Evolution of acoustic signals associated with cooperative parental behavior in a poison frog.

The emergence of complex social interactions is predicted to be an important selective force in the diversification of communication systems. Parental care presents a key social context in which to study the evolution of novel signals, as care often requires communication and behavioral coordination between parents and is an evolutionary stepping-stone toward increasingly complex social systems. Anuran amphibians (frogs and toads) are a classic model of acoustic communication and the vocal repertoires of many species have been characterized in the contexts of advertisement, courtship, and aggression, yet quantitative descriptions of calls elicited in the context of parental care are lacking. The biparental poison frog, Ranitomeya imitator, exhibits a remarkable parenting behavior in which females, cued by the calls of their male partners, feed tadpoles unfertilized eggs. Here, we characterized and compared calls across three social contexts, for the first time including a parental care context. We found that egg-feeding calls share some properties with both advertisement and courtship calls but also had unique properties. Multivariate analysis revealed high classification success for advertisement and courtship calls but misclassified nearly half of egg feeding calls as either advertisement or courtship calls. Egg feeding and courtship calls both contained less identity information than advertisement calls, as expected for signals used in close-range communication where uncertainty about identity is low and additional signal modalities may be used. Taken together, egg-feeding calls likely borrowed and recombined elements of both ancestral call types to solicit a novel, context-dependent parenting response.

Definition of a saxitoxin (STX) binding code enables discovery and characterization of the anuran saxiphilin family.

American bullfrog (Rana castesbeiana) saxiphilin (RcSxph) is a high-affinity "toxin sponge" protein thought to prevent intoxication by saxitoxin (STX), a lethal bis-guanidinium neurotoxin that causes paralytic shellfish poisoning (PSP) by blocking voltage-gated sodium channels (NaVs). How specific RcSxph interactions contribute to STX binding has not been defined and whether other organisms have similar proteins is unclear. Here, we use mutagenesis, ligand binding, and structural studies to define the energetic basis of Sxph:STX recognition. The resultant STX "recognition code" enabled engineering of RcSxph to improve its ability to rescue NaVs from STX and facilitated discovery of 10 new frog and toad Sxphs. Definition of the STX binding code and Sxph family expansion among diverse anurans separated by ∼140 My of evolution provides a molecular basis for understanding the roles of toxin sponge proteins in toxin resistance and for developing novel proteins to sense or neutralize STX and related PSP toxins.

Aquatic Soundscape Recordings Reveal Diverse Vocalizations and Nocturnal Activity of an Endangered Frog.

AbstractAutonomous sensors provide opportunities to observe organisms across spatial and temporal scales that humans cannot directly observe. By processing large data streams from autonomous sensors with deep learning methods, researchers can make novel and important natural history discoveries. In this study, we combine automated acoustic monitoring with deep learning models to observe breeding-associated activity in the endangered Sierra Nevada yellow-legged frog (Rana sierrae), a behavior that current surveys do not measure. By deploying inexpensive hydrophones and developing a deep learning model to recognize breeding-associated vocalizations, we discover three undocumented R. sierrae vocalization types and find an unexpected temporal pattern of nocturnal breeding-associated vocal activity. This study exemplifies how the combination of autonomous sensor data and deep learning can shed new light on species' natural history, especially during times or in locations where human observation is limited or impossible.

Species- and sex-specific chemical composition from an internal gland-like tissue of an African frog family.

Intraspecific chemical communication in frogs is understudied and the few published cases are limited to externally visible and male-specific breeding glands. Frogs of the family Odontobatrachidae, a West African endemic complex of five morphologically cryptic species, have large, fatty gland-like strands along their lower mandible. We investigated the general anatomy of this gland-like strand and analysed its chemical composition. We found the strand to be present in males and females of all species. The strand varies in markedness, with well-developed strands usually found in reproductively active individuals. The strands are situated under particularly thin skin sections, the vocal sac in male frogs and a respective area in females. Gas-chromatography/mass spectrometry and multivariate analysis revealed that the strands contain sex- and species-specific chemical profiles, which are consistent across geographically distant populations. The profiles varied between reproductive and non-reproductive individuals. These results indicate that the mandibular strands in the Odontobatrachidae comprise a so far overlooked structure (potentially a gland) that most likely plays a role in the mating and/or breeding behaviour of the five Odontobatrachus species. Our results highlight the relevance of multimodal signalling in anurans, and indicate that chemical communication in frogs may not be restricted to sexually dimorphic, apparent skin glands.

Antagonistic regulation of homeologous uncx.L and uncx.S genes orchestrates myotome and sclerotome differentiation in the evolutionarily divergent vertebral column of Xenopus laevis.

In anurans, the vertebral column diverges widely from that of other tetrapods; yet the molecular mechanisms underlying its morphogenesis remain largely unexplored. In this study, we investigate the role of the homeologous uncx.L and uncx.S genes in the vertebral column morphogenesis of the allotetraploid frog Xenopus laevis. We initiated our study by cloning the uncx orthologous genes in the anuran Xenopus and determining their spatial expression patterns using in situ hybridization. Additionally, we employed gain-of-function and loss-of-function approaches through dexamethasone-inducible uncx constructs and antisense morpholino oligonucleotides, respectively. Comparative analysis of the messenger RNA sequences of homeologous uncx genes revealed that the uncx.L variant lacks the eh1-like repressor domain. Our spatial expression analysis indicated that in the presomitic mesoderm and somites, the transcripts of uncx.L and uncx.S are located in overlapping domains. Alterations in the function of uncx genes significantly impact the development and differentiation of the sclerotome and myotome, resulting in axial skeleton malformations. Our findings suggest a scenario where the homeologous genes uncx.L and uncx.S exhibit antagonistic functions during somitogenesis. Specifically, uncx.S appears to be crucial for sclerotome development and differentiation, while uncx.L primarily influences myotome development. Postallotetraploidization, the uncx.L gene in X. laevis evolved to lose its eh1-like repressor domain, transforming into a "native dominant negative" variant that potentially competes with uncx.S for the same target genes. Finally, the histological analysis revealed that uncx.S expression is necessary for the correct formation of pedicles and neural arch of the vertebrae, and uncx.L is required for trunk muscle development.

Populating a Continent: Phylogenomics Reveal the Timing of Australian Frog Diversification.

The Australian continent's size and isolation make it an ideal place for studying the accumulation and evolution of biodiversity. Long separated from the ancient supercontinent Gondwana, most of Australia's plants and animals are unique and endemic, including the continent's frogs. Australian frogs comprise a remarkable ecological and morphological diversity categorized into a small number of distantly related radiations. We present a phylogenomic hypothesis based on an exon-capture dataset that spans the main clades of Australian myobatrachoid, pelodryadid hyloid, and microhylid frogs. Our time-calibrated phylogenomic-scale phylogeny identifies great disparity in the relative ages of these groups which vary from Gondwanan relics to recent immigrants from Asia and include arguably the continent's oldest living vertebrate radiation. This age stratification provides insight into the colonization of⁠, and diversification on, the Australian continent through deep time, during periods of dramatic climatic and community changes. Contemporary Australian frog diversity highlights the adaptive capacity of anurans, particularly in response to heat and aridity, and explains why they are one of the continent's most visible faunas.

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.

European common frogs determine migratory direction by inclination magnetic compass and show diurnal variation in orientation.

Animals can use two variants of the magnetic compass: the 'polar compass' or the 'inclination compass'. Among vertebrates, the compass type has been identified for salmon, mole rats, birds, turtles and urodeles. However, no experiments have been conducted to determine the compass variant in anurans. To elucidate this, we performed a series of field and laboratory experiments on males of the European common frog during the spawning season. In field experiments in a large circular arena, we identified the direction of the stereotypic migration axis for a total of 581 frogs caught during migration from river to pond or in a breeding pond. We also found that motivation of the frogs varied throughout the day, probably to avoid deadly night freezes, which are common in spring. The laboratory experiments were conducted on a total of 450 frogs in a T-maze placed in a three-axis Merritt coil system. The maze arms were positioned parallel to the natural migration axis inferred on the basis of magnetic field. Both vertical and horizontal components of the magnetic field were altered, and frogs were additionally tested in a vertical magnetic field. We conclude that European common frogs possess an inclination magnetic compass, as for newts, birds and sea turtles, and potentially use it during the spring migration. The vertical magnetic field confuses the frogs, apparently as a result of the inability to choose a direction. Notably, diurnal variation in motivation of the frogs was identical to that in nature, indicating the presence of internal rhythms controlling this process.

Characteristics of the Integument of Phyllomedusa burmeisteri (Anura, Phyllomedusidae) and Boana semilineata (Anura, Hylidae) Males Can Contribute to Understanding its Relation to Habitat.

The integument of anurans plays vital physiological roles, crucial for understanding the species' survival in their environment. Despite its significance, there are few studies describing the cutaneous morphology of anurans from the Brazilian Atlantic Forest. This study aimed to characterize the integument of Phyllomedusa burmeisteri and Boana semilineata in males using microscopic and histochemical approaches. Histological sections were stained with various dyes, and additional fragments underwent electron microscopy and energy-dispersive X-ray spectroscopy. Results showed different projections on the dorsal and ventral regions of males from these species, without the Eberth-Katschenko layer. Differences in the arrangement of chromatophore cells in regions with varying solar incidence were observed in the spongy dermis. Various gland types were identified, aiding taxonomic differentiation and validation of behavioral data. Both species had seromucous and granular glands, while only P. burmeisteri displayed lipid glands. Histochemical analysis revealed higher production of polysaccharides and proteins, contributing to the integument's moisture and protection. Lipid secretions in P. burmeisteri helped waterproof the integument more effectively against desiccation. This study concludes that analyzing anuran integument provides valuable insights into their behavior, with integument composition potentially influenced by habitat choice among different species.

Lernaea cf. cyprinacea infection in agile frog (Rana dalmatina) tadpoles in Hungary.

The ectoparasitic copepod Lernaea cyprinacea (anchor worm) has more than 100 host species among teleost fishes and affects cyprinids both in fish farms and natural waters. In addition, while L. cyprinacea infection in amphibians has been recorded in Asia, North and South America, there is no data available in the literature on their presence in Europe. In this study, we first reported L. cf. cyprinacea parasitising an anuran tadpole in Europe. Specimens of L. cf. cyprinacea were observed attached to a tadpole of the agile frog (Rana dalmatina), which was caught during fishing of crucian carp fingerlings (Carassius carassius) from a small fish pond in Hungary during the summer of 2012. The infected tadpole was collected from a rearing pond, where juvenile crucian carps were kept. The tadpole was inspected in the laboratory, and digital photos were taken. The parasites were found attached to the body-tail junction and to the leg of the tadpole (at Gosner stage 41). The parasite species was identified as L. cf. cyprinacea based on morphological traits.

Locomotor, ecological and phylogenetic drivers of skeletal proportions in frogs.

Frogs exhibit complex anatomical features of the pelvis, limbs and spine, long assumed to represent specialisations for jumping. Yet frogs employ a wide range of locomotor modes, with several taxa featuring primary locomotor modes other than jumping. Using a combination of techniques (CT imaging and 3D visualization, morphometrics, phylogenetic mapping), this study aims to determine the link between skeletal anatomy and locomotor style, habitat type and phylogenetic history, shedding new light on how functional demands impact morphology. Body and limb measurements for 164 taxa from all the recognised anuran families are extracted from digitally segmented CT scans of whole frog skeletons and analysed using various statistical techniques. We find that the expansion of the sacral diapophyses is the most important variable for predicting locomotor mode, which was more closely correlated with frog morphology than either habitat type or phylogenetic relationships. Predictive analyses suggest that skeletal morphology is a useful indicator of jumping but less so for other locomotor modes, suggesting that there is a wide range of anatomical solutions to performing locomotor styles such as swimming, burrowing or walking.

Osteological and histological comparison of the development of the interphalangeal intercalary skeletal element between hyloid and ranoid anurans.

Some frog species have a unique skeletal element, referred to as the intercalary element (IE), in the joints between the terminal and subterminal phalanges of all digits. IEs are composed of cartilage or connective tissue and have a markedly differ shape than the phalanges. IEs are highly related to the arboreal lifestyle and toe pads. The IE is found only in neobatrachian frogs among anurans, suggesting that it is a novelty of Neobatrachia. IEs are widely distributed among multiple neobatrachian lineages and are found in the suborders Hyloides and Ranoides (the two major clades in Neobatrachia). However, it is unclear whether the IEs found in multiple linages resulted from convergent evolution. Therefore, in this study, we aimed to examine how similar or different the developmental trajectories of the IEs are between Hyloides and Ranoides. To that end, we compared the osteological and histological developmental processes of the IEs of the hyloid frog Dryophytes japonicus and the ranoid frog Zhangixalus schlegelii. Both species shared the same IE-initiation site and level of tissue differentiation around the IE when it began to form in tadpoles, although the IE developments initiated at different stages which were determined by external criteria. These results suggest that similar mechanisms drive IE formation in the digits of both species, supporting the hypothesis that the IEs did not evolve convergently.

Neuroethology of auditory systems: contributions in memory of Albert S. Feng.

Albert (Al) S. Feng (1944 - 1921) was a pioneer in the area of neuroethology of auditory systems. This special issue of the Journal of Comparative Physiology A commemorates his life and work by presenting 15 articles written by friends, students, and colleagues, many of whom have become leading experts themselves in this field. Their contributions not only provide a comprehensive overview of bioacoustics in amphibians and mammals (including bats), but also are intended to inspire a new generation of scientists to advance our understanding of brain mechanisms of acoustic perception.

Descending projections to the auditory midbrain: evolutionary considerations.

The mammalian inferior colliculus (IC) is massively innervated by multiple descending projection systems. In addition to a large projection from the auditory cortex (AC) primarily targeting the non-lemniscal portions of the IC, there are less well-characterized projections from non-auditory regions of the cortex, amygdala, posterior thalamus and the brachium of the IC. By comparison, the frog auditory midbrain, known as the torus semicircularis, is a large auditory integration center that also receives descending input, but primarily from the posterior thalamus and without a projection from a putative cortical homolog: the dorsal pallium. Although descending projections have been implicated in many types of behaviors, a unified understanding of their function has not yet emerged. Here, we take a comparative approach to understanding the various top-down modulators of the IC to gain insights into their functions. One key question that we identify is whether thalamotectal projections in mammals and amphibians are homologous and whether they interact with evolutionarily more newly derived projections from the cerebral cortex. We also consider the behavioral significance of these descending pathways, given anurans' ability to navigate complex acoustic landscapes without the benefit of a corticocollicular projection. Finally, we suggest experimental approaches to answer these questions.

Are frog calls relatively difficult to locate by mammalian predators?

Frogs call in acoustically dense choruses to attract conspecific females. Their calls can potentially reveal their location to predators, many of which are mammals. However, frogs and mammals have very different acoustic receivers and mechanisms for determining sound source direction. We argue that frog calls may have been selected so that they are harder to locate with the direction-finding mechanisms of mammals. We focus on interaural time delay (ITD) estimation using delay-line coincidence detection (place code), and a binaural excitatory/inhibitory (E/I) ITD mechanism found in mammals with small heads (population code). We identify four "strategies" which frogs may employ to exploit the weaknesses of either mechanism. The first two strategies used by the frog confound delay estimation to increase direction ambiguity using highly periodic calls or narrowband calls. The third strategy relies on using short pulses. The E/I mechanism is susceptible to noise with sounds being pulled to the medial plane when signal-to-noise ratio is low. Together, these three strategies compromise both ongoing and onset determination of location using either mechanism. Finally, frogs call in dense choruses using various means for controlling synchrony, maintaining chorus tenure, and abruptly switching off calling, all of which serve to confound location finding. Of these strategies, only chorusing adversely impacts the localization performance of frogs' acoustic receivers. We illustrate these strategies with an analysis of calls from three different frog species.

Male antiphonal calls and phonotaxis evoked by female courtship calls in the large odorous frog (Odorrana graminea).

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.

First Report of Culturable Skin Bacteria in Melanophryniscus admirabilis (Admirable Redbelly Toad).

Melanophryniscus admirabilis is a small toad, critically endangered with a microendemic distribution in the Atlantic Forest in southern Brazil. The amphibian skin microbiome is considered one of the first lines of defense against pathogenic infections, such as Batrachochytrium dendrobatidis (Bd). The knowledge of skin amphibian microbiomes is important to numerous fields, including species conservation, detection, and quantification of environmental changes and stressors. In the present study, we investigated, for the first time, cultivable bacteria in the skin of wild M. admirabilis, and detected Bd fungus by nested polymerase chain reaction (PCR) technique. Skin swab samples were collected from 15 wild M. admirabilis, and the isolation of bacteria was performed by means of different culture strategies. A total of 62 bacterial isolates being Bacillus (n = 22; 34.48%), Citrobacter (n = 10; 16.13%), and Serratia (n = 12; 19.35%) were more frequently isolated genera. Interestingly, all skin samples tested were Bd negative. Some bacterial genera identified in our study might be acting in a synergic relationship and protecting them against the Bd fungus. In addition, these bacteria may play an essential role in maintaining this species in an environment modulated by anthropic actions. This first report of skin cultivable bacteria from M. admirabilis natural population improves our knowledge of skin amphibian microbiomes, contributing to a better understanding of their ecology and how this species has survived in an environment modulated by anthropic action.

Complex Life Histories Alter Patterns of Mercury Exposure and Accumulation in a Pond-Breeding Amphibian.

Quantifying how contaminants change across life cycles of species that undergo metamorphosis is critical to assessing organismal risk, particularly for consumers. Pond-breeding amphibians can dominate aquatic animal biomass as larvae and are terrestrial prey as juveniles and adults. Thus, amphibians can be vectors of mercury exposure in both aquatic and terrestrial food webs. However, it is still unclear how mercury concentrations are affected by exogenous (e.g., habitat or diet) vs endogenous factors (e.g., catabolism during hibernation) as amphibians undergo large diet shifts and periods of fasting during ontogeny. We measured total mercury (THg), methylmercury (MeHg), and isotopic compositions (δ 13C, δ15N) in boreal chorus frogs (Pseudacris maculata) across five life stages in two Colorado (USA) metapopulations. We found large differences in concentrations and percent MeHg (of THg) among life stages. Frog MeHg concentrations peaked during metamorphosis and hibernation coinciding with the most energetically demanding life cycle stages. Indeed, life history transitions involving periods of fasting coupled with high metabolic demands led to large increases in mercury concentrations. The endogenous processes of metamorphosis and hibernation resulted in MeHg bioamplification, thus decoupling it from the light isotopic proxies of diet and trophic position. These step changes are not often considered in conventional expectations of how MeHg concentrations within organisms are assessed.

Septicemia caused by an emerging pathogen, Elizabethkingia miricola, in a laboratory colony of African dwarf frogs (Hymenochirus curtipes).

An outbreak of morbidity and mortality in an African dwarf frog (Hymenochirus curtipes) colony was reported following arrival at an animal research facility. Animals were found dead on arrival or became moribund shortly thereafter, and additional animals showed clinical signs of lethargy, weight loss, and anorexia over the following 3 weeks. Externally, some affected animals presented with multifocal areas of hyperemia in the inguinal and axillary areas and on the limbs, and mottled tan discoloration along the ventral abdomen. Histologically, lesions were consistent with generalized septicemia, characterized by granulomatous meningitis, otitis media, peritonitis (coelomitis), myocarditis and pericarditis, nephritis, pneumonia, and arthritis. Gram staining identified gram-negative rod-shaped bacteria free within tissues and within macrophages. Culture results of coelomic swabs identified moderate to numerous Elizabethkingia miricola. Testing of water from tanks housing affected animals showed elevated levels of nitrites and ammonia, and the presence of Citrobacter, Aeromonas, Pseudomonas, and Staphylococcus spp. cultured from several tank biofilters. E miricola is a newly recognized and rapidly emerging opportunistic pathogen in anurans and has been reported as a cause of septicemia in humans. This report documents the first occurrence of E. miricola septicemia in African dwarf frogs and illustrates the importance of this potential pathogen in the laboratory setting for amphibian research colonies, as well as those individuals directly working with them.

A review of non-destructive biomonitoring techniques to assess the impacts of pollution on reproductive health in frogs and toads.

In anuran amphibians (frogs and toads), evidence linking pollution to population declines is limited, in particular through impaired reproduction. Here we review the evidence for pollutant-induced alterations on reproductive endpoints in wild anurans with a particular focus on the application of non-destructive endpoints including on sex ratios, male reproductive phenotypes (data are too scarce for females) and reproductive outputs (reflective of mating success). Data evidencing alterations in sex ratio in wild anurans are scarce, however, both feminisation and masculinisation in response to pollution have been reported (seven studies). Male nuptial pad morphology and calling behaviour display high sensitivity to pollutant-exposure and are important features determining male breeding success, however there is considerable variation in these endpoints and inconsistencies in the responses of them to pollution are reported in wild anurans. Data for clutch size are insufficient to assess sensitivity to pollutants (five studies only). However, hatch success and offspring fitness (tadpole survival/development) are sensitive to pollution, with clear linkages to population stability. In conclusion, there are a wide range of non destructive measures with good potential for application to assess/monitor reproductive health in wild anurans, however, a greater understanding of pollutant effects on these endpoints is needed. There measures deserve wider application as they are relatively simple and inexpensive to implement, and as they can be applied non-destructively are widely applicable to our declining anuran populations.