Genetic isolation by distance underlies color pattern divergence in red-eyed treefrogs (Agalychnis callidryas)

Investigating the spatial distribution of genetic and phenotypic variation can provide insights into the evolutionary processes that shape diversity in natural systems. We characterized patterns of genetic and phenotypic diversity to learn about drivers of color-pattern diversification in red-eyed treefrogs (Agalychnis callidryas) in Costa Rica. Along the Pacific coast, red-eyed treefrogs have conspicuous leg color patterning that transitions from orange in the north to purple in the south. We measured phenotypic variation of frogs across Pacific sites, with increased sampling at sites where the orange-to-purple transition occurs. At the transition zone, we discovered the co-occurrence of multiple color-pattern morphs. To explore possible causes of this variation, we generated a SNP dataset with RAD sequencing to analyze population genetic structure, measure genetic diversity, and infer the processes that mediate genotype-phenotype dynamics. We investigated how patterns of genetic relatedness correspond with individual measures of color pattern along the coast, including testing for the role of hybridization in geographic regions where orange and purple phenotypic groups co-occur. We found no evidence that color-pattern polymorphism in the transition zone arose through recent hybridization or introgression. Instead, a strong pattern of genetic isolation by distance (IBD) indicates that color-pattern variation was retained through other processes such as ancestral color polymorphisms, ancient secondary contact or generated by novel mutations. We found that color phenotype changes along the Pacific coast more than would be expected from geographic distance alone. Combined, our results suggest the possibility of selective pressures acting on color pattern at a small geographic scale.

Identification of a new myotropic decapeptide from the skin secretion of the red-eyed leaf frog, Agalychnis callidryas

Bradykinin-related peptides (BRPs) family is one of the most significant myotropic peptide families derived from frog skin secretions. Here, a novel BRP callitide was isolated and identified from the red-eyed leaf frog, Agalychnis callidryas, with atypical primary structure FRPAILVRPK-NH2. The mature peptide was cleaved N-terminally at a classic propeptide convertase cleavage site (-KR-) and at the C-terminus an unusual -GKGKGK sequence was removed using the first G residue as an amide donor for the C-terminally-located K residue. Thereafter, the synthetic replicates of callitide were assessed the myotropic activity and showed a significant contraction of balder, with the 0.63 nM EC50 value, more potent than most discovered myotropic peptides. The binding mode was further speculated by molecular docking and stimulation. The result indicated that the C-terminal of callitide might selectively bind to bradykinin receptor B2 (BKRB2). Further investigation of the callitide needs to be done in the future to be exploited as potential future drug leads.

Multimodal mechanosensing enables treefrog embryos to escape egg-predators

ABSTRACTMechanosensory-cued hatching (MCH) is widespread, diverse and important for survival in many animals. From flatworms and insects to frogs and turtles, embryos use mechanosensory cues and signals to inform hatching timing, yet mechanisms mediating mechanosensing in ovo are largely unknown. The arboreal embryos of red-eyed treefrogs, Agalychnis callidryas, hatch prematurely to escape predation, cued by physical disturbance in snake attacks. When otoconial organs in the developing vestibular system become functional, this response strengthens, but its earlier occurrence indicates another sensor must contribute. Post-hatching, tadpoles use lateral line neuromasts to detect water motion. We ablated neuromast function with gentamicin to assess their role in A. callidryas’ hatching response to disturbance. Prior to vestibular function, this nearly eliminated the hatching response to a complex simulated attack cue, egg jiggling, revealing that neuromasts mediate early MCH. Vestibular function onset increased hatching, independent of neuromast function, indicating young embryos use multiple mechanosensory systems. MCH increased developmentally. All older embryos hatched in response to egg jiggling, but neuromast function reduced response latency. In contrast, neuromast ablation had no effect on the timing or level of hatching in motion-only vibration playbacks. It appears only a subset of egg-disturbance cues stimulate neuromasts; thus, embryos in attacked clutches may receive unimodal or multimodal stimuli. Agalychnis callidryas embryos have more neuromasts than described for any other species at hatching, suggesting precocious sensory development may facilitate MCH. Our findings provide insight into the behavioral roles of two mechanosensory systems in ovo and open possibilities for exploring sensory perception across taxa in early life stages.

Multimodal mechanosensing enables treefrog embryos to escape egg-predators

ABSTRACTMechanosensory-cued hatching (MCH) is widespread, diverse, and improves survival in many animals. From flatworms and insects to frogs and turtles, embryos use mechanosensory cues and signals to inform hatching timing, yet mechanisms mediating mechanosensing in ovo are largely unknown. The arboreal embryos of red-eyed treefrogs, Agalychnis callidryas, hatch prematurely to escape predation, cued by physical disturbance in snake attacks. When otoconial organs in the developing vestibular system become functional, this response strengthens, but its earlier occurrence indicates another sensor must contribute. Post-hatching, tadpoles use lateral line neuromasts to detect water motion. We ablated neuromast function with gentamicin to assess their role in A. callidryas’ hatching response to disturbance. Prior to vestibular function, this nearly eliminated the hatching response to a complex simulated attack cue, egg-jiggling, revealing that neuromasts mediate early MCH. Vestibular function onset increased hatching, independent of neuromast function, indicating young embryos use multiple mechanosensory systems. MCH increased developmentally. All older embryos hatched in response to egg-jiggling, but neuromast function reduced response latency. In contrast, neuromast ablation had no effect on timing or level of hatching in motion-only vibration playbacks. It appears only a subset of egg-disturbance cues stimulate neuromasts; thus embryos in attacked clutches may receive uni- or multimodal stimuli. A. callidryas embryos have more neuromasts than described for any other species at hatching, suggesting precocious sensory development may facilitate MCH. Our findings provide insight into the behavioral roles of two mechanosensory systems in ovo and open possibilities for exploring sensory perception across taxa in early life stages.SUMMARYRed-eyed treefrog embryos use both their lateral line and vestibular systems to sense the disturbance cues in egg-predator attacks that inform escape-hatching decisions.

Identification and Rational Design of a Novel Antibacterial Peptide Dermaseptin-AC from the Skin Secretion of the Red-Eyed Tree Frog Agalychnis callidryas

Antibiotic resistance represents a tremendous contemporary clinical challenge. Given this challenge, antimicrobial peptides (AMPs) are regarded as one of the most promising new options for next-generation lead antibiotics. Here, we describe the antibacterial activities of a cationic peptide named DRP-AC4, obtained from frog skin secretion using shotgun cloning. Two modified peptides were derived by substituting the sequence of amino acids to complete the hydrophobic face (DRP-AC4b) and increase net charge (DRP-AC4a), respectively. The activity and cytotoxicity of these two peptides were compared. DRP-AC4a displayed significantly increased potency against bacteria compared to the natural peptide. It should be noted, however, that both analogue peptides demonstrated higher lytic ability than the natural peptide against the membranes of mammalian erythrocytes. At the same time, all three peptides displayed lower hemolytic activity compared to their antibacterial activity. Here, we demonstrate that AMPs have more complex activity mechanisms and faster bactericidal rates than traditional antibiotics, which may be one of the reasons why bacteria do not develop resistance to them. These discoveries provide interesting insights into the discovery and development of novel drugs from natural sources.

How do red-eyed treefrog embryos sense motion in predator attacks? Assessing the role of vestibular mechanoreception

ABSTRACTThe widespread ability to alter hatching timing in response to environmental cues can serve as a defense against threats to eggs. Arboreal embryos of red-eyed treefrogs, Agalychnis callidryas, hatch up to 30% prematurely to escape predation. This escape-hatching response is cued by physical disturbance of eggs during attacks, including vibrations or motion, and thus depends critically on mechanosensory ability. Predator-induced hatching appears later in development than flooding-induced, hypoxia-cued hatching; thus, its onset is not constrained by the development of hatching ability. It may, instead, reflect the development of mechanosensor function. We hypothesize that vestibular mechanoreception mediates escape-hatching in snake attacks, and that the developmental period when hatching-competent embryos fail to flee from snakes reflects a sensory constraint. We assessed the ontogenetic congruence of escape-hatching responses and an indicator of vestibular function, the vestibulo-ocular reflex (VOR), in three ways. First, we measured VOR in two developmental series of embryos 3–7 days old to compare with the published ontogeny of escape success in attacks. Second, during the period of greatest variation in VOR and escape success, we compared hatching responses and VOR across sibships. Finally, in developmental series, we compared the response of individual embryos to a simulated attack cue with their VOR. The onset of VOR and hatching responses were largely concurrent at all three scales. Moreover, latency to hatch in simulated attacks decreased with increasing VOR. These results are consistent with a key role of the vestibular system in the escape-hatching response of A. callidryas embryos to attacks.Red-eyed treefrogs’ hatching responses to predator attacks, vibration playbacks, and egg-jiggling appear when vestibular function develops. Ear development may be a key limiting factor in the onset of mechanosensory-cued hatching.

When and where to hatch? Red-eyed treefrog embryos use light cues in two contexts

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