1 Introduction

Moths of the genus Plutella Schrank, 1802 (Lepidoptera: Plutellidae) are represented by more than 40 species, of which Plutella xylostella (Linnaeus), believed to have originated in the Mediterranean region, is now distributed all over the world. It infests many cruciferous crops and is found in all the states of India (CIE, 1967) and the damage caused by it amounts to more than US $1 billion annually in different countries (Talekar and Shelton, 1993; Mohan and Gujar, 2003). This pest is also known to have developed resistance to almost all groups of insecticides. Santos et al. (2011) in Brazil, Walker et al. (2012) in New Zealand and Gong et al., (2013) in China, reported different levels of resistance to different insecticides in different populations. In India, Cheema et al. (2011) reported that it has become resistant to seven different groups of insecticides including Bacillus thuringiensis.

 

In recent times, modern biotechnological approaches are employed for developing control programs based on studying genetic variation and structure of the population of an insect species (Li et al., 2006; Xie, 2013). Folmer et al., (1994) advocated that molecular markers are powerful tools to determine genetic variation at different population levels. Mitochondrial DNA (mtDNA), particularly COXI region, has gained importance because of maternal inheritance and high evolutionary rate. These characteristics have made mtDNA an appropriate marker for tracing evolutionary relationships. Hebert et al., (2003) advocated the use of mtDNA COXI as a global marker to differentiate various animal species.

 

Diamondback moth is one of very few insect species with extremely high geographic distribution, but not much information is available on the genetic diversity of its populations collected over large areas (Capiro and Tabashnik, 1992; Kim et al., 2003; Ellango et al., 2012; Murthy et al., 2014). Li et al., (2006) studied Chinese and Korean populations that covered an area of ~ 2151600 km² for documentation of genetic variation. In India, one such attempt was made by Ellango et al., (2012) from a very narrow geographical area, covering about ~2500 km² area, by which it is difficult to study diversity properly and Murthy et al., (2014) collected mainly populations from Karnataka and using RAPD markers found genetic diversity in the populations.

 

We studied the genetic diversity of diamondback moth collected over a geographical area spanning ~12250000 km² with the minimum distance within states being 200 km and maximum distance between farthest states being 3500 km using mtDNA molecular marker. We report the genetic variability as deduced by mtDNA COXI gene nucleotide sequence in thirteen populations of P. xylostella collected across the country vis-a-vis COXI sequences available in GenBank.

 

2 Results

2.1 Molecular characterization and development of DNA barcode

Genomic DNA extracted and confirmed on 1% agarose gel from 13 populations of P. xylostella collected from different parts of India was subjected to COXI PCR to obtain a length of 658 bp diagnostic fragments of mitochondrial DNA. All COXI nucleotide sequences representing nine haplotypes, of which five populations formed a haplotype group, were submitted to NCBI-GenBank and accession numbers were obtained for each specimen from 13 populations. As no insertions, deletions or stop codons were observed as 2nd frame of DNA, sequences were chosen from ORF finder for submission to GenBank. Sequences and other specimen information were submitted to Agriculturally Important Insects (AGIMP) project at BOLD Systems and DNA barcodes for each population was developed and process IDs were provided to each specimen. (http://www.boldsystems.org). The details of sequences submitted to GenBank and BOLD Systems and their accession numbers are provided in Table 1.

2.2 Analysis of genetic variation among P. xylostella population

We observed large genetic variability among the Indian P. xylostella populations collected from the vast region ~12250000 km² from sequence analysis nine haplotypes were obtained (out of 39 sequences characterized (3 specimens per location, data not shown), only 13 resulted into haplotype by chance). Populations of Varanasi, Bhubaneswar, Rajahmundry, Solan and Palani, clustered together and formed a haplotype group, designated as NBPx1 (Table 2). Eleven polymorphic sites, four (71, 433, 451 and 541), which were G/A transitions, one T/C transition observed at 154th base, one A/C transversion at 548th base, A/T transversion observed at 3 sites (205, 543 and 599) and G/T transversion at two sites (514 and 540) (Table 3). Nucleotide composition of these 13 sequences ranged from 39.1 to 39.4% in T, 15.0-16.1% in C, 14.7-15.2% in G and 30.01-30.07% in A. The per cent AT was higher as compared to %GC, which was in accordance with invertebrate mitochondrial DNA, AT% at the 1st codon position was 45.2%, at 2nd codon position was 30.3% and at 3rd codon position was 28.55%.

2.3 Phylogenetic analysis

Comparison of populations of P. xylostella from other countries (Figure 1) drawn from NCBI with the Indian populations indicated a high rate of genetic variation in the Indian populations. Two major clades were obtained from maximum likelihood (ML) tree produced and inferred through nearest neighbor interchange (NNI), the first subclade 10 Indian populations were clustered together and one Indian population from Nawanshahr (Punjab) clustered in the 2nd subclade with Canada, Pakistan, China and USA populations, whereas Tirupati (Tamil Nadu) population clustered with Israel and China populations, and Shillong (Meghalaya) population clustered with Australian populations of DBM (Figure 1). Pattern of nucleotide substitution was inferred from the general time reversible with gamma distribution (GTR+G) estimate, the overall transition/transversion bias (R) 3.72 was and the overall average between nucleotide composite distance (d) is 0.06 (Table 4), which does not support towards natural triggers for genetic variability among Indian populations of P. xylostella populations.

3 Discussion

It is important to determine sensitive multiple sequence alignment to the determination of differentiation between populations of insects (Thompson et al., 1994). It is documented that the diamondback moth has the capability to migrate to great distances from its native location. Some evidences have been provided that moths were able to cover a distance of over 3000 km in continuous flight for several days. Considerable variation in some biological traits like biotic potential and tolerance to stresses is known to occur between various geographical locations in P. xylostella. The visualized PCR product obtained in the study contained only discrete single bands, thus indicating that sequences obtained were mitochondrial DNA and not nuclear pseudogenes (Numts), as pseudogenes do not code for a functional protein (Bensasson et al., 2001). The maximum variation between populations collected in the present study was of 7 nucleotides and this determined 9 haplotypes from populations collected from ~12250000 km² area. Similar to this study, sequence divergence from one to seven nucleotides was reported for Bombyx mori (Kim et al., 2000), Lycoriella mali (Bae et al., 2001), P. xylostella (Li et al., 2006) and Pilophorus typicus (Ito et al., 2011). Out of 13 populations collected, nine haplotypes were obtained, of which five formed a separate haplotype group. In a study on 80 populations collected from China-Korea region, Li et al., (2006) recorded 16 haplotypes of P. xylostella. In the present work with populations collected in India, relatively more haplotypes recorded may be due to very large area from which populations were collected. In this study, pattern of nucleotide substitution was inferred from the maximum composite likelihood estimate, the overall transition/transversion bias, R obtained was 3.72 and average composite distance (d) was 0.06 that did not support towards natural triggers for genetic variation among Indian populations of P. xylostella populations. The observations recorded in other countries are mixed and show multiple clusters in the tree, so we finally conclude that genetic variability among P. xylostella populations is not concerned with geographical distribution as also recorded by Li et al., (2006).

 

These differences could probably be induced by local selection pressure due to insecticide usage, host strain variation and cultural practices (Pichon et al., 2006). Due to high fecundity, diamondback moths multiply quickly and become abundant completing many generations per year (Talekar & Shelton, 1993). Furthermore, the distance they migrate can be several thousand kilometres (Lorimer, 1981). Long-distance migration on land and overseas in P. xylostella has been reported by recent radar data (Chapman et al., 2002). The high genetic variability will help species to evolve and adapt at a greatly accelerated pace to different environments and result in rapid evolution of resistance to insecticides In this context, our results obtained from DNA barcoding, phylogenetic analysis and genetic variation studies can be an important aspect to study pest population and possible management of diverse populations.

 

4 Materials and Methods

Populations of P. xylostella were collected in cabbage fields from 10 states during 2013 from the north, north-east, western and southern parts of India at the following locations: Solan (Himachal Pradesh), Nawanshahr (Punjab), Delhi (New Delhi), Varanasi (Uttar Pradesh), Shillong (Meghalaya), Anand (Gujarat), Bhubaneswar (Odisha), Hyderabad (Telangana), Rajahmundry and Tirupati (Andhra Pradesh), Coimbatore, Palani and Oddanchatram (Tamil Nadu) (Table 1). At each of these sites, about 50 DBM larvae were collected and transferred to plastic containers (with mesh lids) containing cabbage leaves. The larvae were transported to the laboratory at NBAIR, Bengaluru and reared on mustard seedlings raised in small ice-cream cups. The adults were identified to species by the characteristic diamond shaped marking on the back. The specimens thus collected and morphologically identified were used for molecular analysis using COXI gene in the Molecular Entomology lab at NBAIR, Bengaluru.

 

4.1 Amplification of mtDNA COXI gene:

DNA was extracted in triplicate from somatic tissues (thorax, legs) rich in mitochondria using Qiagen DNeasy® kit, following the manufacturer’s protocols. The remaining parts of each DBM moth were kept as voucher specimens at NBAIR, Bengaluru, at -70℃. The DNA, thus obtained was subjected to PCR amplification of a 658 bp region near the 5’ terminus of the COXI gene following standard protocol (Hebert et al., 2003). Primers used were: forward primer (LCO 1490 5'-GGTCAACAAATCATAAAGATATTGG-3’), and reverse primer (HCO 2198 5'-TAAACTTCAGGGTGAC CAAAAAATCA-3’). PCR reactions were carried out in flat capped 200 µL volume PCR tubes obtained from M/s Tarsons, Kolkata, India. The PCR reaction consisted of 50 µL reaction volume containing: 5 μL GeNei^TM Taq buffer, 1 μL GeNei^TM 10mM dNTP mix, 1 μL (20 pmol/μL) forward primer, 1 μL (20 pmol/μL) reverse primer, 1 μL GeNei^TM Taq DNA polymerase (1 U/μL), 5 μL DNA (50 ng/μL), and 36 μL sterile water. Thermo cycling consisted of an initial denaturation of 94℃ for 5 min, followed by 30 cycles of denaturation at 94℃ for 1 min, annealing at 55℃ for 1 min, extension at 72℃ for 2 min. PCR was performed using a BioRad C1000™ Thermal Cycler. The amplified products were analyzed on 1.5% agarose gel electrophoresis as described by Sambrook and Russell, (2001). The amplified products were sequenced by M/s Eurofins Analytical Services India Pvt. Ltd., Bangalore. Each specimen PCR sample was bi-directionally sequenced and checked for homology, insertions and deletions, stop codons, and frame shifts by using NCBI-BLAST and ORF finder. All sequences were uploaded to GenBank and the Barcode of Life Database (BOLD, http://www.boldsystems.org).

 

4.2 Data Analysis

The sequences of DBM were aligned using ClustalW with default settings of gap opening penalty 15, and a gap-extension 6.06 in pairwise and 6.06 in multiple alignments and phylogenetic analysis was performed using MEGA 6.0 software. Maximum Likelihood (ML) tree was constructed using a general time reversible (GTR) model with the COXI nucleotide sequences of the Indian populations as per Tamura et al., (2013), two outgroups, viz., Plutella porrectella (KF370678) and Plutella australiana (KF370868) and other COXI deposits available in the NCBI databank. Position wise nucleotide variations in COXI sequence among Indian populations of P. xylostella were inferred as described by Hall, (1999). Transition/ transversion and polymorphism were found among the Indian populations using BioEdit 7.1.3.0 software. The number of base substitutions per site was analysed between all sequences, when homologous sequences from two populations differed by more than one nucleotide eventually considered as haplotypes. The data for all sequences, including out group sequences were obtained from GenBank. 1^st, 2^nd and 3^rd Codon positions were included. All positions containing gaps and missing data were eliminated from the dataset. The A, T, G, C, AT and GC content of all 13 sequences (Indian populations) was obtained using a computer program designed in the Bioinformatics Lab at NBAIR (www.cib.res.in), Bengaluru, India. The AT% at the three codon positions was calculated using the same program. The GenBank accession numbers and Barcode ID for 13 Indian populations of P. xylostella are provided in Table 1 and GenBank accession numbers for P. xylostella from other countries used in the present study are given in Table 2. Sequences and other specimen information are available on BOLD Systems (http://www.boldsystems.org).

 

Author contributions

RO carried out the molecular genetic studies, participated in the sequence analysis, developed DNA barcodes and drafted the manuscript. SKJ conceived the idea and participated in the design of the study and drafted the manuscript. JP participated in the taxonomic identification of specimens into species and helped to draft the manuscript. KSM participated in the collection of the specimens and helped to draft the manuscript. All authors read and approved the final manuscript.

 

Acknowledgements

The authors wish to express their sincere thanks to Dr. Abraham Varghese, the Director, NBAIR, Bengaluru, for providing facilities for the present study. First author is thankful to Jain University, Bengaluru, India, for encouragement. This work is the part of the Ph. D. programme of the first author.

 

References

Bae J.S., Kim I., Kim S.R., Jin B.R., and Sohn H.D., 2001, Mitochondrial DNA sequence variation of the mushroom pest flies, Lycoriella mali (Diptera: Sciaridae) and Coboldia fuscipes (Diptera: Scatopsidae), Applied Entomology and Zoology, 36(4): 451-457

http://dx.doi.org/10.1303/aez.2001.451

 

Bensasson D., Zhang D., Hartl D.L., and Hewitt G.M., 2001, Mitochondrial pseudogenes: evolution’s misplaced witnesses, Trends in Ecology & Evolution, 16(6):314-321

http://dx.doi.org/10.1016/S0169-5347(01)02151-6

 

Capiro M.A., and Tabashnik B.E., 1992, Allozymes used to estimate gene flow among populations of diamondback moth (Lepidoptera: Plutellidae) in Hawaii, Annals of the Entomological Society of America, 21(4): 808–816

 

Chapman J.W., Reynolds D.R., Smith A.D., Riley J.R., Pedgley D.E., and Woiwod I.P., 2002, High-altitude migration of the diamondback moth, Plutella xylostella to the U.K.: a study using radar, aerial netting, and ground trapping, Ecological Entomology, 27(6): 641-650

http://dx.doi.org/10.1046/j.1365-2311.2002.00472.x

 

Cheema H.K., Kang B.K., and Singh B., 2011, Biochemical and molecular basis of insecticide resistance in diamondback moth, Plutella xylostella (Linnaeus): A review, Pesticide Research Journal, 23(2): 123-134

 

C.I.E., 1967, Distribution maps of Plant Pests, No. 32, CAB International, Wallingford, UK

 

Ellango R., Asokan R., Rebijith K.B., Chaitanya B.N., and Mahmood R., 2012, Molecular variations in the diamondback moth, Plutella xylostella (L.) (Hyponomeutidae: Lepidoptera) as inferred from mitochondrial and ribosomal markers, Indian Journal of Entomology, 74(3): 241-245

 

Folmer O., Black M., Hoeh W., Lutz R., and Vrijenhoek R., 1994, DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates, Molecular Marine Biology and Biotechnology, 3(5): 294-299

 

Gong Y.J., Wang Z.H., Shi B.C., Kang Z.J., Zhu L., Jin G.H., and Weig S.J., 2013, Correlation between pesticide resistance and enzyme activity in the diamondback moth, Plutella xylostella, Journal of Insect Science, 13: 135

http://dx.doi.org/10.1673/031.013.13501

PMid:24766444 PMCid:PMC4014041

 

Hall T.A., 1999, BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT, Nucleic Acids Symposium Series No., 41: 95-98

 

Hebert P.D.N., Cywinska A., Ball S.L., and deWaard J.R., 2003, Biological identifications through DNA barcodes, Philosophical Transactions of the Royal Society of London B Biology, 270(1512): 313-321

 

Kim I., Bae J.S., Sohn H.D., Kang P.D., Ryu K.S., Sohn B.H., Jeong W.B., Jin B.R., and Lee K.R., 2000, Genetic homogeneity in the domestic silkworm, Bombyx mori, and phylogenetic relationship between B. mori and wild silkworm, B. mandarina, using mitochondrial COI gene sequences, International Journal of Industrial Entomology, 1(1): 9-17

 

Kim I., Bae J.S., Lee K.S., Kim E.S., Lee H.S., Ryu K.S., Yoon H.J., Jin B.R., Moon B.J., and Sohn H.D., 2003, Mitochondrial COI gene sequence-based population genetic structure of the diamondback moth, Plutella xylostella, Korean Journal of Genetics, 25(2): 155-170

 

Li J., Zhao F., Choi Y.S., Kim I., Sohn H.D., and Jin B.R., 2006, Genetic variation in the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae) in China inferred from mitochondrial COI gene sequence, European Journal of Entomology, 103(3): 605–611

http://dx.doi.org/10.14411/eje.2006.081

 

Lorimer R.I., 1981, Lepidoptera immigrants to Orkney in 1890, Proceedings and transactions of the British Entomological and Natural History Society, London, 14(3): 108-109

Mohan M., and Gujar G.T., 2003, Local variation in susceptibility of diamondback moth to insecticides and role of detoxification enzymes, Crop Protection, 22(3): 495-504

http://dx.doi.org/10.1016/S0261-2194(02)00201-6

 

Murthy K.S., Sannaveerappanavar V.T., and Shankarappa K.S., 2014, Genetic diversity of diamnondback moth, Plutella xylostella L. (Yponomeutidae: Lepidoptera) populations in India using RAPD markers, Journal of Entomology, 11(2): 95-101

http://dx.doi.org/10.3923/je.2014.95.101

 

Pichon A., Arvanitakis L., Roux O., Kirk A.A., Alauzet C., Bordat D., and Legal L., 2006, Genetic differentiation among various populations of the diamondback moth, Plutella xylostella (Lepidoptera: Yponomeutidae), Bulletin of Entomological Research, 96(2): 137-144

http://dx.doi.org/10.1079/BER2005409

PMid:16556334

 

Sambrook J.F., and Russell D.W., 2001, Molecular cloning: A laboratory manual. 3rd Edition, Cold spring Harbor Laboratory Press, Long Island, New York, pp. 2100

 

Santos V.C., de Siqueira H.A., da Silva J.E., and de Farias M.J., 2011, Insecticide resistance in populations of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), from the state of Pernambuco, Brazil, Neotropical Entomology, 40(2): 264-270

http://dx.doi.org/10.1590/S1519-566X2011000200017

PMid:21584410

 

Talekar N.S., and Shelton A.M., 1993, Biology, ecology, and management of the diamondback moth, Annual Review of Entomology, 38:275-301

http://dx.doi.org/10.1146/annurev.en.38.010193.001423

 

Tamura K., Stecher G., Peterson D., Filipski A., and Kumar S., 2013, MEGA6: Molecular evolutionary genetics analysis version 6.0., Molecular Biology and Evolution, 30(12): 2725-2729

http://dx.doi.org/10.1093/molbev/mst197

PMid:24132122 PMCid:PMC3840312

 

Thompson J.D., Higgins D.G., and Gibson T.J., 1994, CLUSTALW: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acids Research, 22(22): 4673-4680

http://dx.doi.org/10.1093/nar/22.22.4673

PMid:7984417 PMCid:PMC308517

 

Walker G.P., Davis S.L., MacDonald F.H., and Herman T.J.B., 2012, Update on diamondback moth (Plutella xylostella) insecticide resistance and vegetable Brassica insecticide management strategy, New Zealand Plant Protection, 65: 114-119

 

Xie S., 2013, The Genome of Diamondback Moth Sequenced, a Model Pest in Lepidoptera, Jiyinzuxue Yu Shengwu Jishu (online), 2: 1-2