Preliminary assessment of a microbound diet as an artemia replacement for mud crab, scylla serrata, megalopa

Preliminary assessment of a microbound dietas an Artemia replacement for mud crab, Scylla serrata, megalopa Crustacean Aquaculture Research Group, Discipline of Aquaculture, School of Ma

Preliminary assessment of a microbound diet

as an Artemia replacement for mud crab,

Scylla serrata, megalopa

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.

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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; Trin˜o 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

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

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 1by 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

a

Based on Kanazawa (1981)

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 the 14

C-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

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 3mg 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

Table 2

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

a

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

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

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 3shows 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 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).

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.

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

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)

culture megalopae % Survival % First stage

crabs

% Survival % First stage

crabs

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