Seedling production of mud crab Scylla serrata has the potential to be an effective tool to support the demand of mud crab due depleted seed stock from the wild. Moreover, there is high consumer demand on the global production of mud crab Scylla serrata from 1999 to 2000. Generally, consumer for mud crab Scylla serrata is Chinese communities which particularly at China, Vietnam, Singapore, Taiwan, Hong Kong and also Malaysia (Liong, 1993). Further, Malaysia aquaculture only practices a growth out of mud crab until the marketable size. Malaysia is currently in progress to develop mud crab industry by not relying on seed stock from Thailand. The crab production is relatively small about 650 tonnes per year (Liong, 1992). In Malaysia, not many studies have been done on mud crab culture. Studies on the larvae culture by Jamari (1992) shows that the high mortality of the megalopa and crablet stages cause by cannibalism among the larvae. Several studies of rearing mud crab larvae had been reported in Indonesia (Marjono and Arifin ,1993), Philippine (Quinitio, 1997) ,Vietnam (Hoang, 1999) and Japan ( Hamasaki,2002 ) that shows the high percentage of survival rate were 3.2 % , 3.7 % ,24 % and 30%-40%. However, the survival rate is still low ranged from 1% to 30% from zoea to megalopa.
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In Malaysia, the production of mud crab has been decreased begin 1995 to 2005 (625 tonnes to 162 tonnes). However, the production of mud crab in other countries such as Indonesia, Philippines and china were increased significantly. Recently, the market demand for mud crabs has exceeded wild harvest. Mostly, mud crabs were imported from India, Sri Lanka, Indonesia and Bangladesh. The present improvised status of mud crab stock in Malaysia is also compounded by other factor. These included a recent increasing interest in soft-shell and growth out mud crab framing .In addition, more conventional culture method were invented such as compact crab farming system for growth out mud crab. Further, this new breeding system has been widely implemented by Perlis state community under the Perlis State Economic Development Corporation.
In addition, the most factors that contribute interest to the mud crab culture is due to high demand and fetches a good price compare to other crabs species such as blue crab (Callinectes sapidus). During this time, the price has increased from RM 10.00/kg in 2009 to RM 30.00/kg in 2010 (Khalidah, 2009).It seem that the increasing of mud crab price has encourage many coastal fishing communities to initiate trials in floating cage, in specially designed earthen ponds and more recently in pen enclosures in mangrove forests.
Other factor that contributed to increase of mud crab farming is the physical characteristic of mud crab itself which is high tolerance to both nitrate and ammonia. High tolerance is beneficial for mud crab culture due to ammonia is often the most limiting factor on closed aquaculture systems. Their high ammonia tolerance may be attributed to various unique physiological responses which may have arisen due to their habitat preferences (mangrove forest). However, the larval survival is low and need to further investigate using alternative approaches. The first studies of crab seed has been done in Malaysia since 1995 by the Inland Fisheries Branch, Department of Agriculture, Sarawak (Tan, 1997) but the survival rates of megalopa are low. Since there is still no commercial-scale hatchery production of S.serrata, all forms of mud crab culture depend on natural seed supply.
Currently, we still do not have any mud crab stock enhancement in Malaysia such as habitat improvement and restocking. However, there are studies has been made by Kosuge(2001) in brief assessment of stock of mud crab Scylla sp. in Matang forest, Malaysia and proposal for resources management. He proposed to the management plan of Matang Mangrove forest by provided separate fishing restrictions area and other indirect effect and basis information for management of mud crab resources.
Berried S.serrata female were caught in open sea by using Trawl net. The broodstock were migrated to the shore for their spawning. The broodstock are commonly scrubbed to remove mud, encrusting algae, infestations and detritus before introducing the wild broodstock into the hatchery. Selection of good quality of broodstock has been discussed by several researchers in term of captive spawning pattern, environment manipulation, fecundity, egg size, egg quality, nutritional status, broodstock husbandry and stress and microbial influence.
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Mating process occur when the female is in soft shelled state just after moulting .While the female in soft shelled, the male of S.serrata were inseminated to the female for mating(Phelan et al,2007). Maturing and spawning in S.serrata species for tropical country shows high incidences of maturation in females appear to be associated with seasonal rain fall. During the spawning, the female mud crab were migrates to the sea to spawn and release their planktotrophic larvae (Vay, 2001). Normally, the duration of eggs to hatch may take 30 to 40 days.
Factor that contribute to hatching success are environmental control such as salinity, temperature and photoperiod. Normally, the broodstock were kept in salinity at range of 30-35 g/L for captive maturation and spawning of S.serrata in the hatchery (Mann et al., 1999).Salinity is a one of the factor that can stimulate the hatching rate of mud crab S.serrata. A high hatching rate (93.6 %) of mud crab at salinity of 35 ppt was reported by Rusli et al. (1994) when incubated at water temperature of 29-30 oC. This report also indicated low hatching rate achieved when incubated at salinity 20 ppt to 30 ppt which is hatching rate dropped to 65. 9%-69.6 % .Moreover, Salinity at 15 ppt were lowered the hatching rate (15.2%) and the larvae died within 4 hours after hatching.
In the mud crab, egg incubation period is stimulated by temperature in the range of 25 oC and 35 oC. A shorts period of egg incubation of broodstock is 10 days were recorded by Hamasaki (2003) .The temperature were varied seasonally as the broodstock were incubated. Different season has different range of temperature and as increasing temperature in the ranges 20.3-30 oC, the egg incubation period were decreased from 30 day to 10 day. In addition, lower temperature in the range of 18- 22°C (Mann et al., 1999) has higher hatch rate and larger egg. It seems that temperature can affect the period of egg incubation and hatch rate of mud crab S.serrata.
In term of fecundity, mud crab at large size has high fecundity (7.98 ± 1.79 million eggs) with crab size between 146 and 181 mm carapace width (Churchill, 2003).To get a good quality egg ,many factors have been study to determine the egg quality of mud crab. Effect of colour egg and diet were observed by Churchill (2003) to determine a good egg quality. However, the egg colour varies at pale yellow, orange and orange red are not an indicator of egg quality due to indifference of hatch rate. In addition, there is no difference on female size and fecundity was reported on egg quality. Moreover, there have been few studies of diet for mud crab broodstock in effects on larval health and reproductive performance. Millamena et al. (2001) highlighted the importance diet feeding for mud crab broodstock on egg development and larval production. Three difference diets were tested (natural food (mussel and fish), mixed diet (natural food and formulated diet) and formulated diet), each diet is capable to improve maturing and spawning of broodstock mud crab S.serrata. However, Broodstock performance and larval quality were improved when fed with mixed diet. In all treatment, all zoea larvae were successful reared to megalopa when fed mixed diet and the female were ablated. In addition, High fecundity and total zoea were obtained when the female are performed eyes stalked ablation method.
Low survival rate of larvae may occur due pathogen and disease from the broodstock. Broodsrtock mud crabs are usually infested with ectoparasite, fouling organism, fungal and bacteria. LeaÅˆo (2002) identified two species of Haliphthoros, namely H. philippinensis and H. milfordensis .Normally, Haliphthoros spp. were founded among broodstock which aborted their eggs prior to hatching. They reported that H. milfordensis was pathogenic to spawned eggs of S.serrata and it was observed the infection rate of H. milfordensis is 2-5 % at two day after inoculation of zoospores with increasing infection rate ( reaching up to 10% ) at five days. While, H. philippinensis is not pathogenic to spawned eggs of S.serrata.
Other research on the ectoparasite is Lagenidium. The parasites may infest the recently hatched zoea of mud crab and ended in mass mortality (Prastowo and Wagimsan, 1996) .According to Zafran et al. (1993), Lagenidium grew best at 35oC and tolerated temperatures from 20-40oC and pH from 4 to 11. Fungus can be killed by exposure to 10 ppm formalin for 24 hours and it safe for zoea compare to 20 ppm formalin for 5 hours.
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To improved survival rate of larval, research on control Lagenidium in mud crab larval rearing has been conducted by Zafran and Taufik (n.d) and Prastowo and Wagiman (1996). Five kinds of fungicide (treflan, malachite green, formalin, potassium permanganate and caltocyn) have been studied in controlling the fungus and their toxicity to mud crab larvae. The minimum effective concentrations of treflan, malachite, formalin and permanganate to inhibit zoospore production were 0.1 ppm, 0.2 ppm 14 ppm and 9 ppm. High survival rate and hatching rate were reported by Prastowo and Wagiman (1996).Healthy zoea were recorded when zoea where treated with mixtures of caltrocyn (1.3 ppm) and treflan (0.02 ppm) in combination with water exchanged at the rate of 50% every three days.
Effect of antibiotics (Penicillin G and Polymixin-B) in feeding treatments (rotifers and Artemia nauplii) has been tested by Kasry (1986).From his experiment, Kasry (1986) found that higher larval survival (52.1 %) at zoea 5 were obtained when the larval fed with combination of antibiotics (35 ppt Penicillin G and 7 ppm Polymixin-B), rotifer and artemia at density of 15 individual per ml.
Previous studies on the luminescent vibriosis were originally reported in culture shrimp (Penaeus vannamei larvae).It is also a devastating disease in crab larvae. The experiment on the vibrios to zoea of mud crab were reported by Parenrengi et al (1993).They conclude that V. catch, V. alginolyticus and V. Parahaemolyticus are pathogenic to zoea but considered moderate compared to V.harveyii (Boer et al. ,1993; Parenrengi et al.,1993). It seems that zoea is very sensitive to luminous bacteria and it may contribute to the high mortality during early larval stage.
Several studies were done in various aspects such as feed, stocking density, feeding protocol, disease and cannibalism to improve the survival of mud crab larvae. Moreover, others species of crab such as Chinese mitten crab (Eriocheir sinensis) in China (Zhang et al. ,1998; Li et al. ,2001), and blue crab (Callinectes sapidus) in Chesapeake Bay (Secor et al. ,2002; Zmora et al. ,2005) has been successful culture at the hatchery scale. Studies on the hatchery scale larval cultures of mud crab were conducted extensively by Australia country begin 1992. Seed stock of mud crab in Australia is dependent on hatchery while the wild seed stock is banned under their management plan in order to control mud crab fisheries. Low survival of mud crab larvae cultures for zoea to megalopa were recorded by Quitino (2001), Fortes (1999) and Nguyen Co Thach (1997) and had survival rate of 10%, 25 % and 1.5% respectively.
Feeding and nutrition
During the growth and moulting of mud crab larvae, the larvae are going through 5 zoea stages from zoea 1 to zoea 5 and metaphases to megalopa and crablet (Phelan et al., 2007). The hatched larvae leads a planktonic life and when they moult to megalopa stage, they migrated to the estuarine area and grow into adult in estuaries as a benthic juveniles (Vay,2001) .Moreover, early zoea stage shows the digestive system is not well develop and lack of enzyme to support the process of breakdown of food particle (Kumlu, 1999). The early zoea is 1 mm long (Phelan et al., 2007) and occupied with forked tail. The forked tail of larvae is used to capture food and transfer to their mouth (Zeng et al., 1991). During the megalopa stage, the pincher are developed and this development can causes the increasing of cannibalism by grasping among the larvae as survival rate is low (1.55-8%) recorded by Quinitio et al. (2004).
Effects of feed and feeding on growth and survival of mud crab larvae have been tested by several researchers. Yunus (1992) and Wang (2005) found that a higher density of rotifer at 60 ind/mL and 40 ind/ml are required to attain higher survival rates. A study on early feeding has been tested by several researchers such as live Artemia, cryst Artemia, copepod and rotifer. They found that, the early larval stage (Z1 and Z2) were physically weak to search food comparing to Z3, Z4, Z5 and megalopa due to physically active searching food. Currently, survival rate during Z1 and Z2 were improved when larvae fed rotifer. Further, decapsulated cyst of Artemia were tested by Jerome et al. (2005) to improve survival larval at early stage but the result were reported poorly. It seem that a high mortality at Z1 and Z2 due to unsuitable diet size where the size of SS type rotifer (147±11 µm) is bigger than size of the mouth opening of Z1 (100 µm) (Setyadi et al. ,n.d).
Instead of live feed, there is a development of microbound diets for larval culture of mud crab S.serrata. Currently, the research on the nutritional requirements of S.serrata is limited. May-halen et al. (2006) tested three different microbound diets (fish meal, dried rotifer and dried artemia).The survival rate of megalopa to crablet stage showing lower survival those fed microbound containing dries rotifer and dried Artemia compare to microbound containing fish meal or squid (46.7% to 60.0%). The highest survival of megalopa to crablet was fed live Artemia (80%).Other studies on microboud diet were tested from zoea 3 to zoea 5, a high survival rate and development rate at the zoea 3 to zoea 5 stage (66%) were recorded for larvae fed the 50%:50% combination of microbound diet and Artemia from his experiment .They concluded that the microbound diet particle has a great potential for both zoea and megalopa diet and it is cost-effective for seedling production of mud crab S.serrata.
Another factor that contributes to mortality in mud crab larva culture is due to poor nutritional, especially in highly unsaturated fatty acids (HUFA) in live food and cannibalism among megalopa and crablet (Truong, 2008). A study conducted by Suprayudi (2004) showed that the effects of Artemia enriched with eicosapentaenoic and docosahexaenoic acid on survival and occurrence of molting failure in megalop larvae .The larvae were fed enriched Artemia with different type of oil. Enriched Artemia appears to be superior to unenriched Artemia. High survival of larvae can be maintained and accelerating intermolt by provided an optimum EPA and DHA at 0.71-0.87% and 0.49-0.72% for larvae feeding. Further, the finding result on the level of EPA and DHA for survival larval were supported by Mann et al. (2001) as his found there were no significant improvement in larval survival as the mud crab larvae fed enriched Artemia at the levels of EPA (39 mg/g) and DHA (15 mg/g) .Moreover , Mann et al. (2001) found that the phospholipid is one of the nutritional component need to study in effect of survival of mud crab larval .Currently ,there are no study on phospholipid in the diet of mud crab larvae.
Another report by Suprayudi et al. (2002) where indicated that survival rate of S.serrata increase with the increase of the total (n-3) highly unsaturated fatty acid (Σ (n-3) HUFA) content of rotifers. From his experiment, Suprayudi et al. (2002) found that the total (n-3) highly unsaturated fatty acid (Σ (n-3) HUFA) in rotifer can be increase from 3-5 mg/g to 7.6-8 mg/g. In term of survival for each larval stage, Suprayudi et al.(2002) found that high mortality through the moult to megalopa to first crab were recorded after fed boosted rotifer at 31 mg/g Σ(n-3) HUFA. Other research on the boosted rotifer indicated that a high mortality at metamorphosis to megalopa with fed boosted rotifer containing Σ(n-3) HUFA levels above 6 mg/g(Hamasaki et al. ,2002 ) .It seem that the specific (n-3) HUFA's may be more important for growth mud crab of mud crab larvae
To improve survival rate of zoea and larvae nutrition, Suprayudi et al. (2004) examined the effect of essential fatty acids (EFA) on the survival, development and bioconversion of fatty acids in mud crab S.serrata larvae. From his experiment, he found that the content of EFA in Artemia were strongly influenced the survival of mud crab larvae compared to rotifers. They found that EFA deficiency were reported at each larvae fed unenriched Artemia, enriched Artemia and enriched rotifer, indicating that DHA was superior to that of EPA. They conclude that mud crab larvae have a limited or negligible capability to convert C18 unsaturated fatty acids to highly unsaturated fatty acids.
Disease of mud crab larvae
Bacteria infestations are major problem for rearing larvae and contribute high mortality. Studies on the bacteria profile in rearing water with Scylla serrata larvae was noted high mortality from zoea 5 to megalopa due to the increase in luminescent bacterial load in the larvae (Quitino, 2001). Further, protozoa can contribute high mortality during egg and larval stages. This protozoon has an ability to interfere with gas exchange that blocked respiratory surfaces of the egg and larvae (Lavilla-Pitogo et al., 2004). Although these organisms do not invade the underlying tissues, they make it difficult for the affected larvae to move and to feed.
Cannibalism behaviour of mud crab larvae indicated high mortality when metamorphosis from megalopa and crablet stage. Study by Juliana (1999) showed that high survival at crablet stage when reared with mud substrate and without shelter. Crablet were tested with different treatment such as coconut leave, mangrove twigs and mud substrate. In addition, asynchronous moulting of mud crab larvae is a one of the factor that contributed to the increased mortality during zoea 4 and zoea 5 (Quinitio, 2001).
Water quality parameter
The optimal condition in rearing mud crab larvae has been conducted in China, Vietnam, South Africa and Australia. Parameters that have been studies are temperature, salinity and total ammonia. The early larvae has a ability to tolerate with range temperature between 25 oC to 30 oC but shorten their development time when reared at a upper temperature ( 29-30 oC) (Dat ,1999; Li et al. ,1999; Quinitio et al. ,1999; Mann et al. ,2001; Quinitio et al. ,2001). In addition, the highest survival rate and growth rate were recorded when the larval reared at higher temperature ranged 28.5 oC to 31 oC and the minimum were recorded when the larval reared at low temperature ranged 22-24 oC (Marichamy, n.d).
The optimal salinity for all larval stages larvae are 27 ppt to 31 ppt (Gui-Zhong, 2005).Other experiment on the optimal salinity for larval rearing was observed by Marichamy (n.d).From his experiment, he found that the optimal salinity for larvae rearing was 35 ppt with the highest production of larval and poor survival when reared at the lower salinity (32-33 ppt).However, high salinity at 36 ppt were not suitable for larval growth. To improve survival of zoea, Baylon (2001) were investigated the effect of salinity on survival and metamorphosis from zoea to Megalopa of the mud crab S.serrata. Each of larval stages (Z1, Z2, Z3, Z4, and Z5) of Scylla Serrata was tested with different salinity (12, 16, 20, 24, 28 and 32 ppt) and different salinity regime. In all treatment, highest metamorphosis to megalopa occurred where salinity was constant at 32 ppt. Further, there is no significant difference on the duration of development of the surviving larvae in all salinity regimes.
Other factor that contributes to high mortality is ammonia. However, there is a few research has been conducted on the ammonia tolerance in mud crab larvae. Churchill (2003) tested concentrations of total ammonia ranged (NH3 and NH4 +) ranged from 25 ppm to 450 ppm at 25 ppm intervals. They concluded that S.serrata larvae are highly tolerated with high levels of nitrogenous waste. From the stress test ammonia, the optimum concentrations of total ammonia for zoea mud crab are 20 and 30 mg/l where the zoea activity was remained normal during total ammonia test. Currently, there is no research has been conducted on the tolerance of Scylla larvae to extremes in pH and oxygen concentration.
Attempt to develop techniques for mud crab (S.serrata larvae) culture system has been made since 1999. Most of the culture systems were adapted from culture shrimp (Penaeus vannamei larvae) system. Currently, there are three different culture system have been used in larvae rearing ranged from exchanged of water system, mesocosm system and green water culture system.
To control water quality during the larvae rearing, exchanged of water were done by either a constant flow-through basis, or by draining or siphoning 50-85% of the tank volume daily and replacing it with clean seawater, or by recirculation a biofilter (100% every 2-3 hours) (Nghia et al. 2001).Under green water culture system ,the water were not exchanged for the first three day .Thereafter, water exchange is slowly increased from 10-20% per day for Z2-Z3 to between 40 and 50% per day at the end of the rearing cycle (Z4-M) (Mann et al. ,1999 ;Quinitio et al. ,2001).
For the mass culture,mesocosm system has been used in Japan where the tank are partially filled with green-water at Z1 (20-25% volume. As the larvae metamorphosis to Z2 and Z3 stages, clean seawater were used instead of green water and the water exchanged on flow-through basis (Hamasaki et al., 2002) during the Z4 and M stages.
To improve water quality on the bottom of tank, dead larvae and uneaten food that accumulate on the tank bottom are daily siphoned out of rearing vessels (Quinitio et al., 2001; Baylon and Failaman, 2001) and care must be taken to avoid siphoning out larvae which have sunk to the bottom of the container. Another problem during larvae rearing is a development of biofilm on tank sides .A high survival were achieved when the biofilm was removed daily (Williams et al., 1998).In addition, improper cleaning the biofilm can release large amounts of bacterial flock into the water column.
Green water culture system is functioned by provide the larvae with microalgae such as Tetraselmis, Skeletonema, Chlorella, Nannochloropsis, Chaetoceros and Isochrysis at densities ranging from 5x104 to 5x105 cells/ml (Djunaidah, et al. ,1998; Dat ,1999; Mann et al. ,1999; Williams et al. ,1999; Zeng and Li ,1999; Mann et al. ,2001; Quinitio et al. ,2001) in order to increases survival and to serve as food for rotifers and Artemia. However, there is not scientific study on the effect of background algae on larval survival and growth of mud crab S.serrata.
Basically, the mud crabs inhabit a turbid estuaries and it seems to require phase. However, there is no significant effect on survival between 12 and 18 hour photoperiods (Nghia et al. ,2001).Further, there is a experiment on the effect of tank colour (black, dark green, maroon, sky blue and white) on larval survival and development of mud crab Scylla serrata .From his experiment, Abed Golam et al.(2005) found that a higher survival and shorter development of zoea were recorded in darker colour background compare to white background .Moreover, the larvae where reared with black background have more efficient feeding as reduced settlement on the bottom of rearing tank. They concluded that background colour was significantly affecting larval survival.