Bangladesh J. Zool. 35(2): 193-203, 2007 STUDIES ON BREEDING AND LARVAL DEVELOPMENT OF RED BELLIED PIRANHA, PYGOCENTRUS NATTERERI KNER, 1858 IN BANGLADESH MD. MIZANUR RAHMAN AND ABU TWEB ABU AHMED Department of Zoology, University of Dhaka, Dhaka-1000, Bangladesh Abstract: The pond reared red bellied piranha, Pygocentrus nattereri Kner, 1858 were spawned through induced breeding from early June to early October 2005. August and September were the best periods for spawning. The red piranha was found to be multiple breeder, i.e. spawned 2-3 times throughout breeding season. It grew rapidly and attained sexual maturity in ponds, but did not breed naturally in confined water. The eggs and sperms were obtained just by stripping through induced spawning and then fertilization was done by artificial insemination. In every experiment of six replicate trials, the females were treated at first with at the rate of mg/kg body weight and at the second time with hormone at the rate of mg/kg of body weight after an interval of nine hours. The males were treated with ( mg/kg body weight) at the time of second injection of the female. The ovulation was recorded after 7-8 hours of the second dose of injection. The fecundity of various sizes of 20 P. nattereri was estimated by gravimetric method. The average total number of eggs laid by individual female (fecundity) was estimated to be 58,340 (34,280-96,000)/kg body weight. The fertilization rate was observed 58-67%. Hatching of the fertilized eggs occurred within 28±3 hours after fertilization at water temperature of 24-27 0 C. The percentage of hatching rate was observed 50-59%. A newly hatched larva was devoid of mouth, pectoral fins and pigment, and measured 8.5 mm in length. The yolk sac was completely absorbed after seven days of hatching. Seventy two hours old larvae started feeding. Twenty one days old larvae gained the shape of an original charachid fish. Key words: Red bellied piranha, Pygocentrus nattereri, breeding, larval development. INTRODUCTION The red bellied piranha, Pygocentrus nattereri Kner, 1858 (Characidae: Characiformes) also called red piranha, originally a resident of Amazon Basin in South America, is one of the exotic species in Bangladesh. It has been a common practice in most Asian countries including Bangladesh to introduce suitable species of fish from one country to another for increasing the overall production of food fishes, utilizing a vacant niche by appropriate species, controlling insect pests, and decorating aquarium by ornamental varieties. In Bangladesh, about 16 species of feed fish have been introduced to serve one or other purposes indicated above (Rahman 2005). Fish traders have introduced P. nattereri in Bangladesh for culture and marketing. They imported this fish in Bangladesh in 2001 from Thailand and China. A number of people is reportedly involved in the culture of the species in
194 Rahman and Ahmed Tongi areas in Dhaka, Trishal, Valuka in Mymensingh, Daudkandi in Comilla and Chandpur, Khulna and Satkhira areas (Rahman 2005). At the early stage, this species resembles Rupchanda and sold in market as Thai Rupchanda. There is a controversial concept among fishery scientists, fish traders, fish farmers and common people about the commercial production and impact of piranha culture in Bangladesh. To overcome the controversial concept and problem of piranha culture it is important to know the vital biological information of this fish species. An understanding of the reproductive mechanism and breeding pattern of fishes is a basic requirement for the successful management of fisheries resources of a country. Importance of the breeding biology of any fish is essential for evaluating the commercial potentialities of its stocks culture practices and actual management of the fishery (Lagler et al. 1962). While studying the breeding potential of a population one of the basic exigencies is to designate the individual of the population in respect to their gonodal conditions (Jhingran 1985). Information on piranha breeding is relatively scarce, because only a few species have been bred in captivity. P. nattereri and Serrsalmatus spilopleura, and only very recently (2002) P. cariba have reproduced in home aquaria, and some other species, like S. rhombeus in large public aquaria (Bennet et al. 1997). Among these species P. nattereri, has been bred most extensively in captivity. The present paper deals with the breeding biology and larval development of P. nattereri. MATERIALS AND METHODS The experiments were conducted during the natural breeding season of P. nattereri from June to October 2005 at the Khaza Matshya Hatchery, Valuka, Mymensingh. The matured male and female fishes were collected from the brood- rearing ponds and were transferred to the cistern (5 m 2 m 1 m) of the hatchery and acclimatized for at least six hours before being used for the experiment. The brood rearing ponds were 0.4 hectare in size and water depth was maintained between 1.8 and 2.2 metres. Supplementary feeding with mustard oil cake and rice bran at the ratio of 1:1 by 4% body weight was provided once a day and inorganic fertilizer, Urea and TSP at the ratio of 2:1 in each fortnight. Male and female brood fishes were identified by their external characteristics, such as swollen light vent, bulging soft abdominal region and smooth pectoral fin of female, and oozing of milt and rough pectoral fin of the male, which are the major criteria for the selection of the female and male breeders (Hussain and Mazid 2001). Both sexes of the fish were of 3-4 years old and
Studies on breeding and larval development of red bellied piranha 195 the weights of male and female breeders were 4-5 kg and 4.2-5 kg, respectively. The length of the male and female breeders varied from 45 to 60 cm and from 50 to 60 cm, respectively. The experiment of induced spawning was designed with six replicate trials throughout the study period. dose for the female consisted of 217 IU ( mg) of suspension per kg body weight of the fish. dose consisted of mg/kg body weight of' the female. The second dose was given after nine hours of the first injection. The males were given single dose at the time of the second injection of the female which was mg/kg body weight of the fishes. The injections were given intra-peritoneally and in few instances, intramuscularly at the soft region at the base of the pectoral fin by a hypodermic syringe fitted with needle. After injection, the spawners were returned to the tank. The males and females in the ratio 1:3 were kept in the same tank. After 6-8 hours, both the males and females were taken out from the tank for stripping. Eggs and milt were collected by stripping method. Fertilization was done by mixing of the eggs and milts (sperms), which were gently stirred with a clean feather to hasten the fertilization of the eggs. The sperms were allowed to remain with the eggs for 5-10 minutes, and then the excess sperms were removed by 3-4 consecutive washes with saline (0.36%) water. The transparent eggs were considered as fertilized ones whereas the opaque eggs were considered as dead eggs. The fertilization rate was calculated by the following formula: Number of fertilized eggs Fertilization rate = 100 Number of total eggs The fertilized eggs were then transferred into a hatching jar, in which an ambient water temperature and dissolved oxygen was maintained through continuous aeration. After 26-28 hours of fertilization, hatchlings started to come out from the egg shell and hatching was completed within another six hours. The rate of hatching was calculated by the following formula: Number of hatchling Hatching rate = 100 Number of total eggs For the estimation of fecundity, 20 ripe females of red piranha were randomly sampled. In the present study, the fecundity of the various sizes of P. nattereri was estimated by the gravimetric method. The fecundity of 20 specimens was computed by the counting of ova in 20 samples of one gram
196 Rahman and Ahmed from each ovary following the method of Sevaraj et al. (1972). The developmental stages of the larvae had been observed at timely interval after hatching until they attained 30 days old larval stages. All the morphometric measurements of the larvae were made on freshly prepared specimens, following the method of McEdward (1984) with slight modification, but the measurements of the different larval stages were done by averaging the data of 10 specimens for each stage. RESULTS AND DISCUSSION It was observed that the pond-reared P. nattereri spawned through induced breeding from the early June to early October when water temperature reaches 24-27 ºC. August and September were recorded as the best period of spawning. Bennet et al. (1997) reported that P. nattereri readily spawned under artificial condition during rainy season from April to May when females are most fertile. Nico and Taphorn (1986) reported that piranhas have a defined, although prolonged, spawning season beginning at the onset of floods. In the present study, all brood fishes were more than three years old. It was found that the fish below three years old were not able to breed in captive condition, but piranha reaches sexual maturity when they are between 18 and 24 months old in natural condition (Merona 1984). During breeding time, it was observed that the fishes (3.6-5.0 kg) spawned 2-3 times at the interval of 10 to 15 days. It was also observed that relatively old fishes spawned within early August to mid August for the first time and then in early September for second time. It was observed that the ripe females and male fishes did not spawn spontaneously so natural fertilization did not occur in pond environment. For this reason mature eggs and sperms were obtained just by stripping through induced spawning and then fertilization was done by artificial insemination. Induced spawning was preformed with and (prepared dose) for the first and second injected dose, respectively. The particulars of the induced spawning are presented in Table 1. The weight of the female breeders varied from 3.6 to 5.0 kg and the weight of the male breeders was 4.0-4.6 kg. The dose to the male breeders and the second doses to the females were applied after nine hours of the first dose given to them. Proper stripping time was found after 7-8 hours of the second dose of injection. Bardach et al. (1972) cited that the accepted dosage of for carp ranges from 700 to 1000 IU/kg of spawners. The present study reveals that comparatively lower amount of hormones are used for the induced breeding of
Studies on breeding and larval development of red bellied piranha 197 P. nattereri. The amount of hormone was found to be about the half of those, which were used in carp fishes. The proper stripping time of P. nattereri was found to be after 7-8 hours after the second dose of injection. Konardt (1968) observed that the time required for ovulation of carp varies from 8 to 13 hours after the final injection depending upon temperature. In the present experiment the time required for stripping was less than that of Konardt (1968). Table 1. Particulars of induced spawning experiment of P. nattereri using and hormones at Khaza Matshya Hatchery, Valuka, Mymensingh. No. of experiment No. of injection Date Time (hr) Weight of breeders (kg) Hormone Doses of injection (mg/kg) Time of ovulati on (hr) Male Female Male Female 1 05.08.2005 05.08.2005 15.00 24.00 5.0 8 2 15.08.2005 16.08.2005 16.30 02.35 4.5 4.5 8 3 20.08.2005 21.08.2005 17.00 02.10 5.0 4 05.09.2005 06.09.2005 15.00 24.00 3.6 8 5 14.09.2005 15.09.2005 14.00 23.20 4.6 4.2 8 6 29.09.2005 29.09.2005 17.00 02.05 5.0 8 The fecundity of 20 female piranha under different body sizes as well as per kg body weight has been shown in Table 2. The minimum estimated number of ova was 1,20,000 from a fish of 46 cm total length and 3.5 kg body weight. The maximum estimated number of ova recorded was 4,80,000 from a fish of 61 cm total length and 5.0 kg of body weight. The fecundity of P. nattereri was estimated to be 58340 (range: 34280-96,000) per kg body weight, while the same for catla, rohu, mrigal and common carp was 150,000-240,000, 112,000-3,03,000, 50,000-250,000 and 90,000-140,000, respectively. Fertilization rate varied from 58 to 67% with an average of 63% in six replicate trials. Hatching rate was not the same in all the trials which varied from 50 to 59% with an average of 55%. The rate of fertilization and hatching of P. nattereri were reported to range from 62 to 72% and 55 to 65%, respectively (Bennet et al. 1997). So, in the present experiment the rate of fertilization and hatching were nearer to that of Bennet et al. (1997).
198 Rahman and Ahmed Table 2. The length, weight and fecundity of female Pygocentrus nattereri. Individual number Body length (cm) Body weight (kg) Total amount of egg (g) Total fecundity (in thousand) Fecundity per kg body weight 1 60 5.0 450 360 72000 2 56 4.5 300 240 53330 3 61 5.0 600 480 96000 4 51 4.0 200 160 40000 5 51 4.0 220 176 44000 6 61 5.0 600 480 96000 7 56 4.5 280 224 49770 8 54 4.0 200 160 40000 9 46 3.5 150 120 34280 10 56 4.0 250 200 50000 11 52 4.5 300 240 53330 12 55 4.5 300 240 53330 13 60 5.0 500 540 80000 14 61 5.0 580 464 92800 15 56 4.5 320 256 56888 16 48 3.5 200 160 45710 17 50 4.0 220 176 44000 18 54 4.0 200 160 40000 19 56 4.5 300 240 53330 20 60 5.0 450 360 72000 Mean fecundity per kg body weight 58340 (34280-96000) Larval Development: The larval development of P. nattereri was observed at 24-27ºC water temperature. The embryo advanced in development, the movement became more and more vigorous. With further twisting movements, the embryo was able to release out from the surrounding membranes. This stage was observed just before hatching and time required for this was 28±3 hours from fertilization. Incubation period varied from 25 to 31 hours. Newly hatched larvae (Fig. 1A): Newly hatched larvae were slender and transparent with no chromatophore. Yolk sac had an anterior bulbous and posterior narrow portion. Mouth was not formed in this stage. Eyes were prominent without pigmentation. The mean length of the newly hatched larvae was mm (7.35-8.60 mm). Six hours old larvae (Fig. 1B): The embryonic fin fold developed in this stage. Yolk sac had a slight dorsal depression just behind the region of its maximum width. Twenty five to twenty eight pre-anal and ten to sixteen post-anal myotomes were present. The posterior margin of the caudal fin was uniformly convex. The average length of the larvae was 8.4 mm (8.2-9.6 mm).
Studies on breeding and larval development of red bellied piranha 199 Twelve hours old larvae (Fig. 1C): Pectoral fin was rudiment and faintly visible. A black spot was seen in the eye which indicated the commencement of pigmentation. Yolk sac reduced in height especially the bulbous portion became elongated. Caudal fin rays were slightly visible. The average length of the larvae measured 8.8 mm (8.7-9.9 mm) Fig. 1. Stages of larval development in P. nattereri. A. Newly hatched; B. 6 hours old; C. 12 hours old; D. 24 hours old. Twenty four hours old larvae (Fig. 1D): Mouth was clearly visible and the position of the mouth was indicated by a slit. Pectoral fin was conspicuous without fin rays. Eyes were more pigmented. Anal depression was well marked. The average length of the larvae was 9.10 mm (8.95-10.15 mm).
200 Rahman and Ahmed Thirty six hours old larvae (Fig. 2E): Pectoral fin well developed but with slightly visible rays. Upper and lower jaws were clearly visible. Anal fin was slightly visible. A few black chromatophores observed on the anterior myotomes and on the dorsal side of the yolk sac up to the tip of notochord and a few on the head. Air bladder was observed which was elliptical in shape. Blood circulation was observed. The average length of the larvae was 9.2 mm (8.98-10.3 mm). Fig. 2. Stages of larval development in P. nattereri. E. 36 hours old, F. 48 hours old, G. 72 hours old, H. 7 days old. Forty eight hours old larvae (Fig. 2F): The average length of the larvae measured 10.5 mm (10.20-10.68 mm). Gill arches were clearly seen and covered by an operculum. Alimentary canal was straight. The ovate region
Studies on breeding and larval development of red bellied piranha 201 of yolk had a dorsal depression above which the air bladder was located. A few chromatophores appeared on the body. Yolk sac very much reduced and thin. Mouth well developed with thick lips. Seventy two hours old larvae (Fig. 2G): Eyes were prominent with black centres. Yolk sac was still discernible below the embryo. The size of air bladder was elliptical. The average length of the larvae was 10.8 mm (9.98-11.35 mm). A few black chromatophores seen along the body. The tip of notochord was sharply upturned. The first indication of forked caudal fin appeared on the body. Alimentary canal was well developed. In this stage the larvae started feeding. Seven days old larvae (Fig. 2H): The average length of the larvae measured 15.8 mm (13.98-17.35 mm). Yolk sac was almost absorbed. Head was separated from the yolk sac. The well indication of dorsal and caudal fin was observed. Mouth was terminal with slightly extended lower lip. Pelvic fin started to develop. Fourteen days old larvae (Fig. 3I): Vertebral spines appeared at the posterior side of the body. Anal fin found to develop rapidly. Eighteen fin rays were in dorsal and anal fins. The larvae started to take small copepod. The average length of the larvae was 20.2 mm (18.98-22.3 mm). Homocercal caudal fins were observed. Twenty one days old larvae (Fig. 3J): In this stage the shape of the larvae was like a characid fish. Minute scales were found at the dorsal side of the body. Twelve to fifteen fin rays were found in pectoral fin. Scattered light black spots were present on the body. The average length of the larvae was 24.8 mm (23.98-26.35 mm). One month old larvae (Fig. 3K): The average length of the larvae was 32.2 mm (30.98-34.3 mm). The ventral side of the body was slightly yellowreddish in colour. Scales were observed all over the body. Comparatively eyes were large. The base of pectoral, pelvic and anal fins was found slightly reddish in colour. As observed earlier by Khan (1943), the newly hatched embryos of carp were seen to be without a mouth, eyes, gill or gill clefts. The embryos of catla (Catla catla) and rohu (Labeo rohita) were similarly under developed without any pigment and almost colourless (Chakrabarti and Murthy 1972). But in the present study, the newly hatched larvae of P. nattereri were slender and eyes were prominent. Chakrabarti and Murthy (1972) observed 26 pre-anal and 12 post-anal myotomes in catla, 26 pre-anal and 14 post-anal myotomes in rohu, 28 pre-anal and 14 post-anal myotomes in mrigal, during the larval stages. More
202 Rahman and Ahmed or less similar phenomenon was observed during the present study, i.e. the larvae of red piranha experienced with 25 to 28 pre-anal and 11 to 16 postanal myotomes. The first feeding time of larvae was observed after 72 hours of hatching which is similar to the larvae of carp fishes. Fig. 3. Stages of larval development in P. nattereri. I. 14 days old, J. 21 days old, K. 30 days old. The success of breeding promises that it is possible to produce stocking materials (seeds) under farm condition. This success is enhancing mass seed
Studies on breeding and larval development of red bellied piranha 203 production and culture practice of this fish. As a result, a number of people have engaged themselves commercially in the culture of this species. It is hoped that this paper will provide necessary information to the fish culturist for the management of this fish and those who are interested in conducting further research on this exotic fish. LITERATURE CITED BARDACH, J.E., RYTHER, J.H. and MCLARNEY, W.O. 1972. Aquaculture- The Farming and Husbandry of Freshwater Martine Organisms. Wiley-Interscience, Sydney-Toronto. 868 pp. BENNET, W.A., CURRIE, R.J. and WAGNER, P.F. 1997. Cold tolerance and potential overwintering of the red-bellied pirhana Pygocentrus nattereri in the United States. Trans. Am. Fish. Soc. 126: 841-849. CHAKRABARTY, R.D. and MURTHY, D.S. 1972. Life history of Indian Major carps Cirrhinus mrigala (Ham.), Catla catla (Ham.) and Labeo rohita (Ham.). J. Inland fish. Soc. India 4: 132-161. HUSSAIN, M.G. and MAZID, M.A. 2001. Genetic Improvement and conservation of carp species in Bangladesh. Bangladesh Fisheris Research Institute, Mymenshing and International Center for Living Aquatic Resources Management, Panang, Malaysia. JHINGRAN, V.G. 1985. Fish and Fisheries of India. Hindustan Publishing Corporation, Delhi. 399 pp KHAN, H. 1943. On the breeding habits and development of an Indian carp, Cirrhina mrigala (Hamilton). Proc. Indian Acad. Sci. 19(1): 1-13. KONARDT, A.G. 1968. Methods of breeding of the Grass Carp, Ctenopharyngodon idella (Val.) and Silver carp, Hypophthalmichthys molitrix (Val.). FAO Fish Report 44(4): 195-204. LAGLER, K.F., BARDARCH, J.E. and MILLER, R.R. 1962. Ichthyology. John Wiley and Sons, Inc., New York. 32p. McEDWARD, L.R. 1984. Morphometric and metabolic analysis of the growth and form of an echinopluteus. J. Exp. Mar. Biol. Ecol. 82: 259-287. MERONA, B. and BITTENCOURT, M.M. 1984. Inter-annual variability of Red Piranha abundance (Pygocentrus nattereri Kner, 1858). In: International Symposium Biology of Tropical Fishes. Manaus, Amazonas. p. 23. NICO, L.G. and TAPHORN, D.C. 1986. Nutrient content of piranha (Characidae, Serrasalminae). Trop. Fish Hobb. 34: 24-57. RAHMAN, A.K.A. 2005. Freshwater Fishes of Bangladesh, 2 nd ed. Zool. Soci. Bangladesh, Dhaka, Bangladesh. 476 pp. SEVARAJ, C., RADHAKRISHNAN, S. and PARAMESWARAN, S. 1972. Notes on the breeding season, fecundity and life history of a minor carp Labeo boggut (Sykes). Central Inland Fisheries Research Institute, Barrckpore 4: 87-97.