Research in Biotechnology, 5(6): 01-06, 2014 ISSN: 2229-791X www.researchinbiotechnology.com

Regular Article Genetic Diversity of Army worm, mauritia Isolated from Kerala,

Mashhoor K.1&4, Lazar K.V.2, Shanas S.3, Ramesh N.1*

1Department of Biotechnology, J.J. College of Arts and Science, Namanasamudram, Pudukkottai, Tamil Nadu, 622404, India 2Department of Environmental Science, Central University of Kerala, Nileswaram, Kasaragod Dist., Kerala, Pin 671314, India 3Division of Entomology, Research Station, Kerala Agricultural University, Thekkekara, Moncompu, Alappuzha, Kerala, 688503, India. 4Department of Biotechnology, E.M.E.A. College of Arts and Science, Kondotti, Malappuram, Kerala, 673638, India *Corresponding Author Email: [email protected]

The army worm Spodoptera mauritia is one of the major pests of paddy which is widely distributed in the Indian subcontinent, East and southern Asia and in the Australian region. Generally the army worms infest paddy crops of less than 20-25 days old. They are gregarious, defoliating the paddy and move from one field to other in large number like an army. The classification of Spodoptera species is mainly based on the structure of the male genitalia, antenna and the colour pattern of the wing. Here we report the partial coding sequence of cytochrome oxidase sub-unit I (COI) sequence of army worm isolated from Kerala, India which is identical to that isolated from Japan. This study highlights the geographical distribution and genetic diversity of army worm in paddy cultivating countries.

Key words: Cytochrome oxidase subunit I, geographical distribution, Spodoptera mauritia, Divergence.

The Army worm, Spodoptera mauritia antenna and the colour pattern of the wing is a gregarious pest which causes severe (Hampson, 1920). The colour pattern of the damage to rice. The larvae of army worm Spodoptera species shows variations that may defoliate the rice crop and move from one leads to the wrong identification. The male field to another field in large number like an genitalia give the reliable taxonomic army devastating the entire crop (Krishnaiah character to identify this species (Chatterjee, et al., 2008). The pest is widely distributed in 1967). The genetic structure of an organism Asia and . In India army worm can provide wealth information to for infestation were reported from Kerala, identification and to study variations within Tamilnadu, Orissa, Bihar, Jharkhand and the species and between the species. The Chhatisgarh (David and Ananthakrishanan, genetic structure of several Spodopteran 2004, Tanwar et al., 2010) species have been reported in many studies The classification of Spodoptera species (Machado et al., 2008; Wan et al., 2011; is mainly based on the structure of the male Nagoshi et al., 2011). The partial coding Mashhoor et al. / Research in Biotechnology, 5(6): 01-06, 2014

sequence of Cytochrome oxidase sub unit I of Laboratories, Inc. California) as per the S. mauritia isolated from Japan and Reunion manufacturer’s instructions. Islands were reported in the NCBI (GenBank The purified PCR product was Accession No. AB733407, AB733409, sequenced from both ends using the forward AB733408, HQ177382, HQ177383 and and reverse primers using the Sanger’s HQ177384). The COI sequence of S. mauritia sequencing method at SciGenom isolated from Japan showed 3.1% divergence Laboratories Ltd., Cochin. The forward and for that isolated from Reunion. The studies reverse sequences were assembled by using on genetic structure of Spodoptera species ClustalW after removing the forward and from India are limited. But there is no report reverse primers and the consensus was taken on the genetic structure analysis of S. mauritia for the analysis. The phylogenetic analysis from India, even though its cause’s major was done using MEGA5 software. economic loss. Hence In this study we have described the genetic structure of S. mauritia Results and Discussion isolated from Kerala, India and also made a The Rice army worm Spodoptera comparative genetic structure analysis of S. mauritia under the family is one of mauritia isolated from Kerala, India with the most serious paddy pests of Kerala. In geographically isolated populations. this study, we have collected various life stages of S. mauritia from the paddy fields of Materials and Methods Kerala. The dark brown adult was 1.7mm in Different life stages of S. mauritia were length. The eggs were spherical and creamy collected from the rice field of Kerala, India in colour and they were covered with gray and sample were stored at -200C until the hairs. The freshly hatched larvae were 2mm DNA was extracted. The genomic DNA was in length with light green with yellowish isolated using GeNei Ultrapure Mammalian white lateral and dorsal stripes. The pupa Genomic DNA Prep Kit (Bangalore GeNei, was dark brown in colour and measures 15 Bangalore) as per the Manufacturer’s mm in length, having two slender apical instruction. The 5’ end of the mitochondrial spines. The mature larva was 4 cm in length, cytochrome oxidase subunit I (COI) gene was light green in colour and two rows of C- amplified using the forward primer with shaped black spots were observed along the DNA sequence 5’ CAT TGG AGA TGA CCA backs. AAT TTA TAA TG -3’ and reverse primer The partial coding sequence of COI with DNA sequence 5’- TGA AAT TAA TCC gene of S. mauritia was PCR amplified using AAA TCC AGG TAA A-3’. The 25 µl PCR the forward primer with DNA sequence of 5’ reaction mixture consisted of 2 nanogram of CATTGGAGATGACCAAATTTA TAATG 3' genomic DNA (1 µl), 1 µl each forward and and the reverse primer with DNA sequence reverse primers at a concentration of 10 µM, of 5’TAA ACT TCA GGG TGA CCA AAA 2.5 µl of dNTPs (2 mM), 2.5 µl 10X reaction AAT CA 3'. The PCR amplification of partial buffer, 0.20 µl Taq polymerase (5 U/µl) and mitochondrial COI gene of S. mauritia yielded 16.8 µl H2O. The PCR profile consisted of an a single product with about 600 bp in size. initial denaturation step of 3 min at 950C, The sequence obtained after removing the followed by 30 cycles of 10 sec at 950C, 45 sec primers used for PCR amplification was at 450C and 45 sec at 720C and ending with a submitted to GenBank (GenBank Accession final phase of 720C for 3 min. The PCR No. KC601856). product was column purified using Mo Bio The nucleotide composition analysis UltraClean PCR Clean-up Kit (Mo Bio showed a similarity in the concentration of

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each nucleotide in the COI sequence of S. isolated from India and S. exigua is the distant mauritia isolated from India and other relative (Fig.1). geographically isolated population. Usage of nucleotides in each position of codon of S. Table 1. Evolutionary divergence between COI mauritia isolated from India is similar to S. sequences of the species S. mauritia isolated mauritia (GenBank Accession Nos. AB733407, from Kerala, India with the different Sopodoptera species indentified from different AB733409, AB733408) isolated from Japan but geographical locations. The GenBank accession it is different from S. mauritia from Reunion numbers are given in parenthesis. islands (GenBank Accession Nos. HQ177382, S. % of HQ177383, HQ177384, HQ177386). The S. No. Name of Species divergence mauritia of Reunion islands showed 0.7-1% 1 S. mauritia (AB733409) variation in the usage of nucleotides in each Japan 0.00% 2 S. mauritia (AB733408) position of codon of S. mauritia isolated from Japan 0.00% India, except in the third position of codon. 3 S. mauritia (AB733407) The sequence divergence analysis Japan 0.00% clearly depicts the evolutionary divergence 4 S. mauritia acronyctoides between the S. mauritia isolated from India (HQ177386) Reunion 0.52% 5 S. mauritia (HQ177384) and other geographically isolated Reunion 3.15% populations. The COI sequence of S. mauritia 6 S. mauritia (HQ177383) isolated from India is identical with that Reunion 3.15% isolated from Japan (GenBank Accession No. 7 S. mauritia (HQ177382) AB733407, AB733409, AB733408). The S. Reunion 3.70% 8 S. cilium (JN988597) 7.40% mauritia isolated from India has 0.52% 9 S. cilium (JN988598) 7.40% divergence with S. mauritia acronyctoides 10 S. albula (JQ567811) 7.68% (GenBank Accession Nos. HQ177386) isolated 11 S. cosmioides (JF854738) 7.72% from Reunion Island and it showed more 12 S. triturata (HM892940) 7.81% than 3% divergence with that of isolated from 13 S. triturata (HM892616) 7.83% Reunion Islands (GenBank Accession Nos. 14 S. exempta (DQ092375) 7.97% HQ177382, HQ177383, HQ177384). Among 15 S. frugiperda (HM136592) 8.01% other Spodoptera sp. used in this study S. 16 S. frugiperda (HM136591) 8.01% cilium, S. albula, S. cosmiodes and S. tritura 17 S. frugiperda (HM136590) 8.01% 18 S. cosmioides (HQ571029) 8.01% showed less divergence with S. mauritia 19 S. albula (JQ535527) 8.27% isolated from India and S. exigua showed 20 S. littoralis (HM756074) 8.29% highest divergence (Table 1). The phylogeny 21 S. ornithogalli (EU768964) 8.32% analysis using Neighbor Joining method 22 S. litura (JQ064571) 8.55% revealed that the S. mauritia evolved two 23 S. latifascia (JQ577384) 8.84% clad. The S. mauritia isolated from India and 24 S. pulchella (HM756076) 9.08% Japan are aligned in a clad and S. mauritia 25 S. pulchella (HM756075) 9.08% acronyctoides (GenBank Accession No 26 S. androgea (GU159412) 9.15% 27 HQ177386) isolated from Reunion formed a S.dolichos (HM756088) 9.44% 28 S. exempta (DQ092376) 9.49% sister clad of it. Other S. mauritia isolated 29 S. dolichos (HM756086) 9.73% from Reunion are arranged in the second 30 S. eridania (HM756083) 10.05% clad. The S. mauritia isolated from India and 31 S. eridania (JQ546665) 10.05% Japan shared a common origin. Among the 32 S. exigua (GU707393) 10.68% other Spodoptera sp. used in this study S. 33 S. exigua (JF415658) 10.68% triturata is the nearest relative of S. mauritia 34 S. exigua (HM914242) 10.68%

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Fig. 2. Phylogenic relationship of S. mauritia isolated from Kerala, inferred by NJ tree method of MEGA5 software. The GenBank accession numbers are given in parenthesis.

DNA sequence based identification The COI sequence of S. mauritia showed close technique has revealed the morphological similarity within the species and considerable and ecological traits of many species during variation between the species. larval stages (Foltan 2005; Smith 2006; The genetic structure analysis of COI Hayashi and Sota 2010). According to Gurney sequence of S. mauritia indicates the et al. (2000) closely related species have 90% similarity in the nucleotide composition and similarity in the standardized DNA sequence variation in the nucleotide usage in the each but distantly related species have less than position of codon of COI gene of the different 90% similarity in the same genes sequence. S. mauritia populations. Variation in

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nucleotides in the codon of COI sequence mutation rate in the COI sequence of S. revealed that the third position in the codon mauritia isolated from India and Japan. of S. mauritia is conserved compared to first The different population of S. mauritia and second position. In the first and second has same concentration of each nucleotide in position the nucleotide ‘T’ composition has COI sequence and the COI sequence is highly high variation in the COI sequence of S. biased towards the nucleotides A and T. But mauritia isolated from India and Reunion in the codon usage the concentration of each compared to other nucleotides and its COI nucleotide in the each position of codon of sequence is highly biased to AT. The shift to may be varied depending upon the AT compositional bias with low ‘G’ in the population. The nucleotide in the third sense and ‘C’ in the template strand may position of the codon of the S. mauritia COI have arisen through directed mutational sequence has little chance for variation. The pressure (Jermiin et al., 1994). S. mauritia isolated from the Reunion has The nucleotide divergence study and high divergence with Indian population and the phylogeny analysis also revealed the S. exigua is highly diverged from S. mauritia. origin of S. mauritia isolated from India. Phylogeneticaly S. triturata is the nearest Generally the genetic variation in the COI relative of S. mauritia among the Spodoptera sequence of the same species will be very low species analysed. averaging 0.43%, while in the different species of the same genus showed 18 fold References higher sequence divergence averaging 7.7% Brown, E.S. and Dewhurst, C.F. 1975. The (Hebert et al., 2010). S. mauritia isolated from genus Spodoptera (: India showed 0-3.7% and 2% average Noctuidae) in Africa and the Near East. intraspecific divergence with that of Bull. Entomol. Res., 65: 221–262. geographically isolated population. The Chatterjee, S.N. 1967. The identity of geographical isolation may be the reason for Spodoptera mauritia Acronyctoides guenee, this high divergence of the S. mauritia Spodoptera pecten Guenee and Spodoptera between the populations. The average abyssinia Guenee (Lepidoptera: interspecific divergence between the Noctuidae) based on a comparative study members of the Spodoptera genus was also of the male and female genitalia, Proc. found high (7%). Most of the members of Natl. Acad. Sci. India, 35: 45-62. genus Spodoptera are polyphagous and they David, B.V. and Ananthakrishnan, T.N. 2004. can feed several plant families (Brown and The swarming caterpillar or armyworm, Dewhurst, 1975; Pogue, 2002). The high Spodoptera mauritia. In “General and interspecific divergence observed among the Applied Entomology” (Second Edition). species of genus Spodoptera may be due to the pp.1184. Tata McGraw-Hill Publishing polyphagy. Even though Japan is an island Co. Ltd, New Delhi. which is separated by over 5000KM from Foltan, P., Sheppard, S.K., Konvicka, M. and India, the S. mauritia isolated from India Symondson, W.O.C. 2005. The showed 100% similarity with that isolated significance of facultative scavenging in from Japan. The similarity of S. mauritia of generalist predator nutrition: detecting these geographically isolated populations decayed prey in the guts of predators (India and Japan) revealed the recent using PCR. Mol. Ecol., 14: 4147–4158. spreading of the S. mauritia to this area from a Gurney, T. jr., Elbel, R., Ratnapradipa, D. and common origin and it also indicates the low Brossard, R. 2000. “Introduction to the molecular phylogeny of . Tested

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studies for laboratory teaching”, (S. J. identify invasive armyworm, Spodoptera Karcher, Eds.), Proceedings of the 21st species in Florida. J. Sci. 11: 154 Workshop/Conference of the Association for Pogue, M.G. 2002. A world revision of the Biology Laboratory Education, 21: 63-79. genus Spodoptera Guenée (Lepidoptera: Hampson, G.F. 1920. Catalogue of the Noctuidae). Mem. Am. Entomol. Soc., 43: 1- Lepidoptera Phalaena in the British 202. Museum. 8. Smith, M.A., Woodley, N.E., Janzen, D.H., Hayashi, M. and Sota, T. 2010. Identification Hallwachs, W. and Hebert, P.D.N. (2006). of elmid larvae (Coleoptera: Elmidae) DNA barcodes reveal cryptic host- from Sanin District of Honshu, Japan, specificity within the presumed based on mitochondrial DNA sequences. polyphagous members of a genus of Entomol. Sci., 13: 417–424. parasitoid flies (Diptera: Tachinidae). Hebert, P.D.N., Deward, J.R. and Landry, J.F. PNAS, 103:3657–3662. 2010. DNA barcodes for 1/1000 of the Tanwar, R.K., Anand Prakash, S.K., Panda, kingdom. Biol. Lett., 6: 359-362. N.C., Swain, D.K., Garg, S.P., Singh, S., Jermiin, L.S., Graur, D., Lowe, R.M. and Kumar, S. and Bambawale, O.M. 2010. Crozier, R.H. 1994. Analysis of directional Rice swarming caterpillar (Spodoptera mutation pressure and nucleotide content mauritia) and its management strategies, in mitochondrial cytochrome b genes. J. Technical Bulletin 24. National Centre for Mol. Evol. 39: 160-73. Integrated Pest Management, New Delhi. Machado, V., Wunder, M., Baldissera, V.D., Wan, X., Li, J., Kim, M.J., Park, H.C., Oliveira, J.V., Fiúza, L.M. and Nagoshi, Kim, S.S. and Kim, I. 2011. DNA R.N. 2008. Molecular Characterization of Sequence Variation of the Tobacco Host Strains of Spodoptera frugiperda Cutworm, Spodoptera litura (Lepidoptera: (Lepidoptera: Noctuidae) in Southern Noctuidae), Determined by Brazil. Ann. Entomol. Soc. Am., 101: 619- Mitochondrial A+T-rich Region and 626. Nuclear ITS2 Sequences. Biochem. Genet., Nagoshi, R.N., Brambila, J. and Meagher, 49: 760-787. R.L. 2011. Use of DNA barcodes to

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