Effects of temperature and salinity on the survival and ...

Aquaculture Research, 2007, 38, 1529^1538

doi:10.1111/j.1365-2109.2007.01810.x

Effects of temperature and salinity on the survival and ˚ l), development of mud crab, Scylla serrata (Forsska larvae Rahmi Nurdiani & Chaoshu Zeng Tropical Crustacean Aquaculture Research Group, School of Marine and Tropical Biology, James Cook University,Townsville, Qld 4811, Australia Correspondence: Dr C Zeng, Tropical Crustacean Aquaculture Research Group, School of Marine and Tropical Biology, James Cook University,Townsville, Qld 4811, Australia. E-mail: [email protected] Present address: Fisheries Faculty, Brawijaya University, Malang, East Java 65145, Indonesia.

Abstract The combined e¡ects of temperature and salinity on larval survival and development of the mud crab, Scylla serrata, were investigated in the laboratory. Newly hatched larvae were reared under 20 1C temperature and salinity combinations (i.e. combinations of four temperatures 25, 28, 31, 34 1C with ¢ve salinities 15, 20, 25, 30, 35 g L 1). The results showed that temperature and salinity as well as the interaction of the two parameters signi¢cantly a¡ected the survival of zoeal larvae. Salinity at 15 g L 1 resulted in no larval survival to the ¢rst crab stage, suggesting that the lower salinity tolerance limit for mud crab larvae lies somewhere between salinity 15 and 20 g L 1. However, within the salinity range of 20^35 g L 1, no signi¢cant e¡ects on survival of zoeal larvae were detected (P40.05). The combined e¡ects of temperature and salinity on larval survival were also evident as at low salinities, both high and low temperature led to mass mortality of newly hatched larvae (e.g. 34 1C/15 g L 1, 34 1C/20 g L 1 and 25 1C/15 g L 1 combinations). In contrast, the low temperature and high salinity combination of 25 1C/35 g L 1 resulted in one of the highest survival to the megalopal stage. It was also shown that at optimal 28 1C, larvae could withstand broader salinity conditions. Temperature, salinity and their interaction also signi¢cantly a¡ected larval development. At 34 1C, the mean larval development time to megalopa under di¡erent salinity conditions ranged from 13.5 to 18.5 days. It increased to between 20.6 and

r 2007 The Authors Journal Compilation r 2007 Blackwell Publishing Ltd

22.6 days at 25 1C. The e¡ects of salinity on larval development were demonstrated by the fact that for all the temperatures tested, the fastest mean development to megalopa was always recorded at the salinity of 25 g L 1. However, a di¡erent trend of salinity e¡ects was shown for megalopae as their duration consistently increased with an increase in salinity from 20 to 35 g L 1. In summary, S. serrata larvae tolerate a broad range of salinity and temperature conditions. Rearing temperature 25^30 1C and salinity 20^35 g L 1 generally result in reasonable survival. However, from an aquaculture point of view, a higher temperature range of 28^30 1C and a salinity range of 20^30 g L 1 are recommended as it shortens the culture cycle.

Keywords: Scylla serrata, salinity, temperature, larval survival, larval development

Introduction Mud crabs of genus Scylla, also known as mangrove crabs, are widely distributed throughout the IndoPaci¢c region (Keenan 1999). They are an important traditional ¢shery resource in the region and, more recently, have become a targeted species for aquaculture (Keenan 1999; Trino & Rodriguez 2002). Among the four mud crab species, Scylla serrata (Forsskl) is the largest one with the widest distribution (Keenan, Davie & Mann 1998). It has been reported that

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E¡ect of temperature & salinity on mudcrab larvae R Nurdiani & C Zeng

S. serrata spends most of its life in brackish, saltwater estuaries or mangrove forests, while mature females migrate o¡shore to spawn and release their larvae (Hill 1994). Despite the lack of direct evidence, it was speculated that migration back to coasts and estuaries occurs during the megalopal stage, before settlement as juvenile crabs (Hill, Williams & Dutton 1982; Baylon, Failaman & Vengano 2001). Mud crab farming has been conducted in Guangdong province, China, for more than a 100 years and over the past 30 years, throughout Asia (Keenan 1999; Williams & Primavera 2001). During the past decade, mud crab farming has rapidly expanded as it provides a feasible alternative to the struggling prawn industry in the region (Keenan 1999; Sheen & Wu 1999; Williams & Primavera 2001; Trino & Rodriguez 2002). However, mud crab aquaculture largely relies on wild-caught juvenile crabs (Keenan 1999; Trino & Rodriguez 2002) for which supply is limited and unreliable. This has become one of the main obstacles to the further expansion of the industry (Keenan 1999). Clearly, to achieve sustainable growth of the mud crab aquaculture industry, the establishment of reliable hatchery techniques is essential. However, despite signi¢cant research e¡orts in this ¢eld (Brick 1974; Hill 1974; Heasman & Fielder 1983; Zeng & Li 1992, 1999; Mann, Asakawa & Pizutto 1999; Baylon et al. 2001; Mann, Asakawa, Pizzutto, Keenan & Brock 2001; Hamasaki, Suprayudi & Takeuchi 2002; Suprayudi, Takeuchi, Hamasaki & Hirokawa 2002; Hamasaki 2003; Baylon, Bravo & Maningo 2004; Zeng, Li & Zeng 2004; Genodepa, Southgate & Zeng 2004a; Ruscoe,Williams & Shelley 2004a; Genodepa, Zeng & Southgate 2004b; Rabbani & Zeng 2005), many problems remain unresolved. This has led to inconsistent and often low larval survival in hatchery operations (Fielder & Heasman 1999; Keenan 1999). Further research is therefore required to better understand larval culture requirements and to improve culture techniques. Temperature and salinity are two of the main environmental parameters that a¡ect aquatic larval survival and development. Both of them can be controlled in a hatchery. While the combined e¡ects of temperature and salinity on larvae of many crab species (Costlow, Bookhout & Monroe 1960, 1962; Christiansen & Costlow 1975; Dawirs 1979; Rothlisberg 1979; Blaszkowski & Moreira 1986; Laughlin & French 1989; Anger 1991; Schuh & Diesel 1995) as well as S. serrata juveniles (Ruscoe, Shelley & Williams 2004b) have been documented, previous

1530


studies on larval mud crabs have focused on the effects of a single factor, i.e. either temperature or salinity (Zeng & Li 1992; Parado-Estepa & Quinito 1999; Baylon et al. 2001; Hamasaki 2003). Although Hill (1974) had conducted an experiment on toleration of newly hatched Zoea I larvae to various temperature and salinity combinations, the trial lasted for only 24 h and larvae were not fed. Because S. serrata is often favoured among other mud crab species for aquaculture due to its fast growth, large size and wide distribution (Baylon et al. 2001;Williams & Primavera 2001), there is clearly a need to investigate the combined e¡ects of temperature and salinity on larval survival and development of S. serrata.

Materials and methods Sources of larvae Mud crab, S. serrata, females with carapace length 14 cm were collected from estuaries around Townsville, Queensland, Australia, using baited traps. They were maintained in the aquarium facility at James Cook University until they spawned. Species identi¢cation was con¢rmed using the criteria outlined by Keenan et al. (1998). Crabs were held in 5000 L outdoor tanks with recirculating seawater (the temperature ranged from 26 to 30 1C, and salinity ranged from 28 to 33 g L 1) and were fed once daily in the evening with squid, mussel or shrimp at approximately 5^8% body weight. Any uneaten feed was removed in the morning. A sand tray was provided to facilitate the attachment of eggs to the pleopod of female crabs at spawning. Larvae used for the present experiment hatched from a spawning on 20 January, 2003. The newly berried crab was disinfected in a100 L formalin bath (formalin concentration 50 mL L 1) for 6 h. The female was then transferred to a 300 L indoor tank for egg incubation and hatching. The incubation tank was provided with a recirculating water supply (exchange rate approximately 1.5 L min 1) subject to mechanical ¢ltration (to1 mm) and ultra-violet treatment. Salinity in the tank was 34.5 1.5 g L 1 while water temperature was maintained at 27 1 1C. Samples of eggs were regularly taken from the female to monitor embryonic development and to predict hatching time. Two days before hatching, the crab was disinfected again with 50 mL L 1 formalin for 6 h. The crab was not fed during the incubation period. Hatching took place on the early morning of 31 January, 2003. Soon after hatching, aeration and

r 2007 The Authors Journal Compilation r 2007 Blackwell Publishing Ltd, Aquaculture Research, 38, 1529^1538



water £ow were turned o¡ to allow larvae to aggregate to the water surface. Actively swimming larvae were then collected by attracting them to a strong light source.

Experiment design and set up Based on the results of previous single factor temperature and salinity experiments on mud crab larvae (Zeng & Li 1992; Parado-Estepa & Quinito 1999; Baylon et al. 2001; Hamasaki 2003), four temperatures, 25, 28, 31 and 34 1C ( 0.5 1C), and ¢ve salinities, 15, 20, 25, 30 and 35 g L 1 ( 1.0 g L 1), were selected for the present experiment. Because the aim of the present study was to identify optimal temperature and salinity combinations for S. serrata larvae, the temperature and salinity range were purposely chosen to be generally within the recognised larval tolerance limits. A 4 5 factorial experimental design that resulted in 20 various combinations of temperature and salinity was adopted. For each temperature and salinity combination, three replicates were set up and each replicate (unit) consisted of 25 larvae reared in a 600 mL beaker ¢lled with 400 mL seawater. A total of 60 culture units were set up initially. More units were later added to separate newly appeared megalopae from the remaining zoeae to reduce incidents of cannibalism. Seawater with desired salinities were made up daily by diluting a source natural seawater (35^ 38 g L 1) with de-chlorinated freshwater. Temperatures were maintained using 12 identical 24.5 L water baths (L W H 5 50 35 14 cm) and immersion heaters. No aeration was provided during larval culture, and a photoperiod of L:D 516 h:8 h was adopted throughout the experiment. Streptomycin sulphate (Sigma-Aldrich, St Louis, Missouri, USA) was added daily to all culture units at 10 mg L 1 to prevent bacterial infection. Larvae were fed a combination of 40 ind. mL 1 s-strain rotifers (Brachionus sp.) and 4 ind. mL 1 Artemia nauplii (unenriched) from Zoea I to Zoea V. At the megalopal stage, they were fed Artemia only at a rate of 8 ind. mL 1. This feeding regimen was derived based on the results of previous studies (Zeng & Li1999; Hamasaki 2003). Microalgae Nannochloropsis sp. was used for rotifer culture while Artemia [GSL AAA, INVE (Thailand), Phichit, Thailand] were hatched daily. During the initial set-up, larvae collected from the hatching tank were ¢rst stocked into several 3.5 L

bowls for acclimatizing to the desired temperature and salinity conditions. They were gradually acclimated to a particular temperature/salinity condition by a stepwise increase/decrease of temperature or salinity to the next level, allowing a 2-h acclimation period at each level. After acclimatization, 25 healthy larvae were randomly selected and transferred to each beaker to complete the set-up. Every morning during the experiment, larvae were transferred with a large bore pipette to new beakers ¢lled with freshly prepared feed and seawater with the desired salinity. Larval mortality and moults were recorded daily during the transfer. Before daily water exchanges, beakers containing fresh seawater were conditioned in water baths to the desired temperatures. Once larvae reached the megalopal stage, to reduce cannibalism, any newly appeared megalopae were transferred to newly setup culture vessels with identical temperature and salinity conditions. A treatment terminated when all larvae had either died or metamorphosed to the ¢rst crab stage.

Data analysis Zoeal larval survival was presented as cumulative survival (%) to each larval stage, which was calculated as the number of larvae that moulted successfully to a particular larvae stage divided by the initial number of larvae in each replicate. Meanwhile, zoeal larval development was expressed as the mean cumulative development time from hatching to each larval stage. Larval survival and development to the megalopal stage under various treatments were analysed with multivariate analysis of variance (MANOVA) as MANOVA has a number of desirable traits for comparing correlated variables compared with performing a series of separate analysis of variance (ANOVAs) (Zar 1999). When any signi¢cant di¡erences (Po0.05) were found, a separate univariate ANOVA was conducted on each of the variables (i.e. survival and development time) (Zar 1999). The Tukey HSD test was performed to detect speci¢c signi¢cant di¡erences among treatments at the 0.05 con¢dence level (Po0.05). Untransformed data were used for analysis as tests showed that data transformation did not improve normality and homogeneity of variance. Statistical analysis was not conducted for each zoeal stage, as those data were not independent. STASTISTICA 6.0 was used for all statistical analysis.


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Results Larval survival The survival of S. serrata larvae to megalopa under 20 di¡erent temperature/salinity combinations is shown in Table 1. Among the four temperatures tested, larvae generally showed higher survival at 25 and 28 1C than at 31 1C. At 34 1C, the highest temperature tested, larval survival was substantially lower than at all the other temperature treatments (Table1; Fig.1). The highest larval survival to the megalopal stage (440%) was recorded at temperature/ salinity combinations of 25 1C/35 g L 1 (54.7%), 25 1C/ 30 g L 1 (52.0%), 25 1C/25 g L 1 (49.3%), 28 1C/ 20 g L 1 (50.7%) and 28 1C/25 g L 1 (42.7%), which were not signi¢cantly di¡erent (P40.05) (Table 1). Statistical analysis showed that larval survival of S. serrata to the megalopa stage was signi¢cantly affected by both temperature (F 5 47.403, d.f. 53, P 5 0.0001) and the interaction of temperature and salinity (F 5 2.869, d.f. 59, P 5 0.0001). At low salinity 15 g L 1, despite the fact that two larvae managed to moult to the megalopal stage at 28 1C, no survival to the ¢rst crab stage was recorded for all the temperatures tested. This suggests that the lower salinity tolerance limit for mud crab larvae lies somewhere between salinity 15 and 20 g L 1. However, within the salinity range of 20^35 g L 1, no signi¢cant e¡ects on larval survival to megalopa were detected (F 5 2.252, d.f. 53, P 5 0.101). Figure 1 shows cumulative larval survival to various stages for all temperature and salinity combinations; the interaction of temperature and salinity was evident.Within the range of temperature and salinity tested, both a low (25 1C) and a high temperature (34 1C) combined with low salinity (15 g L 1) appeared to have the most detrimental e¡ects on early larvae of S. serrata. At 25 1C/15 g L 1, the survival of newly hatched larvae to Zoea II was the lowest (17.3%) among all treatments (Fig. 1a). At the same time, temperature and salinity combinations of 34 1C/15 g L 1 and 34 1C/20 g L 1 led to total larvae mortality at Zoea II and IV stages respectively (Fig. 1d), resulting in the two treatments having the earliest total larval mortality. In contrast, a low temperature of 25 1C, combined with a high salinity of 35 g L 1, resulted in one of the highest larval survival to the megalopal stage (Table 1; Fig.1a). While at temperatures of 25, 31 and 34 1C, a low salinity of 15 g L 1 led to the lowest larval survival among the salinities tested, larval survival fared much better at 28 1C. Actually, larval survival at

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Table 1 Cumulative percentage survival (%) of the mud crab Scylla serrata zoeal larvae (from hatching to megalopal stage) reared under 20 various temperature and salinity combinations Temperature ( 1C) Salinity (g L 15 20 25 30 35

1

)

25 f

0 37.3abcd 49.3ab 52.0ab 54.7a

28

31 ef

2.7 50.7ab 42.7abc 30.1abcd 28.2abcde

f

0 33.3abcd 37.3abcd 28.0bcde 20.0cde

34 0f 0f 13.3def 13.7def 2.8ef

Values with di¡erent superscript letters are signi¢cantly di¡erent (Po0.05).

28 1C/15 g L 1 was not substantially di¡erent from the other salinity treatments until Zoea-IV when mass mortalities started. Nevertheless, two larvae eventually developed to the megalopal stage at 28 1C/15 g L 1, which was the only temperature condition under which larvae managed to reach megalopae with a rearing salinity of 15 g L 1 (Fig. 1b; Table 1). At a high temperature of 34 1C, larval survival declined substantially at Zoea V, leading to the lowest overall zoeal survival among the temperatures tested (Table 1; Fig. 1d). Mass mortalities, and in some cases total mortality, occurred during later zoeal stages, suggesting that their tolerance to temperature and salinity probably becomes narrower (Fig.1).

Larval development As expected, temperature has profound e¡ects on the development of S. serrata larvae (F 5 31.045, d.f. 53, P 5 0.0001). Under the same salinity condition, the cumulative larval development time to each larval stage generally decreased with an increase in temperature (Table 2). The fastest development was recorded for larvae reared at 34 1C/25 g L 1 with the ¢rst appearance of megalopa and the crab on days 12 and 18 respectively (Table 2). At 34 1C, the average larval development time to megalopa ranged from 13.5 to 18.5 days at various salinities. It increased substantially to 20.6^22.6 days at 25 1C (Table 2). Interestingly, except those larvae reared at 28 1C/ 15 g L 1, there was generally no considerable di¡erence in larval cumulative development times to the megalopal stage among those reared at 28 and 31 1C (Table 2). At a temperature and salinity combination of 28 1C/15 g L 1, two larvae developed to megalopae



(a)


Temperature 25°C 15 gL−1 20 gL−1 25 gL−1 30 gL−1 35 gL−1

100 % Survival

80 60 40 20 0 Zoea II

(b)

Zoea III

Zoea IV Larval stages

Zoea V

Megalopa

Temperature 28°C

100 15 gL−1 20 gL−1 25 gL−1 30 gL−1 35 gL−1

% Survival

80 60 40 20 0 Zoea II

Zoea III

Zoea IV Zoea V Larval stages

Megalopa

Temperature 31°C

(c)

% Survival

100 80

15 gL−1 20 gL−1 25 gL−1 30 gL−1 35 gL−1

60 40 20 0 Zoea II

Zoea III


Megalopa

Temperature 34°C

(d)

% Survival

100

15 gL−1 20 gL−1 25 gL−1 30 gL−1 35 gL−1

80 60 40 20 0 Zoea II

Zoea III


Megalopa

Figure 1 Cumulative survival (%) (mean SE) to each zoeal stage of the mud crab Scylla serrata larvae reared under 20 di¡erent combinations of temperature and salinity.

on days 21 and 25, respectively, which was substantially longer than those of larvae reared under other salinities (Table 2). Besides temperature, statistics showed that larval cumulative development time to the megalopal stage

was also signi¢cantly a¡ected by salinity (F 5 3.54, d.f. 53, P 5 0.0254) and the interaction of temperature and salinity (F 5 5.7, d.f. 59, P 5 0.0001). This is evident from the fact that for all the temperatures tested, the shortest average development time to


1533

1534

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

1

3 2 2 2 2

3 3 3 3 3

3 3 3 3 3

5 4 4 4 4

Min.

3 5 5 5 5

6 8 7 6 8

5 7 6 6 5

7 6 7 8 6

Max.

To Zoea II

3.0 3.1 2.8 3.1 3.2

3.8 3.3 3.8 4.0 5.0

3.3 3.4 3.2 3.2 3.3

5.7 4.6 4.5 4.9 5.1

x

0.0 0.2 0.1 0.2 0.1

0.1 0.1 0.2 0.2 0.1

0.1 0.1 0.0 0.0 0.0

0.1 0.1 0.1 0.1 0.2

SE

– 4 4 4 4

6 5 5 6 6

6 5 5 5 5

11 7 7 7 7

Min.

– 8 6 10 7

8 8 9 9 12

11 9 8 9 12

18 10 11 10 11

Max.

To Zoea III

– 4.9 4.8 5.4 5.8

6.9 6.3 6.6 7.2 7.5

6.7 5.8 5.5 5.9 6.5

12.6 7.8 7.9 7.8 8.5

x

– 0.2 0.2 0.2 0.1

0.25 0.1 0.2 0.1 0.6

0.1 0.1 0.1 0.1 0.4

1.1 0.2 0.1 0.1 0.2

SE

– 8 6 7 7

9 8 8 8 8

9 7 7 7 7

16 11 10 10 11

Min.

– 8 10 11 15

10 13 14 17 14

14 15 12 12 13

19 20 15 17 18

Max.

To Zoea IV

– – 7.8 8.5 9.1

9.4 9.3 9.6 9.7 10.0

10.6 8.9 8.3 8.9 9.3

17.3 12.3 11.5 11.8 12.5

x

Only one larva survived. Values with di¡erent superscript letters are signi¢cantly di¡erent (Po0.05). Min., minimum; max., maximum; x, average days required to reach each stage; SE, standard errors.

25 1C 15 g L 20 g L 25 g L 30 g L 35 g L 28 1C 15 g L 20 g L 25 g L 30 g L 35 g L 31 1C 15 g L 20 g L 25 g L 30 g L 35 g L 34 1C 15 g L 20 g L 25 g L 30 g L 35 g L

Treatment

– – 0.3 0.2 0.4

0.3 0.2 0.5 0.4 0.7

0.3 0.1 0.1 0.1 0.2

0.2 0.1 0.2 0.2 0.3

SE

– – 9 10 10

16 11 10 11 11

12 10 10 10 10

24 15 14 14 15

Min.

– – 16 16 18

16 16 18 19 16

20 19 13 15 16

24 23 20 19 23

Max.

To Zoea V

– – 0.3 0.3 0.9

– 0.3 0.7 0.3 1.0

– 12.5 11.8 12.5 13.1 – – 10.9 11.6 12.1

0.6 0.3 0.2 0.2 0.2

– 0.3 0.1 0.3 0.2

SE

14.2 12.0 11.2 11.7 12.1

– 16.8 15.6 15.9 16.8

x

– – 12 13 18

– 15 13 15 15

21 14 14 14 14

– 20 19 19 20

Min.

– –

–

–

18 20 19

20 23 24 19

25 21 21 19 20

28 24 26 28

Max.

To Megalopa

– – 13.5e 16.1de 18.5e

– 16.9bcd 16.0e 16.7cde 16.9bcde

23.0 15.8e 15.3e 15.5e 16.2cde

– 22.6a 20.7abc 20.6abcd 21.4ab

X

2

– – 0.5 0.9 0.4

– 0.1 0.6 0.2 0.7

1.6 0.2 0.3 0.2 0.3

– 0.2 0.4 0.2 0.2

SE

– – 18 25 24

– 22 21 21 22

– 21 20 21 22

– 31 30 29 32

Min.

– –

–

–

–

24 25 25

27 32 33 30

25 26 34 31

36 53 62 48

Max.

To first Crab

– – 20.0 – 24.5

– 22.8 23.2 24.2 26.2

– 23.0 22.7 24.0 25.3

– 31.9 33.0 36.0 35.7

x

– – 1.0 – 0.4

– 0.6 1.0 1.9 1.8

– 0.4 0.5 0.9 1.1

– 0.7 1.5 3.9 1.0

SE

Table 2 Duration (days) of larval development from moment of hatching to the ¢rst crab stage of the mud crab, Scylla serrata, reared under di¡erent temperature and salinity combinations.

E¡ect of temperature & salinity on mudcrab larvae R Nurdiani & C Zeng Aquaculture Research, 2007, 38, 1529^1538




Table 3 Mean duration (days) of megalopal stage of the mud crab Scylla serrata reared under various temperature and salinity combinations. Temperature ( 1C) Salinity (g L

1

)

20 25 30 35

25 9.31 12.27 15.42 14.33

28

0.88ab 1.18bc 3.88c 0.93c

7.17 7.44 8.45 9.08

31

0.22a 0.31a 0.80ab 0.82ab

5.93 7.19 7.50 9.32

34

0.68a 0.48a 1.78a 1.10ab

– 6.46 0.50a – 6.00 0.00a

Values with di¡erent superscript letters are signi¢cantly di¡erent (Po0.05).

megalopae was consistently at salinity 25 g L 1 (Table 2) while longer development times were recorded both at higher and lower salinities, particularly at 35 and 20 g L 1. The interaction of temperature and salinity is illustrated by the di¡erences in development times to megalopa within the salinity range of 20^35 g L 1, which were much smaller at 28 1C (between 15.3 and 16.2 days) and 31 1C (between 16.0 and 16.9 days) than at other temperatures (Table 2). At 25 1C, larvae reared at salinity 15 g L 1 showed substantially longer intermoult durations at each larval stage (Table 2). It is worth noting that unusual cases of prolonged megalopal durations of more than 30 days were recorded for two megalopae reared at 25 1C/30 g L 1. This resulted in both the longest and the second longest overall larval development, i.e. 61 and 62 days, respectively, recorded at 25 1C/30 g L 1, which were more than triple the minimum overall larval development of 18 days registered at 34 1C/ 25 g L 1 (Table 2). The e¡ects of temperature and salinity on larvae development were likewise observed at the megalopal stage (Table 3). While enhanced development at high temperatures is similar to what was found at zoeal stages, megalopal development appears to be enhanced by low salinity. The shortest megalopal duration was consistently recorded at 20 g L 1 for all the temperatures tested, and there was a clear trend towards increased megalopal duration as the salinity increased from 20 to 35 g L 1 (Table 3).

Discussion Our results indicate that the mud crab S. serrata larvae generally tolerate a broad range of salinity and temperature conditions. Among all the temperature and salinity treatments, zoeal larvae survival was comparatively low when reared at a lower salinity of

15 g L 1 and a higher temperature of 34 1C. These conditions are probably unrepresentative of the natural environment for zoeal larvae of the mud crab as it is known that mud crab larvae hatched under o¡shore oceanic conditions (Hill 1994). The present experiment showed that at 15 g L 1, no larvae survived to the ¢rst crab stage at all the temperatures tested. Parado-Estepa and Quinito (1999) and Baylon et al. (2001) also reported that when abruptly transferred from a rearing salinity of 32 g L 1 to 16 g L 1, no Zoea V larvae were able to survive. The low tolerance of zoeae to low-saline conditions could again be explained by the fact that S. serrata females migrate o¡shore to spawn and subsequently release their larvae under oceanic conditions (Hill 1994). A low tolerance of larvae to the high temperature of 34 1C is likely a result of larvae having di⁄culties in keeping up with high metabolic requirements at a high temperature. Previous temperature experiments have generally agreed on the optimal temperature range for rearing larvae of both S. paramamosain (Zeng & Li 1992) and S. serrata (Hamasaki 2003) as between 25 and 30 1C. The present experiment also showed that under a variety of salinity conditions, the highest survival to the megalopal stage for S. serrata was recorded at both 25 and 28 1C. At the same time, our results showed that within the salinity range of 20^ 35 g L 1, larval survival to the megalopal stage was not signi¢cantly a¡ected by salinity. Li, Zeng, Tang, Wang and Lin (1999) likewise suggested a broad salinity range of 23^31g L 1 as being suitable for rearing larvae of S. paramamosain, another mud crab species. The present results showed signi¢cant interaction of temperature and salinity on larval survival. At the optimal temperature of 28 1C, larvae appeared to be capable of withstanding broader salinity conditions. Conversely, low salinity combined with either a low or a high temperature led to mass mortalities at early zoeal stages. The highest mortality (83%) of newly


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hatched larvae was recorded at 25 1C/15 g L 1. Under the same condition, Hill (1974) reported a total mortality of unfed, newly hatched S. serrata larvae within 24 h. In the present experiment, incidents of moultdeath syndrome (MDS), i.e. the phenomenon of larval mortality due to the inability to completely shed its old exoskeleton during moulting, were substantially more often observed at high temperatures during metamorphosis. In particular, MDS was observed to contribute signi¢cantly to high Zoea V mortalities at 34 1C.While nutritional de¢ciencies are generally believed to be the main cause of MDS (Christiansen 1971; Sastry 1983; Larez, Palazon-Fernandez & Bolanos 2000), Zeng and Li (1992) suggested that high temperatures could also trigger high incidents of MDS. They reported that due to MDS, the rate of successful metamorphosis from Zoea V to megalopa declined substantially when temperatures exceeded 30 1C. The results from the present experiment, as well as those from Hamasaki (2003), seem to support such a claim. In the present experiment, incidents of MDS were considerably higher at 34 1C. Similarly, Hamasaki (2003) reported that at 35 1C (salinity 34^35 g L 1), all Zoea V died without any successful moulting to megalopa. After zoeal larvae metamorphosed to megalopae, cannibalism became a major issue in communal culture. With the development of large claws, mud crab megalopae are highly cannibalistic (Zeng & Li 1992). Cannibalism among megalopae was often observed during the present study. Because the numbers of megalopae produced from di¡erent treatments varied substantially, the intensity of cannibalism is likely to be di¡erent among treatments, which compounded the temperature and salinity e¡ects. As a consequence, megalopal survival data are not presented here. Future experiments wherein megalopae are kept individually should be conducted to obtain accurate information on the e¡ects of temperature and salinity on their survival. As expected, temperature had the most profound e¡ects on larval development. For instance, at 25 1C, moulting to Zoea II generally began 1^2 days later than at other higher temperatures. Similarly, at the same salinity of 25 g L 1, overall larval development increased from an average of 20 days at 34 1C to 33 days at 25 1C. A shorter intermoult duration at higher temperatures is likely to be a result of increasing metabolic rate, enzyme activity and hormone levels involved in the moulting process (Passano 1960; Skinner 1985; Larez et al. 2000).

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Besides temperature, larval development of S. serrata was also a¡ected by salinity. The shortest average zoeal development time occurred at salinity 25 g L 1 under all the temperature conditions tested, with longer development durations recorded for both higher and lower salinities. While megalopal duration was likewise a¡ected by salinity, it di¡ered from zoeae; megalopa development was enhanced by low salinity, with the fastest development recorded at 20 g L 1 under all temperature conditions. Ong (1964) similarly reported that at 31g L 1, mud crab megalopae took 11^12 days to reach the ¢rst crab stage while at a lower salinity of 24 g L 1, it took only 7^8 days. Hamasaki (2003) also speculated that the unusually long megalopal duration (15^16 days) recorded in his experiment at 29 1C was due to the high rearing salinity of 34^35 g L 1 used. Present experiment results showed that at 25 1C, larvae generally have high survivorship but long development time. In hatcheries, long larval duration should be avoided because it not only increases production cost (e.g. labour, feed and facility costs) but also substantially increase the risks of potential larval culture crash. Conversely, larval development at 34 1C was the fastest; however, markedly lower survival suggests that it is not recommendable for commercial operations. At 28 and 31 1C, larvae had a relatively short duration, and at the same time, survival was relatively high. The highest survival (440%) occurred when larvae were reared at 28 1C with salinity between 20^25 g L 1. Reasonable survival rates were also recorded at 31 1C with salinity 20^ 25 g L 1. These four temperature/salinity combinations represent the culture conditions that resulted in relatively high larval survivorship with short development times. In summary, S. serrata larvae generally tolerate a broad range of salinity and temperature. A combination of temperature 25^31 1C and salinity 20^ 35 g L 1 resulted in generally reasonable high survival. However, as both the high survival and short development time are the objectives of hatchery operations, a higher temperature of 28^30 1C and salinity 20^30 g L 1 is recommended for S. serrata larval culture as it provides high larval survival and at the same time, shortens the hatchery culture cycle.

Acknowledgments The authors would like to thank Jerome Genodepa, Abed Rabbani and Jongchang Kim for their helps in




maintaining mud crab broodstock and live feed cultures throughout the experiment. We also thank Dr Ross Johnston, school of Marine and Tropical Biology, James Cook University, for his advice with statistical analysis.

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