First confirmed complete incubation of a flapper skate (Dipturus intermedius) egg in captivity

Abstract An egg of the critically endangered flapper skate Dipturus intermedius was successfully incubated to hatching in captivity in what is believed to be a first for the species. Water conditions (temperature, salinity, flow rate) were recorded, with mean water temperatures ranging from a monthly mean of 8.3 ± 1.2 to 13.2 ± 0.3°C and salinity from a monthly mean of 30.5 ± 1.2 to 36.6 ± 2.3 ppt. Hatching occurred after 534 days, suggesting that flapper skate eggs take c. 5700 growing degree‐days to incubate to hatching. The egg's prolonged embryonic development raises concerns about flapper skate eggs' vulnerability to anthropogenic disturbance.


| INTRODUCTION
The flapper skate (Dipturus intermedius) is a large demersal batoid found in the northeast Atlantic (Dulvy et al., 2006). Formerly part of what is known as the "common skate complex," the flapper skate is now known to be genetically distinct from the common blue skate (Dipturus batis; Iglésias et al., 2010;Last et al., 2016). This species complex is listed as critically endangered on the IUCN Red List (Dulvy et al., 2006) due to a reduction in the species' range following a long period of overexploitation (Brander, 1981). The total length of the flapper skate is reported as "up to 230 cm, possibly 285 cm for females," which grow larger than males (Ebert & Stehmann, 2013).
Their egg cases also rank among the largest skate egg cases at 100-144 mm width and 130-235 mm length, excluding the short horns ( Figure 1a; Gordon et al., 2016).
Understanding the developmental biology of long-lived, slowly reproducing species such as skate is important to accurately understand risks and implement appropriate conservation measures across the species' life cycle. What little is known about flapper skate embryonic development is confused by the split of the common skate species complex in 2010 (Iglésias et al., 2010). Literature predating the species complex split describes common skate embryos as taking from 2 to 5 months (Little, 1995) to "possibly years at high latitudes" (Ebert & Stehmann, 2013) to incubate to hatching; skate hatchlings have been reported to measure 21-29 cm long (Ebert & Stehmann, 2013). Detailed studies have been published on the incubation period and size at hatching of other Dipturus species (Concha et al., 2018;Parent et al., 2018), including Dipturus batis (9-10 months; Beard, 1890, Clark, 1922, but to date no comparable data have been available on flapper skate, representing a significant data gap. The influence of temperature on growth rates of individuals and incubation periods of eggs underpins the concept of growing degree-days (GDDs, Neuheimer & Taggart, 2007). GDDs describe egg incubation periods based on the average temperature during incubation, multiplied by the number of days to hatching, and are used to compare incubation periods both within single species at different temperatures (Embody, 2011) and between species (Chezik et al., 2014). Although hatching times among eggs in the same fertilisation batch may vary across a wide range of GDDs, the average number of GDDs is relatively constant for each particular species (Neuheimer & Taggart, 2007). This study aimed at reporting an incubation period for an opportunistically collected flapper skate egg expressed in GDDs.

| MATERIALS AND METHODS
On 4 April 2019, a female flapper skate was captured by a recreational sea angling charter in the northern Sound of Jura, western Scotland (UK). While on deck, the female released a fully developed egg, which was retained in sea water as per the lead author's request and quickly transported 44 km from Ardfern marina to the aquarium facility at the nearby Scottish Association for Marine Science (SAMS) inside a cool box filled with sea water. Due to the opportunistic nature of this sample, the egg was first stored outside in a shaded aquarium tank for 3.5 days before being transferred inside on 8 April 2019 into an insulated and darkened aquarium that became known as the "development tank." A constant amount of sea water was supplied to the tank, which was pumped from the outside environment via a subsand intake (placed 70 cm below the seabed surface at c. 0.3 m below Lowest Astronomical Tide, i.e., within c. 5 m of the sea surface at all times) and allowed to settle. Before entering the development tank, the water passed through a chiller, manually set at 1-2 C below the temperature of the water entering the facility, although due to a fault noted below, this was not maintained throughout the study. An airstone was placed in the tank to aid oxygenation.
In August 2020 the egg was transferred to a new tank (maintaining the same water quality standards) which became known as the "camera tank" to allow for video recording. This tank was kept dark by attaching black corrugated plastic to the outside. The egg was propped up against a rock to allow the camera to be focused on the anterior end of the egg to capture the moment of hatching and was recorded continuously using a macro camera (Axis Communications, model P1357, Stockholm, Sweden) with infrared illuminator (model: IR 70; 940 nm) through a hole in the plastic. Water temperature (using a hand-held mercury thermometer), salinity (using an OPTI Aquatic digital hand-held refractometer followed by a Brix portable refractometer when the OPTI unit malfunctioned on 22 June 2020) and water inflow rate inside both the development tank and the camera tank were recorded several times each week for the duration of the embryonic F I G U R E 1 Images of the skate egg and the hatchling described in this paper. (a) The skate egg. (b) The moment the skate hatchling emerged from the egg (facing right), wings still folded above the body (a video of the hatching is available at https://www.youtube.com/ watch?v=j8Ewgg_INRs). (c) Dorsal view of the hatchling being measured. Note the whip-like extension to the tail (black arrow). (d) A view of the hatchling's underside, showing the vitelline scar and the clasper buds alongside the pelvic fins, indicating it was a male (black arrows) F I G U R E 2 Average monthly temperature and salinity profile experienced by the skate egg. From 24 August 2020, the egg was permanently transferred from the "development tank" to the "camera tank" for constant observation. , Average incoming water temperature ( C); , Average temperature in tank ( C); , Average salinity in tank (ppt) development. Average monthly temperature was calculated to account for seasonal variation in temperature and multiplied by the number of days in the month to calculate GDDs each month. These were added together to calculate the total number of GDDs.
Once weekly, the egg was transferred to a small, clear, plastic container, which was then placed on a light box for up to 5 min to illuminate the egg from below to evaluate embryonic development; care was taken to ensure it remained submerged at all times. The egg case was imaged using a downward-facing mounted camera (Panasonic DMC-FT30, 180 dpi, Tokyo, Japan) following the method used by Musa et al. (2018), before being returned to its tank. Ultrasound scanning of the egg was undertaken opportunistically on four occasions, using three different units: A Sonoscape E1V with a micro-convex 4-13 MHz probe (18 August 2019), an Easi-Scan veterinary scanner with a micro-convex 4.5-8.5 MHz probe (19 November 2019 and 17 April 2020) and a Clarius C3 HD Vet ultrasound with a convex 2-6 MHz probe (9 September 2020). The egg remained submerged during sonographic imaging.

| RESULTS
During the course of development, monthly mean salinity varied between 30.5 ppt (S.D. ± 1.2 ppt) and 36.6 ppt (S.D. ± 2.3 ppt), with a long-term mean of 33.6 ppt (S.D. ± 1.7 ppt), responding to local rain runoff. Monthly mean water inflow rates varied between 11.8 l h -1 (S.D. ± 5.8 l h -1 ) and 44.2 l h -1 (S.D. ± 5.4 l h -1 ) (mean 21.8 l h -1 ; S.D. ± 7.1 l h -1 ) due to periodic debris build-up inside pipes and differences in supply rates between the development tank and the camera tank. Monthly mean water temperatures varied between 8.2 C (S.D. ± 1.2 C) and 13.2 C (S.D. ± 0.5 C); the overall mean water Ultrasound image of egg recorded on 9th Sept 2020, 18 months after laying temperature in the tank was 10.9 C (S.D. ± 1.7 C) (Figure 2). Due to a fault in the chiller's temperature controls, the water temperature in the development tank during September 2019-March 2020 was not 2 C cooler than the temperature of the water entering the aquarium facility as planned but rather 2 C warmer.
The first evidence of a developing embryo, distinct from the yolk sac, was recorded via ultrasound on 18 August 2019 ( Figure 3). As time passed, the embryo assumed a recognisable batoid shape that started to obscure the shrinking yolk sac (from at least January 2020 onwards). The last photograph before hatching was taken on The hatchling emerged from the egg at 22:47 GMT on 19 September 2020, or 534 days (c. 18 months) after being laid, equivalent to 5692 GDDs. The hatchling quickly forced its way through the anterior apron of the egg (within 15 s) and unfurled its wings that were folded dorsally ( Figure 1b) in a similar way to other skate species (e.g., Luer & Gilbert, 1985). The male hatchling measured 279 mm total length and 184 mm disc width and weighed 89 g (Figure 1c). It displayed a bilaterally symmetrical pattern of lighter-coloured spots arranged in rough polygons on the darkbrown dorsal surface, similar to adult flapper skate; the spots appeared less distinct than those observed in adult skate, suggesting ontogenetic changes to dorsal patterning (Benjamins et al., 2018, Figure 1c). The ventral surface was almost black, with white patches on the middle of the wings and on the pelvic fins (Figure 1d). The hatchling also displayed the temporary whip-like extension to the tail (Figure 1c).

| DISCUSSION
Recreational angling presents a unique opportunity to obtain flapper skate eggs close to their natural deposition state. Nonetheless, spontaneous release is rare, which limited the number of eggs collected in this study to one. Although the act of handling the female may have caused the egg to be deposited earlier than would have been expected, the egg appeared to be fully formed and hardened when it was released on to the deck of the vessel, suggesting it was near term.
This study was undertaken opportunistically, and as such, the authors acknowledge lessons learned for future work.
Temperature has been shown to impact the intra-species incubation times of elasmobranch eggs markedly.