Journal of Survey in Fisheries Sciences 5(1) 7-19 2018

Production of tropical oyster seed in hatchery

Fakhrina M.N.1; Christianus A.1*; Ehteshamei F.2

Received: January 2018 Accepted: March 2018

Abstract Oysters have been collected from wild since long time ago for human consumption. Recent development in has allows the production of its seed in hatchery. Factors favoring the oyster production are stocking density, water quality and the availability of live food. These factors can increase the growth and survival of the oyster. Fundamental knowledge on life cycle and biology of oyster is important as it can become the basis for successful development of oyster culture. Due to the limited seed supply, hatchery produce seedlings are important to support the development of oyster industry.

Keywords: Life cycle, Microalgae, Oyster culture, Propagation, Stocking density

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1-Department of Aquaculture, Faculty of Agriculture, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia. 2-Iranian Fisheries Science Research Institute (IFSRI), Agricultural Research Education and Extension Organization (AREEO), P.O. Box: 15745-133. Tehran, Iran. *Corresponding author’s Email: [email protected] 8 Fakhrina et al., Production of tropical oyster seed in hatchery

Introduction on commercial basis are C. iredalei and Little information is available on the C. belcheri. Their spats are usually biology and culture of native oyster in collected from the wild. In recent years, Malaysia. In early 1960’s by the the limited supply of seeds from the Department of Fisheries Malaysia coastal waters of Malaysia causes the favored by a Colombo Plan Expert has decline in the production of these two conducted some studies on oyster. oysters. In order to increase this During mid-seventies, experimental production, the Fisheries Research culture of Crassostrea belcheri was Institute (FRI) and Universiti Sains carried out in Sabah (Chin and Lim, Malaysia (USM) have conducted a 1978). Then, followed by cultured of collaborative research for the artificial flat oyster, Ostrea folium in Pulau propagation of C .iredalei and C. Langkawi (Ng, 1979). The culture belcheri in hatchery. The first system uses polyethylene net as successful spawning and larvae rearing substrate for oyster attachment. In 1988, of C. iredalei was reported in 1990 Department of Fisheries Malaysia with (Wong, 1990). the cooperation of Bay of Bengal Programme (BOBP) started a more aggressive approach for the production of oyster. Under this program, suitable spatfall and areas for culture were studied intensively. Thus, the establishment of oyster production in areas at Sg. Muar (Ng, 1979), Langkawi, Penang, Pangkor and Melaka (Angell, 1988). However, the production of oyster in Malaysia is still very dependent on seedling collected from the natural sources (Ng, 1991). Basically, four genera of oysters considered native to Malaysia are Crassostrea iredalei (Fig. 1a), C. belcheri (Fig. 1b), Ostrea folium,

Downloaded from sifisheriessciences.com at 16:27 +0330 on Sunday October 3rd 2021 [ DOI: 10.18331/SFS2018.5.1.2 ] Saccostrea cucullata and Hyotissa hyotis (Lam and Morton, 2009). Among these oysters, C. iredalei has the highest Figure 1: Oysters species in Malaysia, commercial value due to its taste and Crassostrea iredalei (a) and appearance (Mohd Yatim, 1993). Crassostrea belcheri (b). Oysters have high demand as food in restaurant and steamboat stall. Two Oyster is the most valuable edible mollusks belong to the family . common species cultured by fishermen Journal of Survey in Fisheries Sciences 5(1) 2018 9

The irregular shell and varies shape and C. iredalei (Suzana et al., 2011), depending on its attachment to substrate. often resulted in the misidentification of The upper valve is small and flat while oyster species (Lam and Morton, 2003; the lower valve bigger, convex and Yu et al., 2003; Xia et al., 2008). Thus, slightly excavated. The inside surface the used of molecular technique has of the shell is smooth and chalky white. been applied for phylogenetic and Upper and lower valves are connected taxonomic study of oyster (Xia et al., to each other at the toothless hinge 2008; Wang et al., 2010; Suzana et al., (Rosell, 1990). The colors of scar 2011). depend on species. Internal anatomy showed the position of abductor muscle Maturation and spawning (Fig. 2). Most of the tropical oysters in Malaysia are protandric . This allows it to change from male to female upon reaching adult stage and vice versa (Blanco et al., 1951). Oyster can reach sexual maturity at the size of 50 to 80 mm (Rosell, 1990). Creamy white color indicates of ripe gonad, therefore is ready to spawn. Fertilization occurs externally with sperms being released continuously as dense white streams, Figure 2: Internal anatomy of tropical oyster, Crassostrea iredalei. and eggs released aided by contraction of adductor muscles. A single female According to Visootiviseth (1998), can release millions of eggs. The Crassostrea iredalei has purple-black tropical climate of Malaysia allows adductor muscle scar, whereas white in oysters to spawn throughout the year. C. belcheri (Fig. 3). The peak spawning seasons is between April to June and October to December (Devakie et al., 1993). The temperature and salinity changes during these seasons trigger the oysters to spawn actively.

Downloaded from sifisheriessciences.com at 16:27 +0330 on Sunday October 3rd 2021 [ DOI: 10.18331/SFS2018.5.1.2 ] Artificial propagation can be carried out in hatchery by taking ripe broodstock from grow-out site or from

wild. Broodstock shell are cleaned from Figure 3: Color of scar in Crassostrea iredalei (a) and Crassostrea belcheri (b). barnacle, worm and other organism. Immersion with 10-20 ppm calcium Similarities in the morphological hypochloride for 20 to 30 minutes will structures, such as in C. madrasensis 10 Fakhrina et al., Production of tropical oyster seed in hatchery

further eliminates protozoa and other indicates successful fertilization. Thirty organism on the shells. According to minute after fertilization, the eggs start Ng (1993), selection of spawners is to divide and developed into two-celled based on gonad maturity (Fig. 4). stage. Division of cells increase the weight of eggs. Eggs developed further into multi-celled blastula, gastrula stage and moving trochopores within 24 hours. Trochopores are oval in shape with size of 60 to 80 µm. These trochopores swim with the aid of cilia and long flagellum. Before it become fully-shelled D larvae in 24 hours, the velum will develop as a feeding organ. Whilst the larvae move, the velum will Figure 4: Matured oyster gonad. feed on phytoplankton in the water.

Umbone will develop after thirteen Natural spawning can be conducted by days of fertilization. At umbo stage, the placing oysters in spawning tank umbones are more prominent. Eye- supplied with ultra-violated (UV) larvae stage commences when a small treated running seawater. The release of dark circular dot appear at the center of sperms triggers the female to ovulate. each valve. At pediveliger stage, the As for selective breeding, the foot and gills are developed. Larvae broodstock can be placed in separate will then settle down as soon as they tank, then sperms and eggs are found a suitable substrate. They secrete collected and subsequently fertilized. the cement from the byssal gland at the For both techniques, the fertilized eggs foot for attachment. This cement are collected then placed in incubation hardens rapidly. Upon attachment to tank for 24 hours (Ng, 1993). Artificial substrate, the foot disintegrates, then spawning can be carried out by treating the oyster remains permanently at the the broodstocks with chemical such as selected substrate. serotonin. This chemical triggers the

broodstock to release the sperms and Larvae rearing and spat collection eggs for fertilization (Gibbon and Oysters are an epifaunal species with Castagna, 1984; Velasco et al., 2007). Downloaded from sifisheriessciences.com at 16:27 +0330 on Sunday October 3rd 2021 [ DOI: 10.18331/SFS2018.5.1.2 ] pelagic larval stage and sessile adult.

Life stages include trocophore, umbo, Embryonic and early development D-shape, eye-stage, pediveliger, The pelagic phase of oyster larvae can plantigrade, spat, juvenile and adult last for approximately 20 to 22 days. (Fig. 5). Generally, the growth and The haploid sperm and egg fused when survival of oyster depend on fertilization occurs to form a zygote. temperature. According to Devakie and When the polar body appeared, it Journal of Survey in Fisheries Sciences 5(1) 2018 11

Ali (2000), C. iredalei can tolerate wide µm to 2-5 mm are transferred into tanks range of temperature from 24 to 36ºC. supplied with seawater from estuary. Crassostrea iredalei larvae set at Thus, the juveniles are able to get food temperature range of 10 to 20ºC and from the natural seston in water salinity of 30.2 to 40.1% (Devakie and (Rodhouse et al., 1981). This method Ali, 2000). Southgate and Lee (1998) incur low cost, but with high risk. reported that Saccostrea echinata can Microalgae can be cultured using two survive well when reared at 29ºC. techniques, traditionally with batch mode in carboys or modified technique using semi-continuous mode in alveolar photo-bioreactor (Ponis et al., 2003). Instead of monospecific diet, various studies showed that multispecific diet is better for bivalve. This is because different species of microalgae has Figure 5: Life cycle of tropical oyster. different nutritional profile. Thus, mix diet can complement each other to Stocking density can affect the survival sufficiently support the growth of of larvae. However, high density larvae. In mollusc hatchery, mass culture will increase competition for culture of larvae is hindered due to the space, which may results in the increase limited supply of natural phytoplankton. larvae mortality. D-stage larvae with High concentration of microalgae is size of 50 to 60µm can be stocked at 5 needed in hatchery to support the larvae per mL. Stocking can be reduced optimum growth and survival of further as the larvae grow. molluscs. Common species of Recommended stocking for the larvae microalgae use as food for oyster are as of different species of oyster is as shown in Table 2. shown in Table 1. Selection of microalgae as food for mariculture is based on cell size, shape, Microalgae culture digestibility, and biochemical Microalgae have been used as food in composition (Doroudi et al., 2003; bivalve hatcheries since 1940s (Bruce Martinez- Fernandez et al., 2004). et al., 1940). The development of each According to Chrétiennot-Dinet et al.

Downloaded from sifisheriessciences.com at 16:27 +0330 on Sunday October 3rd 2021 [ DOI: 10.18331/SFS2018.5.1.2 ] bivalve stage is fully dependent on diets (1991), the selected microalgae must of cultured microalgae such as have high nutritional value and can be Chaetoceros calcitrans, Pavlova lutheri, mass cultured without much difficulty. Isochrysis sp. and Thalassiosira Lipids are essential component in the pseudonana (Coutteau and Sorgeloos, food for all aquatic larvae, including 1992). These microalgae are used as bivalves. food for larvae, juvenile and broodstock. In traditional culture, juveniles of 500 12 Fakhrina et al., Production of tropical oyster seed in hatchery

Table 1: Stocking density recommended for the culture of oyster larvae Species Stocking density Size of oyster at Source (larvae/mL) initial stocking (µm) 1,2 or 5 81.5 ± 2.5 Doroudi and Southgate, 2000 Crassostrea iredalei 1 >250 Devakie and Ali, 2000 Pinctada margaritifera 2 81.5 ± 2.5 Doroudi et al., 1999 Pinctada margaritifera 4.1 81.3 ± 0.06 Pinctada margaritifera 2 122.9 ± 0.09 Saccostrea echinata 5 >210 Southgate and Lee, 1998

Table 2: Types of microalgae as food for oyster Class of microalgae Species of microalgae Species of culture Source Prymnesiophyceae Pavlova salina Pinctada margaritifera Martìnez- Pavlova sp. Fernández and Isochrysis sp. clone TISO Southgate, 2007 Bacillariophyseae Chaetoceros muelleri Chaetoceros sp. Skeletonema sp. Prasinophyceae Micromonas pusilla Prymnesiophyceae Isochrysis aff. galbana TISO Crassostrea gigas McCausland et al., Pavlova sp. 1999 Pavlova pinguis Bacillariophyceae C. calcitran S. costatum Chlorophyceae Dunaliella tertiolecta Cryptophyceae Rhodomonas salina Prymnesiophyceae Isochrysis aff. galbana TISO Pinctada margaritifera Doroudi et al., 1999 P. salina Prymnesiophycea Isochrysis galbana Crassostrea iredalei Devakie and Ali, Bacillariophyceae Chaetoceros calcitran 2000 Prymnesiophycea Isochrysis aff. galbana (TISO) Crassostrea gigas Brown and Robert, Pavlova lutheri 2002 Bacillariophyceae C. Calcitran C. Calcitran forma pumilum C. muelleri Chaetoceros sp. Skeletonema costatum Prymnesiophyceae Pavlova lutheri Crassostrea gigas Rico-Villa et al., Isochrysis affinis galbana 2006 Bacillariophyceae C. calcitran forma pumilum Prymnesiophyceae Isochrysis aff. galbana (TISO) Crassostrea gigas Delaporte et al., Bacillariophyceae C. calcitran Ruditapes 2003 Prasinophyceae Tetraselmis suecica philippinarium

Downloaded from sifisheriessciences.com at 16:27 +0330 on Sunday October 3rd 2021 [ DOI: 10.18331/SFS2018.5.1.2 ] Prymnesiophyceae Isochrysis aff. galbana (TISO) Crassostrea gigas Rico-Villa et al., Bacillariophyceae C. gracilis 2009 S. marinoi Bacillariophyceae C. calcitran Pinctada margaritifera Ehteshami et al., C.muelleri 2011 Prymnesiophyceae Isochrysis sp.

Journal of Survey in Fisheries Sciences 5(1) 2018 13

They are source of energy and form microalgae culture contribute to the part of the biological membrane (Arts et cost of the hatchery. The development al., 2009). However, during digestion, of suitable microalgae diet would be a lipids are hydrolysed into primary major consideration to reduce the component called fatty acid. Thus, the operational cost of a hatchery. Several nutritional value of microalgae is very alternative diets proposed are preserved much related to the specific microalgae, bacteria and yeast, composition of fatty acid than lipid microencapsulated and microbound (Webb and Chu, 1982; Delaunay et al., diets, kaolin and silt (Knauer and 1993). The profile of fatty acids differ Southgate, 1999). The most interesting between microalgae. Certain alternative is refrigerated concentrated microalgae are lack of essential fatty microalgae (Ponis et al., 2003). The acid (Brown et al., 1989; Volkman et nutritional value of Pavlova lutheri can al., 1989). For instance, be maintained for 27 days when docosahexanoic acid (22:6(n-3), DHA) preserved at 1-4°C. The efficiency of content in I. galbana is high, while low this storage is because alga will remain in eicosapentanoic acid (20:5(n-3), as living cells when preserved at low EPA). Conversely, C. gracilis is rich in temperature. Tetraselmis suecica after 3 EPA but deficient in DHA (Brown et months of storage, will still contained al., 1997; Ehteshami et al., 2011). level of EPA equivalent to fresh alga Most microalgae used in bivalve (Montaini et al., 1995). Provision of hatcheries contained high nutritional suitable ration will ensure the success value, especially in polyunsaturated of mollusc hatchery. According to fatty acid (PUFA) of the n-3 series EPA Doroudi et al. (1999), food ration at (eicosapentaenoic acid 20: 5n-3) and 20×103 cells/mL for the larvae of DHA (docosahexanoic acid 22:6n-3) Pinctada margaritifera resulted in the respectively (Brown et al., 1997). These 230 µm anterior posterior length of PUFA are essential for marine larvae. While maximum survival of 8% (Kanazawa et al., 1979). In bivalve when fed at 10×103 cells/mL. Even hatcheries, controlled mass culture of though growth is a major factor for the microalgae required manpower and evaluation of physiological condition, economic investment. In fact, survival percentage is still important at microalgae production cost varies from different culture density.

Downloaded from sifisheriessciences.com at 16:27 +0330 on Sunday October 3rd 2021 [ DOI: 10.18331/SFS2018.5.1.2 ] 15 to 85 % of the overall hatchery Algae ration and larvae density management costs depending on scale influence the growth and survival of of production and cultivation methods larvae. The optimum algae ration varies (Urban and Langdon, 1984; Coutteau depending on the larvae age. D-stage and Sorgeloos, 1992; Knaeur and larvae showed high survival and shell Southgate, 1997). Specialized grow when fed with microalgae at 4.5 – equipment and facilities required for 11.5×103 cell/mL, at larvae density of 14 Fakhrina et al., Production of tropical oyster seed in hatchery

3mL-1 (Doroudi and Southgate, 2000). (2007) indicated the possibility of Larvae of 13- 20 day old has maximum combining seven tropical microalgae as survival when fed with microalgae at feed for D-stage larvae of Pinctada 2.5×103 cell/mL. Best shell growth of margaritifera, with the addition of older larvae is when fed at 15- 32×103 diatom at umbo-stage larvae. Feeding cell/mL of microalgae. Doroudi and with Pavlova sp. supports better growth Southgate (2000) suggested the feeding of D-stage larvae, and with the addition of P. margaritifera at 8×103 cell/mL, of diatom it can increase the growth of with density of 3 larvae/mL up to 8-day umbo-stage larvae. Thus, suggesting old. While for 13 to 20 day old larvae, that Pavlova sp. and Pavlova feeding at 25×103 cell/mL, with sp./Chaetoceros muelleri are good diet stocking of less than 2 larvae/mL. for oyster larvae. Therefore, the concentration of Diet composition may affect the microalgae during culture affects the growth and survival of bivalve larvae. growth and survival of larvae (Doroudi Various algae ration has been used in and Southgate, 2000). Since bivalves the oyster larviculture. A mixture of are filter feeders, they require sufficient microalgae species will provide a better food to continue living. Concentration nutrient balance (Ronquillo et al., 2012). of microalgae to be fed to larvae varies Monospecific diet using Tetraselmis depending on species of microalgae and suecica resulted in the lowest growth of larval stage. The minimum requirement mangrove oyster, Crassostrea of microalgae for Crassostrea gigas corteziensis due to the lack of DHA and larvae is 25×103 cell/mL at D-stage low level of Arachidonic acid (20:4(n- (Rico-Villa et al., 2006). 6), AA) and EPA (Rivero-Rodriguez et Fundamentally, microalgae culture al., 2007). Arachidonic acid (AA) also is important to commercial hatchery of is a fatty acid essential for the growth of marine molluscs. According to juvenile clam Tapes sp. (Caers et al., Martinez-Fernandez et al. (2006), the 1999). Ratio of EPA/DHA affects the used of Pavlova sp. and Pavlova salina growth of mollusc. Juvenile oysters fed in diet resulted in the highest shell grow with microalgae with high levels of at D- stage and umbo stage larvae for essential fatty acid (EPA, DHA, AA) Pinctada margaritifera. Pavlova sp. showed better growth compared to and Isochrysis sp. from the group those containing lower level of fatty

Downloaded from sifisheriessciences.com at 16:27 +0330 on Sunday October 3rd 2021 [ DOI: 10.18331/SFS2018.5.1.2 ] Prymnesiophytes are the most preferred acid (Ronquillo et al., 2012). The eye- due to their size and shape which larvae of C. iredalei showed better facilitated better ingestion. Culture of S. setting rate when fed with both echinata has been quite successful Isochrysis galbana and Chaetoceros using diet composed of T-ISO and P. calcitrans. Meanwhile, larvae given I. salina (Southgate and Lee, 1998). galbana alone produce better results Martínez-Fernández and Southgate than C. calcitran alone. The best Journal of Survey in Fisheries Sciences 5(1) 2018 15

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