The majority of reptiles are oviparous, with females burying their eggs in subsurface nests. With few exceptions (e.g., some crocodiles, lizards, and snakes; Shine et al. 1988; Somma 2003), they provide little to no maternal care beyond the selection of a nest site and an attempt to mask its location. Many nests are destroyed as a consequence of biotic (predators, microbial infection, the nesting activity of other females) or abiotic (flooding, drought, unfavorable temperatures) perturbations (Burke et al. 1998; Bernado and Plotkin 2007; Doody 2011). The developing embryos from surviving nests must then, without parental assistance, determine an optimal time for hatching and emergence from the nest. By doing so, they can significantly improve their chance of survival (Doody 2011; Warkentin 2011).

In turtles, eggs deposited in nests are within minutes exposed to oxygen, which breaks developmental arrest (Rafferty and Reina 2014) and initiates the continuation of embryonic development. Embryonic development and incubation duration are highly responsive to temperature variation within a nest. Warmer temperatures will accelerate growth and developmental rates and therefore shorten incubation duration (Deeming and Ferguson 1991; Booth 2018). Under constant temperatures and uniform conditions, turtle eggs should hatch synchronously (Porter 1972; Thompson 1989; Spencer and Janzen 2011). However, in most freshwater turtles, eggs are deposited in layers within shallow subsurface chambers where eggs nearer to the surface are more exposed to the effects of solar radiation, daily temperature fluctuations, and seasonal changes in temperature. As a result, a vertical thermal gradient is created within the chamber where eggs at the top can experience temperatures up to 6°C warmer than eggs at the bottom (Thompson 1989; Telemeco et al. 2016). Because developmental rates are temperature dependent, embryos located in the upper layers should hatch before those below, resulting in hatching asynchrony (Spencer and Janzen 2011). Experimental studies on freshwater turtles, however, indicate that synchrony can still occur because embryos respond to cues from one another that promote coordinated hatching (Warkentin and Caldwell 2009; Doody 2011).

Turtle embryos in the later stages of development are known to exhibit either delayed hatching (until conditions become more favorable) or early hatching (typically, in response to mechanical stimulation or environmental changes; Doody et al. 2001; Spencer et al. 2001; Colbert et al. 2010; Doody 2011). In some freshwater turtles, embryos behind in development (due to cooler incubation conditions in the nest) can “catch up” to embryos ahead in development under controlled laboratory conditions (Spencer et al. 2001; McGlashan et al. 2012). A critical element is that the eggs must be in close physical contact, enabling embryos to respond either by increasing their metabolic rate (McGlashan et al. 2012) or by developing faster without a change in metabolic rate, a process mediated by the thyroid hormone triiodothyronine (McGlashan et al. 2017).

With one exception (Bustard 1973; see below), there have been virtually no experimental studies done to investigate coordinated interactions among embryos in marine turtle nests, as studies have instead centered on posthatching behavior. The following is known. After exiting from the egg, the hatchlings dig their way upward for several days after they hatch (Godfrey and Mrosovsky 1997; Miller et al. 2003). They stop if they encounter warm sand near the surface, heated by the sun. Hatchlings typically emerge en masse, synchronized by the decline in sand temperatures after sunset (Mrosovsky 1968; Hendrickson 1980). Synchronous emergence might also be facilitated by synchronous hatching, but there is a significant time lag (2–7 d) between the 2 processes, making that prospect less likely.

The cues used by marine turtle embryos to initiate hatching are unknown. Sounds, either generated or emitted by late-term embryos prior to or during hatching, have been hypothesized to stimulate development and promote hatching synchrony (Ferrera et al. 2014, 2019; Monteiro et al. 2019); sounds have been shown to do so in birds (Brua 2002) and crocodiles (Vergne and Mathevon 2008). In marine turtles, however, experimental evidence is lacking and sounds might just be byproducts of the effort required to escape from the egg (McKenna et al. 2019).

Because marine turtle nests are much deeper than the nests of most freshwater turtles, the effects of solar radiation are more defuse and depend, to a much greater extent, on species differences in average nest depth, substratum color, seasonal climatic conditions, and the frequency and amount of rainfall (Lolavar and Wyneken 2015). In general, mean temperatures are lower and fluctuate less in deeper nests (Santidrián-Tomillo et al. 2017). These variables interact to determine mean daily nest temperatures, which tend to be similar to ambient substratum conditions up to the last third of the incubation period. At that time, metabolic heat generated by the developing embryos can result in as much as a 7°C rise in nest temperature and a 3°C variation in temperature between eggs in the center of the egg chamber mass and those at the periphery (Kaska et al. 1998; Booth and Astill 2001). Those differences should also result in asynchronous hatching and multiple hatchling emergences over periods spanning several days.

Bustard (1973) was the first to document that a temperature gradient existed between the hotter central and cooler peripheral eggs within a marine turtle nest. At Heron Island, Australia, where he worked, single emergences from green turtle (Chelonia mydas) nests were far more common. He speculated that could only happen if the less advanced and cooler peripheral embryos “caught up” developmentally with the more advanced and warmer embryos in the center. He did an experiment in which eggs incubating at a cooler peripheral temperature were mechanically stimulated to mimic the effect of embryonic movements within the clutch on neighboring eggs. He reported that the stimulated eggs hatched up to 4 d sooner than the nonstimulated controls, a result suggesting that mechanical stimulation promoted faster rates of development and shortened the period of incubation. However, Bustard (1973) presented no statistical or additional quantitative information.

We hypothesized, based upon prior studies done on freshwater (Spencer et al. 2001; Doody et al. 2012; McGlashan et al. 2012) and marine (Bustard 1973) turtles, that if sea turtle embryos in the nest could communicate with one another, the effect would be manifested by an increase in the speed of development, the vigor with which hatching occurred, and the temporal pattern of hatchling emergences from the nest. Our results provide support for each of these hypotheses.

METHODS

Egg Collection, Incubation Conditions, and Measurements. — We collected loggerhead (Caretta caretta) eggs from 50 nests within 12 hrs of deposition at Boca Raton, Florida (26.37°N, 80.13°W) between June and August 2019. Collected eggs were then transported to the laboratory where they were labeled using a no. 2 pencil and positioned appropriately for each experiment in an expanded polystyrene nest box of varying dimensions (see below). Each box containing sterilized (autoclaved) beach sand and was stored inside a large, “crawl-in” incubator where air temperature (31°C ± 1.5°C) and moisture (0.06 ± 0.02 m³/m³) were similar to values recorded in local nests. All nest boxes were equipped with temperature data loggers (HOBO U22-001; accuracy ± 0.21°C; Onset Computer Corp, Bourne, MA) that measured sand temperature every 15 min throughout incubation. Eggs were sprayed with a mist of deionized water twice daily to ensure viability. We used Soil Moisture Smart Sensors (SMC-M005; accuracy ± 0.031 m³/m³; Onset Computer Corp) to measure sand moisture every 15 min and to ensure that it remained within 4%–8% in all nest boxes.

Toward the end of incubation, eggs were inspected twice a day (∼10 hrs apart) for signs of hatching. “Pipping” occurs when a fully developed embryo pierces the top of its eggshell, the first sign of hatching in all experiments because it more accurately determines the completion of incubation than does hatching time (Gutzke et al. 1984). A neonate was considered to have completed the hatching process once it fully escaped from the confines of its egg. We measured incubation duration for each egg in days between the initial deposition date until the first sign of pipping. We measured hatching duration in hours between the first embryo to pip and the last embryo to completely exit its eggshell in a treatment or group. We measured hatching synchrony as the time (hours) between the first embryo to pip and the last embryo to pip within a nest box or treatment group. See below for other details.

Experiment 1: Isolated vs. Grouped Eggs. — The focus of this experiment was to examine the importance of contact between the eggs as a parameter affecting incubation and hatching duration. We created 2 treatment groups from the same clutch: isolated and grouped eggs (McGlashan et al. 2015). To do so, we collected 10 groups of 10 eggs, with each group from 10 different nests for a total of n = 100 eggs. All samples were collected within a 10-d period to assure that each clutch originated from a different female. (Consecutive nests are deposited by the same female at about 14-d intervals; Miller et al. 2003.) Each clutch was placed in its own elongated nest box (91 × 45 × 20 cm, inside dimensions) filled to a level of ∼ 6 cm with moist autoclaved beach sand. One egg was placed in each corner of the box and then isolated by cardboard dividers from the remaining 6 eggs clustered in the center (Fig. 1). All eggs were covered with an additional 2-cm layer of sand. Approximately 2 wks before hatching might occur (at 31°C ± 1.5°C), a small area on the top of each egg was exposed by gently removing the sand so that the onset of pipping could be observed. Nest boxes were thereafter inspected twice daily (in the morning and evening).

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Incubation duration and hatching synchrony were measured. Incubation duration was averaged for each nest box treatment resulting in 10 mean values for the isolated and 10 mean values for the grouped embryos. Similarly, hatching synchrony was averaged in the same way. Comparisons between the 2 treatment groups were done using a 2-factor multivariate analysis of variance (MANOVA) with 1 degree of freedom (Zar 1999). Significance was set at p ≤ 0.05.

Experiment 2: Simulated Nests. — In this experiment, our purpose was to examine embryo-to-embryo interactions in a nest-like simulation. We used 10 groups of 20 eggs, with each group collected from a different nest (n = 200 eggs), and buried each clutch in autoclaved beach sand within a vertically oriented nest box (90 cm tall × 60 cm wide × 30 cm deep) against a thin glass panel (16 mm thick; Fig. 2). Several 1-cm-diameter holes were placed in the side of the box to promote gas exchange during incubation. Eggs in each nest box were clustered together in 4 vertical rows (4, 6, 6, and 4 eggs/column; Fig. 2) to maximize contact both with one another and with the glass, ensuring all would be visible during hatching. Temperature data loggers (HOBO U22-001; accuracy ± 0.2°C, resolution 0.02°C; Onset Computer Corp) were placed at the top, center, and bottom of the egg mass to record temperature changes throughout incubation and hatching (Fig. 2). All nest boxes were maintained at 31°C ± 1.5°C inside the crawl-in incubators described above.

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Ten days before an anticipated pipping date, the nest boxes were moved into an adjacent, windowless laboratory lacking air conditioning (temperature range: 27°C–29°C) for further observations in the absence of visible lighting, as would occur within the egg chamber of a natural nest. An infrared-sensitive game camera (G42 No-Glo Trail Game Camera, model no. STC-G42NG, Stealth Cam^™, Grand Prairie, TX) was placed ∼ 30 cm in front of each nest box and programmed to take a photo every 15 s (using infrared [IR] flash photography). Images were used to determine the first sign of pipping and to record when hatching was completed.

A contact microphone (Luvay Piezo pickup; frequency response 20 Hz–20 kHz) was fixed to the outside of the glass panel using double-stick tape and positioned adjacent to the center of the egg mass. Each microphone was connected to a TASCAM recorder (DR-40X, Teac Corp, Upper Saddle River, NJ) set to record sounds continuously during the last 6 d before hatching was initiated through to the first 3 d after initiation. Sound recordings were analyzed using spectrogram programming software (Raven Pro, Interactive Sound Analysis Software; Cornell Lab of Ornithology, Ithaca, NY).

Mann-Whitney U-tests (Siegel and Castellan 1988) were used to statistically compare incubation and hatching duration among the replicates. Average daily temperatures for the last 3 d of incubation and the first 4 d of hatching were compared using two-tailed t-tests (Zar 1999).

Experiment 3: Separating Correlated Variables: Temperature vs. Mechanical Stimuli. — In Experiment 2, we found consistent differences in both incubation and hatching duration between nests in which only a few eggs hatched and those in which 10 or more eggs hatched. Those differences were also associated with contrasting average nest temperatures. Thus, the outcome of Experiment 2 (when more eggs hatched, incubation and hatching duration were shortened) might have been a consequence of embryo-to-embryo (social) communication, higher temperatures, or some combination of both variables. To distinguish between those alternatives, we designed a third experiment that minimized likely differences in temperature but could potentially accentuate differences in social stimulation between the embryos during development.

To accentuate differences in social stimulation, we established 3 groups, A, B, and C. We then divided 12 eggs from the same nest into 2 groups of 6 eggs each, designated as the A₁ and A₂ eggs. These were placed in separate sterilized, sand-filled nest boxes and incubated for 2 wks at the same (31°C) temperature. Two weeks after incubation started, we paired the A₁ eggs with 6 B eggs from another female's nest that were deposited on the same evening and had also been incubating for 2 wks at 31°C in an adjacent crawl-in incubator. These paired eggs were then maintained at that temperature until they hatched. Because both groups of eggs were at a comparable stage of development, they were expected to provide social stimulation to one another during later development. These “nests” were designated as the “experimental” group (Fig. 3).

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After 2 wks of incubation, the A₂ eggs were also paired with 6 C eggs from a different female, but those eggs were deposited at the beach the previous evening and thus were 2 wks behind in development compared with the A₂ eggs. The C eggs also served as a control for adding 6 eggs to the nest (Fig. 3). These eggs were then also incubated at 31°C until they hatched. Because the C eggs were 2 wks behind the A₂ eggs in development, we anticipated that they would provide less stimulation (if any) to the A₂ eggs.

We hypothesized that if social stimulation enhanced rates of development, then the A₁ eggs should develop faster and hatch sooner than the A₂ eggs. To determine if this happened, we compared the duration of incubation between the A₁ and A₂ eggs using a paired, 1-tailed t-test (Zar 1999). We also determined whether the B and C eggs differed in incubation duration by using a 2-tailed t-test for independent samples (Zar 1999). We hypothesized that the B eggs, stimulated by both their embryonic siblings and the A₁ embryos with which they were paired, might complete development faster than the C eggs, expected to hatch about 2 wks after the A₂ eggs.

Sand temperatures in three experimental and three control nest boxes were recorded for each week of incubation, then compared using a single factor repeated-measures analysis of variance (ANOVA) (Zar 1999) to determine if there were differences between the 2 groups.

RESULTS

Experiment 1: Isolated vs. Grouped Eggs. — A total of 34 of the 40 isolated and 55 of the 60 grouped eggs hatched. There were no statistical differences in the duration of incubation between the isolated eggs (mean ± SD) (47.0 ± 1.23 d, range = 45.3–49.0 d) and the grouped eggs (46.7 ± 1.03 d, range = 45.5–49.0 d; Table 1). There were also no statistical differences in hatching synchrony between the isolated eggs (30.8 ± 16.01 hrs, range = 12–60 hrs) and the grouped eggs (31.2 ± 15.44 hrs, range = 12–52 hrs; Table 1).

Table 1. Multivariate analysis of variance (MANOVA) analysis comparing the duration of incubation (in days) and hatching synchrony (in hours) between isolated vs. grouped eggs incubated at 31°C.^a

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Experiment 2: Simulated Nests. — The number of eggs completing development varied between nests, averaging 11.3 of 20 eggs or a hatching success of 57%. In 3 nests, designated as the low-hatch group, only 1, 2, and 4 eggs hatched, respectively, whereas in the 7 remaining nests (the high-hatch group), 10–18 of the 20 eggs hatched (Table 2).

Table 2. Results from the nest simulation experiment. Data include the number of eggs that hatched (of a total of 20 eggs in each nest), the incubation duration (in days), and the hatching duration (in hours) for each nest.

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In the low-hatch group, the duration of incubation was 48 d (in nest box 1, where only 1 egg hatched) and a mean of 46.6 d in the 2 nest boxes, where 2 and 4 eggs hatched, respectively (Table 2). Among the high-hatch group, the mean incubation period was 44.1 d (SD ranged between 0.46 and 0.81; Table 2). Incubation duration on average was significantly shorter in the high-hatch group (Mann-Whitney U-test, p = 0.02). Hatching duration in the low-hatch group ranged between 88 and 121 hrs with a mean of 101.7 hrs. Hatching duration in the high-hatch group ranged between 58 and 81 hrs with a mean of 68.6 hrs. Hatching duration was significantly shorter in the high-compared with the low-hatch group (Mann-Whitney U-test, p = 0.02).

Incubation temperatures within these nest boxes were measured at 3 different locations (top, center, bottom). There were no significant differences among the 3 locations in any nest boxes, so comparisons between the low- and high-hatch success nest boxes were based upon the data from the temperature loggers placed in the center of the egg mass and restricted to the 3-d period before and 4-d period after hatching occurred (Fig. 4). During days 2–4 posthatching, the high-hatch success nests were significantly warmer (t values ranged between –2.40 and 4.07, p values ranged between < 0.01 and 0.004).

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Sounds recorded from each nest box were classified as either “mechanical” (“taps,” “clicks,” and “scratches”) or “frequency modulated” (FM) emissions (Fig. 5). Mechanical sounds were of short (0.02–0.15 s) duration and consisted of a broad range of frequencies (up to 20 kHz). The FM sounds ranged between 0.14 and 0.2 s in duration and were confined to frequencies below 5 kHz as either 3 or 4 harmonic bands, separated by approximately 100 kHz (Fig. 5). The two sound categories differed in their distribution relative to the time of hatching (Figs. 6 and 7). Mechanical sounds were recorded in the thousands/day, beginning ∼ 5 d before hatching, and steadily increased in number through day 4 past the initiation of hatching (Fig. 6). In contrast, FM sounds were emitted in much lower numbers beginning as early as 6 d before hatching. They reached a peak in daily emission (≤ 120 sounds/d) between 2 d before and the first day after hatching was initiated. The FM sound production was more frequent in the low-hatch success nests until the onset of hatching and more frequent in the high-hatch success nests just after hatching was initiated (Fig. 7). The largest number of FM sounds (215 over 10 d of recordings) came from a nest in which only 1 egg hatched.

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Experiment 3: Separating Correlated Factors. — A total of 53 of the 60 A₁ and 54 of the 60 A₂ eggs hatched. That result enabled us to compare the incubation duration means shown by the 2 groups as 10 matched pairs. However, in both the B and C groups, no eggs hatched in 1 of the 10 nest replicates, reducing the sample size of those groups to n = 9.

The mean (± SD) incubation duration for the A₁ eggs (paired with the B eggs at a comparable stage of development) was 46.8 ± 0.55 d (range = 45.8–47.7 d) whereas the mean for the A₂ eggs (paired with the C eggs, 2 wks behind in development) was 47.4 ± 1.17 d (range = 45.7–49.0 d). The incubation duration of the A₁ eggs was significantly shorter than the A₂ eggs (paired t = –2.33, 9 df, p = 0.023; Fig. 8).

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There were no significant differences in incubation duration between the paired A₁ and B egg groups, in which the latter averaged 47.0 ± 0.73 d (range = 46.2–48.0 d; t = –0.708, p = 0.5). The mean (± SD) incubation duration for the C egg group (46.3 ± 0.51 d; range = 45.7–47.0 d) was significantly shorter than the A₂ group with which it was paired (t = 2.99, p = 0.008), but did not differ statistically from the A₁ group (Fig. 8).

Sand incubation temperatures were compared across 3 pairs of experimental and control nest boxes throughout the 7 wks of incubation. There were no significant differences among their weekly average temperatures (ANOVA F_1,5 = 1.098, p = 0.38).

DISCUSSION

Experimental Approach and Evidence for Embryonic Communication. — Under natural conditions, the duration of incubation in marine turtle nests is significantly affected by both physical (substrate albedo, grain size and moisture content; nest depth; nest temperature; Miller et al. 2003) and biological (parental phenotype; Rafferty et al. 2011; Perrault et al. 2012; Tezak et al. 2020) variables. In this study, we reduced the impact of these variables by incubating eggs in a uniform physical environment and by pooling the data from the eggs produced by different females. At the same time, we altered the embryonic social environment to determine whether differences in social stimulation resulted in changes in development rates and in hatching synchrony (Georges et al. 2008; McGlashan et al. 2018).

Our results provide support for the social stimulation hypothesis and lead to the following conclusions. First, both eggs in isolation or those in groups (Experiment 1) show no statistical differences in either incubation or hatching synchrony, and therefore no evidence that physical contact between small groups of eggs enhanced embryonic communication or influenced rates of development (Table 1). However, these data confirm that when eggs from a marine turtle nest incubate under controlled temperature and moisture conditions, the embryos develop at similar rates and hatch at statistically similar time periods (as hypothesized should happen by Porter 1972). Those data also served to establish a baseline against which any changes associated with additional manipulations of the social environment would become evident.

Experiment 2 revealed that both the duration of incubation and of hatching are shortened when the number of viable embryos in the nest exceeded a certain “facilitation threshold”—in this case, 7 instances in which 10 or more viable embryos hatched in the “nest” (Table 2). Thus, embryonic communication not only stimulated development but appeared as well to also reduce the energetic requirements associated with the hatching process itself. This hypothesis is consistent with the speculations put forth years earlier that groups of hatchlings digging their way out of a nest were more likely to successfully emerge, presumably because they collectively dig more efficiently than do smaller groups (Carr and Hirth 1961). Support for that hypothesis, based upon actual measurement of the energetic costs, has confirmed that on a per-hatchling basis, costs decline as group size increases (Rusli et al. 2016). We hypothesize that an energetically similar process occurs in nests where groups of embryos are actively pipping at the same time.

In Experiment 3, we further explored the relationship between the timing and magnitude of stimulation and its effect on incubation and hatching. Eggs from 2 nests in contact with one another and at a comparable stage of development (the A₁ and B eggs) completed development significantly faster than eggs from the same nest (the A₂ eggs) paired with eggs 2 wks behind in development (the C eggs; Fig. 8). Interestingly, the embryos in the C group also completed development more rapidly while paired with a nest 2 wks ahead in development. That outcome may have occurred because the C group embryos were uniquely exposed to 2 bouts of stimulation: when the A₂ eggs hatched and ∼ 2 wks later, when their siblings hatched. These results provide additional support for the hypothesis that developing embryos can respond to one another days before hatching actually occurs and, in addition, suggest that embryonic communication potentially stimulates developmental rates and reduces incubation duration.

Comparative and Functional Aspects. — In both freshwater and marine turtle nests, temperature gradients occur that should result in different rates of development among the eggs, followed by asynchronous hatching. To the extent that there are benefits to decreasing temporal variation in the hatching process, natural selection should promote the evolution of mechanisms that promote greater control over when hatching occurs (Spencer and Janzen 2011; Warkentin 2011). Doody (2011) considers the topic in some detail and documents the evidence (some of which is anecdotal) that embryos of 17 turtle species (including the green turtles studied by Bustard) modify when they hatch in response to external environmental cues (e.g., environmentally cued hatching). Some species will delay hatching until they detect that conditions are more favorable; hatch earlier than expected in response to degrading environmental conditions (hypoxia) or threats (mechanical stimulation from predators); or synchronize hatching by “catching up” to siblings that are more advanced in development. All three responses should, under natural conditions, reduce the variation among eggs within a clutch in when hatching occurs.

While in all of these examples embryos respond to external stimuli, there is also wide variation within clutches in the temporal pattern of hatching among species. At one extreme is the “explosive” and almost simultaneous hatching shown by pig-nosed turtles (Carettochelys insculpta) that pip within minutes after submersion in water (which induces hypoxia) or mechanical (vibration) cues (Doody et al. 2012); at the other extreme are species such as the Murray River turtle (Emydura macquarii) and painted turtle (Chrysemys picta; Colbert et al. 2010) that respond to siblings more advanced in development in ways that “. . . ensure that hatching within the clutch occurs over a short time period” (i.e., fewer days; Doody 2011; Spencer and Janzen 2011). Thus, the critical element defining synchronous hatching is the “. . . coordinated departure from eggs of fully formed embryos. . .” (i.e., hatchlings; Spencer and Janzen 2011), mediated by mechanisms such as a response to an external stimulus or stimuli that counteract asynchrony due to thermal gradients existing in the nest (Doody 2011). This definition does not specify any strictly defined temporal boundary (e.g., all eggs hatch within a 24, 48, or some other period of hours or days). Accordingly, we demonstrate in this study that loggerhead embryos do, in fact, coordinate through communication with one another and, in the process, reduce the duration of both incubation and hatching (Table 2; Fig. 8). We conclude on that basis that hatching in loggerhead nests is “synchronous,” even though it occurs over several days (Table 2).

Relationships Between Hatching and Emergence in Loggerheads. — In marine turtles, neonates possess an externally extruded, prominent, and vulnerable yolk sac. That energy supply is required to power the lengthy period of offshore migration following emergence from the nest. Hatchlings also retain the folded or “curled” posture assumed inside the egg. Both that yolk sac and their folded posture preclude effective locomotion, and so the turtles require another 2–7 d to unfold, to absorb the yolk sac inside the body cavity, and then to effectively dig their way upward toward the beach surface from the much deeper nests that are characteristic of marine turtles (Godfrey and Mrosovsky 1997; Miller et al. 2003). Thus, in marine turtles there is a prolonged and variable period of development between hatching and nest emergence that acts to temporally disassociate the 2 processes and make it unlikely that synchrony in hatching will be causally related to synchrony in emergence.

In our experiments, and even when the external temperature was tightly controlled at 31°C, the duration of hatching ranged between 58 and 81 hrs with an average of 68.6 hrs (for the nests in which ≥ 10 of 20 eggs in the “nest” hatched; Table 2). In natural nests, in which the mean clutch size exceeds 100 eggs (Van Buskirk and Crowder 1994) and (in South Florida nesting populations) where hatching success on average exceeds 50% of the clutch size (Brost et al. 2015), embryonic stimulation might be greater than we observed and potentially could more effectively shorten the duration of hatching. But even under those circumstances, it is unlikely that in marine turtle nests, synchrony in hatching is directly related to synchrony in emergence.

The evidence from field studies suggests, instead, that a more complex relationship exists in marine turtles between hatching, which can occur at any time, and nest emergence, which normally occurs at night (Salmon and Reising 2014). With few exceptions, emergence is accomplished by groups of hatchlings that are competent at that time to leave the nest and crawl to the sea. However, in some loggerhead nests there is only one (“first”) emergence episode in which all of the hatchlings depart whereas, in other nests, smaller groups of hatchlings emerge over several evenings. In South Florida nesting populations, emergences can occur over as many as 4 evenings (Witherington et al. 1990). Unfortunately, there have been no studies done at our study site that document the proportion of loggerhead nests that show single vs. multiple emergences. Single emergences are by far more common (∼ 90% of all observed; K. Rusenko, pers. comm., August 2019). Additionally, we have no information regarding relationships between when hatching occurs and the timing and frequency of emergence activities. That said, it seems reasonable to hypothesize that in nests where emergences occur over several evenings, the temporal pattern of hatching might be more variable than in nests that show a single emergence.

Evidence in support of that hypothesis comes from field studies showing that loggerhead nests deposited in different geographic areas differ in emergence patterns and that those differences are related to female size, nest depth, and the temperature regimes under which those clutches develop. It is well documented that North Atlantic loggerhead females breeding on the western side of the basin reach sexual maturity at a larger size (86–92.5-cm curved carapace length (CCL; Avens and Snover 2013) than do females nesting to the east, within the Mediterranean Sea, and breeding at rookery sites in Greece, Cyprus, or Turkey. Those females are characteristically smaller (Casale et al. 2018), reaching sexual maturity at 66.5–84.9 cm in CCL (Margaritoulis et al. 2003). There is also a strong positive correlation between female CCL and the depth of her nest (Miller et al. 2003). Females nesting in Florida dig nest chambers in which eggs at the bottom are ∼ 60 cm below the surface, whereas eggs at the top of the nest are ∼ 35 cm below the surface (Carthy 1996). In contrast, nests deposited at beaches in Turkey range between a top depth of 30 cm and a bottom depth of 55 cm below the surface (Kaska et al. 1998). A small sample of nests at Kafalonia, Greece, did not exceed 51 cm in depth (Houghton and Hays 2001).

In the Mediterranean, there are also pronounced thermal differences within, as well as between, individual nests. Nests showing the greatest within-nest temperature differences are also those showing the largest variation in hatchling emergence patterns (Houghton and Hays 2001). Emergence “asynchrony” at Kafalonia, Greece, ranged between 1 and 12 evenings, with a mean of 6 evenings. A similar pattern was reported by Hays et al. (1992) at Cephalonia, Greece, where hatchlings emerged from each of 10 nests on more than 1 night (mean of 8.3 nights, range between 5 and 11 nights), and by Glen et al. (2005) studying the nests deposited at Alagadi Beach, Northern Cyprus, where hatchlings from ∼ 70% of the loggerhead nests emerged over 3 evenings.

In summary, these results reinforce the hypothesis that there is greater variation in the temporal pattern of emergence among Mediterranean than among western Atlantic loggerhead nests. That variation may, in turn, be a function of nest exposure to a more variable thermal environment experienced by eggs located closer to the beach surface. They also suggest that loggerheads may possess a restricted capacity to alter their rates of embryonic development and compensate for these effects, at least compared with the freshwater turtles studied thus far and perhaps, as well, to the green turtles studied by Bustard (1973).

Sound Production. — Recent studies have documented that, toward the end of incubation, sounds are produced by marine turtle embryos and hatchlings. The list of species includes green turtles, Kemp's ridley (Lepidochelys kempi), olive ridley (Lepidochelys olivacea), hawksbills (Eretmochelys imbricata), and leatherbacks (Dermochelys coriacea; Ferrera et al. 2014, 2019; McKenna et al. 2019; Monteiro et al. 2019). In this study, we add loggerheads to the list. What remains a matter of speculation, however, is whether those sounds are functionally significant, serving to coordinate hatching as occurs in other species of reptiles (Vergne and Mathevon 2008) and birds (Brua 2002). These issues remain to be addressed experimentally. Until that is done, we suggest that labeling any of those sounds as “vocalizations” is inappropriate because that term implies that communication occurs when, in fact, there is presently no evidence that it does.

Our results advance our understanding of sound production in two ways. First, the use of contact microphones greatly improves signal-to-noise ratios over those previously illustrated. This procedure should make possible playback experiments using clearer acoustic stimuli to determine whether the sounds influence development. Second, in previous studies, recordings were confined to sampling periods varying in duration and frequency. Our recordings were continuous and enabled us to clarify associations between our two sound categories with events transpiring inside the nest. Thus, as expected, mechanical sounds increased as more embryos hatched whereas FM sounds were clearly associated with the hatching process and declined in occurrence after most of the embryos exited from the egg.

Finally, our more complete record of when sound production began enabled us to be certain that the C eggs, behind in development by 2 wks, would not have produced mechanical stimuli before the A₂ eggs, with which they were paired, began to hatch. What remains to be determined, however, is the relative importance of sound, as well as pressure changes induced by embryonic movements, in affecting developmental rates and coordinated hatching responses among the various species of marine turtles.