DNA Barcoding - A Rapid and Reliable tool for Insect Species Identification

DNA barcoding, a tool of DNA-based taxonomy is in current use to identify known and unknown species on the basis of the pattern of nucleotide arrangement in a fragment of DNA of a particular species. Several researchers have suggested the use of DNA barcoding in taxonomy as a method to achieve rapid species descriptions in the context of the current biodiversity crisis.

DNA barcoding is the use of a short standardized DNA sequence (in insects, a 658 bp fragment of the mitochondrial cytochrome c oxidase ( COX I) gene) to identify and assign unknown specimens to species besides facilitating the discovery of new species. This tool is widely accepted all over the globe from hard-core taxonomists’ to graduate molecular biologists. 

Introduction

Food security is one of the major challenge due to several factors among which emerging and invasive pests are one of the important factor posing threat to agriculture production. Successful management of such invasive pests requires their correct identification as a first and most crucial step in determining the future course of action. Traditionally, identification of pests has been based on morphological diagnoses provided by taxonomic studies. Only experts such as taxonomists and trained technicians can identify taxa accurately, because it requires special skills acquired through extensive experience. As interest in biodiversity has increased in the fields of ecology, evolutionary biology, agriculture and economics, among others, it has become increasingly important to precisely identify species. However, the number of taxonomists and other identification experts has drastically decreased. Consequently, alternative and accurate identification methods that non-experts can use are required. One of the most promising approaches is the use of molecular instead of morphological data for identifying taxa, which has long been a fundamental idea of many biologists (Blaxter 2003). Advances in DNA sequencing technologies have enabled researchers studying biodiversity to conduct simple, cost–effective and rapid DNA analyses. This progress in biotechnology, and the taxonomy crisis itself, played a large role in the creation of DNA barcoding (Jinbo et al., 2011).

After 250 years of Darwin and Linnaeus, a new method called DNA barcoding, a tool of DNA-based taxonomy is in current use to identify known and unknown species on the basis of the pattern of nucleotide arrangement in a fragment of DNA of a particular species (Novotny et al. 2002). Several researchers have suggested the use of DNA barcoding in taxonomy as a method to achieve rapid species descriptions in the context of the current biodiversity crisis (Hebert et al. 2003a, b; Ball and Armstrong, 2006). DNA barcoding is the use of a short standardized DNA sequence (in insects, a 658 bp fragment of the mitochondrial cytochrome c oxidase ( COX I) gene) to identify and assign unknown specimens to species besides facilitating the discovery of new species. Wilson (2012) observed that library barcodes gain their value due to an intimate association, through voucher specimens from where they came, with other data, particularly, Linnaean names, collection localities, and morphology in the form of digital images. This tool is widely accepted all over the globe from hard-core taxonomists’ to graduate molecular biologists. In the recent past, due to the rapid development of techniques in molecular biology, nucleic acid sequencing and analysis of large data, the mtDNA study is becoming more popular. Compared to nuclear markers, mitochondrial markers are more susceptible to the effects of genetic drift (Filipova et al., 2011). As a powerful and widely used molecular marker, mtDNA has been applied in many organisms to determine the genetic variations and structure of population. Mitochondrial DNA has become a major tool of comparative genomics and occupies a significant role in genetic structure of population and molecular variations as it is maternally inherited with no intermolecular genetic recombination with rapid rate of evolution. COI is a protein-coding gene in mtDNA. Due to fast evolution, high polymorphism, easy amplification and sequencing, it has shown valuable information and is a widely used genetic marker for population genetic studies especially intra-specific analysis (Xu et al., 2011).Frequent application of insecticides led to modification and development of genetic resistance by means of genotypic variations in major insect pests. It is therefore necessary to explore the genotypic variations that occur within the population of a species to design and formulate effective insect pest management programmes (Palraju et al., 2018). 

Why CO-1 selected as standard DNA Barcode?

Efficiency in discrimination among the different animal groups and high competency for species identification are the features of standard DNA barcode CO-I. This universal primer can be applied to all animal phyla which were originally designed for marine invertebrates (Folmer et al., 1994). A 648-bp fragment has sufficient information and can be directly sequenced with a sequencer. The alignment procedure is not strenuous because this is a protein-coding region. The errors can be diagnosed by checking whether the obtained sequence is translatable. These are the reasons why the CO-I region was selected as the standard DNA barcode. Hence, DNA barcoding can be a simple but potent tool for non-experts, especially those who customarily identify a large number of samples.

Usefulness of DNA Barcoding

The access to a public reference database of taxa helped in the accurate taxonomic identification of a wide range of species. Thus, DNA barcode can support various scientific domains (e.g. Conservation biology, evolutionary biology etc). DNA barcoding helps to recognize, detect and trace dispersal of patented organisms in biotechnology, either to verify the source organism (e.g. truffles, Rastogiet al., 2007) or assure intellectual property rights for bioresources (Taberletet al., 2007). Molecular data can be rapidly retrieved by using DNA barcoding. However, there is a huge discrepancy for morphological tools, in addition to this it can be time-consuming and in some instances totally confusing (e.g. earthworms, Huang et al., 2007).

For determining the taxonomic identity of damaged organisms orfragments of (e.g. goods, food and stomach extracts). The DNA barcoding tool is likely to be useful in the food industry, forensics and in preventing illegal trade and poaching of endangered species.When there no other means to match adults with immature specimens molecular characterization is necessary(e.g. Coleoptera, Caterino and Tishechkin, 2006). When morphological traits fail to discriminate species then DNA barcoding is the only option available (e.g. field-collected mosquito specimens, Kumar et al., 2007). DNA barcoding as a species identification tool not only attracts the non-specialists but also attracts specialists.To achieve the CBOL objectives, species need to be taxonomically characterized before their deposit in BOLD, which helps researchers to resolve analytical, technical and fundamental issues beforehand.Taxonomy, molecular phylogenetics and population genetics can be brought together and complemented by DNA Barcoding (Hajibabaeiet al., 2007). DNA barcoding can be treated as a ‘formidable tool’ to expedite species discovery and species description.

Limitations of DNA Barcoding

• The under-described part of biodiversity:The shortage of samples across taxa will lead to ‘barcoding gaps’ so sampling quality should be considered with the utmost care during the database construction phase. The selected sample should represent and cover the major part of the existing biodiversity (Wiemer and Fiedler, 2007).
• Inherent risks due to mitochondrial inheritance:The mitochondrial DNA (mtDNA) is maternally inherited so its diversity is strongly linked to female genetic structure. The usage of mitochondrial loci can lead to exaggerate sample divergence and make conclusions about the species status unclear. For instance, inHomonamermerodes (Lepidoptera) mtDNA polymorphism isstructured according to the host plants on which females feed, and the two clades obtained by phylogenetic analyses are artefacts of female nutritional choice (Hulcret al., 2007).
• Nuclear copies of COI (NUMTs):Nuclear mitochondrial DNAs (NUMTs) describe a translocation of any type of cytoplasmic mitochondrial DNA into the nuclear genome of eukaryotic organisms. Across the different species NUMTs differ in number and size. Disturbance due to NUMTs must be seriously considered, in both DNA barcode library construction and further specimen identification.
• Rate of evolution in COI:The rate of genome evolution (mitochondrial or nuclear) is notequal for all living species.The rate of evolution can vary at the ordinal level e.g. Dermapteran insect species (Wirth et al., 1999).
• The intra-specific geographical structure should be taken into account:Species geographical structure, if ignored, it can distort speciesdelineation. Intraspecific divergence canobtain from geographically isolated populations (Hebert et al.,2003a), and hence, it must be taken into account while setting up the DNAbarcode reference database. 

Conclusion

Species identification using short DNA sequenceswill become more reliable when mt-DNA based barcode supplements nuclear barcodes. It will reduce the dependency on a single character and help to identify in cases where mt-DNA behaves differently to nuclear genes. Molecular phylogenetic studies routinely make use of multiple nuclear genes, so it is not a novel idea. In spite of limitations of DNA barcoding, the reported success using barcoding in differentiating species from taxa and to reveal cryptic species is remarkable. Thus, effort should be made to develop nuclear barcodes which can complementmt-DNA based barcodes. DNA barcoding solely can’t help in accurate identification and description of a species. Therefore, Integration of DNA barcoding, morphological and ecological studies will help in achieving accurate identification and description of a species. 

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Contributors : Pratap A Divekar^a and Suresh Nebapure^b

a Division of Crop Protection, ICAR-IIVR, Varanasi-221305 (U.P.) India.

b ICAR-Indian Agricultural Research Institute, New Delhi.

Corresponding mail :  pratapento@gmail.com