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Scylla serrata (Forskal) is well-known being called as a mud crab, belongs to the family Portunidae and strongly related with mangrove environment (Gopurenko, 1999). It is widely distributed and commonly found in most estuarine, mangrove swamps, and shelter bay habitat of the coastal waters (Macnae, 1968).
Estampador (1949) has noted that the genus of Scylla has been considered monotypic, although the morphometric differences have found the existence of more than one species (Sugama, 1999). There are four Scylla species has been recorded which is genetically and morphologically different (Keenan, 1999).
Although inhabit coastal waters mostly in their life cycle, female Scylla has been recorded to migrate offshore during spawning period (Hill, 1994). There are two explanations following this behaviour; the larvae might be intolerance to the low salinity in estuarine habitat and to get more chance in larval dispersal.
Due to the fact that the larval stage took place 3-4 weeks floating in the open sea waters (Brick, 1974), the plaktonic larval phase suggest high dispersal potential and possibility of extensive gene flow between population through passive transport by ocean current (Fratini, 2002). However, Burton (1083) noted that the evident of dispersal capacities cannot dependably determine the genetic structure of natural population of marine invertebrates.
There are two types of mud crab fisheries has been practised in Indonesia, namely wild capture and culture. Since the early 1980s, Scylla serrata has been recognized as a commercially important crab species commodity in Indonesia. In recent year, this fisheries have been developing due to market demand and high value. According to Indonesian fisheries statistics, the crab export production reached 20.713 tonnes in 2008. Despite its importance in generating income to the fisheries sector, however further study about genetic and connectivity of this species in Indonesia was lacking.
This study investigated the phylogeographic distribution of Scylla serrata in Indonesia and further aimed to compare its genetic population structure and connectivity pattern throughout Indo-West Pacific based on the previous existing studies.
Materials and methods
Specimens of Scylla serrata were purchased from 10 different local fish markets throughout Indonesia; including from Sumatera, Java, Kalimantan and Nusa Tenggara Island. These geographic locations are shown in Figure 1. The sites were basically selected based on their presence in the region and level of geographical separation of wild mud crab fishing areas. However, due to the time limit and budget constraints, only samples from 4 major islands (7 Provinces) were chosen in this study. The sample collections were conducted between July to September 2010.
A single cheliped part from each sample specimen was removed and then conserved in 96% ethanol before storing it in a cool temperature until DNA extraction process. The number of samples collected (n) were varied, ranging from 11 to 20 individuals.
Table 1. Collection site and sample size of mudcrab sample examined for this study
Sample size (n)
West Nusa Tenggara
Figure 1. Scylla serrata sampling sites throughout Indonesia island.
A small muscle tissue from each cheliped specimen sample was removed and mixed with 500 µl 5% Chelex, 25 µl 100 mM DTT (Dithiothreitol) and 20 µl Proteinase-K (20mg/ml). The suspension sample containing the muscle tissue was then gently homogenized by incubation machine (Epperdorf Thermomixer Compact) at 750 rpm at 54ËšC and left overnight. The centrifugation 12.500 rpm of the sample was later undertaken for 3 minutes by using Eppendorf Epp Centrifuge Mini Spin 220U-022620151 to separate the supernatant from the chelex, followed by 95ËšC heating for 5 minutes by Thermojet Equibio machine and finally stored in refrigerator at -24ËšC until needed for the next step.
Mitochondrial cytochrome oxidase subunit I (COI) gene of 597-base pair (bp) segment was amplified by polymerase chain reaction (PCR) using two specific primers designed for insect and Scylla spp. sequences, namely: mtd10 5'-T TGA TTT TTT GGT CAT CCA GAA GT-3' (Roehrdanz, 1993) and C/N 2769 5'-TT AAG TCC TAG AAA ATG TTG RGG GA-3' (Gopurenko et al., 1999) which corresponding to 2172 and 2769 locations of Drosophila yakuba mtDNA sequence (Clary and Wolstenholme, 1985).
PCR amplifications were carried out in a MJ Research PTC-200 Thermo Cycler (BC-MJPC200) consisted of 35 cycles, which preceded with denaturation step in 3 minutes and continued by the following cycle profile: 94ËšC denaturation for 30 seconds, 50ËšC annealing for 30 seconds, 72ËšC extension for 5 minutes, and completed with an additional extension step for 5 minutes before storage in 10ËšC. The PCR products were then placed in a refrigerator of -24ËšC.
The PCR products were then screened in gel-electrophoresis in order to see the DNA in the product. Gene ruler TM 0.5 μg/lane, 8 cm length gel was used as a DNA ladder to observe the DNA presence. Electrophoresis was performed in a specific condition of 250V for 35 to 40 minutes.
PCR products were sent to the third party (who, where??????) to be sequenced. The results of the data sequencing were submitted to Genebank (accession numbers…………….). Sequencing results were then edited by using program ……………. and software …………….. was used to align sequences manually by eye. (Fratini, 2002)
Analysis of molecular variance (AMOVA) (Excoffier et al., 1992) were performed in this study to evaluate genetic differentiation between population. The method introduced by Tajima and Nei (1984) was also used in order to estimate FST indices by calculating haplotypic frequencies only or included genetic distances between haplotypes.
Significance levels of pairwise FST were calculated by 10.000 permutation of haplotypes between population. This test was performed under the hypothesis of no differentiation between population occurred. While P value of the test indicated the proportion of permutation which caused an FST value larger or equal to the observed one (Fratini et al., 2001).
To test the correlation between genetic distances with geographic distances, a Mantel test (1967) was performed as applied in ARLEQUIN. Genetic distance was corresponded to the FST value calculated from haplotypic frequencies from each pairwise comparison between localities.