International Research Journal of Biological Sciences ___________________________________ ISSN 2278-3202Vol. 3(9), 51-56, September (2014) Int. Res. J. Biological Sci. International Science Congress Association 51 Karyological Analysis of Mud Crab and Flower Crab of OdishaSatapathy Swagatika and Mohanty Prafulla Kumar PG Department of Zoology, Utkal University, Vani Vihar, Bhubaneswar-751 004, Odisha, INDIAAvailable online at: www.isca.in, www.isca.me Received 29th March 2014, revised 21st May 2014, accepted 22nd June 2014Abstract The present study focuses on the karyologicalanalyses of two commercially important edible crabs namely, Scylla serrata and Portunus pelagicus. Thenumber and characteristics of chromosomes of these two crabs were studied from spermatogonial metaphase of testes tissues. A total of 56 mitotic metaphases were examined in case of Scylla serrata (mud crab). The diploid chromosome number ranged from 99 to 108 with a mode at 106 (2n=106), representing 48.2% of the 56 metaphase compliments. A total of 50 mitotic metaphases were studied in Portunus pelagicus (flower crab or blue crab). The diploid chromosome number ranged from 92 to 101 with a mode at 98 (2n=98), representing 50% of the 50 metaphase compliments. Here sex chromosomes were not detected. Keywords: Crab, karyology, Scylla serrata, Portunus pelagicus. IntroductionScylla serrata is known as Mud crab, the common edible crab of largest size available in Chilika lagoon. In Odisha this is locally named as REDHA KANKADA. Portunus pelagicus is known as flower crab. It represents a valuable component of crustacean fishery. In India, it forms a small scale fishery at different regions along the east and west coasts. In Odisha it is locally named as RANI KANKADA. Karyological studies provides basic information about the number, size and morphology of chromosomes. Since all evolutionary changes have their origin in the chromosomes, any significant alteration in structure, number and behaviour of the chromosomes in the karyotype of a species is also reflected in phenotypic changes which have definite importance in the process of evolution, while morphological features may be influenced by external environmental changes and as such are not so reliable for taxonomic consideration. The crabs are classified under decapod crustaceans, which posses numerous small chromosomes of moreover same shapes which make them difficult to differentiate and count. Great variations in chromosome numbers also have been reported among related species of decapods. For example, two related species of fresh water prawn family Palaemonidae, Macrobrachium rosenbergii and Macrobrachium rude have 118 and 96 chromosomes respectively while two related species of crabs of Grapsidae family; Varuna litterata and Sesarma tetragonum have 146 and 94 chromosomes respectively. Cytogenetic study has immense importance in characterising, classifying and drawing evolutionary relationship among the species. In recent years, chromosomal studies in fishes became a priority area of research3, 4. In general, cytogenetic studies of crustaceans are relatively few and very difficult to perform because their chromosome numbers are large5-12 and they are of small dot and rod shaped. Their shapes are very variable such as metacentric, sub metacentric and acrocentric chromosomes13-15. Karyological studies were undertaken in an attempt to differentiate between two species of mud crabs namely Scyllaserrata and Scylla tranquebarica16. The marine shrimp Penaeus merguiensis from the Persian Gulf17 was studied for understanding the geographical differences in chromosome number. By understanding the basic genetic profile of these crabs, genes of commercial importance can be identified and product quality can be improved in terms of consumer preference and human nutrition. The present paper investigates the karyomorphometrical data of these two edible common crabs as karyotype analysis is the most important step towards the gene manipulation and related genetic engineering. Material and Methods Five adult males, each of Scyllaserrata and Portunus pelagicuswere collected from Satapada, Chilika lagoon (19º 48´ N, 85º 49´E) of Odisha respectively for the present study. After capture, they were transported from the collection sites in live condition in oxygen filled polythene bags and buckets to the laboratory and maintained in aquaria. Testes tissues were dissected out and pretreated with 1% KCl solution and then fixed in freshly prepared Carnoy’s 1:3 acetoalcohol fixative. The fixed material was kept in refrigerator for 2 days. Then the tissues were centrifuged for thrice at 3,000rpm for 10 minutes. The fixative was changed for thrice with an interval of 15 minutes. Slides containing chromosome spreads were prepared from the fixed tissues18. The tissues were stained in 10% Giemsa19 in Sorenson’s phosphate buffer (pH7) for 25 minutes and rinsed in distilled water. The well spread metaphase plates were observed, analysed and photomicrographed at 100X magnification with an Olympus research microscope coupled with digital camera. In the preparation of karyotypes, the print International Research Journal of Biological Sciences ________________________________________________ Vol. 3(9), 51-56, September (2014) International Science Congress Association of chromosomes of well spread metaphase complements were cut along their boundaries and individual chromosomes were matched in pairs considering their length and morphology. The pairs of chromosomes were arranged in serial order accord their decreasing order of lengths as the chromosomes were gradually seriated and grouped according to chromosome morphology. Then karyotyping was done20 and metrical studies were made according to previous workers. 21, 22 Results and Discussion Out of 56 spermatogonial metaphase plates examined in case of Scyllaserrata , 27 plates had 106 chromosomes, while 5 had 105, 2 had 104, 4 had 103, 3 had 102, 2 had 101, 3 had 100, 4 had 99, 1 had 107 and rest 5had 108 chromosomes (table 1 and figure 3(A)). Therefore the diploid chromosome number of this species is 2n=106 and out of 56 cells the modal diploid cells’ percentage was 48.2%. Thus the diploid number of chromosome in this species is 2n =106. This is in conformity with the Metaphase complements (A, B) of (A) (C International Research Journal of Biological Sciences ________________________________________________ International Science Congress Association of chromosomes of well spread metaphase complements were cut along their boundaries and individual chromosomes were matched in pairs considering their length and morphology. The pairs of chromosomes were arranged in serial order accord ing to their decreasing order of lengths as the chromosomes were gradually seriated and grouped according to chromosome and metrical studies 21, 22 . of 56 spermatogonial metaphase plates examined in case of , 27 plates had 106 chromosomes, while 5 had 105, 2 had 104, 4 had 103, 3 had 102, 2 had 101, 3 had 100, 4 had 99, 1 had 107 and rest 5had 108 chromosomes (table 1 and Therefore the diploid chromosome number of this species is 2n=106 and out of 56 cells the modal diploid cells’ percentage was 48.2%. Thus the diploid number of chromosome in this species is 2n =106. This is in conformity with the observations made earlier and at variance with the report where there were 94 chromosomes in the diploid count, 12 less than the present number and 102 chromosomes chromosomes were acrocentric ones varying from rod to dot shapes (figure 1 A and B). The primary spermato metaphase chromosomes presented a circular arrangement. The chromosomes were highly condensed bodies and were almost round. Out of 55 meiotic spermatocytes metaphase plates examined 21 plates had 53 chromosomes, while 7 had 52, 6 had 51, 3 had 50, 2 had 49, 5 had 48, 4 had 47, 2 had 54, 3 had 55 and rest 2 had 56 chromosomes depicted in table 2 and figure 3(B). As the modal haploid cells’ percentage was 38.18% of 55 cells, therefore, haploid chromosome number of this species is n=53. The secondary spe rmatocytes similarly formed smaller circular plates. The chromosomes were round to dot shaped. Thus the chromosome number determined for this species are 2n = 106 in the diploid and n = 53 in the haploid. No chromosomes were observed here as sex chromosom Figure-1 Metaphase complements (A, B) of S. serrata and (C, D) of Portunus pelagicus. (B) (D) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Int. Res. J. Biological Sci. 52 and at variance with the report 7 where there were 94 chromosomes in the diploid count, 12 less than the present number and 102 chromosomes 23. All the chromosomes were acrocentric ones varying from rod to dot shapes (figure 1 A and B). The primary spermato cytic metaphase chromosomes presented a circular arrangement. The chromosomes were highly condensed bodies and were almost round. Out of 55 meiotic spermatocytes metaphase plates examined 21 plates had 53 chromosomes, while 7 had 52, 6 had had 49, 5 had 48, 4 had 47, 2 had 54, 3 had 55 and rest 2 had 56 chromosomes depicted in table 2 and figure 3(B). As the modal haploid cells’ percentage was 38.18% of 55 cells, therefore, haploid chromosome number of this species is rmatocytes similarly formed smaller circular plates. The chromosomes were round to dot shaped. Thus the chromosome number determined for this species are 2n = 106 in the diploid and n = 53 in the haploid. No chromosomes were observed here as sex chromosom es. Portunus pelagicus. International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(9), 51-56, September (2014) Int. Res. J. Biological Sci. International Science Congress Association 53 In Portunus pelagicus, out of 50 spermatogonial metaphase compliments examined 25 cells had 98 chromosomes, 3 cells each had 97 and 96, while 2 had 95, 5 had 94, 3 had 93, 4 had 92, 2 had 99, 1 had100 and rest 2 had 101 chromosomes (table 3 and figure 4(A)). Thus the diploid chromosome number of this species is 2n=98 as the modal diploid cells’ percentage was 50% of 50 cells. The haploid count obtained out of meiotic spermatocyte metaphase compliments revealed 49 chromosomes in 25 cells out of 61 cells studied, while 7 had 48, 6 had 47, 3 had 46, 4 had 45, 6 had 44, 3 had 43, 2 had 50, 3 had 51 and rest 2 had 52 chromosomes. Therefore the haploid chromosome number of this species is n=49 as in 61 cells modal haploid cells’ percentage was 40.98% (table 4 and figure 4 (B)). The chromosomes were isomorphic-acrocentric in nature and were rods to dot in shape (figure 1C and D). The primary spermatocytic division figures were more numerous. In morphology the metaphase I chromosomes were from round to dots. There was no regular pattern for the arrangement of chromosomes. No chromosome was observed which could be considered as the sex- chromosome in respect to behaviour or morphology. The chromosome sizes varied between 1 to 2 µm in these crabs whereas in amphipod crustaceans it varies 5 to 8 µm24. The present comparative karyotypic analysis on Scylla serrataand Portunus pelagicus report the chromosomes to be almost rod and dot (oval to round) shaped. To establish diploid chromosome number in case of large number of small chromosomes, the numbers observed in maximum metaphasic plates is to be considered. Accordingly, the diploid chromosome number in the mud crab Scylla serrata is 106 (figure 2 A and B) while that of flower crab Portunus pelagicusis 98 (figure 2 C and D). The metaphase chromosome counts obtained from testes of Scylla serrata and Portunus pelagicus have diploid chromosome numbers 106 (n=53) and 98 (n=49) respectively. The chromosomes of these two species of decapod crustaceans were studied from the male germ cells. Good metaphase plates were obtained from testes. But from gill, hepatopancreas and ovary tissues suitable metaphase plates were not obtained because fat tissue in advanced stages of sexual maturation was hampering the preparation of suitable metaphase chromosomes. The testicular tissues contain both spermatogonias (mitotic cells) and spermatocytes (meiotic cells) for which diploid and haploid chromosome numbers were obtained respectively. Here a very good chromosomal spread obtained by air drying Giemsa technique. The best Giemsa concentration and time for a suitable metaphase plate were 10% and 25minutes respectively. During dropping of cell suspension the slides also placed on hot plates (43º) 25. But in this study chilled slides give better results than hot slides. The use of different concentrations of colchicine reflected no evident difference in the degree of chromosome condensation. However, greater chromosome condensation was obtained due to longer incubation time. This is a study where good metaphasic plates were observed without treatment with chemicals like colchicine. Since the present paper is restricted to chromosome morphology only, therefore, the results of crossing experiments, which do suggest the existence of sex chromosomes in Portunidae family, are not considered here. (A) (B) (C) (D) Figure-2 Karyotype (A, B) of S. serrata and (C, D) of P. pelagicus International Research Journal of Biological Sciences ________________________________________________ Vol. 3(9), 51-56, September (2014) International Science Congress Association Frequency distribution for diploid and haploid chromosome counts in Frequency distribution for diploid and haploid chromosome counts in Diploid chromosome numbers of No of chromosomes 99 100 101 Cells 4 3 Percentage 7.14 5.35 3.57 Haploid chromosome numbers of No of chromosomes 47 48 49 Cells 4 5 Percentage 7.27 9.09 3.63 Diploid chromosome numbers of No of chromosomes 92 93 Cells 4 3 Percentage 8 6 Haploid chromosome numbers of No of chromosomes 43 44 45 Cells 3 6 Percentage 4.91 9.83 6.55 204060 99101 103 Metaphase relative frequency(%) Diploid Chromosome Number Mitotic spermatogonia in Scylla serrata 204060 9294 96 Metaphase relative frequency(%) Diploid Chromosome Number Mitotic spermatogonia in P.pelagicus ( A (A) International Research Journal of Biological Sciences ________________________________________________ International Science Congress Association Figure-3 Frequency distribution for diploid and haploid chromosome counts in S. Serrata Figure-4 Frequency distribution for diploid and haploid chromosome counts in P. pelagicus Table-1 Diploid chromosome numbers of Scylla serrata 101 102 103 104 105 106 2 3 4 2 5 27 3.57 5.35 7.14 3.57 8.92 48.2 Table-2 Haploid chromosome numbers of Scylla serrata 49 50 51 52 53 54 2 3 6 7 21 2 3.63 5.45 10.9 12.72 38.18 3.63 Table-3 Diploid chromosome numbers of Portunus pelagicus94 95 96 97 98 99 5 2 3 3 25 2 10 4 6 6 50 4 Table-4 Haploid chromosome numbers of Portunus pelagicus 45 46 47 48 49 50 4 3 6 7 25 2 6.55 4.91 9.83 11.47 40.98 3.27 103 105107 Diploid Chromosome Number Mitotic spermatogonia in Scylla serrata 10203040 47495153Metaphase relative frequency(%) Haploid Chromosome Number Meiotic spermatocytes in S.serrata 96 98100 Diploid Chromosome Number Mitotic spermatogonia in P.pelagicus 204060 43454749Metaphase relative frequency(%) Haploid Chromosome Number Meiotic spermatocytes in P.pelagicus A ) (B) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Int. Res. J. Biological Sci. 54 Serrata P. pelagicus 107 108 Total 1 5 56 1.8 8.92 100 55 56 Total 3 2 55 5.45 3.63 100 100 101 Total 1 2 50 2 4 100 51 52 Total 3 2 61 4.91 3.27 100 55 Haploid Chromosome Number Meiotic spermatocytes in S.serrata 51 Haploid Chromosome Number (B) International Research Journal of Biological Sciences ________________________________________________ ISSN 2278-3202 Vol. 3(9), 51-56, September (2014) Int. Res. J. Biological Sci. International Science Congress Association 55 Conclusion Karyological studies have been undertaken in two species belonging to one family portunidae of decapod crustacean collected from Chilika lagoon. The testes material of the adults exclusively served as the source material for the chromosomal studies. Temporary and permanent squash preparations of the testes were stained respectively with lacto-aceto-orcein and Heidenhain’s Iron-haematoxylin and also in air drying Giemsa stain. These three methods were conducted but suitable chromosome plates were obtained in air drying Giemsa technique. Chromosome number, form and size were determined from spermatogonial metaphase counts of these two species. The spermatogonial chromosomes of these species are isomorphic being rod and dot shaped and acrocentric. Minute size and large number of the chromosomes render their counting quite difficult. The diploid chromosome number in Scylla serrata and Portunus pelagicus was ascertain to be 106 and 98 respectively. Acknowledgements The authors are thankful to P.G. Department of Zoology, Utkal University, Vani Vihar, Bhubaneshwar, Odisha for providing facilities. 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