Egg-Clutch Biomechanics Affect Escape-Hatching Behavior and Performance.

Arboreal embryos of phyllomedusine treefrogs hatch prematurely to escape snake predation, cued by vibrations in their egg clutches during attacks. However, escape success varies between species, from ∼77% in Agalychnis callidryas to just ∼9% in A. spurrelli at 1 day premature. Both species begin responding to snake attacks at similar developmental stages, when vestibular mechanosensory function begins, suggesting that sensory ability does not limit the hatching response in A. spurrelli. Agalychnis callidryas clutches are thick and gelatinous, while A. spurrelli clutches are thinner and stiffer. We hypothesized that this structural difference alters the egg motion excited by attacks. Since vibrations excited by snakes must propagate through clutches to reach embryos, we hypothesized that the species difference in attack-induced hatching may reflect effects of clutch biomechanics on the cues available to embryos. Mechanics predicts that thinner, stiffer structures have higher free vibration frequencies, greater spatial attenuation, and faster vibration damping than thicker, more flexible structures. We assessed clutch biomechanics by embedding small accelerometers in clutches of both species and recording vibrations during standardized excitation tests at two distances from the accelerometer. Analyses of recorded vibrations showed that A. spurrelli clutches have higher free vibration frequencies and greater vibration damping than A. callidryas clutches. Higher frequencies elicit less hatching in A. callidryas, and greater damping could reduce the amount of vibration embryos can perceive. To directly test if clutch structure affects escape success in snake attacks, we transplanted A. spurrelli eggs into A. callidryas clutches and compared their escape rates with untransplanted, age-matched conspecific controls. We also performed reciprocal transplantation of eggs between pairs of A. callidryas clutches as a method control. Transplanting A. spurrelli embryos into A. callidryas clutches nearly tripled their escape success (44%) compared to conspecific controls (15%), whereas transplanting A. callidryas embryos into different A. callidryas clutches only increased escape success by 10%. At hatching competence, A. callidryas eggs are no longer jelly-encapsulated, while A. spurrelli eggs retain their jelly coat. Therefore, we compared the hatching response and latency of A. spurrelli in de-jellied eggs and their control, jelly-encapsulated siblings using manual egg-jiggling to simulate predation cues. Embryos in de-jellied eggs were more likely to hatch and hatched faster than control siblings. Together, our results suggest that the properties of parentally produced egg-clutch structures, including their vibration biomechanics, constrain the information available to A. spurrelli embryos and contribute to interspecific differences in hatching responses to predator attacks.

Resumen Los embriones arbóreos de las ranas Phyllomedusinae eclosionan prematuramente para escapar de la depredación de las serpientes, señaladas por las vibraciones en sus posturas durante los ataques. El éxito de escape varía entre especies, desde ∼77% en Agalychnis callidryas hasta sólo ∼9% en A. spurrelli a un día antes de la eclosión espontanea. Ambas especies comienzan a responder a los ataques de serpientes en etapas del desarrollo similares cuando inicia la función mecanosensorial vestibular, lo que sugiere que la capacidad sensorial no limita la respuesta de eclosión en A. spurrelli. Estructuralmente, las posturas de A. callidryas son gruesas y gelatinosas, mientras que las de A. spurrelli son más delgadas y rígidas. Esta diferencia en estructura de la postura podría afectar su movimiento y la propagación de las señales de vibración que perciben los embriones durante los ataques de serpientes. Por lo tanto, investigamos la hipótesis de que las diferencias entre especies en las respuestas de eclosión a los ataques de serpientes se deben a la influencia de la biomecánica de las posturas en las señales disponibles para los embriones. Mecánicamente, las estructuras más delgadas deberían tener frecuencias de resonancia más altas, mayor atenuación espacial y una amortiguación de vibraciones más rápida que las estructuras más gruesas y flexibles. Evaluamos la biomecánica de las posturas mediante la incorporación de acelerómetros pequeños dentro de las posturas de ambas especies, así como la grabación de vibraciones causadas por pruebas de excitación estandarizadas a diferentes distancias del acelerómetro. Los análisis de las vibraciones registradas indican que las posturas de A. spurrelli tienen frecuencias de vibración más altas y una mayor amortiguación de vibraciones que las posturas de A. callidryas. Las frecuencias más altas provocan menos eclosión en A. callidryas, y una mayor amortiguación podría reducir la cantidad de vibración que pueden percibir los embriones. Para probar directamente si la estructura de posturas afecta el éxito de escape en los ataques de serpientes, trasplantamos huevos de A. spurrelli en posturas de A. callidryas y comparamos sus tasas de escape con controles conespecíficos de la misma edad no trasplantados. También realizamos trasplante recíproco en A. callidryas como control de método. El trasplante de embriones de A. spurrelli en posturas de A. callidryas casi triplicó su éxito de escape (44%) en comparación con los controles conespecíficos (15%), mientras que el trasplante de huevos de A. callidryas aumentó el éxito de escape en solo un 10%. Al momento de la eclosión, los huevos de A. callidryas ya no están encapsulados en gelatina, mientras que los huevos de A. spurrelli conservan su cápsula de gelatina. Por lo tanto, comparamos la respuesta de eclosión y la latencia de A. spurrelli en huevos sin gelatina y sus hermanos de control, encapsulados en gelatina, usando movimientos manuales de huevos para simular señales de depredación. Los embriones en huevos sin gelatina tenían más probabilidades de eclosionar y eclosionaron más rápido que los hermanos de control. Nuestros resultados sugieren que las propiedades de las estructuras producidas por padres, como las posturas de huevos, incluida su biomecánica de vibración, restringen la información disponible para los embriones de A. spurrelli y contribuyen a las diferencias interespecíficas en las respuestas de eclosión a los ataques de los depredadores.

Patterns of parental care in Neotropical glassfrogs: fieldwork alters hypotheses of sex-role evolution.

Many animals provide parental care to offspring. Parental sex-roles vary extensively across taxa, and such patterns are considered well documented. However, information on amphibians is lacking relative to other vertebrate groups. We combine natural history observations with functional and historical analyses to examine the evolution of egg care in glassfrogs (Centrolenidae). Parental care was considered rare and predominately provided by males. Our field observations of 40 species revealed that care occurs throughout the family, and the caregiving sex changes across lineages. We discovered that a brief period of maternal care is widespread and occurs in species previously thought to lack care. Using a combination of female-removal experiments, prey-choice tests with egg-eating katydids, and parental disturbance-tolerance assays, we confirm the adaptive benefits of short-term maternal care in wild Cochranella granulosa and Teratohyla pulverata. To examine historical transitions between caregiving sexes, we assembled a molecular phylogeny and estimated ancestral care states using our data and the literature. We assessed patterns indicative of sex-specific constraints by testing whether transitions between the sexes are associated with changes in care levels. Our analyses support that male-only care evolved 2-3 times from female-only care, and this change is associated with substantial increases in care levels - a pattern supporting the hypothesis that male-only care evolved via constraints on maternal expenditure. Many groups of amphibians remain poorly studied, with emerging evidence indicating that care patterns are more diverse than currently appreciated. Natural history remains fundamental to uncovering this diversity and generating testable hypotheses of sex-role evolution.

Risk-induced hatching timing shows low heritability and evolves independently of spontaneous hatching in red-eyed treefrogs.

Plasticity in the timing of transitions between stages of complex life cycles allows organisms to adjust their growth and development to local environmental conditions. Genetic variation in such plasticity is common, but the evolution of context-dependent transition timing may be constrained by information reliability, lag-time and developmental constraints. We studied the genetic architecture of hatching plasticity in embryos of the red-eyed treefrog (Agalychnis callidryas) in response to simulated predator attacks using a series of paternal and maternal half-sibs from a captive breeding colony of wild-collected animals. We compared the developmental timing of induced early hatching across sibships and estimated cross-environment genetic correlations between induced and spontaneous hatching traits. Additive genetic variance for induced early hatching was very low, indicating a constraint on the short-term evolution of earlier hatching timing. This constraint is likely related to the maturation of the hatching mechanism. The most plastic genotypes produced the most extreme spontaneous hatching phenotypes, indicating that developmental range, per se, is not constrained. Cross-environment genetic correlation in hatching timing was negligible, so the evolution of spontaneous hatching in this species has not depended on the evolution of risk-induced hatching and vice versa.

The shape of things to come: linking developmental plasticity to post-metamorphic morphology in anurans.

Development consists of growth and differentiation, which can be partially decoupled and can be affected by environmental factors to different extents. In amphibians, variation in the larval environment influences development and causes changes in post-metamorphic shape. We examined post-metamorphic consequences, both morphological and locomotory, of alterations in growth and development. We reared tadpoles of two phylogenetically and ecologically distant frog species (the red-eyed treefrog Agalychnis callidryas and the African clawed frog Xenopus laevis) under different temperatures with ad libitum food supply and under different food levels at a constant temperature. Low temperature and low food levels both resulted in similarly extended larval periods. However, low temperature yielded relatively long-legged frogs with a lower degree of ossification than warm temperature, whereas low food yielded relatively short-legged frogs with a higher degree of ossification than high food levels. Such allometric differences had no effect on locomotor performance of juveniles. Our results provide a basis for understanding the relationship between growth, differentiation and post-metamorphic shape in anurans and help explain many of the discrepancies reported in previous studies.

Genetic variation in pathogen-induced early hatching of toad embryos.

Accelerated hatching is one of few defences available to embryos, and is effective against many egg-stage risks. We present the first analysis of genetic variation in hatching plasticity, examining premature hatching of American toad embryos in response to pathogenic water moulds. We reared eggs from half- and full-sib families in the presence and absence of water mould. Hatching age and hatchling size showed low cross-environment genetic correlations, suggesting that early-induced hatching can evolve largely independently of spontaneous hatching. We found less phenotypic and additive genetic variation for early-induced hatching than spontaneous hatching, and a stronger correlation between egg and induced hatchling sizes. Directional selection by the pathogen may have eroded variation in early-induced hatching, pushing it against the constraint of hatching gland development. Later hatching has a second, muscular component. This pattern of variation may characterize defences based on developmental transitions, although other inducible defences show more variation in induced phenotypes.

Do prostaglandins regulate external gill regression in anurans?

Although the endocrinological mechanism controlling regression of the internal, larval gills of anurans (frogs and toads) is well understood, the mechanism regulating loss of the external, embryonic gills is not known. Based on the homology of the mammalian ductus arteriosus with a portion of the amphibian branchial arches, and the regulation of blood flow in the mammalian ductus by prostaglandins of the E family (PGEs), we hypothesized that anuran external gill loss is also regulated by PGEs. To test this hypothesis, we topically applied both PGE2 and a synthetic analogue of PGE1, misoprostol, to embryos and young hatchlings of the red-eyed treefrog, Agalychnis callidryas. Both agents accelerated external gill regression. Furthermore, misoprostol overrode the inhibitory effect of hypoxia on gill regression in hatchlings and induced rapid loss of external gills in embryos, which normally maintain the gills until hatching. These observations support the hypothesis that PGEs regulate anuran external gill loss. The specific site of action for prostaglandins within the gills is not known; however, PGEs are secreted in the oral mucus of tadpoles, and this could be a natural topical source for these agents. PGEs offer a tool for manipulation of external gills and should facilitate tests of the physiological importance of these structures.

Environmental and developmental effects on external gill loss in the red-eyed tree frog, Agalychnis callidryas.

I examined the effects of development, hatching, and oxygen availability on external gill loss in red-eyed tree frogs, Agalychnis callidryas. Under natural conditions, the arboreal embryos maintained large external gills until hatching, which occurred from 5-8 d after oviposition. At hatching, when tadpoles entered the water, external gills began to regress. In older hatchlings this process was extremely rapid. Gill circulation was lost on average within 16 min and sometimes within 5 min. Gills often regressed completely in under 2 h. Younger hatchlings reduced gill circulation, shortened and adducted their gills, then resumed normal circulation for some time after hatching; half had completely lost external gills within 24 h. Experimentally increasing the area of egg surface exposed to the air induced loss of external gills in unhatched embryos. Older hatchlings in hypoxic water without access to air maintained their external gills. This suggests that loss of external gills is a response to increased oxygen availability, rather than a response to hatching per se. Extended maintenance of external gills by large, late-hatching embryos may facilitate continued rapid development in closely packed eggs.

Adaptive plasticity in hatching age: a response to predation risk trade-offs.

The life histories of many animals are characterized by niche shifts, the timing of which can strongly affect fitness. In the tree frog Agalychnis callidryas, which has arboreal eggs, there is a trade-off between predation risks before and after hatching. When eggs are attacked by snakes, tadpoles escape by hatching rapidly and falling into the water below. Eggs not attacked by snakes hatch later, when newly emerged tadpoles are less vulnerable to aquatic predators. Plasticity in hatching allows embryos to use immediate, local information on risk of mortality to make instantaneous behavioral decisions about hatching and the accompanying shift from arboreal to aquatic habitats.