Effects of temperature, stocking density and diet ... - Wiley Online Library

Aquaculture Research, 2006, 37, 398^408

doi:10.1111/j.1365-2109.2006.01447.x

Effects of temperature, stocking density and diet on the growth and survival of juvenile Mithraculus forceps (A. Milne Edwards, 1875) (Decapoda: Brachyura: Majidae) Gil Penha-Lopes1,2, Andrew L Rhyne2, Junda Lin2 & Luis Narciso1 1

Laborato¤rio Mar|¤ timo da Guia, IMAR, Faculdade de CieŒncias da Universidade de Lisboa Estrada do Guincho, Cascais, Portugal

2

Department of Biological Sciences, Florida Institute of Technology, FL, USA

Correspondence: G Penha-Lopes, Laborato¤rio Mar|¤ timo da Guia, IMAR, Faculdade de CieŒncias da Universidade de Lisboa Estrada do Guincho, 2750-642 Cascais, Portugal. E-mail: [email protected]

Abstract

Introduction

Mithraculus forceps (A. Milne Edwards) has demonstrated a great potential for ornamental aquaculture and the present study tests the e¡ects of temperature, stocking density and diet on the survival and growth of M. forceps juveniles. For 28 days post metamorphosis (DPM), the newly metamorphosed juveniles were reared at two temperatures (25 0.5 or 28 0.5 1C), stocked at ¢ve densities (1,5,15,30 or 60 crabs ring 1; approximately 226,1132,3395,6791or13581crabs m2 respectively) and fed with commercial pellets (CP), microalgae (Amphora spp.), live newly hatched Artemia nauplii (NHA), frozen Artemia nauplii (FNHA), or combinations of each of these diets with NHA. At the end of the temperature experiment, carapace width of the crabs cultured at 28 1C was signi¢cantly larger than the crabs reared at 25 1C and average intermolt period was signi¢cantly shorter. Increased stocking density had a negative e¡ect on survivorship and growth. Survivorship at the end of the diet experiment was signi¢cantly di¡erent between the crabs not fed, fed with CP and Amphora and the crabs fed with the other diets. Between the diet treatments, the crabs fed with NHA1Amphora were signi¢cantly larger than the ones fed with NHA1FNHA, NHA, FNHA and NHA1CP, and these in turn larger than ones fed with Amphora.

While the freshwater segment of the aquarium industry relies heavily on aquaculture raised animals, more than 90% of all the marine ornamental species traded are collected in the wild, especially from coral reefs (Tlusty 2002; Moe 2003). Aquaculture has been recognized as the best solution to minimize wild harvest on coral reef ecosystems, while allowing a sustainable growth of the marine aquarium industry (Tlusty 2002; Calado, Narciso, Arau¤jo & Lin 2003). Marine ornamental crabs are very popular in the aquarium trade. Several species of hermit crabs (e.g. Calcinus spp., Clibanarius spp., Dardanus spp., and Paguristes spp.), porcelain crabs (e.g. Porcellana spp., Petrolisthes spp. and Neopetrolisthes spp.), sponge crabs (Dromnia spp.), boxer crabs (Lybia spp.) and spider crabs (e.g. Mithraculus spp. and Stenorhyncus spp.) are among the most traded species (Calado et al. 2003). Some of these species, such as the hermit crabs, porcelain crabs and sponge crabs, are popular because of their dazzling coloration or associative behaviour, while others (e.g. spider crabs) perform a speci¢c function on the reef aquarium (Calado et al. 2003). Mithraculus forceps (A. Milne Edwards) and especially M. sculptus (Lamark) are very popular and heavily traded in the marine aquarium industry for their ability to control nuisance algae in the aquarium tanks. In a previous study, M. forceps demonstrated a much better aquaculture potential than M. sculptus (Rhyne, Penha-Lopes & Lin 2005), and a larval rearing protocol was later developed for the

Keywords: Mithraculus forceps, juvenile culture, temperature, stocking density, diet, survival, growth

398

r 2006 The Authors. Journal Compilation r 2006 Blackwell Publishing Ltd

former species (Penha-lopes, Rhyne, Lin & Narciso 2005). Mortality during larval rearing is usually related to the inappropriate rearing system used (Rhyne & Lin 2004; Calado, Figueiredo, Rosa, Nunes & Narciso 2005) or improper nutrition (Felder, Martin & Goy 1985; Rodriguez, Medina & Arrue 1990). In aquacultured juvenile crabs, cannibalism (Trino, Millamena & Keenan 1999; Sainte-Marie & Lafrance 2002), food availability (quantity and quality) (Hartnoll 1982; Sheen 2000) and overcrowding stress (Wilber & Wilber 1991; Dittel, Hines, Ruiz & Ru⁄n 1995) seem to be the major factors in£uencing survivorship. In addition to survivorship, growth rate is another factor a¡ecting the crabs’ potential for aquaculture. Crustacean growth is a function of two principal components: the size increment after molting (intermolt increment) and intermolt period, estimated as the mean duration that each crab takes to molt (Kurata1962; Hartnoll1982). Intrinsic (Hartnoll1982) and environmental factors, such as temperature (Kurata 1962; Mauchline 1976), food availability (Hartnoll 1982; Wilber & Wilber 1989), holding space (Wilber & Wilber 1989), stocking density (Baliao, Rodrigues & Gerochi 1981) and social conditions (Bliss & Boyer 1964; Cobb & Tamm 1974) are known to decrease growth in crustaceans, a¡ecting one or both of the growth components, when sub-optimal conditions are provided. The present study investigates the e¡ects of temperature, stocking density and diets on survival and growth of early juveniles of M. forceps in an e¡ort to develop juvenile rearing protocols.

Materials and methods Ovigerous females of M. forceps were identi¢ed according to Coelho and Torres (1990) and collected from Porites spp. coral £ats in Florida Bay (north of Grassy Key), Florida and transported in aerated 18 L buckets to Florida Institute of Technology’s Vero Beach Marine Laboratory. Larval rearing followed the protocols developed by Penha-Lopes et al. (2005). The early juveniles metamorphosed on the ninth day post hatching (DPH) were used in the experiments. A rearing system designed by Tunberg and Creswell (1988) and modi¢ed by Penha-Lopes et al. (2005) was used. This system consists of large, shallow recirculating water tables supplied with approximately 20 L min 1 of ¢ltered seawater (5 mm). The juvenile crabs were placed in 7.5 cm diameter and 5 cm height

Rearing of juvenile Mithraculus forceps G Penha-Lopes et al.

200 Number of molts


150

II

III

I

IV

100 50 0 0.8

1.3

1.8 2.3 2.8 Crab instar width (mm)

Figure1 Frequency distribution of carapace width (CW, measured on exuviae) among the stocking density experiment. They are grouped by 0.1mm size classes and crab intars are identi¢ed by the roman numerals above.

polyvinyl chloride (PVC) rings, maintained in suspension in the water column. Each PVC ring was gravity fed with 0.2 L min 1 of ¢ltered seawater (5 mm) from a head tank through a 2 mm inlet positioned in the middle of the PVC ring. A 200 mm screen attached to the bottom of the PVC ring, combined with a complete water exchange in the system every 3 h, assured the maintenance of water quality while retaining the Artemia nauplii in each ring. Salinity of 35, pH of 8.0^8.2 and a photoperiod of14 light:10 dark were maintained. Ammonia, nitrite and nitrate were sustained below critical levels. At each feeding period (8:00 and 18:00 hours), the leftover food was removed from each PVC ring. Every morning, dead crabs were removed, molts were measured (carapace width (CW)) and preserved in 5% formalin for developmental stage (instar) determination. Monitoring crab molting, mortality, and the observation of di¡erent sizes for di¡erent crab instars (see Fig.1) was also helpful to insure the correct stage determination. Carapace width of crabs and molts were measured to the nearest 0.01mm under a stereo microscope (OlympusR, model SZ6045TR, Olympus Optical, Tokyo, Japan) with a calibrated micrometer eyepiece. Experiment 1: temperature To test the e¡ect of temperature on juvenile growth, 30 newly metamorphosed crabs were reared individually at each of the two temperature treatments: 25 0.5 and 28 0.5 1C. For 28 days, the juveniles were fed with newly hatched Artemia nauplii (NHA) in excess (7 nauplii mL 1 for the ¢rst 2 weeks and 10 nauplii mL 1 for the last 2 weeks of the experiment) twice daily. carapace width at the end of the

r 2006 The Authors. Journal Compilation r 2006 Blackwell Publishing Ltd, Aquaculture Research, 37, 398^408

399



experiment and of each crab instar (by measuring the molts), intermolt increment (I. Incr., ratio of the CW of consecutive molts: CW(n11)/CWn) and intermolt period (I. Per., duration between two successive molts) were compared between the treatments till crab instar IV.

thawed in seawater before being fed to the crabs at 5 nauplii mL 1 for the ¢rst 2 weeks and 8 nauplii mL 1 for the last 2 weeks of the experiment. The commercial food (200^400 mm pellets) was produced by Bonney, Laramore & Hopkins, Vero Beach, FL, USA. The Amphora spp. was produced by AlgaGen,Vero Beach, FL, USA every week and stored at 11 1C, ensuring that the nutritional quality of the microalgae would be optimal. The same concentration of the algae was provided to each of the six replicates for each diet treatment. For 28 days, the newly metamorphosed crabs were fed in excess twice a day and at each feeding the leftover food was removed. Survivorship, CW of crab instars, CW at the end of the experiment, and molting periods of each instar were also compared between the treatments. When more than one crab was reared in one PVC ring, individual I. Per. and I. Incr. could not be measured directly. Instead, the I. Per. Incr. was estimated as the mean period that newly metamorphosed crabs (day 0 of instar I) from each replicate took to molt to a speci¢c instar stage (II, III, IVor V). The molting period (IÎI, IÎII, IÎVor I^V) can indicate the time after which crab density in£uence signi¢cantly the molting process. By measuring each crab instars molt and comparing the mean size between the treatments, we have an indication of the stocking density e¡ect on the molt increment. If a given crab instar stage has di¡erent CWat di¡erent stocking densities, we can deduce that molt increment was a¡ected.

Experiment 2: stocking density To test the e¡ect of initial stocking density on the survival and growth of newly metamorphosed crabs,1,5, 15, 30 or 60 juvenile crabs were stocked in each PVC ring (equivalent to approximately 225, 1130, 3390, 6790 or 13580 crabs m 2 respectively). For 28 days, the juveniles were fed with NHA in excess (10 nauplii mL 1 for the ¢rst 2 weeks and 20 nauplii mL 1 for the last 2 weeks of the experiment) twice a day. Water temperature was maintained at 28 0.5 1C (best result from experiment 1). Thirty replicates were used for stocking density of 1crab ring 1, ten replicates for 5 crabs ring 1, and ¢ve replicates for higher stocking densities (15, 30 and 60 crabs ring 1). Survivorship, CWof crab instars (by measuring the molts collected), CW at the end of the experiment, and molting periods (estimated as the mean period that crabs, from each replicate tank, took from metamorphosis to speci¢c crab stage) of each instar were also compared between the treatments. Experiment 3: diets To reduce possible cannibalism, and maintain a su⁄cient number of crabs per ring for statistical analysis, ¢ve juvenile crabs were stocked in each PVC ring (approximately 1130 crabs m 2). To test the e¡ect of di¡erent diets on the survival and growth, early juvenile crabs were fed with one of the following eight diets: Control (not fed) NHA Frozen NHA (FNHA) Commercial pellets (CP) Amphora spp. (Amphora) NHA1FNHA NHA1CP NHA1Amphora The Artemia nauplii used were the Great Salt Lake Strain (Brine Shrimp Direct). The FNHA treatment was prepared 48 h before the experiment and newly hatched Artemia nauplii (from the same batch) were immediately frozen at 30 1C. Every day FNHA was

400

Statistical analysis At a stocking density of 1crab ring 1, the survival per replicate is either 100% or 0%. For this reason the survivorship was not statistically compared between the temperature treatments or between stocking density of 1crab ring 1 and the other stocking densities. A Student’s t-test was used to compare I. Per., I. Incr. and CW of each crab instar, and CW at the end of the temperature experiment, between the treatments. To compare the I. Per. and I. Incr. of each juvenile instar within each temperature, a one-way analysis of variance (ANOVA) was used. A one-way ANOVA was also used to compare instar survival, molting periods, crab instar CWand crab CWat the end of the experiment, when reared at di¡erent stoking densities and diets. When ANOVA assumptions, homogeneity of variances and normal distribution of residuals, were not met, the data was transformed (log or square root) and tested again. Equivalent non-para-




metric Kruskal^Wallis test was used when transformation failed to meet the ANOVA assumptions. A Tukey’s or Dunn’s multi-comparisons test was used if ANOVA or Krustal^Wallis respectively, showed signi¢cant treatment e¡ect. All percentage data were arcsine transformed before being tested with ANOVA. All the results were considered statistically signi¢cant at 0.05 and highly signi¢cant at 0.01 levels (Sokal & Rohlf 1995). Analysis was performed using STATISTICA 7.0 and EXCEL 2000.

Table 1 Average ( SD) carapace width (CW), intermolt increment (I. Incr.), intermolt period (I. Per.) of Mithraculus forceps ¢rst four juvenile instars reared at two di¡erent temperatures: 25 or 28 1C

25 1C

28 1C

Results Experiment 1: temperature

Crab survivorship (%)

Overall survival at the end of the experiment was higher than 60% in both treatments and the majority of the mortality (485%) occurred during molting to the second crab instar in both temperature treatments (Fig. 2). Although CW of each crab instar was not signi¢cantly di¡erent between the temperature treatments (Table 1), after 28 days post metamorphosis (DPM), CW of the crabs cultured at 28 1C was signi¢cantly (Po0.01) wider (4.28 0.67 mm) than the ones reared at 25 1C (3.45 0.22 mm) and developed faster (Fig. 2). The I. Incr. was on average about 30% of previous instar size (Table 1). At 25 1C, there is no signi¢cant di¡erence in I. Per. between the crab instars, while at 28 1C I. Per. of the ¢rst three crab instars were signi¢cantly shorter than that of the last instar (Table 1). 25 °C

100 80 60 40 20 0

Crab survivorship (%)

0

5

10

15 DPM

20

25

30

20

25

30

28 °C

100 80 60 40 20 0 0

5

10

Instar I Instar IV

15 DPM Instar II Instar V

Instar III Instar VI

Figure 2 Percentage survivorship and instar stage of juvenile Mithraculus forceps reared at two temperatures: 25 and 28 1C. DPM, days post metamorphosis.

Crab Instar

CW (mm)

I II III IV I II III IV

1.10 1.50 1.96 2.67 1.14 1.49 1.93 2.55

0.05 0.07 0.12 0.12 0.03 0.10 0.13 0.22

I. Incr. (%) 36.7 28.0 31.0 32.2 31.7 29.8 30.9 29.6

I. Per. (days) 4.5 4.5 5.9 3.7 9.5 5.5 10.5 6.6

5.8 6.4 6.5 6.8 4.7 4.8 5.0 6.6

0.91 1.11 0.81 0.6 0.7a,2 1.0a,2 0.9a,2 1.2b

Di¡erent letters indicate signi¢cant di¡erence ^ Po0.05 ^ within the temperature treatment; di¡erent numbers indicate signi¢cant di¡erence ^ Po0.05 for the same crab instar between the temperature treatments.

For the ¢rst three instars, the intermolt periods were signi¢cantly longer at 25 1C than at 28 1C; but no signi¢cant di¡erence in the intermolt period for instar IV was observed between the two temperatures (Table 1).

Experiment 2: stocking density Survival (Tables 2 and 3) and growth (Tables 2, 4 and 5) of M. forceps in general decreased with increasing density. At the end of the experiment, survival of the crabs stocked at a density of 60 crabs ring 1 was signi¢cantly lower than the ones stocked at 5 and 15 crabs ring 1 (Table 2). During the ¢rst crab instar, all the treatments showed high survival rates (485%) and signi¢cant di¡erence was only observed between the lowest densities (5 and 15 crabs ring 1) and the density of 60 crabs ring 1 at crab instars II, III and IV (Table 3). Carapace width at the end of the experiment decreased signi¢cantly with increasing stocking density, except between 30 and 60 crabs ring 1 (Table 2). At instar II, CW of the crabs stocked at 60 crabs ring 1 was already signi¢cantly smaller than the ones stocked at 5 and15 crabs ring 1 (Table 4). At instar III, CW of the crabs was signi¢cantly di¡erent between all the treatments, except between 1 and 5 crabs ring 1 and between 15 and 30 crabs ring 1. At instar IV, signi¢cant di¡erence was only found between the two lowest densities (1 and 5 crabs ring 1) and the two highest ones (30 and 60 crabs ring 1) (Table 4).


401



Table 2 Average ( SD) carapace width (CW, mm), survival and developmental stage of Mithraculus forceps crabs at the end of the experiment, stocked at ¢ve densities: 1, 5, 15, 30 or 60 crabs ring 1 (225, 1130, 3395, 6790 or 13580 crabs m 2 respectively) Development (28 DPM) Density (ring 1)

Survival (%)

CW (mm)

1 5 15 30 60

76.7 80.0 78.3 57.5 37.1

4.32 3.29 2.83 2.61 2.62

10.7a 13.4a 13.4a,b 7.0b

0.73a 0.71b 0.45c 0.33d 0.36d

Instar V (%)

Instar VI (%)

21.80 65.30 91.20 100.00 100.00

78.20 44.70 8.20 0.00 0.00

Di¡erent letters indicate signi¢cant di¡erence ^ Po0.05 ^ between the density treatments; di¡erent letters with indicate highly signi¢cant di¡erence ^ Po0.01 ^ between the density treatments. DPM, days post metamorphosis.

Table 3 Average ( SD) survivorship at the end of each crab instar of Mithraculus forceps crabs stocked at four densities (5,15, 30 or 60 crabs ring 1 ^ 1130, 3395, 6790 or 13580 crabs m 2 respectively) fed with eight di¡erent diets Instar I Density (ring 1) 5 15 30 60 Diet NHA Amphora FNHA CP NHA1Amphora NHA1FNHA NHA1CP Control

Instar II

Instar III (%)

Instar IV (%)

92.5 98.3 88.3 89.2

10.4% 3.3% 6.4% 2.2%

87.5 93.3 72.5 67.1

9.9% a 5.4% a 5.7%a,b 8.0%b

82.5 86.7 70.0 54.6

7.1a 5.4a 7.2a,b 13.6b

80.0 78.3 57.5 37.1

90.0 86.7 86.7 63.3 93.3 90.0 83.3 82.9

11.0 10.3 9.8 15.1 11.0 11.0 15.0 16.0

90.0 66.7 83.3 53.3 80.0 70.0 76.7 23.3

11.0a 16.3a,c 15.1a,c 20.7b,c 12.6a,c 11.0a,c 15.1a,c 15.1b

83.3 36.7 66.7 33.3 70.0 66.7 73.3

–

8.2a 23.3b 16.3 27.3b 11.0 10.3 10.3

80.0 – 66.7 – 63.3 63.3 70.0 –

10.7a 12.6a 13.4a,b 7.0b 12.6 16.3 15.1 8.2 10.9

Di¡erent letters indicate signi¢cant di¡erence ^ Po0.05 ^ between the treatments; di¡erent letters with indicate highly signi¢cant di¡erence ^ Po0.01 ^ between the treatments. NHA, newly hatched Artemia nauplii; FNHA, frozen Artemia nauplii; CP, commercial pellets.

There was no signi¢cant di¡erence in molting period between the ¢ve stocking densities during the ¢rst two instars (Table 5). From the molting period instar IÎV, there was a signi¢cant di¡erence between the lowest stocking density (1crabs ring 1) and the two highest ones. On molting period instar I^V, the two lowest stocking densities were signi¢cantly shorter when compared with all the other stocking densities (Table 5).

Experiment 3: diet Survival at the end of the experiment was signi¢cantly di¡erent between the crabs not fed, fed with

402

CP and Amphora, and the crabs fed with the other diets (Table 6). While mortality in the CP and Amphora treatments, as well as in the control group, occurred during all juvenile instars at a high rate, in the other diet treatments mortality rates were much lower and generally occurred at the ¢rst and second juvenile instars (Table 3 and Fig. 3). At instar II signi¢cant di¡erences in CW were already found between the crabs fed with di¡erent diets (Table 4). The mixed diet of NHA1Amphora resulted in the largest CW, whereas the crabs fed with CP had the smallest dimensions of all the diet treatments (Table 4). At instar III and IV, the mixed diet of NHA1Amphora continued to produce the largest crabs while Amphora diet resulted in the smallest




crabs (Table 4). At the end of the experiment, crabs fed with NHA1Amphora were signi¢cantly larger than the ones fed with NHA1FNHA, NHA, FNHA and NHA1CP, and these crabs in turn were larger

than the ones fed with Amphora. The crabs from the control group were signi¢cantly smaller than the crabs from all the diet treatments at every instar stage. The molting period was a¡ected by the diet early on. At the ¢rst molting period, crabs fed with NHA molted to instar II in a shorter period of time than the ones fed with only Amphora and CP (Table 5). At instars II and III, crabs fed with NHA1Amphora molted faster than the crabs in all the other treatments, followed by the ones fed with NHA1FNHA, NHA and FNHA. The ones fed with NHA1CP, CP and Amphora took a longer time to molt to instar III and IV. When fed with NHA1Amphora the crabs molted to instar V faster than the ones fed with NHA1CP, NHA1FNHA, NHA, and FNHA (Table 5). While in theAmphora treatment all the crabs reached the instar IV stage, in the CP treatment some crabs developed to instar V and in the rest of the diet treatments instar VI was reached by at least some crabs. In the control group the crabs did not molt past crab instar III (Table 6 and Fig. 3).

Table 4 Average ( SD) crab instar carapace width (mm) of Mithraculus forceps crabs stocked at ¢ve densities (1,5,15,30 or 60 crabs ring 1 ^ 225, 1130, 3395, 6790 or 13580 crabs m 2 respectively) and fed with eight di¡erent diets Instar II Density (ring 1) 1 5 15 30 60 Diet NHA Amphora FNHA CP NHA1Amphora NHA1FNHA NHA1CP Control

Instar III

Instar IV

1.49 1.50 1.49 1.46 1.44

0.11a,b 0.08a 0.08a 0.09a,b 0.10b

2.08 2.05 1.94 1.96 1.84

0.11a 0.11a 0.11b 0.11b 0.13c

2.53 2.45 2.40 2.33 2.30

0.23a 0.20a 0.20a,b 0.16b 0.18b

1.48 1.44 1.44 1.37 1.54 1.47 1.40 1.17

0.11a 0.07a 0.08a 0.05b 0.06c 0.05a 0.08a 0.07d

1.95 1.77 1.90 1.87 2.03 1.95 1.83 1.57

0.13a 0.14b 0.07a 0.08c 0.10d 0.13a 0.09b 0.17e

2.45 2.00 2.44 2.38 3.65 2.43 2.36

–

0.19a 0.24b 0.17a 0.12a 0.18c 0.24a 0.14a

Discussion Di¡erent letters indicate signi¢cant di¡erence ^ Po0.05 ^ between the treatments; di¡erent letters with indicate highly signi¢cant di¡erence ^ Po0.01 ^ between the treatments. NHA, newly hatched Artemia nauplii; FNHA, frozen Artemia nauplii; CP, commercial pellets.

Experiment 1: temperature Hartnoll and Bryant (2001) demonstrated that the effect of temperature on the intermolt increment was

Table 5 Average ( SD) molting period (days) of Mithraculus forceps crabs stocked at ¢ve densities (1, 5, 15, 30 and 60 crabs ring 1 ^ 225, 1130, 3395, 6790 and 13580 crabs m 2 respectively) and fed with eight di¡erent diets Molting period (days) Instar IÎI

Instar IÎII

Instar IÎV

Instar I^V

1

Density (ring ) 1 5 15 30 60 Diet NHA Amphora FNHA CP NHA1Amphora NHA1FNHA NHA1CP Control

4.63 4.42 4.53 4.76 4.79

0.65 0.83 0.72 0.86 0.77

4.40 5.35 4.90 5.74 4.79 5.11 5.24 5.40

0.65a 1.26b 0.62 1.15b 0.57 0.88 0.83 0.88b

9.46 9.13 9.32 9.63 9.72

1.28 1.20 1.11 1.31 1.10

14.45 14.69 15.24 15.80 15.68

1.50a 1.25a,b 1.42a,b 1.62b 1.46b

20.08 20.81 22.52 22.49 21.91

1.38a 1.17a 2.22b 2.23b 1.59b

9.04 11.76 9.67 11.29 8.46 8.82 10.13 11.67

0.84a,c 1.09b 0.86c,d 1.61b 0.98a 0.88a,c 1.06d 1.22b

14.07 21.47 15.39 19.75 13.23 14.74 15.20

–

0.80a 2.91b 1.14c,e 0.75b 0.92c 1.48c 1.47d,e

21.12 – 22.30 – 19.75 20.68 20.44 –

1.99a 2.25b 0.99c 1.29a 0.70a

Di¡erent letters indicate signi¢cant di¡erence ^ Po0.05 ^ between the treatments; di¡erent letters with indicate highly signi¢cant di¡erence ^ Po0.01 ^ between the treatments. NHA, newly hatched Artemia nauplii; FNHA, frozen Artemia nauplii; CP, commercial pellets.


403



Table 6 Average ( SD) carapace width (CW, mm), survival and developmental stage of Mithraculus forceps crabs at the end of the experiment, fed with seven di¡erent diets or not fed (control) (mean SD) Diet

Survival (%)

NHA Amphora FNHA CP NHA1Amphora NHA1FNHA NHA1CP Control

78.9 32.5 60.0 30.3 61.1 60.1 67.1 16.7

CW (mm)

10.6a 8.1b 17.9a 10.3b 16.1a 11.0a 15.0a 15.1b

3.15 2.27 2.92 2.75 3.55 3.19 2.85 1.57

Development (28 DPM)

0.69a 0.24b 0.24a,c 0.28c 0.68d 0.58a 0.58c 0.17e

83.3% 100.0% 90.0% 50.0% 57.9% 81.0% 63.2% 100.0%

instar instar instar instar instar instar instar instar

V IV V IV V V V III

16.7% instar – 10.0% instar 50.0% instar 42.1% instar 19.0% instar 36.8% instar –

VI VI V VI VI VI

Di¡erent letters indicate signi¢cant di¡erences ^ Po0.05 ^ between the treatments. NHA, newly hatched Artemia nauplii; FNHA, frozen Artemia nauplii; CP, commercial pellets.

Number of crabs 15 DPM

20

25

NHA+ Amphora

35 30 25 20 15 10 5 0 0

30

5

10

5

10

35 30 25 20 15 10 5 0

15 DPM

20

25

5

10

15 DPM

20

25

0

0

5

10

15 DPM

20

25

30

5

10

25

30

15 DPM

20

25

30

20

25

30

20

25

NHA+ CP

35 30 25 20 15 10 5 0 0

30

CP

35 30 25 20 15 10 5 0

20

35 30 25 20 15 10 5 0

30

FNHA

0

15 DPM NHA+ FNHA

Amphora

Number of crabs

Number of crabs

10

35 30 25 20 15 10 5 0 0

Number of crabs

5

Number of crabs

Number of crabs

0

Number of crabs

Number of crabs

NHA 35 30 25 20 15 10 5 0

5

10

15 DPM Control

35 30 25 20 15 10 5 0 0

5

10

Instar 1

Instar 2

Instar 3

Instar 4

Instar 5

Instar 6

15 DPM

30

Figure 3 Mithraculus forceps crabs daily survival (absolute number) and stage development at each of the eight diet treatments.

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related to the absence (indeterminate species) or presence (determinate species) of the terminate molt in the crabs. Mithraculus forceps is a determinate species, as are all majid crabs (Hartnoll1982), and the results obtained in this study agree with the pattern exhibited by the majority of the determinate species (Hartnoll & Bryant 2001). Thus, a shorter intermolt period, in spite of a similar molt increment, is responsible for superior growth of juveniles of this species at the higher temperature. In our study, the mean increment was about 30% at both temperatures, a value very close to the average molt increment of several crustacean species (Hartnoll 1982). However, it is known that marine decapods reared in captivity have lower percent molt increment than that in the wild (Anger 2001).

more active, mortality increased. This indicates that the crabs could be reared at higher densities in early juvenile stages. Although the complete development till commercial size was not accomplished, our study suggests that for the 28 DPM the highest stocking density tested, 60 crabs ring 1 (13580 crabs m 2), maximized juvenile production. However, when high survival rate is more important, lower stocking density should be adopted. Growth was strongly a¡ected by stocking density at the present study. The lower the stocking density, the larger the crabs obtained. It has already been demonstrated that culturing crabs at high densities can afect intermolt period and/or intermolt increment (e.g. Wilber & Wilber 1989; Sainte-Marie & Lafrance 2002). In our study, both of these factors were also a¡ected when the crabs were stocked at di¡erent densities. Molting period increased while intermolt increment decreased with increasing density. At high stocking densities (30 and 60 crabs ring 1), the high mortalities may reduce the density e¡ect on growth during the experiment, as well as may a¡ect the calculation of the molting period, especially if these mortalities are con¢ned to the crabs that molt later, resulting in shorter molting periods. The smaller crab instars at higher stocking densities could potentially result from several non-exclusive processes. The reduced instar sizes by a decrease in food intake at high densities could result from competition for food among the crabs (Sainte-Marie & Lafrance 2002). Although food was never limiting, reduced food intake may also result from appetite suppression because of behavioural conditioning or feeding inhibition caused by agonistic interactions (Cobb,Tamm & Wang1982). Trino et al. (1999) also reported less e⁄cient food conversion rate with increasing stock density, when culturing several mud crab species (Scylla). Other factors such as stress, interactions and sub-lethal injuries (e.g. limb autotomy) may also a¡ect molt increment by reducing the energy available to growth (Sainte-Marie & Lafrance 2002). Although interactions were not observed during the day, the crabs are generally more active during the night (Reigada & Negreiro-Fransozo 2001). In the present study, most of the dead crabs were found when checked in the morning. Sub-lethal injuries did not seem to be the major cause because of the low incidents of limb loss observed among the living and dead crabs. Therefore, the high interaction between the crabs and the consequent physiological stress appear to be the main causes of the lower growth rate observed at high culture densities.

Experiment 2: stocking density In aquaculture, high rearing densities are often employed to maximize culture e⁄ciency, although they are usually associated with lower survival and growth rates and/or a delay in juvenile development (Sainte-Marie & Lafrance 2002). In this experiment, cannibalistic behaviour was not observed and dead crabs did not present any indication of cannibalism (usually indicated by small opening in the anterior ventral part) (Wilber & Wilber 1991). At the stock density of1crab ring 1, no interaction between crabs is possible and survival and growth are expected to be maximum. At stock densities of 5 and 15 crabs ring 1, where these interactions were possible, high survival was also obtained, indicating that interactions at this density did not a¡ect survival signi¢cantly. Poor survival rates at the higher densities may be associated with intensive agonistic behaviour among the crabs (Sainte-Marie & Lafrance 2002). It might be true that at higher stocking densities, a smaller prey/predator density ratio and lower prey concentration was probably obtained in the tanks (because of the higher consumption by the total number of crabs in the tanks with higher stocking concentration). Nevertheless, the high Artemia nauplii density observed in the rings stocked with 60 crabs, at the end of each feeding period, was not very di¡erent from the others (personal observation), showing that the food concentration used was not a limiting factor. During the ¢rst juvenile instar, a high survival was observed at all densities, because of the low crab activity and mobility. However, when the crabs became


405



Using survival and growth rate as an evaluation criteria for optimal culture conditions, low densities such as 15 crabs ring 1 (3390 crabs m 2) are recommended. But if the objective is to maximize production, a higher rearing density can be used.

2004), because of the low amount of lipids and proteins, which are the major reserves for decapod crustaceans during molting (Anger 2001). The water £ux in the ring could increase the dissolution of the diet (or speci¢c components) in the water, considered one of the main problems while developing a nutritionally adequate and stable diet (Sudaryono, Hoxey, Kailis & Evans 1995). Newly hatched Artemia nauplii has been demonstrated to be an excellent diet for both M. forceps larvae (Penha-Lopes et al. 2005; Rhyne et al. 2005) and juveniles (present study). The crabs were able to prey on NHA in the water column, as well as to catch or grasp food lying on the bottom of the PVC ring (FNHA, CP and Amphora). Frozen NHA nauplii performed equally well in terms of survival and growth rates for M. forceps juveniles. Instead of enriching NHA nauplii everyday, it is possible to do it once at a large scale and freeze the Artemia nauplii, improving production e⁄ciency and reducing production costs. As in many other studies (Thompson et al. 2002; Buck et al. 2003), the present one demonstrates that mixed diets (e.g. NHA1Amphora) improve crustacean growth. Mixed diets are assumed to be nutritionally more complete, as well as reduce animal interactions, by forcing the animals to switch feeding behaviour (Barkai, Davis & Tugwell 1996; Dubber et al. 2004).

Experiment 3: diet Inadequate diet has been found to decrease survival and growth in several decapod species (Hartnoll 1982; Buck, Breeda, Penningsa, Chasec, Zimmerc & Carefoot 2003; Dubber, Branch & Atkinson 2004). Mithraculus forceps early juveniles were able to survive and grow on all the diets supplied. However, their survival and growth rates were a¡ected by the di¡erent diets. When cultured in ‘starved’ conditions (control group), M. forceps early juveniles displayed intensive cannibalism. This predatory behaviour upon conspeci¢cs may explain the presence of some crabs at the end of the experiment, as well as the increase in the juvenile’s CW throughout the experiment. The molting period of juvenile instars increased signi¢cantly, as expected when crustaceans have access to limited food (Hartnoll 1982). The microalgae of the genus Amphora have been used for decades as a primary diet, a supplement, or even as a bio¢lter (e.g. ammonium and phosphate) in hatcheries and culture facilities of ¢sh, shell¢sh and shrimp (Thompson, Abreu & Wasielesky 2002; Lee, Rodriguez, Neori, Zmora, Symons & Shpigel 2004). In our study, when the crabs were fed only with Amphora spp., they were able to survive and grow, but with high mortality (majority because of cannibalism ^ personal observation) and the lowest growth rate (longest molting periods and smaller crab instars) of all the diet treatments. However, Amphora proved to be a good food supplement, by increasing the molt increment and decreasing the molting period, when supplied with NHA. Commercial feeds have several advantages over live feed: easier manipulation, transport and storage; availability all year round; as well as the reduced disease transmission risk (Cowey & Sargent 1972). But the CP diet alone (as well as Amphora diet alone) resulted in lower survivorship when compared with the other diet treatments. As no cannibalism was observed in the dead crabs from the CP treatment, the high mortality indicates that CP is not an appropriate diet for the M. forceps juveniles. Some commercial shrimp feed pellets have already been demonstrated to be inappropriate for other decapods (Dubber et al.

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Conclusions In this study, we have successfully developed protocols for mass juvenile rearing for M. forceps. Survival rates higher than 75% after 28 DPM were obtained at a high stocking density (equivalent to 3395 crabs m 2) when fed with newly hatched Artemia nauplii (10^20 mL 1) and at 28 1C. The development of e⁄cient culture methodologies for M. forceps juveniles will allow aquaculture-raised individuals to compete with wild caught animals in the aquarium trade market, reducing Mithraculus spp. collection from natural systems and promoting a sustainable industry. Before commercial production of M. forceps can be realized, the conditions for maturation, mating and juvenile rearing (to market size), as well as maintaining/enhancing the attractive coloration need to be studied. Acknowledgments The authors would like to thank Eric Pedersen and Ronny Boggess for help in collecting Mithraculus for-




ceps ovigerous females and Eric Stenn of AlgaGen LLC for providing Amphora spp.We would also like to thank the Fundacao Calouste Gulbenkian, Portugal, (# 40739), European Social Fund and Portuguese Government (# 1/3.2/PRODEP/2003), and PADI Aware Foundation USA for their ¢nancial support. The authors are grateful to Joana Figueiredo and the reviewers for making valuable suggestions during the review process.

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