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

Abstract

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

Keywords: Agalychnis callidryas; Anuran; Diel timing; Embryo behavior; Hatching; Hatching synchrony; Phenotypic plasticity; Photoperiod; Phototaxis.

Figure 1. Experimental methods.

For the hatching timing experiment, we made three light-proof compartments (A), each with a different photoperiod. We split each Agalychnis callidryas egg clutch into three groups of embryos, held in a petri dish over dechlorinated tap water (B). We placed dishes of embryos within plastic bins in each compartment (C). To observe hatching orientation underwater, we submerged whole egg clutches (D, F) and individual embryos in glass cups (E, G). The surface exposure of eggs was similar but embryos’ visual environments differed (F, G). In the hatching phototaxis experiment, we placed A. callidryas eggs in close-fitting tubes in the dividing wall of half-dark cups (H, I). Schematics are not to scale; compartments were longer than in A, aquaria larger than in D, E, and half-dark cups larger than in H (see I).

Figure 2. Hatching timing of Agalychnis callidryas embryos in three light environments.

Data are proportion hatched for embryos in each treatment, recorded every 2 h (N = 11 clutches, each split across treatments). Boxplots show median, interquartile range (IQR), and extent of data to ±1.5 × IQR (box and whiskers), and outliers. Grey background sections indicate dark periods in the photoperiod treatment, and the corresponding periods of time in constant dark and constant light treatments.

Figure 3. Timing and synchrony of hatching of Agalychnis callidryas embryos in three light environments, for N = 11 clutches, each split across treatments.

(A) Hatching at nightfall. Data are mean proportion of embryos that hatched between 16:00 and 20:00 h at age 5 d, ±SE across sibships. Hatching was more concentrated in this 4-h period when it included the onset of darkness; different letters indicate significant differences at α = 0.05. (B) Hatching synchrony within egg masses, measured as the highest proportion of embryos hatched in any 2-h period. Embryos within masses hatched more synchronously in the photoperiod treatment. (C) Synchrony across egg masses (sibships). Data are number of clutches whose peak of hatching occurred in each 4-h time period. Grey bars indicate dark periods of photoperiod treatment. Hatching was more synchronous across sibships in the photoperiod treatment. (D) Delayed hatching. Proportion of embryos that hatched after age 6 d. More embryos hatched late in development in the continuous light treatment.

Figure 4. Hatching direction of Agalychnis callidryas embryos submerged in hypoxic water with one side illuminated (phototaxis experiments) or neither side illuminated (control).

In the absence of light, there was no evidence of side-bias due to the structure of the half-dark cups. Under two illumination intensities more embryos hatched towards the light. Data are number of embryos exiting their egg, and egg-holding tube, in each direction.