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Sep 7, 2010 - and wild mud crabs, Scylla Serrata, and the effects of conditioning. Lee Parkes • Emilia T. Quinitio • Lewis Le Vay. Received: 7 April 2010 ...
Aquacult Int (2011) 19:361–380 DOI 10.1007/s10499-010-9372-1

Phenotypic differences between hatchery-reared and wild mud crabs, Scylla Serrata, and the effects of conditioning Lee Parkes • Emilia T. Quinitio • Lewis Le Vay

Received: 7 April 2010 / Accepted: 24 August 2010 / Published online: 7 September 2010 Ó Springer Science+Business Media B.V. 2010

Abstract Hatchery-reared animals for stock enhancement should be competent to survive and grow at rates equivalent to those of wild conspecifics. However, morphological differences are often observed, and pre-conditioning steps may be required to improve the fitness of hatchery-reared juveniles prior to release. In the present study, hatchery-reared Scylla serrata juveniles were reared either individually (HR-solitary) or groups in tanks (HR-communal), the latter group being exposed to intraspecific competition and foraging for food. After 21 days, both groups were compared to similar size wild-caught juveniles in terms of morphometric measurements of carapace spination, abnormalities and carapace colouration. There were some limited significant differences between HR-communal crabs and HR-solitary crabs in terms of length of 8th and 9th lateral spines and in body-weightcarapace width ratio, but both treatments differed from wild crabs, which were heavier and had longer carapace spines for their size. In contrast, both HR treatments exhibited common abnormalities including deformities in the shape of the abdomen, in particular occurrence of an asymmetrical telson or a deeply folded telson. In all cases, abnormalities persisted through moulting. Initially, carapace colour differed in all measures of colour between HR and wild crabs. However, these differences reduced after a period of 4–8 days of conditioning on coloured tank backgrounds or dark sand or mud backgrounds, without moulting. Similarly, hatchery-reared crabs exhibited very limited burying behaviour on first exposure to sediment, but this increased to levels observed in wild crabs within 2–4 days. Thus, short-term conditioning of hatchery-reared crabs on dark sediments may be effective in increasing predator avoidance and survivorship in released animals, and present results suggest that this can be achieved after relatively short periods of 1 week or less.

L. Parkes  L. Le Vay (&) School of Ocean Sciences, College of Natural Sciences, Bangor University Menai Bridge, Anglesey LL59 SAB, UK e-mail: [email protected] E. T. Quinitio Aquaculture Department, Southeast Asia Fisheries Development Center, 5021 Tigbauan, Iloilo, Philippines

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Keywords Abnormality  Conditioning  Mud crab  Pigmentation  Scylla  Stock enhancement

Introduction Mud crabs are a valuable component of many coastal fisheries in southeast Asia (Overton et al. 1997; Keenan 1999; Le Vay 2001; Walton et al. 2006). All size classes of crabs, from juveniles for pond culture to mature crabs for premium markets, are subject to heavy fishing pressure in Asia. Declines in fishery landings have been attributed to over-exploitation and environmental degradation, including siltation, pollution and loss of mangrove areas, in which mud crabs are closely associated (Angell 1992; Walton et al. 2006). Recent advances in Scylla spp. culture include the development of broodstock technology (Millamena and Quinitio 2000), mass seed production (Heasman and Fielder 1983; Hamasaki et al. 2002; Quinitio et al. 2001; Nghia et al. 2007), development of nursery technology (Rodriguez et al. 2001; Ut et al. 2007) and the closure of the life cycle of Scylla serrata (Quinitio et al., in press). Commercial hatchery production is now becoming a reality (Fushimi and Watanabe 1999; Wang et al. 2005) and will reduce dependence on wild stocks and increase supply of juveniles for pond culture. The cost-effective production of juvenile crabs also opens up the potential for stock enhancement based on hatchery releases; mud crabs are attractive species for stock enhancement in mangrove fisheries due to their post-recruitment habitat fidelity and rapid growth rates. This has been confirmed in initial trials that have demonstrated high recapture rates and positive effects on yield in localised artisanal fisheries (Le Vay et al. 2008; Lebata et al. 2009). Lebata et al. (2009) reported that survival to recapture of hatchery-reared mud crabs that had been held in ‘‘conditioning’’ earthen brackish water nursery ponds for a month prior to release was almost four times greater than for crabs that had not been conditioned before release. Similarly, in pond-rearing trials, hatchery-reared Scylla paramamosain may be out-competed by wild-collected seed crabs (Ut et al. 2007). This is consistent with the observation in a range of species that hatchery-rearing exposes animals to artificial selective pressures and developmental cues that may result in divergence from wild phenotypes (Fleming et al. 1994; Secor and Houde 1998). Both the proportion of the life cycle and the number of generations spent in artificial environments can be important in determining the degree of divergence (Fleming et al. 1994; Berejikian 1995; Davis et al. 2004). Differences between hatchery-reared and wild invertebrates have recently been reviewed by Le Vay et al. (2007) and may include colouration and pigmentation, claw morphology, spine development, shell thickness and behaviour (e.g. Ray et al. 1994; Stoner and Davis 1994; Davis et al. 2004, 2005a). Identifying differences between hatchery-reared and wild organisms allows research to focus upon assessment of their significance and improvement of survivorship through development of appropriate conditioning steps (Young et al. 2008). Many morphological differences between hatcheryreared and wild animals have been observed to be plastic, so that introduction of conditioning prior to release can significantly increase survivorship following release into the wild (Stoner and Davis 1994; Davis et al. 2004, 2005b; Lebata et al. 2009). Hence, the success of release programmes can be improved by the ability of hatchery managers to manipulate the hatchery and nursery environments to condition juveniles, reducing divergences from typical wild-type morphology and encouraging behavioural traits that may enhance survival after release. The present study investigated morphological and

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behavioural differences between wild and hatchery-reared S. serrata juveniles. Specifically, the study determines the type and frequency of abnormalities between hatcheryreared and the wild crabs and the effects of rearing conditions and short-term conditioning treatments.

Materials and methods Sources of hatchery-reared and wild juvenile crabs Hatchery-reared Scylla serrata juveniles were reared from wild-caught broodstock females collected from Samar, Eastern Visayas, Philippines. Larval production was conducted in the crustacean hatchery of the Aquaculture Department (AQD) of the Southeast Asian Fisheries Development Centre (SEAFDEC) in Tigbauan, Iloilo, Philippines, following standard methods for spawning and larval rearing developed by Quinitio and ParadoEstepa (2003). Several hundred wild S. serrata juveniles were sourced from seed collectors in Samar and transported to SEAFDEC/AQD. To reduce family effects, wild crabs were obtained from several areas and hatchery-reared crabs from several batches of larvae. Conditioning treatments for hatchery-reared crabs, group- and individual-rearing Juveniles, raised from four batches of larvae, were grown in the hatchery until reaching a size of 2–5 cm internal carapace width (ICW, measured as the distance across the carapace between the bases of 8th and 9th anterolateral spines). Each female produced 10,000–16,000 juveniles that were combined and mixed as the source of hatchery-reared animals that were divided into two conditioning treatments, held in 10-ton concrete tanks. Individually held hatchery-reared crabs (HR-solitary) were reared separately in perforated cylindrical, transparent, plastic containers (25 cm diameter 9 10 cm height). These were considered unconditioned since they were not exposed to competition, predation or foraging for food. Communally held (HR-communal) crabs were reared in free-ranging groups within each tank, provided with black nylon netting as refuge, over a bare concrete tank floor. These crabs were exposed to conditioning through intra-specific competition, agonistic behavioural interaction and foraging for food but were not exposed to predators. In both treatments, crabs were maintained at a maximum stocking density of 60 per tank for a minimum of 21 days and two moults prior to measurement and were fed fish or mussel to satiation twice daily. Water temperature and salinity in the tanks ranged from 26 to 29°C and from 29 to 32 psu, respectively. At least 50% water was exchanged every 2 days. Crabs were exposed to natural photoperiod and light intensity throughout the experiment. Pond-reared crabs used in experiments investigating the conditioning of burying behaviour were hatchery-reared animals, as above, and transferred to earthen ponds at the Dumangas Brackishwater Station of SEAFDEC/AQD, at 30–55 days posthatching. They were reared in ponds for upto 30 days before the experiments, being exposed to muddy pond sediment and to intraspecific competition but not to predators. Morphometric analysis Morphometric data were collected from 166 juvenile crabs in the intermoult stage: 55 HRsolitary (2.50–5.26 cm ICW), 55 HR-communal (2.28–4.9 cm ICW) and 56 wild crabs

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Fig. 1 Morphometric measurements taken from HR-communal, HR-solitary and wild Scylla serrata juveniles (adapted from Overton et al. 1997; Keenan et al. 1998). CW carapace width, ICW internal carapace width, PWC posterior width of carapace, CL carapace length, FW frontal width, FMSH height of frontal median spines, DFMS distance between frontal median spines, SW sternum width, AW width of 1st abdominal segment, 3PL 3rd pereiopod propodus length, 5PL 5th pereiopod dactyl length, 5PW 5th pereiopod dactyl width. Ratios used in discriminant function analysis were a carapace: 9LSH/ICW (where 9LSH = CW - ICW/2), ICW/8CW, CL/ICW, PWC/ICW, b frontal spines: PWC/FW, FW/ICW, FMSH/ FW, FMSH/DFMS, DFMS/FW, DFLS/FW, DFMS/DFLS, c abdomen and sternum: SW/ICW, AW/SW, d 3rd pereiopod: 3PML/ICW, e 5th pereiopod: 5PW/5PL

(2.86–5.19 cm ICW). Morphological measurements were determined following Overton et al. (1997) and Keenan et al. (1998), as shown in Fig. 1. Morphometric characters were measured with vernier calliper to the nearest 0.05 mm. Only intact crabs, with all limbs, were measured. Observation of morphological abnormalities All crabs sampled for morphometric analysis were also examined for occurrence of morphological abnormalities. Type and per cent frequency of abnormalities for HRcommunal, HR-solitary and wild crabs were noted. Those crabs with recorded abnormalities were isolated in perforated plastic containers (as used of HR-solitary crabs) and monitored until the crabs had undergone at least one moult to determine persistence.

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Fig. 2 Sampling points for colour hue, saturation and brightness on the carapace of juvenile Scylla serrata

Conditioning effects on colouration Intermoult crabs of similar size (ICW = 3.74–4.84 cm for HR-solitary, 3.44–4.90 cm for HR-communal and 3.95–4.86 cm for wild crabs) were selected for the quantification of colour differences between treatments (n = 9 for each treatment). Carapace colour did not differ between male and female crabs based on preliminary assessment; therefore, sex was not included as a variable. Differences in the colour of carapace were quantified using digital photography (Fuji Finepix S700 digital camera at 6 megapixels resolution) on a white background. Lighting and camera settings were standardised to minimise differences between photographs. Each image was analysed following the methodology of Davis et al. (2005a). Adobe Photoshop 7.0 was used to quantify three measures of colour, hue, saturation and brightness at ten points across the carapace of each crab (Fig. 2). The means of values at the ten points were taken to obtain one value per colour measurement per crab. The central anterior region of the carapace was avoided for colour analysis as lighting in the laboratory caused glare and may therefore misrepresent the colour of the carapace. Nine round plastic 2-l containers (25 cm width 9 12 cm height), each holding a single crab, were allocated to each treatment as follows: black basins with 2 cm of mud collected from brackish water ponds to represent typical substrate in mangroves, white basins with 2 cm of white sand, blue basins without any substrate, representing typical hatchery tanks, blue basins with 2 cm of fine grey sand representing substrate used in open nursery tanks. Each container was moderately aerated and covered with perforated plastic sheet to prevent the escape of crabs. Fifty per cent of the water was replaced daily. The ranges of water temperature and salinity were 27–30°C and 28–30 psu, respectively. A natural photoperiod of 12 h light:12 h dark was maintained. The containers were arranged in a randomised block design, with three replicates for each crab source (HR-solitary, HRcommunal and wild). The hue, saturation and brightness for each group of crabs were assessed prior to stocking them in the containers and labelled as day 0, and 2, 4, 6, 8, 10, 14 and 18 days after stocking in each treatment (n = 9 per treatment). Burying behaviour The burying efficiency (proportion of the dorsal surface of a crab covered with substrate after one burying attempt) and frequency of burying were compared between HR-solitary (3.42–4.62 cm ICW), HR-communal (3.21–4.81 cm ICW) and pond-reared (3.52–5.04 cm ICW) juvenile S. serrata. A glass aquarium (25 9 10 9 20 cm) was layered with 3 cm of

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fine, grey beach sand and filled with 2 l of seawater (28–30°C, 29–31 psu). Five crabs from each treatment were introduced separately into the centre of the experimental aquarium. The behaviour of each crab immediately on contacting the sediment was observed, with the proportion of the carapace dorsal surface area that was covered by substrate and the time taken to settle were recorded. The proportion of the dorsal surface covered by substrate was scored following the methodology of Gibson and Robb (1992): 0, no sand particles on any of the dorsal surfaces; 1, some sand on the dorsal surface but \25% covered; 2, 25–50% covered; 3, 50–75% covered; 4, 75–95% covered; 5, [95% covered. After initial burying efficiency was observed in the experimental aquarium, each crab was transferred to one of 15 cylindrical, plastic containers (2.5-l volume, 25 9 12 cm), each with a layer of 3 cm of fine grey sand. All containers were lightly aerated and 50% of the water was replaced daily, ambient water temperature ranged from 27 to 30°C, 28–30 psu salinity, and throughout the experiment, a natural photoperiod of 12 h of light and 12 h of dark was maintained. Repeated measures of burying efficiency were scored after 1, 2, 3 and 4 days of being exposed to substrate. All burying efficiency observations were made in the middle of the day, between 1,200 and 1,300 h, using natural light. Statistical analysis Morphometric data were standardised as 15 separate ratios (Fig. 1), some of which have been used to describe morphological variation in Scylla species (Overton et al. 1997; Keenan et al. 1998). All morphometric data were assessed using discriminant function analysis (DFA) following the method of Keenan et al. (1998) to determine differences between the treatments, followed by one-way analysis of variance (ANOVA) and post hoc tests. Where data met the required assumptions, differences in hue, saturation or brightness between the three crab sources (HR-communal, HR-solitary and wild) after exposure to each treatment were determined by two-way ANOVA, followed by Tukey’s pairwise tests of differences between means. Otherwise, the non-parametric Scheirer–Ray–Hare (SRH) test was applied. Burying efficiency was compared between pond-reared, HR-communal and HR-solitary crabs using the Scheirer–Ray–Hare (SRH) test.

Results Morphometrics The morphometric data revealed clear differentiation between the three sources of S. serrata, with 15 ratios accounting for 75.8% discrimination between treatments. Six of the 15 ratios used for discriminant function analysis contributed significantly to discrimination between the three treatments, 9LSH/ICW, ICW/8CW, DFMS/FW, FW/ICW, FMSH/FW and PWC/FW, accounting for 72% of the differences among sources (Table 1). The other 9 ratios accounted for only 3.8% of the discrimination between the three treatments. Wild crabs exhibited marked morphometric differences from both HR treatments, while there were only small marginal differences between the two HR treatments. The most significant differences were in the 9LSH/ICW and ICW/8CW ratios (Fig. 3). When only these two ratios were included in the model for DFA analysis, there was still 63% discrimination between the three treatments, reflecting significantly longer 8th and 9th lateral spines in wild crabs than HR-solitary and HR-communal crabs, respectively (Table 1). Wild crabs were also heavier for their size than hatchery-reared crabs; mean

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0.385 ± 0.031

0.634 ± 0.039

55

53

55

55

55

55

DFMS/FW*

PWC/FW*

FMSH/FW*

FW/ICW*

5PW/5PL

55

DFLS/FW

0.143 ± 0.017

0.332 ± 0.014

0.447 ± 0.136

0.924 ± 0.138

12,363

-225,853

-82,231

-13,069

-30.413

476

6,675

9,102

55

55

55

55

55

55

55

55

55

55

55

55

55

55

55

0.141 ± 0.016

0.330 ± 0.018

0.456 ± 0.098

0.980 ± 0.104

0.533 ± 0.163

0.551 ± 0.039

0.704 ± 0.038

0.643 ± 0.090

0.396 ± 0.040

0.762 ± 0.067ab

0.048 ± 0.011b

0.435 ± 0.018a

0.138 ± 0.021b

0.963 ± 0.009

a

0.019 ± 0.005a

-225,932

-82,342

-13,042

-30.42

474

6,696

9,108

12,378

27,047

32,765

45,587

47,723

173,527

8,174,691

14,893,608

Linear discrimination function

55

55

55

55

55

55

55

55

52

55

55

55

55

55

55

N

Wild

0.154 ± 0.110

0.348 ± 0.023

0.457 ± 0.091

0.912 ± 0.081

0.500 ± 0.033

0.544 ± 0.014

0.697 ± 0.023

0.661 ± 0.024

0.398 ± 0.030

0.813 ± 0.053b

0.064 ± 0.012c

0.429 ± 0.021b

0.140 ± 0.110b

0.945 ± 0.023b

0.030 ± 0.015b

Mean ± SD

-225,764

-82,150

-13,060

-30,406

470

6,668

9,090

12,377

27,041

32,718

45,785

47,644

173,326

8,173,918

14,892,413

Linear discrimination function

Ratios marked * are those that contribute most significantly to separation of crab sources, and different superscripts denote significant differences between mean values within rows (ANOVA and appropriate post hoc test, P \ 0.05). Full descriptions of acronyms are given in Fig. 1

54

55

55

FMSH/DFMS

0.518 ± 0.053

55

3PML/ICW

DFMS/DFLS

PWC/ICW

0.546 ± 0.028

55

SW/ICW

0.695 ± 0.033

55

55

32,727

0.765 ± 0.042a

AW/SW

45,785

0.058 ± 0.018a

CL/ICW

47,655

0.435 ± 0.019a

27,036

173,362

8,175,359

0.131 ± 0.180a

0.968 ± 0.015

a

CW/8CW*

14,893,608

0.017 ± 0.008a

55

9LSH/ICW*

Mean ± SD

N

Linear discrimination function

N

Mean ± SD

HR solitary

HR communal

Table 1 Standardised morphometric ratios for Scylla serrata juveniles from two hatchery treatments and wild crabs, in rank order of linear discriminant function score

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Fig. 3 Graphical representation of discriminant function scores for a ratios 9LSH/ICW and DFMS/FW that ranked first and third and b CW/8CW and DFMS/ FW that ranked second and third, respectively, among those ratios that most significantly contributed to differentiation between three treatments of juvenile Scylla serrata. Light shaded circle HR-solitary, plus sign HR-communal, open circle wild

BW:CW ratios were significantly higher for wild crabs (3.46 ± 0.08 g cm-1) than for HRsolitary crabs (2.78 ± 0.15 g cm-1), which in turn were significantly higher than for HRcommunal crabs (1.83 ± 0.10 g cm-1) (Mann–Whitney U test, P \ 0.001). Abnormalities Wild S. serrata juveniles exhibited fewer morphological abnormalities (3.7%) than either HR-communal or HR-solitary crabs (7.3 and 14.6%, respectively), while there was no clear indication of differences between the HR treatments. The only abnormality observed in any wild crab was light pigmentation on part of the carapace and occurrence of an extra spine adjacent to the 8th carapace spine (Fig. 4). The most common abnormalities observed in HR crabs were deformities in the shape of the abdomen, in particular asymmetrical telsons, which varied in the degrees of severity (Fig. 5) and folding of the telson, sometimes causing a depression in the sternum (Fig. 6). Some crabs exhibited abnormal pigmentation (Fig. 7), usually patches of opaque white occurring on the carapace. All abnormally pigmented patches or lines originated between the frontal median spines but the brightness and size of the pigmentation varied greatly between individuals. In all cases, abnormalities persisted through moulting. Differences in colouration between hatchery-reared and wild crabs The carapace colour, hue, saturation and brightness of HR-communal crabs, initially raised in fibreglass tanks in communal condition differed significantly from the wild crabs.

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Fig. 4 Wild Scylla serrata juvenile exhibiting morphological abnormalities including a an extra spine on the 8th carapace spine b abnormal pigmentation at the posterior left quadrant of the carapace

Fig. 5 Hatchery-reared Scylla serrata juvenile with persistently abnormal skewed telson a 2 days prior to moulting and b the same crab 3 days after moulting

Compared to wild crabs, HR-communal crabs were more blue/green in hue, lighter (increased brightness) and less pigmented (lower saturation). There was also greater variation in brightness, suggesting increased variation in pigmentation across the carapace (Fig. 8). Initially, the colouration of HR-solitary crabs more closely resembled that of wild crabs, than did the HR-communal crabs. Carapace brightness of HR-solitary crabs (17.90 ± 0.90%) did not differ significantly from that of HR-communal crabs

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Fig. 6 Example of hatcheryreared Scylla serrata juvenile with persistently abnormal telson that folds into the sternum causing a deep indentation and the telson not meeting with the anterior end of the abdominal groove

Fig. 7 Example of persistent abnormal pigmentation in hatchery-reared Scylla serrata juvenile with a exoskeleton following moulting and b the same crab 2 days after moulting

(17.69 ± 1.10%). Crabs from both HR treatments had significantly lighter carapaces than wild crabs (9.24 ± 0.50%). Hue and saturation did not differ significantly between HRsolitary and wild crabs, both having a greater (more yellow) hue (42.56 ± 2.44° and 48.29 ± 1.73°, respectively) and greater saturation (41.42 ± 2.03% and 46.31 ± 2.41%, respectively) than HR-communal crabs (77.10 ± 3.33° and 28.54 ± 1.80%). HR-solitary crabs exhibited significantly greater variation in the brightness of the carapace (4.64 ± 0.36%) than the wild crabs (2.64 ± 0.26%) but less variation in brightness than the HR-communal crabs (5.73 ± 0.26%). Effects of colour conditioning Significant changes in carapace colouration occurred after only short periods (4–8 days) of exposure to a substrate, and colouration of crabs from the three sources generally converged after 4–8 days of exposure to a particular substrates and backgrounds. Blue hatchery tanks without sediment Hue, brightness and saturation in wild crabs did not alter significantly from their original colour at any point over the 18-day experiment (Fig. 9). By day 10, crabs from all

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Fig. 8 Differences in carapace colouration in hatchery-reared and wild juvenile Scylla serrata: a hue (hues of 20°, 40° and 80° represent orange, yellow and green, respectively), b brightness, c saturation and d variation in brightness. Light shaded circle HR-communal, dark shaded circle HR-solitary and open circle wild. N = 9 for each treatment, values are means (±SD), different superscripts indicate significant difference between crab conditions (ANOVA, Tukey’s post hoc test, P \ 0.05)

treatments converged to similar levels of carapace hue, equating to a yellow/green colour (HR-communal—49.37 ± 1.53°, HR-solitary—39.9 ± 0.81° and wild—39.43 ± 0.93°). HR-solitary crabs became darker within 6 days and converged with the brightness of wild crabs. HR-communal crabs were significantly lighter than wild crabs throughout the experimental period apart from 4 to 6 days. In HR-communal crabs, saturation did not significantly alter at any point during the experimental period. In wild crabs, saturation fluctuated over the 18-day experiment, increasing by 21.8% between 0 and 6 days and decreasing by 10 days to a saturation not significantly different from the original value (Fig. 10).

Dark mud substrate In none of the three treatments, did crabs deviate significantly from their original hue (Fig. 11). HR-solitary and wild crabs had significantly less hue (more yellow) than HRcommunal crabs for the entire experimental period. HR-communal and HR-solitary crabs had significantly brighter carapaces than wild crabs initially but after 4 days the brightness of HR-communal crabs converged with that of wild crabs, while that of HR-solitary crabs remained significantly brighter throughout the experiment. Thereafter, brightness of HRsolitary crabs (10.57 ± 1.39%) and HR-communal crabs (15.44 ± 1.95%) remained fairly constant for the remainder of the experimental period. HR-solitary and wild crabs were

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Fig. 9 Effect of blue hatchery tank background, without sediment, on three aspects of crab carapace colouration in hatchery-reared and wild juvenile Scylla serrata: a hue (hues of 20°, 40° and 80° represent orange, yellow and green, respectively) and b brightness and c saturation of Light shaded circle HRcommunal, dark shaded circle HR-solitary and open circle wild Scylla serrata juveniles. N = 9 for each treatment, values are means (±SD), different superscripts indicate significant differences (ANOVA, P [ 0.05, Tukey’s post hoc test)

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Fig. 10 Effect of fine, dark grey mud substrate on three aspects of crab carapace colouration in hatcheryreared and wild juvenile Scylla serrata: a hue (hues of 20°, 40° and 80° represent orange, yellow and green, respectively) and b brightness and c saturation of Light shaded circle HR-communal, dark shaded circle HR-solitary and open circle wild Scylla serrata juveniles. N = 9 for each treatment, values are means (±SD), different superscripts indicate significant differences (ANOVA, P [ 0.05, Tukey’s post hoc test)

similar in colour saturation for the entire experimental period, crabs in both treatments being significantly more saturated than the HR-communal crabs, which therefore appeared paler than the deeply saturated wild and HR-solitary crabs.

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Fig. 11 The effect dark sand substrate on 3 aspects of crab carapace colouration in hatcheryreared and wild juvenile Scylla serrata: a hue (hues of 20°, 40° and 80° represent orange, yellow and green, respectively), b brightness and c saturation on Light shaded circle HRcommunal, dark shaded circle HR-solitary and open circle wild Scylla serrata. N = 9 for each treatment, values are means (±SD), different superscripts indicate significant differences (ANOVA, P [ 0.05, Tukey’s post hoc test)

Dark sand substrate HR-communal crabs exhibited a significant shift in hue after 8 days, from 71.68 ± 0.07 (green/blue) to 51.18 ± 0.07 (yellow/green) (Fig. 11). The carapace hue of the HRcommunal crabs did not significantly differ from that of wild or HR-solitary crabs after 10 days. Crabs in both HR treatments became darker over the first 4–6 days, converging to the levels of brightness to those observed in wild crabs, though HR-solitary crabs remained

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significantly brighter throughout the experimental period. Crabs in all three treatments converged to similar levels of colour saturation, with no significant differences between treatments after 10 days. Burying behaviour When introduced into the experimental aquaria, all HR-communal and pond-reared crabs swam to the bottom of the tank and the majority attempted to bury themselves immediately. Others explored the surface of the substrate for not more than 35 s prior to burying, while a few crabs made no attempt to burrow. On contact with the sediment, both HRcommunal and pond-reared crabs forced the posterior of the carapace into the sediment and use their chelae to drive the posterior of the carapace beneath the substrate, in a series of thrusts. Once 50–60% of the carapace was buried, the 5th pair of legs was used to flick sand over the anterior of the carapace. The majority of crabs left only the tips of the frontal spines, eyes and antennae exposed from the substrate. The mean time taken to complete the first burial attempt was 21.8 ± 10.6 s for HR-communal crabs, compared to 12.9 ± 9.0 s for pond-conditioned crabs. In contrast, most HR-solitary crabs explored the surface of the substrate for 33.6 ± 19.3 s before making a burying attempt. Initial burying attempts were often unsuccessful, as crabs would initially use their chelipeds to flick sand up into the water column, instead of driving the posterior of the carapace into the sediment. Crab burying scores differed significantly initially between treatments (Fig. 12), with a significant interaction between number of days exposed to sediment and treatment. There was no significant difference in burial efficiency between pond-reared and HR-communal crabs throughout the experiment, with an initially significantly lower score for HR-solitary crabs (2.0 ± 0.52). After 24 h, the burial efficiency of HR-solitary crabs increased and scores were not significantly different from those of the other treatments, and thereafter crabs from the three treatments exhibit similar burial efficiency.

Discussion The most consistent morphological differences between treatments observed in the present study were the shorter 8th and 9th carapace spines in hatchery-reared compared to wild

Fig. 12 Semi-quantitative burying scores for Scylla serrata juveniles from three treatments: filled triangle HR-communal, dark shaded circle HR-solitary and open circle pond-reared. N = 5 for each point, values are means ± standard deviations, different letters indicate a significant difference between treatments (ANOVA, P \ 0.05). See ‘‘Methods’’ text for description of score levels

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crabs, which were shorter in HR crabs compared with wild crabs. The mean ratios of the height of the 8th and 9th carapace spines of wild crabs in this study are comparable to ratios reported by Keenan et al. (1998) for wild S. serrata, whereas the relative length of the 9th carapace spines of hatchery-reared crabs was significantly shorter than that in wild crabs in both the present study and Keenan et al. (1998). Similar observations have been made in the blue crab Callinectes sapidus, where hatchery-produced crabs were found to have smaller lateral carapace spines than wild conspecifics (Davis et al. 2004), and in molluscs; both Stoner and Davis (1994) and Ray et al. (1994) reported truncated and fewer spines in hatchery-produced queen conch Strombus gigas, when compared to wild specimens. However, the morphological characteristics were found to be plastic in these cases and could be altered by transferring hatchery-produced animals into the natural environment (Stoner and Davis 1994; Ray et al. 1994; Davis et al. 2004, 2005a). Exposure to predators in both artificial and natural environments has been documented to influence morphological development in other species (Lively 1986; Trussel 2000). Conversely, the presence of predators has been documented to cause phenotypic responses, including the increase in dorsal spine length in Daphnia species (Grant and Bayly 1981; Krueger and Dodson 1981; Herbert and Grewe 1985) and lateral spine length in C. sapidus (Davis et al. 2004; Young et al. 2008), though in the latter case this may not be the only controlling factor (Davis et al. 2005a). In the present study, HR-communal S. serrata did not develop longer spines than HR-solitary crabs. Thus, it appears that intraspecific interactions are not a factor controlling the length of the 8th and 9th carapace spines, at least under the rearing conditions tested, but may be influenced by exposure to predators in natural habitats. Ut et al. (2007) reported that high stocking densities for Scylla paramamosain in nursery tanks resulted in low survival but increased growth and apparent fitness in those that survived. In the present study, frontal median spine (FMSH) length differed between all rearing treatments, wild crabs possessing the longest and HRsolitary crabs having the shortest FMS. Thus, intra-specific interaction appears to have had some effect on spine morphology. The release of hatchery-produced crabs with reduced spines into a natural environment may result in high levels of predation and subsequently low survival after release. Conversely, Davis et al. (2005b) observed a reduction in the level of predation on C. sapidus with elongated carapace spines, leading to increased survival in the wild. In the present study, wild crabs were relatively heavier than hatchery-reared crabs having the same size range. Similar results were observed by Ut et al. (2007) where hatchery-produced and wild S. paramamosain were compared. Differences in body weight with size are likely symptomatic of a combination of nutritional and environmental differences between rearing conditions or due to substantially greater levels of selective mortality acting upon wild crabs (Fleming et al. 1994; Miyazaki et al. 2000). Body weight or body mass is often used as an indication of condition (Baird 1958); a low body weight relative to body size equates to an animal in poor condition and thus less fit (in terms of survival) in comparison with an animal with a relatively greater body weight. Markrecapture studies of Scylla olivacea in mangrove systems have shown that a proportion of juvenile crabs gain no weight over periods of up to 1 month, indicating that food supply can be limiting and suggesting that energy reserves in released juveniles may be critical to survival (Lebata 2006). Abnormalities may be considered as non-plastic phenotypic differences between hatchery and wild animals and are commonly reported in many aquatic species, e.g. abnormal lateral lines in turbot Scopthalmus maximus (Ellis et al. 1997), abnormal spination, shape of the abdomen and carapace in Scylla spp. (Fuseya and Watanabe 1999),

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skeletal abnormalities in marine fish larvae (Cahu et al. 2003) and abnormal pigmentation, particularly albinism in flatfishes (Bolker and Hill 2000; Fairchild and Howell 2004). The present study demonstrates elevated frequency and variation of morphological abnormalities in hatchery-reared juvenile S. serrata in comparison with wild juveniles. Differences in frequency and type of abnormalities between hatchery-produced and wild crabs may be due to nutritional differences between the hatchery diet and natural prey. For example, Bolker and Hill (2000) reported 97% abnormality occurrence in Japanese flounder reared on Artemia alone, but this reduced to 2% when larvae were fed a variety of zooplankton. Enriching larval feeds with fatty acids and vitamins has also been reported to reduce abnormal pigmentation in flatfish (see review by Bolker and Hill 2000). In the present study, abnormally pigmented areas of the carapace were relatively common in hatchery-reared crabs, all stemming from the frontal lobe, but were not present in wild crabs. The development of non-pigmented patches on crabs may have significant effects on survival after release, as cryptic abilities might be reduced increasing susceptibility to predation. A lack of cryptic colouration has been documented to increase predatory-induced mortality in salmonids (Donnelly and Whoriskey 1991) and in flatfish (Ellis et al. 1997; Bolker and Hill 2000). In the present study, all initial measures of colour differed between HR-communal and wild crabs and in terms of brightness between HR-solitary and wild crabs. Variation in brightness differed between all rearing treatments, with HR-communal crabs varying most and wild crabs varying least, suggesting uniformity in the colour of the carapace in wild crabs. This is in contrast with the findings of Davis et al. (2005b) where carapace brightness was uniform in hatchery-produced C. sapidus with higher variance in brightness in wild crabs. The effect of variable pigmentation on survival is not clear, and in some species it may be advantageous (Palma and Steneck 2001). Overall carapace colour was plastic and significant adaptation occurred within 4–8 days of exposure to a change in background and without moulting being required. All measurements of colour altered at some point during conditioning trials. Hence, it appears that S. serrata are able to detect hue, saturation and brightness of their habitat and alter their carapace colour accordingly. HRsolitary crabs altered their hue, saturation and brightness of the carapace to similar levels as in wild crabs, whereas HR-communal crabs could not be as effectively conditioned. This difference is not easily explained from the present results and requires further investigation. One of the main functions of burying behaviour in epibenthic species is avoidance of predation (Gibson and Robb 1992), primarily by increasing cryptic camouflage (Ellis et al. 1997). Impaired burying behaviour in hatchery-reared invertebrates has been observed in penaeid shrimp Marsupenaeus japonicus (Kurata 1986; cited in Kitada 1999), crabs C. sapidus (Davis et al. 2004) and conch Strombus gigas (Stoner and Davis, 1994), while slow responses to predators have been observed in hatchery-reared red abalone Haliotis rufescens (Schiel and Welden, 1987). As has been reported in both crabs (Young et al. 2008) and fish (Ellis et al. 1997), burying behaviour of hatchery-reared juveniles improved to be equivalent to that in wild conspecifics after a period of conditioning through exposure to sediment. In the present study, the control group comprised hatchery-reared crabs that had been held in earth ponds for 1 month prior to the experiment, and the initial difference in burial behaviour in this group compared to naive hatchery-reared crabs kept in tanks was very marked. The current study cannot identify the mechanism for improvement in burial behaviour over time that may result from learning or delayed development of an intrinsic behavioural response to presence of sediment. However, it is clear that several days are required for normal burying behaviour to be achieved.

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Conclusions Lebata et al. (2009) conducted stock enhancement trials with Scylla spp. sourced from three different rearing environments and released into mangrove habitat in Bugtong-Bato, Aklan, Philippines. They observed that crabs reared in communal ponds for 1 month attained greater post-release survival than crabs reared in a HR-solitary regime similar to that used here and both HR groups of crab attained lower post-release survivorship than wild crabs. The conditioning period of S. serrata juveniles in ponds resulted in a mean post-release recovery rate of 31% compared to 10.2% in non-conditioned crabs (Lebata et al. 2009). Our present results indicate that for several days post-release the non-conditioned juveniles would have been unable to bury normally in mangrove sediments, while being conspicuous to predators due to their brighter colouration and having reduced spination to protect against predation. Together these effects might explain the apparent lower survivorship in this treatment. However, the timescale of changes in behaviour and colouration observed in the present experimental study suggests that conditioning may be reduced to a period of a few days, and this might be achieved in sediment-covered tanks rather than ponds, greatly facilitating and reducing the cost of production of crabs for release. Some of the present results are less easily interpreted and would benefit from further research, particularly the variable effect of rearing treatment on the ability of crabs to adapt to background colour. Acknowledgments This study was supported by a European Social Fund (ESF) grant to L. Parkes. The first author acknowledges the assistance of the SEAFDEC/AQD Crustacean Hatchery staff for the experimental set-up and animal husbandry and Dr. Fe Estepa for his help with the statistical analysis.

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