Herpetology Notes, volume 13: 425-428 (2020) (published online on 26 May 2020)

781 days in the egg: Prolonged incubation time in Calumma parsonii parsonii (Cuvier, 1824) resulting in a healthy juvenile and revealing circumstantial evidence for sperm retention in this species

Alexandra Laube1,*, Thorsten Negro1, and Andreas Augustin2

Parson’s chameleons are not only the largest distilled water by the third author and bedded in plastic chameleons of Madagascar (Kalisch, 1994), they are boxes on moistened perlite. Only five eggs of the entire also the longest-lived species of all known chameleons clutch showed a bright white surface and were thought (Tessa et al., 2017). Calumma parsonii parsonii (Cuvier, to be fertilised. However, all eggs were left at room 1824) has been bred in captivity for years (Abate, 1997, temperature around 20-22 °C for four weeks. Many 1998; Pietschmann, 1997; Tröger, 1997). In artificial infertile eggs shrunk, turned yellow and were removed incubation, periods between egg-laying and hatching from the boxes. usually range from 400 to 660 days (Kalisch, 1994; Hereafter, the remaining eggs were transferred Masurat, 2005). A similarly long incubation period is not in their boxes with substrate to a Herp Nursery II known from any other reptile species. During incubation, incubator (Lucky Reptile, August Jeanmaire Str. 12, the eggs pass a diapause (developmental arrest following 79183 Waldkirch, Germany). The temperature was oviposition) and a cold torpor (temporary suspension in held constantly at 23-24 °C, the substrate moisture was later developmental stages) (Ewert, 1991; Pietschmann, maintained by regularly dripping distilled water on the 1997; Tröger, 1997; Masurat, 2005). In Madagascar, eggs. In February 2017, the eggs were cooled down daily these periods of diapause are associated with the cooler by 1 °C until they reached an incubation temperature of dry season (Abate, 1997; Schmidt et al., 2010). We 13-14 °C. This diapause was maintained for four weeks. hereby report on the successful hatching of Calumma Afterward, the eggs were gradually warmed up again parsonii parsonii after a prolonged incubation time of by 1 °C daily up to a temperature of 23-24 °C. A cold 781 days. torpor according to the same scheme as described for In August 2015 mating between a male and a female the diapause in the previous year followed in February C. p. parsonii, was observed by the third author. 2018. At that time, only the five white eggs of the clutch These individuals were both captive bred in Germany. were left. All the others had collapsed little by little. Throughout the following year, the female did not mate The collapsed eggs were opened and assessed as being anymore and showed defensive behaviour towards infertile. several males. All mating attempts were carried out Finally, three of the remaining eggs did not hatch but under the supervision of the third author. Apart from did not shrink either, and subsequently remained in the failed mating efforts, males and female were kept the incubator. On 12 October 2018, these three eggs separately. The female deposited a clutch of 69 eggs were handed over to the first and second author, who on 28 January 2017. These were carefully cleaned with transported them 650 km by car and transferred them to another Herp Nursery II incubator. A device able to measure the heartbeat of reptile embryos, a Buddy Mk2 Egg Monitor (Avitronics, Rose in Valley, Lower Hugus Road, Threemilestone, TR 6BD Cornwall, United Kingdom), revealed live embryos in two of the three 1 Madcham.de, Am Damm 5, 55262 Heidesheim am Rhein, Germany. eggs. The Egg Monitor was originally developed for 2 Moorstr. 14, 21261 Welle, Germany. birds but is also suitable for measuring the embryonic * Corresponding author. E-Mail: heartbeat in reptile eggs (Sartori et al., 2015). The egg [email protected] without an embryonic heartbeat was opened by the first 426 Alexandra Laube et al.

Figure 1. Two eggs of Calumma parsonii parsonii on perlite Figure 2. Calumma parsonii parsonii juvenile that hatched after 740 days of incubation. The right egg is shrinking, after an incubation period of 781 days, at the age of 18 days, probably due to the death of the malformed embryo inside. on the finger of the first author.

author and turned out to be infertile. The incubation of In the past, there have been isolated cases of unusually the remaining two eggs was continued at 23-24 °C. In long incubation periods in captivity, for example February 2019, the eggs experienced another cold torpor. in Furcifer minor (Fitzinger, 1843) with successful During this cold torpor, one egg shrunk and began to hatching of three juveniles after about 700 days (Giering, mold (Fig. 1). The egg was opened on the 740th day of personal communication), in Furcifer lateralis (Gray, incubation by the first author after the Egg Monitor did 1831) with 615 days (Schwarz, 2007) and in Furcifer not show any heartbeat. The dead embryo inside the oustaleti (Mocquard, 1894) with one surviving juvenile egg had a malformed, rigid spine which probably had after more than 1000 days (Hellendrung, personal prevented the chameleon from hatching. The yolk sac of communication). The exact cause of the prolonged this chameleon was extremely small and residual. incubation periods remains unknown. In several On 19 March 2019, the incubator failed. As a result cases, as told by the breeders, unsuitable incubation of this failure, the remaining egg was exposed to a conditions may have been involved. It is conceivable temperature increase from 13 °C to the ambient room that largely neglected incubation factors such as air temperature of 22 °C in a time frame of a few hours. On 20 March 2019, a very active juvenile C. p. parsonii hatched from the egg after a period of 781 days since oviposition. The juvenile hardly carried traces of a yolk sac and ate fruit flies after a few hours. The chameleon is still alive and thriving at the time of this publication (Figs. 2 and 3). According to current literature research, this observation describes the longest incubation period of C. p. parsonii ending with a successful hatching. There are no studies from Madagascar on unusually long incubation periods yet. In theory, it may be possible in the wild that unfavourable conditions prevent eggs from hatching. However, due to strong predation pressures by snakes and egg-eating lizards, it is unlikely that eggs will remain undiscovered and undamaged in the soil over such a long time (Hasegawa et al., 2009; Knoll et Figure 3. Calumma parsonii parsonii juvenile that hatched al., 2009). after incubation of 781 days, at the age of six months. 781 days in the egg: Prolonged incubation time in Calumma parsonii parsonii 427 pressure, substrate type, the amount of egg contacting Chameleon information network 29: 17–25. the substrate or a certain amount of precipitation may Andrews, R.M., Díaz-Paniagua, C., Marco, A., Portheault, A. contribute to hatching success. If one of these factors (2008): Developmental arrest during embryonic development of the common chameleon (Chamaeleo chamaeleon) in Spain. lacked in the herein above mentioned case or was not Physiological and Biochemical Zoology 81: 336–344. available in sufficient quality, it may have delayed Atsatt, S.R. (1953): Storage of sperm in the female chameleon hatching until the extremely rapid rise in temperature Microsaura pumila pumila. Copeia 1: 59. by incubator failure. Ballen, C., Shine, R., Olsson, M. (2915): Developmental plasticity Diapause and cold torpor in reptiles are known to in an unusual animal: The effects of incubation temperature on provide eggs a mechanism to respond to changing behavior in chameleons. Behaviour 152: 1307–1324. environmental conditions during embryonic Díaz-Paniagua, C., Cuadrado, M. (2002): Influence of incubation conditions on hatching success, embryo development and development (Andrews et al., 2008; Ballen et al., 2015). hatchling phenotype of common chameleon (Chamaeleo Due to the developmental arrest, the juveniles hatch in chamaeleon) eggs. Amphibia-Reptilia 24: 429–440. synchrony with the arrival of the rainy season, when Eckhardt, F.S. (2018): Triplet from a single egg in the Panther the climatic conditions and the abundance of insects Chameleon (Furcifer pardalis). Herpetology Notes 11: 777– allow a higher chance of survival (Díaz-Paniagua and 779. Cuadrado, 2003; Andrews et al., 2008; Karsten et al., Ewert, M. (1991): Cold torpor, diapause, delayed hatching and 2008). Hatching synchronisation probably failed in our aestivation in reptiles and birds. In: Egg incubation, its effects case due to the lack of other healthy hatchlings. on embryonic development in birds and reptiles, p. 173–192. Ferguson, M.W. and Deeming, D.C., Ed., Cambridge, United Many reptiles are capable of sperm retention (Ewert, Kingdom, Cambridge University Press. 1991; Sever, 2002; Holt, 2011). Some turtles and snakes Hasegawa, M., Mori, A., Nakamura, M., Mizuta, T., Asai, S., are even known to have special sperm storage tubules Ikeuchi, I., Rakotomanana, H., Okamiya, T., Yamagishi, S. within the oviduct (Sever, 2002). In chameleons, there (2009): Consequence of inter class competition and predation is evidence of sperm retention in genera Bradypodion on the adaptive radiation of lizards and birds in the dry forest of (Atstatt, 1953; Tolley et al., 2014), Trioceros (Lim, western Madagascar. Ornithological Science 8: 55–66. 1982), Furcifer (Saint-German, 1962) and Chamaeleo Kalisch, K. (1994): Chameleon profile of Chamaeleo parsonii (Cuvier, 1824). Chameleon information network 12: 8–19. (Saint-German, 1962). No specialised sperm retention Karsten, K.B., Andriamandimbiarisoa, L.N., Fox, S.F., Raxworthy, structures have been found in chameleons yet (Tolley C.J. (2008): A unique life history among tetrapodes: An annual et al., 2014). However, sperm could be detected in chameleon living mostly as an egg. Proceedings of the National epithelial folded walls of the posterior oviduct in adult Academy of Sciences of the United States of America 105: female chameleons (Saint-Germain, 1962; Tolley et al., 8980–8984. 2014). Knoll, A., Glaw, F., Köhler, J. (2009): The Malagasy snake There was no evidence of sperm retention in the genus Pseudoxyrhopus ambreensis preys upon chameleon eggs by shell slitting. Herpetology Notes 2: 161–162. Calumma until this study. Since the female Parson’s Lin, J.-Y. (1982): Sperm retention in the lizard Chamaeleo Chameleon was last mated in 2015 and laid eggs five hoehnelli. Copeia 1982: 488–489. times until the end of 2019 without any further mating, Masurat, G. (2005): Vermehrung von Chamäleons. First edition. producing fertilized eggs in 2016, 2017, 2018 and Offenbach, Germany, Herpeton, Verlag Elke Köhler. 2019, sperm retention in the oviduct can be assumed. Pietschmann, J. (1997): A brief account of additional hatching of Parthenogenesis has not yet been described in the Calumma parsonii parsonii in Germany. Chameleon information species and appears unlikely. Another argument in network 25: 38. Rafferty, A.R., Reina, R.D. (2012): Arrested embryonic favour of sperm retention is that the number of fertilised development: a review of strategies to delay hatching in egg- eggs decreased and the number of malformed embryos laying reptiles. Proceedings of the Royal Society B 279: 2299– increased with each additional clutch of eggs, probably 2308. due to declining sperm quality. In the first clutch of 55 Risley, T. (1997): Preliminary observations on a small population of eggs laid by the Parson’s female, 20 healthy juveniles Chamaeleo [Calumma] parsonii parsonii in eastern Madagascar. hatched, none of them being malformed. Chameleon information network 25: 25–28. Saint-Girons, H. (1962): Présence de réceptacles séminaux chez les Caméléons. Beaufortia Series of miscellaneous publications 9: References 165–172. Abate, A. (1997): A report of successful captive hatching of C. p. Sartori, M.A., Taylor, E.W., Abe, A.S., Crossley, D.A. (2015): An parsonii in the United States. Chameleon information network appraisal of the use of an infrared digital monitoring system 25: 37–38. for long-term measurement of hear rate in reptilian embryos. Abate, A. (1998): Reports from the field: Parson’s chameleon. Comparative Biochemistry and Physiology 188: 17–21. 428 Alexandra Laube et al.

Schmidt, W., Tamm, K., Wallikewitz, E. (2010): Chamäleons – Drachen unserer Zeit. Fifth edition. Münster, Germany, Natur und Tier Verlag. Schwarz, M. (2007): 615 Tage! Inkubationsdaten zu Furcifer lateralis. Chamäleo Mitteilungsblatt 35: 9–10. Sever, D.M., Hamlett W.C. (2002): Female sperm storage in reptiles. Journal of Experimental Zoology 292: 187–199. Tessa, G., Glaw, F., Andreone, F. (2017): Longevity in Calumma parsonii, the world’s largest chameleon. Experimental Gerontology 89: 41–44. Tolley, K.A., Chauke, L.F., Jackson, J.C., Feldheim, K.A. (2014): Multiple paternity and sperm storage in the Cape Dwarf Chameleon (Bradypodion pumilum). African Journal of Herpetology 63: 47–56. Tröger, M. (1997): Remarks about the long-term captive care and first steps towards captive breeding of Calumma parsonii. Chameleon information network 25: 29–36.

Accepted by Benjamin Tapley