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.

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.

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.

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.

Tree frogs (Polypedates dennysi) landing on horizontal perches: the effects of perch diameter.

Secure landing is indispensable for both leaping animals and robotics. Tree frogs, renowned for their adhesive capabilities, can effectively jump across intricate 3D terrain and land safely. Compared with jumping, the mechanisms underlying their landing technique, particularly in arboreal environments, have remained largely unknown. In this study, we focused on the landing patterns of the tree frog Polypedates dennysi on horizontally placed perches, explicitly emphasizing the influence of perch diameters. Tree frogs demonstrated diverse landing postures, including the utilization of: (1) single front foot, (2) double front feet, (3) anterior bellies, (4) middle bellies, (5) posterior bellies, (6) single hind foot, or (5) double hind feet. Generally, tree frogs favoured bellies on slimmer targets but double front feet on large perches. Analysis of limb-trunk relationships revealed their adaptability to modify postures, including body positions and limb orientations, for successful landing. The variations in the initial landing postures affected the subsequent landing procedures and, consequently, the dynamics. As the initial contact position switched from front foot back to the hind foot, the stabilization time decreased at first, reaching a minimum in middle belly landings, and then increased again. The maximum vertical forces showed an inverse trend, whereas the maximum fore-aft forces continuously increased as the initial contact position switched. As the perch diameter increased, the time expended dropped, whereas the maximum impact force increased. These findings not only add to our understanding of frog landings but also highlight the necessity of considering perch diameters and landing styles when studying the biomechanics of arboreal locomotion.

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.

Male Cope's gray treefrogs (Hyla chrysoscelis) in amplexus have elevated and correlated steroid hormones compared to solitary males.

Gonadal steroid hormones are typically elevated during the breeding season-a finding known as an associated reproductive pattern. Though less studied, there is also evidence, in both sexes, for elevated adrenal/interrenal steroids, including acute elevations on the day of mating. I investigated gonadal and interrenal steroids in wild male Cope's gray treefrogs at breeding aggregations. I collected blood from males found in amplexus with female mates (amplexed males) and males sampled at the same time and location that were actively advertising vocally and without a mate (solo males). Concentrations of plasma corticosterone, testosterone, and 17ß-estradiol (CORT, T and E2, respectively) were validated and measured. These two categories of males differed in four ways: (1) amplexed males exhibited significantly elevated concentrations of all three steroids compared to solo males (CORT: +347 %; T: +60 %; and E2: +43 %); (2) these hormone profiles alone accurately predicted male mating category with ca. 83 % accuracy using a discriminant function analysis; (3) amplexed males exhibited significant between-hormone correlations (T and E2 were positively correlated and CORT and E2 were negatively correlated) whereas no correlations were found in solo males; (4) amplexed males showed a negative correlation with CORT concentration and the time of night, whereas no such pattern was present in solo males. These findings suggest an acute and strong coactivation of the interrenal and gonadal axes that could drive phenotypic integration during this fitness-determining moment. I discuss these findings and suggest experiments to determine causation, including the role of motor behavior driving endocrine states and the role of female selection on endocrine profiles.

Dark or light? Preference of anuran tadpoles to background illumination in response to food and predators.

Background illumination and its intensity are crucial factors in visual interaction among organisms. In the present study, we used tadpoles of eight sympatric anuran species (Duttaphrynus melanostictus, Microhyla ornata, Uperodon globulosus, Kaloula taprobanica, Euphlyctis cyanophlyctis, Fejervarya orissaensis, Polypedates maculatus, and Polypedates teraiensis) to examine species-specific responses towards background illumination (shade and light), combined with four treatments [without food and predator (F-P-), with food and without predator (F+P-), without food and with predator (F-P+), and with food and with predator (F+P+)], with four coloured backgrounds (green, red, brown, and black) during different hours of a day (early morning, late morning, noon, and afternoon). Anuran tadpoles choose shaded sides over lighter sides, as such microhabitats are used as an effective means of camouflage, but most of the M. ornata tadpoles preferred the opposite. The observations were similar for red, green, and brown backgrounds simulating different natural conditions. The developmental stage and co-occurrence of tadpoles of multiple species do not affect their behavioural responses. These results draw attention to the importance of low illuminated/shaded backgrounds for behavioural responses of anuran tadpoles to environmental changes.

Baseline corticosterone levels in spadefoot toads reflect alternate larval diets one year later.

Early-life environmental variation can influence later-life physiology, such as the regulation of glucocorticoids. However, characterizing the effects of environmental factors on hormone regulation can be hampered when assessing animals that are small and require destructive sampling to collect blood. Using spadefoot toads (genus Spea), we evaluated whether waterborne corticosterone (CORT) measures could be used as a proxy for plasma CORT measures, detect stress-induced levels of CORT, and detect larval diet-induced changes in CORT regulation after metamorphosed individuals were maintained for 1 year under common garden conditions. We found that waterborne CORT measures were correlated with plasma CORT measures and could be used to detect stress-induced levels of CORT. Further, larval diet type significantly influenced baseline plasma CORT levels 1-year post-metamorphosis: adults that had consumed live prey as larvae had higher plasma CORT levels than adults that had consumed detritus as larvae. However, waterborne measures failed to reflect these differences, possibly due to low sample size. Our study demonstrates the utility of the waterborne hormone assay in assessing variation in baseline and stress-induced CORT levels in adult spadefoots. However, resolving more subtle differences that arise through developmental plasticity will require larger samples sizes when using the waterborne assay.

Sperm parameters following hormonal induction of spermiation in an endangered frog [the alpine tree frog] (Litoria verreauxii alpina).

CONTEXT: With global amphibian biodiversity rapidly declining, improving reproductive technology outcomes has become essential. Captive breeding programs have struggled because amphibian breeding physiology often requires specific environmental cues that reproductive technologies can circumvent. AIMS: This study tests the efficiency of hormonal induction by evaluating sperm quality in the endangered Litoria verreauxii alpina . METHODS: We assessed the effects of exogenous hormones - gonadotrophin-releasing hormone agonist (GnRH-a, Lucrin), and human chorionic gonadotrophin (hCG, Chorulon) - on sperm quality. KEY RESULTS: Hormone induction with hCG showed high efficacy while GnRH-a yielded a low response in producing sperm. Sperm quantity was affected by time post injection, with the greatest quantity at 1h post injection. Sperm quality was also affected by time, where the sperm head size decreased by 11% at 7h post injection. CONCLUSIONS: Based on the results from this study, we recommend that that sperm be collected soon after induction, and not more than 4h post induction in L. v. alpina . More work needs to be completed before recommending an optimal hormone induction method and dose, but 120IU of hCG per male was successful for inducing spermiation. IMPLICATIONS: This study represents a useful starting point for developing assisted reproductive techniques for non-model organisms.

How signals interact in multimodal displays: Insights from a robotic frog.

IN FOCUS: Caldart, M. V., M. B. dos Santos & G. Machado (2021). Function of a multimodal signal: a multiple hypothesis test using a robot frog Journal of Animal Ecology, https://doi.org/10.1111/1365-2656.13620. Animals can communicate using signals perceived by different sensory systems, and many combine multiple sensory modalities in their display repertoires. Why these multimodal displays evolve and how they function to transmit information between individuals are crucial questions in behavioural and evolutionary research. Most empirical studies addressing these questions assume, even if implicitly, that signals of different modalities have independent effects on receiver responses. Nonetheless, the potential for interactions between signals as an explanation for the function of multimodal displays has been recognized for over two decades. Caldart et al. (2021) use a robotic frog and a receiver-based approach to test four alternative hypotheses for the function of multimodal (acoustic + visual) displays in the stream-dwelling frog Crossodactylus schmidti. Their results lend support to an inter-signal interaction mechanism, whereby inclusion of visual signals modifies the context in which an acoustic display is interpreted. In contrast, the results in Caldart et al. (2021) are less consistent with the hypotheses that emphasize the quality-related information encoded in different signal modalities and a hypothesis that focuses on signal transmission across heterogeneous environments. These results showcase how experimental manipulation of different signal modalities and tests of multiple alternative hypotheses are key to clarifying the function of multimodal displays.

Response of hepatic biomarkers in Physalaemus nattereri (Anura) to different benzo(α)pyrene exposure routes.

For over 40 years, anurans have been used as a study model to assess the adverse effects of benzo(α)pyrene (BαP), which include genotoxic, hepatotoxic, and immunotoxic effects. In these studies, BαP is administered cutaneously or by injection, with no comparison between two or more routes. The purpose of this study is to assess whether the effect of BαP is influenced by its route of administration, using the response of hepatic biomarkers of Physalaemus nattereri. Specimens (n = 108) were collected and divided into three experimental treatments (cutaneous, injection, and oral) and three experimental times (one, three, and seven days). Specimens received 0.02 ml of pure mineral oil (control) or mineral oil containing 2 mg/kg of BαP. The BαP causes changes in morphological (melanin, hemosiderin, lipofuscin, and mast cells) and biochemical (superoxide dismutase and glutathione S-transferase) hepatic biomarkers. Compared to biochemical, morphological biomarkers underwent a greater number of significant changes due to the treatment with BαP. The route of exposure alters the effects of BαP, mainly seen in morphological biomarkers, especially the pigments melanin, hemosiderin, and lipofuscin. In these pigments, the effect of the exposure pathway changes according to the analyzed biomarker, and the exposure time modulates the exposure pathway effect. These results are unprecedented for anurans and contribute to the field of herpetology and ecotoxicology.

The peptide secreted at the water to land transition in a model amphibian has antioxidant effects.

In addition to the morphophysiological changes experienced by amphibians during metamorphosis, they must also deal with a different set of environmental constraints when they shift from the water to the land. We found that Pithecopus azureus secretes a single peptide ([M + H]+ = 658.38 Da) at the developmental stage that precedes the onset of terrestrial behaviour. De novo peptide and cDNA sequencing revealed that the peptide, named PaT-2, is expressed in tandem and is a member of the tryptophyllins family. In silico studies allowed us to identify the position of reactive sites and infer possible antioxidant mechanisms of the compounds. Cell-based assays confirmed the predicted antioxidant activity in mammalian microglia and neuroblast cells. The potential neuroprotective effect of PaT-2 was further corroborated in FRET-based live cell imaging assays, where the peptide prevented lipopolysaccharide-induced ROS production and glutamate release in human microglia. In summary, PaT-2 is the first peptide expressed during the ontogeny of P. azureus, right before the metamorphosing froglet leaves the aquatic environment to occupy terrestrial habitats. The antioxidant activity of PaT-2, predicted by in silico analyses and confirmed by cell-based assays, might be relevant for the protection of the skin of P. azureus adults against increased O2 levels and UV exposure on land compared with aquatic environments.

Concerted evolution reveals co-adapted amino acid substitutions in Na+K+-ATPase of frogs that prey on toxic toads.

Although gene duplication is an important source of evolutionary innovation, the functional divergence of duplicates can be opposed by ongoing gene conversion between them. Here, we report on the evolution of a tandem duplication of Na+,K+-ATPase subunit α1 (ATP1A1) shared by frogs in the genus Leptodactylus, a group of species that feeds on toxic toads. One ATP1A1 paralog evolved resistance to toad toxins although the other retained ancestral susceptibility. Within species, frequent non-allelic gene conversion homogenized most of the sequence between the two copies but was counteracted by strong selection on 12 amino acid substitutions that distinguish the two paralogs. Protein-engineering experiments show that two of these substitutions substantially increase toxin resistance, whereas the additional 10 mitigate their deleterious effects on ATPase activity. Our results reveal how examination of neo-functionalized gene duplicate evolution can help pinpoint key functional substitutions and interactions with the genetic backgrounds on which they arise.

Estrogenic Modulation of Retinal Sensitivity in Reproductive Female Túngara Frogs.

Although mate searching behavior in female túngara frogs (Physalaemus pustulosus) is nocturnal and largely mediated by acoustic cues, male signaling includes visual cues produced by the vocal sac. To compensate for these low light conditions, visual sensitivity in females is modulated when they are in a reproductive state, as retinal thresholds are decreased. This study tested whether estradiol (E2) plays a role in this modulation. Female túngara frogs were injected with either human chorionic gonadotropin (hCG) or a combination of hCG and fadrozole. hCG induces a reproductive state and increases retinal sensitivity, while fadrozole is an aromatase inhibitor that blocks hCG-induced E2 synthesis. In an analysis of scotopic electroretinograms (ERGs), hCG treatment lowered the threshold for eliciting a b-wave response, whereas the addition of fadrozole abolished this effect, matching thresholds in non-reproductive saline-injected controls. This suggests that blocking E2 synthesis blocked the hCG-mediated reproductive modulation of retinal sensitivity. By implicating E2 in control of retinal sensitivity, our data add to growing evidence that the targets of gonadal steroid feedback loops include sensory receptor organs, where stimulus sensitivity may be modulated, rather than more central brain nuclei, where modulation may affect mechanisms involved in motivation.

Dual skin functions in amphibian osmoregulation.

August Krogh's studies of the frog identified the respiratory function of the skin in 1904 and the osmoregulatory function of the skin in 1937. It is the thesis of my review that the osmoregulatory function of the skin has evolved for meeting quite different demands. In freshwater the body fluid homeostasis is challenged by loss of ions to the environment. This is compensated for by active ion uptake energized by the sodium-pump ATPase and the V-type proton pump ATPase. I conclude that Krogh's astonishing observation of cutaneous chloride uptake from µM concentrations of NaCl is compatible with the free energy changes of ATP hydrolysis catalyzed by the sodium­potassium pump ATPase and the V-type proton pump ATPase operating in series, and in parallel with experimentally verified vanishingly small leak fluxes. On land the frog is challenged by evaporative water loss through the highly water permeable skin, similar to the water permeable conducting airways of terrestrial vertebrates including man. The epithelia serving respiratory gas exchanges are heterocellular and have molecular, structural and functional properties in common. The cutaneous surface liquid of amphibians evolved for protecting the skin epithelium from desiccation like the airway surface liquid of the lung. Published studies of ion transport mechanisms of acinar cells and the two types of epithelial cells, lead to the hypothesis that subepithelial gland secretion, evaporative water loss, and ion reabsorption by the epithelium regulate composition and volume of the cutaneous surface liquid.

Geographical range evolution of the genus Polypedates (Anura: Rhacophoridae) from the Oligocene to present.

Currently, the genus Polypedates comprises 26 species distributed in South, Southeast, and East Asia. Because of their relatively low dispersal capability and intolerance to seawater, this genus is ideal for the study of terrestrial range evolution that extends into the island archipelagos of southeastern Asia. In this study, based on data compiled for Polypedatesfrom previous studies and partial mitochondrial and nuclear genes collected in this study, we performed systematic biogeographical analysis. We confirmed a Sundaland origin for the extant genus and showed northward dispersal into mainland Southeast Asia and Asia, which coincided with the timing of paleoclimatic change from the Oligocene to Middle Miocene. Climate fluctuations had a profound impact on species diversification within the genus Polypedates. Furthermore, the Red River did not mediate species exchange between Southeast Asia and mainland Asia until the end of the Miocene, with the sudden onset of northward dispersal in several clades independently at that time. Alternatively, the lineage of widespread insular P. leucomystaxstrongly supports the hypothesis of terrestrial connection between island archipelagos of Southeast Asia during the Mid-Pleistocene paleoclimate fluctuations. Our biogeographical analysis also supports the recent introduction of P. leucomystax to the Philippines and Ryukyus, as previously suggested.

Background choice and immobility as context dependent tadpole responses to perceived predation risk.

The association of immobility and camouflage is widespread as a defensive mechanism in prey from varied taxa. However, many experiments assessing the reaction of prey to predator cues are conducted under artificial laboratory conditions. In a previous experiment we observed the tadpoles of Ololygon machadoi (Hylidae) to respond to predator visual and/or chemical cues by choosing backgrounds that improve their disruptive properties, but detected no associated reduction of movement. Here we experimentally demonstrate this response in the species' natural habitat, on backgrounds where the tadpoles are likely to achieve their best camouflage. We also tested whether previous experiences could influence both background choice and immobility in O. machadoi tadpoles. These novel experimental results suggest that a defensive behavior-i.e., reduction of movement-in these tadpoles is more strongly expressed under the natural conditions where they evolved, compared to laboratory conditions where prey and predator were brought into closer contact. Besides, previous experiences are likely to play an important role in expressed defensive responses.

B1CTcu5: A frog-derived brevinin-1 peptide with anti-tuberculosis activity.

Tuberculosis (TB) is a devastating infectious disease that causes a high rate of mortality. Drugs with new modes of action are needed to overcome this scenario. Cationic antibacterial peptides can serve as a potential alternative to existing TB drugs as they target the entire bacterial membrane for activity, thereby reducing the probability of development of drug resistance. In this study, we report anti-tuberculosis activity of B1CTcu5, a peptide that belongs to brevinin-1 family of antimicrobial peptides. This peptide possesses potent in vitro inhibitory activity against M. tuberculosis at 12.5 µg/mL but was not active against M. smegmatis. B1CTcu5 successfully eliminated intracellular mycobacteria without inducing cytotoxicity to the human macrophages at the concentrations tested. This peptide can be used as a template to design peptide-based anti-tubercular agents.

Mechanisms for Color Convergence in a Mimetic Radiation of Poison Frogs.

In animals, bright colors often evolve to mimic other species when a resemblance is selectively favored. Understanding the proximate mechanisms underlying such color mimicry can give insights into how mimicry evolves-for example, whether color convergence evolves from a shared set of mechanisms or through the evolution of novel color production mechanisms. We studied color production mechanisms in poison frogs (Dendrobatidae), focusing on the mimicry complex of Ranitomeya imitator. Using reflectance spectrometry, skin pigment analysis, electron microscopy, and color modeling, we found that the bright colors of these frogs, both within and outside the mimicry complex, are largely structural and produced by iridophores but that color production depends crucially on interactions with pigments. Color variation and mimicry are regulated predominantly by iridophore platelet thickness and, to a lesser extent, concentration of the red pteridine pigment drosopterin. Compared with each of the four morphs of model species that it resembles, R. imitator displays greater variation in both structural and pigmentary mechanisms, which may have facilitated phenotypic divergence in this species. Analyses of nonmimetic dendrobatids in other genera demonstrate that these mechanisms are widespread within the family and that poison frogs share a complex physiological "color palette" that can produce diverse and highly reflective colors.