For Review Only

Relationship between the Carapace Width and Body Weight

Increments and the Confirmation of Stage 1 Ovary after the Molting of Immature Orange Mud Crabs, Scylla olivacea

(Herbst, 1796), in Captivity

Journal: Songklanakarin Journal of Science and Technology

Manuscript ID SJST-2017-0375.R1

Manuscript Type: Original Article

Date Submitted by the Author: 16-Mar-2018

Complete List of Authors: Adnan, Amin-Safwan; Universiti Malaysia Terengganu, Institute of Tropical Aquaculture Harman, Muhd-Farouk; Universiti Malaysia Terengganu, Institute of Tropical Aquaculture; Impact Assessment Research Division, Fisheries Research Institute Batu Maung, Department of Fisheries Ismail, Nurul-Hasyima; Universiti Malaysia Terengganu, Institute of Tropical Aquaculture Mahsol, Hairul Hafiz; Universiti Malaysia Terengganu, Institute of Tropical

Aquaculture; Universiti Malaysia Sabah, Institute of Tropical Biology and Conservation Ikhwanuddin, Mhd; Universiti Malaysia Terengganu, Institute of Tropical Aquaculture; Universiti Malaysia Terengganu, School of Fisheries and Aquaculture Sciences

Keyword: Molting, <i>S. olivacea</i>, carapace width, body weight, maturation

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

For Review Only

Relationship between the Carapace Width and Body Weight Increments and the

Confirmation of Stage 1 Ovary after the Molting of Immature Orange Mud Crabs,

Scylla olivacea (Herbst, 1796), in Captivity

Adnan Amin-Safwan1, Harman Muhd-Farouk1, 2*, Nurul Hasyima-Ismail1, Hairul Hafiz

Mahsol1,3, and Mhd Ikhwanuddin1*

1Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, 21030 Kuala Nerus,

Terengganu, Malaysia

2Impact Assessment Research Division, Fisheries Research Institute Batu Maung,

Department of Fisheries, 11960 Batu Maung, Pulau Pinang, Malaysia

3Institute for Tropical Biology and Conservation, Universiti Malaysia Sabah, Jalan UMS,

88400 Kota Kinabalu, Sabah, Malaysia

*Corresponding Authors: Mhd Ikhwanuddin, Email: ikhwanuddin@umt.edu.my and

Harman Muhd-Farouk, Email: mhd.farouk@yahoo.com

Page 3 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

1

Relationship between the Carapace Width and Body Weight Increments and the 1

Confirmation of Stage 1 Ovary after the Molting of Immature Orange Mud Crabs, Scylla 2

olivacea (Herbst, 1796), in Captivity 3

4

Abstract 5

This study describes the relationships between the carapace width (CW) and body weight 6

(BW) increment and the confirmation of Stage 1 ovary after the molting of immature orange 7

mud crabs, Scylla olivacea, in captivity. Morphological coloration and histological assessments 8

were done on 165 immature female S. olivacea. Healthy crabs were sampled from the Setiu 9

Wetlands in the coastal waters of Terengganu on the Malaysian Peninsula from July to 10

September 2015. Thirty crabs were sacrificed for a preliminary study as a standard (control) in 11

which the gonads (if available) were dissected for histological study. The remaining crabs 12

(n=135) were selected for subsequent analysis (limb autotomy). Compared to the controls, the 13

molted crabs generally did not produce any difference in the stage of the ovaries (remaining in 14

Stage 1) but were observed to have larger oocytes. This demonstrated that the limb autotomy 15

technique may activate hormone regulation, thus triggering vitellogenesis in the mud crab. There 16

were also positive correlations between CW and BW (p=0.001; p<0.01) and significant 17

differences through regression analysis (ρ=0.002; ρ<0.01) with the equation y=2.61x + 6.27 18

(R2=0.069). These results can be useful for developing baseline data for further crab 19

management in Malaysia. 20

21

Keywords: Molting, S. olivacea, carapace width, body weight, maturation 22

Page 4 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

2

23

1.0 Introduction 24

Mud crabs (Scylla olivacea) have recently become the most important and valuable 25

fisheries species in Malaysia along with two other species, S. tranquebarica and S. 26

paramamosain (Azra & Ikhwanuddin, 2016; Ikhwanuddin, Bachok, Hilmi, Azmie, & Zakaria, 27

2010a; Ikhwanuddin, Bachok, Mohd-Faizal, Azmie, & Abol-Munafi, 2010b; Ikhwanuddin, 28

Azmie, Juariah, Zakaria, & Ambak, 2011). They are widely distributed around the South China 29

Sea and extend into the Indian Ocean and the western Pacific (Ikhwanuddin et al., 2011; Keenan, 30

Davie, & Mann, 1998). The demand for this species has increased recently due to its quality, 31

including high meat yield (Rattanachote & Dangwatanakul, 1992), large size (Ikhwanuddin, Nur-32

Atika, Abol-Munafi, & Muhd-Farouk, 2014) and rapid growth during culture (Millamena & 33

Quinitio, 1999). Recently, the production of mud crabs has become an important import 34

commodity (Ikhwanuddin et al., 2014), leading to many great opportunities in crab farming 35

(Begum, Shah, Mamun, & Alam, 2009), as they are in high demand for all size classes, including 36

mature females for premium market activities as well as soft-shell production for food materials 37

(Hungria, Tavares, Pereira, Silva, & Ostrensky, 2017; Marichamy & Rajapackiam, 2001). The 38

high demand in local markets for large-size crabs, together with the local fisheries practice of 39

selling all sizes of mud crabs (Waiho, Fazhan, & Ikhwanuddin, 2016), prompted the present 40

study of the size and carapace width increments of mud crabs. 41

A morphometric analysis based on Cadrin (2000) provides a powerful complement to 42

genetic and environmental stock identification approaches and weight-width relationships in 43

populations and is important for estimating the population size of a stock for exploitation 44

purposes (Oluwatoyin, Akintade, Edwin, & Victor, 2013). According to Atar and Secer (2003), 45

Page 5 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

3

the weight increment and length-width ratio are widely used in a given geographic region for 46

identifying the growth and formation of a species. Many investigations based on Oluwatoyin et 47

al. (2013) have studied the length-weight relationships of fin-fishes, but such information about 48

portunid species is still scarce (Sukumaran & Neelakantan, 1997). The length-width relationship 49

is regarded as the most suitable measurement for evaluating fish (Dulcic & Kraljevic, 1996; 50

Jones, Petrell, & Pauly, 1999; Petrakis & Stergiou, 1995; Stickney, 1972) and crustacean 51

(Ikhwanuddin et al., 2010a; Sukumaran & Neelakantan, 1997; Tabash, 2001; Villasmil, 52

Mendoza, & Ferrer, 1997) populations. Moreover, there is little information on carapace width 53

and body weight increments and estimation equations after the crabs have molted. There has also 54

been disagreement about the ovarian maturation stage changes after each molt (International 55

Workshop on Portunid Crabs Aquaculture and Sustainable Fisheries 2016, Universiti Malaysia 56

Terengganu, Terengganu, Malaysia). 57

In this study, the relationships between carapace width and body weight were analyzed 58

and presented. We aimed to determine the sizes of and relationships between the carapace width 59

and body weight increments after immature female mud crabs, S. olivacea, molted. In addition, 60

the ovarian maturation stages after the immature crabs molted were also determined to confirm a 61

baseline as a starting point for future research. 62

63

2 Methodology 64

2.1 Sampling 65

A total of 165 immature female S. olivacea with carapace widths (CW) less than 9.06 cm 66

(Ikhwanuddin et al., 2010a, b) were sampled from Setiu Wetlands Mangrove Forest, 67

Page 6 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

4

Terengganu, Malaysia (5°31′23.1″N 102°55′56.1″E) from July to September 2015. The crabs 68

were transported alive to the Institute of Tropical Aquaculture Marine Hatchery, where their 69

morphological characteristics were determined following the methods of Keenan et al. (1998). 70

The CW and BW of each crab were measured using a six-inch liquid crystal display (LCD) 71

digital Vernier caliper (accuracy, 0.01 cm; Kingsmart brand, Hong Kong) and a digital electronic 72

balance (accuracy: 0.01 g; Shimadzu model, Japan) and were labeled with a cable tie tag (Nylon, 73

3 x 150 mm). The CW distance was measured between the 9th

antero-lateral spines of the mud 74

crabs’ carapaces. 75

76

2.2 Induced molting in immature crabs 77

Thirty immature crabs were randomly chosen as a control, while the remaining 135 crabs 78

were used for subsequent analyses. To induce molting, limb autotomy was applied (Amin-79

Safwan, Muhd-Farouk, Nadirah, & Ikhwanuddin, 2016; Nadiah, Ikhwanuddin, & Abol-Munafi, 80

2012) to 135 immature crabs by cutting off the chelipeds and walking legs while leaving the 81

pleopods (swimming legs) for the crab’s movement. The autotomized crabs were then cultured in 82

fiberglass tanks (320 cm x 138 cm x 60 cm) with an ambient salinity (28-32 ppt), temperature 83

maintained (27-29°C) using a heater, moderate aeration, ambient light intensity and 100% water 84

exchange every two days. The crabs were fed with chopped yellowstripe scad, Selaroides 85

leptolepis, at 10% of their body weight twice daily (at 0900 and 1700 h) for observation of the 86

molting event. The culture period ended after all the autotomized crabs had successfully molted. 87

88

2.3 Experimental design 89

Page 7 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

5

Thirty immature crabs were randomly chosen and dissected to be used for the control 90

data for external morphology and histological assessment. A total of 135 immature crabs were 91

administered the limb autotomy technique to induce molting. Once the crabs molted and their 92

carapace had fully hardened (on average in 7 days), their BW and CW were measured again to 93

determine the increment size. Next, all the molted crabs with fully hardened carapaces were 94

dissected to determine the ovarian maturation stages (based on coloration), and a small portion of 95

the ovarian lobes was fixed in Davidson’s solution (24 h) for histological assessment and 96

confirmation of the ovary stage. Davidson’s solution was chosen as the fixative in the present 97

study as it is the most suitable medium for crab tissues (Muhd-Farouk, Amin-Safwan, & 98

Ikhwanuddin, 2016a). 99

100

2.4 Histological assessments 101

The histological study was based on the standard histological procedure following Muhd-102

Farouk, Jasmani, and Ikhwanuddin (2016b). Tissue processing of a small portion of ovarian 103

lobes that had been fixed in Davidson’s solution (24 h) and continued with 70% alcohol 104

(overnight) was done in the Automatic Tissue Processor for 18 ± 1 h at 60°C to infiltrate the 105

fixed tissue samples. After the processing and hydration of the tissue was complete, wax 106

impregnation was performed by embedding it in a paraffin wax to form a solid block for easier 107

handling. The solid block was then sectioned into 5 µm sections using a rotating microtome 108

(Leica RM2135). The sections were placed on the paraffin section in a water bath (temperature 109

maintained at 40-45°C) to allow them to expand. After the sections were fully expanded, a 110

microscope slide was held at an angle and slid under one or two well-formed tissue sections (the 111

fishing step). After the fishing step was complete, the microscopic slides were dried on a hot 112

Page 8 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

6

plate (40°C) overnight. The samples were then stained with modified haematoxylin-eosin 113

(H&E). The diameters of 100 oocytes from each crab were measured using an Advanced 114

Research Microscope (Nikon Eclipse 80i, Japan) together with NIS-Elements D 2.30 software. 115

116

2.5 Data analysis 117

The CW and BW increment sizes were measured and recorded. The external morphology 118

of the ovaries was photographically recorded, and the oocyte structures and diameter sizes were 119

histologically measured and recorded. The collected data were analyzed using statistical 120

correlation (to determine the strength of a relationship with available statistical data) and 121

regression analysis (for estimating the relationships among the variables) through the Statistical 122

Package for the Social Sciences (SPSS) software (version 22.0 for Windows; SPSS Inc., 123

Armonk, NY: IBM Corp.). The data are shown as the means ± standard deviations (SD). 124

125

3 Results 126

3.1 External morphology and histological assessment 127

Morphologically, all 30 immature female crabs dissected for control data were in Stage 1 128

ovary (Figure 1a). The ovary was very small, thin and very hard to differentiate from the 129

digestive gland. Ovaries were seen as a strand- and ribbon-like tissue structure. However, the 130

result for 135 crabs that underwent induced molting by limb autotomy techniques showed 131

development after the crab had molted, but remained in Stage 1 ovary with increased ovary 132

volume (Figure 1b). 133

Table 1 shows the mean oocyte diameter for the control and treatment (autotomized crab) 134

of S. olivacea in this study. Regarding the histological assessment, the mean oocyte diameter for 135

Page 9 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

7

the control crabs was 25.67 ± 4.38 µm, which was referred to as Stage 1 ovary (Figure 1c). The 136

mean oocyte diameter after the crabs molted showed an increment in oocyte sizes, but still 137

maintained a Stage 1 ovary with 34.76 ± 12.13 µm. The oocyte, follicle cells, nucleus and 138

oogonia were still present (Figure 1d). 139

140

3.2 Size of Carapace Width and Body Weight Increments 141

Table 2 shows the mean, standard deviation, and highest and lowest recorded CW and 142

BW increments of S. olivacea in this study. Figures 2 and 3 show the frequency of CW and BW 143

increments in S. olivacea. On average, the CW and BW increments were 0.816 ± 0.27 cm and 144

8.395 ± 2.72 g, respectively. 145

146

3.3 Correlation and Regression Analyses 147

Table 3 shows the correlation analysis in this study. There was a positive correlation 148

between CW and BW (ρ=0.001; ρ<0.01). As CW increased, BW also significantly increased in 149

S. olivacea. The regression analysis (Table 4) showed a significant difference (ρ=0.002; ρ<0.01), 150

with y=2.61x + 6.27 (R2=0.069) (Figure 4) between CW and BW in S. olivacea. 151

152

4 Discussion 153

Crustaceans undergo molting (ecdysis) numerous times throughout their lives (Ryer, 154

Montfrans, & Moody, 1997). The molting event, a complex, energy-demanding process, is a 155

difficult and stressful time in the life of crustaceans and is the most vulnerable time for 156

Page 10 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

8

cannibalization. A crab molts 20 to 25 times during its life (Gaude & Anderson, 2011), normally 157

for growth, ovarian maturation, mating, and stressful conditions, and molting is also influenced 158

by several environmental factors. On the basis of previous studies of spiny crab species by Atar 159

and Secer (2003) and Oluwatoyin et al. (2013), the length measurement of crabs is said to be 160

difficult, and during attempts to measure them, either the extremities of the crab can be broken or 161

the investigator can be injured by the crab. In addition, determinations of the size of the 162

increment after crabs molted in the wild are still scarce. Only by rearing the crabs in captivity 163

can the size of the increments be confirmed. It is therefore convenient to estimate and convert 164

into width (length) when only the weight is known or vice versa (Atar & Secer, 2003; 165

Czerniejewski & Wawrzyniak, 2006; Josileen, 2011; Oluwatoyin et al., 2013). These 166

relationships are often used to calculate the standing stock biomass, condition indices, 167

ontogenetic changes and several aspects of fish or crustacean population dynamics (Atar & 168

Secer, 2003; Oluwatoyin et al., 2013). Moreover, according to Sukumaran and Neelakantan 169

(1997), body weight, total length, carapace width and carapace length are the most frequently 170

used dimensions, especially in the field of crustacean studies. 171

A previous study by Smith (1990) on the blue crab, Callinectes sapidus, found that this 172

species can regenerate almost 90% of its normal limb length in the first molt and nearly 100% in 173

the second molt after the loss of a single cheliped. Josileen and Menon (2005) stated that blue 174

swimming crabs, Portunus pelagicus, were able to regenerate 90% of their normal size in the 175

next molt, supporting a previous study by Smith (1990), and this does not affect the molt 176

increment or the molt interval. Our study recorded a similar result, with 85% regeneration of 177

their normal size; however, the chelipeds’ size became smaller than before. This may due to the 178

multiple limb loss from the limb autotomy technique used in our study, which affected the rate of 179

Page 11 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

9

the chelipeds’ growth. Bennett (1973) and Kuris and Mager (1975) noted that the molt increment 180

decreases proportionally with the increasing numbers of limbs lost, thus supporting our 181

assessment of the cause of the decreasing size of mud crab chelipeds. 182

Our study showed that as the crabs molted, both the carapace width (CW) and body 183

weight (BW) increased. These results were supported by a strong positive correlation (ρ=0.001; 184

ρ<0.01) between CW and BW. The regression analysis showed a significant result (ρ=0.002; 185

ρ<0.01) with y=2.61x + 6.27. However, our value of R2

=0.069 showed a very weak coefficient 186

of determination compared to previous studies (Atar & Secer, 2003; Oluwatoyin et al., 2013; 187

Sudha Devi & Smija, 2015) on crabs. On average, the CW increment was 0.816 ± 0.27 cm, with 188

the highest and the lowest increments recorded of 1.54 cm and 0.30 cm, respectively. 189

Meanwhile, the average BW increment was 8.395 ± 2.72 g, with the highest and lowest 190

increments recorded at 19.0 g and 3.2 g, respectively. Our findings proved that as female crabs 191

molted, their carapace size, body weight and ovary increased in size, similar to the results 192

recorded by Josileen and Menon (2005) for P. pelagicus. However, their study did not include 193

ovarian development. By focusing on the CW-BW relationship, the equation obtained can be 194

used to determine the size of the in situ population in an area and thus can predict the population 195

there. 196

Concerning ovarian maturation, four ovarian stages have been recorded on the basis of 197

recent studies (Amin-Safwan et al., 2016; Azmie, Azra, Noordiyana, Abol-Munafi, & 198

Ikhwanuddin, 2017; Ikhwanuddin et al., 2014; Muhd-Farouk et al., 2016b) on the same species, 199

S. olivacea. However, no study has been done on the ovarian maturation stages after immature 200

crabs molted, and the biological information about mud crabs is scarce. There is also confusion 201

among crab breeders and researchers about the ovarian maturation after immature crabs molted 202

Page 12 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

10

(International Workshop on Portunid Crabs Aquaculture and Sustainable Fisheries 2016, 203

Universiti Malaysia Terengganu, Terengganu, Malaysia). Some of them suggested that the crabs 204

have advanced to the next stage, while others suggested that the cycle of ovarian maturation 205

started again (Personal communications: Amirul, 2016; Aziz, 2015). Therefore, there is an urgent 206

need for the present study focusing on the ovarian stage after immature crabs have molted, which 207

we claim is the first report on immature crab ovarian development after a molting event occurs. 208

Our study recorded that ovarian development was present as the crabs molted; however, 209

they remained in Stage 1 ovary. The external morphology showed that the molted crabs’ ovaries 210

were much more developed compared to the control ovary, which was very small, thin and very 211

difficult to differentiate from the digestive gland. Moreover, the ovaries were seen as strand- and 212

ribbon-like tissue structures both before and after the molting event. To confirm the ovarian 213

maturation stages, a histological assessment was done to obtain more accurate results (Madlen, 214

Khadiga, & Montaser, 2012). From a histological perspective, the ovarian maturation of S. 215

olivacea after the molting event was confirmed as Stage 1 ovary with a mean oocyte diameter of 216

34.76 ± 12.13 µm. The oocyte, follicle cells, nucleus and oogonia were present. Compared to the 217

control crabs with a mean oocyte diameter of 25.67 ± 4.38 µm, the oocyte diameter after the 218

molting event showed an increase in size, thus demonstrating the occurrence of ovarian 219

development after crab molting. The ovarian development could have resulted from the limb 220

autotomy technique, which leads to the activation of hormone regulation and vitellogenesis. 221

However, a further in-depth study is needed as our understanding of vitellogenesis, particularly 222

in the mud crab, is still limited. Moreover, a better understanding of hormone regulation after 223

limb autotomy is performed is crucial as this technique is currently one of the favorite practices 224

in soft-shell production. Nevertheless, our result shows that the ovary remained in Stage 1, and 225

Page 13 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

11

this information can be used as a new finding about mud crab biology. In addition, this 226

information can become a new guideline and an indicator for future research perspectives. 227

Temperature and the quality and quantity of food are the most important environmental 228

factors that can affect molting and growth in crustaceans (Josileen & Menon, 2005). In this 229

study, these two parameters have been maintained as well as possible. The temperature was 230

maintained in the range of 27-29°C throughout the study period, similar to the temperature range 231

of a previous study done by Josileen and Nemon (2005) on P. pelagicus, while feeding 10% of 232

the body weight twice daily (0900 and 1700 h). Compared to these parameters, salinity and light 233

intensity have little effect on the molting event and inter-molt period of S. olivacea in captivity. 234

These findings were supported by Hartnoll (1982) and Josileen and Menon (2005), who 235

concluded that there was no significant change in the inter-molt period with regard to salinity in 236

several species of crustaceans. 237

It is recommended that further studies are needed on CW-BW as the equation in this 238

study showed a weak coefficient of determination. It is also suggested that male crabs and a 239

higher number of samples be included in future studies. In addition, we also suggest that further 240

studies focus more on limb autotomy, as we believe that this technique could trigger some 241

hormones that activate vitellogenesis and growth in crustaceans. Further study is thus crucial to 242

fully understand hormone regulation and the vitellogenesis mechanism after limb autotomy is 243

introduced. With a complete understanding of the CW-BW relationship and the ovarian 244

maturation stages, the data obtained can be used as an important baseline for future mud crab 245

resource management in that particular area. 246

247

Page 14 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

12

5 Conclusion 248

This study showed that there was a positive and strong correlation between CW and BW 249

(ρ=0.001; ρ<0.01) and that the mean CW and BW increments of female S. olivacea were 0.816 ± 250

0.27 cm and 8.395 ± 2.72 g. The regression analysis showed a significant difference (ρ=0.002; 251

ρ<0.01) with an estimation equation of y=2.61x + 6.27 for BW and CW. However, the 252

coefficient of determination (R2

=0.069) showed a very weak estimation value in this study. We 253

recommend that further studies are needed to obtain a strong coefficient of determination, as the 254

R2 value is important to ascertain how strong a linear relationship is between the variables 255

determining the population in a particular area. Meanwhile, both the external morphological 256

assessment and the histology have shown that after immature crabs molted, the ovaries remained 257

in Stage 1. However, this study may have demonstrated that vitellogenesis was activated by 258

hormone regulation during the performance of limb autotomy and thus can be used as the 259

baseline for future studies. 260

261

Acknowledgements 262

This research was funded by the Ministry of Higher Education under the Niche Research Grant 263

Scheme (NRGS) Vote No. 53131. Our greatest appreciation goes to the Institute of Tropical 264

Aquaculture, Universiti Malaysia Terengganu and to all the people who were directly or 265

indirectly involved during this study. We thank Mohamad N Azra and Mardhiyyah Mohd Pauzi 266

for providing valuable comments and an English revision of an earlier draft of the manuscript. 267

268

References 269

Page 15 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

13

Amin-Safwan, A., Muhd-Farouk, H., Nadirah, M., & Ikhwanuddin, M. (2016). Effect of water 270

salinity on the external morphology of ovarian maturation stages of orange mud crab, 271

Scylla olivacea (Herbst, 1796) in captivity. Pakistan Journal of Biological Sciences, 19, 272

219-226. doi:10.3923/pjbs.2016.219.226 273

Atar, H. H., & Secer, S. (2003). Width/Length relationships of blue crab (Callinectes sapidus, 274

Rathbun, 1896) population living in Beymelek Lagoon Lake. Turkish Journal of 275

Veterinary and Animal Sciences, 27, 443-447. Retrieved from 276

http://journals.tubitak.gov.tr/veterinary/issues/vet-03-27-2/vet-27-2-24-0203-12.pdf 277

Azmie, G., Azra, M. N., Noordiyana, M. N., Abol-Munafi, A. B., & Ikhwanuddin, M. (2017). 278

Ovarian morphological development and fatty acids profile of mud crab (Scylla olivacea) 279

fed with various diets. Aquaculture, 468, 45-52. doi:10.1016/j.aquaculture.2016.09.038 280

Azra, M. N., & Ikhwanuddin, M. (2016). A review of maturation diets for mud crab genus Scylla 281

broodstock: Present research, problems and future perspective. Saudi Journal of 282

Biological Science, 23, 257-267. 283

Begum, M., Shah, M. M. R., Mamun, A. A., & Alam, M. J. (2009). Comparative study of mud 284

crab (Scylla serrata) fattening practices between two different systems in Bangladesh. 285

Journal of Bangladesh Agricultural University, 7, 151-156. 286

Bennett, D. B. (1973). The effect of limb loss and regeneration on growth of the edible crab, 287

Cancer pagurus Linnaeus, 1758. Journal of Experimental Marine Biology and Ecolology, 288

13, 45-53. doi:10.1016/0022-0981(73)90045-2 289

Page 16 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

14

Cadrin, S. X. (2000). Advances in morphometric identification of fishery stocks. Reviews in Fish 290

Biology and Fisheries. 10, 91-112. Retrieved from 291

https://link.springer.com/content/pdf/10.1023%2FA%3A1008939104413.pdf 292

Czerniejewski, P., & Wawrzyniak, W. (2006). Body weight, condition, and carapace width and 293

length in the Chinese mitten crab (Eriocheir sinensis, H. Milne-Edwards, 1853) collected 294

from the Szczecin Lagoon (NW Poland) in spring and autumn 2001. Oceanologia, 48 (2), 295

275-285. Retrieved from http://agro.icm.edu.pl/agro/element/bwmeta1.element.agro-296

article-b60e8d91-1248-4ccb-9888-f89e1e10a0e2 297

Dulcic, J., & Kraljevic, M. (1996). Weight-Length Relationship for 40 Fish Species in the 298

Eastern Adriatic (Croatian Waters). Fisheries Research, 28, 243-251. doi:10.1016/0165-299

7836(96)00513-9. 300

Gaude, A. R., & Anderson, J. A. (2011). Soft Shell Crab Shedding Systems. Southern Regional 301

Aquaculture Center, 1-6. 302

Hartnoll, R. G. (1982). Growth. In: The Biology of Crustacea. Embryology, morphology and 303

genetics, L.G. Abele, editor. Academic Press Vol. 2, New York, pp 111-185. ISBN 0-12-304

106402-6. xxi. 305

Hungria, D. B., Tavares, C. P. D. S., Pereira, L. A., Silva, U. D. A. T. D., & Ostrensky, A. 306

(2017). Global status of production and commercialization of soft-shell crabs. 307

Aquaculture International, 1-14. 308

Page 17 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

15

Ikhwanuddin, M., Azmie, G., Juariah, H. M., Zakaria, M.Z., & Ambak, M. A. (2011). Biological 309

information and population features of mud crab, genus Scylla from mangrove areas of 310

Sarawak, Malaysia. Fisheries Research. 108, 299-306. doi:10.1016/j.fishres.2011.01.001. 311

Ikhwanuddin, M., Bachok, Z., Hilmi, M. G., Azmie, G., & Zakaria, M. Z. (2010a). Species 312

diversity, carapace width-body weight relationship, size distribution and sex ratio of mud 313

crab, genus Scylla from Setiu Wetlands of Terengganu coastal waters, Malaysia. Journal 314

of Sustainability Science and Management, 5, 97-109. 315

Ikhwanuddin, M., Bachok, Z., Mohd-Faizal, W. W. Y., Azmie, G., & Abol-Munafi, A. B. 316

(2010b). Size of maturity of mud crab Scylla olivacea (Herbst, 1796) from mangrove 317

areas of Terengganu coastal waters. Journal of Sustainability Science and Management, 318

5, 134-147. 319

Ikhwanuddin, M., Nur-Atika, J., Abol-Munafi, A. B., & Muhd-Farouk, H. (2014). Reproductive 320

biology on the gonad of female orange mud crab, Scylla olivacea (Herbst, 1796) from the 321

west coastal water of peninsular Malaysia. Asian Journal of Cell Biology, 9, 14-22. 322

doi:10.3923/ajcb.2014.14.22 323

Jones, R. E., Petrell, R. J., & Pauly, D. (1999). Using Modified Length-Weight Relationship to 324

Assess the Condition of Fish. Aquaculture Engineering, 20, 261-276. Retrieved from 325

https://pdfs.semanticscholar.org/0b81/df4081970a3933aa59f5dd2cb44995fea004.pdf 326

Josileen, J. (2011). Morphometric and length-weight relationship in the blue swimmer crab, 327

Portunus pelagicus (Linnaeus, 1758) (Decapoda, Brachyura) from the Mandapam Coast, 328

India. Crustaceana, 84 (14), 1665-1681. doi: 10.1163/156854011X607060 329

Page 18 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

16

Josileen, J., & Menon, N. G. (2005). Growth of the blue swimming crab, Portunus pelagicus 330

(Linnaeus, 1758) (Decapoda, Brachyura) in captivity. Crustaceana, 78(1), 1-18. 331

doi:10.1163/1568540054024556 332

Keenan, C. P., Davie, P. J. F., & Mann, D. L. (1998). A revision of the genus Scylla De 333

Haan, 1833 (Crustacea: Decapoda: Brachyura: Portunidae). Raffles Bulletin of 334

Zoology, 46, 217-245. Retrieved from 335

https://www.researchgate.net/profile/Peter_Davie/publication/256107287_A_revision_of336

_the_genus_Scylla_De_Haan_1833_Crustacea_Decapoda_Brachyura_Portunidae/links/0337

c960521c815370d62000000/A-revision-of-the-genus-Scylla-De-Haan-1833-Crustacea-338

Decapoda-Brachyura-Portunidae.pdf 339

Kuris, A. M., & Mager, M. (1975). Effect of limb regeneration on size increase at molt of the 340

shore crabs Hemigrapsus oregonensis (Dana, 1851) and Pachygrapsus crassipes (Randall, 341

1839). Journal of Experimental Zoology, 193, 105-112. doi:10.1002/jez.1401930311 342

Madlen M. H., Khadiga M. S., & Montaser, M. S. H. (2012). Morphological and histological 343

studies on the embryonic development of the freshwater prawn, Macrobrachium 344

rosenbergii (Crustacea, Decapoda). The Journal of Basic & Applied Zoology, 65 (3), 345

157-165. 346

Marichamy, R., & Rajapackiam, S. (2001). The aquaculture of Scylla species in India. Asian 347

Fisheries Science Journal, 14, 231-238. Retrieved from 348

http://eprints.cmfri.org.in/id/eprint/5651 349

Page 19 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

17

Millamena, O. M., & Quinitio, E. T. (1999). Reproductive performance of pond-sourced Scylla 350

serrata fed various broodstock diets. Proceedings of the International Scientific Forum of 351

Mud Crab Aquaculture and Biology, Darwin, Australia, April 21-24, 1997, 114-117. 352

Muhd-Farouk, H., Amin-Safwan, A., & Ikhwanuddin, M. (2016a). Effect of different fixatives 353

on different organs of orange mud crab, Scylla olivacea (Herbst, 1796). International 354

Conference on Marine Science and Aquaculture, Kota Kinabalu, Sabah, Malaysia, March 355

23-24, 2016. 356

Muhd-Farouk, H., Jasmani, S., & Ikhwanuddin. M. (2016b). Effect of vertebrate steroid 357

hormones on the ovarian maturation stages of orange mud crab, Scylla olivacea (Herbst, 358

1796). Aquaculture, 451, 78–86. doi:10.1016/j.aquaculture.2015.08.038 359

Nadiah, W. N., Ikhwanuddin, M., & Abol-Munafi, A. B. (2012). Remarks on the mating 360

behaviours and success of blue swimming crab, Portunus pelagicus (Linnaeus, 1766) 361

through the induction of limb autotomy technique. Journal of Animal and Veterinary 362

Advances. 11 (8), 1149-1157. 363

Oluwatoyin, A., Akintade, A., Edwin, C., & Victor, K. (2013). A study of length-weight 364

relationship and condition factors of West African blue crab (Callinectes pallidus) from 365

Ojo Creek, Lagos, Nigeria. American Journal of Research Communication, 1(3), 102-366

114. 367

Petrakis, G., & Stergiou, K. I. (1995). Weight-length relationship for 33 fish species in Greek 368

Waters. Fisheries Research, 21, 465-469. doi:10.1016/0165-7836(94)00294-7 369

Page 20 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

18

Rattanachote, A., & Dangwatanakul, R. (1992). Mud Crab, Scylla serrata (Forskal) Fattening in 370

Surat Thani Province. In The Mud Crab, C.A. Angell, editor. Queensland Department of 371

Primary Industries, Queensland, Australia, pp 171-177. 372

Ryer, C. H., Montfrans, J. V., & Moody, K. E. (1997). Cannibalism, refugia and the molting blue 373

crab. Marine Ecology Progress Series. 147, 77-85. Retrieved from 374

http://www.jstor.org/stable/24857346 375

Smith, L. D. (1990). Patterns of limb loss in the blue crab, Callinectes sapidus Rathbun, and the 376

effects of autotomy on growth. Bulletin of Marine Science. 46: 23-35. Retrieved from 377

http://www.ingentaconnect.com/content/umrsmas/bullmar/1990/00000046/00000001/art0378

0003#expand/collapse. 379

Stickney, R. R. (1972). Length-Weight relationships for several fishes and invertebrates in 380

Georgia coastal waters with condition factors for fish species. Skidaway Institute of 381

Oceanography Savannah, Georgia. 382

Sudha Devi, A. R., & Smija, M. K. (2015). Growth and moulting pattern in the freshwater crab 383

Travancoriana schirnerae. International Journal of Fisheries and Aquatic Studies, 3(2), 384

391-396. 385

Sukumaran, K. K., & Neelakantan, R. 1997. Length weight relationship in two marine portunid 386

crabs Portunus sanguinolentus (Herbst) and Portunus pelagicus (Linnaeus) from the 387

Kamalaka coast. Indian Journal of Marine Science, 26, 39-42. Retrieved from 388

http://eprints.cmfri.org.in/id/eprint/8255 389

Page 21 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

19

Tabash, F. A. (2001). Assessment and ecological characterization of the Blue Crab (jaiba, 390

Callinectes arcuatus) in the Gulf of Nicoya, Costa Rica. 391

www.una.ac.cr/biol/unalaw/english /crab.htm. [January 2, 2017]. 392

Villasmil, L., Mendoza, J., & Ferrer, O. J. M. (1997). Growth and mortality of the blue crab 393

Callinectes sapidus (Rathbun, 1896) in Lake Maracaibo. Ciencia (Maracaibo), 5, 7-15. 394

Waiho, K., Fazhan, H., & Ikhwanuddin, M. (2016). Size distribution, length-weight relationship 395

and size at the onset of sexual of the orange mud crab, Scylla olivacea, in Malaysian 396

waters. Marine Biology Research, 12(7), 726-738. doi:10.1080/17451000.2016.1200726 397

398

399

400

Page 22 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

20

Appendix

Carapace width and body weight increment of S. olivacea that being introduced with limb autotomized technique. All crabs molted

successfully and showed Stage 1 ovary (n=135).

Carapace Width Body Weight

No. of crab Before (cm) After (cm) Size of Increment (cm) Before (g) After (g) Size of Increment(g)

1 7.71 8.91 1.20 103.70 110.50 6.80

2 6.81 7.49 0.68 62.50 69.80 7.30

3 7.11 8.02 0.91 79.10 88.30 9.20

4 7.61 8.65 1.04 78.50 86.70 8.20

5 6.74 7.35 0.61 60.30 68.70 8.40

6 7.74 8.35 0.61 68.90 78.40 9.50

7 8.33 9.37 1.04 86.70 96.40 9.70

8 9.02 10.56 1.54 102.60 110.80 8.20

9 6.92 7.61 0.69 75.60 84.30 8.70

10 7.74 8.44 0.70 80.30 89.70 9.40

11 7.76 8.47 0.71 75.10 83.60 8.50

12 7.04 7.57 0.53 68.30 79.90 11.60

13 8.14 9.33 1.19 98.80 110.50 11.70

14 7.19 7.95 0.76 79.60 86.50 6.90

15 8.14 9.33 1.19 90.50 99.60 9.10

16 7.47 8.17 0.70 79.40 84.60 5.20

17 7.59 8.28 0.69 86.30 94.70 8.40

18 7.39 7.82 0.43 69.60 78.60 9.00

19 6.85 7.59 0.74 68.30 79.40 11.10

20 7.66 8.34 0.68 85.90 98.40 12.50

21 7.86 9.35 1.49 102.20 121.20 19.00

22 6.81 7.13 0.32 67.90 75.40 7.50

23 7.54 8.63 1.09 78.30 84.70 6.40

24 7.17 7.98 0.81 71.90 79.40 7.50

25 6.42 7.56 1.14 57.30 69.90 12.60

Page 23 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

21

26 6.44 7.45 1.01 51.80 63.80 12.00

27 7.95 8.96 1.01 67.70 76.80 9.10

28 6.68 7.32 0.64 55.10 67.90 12.80

29 6.98 7.76 0.78 69.40 80.30 10.90

30 7.17 8.34 1.17 72.90 83.40 10.50

31 6.45 7.38 0.93 52.00 63.70 11.70

32 6.56 7.31 0.75 54.10 63.70 9.60

33 6.19 7.06 0.87 44.30 52.80 8.50

34 7.27 8.07 0.80 77.00 85.70 8.70

35 6.85 7.64 0.79 66.10 75.90 9.80

36 6.59 7.31 0.72 57.10 65.60 8.50

37 7.86 8.54 0.68 63.80 71.50 7.70

38 6.84 7.96 1.12 65.50 76.30 10.80

39 6.88 7.56 0.68 63.40 75.10 11.70

40 6.33 6.92 0.59 51.20 56.50 5.30

41 6.52 7.42 0.90 51.60 57.40 5.80

42 7.61 8.76 1.15 77.30 86.90 9.60

43 7.75 8.41 0.66 95.40 101.20 5.80

44 8.73 9.52 0.79 136.80 148.80 12.00

45 8.76 9.45 0.69 115.80 128.50 12.70

46 8.21 9.18 0.97 109.60 124.30 14.70

47 8.11 8.41 0.30 98.60 101.20 2.60

48 7.12 8.35 1.23 65.40 70.80 5.40

49 8.12 9.52 1.40 139.80 148.80 9.00

50 7.71 8.55 0.84 107.80 111.00 3.20

51 7.81 8.63 0.82 76.90 88.30 11.40

52 7.90 8.01 0.11 68.30 79.70 11.40

53 5.90 6.97 1.07 50.60 60.40 9.80

54 6.81 6.93 0.12 48.60 56.80 8.20

55 8.61 9.66 1.05 114.70 126.50 11.80

56 6.85 7.59 0.74 75.30 84.30 9.00

57 5.95 6.56 0.61 64.20 69.70 5.50

58 9.09 9.79 0.70 88.50 97.80 9.30

Page 24 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

22

59 8.63 9.22 0.59 76.10 85.30 9.20

60 6.26 6.91 0.65 75.40 80.60 5.20

61 7.91 8.52 0.61 73.40 79.60 6.20

62 5.92 6.82 0.90 58.90 64.90 6.00

63 8.28 8.92 0.64 86.70 94.30 7.60

64 5.85 6.63 0.78 63.20 68.50 5.30

65 5.53 6.06 0.53 59.60 68.70 9.10

66 5.45 6.09 0.64 58.50 71.30 12.80

67 9.56 10.24 0.68 91.40 101.3 9.90

68 5.72 6.28 0.56 65.20 71.30 6.10

69 6.52 7.13 0.61 64.10 71.40 7.30

70 6.41 7.14 0.73 64.60 73.10 8.50

71 9.20 9.52 0.32 92.30 95.60 3.30

72 7.07 7.52 0.45 84.10 89.60 5.50

73 6.36 6.63 0.27 64.80 73.80 9.00

74 8.44 8.95 0.51 75.40 81.20 5.80

75 9.15 9.74 0.59 98.60 107.40 8.80

76 7.91 8.84 0.93 69.70 76.30 6.60

77 6.56 7.28 0.72 59.60 71.40 11.80

78 5.85 6.56 0.71 55.80 67.20 11.40

79 8.26 9.02 0.76 75.10 79.50 4.40

80 8.52 9.44 0.92 76.30 88.10 11.80

81 8.15 8.86 0.71 70.50 76.80 6.30

82 7.36 8.55 1.19 66.50 73.30 6.80

83 7.52 7.78 0.26 67.40 76.10 8.70

84 8.48 9.64 1.16 68.90 77.40 8.50

85 7.32 8.27 0.95 57.80 68.50 10.70

86 7.47 8.14 0.67 58.30 64.70 6.40

87 7.82 8.74 0.92 60.30 67.10 6.80

88 7.08 7.69 0.61 56.40 61.70 5.30

89 7.24 8.58 1.34 57.10 61.20 4.10

90 8.15 8.77 0.62 71.50 77.80 6.30

91 8.07 9.21 1.14 65.30 69.80 4.50

Page 25 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

23

92 7.77 8.84 1.07 59.80 64.60 4.80

93 7.33 8.02 0.69 58.20 63.10 4.90

94 6.83 7.85 1.02 55.80 67.20 11.40

95 7.32 8.39 1.07 61.70 68.50 6.80

96 6.76 7.35 0.59 56.90 61.60 4.70

97 7.53 8.42 0.89 60.70 67.90 7.20

98 7.63 8.56 0.93 61.80 68.10 6.30

99 7.49 7.95 0.46 59.60 65.30 5.70

100 7.68 8.09 0.41 61.80 66.70 4.90

101 7.76 8.59 0.83 64.30 71.20 6.90

102 8.58 9.54 0.96 72.40 81.20 8.80

103 7.93 8.82 0.89 65.30 69.60 4.30

104 7.84 8.49 0.65 63.10 69.80 6.70

105 7.14 8.24 1.10 60.80 68.50 7.70

106 7.84 8.98 1.14 72.90 83.60 10.70

107 7.83 8.54 0.71 70.60 79.50 8.90

108 7.93 8.98 1.05 75.40 85.20 9.80

109 7.24 8.36 1.12 68.90 77.90 9.00

110 7.26 7.75 0.49 69.70 76.50 6.80

111 7.74 8.33 0.59 70.80 77.30 6.50

112 7.35 8.07 0.72 69.80 74.30 4.50

113 7.13 7.83 0.70 66.40 75.10 8.70

114 7.68 8.38 0.70 74.30 83.10 8.80

115 7.07 7.73 0.66 66.70 71.80 5.10

116 7.44 8.22 0.78 69.50 76.30 6.80

117 8.44 9.35 0.91 79.60 87.90 8.30

118 8.05 9.26 1.21 77.80 88.40 10.6

119 8.87 9.84 0.97 88.30 92.10 3.80

120 7.16 7.95 0.79 68.30 74.80 6.50

121 6.27 6.65 0.38 59.70 68.30 8.60

122 6.63 7.39 0.76 68.10 78.90 10.8

123 6.56 7.29 0.73 67.10 71.70 4.60

124 7.86 8.48 0.62 75.90 79.90 4.00

Page 26 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

24

125 8.26 9.43 1.17 69.60 78.90 9.30

126 7.69 8.66 0.97 73.20 84.30 11.10

127 7.84 8.78 0.94 68.30 76.90 8.60

128 8.05 8.68 0.63 73.20 85.10 11.90

129 8.39 9.44 1.05 72.40 83.30 10.90

130 7.61 8.61 1.00 66.70 78.10 11.40

131 7.62 8.55 0.93 68.90 76.30 7.40

132 8.90 9.90 1.00 76.90 89.30 12.40

133 8.35 9.44 1.09 63.20 75.10 11.90

134 7.43 8.82 1.39 65.20 75.10 9.90

135 8.67 10.17 1.50 77.80 89.20 11.40

Page 27 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

Table 1. Mean oocyte diameter of S. olivacea before (control) and after the molt

(induced by the limb autotomy).

Mean oocyte diameter (µm)

Standard deviation n

Before (Control)

25.67

4.38

30

After (Introduced to limb autotomized technique)

34.76

12.13

135

Page 28 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

Table 2. Mean, standard deviation, highest and lowest carapace width and body weight

increments recorded after the crabs molted (n=135).

Mean Standard deviation

Highest increment

Lowest increment

CW (cm)

0.816

0.27

1.54

0.30

BW (g) 8.395 2.72 19.00 3.20

Page 29 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

Table 3. Correlation analysis for carapace width and body weight increments of S.

olivacea (n=135).

CW BW

CW Pearson Correlation 1 0.263**

Sig. (1-tailed) 0.001 N 135 135

BW Pearson Correlation 0.263** 1

Sig. (1-tailed) 0.001

N 135 135

Page 30 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

Table 4. Regression analysis for carapace width and body weight increments of S.

olivacea (n=135).

Model Sum of Squares df

Mean Square F Sig.

1 Regression 68.665 1 68.665 9.897 0.002b

Residual 922.702 133 6.938

Total 991.366 134

Page 31 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

Figure 1. (a) External morphology of Stage 1 ovary before the limb autotomy, (b)

External morphology of Stage 1 ovary after the crab molted, (c) Histological assessment

of Stage 1 ovary before the crab’s molt, (d) Histological assessment of Stage 1 ovary

after the crab molted. O: oocyte, F: follicle cell, N: nucleus, Og: oogonia.

Page 32 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

Figure 2. Frequency of carapace width increments of S. olivacea (n=135).

(cm)

Page 33 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

Figure 3. Frequency of body weight increments of S. olivacea (n=135).

(g)

Page 34 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960

For Review Only

Figure 4. Regression analysis for carapace width and body weight increments of S.

olivacea with the equation y=2.61x + 6.27 (R2 =0.069) (n=135).

(cm)

(g)

Page 35 of 35

For Proof Read only

Songklanakarin Journal of Science and Technology SJST-2017-0375.R1 Ikhwanuddin

123456789101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960