Insights into the evolution of photoreceptor oil droplets in frogs and toads.

Photoreceptor oil droplets (ODs) are spherical organelles placed most commonly within the inner segment of the cone photoreceptors. Comprising neutral lipids, ODs can be either non-pigmented or pigmented and have been considered optically functional in various studies. Among living amphibians, ODs were only reported to occur in frogs and toads (Anura), while they are absent in salamanders and caecilians. Nonetheless, the limited understanding of their taxonomic distribution in anurans impedes a comprehensive assessment of their evolution and relationship with visual ecology. We studied the retinae of 134 anuran species, extending the knowledge of the distribution of ODs to 46 of the 58 currently recognized families, and providing a new perspective on this group that complements the available information from other vertebrates. The occurrence of ODs in anurans shows a strong phylogenetic signal, and our findings revealed that ODs evolved at least six times during the evolutionary history of the group, independently from other vertebrates. Although no evident correlation was found between OD occurrence, adult habits and diel activity, it is inferred that each independent origin involves distinct scenarios in the evolution of ODs concerning photic habits. Furthermore, our results revealed significant differences in the size of the ODs between nocturnal and arrhythmic anurans relative to the length of the cones' outer segment.

Flexible oviposition behavior enabled the evolution of terrestrial reproduction.

Among vertebrates, nearly all oviparous animals are considered to have either obligate aquatic or terrestrial oviposition, with eggs that are specialized for developing in those environments. The terrestrial environment has considerably more oxygen but is dry and thus presents both opportunities and challenges for developing embryos, particularly those adapted for aquatic development. Here, we present evidence from field experiments examining egg-laying behavior, egg size, and egg jelly function of 13 species of Central and South American treefrogs in the genus Dendropsophus, which demonstrates that flexible oviposition (individuals laying eggs both in and out of water) and eggs capable of both aquatic and terrestrial development are the likely factors which enable the transition from aquatic to terrestrial reproduction. Nearly half of the species we studied had previously undescribed degrees of flexible oviposition. Species with obligate terrestrial reproduction have larger eggs than species with aquatic reproduction, and species with flexible reproduction have eggs of intermediate sizes. Obligate terrestrial breeding frogs also have egg masses that absorb water more quickly than those with flexible oviposition. We also examined eight populations of a single species, Dendropsophus ebraccatus, and document substantial intraspecific variation in terrestrial oviposition; populations in rainy, stable climates lay fewer eggs in water than those in drier areas. However, no differences in egg size were found, supporting the idea that the behavioral component of oviposition evolves before other adaptations associated with obligate terrestrial reproduction. Collectively, these data demonstrate the key role that behavior can have in facilitating major evolutionary transitions.

Longer days, larger grays: carryover effects of photoperiod and temperature in gray treefrogs, Hyla versicolor.

Environmental conditions like temperature and photoperiod can strongly shape organisms' growth and development. For many ectotherms with complex life cycles, global change will cause their offspring to experience warmer conditions and earlier-season photoperiods, two variables that can induce conflicting responses. We experimentally manipulated photoperiod and temperature during gray treefrog (Hyla versicolor) larval development to examine effects at metamorphosis and during short (10-day) and long (56-day) periods post-metamorphosis. Both early- and late-season photoperiods (April and August) decreased age and size at metamorphosis relative to the average-season (June) photoperiod, while warmer temperatures decreased age but increased size at metamorphosis. Warmer larval temperatures reduced short-term juvenile growth but had no long-term effect. Conversely, photoperiod had no short-term carryover effect, but juveniles from early- and late-season larval photoperiods had lower long-term growth rates than juveniles from the average-season photoperiod. Similar responses to early- and late-season photoperiods may be due to reduced total daylight compared with average-season photoperiods. However, juveniles from late-season photoperiods selected cooler temperatures than early-season juveniles, suggesting that not all effects of photoperiod were due to total light exposure. Our results indicate that despite both temperature and photoperiod affecting metamorphosis, the long-term effects of photoperiod may be much stronger than those of temperature.

Not so cryptic-differences between mating calls of Hyla arborea and Hyla orientalis from Bulgaria.

Anurans are among the most vocally active vertebrate animals and emit calls with different functions. In order to attract a mate, during the breeding season male frogs produce mating calls which have species-specific structure and parameters, and have been successfully used to resolve issues in taxonomy and phylogenetic relations. This is particularly useful when closely related taxa are concerned, as many species are morphologically almost identical, but still their status is well-supported by molecular and genetic data, suggesting the existence of mechanisms for reproductive isolation. Such is the case for treefrogs from the Hyla arborea group, which are now recognized as several distinct species. The present study aims to establish differences in call parameters between the European tree frog, Hyla arborea, and the Eastern tree frog, Hyla orientalis, which both occur on the territory of Bulgaria. Using autonomous audio loggers, calls from six sites (three in the range of H. arborea and three in the range of H. orientalis) were recorded between 7 p.m. and 12 a.m. during the breeding season in 2020-2023. The following parameters in a total of 390 mating calls were analyzed: call count, pulse count, call series duration, call period, peak (dominant) frequency, entropy. Results indicated that sites formed two distinct groups, which corresponded to the known distribution ranges of H. arborea and H. orientalis. The first two components of the PCA explained 71% of the total variance, with variables call count, call series duration, peak frequency and entropy being most important for differentiation between the sites. This study presents the first attempt to differentiate between the calls of these two sister taxa, which both fall within the "short-call treefrogs" group, and results are discussed in terms of known data for mating calls in Hyla sp., as well as limitations and future perspectives.

Transcription factor roles in the local adaptation to temperature in the Andean Spiny Toad Rhinella spinulosa.

Environmental temperature strongly influences the adaptation dynamics of amphibians, whose limited regulation capabilities render them susceptible to thermal oscillations. A central element of the adaptive strategies is the transcription factors (TFs), which act as master regulators that orchestrate stress responses, enabling species to navigate the fluctuations of their environment skillfully. Our study delves into the intricate relationship between TF expression and thermal adaptation mechanisms in the Rhinella spinulosa populations. We sought to elucidate the dynamic modulations of TF expression in prometamorphic and metamorphic tadpoles that inhabit two thermally contrasting environments (Catarpe and El Tatio Geyser, Chile) and which were exposed to two thermal treatments (25 °C vs. 20 °C). Our findings unravel an intriguing dichotomy in response strategies between these populations. First, results evidence the expression of 1374 transcription factors. Regarding the temperature shift, the Catarpe tadpoles show a multifaceted approach by up-regulating crucial TFs, including fosB, atf7, and the androgen receptor. These dynamic regulatory responses likely underpin the population's ability to navigate thermal fluctuations effectively. In stark contrast, the El Tatio tadpoles exhibit a more targeted response, primarily up-regulating foxc1. This differential expression suggests a distinct focus on specific TFs to mitigate the effects of temperature variations. Our study contributes to understanding the molecular mechanisms governing thermal adaptation responses and highlights the resilience and adaptability of amphibians in the face of ever-changing environmental conditions.

Hotspot shelters stimulate frog resistance to chytridiomycosis.

Many threats to biodiversity cannot be eliminated; for example, invasive pathogens may be ubiquitous. Chytridiomycosis is a fungal disease that has spread worldwide, driving at least 90 amphibian species to extinction, and severely affecting hundreds of others1-4. Once the disease spreads to a new environment, it is likely to become a permanent part of that ecosystem. To enable coexistence with chytridiomycosis in the field, we devised an intervention that exploits host defences and pathogen vulnerabilities. Here we show that sunlight-heated artificial refugia attract endangered frogs and enable body temperatures high enough to clear infections, and that having recovered in this way, frogs are subsequently resistant to chytridiomycosis even under cool conditions that are optimal for fungal growth. Our results provide a simple, inexpensive and widely applicable strategy to buffer frogs against chytridiomycosis in nature. The refugia are immediately useful for the endangered species we tested and will have broader utility for amphibian species with similar ecologies. Furthermore, our concept could be applied to other wildlife diseases in which differences in host and pathogen physiologies can be exploited. The refugia are made from cheap and readily available materials and therefore could be rapidly adopted by wildlife managers and the public. In summary, habitat protection alone cannot protect species that are affected by invasive diseases, but simple manipulations to microhabitat structure could spell the difference between the extinction and the persistence of endangered amphibians.

A New Method for Low Density Distribution Modeling and Near Threatened Species: The Study Case of Plectrohyla Guatemalensis.

We introduce a model that can be used for the description of the distribution of species when there is scarcity of data, based on our previous work (Ballesteros et al. J Math Biol 85(4):31, 2022). We address challenges in modeling species that are seldom observed in nature, for example species included in The International Union for Conservation of Nature's Red List of Threatened Species (IUCN 2023). We introduce a general method and test it using a case study of a near threatened species of amphibians called Plectrohyla Guatemalensis (see IUCN 2023) in a region of the UNESCO natural reserve "Tacaná Volcano", in the border between Mexico and Guatemala. Since threatened species are difficult to find in nature, collected data can be extremely reduced. This produces a mathematical problem in the sense that the usual modeling in terms of Markov random fields representing individuals associated to locations in a grid generates artificial clusters around the observations, which are unreasonable. We propose a different approach in which our random variables describe yearly averages of expectation values of the number of individuals instead of individuals (and they take values on a compact interval). Our approach takes advantage of intuitive insights from environmental properties: in nature individuals are attracted or repulsed by specific features (Ballesteros et al. J Math Biol 85(4):31, 2022). Drawing inspiration from quantum mechanics, we incorporate quantum Hamiltonians into classical statistical mechanics (i.e. Gibbs measures or Markov random fields). The equilibrium between spreading and attractive/repulsive forces governs the behavior of the species, expressed through a global control problem involving an energy operator.

Benefits and costs: Understanding the influence of heavy metal pollution on environmental adaptability in Strauchbufo raddei tadpoles through an energy budget perspective.

Understanding the impact of environmental pollution on organismal energy budgets is crucial for predicting adaptive responses and potential maladaptation to stressors. However, the regulatory mechanism governing the trade-off between energy intake and consumption remains largely unknown, particularly considering the diverse adaptations influenced by exposure history in realistic field conditions. In the present study, we conducted a simulated field reciprocal transplant experiment to compare the energy budget strategies of Strauchbufo raddei tadpoles exposed to heavy metal. The simulated heavy metal concentrations (0.29 mg/L Cu, 1.17 mg/L Zn, 0.47 mg/L Pb, 0.16 mg/L Cd) mirrored the actual environmental exposure concentrations observed in the field habitat. This allowed for a comparison between tadpoles with parental chronic exposure to heavy metal pollutants in their habitat and those without such exposure. Results revealed that under heavy metal exposure, tadpoles originating from unpolluted areas exhibited heightened vulnerability, characterized by reduced food intake, diminished nutrient absorption, increased metabolism cost, reduced energy reserves, and increased mortality rates. In contrast, tadpoles originating from areas with long-term heavy metal pollution demonstrated adaptive strategies, manifested through adjustments in liver and small intestine phenotypes, optimizing energy allocation, and reducing energy consumption to preserve energy, thus sustaining survival. However, tadpoles from polluted areas exhibited certain maladaptive such as growth inhibition, metabolic suppression, and immune compromise due to heavy metal exposure. In conclusion, while conserving energy consumption has proven to be an effective way to deal with long-term heavy metal stress, it poses a threat to individual survival and population development in the long run.

Dehydration followed by restraint sustains high circulating corticosterone and improves immunity in toads.

Amphibians are suffering population declines due to a variety of factors such as increased ultraviolet radiation, climate change, habitat loss, pathogens, and pollution, or a combination of these. Such changes are associated with a reduction in the availability of water, exposing these animals to a greater risk of desiccation. In this context, understanding how dehydration can modulate the hypothalamic-pituitary-interrenal axis (HPI) and the immune response is an imperative question to predict how stressors can affect amphibian species. We investigated whether dehydration promotes long-lasting effects on toads' ability to respond to a consecutive stressor (restraint) even if the toads are allowed to rehydrate, as well as its effects on the immune function. We also tested the hypothesis that the toads showing more severe dehydration would exhibit lower responsiveness to restraint challenge, even if the animals were allowed to rehydrate. Individuals of R. ornata were dehydrated mildly and severely. Thereafter, they were submitted to a restraint stress challenge for 1 and 24 h. Our results show that dehydration increased hematocrit and CORT in R. ornata toads. The restraint induced an acute stress response in fully hydrated toads (increased CORT and neutrophil: lymphocyte ratio). Otherwise, restraint in moistened cloth bags allowed full rehydration in previously dehydrated toads and did not induce an additional increase in CORT, but those toads sustained elevated CORT up to 24 h of restraint. Also, these animals showed increased neutrophil: lymphocyte ratio and the phagocytic activity of blood cells, even when they rehydrated during restraint. These results point to a continuous activation of the HPA during dehydration and subsequent restraint, even when they recovered from the dehydration state. Also, acute stressors seem to promote immune cell redistribution and augmentation of immune cellular function in R. ornata toads.

Testing the efficiency of passive acoustic monitoring and active monitoring in anuran community in lotic environments in Itinguçu State Park, Peruíbe-SP.

The following work presents the first study applying the passive acoustic monitoring of anurans at lotic environments for a long time. This study aims to test the efficiency of the passive acoustic monitoring method and active monitoring in detecting anurans in lotic environments of Itinguçu State Park. Specifically, we tested whether species richness differs when comparing active and passive monitoring surveys. Therefore, this study aims to test the efficiency of the passive acoustic monitoring method and active monitoring in detecting anurans in lotic environments of the Itinguçu State Park. The passive acoustic monitoring period was 72 uninterrupted hours at each collection point with intervals of 45 days. Finally, species richness was calculated, and the efficiency of the methods was compared in different scenarios. Our results demonstrated that the park has species that vocalize day and night, but most at night, there is overlapping acoustic niche; waterfall environments harm the quality of recordings; and in lotic environments the active monitoring method was more efficient than the passive acoustic monitoring in all sampling scales. Although the passive acoustic monitoring was not as efficient in a low temporal scale, it tends to increase in efficiency with longer sampling duration.

Differential Mitochondrial Genome Expression of Four Hylid Frog Species under Low-Temperature Stress and Its Relationship with Amphibian Temperature Adaptation.

Extreme weather poses huge challenges for animals that must adapt to wide variations in environmental temperature and, in many cases, it can lead to the local extirpation of populations or even the extinction of an entire species. Previous studies have found that one element of amphibian adaptation to environmental stress involves changes in mitochondrial gene expression at low temperatures. However, to date, comparative studies of gene expression in organisms living at extreme temperatures have focused mainly on nuclear genes. This study sequenced the complete mitochondrial genomes of five Asian hylid frog species: Dryophytes japonicus, D. immaculata, Hyla annectans, H. chinensis and H. zhaopingensis. It compared the phylogenetic relationships within the Hylidae family and explored the association between mitochondrial gene expression and evolutionary adaptations to cold stress. The present results showed that in D. immaculata, transcript levels of 12 out of 13 mitochondria genes were significantly reduced under cold exposure (p < 0.05); hence, we put forward the conjecture that D. immaculata adapts by entering a hibernation state at low temperature. In H. annectans, the transcripts of 10 genes (ND1, ND2, ND3, ND4, ND4L, ND5, ND6, COX1, COX2 and ATP8) were significantly reduced in response to cold exposure, and five mitochondrial genes in H. chinensis (ND1, ND2, ND3, ND4L and ATP6) also showed significantly reduced expression and transcript levels under cold conditions. By contrast, transcript levels of ND2 and ATP6 in H. zhaopingensis were significantly increased at low temperatures, possibly related to the narrow distribution of this species primarily at low latitudes. Indeed, H. zhaopingensis has little ability to adapt to low temperature (4 °C), or maybe to enter into hibernation, and it shows metabolic disorder in the cold. The present study demonstrates that the regulatory trend of mitochondrial gene expression in amphibians is correlated with their ability to adapt to variable climates in extreme environments. These results can predict which species are more likely to undergo extirpation or extinction with climate change and, thereby, provide new ideas for the study of species extinction in highly variable winter climates.

Lack of glucocorticoid flexibility is indicative of wear-and-tear in Hyla versicolor tadpoles from agricultural environments.

In habitats where stressors are frequent or persistent, it can become increasingly difficult for wildlife to appropriately match their endocrine responses to these more challenging environments. The dynamic regulation of glucocorticoid (GC) hormones plays a crucial role in determining how well individuals cope with environmental changes. Amphibians exposed to agricultural stressors can dampen aspects of their GC profile (baseline, agitation, recovery, stress responsiveness, and negative feedback) to cope in these stressful environments, but this dampening can lead to reductions in an individual's reactive scope and a loss of endocrine flexibility. Organic agriculture could potentially limit some of these effects, however, little is known about how amphibians respond physiologically to organic agricultural environments. We compared GC profiles of Hyla versicolor tadpoles from three treatments: natural ponds (<5% agriculture within 500m), ponds near organic farms, and ponds near conventional farms. We hypothesized that tadpoles would cope with agricultural habitats by dampening stress responsiveness and exhibiting more efficient negative feedback and that the magnitude of these changes in response would differ based on agricultural method. We found that tadpoles from conventional and organic ponds were less likely to downregulate GCs via negative feedback after stressor exposure than tadpoles from natural ponds. For agricultural tadpoles that did downregulate GCs after the stressor, we found lower stress responsiveness and faster downregulation to baseline corticosterone than tadpoles from natural ponds. These results point to an accumulation of wear-and-tear, leading to an overall reduction in reactive scope and limited GC flexibility in our agricultural tadpoles. Regardless of agricultural method used, agricultural tadpoles exhibited the same patterns of GC response, indicating that current efforts to incentivize farmers to switch to organic farming methods may not be sufficient to address negative agricultural impacts on amphibians.

Acute hypoxia exposure rapidly triggers behavioral changes linked to cutaneous gas exchange in Lake Titicaca frogs.

Ventilation is critical to animal life-it ensures that individuals move air/water across their respiratory surface, and thus it sustains gas exchange with the environment. Many species have evolved highly specialized (if not unusual) ventilatory mechanisms, including the use of behavior to facilitate different aspects of breathing. However, these behavioral traits are often only described anecdotally, and the ecological conditions that elicit them are typically unclear. We study one such "ventilation behavior" in Lake Titicaca frogs (Telmatobius culeus). These frogs inhabit high-altitude (i.e., low oxygen) lakes in the Andean Mountains of South America, and they have become textbook examples of cutaneous gas exchange, which is essentially breathing that occurs across the skin. Accordingly, this species has evolved large, baggy skin-folds that dangle from the body to increase the surface area for ventilation. We show that individuals exposed to acute hypoxic conditions that mirror what free-living individuals likely encounter quickly (within minutes) decrease their activity levels, and thus become very still. If oxygen levels continue to decline, the frogs soon begin to perform push-up behaviors that presumably break the low-oxygen boundary layer around skin-folds to increase the conductance of the water/skin gas exchange pathway. Altogether, we suspect that individuals rapidly adjust aspects of their behavior in response to seemingly sudden changes to the oxygen environment as a mechanism to fine tune cutaneous respiration.

Traffic Noise Impacts Glucocorticoid Response, Activity, and Growth in Two Species of Tadpoles.

There is a large body of evidence linking increased noise to negative health effects for animals. Anthropogenic noise induces behavioral and physiological reactions across a range of taxa and increased traffic noise affects glucocorticoid (GC) hormones associated with the stress response in amphibians. GCs help to maintain homeostasis while balancing energetic trade-offs between reproduction, growth, and activity. Stressors during early development can impact fitness at later life stages. We measured growth, activity, and GCs in response to high levels of traffic noise in two tadpole species that differ in life history: Acris crepitans and Rana berlandieri. We predicted that earlier exposures to traffic noise will slow down the development and alter the behavior and GC concentrations differently than later exposures. Subjects were initially either exposed to natural levels of traffic noise for 8 days (early exposure) or a white noise control (later exposure), then the treatment was switched. Activity was measured via focal sampling and tadpoles were categorized as active if movement was detected. Tadpoles exposed to white noise initially maintained mass and activity throughout the experiment and early exposure to traffic noise had a greater impact on mass, activity, and GCs. Tadpoles exposed to traffic noise initially lost mass, with A. crepitans regaining mass but not R. berlandieri. When exposed earlier to traffic noise, R. berlandieri increased movement when shifted to the white noise treatment while A. crepitans did not significantly change activity. Acris creptians had higher corticosterone release rates compared to R. berlandieri, and in both species, release rates were higher for tadpoles exposed to noise earlier. The longer-lived R. berlandieri allocated more of their energetic resources into activity, while the shorter-lived A. crepitans allocated energy toward growth. Rana berlandieri and A. crepitans utilized different coping strategies to contend with early exposure to traffic noise, potentially due to differences in life histories. Our findings suggest that these tadpoles employ different coping mechanisms to modulate stress responses in noise-polluted environments, and these mechanisms could influence their fitness later in life. Further study is needed to understand the impact in more sensitive tadpole species.

Contrasting nidification behaviors facilitate diversification and colonization of the Music frogs under a changing paleoclimate.

In order to cope with the complexity and variability of the terrestrial environment, amphibians have developed a wide range of reproductive and parental behaviors. Nest building occurs in some anuran species as parental care. Species of the Music frog genus Nidirana are known for their unique courtship behavior and mud nesting in several congeners. However, the evolution of these frogs and their nidification behavior has yet to be studied. With phylogenomic and phylogeographic analyses based on a wide sampling of the genus, we find that Nidirana originated from central-southwestern China and the nidification behavior initially evolved at ca 19.3 Ma but subsequently lost in several descendants. Further population genomic analyses suggest that the nidification species have an older diversification and colonization history, while N. adenopleura complex congeners that do not exhibit nidification behavior have experienced a recent rapid radiation. The presence and loss of the nidification behavior in the Music frogs may be associated with paleoclimatic factors such as temperature and precipitation. This study highlights the nidification behavior as a key evolutionary innovation that has contributed to the diversification of an amphibian group under past climate changes.

Combined effect of seasons and life history in an anuran strengthens the response and relationship between their physiology and gut microbiota.

Gut microbiota impact host physiology, though simultaneous investigations in ectothermic vertebrates are rare. Particularly, amphibians may exhibit more complex interactions between host physiology and the effects of gut microbiota due to the combination of seasonal changes and complex life histories. In this study, we assessed the relationships among food resources, gut bacterial communities, and host physiology in frogs (Phelophylax nigromaculatus), taking into account seasonal and life history variations. We found that food sources were not correlated with physiological parameters but had some relationships with the gut bacterial community. Variations in gut bacterial community and host physiology were influenced by the combined effects of seasonal differences and life history, though mostly driven by seasonal differences. An increase in Firmicutes was associated with higher fat content, reflecting potential fat storage in frogs during the non-breeding season. The increase in Bacteroidetes resulted in lower fat content in adult frogs and decreased immunity in juvenile frogs during the breeding season, demonstrating a direct link. Our results suggest that the gut microbiome may act as a link between food conditions and physiological status, and that the combined effect of seasons and life history could reinforce the relationship between gut microbiota and physiological status in ectothermic animals. While food sources may influence the gut microbiota of ectotherms, we contend that temperature-correlated seasonal variation, which predominately influences most ectotherms, is a significant factor.

Predation cues induce predator specific changes in olfactory neurons encoding defensive responses in agile frog tadpoles.

Although behavioural defensive responses have been recorded several times in both laboratory and natural habitats, their neural mechanisms have seldom been investigated. To explore how chemical, water-borne cues are conveyed to the forebrain and instruct behavioural responses in anuran larvae, we conditioned newly hatched agile frog tadpoles using predator olfactory cues, specifically either native odonate larvae or alien crayfish kairomones. We expected chronic treatments to influence the basal neuronal activity of the tadpoles' mitral cells and alter their sensory neuronal connections, thereby impacting information processing. Subsequently, these neurons were acutely perfused, and their responses were compared with the defensive behaviour of tadpoles previously conditioned and exposed to the same cues. Tadpoles conditioned with odonate cues differed in both passive and active cell properties compared to those exposed to water (controls) or crayfish cues. The observed upregulation of membrane conductance and increase in both the number of active synapses and receptor density at the postsynaptic site are believed to have enhanced their responsiveness to external stimuli. Odonate cues also affected the resting membrane potential and firing rate of mitral cells during electrophysiological patch-clamp recordings, suggesting a rearrangement of the repertoire of voltage-dependent conductances expressed in cell membranes. These recorded neural changes may modulate the induction of an action potential and transmission of information. Furthermore, the recording of neural activity indicated that the lack of defensive responses towards non-native predators is due to the non-recognition of their olfactory cues.

Elevated ammonia cues hatching in red-eyed treefrogs: A mechanism for escape from drying eggs.

Egg dehydration can kill terrestrial frog embryos, and this threat is increasing with climate change and deforestation. In several lineages that independently evolved terrestrial eggs, and retained aquatic tadpoles, embryos accelerate hatching to escape from drying eggs, entering the water earlier and less developed. However, the cues that stimulate drying-induced early hatching are unknown. Ammonia is a toxic, water-soluble metabolic waste that accumulates within eggs as embryos develop and concentrates as eggs dehydrate. Thus, increasing ammonia concentration may be a direct threat to embryos in drying eggs. We hypothesized that it could serve as a cue, stimulating embryos to hatch and escape. The embryos of red-eyed treefrogs, Agalychnis callidryas, hatch early to escape from many threats, including dehydration, and are known to use mechanosensory, hypoxia, and light cues. To test if they also use high ammonia as a cue to hatch, we exposed stage-matched pairs of hatching-competent, well-hydrated sibling embryos to ammonia and control solutions in shallow water baths and recorded their behavior. Control embryos remained unhatched while ammonia-exposed embryos showed a rapid, strong hatching response; 95% hatched, on average in under 15 min. This demonstrates that elevated ammonia can serve as a hatching cue for A. callidryas embryos. This finding is a key step in understanding the mechanisms that enable terrestrial frog embryos to escape from egg drying, opening new possibilities for integrative and comparative studies on this growing threat.

Protocol for the controlled reproduction of the edible frog Aubria subsigillata in aquaculture hatcheries in the Republic of Benin.

Context Aubria subsigillata is such a highly valued, edible species for the citizens of Benin that over exploitation has led to a rarefaction of wild populations. Aims The aim of captive breeding is to develop breeding protocols and farming practices for the species which will reduce hunting pressure on wild populations. Methods The methodology consisted of determining the concentration of ovulatory hormone and its method of injection into the breeding stock, followed by in vitro fertilisation of the unfertilised eggs of the females by the spermic urine of the males to determine the optimum injection method, hormone concentration for ovulation and sperm collections, and the development of in vitro fertilisation protocols using gametes obtained via the aforementioned methodologies. Key results Results indicated that 0.2IU/g concentration of gonadotropin-releasing hormone agonist administered intrafemorally enabled spontaneous release of spermic urine and ova in the breeding animals. The latency time between injection and collection of gametes was 13h in males and 27h in females at a temperature of 28.5°C. Females laid an average of 172 eggs weighing 1mg mass. Conclusions Aubria subsigillata is a frog that reproduces using stimuli (hormone), and in vitro fertilisation resulted in a high rate of fertilised eggs. Implications Artifical reproduction in A. subsigillata is carried out in five phases: (1) selection of mature broodstock; (2) hormonal injection; (3) gamete collection; (4) in vitro fertilisation; and (5) incubation. However, work should continue on improving the egg hatching rate.

Single cell RNA analysis uncovers the cell differentiation and functionalization for air breathing of frog lung.

The evolution and development of vertebrate lungs have been widely studied due to their significance in terrestrial adaptation. Amphibians possess the most primitive lungs among tetrapods, underscoring their evolutionary importance in bridging the transition from aquatic to terrestrial life. However, the intricate process of cell differentiation during amphibian lung development remains poorly understood. Using single-cell RNA sequencing, we identify 13 cell types in the developing lungs of a land-dwelling frog (Microhyla fissipes). We elucidate the differentiation trajectories and mechanisms of mesenchymal cells, identifying five cell fates and their respective driver genes. Using temporal dynamics analyses, we reveal the gene expression switches of epithelial cells, which facilitate air breathing during metamorphosis. Furthermore, by integrating the published data from another amphibian and two terrestrial mammals, we illuminate both conserved and divergent cellular repertoires during the evolution of tetrapod lungs. These findings uncover the frog lung cell differentiation trajectories and functionalization for breathing in air and provide valuable insights into the cell-type evolution of vertebrate lungs.