs (Agalychnis callidryas) hatch early to escape from several egg-stage risks but otherwise hatch later, improving larval survival with predators. We reconstructed a phylogeny for Agalychnis and related genera based on three mitochondrial and four nuclear genes. We quantified onset of hatching competence, spontaneous hatching timing, responses to egg-stage risks, and costs of premature hatching in Agalychnis and Pachymedusa. We also assessed hatching plasticity in a basal phyllomedusine, Cruziohyla calcarifer. The capacity to hatch ∼30% before the spontaneous hatching age appears ancestral for phyllomedusines, with little change over ∼34—50 million years among the species examined. A strong hatching response to flooding, with no mortality of hatching-competent eggs, is similarly ancient and conserved. Premature hatchlings of Agalychnis and Pachymedusa are more vulnerable to fish predation than are full-term hatchlings, indicating a conserved risk trade-off across hatching that would make plasticity advantageous. In contrast, the hatching response to snake attack has undergone major changes at least twice in the Agalychnis—Pachymedusa clade, with two species showing substantially lower escape success than the others. Responses to different threats have thus evolved independently. The capacity for switch point plasticity may be evolutionarily more stable than the response to individual stage-specific threats.