Journal of Aquariculture & Aquatic Sciences Article

This article is written by J.G. West, and S. Carter, Aquarium Division, Taronga Zoo, P.O. Box 20, Mosman, Sydney Australia 2088. From the Journal of Aquariculture and Aquatic Sciences Volume 5, No. 4, pages 111-117.

ABSTRACT

The breeding biology of the epaulette shark (Hemiscyllium ocellatum (Bonnaterre)) was studied over a 429 day period. Observations were recorded on the mating and egg laying behavior of three breeding groups, in captivity. Embryonic development and juvenile growth rates were also studied. Embryonic development averaged 130 days from deposition of the egg to hatching. Over the six month period, the length of the young increased by an average of 16 mm (a 17.6 percent increase) and the weight increased by an average of 4 g (a 29.9 percent increase). Weight decreased in the first five weeks and length decreased in week 5. Egg viability was found to be 65.9 percent. There were 27 (9.4 percent) hatchings of which 10 (37.04 percent) juveniles survived the six month period.

INTRODUCTION

The epaulette shark, Hemiscyllium ocellatum (Bonnaterre, 1788) is a common, small, harmless tropical bottom dwelling shark from the Western South Pacific, New Guinea and Northern Australia (Compagno, 1984. Dingerkus and DeFino, 1983). Named for the prominent large dorsal dark brown spot on its back, they are normally compatible with most species of fishes and make extremely good display specimens in an aquarium. Taronga Zoo Aquarium has displayed this species for over 60 years.

Whitley (1967) reported observations made by Taronga Zoo Aquarium staff on this oviparous species, but little is known of the egg devel- opment and early juvenile stages of the epaulette shark. The aim of this paper is to report the findings of a breeding program at Taronga Zoo Aquarium. The areas covered: 1) the development of the egg; 2) development of the embryo; 3) hatching period; 4) juvenile growth rates. Three breeding groups were set up to produce eggs on a continuous basis.

METHODS AND MATERIALS

Epaulette sharks have randomly bred at the aquarium for many years. Initially two adult females (60-65 cm total length (TL)) and one adult male (72 cm TL) were removed from display to become one of the breeding groups. These specimens had been in captivity for 4-5 years in a multi-species tank and were originally collected on the Great Barrier Reef off Townsville, Australia.

Two additional males (60-65 cm TL) and three females (58-68 cm TL) were obtained from the Great Barrier Reef around Cairns by local fish collectors. Two additional breeding groups were established with sex ratios of 1:2 and 1:1. Data recorded on all eggs included date of depo- sition, date hatched or date considered infertile. Data recorded for selected eggs included dimensions and weight.

The three breeding groups were set up in separate 565 L all glass aquariums. Each aquarium was supplied by continuous natural sea water (open system) at 25 degrees C. The water replacement rate for each aquarium was 4 L per minute (total exchange rate of 10 times per day). Each tank incorporated a sub-gravel biological filter enabling closed system capability. The sharks were fed every second day with fish prawns and squid. An additional multi-vitamin supplement (Avi-Drops, for birds) was inserted into small whole fish once a week.

Eggs were deposited at night, usually two at a time (on 6 occasions three eggs were found). Each egg was assigned a number derived from the tank number, a letter indicating egg one (A). two (B) or three (C) from that tank plus the day, month and year (i.e., 1A110288 = tank one, first egg recovered on 11th February 1988). A sub-sample of eggs from each breeding tank was measured and total weights recorded. The egg was blotted dry, using paper towels and weight immediately.

The eggs were transferred from the breeding tank to an egg development tank. Each egg was suspended by a length of fishing line threaded through one of the corners of the egg case (with the identification number attached). This tank was 60 L in volume and supplied from the same water source as the breeding tanks at 0.5 L per minute (total exchange rate of 12 times per day). The suspended eggs where inspected daily for signs of fungus developing on the egg cases. A number of eggs were dissected at known time intervals and their embryonic development recorded.

The hatchlings were weighed and measured and tattooed (with Indian ink) as they were too small to have a tag attached without extensive damage. The tattoos were combinations of dots on the dorsal surface of the pectoral, pelvic and caudal fins. The hatchlings were transferred to a 280 L aquarium containing P.V.C. pipes and some rocks for the pups to hide in and under. They were fed varied assortments of finely chopped fish, prawn, mussel and brine shrimp daily. A multi-vitamin supplement was added once a week (as used for the adults).

Weekly length and weight measurements were taken from the first eight weeks and from then on at monthly intervals. All weights were taken on a Mettler P162N electronic balance to the nearest ).0.1 g. The length was recorded as mm total length using a fish measuring board technique. Each shark was measured three times and the mean length calculated.

RESULTS

Mating. The act of mating appears to be instigated by the female. Several times the females were seen grasping the male's fins with their mouths. Consequently the male dorsal and pectoral fins were damaged quite extensively at the edges.

Mating was observed several times and a brief description of one event follows. The sequence is described as the male grasped the female's right pectoral fin (Fig.1 a,b): however, there appears to be no particular preference to right or left in other observed matings. The male grasped the female's right pectoral fin and swallowed the fin until he was firmly holding the fin base. He then curved his body up and over the female, bringing his underbelly in contact with the female's side. His claspers are held at right angles to the normal. Once the male had hold of the female he would not release her, often despite being dragged around the tank.

Figure 1. During mating the male grasps the female's pectoral fin and lies alongside her: a) doral view; b) ventral view; c) detail showing position of male's claspers during copulation. (see original article)

The male tried to ease his anal fin under the female's body, but she was often seen to swim with her back arched, holding her cloaca against the substrate. During this time, the male's pelvic finds and claspers were directed over the female's back. When the male finally trapped the female's right pelvic fin between his right pelvic and left pectoral finds, he slid his right pelvic fin further across and flattened the female's left pelvic fin against her body, exposing the cloaca. The left clasper then slid along the rear edge of the female's right pelvic and was thrust into the cloaca (Fig. 1 c.). The clasper is held firmly in place as the coupled animals often vigorously rolled several times while joined. In this particular instance, the coupling too several minutes from when the male first grasped the female's fin. The time from penetration to the first sign of semen leaking from the cloaca was 45 seconds. It was another 45 seconds before the sharks separated. On separation the male's clasper was still flared at the distal end. On several occasions after mating, there were traces of blood on the claspers.

Figure 2. Total Mean Weight v. Time (see original article)

Egg Deposition. Observations have been made on two occasions of the female in the process of egg deposition. The process was similar in both cases, only the amount of time involved differed. The tail was raised, arching the back and exposing the cloaca. The diametrically opposed pectoral fins were undulated repeatedly and rapidly, causing the body to move with a whipping motion. During this activity, the egg was gradually expelled from the cloaca. The activity occurred in bursts until the egg was half expelled. Then continued in one long period until the egg was fully expelled and continued for several seconds afterwards. The egg was presented blunt end first. The female then lay quietly on the bottom. Respiratory rate was elevated from the normal resting rate of 30 beats per minute to 140/minute in one case. The total time for the laying activity was three minutes on one case and 90 seconds in the other.

The Egg. Over a period of 429 days, the five females laid a total of 287 eggs. When removed from the breeding tank, 98 (34.15 percent) of these egg cases were found to be empty. The remaining 189 eggs were moved to the hatching tank.

The eggs were brown, ellipsoid shaped and often wrapped entirely in a mass of fine, soft, sticky fibers. The eggs ranged in length from 82-110 mm (x=96.8 +/- 4.0, N=53), in width from 35-46mm (x=41.5 +/- 2.2) and in thickness from 19.0-25.0 mm (x=21.8 +/- 1.7). The weights of the eggs varied from 23.85-35.14 grams (x=29.9 +/- 2.2).

The casing was faintly translucent and when held up to strong light, the yolk was faintly visible. The casing was composed of two distinct layers, an external fibrous outer layer and a clear brown inner layer. The casing was flexible but very tough. After laying, the embryo developed inside the egg, completely isolated from the external environment. After 25-34 days, the end of the egg developed slits and the embryo's move- ments created water currents through the egg. The embryo reached full development in 124-142 days (x=130.1 N=23) at which time the splits at the end of the egg enlarged to permit the escape of the juvenile shark.

Records for 160 of these eggs show that 49 (30.6 percent) developed fungus within a few days. Within the next 21 days a further 14 (8.7 percent) died. Between 21 and 42 days after deposition, when the egg cases split to allow for water circulation, 28 (17.5 percent) of eggs died. Another 21 (13.13 percent) survived the egg splitting but did not result in live hatchings and 26 (16.25 percent) eggs resulted in hatchlings and 22 (13.75 percent) viable eggs were still in the hatching tank.

Egg deposition rates in the three tanks showed similar rates in Tank 1 and Tank 2 (127 and 116 eggs respectively) with one male and two females in each. Tank 3 had a much lower rate (44 eggs), but only had a single male and female. The female in Tank 3 came in as a sub-adult and started her breeding cycle later in the program. Viability rates varied quite widely between the tanks (see Table).

The Hatchlings. Of the 287 eggs deposited during the 429 days of the program 27 (9.41 percent) resulted in live hatchlings. Only 10 (3.74 percent) of the hatchlings survived six months.

Records are available for 23 cases. Death by natural causes can be assigned to 5 (18.51 percent) of the juveniles. These animals continually lost condition from hatching and were found dead in the tank. Unnatural causes attributed to the loss of 12 (44.44 percent) juveniles. In all of these cases, the juvenile had jumped from the tank. Again 5 (18.51 percent) of the individuals that jumped from the tank had been continually losing weight, the other 3 (11.11 percent) appeared healthy. The remaining 4 (14.81 percent) pups died at birth, due to the aquarium water supply being contaminated from fresh water from heavy rain.

Growth rates show a gradual reduction in weight from birth (x=12.80 g, N=-23) for the first five weeks to a low of 11.64 g. After this low, the weights slowly increased (Figure 3). Increase in length was gradual, from a mean hatch length of 150 mm to 176 mm at 24 weeks (Figure 2).

Development within the Egg. At egg deposition, the yolk appeared a pale creamy green acellular mass, suspended in clear amniotic fluid. The yolk measure approximately 30 mm across and had no obvious outer membrane. The mass appeared to hold together due to differing densities of the yolk and amnion. The yolk mass constituted approximately 43 percent of the total egg mass by weight.

Within two hours of deposition, the embryo appeared as a small (1.5 mm) pale yellow spot, with a darker spot on the dorsal edge. Within two days, the embryo had developed to a stage that it has lifted free of the yolk surface. A network of veins had started to appear over the dorsal surface of the yolk, radiating from the embryo's attachment point. The majority of the veins appeared colorless, although some of the large veins started to take on a pinkish tinge. The embryo was 5-6 mm in length and was very active, flexing its body continuously. Closer examination revealed the gill slits starting to develop, the buccal cavity deeply invaginated and the hollow body cavity clearly visible. The orbit of the eye was quite distinct.

Over the next ten days the embryo grew to a length of approximately 12 mm. The buccal cavity had started to close around to form a distinct mouth. The cartilaginous skull was starting to be laid down and the nares were becoming apparent, ventral to the well developed orbit. The orbit had an apparent extension dorso-ventrally towards the position of the spiracle. The five gill slits were fully developed. A distinct thickening of material along the line of the spine was clearly evident, was the gradual development of faint muscle differentiation.

After approximately 21 days, when the embryo was 25 mm long and the vascular net had spread over the whole yolk surface. External filamentous gills had started to develop through the gill slits and the fins were apparent along the body profile. The heart was developed enough to be easily visible and the medullary swelling was clearly visible.

The eye started to develop over the next few days. The vascular ring in the orbit had appeared and the first sight o eye pigment occurred. The embryo was now large enough for the myotomes to be easily differentiated with the naked eye and were starting to develop a faint pink color. The external gill filaments were now 103 mm in length and the embryo was still in a constant state of movement.

By approximately day 28, the egg casing had split, allowing the influx of water. At this stage, the embryo had developed a distinctly pink color as the circulatory system continued its development. The external gill filaments had reached approximately 8-10 mm in length. Vascularization was more heavily concentrated around the eye, the brain, the snout, the gills, heart and the ventral blood vessel running the length of the body. Eye pigment had developed enough to see the future shape of the iris.

External developed appeared to slow over the next 14 days. The external gill filaments continued to grow, reaching a length of approximately 15 mm. The body developed a richer pink color. The vascular net was spreading through the gut region and developing more in the brain. The overall length increased only about 5 mm, to approximately 60 mm. The external gill filaments were resorbed completely by the time the embryo reached 85 mm in length.

After 60 days, the embryo measure between 188 and 122 mm. The circulatory system seemed to be fully developed. There are heavy concentrations of blood vessels in the snout, brain, surrounding the eye, gills and heart region. The gut wall had developed very distinct blood vessels as had the fins. Apart from the slightly swollen appearance of the gill region, the shark closely resembled the adult in shape. Coloration of the skin was starting to develop. The dark banding was starting to appear along the dorsal surface, accompanied by some very faint spots. The distinctive epaulette spot was not visible.

At 90 days, the shark was fully developed replica of the adult. The embryo's length was almost the same as the hatchling (95-98 percent). The yolk had reduced to approximately 12 percent of its original volume.

DISCUSSION

The Egg. Eggs appear to be laid in clusters of two to three throughout the year at 25 degrees C. Several eggs were laid in each tank within a few days, then there is a quiet period for two to four weeks before another group was laid in each tank within a few days, then there is a quiet period for two to four weeks before another group was laid. It appears that each female lays a pair of eggs at a time. At this point it is unknown how much time passes between mating and laying of eggs. An influx of fresh water into the open water system in the beginning of May caused an obvious upset in the regular patter of laying. The female in Tank 3 laid her first egg of 19 Feb. 1988. This egg was extremely small (60 mm long) and contained no yolk. Several more eggs were laid without any yolk until 3 March 1988. An egg laid on 4 March 1988 was successfully hatched on the 7 July 1988 but died seven weeks later.

A closer inspection of laying rates between the three tanks shows some marked differences (Table 1). Tank 3 with a single female laid 44 eggs and had a viability rate of 56.8 percent. This female was younger and had only started to lay. Tank 2 had a viability rate of 73.3 percent and Tank 1, 62.2 percent. On average, 34.1 percent of the eggs were empty. The empty cases were found to be crushed and bear what appeared to be teeth marks. the adult sharks seemed to be eating the eggs soon after deposition. Whitley (1967) reported it to be the male eating the eggs. Late in the study the male in Tank 1 was observed eating eggs.

Survival to hatching was also very different between tanks. Tank 3, with a single young female in her first year of breeding, had only one successful hatchling. This died after seven weeks. Tank 1 had seven hatchlings and Tank 2 had 15. We could find no definite reason for this large difference in the numbers of survivors. From observations during feeding, the male in Tank 2 was more active feeder and one of the females was a far more aggressive and active animal than any of the others in this program.

Overall, the mortality rates of the eggs varied with time. Contributing to the high mortality rates of the eggs varied with time. Contributing to the high mortality was the act that 34 percent of the eggs were empty. The next most common time of high mortality was the first few days after deposition. These eggs were more than likely infertile, with no mechanism for defense against fungal attack.

Another time period with high incidence of death occurs when the egg splits to allow the circulation of water. This ingress of water also carries with it fungi and if the embryo was dead a fungal infection quickly develops. Unfortunately the fungus develops inside the egg before being seen on the exterior. This makes it impossible to remove dying eggs to observe the embryo and its state of decay. Generally, the contents are a white milky fluid with a rather overpowering aroma.

The fresh water influx in May 1988 lowered the salinity of the tanks from 34 ppt to 15 ppt and accounted for the demise of eight of the eggs. The majority of these eggs were close to hatching. On opening the embryos, all appeared to have literally exploded inside the egg capsule, no doubt due to the change in osmotic potential. We cannot say how many of the other eggs in the hatching tank at the time were directly affected by the water to cause mortality.

The embryo developed inside the egg for an average of 130 days. This is almost twice the incubation time of 7-80 days reported for the tawny catshark, Chiloscyllium griseum Muller and Henle (Dral, 1981). We found that 11 of the embryos survived the influx of fresh water to hatching. However, only three of these hatchlings survived the full six months of the program. Several of the young were gross necropsied and no apparent cause of death was observed. Future autopsies should include full histological testing.

Growth. The mean length of the hatchlings at birth was 150 mm (Table 2, Figure 2) and the mean weight was 12 g (Table 3, Figure 3). Chiloscyllium griseum measures approximately 110 mm at birth (Dral, 1981). Length of H. ocellatum steadily increased over the first four weeks. Weight slowly decreased over the same period. At week five there was a bottoming out in weight loss and sudden negative length gain.

One possible reason for the slow drop in weight is the depletion of fat reserves laid down when receiving nourishment from the yolk. It was only after five weeks that they started to fully utilize their gut to digest the food being offered. Condition slowly improved over the next five months.

Unfortunately, there was some bias introduced into the measured weights by the feeding habits of the young sharks. Often when they were weighed they had obvious distention of the belly from a recent meal. The weights of the surviving animals were compared to the total population and it was found that there was little difference in the overall growth rates.

The apparent reduction in mean length during week 5 could possible be explained in two ways: 1) there was significant error in measuring involved; or 2) the apparent shrinkage of the young can be related to the corresponding reduction in weight. Shrinkage in juvenile teleosts from starvation has been reported (Blaxter, 1969).

From observations of epaulette sharks already on display, it appears that growth is quite slow. One sub-adult female was observed to grow from 310 mm to 340 mm over a six month period.

ACKNOWLEDGEMENTS

We acknowledge the assistance of Mr. Michael McGuiness, Mr. Paul Watts, Ms. Penny Wheeler (current staff members), Mr. Simon Garnett and Mr. John Hoey (past staff members) in setting up and gathering the data required. We are very grateful to Dr. Walter Ivantsoff (Macquarie University.) and Dr. John Paxton (Australian Museum) and Ms. Lesley Gidding for their review of and constructive suggestions for this paper.

REFERENCES

Blaxter, J. H. S. 1969. Development: eggs and larvae in fish physiology. vol. 3. Edited by W. S. Hoar and D. J. Randall. Academic Press, New York and London, pp. 177-252.

Compagno, L. J. V. 1984. FAO species catalogue. Vol. 4. Sharks of the world. An annotated and illustrated catalogue of sharks species known to date. Part 1 Hexanchiformes to Lamniformes. FAO Fish. Synop. (125) Vol. 4, Pt. 1:249 pp.

Dingerkus, G. and T. DeFino. 1983. A revision of Orectolobiform shark family Hemiscyllidae (Chondrichthyes, Selachii). Bulletin of the American Museum of Natural History, 176: 1-93.

Dral, A. J. 1981. Reproduction en aquarium du Requin de fond tropical Chiloscyllium griseum Mull. et Henle (Orectolobides). Rev. fr. Aquariol. 7:99-104. 83

Whitley, G. P. 1967. Sharks of the Australian Region. Australian Zoologist, 14:173-188.