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1 This article was downloaded by: [Sultan Qaboos University] On: 30 November 2014, At: 19:48 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Zoology in the Middle East Publication details, including instructions for authors and subscription information: Emergence pattern of the Green Turtle, Chelonia mydas, hatchlings under laboratory and natural conditions Ibrahim Y. Mahmoud a, Abdulaziz Y. AlKindi a, Taher A. Ba-Omar a, Sultan Al-Siyabi a, Saif N. Al-Bahry a, Abdul Qader Elshafie a & Charles S. Bakheit b a Biology Department, College of Science, Sultan Qaboos University, P.O. Box 36, Al-Khod 123, Muscat, Sultanate of Oman b Mathematic and Statistics Department, College of Science, Sultan Qaboos University, P.O. Box 36, Al- Khod 123, Muscat, Brazil Published online: 28 Feb To cite this article: Ibrahim Y. Mahmoud, Abdulaziz Y. AlKindi, Taher A. Ba-Omar, Sultan Al-Siyabi, Saif N. Al-Bahry, Abdul Qader Elshafie & Charles S. Bakheit (2005) Emergence pattern of the Green Turtle, Chelonia mydas, hatchlings under laboratory and natural conditions, Zoology in the Middle East, 35:1, 19-28, DOI: / To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the Content ) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views

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3 Emergence pattern of the Green Turtle, Chelonia mydas, hatchlings under laboratory and natural conditions by Ibrahim Y. Mahmoud, Abdulaziz Y. AlKindi, Taher A. Ba-Omar, Sultan Al-Siyabi, Saif N. Al-Bahry, Abdul Qader Elshafie, and Charles S. Bakheit Abstract. Green Turtle eggs were collected at random from 5 different nests from the Ras Al- Hadd Reserve in Oman. They were incubated 16hr after oviposition at constant temperatures set at C for female producers and at for male producers. Standard histological procedure of the gonads was performed for sex determination. Despite the constant temperatures set in the incubators, there were differences among the incubated eggs. This is similar to the natural condition, where an asynchronous pattern of emergence is common. Pipping occurs when the eggshell has a slit and hatching is when the head and one flipper are outside the eggshell. Emergence occurs when the hatchling is completely free from the extraembryonic membranes and eggshell. There was no correlation between the pipping-hatching and hatching-emergence intervals in the female, but in males the two intervals were significantly correlated (P<0.01). Hatchling weights were: g (±0.83) for females and g (±0.58) for males. In both sexes, hatchling weight did not influence the duration of the two intervals. The duration between pipping emergence was significantly (P<0.01) longer in females than in males. Natural and incubator observations showed that the hatchlings remained in the nest after emergence for at least 48hr until the umbilical swelling had subsided before attempting their emergence escape. While the hatchlings remain inside the eggshell after pipping attached to chorioallontoic/amniotic membranes, they are occasionally vulnerable to injury by nesting turtles, micro-organisms or early-emerged hatchlings inside the nest. Kurzfassung. Im Ras Al-Hadd-Schutzgebiet im Oman wurden die Eier von Suppenschildkröten nach dem Zufallsprinzip von fünf verschiedenen Nestern gesammelt. Sie wurden über 16 Stunden nach der Eiablage unter konstanten Temperaturen bebrütet, und zwar mit C zur Produktion von Weibchen und mit C zur Produktion von Männchen. Zur Geschlechtsbestimmung wurden histologische Standardmethoden angewandt. Trotz der konstanten Temperaturverhältnisse ergaben sich bei den bebrüteten Eiern Unterschiede: Dies entspricht natürlichen Bedingungen, wo ein asynchrones Schlüpfmuster gewöhnlich ist. Als pipping wird der Zeitpunkt bezeichnet, wenn der erste Riss in der Eischale erkennbar ist, als hatching, wenn sich der Kopf und das erste Gliedmaß außerhalb der Eischale befinden, und als emergence, wenn das Jungtier vollständig frei von extraembryonalen Membranen und der Eischale ist. Bei Weibchen bestand keine Korrelation in den Intervallen zwischen pipping-hatching und hatching-emergence, doch waren beide Intervalle bei Männchen signifikant korreliert (P<0.01). Das Gewicht der Schlüpflinge betrug bei Weibchen 28,22 g (±0.83), bei Männchen 25,74 g (±0.58). In beiden Geschlechtern war das Gewicht unabhängig von den beiden Intervallen. Der vollständige Schlüpfprozess dauerte bei Weibchen signifikant (P<0.01) länger als bei Männchen. Beobachtungen in der Natur und im Labor zeigen, dass Schlüpflinge mindestens 48 Stunden im Nest bleiben, bis der Dottersack so weit resorbiert ist, dass sie ihre Flucht ins Meer wagen können. Zwar bleiben die Schlüpflinge nach dem Platzen der Eischale zunächst noch mit der Allontois bzw. dem Chorion verbunden, doch sind sie im Nest Gefahren durch nistende Alttiere, Mikroorganismen und früh schlüpfenden Jungtiere ausgesetzt. Key words. Green turtle, marine turtles, hatchlings, pipping, emergence, Oman, Middle East. Zoology in the Middle East 35, 2005: ISSN Kasparek Verlag, Heidelberg

4 20 Zoology in the Middle East 35, 2005 Introduction At the end of the incubation period and before emergence, marine turtle hatchlings undergo some morphological and behavioural adjustments during two brief intervals. These are pipping-hatching and hatching-emergence. During these two periods, the turtles remain inside the eggshell in order to close of the umbilical area and straighten the plastron as well as to absorbe the remnants of the yolk sac (KASKA & DOWNIE 1999, MILLER 1985, GODFREY & MROSOVSKY 1997). The duration of incubation depends on the temperature of the nest chamber, where sexual differentiation takes place during the middle third of incubation: lower temperatures produce males while higher temperatures produce females (YNTEMA & MROSOVKY 1982, RAYNAUD & PIEAU 1985, KASKA et al. 1998). Previous studies have focused only on the phase of the emergence of hatchlings onto the surface relative to the thermal cues (HENDRICKSON 1958, BUSTARD 1967, 1972, WITHERINGTON et al. 1990, GYURIS 1993) and also to the moisture (MCGEHEE 1990). The major aim of this investigation was to observe the morphological changes which occur specifically in the umbilical region of the Green Turtle (Chelonia mydas) immediately after hatching. These changes involve the detachment of the hatchling from its extraembryonic membranes and the eggshell, which may take a little over 3 days before the hatchling finally emerges from its nest onto the beach surface. Knowledge of the emergence intervals under laboratory and natural conditions may be of value in the overall understanding of the hatchlings behaviour during a brief critical period between pipping and emergence. Material and methods Eggs were collected randomly within a 1 km area from 5 different nests immediately after oviposition from the Ras Al-Hadd Reserve in Oman (22 32 N, E to N, E) in January and May Each nest was excavated, and some of the eggs were transferred in plastic buckets containing sand from Ras Al-Hadd to the Department of Biology at Sultan Qaboos University (SQU) where they were placed in the incubators with constant temperature ranges approximately 16 hr after oviposition. The clutch size from each of the five nests was as follows: nest one 100, nest two 93, nest three 105, nest four 98 and nest five 95 eggs. Clutch one and two were collected during January 2003, and the others during May of the same year. After hatching, 20 hatchlings were selected at random from each clutch. The rest were used for another study. Five nests were chosen rather than a single nest because some differences in embryological development may occur between the clutches or even within a single clutch. For the same reason, all the eggs were used from each nest rather than a portion of the eggs. Each egg was incubated singly in a 600 ml plastic container according to the method of MROSOVSKY (1988), with some modifications. Each container had a plastic cover 10 cm in diameter. The upper part of the container was punched (eight holes in each container, 2.5 mm in diameter). Each egg was placed at the centre of the container on top of a foam sponge surrounded by vermiculite and 65 ml of distilled water was added to moisten the foam. An additional 65 ml of distilled water was added to the vermiculite on day 20 of the incubation. The eggs were placed in Precision Scientific (Thermo Forma, Marietta, OH, USA) incubators adjusted to run at different temperatures. Uncovered containers containing distilled water were placed at the bottom of the incubators in order to provide humidity for the eggs (MROSOVSKY 1988).

5 Reptilia 21 Fig. 1. Green Turtle (Chelonia mydas): Left: pipping; right: hatching, Tab. 1. Pipping-hatching and hatching-emergence intervals in the Green Turtle, Chelonia mydas. Hatchling weights were recorded shortly after emergence. Interval Sex N Duration in hours Mean (± SE) Pipping-hatching (± 1.21) (± 1.04) Hatching-emergence (± 3.33) (± 2.17) Weight (g) Mean (± SE) : (± 0.83), N = 29 : (± 0.58), N = 64 Hatchlings were sacrificed and the gonads were dissected along with the adjacent kidneys. They were fixed in 10% buffer formalin and then processed for light microscope study according to the method of YNTEMA & MROSOVSKY (1980). Emergence of hatchlings on the beach surface The hatchlings at Ras Al-Hadd were observed during January and May 2003, and were counted from each nest. A total of 5 nests was observed. The hatchlings usually emerged during early evening, at night or early morning from usually saucer-shaped depressions. The temperature on the sand surface was recorded during emergence. When each depression was found to contain hatchlings, it was marked in order to follow up the future activities of the hatchlings. A small mesh net was placed loosely over each depression with stones laid on the edges of the net. Four visits were made to the nests with approximately a 14 hr interval between visits to each nest. Each visit to a nest lasted about two hours. During the second visit, we observed emergence activity in all 5 of the nests. Each nest was visited again for the third time and during this time only two nests had hatchlings while the other three nests were completely untraceable because of excavations by the nesting turtles. At first, we did not attempt to excavate these nests for the presence of hatchlings, to avoid any disturbance to the nests and to keep them under natural conditions. However, during the fourth and the final visit, the nests were excavated and examined for the presence of live and dead hatchlings and for the unhatched eggs.

6 22 Zoology in the Middle East 35, 2005 Tab. 2. Green Turtle hatchlings observed at 5 sites in the Ras Al-Hadd Reserve, Oman. Each site was visited four times. During the third visit, sites 2, 4 and 5 were destroyed by nesting turtles (*). During the fourth visit 8 hatchlings were in site 1 and 1 in site 3. At the end of the fourth visit, the remaining two nests were excavated. Site no. No. of hatchlings visit 1 visit 2 visit 3 visit 4 Unhatched eggs Excavation of nests Live hatchlings Dead hatchlings * * * * * * * * * * * * * * * Examination of the umbilical area The rangers at Ras Al-Hadd Reserve collect hundreds of stray hatchlings daily, which take the wrong direction from the sea as they are attracted to the village lights. The number of these hatchlings increases significantly during the peak period, especially during July - October. These hatchlings are taken to the sea on the same or the following day. A total of 2561 stray hatchlings was examined over a span of 5 years for the presence or absence of a swelling in the umbilical area of the plastron. The results were recorded. Statistical analysis All analyses were carried out using statistical package, SPSS (version 10). P values below 0.05 were considered statistically significant. Results Under laboratory conditions Eggs incubated at C produced females while eggs incubated at C produced males. Histologically, females showed a well-developed cortex while males showed a welldeveloped medulla as in YNTEMA & MROSOVSKY (1980). Pipping occurred when the eggshell had a large crack (Fig. 1). During the pipping-hatching interval, the chorioallantoic/amniotic membranes were still associated with the hatchling (Fig. 1, 3). There was a swelling in the umbilical region and also the remnants of the yolk sac when the hatchlings first emerged from the eggshell. Limited movements of the hatchlings were observed. Hatching occurred when the head and one front flipper were out of the eggshell (Fig. 1). Hatchling movements increased and there was still some association with the chorioallantoic/amniotic membranes (Fig. 3). Once the hatchlings were completely out of the eggshells, the umbilical region was still with a slight swelling (Fig. 3). The plastron gradually became flat and straight, and the swelling in the umbilical region gradually subsided. The hatchlings became very active and sometimes they pushed up the container lid. These movements may be similar to the natural conditions when they begin their attempts to emerge from the nest onto the beach surface.

7 Reptilia TIME (hours) Pipping-Hatching Interval Hatching-Emergence Interval FEMALE (n=21) MALE (n=39) Fig. 2. Boxplots of pipping-hatching and hatching-emergence intervals in hatchlings of the Green Turtles, Chelonia mydas, incubated in the laboratory under constant temperatures. Boxes represent the middle 50% of the observations. Horizontal lines inside the boxes represent the median. Observations beyond the vertical lines are outliers. The duration of both pipping-hatching and hatching-emergence under laboratory conditions was significantly longer in females than in males (P<0.01) (Tab. 1, Fig. 2). There is no correlation between pipping-hatching and hatching-emergence intervals in females but in males there was a significant correlation (P<0.01). The mean hatchling weight after emergence for female hatchlings was (28.22±0.83g, n=29) and for male hatchlings was (25.74±0.59 g, n=64). There was no significant difference between the weights of both sexes (Tab. 1). In both sexes, there is no correlation between the two intervals and the hatchling weights. Weight therefore has no influence on the two intervals. Under natural conditions During the initial and second visits to each of the five nests on Ras Al-Hadd Reserve, there was a wide difference between the numbers of hatchlings in the 5 nests observed (Tab. 2). During the third visit to the nests, hatchlings were present in 2 of the 5 nests; the other three had been destroyed by the excavating nesting turtles. During the fourth visit, hatchlings were found in the two remaining nests. When the two nests were excavated, there were unhatched eggs, dead hatchlings and empty shells (Tab. 2). When the five nests were first found, it is possible that some of the hatchlings had already emerged before the first visit to the sites, or that some of them later escaped under the edge of the net. So, the number of hatchlings may not represent the total number of hatchlings for each site. However, based on the number of emerged hatchlings and unhatched eggs in each of the two remaining nests, the totals may represent the major segment of the clutch. These results indicate that the hatchlings emerged

8 24 Zoology in the Middle East 35, 2005 in groups over several nights rather than all at one time. Field observations indicate that the total emergence in one clutch may take 4-5 nights. The sand temperature on the surface (ranges and means) for January and May during the visits was: C ( ); C ( ), respectively. Based on 2561 stray hatchlings over a period of 5 years, 96% of these hatchlings had a flat to slightly raised umbilical area. The other 4% had a swollen umbilicus (height mm), which is the result of premature emergence caused by the excavating nesting turtles. Discussion This study reports on the morphological and behavioural changes in the Green Turtle during the pipping-hatching and hatching-emergence intervals. It appears that hatchlings go through a crucial and sensitive period before their final detachment from the eggshells. While inside the eggs, the hatchlings are vulnerable to injury by movements of early-emerged hatchlings in the nest or by adult turtles excavating their nests. Observing these changes in hatchlings under laboratory conditions has given us valuable information on this transitional period of development. The pattern of emergence was also observed in nature and was compared with that under laboratory conditions. Under laboratory conditions, both pipping-hatching and hatching-emergence intervals were longer in female hatchlings than in males. It is unclear why female hatchlings remained attached to the eggshell membrane longer than male hatchlings. Despite the constant temperatures set in the incubators, there were still some differences in the incubation time among the eggs. Under natural conditions, emergence asynchrony is common as the hatchlings emerged in groups over several nights rather than all at one time. In addition, field observations indicated that the total emergence in one clutch may take 4-5 nights. This is similar to the emergence pattern displayed by the Loggerhead Turtle, Caretta caretta (CHRISTENS 1990, HAYS et al. 1992, HOUGHTON & HAYS 2001). It has been suggested that under natural conditions among large sea turtles such as the Green Turtles, thermal variations within the nests are minimal (CARR & HIRTH 1961). It would therefore be expected that under such conditions all the eggs would hatch at the minimal incubation time. However, based on the present field and laboratory data, asynchronous emergence is the common practice in the Green Turtles at Ras Al-Hadd. On the other hand, thermal variations also occur in the nests of sea turtles that dig shallow nest chambers, such as the Loggerhead Turtle (HOUGHTON & HAYS 2001). HAYS et al. (1992) suggested that if asynchrony is marked, it may be related to wide thermal variations within the nests. It would thus be beneficial for the hatchlings to emerge separately in small groups over several nights, rather than waiting for all to emerge at the same time. In this study, the sand temperatures varied with the month during which they were taken, and there is no specific temperature range for the emergence of hatchlings to the surface as indicated in previous studies. In the data gathered during the last five years, there was substantial evidence that the umbilical region is detached from the eggshell and its associated extra-embryonic membranes after hatching. There was approximately a hr waiting period before the hatchlings emerged on to the beach surface. During this time, there was a significant decrease in the umbilical swelling. This is based on our data, which showed that 96% of the hatchlings had a flat to slightly raised umbilical region. This is consistent with the laboratory data, which

9 Reptilia 25 Fig. 3. Top: Just before emergence with chorioalantoic-amniotic membranes still attached to the umbilical area; Middle: Immediately after emergence with a swelling in the umbilical area; Bottom: the swelling has subsided.

10 26 Zoology in the Middle East 35, 2005 showed that the hatchlings had an umbilical swelling soon after they emerged from the eggshell and freed themselves from the extra-embryonic membranes, but in the incubator it took about 48 hr for the umbilical swelling to subside. Field observations showed that the Loggerhead Turtle hatchlings remained inside the nest for about 4.1 days (range 4-7) before they made their move on to the surface of the beach (GODFREY & MROSOVSKY 1997). These authors also estimated the time between pipping and hatching to be 0.8 days, which brings the total time to approximately 5 days. HENDRICKSON (1958) and CARR & OGREN (1960) have estimated the time between hatching and emergence of the Green Turtles to be about 4 and 7 days, respectively. In the laboratory study the total time between pipping and emergence was 3.38 days for females and 2.73 days for males. For the field study, the total time was estimated to be about 4 and 5 days. The reason for the discrepancy between pippinghatching and hatching-emergence intervals under laboratory and natural conditions is that natural hatchlings would require additional time to climb from their nests on to the beach surface. Moreover, the time of emergence under laboratory conditions was recorded as soon as the hatchlings detached from the eggshell. With reference to the temperature during emergence on to the surface, HAYS et al. (1992) indicated that there is no fixed temperature that can initiate emergence. GYURIS (1993) suggested that the temperature gradient in the top 10 cm of the sand column rather than absolute temperature is the main factor in emergence. However the specific thermal cue during emergence is still unclear (WITHERINGTON et al. 1990). There is a possible advantage for the hatchlings to emerge at night, to escape heat stress and predators (HENDRICKSON 1958, BUS- TARD 1972). When the hatchlings left the eggshells in the incubator, they at first maintained limited movements for at least hr. After that they increased their activities, moving inside the containers, and after one day they occasionally tried to push up the container lids with their heads. Perhaps these movements are similar to the field movements when they try to climb on to the surface. When the hatchlings became active in the containers, the umbilical swelling was already starting to subside. Linked with this, the majority of hatchlings when seen on the beach also had a flat umbilicus with a significant decrease in the swelling. Based on these observations, it appears that the hatchlings remained inactive soon after detaching from the eggshell and before making their final emergence attempt on to the beach surface. Although the data indicate that the Green Turtle hatchlings at Ras Al-Hadd are predominantly asynchronous during emergence, we cannot rule out the possibility that occasional emergence en masse may take place. The lack of correlation between hatchling weight and the time spent inside the eggshell is a clear indication that hatchling weight is not a factor that determines the time spent inside the eggshell. HAYS et al. (1992) also reported that hatchling size was not a factor in temporal emergence. Acknowledgements. The authors would like to acknowledge the help of the Ministry of Regional Municipalities, Environment and Water Resource for providing transportation and also the valuable assistance in the field of Mr Rashid AL-AAMRI, the chief-ranger of Ras Al-Hadd Reserve, and his staff.

11 Reptilia 27 References BUSTARD, H. R. (1967): Mechanism of nocturnal emergency from the nest in Green Turtle hatchlings. Nature 214: 317. BUSTARD, H. R. (1972): Sea turtles: their natural history and conservation. NewYork. CARR, A. & L. OGREN (1960): The ecology and migrations of sea turtles. 4. The Green Turtle in the Caribbean Sea. Bulletin of the American Museum of Natural History 121: CARR, A. F. & H. F. HIRTH (1961): Social facilitation in green turtle siblings. Animal Behavior 9: CHRISTENS, E. (1990): Nest emergence lag in loggerhead sea turtles. Journal of Herpetology 24: GODFREY, M. H. & N. MROSOVSKY (1997): Estimating the time between hatching of sea turtles and their emergence from the nest. Chelonian Conservation & Biology 2: GYURIS, E. (1993): Factors that control the emergence of turtle hatchlings from the nest. Wildlife Reserve 20: HAYS, G. C., J. R. SPEAKMAN & J. P. HAYES (1992): The pattern of emergence by loggerhead turtle (Caretta caretta) hatchlings on Cephalonia, Greece. Herpetology 48: HENDRICKSON, J. R. (1958): The green turtle, Chelonia mydas (Linn.), in Malaya and Sarawak. Proceedings of the Zoological Society of London 130: HOUGHTON, J. D. R. & G. C. HAYS (2001): Asynchronous emergence by loggerhead turtle (Caretta caretta) hatchlings. Naturwissenschaften 88: KASKA, Y., R. DOWNIE, R. TIPPETT & R. W. FURNESS (1998): Natural temperature regimes for loggerhead and green turtle nests in the Eastern Mediterranean. Canadian Journal of Zoology 76: KASKA, Y. & R. DOWNIE (1999): Embryonic development of sea turtles in the Mediterranean. Zoology in the Middle East 19: MCGEHEE, M. A. (1990): Effects of moisture on eggs and hatchlings of loggerhead sea turtles (Caretta caretta). Herpetologica 46: MILLER, J. D. (1985): Embryology of Marine Turtles. p In: C. GANS, R. G. NORTHCUTT & P. ULINSKY (Eds.), Biology of the Reptilia. Vol. 14. London & New York. MROSOVSKY, N. (1988): Pivotal temperatures for loggerhead turtles (Caretta caretta) from northern and southern nesting beaches. Canadian Journal of Zoology 66: RAYNAUD, A. & C. PIEAU (1985): Embryonic development of the genital system. p In: C. GANS & F. BILLENT (Eds.), Biology of Reptilia. Vol. 15. New York. WITHERINGTON, B. E., K. A. BJORNDAL & C. M. MCCABE (1990): Temporal pattern of nocturnal emergence of loggerhead turtle hatchlings from natural nests. Copeia 1990: YNTEMA, C. L. & N. MROSOVSKY (1980): Sexual differentiation in hatchling loggerhead (Caretta caretta) incubated at different controlled temperatures. Herpetologica 36: YNTEMA, C. L. & N. MROSOVSKY (1982): Critical periods and pivotal temperatures for sexual differentiation in loggerhead sea turtles. Canadian Journal of Zoology 60: Authors addresses: Prof. Ibrahim Y. Mahmoud, Dr Abdulaziz Y. AlKindi, Dr Taher A. Ba-Omar, Mr Sultan Al-Siyabi, Dr Saif N. Al-Bahry and Dr Abdul Qader Elshafie, Biology Department, College of Science, Sultan Qaboos University, P.O. Box 36, Al-Khod 123, Muscat, Sultanate of Oman. Dr Charles S. Bakheit, Mathematic and Statistics Department, College of Science, Sultan Qaboos University, P.O. Box 36, Al-Khod 123, Muscat, Sultanate of Oman. contact: taher@squ.edu.om.

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