{'fraction_non_alphanumeric': 0.02898886827458256, 'fraction_numerical': 0.01893939393939394, 'mean_word_length': 4.8538461538461535, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Genetic homogeneity in brown planthopper , Nilaparvata lugens ( Stål ) as revealed from mitochondrial cytochrome oxidase

CURRENT SCIENCE, VOL. 119, NO. 6, 25 SEPTEMBER 202

households cannot afford it and hence, they continue to remain dependent on dry wood collected from the sanctuary.
In conclusion, we strongly recommend the plantation of fast-growing firewood species, such as Dalbergia sissoo, Acacia auriculiformis, Gmelina arborea and Ailanthus excelsa in the villages adjoining the sanctuary and along the boundary of the tea plantations to meet the fuelwood needs of the people. Additionally, the use of alternative fuel sources, such as LPG, should be encouraged under government schemes, such as the Pradhan Mantri Ujjwala Yojana. Previous attempts in this direction have, however, failed because of the significant cost involved in refilling gas cylinders. Finally, appropriate alternative livelihood options need to be urgently considered for those involved in the commercial harvest of NTFP. An example of such options includes the training and recruitment of members of the local communities as forest guides by government agencies and nongovernmental organizations, as the sanctuary attracts a significant number of wildlife tourists every year. These individuals can also regularly be employed to monitor the flora and fauna of the sanctuary, important indicators of the health of this unique, though increasingly threatened, lowland rainforest patch of northeastern India.
Brown planthopper, Nilaparvata lugens (Stål) is a seasonal migrant pest in North India. The present study analyses the genetic diversity of N. lugens by employing a partial fragment of the mitochondrial gene encoding cytochrome oxidase I (COI) using samples from 16 different localities of India. Total of 16 fulllength COI gene sequences generated from this study with 16 COI gene sequences retrieved from GenBank were analysed for genetic differentiation and haplotypes of N. lugens populations in order to determine the genetic structure. Based on the partial COI gene, high genetic homogeneity was detected in N. lugens populations of India and they form a single genetic group. The Tajima's D test and Fu's F test also support our result, and indicate recent population expansion, while the phylogenetic tree suggests that geographically distinct populations of N. lugens do not exist in India.
THE brown planthopper (BPH), Nilaparvata lugens (Stål) is one of the serious pests of rice causing an annual yield loss worth US$ 1.06 billion in Southeast Asia 1 . The major damage symptom of N. lugens in rice involves yellowing and drying of plants in patches, also known as 'hopper burn' 2 , due to phloem sapping. N. lugensis known to transmit rice virus diseases such as rice ragged stunt (RRSV) and rice grassy stunt (RGSV) in India 3,4 . Due to extremely low winter temperatures in North India and the absence of rice crop, N. lugens is not expected to survive in the North Indian states, but it infests the new rice crop every year 5 . Further, there are no alternate hosts of N. lugens in North India on which it can survive to complete its life cycle and to act as the nucleus population of the pest for infestation in the next season of rice. Migration of N. lugens during November to West Bengal 6 , and two population peaks of the pest and associated insect predators were observed in net catches placed 150 m above ground level during May and October respectively, in the state, indicating the migratory behaviour of the pest 7 . N. lugens does not overwinter in temperate Asian countries such as Korea, Japan and northern China. It annually migrates into Korea and Japan during the rainy season from northern Vietnam and southern China where rice is cultivated throughout the year. It has been reported that northern Philippines is also the source for N. lugens migration to Japan and Taiwan 8,9 . Therefore, it is possible that N. lugens must be migrating to North India from the southern or eastern parts of the country during May-July, immediately after the harvest of rabi rice crop in these regions, with the aid of southwest monsoon winds 10 . N. lugens has developed resistance to important groups of insecticides, including organophosphates and neonicotinoids, particularly imidacloprid 11 and has shown distinct virulence reactions to some rice varieties 12 . 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.
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 [13][14][15] . The population genetic structure of various species such as finless porpoises 16 , Reticlitermes 17 , Amrasca biguttula biguttula 18 , etc. has been revealed using mitochondrial DNA sequences 16,17 . In this study we examined genetic diversity among N. lugens populations that were collected from 16 different geographical regions of India, along with 16 sequences retrieved from NCBI, including samples from India, Pakistan and Bangladesh.
N. lugens populations were collected from 15 different states of India using sweep net during 2017-18 and 2018-19 rainy seasons ( Table 1). The samples were transferred to glass vials containing 70% ethanol and the date and place of collection were noted. Further, samples were studied under a microscope for identification. The N. lugens populations were stored at -20°C in the laboratory for DNA extraction and further analysis.
DNA was extracted from individual N. lugens using DNAeasy Blood and Tissue Kit (Qiagen GmbH, Germany), following the manufacture's protocol. 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. 19 specific to COI. The PCR composition consists of (per 25 μl) Taq DNA polymerase (Fermentas Inc., USA) were 2.5 μl of 10× PCR buffer with 2 μl of 25 mM MgCl 2 , 0.5 μl of 10 mM dNTPs, 0.5 μl each of forward and reverse primer, IU of Taq, and 17 μl of UltraPure water (Invitrogen, Thermo Fisher Scientific, USA). Thermal cycling was performed with the following conditions: pre-denaturation for 5 min at 94°C followed by 35 cycles of 30 sec at 94°C (denaturation), 40 sec at 47°C (annealing) and 40 sec at 72°C (extension), with a final extension for 8 min at 72°C. PCR products were visualized on 1% TAE-agarose gel electrophoresis (Figure 1). Single bands were purified using a QIAquick PCR purification kit (Qiagen GmbH, Germany). Purified PCR products were sequenced directly using an automated sequencer (ABI prism® 3730 XL DNA analyzer; Applied Biosystems, USA) at Scigenomics Lab, Cochin, India. To verify the authenticity of the COI sequences obtained from this study, they were cross-checked with the established reference sequences of GenBank. The sequences were analysed carefully and submitted to NCBI (Table 1).
All the sequences were arranged in FASTA format for analysis using MEGA 6.0 software. Totally 32 sequences were used in diversity analysis (16 sequences of mitochondrial COI gene of N. lugens generated in this study and the remaining 16 sequences were retrieved from NCBI, including sequences of N. lugens from different localities of India, Pakistan and Bangladesh). The sequences were subjected to multiple alignments using Clustal W (ref. 20). The phylogenetic neighbor-joining tree based on Kimura-2-parameter 21 distance was  1, HM160144.1). The mean total nucleotide composition in the sequences was found to be A 31%, T 36.2%, G 12.8% and C 20%. The average AT content accounted for 67.2% and GC for the rest 32.8% of the sequences. The bias towards AT content (67.2%) is a general feature of the COI region in arthropods. The maximum pairwise genetic distance was found to be 0.7%, and there existed only one segregating site. Further, test statistics were generated for the 32 COI sequences ( Table 2). A total of four haplotypes were observed with nucleotide diversity as 0.00491 and haploid diversity as 0.7319, indicating lower nucleotide diversity and higher haplotype diversity.
The phylogenetic neighbor-joining tree was constructed based on 32 sequences using MEGA 6.0 software 22 . The  phylogenetic tree had a total branch length of 0.33077592 base substitutions per site (Figure 2). The N. lugens populations from India, Bangladesh and Pakistan clustered in four different clades and are clearly depicted in the tree. There was no clustering of sequences from the same geographical region. However, samples from the same locality grouped together, except in case of New Delhi and Umiam, Meghalaya samples. Further, samples from different localities of India including the southern, eastern and northern populations, were mixed in different clades without forming clades of geographical distinction ( Figure 2). N. lugens is a serious migratory pest of rice in tropical and temperate Asia. It is widely distributed across the region: in the west from Pakistan to northeastern Australia and in the south from the Malay Archipelago to New Guinea and the Solomon Islands to central Japan. It is a serious pest of rice in India, Bangladesh, Japan, China, Philippines, South Korea, Sri Lanka, Taiwan, Indonesia, Thailand and Vietnam 26 . In India it is also migratory in nature, infesting the new rice crop in North India every year and disappearing after November due to freezing temperatures in winter. However, information on genetic diversity of this pest is lacking in India.
The genetic variation in N. lugens populations based on 32 sequences of the COI gene in the present study indicated high genetic homogeneity. The maximum genetic distance among the populations was found to be 0.7%, which is below the limit of 2% generally considered for species delineation 18,27 . The COI sequence analysis of cotton leafhopper, Amrasca biguttula biguttula collected from cotton-growing areas of India suggested the presence of single species in the country 18 . Similar reports of genetic variation of 0.3-1.2% was reported among populations of tea green leafhoppers, Empoasca onukii and Jacobiasca formosana from China, Japan and Taiwan based on 16SrDNA and COI, corroborating the hypothesis that geographically separated populations of the pest represent a single species 27 . On the other hand, high haplotype and low nucleotide diversity observed in N. lugens in India suggest rapid demographic expansion of the pest from a small effective population size; similar results were reported earlier in Bicyclus anynana 28 . The negative values of Tajima's D and Fu's F tests in the present study indicate recent population expansion that has also been observed in Bicyclus anynana 28 and in North Indian cotton leafhopper 18 . On the other hand, the phylogenetic tree did not depict any clustering of sequences based on geographic distinction such as North, North East and South Indian populations of N. lugens. However, samples from the same locality clustered together, except for a few sub-populations. The sub populations of N. lugens of India, Pakistan and Bangladesh were mixed in different clades without forming distinct clades. This result clearly indicates that N. lugens populations from different regions of India formed a single genetic group. N. lugens is known to migrate during two-seasons of the year: once in May when the southwest monsoon aids migration and another during October to November in eastern India due to harvest of rice crop 7 . This migration might be responsible for gene flow among the N. lugens populations that helped in maintaining their genetic homogeneity. 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 or good dispensers such as Monarch butterflies Danaus plexippus 29 , bumblebee Bombus terrestris 30 , dragonflies Anax junius 31 and cotton leafhopper A. biguttula biguttula 18,32 .
From the present study, it can be concluded that owing to the migratory nature of N. lugens, genetic homogeneity was maintained among the populations in India with less genetic variability. It is not possible to pinpoint the geographic source of N. lugens migration to northern India based on this level of variability. However, the future work with other molecular markers may help find the migration source of the pest. Further, studies are essential to explore the offseason survival and migratory pathway of N. lugens in India that will be helpful in designing regional pest management strategies for its suppression.