mitochondrial cytochrome oxidase I (COI) gene

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The present chapter summarizes the research reported in the previous chapters. The chapter capsules the findings of the problem studied and briefly outlines the conclusion of the study.

Rice is one of the dominant grain crops in the world, with about 80% of the world’s population dependent on it as a major source of food. In recent years, the production of rice has drastically fallen due to insect pest attack. The pest causes great damage by attacking their leaf, roots, stem and even the young grains when they are in milk stage. The successful control of any pest is based on the correct identification of distinct insect populations.

There have been several research work carried out for correct identification of distinct insects based on conventional and modern method. Many factors affect the conventional mode of identification of rice pests. These are detrimental in designing an Integrated Pest Management (IPM) strategy. Some of the species of insects pests show a high degree of similarity with the members of the same genus or other related groups particularly in immature and adult stages. Therefore the identification of insects in the immature stage using conventional method is a tedious process. To overcome this problem, the use of DNA barcoding technique as a modern method is proved to be more beneficial. The DNA barcoding when coupled with traditional taxonomic method is a valuable tool which acts as a fundamental technique in revealing hidden diversity.

The DNA barcoding has become a dominant technique for generating comparative molecular data. The DNA sequence of mtDNA has been determined for a large number of organism and individuals and comparison of those DNA sequences represents a main stay of phylogenetics, in that it allows the biologists to elucidate the evolutionary relationships among species. The mtDNA has been used in studying the evolutionary relationship among various taxa owning to its conserved protein coding regions, high variability in non-coding sequences, and lack of recombination. The sequence divergence accumulates more rapidly in mtDNA than in nuclear DNA owing to a faster mutation rate and lack of repair system, meaning that it often contains high levels of informative variation. The present study has been concerned with the molecular barcoding and phylogeny analysis of hoppers (Hemiptera: Auchenorrhyncha) associated with paddy agro ecosystem of Kerala using mtDNA cytochrome oxidase subunit I (COI)

This study was aimed to develop the mitochondrial cytochrome oxidase I (COI) gene of ten species of hoppers collected from paddy fields of different geographic locations in Kerala. The experimental insect Nilaparvata lugens, Cofana spectra, Nephotettix virescens, Reciliadorsalis, Nephotettix malayanus, Nephotettix nigropictus, Sogatella furcifera, Nisia carolinensis, Thaia subrufa and Proutista moesta were collected from paddy fields Dhoni-Palakkad district, Meppayar-Kozhikode, Ponnani -Malappuram, Kuttanad-Alappuzha, Kabani –Wayanad, Kole fields – Thrissur, Kaipad-Kannur and Pokkali-Ernakulam. The major objectives of the present work are (1) developing a mtDNA COI gene based DNA barcode for identification of hopper pests of paddy, (2) to analyse the genetic divergence within and between the species of leafhopper and planthopper pest of paddy, (3) to determine the phylogenic status of hopper pests of paddy.

To achieve these objectives the genomic DNA extraction, barcoding and phylogeny analysis were done. The collected samples were identified using conventional taxonomic method. The genomic DNA from the identified insects was isolated.

The partial coding region of mitochondrial cytochrome oxidase subunit I (COI) gene of each organism was amplified separately using different sets of forward and reverse primers. The PCR amplifications were visualized on 2.0% agarose gel using ethidium bromide staining (Fig. 71), and the images were captured using a gel documentation system. The PCR products were then column purified using GeneJET PCR purification kit (Fermentas Life Sciences) as per manufacturer’s instruction and sequenced from both ends using Sanger’s sequencing (ABI 3730XL automated sequencer) method at SciGenome Laboratories Ltd. The forward and reverse sequences were assembled using ClustralW. After removing the forward and reverse primer sequences the consensus was taken for analysis. The final sequence was searched for its sequence similarity using BLAST programme of NCBI. The phylogenetic analysis was done using MEGA6 software.

The present study using the mitochondrial COI gene of the ten species revealed the molecular phylogeny of the species of hoppers the pest of paddy.

In the species N. lugens the species within India showed variation in the composition of nucleotide. The species (GenBank accession KJ796483) isolated from Kerala differs from that of the species isolated from Karnataka (KC858992) and China (AB572318, KC476395 and KC476394).

In the species C. spectra the intra species evolutionary divergence calculated shows 1% divergence with C. spectra isolated from Australia. There is no intra species peptide divergence between C. spectra (GenBank accession KJ186109) isolated from Kerala and that from Australia region. This peptide similarity of the species of the two fauna indicates their common origin from Gondwana land.

The N. virescens, N. malayanus and N. nigropictus isolated from Kerala (GenBank accession LM994675; LN606698; KM216270) yielded a product of 647 bp, 313 bp and 559 bp respectively (Fig. 71). The NCBI BLAST tool revealed that the species N. virescens shows 93% nucleotide identities with N. virescens (Sequence ID: BIPR006-1), whereas N. malayanus shows 91% identities with N. virescens (Sequence ID: HM160144.1), and N. nigropictus isolates from Kerala shows 89% identities with that of N. virescens ( Sequence ID. HM160144.1). This inter and intra species divergences may be due to the geographical isolation of these species.

Fig. 71. Gel electropherogram indicating PCR amplified product of 10 hopper species isolated, visualised by ethidium bromide staining. Markings (a). L indicates molecular marker with sizes indicated on the left. (b) S1 - S10 indicates 10 species of hoppers.

It is interesting to note that the reference sequences from the BLAST analysis are those from temperate zones and high altitude, which are from China and India. The BLAST result revealed that there is no 100% similarities in both BLASTn and BLASTp. This revealed that the resultant sequences of N. virescens, N. nigropictus and N. malayanus are novel.

The T. subrufa species isolated from kole fields in Thrissur give a product length of 507 bp. (GenBank accession LC005702), N. carolinesis species isolated form Kuttanad, Alappuzha yielded a product of 305 bp (GenBank accession LC015451) and the whitebacked planthopper S. furcifera isolated from Dhoni, Palakkad yielding a product of 322bp (GenBank accession LC005703). The sequence homology search with the NCBI BLAST tool reveals that no sequences for the COI genes for T. subrufa, N. carolinesis and for S. furcifera genus are available in the database, and maximum homology was shown to T. subrufa is Anzygina sp. (Sequence ID: gb|KF226761.1) isolated from Barrow Island, Western Australia. The species displays an identity of 82%. The NCBI BLAST result for N. carolinesis species showed 80% identities to Caliscelidae sp. isolated from Australia whereas the species S. furcifera shows maximum identities of 84% with Nesophyla sp. (Sequence ID: emb|HF9686658.1) isolated from Berkeley, USA. This sequence result of partial cytochrome oxidase genes shows that these sequences are novel one.

NJ tree method revealed the phylogeny of P. moesta isolated from Kerala (GenBank accession LN681351). The Siphanta patruelis species showed to be nearest relative and from the same Clade of the P. moesta isolated from Kerala. The NCBI BLAST revealed with an maximum identities of 83%. The sequence isolated for P. moesta is a novel one.

Comparison of sequence obtained with that of nucleotide BLAST result of NCBI revealed that the R. dorsalis isolated from Kerala (GenBank accession LN681350) is 98% identical to Cicadellidae sp. isolated from Maharashtra (Accession No. HF968658, HF968651 and HF968655). The BLAST result of NCBI shows that the sequence is a novel one.

The overall COI phylogenetic relationships of hemiptera (Auchenorrhyncha) paddy pests of Kerala using NJ tree showed the inter and intra species divergence (Fig.72). The insect in the Cicadellidae family were aligned in a single clad and all the hopper pests originated from a common ancestor. The clades are nested together and Sogatella furcifera and Nilaparvatha lugens of the Delphacidae family forms an outgroup of the clad of the Proutista moesta. This reveals the distinction between the DNA sequence evolving randomly and one evolving under a non-random process, including directional selection, demographic expansion, and genetic introgression. The average interspecific COI divergence in N. nigropictus and N. carolinensis is found to be 0.35% and between N. nigropictus and N. lugens it is found that 1.16%. Form the data itself it can see a clear differences between the different families itself.

Nisia carolinensis in the Meenoplidae family were aligned as nearest relative of Thaia subrufa which comes under Cicadellidae family, pests of paddy. Nisia carolinensis and Thaia subrufa are the two descendents that split from the same node and are closest relatives. They orginates form a common ancestor. Proutista moesta is found to be evolved as an outgroup of the Nisia carolinensis.

Fig. 72. Phylogenetic relationship of different Auchenorrhynchan pests isolated from Kerala

The present study revealed that most of the insect paddy pests show considerable variations within related species. Therefore the COI sequence can be used as effective tool for the identification of the insect paddy pest at any stage of life. Polyphagosity of insects (insect feed on more than one species of plant) may cause sequence divergences due to adaptive radiation. The geographical isolation and time of continent separation also have an effect on the degree of sequence divergence in the population. The DNA barcode developed for paddy pests in this study can be used to identify them at any stage of their life and to evaluate genetic variations in the pest population which will help to design better strategies to develop Integrated Pest Management (IPM) system.