www.elsevier.com/locate/aqua-online

Aquaculture 236 (2004) 497–509

Preliminary assessment of a microbound diet

as an Artemia replacement for mud crab,

Scylla serrata, megalopa

Jerome Genodepa1, Chaoshu Zeng*, Paul C. Southgate

Crustacean Aquaculture Research Group, Discipline of Aquaculture, School of Marine Biology and Aquaculture,

James Cook University, Townsville, Queensland 4811, Australia

Received 17 December 2003; received in revised form 13 February 2004; accepted 13 February 2004

Abstract

As an important step toward development of a formulated diet for hatchery culture of the mud

crab, Scylla serrata, this paper reports on laboratory experiments to assess the potential of a

microbound diet (MBD) as a replacement for Artemia nauplii fed to megalopal larvae of S. serrata.

The effects of different proportions of dietary MBD and Artemia on survival and moulting success of

megalopa to the crab stage were investigated. In the first experiment, megalopae were reared

communally and fed either 100% MBD, 100% Artemia or different combinations of the two

(75%:25%, 50%:50%, 25%:75%). The experiment was terminated when all larvae had either

metamorphosed or died. Larvae fed a combination of 25% MBD and 75% Artemia consistently

showed the highest survival among all treatments throughout the experiment. Survival of larvae fed

100% MBD was the lowest early in the experiment but improved to become the second highest

toward the end of the culture period. Overall survival of larvae fed 100% MBD did not differ

significantly from that of larvae fed 100% Artemia. Moulting to the crab stage began on day 7 for

larvae in the treatment receiving a 50%:50% combination of MBD and Artemia. On day 8, all larvae

in treatments receiving greater than 25% MBD had some first stage crabs. Larvae fed Artemia only

were the last to moult to the juvenile crab stage, but moulting occurred simultaneously on day 10.

Because of cannibalism observed in the first experiment, a second experiment was conducted where

megalopae were reared individually and fed either 100% Artemia or 100% MBD. Ninety percent of

larvae from both treatments successfully moulted to the crab stage. Again, megalopae fed MBD

began moulting 1 day ahead of those fed Artemia. The results of these studies show that the MBD

used contained all necessary nutrients to sustain successful moulting of S. serrata megalopae to the

0044-8486/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.aquaculture.2004.02.007

* Corresponding author. Tel.: +61-7-47816237; fax: +61-7-47814585.

E-mail address: Chaoshu.Zeng@jcu.edu.au (C. Zeng).1 Current address: Institute of Aquaculture, College of Fisheries and Ocean Sciences, University of the

Philippines in the Visayas, Miag-ao, Iloilo 5023, Philippines.

J. Genodepa et al. / Aquaculture 236 (2004) 497–509498

crab stage. The acceptability of MBD by S. serrata larvae suggests significant potential for using the

MBD in future experiments to investigate larval nutritional requirements of this commercially

important crab species. Indeed, the more rapid moulting of larvae fed MBD in both experiments

suggests that the MBD may have contained certain beneficial nutrients that were not provided by

Artemia alone. The fact that no significant differences in survival between megalopae fed 100%

MBD and those fed 100% Artemia in both communal and individual rearing experiments suggests

that total replacement of Artemia with MBD is possible for S. serrata megalopae. This could result in

substantial savings in operating costs for S. serrata hatcheries.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Mud crab; Scylla serrata; Megalopa; Microbound diet; Artemia replacement

1. Introduction

Mud crabs of the genus Scylla are commercially important in many Indo-Pacific nations

(Keenan, 1999) and there is growing interest in mud crab farming in the Indo-Pacific

region (Keenan, 1999; Sheen and Wu, 1999; Trino and Rodriguez, 2002). At present, mud

crab aquaculture is based primarily on wild caught juveniles and sustainable growth of the

mud crab aquaculture industry is only likely through the development of appropriate

hatchery techniques. However, despite significant research efforts in this field (e.g. Brick,

1974; Hill, 1974; Heasman and Fielder, 1983; Marichamy and Rajapackiam, 1992;

Hamasaki, 1993, 2003; Mann et al., 1999, 2001; Zeng and Li, 1999; Baylon et al.,

2001), hatchery culture of mud crabs is currently inconsistent and not yet commercially

viable (Keenan, 1999). The major problems experienced in mud crab hatcheries include

substantial variations in larval viability at hatch, mortality due to build-up of pathogenic

microorganisms in the culture system, lack of understanding of larval nutritional require-

ments, ‘moulting-death-syndrome’ phenomenon and cannibalism during later larval stages

(Fielder and Heasman, 1999; Keenan, 1999; Kim and Zeng, Liessmann and Zeng,

unpublished data).

Among these problems, ‘moult-death-syndrome’ occurs mainly during moulting from

Zoea V to megalopa stage, and sometimes from megalopa to first crab stage. It refers to

the phenomenon of high mortality resulting from the inability of crab larvae to

completely shed their old exoskeleton during moulting. It is believed that inappropriate

nutrition is the probable cause of such mortality (Williams et al., 1999; Hamasaki et al.,

2002). Larval nutrition has an important role in maximising larval growth and survival in

mud crab hatcheries. Mud crab larvae are carnivorous; they readily accept rotifers and

Artemia nauplii, which have become standard hatchery feeds (e.g. Brick, 1974; Heasman

and Fielder, 1983; Mann et al., 1999, 2001; Zeng and Li, 1999; Hamasaki, 2003).

However, development of a suitable formulated diet to replace live foods in mud crab

larval production is an important goal considering the problems that may be experienced

when live foods are used. For example, rotifers and Artemia can be deficient in highly

unsaturated fatty acids, which are widely accepted as being essential nutrients for marine

organisms (Liao et al., 1990; Sorgeloos et al., 1991; Navarro et al., 1995). Live feeds are

also known to vary in their nutritional compositions depending on source, age and

J. Genodepa et al. / Aquaculture 236 (2004) 497–509 499

culture technique (Sorgeloos et al., 1986; Tucker, 1992) and they are costly to produce

(Rodgers and Barlow, 1987; Kanazawa, 1990; Pearce, 1991; Rimmer and Rutledge,

1991). Furthermore, live feeds provide vectors for introduction of disease into culture

systems (Person-Le Ruyet, 1990) and this appears to be a particular problem for mud

crab larval culture (Fielder and Heasman, 1999). Early larvae of mud crabs are extremely

sensitive to a build-up of pathogenic bacteria in the culture water and there is evidence

that the main sources of harmful bacteria in mud crab larval cultures are live feeds

(Liessmann and Zeng, unpublished data). As well as offering solutions to some of these

problems (Southgate and Partridge, 1998), suitable formulated diets would also provide a

tool for investigating the nutritional requirements of mud crab larvae, and a potential

means to address problems such as ‘‘moult-death syndrome’’, experienced in mud crab

hatcheries.

Research in this laboratory has shown that microbound diets (MBD), composed of

ingredients encapsulated within a polysaccharide or proteinaceous matrix, are readily

ingested by Scylla serrata larvae. This observation prompted a series of laboratory studies

aimed at eventual development of a formulated diet for S. serrata larvae (Genodepa,

2004). In a previous paper, we reported on experiments using 14C-labelled rotifers as

components of MBD fed to mud crab larvae (Genodepa et al., 2004). Ingestion rates of

MBD by S. serrata larvae were used to determine optimum size range and optimum

rations for MBD fed to different larval stages (Genodepa et al., 2004). As a further step

towards developing a formulated feed that can be used in mud crab hatcheries, it is

necessary to determine the extent to which MBD can be used as a replacement for live

foods over longer term rearing experiments. Total replacement of live foods with

formulated diets has been demonstrated for penaeids (Jones et al., 1987, 1979) and, more

recently, for the freshwater prawn Macrobrachium rosenbergii from 5th stage larvae

onward (Kovalenko et al., 2002). However, it has not been achieved for other caridean

prawn and homarid lobster larvae (Jones et al., 1993) and we are unaware of any similar

research with mud crab (Scylla sp.) larvae.

This paper reports results of laboratory rearing experiments to assess the potential of

MBD as a replacement for Artemia as food for megalopae of S. serrata. It specifically

investigated the effect of different proportions of dietary MBD and Artemia on survival

and moulting success of megalopa to crab stage.

2. Materials and methods

2.1. Source of larvae

Mud crab, S. serrata, spawners were collected from estuaries around Townsville,

north Queensland, Australia, using baited traps. They were maintained in the aquarium

facility at James Cook University until they spawned. Species identity was confirmed

using the criteria outlined by Keenan et al. (1998). Crabs were held in 5000 l outdoor

tanks with re-circulating seawater (temperature ranged from 26 to 30 jC, salinity from

28xto 36x) and fed once daily with squid, mussel and shrimp at a rate of 5–8% body

weight.

J. Genodepa et al. / Aquaculture 236 (2004) 497–509500

Berried crabs were disinfected by bathing them in a 100 l tank for 6 h with formalin

added to the static seawater at a concentration of 50–80 Al l� 1. After disinfection, crabs

were transferred to a 300-l indoor tank for egg incubation and hatching. The tank was

provided with re-circulating water supply (at an exchange rate of 1.5 l min� 1) subject to

mechanical filtration (to 1 Am) and UV treatment. Salinity in the incubation tank ranged

from 32xto 36xwhile temperature ranged from 27 to 29 jC.Vigorously swimming newly hatched larvae were collected by attracting them to a

strong light source. They were stocked into static rearing tanks (150 and 300 l) at a density

of 100–150 larvae l� 1. The newly hatched larvae were directly transferred from the

hatching salinity (32–36x) to 20–22x, which was the salinity used for rearing Zoea I.

The salinity in the larval tanks was gradually increased to 25–28xas the larvae

developed (Genodepa, 2004; Genodepa et al., 2004).

The larvae were fed rotifers (Brachionus sp.) and Artemia nauplii (INVE). Rotifers

were introduced into larval rearing tanks only once (at a rate of 40–60 individuals ml� 1)

on the first day of larval culture. Rotifer populations were then maintained in the larval

rearing tanks by daily addition of microalgae (Nannochloropsis sp). As the larvae grew

older, the rotifer density was gradually reduced to a negligible level by the time the larvae

had reached the Zoea V stage. Artemia nauplii were first introduced into larval rearing

tanks (at the density of 0.5 individuals ml� 1) on the second day after larvae moulted to

the Zoea II stage. Their density was gradually increased to 3–5 individuals ml� 1 by the

time the larvae reached the Zoea V stage. Daily water exchange in larval culture tanks

ranged from 20–25% between Zoea I and Zoea II stages to 30–50% from Zoea III

onward.

2.2. Diet preparation

The ingredient composition of the MBD used in the feeding experiments is shown in

Table 1. Except for a few recent publications on fatty acid requirements of mud crab larvae

(Kobaysahi et al., 2000; Suprayudi et al., 2004), little is known about their nutritional

requirements. Hence, diet formulation was based on the known requirements of adult mud

Table 1

Composition of the microbound diet (MBD) fed to mud crab, S. serrata, megalopae

Ingredient % Dry weight

Squid meal 39.7

Rotifer 39.7

Fish oil 5.0

Corn oil 1.0

Lecithin 3.0

Cholesterol 1.0

Dibasic calcium phosphate (DCP) 0.6

Mineral mixa 4.0

Vitamin mixa 3.0

Zein (binder) 3.0

Total 100

a Based on Kanazawa (1981).

J. Genodepa et al. / Aquaculture 236 (2004) 497–509 501

crabs and the compositions of diets used in previous studies with crustaceans. For

example, the levels of cholesterol, vitamins and minerals were based on the studies of

Kanazawa (1981), Millamena and Quinitio (2000) and Sheen (2000) while the level of

dietary lecithin was based on that used by Kanazawa (1990). The inclusion and

proportions of the oil used (fish oil and corn oil) was based on that used by Castell et

al. (1989).

In prior experiments, 14C-labelled rotifers were used as a major ingredient for MBD to

enable quantitative detection of MBD ingestion and retention rates by mud crab larvae

(Genodepa, 2004; Genodepa et al., 2004). These MBD were well ingested and assimilated

by S. serrata larvae (Genodepa, 2004; Genodepa et al., 2004). On this basis, this diet

composition was maintained for the MBD used in this study with the exception that the14C-labelling procedure of rotifers was omitted. Rotifers used for production of the MBD

for current experiments were first collected on a 53-Am mesh, washed with distilled water

and oven dried at 40 jC for 12 h. They were then incorporated into MBD on dry weight

basis (Genodepa et al., 2004).

The MBD was prepared by combining and thoroughly mixing the dry ingredients and

moist ingredients in separate containers. Both were then combined and mixed thoroughly.

Finally, the binder (zein), which was prepared previously by dissolving it in water, was

added to the diet mixture and the complete diet was thoroughly mixed, spread thinly in an

aluminium dish and oven-dried at 50 jC for 72 h. It was then ground using a mortar and

pestle and sieved to 400–600 Am, the most desirable particle size range for megalopa of S.

serrata (Genodepa et al., 2004).

2.3. Megalopa feeding experiments

Mud crab larvae go through five zoeal stages before they metamorphose to become

megalopae. This takes approximately 15–20 days under the culture conditions specified

above. The megalopae used in the current MBD feeding experiments had been fed

rotifers and Artemia up to the start of the experiment. After metamorphosis, megalopae

are morphologically very different from zoeal larvae. Newly moulted megalopae often

actively swim on or near the surface of water, they can easily be picked up individually

using a large bore pipette. To ensure that larvae used in the feeding trials were at the

similar status of the molting cycle, the first batch of larvae (normally small number) that

moulted to the megalopa stage were removed from the culture tanks in the evening; the

batch of larvae that moulted the following day (usually the majority of larvae moult on

this day) were used for the experiments. They were collected and transferred to a

separate container in the afternoon but experiments were not established until the

next day. Experiments were purposely started the day after metamorphosis to the

megalopa stage because megalopae often show high mortality immediately following

metamorphosis.

In the first experiment, 2-day-old megalopae (the day of zoeal metamorphosis to

megalopae was designated as day-1) were stocked into tall conical-based (45j taper)

plastic culture vessels containing 1 l of seawater. They were stocked at a density of 12

larvae l� 1 and were fed a diet of either MBD or Artemia exclusively, or various

combinations of the two (Table 2). All diets were fed on the same dry weight basis.

Table 2

The proportions of Artemia and microbound diet (MBD) fed to S. serrata megalopa in the dietary treatments of

the first experimenta

Treatment Artemia l� 1 MBD (mg l� 1)

100% MBD 0 16.30

75% MBD+25% Artemia 1250 12.22

50% MBD+50% Artemia 2500 8.15

25% MBD+75% Artemia 3750 4.07

100% Artemia 5000 0

a The 100% ration of Artemia was equal to feeding larvae with 5 individuals ml� 1 Artemia. The 100% ration

of MBD was 16.3� 10� 3 mg ml� 1, based on a mean individual Artemia dry weight of 3.26� 10� 3 mg.

J. Genodepa et al. / Aquaculture 236 (2004) 497–509502

The 100% Artemia ration was 5 individuals ml� 1, and the 100% MBD ration was

calculated on the basis of an Artemia dry weight of 3.26� 10� 3 mg individual� 1 (Table

2). All dietary treatments had five replicates. Megalopae were fed twice daily at 0700 and

1900 h. Each time a full ration (100% ration) was given after a 100% water exchange.

Survival and moulting of larvae was recorded daily during water exchange. Larvae that

moulted to the crab stage during the experiment were removed and placed into a separate

container. The experiment was terminated when all megalopae used in the experiment had

either moulted to the crab stage or died.

Cannibalism occurred in the first experiment under the condition of communal culture.

To eliminate the possibility that nutrients derived from cannibalism may have influenced

successful moulting of megalopae fed MBD, a second experiment was carried out with

megalopae reared individually. Twenty 2-day-old megalopae were stocked individually

into 20� 500 ml round flat-bottomed plastic containers. The containers were filled with

150 ml 1 Am filtered and UV treated seawater. Half of the megalopae were fed MBD only,

while the other half were fed Artemia only. Both diets were fed at the same ration as the

100% Artemia and 100% MBD treatments in the first experiment (Table 2). Feeding, water

exchange and monitoring of survival and development of the megalopae were conducted

as described previously for the first experiment.

2.4. Statistical analysis

Daily percentage survival and the number of megalopae that had moulted to the crab

stage among different treatments in the first experiment were compared using one-way

analysis of variance (ANOVA). Specific differences among treatments were determined

using Duncan’s multiple range test at the 0.05 level of significance. Survival and moulting

in the second experiment were compared using the paired sample t-test. All statistical

analyses were performed using SPSS for Windows, version 10.0.

3. Results

Survival of megalopae in the first experiment from the day following stocking (day 3)

is shown in Fig. 1. Overall survival, as well as survival from day 9 onward, did not

Fig. 1. Mean daily survival (F S.E., n= 5) of mud crab, S. serrata, megalopae fed with different combinations of

zein microbound diet (MBD) and Artemia. Columns on each day with the same superscripts are not significantly

different (P>0.05).

J. Genodepa et al. / Aquaculture 236 (2004) 497–509 503

differ significantly among treatments, but larvae fed the combination of 25% MBD and

75% Artemia consistently had the highest survival throughout the experiment. Survival

of larvae in the 100% MBD treatment was the lowest from day 3 to day 6 but improved

to become the treatment with second highest survival towards the end of the culture

period (from day 9 onward). Survival of larvae in the treatment receiving 100% MBD

did not differ significantly from that of larvae receiving 100% Artemia throughout the

culture period except on day 6. For the various combinations of MBD and Artemia,

survival from day 3 to day 8 was generally lower in treatments receiving a greater

proportion of MBD as compared to those receiving more Artemia (often significantly,

see Fig. 1).

The proportion of megalopa and crab stage within the different treatments from day 6 to

day 11 is shown in Fig. 2. Moulting to the crab stage began on day 7 in the dietary

treatment consisting of a 50%:50% combination of Artemia and MBD, but the majority of

the moults occurred between day 8 and day 10. On day 8, treatments fed with 100%, 75%,

and 50% MBD had some first stage crabs present but there were no crab in treatments

receiving either 25% or 0% MBD (i.e. 100% Artemia) at this stage. On day 9, all

treatments receiving MBD had some first stage crabs present, but still no crabs were

present in the treatment fed Artemia only. Megalopae fed Artemia only were the last to

moult but they moulted simultaneously on day 10. Megalopa that did not moult on day 10

died the following day.

Table 3 shows daily survival and moulting of megalopae in the second experiment. In

treatments receiving either 100% Artemia or 100% MBD, 90% of the megalopae

successfully moulted to the crab stage. Moulting of megalopae fed MBD started 1 day

ahead of those fed Artemia, but moulting was again more synchronous in the latter

Fig. 2. Mean survival and proportion of megalopa and first stage crab from day 6 to day 11 in treatments fed with

different combinations of zein microbound diet (MBD) and Artemia. M and C1 stand for megalopa and first stage

crab, respectively.

J. Genodepa et al. / Aquaculture 236 (2004) 497–509504

Table 3

Daily percent survival and percentage of the first crab stage present in Experiment 2 in which S. serrata

megalopae were reared individually and fed either Artemia or microbound diet (MBD)

Days of Age of Artemia MBD

culture megalopae% Survival % First stage

crabs

% Survival % First stage

crabs

1 3 100 0 100 0

2 4 100 0 100 0

3 5 100 0 100 0

4 6 100 0 100 40

5 7 100 40 100 60

6 8 100 70 100 60

7 9 90 80 100 80

8 10 90 90 100 80

9 11 90 90 100 90

10 12 90 90 90 90

J. Genodepa et al. / Aquaculture 236 (2004) 497–509 505

treatment. The time between the first and the last moult was 5 days for larvae fed MBD but

only 3 days for those fed Artemia.

4. Discussion

The results of the second experiment in which megalopae were reared individually,

showed that an equally high proportion (90%) of megalopae successfully moulted to the

crab stage when fed exclusively either Artemia or MBD. The results highlight a number of

important findings: (1) MBD used in the present study contained all nutrients required to

sustain successful moulting of S. serrata megalopae to crab stage; (2) they dispel the

doubts stemming from the first (communal rearing) experiment about whether successful

moulting of megalopae fed MBD alone relied on nutrients derived from cannibalism; (3)

they show that complete replacement of Artemia with MBD for S. serrata megalopa is

possible.

Total replacement of live feeds with formulated diets for crustacean larvae has been

demonstrated for penaeid prawns (Jones et al., 1979, 1987). More recently, Kovalenko et

al. (2002) reported that a high moisture, semi-purified MBD containing alginate can be

used to totally replace Artemia for the freshwater prawn M. rosenbergii from 5th larval

stage onward. However, development rates of larvae fed MBD alone were significantly

slower than those fed with Artemia in two repeated trials which used larvae from different

females. Furthermore, although differences between larval survival were not significant in

the first trial, survival of larvae fed with MBD was significantly lower than those fed

Artemia in the second trial (Kovalenko et al., 2002).

In contrast, in our experiments, the earlier occurrence of moulting of megalopae to the

crab stage in treatments receiving >50% MBD in both experiments suggests that the MBD

had certain beneficial nutrients that were not provided by Artemia. On the other hand, the

less synchronous moulting of megalopae fed MBD perhaps reflects variation in the ability

of megalopae to adjust from Artemia to the MBD diet. The megalopa used in the

J. Genodepa et al. / Aquaculture 236 (2004) 497–509506

experiments were reared on Artemia from late Zoea II. For treatments where MBD was

offered, the megalopae went through a weaning process during the early phase of the

experiment. This process would have been particularly abrupt for larvae in the treatment

receiving only MBD. The relatively low survival of megalopae fed 100% MBD during the

first 4 days of the first experiment and decreasing survival with increasing MBD ration

during the earlier days of the first experiment suggests significant stresses imposed by the

weaning process on the megalopal larvae; this may have facilitated cannibalism incited by

starvation under the communal culture condition. Interestingly, survival of megalopae

appeared unaffected by the weaning process in the second experiment. This can probably

be explained by the fact that in this experiment, megalopae were reared individually and,

without the option of cannibalism, may have weaned to MBD more rapidly and more

effectively.

The consistently high survival of megalopal larvae fed the combination of 25% MBD

and 75% Artemia suggests that while both MBD and Artemia were adequate feeds on

their own, a combination of the two (in an appropriate proportion) may give improved

results. The benefit of combining different types of foods for mud crab larvae has also

been shown in previous studies. For example, Williams et al. (1999) found Artemia to be

a better diet for mud crab megalopa than Acetes shrimp and mud worm (Marphysa sp.);

however, a combination of mud worm and Artemia gave the highest survival. Quinitio et

al. (1999) found that while mud crab larvae fed with a microencapsulated larval shrimp

diet did not survive beyond the Zoea II, a combination of natural food and the

microencapsulated diet resulted in significantly higher survival to the megalopa stage

compared to that of larvae fed natural food alone. An appropriate combination of MBD

and Artemia fed to S. serrata larvae is also likely to reduce the stress encountered by

larvae during the weaning process.

Survival percentages reported for megalopal larvae of mud crabs are hugely variable

and range from zero or near zero to up to more than 90%, depending on conditions (e.g.

Hamasaki, 1993; Williams et al., 1999; Zeng and Li, 1999; Hamasaki et al., 2002).

However, taking into consideration that a survival range of 30 to 50% was most often

reported (e.g. Hamasaki, 1993; Williams et al., 1999; Zeng and Li, 1999; Hamasaki et al.,

2002), the survival of megalopae in the first experiment was lower than expected. This is

probably due to the type of rearing container used. Tall, cone-shaped culture vessels were

used in the first experiment on the basis that this is likely to help continuous suspension of

the MBD in the water column; however, during the experiment, megalopae were often

observed to settle and aggregate in the very narrow area at the base of the culture vessels

where faeces and debris also accumulated. With newly developed claws, congestion of the

megalopae at the narrow base of the culture vessels appeared to facilitate high rates of

cannibalism. Such conditions are also likely to have caused serious stress to larvae and

infection by pathogenic micro-organisms that may have contributed to the high mortality

observed in the experiment. In the second experiment, where larvae were reared individ-

ually in flat-bottomed culture vessels, survival of megalopa to crab stage reached 90%.

Megalopa survival was not significantly different when fed either MBD or Artemia

alone in both the first and second experiment. In particular high larval survival in the

second experiment clearly showed that the MBD tested was a suitable substitute for

Artemia for S. serrata Megalopae. Partial or complete replacement of live feeds with

J. Genodepa et al. / Aquaculture 236 (2004) 497–509 507

artificial feeds offers considerable cost saving for crustacean hatcheries (Jones et al.,

1993). Rearing of megalopae is considered the most costly part of mud crab larval culture

due to their high consumption rates of Artemia (Zeng, 1998) and the longer duration of the

megalopa stage when compared to the Zoeal larval stages (7–10 vs. 3–4 days). The

possibility of replacing Artemia with a formulated diet at this stage would therefore be

expected to result in significant cost savings in mud crab hatchery culture. Furthermore,

Bautista et al. (1989) highlighted other potential advantages of replacing live food

organisms with artificial diets in crustacean hatcheries. They included: (1) reduced levels

of technical skill required for hatchery operation; (2) reliability in supply of a nutritionally

complete larval feed; (3) reduced potential for contamination and disease introduction; and

(4) simplified hatchery design and reduced capital costs which provide impetus for small-

scale hatchery development.

The high acceptability of the MBD tested in this study suggests that in future

experiments, by further defining chemical composition of ingredients and subsequent

manipulation of diet compositions, it could serve as a useful tool for investigating the

nutritional requirements of S. serrata larvae and would provide a potential means to

address problems such as ‘‘moult-death syndrome’’. Clearly, the easy manipulation of the

dietary content of MBD offers huge potential for such research, which is likely to improve

our knowledge of the nutrient requirements of S. serrata larvae and, in turn, lead to more

efficient cost-effective and reliable hatchery production of this species.

References

Bautista, M.N., Millamena, O.M., Kanazawa, A., 1989. Use of kappa-carrageenan microbound diet (C-MBD) as

feed for Penaeus monodon larvae. Mar. Biol. 103, 169–173.

Baylon, J.C., Failaman, A.N., Vengano, E.L., 2001. Effect of salinity on survival and metamorphosis from zoea

to megalopa of the mud crab Scylla serrata Forskal (Crustacea: Portunidae). Asian Fish. Sci. 14, 143–151.

Brick, R.W., 1974. Effects of water quality, antibiotics, phytoplankton and food on survival and development of

larvae of Scylla serrata (Crustacea: Portunidae). Aquaculture 3, 231–244.

Castell, J.D., Kean, J.C., D’Abramo, L.R., Conklin, D.E., 1989. A standard reference diet for crustacean nutrition

research. I: Evaluation of two formulations. J. World Aquac. Soc. 20, 93–99.

Fielder, D.R., Heasman, M.P., 1999. Workshop 2: larval rearing and nursery production. In: Keenan, C.P.,

Blackshaw, A. (Eds.), Mud Crab Aquaculture and Biology. ACIAR Proceedings, vol. 78. ACIAR, Canberra,

Australia, pp. 209–214.

Genodepa, J., 2004. Development of an artificial diet for larvae of the mud crab, Scylla serrata. MSc thesis,

School of Marine Biology and Aquaculture, James Cook University, Townsville, Australia, 110 pp.

Genodepa, J., Southgate, P.C., Zeng, C., 2004. Diet particle size preference and optimal ration for mud crab,

Scylla serrata, larvae fed microbound diets. Aquaculture 230, 493–505.

Hamasaki, K., 1993. Mass seed production of mud crab. SuisanNoKenkyu 12, 103–115 (in Japanese).

Hamasaki, K., 2003. Effects of temperature on the egg incubation period, and survival and developmental period

of larvae of the mud crab Scylla serrata (Forskal) (Brachyura: Portunidae) reared in the laboratory. Aqua-

culture 219, 561–572.

Hamasaki, K., Suprayudi, S.M., Takeuchi, T., 2002. Effects of dietary n-3HUFA on larval morphogenesis and

metamorphosis to megalops in the seed production of the mud crab, Scylla serrata (Brachyura: Portunidae).

Suisanzoshoku 50, 333–340.

Heasman, M.P., Fielder, D.R., 1983. Laboratory spawning and mass rearing of the mangrove crab, Scylla serrata

(Forskal), from first zoea to first crab stage. Aquaculture 34, 303–316.

Hill, B.J., 1974. Salinity and temperature tolerance of zoea of portunid crab Scylla serrata. Mar. Biol. 25, 21–24.

J. Genodepa et al. / Aquaculture 236 (2004) 497–509508

Jones, D.A., Kanazawa, A., Rahman, S.A., 1979. Studies on the presentation of artificial diets for rearing the

larvae of Penaeus japonicus Bate. Aquaculture 17, 33–43.

Jones, D.A., Kurmaly, K., Ashard, A., 1987. Penaeid shrimp hatchery trials using microencapsulated diets.

Aquaculture 64, 133–146.

Jones, D.A., Kamarudin, M.S., Le Vay, L., 1993. The potential for replacement of live feeds in larval culture.

J. World Aquac. Soc. 24, 199–210.

Kanazawa, A., 1981. Penaeid nutrition. In: Pruder, G.D., Langdon, C., Conklin, D. (Eds.), Proceedings of the

Second International Conference on Aquaculture Nutrition: Biochemical and Physiological Approaches to

Shellfish Nutrition. Louisiana State University, Baton Rouge, LA, USA, pp. 87–105.

Kanazawa, A., 1990. Microparticulate feeds for Penaeid larvae. Advances in Tropical Aquaculture Work-

shop, Feb. 20–Mar. 4, 1989, Tahiti, French Polynesia, AQUACOP IFREMER. Actes de Colloque, vol.

9, pp. 395–404.

Keenan, C.P., 1999. Aquaculture of the mud crab, genus Scylla—past, present, future. In: Keenan, C.P., Black-

shaw, A. (Eds.), Mud Crab Aquaculture and Biology. ACIAR Proceedings, vol. 78. ACIAR, Canberra,

Australia, pp. 9–13.

Keenan, C.P., Davie, F., Mann, D.L., 1998. A revision of the genus Scylla de Haan, 1833. Raffles Bull. Zool. 46,

217–245.

Kobaysahi, T., Takeuchi, T., Arai, D., Seikya, S., 2000. Sustainable dietary levels of EPA and DPA for larval mud

crab during the Artemia feeding period. Nippon Suisan Gakkaishi 66, 1006–1013 (in Japanese with English

abstract).

Kovalenko, E.E., D’Abramo, L.R., Ohs, C.L., Buddington, R.K., 2002. A successful microbound diet for the

larval culture of freshwater prawn Macrobrachium rosenbergii. Aquaculture 210, 385–395.

Liao, I.C., Kanazawa, A., Su, M., Liu, K.F., Kai, H., 1990. Studies on artificial microbound diets for larval grass

prawn, Penaeus monodon. In: Hirano, R., Hanyu, I. (Eds.), Second Asian Fisheries Forum. Asian Fisheries

Society, Manila, Philippines, pp. 337–340.

Mann, D., Asakawa, T., Pizzutto, M., 1999. Development of a hatchery system for larvae of the mud crab Scylla

serrata at the Bribie Island Aquaculture Research Centre. In: Keenan, C.P., Blackshaw, A. (Eds.), Mud Crab

Aquaculture and Biology. ACIAR Proceedings, vol. 78. ACIAR, Canberra, Australia, pp. 153–158.

Mann, D., Asakawa, T., Pizzutto, M., Keenan, C.P., Brock, I.J., 2001. Investigation of an Artemia based diet for

larvae of the mud crab Scylla serrata. Asian Fish. Sci. 14, 175–184.

Marichamy, R., Rajapackiam, S., 1992. Experiments on larval rearing and seed production of the mud crab Scylla

serrata (Forskal). In: Angel, C.A. (Ed.), The Mud Crab: A Report on the Seminar Convened in Surat Thani.

Bay of Bengal Programme, Madras, India, pp. 135–141.

Millamena, O., Quinitio, E., 2000. The effects of diets on the reproductive performance of eyestalk ablated and

intact mud crab Scylla serrata. Aquaculture 181, 81–90.

Navarro, J.C., McEvoy, L.A., Amat, F., Sargent, J.R., 1995. Effect of diet on fatty acid composition of body

zones in the larvae of sea bass Dicentrachus labrax: a chemometric study. Mar. Biol. 124, 177–183.

Pearce, M.G., 1991. Improved growth rates of hatchery reared barramundi. Austasia Aquac. 5, 17–18.

Person-Le Ruyet, J., 1990. Early weaning of marine fish larvae onto microdiets: constraints and perspectives.

Advances in Tropical Aquaculture. IFREMER Actes de Colloque, vol. 9, pp. 625–642.

Quinitio, E.T., Estepa, F.P., Alava, V., 1999. Development of hatchery techniques for the mud crab Scylla serrata

(Forskal): comparison of feeding schemes. In: Keenan, C.P., Blackshaw, A. (Eds.), Mud Crab Aquaculture

and Biology. ACIAR Proceedings, vol. 78. ACIAR, Canberra, Australia, pp. 125–130.

Rimmer, M.A., Rutledge, W.P., 1991. Pond rearing of barramundi larvae. Austasia Aquac. 5, 19–20.

Rodgers, I.J., Barlow, C.G., 1987. Better nutrition enhances the growth of barramundi larvae. Aust. Fish. 46,

30–32.

Sheen, S.-S., 2000. Dietary cholesterol requirement of juvenile mud crab Scylla serrata. Aquaculture 189,

277–285.

Sheen, S.-S., Wu, S.-W., 1999. The effects of dietary lipid levels on the growth response of juvenile mud crab

Scylla serrata. Aquaculture 175, 143–153.

Sorgeloos, P., Lavens, P., Leger, P., Tackaert, W., Versichelel, W., 1986. Manual for the Culture and Use of Brine

Shrimp Artemia in Aquaculture. FAO, Gent, Belgium.

Sorgeloos, P., Lavens, P., Leger, P., Tackaert, W., 1991. State of the art in larviculture of fish and shelfish.

J. Genodepa et al. / Aquaculture 236 (2004) 497–509 509

In: Lavens, P., Sorgeloos, P., Jaspers, E., Ollevier, F. (Eds.), Larvi ’91—Fish and Crustacean Larvicul-

ture Symposium. Special Publication - European Aquaculture Society, vol. 15. ACIAR, Gent, Belgium,

pp. 3–5.

Southgate, P.C., Partridge, G.J., 1998. Development of artificial diets for marine finfish larvae: problems and

prospects. In: De Silva, S.S. (Ed.), Tropical Mariculture. Academic Press, London, pp. 151–170.

Suprayudi, M.A., Takeuchi, T., Hamsaki, K., 2004. Essential fatty acids for larval mud crab Scylla serrata:

implications of lack of the ability of bioconvert C18 unsaturated fatty acids to highly unsaturated fatty acids.

Aquaculture 231, 403–416.

Tucker Jr., J.W., 1992. Feeding intensively cultured marine fish larvae. In: Allan, G.L., Dall, W. (Eds.), Proceed-

ings of the Aquaculture Nutrition Workshop, NSW Fisheries, Brackish Water Fish Culture Research Station,

Salamander Bay, Australia, pp. 129–146.

Trino, A.V., Rodriguez, E.M., 2002. Pen culture of mud crab Scylla serrata in tidal flats reforested with

mangrove trees. Aquaculture 211, 125–134.

Williams, G.R., Wood, J., Dalliston, B., Shelley, C., Khu, C.M., 1999. Mud crab (Scylla serrata) megalopa larvae

exhibit high survival rates on Artemia-based diets. In: Keenan, C.P., Blackshaw, A. (Eds.), Mud Crab

Aquaculture and Biology. ACIAR Proceedings, vol. 78. ACIAR, Canberra, Australia, pp. 131–137.

Zeng, C., 1998. Effects of diet density on feeding rates of larvae of the mud crab Scylla paramamosain from

hatching through metamorphosis. Extended Abstract of International Forum on the Culture of Portunid Crabs,

Boracay, Philippines, 27–28.

Zeng, C., Li, S., 1999. Effects of density and different combinations of diets on survival, development, dry weight

and chemical composition of larvae of the mud crab Scylla paramamosain. In: Keenan, C.P., Blackshaw, A.

(Eds.), Mud Crab Aquaculture and Biology. ACIAR Proceedings, vol. 78. ACIAR, Canberra, Australia, pp.

159–166.