Indian Journal of Experimental Biology Vol. 57, December 2019, pp. 923-930

Spatial genetic variation in South Indian larval populations of legume pod borer, Geyer (: )

S Sambathkumar1*, C Durairaj1, S Mohankumar2, B Preetha2, R Aravintharaj2 & N Ganapathy1 1Department of Agricultural Entomology; 2Department of Plant Biotechnology, Tamil Nadu Agricultural University, Coimbatore-641 003, Tamil Nadu, India

Received 27 February 2017; revised 15 February 2019

The Legume pod borer Maruca vitrata (Fabricius) is a serious pest in most common pulses affecting their yield. In the present investigation, genetic diversity among larval population of Maruca vitrata from five locations of India was assessed using 23 RAPD primes and 6 SSR primers. RAPD primers produced 364 (98.9% polymorphism) polymorphic alleles with the mean number of 18.2 alleles/primer while SSR primers produced 45 polymorphic alleles with 68% polymorphism. The mean number of alleles detected per primer ranged from 4 to 46 and 5 to 12 with RAPD and SSR primers, respectively in different larval samples. Dendrogram constructed using similarity index values for RAPD and SSR primers differentiated the larval samples. In all three types of Maruca larval samples, Polymorphism information content (PIC) value observed for RAPD primers ranged from 0.019 (OPC 08) to 0.375 (OPAF 12). Similarly, SSR primers showed PIC value of 0.061 (C32008E) to 0.781 (C3393E,1) irrespective of larval samples.

Keywords: Polymorphism, Pulses, RAPD Primer, SSR Primer

Legume pod borer, Maruca vitrata (Geyer) for M. vitrata. Existence of different species of (Lepidoptera: Crambidae), is considered as the most M. vitrata is already reported10,11 and it is hard to serious pest of food legumes in tropical Asia, identify and distinguish the correct species using sub-Saharan Africa, South America, North America, morphological characters because of their tough Australia and the Pacific1. It is already reported on species complex12. Apart from M. vitrata, other species 45 different host plant species, especially on legumes viz., M. amboinalis (Felder), M. aquitilis (Guérin- in tropical Asia and sub-Saharan Africa2,3. The larvae Méneville), M. bifenestralis (Mabille), M. fuscalis feed upon flowers and pods of leguminous plants by (Yamanaka), M. nigroapicalis (Joannis), M. simialis webbing on them and cause significant yield loss to (Snellen) and M. Testulalis (Geyer) were also reported legumes4-6. Hence, M. vitrata is can be considered as a and M. testulalis was found to be synonymous with major emerging threat to legume production in many M. vitrata. M. amboinalis and M. nigroapicalis were developing and underdeveloped nations. In India, observed in the Indo-Malaysian and Tonkin area, the M. vitrata is one of the major pests of pulses, especially most probable centre of origin for the Maruca13,14. during flowering and pod formation stages. At present, However, in India, only the incidence of M. vitrata is chemicals are mainly used to control this pest, and in reported so far. We have recently reported the many occasions it is not effective in managing, detoxifying enzyme profile and developmental particularly the larval stages. This might be attributed to flexibility that M. vitrata possess to overcome pesticides the construction of webbing around the larvae which in fields by analyzing gut hydrolytic enzymes15. In shields them from insecticide sprays, existence of consideration of above issues, an attempt was made to resistance strains of this species7,8 and high cost of plant study the genetic variation in different populations of M. protection chemicals9. vitrata collected from different parts of pulses growing Lack of sufficient population genetic data on regions in South India. DNA sequences, molecular markers, and population structure hinders the development of IRM protocols Material and Methods

Isolation of genomic DNA —————— *Correspondence: Larvae of M. vitrata collected from different E-mail: [email protected] locations of South India (Table 1), such as 924 INDIAN J EXP BIOL, DECEMBER 2019

Table 1—Geographic details of Maruca vitrata larval collection Table 2—List of RAPD primers used for M. vitrata Location State Latitude and Longitude S. No. Primer Sequence (5'-3') TNAU, Coimbatore Tamil Nadu 11.0144120N and 1. OPA 01 CAGGCCCTTC 76.9354510E 2. OPA 07 GAAACGGGTG KAU, Vellanikkara, Kerala 10.5474690N and 3. OPA 08 GTGACGTAGG Thrissur 76.2833450E 4. OPA 13 CAGCACCCAC Lam, Guntur Andra Pradesh 16.3693280N and 5. OPA 15 TTCCGAACCC 80.4270880E 6. OPA 20 GTTGCGATCC Kollegal, Karnataka 12.1537100N and 7. OPAF 12 GACGCAGCTT Chamrajnagar 77.111100E 8. OPAF 13 CCGAGGTGAC Aurangabad Maharashtra 19.9063760N and 9. OPAF 14 GGTGCGCACT 75.3465560E 10. OPAG 10 ACTGCCCGAC 11. OPBB 03 TGGCGCACAC Coimbatore, Thrissur, Guntur, Chamrajnagar, and 12. OPBB 13 CCTACCGTGG Aurangabad. DNA was individually extracted from 16 13. OPBB 15 AAGTGCCCTG larva of M. vitrata using modified CTAB method . 14. OPC 02 GTGAGGCGTC Agarose gels (0.8%) were used to analyze the 15. OPC 04 CCGCATCTAC quality of DNA samples. After the run, the gel was 16. OPC 08 TGGACCGGTG examined and documented using gel documentation 17. OPC 15 GACGGATCAG (Bio-Rad, USA). The quantification of DNA was 18. OPE 04 GTGACATGCC done by using Nano drop Spectrophotometer 19. OPE 08 TCACCACGGT (ND – 1000). The absorbance for all samples was 20. OPE 15 ACGCACAACC measured at 260 to 280 nm. Based on the quantity 21. P 210 AAATGCGGCA of DNA, dilutions were made in TE buffer to a final 22. P 225 AGTGGTCGCG concentration of 50 ng/µL and stored in -20°C for 23. P 443 GCCGTGATAG further use. 24. S 34 GATAGCCGAC

RAPD primers analysis of M. vitrata Table 3—List of SSR primers used for M. vitrata Annealing Isolated larval DNA samples of M. vitrata were Loci Sequence (5'-3') subjected to Random Amplified Polymorphic DNA temp. E O (RAPD) primer analysis using RAPD primer sets C32008 F-(MAX)AAAAAGCGCTTATATGTTTGTTATAGT 57 C R-GAAATTTTTAACGGAGATACAATCA (Table 2) OPERON random primers according to A O Williams et al.17 and the variation with respect to 7_02K06 F-(FAM)ATTTGTCAGAATGGTATCTTACGT 52 C R-CCTCTGGGTCATAATTATATTGTTCA genomic DNA was confirmed by PCR amplification. E,1 O C3393 F-(ROX)AGACCCCCAAAGTGGAGAA 55 C RAPD analysis and PCR conditions R-ACGTTCACGAACCTCCTGTT PCR mixtures (25 μL volume) contained 2 μL of C0444E,1 F-(FAM)AAAGGAACTACGCCGTCAGG 57O C template DNA (50-150 ng/µL) followed by 2.5 μL R-GTTGAGCGATCTTGGCACAG E 10×PCR buffer, 0.5 μL of MgCl2, 1.0 μL dNTP C0241 F-(TAM)GACGAAACAAGGCCTACCAG mixtures (200 M/L each of dATP, dCTP, dGTP and R-GGTACTTCYGACGTTGTTCG dTTP), 1.0 μLeach of RAPD forward and reverse C0325E F-(ROX)CGAAAAGAAACACCGCTCTG primer, 0.5 μL Taq DNA polymerase (1.5U), and R-CAGTCTGTTCAGWCTCTTCAGTGG

16.5 μL distilled water. PCR amplification was documented in image documentation system performed in thermocycler (Bio-Rad DNA engine) (Bio-Rad USA). programmed for initial denaturation at 94°C for 5 min; 40 cycles of denaturation at 94°C for 1 min, SSR primer analysis of M. vitrata annealing at 40°C for 1 min, extension at 72°C for Six SSR primers (Table 3) were used to assess the 2 min and a final extension step at 72°C for 7 min. genome of larval populations of M. vitrata from five 18 Reaction tubes were held at 4°C prior to visualization different locations of South India . The PCR reaction of PCR products in a 2% agarose gel stained with mixture (25 μL) contained 25 μL of DNA, 2.5 μL of 0.5 µg/mL ethidium bromide. Finally, the amplified 10 × PCR buffer, 10.5 μL of MgCl2 (1.2 M), 1.0 μL PCR products were viewed under UV light and each dNTPs (mixture of dATP, dCTP, dGTP and SAMBATHKUMAR et al.: SPATIAL GENETIC VARIATION IN LARVAL POPULATIONS OF MARUCA VITRATA 925

dTTP), 1.0 μL each of forward and reverse primers Data analysis and 0.5 μL of 1.25 U Taq DNA polymerase enzyme Clearly resolved, unambiguous polymorphic bands (Fermentas, USA). The DNA was amplified using the obtained out of RAPD and SSR primers analysis were following conditions as Initial denaturation at 94°C scored either absent (0) or present (1) with the help of for 2 min, followed by 40 cycles of denaturation at Alpha Ease® FC Software (Source: Protein Simple, 94°C for 1 min, annealing of primer at 52 to 57°C for California, USA). Allele scoring was done using the 1 min (Table 3), extending primer at 72°C for 1 min 100 bp primer lane as a reference. The sizes of the and final extension at 72°C for 2 min. alleles are approximated by comparing with the band The amplified bands were separated by denaturing migration distance of the standard 100 bp ladder. The polyacrylamide (6%) gel electrophoresis in TBE total number of fragments , polymorphic fragments , buffer and photographed. It was prepared from the per cent polymorphism and the Nei̓ ’s genetic distance stock solution (acrylamide 29.0 and 1.0 g and distilled (GD) were calculated. The polymorphism information water) by adding 20 mL of polyacrylamide stock content (PIC) values are calculated for each RAPD solution, 10 mL of 10 × TBE buffer, 900 µL of and SSR primers using the following algorithm: ammonium per sulphate (APS) and 100 µL of tetra PIC = 1-f 2i = 1 where f 2 is the frequency of ith methyl ethylene diamine (TEMED). The gel was i i allele21 immediately poured in to the glass plates immediately after adding TEMED (to avoid solidification) without The binary data scored was used to construct a any air bubbles. The gel was pre-run for about 20 min dendrogram. Similarity matrix was generated using before loading the sample and maintained at 70 W for the SIMQUAL programme of NTSYS-pc software, 22 2 h. After electrophoresis, the gel was separated from version 2.02 . The cluster analysis and dendrograms the plates and treated for 15 min in fixation solution were implemented on the basis of the similarity (double distilled water and 0.5% v/v acetic acid) with coefficients and genetic distances using the UPGMA gentle shaking, the gel was washed twice with (un weighed pair group method arithmetic average) 23 distilled water for 2 min. The gel was stained in method . staining solution (0.1% w/v silver nitrate), the gel was Results washed 2-times with distilled water for 2 min, and then the gel was transferred to developing solution Analysis of genetic diversity in M. vitrata using RAPD primers (1.5% w/v sodium hydroxide, 0.3% formaldehyde) to The RAPD primer profile of Maruca larvae develop the silver-staining DNA bands. The reactions collected from different locations of India generated were stopped by using stopper solution (double by 20 primers (in series OPA, OPAF, OPAG, OPBB, distilled water and 0.5% v/v acetic acid). The bands OPC, OPE and few specific primers) are given in were then visualized by silver staining19,20, dried Fig. 1) for illustration. All primers produced overnight and photographed. The size of individual unambiguous scorable alleles. A total of 364 DNA fragments was compared with a co-migrating polymorphic alleles were obtained out of 368 total 100 bp DNA ladder (MBI Fermentas). alleles produced by RAPD primers (Table 4). The size

Fig. 1 — RAPD primer profiles of Maruca vitrata larvae from different locations of India. [Lane L, 1 kb ladder; and Lanes 1 to 5: Larva of Coimbatore, Tamil Nadu; Thrissur, Kerala; Mysore, Karnataka; Guntur, Andhra Pradesh; and Aurangabad, Maharashtra, respectively] 926 INDIAN J EXP BIOL, DECEMBER 2019

Table 4—RAPD primer profile of M. vitrata larvae collected from Table 5—Similarity matrix of M. vitrata larvae collected from different locations and per cent polymorphism observed different locations of India with RAPD primers Primer No. of No. of poly No. of mono Poly PIC State TN KL KA AP MH alleles morphic morphic morphism value Tamil Nadu 1.000 alleles alleles (%) Kerala 0.484 1.000 OPA 01 13 13 0 100.0 0.101 Karnataka 0.513 0.606 1.000 OPA 07 14 14 0 100.0 0.082 Andhra Pradesh 0.477 0.577 0.613 1.000 OPA 08 16 16 0 100.0 0.102 Maharashtra 0.495 0.581 0.602 0.645 1.000 OPA 13 19 19 0 100.0 0.091 OPA 15 11 11 0 100.0 0.124 [TN, Tamil Nadu; KL, Kerala; Ka, Karnataka; AP, Andhra OPAF 12 4 4 0 100.0 0.250 Pradesh; MH, Maharashtra] OPAF 13 10 10 0 100.0 0.180 OPAF 14 10 10 0 100.0 0.200 OPAG 10 28 26 2 92.9 0.056 OPBB 03 5 5 0 100.0 0.280 OPBB 13 13 13 0 100.0 0.089 OPC 02 30 30 0 100.0 0.069 OPC 04 23 23 0 100.0 0.078 OPC 08 25 25 0 100.0 0.056 OPE 04 27 27 0 100.0 0.048 OPE 15 22 22 0 100.0 0.066 P 210 24 24 0 100.0 0.056 P 225 13 13 0 100.0 0.148 P 443 46 46 0 100.0 0.027 S 34 15 13 2 86.7 0.029 Total 368 364 4 - - Mean 18.4 18.2 2 98.9 - of the amplicons ranged 0.16-2.54 kb. Among the primers used, P 443 produced the highest number of fragments (46) while OPAF 12 produced the lowest number of four fragments. All the primers except Fig. 2 — Dendrogram of larvae of M. vitrata collected OPAG 10 (92.9%) and S 34 (86.7%) exhibited 100% from different pulses based on 20 RAPD primers constructed polymorphism. The maximum PIC value of 0.280 was using UPGMA derived from OPBB 03 compared to 0.027 in P 443. and F and sub cluster F, finally differentiated into G Similarity index and H. Cluster E comprised of populations from Out of ten combinations of the Maruca larvae Karnataka and obtained 61.2% similarity with collected from different locations of India, the highest subcluster F. Highest similarity (64.6%) was observed similarity index values of 0.645 was obtained between between larval populations of Andhra Pradesh larvae collected from Andhra Pradesh and Maharashtra (subcluster G) and Maharashtra (subcluster H). (Table 5). The minimum similarity index was observed between Tamil Nadu and Andhra Pradesh (0.477). The Analysis of genetic diversity in M. vitrata using SSR primers mean similarity index was 0.559. The SSR profile of larvae collected from different locations of India showed that four primer pairs E,1 E E E Cluster analysis (C0444 , C0241 , C0325 and C32008 ) showed An UPGMA dendrogram of Maruca larvae variation by presence of alleles and they were found collected for five locations of India using 20 RAPD to be completely polymorphic (Fig. 3). In total, there primers (Fig. 2) revealed the existence of two principal were 45 alleles detected across the five loci with the clusters (A and B) and two sub clusters in B (C and D). maximum number of 12 alleles obtained per locus per The larval populations form Tamil Nadu (cluster A) location in primers C32008E and C0325E and the and Kerala (sub cluster C) showed similarity of minimum of 5 alleles in C0444E,1 with the mean of 48.9 and 58.9% with cluster B and sub cluster D, 9 alleles. Of 45 alleles detected, 25 were polymorphic. respectively. Subcluster D again divided into clusters E Primer 7_02K06A showed complete monomorphism SAMBATHKUMAR et al.: SPATIAL GENETIC VARIATION IN LARVAL POPULATIONS OF MARUCA VITRATA 927

Fig. 4 — Dendrogram of larvae of M. vitrata collected from Fig. 3 — SSR primer profiles M. vitrata larvae from different different locations of India based on five SSR primers constructed locations of India. [Lane L,100 bp ladder; and Lanes 1 to 5: Larva using UPGMA of Coimbatore, Tamil Nadu; Thrissur, Kerala; Mysore, Karnataka; Guntur, Andhra Pradesh; and Aurangabad, Table 7 — Similarity matrix of M. vitrata larvae collected from Maharashtra, respectively] different locations of India with SSR primers State TN KL KA AP MH Table 6—Polymorphism information content for SSR primers Tamil Nadu 1.000 amplified with larvae of M. vitrata collected from different Kerala 0.636 1.000 locations of India Karnataka 1.000 0.636 1.000 Primer No. of No. of poly No. of mono Poly PIC Andhra Pradesh 0.727 0.727 0.727 1.000 alleles morphic morphic morphism value alleles alleles (%) Maharashtra 0.727 0.545 0.727 0.818 1.000 C32008E 12 7 5 58.3 0.173 [TN, Tamil Nadu; KL, Kerala; KA, Karnataka; AP, 7_02K06A 5 0 5 Monomorphic 0.000 Andhra Pradesh; MH, Maharashtra] E,1 C0444 5 5 0 100.0 0.520 subclusters (E and F) and subcluster E ultimately C0241E 11 6 5 54.5 0.483 divided into subclusters G and H. The larvae collected C0325E 12 7 5 58.3 0.347 from Karnataka (subcluster F) showed a similarity Total 45 25 20 - - of 51.1% to the subcluster E. The sub clusters G and Mean 9 5 4 68.0 - H comprised of larval populations from Tamil Nadu with the larvae of different locations. The size of the and Kerala, respectively and had the highest similarity amplicons ranged 107-410 bp. The PIC value ranged of 80.1%. 0.173-0.520 in C32008E and C0444E,1, respectively (Table 6). Discussion Assessment of genetic diversity and identification Similarity Index of variation across the population give an idea Similarity index values obtained from each pair wise about evolution and migratory pattern of the species. comparison of larvae collected from five locations of The present study showed the presence of 364 and India (Table 7) showed the highest value of 1.000 45 polymorphic alleles (mean= 9 alleles) with RAPD between Tamil Nadu and Karnataka followed by 0.818 and SSR primers, respectively in M. vitrata populations between larvae collected from Andhra Pradesh and from five different locations of India. This is in Maharashtra. The lowest similarity index was observed conformity with the findings of Agunbiade et al.18 between Kerala and Maharashtra (0.545). The mean who obtained 38 alleles with mean of 6 alleles per similarity index was 0.727. loci when using the same primes on larval populations

Cluster analysis of M. vitrata collected from different parts of Africa. UPGMA dendrogram obtained from larvae of It was also recorded that C32008E yielded maximum M. vitrata collected from five locations of India of 10 alleles. High similarity coefficients recorded in using five SSR primers (Fig. 4) showed the presence larval samples between Tamil Nadu and Karnataka of two major clusters (A and B) and two subclusters (1.000) and followed by Andhra Pradesh and in B (C and D). The sub clusters C and D comprised Maharashtra (0.818) showed their less genetic the larval populations from Andhra Pradesh and diversity. Similarly, Zhou et al.24 observed the Maharashtra and exerted 100% similarity between existence of 99% polymorphism in Helicoverpa them. Cluster A further differentiated into two collected across six locations in eastern Mediterranean 928 INDIAN J EXP BIOL, DECEMBER 2019

region with RAPD primers. Zang-Ying et al.25 putative subspecies of M. vitrata which cannot be investigated genetic diversity among eleven differentiated based on morphological characters. geographic populations of Conogethes punctiferalis Various environmental factors and temporal Guenée using ISSR primers and recorded 99.05% barriers due to cropping pattern may play a key role in polymorphism with the genetic distance of 0.0059 to isolating Maruca populations, which resulted in high 0.0237 among the population. In West Africa, amount of genetic variability among geographically Margam et al.3 reported the existence of genetic different populations in India. Intermating of subdivision using single nucleotide polymorphism geographically distant populations and migration (SNP) studies of M. vitrata. followed by isolation would result in high genetic In the present investigation, the average number of variability within a population. This may help the polymorphic fragments was 18 and polymorphism species to evolve and adapt faster to different level of nearly 100% across the locations. The environments and rapid evolution of resistance to dendrogram generated from the similarity matrix various biotic and abiotic stresses including throws light on the interrelationships of populations insecticides. Scientific evidences for the higher collected from Andhra Pradesh and Maharashtra as genetic variation in M. vitrata are already reported in well as Tamil Nadu and Kerala. Clustering analysis African countries18,45,46. The seasonal flowering revealed the existence of two major groups and close patterns of different legume hosts across India may proximity between populations of Andhra Pradesh also influence both active and passive migration and and Maharashtra and they are highly deviated from establishment of pest species and thereby it could Maruca populations of Tamil Nadu. This variation locate ideal conditions for feeding and reproduction might be due to the topographical barriers, lesser gene and thus leading to maintenance of ideal population in flow and poor passive disposal among studied subsequent generations. Sometimes, these might end populations18 Existence of Microsatellite null alleles up with poor population growth due to inadequate in certain species is also believed to be the major mating populations and various rates of larval cause for polymorphism. They are commonly found development. Host plant availability, suitability and among a wide range of taxa but have a particularly weather parameters like rainfall and temperature also high incidence among species of Lepidoptera26, influence the movement of species and make them Diptera27 and Orthoptera28. Using the similar restricted in the same locations18. During these techniques, higher genetic variation was detected in circumstances, the survival might be supported by Nebria gregaria Fischer von Waldheim29, Culex sparse alternate host plants. This is corroborating to quinquefasciatus Say30, Aedes aegypti Linnaeus31, the findings of Bottenberg et al.45 who reported Trypanosoma cruzi Chagas32 and Rhynchophorus the off season survival of M. vitrata on alternate ferrugineus Olivier33. Similarly, large geographic host plants within the more humid southern regions distance may also results in wider genetic distances as and in migrated populations in northern regions of in the case of highly mobile populations such as, Africa. During the seasonal migration, the population Chazara briseis Linnaeus34 and Hesperia dacotae of M. vitrata finds favourable conditions for Skinner35, Rhynchoporus ferrugineus (Olivier)36, multiplying on a succession of different host plants, Bombyx mori37 and Chironomus plumosus38. which may result in temporal mating barriers due RAPD primers also help in identification and to differential larval maturation rates on the alternate differentiation of closely related subspecies and host plants. populations within a species28,39. In consonance to the present findings, RAPD differences have also been Conclusion found between geographically isolated populations of Present study clearly revealed the existence of Aedes aegyptii40, Mediterranean fruit fly, Ceratitis 98.9% polymorphism among larval population of capitata Wiedemann41, Pine bast scales Matsucoccus Maruca vitrata from five locations of India when matsumurae (Kuwana)42 and Argentine stem weevil, assessed using RAPD primes whereas, SSR primers Listronotes bonariensis Kuschel43. According to produced 68% polymorphism. Hence, it is clear that Malini Periasamy et al.44 mitochondrial COI primer the spatial variation brought the genetic diversity analysis on larval populations from Asia and sub- within the M. vitrata species. It may further be Saharan Africa resulted in the existence of two extrapolated to find out the genetic insights of SAMBATHKUMAR et al.: SPATIAL GENETIC VARIATION IN LARVAL POPULATIONS OF MARUCA VITRATA 929

M. vitrata with additional geographic populations in 9 Arodokoun DY, Tamo M, Cloutier C & Brodeur J, India. However, it is helpful in finding out the level of Larval parasitoids occurring on Maruca vitrata Fabricius (Lepidoptera: Pyralidae) in Benin, West Africa. Agric variation across the collected South Indian larval Ecosyst Environ, 113 (2006) 320. populations of M. vitrata. Further, these may also 10 Malini P, Schafleitner R, Krishnan M & Srinivasan R, aid in the implementation of effective IPM and Phylogeographical structure in mitochondrial DNA of IRM strategies. legume pod borer (Maruca vitrata) population in tropical Asia and sub-Saharan Africa. PLoS ONE, 10(4) (2015) Acknowledgement e0124057. 11 Brownlie JC, Cass BN, Riegler M, Witsenburg JJ, The first author is thankful to the Department of Iturbe-Ormaetxe I, Mcgraw EA & O’Neill SL, Evidence Science and Technology, Govt. of India for the for metabolic provisioning by a common invertebrate INSPIRE fellowship. Laboratory facilities rendered endosymbiont, Wolbachia pipientis, during periods of by the Molecular Ecology Laboratory, Department of nutritional stress, PLoSPathog, 5 (2009) Plant Biotechnology TNAU, Coimbatore, Tamil Nadu 12 Brownlie JC & Johnson KN, Symbiont mediated protection in insect hosts, Tr Microbiol, 17 (2009), 348–354. is sincerely acknowledged. 13 Rose HS & Singh AP, Use of internal reproductive organs in the identification of Indian species of the genus Maruca Conflict of Interest Walker (Pyraustinae: Pyralidae: Lepidoptera). J Adv Zool, The authors declare is no conflict of interests. 10(2) (1989) 99. 14 Kirti JS & Gill NS, Taxonomic studies on Indian species of References Genus Maruca Walker (Lepidoptera: Pyralidae: Pyraustinae), Zoos’ Print J, 20(7) (2005) 1930. 1 Sharma HC, Bionomics, host plant resistance, and management of the legume pod borer, Maruca vitrata-a 15 Sambathkumar S, Durairaj C, Mohankumar S, Preetha B, review. Crop Prot, 17 (1998) 373. Aravintharaj R, Ganapathy N & Surendran R, Variation in the gut hydrolytic enzymes of legume pod borer, Maruca 2 Malini P, Srinivasan R, Lin MY, Yule S & Krishnan M, vitrata (Fabricius) feeding on different pulses. Indian J Exp A revision on the diversity within the legume pod borer Biol, 57 (2019) 239. (Maruca spp.), its host plants and natural enemies. Bang J Entomol, 24 (2) (2014) 1. 16 Doyle JJ & Doyle LL, A rapid DNA isolation procedure for small quantities of fresh leaf tissue, Phytochemistry, 19 3 Margam VM, Coates BS, Ba MN, Sun W, Binso-Dabire CL (1987) 11. & Baoua I, Geographic distribution of phylogenetically- distinct legume pod borer, Maruca vitrata (Lepidoptera: 17 Williams JGK, Kubelik AR, Livak KJ, Rafalski JA & Pyraloidea: Crambidae). Mol Biol Rep, 38 (2011) 893. Tingey SV, DNA polymorphisms amplified by arbitrary primers are useful as genetic markers, Nucleic Acids Res, 18 4 Chinh NT, Dzung DT, Long TD, Tam HM & Ramakrishna A, (1990) 6531-6535. Legumes in Viet Nam: constraints and opportunities. In: Legumes in rice-based cropping systems in tropical Asia: 18 Agunbiade TA, Coates BS, Kim KS, Forgacs, D, constraints and opportunities, (Ed. Gowda CLLRA, Rupela Margam VM, Murdock LL, Ba MN, Binso-Dabire CL, OP & Wani SP, ICRISAT, Hyderabad, India), 2000, 111. Baoua I, Ishiyaku MF, Tamo M & Pittendrigh BR, The spatial genetic differentiation of the legume pod borer, 5 Soeun MSM, Legumes in rice-based cropping systems in Maruca vitrata F. (Lepidoptera: Crambidae) populations in Cambodia: constraints and opportunities. In: Legumes in West Africa, Bull Entomol Res, (2012) 1 rice-based cropping systems in tropical Asia: constraints and opportunities, (Ed. Gowda CLLRA, Rupela OP & Wani SP, 19 Bassam BJ, Caetano-Anolles G & Gresshoff PM, Fast and ICRISAT, Hyderabad, India), 2001, 4. sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem, 196 (1991) 80. 6 Ulrichs C, Mewis I, Schnitzler WH & Burleigh JR, Effectivity of synthetic insecticides against Marucavitrata F 20 Chalhoub BA, Thibault S, Laucou V, Rameau C & and the parasitoid Bassus asper Chou & Sharkey in the Hofte H, Silver staining and recovery of AFLP (TM) Philippines. Mitteilungen der Deutschen Gesellschaft fur amplification products on large denaturing polyacrylamide allgemeine und angewandte Entomologie [German]. Comm gels. Biotechniques, 22 (1997) 216. German Soc General Appl Entomol, 13 (2001a) 279 21 Hildebrand CE, Torney DC & Wagner RP, Informativeness 7 Ekesi S, Insecticide resistance in field populations of the of Polymorphic DNA Markers. Los Alamos Sci, 20 (1992): legume pod borer MarucavitrataFabricius in Nigeria. Int J 100. Pest Mgmt, 45 (1999) 57 22 Rohlf FJ, NTSYS-pc Numerical and Multivariate 8 Ulrichs C, Mewis I, Schnitzler WH & Burleigh JR, Analysis System- Version 2.02.Exeter Publications Setauket, Parasitoids of the bean podborer, Maruca vitrata F. New York (1998) (Lepidoptera: Pyraustinae), a pest of Vigna sesquipedalis in 23 Sneath PHA. & Sokal RR, Numerical Taxonomy: the the Philippine lowlands, Mitteilungen der Deutschen Principles and Practice of Numerical Classification, Gesellschaft fur allgemeine und angewandte Entomologie San Francisco, California, (1973) p.573 [German]. Comm German Soc General Appl Entomol, 13 24 Zhou X, Faktor O, Applebaum SW & Coll M, Population (2001b) 283. structure of the pestiferous Helicoverpa armigera in the 930 INDIAN J EXP BIOL, DECEMBER 2019

eastern Mediterranean using RAPD analysis. Heredity, 85 36 El-Mergawy RAAM, Al Ajlan AM, Abdallah NA, Nasr MI (2000) 251. & Silvain JF, Determination of different geographical 25 Zang-Ying, Li-Jing, Wang-Zhen-Ying & He-Kang-Lai, populations of Rhynchophorus ferrugineus (Olivier) Genetic diversity of Conogethes punctiferalis (Guenee) (Coleoptera: Curculionidae) using RAPD-PCR. Int J Agric (Lepidoptera: Crambidae) populations from different Biol, 13 (2011) 227. geographic regions of China. Acta Entomologica Sinica, 37 Srivastava PP, Vijayan K, Awasthi AK, Kar PK, Thangavelu K 53(9) (2010) 1022. & Saratchandra B, Genetic analysis of silkworms Bombyx mori 26 Meglécz E, Petenian F, Danchin E, Coeur D’Acier A, through RAPD markers. Indian J Biotechnol, 4 (2005) 389. Rasplus J-Y, Faure E, High similarity between flanking 38 Gunderina LI, Kiknadze II, Istomina AG & Batler M, regions of different microsatellites detected within each of Geographic differentiation of genomic DNA of Chironomus two species of Lepidoptera, Parnassius Apollo and plumosus (Diptera: Chironomidae) in natural holarctic Euphydryas aurinia. Mol Ecol, 13 (2004) 1693. populations. Genetika, 45 (2009) 64. 27 Lehmann T, Besansky NJ, Hawley WA, Fahey TG, Kamau L 39 Sebastiani F, Meiswinkel R, Gomulski LM, Guglielimino CR, & Collins FH, Microgeographic structure of Anopheles Mellor PS, Malacrida AR & Gasperi G, Molecular gambiaein western Kenya based on mtDNA and differentiation of the Old World Culicoides imicola microsatellite loci. Mol Ecol, 6 (1997) 243. species complex (Diptera: Ceratopogonidae), inferred using 28 Chapuis MP, Loiseau A, Michalakis Y, Lecoq M & random amplified polymorphic DNA markers. Mol Ecol, Estoup A, Characterization and PCR multiplexing of 10 (2001) 1773. polymorphic microsatellite loci for the locust Locusta 40 Kambhampati S, Black WC & Rai KS, Random amplified migratoria. Mol Ecol Notes, 5 (2005) 554. polymorphic DNA of mosquito species and populations 29 Clarke TE, Levin DB, Kavanaugh DH & Reimchen TE, (Diptera: Culicidae): technique, statistical analysis and Rapid evolution in Nebria gregaria group (Coleoptera: applications. J Med Entomol, 29 (1992) 939. Carabidae) and the paleogeography of the Queen Charlotte 41 Haymer DS & Mc Innis DO, Resolution of population of the Islands. Evolution, 55 (2001) 1408 Mediterranean fruit fly at the DNA level 7using random primers 30 Sharma AK, Mendki MJ, Tikar SN, Chandel K, Sukumaran D, for the polymerase chain reaction. Genome, 37 (1994) 244. Parashar BD, Veer V & Agarwal and Prakash OPS, Genetic 42 Mendel Z, Nestel D & Gafny R, Examination of the origin variability in geographical populations of Culex of the Israeli population of Mastsucoccus josephi quinquefasciatus Say (Diptera: Culicidae) from India based (Homoptera: Matsucoccidae) using random amplified on random amplified polymorphic DNA analysis. Acta Trop, polymorphic DNA – polymerase chain reaction method. 112 (2009) 71 Ann Entomol Soc Am, 87 (1994) 165. 31 Hiragi C, Simões, K, Martins E, Queiroz P, Lima L & 43 Williams CL, Goldson SL, Baird DB & Bullock DW, Monnerat R, Genetic variability in Aedes aegypti (L.) Geographical origin of an introduced insect pest, Listronotus (Diptera: Culicidae) populations using RAPD markers. bonariensis (Kuschel), determined by RAPD analysis. Neotrop Entomol, 38 (2009) 542 Heredity, 72 (1994) 412. 32 Lala ERP, Andó MH, Zalloum L, Bértoli M, de Oliveira 44 Malini Periasamy, Roland Schafleitner, Krishnan Machado Dalalio M, Silveira TGV, Gomes M L, Guedes TA Muthukalingan, Srinivasan Ramasamy, Phylogeographical & De Araújoe SM, Trypanosoma cruzi: Different methods of structure in mitochondrial DNA of legume pod borer data analysis to evaluate the genetics–biology relationship. (Maruca vitrata) Population in Tropical Asia and Sub- Exp Parasitol, 123 (2009) 173. Saharan Africa. PLoS ONE, 10 (4) (2015) 24 33 Gadelhak GG & Enan MR, Genetic diversity among 45 Bottenberg H, Tamò M, Arodokoun D, Jackai LEN, populations of red palm weevil, Rhynchophorus ferrugineus Singh BB & Youm O, Population dynamics and migration Olivier (Coleoptera: Curculionidae), determined by random of cowpea pests in northern Nigeria: implications amplified polymorphic DNA polymerase chain reaction for integrated pest management. In: Advances in (RAPD-PCR). Int J Agric Biol, 7 (2005) 395. Cowpea Research, (Ed. Dashiell KE, Jackai LEN, Singh BB, 34 Johannesen J, Schwing U, Seufert W, Seitz A & Veith M, Mohan Raj DR, International Institute of Tropical Analysis of gene flow and habitat patch network for Chazara Agriculture Ibadan and Japan International Research Center briseis (Lepidoptera: Satyridae) in an agricultural landscape. for Agricultural Sciences, Tsukuba), 1997, 271. Biochem Syst Ecol, 25 (1997) 419. 46 Ba NM, Margam VM, Dabiré LCB, Sanon A, McNeil JN, 35 Britten HG & Glasford JW, Genetic population structure of Murdock LL & Pittendrigh BR, Seasonal and regional the Dakota Skipper (Lepidoptera: Hesperia dacotae): A distribution of the cowpea pod borer Maruca vitrata North American native prairie obligate. Conser Genet, 3 (Lepidoptera: Crambidae) in Burkina Faso. Int J Trop Insect Sci, (2002) 363. 29 (2009) 109.