Embryonic and larval development of critically endangered riverine catfish Rita rita

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1 Eurasia J Biosci 5, (2011) DOI: /ejobios Embryonic and larval development of critically endangered riverine catfish Rita rita Research Note Muhammad Fazlul Awal Mollah 1, Khanam Taslima 1*, Harunur Rashid 2, Zakir Hossain 1, Muhammad Nasif Sarowar 1, Muhammad Rakibul Kabir Khan 3 1Fisheries Biology and Genetics Department, Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh, Bangladesh 2Fisheries Management Department, Faculty of Fisheries, Bangladesh Agricultural University, Mymensingh, Bangladesh 3Surgery and Obstetrics Department, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh, Bangladesh *Corresponding Author: taslima_bau@yahoo.com Abstract The present study was carried out to investigate the embryonic and larval development of freshwater catfish Rita rita. The mature eggs and sperms were collected by using artificial insemination technique and fertilized eggs were incubated in mini circular hatchery with provision of continuous water supply. The fertilized eggs were transparent, demarsal, spherical, non-adhesive and brownish in colour with a diameter ranging between 1.3 to 1.6 mm. First cleavage occurred within min post-fertilization at temperature of 28±1 C. Hatching started 22 h post-fertilization and completed within 24 h at the same temperature range. Newly hatched larvae were 2.0 mm in length devoid of mouth and pigmentation and started feeding within h post-hatching. To date, this is the first time the early embryonic and larval development of freshwater catfish R. rita is described. Thus the findings of the present study provide valuable information that may help establishing the large scale seed production technique of Rita. Keywords: Embryo development, larva, metamorphosis, Rita rita. Mollah MFA, Taslima K, Rashid H, Hossain Z, Sarowar MN, Khan MRK (2011) Embryonic and larval development of critically endangered riverine catfish Rita rita. Eurasia J Biosci 5: DOI: /ejobios INTRODUCTION Bangladesh is a land of high potential water resources where inhabits 260 freshwater fish species, 24 freshwater prawns, 475 marine fish species, 36 marine and brackish water shrimps and 16 exotic species which supplying around 58% of animal protein (Anonymous 2010). At least 55 species of freshwater catfishes belonging to 35 genera have been recorded in Bangladesh (Rahman 2005). Rita rita is one of them popularly known as Rita in Bangladesh and also in India. The fish being greenish brown above, brownish white below with an extremely slimy body which prefers muddy to clean water as their habitat. 54 fish species of inland water of Bangladesh are considered as either in endangered or critically endangered situation. Rita rita (Hamilton, 1822) is categorized as critically endangered fish (Anonymous 2000). Once the abundance of this fish was very rich in Afghanistan, Pakistan, India, Nepal, Bangladesh and Mayanmar (Tripathi 1996). But the availability of this fish is decreasing day by day due to the ecological changes of the breeding ground. As a result breeding grounds are losing their suitability to be used by the species posing a great threat of extinction and till now it is not possible to collect the fry of this fish from the river system where they breed naturally. Although Rita is a tasty, commercially important, critically endangered catfish of Indian subcontinent, published reports on its induced breeding and rearing are quite scanty and no reports are readily available on the Received: July 2011 Accepted: October 2011 Printed: November 2011 EurAsian Journal of BioSciences,

2 developmental biology and larvae rearing technique of this species. Earlier attempts have been made to study its length-weight (Lal and Dwevedi 1969), age and growth (Devi et al. 1990), environmental impact assessment (Mukhopadhyay 1994), morphometric characters (Devi et al. 1991), food (Devi et al. 1992), maturity and fecundity (Saxena 1972), structure of the skin (Mital 1968) and reproductive biology of R. rita (Amin et al. 2008). Recent success in induction of breeding of R. rita was performed by using PG ( 2008). Considering the enormous importance of Rita fish, early life history information is an essential requirement for optimization of large scale seed production, culture and management. The present work is the first ever preliminary attempt and is expected to serve as a basis for further and more intensive future studies. An attempt was made to conduct this study to investigate and to provide the detailed information of the embryonic and larval development of critically endangered fish (R. rita). This experimental works were carried out in the Mini Hatchery cum Breeding Complex and the laboratory of the Department of Fisheries Biology and Genetics under the Faculty of Fisheries, Bangladesh Agricultural University (BAU) Mymensingh. The collected mature broods of Rita rita (Hamilton, 1822) were induced to spawn through hypophysation previously described by Mollah et al. (2008). After that, the eggs were fertilized by the normal milt. For collection of milt, the testes from male were dissected out from its body cavity and were crushed by scissor. Fertilized eggs were washed several times with sterile distilled water and finally transferred to and spread as homogeneously as possible in mini circular hatchery (50 L capacity) with continuous water supply throughout the incubation phase. A set of fertilized eggs were collected very carefully from the mini plastic circular hatchery where they were in constant motion by using a 111 MATERIAL AND METHODS dropper and siphoning process. After that, it was observed under the microscope (Olympus CX41) fitted with a software (Magnus MIPS- Microsoft Image Processing System). Each sample was observed four times to identify the developmental stages (Haniffa et al. 2003). Fertilized eggs were collected up to morula stage every 15 min and then every one hour interval until hatching and every four hours for the next. The hatching of fertilized eggs was completed within 24 h. Formalin (5%, v/v) was used for the immobilization of the larvae during study periods. The diameters of the eggs were measured by using an eye piece micrometer. The size of the larvae was measured by placing on the petri dish placed on a 1 mm graph paper (Miah et al. 2009). The whole experimental works were done at a temperature of 28±1 C. RESULTS Embryonic development The embryonic period starts when the egg is fertilized by a sperm and ends when the embryo has attained the generalized organ systems as they appear in common in all the fish (Table 1). Unfertilized and fertilized eggs The unfertilized eggs of R. rita were opaque, demarsal, spherical and whitish in colour measuring 1.0 to 1.3 mm in diameter (Fig. 1a). While the fertilized eggs were transparent, demarsal, spherical, nonadhesive and brownish colour (Fig. 1b). Immediately after fertilization the diameter of the egg increased owing to slight swelling of the egg which ranged between 1.3 to 1.6 mm. Fertilized eggs had a reddish spot (blastodisc) and readily recognizable with the naked eye (Fig. 1c). Formation of embryo The first cleavage that divided the blastodisc into two blastomeres was occurred within 25 to 30 min of post-fertilization (Fig. 1d). The cleavage was typically meroblastic. While the second and third cleavage (4 and 8 cell stage) was appeared for 60 min and 90 min after fertilization respectively (Fig. 1e and EurAsian Journal of BioSciences, 2011

3 Table 1. Brief descriptions of the embryonic developmental stages of R. rita. 1f), the 16 and 32 cell-stages were observed between 130 min and 160 min postfertilization, respectively (Fig. 1g and 1h). Gradually the successive cleavages were found, but the blastomeres were decreased in size. The morula stage was completed within the duration of 2.75 to 3.30 h after fertilization (Fig. 1i and 1j). During 4.0 to 4.3 h of post-fertilization, the top of the blastoderm started spreading over the yolk sphere formed a thin layer and the embryo reached the blastula stage (Fig. 1k and 1l). After 6.0 h of fertilization, the blastoderm flattened down onto the yolk-sphere. The outer surface followed the curvature of the yolk sphere resulted a dome shape structure (Fig. 1m). The blastoderm began to expand (30% epiboly, about 1/4 of the yolk sphere) over the upper surface of the yolk sphere during 7.30 h of post fertilization (Fig.1n). Within 9.30 h of post-fertilization, more than half a portion of the yolk (50% epiboly, Fig.1o) was invaded by the embryonic shield reached the late gastrula stage (Fig.1p). Then after h of fertilization, the head (rudimentary brain) was made anteriorly and the rudimentary tail was created posteriorly in the distinct embryonic body (Fig.1q). During EurAsian Journal of BioSciences,

4 13.30 h post-fertilization, some of the mesodermal cells divided the body of the embryo in a number of somites and also visibly showed optic cups. Antero-posterior end was distinguished, the cephalic portion became broader and anterior end formed the head fold, and posterior end formed the tail fold (Fig.1r). After that, h old embryo occupied more than three fourths of the egg peripherical space and mesodermal somites increased in number and became more distinct and the pigmentation was developed in the somites area. The tubular heart was appeared underneath the head and the heart actively started the blood circulation (Fig.1s). Before hatching (Fig.1t), the embryo started twisting movement inside the egg and continuously beat the egg shell by the caudal region especially around the middle part of the body. This movement gradually became vigorous and the egg capsules were weakened. By rupturing the egg capsule, the middle part of the embryo gradually disconnected from the egg capsule. The egg membrane was broken down from the caudal region and the larva firstly emerged with its tail portion. At least 1-2 h was required for complete emerging of the hatchling because the entire embryo did not hatch out at a time. Larval development A larva (Latin; plural larvae) is a young form of animal with indirect development going through or undergoing metamorphosis (for example, insects, amphibians or cnidarians). The larva looks completely different from the adult form (Table 2). Hatchling Newly hatched larvae were straight, slender, transparent, laterally compressed body and gradually tapering towards the tail. The larvae were brownish-colour and 2.0 mm in length. The hatchlings had unpigmented eyes and devoid of distinct mouth and fins. The head was very small and the yolk sac was initially oval-shape with pale-brownish colour, while numerous small chromatophores at the dorsal region gave it a dark appearance. The larvae could not swim and floated passively on the water and irregularly appeared upside 113 down movement. The numbers of body somites ranged between 40 and 46. The whole hatching experimental works were done at temperature of 28±1 C (Fig. 2a). 8 h old larva Average length of the larvae was found about 2.1 mm and brownish in colour. Mouth was not yet developed but only a conspicuous depression identified the position of mouth. A depression at the posterior end of the yolk sac was visible which was identified as anal pore. Heart became more distinct. Circulation of body fluid was continued and barbels had not yet developed (Fig. 2b). 16 h old larva Sixteen hours after hatching, the larvae were found of 2.2 mm in length. At this stage, yolk sac was partially reduced and gradually became elongated. Heart and brain were clearly distinct. A tube like structure within the body was represented the alimentary canal (Fig. 2c). 24 h old larva One day old larva with reduced yolk sac was approximately 2.5 mm length. Eye spot with a dark pigmented area was appeared on the anterior part of the head. Barbels were found in the forms of tiny knobs. Pectoral fin buds were seen. Mouth was not opened and the anal opening was still closed. Pigmentation was gradually extended all over the body and the blood circulation system was fully developed (Fig. 2d). 36 h old larva The average length of 36 h old larva was 2.8 mm in length. Dark pigmented eyes with spherical shape were visible. Distinct heart was visible and functioned actively. Reddish blood was seen around the heart region. Fully formed upper and lower jaws were visible and gills were covered by the operculum. Mouth was formed as an opening and the anal pore was also opened and caudal fin bud was formed. Vigorous movements of the larvae were seen (Fig. 2e). 48 h old larva Two days old larva was 3.0 mm in length and partial yolk sac was presented. Dorsal region became dark due to the formation of numerous chromatophores and the EurAsian Journal of BioSciences, 2011

5 Fig. 1. Embryonic developmental stages of R. rita. (a) unfertilized egg, (b) fertilized egg, (c) blastodisc and (d) 2 cell stage, (e) 4 cell stage, (f) 8 cell stage, (g) 16 cell stage (h) 32 cell stage (i) early morula stage and (j) late morula stage, (k) early blastula stage, (l) late blastula stage, (m) pre- early gastrula stage (n) 30% epiboly (o) mid gastrula stage and (p) late gastrula stage, (q) head and tail bud formation, (r) somite stage, (s) h old embryo and (t) just before hatching. (EM- Egg membrane, VM- Vitelline membrane, PS- Perivitelline space, Y- Yolk, BD- Blastoderm, BM- Blastomere, AP- Animal pole, EVL- Enveloping layer, VP- Vegetal pole, YSL- Yolk syncytial layer, DBL- Dorsal blastoporal lip, VBL- Ventral blastoporal lip, H- Head portion, T- Tail portion, YS- Yolk sac) EurAsian Journal of BioSciences,

6 Table 2. Summary of the larval developmental stages of R. rita. transparent ventral region was visible. Pectoral fin folds became distinct and the rudimentary rays developed in the caudal fin. Barbels became elongated and mouth was well formed. The alimentary tract became short, straight and distinct and a small pouch like stomach was formed. Blood circulation was well observed in the head, heart and tail region. At the end of this stage, yolk sac was absorbed to half size and the larvae were found smoothly swimming and feeding exogenously (Fig. 2f). R. rita is a critically endangered fish species and their culture technique in controlled environment is not practiced. To protect this species and develop their cultural technique, it is essential to assemble information about embryonic and larval development of this critically endangered fish. But there are no reports about the embryonic and larval development of this species. Therefore, the aim of the present study potentially investigated and provided detailed information about the embryonic and larval development of R. rita species. The eggs of R. rita reached rapidly in their embryonic and larval stages. The fertilized eggs of Rita are non-adhesive but adhesiveness of eggs is the special character found in other catfishes such as Claries gariepinus (Khan and Mollah 1998, Osman et al. 2008), C. batrachus (Bruton 1979), 115 DISCUSSION Mystus montanus (Arockiaraj et al. 2003) and Pangasius sutchi (Islam 2005). The diameter of the fertilized eggs ranged between 1.0 and 1.3 mm. In similar study, some researchers showed different egg size of C. gariepinus (Osman et al. 2008). These differences might be attributed to the species variation and brood size (Puvaneswari et al. 2009). First cleavage of eggs of Rita was observed within min post-fertilization at the water temperature of C, but a discrepancy of time was observed in case of C. gariepinus, Mystus cavasius which was within min post-fertilization reported from Khan and Mollah (1998) and Rahman et al. (2004) at 28.5 and 26 C respectively. This variation might be due to species difference and other environmental factors. Just 1-2 h before hatching, the embryo of R. rita showed twisting movements inside the egg capsule which was also found in other fishes reported by Puvaneswari et al. (2009), Khan and Mollah (1998) and Osman et al. (2008). In the present study, hatching began after 22 h post-fertilization and completed within 24 h at C which is similar to other findings reported from Puvaneswari et al. (2009). Another report from Mollah and Tan (1982) showed that in Clarias macrocephalus, incubation period was 22 h at 30 C and 34 h at 25 C but no hatching was observed at 20 C. Time requirement of egg hatching was inversely related to their incubation temperature (Rana 1990). Length of the newly hatched larvae of this species was around 2.0 mm EurAsian Journal of BioSciences, 2011

7 Fig. 2. Larval developmental stages of R. rita. (a) newly hatched larva, (b) 8 h old larva, (c) 16 h old larva and (d) 24 h old larva, (e) 36 h old larva and (f) 48 h old larva. (M- Myomere, PFB- Pectoral fin bud, E- Eye, YS- Yolk sac, UJ- Upper jaw, LJ- Lower jaw, CFB- Caudal fin bud, B- Barbel, MO- Mouth opening, V- Vertebra) which is in the ranges of the findings of Khan and Mollah (1998). In the present study, larvae were started to feed at 48 h posthatching which is similar to other previously reported studies stated by Puvaneswari et al. (2009). R. rita was selected for this study because it is already declared as critically endangered fish, so an attempt should be taken to protect this species from being extinct. Unfortunately there is no remarkable information on the embryonic and larval development of this fish, but this unique research work will act as base line for further study. ACKNOWLEDGEMENTS The author takes the privilege to acknowledge BARC (Bangladesh Agricultural Research Council) authorities for providing financial assistance to conduct this research work. REFERENCES Amin MR, Mollah MFA, Nasrin S, Sayeed, MA (2008). Some aspects of the reproductive biology of Rita rita (Hamilton). Journal of Bangladesh Agricultural University 6(1): EurAsian Journal of BioSciences,

8 Anonymous (2000) International Union for Conservation of Nature, Bangladesh. Bangladesher Bipanno Prani, IUCN Bangladesh, The World Conservation Union, Dakka. Anonymous (2010) Department of Fisheries, Bangladesh, Ministry of Fisheries and Livestock, Dakka. Arockiaraj AJ, Haniffa MA, Seetharaman S, Singh SP (2003) Early development of a threatened freshwater catfish (Mystus montanus). Acta Zoologica Taiwanica 14(1): Bruton MN (1979) The breeding and early development of Clarias gariepinus (Pisces: Clariidae) in lake Sibaya South Africa, with a review of breeding in species of the subgenus Clarias (Clarias). Trans Zoological Society of London 35: Devi NT, Khumar F, Siddiqui MS (1991) Observations on the morphometric characters of the catfish Rita rita (Ham.) of the river Yamuna. Journal of Inland Fisheries Society of India 23(1): Devi NT, Siddiqui MS, Anwar S (1990) The age and growth of catfish Rita rita (Ham.) from the river Yamuna in north India. Journal of Indian Fisheries Association 20: Devi NT, Anwar S, Siddiqui MS (1992) On the food of the catfish Rita rita (Ham.) from the river Yamuna in north India. Journal of Inland Fisheries Society of India 24(1): Hanifa MA, Nagarajan M, Marimuthu K, Jesu Arockiaraj A (2003) Embryonic and larval development of spotted murrel, Channa punctatus (Bloch). Indian Journal of Fisheries 50: Islam A (2005) Embryonic and larval development of Thai pangas (Pangasius sutchi). Development, Growth and Differentiation 47: 1-6. Khan MMR, Mollah MFA (1998) Embryonic and larval development of African catfish, Clarias gariepinus (Burchell). Bangladesh Journal of Fisheries 21(1): Lal MS, Dwevedi AS (1969). Studies on the Fishery and Biology of a freshwater Teleost, Rita rita. 1. Racial studies. Indian Journal of Zoology 9(2): Miah MI, Harun MS, Rahman MM, Haque MR, Hossain MA (2009) Study on the embryonic and larval development of an endangered species of bata (Labeo bata). International Journal of Sustainable Crop Production 4(1): Mital AK 1968). Studies on the structure of the skin of Rita rita (Ham.) (Bagridae, Pisces) in relation to its age and regional variations. Indian Journal of Zoology 9(2): Mollah MFA, Amin MR, Sarowar MN, Muhammadullah (2008) Induceed breeding of the riverine catfish Rita rita. Journal of Bangladesh Agricultural University 6(2): Mollah MFA, Tan ESP (1982) Effects of incubation temperature on the hatching of catfish (Clarias macrocephalus) eggs with an illustration on the larval stages. Malayan Nature Journal 36: Mukhopadhyay MK, Vass KK, Mitra K, Bagchi MM, Biswas DK (1994). Environmental impact assessment on the test fish Rita rita in the river Ganga in west Bengal. Journal of Inland Fisheries Society 26(1): Osman AGM, Wiertz S, Mekkawy IA,Verreth J, Kirschbaum F (2008) Early development of the African catfish Clarias gariepinus focusing on the ontogeny of selected organs. Journal of Applied Ichthyology 24: Puvaneswari S, Marimuthu K, Karuppasamy R, Haniffa MA (2009) Early embryonic and larval development of Indian catfish, Heteropneustes fossilis. EurAsian Journal of BioSciences 3: EurAsian Journal of BioSciences, 2011

9 Rahman AKA (2005) Freshwater fishes of Bangladesh. Zoological Society of Bangladesh, Dhaka. Rahman MR, Rahman MA, Khan MN, Hussain MG (2004) Observation on the embryonic and larval development of Silurid catfish, Gulsha (Mystus cavasius). Pakistan Journal of Biological Science 7(6): Rana KJ (1990) Influence of incubation temperature on Oriochromis niloticus (L.) eggs and fry. Gross embryology, temperature tolerance and rates of embryonic development. Aquaculture 87: Saxena RK (1972). Studies on the maturity and fecundity of Rita rita (Ham.) of Ganga river system. Journal of Inland Fisheries Society of India 4: Tripathi SD (1996) Present status of breeding and culture of catfishes in South Asia. Aquaculture Living Resources 9: Kritik Olarak Tehlike Altinda Olan Dere Kedi Baligi Rita rita'nin Embriyonik ve Larval Gelisimi Özet Bu çalisma, tatli su kedi baligi Rita rita'nin embriyonik ve larval gelisimini incelemek amaciyla gerçeklestirildi. Olgun yumurta ve spermler yapay döllenme teknigi kullanilarak toplandi ve döllenmis yumurtalar sürekli su kaynagina sahip mini dairesel kuluçkahanede inkübe edildi. Döllenmis yumurtalar; seffaf, demarsal, dairesel, yapiskan olmayan, renkleri kahverengimsi idi ve çaplari mm arasinda degismekteydi. Ilk bölünme, döllenmeyi takibeden dak. içerisinde 28±1 C'de gerçeklesti. Yumurtadan çikma islemi, ayni sicakliklarda, döllenmeden 22 s sonra basladi ve 24 s içerisinde tamamlandi. Yumurtadan yeni çikan larvalar, 2.0 mm uzunlugunda, agizdan ve pigmentten yoksundu ve beslenme, yumurtadan çikistan s sonra basladi. Tatli su kedi baligi R. rita'nin erken dönem embriyonik ve larval gelisimi ilk kez tanimlanmaktadir. Bu yüzden bu çalismanin bulgulari, büyük ölçekli Rita yavru balik üretimi gerçeklestirilmesi için kiymetli bilgiler saglamaktadir. Anahtar Kelimeler: Embriyo gelisimi, larva, metamorfoz, Rita rita. EurAsian Journal of BioSciences,