SPECIES COMPOSITION, REPRODUCTION, AND BODY SIZE OF MUD CRABS, SCYLLA SPP., CAUGHT IN URADO BAY, JAPAN

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1 JOURNAL OF CRUSTACEAN BIOLOGY, 32(5), , 2012 SPECIES COMPOSITION, REPRODUCTION, AND BODY SIZE OF MUD CRABS, SCYLLA SPP., CAUGHT IN URADO BAY, JAPAN Cynthia Yuri Ogawa 1, Katsuyuki Hamasaki 1,, Shigeki Dan 2, Yasuhiro Obata 2, and Shuichi Kitada 1 1 Department of Marine Biosciences, Tokyo University of Marine Science and Technology, Konan, Minato, Tokyo , Japan 2 Tamano Laboratory, National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, Tamano, Okayama , Japan ABSTRACT Species composition, reproduction and body size of mud crabs, genus Scylla de Haan, 1833, were investigated using gill nets from October 2008 to October 2009 in Urado Bay, Japan. Three mud crab species were identified in the area, with S. paramamosain Estampador, 1949 being the dominant species (74% of the catch), followed by S. serrata (Forskål, 1775) (23%) and S. olivacea (Herbst, 1796) (3%). We found temporal changes in species composition with abundances of S. serrata increasing towards the summer season. Ovigerous females of S. paramamosain and S. olivacea occur between January and October, peaking in the warm (May-July) and rainy (June-July) seasons, but we found no berried females of S. serrata. The female-biased sex ratios of S. paramamosain and S. serrata, and the larger body size of females during the period from autumn (November) to early spring (April), suggest that large gravid females may have migrated offshore before the spawning season. The mean body size of females and males of S. paramamosain and S. serrata tended to increase and the mating activity was high between May and October, showing the moulting and growth season. KEY WORDS: mating, maturity, mud crabs, offshore migration, sex ratio DOI: / X INTRODUCTION Mud crabs, genus Scylla de Haan, 1833, are a group of four species that inhabit littoral zones of shallow estuaries and coastal waters throughout the Indo-Pacific region (Keenan et al., 1998; Keenan, 1999). They are an important component of artisanal fisheries in many countries over their area of occurrence. There is a great demand for consumption and the price of mud crabs in local and export markets is increasing. Therefore, mud crab aquaculture and fishery have gained worldwide importance; however, the mud crab aquaculture industry has developed with a dependence on wild-caught juveniles (Keenan and Blackshaw, 1999; Le Vay, 2001; Fielder and Allan, 2004), because reliable larviculture technologies have not yet been developed (Fielder and Allan, 2004; Le Vay et al., 2008). Thus, in many regions, fishing pressure that targets all size classes from juvenile to adults has increased (Le Vay et al., 2001; Walton et al., 2006), resulting in decreased sizes and abundances (Kosuge, 2001; Lebata et al., 2007). Fishing regulations that prohibit the capture of immature crabs are indispensable for sustainable utilisation of mud crab resources (Overton and Macintosh, 2002; Walton et al., 2006; Hamasaki et al., 2011a). Although mud crabs are typically associated with mangroves in subtropical and tropical areas (Keenan et al., 1998; Le Vay, 2001; Walton et al., 2006; Lebata et al., 2007), their distribution extends to temperate zones in Japanese waters where no mangroves grow (Ito, 2000; Imai et al., 2004; Obata et al., 2006). Japanese temperate waters are the northern limit of the distribution of mud crab species. They support commercially important fisheries on a local scale in small bays and inlets, such as Hamana Bay in Shizuoka Prefecture and Urado Bay in Kochi Prefecture (Fig. 1) (Hamasaki and Kitada, 2008; Hamasaki et al., 2011b). However, little is known about the biology of each mud crab species in Japanese temperate waters. The aim of this study was to examine the species diversity and composition, reproduction and the size of mud crab species in the commercial catch in Urado Bay, which is a shallow semi-enclosed bay of 7 km 2 with some rivers, located in the southern central area of Shikoku Island, Japan (Fig. 1). MATERIALS AND METHODS Crab Collection and Measurement In Urado Bay, the mud crab fishery use gill nets with a stretched mesh size of 15 cm. We employed two fishers to record the following data on a daily basis from October 2008 to October 2009: the species, sex and carapace width (CW) including the anterolateral spines to the nearest 1 mm of each specimen of mud crab captured, as well as the occurrence of ovigerous females and mating pairs. Mud crab males can mate soft-shelled females just after moulting. In the present study, no distinction between pre- and post- Corresponding author; hamak@kaiyodai.ac.jp The Crustacean Society, Published by Brill NV, Leiden DOI: / X649787

2 OGAWA ET AL.: COMPOSITION, REPRODUCTION, AND BODY SIZE OF SCYLLA SPP. 763 Fig. 1. Map showing Urado Bay, Kochi Prefecture, Shikoku Island, Japan. The location of Hamana Bay is indicated. copulatory embrace was made, and all males and females caught in an embrace were treated as a mating pair. Species identification was performed according to Keenan et al. (1998) and Keenan (1999). Previous observations suggested that Scylla paramamosain Estampador, 1949 is the dominant species in Urado Bay (Obata et al., 2006). Therefore, to assess the gonadal development of this species, monthly sampling was performed from October 2008 to September 2009 and a total of 128 females and 128 males were obtained from the fishers. All females had no egg mass on their pleon. Each crab was humanely sacrificed by being dipped in ice-cold seawater, and the CW was measured using a vernier calliper to the nearest 0.1 mm. To identify the maturity stage of crabs, the widest portion of the fifth pleomere (AW) of females, and the highest point of the crushing chela (CH) excluding the dorsal spines of males, were measured. After body measurements, crabs were dissected to remove the gonads which were weighed to the nearest 0.1 g. Data Analysis From the fishery data collected, the following data were summarised on a monthly basis: species composition, percentage of the number of each crab species to the total number of crabs; size (CW) frequency distribution and mean values of CW for each sex; sex ratio, ratio of the number of males to the total number of crabs; ovigerous rate, percentage of ovigerous females to the total number of mature females >105 mm CW (smallest ovigerous female); mating rate, percentage of the number of mating pairs to the total number of females. The gonado-somatic index (GSI) was calculated as GSI = (GW)/(CW 3 ) 10 6, where GW is gonad weight and CW is carapace width. The AW and CH were plotted against CW to establish the increase in their values that deternined maturity (Walton et al., 2006). The difference in the mean body sizes between species was assessed with a pairwise t-test without assuming equal variances based on Welch s method. The monthly mean values of CW and GSI were compared with one-way analysis of variance (ANOVA) without assuming equal variances based on Welch s method. A binomial test was used to test the null hypothesis for sex ratio in each month (H 0 ; sex ratio = 0.5). The relationship between the body size of male and female crabs in mating pairs was evaluated by a Pearson s correlation coefficient. All statistical analyses were performed using the R language (R Development Core Team, 2011), and the level of significance was assessed at α = RESULTS Three species of mud crab, S. paramamosain Estampador, 1949, S. serrata (Forskål, 1775) and S. olivacea (Herbst, 1796), were identified in the fishing activity in Urado Bay. The total number of crabs caught by the two fishers was Scylla paramamosain was the dominant species, accounting for 74% of the total catch, followed by S. serrata (23%) and S. olivacea (3%). Abundances of S. paramamosain fluctuated seasonally between 48% in July (summer) and 100% in February (winter), i.e., the other two species, especially S. serrata, increased towards the summer season (Fig. 2). TheCWofmostoftheS. paramamosain, S. serrata, and S. olivacea was between 90 and 160 mm, 100 and 200 mm, and 70 and 140 mm, respectively (Fig. 3), showing

3 764 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 5, 2012 Fig. 2. Species composition as a percentage (%) of each mud crab species towards the total number of crabs per month. Secondary y-axis corresponds to the total number of crabs. that S. serrata was the largest and S. olivacea the smallest (pairwise t-test; P<0.0001). There were no evident modal progressions in the monthly size-frequency distributions for S. paramamosain and S. serrata (see on-line supplementary material; Fig. S1). However, significant seasonal fluctuation was detected in the mean CW values of the females and males (ANOVA; female S. paramamosain, F 12,192.3 = 14.79, P<0.0001; male S. paramamosain, F 12,87.4 = 6.84, P<0.0001; female S. serrata, F 11,60.4 = 4.00, P<0.001; male S. serrata, F 10,42.4 = 2.37, P = 0.025) (Fig. 4). The mean CW value of females of S. paramamosain was large at mm between November and April; then it decreased to 116 mm in May and increased again to 126 mm in October. The mean CW value of males of S. paramamosain fluctuated around mm until June; then it increased to around mm in August. A similar trend of the Fig. 4. Monthly mean values of carapace width (CW) of males and females. A, Scylla paramamosain; B, S. serrata. Error bar represent the standard error of the mean. mean CW change was observed for females and males of S. serrata. Monthly changes in the sex ratio were also observed for S. paramamosain and S. serrata (Fig. 5). Overall trends showed that the proportion of males largely decreased from October to January/March, and then it linearly increased towards May. Analyses of seasonal changes in the CW and sex ratio were not performed for S. olivacea due to the low catch of this species. A total of 32 ovigerous females of S. paramamosain and 15 of S. olivacea were captured. No berried S. serrata were Fig. 3. Carapace width (CW) distribution frequency of females (a) and males (b) of Scylla spp. Fig. 5. Sex ratio, as the ratio of the number of males to the total number of crabs per month for Scylla paramamosain and S. serrata. Marks in the graph ( for S. paramamosain and + for S. serrata) show a month when the null hypothesis (H 0 ; the sex ratio = 0.5) was rejected by the binomial test.

4 OGAWA ET AL.: COMPOSITION, REPRODUCTION, AND BODY SIZE OF SCYLLA SPP. 765 Fig. 6. Occurrence of ovigerous females of Scylla paramamosain and S. olivacea represented as a percentage (%) of ovigerous versus the total number of mature females (> smallest ovigerous female) per month. recorded. Ovigerous females of S. paramamosain occurred from January to October, peaking from May to July (Fig. 6). Ovigerous females of S. olivacea were found from March to September. The smallest ovigerous female captured for both species was mm CW and the largest S. olivacea was mm CW while the largest S. paramamosain was mm CW. Mating pairs were observed for all three species: 116, 18, and 2 pairs for S. paramamosain, S. serrata, and S. olivacea, respectively. Moreover, an interspecific copulatory pair was found between a female S. serrata (120 mm CW) and a male S. paramamosain (145 mm CW). Although the mating rate was higher in S. paramamosain than S. serrata, similar seasonal changes were observed in the occurrence of mating pairs for both species; almost all mating pairs were recorded from May to October (Fig. 7). Mating pairs of S. olivacea were found in May and October. The relationship between the body size of males and females caught in a couple indicated for all three species that mating tended to occur in a male with a smaller female and the female body size tended to increase with increasing male body size (Pearson s correlation coefficient; S. paramamosain, r = 0.322, P < 0.001; S. serrata, r = 0.586, P = 0.011) (Fig. 8). Based on the pleon shape of 128 females of S. paramamosain, 16 crabs with narrow pleonal flaps were classified as immature ( mm CW) and 112 crabs with Fig. 8. Relationship between the carapace width (CW) of males and females for Scylla paramamosain, S. serrata and S. olivacea caught in a mating pair. The line represents the equal sizes between mating pairs. broad U-shaped pleonal flaps as mature ( mm CW), while the relationship between CW and CH values was not able to identify morphologically mature males (Fig. 9). The GSI values of mature females ranged between 0.30 and and significantly fluctuated with month (ANOVA; F 11,32.8 = 18.03, P<0.0001) (Fig. 10). The GSI of females began to increase from October and remained high from December to April. Females with low GSI values occurred from April (mainly May) to September; however, higher GSI values were recorded from July to September (mainly in July). The GSI values of males, whose maturity stage could not be determined, ranged between 0.32 and 4.66, and they did not Fig. 7. Occurrence of mating pairs of Scylla paramamosain and S. serrata as a percentage (%) of the number of mating pairs to the total number of females per month. Fig. 9. Relationships between carapace width (CW) and the fifth pleomere width (AW) of females (a) and chela height (CH) of males (b) of Scylla paramamosain.

5 766 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 5, 2012 Fig. 10. Gonado-somatic index (GSI) of females (a) and males (b) of Scylla paramamosain. The line represents the change in mean value of each month. largely fluctuate with month compared with the GSI values of females (ANOVA, F 11,23.6 = 2.63, P = 0.024) (Fig. 10). DISCUSSION In Urado Bay, S. paramamosain was dominant, followed by S. serrata and S. olivacea, as previously reported in this bay (Obata et al., 2006) and Hamana Bay, Japan (Ito, 2000). Thus, the predominance of S. paramamosain over the other two mud crab species is common at their northern limit of distribution. Although similar dynamics were observed in the mean CW value, sex ratio and mating rate between S. paramamosain and S. serrata, the species composition changed seasonally in Urado Bay. Increased abundance of S. serrata towards the summer season may be related to temperature adaptation of this species because the estimated lower critical temperatures for egg development of S. serrata are known to be higher than those of S. paramamosain (Hamasaki, 2002, 2003). Body sizes of the three mud crab species caught in Urado Bay generally agreed with those recorded from subtropical and tropical areas (Overton and Macintosh, 2002; Hamasaki et al., 2011a; Ogawa et al., 2011): S. serrata is the largest followed by S. paramamosain and S. olivacea. Increased mean CW values and higher mating activity from May to October suggest that this period is the moulting and growth season for S. paramamosain and S. serrata. Ovigerous females of S. paramamosain and S. olivacea were caught in Urado Bay, peaking in the warm (May-July) and rainy (June-July) seasons (Takeda, 1957). The estuarine conditions in the spawning season of temperate mud crab populations coincided with those of tropical and subtropical populations summarized by Le Vay (2001). The highly variable GSI values in/after July may indicate gonadal development of recently moulted mature females and/or rematuration of spawned females of S. paramamosain. Similar temporal changes in GSI values and occurrence of ovigerous females were known for the portunid crab Portunus trituberculatus (Miers, 1876) in Japanese temperate waters (Imai et al., 1998; Hamasaki et al., 2004). It has been reported for mud crab species that few ovigerous females are found in estuarine habitat, and that females migrate offshore to spawn (Hyland et al., 1984; Hill, 1994; Le Vay, 2001; Koolkalya et al., 2006; Hamasaki et al., 2011a). In Urado Bay, however, berried females were observed for S. paramamosain and S. olivacea, but not for S. serrata. Even though female S. paramamosain can spawn in the bay, the female-biased sex ratios and the larger mean CW values of females during the period from autumn (November) to early spring (April), suggest increased offshore migration by large gravid females that had developed ovaries before the spawning season. In S. serrata, the absence of berried females and population dynamics similar to S. paramamosain strongly suggest offshore migration from the bay by females. For S. olivacea, the lack of data on the sex ratio and mating rate because of the small number caught did not allow us to infer an offshore migration by females, although the higher ovigerous rate (100%) of this species may indicate that most females laid eggs in the bay. Hill (1994) suggested two possible reasons for an offshore spawning migration by Scylla females: intolerance of larvae to estuarine conditions, especially low salinity, and improved dispersal of larvae. Larval culture experiments have demonstrated that the appropriate salinity for rearing larvae was >25 for S. paramamosain (Ogawa et al., unpublished data) and S. olivacea (Baylon, 2011). We did not observe the egg developmental stage of berried females; therefore, it is not known whether the larvae hatched inside the bay. If hatching occurs in the bay, it can be expected from the egg incubation period (Hamasaki, 2002; Ates et al., 2011) and water temperatures (Takeda, 1957), that the main hatching season would occur after June. The depth in Urado Bay is commonly 10 m and the seawater layer with >25 was found under 2 m depth (Yamamoto and Imai, 1956; Kimura et al., 1997). Therefore, in Urado Bay, estuarine conditions with moderate low salinity may allow females of S. paramamosain and S. olivacea to spawn and possibly hatch their eggs inside the bay. According to Keenan et al. (1998), S. serrata is the most widespread and dominant species in oceanic conditions with full salinity (>34 ). Dan and Hamasaki (2011) suggested that the appropriate salinity for rearing S. serrata larvae was Larval salinity adaptation may stimulate S. serrata females to spawn outside the bay. Morphological maturity of females in S. paramamosain samples occurred from mm CW. It was similar to the

6 OGAWA ET AL.: COMPOSITION, REPRODUCTION, AND BODY SIZE OF SCYLLA SPP. 767 smallest size of ovigerous females (105.0 mm CW) of this species. For males of S. paramamosain, maturity size could not be determined based on chela morphology because of lack of smaller specimens. However, body sizes of mating pairs may indicate the size at functional maturity of male crabs (S. paramamosain 95 mm CW; S. serrata 140 mm CW). The sizes of immature and mature individuals generally overlapped in mud crab species (Overton and Macintosh, 2002; Walton et al., 2006; Hamasaki et al., 2011a; Ogawa et al., 2011); e.g., morphologically pubertal moulting occurred between and mm CW and 98.4 and mm CW for females and males of S. paramamosain, respectively, in Don Sak, Bandon Bay, Thailand (Hamasaki et al., 2011a). The size at sexual maturity is important as the basis for setting a minimum landing size as a fishing regulation and the variation in size at sexual maturity has been shown to occur not only between species, but also between populations of the same mud crab species (Walton et al., 2006; Hamasaki et al., 2011a). Therefore, further studies are needed to determine the size at sexual maturity for mud crab species at the northern limit of their distribution. In the present study, a copulatory pair between a female S. serrata and a male S. paramamosain was recorded. This suggests that natural hybrids may occur between different mud crab species. Indeed, Imai and Takeda (2005) demonstrated by genetic and histological analyses using a specimen collected in Wakayama Prefecture, Japan, the existence of a natural hybrid male mud crab having a sterile testis that appears to have been produced by cross-breeding between a female S. olivacea and a male S. serrata. Thus, a natural hybrid mud crab has been found; however, its occurrence may be less common because of the very low frequency of interspecific copulatory pairs (0.7% in all mating pairs). The results of this study give evidence that there is a synchronism between reproductive events. The mating season is evidenced by the presence of mating pairs, followed by the increase in the GSI values resulting in the spawning season and the occurrence of ovigerous females. Furthermore, reproductive activities influenced the body size of the catch in the bay. However, actual evidence of migration and the hatching places of females are lacking. Therefore, to better understand the complete reproductive cycle of mud crab species at the northern limit of their distribution, further studies such as mark-recapture experiments and offshore sampling of crabs are needed. ACKNOWLEDGEMENTS We gratefully acknowledge the help of H. Nagano for collecting fishery data and crab samples during the surveys. 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8 OGAWA ET AL.: COMPOSITION, REPRODUCTION, AND BODY SIZE OF SCYLLA SPP. S1 Fig. S1-1. Size-frequency distributions of Scylla paramamosain males and females in monthly catches by gill nets from October 2008 to October 2009 in Urado Bay, Kochi Prefecture, Japan. Abbreviations: nf, number of females; nm, number of males.

9 S2 JOURNAL OF CRUSTACEAN BIOLOGY, VOL. 32, NO. 5, 2012 Fig. S1-2. Size-frequency distributions of Scylla serrata males and females in monthly catches by gill nets from October 2008 to October 2009 in Urado Bay, Kochi Prefecture, Japan. Abbreviations: nf, number of females; nm, number of males.