Influence of Salinity on Survival and Molting in Early Stages of Three Species of Scylla Crabs

The Israeli Journal of Aquaculture - Bamidgeh, IIC:63.2011.631, 6 pages The IJA appears exclusively as a peer-reviewed on-line open access journal at http://www.siamb.org.il Sale of IJA papers is strictly forbidden. Influence of on Survival and Molting in Early Stages of Three Species of Scylla Crabs Fe D. Parado-Estepa* and Emilia T. Quinitio Aquaculture Department, Southeast Asian Fisheries Development Center, Tigbauan, Iloilo 5021, Philippines (Received 22.8.10, Accepted 7.11.10) Key words: mud crab, Scylla, nursery, salinity, molting, early crab stage Abstract Early instars of three mud crab Scylla species were reared in different salinities and survival and growth were compared. Scylla olivacea were reared in salinities of 12, 16, 20, 24, and 32 g/l (control). Scylla serrata and S. tranquebarica were reared in salinities of 8, 16, 20, 24, and 32 g/l (control) After 75 days, survival of S. olivacea and S. serrata was not affected by salinity but survival in S. tranquebarica was significantly higher in 8-20 g/l than in 24 and 32 g/l. The molt interval was shorter in S. olivacea, and more animals attained the fifth molt, than in the other species. The molt interval was shorter in 12-20 g/l than in 24 and 32 g/l for S. olivacea, did not vary among test salinities for S. serrata, and was shortest in 20 and 24 g/l in S. tranquebarica where fewer animals attained a fourth molt in 32 g/l than in 8-20 g/l. The molt increment was influenced by salinity only in S. olivacea. At the end of the test, all three species exhibited lower internal carapace width and mean body weights in 32 g/l. Among the three species, S. serrata was most versatile in tolerating a wide range of salinities during nursery culture. * Corresponding author. E-mail: fpestepa@seafdec.org.ph

2 Parado-Estepa and Quinitio Introduction Techniques for producing juvenile mud Scylla crabs for stocking in growout ponds have recently been developed (Mann et al., 1999; Quinitio et al., 1999; Quinitio and Parado- Estepa, 2003). Zoeae are reared to the megalopa stage in the hatchery and transferred during the late megalopa stage to net cages in earthen ponds (Rodriguez et al., 2001, 2007; SEAFEC/AQD et al., 2010) or reared continuously in tanks. Rearing of megalopa (first crab instar stage) to a size that can be stocked in growout ponds constitutes the nursery phase. may greatly affect survival of aquatic organisms. The effect of salinity has been studied in Scylla larvae (Hill, 1974; Parado-Estepa and Quinitio, 1999; Baylon et al., 2001), juveniles and adults (Davenport and Wong, 1987; Chen and Chia, 1996a, 1997) but there are few studies on the optimal salinity for early crab stages (Jantrarotai et al., 2002; Ruscoe et al., 2004). In the Philippines, the genus Scylla (family Portunidae) consists of three species (Estampador, 1949) although, in the past, there was insufficient evidence to distinguish among them (Stephenson and Campbell, 1960). Advanced techniques confirm that Scylla consists of more than one species (Fuseya and Watanabe, 1996; Overton et al., 1997; Keenan et al., 1998) and the genus was revised, resulting in four species: S. serrata, S. tranquebarica, S. paramamosain, and S. olivacea, based on morphometric, allozyme, and mitochondrial DNA analysis (Keenan et al., 1998). As the name suggests, mud crabs are found in mangroves, but the four species may have natural habitats that differ in salinity traits (Ng, 1998) and they may differ in salinity tolerance (Keenan et al., 1998). Since all these species have high economic value, this study compared survival and growth of S. olivacea, S. tranquebarica, and S. serrata grown in different salinities to determine the most suitable salinity for nursery culture of each species. Materials and Methods Larvae of S. olivacea, S. tranquebarica, and S. serrata were reared to the megalopa stage in separate 12-ton tanks using methods described by Quinitio and Parado-Estepa (2003). Crab instar 1, selected a day after the start of metamorphosis to megalopae in the hatchery tank, were stocked in 500-ml plastic cups (3 inch diameter), perforated to allow water to flow freely in and out of the container. The instars were stocked at 1 individual/cup to avoid cannibalism. Five cups were placed in a 12-l aerated plastic tub filled with water of the test salinity, and maintained at 29±1 C. Since the larvae of the three species were not of the same age, experiments were completely randomized for each species. Tested salinities were 12, 16, 20, 24, and 32 g/l for S. olivacea and 8, 16, 20, 24, and 32 g/l for S. tranquebarica and S. serrata. Each tub of five animals constituted one replicate and each salinity was replicated three times. Desired salinities were obtained by diluting filtered sea water (ambient salinity 32 g/l) with fresh water. Ammonia, nitrate, nitrite, and ph were measured weekly and kept within tolerable levels by total daily replacement of water. Animals were fed fresh mussel meat ad libitum. Each animal was monitored daily for mortality and molting. The presence of an exuvia indicated that molting had occurred. Each animal was weighed and internal carapace widths (Overton et al., 1997) were measured two days after molting to make sure that the animals were truly in the intermolt stage. Exuviae were retrieved, measured, and compared to the previous exuvia of the corresponding crab instar to determine if a molting had occurred but was not noted, e.g., if the old exoskeleton was ingested after exuviation. Survival was determined as the percent of animals in each tub that survived to the end of the test (75 days). Molt interval was determined as the number of days between two consecutive molts. Molt increment was determined as the difference in size before and after molt. Body weights and internal carapace widths were compared among salinities at the end of the experiment. Molt intervals and increments between stocking and the first molt (M1) were not used in the analysis, since the first molting may have been caused by handling-related stress.

Molting of early crab stages of three Scylla species in different salinities 3 Data on survival and percent individuals reaching the fifth molt were subjected to arcsin transformation to normalize data. Other data were subjected to Bartlett s test for homogeneity or Levene s test, before one-way analysis of variance (ANOVA) for each species. Results Survival of S. olivacea and S. serrata crab instars was not affected by salinity but survival of S. tranquebarica was significantly lower (p<0.03) in 24 and 32 g/l than in 8-20 g/l (Table 1). For all three species, growth, as reflected by final body weight and internal carapace width, was significantly lower in 32 g/l. Table 1. Survival and growth of three species of Scylla mud crab larvae after 75 days rearing in different salinities. Survival Molt intervals of S. olivacea were similar (p>0.05) among salinities from the first (M1) to the second (M2) molt (Table 2) but fewer animals (p<0.02) underwent a fifth molt within the experimental period when stocked in 24 or 32 g/l (Table 3). In S. serrata, there were no significant differences in molt interval but fewer (p<0.02) animals underwent a fourth molt in 32 g/l than in the other treatments. In S. tranquebarica, there were no differences in molt interval from M1 to M4 but the molt interval from M4 to M5 was significantly shorter (p<0.05) in 20 and 24 g/l than in other salinities; the same percent of survivors reached the fifth molt in all salinities. In S. olivacea, the change in size from one molt to the next (molt increment) appeared to be affected by salinity after the second molt (Table 4). but there were no significant differences in S. serrata or S. tranquebarica. Scylla olivacea - 100.00±0.00 100.00±0.00 91.67±5.89 93.33±4.71 100.00±0.00 Scylla serrata 80.00±11.54-91.67±8.33 100.00±0.00 85.00±7.64 70.00±10.00 Scylla tranquebarica 100.00±0.00 b - 86.67±13.33 b 93.33±6.67 b 60.00±10.00 a 53.33±13.33 a Body weight (g) Scylla olivacea - 0.92±0.05 c 1.03±0.08 c 0.65±0.07 b 0.69±0.05 b 0.45±0.05 a Scylla serrata 0.79±0.05 b - 0.77±0.05 b 0.87±0.06 b 0.83±0.06 b 0.37±0.05 a Scylla tranquebarica 1.21±0.03 b - 1.18±0.03 b 1.16±0.02 b 1.29±0.03 b 0.88±0.04 a Internal carapace width (cm) Scylla olivacea - 1.74±0.04 cd 1.81±0.04 d 1.55±0.06 b 1.61±0.04 bc 1.32±0.04 a Scylla serrata 1.58±0.05 b - 1.60±0.04 b 1.65±0.05 b 1.58±0.06 b 1.24±0.07 a Scylla tranquebarica 1.12±0.00 b - 1.08±0.00 b 1.08±0.00 b 1.07±0.00 b 0.96±0.00 a Table 2. Number of days (molt interval) between molts of three species of Scylla mud crab larvae during 75 days rearing in different salinities. Scylla olivacea Molt 1 and 2-6±0.4 6±0.2 5±0.3 6±0.5 5±0.5 Molt 2 and 3-8±0.5 a 8±0.5 a 7±0.6 a 9±0.6 ab 10±0.8 b Molt 3 and 4-9±0.8 a 10±0.5 a 10±0.9 a 14±1.5 b 13±1.3 b Molt 4 and 5-11±0.8 14±1.1 13±0.9 12±0.5 13±1.3 Scylla serrata Molt 1 and 2 10±0.9-8±0.9 7±0.5 8±1.0 10±1.7 Molt 2 and 3 10±1.2-10±1.4 12±1.0 11±1.2 13±2.6 Molt 3 and 4 13±1.2-14±1.4 12±0.8 15±2.6 15±1.5 Molt 4 and 5 13±1.7-16±1.5 16±1.6 16±2.4 15±0.5 Scylla tranquebarica Molt 1 and 2 6±0.5-6±0.5 5±0.3 7±0.8 6±0.5 Molt 2 and 3 7±1.0-9±1.4 6±0.3 7±0.7 9±1.1 Molt 3 and 4 12±0.5-15±0.5 12±0.6 10±0.6 10±0.8 Molt 4 and 5 19±2.5 b - 18±2.2 b 12±1.4 a 16±2.4 a 20±2.4 b

4 Parado-Estepa and Quinitio Table 3. Mean survival (%) of three Scylla species that underwent at least five molts within the duration of the tests. Scylla olivacea - 86.67±6.67 c 91.67±8.33 c 70.00±5.00 bc 48.33±16.41 ab 33.33±13.33 a Scylla serrata 93.33±6.65 b - 82.21±9.67 b 86.65±6.65 b 75.00±0.00 ab 35.00±15.00 a Scylla tranquebarica 76.67±14.52-82.22±9.69 86.67±6.67 83.33±16.67 91.67±8.33 Table 4. Molt increments (mm) of Scylla mud crab larvae reared in different salinities. Scylla olivacea Molt 1 to 2-2.29±0.18 2.27±0.11 2.59±0.39 2.49±0.46 2.42±0.37 Molt 2 to 3-3.10±0.15 b 3.38±0.13 b 2.16±0.27 a 2.24±0.29 a 2.33±0.19 a Molt 3 to 4-3.28±0.09 b 3.41±0.26 b 3.17±0.19 b 2.77±0.16 ab 2.46±0.20 a Molt 4 to 5-3.40±0.14 3.23±0.19 3.31±0.32 3.17±0.35 2.58±0.24 Scylla serrata Molt 1 to 2 1.88±0.19-2.50±0.23 2.65±0.23 2.00±0.22 2.23±0.38 Molt 2 to 3 2.72±0.35-3.26±0.39 3.01±0.35 3.15±0.44 2.74±0.39 Molt 3 to 4 2.83±0.23-3.40±0.26 3.25±0.45 3.00±0.46 2.33±0.00 Molt 4 to 5 Scylla tranquebarica Molt 1 to 2 1.88±0.27-2.59±0.34 1.77±0.10 2.12±0.21 2.55±0.35 Molt 2 to 3 2.52±0.35-3.31±0.50 2.88±0.45 2.94±0.40 1.91±0.20 Molt 3 to 4 3.79±0.70-3.11±0.42 2.17±0.21 2.69±0.28 2.42±0.55 Molt 4 to 5 2.65±0.41-2.11±0.26 3.41±0.51 2.96±0.26 2.98±0.50 Discussion Although survival in the early crab stages of S. olivacea was not affected by salinity, this species exhibited protracted molt intervals in 32 g/l, so fewer animals were able to undergo the fourth molt. In addition, a lower molt increment was attained in this salinity. Since crustacean growth comprises molt interval and increment, the lengthening of the former and reduction of the latter as exhibited by S. olivacea reared in 32 g/l ultimately resulted in poor growth performance. The intermediate growth attained in 20 and 24 g/l was due to the longer molt interval between M2 and M3. The best growth was in 12 and 16 g/l as reflected in the final body weight and internal carapace width. Our results agree with Ruscoe et al. (2004) for S. serrata crab instar 2 reared in different combinations of salinity (0, 5, 10, 20, 30, and 40 g/l) and temperature (20, 25, 30, and 35 C) where survival was not affected by salinity but the weight-specific growth rate was best at the lower salinities of 10-20 g/l and 30 C. As early as the megalopa stage, S. serrata seems to perform better at lower salinities. After 18 days, megalopa survival was higher in 26 g/l (40%) than in 34 g/l (26%) water (Quinitio et al., 2001). The poor growth at 32 g/l indicates that, at this salinity, much energy may be expended on osmoregulation. However, survival at this salinity was comparable to survival in lower salinities. Scylla serrata adults are euryhaline and can tolerate a wide salinity range of 2-42 ppt (Davenport and Wong, 1987). At salinities higher than 33.4 g/l, adult (about 200 g) S. serrata osmoconform (Chen and Chia, 1997). The isosmotic point is equivalent to 33.4 g/l and isoionic points are equivalent to 21-24 g/l (except for Ca +2 which was at 33 g/l), suggesting that S. serrata is a more efficient hyperosmotic than hyposmotic regulator. Although isosmotic values may change with the life stage, this finding suggests that S. serrata is better adapted to lower salinities and had to hyporegulate osmolality or ionic contents of its intercellular fluids in high salinity, expending energy that could have been directed towards growth. The lowest oxygen uptake in juveniles (0.49 g) is in 25 g/l and nitrogen metabolism changes from

Molting of early crab stages of three Scylla species in different salinities 5 ammoniotelism to ureotelism at higher salinities (Chen and Chia, 1996b). In adults, the shift to ureotelism occurs at 44 g/l (Chen and Chia, 1996a). However, since the cited investigations were done prior to revision of the genus (Keenan et al., 1998), it is uncertain whether the tested species was the same as that used in this paper. The best growth and survival were obtained in 12-16 g/l in S. olivacea and in 8-20 g/l in S. tranquebarica. Scylla serrata survived and grew well in a wider range of salinities (8-24 g/l) than S. olivacea or S. tranquebarica. In India, 23% of S. serrata survived and attained market size even after exposure to salinity levels of 2-10 g/l for one month, then 0 g/l for another month during the rainy season, and 23-28 g/l during the summer (Quinitio and Samraj, 2007). Scylla olivacea and S. tranquebarica are associated with mangrove forests and coastlines inundated with sea water of reduced salinity while S. serrata is associated with mangrove forests with fully saline ocean water and can tolerate reduced salinity (Keenan et al., 1998). This high degree of adaptability to salinity may be one of the reasons why S. serrata is the most widely distributed Scylla species (Keenan, 1997; Le Vay, 2001). It can be found at the East African coast (South Africa, Mauritius, Yemen), throughout Australia (Northern Territory, Moreton Bay) and North Asia (Japan, Philippines, Taiwan), to the eastern Pacific Ocean (Fiji, Solomon Islands, New Caledonia). Scylla olivacea and S. tranquebarica appear to be centralized in the South China Sea (Keenan, 1997). The difference in response to salinity indicates that division of the genus into the present species is justified. Results of this study can be used in selecting sites for nursery culture of specific mud crab species in ponds since the recommended levels are based primarily on monitored salinity levels in the pond site during good production runs (Rodriguez et al., 2001; Quinitio and Parado-Estepa, 2003; SEAFDEC/AQD et al., 2010). Existing pond areas with wide salinity fluctuations may produce better yields when culturing S. serrata rather than S. olivacea or S. tranquebarica. In more controllable conditions, such as tanks, salinity can be manipulated to suit the intended species. Acknowledgements This study was supported by the European Commission (INCO-DC) under project grant no. ICA4-CT-2001-10022 entitled Culture and Management of Scylla Species and SEAFDEC/AQD with contribution no. 2011-04. The authors wish to thank Mr. Tonymer Castillo and Mr. Federico Ledesma for invaluable assistance in collecting samples and in the conduct of the experiments, the Crustacean Hatchery staff especially Jennette de Pedro and Quirico Ganon for helping with the larval rearing, and Dr. Jurgenne Primavera for the encouragement to work in the project. References Baylon J.C., Failaman A.N. and E.L. Vengano, 2001. Effect of salinity on survival and metamorphosis from zoea to megalopa of the mud crab Scylla serrata Forskal (Crustacea: Portunidae). Asian Fish. Sci., 14:143-151. Chen J.C. and P.G. Chia, 1996a. Oxygen uptake and nitrogen excretion of juvenile Scylla serrata at different temperature and salinity levels. J. Crust. Biol., 16:437-442. Chen J.C. and P.G. Chia, 1996b. Hemolymph ammonia and urea and nitrogenous excretions of Scylla serrata at different temperature and salinity levels. Mar. Ecol. Prog. Ser., 139:119-125. Chen J.C. and P.G. Chia, 1997. Osmotic and ionic concentrations of Scylla serrata (Forsskal) subjected to different salinity levels. Comp. Biochem. Physiol. A, 117:239-244. Davenport J. and T.M. Wong, 1987. Responses of adult mud crabs (Scylla serrata) (Forskal) to salinity and low oxygen tension. Comp. Biochem. Physiol. A, 86:43-47. Estampador E.P., 1949. Studies on Scylla (Crustacea: Portunidae). I: Revision of the genus. Phil. J. Sci., 78:95-108. Fuseya R. and D. Watanabe, 1996. Genetic variability in the genus Scylla (Decapoda: Portunidae). Fish. Sci., 78:95-109. Hill B.J., 1974. and temperature tolerance of zoea of the portunid crab Scylla serrata. Mar. Biol., 25:21-24.

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