Mitochondrial DNA Barcoding

Abstract

Hoppers are economically important insects, causing serious damage to paddy. These insect falls under the family of Hemiptera, which include diverse groups of insects. The identification of these insects is difficult because of the morphological similarity of some species. Molecular techniques, mainly of those based on the DNA sequencing can improve the reliability of insect identification.

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The present study analyses the partial coding region of the mitochondrial cytochrome oxidase subunit I gene of ten hopper paddy pest species Nilaparvata lugens, Cofana spectra, Nephotettix virescens, Recilia dorsalis, Nephotettix malayanus, Nephotettix nigropictus, Sogatella furcifera, Nisia carolinensis, Thaia subrufa and Proutista moesta. The composition of nucleotide analysis revealed that, the composition of nucleotides and their position in each codon vary between the species. The phylogenetic analysis using NJ tree clearly revealed the phylogenetic position of the Hemipteran-Auchenorrhynchan paddy pest from Kerala isolated for this study.

The barcoding region of mitochondrial genome

The barcoding region is a gene segment within a protein-coding region of the mitochondrial genome. Protein-coding regions of DNA have specific constraints that can be useful or detrimental to this application. First of all, since a change in nucleotides will often have an effect on the amino acids and hence the protein that is produced, sequences can only experience limited changes. Fortunately, the third positions of codons are not under strong selection to remain constant because of the redundancy of the amino acid coding system. Therefore, one-third of the nucleotide sites have a higher potential to change once species diverge. Another advantage to using protein-coding regions instead of genes encoding RNA is the relative rarity of indels (Hebert et al., 2003). In protein coding genes indels are partially constrained by the necessity of avoiding frame shifts.

The points of interest of utilizing the COI for barcoding the mitochondrial genome is frequently connected with well-conserved primers and the COI gene is especially rich in highly conserved primers. Hebert et al. (2003) report that the primers have been functional with “representatives of most, if not all, animal phyla.” This gene has the added advantage of both being rapid enough (at silent sites) to differentiate between phylogeographic groups within a species and slow enough (at amino acid replacement sites) to determine deeper phylogenetic relationships (Hebert et al., 2003).

Mitchell (2008) and Rock et al. (2008) realized that recently diverged species that lack fixed differences within the barcoding sequence lead to uninformative knowledge and be problematic for any applications of barcoding.

Applications of barcoding

Species identifications are the most widely accepted of the potential applications of barcoding. A database of all COI barcoding sequences helps to makes the future specimen identification easier (Hebert et al., 2003). The upcoming databases from different areas become part of a global bio identification system (GBS), designed to help solve many of the problems associated with morphological taxonomy and help reduce misidentifications (Hebert et al., 2003). The utilization of DNA barcodes to highlight areas of customary taxonomy that ought to be revaluated has been gaining popularity in recent years (Kerr et al., 2009; Packer et al., 2009). Some controversy has also surrounded proposals to utilize DNA barcodes for identification of new species. Rubinoff (2006a) discouraged “the sole use of mtDNA to identify (discover) new species and comprehend worldwide biodiversity.” One suggested requisition for DNA barcoding is to have the capacity to dependably perceive snake venom so scientists endeavouring to design antivenoms could be certain that they have venom from the correct snake (Pook and McEwing, 2005).

The identification of the larval stage of insects that are responsible for destroying crops is a major problem. Barcodes could be used to determine which pest is plaguing a farmer since the organisms’ DNA remains constant throughout its lifetime and the adult stages are usually more easily identified. Once the type of pest is quickly identified, the farmer could proceed with treatment more rapidly and lose fewer crops (Mitchell, 2008). Phenotypic differences in the life stages of an organism are also a problem within fisheries. These important food production operations could benefit greatly from DNA barcoding (Rock et al., 2008).

The rapid identifications provided by DNA barcoding could also be beneficial for managing invasive species. This technology would be especially useful at commercial ports and national borders, where a speedy identification of taxa could result in swift action that could prevent the spread of the invasive species (Mitchell, 2008).

Phylogenetic analysis

Species identification process through barcoding requires the assignment of taxa to clusters on a tree based on neighbor-joining phylogenetic analysis (Meier et al., 2006). Will and Rubinoff (2004) criticize tree diagrams in Hebert et al. (2003) because the diagrams do not agree with any existing hypothesized phylogenies and acknowledge that they avoid using the term “phylogeny” in favor of “profile.” Another problem with the studies of Hebert et al. (2003) was their utilization of phenetics with some phylogenetic methods, meaning their results are based solely on similarities. Rubinoff (2006b) states that “barcoding is not proposed to and does not give evolutionary information about taxa; rather, it is planned almost as a system for ‘yes’ or ‘no’ identification based on predetermined units”. Notwithstanding all the issues, Hebert et al., (2003) maintain that their barcoding study was mostly successful at identifying species

Taxonomy, barcoding and integration

Taxonomy, is the science of assigning names to species and higher taxa, is crucial to other fields of science. As, the number of taxonomists is in decline and there are already insufficient numbers of specialists in this field to handle the existing workload (Rubinoff, 2006a; Hebert et al., 2003; Packer et al., 2009). Notwithstanding the huge number of taxonomists, a significant time investment is required inorder to describe all existing species if the conventional methods are applied (Meier et al., 2006; Packer et al., 2009). The necessities for an alternative system is progressively that might take a portion of the strains off from the taxonomists are increasingly, so that they can concentrate on different areas of systematics instead of performing species identifications (Will and Rubinoff, 2004; Packer et al., 2009).

Barcoding can utilize the expertise of current as well as former taxonomists, since previously identified museum specimens should be used to produce barcodes whenever possible. The barcode database will help preserve taxonomic information in a novel format and allow laboratories without morphology experts to identify relevant species (Hebert et al., 2003).

The concurrent use of mtDNA sequences in spite of morphology to resolve difficult species identifications has gained its notoriety (Mitchell, 2008; Pages et al., 2009). The usage of microgenomic identification systems for viruses, bacteria, and protists were acknowledged in the works of Lewis and Lewis 2005; Hebert et al., 2003 and Abriouel et al., 2008.

Arthropod heterogeneity in rice agro-ecosystem

Irrigated rice fields, being agronomically managed wetland ecosystems with a high degree of environmental heterogeneity operating on a short temporal scale, harbour a rich and varied fauna (Heckman, 1979). The fauna is dominated by micro, meso and macro invertebrates (especially arthropods) inhabiting the soil, water and vegetation sub-habitats of the rice fields. The terrestrial arthropod community in rice fields consists mainly of insects and spiders which largely inhabit the vegetation (rice plants and weeds), and the soil surface. The occurrence of terrestrial arthropods in the rice ecosystem is mainly influenced by the rice plants. The different communities of terrestrial arthropods in the rice field include rice pests, their natural enemies (predators and parasitoids) and other non-rice pest insects that inhabit or visit the vegetation. The composition of the arthropod communities is known to change with the growth of the rice crop (Heong et al., 1991).

Dale (1994) in his comprehensive account on the biology and ecology of insect pests of rice states that over 800 species of insects damage the rice plant in few ways, in spite of the fact that majority of them cause minor damage. Despite the fact that the species composition of terrestrial arthropod pests and natural foes in rice fields all around the world is moderately well documented, there are just a couple of studies that examine the overall terrestrial arthropod community in rice fields. The work carried out by Heong et al. (1991) in the Philippines provides an insight into the arthropod communities and their guild structure in irrigated rice fields. Settle et al. (1996) demonstrated the existence of a mechanism in tropical irrigated rice systems that support high levels of natural biological control. Studies conducted in Sri Lanka, on terrestrial arthropods in rice fields are confined to surveys documenting the distribution of major rice insect pests and their natural enemies (Kobayashi et al., 1991; Kobayashi et al., 1995), while no attempts have been made to document the molecular structure and diversity of terrestrial arthropod communities in rice fields. Such a study carried out over successive rice cultivation cycles would provide useful information for the development of effective and safe integrated rice pest management strategies.