DNA Barcoding In Mosquitoes

By using DNA barcoding and nuclear genome analysis, correct identification of mosquito species can be done for effective control techniques.

DNA barcoding is a system that is used for quick and precise identification of different organisms, either prokaryotes or eukaryotes, and their species. It has created a strong ecological system that can only be accessed by a short sequence of DNA instead of a whole organism with very little ambiguity in identification.

The short DNA palindrome working as a marker is produced from a basic region of the genome. This marker is different for different species like cytochrome oxidase 1 (CO1) is used in animals, Internal Transcribed Spacer factor (ITS) for fungi, while matK and rbcL are used for the identification of various plant species.

DNA barcoding is a vast science used in various fields of research such as agriculture, medicine, aquaculture, maintaining natural resources, protecting endangered species, and identifying the origin of different unknown species.

The DNA barcoding consists of two basic steps:

1. Creating the DNA reference library of various known species
2. Matching of DNA barcode sequence of an unknown sample against the barcode reference library for accurate identification

DNA barcoding uses the genome information of one or more genes to identify all species of life, while genomics is the inverse of barcoding and describes one or a few selected species and their function and interaction among all genes.

Steps of DNA Barcoding

DNA barcoding consists of the following basic steps:

1. DNA extraction/isolation from the sample
2. PCR amplification of the target DNA barcode
3. DNA sequencing
4. Sequence Analysis or comparison against the barcode reference library and identification of the species

DNA Barcoding in Mosquitoes

Correct identification of the mosquito species remained a dynamic factor in the implementation of effective control techniques for the mosquitoes. Conventionally, the identification of the mosquito fauna was done on the morphological characteristics, stereoscope, cytogenetics, and iso-enzyme markers of the species.

Taxonomic classification of mosquitoes is mainly dependent on the external structures of the adult female and the fourth larval instar. But in this, the handling and storing of the required specimens must carefully proceed without any damage to the external body features.

The traditional techniques of mosquito identification were not so efficient due to the genetic variation, phenotypic plasticity, responsiveness, and due to diversity among the species. Due to this reason, many species of public health importance remained unidentified, and vector control strategies were ineffective.

This gap in species recognition can be overcome using molecular-level identification like DNA barcoding and nuclear genome analysis. This method has classified species and subspecies of Culicidae with a good capability of identification.

Molecular Characterization of Mosquitoes

The importance of mosquitoes as a vector of many diseases that drives disease highlights the significance of detailed, specific, and similar methods of taxonomic group identification of mosquito species.

Several molecular targets have been used to study mosquitoes since the start of PCR in the 1980s, though its application in identifying mosquitoes was practiced in the early 1990s.

The use of mitochondrial DNA (mtDNA) cytochrome oxidase 1 (COI or COX1) and the internal transcribed spacer 2 (ITS2), a region in the nuclear ribosomal subunit of DNA, were used as DNA barcode targets for the identification of mosquito species is now escalating for discriminating mosquitoes.

Molecular methods were used to explain problems arising from the identification of organisms based on morphology. As morphology-based taxonomy failed in providing the exact data regarding species and subspecies. This failure of conventional taxonomy led scientists to shift from conventional taxonomy to molecular taxonomy based on DNA specifically DNA barcoding using different markers such as COI, ITS, 16S, etc.

DNA barcoding has rapidly gained much popularity and attention in the molecular identification of mosquito species in variable contexts.

In this technique, a small but standard fragment of DNA is used that helps in the specific sequence identification. Sequence analysis is performed, a neighbour-joining tree or maximum likelihood tree is constructed, and phylogenetic analysis is performed to check the ancestral history of mosquitoes or of other organisms.

COI is the most common, conserved, and accurate gene in terms of amino acid sequences as compared to the other markers of the DNA. The use of a single marker of DNA is less time-consuming and relatively inexpensive than the barcodes of three or more DNA markers (COI, 16S rDNA, and ITS2).

For mosquito and disease control programs, accurate identification of mosquito species is an essential tool. However, this taxonomic study is complicated by the difficulties in morphologically identifying some species of mosquitoes.

In the DNA barcoding technique, for the identification of mosquito species, variation of a partial sequence of the cytochrome c oxidase unit I (COI) gene is used which not only facilitates the discovery of cryptic diversity, but also monitor the invasive species of mosquitoes. The combination of DNA barcoding and morphology acts as an effective tool for the identification of invasive mosquito species.

This article is jointly written by Maria Kausara, Kashif Hussaina, Muhammad Sohail Sajida, Talha Khanb from a Department of Parasitology, University of Agriculture Faisalabad, Pakistan; and b Department of Applied Chemistry, Government College University Faisalabad, Pakistan