Heartbeat, embryo communication and hatching synchrony in snake eggs

Communication is central to life at all levels of complexity, from cells to organs, through to organisms and communities. Turtle eggs were recently shown to communicate with each other in order to synchronise their development and generate beneficial hatching synchrony. Yet the mechanism underlying embryo to embryo communication remains unknown. Here we show that within a clutch, developing snake embryos use heart beats emanating from neighbouring eggs as a clue for their metabolic level, in order to synchronise development and ultimately hatching. Eggs of the water snake Natrix maura increased heart rates and hatched earlier than control eggs in response to being incubated in physical contact with more advanced eggs. The former produced shorter and slower swimming young than their control siblings. Our results suggest potential fitness consequences of embryo to embryo communication and describe a novel driver for the evolution of egg-clustering behaviour in animals.

that younger eggs respond to the perceived heart rates of older eggs as a proxy for metabolic levels (heart rates steadily increase throughout the incubation period in water snakes 15 ; Fig. 1). For this purpose 6 freshly laid snake clutches (77 eggs) were split into 12 half clutches of which 6 were incubated in close contact with slightly older eggs and the remaining 6 were used as controls. We recorded heart rates for each egg throughout the incubation period, hatching success and hatching phenotypes of the newly hatched snakes.

Results
Experimental design. Eggs (hereafter called "younger" eggs) mixed with older eggs (i.e. born 5.8 ± 1.7 days earlier on average) were consistently exposed, from incubation day 17 onwards, and until older eggs hatched, to higher heart rates (on average 92.73 ± 5.23 bpm; Fig. 1) than they would have experienced if not mixed (i.e. control eggs averaged 84.76 ± 7.68 bpm over the same timeframe-F 1, 72 = 85.51, P < 0.0001 -see Fig. 2).
Younger eggs versus control eggs. A repeated measure ANOVA with "clutch of origin" and "treatment" as factors and 8 successive heart rates as the repeated measure through time yielded a significant result Figure 1. Six freshly laid clutches of the water snake Natrix maura were split into 12 half clutches: 6 half clutches were incubated in close contact with slightly older eggs (mixed) and the remaining 6 half clutches were used as controls. Heart rates (Y axis) were recorded for each egg throughout the incubation period (X axis). Heart rates naturally increase during the incubation, therefore eggs mixed with older eggs were consistently exposed, from incubation day 17 onwards, and until older eggs hatched (black arrow) to higher heart rates (on average 89.81 ± 3.92 bpm) than they would have experienced if not mixed (i.e. control eggs averaged 82.52 ± 3.39 bpm). Means ± SE are plotted. (Wilks lambda = 0.53, F 8, 46 = 5.08, P < 0.00015). There was no significant interaction between the two factors (F 7, 371 = 0.81, P = 0.58) but a significant effect of treatment (F 1, 53 = 21.36, P < 0.0001): younger egg heart rates averaged 90.47 ± 2.69 bpm throughout the incubation versus 87.27 ± 2.88 bpm in (control) sibling eggs.

Discussion
This simple experiment demonstrated that within a clutch, snake embryos are able to alter heart rates (i.e. metabolic levels) in response to the perceived heart beats of surrounding embryos. Eggs incubated in contact with older eggs, that is, exposed to higher heart rates, responded by accelerating development and hatched half a day earlier than control eggs. This result (i) highlights a mechanism (heart beats) by which intra-clutch hatching synchrony may be achieved in snakes 13 and (ii) is in accordance with the catch-up hypothesis 7 where turtle eggs from the (relatively colder) bottom of the nest accelerated their development in an attempt to synchronously hatch with eggs from the (relatively warmer) top of the nest. Faster development and early hatching altered yolk assimilation (young snakes were born shorter, but with higher fat storage) and came at a cost: swimming performance was reduced in early hatchers compared to sibling control snakes.
Egg-clustering and communal nesting behaviour in reptiles and indeed in most egg laying species, were traditionally assumed to have evolved primarily as anti-predatory tactics (from extinct dinosaurs 25 through to insects 26 , spiders 27 , cephalopods 28 , fish 29 to amphibians 30 ). Recent studies also reported direct benefits of egg clustering and communal nesting to offspring phenotypes through hydric modifications of incubation conditions 24 or through securing and maintaining egg position 12 . Our results add a novel dimension to the evolution of egg-clustering and communal nesting in the animal kingdom in the form of embryo to embryo communication: the kind of information exchanged (metabolic rate, stress, sex ratio, relatedness, clutch size etc.) may convey important information about the quality of the natal environment as well as an estimate of forthcoming competition amongst mates, conspecifics and heterospecifics. As such, long term effects of embryo to embryo communication (personality, dispersal) are a new, yet fascinating field to be researched at a traditionally overlooked life-stage.

Material and Methods
Experimental design. Gravid females were captured along the banks of the Lez River in South-West Ariège, France, in June and July 2014. A total of 77 eggs were obtained from 6 clutches between 14/07/2014 and 07/08/2014 (mean clutch size = 12.8 ± 5.9 eggs). Eggs were collected within 12 hours post laying. Eggs were individualised by gently rupturing the natural contact areas. This procedure greatly reduced the possibility of chemical communication amongst eggs; although there is no evidence that eggs may exchange chemical substances such as steroid hormones (Spencer & McGlashan, pers. com.). Eggs were individually marked for identification purposes with a pencil using a letter (coding for clutch of origin) and a number (egg number within each clutch). Egg mass influences both embryo metabolism and hatching phenotype 15 , so eggs were allocated to two treatments using a split clutch design: eggs were numbered within each clutch from heaviest to lightest and evenly reunited into two half-clutches. This ensured consistency in egg mass across treatments (two-factors Nested ANOVA with female nested into half-clutch: F 1, 65 = 0.30, P = 0.59). Six of the twelve half clutches (N = 6.67 ± 3.01 eggs on average) were merged and mixed with older eggs (N = 6.50 ± 2.26 eggs on average) obtained from another 6 clutches laid a few days earlier (between 09/07/2014 and 03/08/2014) to generate 6 experimental mixed clutches (average difference in age between eggs was 5.8 ± 1.7 days (span: 4 to 9 days). The remaining half clutches (N = 6.2 ± 2.9 eggs on average) were incubated along with the 6 experimental mixed clutches in an Aqualytic ® incubation chamber set at a constant 28 °C. Eggs were placed in a plastic container (one clutch per container; 20 cm × 15 cm × 5 cm) on a 2 cm layer of vermiculite.
Egg and hatchling measurements. Eggs were measured in mass to the nearest 0.01 g using a digital scale within 12 hours of oviposition. Eggs were individually placed into small jewellery bags (5 × 4 cm, made of fine mesh material) a few days prior to hatching. This ensured juvenile snakes could be matched to their egg shell when multiple births occurred at the same time, while maintaining physical contact amongst eggs. Hatchlings were measured in body mass ( ±0.01 g) and snout-vent length ( ±0.1 mm) within 12 hours of emergence. Siblings were housed together in plastic boxes (15 cm × 10 cm × 5 cm) with a water dish, shelter and paper towel as substrate. A Body Condition Index (BCI) was calculated for each snake, using the residual values of the linear least-squares regression of Log (birth body mass) against Log (birth snout-vent length). All tests were performed on all hatchlings.
Heart rate measurements. We measured embryo heart rates using the Buddy ® digital egg monitor (MK2, Avitronics) under the standardised protocol described for eggs 12,15 . The Buddy ® system works by shining an infrared beam onto the surface of the egg, detecting minute distortions caused by embryonic heart beats. The Buddy ® monitor was left inside the incubator at all times to prevent temperature variation during heart rate readings. Each egg was gently placed onto the sensor pad for heart rate reading (a stable reading was obtained after Scientific RepoRts | 6:23519 | DOI: 10.1038/srep23519 approximately 30 seconds) and then returned to its clutch. Embryo heart rates were measured at incubation day 10, 20 and 30, and then every two days until hatching.
Swimming performance. A high definition wide angle digital camera (25 fps) was fitted above a linear 300 cm × 40 cm × 50 cm swimming track and used to record trials (recording section of 120 cm). The tank was filled with 10 cm of water adjusted to 25 °C using a reverse-cycle water chiller (TECO ® TC15). The video was then edited on a computer and swimming speed calculated over the first five lengths of the track swum by each snake. The fastest performance was retained for each of the five lengths swum, and a fastest overall performance for the entire swimming test (the first length was usually the fastest).
All experimental protocols were approved by the Préfecture de l' Ariège, which provided capture, breeding, experimentation, release and ethics permits (Arrété #2012-11). All experiments were carried out in accordance with the approved guidelines. All females were returned to their exact site of capture within two weeks of egg-laying. Once tests were completed, young snakes were given their first meal (small dead minnows ranging from 0.5 g to 1 g; supplied by Armorvif ® ) prior to being released at the maternal capture site.

Data analysis.
Assumptions for normality of the data and equality of variances were tested on all variables (Lilliefors and Levene's tests). Means ± standard deviations are given unless otherwise stated.