Molecular identification of mosquito vectors using genomic DNA isolated from eggshells, larval and pupal exuvium

Correct and precise identification of mosquito vectors is important in many respects including development of vector control strategies. Conventional identification methods have limitations for sibling and closely related species of mosquitoes, stage and quality of the specimen used and this could be overcome by DNA-based identification methods using molecular markers such as nuclear ribosomal internal transcribed spacer (ITS) which do not demand intact or undamaged specimen. Genomic DNA is usually isolated from whole mosquito, legs, wings etc. Alternate sources for genomic DNA isolation such as eggshells, larval and pupal exuviae were explored in this study by amplifying the ITS markers. Standardization of genomic DNA extraction and ITS amplification were carried out with laboratory specimens. The same was applied to specimens collected from the field. The results show that PCR amenable genomic DNA could be isolated from fresh exuviae collected in the laboratory and not from older and/or field specimens. But exuviae of larvae and/or pupae collected in the field reared to adulthood in the laboratory yielded PCR amenable genomic DNA. The results also revealed that the ITS2 marker could very well differentiate Aedes aegypti and Aedes albopictus by producing amplicons of ~330 bp and ~520 bp, respectively. The genomic DNA from these alternate sources also supported the species-specific PCR to distinguish the Culex vishnui subgroup mosquitoes.