Indian Journal of Geo-Marine Sciences Vol. 44(4), April 2015, pp. 609-613

Studies on the embryonic development of pharaoh’s pharaonis Ehrenberg, 1831 under laboratory conditions

Deepak Samuel.V* 1,2 & Jamila Patterson1 1Suganthi Devadason Marine Research Institute, 44, Beach Road, Tuticorin 628 001, India. 2National Centre for Sustainable Coastal Management (NCSCM), Ministry of Environment & Forests, Govt. of India, Koodal Building, Anna University Campus, Chennai 600 025, India. *[E-mail: [email protected]]

Received 6 June 2013; revised 9 July 2013

Pharaoh’s cuttlefish Sepia pharaonis was raised from laboratory spawned eggs in fiber glass tanks. This work was attempted to understand and document day wise embryonic developmental changes for easy identification. Hatching process took 13 days for a mini adult-like hatchling (paralarva) to squeeze out of the eggs. Digital microphotographs and line drawings are presented for the stages of development.

[Keywords: Sepia pharaonis, Pharaoh’s cuttlefish, embryonic development, life cycle, laboratory rearing]

Introduction funded by DANIDA, Denmark. Spawning was Embryonic development is a continuous process initiated when the female started to deposit her eggs and the identification of a particular developmental on the nylon net provided that was suspended to a stage is still arbitrary1. Characteristic changes take stone. Brood stock had 96 eggs deposited in 3 place within the embryo till the time of hatching. This clusters. The day at which the eggs were deposited is possible only if the physical, chemical and the was considered day one. Spawned eggs were biological parameters are in synchrony with one separated from the substrata and transferred to 150 another. Laboratory conditions should be identical to litres capacity fiberglass tanks. Eggs were removed the natural conditions for successful development and from the cluster individually and suspended in hatching. Numerous zoologists have studied the perforated plastic baskets of 16 cms diameter at the embryonic phase of the cephalopod life cycle since base and 20 cms at the brim region. Water the middle of the nineteenth century. For temperature in the rearing tanks did not show notable embryological studies, cephalopod eggs have always variation and was maintained at 28 ± 0.5 °C been of particular interest because they are very large throughout the experiment. Salinity was maintained at and permit observation of many details of 36 ppt and constantly monitored with the help of a embryogenesis in vivo at low microscopic refractometer. The entire study period ranged for 13 magnifications2. In the present study, embryonic days. development of Sepia pharaonis was observed from Regular observation was made on the spawned day 1(time from when the eggs were deposited) till eggs from day 1 till the day of hatching. Eggs were the day of hatching under laboratory conditions. The measured for their length, width, size of the embryo formation of different organs like eyes, arms, ink sacs and yolk in order to understand the growth differences and were clearly recorded in the yolk and the embryo. All distinctive changes corresponding to the day of their appearance on were observed carefully under a dissection embryo. microscope. Embryos were preserved everyday in Materials and Methods 90% alcohol and were later photographed with the Laboratory-spawned eggs were studied daily for help of a microscope mounted with NIKON E 990 the post- spawning development. The study was Digital camera with a resolution of 4 megapixels. conducted during July 2002 as an ongoing initiative Certain characteristic changes were also recorded as of Tropical Marine Mollusc Programme (TMMP) 610 INDIAN J. MAR. SCI., VOL. 44, NO. 4, APRIL 2015

line drawings. this seems to be the growing edge of the layer of ectoderm. Results Anal knoll, shell gland, primordia of anterior A total of 96 eggs were laid by the wild female in 3 funnel fold and primordia of posterior funnel fold clusters on the nylon net that was suspended by a start to appear by day 3. At day 4, appearance of the stone. The egg capsules were transparent, soft, primordia of arms, tentacles and characteristic eyes gelatinous and bulbous in shape and measured 21 x 14 start appearing (Fig.3). A clear albuminous fluid mm size. All eggs have a forked basal stalk and these called the Perivitelline fluid fills the space between stalks were attached together as a cluster. Incubation the eggshell and the yolk. This fluid is not seen at the period was for 13 days during which each egg capsule initial stages of development but from the fourth day became larger, transparent and fragile growing of development, the fluid bathes the developing maximum in size at the time of hatching. At the time embryo. At day 6, gills and anal knoll becomes more of deposition, the eggs measured 21 x 14 mm in size prominent with the ink sac (IS) seen as a spot on the and during the time of hatching they were 24 x 17 mm ventral side of the (Fig. 4). Chromatophores in size (Table. 2). start appearing from day 8 as scattered spots (Figs. 5 Once the eggs were laid, changes by gastrulation and 6) throughout the body. Cuttlebone prevents the were noticed. Pre-organogenesis began from day 1 translucent mantle from displaying the ink sac which where the blastoderm formation was visible, further is a major drawback for tracing the ink sac at metamorphosing into two layers by means of a development. It is possible to see a black spot (ink complex process called “Gastrulation“. This further sac) only on the ventral side of the animal (Figs. 7 and developed into visible primordium of the shell gland 8). A miniature adult-like appearance can be noticed (PSG) and primordium of the optic vesicle (PO) (Fig. on day 10 and during day 11, pigmentation becomes 1). At day 2, separation of the blastoderm into more prominent, retina is black in colour, fins more ectodermal and mesodermal germ layers takes place. widely split near the tip of the cuttlebone spine area Blastoderm covers one tenth of the egg size and (Fig. 9). Premature hatching is witnessed when a slowly increases. After the completion of this mechanical shock is transmitted to the eggs. Usually process, organogenesis begins. A clear oval mantle premature are weak and are easily susceptible slowly starts to develop towards the end of day 2 (Fig. to bacterial infection. 2). Sl.No Author Year Species Incubation Days

1. Nabhitabhata 1997 Sepiella inermis 8 – 19 days 2. Sakai and Brunetti 1997 Illex argentinus 6 – 7 days 3. Segawa et al., 1998 Loligo forbesi 68 – 75 days 4. Sakai et al., 1998 Illex argentinus 340 h 5. Nabhitabhata and Nilaphat 1999 Sepia pharaonis 9 – 25 days 6. Sakai et al., 1999 Illex argentinus 40 – 60 days 7. Pringennies et al., 2000 Loligo duvauceli 3 – 4 weeks 8. Villanueva 2000 Loligo vulgaris 1 – 15 days 9. Samuel and Patterson 2002 Sepioteuthis lessoniana 18 – 20 days 10. Anil et al., 2005 Sepia pharaonis 15 days

Table.1. Embryonic studies by various authors

The yolk is also considerably reduced with an As the embryo grows, the space inside the eggshell increase in the size of the developing embryo. enlarges and the shell itself is stretched and pushed Premature hatchlings often swim with little yolk out. It hollows out a place for itself in the surrounding followed by inking for a few seconds. Ink gland was gelatinous matter but both the outer and inner surfaces found to be functional even in the embryos, just of the eggshell are well marked and clearly visible. before hatching. Any mechanical shock to the culture The blastoderm occupies a considerable area while tanks results in inking within the egg capsule. The th two parallel lines start appearing around the yolk. embryo attained maximum size of 11 mm on the 13 This takes place during the latter part of the day and day with a decrease in yolk to 2 mm (Table. 2). SAMUEL AND PATTERSON: EMBRYONIC DEVELOPMENT OF SEPIA PHARAONIS 611

During the tenth day, the pigmentation becomes more yolk. At day 10, the arm 4 appears as two faint prominent. The chromatophores that initially swellings (Fig. 9) on the base of the fused tentacles, appeared to be pale became darker leading to a bluish visible only from the ventral side. Branchial hearts is pink pigmentation all over the body towards the final visible along with heart – gill complex. stages. Bluish pink pigmentation is characteristic for

Sepia pharaonis when compared to other Discussion (Fig. 10). Details in development help us to distinguish the The first arm starts to develop slowly followed by differences between closely related species of the second and the third arms (Fig. 9). The tentacles . Some important works carried out for and the sucker primordia were visible from the eighth the embryonic development of cuttlefishes and squids day onwards and the arms were short overlying the are tabulated in table 1. Embryonic development was 612 INDIAN J. MAR. SCI., VOL. 44, NO. 4, APRIL 2015

documented, as it was possible to hatch the cuttlefish over the eye. Respiration was visible the naked eye normally under laboratory conditions. The duration of (pulsation transmitted by the branchial hearts) from embryonic development is always temperature day 6 onwards (Fig.4). Anal papillae become more dependent between species. Incubation period ranges prominent in day 11 embryos where the embryo between 9 to 25 days (average of 14.3 days) at about measured 10 mm with a reduced yolk of 3 mm 28 C for Sepia pharaonis3 and 160 days at 12C for (Table–1). Sepia apama4. Laboratory rearing experiments carried The yolk sac in the cephalic region is reduced to 2 out in India for Sepia pharaonis was 12 days for mm just before hatching. In the same species the incubation and 7 days for hatching5. In the present external yolk sac was seen rarely observed and, when study it took 13 days for the paralarva to hatch out present, was shed immediately5. The buccal mass and from their eggs. It is possible for the species fins become functional with an increase in the size of inhabiting the tropical waters to have short incubation caecum and the stomach. External yolk is almost days when compared to their temperate counter parts. absorbed and remains as only the yolk sac envelop. Presence of a basal stalk on each egg is for the Some hatchlings possessed external yolk sac even attachment to different substrates. If the substratum is after hatching while they are just lost after hatching in nylon net or a weed, the eggs are individually the case of Loligo forbesi1. At the time of hatching, deposited to different sites instead of attaching as a embryos measured 12 mm overall length and a dorsal cluster to just one. The forked base of the stalk is mantle length of 6 mm. Females in the natural habitat ideal for attachment of eggs to the substrate when the usually lay their eggs in suggesting the occurrence of female starts depositing her eggs. The eggs start to chemical attraction exerted by the freshly spawn of develop by leaving a large yolk mass and a cap- eggs9. A female cuttlefish investigates the substrate shaped “disco- blastula” covering the animal pole of before she starts laying her eggs in order to avoid the egg6. them from being swept away from currents or Throughout development, the capsule dimensions are damaged by curious predators. Basically, seaweeds enlarged by absorption of water into the perivitelline and rocks are chosen by female Pharaoh’s cuttlefish space by a combination of increasing the surface area for the deposition of its eggs. and decreasing the thickness of the capsule4. The eggs become more transparent by the thinning of the outer Days Egg length Egg Embryo Yolk size membrane and small damage or rupture on the egg (mm) width size (mm) (mm) (mm) surface resulted in the puncturing of the egg. This 1 21 14 -- leads to the Perivitelline fluid to drain and the 2 22 14 2 4 ultimately to death of the embryo. Reduction in yolk 3 22 15 4 4 is another character to confirm the development of the 4 22 16 5 6 embryo7 because the yolk is used by the developing 5 22 16 6 6 6 22 16 6 6 embryo. Reduction in the yolk size commence from 7 22 16 8 5 day 7 onwards in the present study. Maximum yolk 8 23 17 8 5 size was measured to be 6 mm from day 4 to day 6 9 23 17 9 5 and was reduced to 2 mm on the twelfth day till 10 24 17 9 4 11 24 17 10 3 hatching time (Table. 1). Nearly all chromatophores 12 24 17 11 2 in the embryo begin as yellow and transform into a 13 24 17 11 2 darker (red-brown) pigment with time8. In the present study we noticed that the pigmentation color was Table. 1. Embryonic development of Sepia pharaonis bluish pink for S. pharaonis which grew darker with development (Figs. 9 and 10). Dark Present study documents daily changes in the pattern on both dorsal and ventral head remain developing embryo of Pharaoh’s cuttlefish Sepia unchanged till hatching. We observed from the eighth pharaonis under laboratory conditions. Commercial day that the proportions among some internal organs exploitation has resulted in the large loss of flora and such as internal yolk sac, mid gut, stomach, caecum fauna in the coastal regions of southern India mainly and ink sac change rapidly in S. pharaonis. An by the operation of bottom trawling. Since important process is the formation of a primary lid cephalopod egg capsules occur as by catch, they can SAMUEL AND PATTERSON: EMBRYONIC DEVELOPMENT OF SEPIA PHARAONIS 613

be sorted out and used for incubation and hatching in cuttlefish, Sepia pharaonis, Ehrenberg, 1831. 1999. Phuket. the laboratory. The paralarva after a few days of Mar. Biol. Cent. Spl. Publn. No.19 (1): 25-40. 4. Cronin, E. R and R. S. Seymour. Respiration of the eggs of rearing can be released back into the environment and the giant cuttlefish Sepia opama. 2000. Mar. Biol. 136: 863- even sea ranching programmes could be attempted. 870. 5. Anil M. K, Joseph Andrews and C. Unnikrishnan. Growth, Acknowledgement behavior, and mating of Pharaoh cuttlefish (Sepia pharaonis Israeli Journal of Authors are thankful to Prof. Jorgen Hylleberg, Ehrenberg) in captivity. 2005. Aquaculture: 57(1) 25 -31. Tropical Marine Mollusc Programme (TMMP) 6. Boletzky, S.V. Juvenile behaviour. (Boyle P R., DANIDA, Denmark for the financial assistance and Ed.).Cephalopod Life Cycles, Vol. II. 1990. Comparative the Director, Suganthi Devadason Marine Research Reviews. 45-60. London, ENGLAND: Academic Press, Inc. Institute (SDMRI), Tuticorin, India, for the facilities 7. Samuel, V. Deepak and Jamila Patterson. Intercapsular embryonic development of the big-fin squid Sepioteuthis and support. lessoniana (Loliginidae). 2002. Indian. J. Mar. Sciences. Vol. 31(2): 150-152. References 8. Packard, A. Size and distribution of chromtophores during 1. Segawa, S., W. T. Yang, H. J. Marthy and R. T. Hanlon. post-embryonic development in cephalopods. 1980. Viet et Illustrated embryonic stages of the eastern Atlantic squid Milieu 35(3/4): 285-298. Loligo forbesi. 1998. The Veliger. 30 (3): 230-243. 9. Zatylny, C, J. Gagnon, E. Boucaud-Camou and J. Henry. 2. Boletzky, S. V. Embryonic phase. In: Boyle P.R (ed) ILME: water borne pheromonal peptide released by the eggs Cephalopod life cycles. Vol.2. 1987. Academic Press London of Sepia officinalis. 2000. Biochemical and Biophysical pp 5-31. research communication. 275: 207-222. 3. Nabhitabhata, J and P. Nilaphat. Life cycle of cultured