AQUACUL ACULTURE GENETICS RESEARCH IN BANGLADESH ADESH M.G. Hussain and M.A. Mazid Bangladesh Fisheries Research Institute Mymensingh 221, Bangladesh HUSSAIN, M.G. and M.A. MAZID. 21. Aquaculture genetics research in Bangladesh, p. 7-14. In M.V. Gupta and B.O. Acosta (eds.) Fish genetics research in member countries and institutions of the International Network on Genetics in Aquaculture. ICLARM Conf. Proc. 64, 179 p. ABSTRACT Aquaculture productivity has declined in recent years in Bangladesh. The reasons for this are habitat degradation, loss of genetic diversity and stock deterioration in hatchery populations due to poor broodstock management and inbreeding. In consonance with the government s plans to rehabilitate inland fisheries in the country, the Bangladesh Fisheries Research Institute through its Freshwater Station, initiated fish genetics research in 1986. Making effective breeding plans for commercially important carps and other fish species is an important research area for the development of genetically improved broodstock. A new dimension of this research has begun with the involvement of international agencies such as the International Center for Living Aquatic Resources Management, Australian Centre for International Agricultural Research/Commonwealth Scientific and Industrial Research Organization and Institute of Aquaculture, University of Stirling, Scotland. The major research programs being implemented are: genetic stock improvement of endemic (Catla catla and Labeo rohita) and exotic (Barbodes gonionotus) carps, catfish (Heteropneustes fossilis) and genetically improved GIFT strain Nile tilapia (Oreochromis niloticus); population genetics of an anadromous clupeid, hilsa shad (Tenualosa ilisha); and genetic conservation of some endemic threatened fish species. This paper presents the highlights of these research programs in Bangladesh and future plans. Introduction Stock deterioration in hatchery populations caused by poor broodstock management and inbreeding has been observed in recent years in Bangladesh. Retarded growth, reduction in reproductive performance, morphological deformities, increased incidence of diseases and mortalities of hatcheryproduced seed have been reported. This has resulted in deterioration of carps and barb seed quality. There is widespread concern that stocking of such genetically poor quality seed in floodplains and related open water bodies for fishery enhancement might cause serious feral gene introgression in the wild stocks. To avoid loss of genetic diversity and inbreeding depression in hatchery populations, the development of improved broodstocks through implementation of effective breeding plans for commercially important carps and other fish species has been identified by Aquaculture Genetics Research in Bangladesh 7
the Bangladesh Fisheries Research Institute (BFRI) as an important area for research. Research was initiated with technical and financial support from the International Center for Living Aquatic Resources Management (ICLARM); Australian Centre for International Agricultural Research (ACIAR)/Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia; and Institute of Aquaculture (IOA), University of Stirling, Scotland. Genetic evaluation of improved GIFT strain Nile tilapia (Oreochromis niloticus) was undertaken during 1994-1996. Genetic stock improvement of two selected carp species, silver barb (Barbodes gonionotus) and catla (Catla catla), has been in progress since January 1997 under the auspices of ICLARM. A collaborative research program with IOA to produce all-female population of B. gonionotus, through gynogenesis and sex reversal, has been initiated. The population genetic structure of hilsa shad (Tenualosa ilisha) in Bangladesh is being investigated in collaboration with ACIAR/CSIRO, Australia. Other programs being implemented include the selective breeding of rohu (Labeo rohita), further genetic selection and development of all-male population of GIFT strain O. niloticus, genetic manipulation in catfish (Heteropneustes fossilis) and genetic conservation of endangered fish species like Labeo gonius, Cirrhinus reba, Channa striata and others. Freshwater Fish Species Used for Artificial Seed Production Endemic carps and other fish species There are at least 13 species of carps and other species under six genera inhabiting Halda and Padma-Brahmaputra River systems. Of these species, eight species of carps and barbs are used for artificial seed production in hatcheries (Table 1). Breeding techniques have been developed for the other five species: Clarias batrachus, Ompok pabda, Mystus cavasius, Anabas testudineus, and H. fossilis. Exotic carps and other fish species Several exotic fish species have been introduced in Bangladesh since 196. Such introductions were not properly recorded. Table 2 lists the introduced fish species which are bred in hatcheries for seed production. Genetics Research in Progress Carp species Breeding plans for stock improvement of catla and rohu Land races of C. catla and L. rohita were collected from Halda, Jamuna and Brahmaputra river systems, and were investigated for differences in extrinsic genetic traits by means of morphological and growth assessment. During the spawning season of 1999, two wild L. rohita stocks (from Jamuna and Brahmaputra Rivers) were mated with existing hatchery stocks to produce three crossbreds (i.e., hatchery x Jamuna ; Jamuna x Brahmaputra ; and Brahmaputra x Jamuna ) and three purebred lines (i.e., hatchery x hatchery ; Jamuna x Jamuna ; and Brahmaputra x Brahmaputra ). With all these lines, selective breeding and line crossing program will be continued, which will result in the development of genetically improved strain, Table 1. Endemic fish species of Bangladesh (Hussain 1998). Family Species Common name Local name Cyprinidae Labeo rohita Rohu Rui Catla catla Catla Katla Cirrhinus mrigala Mrigal Mrigal Labeo calbasu Calbashu Kalibaush Labeo bata Bata Bata Labeo gonious Gonious Gonnia Puntius sarana Barb Sarpunti Tor putitora Tor mahseer Mahashoal Claridae Clarias batrachus Asian catfish Magur Siluridae Ompok pabda Pabda Pabda Bagridae Mystus cavasius Gulsha Golsha Anabantidae Anabas testudineus Climbing perch Koi Heteropneustidae Heteropneustes fossilis Asian catfish Shingi 8 Fish Genetics Research in Member Countries and Institutions of INGA
Table 2. Exotic fish species used for artificial seed production in Bangladesh (Hussain 1998). Family Species Common name Country Year introduced Cyprinidae Ctenopharyngodon idella Grass carp Hongkong 1966 Nepal 1979 Japan 197 Mylopharyngodon piceus Black carp China 1983 Hypophthalmichthys molitrix Silver carp Hong Kong 1969 Aristichthys nobilis Bighead carp Nepal 1981 Cyprinus carpio var. communis Common carp China 196 Vietnam 1995 Cyprinus carpio var. specularis Mirror carp Nepal 1979 Hungary 1996 Barbodes gonionotus Silver barb Thailand 1987,1994 Tor putitora Mahseer Nepal 1991 Schilbedae Pangasius sutchi Thai pangas Thailand 199 Claridae Clarias gariepinus African catfish Thailand 1991 Cichlidae Oreochromis niloticus Nile tilapia Thailand 1974 1987 O. mossambicus x O. niloticus Red tilapia Thailand 1988 O. niloticus GIFT strain Philippines 1994, 1996 with better cultivable traits. Genetic evaluation in terms of growth and other relative performances will be undertaken in nursery and grow-out systems. Phenotypic differences among wild stocks of L. rohita The wild stocks of L. rohita from different river systems of Bangladesh were compared with hatchery stocks for phenotypic differences. Variations were observed in some meristic characters (Table 3). There were similarities between hatchery and Halda River stocks, particularly in the number of pectoral, anal and ventral fin rays, lateral line scales and vertebrae. Allozyme analysis and comparison of growth performance are in progress. Interspecific hybridization between endemic and exotic barbs Interspecific hybridization between two barb species, P. sarana (endemic) and B. gonionotus (exotic) was undertaken by Begum (1996). Fertilization (55-62%) and hatching (19-25%) of reciprocal hybrids were significantly lower (P<.1 and P<.5, respectively) than those of the control groups fertilization (73- Table 3. Meristic characters (range and average) of L. rohita, collected from hatcheries and different river systems of Bangladesh (figures in parentheses are the coefficient of variation). No. of Stock Dorsal fin rays Pectoral fin rays Ventral fin rays Anal fin rays Lateral line scales Vertebrae Hatchery (n=75) 13-15 14.2 ±.5 (3.2) 14-17 15.5 ± 1 (6.5) 8-1 9. ±.2 (2.3) 6-8 6.9 ±.3 (4.8) 4-43 41.4 ± 1 (1.4) 31-33 32.9 ±.4 (1.3) Brahmaputra R. (n=25) 13-15 14.1 ±.5 (3.5) 17-18 17.8 ±.4 (2.1) 9 9. ± (.) 7-8 7.2 ±.4 (5.7) 41-42 41.6 ±.5 (1.2) 33-34 32.2 ±.4 (1.3) Halda R. (n=5) 13-15 13.9 ±.4 (3.) 16-18 16.8 ±.5 (2.9) 8-1 8.9 ±.3 (3.5) 6-8 7.2 ±.7 (1.9) 4-43 41.6 ±.6 (1.5) 3-35 33. ±.5 (1.6) Jamuna R. (n=25) 14-15 14.6 ±.5 (3.4) 17-18 17.9 ±.3 (1.7) 9 9. ± (.) 7 7. ± (.) 41-42 41.8 ±.4 (.9) 33 33. ± (.) Aquaculture Genetics Research in Bangladesh 9
81%) and hatching (49-56%). Both control groups produced significantly higher (P<.5) percentage of normal embryos (52-59%) compared with both hybrids (23-24%). No variations were found between the two control and hybrid groups. Genetic improvement of B. gonionotus B. gonionotus was first introduced from Thailand in 1977, and subsequently in 1994 wild germplasm was introduced from Thailand and Indonesia with the help of ICLARM. Studies are in progress to develop a genetically improved and allfemale population of B. gonionotus using these three founder stocks through selective breeding and genetic manipulation techniques under projects supported by ICLARM and the Department for International Development (UK), respectively (Hussain 1997). For genetic improvement of B. gonionotus through selective breeding and line crossing techniques, germplasm obtained from Thailand and Indonesia and existing local stocks were used. The representative breeders of F 1 generations of the three unrelated stocks were crossed through 3 x 3 diallele crossing to produce nine genetic groups. The rationale of this crossing scheme is to form a heterogeneous outbred base population for the breeding program. For each of the reciprocal crosses, five to eight pairs were mated separately and the best three progeny (larvae) groups were selected to make 18 F 1 full sib progeny families which were then communally stocked by mixing 125 and/or 695 larvae from each family. Subsequently, they were transferred to communal grow-out ponds where stocking density was maintained at.75 to 1. fish/m 2 (Fig. 1). A separate growth trial experiment was conducted for eight months with communal F 1 crossbred and existing purebred (Bangla x Bangla) strain in four chambered earthen ponds with two replicates at a stocking density of 3 fish/m 3. At harvest, the maximum and minimum average weight gains of communal crossbred groups were 77.26 g and 49.21 g, respectively, whereas those for the control groups were 61.47 g and 56.13 g, respectively (Table 4). The data of monthly mean weight showed superior growth rate in crossbreeds but was not significantly different (P>.5). During the spawning season of 1998, 2% of largest females (at least 15 pairs) were selected from mature communal F 1 crossbreeds and used in the production of F 2 generation (Fig. 2). A comparative growth evaluation of F 2 crossbred and control groups (Bangla x Bangla) in nursery and grow-out systems indicated that the former had higher Mating of 3-4 pairs of breeders for each 6 reciprocal crosses Incubation of eggs in separate funnel units Growth evaluation of communal stock with control group Communal stocking with 695 larvae per pair and rearing in nursery / rearing ponds Communal stocking with 125 larvae per pair and rearing in nursery / rearing ponds Communal stocking of fingerlings in grow-out ponds Communal stocking of fingerlings in grow-out ponds Mass selection of at least 2% fish as broodstock for further selective breeding Mass selection of at least 2% fish as broodstock for further selective breeding Fig.1. Design for mating and production of F 1 broodstock from the base population derived from reciprocal cross of B. gonionotus. 1 Fish Genetics Research in Member Countries and Institutions of INGA
Table 4. Comparison of performance between crossbred and control groups of B. gonionotus. Month Average Weight (g) Communal F 1 hybrid Control group Initial October November December January February March April May Group 1 3.86 ± 1.4 17.41 ± 3.7 25.17 ± 6.4 32.5 ± 17.89 4.97 ± 1.13 43.56 ± 1.6 46.25 ± 14.57 55.24 ± 12.47 77.26 ± 29.49 Group 2 3.86 ± 1.4 16.58 ± 4.9 21.53 ± 4.14 23.79 ± 7.6 33.42 ± 9.6 36.11 ± 8.89 39.76 ± 12.1 45.4 ± 11.62 49.21 ± 13.56 Group 1 3.49 ±.88 2.1 ± 3.94 24.2 ± 4.77 28.48 ± 5.11 36.18 ± 11. 4.23 ± 9.75 42.56 ± 13.45 45.6 ± 14.49 61.47 ± 14.5 Group 2 3.49 ±.88 13.43 ± 4.1 17.68 ± 3.33 21.92 ± 8.51 32.7 ± 9.24 35.58 ± 11.22 39.37 ± 12.18 48.8 ± 11.71 56.13 ± 13.96 growth in terms of weight at an early age than the latter, but this was not statistically significant. In grow-out ponds, the crossbred group showed significantly higher growth (7-8%). During the spawning season of 1999, 15% of largest females and males were selected from the mature F 2 crossbred progenies and used them (182 pairs) for the generation of F 3 progenies. At the same time, 78 pairs of nonselected control (existing Bangla x Bangla) breeders were separately used to produce control progeny group. The on-station comparative growth evaluation trial data revealed that the average genetic gain in terms of body weight achieved by the selected group (F 3 ) was 22-25% over nonselected control group. Like other cyprinids, female B. gonionotus has better growth than males; therefore, a program for mass production of all female population through gynogenesis and sex reversal was initiated in 1995 in collaboration with the University of Stirling, Scotland, and the National Aquaculture Genetic Research Institute (NAGRI), Thailand. The protocol for such approach was to produce neomale (phenotypic male having genotype) Pool breeding of at least 15 pairs of mass selected breeders in several batches (25-3 pairs per batch) Pool breeding of at least 5 pairs of nonselected breeders (existing stock) Hatching of eggs Hatching of eggs Nursing of early fry at a stocking density of 25/m 2 Evaluation for growth in partitioned ponds Nursing of early fry at a stocking density of 25/m 2 Rearing of progenies in grow-out ponds On-station and on-farm evaluation of growth performance of progenies between selected and nonselected control pairs Rearing of progenies in grow-out ponds Sale of hatchlings/fry to interested grow-out farmers Fig. 2. Design for mating of selected broodstock and production of F 2 generation of B. gonionotus and their evaluation for growth. Aquaculture Genetics Research in Bangladesh 11
through feeding androgen hormone treated feed to the gynogenetic fish and then crossing neomales with normal females for mass production of all female seeds. Neomales of B. gonionotus were imported from NAGRI for mating them with females belonging to wild germplasm of Thailand and Indonesia (introduced in 1994 for selective breeding program). Production of additional batches of neomale through gynogenesis and sex reversal was initiated (Pongthana et al. 1998). The putative monosex female progeny groups produced were tested on-station and on-farm. Putative monosex females produced from newly produced neomales and mixed sex control fish attained weights of 55-15 g in on-station trials, while 4-8 g in on-farm trials. The sex ratio in the harvested neomale progenies varied from 5% to 9% female. The male individuals present in the neomale progenies could be of second generation neomale or there might have been some complexities in the sex determination, Y systems. Sexual dimorphism for weight in B. gonionotus was initially done through sampling of different populations from different culture systems. The sexual dimorphism indices (ratio of mean weight of females to mean weight of males, which is expressed as percentage) for weight in B. gonionotus at the age of one year class ranged between 1.1 and 1.7 in different populations and culture systems. Proximate composition of the carcass of female and male fish indicated insignificant differences (P>.5) in moisture, protein and ash content, except fat (Azad 1998). Catfish species Genetic manipulation in H. fossilis Meiotic gynogenesis was induced in H. fossilis by application of cold shock to the eggs that were fertilized with UV-irradiated sperm. Optimal irradiation was achieved when the sperm suspension with a concentration of 1 x 1 8 sperm ml -1 in a physiological saline (.85) solution was exposed for 2.5 min to an intensity of UV at 25 µw cm -2. Cold shock was applied to eggs 3-7 min after fertilization for a duration of 5-25 min at a temperature range of 2-6 o C (Table 5). Apart from gynogens, haploid, triploid and normal diploid control groups were maintained simultaneously to ascertain the functions of irradiation, cold shock and quality of germ cells used. The haploids were produced by using irradiated sperm but without cold shock and the triploids were produced through cold shock of eggs fertilized with normal sperm. The status of ploidy-haploid, diploid normal/gynogen and triploid were assessed through karyological investigation which revealed the chromosomal number (N=28, 2N=56 and 3N=84). Diameter and volume of erythrocytes along with nucleus from normal diploid, gynogen and triploid individuals of two months of age were compared to assess their ploidy status. All values of these measures in triploid were found to be Table 5. Effect of cold shock to a developing embryo at various temperatures and duration on ploidy manipulations in H. fossilis. (Sperm irradiated at 25 µw cm -2 with a concentration of 1.1 8 sperm ml -1 ). Temperature/ Duration Observation Meiotic gynogens (%) Haploid control (%) Triploid control (%) Diploid control (%) 2 o C for 1 min; 3-7 min after fertilization Fertilization Hatching Survival (3 days) Induction rate 85-96 33-69 59-9 94-1 83-97 78-1 1 91-98 45-79 58-84 94-1 - - 87-1 27-68 - 83 2 o C for 15 min; 3-7 min after fertilization Fertilization Hatching Survival (3 days) Induction rate 83-95 32-56 36-76 95-1 83-97 78-1 1 82-97 4-73 23-4 22-41 87-1 27-68 - 83 4 o C for 1 min; 3-7 min after fertilization Fertilization Hatching Survival (3 days) Induction rate 73-9 9-14 - 1-3 83-97 78-1 1 91-97 24-63 7-11 87-1 27-68 - 83 4 o C for 15 min; 3-7 min after fertilization Fertilization Hatching Survival (3 days) Induction rate 72-91 11-23 15-28 21-32 83-97 78-1 1 86-96 22-56 16-28 17-22 87-1 27-68 - 83 12 Fish Genetics Research in Member Countries and Institutions of INGA
significantly higher (P<.5) than that of corresponding normal diploid and gynogenetic diploid, but insignificant differences (P>.5) were found between the last two groups. A slow development of embryo and also delayed hatching were observed in gynogenetic and triploid groups (Gheyas 1998). Production of all-male population of GIFT strain O. niloticus Administration of androgen hormone 17 alphamethyltestosterone (MT) orally to the fry of O. niloticus from first feeding stage to 4 days at different doses ranging from 6 to 1 µg/g of feed in hapas, pond and aquarium resulted in 75-95% males. Seven to 1-day old fry of GIFT strain O. niloticus, when administered orally with MT hormone at a dose of 6 and 1 µg/g of feed and SRT-99 (MT 1%) feed obtained from the Philippines for 3 weeks resulted in 95-1% males (n=5) in the batch that were administered with a dose of MT 1 µg/g feed whereas 85% males were obtained in the batch fed with MT 6 µg/g feed. SRT- 99 hormone treated feed also resulted in 95-1% males (n=5). Population genetic structure of hilsa shad, Tenualosa ilisha The population genetic structure of T. ilisha which constitutes 25% of the total fish production in Bangladesh was investigated using starch gel electrophoresis. The research was done in collaboration with ACIAR/CSIRO, Australia. Fish samples were collected from different geographical areas within and beyond Bangladesh (Table 6). Starch gel electrophoresis of different enzymes of samples of various tissues was initially attempted to investigate allozyme variation. Five loci - Idh-l, Idhm, Mdh-l, Mdh-m and Pgm - were polymorphic over the six locations studied. Levels of variation were low but significant differences in allele frequencies were detected among Kuwait, Bangladesh and Indonesia (Table 7). However, allozyme data, so far, cannot discriminate genetically distinct stocks of hilsa within Bangladesh (Hussain et al. 1998). Conservation of endangered carp species The International Union for the Conservation of Nature, Bangladesh (1998) documented 11 cyprinid species as critically or fairly endangered in the country (Table 8), indicating a need for the development of artificial breeding techniques for the conservation of the carp s gene pool. Attempts were made to develop artificial propagation techniques for Tor putitora and Puntius sarana, and very recently for L. gonius and C. reba. Future Research Plans Following are plans for aquaculture genetics research in Bangladesh: Genetic characterization of wild land races of C. catla and L. rohita; Initiation of genetic stock improvement of C. catla and L. rohita using selective breeding and Table 6. Locations and sample sizes used in the electrophoretic study. Location Kuwait Bangladesh Khulna Aricha Ghat Chandpur Cox s Bazar Indonesia Table 7. Results of G statistics tests (similar to contingency 2 tests) for homogeneity of alleles. H o : all alleles are drawn from the same homogeneous population. Comparisons Within Bangladesh Kuwait vs. Bangladesh vs. Indonesia Kuwait vs. Indonesia Kuwait vs. Bangladesh Indonesia vs. Bangladesh P NS P<.1 P<.1 P<.1 P<.1 Number 47 65 7 69 82 33 Significant loci None Mdh-m, Pgm, Idh-l Idh-1 Mdh-m, Pgm Mdh-m, Pgm, Idh-l Table 8. List of endangered carp and barb species of Bangladesh. Scientific name Labeo nandina L. boga L. gonius L. bata L. pangusia L. calbasu Cirrhinus reba Puntius sarana P. ticto Tor tor T. putitora Local name Nandina Bhangan Ghonia Bata Ghora maach Kalbaus Laachu, bata Sarpunti Tit punti Mahashol Mahashol Critically endangered Fairly endangered Aquaculture Genetics Research in Bangladesh 13
line crossing techniques; Evaluation of growth performance of F 4 putative genetically improved B. gonionotus; Genetic sex determination and production of allfemale population of B. gonionotus through gynogenesis and sex reversal; Continuation of selection of GIFT strain O. niloticus for further genetic improvement; Optimization of sex reversal technique for GIFT strain O. niloticus; Continuation of genetic manipulation in H. fossilis, particularly production of all-female population through inducing homogametic neomales; and Continuation of population genetics study of T. ilisha. References Azad, M.A.K.1998. A preliminary study on the sexual dimorphism for weight of silver barb, Puntius gonionotus Bleeker. Bangladesh Agricultural University, Mymensingh, Bangladesh. 97 p. M.S. thesis. Begum, S. 1996. Studies on the hybridization between Puntius sarana and Puntius gonionotus and their embryonic development. Bangladesh Agricultural University, Mymensingh, Bangladesh. 8 p. M.S. thesis. Gheyas, A.A. 1998. Studies on cold shock induced gynogenesis and artificial breeding performance in Heteroneustes fossilis Bloch. Bangladesh Agricultural University, Mymensingh, Bangladesh. 132 p. M.S. thesis. Hussain, M.G. 1997. Current status of carp genetic research and breeding practices in Bangladesh, annex 2.1. In M.V. Gupta, M.M. Dey, R. Dunham and G. Bimbao (eds.) Proceedings of the Collaborative Research and Training on Genetic Improvement of Carp Species in Asia, 26-29 July 1997, Central Institute of Freshwater Aquaculture, Bhubaneswar, India. ICLARM Work. Doc. 1, 12 p. (Unpublished). Hussain, M.G. 1998. Broodstock management and breeding plans to control negative selection and inbreeding in hatchery stocks: BFRI concepts. Paper presented at the Workshop on Broodstock Management for Genetic Maintenance and Improvement, 27-28 April 1998, Dhaka, Bangladesh. Hussain, M.G., J.P. Salini, M.S. Islam and M.A. Mazid. 1998. Genetic structure of hilsa shad population: preliminary results using starch gel allozymes. In M.A. Mazid and S.J.M. Blaber (eds.) Proceedings of Hilsa Research Project Workshop. (In press). IUCN, Bangladesh. 1998. List of threatened animals of Bangladesh. Paper presented at the Special Workshop on Bangladesh Red Book of Threatened Animals, 22 February 1998, Dhaka, Bangladesh. Pongthana, N., D.J. Penman, P. Baoprasertkul, M.G. Hussain, M.S. Islam, S.F. Powel and B.J. McAndrew. 1998. Monosex female production in the silver barb (Puntius gonionotus Bleeker). Aquaculture 173: 247-256. 14 Fish Genetics Research in Member Countries and Institutions of INGA