ISSN Original Article Simulated breeding of Indian Pearl mussel, Lamellidens marginalis (L.) in laboratory condition

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Available online at http://www.urpjournals.com International Journal of Research in Fisheries and Aquaculture Universal Research Publications.

Janaki Ram 3 1 Central Inland Fisheries Research Institute, Barrackpore, Kolkata - 700 120 2 Central Inland Fisheries Research Institute, Regional Centre, Guwahati-781006 3 Centre Institute of

1 Available online at International Journal of Research in Fisheries and Aquaculture Universal Research Publications. All rights reserved ISSN Original Article Simulated breeding of Indian Pearl mussel, Lamellidens marginalis (L.) in laboratory condition B. K. Behera 1, *D. K. Meena 1, Pronob Das 2 and K. Janaki Ram 3 1 Central Inland Fisheries Research Institute, Barrackpore, Kolkata Central Inland Fisheries Research Institute, Regional Centre, Guwahati Centre Institute of Freshwater Aquaculture, Bhubaneswar, Orissa *Corresponding author: dkmeenafnb@gmail.com Received 10 September 2014; accepted 01 October Abstract Accomplishment in seed production of the freshwater mussel, Lamellidens marginalis largely depends on a number of intrinsic and extrinsic factors. In the present study, an attempt was made to elucidate the mode of reproduction, spawning, larval cycle, and juvenile production in captive conditions and to find out the fish host-mussel larval relationship. It was observed that L. marginalis is a prolific breeder and spawns round the year with a peak spawning activity from July to September. It is evident from the present investigation that life history of L. marginalis passes through various critical stages including development of glochidium larvae which have mandatory parasitic existence and their host specificity for being juveniles to adulthood. Result of the experiment suggested that Glossogobius giuris showed highly significant avg. no. of glochidium larvae infected per fish,54±2.0 ( p <0.05) followed linearly in increasing order by Catla catla, 25±1.8 and Labeo rohita, 22± 1.2,proved to be the best suited fish hot species for the attachment, parasitism and recovery of the glochidium larvae and juveniles. It has been observed that a glochidium larva takes days for appropriate fish host to reach the juvenile stage at ambient temperature range of C. However, the duration of larval metamorphosis is different with respect to different fish hosts tried. The glochidium larvae infestation was only recorded on the fins of the experimental fish Universal Research Publications. All rights reserved Key words: Lamellidens marginalis, Glochidium larvae, Freshwater mussel. Introduction Freshwater pearl culture is one of the emerging areas in aquaculture sector. Realizing the scope and the importance of Inland Pearl culture in India Central Institute of Freshwater Aquaculture, Fisheries organization took the lead and evolved an indigenous system of culturing pearl through common freshwater pearls, Lamellidens marginalis, Lamellidens corrianus and Parreysia corrugate [6, 8]. The availability of pure mussel seed on mass scale assumes critical importance for realizing potential benefits of this important group of aquatic organisms. Aquatic environment is dynamic and ever changing to play the key role in dispersal and distribution of mussel population and the success or failure of a brood in reaching its appropriate destinations. It is pertinent to note that a majority of freshwater mussels have indirect larval development with a mandatory parasitic glochidium stage, making seed production under controlled condition more difficult[ 9,10]. Considering the above facts, an attempt was made to examine eggs of juvenile stage in life history of freshwater mussels in captive conditions and to find out the fish host mussel larval relationship and target organs of the host for larval attachment. Material and Methods Experimental Setup Round plastic tubs of 201 capacities were employed for brood mussel rearing and rectangular FRP tanks (100cm X 50CM X 45CM) were used for rearing different fish host species. All the FRP tanks and plastic tubs were cleaned with mild detergents and disinfected with 2 % KMnO 4 solutions as a prophylactic measure before starting the experiments. The test fishes such as Cirrhinus mrigala, Labeo rohita, Glossogobius giuris, Catla catla, Hypophthalmicthys molitrix, Cyprinus carpio and Ctenopharyngodon idella ranging 7-15cm in size were collected and disinfected with 0.5 KMnO 4 prior to the experimentation. These fish species were released in equal numbers (30 no. / tank) into fish holding tanks (1001) filled 145

2 with aged tap water. Live healthy mussels, L. marginalis of 8-10 cm in shell length were collected from natural water bodies and cleaned thoroughly with tap water to remove mud, epiflora and fauna of the shell surface. These mussels were kept in bucket without water for about 1 hour and the resultant physical stress conditions induced the spawning activity [7, 4]. These mussels were reared in the plastic 20 mussels/ tub (201) containing aged tap water. The eggs and glochidium larvae were collected by pipetting. Both experimental fish tanks and mussels spawning tubs were aerated continuously. Pelleted feeds of 1 cm diameter were 2% of body weight on alternate days to the test fishes, C. mrigala, L. rohita, H. molitrix. C. carpio, C. idella. The G. giuris and C. catla were fed ad libetum with zooplankton while the experimental mussels were not fed during the rearing. Hatching and Collecting of mussel larvae Mussels were removed from the tubs after release of eggs and glochidium larvae. Aeration was suspended in mussel spawning tubs till collection of larvae and egg. After settlement, excess water in spawning tubs was carefully siphoned out without disturbing settled mussel larvae and eggs. The whitish fertilized eggs were observed to be agglutinated in a mass of eggs and were collected by pipetting carefully from bottom of mussel spawning tubs. Larval numbers were estimated by making small volume of collected glochidium larvae with water from the spawning tub 1 litre in glass jar. Then, 10 random samples of thoroughly mixed larval suspension agglutinated in a mass of eggs and were collected by pipetting carefully from bottom of mussel spawning tubs. Larval numbers were estimated by making small volume of collected glochidium larvae with water from the spawning tub up to 1 litre in a glass jar. Then, 10 random samples of thoroughly mixed larval suspension were taken from the jar and each sample was counted under a binocular stereo zoom microscope and the total number of larvae in the sample was computed by using the following formula [1]. Screening and observation of larval stages in fish host Fish host were depurated for a period of 30 days in tanks to avoid earlier glochidium infection before start of experiments. After ascertaining that the experimental fishes are devoid of mussel larval infection, approximately 3000 numbers of newly collected glochidium larvae were transferred into fish holding tanks. Seven days later the introduction of mussel larvae, followed by one fish from each tank was sacrificed to detect infection rate and for this different parts of the body like skin, gills and fins for glochidium larval attachment were examined. Observation was continued for 1 month to distinguish different stages of larval metamorphosis in fish host. Gills and fins were removed from sacrificed fish and observed individually under the microscope. Observation of Juveniles and enumeration Bottom sediments from each experimental tank was collected at regular intervals and examined under the microscope to record the presence of juveniles and their developmental stages. Microphotographs were taken using a Leitz DMIL microscope of fertilized eggs, glochidium, developing glochidium in fish host and juvenile mussels after metamorphosis. Results In the current investigation, it was observed that the freshwater mussels, L. marginalis is a prolific breeder and it spawns round the year with a peak spawning activity from July to September. Testis of mature mussel was white to cream in colour while ovary was dull red to conspicuously pinkish and the fertilization was observed to be internal. Sperms of L. marginalis were elliptical with a rudimentary tail part. Mature ovum was large compared to spermatocyte, round and filled with finely granular cytoplasm with avescicular nucleus. The fertilized eggs were agglutinated in various shapes and sizes and the eggs undergo further development in about 24 to 48h to reach glochidium larval stage (Fig: 1 & 2). Fig. 1 An encapsulated glochidium larva in the fin tissue of the fish host Fig. 2 Glochidium larvae of the pearl mussel, L. marginalis Glochidium larvae expelled into ambient water from the gill marsupial of spawning females through exhalent siphon. Initially the glochidium larvae was D shaped and minute in size (0.22mm) with two shell valves. The shell valves were thin, brittle, brown and translucent with pitted appearance on surface. On both of valves, tiny spines were present in the interior, towards ventral aspect of the shells. The larva was provided with a long convoluted tube. Larva was active in first 12 hrs after hatching by constantly opening and closing the shell valves and is normally benthic in nature with very much restricted swimming movement. When the larva was in contact with fish host, instantly clings to peripheral margins of fins by employing spine like structures. In due course of time, larva enters into subcutaneous space of the fin and gradually loos it s D shape and become oval. Finally on 9 th & 10 th day, the larva become completely round and encapsulated (Fig: 3) inside fin tissue referred as advance stage. The long convoluted tube penetrates into nearby blood capillary and there by the larva derives nourishment from fish host. It has been observed that larva spends nearly days for metamorphosis and the juvenile mussel, a miniature adult in appearance and function dropped out of the host at the bottom of the experimental tank and started growing further. Recently metamorphosed juvenile mussel is small and the shells are comparatively thicker with greenish brown coloration (Fig: 4). 146

Fig.3 Embryonic development of the fertilized egg of the mussel Fig. 4 Recently metamorphosed juvenile mussel Table: 1- Result for fish host mussel larval parasitic relationship studies Total no.

3 Fig.3 Embryonic development of the fertilized egg of the mussel Fig. 4 Recently metamorphosed juvenile mussel Table: 1- Result for fish host mussel larval parasitic relationship studies Total no. of Av. no. of Av. no. of Total no. of Av. no. Fish Fish host glochidium glochidium glochidium juveniles of juveniles tank species larvae released larvae released larvae infected recovered per recovered per no per fish tank per fish per fish fish tank fish T1 C. mrigala ±1.2 c T 2 L. rohita ±1.4 c T 3 G. giuris ±2.0 a T 4 C. catla ±1.8 b T 5 H. molitrix ±1.2 d T 6 C. carpio ±1.0 e 80 4 T 7 C. idella ±1.6 e 90 4 Values with a common superscript in the same column with the different letter index differ significantly (P< 0.05); Mean± S.E. Table: 2- Physico-chemical parameters of water in experimental tanks and mussel spawning tubs recorded during experimental period S.No. Parameters Range 1 Temperature 28 C - 31 C 2 ph Dissolved Oxygen (ppm) Total alkalinity (ppm) Total hardness (ppm) Calcium harness (ppm) Ammonical- nitrogen (ppm) Juvenile mussel was capable of restricted movements with periodic closing and opening of two shell valves and readily accepted natural food like Chlorella for growth and further development into adult. An average number of 22 ± 1.2 glochidium larvae infested the host fish C. mrigala. In case of L. rohita and G. giuris, the average glochidium infestation was 21 ± 1.4 and 54 ± 2.0 respectively, while an average of 25 ± 1.8 glochidium larvae were infefested on the fish host, C. catla. As far as the exotic carps are concerned it was observed that in H. molitrix, C. idella and C. carpio, the average rate of infestation was 9 ± 1.2, 7 ± 1.6 and 6 ± 1.0 respectively. It can also be observed that, G. giuris harbored maximum infection of the glochidium larvae followed by C. catla and L. rohita (Table: 1). The average rate of juvenile recovery was 23 in G. giuris, 12 in C. mrigala and 10 in L. rohita. In case of C. catla, H. molitrix, C. carpio and C. idella, the average rate of juvenile recovery was 10, 5, 4 and 4 respectively (Table: 1) From these investigations under controlled conditions it is clear that among the different fish hosts tried G. giuris proves to be the ideal fish host for production of maximum number of juveniles followed by C. mrigala and L. rohita. It could be observed from these experiments as trials that though the initial numbers of infestations by the larvae were more, the progression to advanced larval stage and juvenile stages were by far limited. Present preliminary experiments have also indicated that duration of larval metamorphosis was different with different fish hosts tried. Observations were also made on host specimens to record incidence of glochidium in different organs. Tissues such as skin, gill and different fins like dorsal, pectoral, pelvic, anal and caudal fins were microscopically examined minutely to ascertain the suitable target organ for glochidium larval attachment. It was observed that skin and gills were not target organs for attachment of glochidium larval infestation. Glochidium larvae were recorded only on different fins of experimental fish. However, comparatively more number of glochidium larvae was recorded on pelvic, anal and caudal fins 147

4 followed by pectoral fin in all the fish host species except G. giuris. In G. giuris, it was observed that nearly all the fins were uniformly infested with glochidium larvae. Physico-chemical parameters of water in fish host rearing tank and mussels spawning tubs during the experimental period (Table: 2). Discussion As L. marginalis is unisexual [11], the secondary sexual characters are not clearly defined [10]. Microscopic analysis of the gonad of 95 specimens of Anodonta cygnea revealed that 18 were harmaphrodites [2]. It was also recorded that some individuals of Lamellidens thwaitesii were hermaphroditic in nature [3]. In the present investigations sexual dimorphism has not been recorded in L. marginalis. Earlier report shows that peak spawning season of L. marginalis is during August to September [5]. It has been recorded that the species Hyriopsis shlegelli spawns from April to August [10]. However, in Washboard mussel Melalonaias nervosa peak spawning activity occurs during August to October [12]. The present reported investigations are though short term in nature (May to October), general studies made on maturation and spawning activity of L. marginalis indicated that peak spawning season corresponds with monsoon season (July to September). It was reported that size of glochidium of freshwater mussels in general ranges between 0.1 and 0.5 mm [10]. The larval size of the H. shlegelli is reported to be 0.3 mm [9]. In the current study the size of the glochidium larva of L. marginalis was observed to be 0.22 mm. Kotpal, reported that the glochidium larvae of L. marginalis taken about 10 weeks for completion of metamorphosis to attain juvenile stage [10]. However, in the present investigation it has been observed that larva takes days of duration to reach the juvenile mussel stage. Some species of mussels require certain species of fish as a specific host, while in other mussel species several kinds of fish will serve as hosts [10]. It was earlier reported that three fish species, Green sun fish (Lepomis cyanellus), Black bull head (Ictalurus melas) and Channel catfish (Ictalurus punctatus) act as fish hosts for glochidium stage of M. nervosa in host specificity studies [12]. It was mentioned that mussel larvae requires days for larval metamorphosis at 17 o C. In another trial it was shown that the glochidium required 56 days for larval metamorphosis in Channel catfish, I. punctatus, White suckers (Catastomus commersoni) and Yellow perch (Perca flavescens) retained glochidium up to days, however no juveniles were produced [12]. In contrast the present study showed that fins of fish G. giuris were infected by glochidium larvae at an average of 54±2.0 per fish and after duration of days an average number of 23 juveniles per fish were recovered at o C. In a similar way Cirrihinus mrigala were infected at an average of 22±1.4 per fish resulting in 12 numbers of juveniles. In case of fish host L. rohita, the average larval infection rate was 21±1.4 per fish and the juvenile recovery was 10 per fish host. C. catla were infected by glochidium larvae at an average of 25±1.8 per fish resulting 10 numbers of juvenile mussels.the average infection rate in case of H. molitrix was 9±1.2 per fish which yielded an average of 5 juveniles per fish when, C. carpio was infected with an average of 6±1.0 glochidium larvae per fish giving an average 4 juveniles per fish. Similarly, fins of the fish C. idella were infected by glochidium larvae at an average of 7: t 1.6 per fish and after duration of days an average of 4 juveniles were produced at o C. During the present studies, a maximum number of glochidium larva (144) attached on the host species G. giuris of the size of 8.2 cm in length was recorded. Size of the fish host vis-a-vis the density of glochidium infection assumes significance in the future mussel breeding and seed production technology development. However, further detailed studies are required to establish the precise host related factors influencing the mussel larval metamorphosis. It has been stated that when glochidium larvae come in contact with proper host, it clings to the skin, gill and fins by closing their hooked valves [11]. It was also reported that in case of H. schlegelli, glochidium larvae clamp to gill filaments of fish being brought through respiratory water current [10]. In the present studies, infection of glochidium larvae in the gills and skin of the fish host were not recorded while glochidium could only be seen on different fins of fish host. It has been observed that glochidium larvae do possess certain specificity for infection on fins such as pelvic, anal and caudal fins. The reason for this selective infection on fins could be that these fins normally come in contact with sediment on which benthic glochidium larvae are naturally dispersed. In G. giuris, nearly all the fins were uniformly infested with the larvae, the reason may be due to G. giuris is not an active swimmer which remains dormant and attached to the bottom substratum as compared to other test fishes. Therefore, glochidium larvae get more chance for attachment to all the fins of G. giuris. Further, the Glossogobius giuris has long anal fin and large disc-shaped pelvic fins compared to other short finned test fishes, which increases the chance of infestation area on fins. Conclusion From the present investigation, it may be concluded that among all fish species screened for host specificity study, G. giuris was found to be the ideal fish host followed by C. mrigala and L. rohita. These studies have tremendous application vale in commercial mussel seed production technology not only to cater the needs of pearl industry for pearl seed mussels but also in rehabilitation of endangered freshwater mussel populations in the natural environment. Reference 1. B.K. Behera, Breeding of selected species of Indian Freshwater pearl mussel, Lamellidens marginalis (L.). M.F.Sc. Thesis, (1996) pp C. Ghosh, K.C. Ghosh, Reproductive system and gonadial activities in Lamellidens marginalis. The Veligar, (1972) 14: C.A. Woody, B. Holland, Reproductive characteristics of population of Washboard mussel, Megalonaios nervosa (Refinesque) in the upper Mississippi River. J. Freshwater Ecol. (1993) 8 (1): E. I. Thomas, The bionomics, anatomy and development of the freshwater mussel, Lamellidens 148

5 marginalis (Lamarck). The Ann. Zool. (1974) 10(4): H.H. Bloomer, A note on the sex of Anodonta, J. Conch. Lond. (1933). 19: H.H.Bloomer, A note on the anatomy of Lamellidens marginalis Lamarck and Lamellidens thwaitesii Lea, Porc. Malac. Soc. (1930) 19 (6): K. Janaki Ram, G.R.M. Rao. Effect of the thermal shock on spowning of the common pond snail, Vipiparur bengalensis. Ind. J. Expt. Biol. (1987) 25 (7): K. Janaki Ram, S.D. Tripathy, A manual on freshwater pearl culture. Manual series, (1992) 1: K. Janaki Ram, Studies on culture pearl production from freshwater mussels, Lamellidens marginalis. Curr. Sci. (1989) 58 (8): R.L. Kotpal, Reproductive system of freshwater mussels. Mullusca, (1995) S. Dharmaraj, T.S. Velayudhan, A. Chellam, A.C.C.Victor, C.P. Gopinathan, Hatechery production of pearl oyster spat, Pinctada fucata. CMFRI special publication, (1991) 49: T. Kafuku, H.lkenoue, Modern methods of aquaculture in Japan. Dev Aquacul Fish Sci. (1983) 11: Source of support: Nil; Conflict of interest: None declared 149

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!#$%&'() Mola mola *+,+-./ Mola mola 2008 Summary A study on the reproductive biology of ocean sunfish Mola mola Toshiyuki akatsubo 1. Introduction Ocean sunfish, Mola mola, which belongs to the family Molidae in the order Tetraodontiformes,