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Last Layout Final Indian J. Fish., 51(4) : 389-399, Oct.-Dec., 2004 389 Genetic relationship between the genus Helice and three grapsid crab species MD. YOUNUS MIA, REIKO FUSEYA* AND SEIICHI WATANABE* Bangladesh Fisheries Research Institute, Brackishwater Station, Paikgacha, Khulna-9280, Bangladesh *Department of Aquatic Biosciences, Tokyo University of Fisheries, 4-5-7 Konan, Minato-ku, Tokyo 108-8477, Japan ABSTRACT Horizontal starch gel electrophoresis was used to analyse the genetic variation and relationship between the mud-flat crab of genus Helice and three other grapsid crabs, Chiromantes dehaani, Eriocheir japonica and Hemigrapsus penicillatus. Twenty five allozyme loci of 15 enzymes were investigated. The reaction of phosphoglucomutase (PGM) was found in those crabs except for E. japonica. Gene replacement was found at six loci, ALP*, FH-1*, G3PDH-1*, HK-1*, MPI-1* and MPI-2*. The average proportion of polymorphic loci and heterozygosity were 0.058 and 0.005 in Helice species, 0.091 and 0.023 in C. dehaani, 0.050 and 0.009 in E. japonica and 0.040 and 0.002 in H. penicillatus. Genetic analysis showed that Neis genetic distance was 0.14196 between the genus Helice and H. penicillatus and 1.23512 between C. dehaani and E. japonica. Introduction Rathbun 1929 are the known species under this genus (Sakai, 1976). The crabs of the genus Helice are the members of the family Grapsidae Electrophoretically detectable (Decapoda). There are many species allozyme genes are useful for analyzing under this family. Within this family genetic divergence among subspecies most of the species inhabit coastal believed to be closely related on the basis waters, though some species like of morphological traits, species and Eriocheir japonica, Chiromantes genera of a variety of organisms. In crab dehaani, C. haematocheir are found in species Takano et al. (1997) reported the estuary to upstream (Sakai, 1976). The genetic divergence between two estuarine crab of genus Helice consists sympatric forms (I and II) of the of four species. Helice tridens latimera estuarine grapsid crab, Hemigrapsus Parisi 1918, H. t. tridens De Haan 1835, penicillatus using isozyme gene markers. H. leachi Hess 1865, and H. t. wuana Fuseya and Watanabe (1996) suggested MD. Younus Mia et al. 390 that the genus Scylla includes at least taken from the legs and stored at 800C. three species based on isozyme analysis The tissues to be analysed for allozymes and Gao and Watanabe (1998) evaluated were homogenized with approximately the level of genetic divergence between equal volumes of the fragments of frozen the Japanese mitten crab, Eriocheir muscle and distilled water. Then, the japonica and the chinese mitten crab, E. homogenates were centrifuged at 12000 sinensis. rpm for 12 minutes at 40C and the supernatant was absorbed onto filter The aim of this study was to paper and used for electrophoresis. estimate the degree of genetic divergence Horizontal starch gel electrophoretic among the former three species of Helice techniques, buffer systems and staining compared to Chiromantes dehaani, procedure followed the method of the Eriocheir japonica and Hemigrapsus Japan Fisheries Resource Conservation penicillatus as out-group species by Association (1989) and Pasteur et al. genetic distance, number of polymorphic (1988). A total of 15 enzymes were loci and number of gene replacements. surveyed; the name, numbers and Materials and methods abbreviations of the enzymes followed Shaklee et al. (1990) (Table 2). Horizontal starch gel electrophoresis was used in order to estimate the degree The allele frequencies and observed heterozygosities (H ) for each locus were of genetic differentiation. A total of 59 o H. t. latimera, 230 H. t. tridens, 176 H. determined by direct census of the leachi, 25 C. dehaani, 43 H. penicillatus population data. Mean heterozygosity (H ) within population was estimated and 16 E. japonicus were collected (Table e 1). All samples were kept at 200C prior from average values across all samples to electrophoresis analysis. Muscle was (Nei, 1987). A locus was considered to be TABLE 1: Samples used for electrophoretic analysis in the genus Helice and three grapsid crabs Location Species Number Date Okinawa Helice tridens latimera 57 Mar-97 * H. leachi 40 Mar-97 * Ishigaki H. tridens latimera 2 Nov-98 * H. leachi 26 Nov-98 * Chiba H. t. tridens 76 Jun-97 * Wakayama H. t. tridens 19 Aug-99 Ehime H. t. tridens 33 Nov-98 * Fukuoka H. t. tridens 52 Nov-98 * H. leachi 57 Nov-98 * Kagoshima H. t. tridens 50 Aug-97 * Ogasawara H. leachi 53 Oct-98 * Miyagi Chiromantes dehaani 25 Oct-98 Tokyo Hemigrapsus penicillatus 43 Sep-99 Ogasawara Eriocheir japonica 16 Oct-98 *Samples used in the study of Mia et al. (1999) Genetic relationship between grapsid crabs 391 TABLE 2: Names, numbers and abbreviations of enzymes and buffers used for electrophoresis Enzyme name (abbreviation) Enzyme number Buffer Aspartate aminotransferase (AAT) 2.6.1.1 CAPM-7 Alkaline phosphatase (ALP) 3.1.3.1 CAPM-7 Esterase (EST) 3.1.1 CAPM-7 Fumarate hydratase (FH) 4.2.1.2 CT-8N Glycerol-3-phosphate dehydrogenase (G3PDH) 1.1.1.8 CAPM-7 Glucose-6-phosphate isomerase (GPI) 5.3.1.9 CT-8N Hexokinase (HK) 2.7.1.1 CT-8N Isocitrate dehydrogenase (IDHP) 1.1.1.42 CT-8 Leucine aminopeptidase (LAP) 3.4.11 CT-7 Lactate dehydrogenase (LDH) 1.1.1.27 CT-8 Malate dehydrogenase (MDH) 1.1.1.37 CAPM-7 Mannose-6-phosphate isomerase (MPI) 5.3.1.8 CT-7 Phosphogluconate dehydrogenase (PGDH) 1.1.1.44 CAPM-7 Phosphoglucomutase (PGM) 2.7.5.1 CAPM-7 Superoxide dismutase (SOD) 1.15.1.1 CT-8N polymorphic when the frequency of the Electrophoretic patterns and most common allele was less than or banding positions of the six species were equal to 0.99 at one or more localities compared on the same gel in order to (Nei, 1987). In order to estimate the analyse the allelic differences among degree of genetic divergence among the them. Electrophoresis revealed 25 loci samples, the genetic distance between encoding the 15 investigated enzymes in samples was calculated using Neis Helice species and Hemigrapsus formulae (Nei, 1972), and dendrogram penicillatus. Twenty-two loci coded by 15 from the matrix of genetic distances were enzymes and twenty loci coded by 14 constructed using the neighbor-joining enzymes were investigated in method (Saitou and Nei, 1987). Chiromantes dehaani and Eriocheir japonica, respectively (Table 3). Bands Results for the enzyme of phosphoglucomutase Isozymic bands were compared on (PGM) was not appeared in E. japonica. the same gel in order to identify common Eight loci, AAT-1*, AAT-2*, GPI*, IDHP*, or different alleles between the genus MDH-1*, MDH-2*, PGDH* and LDH* in Helice and three other grapsid crab Helice species, two loci GPI* and IDHP* species. Bands that appeared at the same in C. dehaani, one locus AAT-2* in E. position on the same gel were assumed japonica and also one locus GPI* in H. to be controlled by the same allele, while penicillatus exhibited polymorphism those that appeared at different positions (Fig. 1). The genotypes of each species were controlled by different alleles. The are shown in Table 3. allele frequencies estimated for the 25 Five enzymes, namely alkaline loci in Helice species and Hemigrapsus phosphatase (ALP), glucose-6-phosphate penicillatus, 22 loci in Chiromantes isomerase (GPI), isocitrate dehaani and 20 loci in Eriocheir japonica dehydrogenase (IDHP), lactate are summarized in Table 3. MD. Younus Mia TABLE 3: Allele frequency, proportion of polymorphic loci and heterozygosity in the genus Helice and three other grapsid crab species. Species H. t. latimera H. t. tridens H. leachi C. dehaani E. japonica H. penicillatus Locality Okinawa Ishigaki Chiba Wakayama Ehime Fukuoka Kagoshima Fukuoka Okinawa Ishigaki Ogasawara Miyagi Ogasawara Tokyo Locus Allele 57 2 76 19 33 52 50 57 40 26 53 25 16 43 AAT-1* *a 0 0 0 0 0 0.019 0 0 0 0 0 1 0 0 *b 1 1 1 1 1 0.981 1 1 1 1 1 0 1 1 AAT-2* *a 0 0 0 0 0 0.019 0 1 1 1 0 0 0 0 et al. *b 1 1 1 0.895 1 0.981 1 0 0 0 0.991 0 0.906 0 *c 0 0 0 0.105 0 0 0 0 0 0 0.009 1 0.063 1 *d 0 0 0 0 0 0 0 0 0 0 0 0 0.031 0 ALP* *a0 000 00 0 00000 10 *b1 111 11 1 11111 01 EST-1* *a1 111 11 1 11111 11 EST-2* *a1 111 11 1 11111 11 FH-1* *a1 111 11 1 11110 11 *b0 000 00 0 00001 00 FH-2* *a1 111 11 1 11110 01 G3PDH-1* *a1 111 11 1 11110 11 *b0 000 00 0 00001 00 G3PDH-2* *a1 111 11 1 11110 01 GPI* *a 0.009 0 0 0 0 0.01 0.02 0 0 0 0 0 0 0 *b 0.851 1 0.99 0.974 0.97 0.962 0.95 0 0 0 0 0 0 0 *c 0.14 0 0.01 0.026 0.03 0.01 0.02 0 0 0 0 0 0 0 *d 0 0 0 0 0 0.019 0.01 0.009 0 0 0 0 1 0 *e 0 0 0 0 0 0 0 0.982 0.975 1 1 0.64 0 0.023 *f 0 0 0 0 0 0 0 0.009 0.025 0 0 0.36 0 0.977 HK-1* *a1 111 11 1 11110 11 *b0 000 00 0 00001 00 HK-2* *a1 111 11 1 11110 01 IDHP* *a 0.018 0 0 0 0 0 0 0 0 0 0 0 0 0 *b 0.982 1 0.99 1 1 0.981 0.99 0 0 0 0 0 1 0 *c 0 0 0.01 0 0 0.019 0.01 1 0.988 1 1 0.98 0 1 *d 0 0 0 0 0 0 0 0 0.013 0 0 0.02 0 0 392 Continued..
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