Genetic Structure and haplotype diversity in South Indian Populations of Brown Planthopper, (Nilaparvata lugens) using mtCOI

Genetic Structure and haplotype diversity in South Indian Populations of Brown Planthopper, (Nilaparvata lugens) using mtCOI | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Genetic Structure and haplotype diversity in South Indian Populations of Brown Planthopper, (Nilaparvata lugens) using mtCOI Madhukumar H, Dr. Sujay Hurali, Dr. V Chinna Babu Naik, Dr. Basavaraj S Kalmath, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8685733/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 7 You are reading this latest preprint version Abstract The brown planthopper Nilaparvata lugens (Stal) is a major pest of rice, causes high economic losses by reducing yield. In India, most populations of N. lugens could not be managed probably due to high genetic variation in populations. Hence, the study was conducted to investigate the genetic diversity of N. lugens in south Indian populations. A total of eight populations were collected from Karnataka, Telangana, Andhra Pradesh, Tamil Nadu and Kerala. DNA extraction, PCR amplification and sequencing done using the COI gene primer. The study identified eight haplotypes having high haplotype diversity and nucleotide diversity along with two polymorphic sites and two mutations. Neutrality tests (Tajima’s D, Fu’s Fs) indicated no significant deviation from neutrality. The high genetic diversity highlights the adaptive potential of N. lugens to environmental shifts, host resistance and management practices. Such adaptability makes N. lugens a serious threat to rice cultivation, as it can overcome existing control measures. The findings emphasize the importance of continuous molecular monitoring to track evolutionary dynamics in developing effective and sustainable pest management strategies. Genetic diversity Haplotype Nilaparvata lugens Population Figures Figure 1 Figure 2 Figure 3 1. Introduction The brown planthopper (BPH), Nilaparvata lugens , is a major pest of rice in many Asian countries. It damages plants directly by sucking phloem sap and indirectly by transmitting rice ragged stunt and grassy stunt viruses. Once a minor pest, BPH became a serious threat to rice production during the 1970s. Both nymphs and adults infest rice at all growth stages, causing yellowing, drying, and hopper burn. Severe infestations result in lodging and yield losses ranging from 10–70%. N. lugens has developed resistance to important groups of insecticides, including organophosphates and neonicotinoids, particularly imidacloprid and has shown distinct virulence reactions to some rice varieties. Mitochondrial cytochrome oxidase I (COI) is a widely used marker for understanding population structure, species identification and phylogenetic relationships of insects because of its rapid evolutionary rate, maternal inheritance and lack of introns. Mitochondrial genes have served as molecular markers in the insect’s population genetic studies (Liu et al ., 2010). In this study we examined genetic diversity among N. lugens populations that were collected from eight different geographical regions of South India, along with ten sequences retrieved from NCBI, including samples from India, Indonesia Thailand. Understanding this pest genetic diversity is essential for developing sustainable pest management strategies, including breeding for durable resistance and predicting the emergence of virulent biotypes. As climate change and agricultural practices continue to influence pest dynamics, continued research into the genetic makeup of the brown planthopper remains crucial for securing global rice production and food security (Wang et al ., 2015). Therefore, the present study was undertaken to evaluate the molecular genetic structure and diversity of N. lugens , which is essential in designing effective management strategies for its suppression. 2. Material and method Present investigation on genetic diversity of different N. lugens population of Southern states of India were carried out during 2024-25 at Agricultural Research Station, Gangavathi. The details of the material used and methods followed are described below. The genetic diversity of N. lugens populations collected from major rice-growing regions of South India-Karnataka (Gangavathi, Mandya, Mugad and Bramhavara) Andhra Pradesh (Maruteru), Telangana (Hyderabad), Tamil Nadu (Coimbatore) and Kerala (Pattambi) (figure 1) was characterized using mitochondrial cytochrome oxidase subunit I (mtCOI) markers (Folmer et al., 1994). Genomic DNA was extracted from pooled samples of ten female adults per location using the CTAB method (Saghai-Maroof et al., 1984). The universal barcode primer used in the present study (LCO-1490-5 / - GGT CAA CAA ATC ATA AAG ATA TTG G-3 / ; HCO-2198 -5 / TAA ACT TCA GGG TGA CCA AAA AAT CA -3 / ) has been described by Folmer et al. specific to COI. The PCR amplification of the mtCOI gene was performed in a 50 µl reaction mixture containing 25 µl 2X Taq Mix, 5 µl template DNA, 2.5 µl each of forward and reverse primers and sterile water. Thermal cycling was performed with the following condition: pre denaturation at 95 °C for 3 min followed by 35 cycles of 30 seconds at 95 °C (denaturation), 15 seconds at 54 °C (annealing) and 30 seconds at 72 °C (extension), followed by a final extension at 72 °C for 7 min. Amplified PCR products were visualized on 1% TAE-agarose gel electrophoresis using ethidium bromide under UV light (figure 2). PCR products were sequenced bidirectionally at Eurofins Genomics (Bangalore). Sequences were assembled using BioEdit 7.2 and validated through BLAST. To verify the authenticity of the COI sequences obtained from this study, total 18 sequences were used in the diversity analysis (eight sequences of mitochondrial COI gene of N. lugens generated in this study and remaining ten sequences were retrieved from NCBI, including sequences of N. lugens from different localities of India, Indonesia, Thailand). they were cross-checked with the established reference sequences of GenBank. The sequences were analysed carefully and submitted to NCBI (Table 1) and GenBank for accession numbers. Genetic diversity parameters (haplotype and nucleotide diversity, polymorphic sites, Tajima’s D and Fu’s Fs) were analyzed using DnaSP 6 and phylogenetic relationships were inferred in MEGA 11 using the Maximum Likelihood method. 3. Results Adults of N. lugens were collected from eight different localities of southern states of India. Totally 18 sequences of N. lugens were used for the analysis, The mean total nucleotide composition in the sequences was found to be A 34.7%, T 32.9%, G 16.5% and C 15.9%. Amplification of the mtCOI gene produced a consistent amplicon length of approximately 710 base pairs (Fig. 2). BLAST analysis confirmed 99-100 per cent similarity of N. lugens sequences with previously reported sequences from India, Thailand and Indonesia, validating species identity. GenBank accession numbers for both newly obtained and reference sequences are presented in Table 1. Descriptive analysis of genetic diversity based on mtCOI sequences revealed eight distinct haplotypes among the eight populations studied. The haplotype diversity (h) within southern India was 1.0, higher than the overall Indian population average (0.7606), indicating greater intra-regional variability. The average number of nucleotide differences (k) was 320.29 and the nucleotide diversity (π) was 0.69, both reflecting moderate to high genetic diversity among the southern populations (Table 2). High haplotype and nucleotide diversity in southern Indian N. lugens populations suggest the existence of multiple haplotypes and potential demographic expansion from smaller ancestral populations. A total of eight unique haplotypes were identified across all regions. Haplotype 1 was shared across 10 populations, whereas haplotypes 2-9 were population-specific: Gangavathi (Haplotype 2), Mandya (Haplotype 3), Mugad (Haplotype 4), Bramhavara (Haplotype 5), Coimbatore (Haplotype 6), Pattambi (Haplotype 7), Maruteru (Haplotype 8) and Hyderabad (Haplotype 9). These results highlight the genetic differentiation of N. lugens across geographically distinct southern states of India. (Table 3). Neutrality tests using Tajima’s D and Fu’s Fs were employed to infer population dynamics. The Tajima’s D value for mtCOI was –0.77955 (non-significant), while the overall Tajima’s D was -0.68886, both negative, indicating an excess of low-frequency polymorphisms, consistent with population expansion or purifying selection. The positive Fu’s Fs value (6.956) for mtCOI suggests a deficiency of alleles, possibly resulting from a recent population bottleneck. Conversely, a positive overall Fu’s Fs value (10.231) across all populations suggests a smaller number of alleles consistent with a recent expansion or genetic hitchhiking (Table 4). Phylogenetic analysis of mtCOI sequences was conducted using the Neighbor-Joining method based on the Maximum Composite Likelihood model.The tree was constructed using eight sequences from the present study and ten reference sequences retrieved from NCBI. The N. lugens populations clustered into four major clades, indicating genetic differentiation among regions.Clade I included populations from Pune, Cuttack, Pantnagar, Hyderabad, Tirupati and international locations such as Indonesia, Thailand, New Delhi, Bengaluru, Mahabubnagar and Gangavathi, suggesting strong genetic similarity and possible gene flow among these regions.Clade II consisted of Mandya and Mugad populations from Karnataka, forming a distinct branch with moderate bootstrap support.Clade III grouped Bramhavara (Karnataka) and Coimbatore (Tamil Nadu), indicating a closer genetic relationship between these geographically adjacent regions.Clade IV encompassed Maruteru (Andhra Pradesh), Pattambi (Kerala) and Hyderabad (Telangana), forming a separate genetic cluster, suggesting regional differentiation. (Fig. 3). The phylogenetic structure indicates that N. lugens populations in southern India are genetically diverse and exhibit regional differentiation while maintaining genetic connections with populations from Southeast Asia. The presence of unique haplotypes and high nucleotide diversity in some populations (e.g., Gangavathi and Coimbatore) suggest adaptation to local ecological conditions and possibly independent evolutionary trajectories. The mixing of Indian and foreign haplotypes reflects the migratory ability of N. lugens and the potential role of wind-aided long-distance dispersal. 4. Discussion The genetic variation in N. lugens populations based on 18 sequences of the COI gene in the present study demonstrates that southern Indian N. lugens populations harbor significant genetic variation, contributing to their adaptability and potential resistance evolution. The present study was indicated that, high haplotype and nucleotide diversity in southern parts of India with rapid demographic expansion of the pest from a small effective population size. Similar report were observed in North Indian cotton leafhopper with higher haplotype diversity and lower nucleotide diversity (Kranthi et al ., 2018). The COI sequence of higher haplotype diversity and lower nucleotide diversity in Chlorops oryzae populations from China (Li et al ., 2022). Chang et al . (2016) examined eight populations of Leucinodes orbonalis from the Philippines revealed both low haplotype and nucleotide diversities, which suggests a recent colonization or founder effect. On other hand, the negative values of Tajima’s D and positive value Fu’s Fs test indicate that there is an excess of rare mutations (excess of rare polymorphism) in the populations. These observations were noted in N. lugens from India (Srinivasa et al. , 2020), North Indian cotton leafhopper (Kranthi et al ., 2018) and Whitefly populations from Pakistan (Ashfaq et al ., 2014). The neutrality tests for the seven of populations gave negative Tajima’s D value which is statistically non-significant indicating low-frequency polymorphism among these populations (Chatterjee et al ., 2019). Whereas, the phylogenetic tree constructed based on populations of N. lugens of southern parts of India and other retrieved from GenBank were mixed in 4 different clades could be due to migratory nature of N. lugens. It has been previously observed that sufficient gene flow among populations of the same species could slow down or prevent geographic differentiation and result in small population size over larger areas, as commonly observed in migratory insects like, N. lugens from India (Srinivasa et al. , 2020) or good dispensers such as monarch butterflies, Danaus plexippus (Brower and Boyce, 1991), bumblebee, Bombus terrestris (Estoup et al ., 1996), dragonflies, Anax junius (Freeland et al ., 2003) and cotton leafhopper, A. biguttula biguttula (Akmal et al ., 2018). The current research implies that genetic diversity was preserved among the populations in south India with genetic heterogeneity since N. lugens is a migratory species. This degree of heterogeneity makes it impossible to determine the geographic origin of N. lugens migration to South India. Future research using additional molecular markers, however, might be able to identify the pest's migration source. In order to develop regional pest management methods for the reduction of N. lugens , more research is necessary to understand its migratory track and offseason survival in India. The findings suggest strong genetic diversity, local adaptation and possible long-distance migration, emphasizing the need for continued monitoring to inform pest management strategies. Declarations Acknowledgements: We thank the University of Agricultural Sciences, Raichur, for providing the facilities required to conduct this experiment. We are also grateful to the Division of Rice Entomology and Pathology, AICRP on Rice, Agricultural Research Station (ARS), Gangavathi, for providing laboratory facilities. Funding : No funding. Declarations Conflict of interest : The authors declare no conflict of interest Author Contribution : All authors contributed to the study conception and design. Madhukumar conducted experiment. Sujay Hurali and Netra helped in manuscript writing and data analysis. Baradiprasad, Chinna Babu and Mahantashivayogayya helped in analysis and manuscript correction. All authors approved the manuscript. Data Availability The data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Akmal M, Freed S, Dietrich CH, Mehmood M, Razaq M (2018) Patterns of genetic differentiation among populations of Amrasca biguttula biguttula (Shiraki) (Cicadellidae: Hemiptera). Mitochondrial DNA A , 2018, 29(6), 897–904 Ashfaq M, Hebert PD, Mirza MS, Khan AM, Mansoor S, Shah GS, Zafar Y (2014) DNA barcoding of Bemisia tabaci complex (Hemiptera: Aleyrodidae) reveals southerly expansion of the dominant whitefly species on cotton in Pakistan. PlOS one. , 2014, 9(8), 104–485 Brower AVZ, Boyce TM (1991) Mitochondrial DNA variation in monarch butterflies. Evol. , 1991, 45, 1281–1286 Chang JC, Ponnath DW, Ramasamy S (2016) Phylogeographical structure in mitochondrial DNA of eggplant fruit and shoot borer, Leucinodes orbonalis Guenée (Lepidoptera: Crambidae) in South and Southeast Asia. Mitochondr. DNA Part A , 2016, 27(1), 198–204 Chatterjee M, Yadav J, Vennila S, Shashank PR, Jaiswal N, Sreevathsa R, Rao U (2019) Diversity analysis reveals genetic homogeneity among Indian populations of legume pod borer, Maruca vitrata (F.). Biotech. , 2019, 9(9), 319 Estoup A, Solignac M, Cornuet JM, Goudet J, Scholl A (1996) Genetic differentiation of continental and island populations of Bombus terrestris (Hymenoptera: Apidae) in Europe. Mol Ecol 5(4):19–31 Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplifiation of mitochondrial cytochrome c oxidase subunit 1 from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3(2):294–299 Freeland JR, May M, Lodge R, Conrad K (2003) F.,Genetic diversity and widespread haplotypes in a migratory dragonfly, the common green darner Anax junius . Ecol Entomol 28:413–421 Kranthi S, Ghodke AB, Puttuswamy RK, Mandle M, Nandanwar R, Satija U, Pareek RK, Desai H, Udikeri SS, Balakrishna DJ, Hugar BM (2018) Mitochondria COI-based genetic diversity of the cotton leafhopper Amrasca biguttula biguttula (Ishida) populations from India. Mitochondr DNA Part A 29(2):228–235 Liu JN, Gui FR, Li ZY (2010) Genetic diversity of the planthopper, Sogatella furcifera in the greater mekong subregion detected by inter-simple sequence repeats (ISSR) markers. J Insect Sci 10(1):52 Li X, Wu S, Xu Y, Liu Y, Wang J (2022) Population genetic structure of Chlorops oryzae ( Diptera, Chloropidae ) in China. Insects 13(4):327 Srinivasa N, Chander S, Chandel RK (2020) Genetic homogeneity in brown planthopper, Nilaparvata lugens as revealed from mitochondrial cytochrome oxidase I. Curr Sci 119(6):1045–1050 Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard R (1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location and population dynamics. Proc. Natl. Acad. Sci. , 81(24), 8014–8018 Wang Y, Zhou DR, Wang XY, Zhang L, Li YH (2015) Genetic differentiation and population structure of Nilaparvata lugens in East Asia inferred from mitochondrial COI gene sequences. J Asia-Pac Entomol 18(4):789–794 Tables Tables 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Tables.docx Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 06 May, 2026 Reviews received at journal 10 Apr, 2026 Reviewers agreed at journal 07 Apr, 2026 Reviewers invited by journal 06 Apr, 2026 Editor assigned by journal 29 Jan, 2026 Submission checks completed at journal 29 Jan, 2026 First submitted to journal 24 Jan, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8685733","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":620595555,"identity":"4c1fafe2-a3b5-4e5d-b52a-320acc817919","order_by":0,"name":"Madhukumar H","email":"","orcid":"","institution":"University of Agricultural Sciences Raichur","correspondingAuthor":false,"prefix":"","firstName":"Madhukumar","middleName":"","lastName":"H","suffix":""},{"id":620595558,"identity":"b29aff39-e1ba-44eb-91a8-11a5236909d5","order_by":1,"name":"Dr. Sujay 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collections of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eN. lugens\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e for genetic diversity studies\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-8685733/v1/cb6ca5aa4921b1fe85e45737.png"},{"id":106961178,"identity":"266c6f71-f44a-4cdf-a0e4-d70741fef9af","added_by":"auto","created_at":"2026-04-15 09:24:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":5713,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eN. lugens \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edetection based on the fragment (~710 bp) of the amplified mtCOI\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDNA using agarose gel electrophoresis\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"placeholderimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8685733/v1/f7510fbcbfd53df4e408f61f.png"},{"id":106901390,"identity":"bbefa0e4-6297-4ed3-9d94-73b3d43668da","added_by":"auto","created_at":"2026-04-14 14:59:39","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":91028,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular phylogenetic analysis of mtCOI gene inferred by using neighbour- joining method based on maximum composite likelihood method\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-8685733/v1/9738bd3d02c89373526e4944.png"},{"id":109067364,"identity":"560d31d2-ef5c-4c96-a0ba-2d06175c35a7","added_by":"auto","created_at":"2026-05-12 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Introduction","content":"\u003cp\u003eThe brown planthopper (BPH), \u003cem\u003eNilaparvata lugens\u003c/em\u003e, is a major pest of rice in many Asian countries. It damages plants directly by sucking phloem sap and indirectly by transmitting rice ragged stunt and grassy stunt viruses. Once a minor pest, BPH became a serious threat to rice production during the 1970s. Both nymphs and adults infest rice at all growth stages, causing yellowing, drying, and hopper burn. Severe infestations result in lodging and yield losses ranging from 10\u0026ndash;70%. \u003cem\u003eN. lugens\u003c/em\u003e has developed resistance to important groups of insecticides, including organophosphates and neonicotinoids, particularly imidacloprid and has shown distinct virulence reactions to some rice varieties. Mitochondrial cytochrome oxidase I (COI) is a widely used marker for understanding population structure, species identification and phylogenetic relationships of insects because of its rapid evolutionary rate, maternal inheritance and lack of introns. Mitochondrial genes have served as molecular markers in the insect\u0026rsquo;s population genetic studies (Liu \u003cem\u003eet al\u003c/em\u003e., 2010).\u003c/p\u003e \u003cp\u003eIn this study we examined genetic diversity among \u003cem\u003eN. lugens\u003c/em\u003e populations that were collected from eight different geographical regions of South India, along with ten sequences retrieved from NCBI, including samples from India, Indonesia Thailand. Understanding this pest genetic diversity is essential for developing sustainable pest management strategies, including breeding for durable resistance and predicting the emergence of virulent biotypes. As climate change and agricultural practices continue to influence pest dynamics, continued research into the genetic makeup of the brown planthopper remains crucial for securing global rice production and food security (Wang \u003cem\u003eet al\u003c/em\u003e., 2015). Therefore, the present study was undertaken to evaluate the molecular genetic structure and diversity of \u003cem\u003eN. lugens\u003c/em\u003e, which is essential in designing effective management strategies for its suppression.\u003c/p\u003e"},{"header":"2. Material and method","content":"\u003cp\u003ePresent investigation on genetic diversity of different \u003cem\u003eN. lugens\u003c/em\u003e population of Southern states of India were carried out during 2024-25 at Agricultural Research Station, Gangavathi. The details of the material used and methods followed are described below.\u0026nbsp;The genetic diversity of \u003cem\u003eN. lugens\u003c/em\u003e populations collected from major rice-growing regions of South India-Karnataka (Gangavathi, Mandya, Mugad and Bramhavara) Andhra Pradesh (Maruteru), Telangana (Hyderabad), Tamil Nadu (Coimbatore) and Kerala (Pattambi) (figure 1) was characterized using mitochondrial cytochrome oxidase subunit I (mtCOI) markers (Folmer \u003cem\u003eet al.,\u003c/em\u003e 1994). Genomic DNA was extracted from pooled samples of ten female adults per location using the CTAB method (Saghai-Maroof \u003cem\u003eet al.,\u003c/em\u003e 1984). The universal barcode primer used in the present study (LCO-1490-5\u003cstrong\u003e\u003csup\u003e/\u003c/sup\u003e\u003c/strong\u003e- GGT CAA CAA ATC ATA AAG ATA TTG G-3\u003csup\u003e/\u003c/sup\u003e; HCO-2198 -5\u003csup\u003e/\u0026nbsp;\u003c/sup\u003eTAA ACT TCA GGG TGA CCA AAA AAT CA -3\u003csup\u003e/\u003c/sup\u003e) has been described by Folmer et al. specific to COI. The PCR amplification of the mtCOI gene was performed in a 50 µl reaction mixture containing 25 µl 2X Taq Mix, 5 µl template DNA, 2.5 µl each of forward and reverse primers and sterile water. Thermal cycling was performed with the following condition: pre denaturation at 95 °C for 3 min followed by 35 cycles of 30 seconds at 95 °C (denaturation), 15 seconds at 54 °C (annealing) and 30 seconds at 72 °C (extension), followed by a final extension at 72 °C for 7 min.\u003c/p\u003e\n\u003cp\u003eAmplified PCR products were visualized on 1% TAE-agarose gel electrophoresis using ethidium bromide under UV light (figure 2).\u0026nbsp;PCR products were sequenced bidirectionally at Eurofins Genomics (Bangalore). Sequences were assembled using BioEdit 7.2 and validated through BLAST. To verify the authenticity\u0026nbsp;of\u0026nbsp;the\u0026nbsp;COI\u0026nbsp;sequences\u0026nbsp;obtained\u0026nbsp;from\u0026nbsp;this study, total 18 sequences were used in the diversity analysis (eight sequences of mitochondrial COI gene of \u003cem\u003eN. lugens\u003c/em\u003e generated in this study and remaining ten sequences were retrieved from NCBI, including sequences of \u003cem\u003eN. lugens\u003c/em\u003e from different localities of India, Indonesia, Thailand). they were cross-checked with the established reference sequences of GenBank. The sequences were analysed carefully and submitted to NCBI (Table 1) and GenBank for accession numbers. Genetic diversity parameters (haplotype and nucleotide diversity, polymorphic sites, Tajima’s D and Fu’s Fs) were analyzed using DnaSP 6 and phylogenetic relationships were inferred in MEGA 11 using the Maximum Likelihood method.\u003c/p\u003e"},{"header":"3. Results","content":"\u003cp\u003eAdults of \u003cem\u003eN. lugens\u0026nbsp;\u003c/em\u003ewere collected from eight different localities of southern states of India. Totally 18 sequences of \u003cem\u003eN. lugens\u0026nbsp;\u003c/em\u003ewere used for the analysis, The mean total nucleotide\u0026nbsp;composition\u0026nbsp;in\u0026nbsp;the\u0026nbsp;sequences\u0026nbsp;was\u0026nbsp;found\u0026nbsp;to\u0026nbsp;be A 34.7%, T 32.9%, G 16.5% and C 15.9%. Amplification of the mtCOI gene produced a consistent amplicon length of approximately 710 base pairs (Fig. 2). BLAST analysis confirmed 99-100 per cent similarity of \u003cem\u003eN. lugens\u003c/em\u003e sequences with previously reported sequences from India, Thailand and Indonesia, validating species identity. GenBank accession numbers for both newly obtained and reference sequences are presented in Table 1.\u003c/p\u003e\n\u003cp\u003eDescriptive analysis of genetic diversity based on mtCOI sequences revealed eight distinct haplotypes among the eight populations studied. The haplotype diversity (h) within southern India was 1.0, higher than the overall Indian population average (0.7606), indicating greater intra-regional variability. The average number of nucleotide differences (k) was 320.29 and the nucleotide diversity (π) was 0.69, both reflecting moderate to high genetic diversity among the southern populations (Table 2). High haplotype and nucleotide diversity in southern Indian \u003cem\u003eN. lugens\u003c/em\u003e populations suggest the existence of multiple haplotypes and potential demographic expansion from smaller ancestral populations.\u003c/p\u003e\n\u003cp\u003eA total of eight unique haplotypes were identified across all regions. Haplotype 1 was shared across 10 populations, whereas haplotypes 2-9 were population-specific: Gangavathi (Haplotype 2), Mandya (Haplotype 3), Mugad (Haplotype 4), Bramhavara (Haplotype 5), Coimbatore (Haplotype 6), Pattambi (Haplotype 7), Maruteru (Haplotype 8) and Hyderabad (Haplotype 9). These results highlight the genetic differentiation of \u003cem\u003eN. lugens\u003c/em\u003e across geographically distinct southern states of India. (Table 3).\u003c/p\u003e\n\u003cp\u003eNeutrality tests using Tajima’s D and Fu’s Fs were employed to infer population dynamics. The Tajima’s D value for mtCOI was –0.77955 (non-significant), while the overall Tajima’s D was -0.68886, both negative, indicating an excess of low-frequency polymorphisms, consistent with population expansion or purifying selection. The positive Fu’s Fs value (6.956) for mtCOI suggests a deficiency of alleles, possibly resulting from a recent population bottleneck. Conversely, a positive overall Fu’s Fs value (10.231) across all populations suggests a smaller number of alleles consistent with a recent expansion or genetic hitchhiking (Table 4).\u003c/p\u003e\n\u003cp\u003ePhylogenetic analysis of mtCOI sequences was conducted using the Neighbor-Joining method based on the Maximum Composite Likelihood model.The tree was constructed using eight sequences from the present study and ten reference sequences retrieved from NCBI. The \u003cem\u003eN. lugens\u003c/em\u003e populations clustered into four major clades, indicating genetic differentiation among regions.Clade I included populations from Pune, Cuttack, Pantnagar, Hyderabad, Tirupati and international locations such as Indonesia, Thailand, New Delhi, Bengaluru, Mahabubnagar and Gangavathi, suggesting strong genetic similarity and possible gene flow among these regions.Clade II consisted of Mandya and Mugad populations from Karnataka, forming a distinct branch with moderate bootstrap support.Clade III grouped Bramhavara (Karnataka) and Coimbatore (Tamil Nadu), indicating a closer genetic relationship between these geographically adjacent regions.Clade IV encompassed Maruteru (Andhra Pradesh), Pattambi (Kerala) and Hyderabad (Telangana), forming a separate genetic cluster, suggesting regional differentiation. (Fig. 3).\u003c/p\u003e\n\u003cp\u003eThe phylogenetic structure indicates that \u003cem\u003eN. lugens\u003c/em\u003e populations in southern India are genetically diverse and exhibit regional differentiation while maintaining genetic connections with populations from Southeast Asia. The presence of unique haplotypes and high nucleotide diversity in some populations (e.g., Gangavathi and Coimbatore) suggest adaptation to local ecological conditions and possibly independent evolutionary trajectories. The mixing of Indian and foreign haplotypes reflects the migratory ability of \u003cem\u003eN. lugens\u003c/em\u003e and the potential role of wind-aided long-distance dispersal.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe genetic variation in \u003cem\u003eN. lugens\u003c/em\u003e populations based on 18 sequences of the COI gene in the present study demonstrates that southern Indian \u003cem\u003eN. lugens\u003c/em\u003e populations harbor significant genetic variation, contributing to their adaptability and potential resistance evolution. The present study was indicated that, high haplotype and nucleotide diversity in southern parts of India with rapid demographic expansion of the pest from a small effective population size. Similar report were observed in North Indian cotton leafhopper with higher haplotype diversity and lower nucleotide diversity (Kranthi \u003cem\u003eet al\u003c/em\u003e., 2018). The COI sequence of higher haplotype diversity and lower nucleotide diversity in \u003cem\u003eChlorops oryzae\u003c/em\u003e populations from China (Li \u003cem\u003eet al\u003c/em\u003e., 2022). Chang \u003cem\u003eet al\u003c/em\u003e. (2016) examined eight populations of \u003cem\u003eLeucinodes orbonalis\u003c/em\u003e from the Philippines revealed both low haplotype and nucleotide diversities, which suggests a recent colonization or founder effect. On other hand, the negative values of Tajima\u0026rsquo;s D and positive value Fu\u0026rsquo;s Fs test indicate that there is an excess of rare mutations (excess of rare polymorphism) in the populations. These observations were noted in \u003cem\u003eN. lugens\u003c/em\u003e from India (Srinivasa \u003cem\u003eet al.\u003c/em\u003e, 2020), North Indian cotton leafhopper (Kranthi \u003cem\u003eet al\u003c/em\u003e., 2018) and Whitefly populations from Pakistan (Ashfaq \u003cem\u003eet al\u003c/em\u003e., 2014). The neutrality tests for the seven of populations gave negative Tajima\u0026rsquo;s D value which is statistically non-significant indicating low-frequency polymorphism among these populations (Chatterjee \u003cem\u003eet al\u003c/em\u003e., 2019).\u003c/p\u003e \u003cp\u003eWhereas, the phylogenetic tree constructed based on populations of \u003cem\u003eN. lugens\u003c/em\u003e of southern parts of India and other retrieved from GenBank were mixed in 4 different clades could be due to migratory nature of \u003cem\u003eN. lugens.\u003c/em\u003e It has been previously observed that sufficient gene flow among populations of the same species could slow down or prevent geographic differentiation and result in small population size over larger areas, as commonly observed in migratory insects like, \u003cem\u003eN. lugens\u003c/em\u003e from India (Srinivasa \u003cem\u003eet al.\u003c/em\u003e, 2020) or good dispensers such as monarch butterflies, \u003cem\u003eDanaus plexippus\u003c/em\u003e (Brower and Boyce, 1991), bumblebee, \u003cem\u003eBombus terrestris\u003c/em\u003e (Estoup \u003cem\u003eet al\u003c/em\u003e., 1996), dragonflies, \u003cem\u003eAnax junius\u003c/em\u003e (Freeland \u003cem\u003eet al\u003c/em\u003e., 2003) and cotton leafhopper, \u003cem\u003eA. biguttula biguttula\u003c/em\u003e (Akmal \u003cem\u003eet al\u003c/em\u003e., 2018).\u003c/p\u003e \u003cp\u003eThe current research implies that genetic diversity was preserved among the populations in south India with genetic heterogeneity since \u003cem\u003eN. lugens\u003c/em\u003e is a migratory species. This degree of heterogeneity makes it impossible to determine the geographic origin of \u003cem\u003eN. lugens\u003c/em\u003e migration to South India. Future research using additional molecular markers, however, might be able to identify the pest's migration source. In order to develop regional pest management methods for the reduction of \u003cem\u003eN. lugens\u003c/em\u003e, more research is necessary to understand its migratory track and offseason survival in India. The findings suggest strong genetic diversity, local adaptation and possible long-distance migration, emphasizing the need for continued monitoring to inform pest management strategies.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements:\u003c/strong\u003e We thank the University of Agricultural Sciences, Raichur, for providing the facilities required to conduct this experiment. We are also grateful to the Division of Rice Entomology and Pathology, AICRP on Rice, Agricultural Research Station (ARS), Gangavathi, for providing laboratory facilities.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: No funding.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclarations Conflict of interest\u003c/strong\u003e: The authors declare no conflict of interest\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e: All authors contributed to the study conception and design. Madhukumar conducted experiment. Sujay Hurali and Netra helped in manuscript writing and data analysis. Baradiprasad, Chinna Babu and Mahantashivayogayya helped in analysis and manuscript correction. All authors approved the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAkmal M, Freed S, Dietrich CH, Mehmood M, Razaq M (2018) Patterns of genetic differentiation among populations of \u003cem\u003eAmrasca biguttula biguttula\u003c/em\u003e (Shiraki) (Cicadellidae: Hemiptera). \u003cem\u003eMitochondrial DNA A\u003c/em\u003e, 2018, 29(6), 897\u0026ndash;904\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAshfaq M, Hebert PD, Mirza MS, Khan AM, Mansoor S, Shah GS, Zafar Y (2014) DNA barcoding of \u003cem\u003eBemisia tabaci\u003c/em\u003e complex (Hemiptera: Aleyrodidae) reveals southerly expansion of the dominant whitefly species on cotton in Pakistan. \u003cem\u003ePlOS one.\u003c/em\u003e, 2014, 9(8), 104\u0026ndash;485\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrower AVZ, Boyce TM (1991) Mitochondrial DNA variation in monarch butterflies. \u003cem\u003eEvol.\u003c/em\u003e, 1991, 45, 1281\u0026ndash;1286\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang JC, Ponnath DW, Ramasamy S (2016) Phylogeographical structure in mitochondrial DNA of eggplant fruit and shoot borer, \u003cem\u003eLeucinodes orbonalis\u003c/em\u003e Guen\u0026eacute;e (Lepidoptera: Crambidae) in South and Southeast Asia. \u003cem\u003eMitochondr. DNA Part A\u003c/em\u003e, 2016, 27(1), 198\u0026ndash;204\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChatterjee M, Yadav J, Vennila S, Shashank PR, Jaiswal N, Sreevathsa R, Rao U (2019) Diversity analysis reveals genetic homogeneity among Indian populations of legume pod borer, \u003cem\u003eMaruca vitrata\u003c/em\u003e (F.). \u003cem\u003eBiotech.\u003c/em\u003e, 2019, 9(9), 319\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEstoup A, Solignac M, Cornuet JM, Goudet J, Scholl A (1996) Genetic differentiation of continental and island populations of \u003cem\u003eBombus terrestris\u003c/em\u003e (Hymenoptera: Apidae) in Europe. Mol Ecol 5(4):19\u0026ndash;31\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFolmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplifiation of mitochondrial cytochrome c oxidase subunit 1 from diverse metazoan invertebrates. Mol Mar Biol Biotechnol 3(2):294\u0026ndash;299\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFreeland JR, May M, Lodge R, Conrad K (2003) F.,Genetic diversity and widespread haplotypes in a migratory dragonfly, the common green darner \u003cem\u003eAnax junius\u003c/em\u003e. Ecol Entomol 28:413\u0026ndash;421\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKranthi S, Ghodke AB, Puttuswamy RK, Mandle M, Nandanwar R, Satija U, Pareek RK, Desai H, Udikeri SS, Balakrishna DJ, Hugar BM (2018) Mitochondria COI-based genetic diversity of the cotton leafhopper \u003cem\u003eAmrasca biguttula biguttula\u003c/em\u003e (Ishida) populations from India. Mitochondr DNA Part A 29(2):228\u0026ndash;235\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu JN, Gui FR, Li ZY (2010) Genetic diversity of the planthopper, \u003cem\u003eSogatella furcifera\u003c/em\u003e in the greater mekong subregion detected by inter-simple sequence repeats (ISSR) markers. J Insect Sci 10(1):52\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi X, Wu S, Xu Y, Liu Y, Wang J (2022) Population genetic structure of \u003cem\u003eChlorops oryzae (\u003c/em\u003eDiptera, Chloropidae\u003cem\u003e) in\u003c/em\u003e China. Insects 13(4):327\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSrinivasa N, Chander S, Chandel RK (2020) Genetic homogeneity in brown planthopper, \u003cem\u003eNilaparvata lugens\u003c/em\u003e as revealed from mitochondrial cytochrome oxidase I. Curr Sci 119(6):1045\u0026ndash;1050\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaghai-Maroof MA, Soliman KM, Jorgensen RA, Allard R (1984) Ribosomal DNA spacer-length polymorphisms in barley: Mendelian inheritance, chromosomal location and population dynamics. \u003cem\u003eProc. Natl. Acad. Sci.\u003c/em\u003e, 81(24), 8014\u0026ndash;8018\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang Y, Zhou DR, Wang XY, Zhang L, Li YH (2015) Genetic differentiation and population structure of \u003cem\u003eNilaparvata lugens\u003c/em\u003e in East Asia inferred from mitochondrial COI gene sequences. J Asia-Pac Entomol 18(4):789\u0026ndash;794\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTables 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-tropical-insect-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtis","sideBox":"Learn more about [International Journal of Tropical Insect Science](http://link.springer.com/journal/42690)","snPcode":"42690","submissionUrl":"https://www.editorialmanager.com/jtis/default2.aspx","title":"International Journal of Tropical Insect Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Genetic diversity, Haplotype, Nilaparvata lugens, Population","lastPublishedDoi":"10.21203/rs.3.rs-8685733/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8685733/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe brown planthopper \u003cem\u003eNilaparvata lugens\u003c/em\u003e (Stal) is a major pest of rice, causes high economic losses by reducing yield. In India, most populations of \u003cem\u003eN. lugens\u003c/em\u003e could not be managed probably due to high genetic variation in populations. Hence, the study was conducted to investigate the genetic diversity of \u003cem\u003eN. lugens\u003c/em\u003e in south Indian populations. A total of eight populations were collected from Karnataka, Telangana, Andhra Pradesh, Tamil Nadu and Kerala. DNA extraction, PCR amplification and sequencing done using the \u003cem\u003eCOI\u003c/em\u003e gene primer. The study identified eight haplotypes having high haplotype diversity and nucleotide diversity along with two polymorphic sites and two mutations. Neutrality tests (Tajima\u0026rsquo;s D, Fu\u0026rsquo;s Fs) indicated no significant deviation from neutrality. The high genetic diversity highlights the adaptive potential of \u003cem\u003eN. lugens\u003c/em\u003e to environmental shifts, host resistance and management practices. Such adaptability makes \u003cem\u003eN. lugens\u003c/em\u003e a serious threat to rice cultivation, as it can overcome existing control measures. The findings emphasize the importance of continuous molecular monitoring to track evolutionary dynamics in developing effective and sustainable pest management strategies.\u003c/p\u003e","manuscriptTitle":"Genetic Structure and haplotype diversity in South Indian Populations of Brown Planthopper, (Nilaparvata lugens) using mtCOI","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-04-14 14:59:35","doi":"10.21203/rs.3.rs-8685733/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-05-06T08:10:28+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-10T08:38:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"170757642907061044349043410756263323657","date":"2026-04-07T06:28:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-06T15:56:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-29T08:27:33+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-01-29T08:24:38+00:00","index":"","fulltext":""},{"type":"submitted","content":"International Journal of Tropical Insect Science","date":"2026-01-24T09:58:43+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"international-journal-of-tropical-insect-science","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jtis","sideBox":"Learn more about [International Journal of Tropical Insect Science](http://link.springer.com/journal/42690)","snPcode":"42690","submissionUrl":"https://www.editorialmanager.com/jtis/default2.aspx","title":"International Journal of Tropical Insect Science","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"0842ba32-a604-4378-9c3c-9d8769adcfad","owner":[],"postedDate":"April 14th, 2026","published":true,"recentEditorialEvents":[{"type":"decision","content":"Revision requested","date":"2026-05-06T08:10:28+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[],"tags":[],"updatedAt":"2026-05-06T08:26:37+00:00","versionOfRecord":[],"versionCreatedAt":"2026-04-14 14:59:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8685733","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8685733","identity":"rs-8685733","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}