Agalychnis Callidryas Research Articles

Novel dermaseptin, adenoregulin and caerin homologs from the Central American red-eyed leaf frog, Agalychnis callidryas, revealed by functional peptidomics of defensive skin secretion

By integrating systematic peptidome and transcriptome studies of the defensive skin secretion of the Central American red-eyed leaf frog, Agalychnis callidryas, we have identified novel members of three previously described antimicrobial peptide families, a 27-mer dermaseptin-related peptide (designated DRP-AC4), a 33-mer adenoregulin-related peptide (designated ARP-AC1) and most unusually, a 27-mer caerin-related peptide (designated CRP-AC1). While dermaseptin and adenoregulin were originally isolated from phyllomedusine leaf frogs, the caerins, until now, had only been described in Australian frogs of the genus, Litoria. Both the dermaseptin and adenoregulin were C-terminally amidated and lacked the C-terminal tripeptide of the biosynthetic precursor sequence. In contrast, the caerin-related peptide, unlike the majority of Litoria analogs, was not C-terminally amidated. The present data emphasize the need for structural characterization of mature peptides to ensure that unexpected precursor cleavages and/or post-translational modifications do not produce mature peptides that differ in structure to those predicted from cloned biosynthetic precursor cDNA. Additionally, systematic study of the secretory peptidome can produce unexpected results such as the CRP described here that may have phylogenetic implications. It is thus of the utmost importance in the functional evaluation of novel peptides that the primary structure of the mature peptide is unequivocally established – something that is often facilitated by cloning biosynthetic precursor cDNAs but obviously not reliable using such data alone.

EVOLUTION OF ADAPTIVE PLASTICITY: RISK‐SENSITIVE HATCHING IN NEOTROPICAL LEAF‐BREEDING TREEFROGS

Adaptive plasticity at switch points in complex life cycles (e.g., hatching, metamorphosis) is well known, but the evolutionary history of such plasticity is not. Particularly unclear is how a single plastic response (e.g., shifts in hatching timing) evolves to respond to different threats and cues (e.g., abiotic and biotic). We conducted a comparative phylogenetic study of hatching plasticity in a group of frogs with arboreal embryos to determine when risk‐accelerated hatching evolved in the clade, whether responses to two common egg‐stage risks (snake predation and flooding) evolved independently, and whether the overall capacity for hatching plasticity was evolutionarily more conservative than responses to specific cues. Red‐eyed treefrogs (Agalychnis callidryas) hatch early to escape from several egg‐stage risks but otherwise hatch later, improving larval survival with predators. We reconstructed a phylogeny forAgalychnisand related genera based on three mitochondrial and four nuclear genes. We quantified onset of hatching competence, spontaneous hatching timing, responses to egg‐stage risks, and costs of premature hatching inAgalychnisandPachymedusa. We also assessed hatching plasticity in a basal phyllomedusine,Cruziohyla calcarifer. The capacity to hatch ∼30% before the spontaneous hatching age appears ancestral for phyllomedusines, with little change over ∼34–50 million years among the species examined. A strong hatching response to flooding, with no mortality of hatching‐competent eggs, is similarly ancient and conserved. Premature hatchlings ofAgalychnisandPachymedusaare more vulnerable to fish predation than are full‐term hatchlings, indicating a conserved risk trade‐off across hatching that would make plasticity advantageous. In contrast, the hatching response to snake attack has undergone major changes at least twice in theAgalychnis–Pachymedusaclade, with two species showing substantially lower escape success than the others. Responses to different threats have thus evolved independently. The capacity for switch point plasticity may be evolutionarily more stable than the response to individual stage‐specific threats.

Mating Patterns of Red‐Eyed Treefrogs, Agalychnis callidryas and A. moreletii

AbstractDeviations from random mating in frogs are often explained by two different size‐based patterns. The large‐male mating advantage predicts that males found in amplexus with females will be larger on average than non‐amplectant males, whereas size‐assortative mating predicts that males and females found in amplexus will maintain an optimal size ratio. Both these pairing patterns are consistent with a female mating preference for larger males, or for males of a given size relative to the choosy female. I examined pairing patterns of two species of Neotropical hylids, Agalychnis callidryas and A. moreletii for three consecutive breeding seasons in Belize, Central America to evaluate whether mating behavior was influenced by either a large‐male mating advantage or size‐assortative mating. For each species, I compared size traits between amplectant and non‐amplectant males, and within amplectant pairs, and I quantified fertilization success for each amplectant pair. For both species I found evidence of deviations from random mating by size, but the nature of the deviations varied between species and among years. The proportion of eggs fertilized was consistently high among years for both species and there was no relationship between fertilization success and the size ratio of amplectant pairs. These data are consistent with female mate preference, but a role for male–male competition cannot be excluded. My findings suggest that mating patterns may be density‐dependent and that the nature and intensity of sexual selection may be increased by extreme environmental conditions.

Spatial and temporal patterns of phenotypic variation in a Neotropical frog

AbstractAim Studies of the spatial and temporal patterns of phenotypic diversity help to elucidate the fine‐scale evolutionary and ecological mechanisms underlying geographical differentiation. The red‐eyed tree frog, Agalychnis callidryas, is a widespread Neotropical frog that exhibits a broad range of polymorphism of coloration and flank‐stripe pattern. The goal of this study was two‐fold: first, to investigate the stability of polymorphisms over a 38‐year period; and second, to evaluate biogeographical hypotheses of diversification between lower Central American populations through quantification of phenotypic diversity on a fine geographical scale.Location This study was conducted at 12 sites across four biogeographical regions in Costa Rica and Panama.Methods We quantified colour, categorized flank‐stripe pattern from digital photos taken during field sampling, and measured body size for each individual. We compared the regional frequency of each flank‐stripe pattern in 2005 with the frequency distribution from a previous study of the same sites in 1967 using logistic regression analyses. We determined the geographical signal of leg coloration by employing linear discriminant function analyses to generate a classification matrix based on covariance similarities, and by comparison of the average hue values within and between regions.Results We found a temporal shift in the frequency of flank‐stripe patterns in three of four regions over 38 years. Based on measures of leg coloration, the frequency distribution of flank‐stripe patterns and body size, we conclude that A. callidryas populations are easily distinguishable at a regional scale.Main conclusions Agalychnis callidryas exhibits regional differentiation in all phenotypic traits measured in this study, supporting the role of three major biogeographical barriers to gene exchange. We found evidence of a putative contact zone between polytypic regions in Costa Rica. In addition, we report temporal instability of the relative frequency of stripe patterns located on the flanks. The ecological and evolutionary mechanisms that may underlie this variation include sexual selection and avoidance of predators.

To hatch and hatch not: similar selective trade-offs but different responses to egg predators in two closely related, syntopic treefrogs

Risk-sensitive hatching is adaptive for species facing a trade-off between egg-stage and post-hatching risks, and environmental variation in one or both stages. Such plasticity has been found in amphibians, fishes, reptiles and spiders, with red-eyed treefrogs (Agalychnis callidryas) being the best-studied case. We assessed hatching plasticity and egg- and larval-stage risks in a closely related, syntopic species, the gliding leaf-frog (Agalychnis spurrelli). We found a lower hatching response to egg-eating snakes in A. spurrelli (9-28% of embryos escaped) than in A. callidryas (59-80% escaped). Levels of snake predation were similarly high for clutches of both species monitored at a pond in Costa Rica, and in fish predation experiments early-hatched A. spurrelli tadpoles were more vulnerable than later hatchlings, as has been shown for A. callidryas. A. spurrelli thus face a risk trade-off similar to A. callidryas, and likely would benefit from predator-induced hatching; their lower responsiveness to snakes appears nonadaptive. A. spurrelli embryos showed a stronger hatching response (57% hatched in 1 h) to submergence underwater than to snake attacks even though submergence is a less frequent risk. This suggests they have a greater capacity for early hatching than is expressed in the context of snake attacks, but have much lower sensitivity to snake cues than to flooding cues. Development in A. spurrelli is accelerated compared to syntopic A. callidryas, and spontaneous hatching is earlier and more synchronous. This is congruent with predictions based on selection by egg predators in the absence of a strong escape hatching response.

Flexible information sampling in vibrational assessment of predation risk by red-eyed treefrog embryos

Prey assessing risk may miss cues and fail to defend themselves, or respond unnecessarily to false alarms. Error rates can be ameliorated with more information, but sampling predator cues entails risk. Red-eyed treefrogs have arboreal eggs and aquatic tadpoles. The embryos use vibrations in snake attacks to cue behaviorally mediated premature hatching, and escape, but vibrations from benign sources rarely induce hatching. Missed cues and false alarms are costly; embryos that fail to hatch are eaten and hatching prematurely increases predation by aquatic predators. Embryos use vibration duration and spacing to inform their hatching decision. This information accrues with cycles of vibration, while risk accrues over time as snakes feed. We used vibration playback experiments to test if embryos adjust sampling of information based on its cost, and measured latency to initiate hatching in videotaped snake attacks. Embryos did not initiate hatching immediately in attacks or playbacks, and the delay varied with the rate at which information accrued. Embryos started hatching sooner in response to stimuli with shorter cycles but sampled fewer cycles (less information) of longer-cycle stimuli before hatching. This flexible sampling is consistent with embryos balancing a trade-off between the value and cost of information.

Bad vibrations: Mechanosensory risk assessment by red-eyed treefrog embryos

Red-eyed treefrogs, Agalychnis callidryas, lay gelatinous egg masses on vegetation overhanging ponds; tadpoles fall into the water upon hatching. Embryos hatch up to 30% prematurely in attacks by egg-eating snakes, but not in other intense but benign disturbances, such as storms. We used modal analysis and vibration recordings from clutches in natural disturbances to characterize the vibrational environment of embryos, and playbacks to assess if vibrations cue hatching, and how vibration characteristics affect hatching behavior. Escape hatching can be induced by snake vibrations, without actual snakes, and embryos distinguish between the vibrational patterns of snake attacks and rainstorms. Benign and dangerous disturbances overlap in individual temporal and frequency characteristics, but can be distinguished using combinations of features. Both frequency and temporal characteristics of vibrations affect the hatching response. Embryos use at least two temporal pattern elements—disturbance duration and interdisturbance interval—and respond to a limited range of low frequencies. These are all necessary elements of a composite cue. Moreover, higher frequencies, found in rainstorms but not attacks, reduce hatching. This is the most detailed examination of mechanisms underlying embryo decision making to date, and a demonstration of the importance of vibrational information in predator-prey interactions. [Work supported by NSF IBN-0234439.]

Temporal pattern cues in vibrational risk assessment by embryos of the red-eyed treefrog, Agalychnis callidryas

The embryos of red-eyed treefrogs, Agalychnis callidryas, use vibrations transmitted through their arboreal egg clutch to cue escape hatching behavior when attacked by egg-eating snakes. Hatching early increases the risk of predation in the water, so embryos should avoid it unless they are in danger. We exposed egg clutches to intermittent vibrations with different combinations of vibration duration and spacing to examine the role of simple temporal pattern cues in the escape hatching response. Stimuli were bursts of synthetic white noise from 0 to 100 Hz, including the range of frequencies with substantial energy in snake attacks, and had approximately rectangular amplitude envelopes. Embryos hatched in response to a small range of temporal patterns and not in response to many others, rather than hatching to most vibrations except for certain patterns perceived as safe. Neither cycle length nor duty cycle predicted hatching response, except at extreme values where no hatching occurred; the highest energy stimuli elicited little or no hatching. Both vibration duration and inter-vibration interval strongly affected the hatching response. The highest levels of hatching were to durations of 0.5 s combined with intervals of 1.5-2.5 s, and hatching decreased gradually with increasing difference of either duration or interval from these most effective stimuli. Vibration duration and interval appear to function as two necessary elements of a composite cue, rather than as redundant cues. This increases response specificity and reduces the range of stimuli that elicit hatching, likely reducing the chance of hatching unnecessarily in a benign disturbance. Vibration-cued hatching in A. callidryas embryos offers an opportunity to experimentally assess the behavioral decision rules underlying an effective and costly anti-predator defense.

EMBRYOS SENSE SEISMIC EVETNTS

Karen Warkentin knew she'd have some interesting questions to answer when she saw a hungry snake attacking a clutch of near-term red-eyed treefrog eggs in the lab. As the predator started tucking in to the eggs, tiny frog embryos began tumbling from the clutch, even though they should have waited another 2 days before hatching. Warkentin eventually discovered that the tadpoles were making a tough decision: to escape the snake by fleeing to the water, even though they are much more vulnerable to aquatic predators at such an early age. Intrigued by the youngster's decision, Warkentin was curious to find out which cues had triggered their evacuation. Warkentin began to suspect that vibrations, generated by the snake's assault, prompted the treefrog's bid for freedom, but why didn't other less sinister vibrations send the youngsters tumbling free too? Curious to know how the embryos distinguished a life-threatening attack from vibrations caused by rain or rustling leaves,Warkentin and her student Michael Caldwell decided to see what makes vibration sequences scary for red-eyed treefrog embryos(p. 1376).Travelling to the Smithsonian Tropical Research Institute at Gamboa,Panama, Warkentin and Caldwell collected frogspawn from trees growing over a local pond. Back in the lab, the team waited until the eggs were 5 days old before attaching a vibrating probe to the clutch to shake the embryos up. Teaming up with Gregory McDaniel, a vibrations engineer, Warkentin designed 32 white noise vibration patterns, with bursts of vibration ranging from 0.5–20 s interspersed with gaps ranging from 0.5–100 s. Exposing egg clutches to the vibrations, the team recorded how many embryos were scared enough to hatch during the following 10 minutes.Analysing the embryo's escapology, Warkentin realised that the frogs weren't responding to the percentage of time filled with vibration or the length of the time cycle that the pattern repeated over. However, the vibration duration and gap between vibration bursts had a profound effect on the embryo's desire to hatch; 0.5 s bursts combined with 1.5-2.5 s gaps were very scary, with three quarters of the embryos deciding to take their chances in the water, but combining a scary 0.5 s burst with a lengthy gap wasn't at all scary. `Vibration duration and interval appear to function as two necessary elements of a composite cue' says Warkentin. The team also realised that the embryos sometimes waited for up to a minute after the vibrations started before beginning to hatch. Warkentin explains that the treefrog eggs are secured with jelly and so are quite tough for the snake to tear loose,giving the embryos enough time to sample several vibration cycles before making their life or death decision.So how do the embryos sense these seismic events? Warkentin isn't sure. She explains that it is possible that the embryo's lateral line neuromasts pick up the vibrations, or that the embryos simply sense the signal by sloshing around within their capsules. But that is one of the unresolved questions that will keep her returning to Panama for years to come.

OPPOSITE SHIFTS IN SIZE AT METAMORPHOSIS IN RESPONSE TO LARVAL AND METAMORPH PREDATORS

Predation risk can cause organisms to alter the timing of life history switch points. Theory suggests that increased risk in an early life stage should select for switching earlier and smaller, while increased risk in the subsequent stage should select for switching later and larger. This framework has frequently been applied to metamorphosis in amphibians, with mixed results. Few studies examining the effect of larval predation risk on metamorphosis have observed the predicted pattern, and no studies, to our knowledge, have examined the effect of increased risk during and after metamorphosis on the timing of this switch point. Here we examine the effect of larval and post-metamorphic predation risk on metamorphosis in the red-eyed treefrog, Agalychnis callidryas. We raised tadpoles in the presence or absence of cues from caged water bugs fed larvae and cues from spiders fed emerging metamorphs. Water bugs are effective larval predators, while spiders are poor larval predators but prey on metamorphs. Furthermore, since spiders forage on the water surface it is possible that tadpoles could assess future risk from this predator. Predators induced opposite shifts in life history. Tadpoles emerged smaller and less developed in response to water bugs, but later and larger in response to spiders. Interestingly, predator effects on larval duration were not independent; tadpoles delayed emerging in response to spiders, but only in the absence of water bugs.

Development of red-eyed treefrog eggs affects efficiency and choices of egg-foraging wasps

The arboreal eggs of red-eyed treefrogs, Agalychnis callidryas, are preyed on by social wasps, Polybia rejecta. Embryos hatch rapidly up to 30% before their typical undisturbed hatching age when attacked by wasps, so many embryos escape. We hypothesized that the escape ability of older embryos would reduce wasp foraging success or efficiency, and that wasps would therefore prefer to prey on younger eggs. To characterize wasp foraging abilities and preferences, we videotaped the behaviour of individual foragers at an outdoor feeding station. We presented wasps with pairs of clutches differing in age by 1 day, from newly laid versus 1 day through 4 versus 5 days. Eggs become competent to hatch at 4 days. Wasps foraged more efficiently and successfully on older eggs than on younger ones. Among not-yet-hatchable clutches, wasps preferred older eggs, but there was no evidence for discrimination between late unhatchable eggs and hatchable eggs (3–5 days), and wasps readily attacked embryos likely to escape. The thick egg jelly of younger clutches makes it difficult for wasps to break into eggs. The poorly developed embryos also disintegrate easily, making them harder to carry. Thus, at least when wasps have experience with a range of egg stages, they kill few young eggs. Older eggs frequently escaped by hatching, so the heaviest mortality fell on intermediate developmental stages, both at the feeding station and at a natural breeding site. Structural variation in egg clutches and developmental changes in embryos may affect interactions between other small, mobile predators and amphibian eggs.

Development, Surface Exposure, and Embryo Behavior Affect Oxygen Levels in Eggs of the Red‐Eyed Treefrog,Agalychnis callidryas

Oxygen stress can slow development, induce hatching, and kill eggs. Terrestrial anamniote embryos face a potential conflict between oxygen uptake and water loss. We measured oxygen levels within eggs to characterize the respiratory environment for embryos of the red-eyed treefrog, Agalychnis callidryas, a Neotropical frog with arboreal egg masses and plastic hatching timing. Perivitelline oxygen partial pressure (Po2) was extremely variable both within and among eggs. Po2 increased with air-exposed surface of the egg and declined over the developmental period before hatching competence. Through the plastic hatching period, however, average Po2 was stable despite continued rapid development. Development was synchronous across a wide range of perivitelline Po2 (0.5-16.5 kPa), and hatching-competent embryos tolerated Po2 as low as 0.5 kPa without hatching. The variation in Po2 measured over short periods of time within individual eggs was as great as that measured across development or surface exposure, including sharp transients associated with embryo movements. There was also a strong gradient of Po2 across the egg from superficial to deep positions. Ciliary circulation of fluid within the egg is clearly insufficient to keep it mixed. Embryos may maintain development under hypoxic conditions by strategic positioning of respiratory surfaces, particularly external gills, to exploit the patchy distribution of oxygen within their eggs.

How do embryos assess risk? Vibrational cues in predator-induced hatching of red-eyed treefrogs

I examined the role of vibrations in predator-induced early hatching of red-eyed treefrogs, Agalychnis callidryas. The arboreal eggs of A. callidryas hatch up to 30% early if attacked by egg-eating snakes. This induced hatching is a behavioural response that occurs once snakes begin physically disturbing the clutch, and is sufficiently rapid to allow most embryos to escape. Other intense but benign disturbances, such as tropical storms, do not induce such hatching. I used a miniature accelerometer to record vibrations in egg clutches during snake attacks and rainstorms, and analysed the recordings to identify parameters that distinguished disturbance types. Snake-induced vibrations were on average longer, more widely spaced, and of lower frequency than rain-induced vibrations. I performed three sets of vibration playbacks to examine the hatching response of embryos to different vibration patterns. (1) Playbacks of recorded snake attacks elicited more hatching than did rain recordings. (2) I edited snake and rain recordings by moving periods of stillness to clump together rain vibrations and extend intervals, and divide snake vibrations into shorter, more tightly spaced bits. In playbacks, clumped rain elicited more, and divided snake vibrations less hatching than did the original recordings. (3) Bursts of white noise with a constant vibration-to-interval ratio but different cycle durations elicited different levels of hatching. Vibrations alone were sufficient to induce early hatching, without chemical or visual cues from predators. Embryos also clearly distinguished among different vibration patterns and used cues in the gross temporal pattern in making their hatching decision.

Hatching timing, oxygen availability, and external gill regression in the tree frog, Agalychnis callidryas.

The physiological role of the embryonic external gills in anurans is equivocal. In some species, diffusion alone is clearly sufficient to supply oxygen throughout the embryonic period. In others, morphological elaboration and environmental regulation of the external gills suggest functional importance. Since oxygen stress is a common trigger of hatching, I examined the relationships among hatching timing, oxygen stress, and external gill loss. I worked with the red-eyed tree frog, Agalychnis callidryas, a species with arboreal eggs and aquatic tadpoles in which gill regression is associated with hatching, and hatching timing affects posthatching survival with aquatic predators. Both exposure to a hypoxic gas mixture and submergence in water, a natural context in which hypoxic stress can occur, induced early hatching. Exposure to hyperoxic gas mixtures induced regression of external gills, and subsequent exposure to air induced early hatching. Prostaglandin-induced external gill regression also induced hatching, and this effect was partially ameliorated by exposure to hyperoxic gas. Together, these results suggest that external gills enhance the oxygen uptake of embryos and are necessary to extend embryonic development past the onset of hatching competence.

EGG-KILLING FUNGUS INDUCES EARLY HATCHING OF RED-EYED TREEFROG EGGS

Pathogens can cause substantial mortality of amphibian eggs. If the timing of hatching is phenotypically plastic, embryos could escape from otherwise lethal infections by hatching early. We tested this with the arboreal eggs of red-eyed treefrogs, Agalychnis callidryas. A filamentous ascomycete (Dothideales: Phaeosphaeriaceae) was present on ∼7% of egg clutches collected from a pond in the rain forest in Panama and, when present, killed 40% of the eggs, on average. Inoculation experiments confirmed that the fungus attacked and killed healthy embryos, establishing that this fungus is a pathogen of A. callidryas eggs. As predicted from life history theory, embryos hatched earlier from both naturally infected and inoculated clutches than from fungus-free control clutches. Within infected clutches, live embryos in contact with fungal hyphae hatched before those embryos not in contact with the fungus. Accelerated hatching allowed embryos to survive that otherwise would have been killed, and tadpoles hatched from infected clutches were themselves uninfected. Red-eyed treefrog embryos also hatch early if attacked by predators, apparently in response to vibratory cues. Because fungal infection provides no vibratory stimuli, embryos must respond to different cues in fungus-induced hatching than in predator-induced hatching. The behavioral decision of when to hatch is complex and merits further investigation. Our study indicates that pathogens can influence the timing of life history transitions, as do other stage-specific risks.

Egg-Killing Fungus Induces Early Hatching of Red-Eyed Treefrog Eggs

Egg-Killing Fungus Induces Early Hatching of Red-Eyed Treefrog Eggs

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.

Wasp predation and wasp-induced hatching of red-eyed treefrog eggs

Eggs often suffer high levels of predation and, compared with older animals, embryos have few options available for antipredator defence. None the less, hatchlings can escape from many predators to which eggs are vulnerable. I studied early hatching as an antipredator defence of red-eyed treefrog embryos, Agalychnis callidryas, in response to egg predation by social wasps (Polybia rejecta). Red-eyed treefrogs attach their eggs to vegetation overhanging water, where they are exposed to arboreal and aerial predators. Wasps attacked half the egg clutches and killed almost a quarter of the eggs I monitored at a natural breeding site in Panama. Hatching tadpoles fall into the water, where they face aquatic predators. As predicted from improved survival of older hatchlings with aquatic predators, most undisturbed eggs hatched relatively late. However, many younger embryos directly attacked by wasps hatched immediately. Embryos attacked by wasps hatched as much as a third younger than the peak undisturbed hatching age, and most hatching embryos escaped. Thus hatching is an effective defence against wasp predation, and plasticity in hatching stage allows embryos to balance risks from stage-specific egg and larval predators. Wasp-induced hatching is behaviourally similar to the snake-induced hatching previously described in A.callidryas, but occurs in fewer eggs at a time, congruent with the scale of the risk. Individual embryos hatch in response to wasps, which take single eggs, whereas whole clutches hatch in response to snakes, which consume entire clutches. Embryos ofA.callidryas thus respond appropriately to graded variation in mortality risks.

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.

Effects of hatching age on development and hatchling morphology in the red-eyed tree frog, Agalychnis callidryas

The red-eyed treefrog, Agalychnis callidryas, lays eggs on leaves overhanging ponds. Tadpoles hatch and enter the water at different ages, and late-hatched tadpoles survive aquatic predators better than do early-hatched tadpoles. Here I assess developmental consequences of hatching age through: (1) a morphological study of embryos and tadpoles through the plastic hatching period; (2) a behavioural assay for an effect of hatching age on feeding; and (3) a field experiment testing the effect of hatching age on growth to metamorphosis. Substantial development of feeding, digestive, respiratory and locomotor structures occurs in embryos over the plastic hatching period. Hatchling morphology thus varies with age, with consequences for behaviour and predation risk. Hatched tadpoles develop faster than embryos, and early-hatched tadpoles feed before late-hatched tadpoles. After all tadpoles have hatched, the effect of hatching age on size decreases. I found no evidence for an effect of hatching age on size at metamorphosis and only weak evidence for an effect on larval period. Hatching age affects the sequence of developmental change: early-hatched tadpoles lose external gills while otherwise more developed embryos maintain them. Plasticity in external gill resorption may be adaptive given differences in the respiratory environments of embryos and tadpoles. Early-hatched tadpoles also diverge from embryos in shape, growing relatively smaller tails. The study of functional morphology and developmental plasticity will contribute to understanding hatching as an ontogenetic niche shift.