To hatch and hatch not: does heterochrony in onset of vestibular mechanosensing explain species differences in escape-hatching success of Agalychnis embryos in snake attacks?

doi:10.21203/rs.3.rs-3218619/v1

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To hatch and hatch not: does heterochrony in onset of vestibular mechanosensing explain species differences in escape-hatching success of Agalychnis embryos in snake attacks?

https://doi.org/10.21203/rs.3.rs-3218619/v1

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published 15 Dec, 2023

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Phyllomedusid treefrogs hatch prematurely to escape egg predation, but escape success varies among species. Snake attacks elicited 55% less escape-hatching in spontaneously hatching Agalychnis spurrelli than in less developed A. callidryas. Agalychnis callidryas use their vestibular system and, secondarily, their lateral line to sense physical disturbances that cue hatching. Since A. spurrelli develop faster, we hypothesized that heterochronic shifts in the onset timing of vestibular mechanosensory function, relative to hatching ability, might explain their lower escape response to mechanosensory cues. To test this, we compared onset timings of hypoxia- and mechanosensory-cued hatching (MCH) and vestibular mechanosensory function in developmental series of both species. Across species, most sibships began responding to each cue at the same developmental stage and vestibular function development, measured by the vestibulo-ocular reflex (VOR), was similar. These results demonstrate that low escape-hatching success in A. spurrelli is not caused by a delay in the onset of vestibular mechanosensory function. MCH onset in A. spurrelli was associated with VOR, but with a higher threshold than in A. callidryas. The absence of MCH before strong vestibular function suggests multimodal mechanosensing may not contribute to antipredator responses of A. spurrelli embryos. Our observations of higher escape success of larger clutches in snake attacks and hatching complications in flooded A. spurrelli suggest that differences in clutch and egg capsule structure may contribute directly to species differences in escape-hatching success. Moreover, hatching complications in A. spurrelli may select against false alarms, increasing the stringency of hatching decision rules.

Adaptive embryo behavior

Environmentally cued hatching

Heterochrony

Ontogeny

Phenotypic plasticity

Predation

Hatching is an essential life-stage transition during development. For some species, it also functions as an effective defense against egg-stage risks. However, the causes for variation in environmentally cued hatching responses are poorly understood. We assessed why two closely related treefrogs exhibit different escape-hatching responses to snake attacks. Comparing the onset of induced-hatching responses and vestibular function in Agalychnis callidryas and A. spurrelli revealed that the primary mechanism for mechanosensing and its developmental timing are conserved within this group, but the sensory function threshold for behavioral response differs. It also revealed that egg-clutch properties, including egg-clutch size and individual egg structure, affect the escape hatching response. This motivates further research assessing the role of egg-clutch properties and their biomechanics in embryo escape-hatching responses.

Hatching is a developmental event and essential embryo behavior that mediates a critical transition between life stages and environments. Its timing is often cued by environmental conditions and variation in either egg or post-hatching environments can alter the optimal timing or developmental stage for hatching (Warkentin 2011a). Plastic changes in hatching timing in response to environmental cues (i.e., environmentally cued hatching) are widespread and often adaptive, allowing embryos to adjust their hatching timing to escape threats or exploit new opportunities (Warkentin 2011a; Warkentin 2011b; Du and Shine 2022). However, the causes for variation in cued hatching responses, even across closely related species, are poorly understood.

Environmentally cued hatching requires mechanisms for sensing environmental cues and performing hatching behaviors, and the onset timing of either mechanism may limit when and how well animals respond to hatching-inducing cues. Interspecific variation in the onset timing of cued-hatching mechanisms may occur as a result of an overall change in the development rate of embryos (e.g., effects of temperature on development; Güell and Warkentin 2018a), altering the timing but not developmental stage of hatching mechanism development. It may also occur as a result of changes in the sequence of developmental events (i.e., heterochrony) that shift the onset timing of hatching mechanisms to an earlier or later developmental stage (Gould 1977; Alberch et al. 1979; Raff and Wray 1989; Reilly et al. 1997). Here, we assess whether a heterochronic shift in the onset timing of vestibular mechanosensing explains the differences in escape hatching success during snake attacks between two closely related treefrogs.

Phyllomedusid treefrogs are a particularly well-suited system in which to investigate interspecific variation in hatching plasticity. These arboreal amphibians lay eggs on plants that overhang forest pools and embryos hatch into the water below (Duellman 1968; Duellman 1970). Comparative studies found that all tested species show the capacity to hatch up to ~ 30% prematurely in response to egg-stage threats and similarly effective escape-hatching responses to hypoxia cues when flooded (Gomez-Mestre et al. 2008). In contrast, embryos show striking differences in their escape success in snake attacks; while most species show a strong, effective escape response to mechanosensory cues in attacks, others have much lower escape success and most embryos show no signs of attempting to hatch in video recordings of attacks (Gomez-Mestre and Warkentin 2007; Gomez-Mestre et al. 2008). The causes of these differences in embryo behavior are unclear. However, rates of embryonic development also vary across species, as do egg mass structures and adult reproductive strategies; notably, these all differ strongly between species with high vs. low embryo escape-success in snake attacks (Gomez-Mestre and Warkentin 2007; Gomez-Mestre et al. 2008).

To assess a potential cause for the differences in embryo behavior within this group we examined the two best-studied species with the largest difference in escape success in snake attacks, Agalychnis callidryas and A. spurrelli. Compared to A. callidryas, the faster-developing A. spurrelli has been reported to have particularly low escape success in snake attacks (9–28% vs. 59–80%; range of means across 4th − 5th and 5th − 7th nights after oviposition in A. spurrelli and A. callidryas, respectively), despite sharing the same reproductive periods, breeding ponds, oviposition sites, and predation pressures (Gomez-Mestre and Warkentin 2007). However, the earliest escape hatching that Gomez-Mestre and Warkentin (2007) report for A. callidryas at Estación Sirena, on Costa Rica’s Osa Peninsula, based on data collected in the early 1990s is later than more recent data collected from a well-studied population in Gamboa, Panama (Warkentin 2000; Warkentin et al. 2017; Almanazar and Warkentin 2018), beginning at age 5 vs. 4 days, respectively. Therefore, before testing a potential cause for differences in escape-hatching success between these species, we first determined the undisturbed hatching timing and hatching response to snake attacks for both A. spurrelli and A. callidryas at our field site in Piro, on Costa Rica’s Osa Peninsula. Moreover, as egg clutch size varies considerably, ranging from 12–231 eggs for A. spurrelli (76.8 ± 64.5; mean ± SD here and in text throughout) and from 10–101 eggs for A. callidryas (40.5 ± 15.5) (Gomez-Mestre and Warkentin 2007), we also assessed the role of clutch size on escape-hatching success in both species. Assuming snakes cannot consume an entire clutch in one bite, embryos that are not directly attacked may have more time to escape from larger clutches.

In A. callidryas, the earliest induced hatching occurs in response to strong hypoxia cues, whereas predator-induced, mechanosensory-cued hatching (MCH) begins later, limited by sensory system development (Warkentin et al. 2017; Jung et al. 2019; Jung et al. 2020). Multiple sensory mechanisms contribute to MCH in A. callidryas (Jung et al. 2020). The vestibular system of the inner ear is the predominant motion-detecting system across vertebrates, and in A. callidryas it appears crucial for embryos to perceive motion cues and assess risk in snake attacks on their clutch before direct snake contact with their egg. Jung et al. (2019) found a strong correlation between the developmental onset of MCH and the vestibulo-ocular reflex (VOR), an indicator of vestibular function. The lateral line system also contributes to MCH in A. callidryas; it develops before the onset of vestibular function and seems sufficient to mediate a weaker hatching response in directly attacked eggs (Jung et al. 2020).

The fact that some A. spurrelli embryos hatch in snake attacks means they have a mechanism—presumably mechanosensory—to detect attacks, as well as the ability to hatch. The difference in their response to snakes, compared to A. callidryas, suggests that something about their cue sensing or decision rules differ. We hypothesized that A. spurrelli embryos may lack multimodal mechanosensing, or develop different modalities of mechanosensing at different times, compared to A. callidryas. Overall, A. spurrelli embryos develop more rapidly than A. callidryas, but their acceleration of development may be uneven. If vestibular system development is less accelerated than other processes, A. spurrelli may have a relatively longer period of limited mechanosensory perception, when only the lateral line system is functional, after the onset of hatching ability. Moreover, the lateral line appears to be stimulated only by complex mechanosensory cues from direct attacks, not motion-only cues (Jung et al. 2020). Since many embryos in large egg masses receive only motion cues while snakes attack their spatially distant siblings, a developmental delay in the onset of vestibular mechanosensing, relative to the onset of hatching ability, might explain A. spurrelli’s low escape success with predators.

Therefore, as a first step in determining the mechanistic cause for differences in escape-hatching success between species we assessed the onset of hatching ability, vestibular function, and mechanosensory-cued hatching in A. spurrelli and compared it to that of A. callidryas. To do this we exposed a developmental series of A. spurrelli and A. callidryas embryos to hypoxia and mechanosensory cues and compared the onset of their hatching responses. To assess the onset of vestibular mechanosensory function in A. spurrelli we measured the roll-induced vestibulo-ocular reflex (VOR) of embryos—in which eyes roll counter to body roll based on vestibular sensory input—before and after the onset of MCH and compared their VOR development to previously reported measurements in A. callidryas. We also assessed if vestibular function development predicts MCH response in A. spurrelli.

Egg clutch collection and care

We collected A. spurrelli and A. callidryas egg clutches at age 0–3 days from Shampoo Pond (8°24'55"N, 83°20'45"W) on Costa Rica’s Osa Peninsula between 22 May and 14 August in 2018 and between 27 June and 3 July in 2021. We brought clutches on their leaves to an ambient temperature and humidity laboratory at Osa Conservation’s Piro Biological Station and maintained them on plastic cards over aged tap water in cups within large plastic bins (egg humidors) with mesh windows in the lids to allow ventilation. Clutches and humidors were misted frequently with rainwater to maintain healthy levels of egg and clutch hydration (Salica et al. 2017; González et al. 2021). We also removed unfertilized and dead eggs at setup to reduce the risk of fungal infection. We examined all experimental embryos to ensure they were morphologically normal and in developmental synchrony with siblings in their clutch when tested; any found to be less developed or morphologically abnormal were not used in experiments or included in analyses. It was not possible to record data blind because our study involved visibly different focal animals of different species and ages. In contrast to A. callidryas which lays most of its eggs between 10:00 and 2:00 h (Warkentin 2002; Warkentin et al. 2005), most A. spurrelli eggs are laid between midnight and 6:00 h (BAG personal observation); for ease of comparison and consistency between species embryo ages were assigned starting from midnight of their oviposition night (Warkentin 2002; Warkentin et al. 2005). Thus, comparisons of embryo ages reflect conservative estimates of species differences. All hatched tadpoles were returned to their pond at the end of the experiment. This research was conducted under BU IACUC protocol 18 − 003 and permits from the Costa Rican Ministerio de Ambiente y Energía (MINAE) and the Sistema Nacional de Áreas de Conservación (SINAC) (ACOSA-INV-048-18, INV-ACOSA-055-18, ACOSA-DASP-OI-R-019-2021).

Spontaneous hatching timing

We assessed the spontaneous hatching timing of A. callidryas (N = 39 clutches) and A. spurrelli (N = 32 clutches) using egg masses collected in the field at age 0 days and maintained in humidor bins as described above. We checked clutches every 12 hours at 06:00 and 18:00 h until all eggs hatched. At each check, we counted the number of unhatched eggs on each clutch and/or the number of hatched tadpoles in each cup, staged embryos according to Warkentin (2017), and carefully misted the clutches and their humidors to maintain humidity without disturbing the embryos (Salica et al. 2017; González et al. 2021). We stopped staging embryos at stage 29, once embryos became responsive to physical disturbance cues (Gomez-Mestre and Warkentin 2007; Warkentin et al. 2017; this study, Fig. 1), to reduce risk of inducing hatching while conducting our checks. We assigned hatching age based on when we first observed each tadpole in the water; i.e., grouping together all individuals hatched since the last check. We conducted these experiments from 22 May to 2 June 2018 with A. callidryas and from 26 May to 17 June 2018 with A. spurrelli.

Escape from egg-predation by snakes

To assess escape-hatching success rates for A. spurrelli and A. callidryas at our field site, we experimentally exposed egg clutches of both species to attacks by cat-eyed snakes, Leptodeira ornata (formerly L. septentrionalis, Barrio-Amorós 2019; Torres-Carvajal et al. 2020) following methods by Gomez-Mestre and Warkentin (2007) and Warkentin (1995). Because A. spurrelli develop faster than A. callidryas and show escape-hatching responses to snakes during two nights, including the night when most embryos hatch spontaneously (Gomez-Mestre and Warkentin 2007; this study, Fig. 1), we performed both age- and stage-matched comparisons of escape-hatching success between species. We tested 28 and 26 A. spurrelli clutches on the 4th and 5th night after oviposition, respectively, and 20 A. callidryas clutches on the 5th night after oviposition (i.e., age 5.75–6.25 days, Fig. 1A). We collected snakes at night by hand from Shampoo Pond and housed them in individual mesh cages with ample leaf litter, branches, and a shallow tray of water, under ambient temperature, humidity, and photoperiods. Test-clutches were taped to bricks by the morning at age 3 days, before embryos became responsive to mechanosensory cues (Gomez-Mestre and Warkentin 2007; Warkentin et al. 2017; this study), and kept in egg humidors until testing. We began egg-predation trials at 18:00 by carefully placing clutches, attached to their bricks, in the tray of water to catch hatchlings, within the snake cages. We then checked the clutches and snakes frequently (every 15–30 minutes) using dim red light to count any eggs that hatched spontaneously, or due to our setup procedure, prior to snake attacks in order to exclude them from our analysis. We calculated escape success by determining the proportion of normally developed eggs present at the start of attacks that hatched during attacks, for each clutch. In most trials all eggs were either eaten or hatched. However, when snakes left unhatched eggs at the end of the trial, we included them in the total number of initial eggs that could have hatched in our calculations. Trials with A. spurrelli were conducted from 3–19 August 2018 using five different L. ornata individuals, and trials with A. callidryas were conducted from 2–8 July 2021 using four different L. ornata. We conducted up to four trials simultaneously with different snakes and offered individual snakes multiple test-clutches a night, consecutively, when feasible.

Onset of hypoxia-cued hatching

We began hypoxia testing at developmental stage 24 (sensu Warkentin 2017), when the iris edge becomes distinct from the lens at top and sides but melanophores on the dorsal yolk do not yet reach halfway down the sides. Every 3 h, we tested 3 siblings per clutch, using new individuals each time, until one of them hatched. We repeated this procedure for 12 A. callidryas and 59 A. spurrelli clutches collected between 6 June to 20 July 2018 from multiple breeding events. We presented eggs with a strong hypoxia cue by submerging them in degassed tap water that had been boiled for at least 10 minutes and allowed to cool in sealed glass bottles (Warkentin et al. 2017; Güell and Warkentin 2018b; Güell et al. 2022), using hypoxic water from newly opened bottles at each testing period. We gently removed eggs from their clutches and placed them in small glass jars, filled the jars with hypoxic water, and sealed them for the entirety of the experiment. We continuously observed embryos during 40-minute trials to record hatching occurrence, timing (latency), and any complications we observed in the process, then returned unhatched eggs to air and hatched tadpoles to air-saturated water. Hatchlings were photographed in dorsal view next to a ruler for scale using a Canon EOS 5D Mark III camera and 100 mm macro lens and measured using ImageJ (Schneider et al. 2012). Because siblings were tested concurrently in groups of three, we measured hatchling length and hatching latency for 1 to 3 hypoxia-cued hatchlings per clutch, at the same developmental time point.

Onset of mechanosensory-cued hatching

To ensure we captured the earliest possible hatching from each clutch, we excluded clutches if embryos hatched in response to the mechanosensory stimulus in our first test, without an initial non-hatching time point; this occurred only when we began after stage 27. Therefore, we began all mechanosensory testing at developmental stage 27 (Warkentin 2017), when the cornea is clear and well separated from the lens and the yolk blood vessel enters medially below the heart, within the edges of cement glands in frontal view. For 12 A. callidryas clutches and 34 A. spurrelli clutches, we tested 3 new individuals per clutch simultaneously every three hours, and we stopped testing each clutch after the first embryo hatched. All A. callidryas clutches tested for mechanosensory-cued hatching had previously demonstrated a response to hypoxia. For A. spurrelli, 15 out of 34 clutches tested for mechanosensory-cued hatching had previously been tested under hypoxia, and demonstrated a hatching response to it, but egg mass size and the number of sequential test periods required meant that we could not test all clutches for responses to both cues. We presented eggs with a complex multimodal mechanosensory stimulus by jiggling them using a blunt metal probe in small hexagonal weight boats with a drop of water. This stimulus includes tactile cues, whole egg motion, and both linear and rotational acceleration (Warkentin et al. 2017; Jung et al. 2019; Güell et al. 2022). We alternated 15 s of jiggling with 15 s of rest for 5 minutes, or until the first egg hatched, and recorded the occurrence and timing (latency) of hatching. Hatchlings were photographed and measured as in hypoxia tests.

Onset of vestibular function

In order to assess the timing of vestibular function onset and its congruence, or lack thereof, with the onset of mechanosensory-cued hatching in A. spurrelli, we measured the vestibulo-ocular reflex (VOR) for a subset of A. spurrelli individuals (N = 69 from 48 clutches) immediately after they were tested for mechanosensory-cued hatching, following methods from Jung et al. (2019). We compared these data with VOR measurements and hatching responses of A. callidryas reported in Jung et al. (2019, see 'series III': doi:10.5061/dryad.p2ngf1vm7). We measured the VOR of both A. spurrelli individuals that failed to hatch in response to mechanosensory cues (N = 22, up to 3 individuals per clutch at different time points, from 14 clutches) and those that hatched (N = 47, 1 individual per clutch at the onset of hatching). Most unhatched individuals were measured 3 h before a sibling hatched, and no VOR measurements were more than 12 h before hatching began in the clutch. For 24 hatched individuals, each the first in their clutch to respond to mechanosensory cues, we have no VOR measurements from younger, unhatched siblings. Our data includes paired VOR measurements, from younger unresponsive and older mechanoresponsive siblings, from 13 clutches. Embryos who did not hatch in response to jiggling were manually decapsulated to enable VOR measurements. We used a custom-built, Arduino-based, portable tadpole rotator (Jung et al. 2019) to hold each individual in a chamber filled with aged tap water facing a Canon EOS 80D or EOS 5D Mark III camera with a MP-E 65mm lens. We rotated them 180 degrees about their body axis in each direction in 15° increments and photographed them at each position immediately after their bodies and eyes stopped moving (see Movie 1 in Jung et al. 2019). We then measured right and left eye and body axis angles using ImageJ (Schneider et al. 2012) and constructed VOR curves for each individual using a sine-fitting function in Python (version 2.7.9, Build 1, Python Software Foundation). VOR amplitudes were calculated following methods from Jung et al. (2019) and averaged across left and right eye measurements for analysis.

Statistics

We used generalized linear mixed models (GLMM; 'lme4' package; Bates et al. 2015) with clutch as a random effect, followed by likelihood ratio tests of nested models, to determine the effects of age, species, and their interaction on the timing (binomial GLMM) and developmental stage (LMM) of spontaneous hatching. We used a similar approach (binomial GLMM) to test for effects of age, clutch size, and their interaction on escape hatching success in egg-predation experiments. We used the same model to reanalyze the escape-hatching data from Gomez-Mestre and Warkentin (2007) and test for effects of age, clutch size, and their interaction within each species. In these binomial GLMMs, the response variable was the number of tadpoles hatched, of the initial number of test embryos per clutch. In experiments testing the onset of cued hatching, we tested for effects of stimulus (hypoxia or mechanosensory), species, and their interaction on hatchling size (LMM) and latency to hatch (Gamma GLMM). We used Wilcoxon rank sum tests to non-parametrically compare age and stage at hatching response onset for each stimulus type between species. Since we found complete separation of developmental stage as a predictor variable for the binomial hatching response in mechanosensory-cued hatching tests in A. spurrelli—where no stage 28 individuals hatched, and all stage 30 individuals hatched—we used a binomial GLMM with clutch as a random effect to determine the effects of VOR amplitude, species, and their interaction on the hatching response and excluded stage from our model. We used a LMM to assess the effects of species, stage, and their interaction on VOR amplitude. We then compared the effects of species, hatching response, and their interaction on VOR amplitude between the comparable subsets of embryos from our A. spurrelli data and the A. callidryas data collected by Jung et al. (2019, see 'series III': doi:10.5061/dryad.p2ngf1vm7) that included the last individual that failed to hatch and the first one that did hatch from each clutch in response to jiggling. We used likelihood ratio tests of nested models and post hoc comparisons of estimated marginal means (EMMs) with Bonferroni corrections ('emmeans' package, Lenth 2023) to determine within-stage, -species, and -hatching response effects. We used type-II F tests (‘car’ package; Fox and Weisberg 2019) to determine the effect of VOR amplitude and hatching complications on latency to hatch. All statistical tests were performed in the R statistical environment (R Core Development Team 2021) using RStudio (version 4.1.1; https://www.rstudio.com/products/rstudio/).

Spontaneous hatching timing

Spontaneous hatching was substantially more synchronous and occurred earlier in age in A. spurrelli compared to A. callidryas (Fig. 1A). Hatching in A. spurrelli began on the 4th afternoon after oviposition, before eggs reached 4.75 days, while in A. callidryas hatching began on the 4th night after oviposition when eggs were age 4.75–5.25 days (Fig. 1A). Peak spontaneous hatching for A. spurrelli occurred on the 5th night after oviposition (83% of embryos hatched) while in A. callidryas it occurred on the 6th night after oviposition (70% of embryos hatched). The mean hatching age was 6.22 ± 0.85 and 7.15 ± 0.85 days for A. spurrelli and A. callidryas, respectively (Fig. 1A). Both age (χ² = 52347, P < 2.2e-16) and species (χ² = 1775.7, P < 2.2e-16) were strong predictors of spontaneous hatching in our mixed model. Spontaneous hatching occurred earlier in chronological age in A. spurrelli, compared to A. callidryas (age × species interaction: χ² = 264.58, P < 2.2e-16). Embryos of A. spurrelli also developed faster than those of A. callidryas, reaching stage 25 at least half a day earlier (3.25 vs. 3.75 days) and stage 29 at least a full day earlier (3.75 vs. 4.75 days, Fig. 1B); developmental stage varied by age, species, and their interaction in our mixed model (age: χ² = 1641, P < 2.2e-16; species: χ² = 297.84, P < 2.2e-16; interaction: χ² = 197.21, P < 2.2e-16).

Escape from egg-predation by snakes

Escape-hatching success was considerably lower in A. spurrelli embryos on both the 4th and 5th night after oviposition, the night before and night of peak spontaneous hatching, compared to A. callidryas embryos on the 5th night after oviposition, the night before their peak of spontaneous hatching (F_1,46=313.45, P < 2.2e-16; F_1,44=136, P = 4.74e-15, respectively, Fig. 2A). In our A. spurrelli data, both age (χ² = 5.498, P = 0.019) and clutch size (χ² = 8.429, P = 0.004) were strong predictors of escape-hatching success, with no significant interaction (χ² = 1.796, P = 0.18); older embryos and larger clutches had higher escape success (Fig. 2B). Escape-hatching success did not vary with clutch size in A. callidryas (P > 0.1). In re-analyzing the data from Gomez-Mestre and Warkentin (2007), we found that clutch size had a strong effect on escape-hatching success for both A. callidryas and A. spurrelli (χ² = 14.161, P = 0.0001; χ² = 11.497, P = 0.0007; Fig. 2C). There was no significant age × clutch size effect in either species (both P > 0.7).

Onset of hypoxia- and mechanosensory-cued hatching

Hatchlings at the onset of hypoxia-cued hatching were significantly smaller than those at the onset of mechanosensory-cued hatching (χ² = 235.38, P < 2.2e-16, Fig. 3) and A. spurrelli hatchlings were smaller than A. callidryas ones (χ² = 17.843, P = 2.4e-05), but there was no cue type × species interaction effect on hatchling size (P > 0.1). Embryos of A. spurrelli began hatching in response to both hypoxia (W = 702, P = 5.38e-08) and mechanosensory cues (W = 339.5, P = 0.0006) at earlier chronological ages compared to those of A. callidryas (hypoxia: 3.25 ± 0.17 vs. 3.78 ± 0.14 days; mechanosensory: 4.41 ± 0.23 vs. 4.68 ± 0.11 days; Fig. 4A). However, embryos of both species began hatching in response to hypoxia at developmental stage 25, except for 1 clutch of each species that began at stage 26 (W = 353.5, P = 1; 25.08 ± 0.28 vs. 25.08 ± 0.29; Fig. 4B). With mechanosensory cues, in most clutches of both species embryos began hatching at stage 29. However, in one A. callidryas clutch the first individual hatched at stage 28 and in 29% of A. spurrelli clutches (10 of 34) embryos did not hatch until stage 30, resulting in a significant effect of species on the stage at onset of MCH (W = 132, P = 0.016, Fig. 4B). Focusing on clutches for which we know the onset timing of both hypoxia-cued and mechanosensory-cued hatching (N = 15 A. spurrelli and 12 A. callidryas clutches), the proportionate delay until mechanosensory-cued hatching was slightly, but not significantly, longer in time for A. spurrelli embryos than A. callidryas embryos (27 ± 0.05% vs. 24 ± 0.04% later; W = 59, P = 0.1343). In developmental terms, A. spurrelli passed through more stages between the onset of responses to the two cues (4.27 ± 0.46 vs. 3.83 ± 0.39 developmental stages; W = 55, P = 0.02).

Latency to hatch

Latency to hatch for both A. spurrelli and A. callidryas embryos at the onset of each response type was longer with hypoxia cues than with mechanosensory cues (χ² = 164.04, P < 2.2e-16). The main effect of species was only marginally significant (χ² = 6.061, P = 0.048), but we found a significant cue type × species interaction effect (χ² = 5.949, P = 0.015, Fig. 5). Latency to hatch of A. spurrelli embryos, compared to A. callidryas embryos, was slightly but nonsignificantly shorter for hypoxia-cued hatching (18.43 ± 1.06 vs. 20.61 ± 1.9 min, post hoc from GLMM P = 0.55) but it was considerably longer for mechanosensory-cued hatching (3.67 ± 0.27 vs. 2.66 ± 0.32 min, P < 0.047).

Ontogeny of VOR amplitude and the hatching response

VOR amplitude and species were strong predictors of the hatching response of embryos to egg jiggling (GLMM, main effects of VOR: χ² = 50.483, P = 1.091e-11; species: χ² = 10.243, P = 0.006), but there was no interaction effect (χ² = 2.4102, P = 0.1205). Post hoc analysis showed that A. callidryas embryos were more likely to hatch at lower VOR amplitudes than A. spurrelli (P = 0.0085, Fig. 6A). VOR amplitude varied with stage and marginally with species (LMM, main effects of stage: χ² = 107.3, P < 2.2e-16; species: χ² = 8.6377, P = 0.03452), but not by their interaction (χ² = 5.5851, P = 0.06126). In both species, VOR amplitude increased from stage 28 to stage 30 (A. spurrelli: 11.2 ± 4.7° vs. 39 ± 9.1°, P < 0.0001; A. callidryas: 18.8 ± 7.0° vs. 31.6 ± 3.8°, P = 0.0044, Fig. 6B). Compared to A. callidryas, VOR amplitude was lower in A. spurrelli at stage 28 and higher at stages 29 and 30; however, it did not differ significantly between species at any single stage (all pairwise comparisons P > 0.1, Fig. 6B).

The VOR of the first embryos to hatch from each clutch was over three times higher in A. spurrelli than in A. callidryas (37.2 ± 10.2° vs. 11.3 ± 6.5°, N = 47 and 12, post hoc from LMM: P < 0.0001, Fig. 6C). The oldest unresponsive embryos from each clutch also had considerably higher VOR in A. spurrelli than in A. callidryas (26.9 ± 2.7° vs. 8.8 ± 5.7, N = 22 and 12, P = 0.0001, Fig. 6C). In A. spurrelli no embryos with a VOR less than 19.80° hatched in response to jiggling. We found complete separation of developmental stage as a predictor of mechanosensory-cued hatching in A. spurrelli; no embryos hatched to egg-jiggling cues at stage 28, some hatched at stage 29, and all hatched at stage 30 (Fig. 6A). Among A. spurrelli embryos that hatched, there was no effect of VOR amplitude on latency to hatch (F_1,45=0.582, P = 0.45).

Hatching complications

In A. spurrelli 15.4% of embryos that attempted to hatch in hypoxia-cued hatching experiments experienced hatching complications (N = 10 of 65, from 8 clutches at age 3–3.25 days, all stage 25). Siblings from two clutches experienced hatching complications at two consecutive testing points. Affected embryos created and partially exited through ruptures in their vitelline membrane but remained trapped within their external jelly capsule (Fig. 7). Half of these embryos failed to hatch and were rescued. Latency to hatch was longer for those that did hatch, compared to embryos that did not experience complications (N = 5 vs. 55, χ² = 7.99, P = 0.0047; 29.1 ± 10.7 vs. 18.0 ± 8.1 min). Such hatching complications never occurred in A. callidryas.

Under egg predation by snakes, the escape success of Agalychnis embryos varies among species. Both A. spurrelli and A. callidryas are capable of rapid mechanosensory-cued hatching (MCH), yet A. spurrelli show much lower escape rates even during their night of peak spontaneous hatching. Here, we reject the hypothesis that a sensory constraint due to developmentally later onset of vestibular mechanosensory function in A. spurrelli can explain this difference. Instead, a different sensory constraint or a difference in decision rules may explain this difference in embryo escape performance. Moreover, we found that clutch size and egg capsule structure affect escape-hatching success. This motivates research to assess the biomechanics of different egg mass structures and their effects on hatching in snake attacks.

Vestibular mechanosensing contributes to MCH in A. spurrelli

The low escape success of A. spurrelli in our snake predation experiments does not simply reflect constraints on hatching or mechanosensing ability, as most A. spurrelli sibships began hatching to egg-jiggling cues at the same developmental stage as A. callidryas (Fig. 4B). We found a strong correlation between VOR and MCH in A. spurrelli; individuals with stronger vestibular function (higher VOR) were more likely to hatch and embryos did not hatch below a VOR threshold of 19.8° (Fig. 6A). Developmental stage was also highly predictive of MCH in A. spurrelli; no stage 28 embryos hatched while all stage 30 embryos did, and VOR amplitudes were higher in more developed embryos (Fig. 6A). These results suggest that vestibular mechanosensory function contributes strongly to MCH in A. spurrelli, as in A. callidryas (Jung et al. 2019).

Species similarities in the onset of MCH and vestibular mechanosensing

The association we found between vestibular function and MCH in A. spurrelli is largely, but not entirely, consistent with findings in A. callidryas (Jung et al. 2019; Jung et al. 2020). Across species, stage-matched embryos had comparable VOR amplitudes (Fig. 6B), rejecting our hypothesis that vestibular development timing differs. In both species, embryos with higher VOR amplitudes were more likely to hatch in response to egg-jiggling cues (Fig. 6A) supporting a key role for the vestibular system in MCH. However, A. callidryas embryos, including those at later developmental stages after the onset of MCH, were more likely to hatch at lower VOR amplitudes (Fig. 6A). Moreover, the first embryos to hatch—as well as the oldest unresponsive ones—had lower VOR amplitudes in A. callidryas than in A. spurrelli (Fig. 6C). Thus, A. spurrelli have considerably higher VOR thresholds for a response to egg-jiggling cues, compared to A. callidryas, despite their similarity in developmental onset of both MCH and vestibular mechanosensing. We also found no evidence of jiggling-cued hatching in A. spurrelli with no detectable VOR, or a VOR below our threshold of 19.8°, whereas 11% of tested A. callidryas embryos (7 of 66) hatched with no detectable VOR, and 23% hatched with VOR amplitudes < 10° (Jung et al. 2019). This suggests A. spurrelli’s low escape success in snake attacks might be due to either a different mechanosensory constraint or difference in decision rules, rather than a vestibular development constraint.

A lack of lateral line input may explain A. spurrelli’s weak MCH response

In A. callidryas at the onset of vestibular function, lateral line input substantially increases MCH to egg-jiggling cues; neither lateral line nor vestibular input alone enables strong responses, suggesting multimodal mechanosensing is critical for effective antipredator defenses (Jung et al. 2020). Thus, it is possible that A. spurrelli’s low hatching response to snakes might be due to later lateral line development. The VOR of A. spurrelli at the onset of hatching in our egg-jiggling experiments was similar to that of A. callidryas at the onset of hatching responses to tine-based vibration playbacks (mean ± SD: 37.15 ± 1.50° vs. 35.92 ± 0.95°; minimum:19.80° vs. 21.38° in A. spurrelli and A. callidryas, respectively) (Jung et al. 2019). These vibration playbacks produce substantial whole-egg motion, providing vestibular stimulation, and a subset of eggs also receive direct contact cues, and thereby some lateral line stimulation, from the tines (Warkentin et al. 2022). In contrast, egg-jiggling produces a more complex, multimodal stimulus that seems to provide considerable lateral line input (Jung et al. 2020). The similarity of VOR threshold for jiggled A. spurrelli and A. callidryas in vibration playbacks suggests that examining lateral line development and its relation to MCH in A. spurrelli would be informative.

Egg and clutch properties affect escape success

Escape-hatching success for A. spurrelli clutches in our snake predation experiments varied more than previously found, 0–47% and 0–62% for embryos tested on the 4th and 5th night after oviposition, respectively vs. 0–22% and 0–46% reported by Gomez-Mestre and Warkentin (2007). Our study might reflect more of the natural variation in escape-hatching rates, since we tested more clutches than did Gomez-Mestre and Warkentin (2007) (N = 28 and 26 vs. 8 per age). However, we found higher escape rates in larger clutches in A. spurrelli in both studies and in A. callidryas in the earlier work (Fig. 2B, C). Thus, our testing of only smaller, more tractable A. spurrelli egg masses could even have resulted in an underestimate of maximum escape success in our study population. The positive relationship between clutch size and escape-hatching success across sites and years in A. spurrelli suggests that egg clutch size and structure may play a key role in embryos’ hatching response. This effect also occurs in A. callidryas (Fig. 2C) but may be detectable only with larger sample sizes (Fig. 2C, N = 49 vs. Figure 2B, N = 20). Whether whole clutch traits affect embryo risk assessment, information acquisition, and/or the hatching process will require further investigation.

The structure of egg capsules also appears to affect the hatching process in A. spurrelli. Each embryo is fully encapsulated by a tough jelly layer that surrounds its vitelline membrane throughout development. During our hypoxia-cued hatching experiments, we noticed several A. spurrelli embryos emerging part-way through the vitelline membrane but remaining stuck within an intact jelly capsule after attempting to hatch (Fig. 7). This hatching complication delayed full escape by more than 10 minutes in embryos that managed to exit, and it resulted in failed hatching attempts in half of the affected embryos. Embryos experienced severe body compression while stuck part-way through their membrane (Fig. 7) and presumably would have died had we not rescued them after the experiment. Embryos also experienced this hatching complication in egg-jiggling experiments at later developmental stages and all required rescue (Güell 2023). If hatching complications compromise the speed or success of hatching attempts, and even cause embryo mortality, this would add an egg-stage cost of false alarms to the known larval-stage costs (i.e., increased mortality to aquatic predators, Gomez-Mestre and Warkentin 2007), thereby increasing selection against unnecessary early hatching in A. spurrelli. Thus, effects of egg structure on the hatching process and incidence of hatching complications may contribute to lower escape-hatching success of A. spurrelli during snake attacks either directly, by slowing hatching of some embryos, or indirectly, by selecting for more stringent decision rules to avoid false alarms.

Other factors selecting against early hatching in A. spurrelli

Selection by aquatic predators favors later hatching in both A. spurrelli and A. callidryas, but relative hatchling survival appears substantially lower for 1-day premature A. spurrelli than for 1-day premature A. callidryas (Warkentin 1995; Warkentin 1999; Gomez-Mestre and Warkentin 2007). The cost of larval-stage predation may be stronger in A. spurrelli if early hatching affects their ability to avoid or escape aquatic predators more than in A. callidryas. Consistent with the latter, we found that A. spurrelli hatchlings were smaller than A. callidryas ones at the onset of hypoxia- and mechanosensory-cued hatching responses (Fig. 3), suggesting that larval size may contribute to lower hatchling survival. Agalychnis spurrelli’s highly synchronous explosive breeding (Güell and González 2019; Güell et al. 2019; Güell & Warkentin in prep) and hatching timing (Fig. 1) could also select against early hatching, or for hatching synchrony, by reducing predation at egg- and/or larval-stages via predator swamping effects (Ims 1990; Santos et al. 2016). A previous attempt to find evidence for predator-swamping in a much smaller population of A. spurrelli at Estación Sirena, on Costa Rica’s Osa Peninsula was unsuccessful (Gomez-Mestre and Warkentin 2007). However, because predator-swamping is a density-dependent effect, it is presumably only detectable at a minimum threshold of population size and therefore may be worth testing for again in a larger explosively-breeding population of A. spurrelli.

Is climate change affecting spontaneous hatching timing and escape success?

The spontaneous hatching timing of A. callidryas in our study was more synchronous and peaked a full day earlier (at age 6.75–7.25 days) than was reported from Corcovado in the 1990’s (at age 7.75–8.25, Gomez-Mestre and Warkentin 2007, Fig. 1). Agalychnis callidryas also began hatching to mechanosensory cues earlier in our study, at age 4 days (Fig. 4A). We tested several A. callidryas clutches at age 4 days in pilot trials of our egg-predation experiment and found that most clutches were mechanoresponsive. In contrast, predator-induced hatching did not begin in A. callidryas from Corcovado until age 5 days in the 1990’s (Gomez-Mestre and Warkentin 2007). An acceleration in the overall developmental rate of embryos, due to effects of climate change and higher temperatures at egg laying sites on the Osa Peninsula compared to the early 1990’s (i.e., temperature-induced heterokairy; see Güell and Warkentin 2018a), may explain a forward shift in both spontaneous hatching timing and the onset of mechanosensory-cued hatching by A. callidryas, as well as greater spontaneous hatching synchrony. Indeed, the Osa Peninsula has warmed considerably since the early 1990’s: Mean annual temperatures have increased at 0.18°C per decade (with respect to the 1981–2010 average) (NOAA National Centers for Environmental Information 2022). Our findings of higher escape-hatching success of embryos from both species on the night before their peak of spontaneous hatching, compared to age-matched embryos tested in Corcovado in the 1990s (4d A. spurrelli: ~15% vs. 9%; 5d A. callidryas: ~78% vs. 60%) are also consistent with slightly faster development under the current warmer temperatures.

Differences in decision rules may affect escape-hatching success

In addition to snake attacks, the terrestrial embryos of both A. spurrelli and A. callidryas experience physical disturbance during rainstorms which cause frequent yet benign vibrations in egg clutches. In A. callidryas, embryos use information from several vibration properties to discriminate among these cues and assess risk (Warkentin et al. 2006; Warkentin et al. 2007; Caldwell et al. 2009; Warkentin and Caldwell 2009; Caldwell et al. 2010; Jung et al. 2022). In vibration playbacks experiments, low frequencies found in snake attacks induce hatching, while higher frequencies found in rainstorms elicit little or no hatching and reduce the hatching response to concurrently presented low frequencies (Caldwell et al. 2009; Caldwell et al. 2010). The temporal patterns of vibrations also matter; information from the duration and intervals between vibrations combine to affect context-dependent hatching responses (Warkentin 2005; Warkentin et al. 2006; Warkentin and Caldwell 2009; Jung et al. 2022). Thus, differences in evolved decision rules and hatching thresholds in response to any of these vibration properties may also explain variation in hatching responses to snake attacks between species. Analyses of vibrations produced by snakes and other natural disturbances in A. spurrelli clutches, in conjunction with playback experiments to examine vibration-cued risk assessment could elucidate the vibrational information and decision rules that affect embryo hatching responses in nature.

The developmental consistencies we found in the timing of onset of hypoxia-cued hatching, mechanosensory-cued hatching, and vestibular mechanosensory function in A. spurrelli and A. callidryas reject our hypothesis of a vestibular development constraint as a possible explanation for A. spurrelli’s poor escape performance in snake attacks. Thus, something else must cause the difference in escape success between species. Our finding that A. spurrelli’s poor hatching response continues through the night of spontaneous hatching, well after the development of strong vestibular mechanoreception, also supports this interpretation (Fig. 2A, 4). Our results suggest that differences in the physical structures around embryos may contribute to differences in escape-hatching success between species. The positive relationship between clutch size and escape success (Fig. 2B, C) and the high incidence of hatching complications in A. spurrelli due to egg-capsule structure (Fig. 7) suggest a role of clutch and egg structure on escape success. The potentially fatal risk of hatching complications may also have increased selection against hatching early in A. spurrelli and changed their decision rules compared to A. callidryas. The present study motivates future research to assess the role of the lateral line in MCH in A. spurrelli and the effects of clutch and egg structure on vibration biomechanics and escape-hatching success.

Conflict of interest: The authors have no relevant financial or non-financial interests to declare.

Ethics approval: All applicable international, national, and institutional guidelines for the use of animals were followed. This research was conducted under BU IACUC protocol 18-003 and permits from the Costa Rican Ministerio de Ambiente y Energía (MINAE) and the Sistema Nacional de Áreas de Conservación (SINAC) (ACOSA-INV-048-18, INV-ACOSA-055-18, ACOSA-DASP-OI-R-019-2021).

Funding: This work was supported by the National Science Foundation (DGE-1247312 to BAG and IOS-1354072 to KMW), Sigma Xi (G2018031596022314 to BAG), and Boston University.

Data availability: The data collected for and analyzed in this paper is available on Dryad at https://doi.org/10.5061/dryad.cnp5hqc8x

Acknowledgments: We thank Adrian Tanner and J. Gregory McDaniel for helping design and build the tadpole rotator, Sonia M. Pérez Arias for developing the python code to construct VOR curves, Katherine González for field assistance, Elena K. Gomez for measuring images to calculate VOR, and Osa Conservation for laboratory space and logistical support. We also thank members of the BU Egg Science Research Group for feedback that improved this manuscript at many stages of its development.

Author contributions: Brandon A. Güell: Conceptualization, Data curation, Formal Analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. Karen M. Warkentin: Conceptualization, Funding acquisition, Methodology, Project administration, Resources, Validation, Writing – review & editing.

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To hatch and hatch not: does heterochrony in onset of vestibular mechanosensing explain species differences in escape-hatching success of Agalychnis embryos in snake attacks?

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Journal Publication

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Abstract

Figures

Significance Statement

INTRODUCTION

MATERIALS AND METHODS

Egg clutch collection and care

Spontaneous hatching timing

Escape from egg-predation by snakes

Onset of hypoxia-cued hatching

Onset of mechanosensory-cued hatching

Onset of vestibular function

Statistics

RESULTS

Spontaneous hatching timing

Escape from egg-predation by snakes

Onset of hypoxia- and mechanosensory-cued hatching

Latency to hatch

Ontogeny of VOR amplitude and the hatching response

Hatching complications

DISCUSSION

Species similarities in the onset of MCH and vestibular mechanosensing

Egg and clutch properties affect escape success

Is climate change affecting spontaneous hatching timing and escape success?

Differences in decision rules may affect escape-hatching success

Conclusions

Declarations

References

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Journal Publication

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