Black tiger shrimp Penaeus monodon from 19 locations of Andaman and Nicobar Islands were studied for genetic

differentiation and variability using allozyme electrophoretic analysis. Thirty two presumed loci were consistently expressed from 25 enzymes and a general protein, and these were also temporaly stable during the period 2002-2005. The percentage of polymorphic loci, mean number of alleles, mean effective number of alleles, observed homozygosity, expected homozygosity, observed heterozygosity, expected heterozygosity, average heterozygosity, gene flow (Nm), Fis, Fit, Fst,

Rare alleles, monomorphic loci, diagnostic loci, genetic identity, genetic distance and establishment of breeding boundaries were documented in all tested populations, and between the population of Andaman group and Nicobar group of islands. Colonization of P. monodon population due to the decrease of genetic diversity has been formed from north Andaman to south Andaman. As a result, two insular populations i.e., A (Andaman group of islands) and B (Nicobar group of islands) were differentiate

Madhu, K

Madhu, Rema

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145Genetic differentiation of black tiger shrimp Penaeus monodon Fabricius(Crustacea: Decapoda) in Andaman  and Nicobar islandsK. MADHU AND REMA MADHUCentral Marine Fisheries Research Institute, P. B. NO. 1603Ernakulam  North P. O.,  Kochi - 682 018, Kerala, Indiae-mail: kmadhu30@rediffmail.comABSTRACTBlack tiger shrimp Penaeus monodon from 19 locations of Andaman and Nicobar Islands were studied for geneticdifferentiation and variability using allozyme electrophoretic analysis. Thirty two presumed loci were consistently expressedfrom 25 enzymes and a general protein, and these were also temporaly stable during the period 2002-2005. The   percentageof polymorphic loci, mean number of alleles, mean effective number of alleles, observed homozygosity, expectedhomozygosity, observed heterozygosity, expected heterozygosity, average heterozygosity, gene flow (Nm), Fis, Fit, Fst,Rare alleles, monomorphic loci, diagnostic loci, genetic identity, genetic distance and establishment of breeding boundarieswere documented in all tested populations, and between the population of Andaman group and Nicobar group of islands.Colonization of P. monodon  population due to the decrease of genetic diversity has been formed from north Andaman tosouth Andaman. As a result, two insular populations i.e., A (Andaman group of islands) and B (Nicobar group ofislands) were differentiated.Keywords: Alleles, Allozyme, Genetic differentiation, Heterozygosity, Penaeus monodonPenaeus monodon has a broad geographic range intropical and subtropical coastal waters from Africa throughIndia, China to south-east Asia to Australia (Greyet al., 1983). The increase in commercial fishing ofP. monodon has stimulated interest in its stock identification,distribution and biology. Among the available techniquesfor molecular genetics studies, the allozyme electrophoresisdata still presents the most reliable and powerful methodfor the identification of genetically distinct group of fishes(Ridgeway, 2005). Though many reports emphasized thatpenaeid prawns have low levels of genetic variation incomparison to other organisms (Nelson  and  Hedgecock,1980), population differentiation in several penaeid specieshas been reported (Lester, 1979; Mulley and Latter, 1981;Benzie et al., 1992; Klinbunga et al., 1999; Klinbunga etal., 2001; Tassanakajon et al., 2004).Samples (10-20 individuals per collection per site) ofboth sexes and tissues of tiger prawn P. monodon (130 to160 mm total length) were collected from trawlers at 19sites in Andaman and Nicobar islands. Specimens werefrozen immediately in dry ice, transported to the laboratoryand stored at – 80 °C until further analysis. The musclefrom the first abdominal segment, hepatopancreas and eyesalong with stalk (100 mg each) were thawed and  groundwith 1000 μl of double distilled water and the homogenatewas centrifuged at 10,000 rpm for 20 minutes at 4 ºC. Dropsof supernatant were frozen in liquid nitrogen to form beadsIndian J. Fish., 58(2) : 145-148, 2011and stored at -80 °C until electrophoretic analysis. Thesupernatants after thawing, were subjected topolyacrylamide gel electrophoresis (PAGE) with a 30minutes pre-run at 20 V and final electrophoresis at 3°C(100 V, 90 and 180 minutes) with different buffer systems.The enzymes and proteins were visualized by using standardhistochemical stain recipes (Table 1). The locus and allelicnomenclature  (Allendorf  and  Utter, 1979) were labeled(Shaklee et al., 1990) according to their migration distancerelative to that of the most common allele which wasassigned a value of 100. The temporal changes in allelicfrequency of all the tested populations (19 sites) werecarried out between the years 2002-2005 to assess the stablepolymorphic loci which were considered to assess thegenetic differentiation of the candidate species. ThePOPGEN 32 package and PHYLIP Version 3.5 were usedfor the analysis.Out of the 41 enzymes studied, only 25 enzymes andthe general protein loci consistently expressedpolymorphism. Thirty two loci  were  polymorphic(Table 1) and  none of these loci showed significantdeviations from Hardy-Weinberg. The enzyme systems suchas aconitase 4.2.1.3 (ACO), adinosine deaminase 3.5.4.4(ADA), creatin kinase 2.7.3.2 (CK), diaphorase 1.6.4.3(DIA), β-galactosidase 3.2.1.23. (GAL), glyoxalase - I4.4.1.5 (GLO), glutamate dehydrogenase 1.4.12 (GLD),guanine deaminase 3.5.4.3 (GDA), fumarase  4.2.1.2Note146(FUM), malic enzyme NADP + 1.1.1.40 (MEP), peptidase(PEP), phospho gluconate dehydrogenase 1.1.1.43 (PGD),phosphohexose isomerase 5.3.1.9 (PHI), purine nucleosidephosphorylase 2.4.2.1 (NP) and  pyruvatekinase 2.7.1.40.(PK) were not scorable because of either inadequate stainingor interrupted expression of alleles and  were  not consideredfor further analysis.The genetic variability of P. monodon are presented inTable 2. In all tested populations of P. monodon, the lowestpercentage of polymorphism was observed from southAndaman (31.25%) and the highest value was from Nicobargroup of islands (53.12%). The trend of polymorphism inNicobar group of islands  decreased from Car Nicobar toCampbell Bay whereas in the case of Andaman group ofislands, it  increased from south  to north Andaman. A similarpattern of reducing genetic diversity was displayed in themean number of alleles per locus. In addition to these,genetic identity and genetic distance, temporal unstable loci,monomorphic loci, diagonostic loci, rare alleles andestablishment of  breeding  boundaries were documented.Though loci such as  AK-1*, MPI-1*,  PGM-3*, GPI-2*,  ACP-2*, ADH-3*, AK-2*, AOK-2*, AO-3*, SDH-2*,XDH-1*, PROT-7*, AOK-2*, AO-3*,  LDH-2*, LAP-1*,MDHP-2*, PGM-3* showed significant polymorphisms(p<0.05),  these loci have not been considered as  thesewere not consistently expressed (temporal unstable loci).The loci such as AAT-2*, ACP-3*, ADH-1*, AKP-1*, AO-2*, ALD-1*, AAT-3*, AOK-1*, G3PDH-1*&3*, G6PDH-1*,GPI-1*,  HK-1*, IDH-1,2*, LDH-1, LAP-3*, ME-1,2*,ODH-1*, PGDH-1*, PROT-1*,4*,5*&8* wereconsistently monomorphic. The expression of the diagnosticloci and alleles: EST-3*, G3PDH-2*, GPI-3*, IDH-3*,MDHP-1* from Andaman waters  and   AAT-1*, ADH-2*,G6PDH-2*, LAP-2*, MDH-1*, ME-3*, MPI-2*, PGDH-2* PGM-1*,2*, SDH-1*, SOD-1*, TR-1* from Nicobargroup of islands were obtained. The number of rare allelesalso reduced from Nicobar to Andaman group of islandswith an average of 12 in the Nicobar populations, 8 in thesouth Andaman populations and 4 in the middle and northAndaman populations. The frequencies of rare allelesoccurring in the Nicobar group populations were higherthan those occurring in the Andaman islands.Heterozygosity in penaeid relatives is lower than inother animal groups (Nelson and Hedgecock, 1980). TheK. Madhu and Rema MadhuTable  1.  List of polymorphic enzymes and electrophoretic conditions  employed  for population  genetic  studies in  P. monodonEnzymes EC No. Polymorphic Loci Tissue Buffer systemsAspartate aminotransferase 2.6.1.1. AAT-1* M/E/H TC/TG/ TMEAcid phosphatase 3.1.3.2. ACP-1* M//H PCAlcohol dehydrogenase 1.1.1.1. ADH-2* M/E/H TG/  TBEAdenylate kinase 2.7.4.3. AK- 3* M/E/H TGAlkaline phosphatase 3.1-3.1. AKP-2* M TG/TVB/ TBEAldolase 4.1.2.13. ALD-2* M/E/H TGAldehyde oxidase 1.23.1. AO-1* M/E/H TG/TCLEsterase 3.1.1.- EST-1*,2*,3* H/M TG/ TCL/ LiOHGlycerol-3-phosphate dehydrogenase 1.1.1.8. G3 PDH-2* M/E TG/ TMEGlucose 6 phosphate dehydrogenase 1.1.1.49. G6PDH-2* M/E/H TG /TCLGlucose-6-phosphate isomerase 5.3.1.9. GPI-3* M/E TCL /TVBHexokinase 2.7.1.1. HK-2* M /H TC/ TBE/ TCBIsocitrate dehydrogenase 1.1.1..42. IDH-3* M/E/H TG/ TRICLeuicine aminopeptidase 3.4.1.1.1. LAP-2* M/E/H TG/TCB/ TGCMalatate dehydrogenase 1.1.1.37. MDH-1* M TM / TMEMalatate dehydrogenase 1.1.1.40. MDH P-1*3* M CAEA/ TMEMalic enzyme NAD+ 1.1.1.39. ME-3* M/H TG/TM/ TBEManos 6 phosphate isomerse 5.3.1.8. MPI-2* M/H TVB/TCOctanol dehydrogenase 1.1.1.73. ODH-2* E/H TG/TC/ TGCPhosphogluconate dehydrogenase 1.1.1.44. PGDH-2* M/H TG/ TRICPhosphoglucomutase 5.4.2.2. PGM-1*2* M/E TCE / TMgPSorbitol dehydrogenase 1.1.1.14. SDH-1* M/H TG/TC/TCESuperoxide dismutase 1.15.1.1. SOD-1* M/E TG/  TBETetrasolium reductase TR-1* M/H/E TG/ TVB/TCLXanthin dehydrogenase 1.2.1.37. XDH-2* M/E TG/TCE/TCGeneral protein 3.1.1.X PT-2*3*, 6* M/H/E TG/TCE/TCTotal presumed loci = 32147mean heterozygosity for P. monodon   from Andaman andNicobar group of islands in the present study is similar tothat of other reports (Mulley and Latter, 1980; Redfieldet al., 1981). The low average heterozygosity in Andamangroup of population may be due to local differentiation,which may also reflect the effects of isolation and increasedgenetic distances from the other populations (Nicobar groupof islands). The loci such as AAT-1, ADH-2, G6PDH-2,LAP-2, MDH-1, ME-3, MPI-2, ODH-2, PGM-1, PGM-2,PGDH-2,   SDH-1,  SOD-1 and  TR-1 were  polymorphicfrom Nicobar group of islands and  monomorphic in theAndaman group of islands. Similarly the loci ALAT*, GPI*,IDHP* were heterozygous in P. monodon from Gulf ofThailand  whereas these loci were homozygous from  theAndaman sea (Sodsuck, 1996). The variation in expressionof loci  may be due to the spatial structuring of a speciesand effect of adaptive fitness which in turn can alter thegene pool (Wright, 1978; Nelson and Hedgecock, 1980).Though the mean  values of Fis,  Fit and  Fst  from over allpopulations in Anadaman and  Nicobar group of islandswere lower (Table 2.) compared to other aquatic animals,inter-population differentiation were observed due to thesignificant contribution of high frequency alleles (locationspecific). The genetic variation from Nicobar group andAndaman group of islands was also confirmed with Fstatistics. Pair-wise comparison of gene frequenciesbetween populations using chi-square confirmed the generalpattern of variation in all tested populations Comparisonrevealed significant  heterogeneity between Andaman andNicobar group of islands (p<0.01 and  p<0.05). Genefrequencies of all loci in these populations were alsosignificantly different (p<0.01and  p<0.05). The stockstructure of P. monodon showed that Andaman populationwas significantly different from that of the Gulf of Thailand(Tassanakajon et al., 2004). In the present study, populationdifferentiation noticed between Andaman and Nicobargroup of islands can be attributed to the prevention of adultspawning migration as a result of  hindrance from ten degreechannel.    Seasonal currents channel of the Andaman sea(Subramanian, 1977) or  past biogeographical barriersoccurring as a result of changes in the sea level as reportedin P. latisulcatus  from western and southern Australia  thanthe northern populations (Mulley and Latter, 1981)influenced its larval dispersal.The genetic diversity data of  P. monodon revealed  thatit has decreased from north Andaman to middle Andamanbut remarkable decrease was observed between the locationsof south Andaman and Car Nicobar. The colonization of thecandidate species might have been generated from Nicobarand Andaman group of islands due to the effects of currentsand founder effects of the population. Loss of geneticvariation was noticed in Andaman population which alsohad increased frequencies of rare alleles. This suggestscolonization by a relatively small number of individuals andhighly restricted dispersal between the northern, middle andsouthern region. The genetic distinction of the Nicobarpopulation of P. monodon may reflect an isolated populationon the periphery of the species range. Such peripheralpopulations often show reduced genetic diversity and shiftsin gene frequency (Baker et al., 1990) and  the geneticallydiscrete stocks represent the best adapted genotypes for eachparticular area (MacLean and Evans, 1981). Thus the presentstudy indicates that the stock of P. monodon from north,middle and south Andaman are of the same breedingpopulation. Although these populations showed few geneticvariations, data have not been considered as these differenceswere not significant. However, there exists significantgenetic differentiation  between the Andaman and Nicobarpopulations as demonstrated by the cluster analysis.(Fig. 1).  It is therefore  suggested that two insular populationof P. monodon existed in the waters of Nicobar and Andamanregions which should be managed as two separatebroodstocks for  captive breeding in future.Genetic differentiation of Penaeus monodon from Andaman and Nicobar islandsTable  2.   Population differentiation of P. monodon  from Andaman and Nicobar islandsMajor LocationsNorth Andaman Middle Andaman South Andaman NicobarPercentage of polymorphic loci 46.88 40.62 31.25 53.12Mean number of alleles 1.7812 1.5312 1.500 2.1250Mean effective number of alleles 1.1246 1.0759 1.1376 1.2962Observed  homozygosity 0.9108 0.9505 0.9146 0.8112Expected  homozygosity 0.9065 0.9400 0.9069 0.8197Observed heterozygosity 0.0892 0.0495 0.0854 0.1888Expected heterozygosity 0.0935 0.0600 0.0931 0.1803Average heterozygosity 0.0859 0.0553 0.0884 0.1750Gene flow (Nm) 2.3474 2.9079 5.1632 6.1228Mean Fis -0.0575 0.1042 0.0365 -0.0935Mean Fit 0.0443 0.1751 0.0810 -0.0506Mean Fst 0.0962 0.0792 0.0462 0.0392148AcknowledgementsThe authors are highly indebted to the Indian Councilof Agricultural Research for granting the A.P. Cess FundScheme. The authors are also thankful to the Director,Central Agricultural Research Institute, Port Blair, Andamanand Nicobar islands for his valuable support.ReferencesAllendorf,  F. W. and  Utter,  F.  M. 1979.  Population genetics. In:Hoar, W. S., Randall, D. J., Brett, J. R. (Eds.), Fish Physiol.,Academic Press, New York, 8, pp. 407-454.Baker, A. J., Dennison, M. D., Lynch, A. and Le Grand, G. 1990.Genetic divergence in peripherally isolated populations ofchaffinches in the Atlantic Islands. Evolution  44: 981-999.Benzie, J. A. H., Frusher, S. and Ballment, E. 1992. Geographicalin Allozyme frequencies of populations of Penaeus monodon(Crustacea: Decapoda ) in Australia. Aust. J. Mar.  Freshwat.Res.,  43, 715- 725.Forbes, A. T., Demetriades, N. T., Benzie, J. A. H. and Ballment,E. 1999. Allozyme frequencies indicate little geographicvariation among stocks of giant tiger prawn Penaeusmonodon in the south-west Indian Ocean. S. African J. Mar.Sci., 21: 271–277.Grey, D. L., Dall, W. and Baker, A. 1983. A Guide to the AustralianPenaeid Prawns. Northern Territory Government : Darwin.,pp. 1-140.Klinbunga, S., Penman, D. J, Mc Andrew, B. J. and Tassanakajon,A. 1999. Mitochondrial DNA diversity in three populationsof the giant tiger shrimp Penaeus monodon. Mar.Biotechnol., 1: 113-121.Fig. 1. Dendrogram Based Nei's (1978) showing the geneticrelationships  (genetic distance)  among  A & N populationof P. monodon: (Method = UPGMA clustering method)Klinbunga, S., Siludfai, D.,  Wudthijinda,  W., Tassanakajon, A.,Jarayabhand, P. and  Menasveta, P. 2001. GeneticHeterogeneity of the Giant Tiger Shrimp (Penaeus monodon)in Thailand Revealed by RAPD and Mitochondrial DNARFLP Analyses. Mar.  Biotechnol.,  3: 428-438.Lester,  L. J. 1979. Population genetics of penaeid shrimp fromthe Gulf of Mexico. J. Hered., 70: 175-180.MacLean,  J. A. and Evans,  D. O. 1981. The stock concept,discreteness of fish stocks, and fisheries management. Can.J.  Fish. Aquat.  Sci., 38: 1889-1898.Mulley,  J. C. and Latter,  B. D. H. 1980. Genetic variation andevolutionary relationships within a group of thirteen speciesof penaeid prawns. Evolution,  34: 904-916.Mulley, J. C. and Latter  B. D. H. 1981a. Geographic differentiationof tropical Australian prawn populations. Aust. J. Mar.Freshwat. Res., 32: 897-906.Nei,  M. 1978. Estimation of average heterozygosity and geneticdistance from a small number of individuals. Genetics,89:583-90.Nelson, K. and Hedgecock,  D. 1980.  Enzyme polymorphismand adaptive strategy in the decapod Crustacea. Am. Natural.,116:238-280.Redfield, J. A., Hedgecock, D., Nelson, K. and  Salini, J. 1981.Low heterozygosities in tropical marine crustaceans ofAustralia and the trophic stability hypothesis. Mar. Biol.Lett.,  1: 303-313.Ridgeway, T. 2005. Allozyme electrophoresis still represents apowerful technique in the management of coral reefs.Biodivers. Conserv., 14(1):135-149.Shaklee, J. B., Allendorf, F. W., Morizot, D. C. and Whitt, G. S.1990. Gene nomenclature for Protein-Coding Loci in Fish.Trans. Amer. Fish. Soc.,  119: 2-15.Siludja, D.,  Klinbunga, S., Tassanakajon, A., Jarayabhand, P. andMenasveta, P. 2000. Genetic diversity and mtDNA markersof the giant tiger shrimp (Penaeus monodon) in Thailand.26th Congress on  Science  and  Technology  of  Thailand.Sodsuk, S.1996. Genetic differentiation and population structureof Penaeus monodon in Thailand. Technical  Paper ofNational Aquaculture Genetics Research Institute,Department of  Fisheries, Thailand.  12: 23.Subramanian, H.1977. Sea surface currents in the Arabian seaand the Bay of Bengal. In: Marine Biology. Vijayapublications, p.140-144.Tassanakajon, A., Pongsomboon, S.,  Jarayabhand, P., Klinbunga,S. and Boonsaeng, V. 2004. Genetic structure in wildpopulations of black tiger shrimp (Penaeus monodon ) usingrandomly amplified polymorphic DNA analysis. J. Mar.Biotechol., 6: 249-254.Wright, S. 1978. Evolution and Genetics of Populations. In:Variability within and among Populations. University ofChicago Press, Chicago, p. 562.K. Madhu and Rema MadhuDate of Receipt : 19.03.2008Date of Acceptance : 21.04.2011

Genetic differentiation of black tiger shrimp Penaeus monodon Fabricius (Crustacea: Decapoda) in Andaman and Nicobar islands

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Genetic differentiation of black tiger shrimp Penaeus monodon Fabricius (Crustacea: Decapoda) in Andaman and Nicobar islands

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