Molecular identification of mosquitoes from direct and indirect sources of DNA

Insects are pivotal to life on Earth as they aerate soil, pollinate plants as well as control pests; contributions known as ecosystem services. Insects can also be a cause of concern, particularly those that are vectors for disease such as some of the 3500 species of mosquitoes that occur worldwide. Across Europe, cases of mosquito borne diseases have been recorded and are associated with changes in climate, land use, urbanisation, trade and travel. It is important to monitor mosquitoes to create informed mitigation measures in advance of potential disease outbreaks. Identification of mosquitoes typically involves morphological identification of larvae and adults which requires a high level of skill and expertise, particularly for similar species, resulting in potential ambiguity of species identification. DNA-based identification methods can overcome some of these difficulties, while also having the potential to be applied to environmental samples containing traces of mosquito DNA. In this study, we demonstrate the application of DNA technology to identify mosquitoes directly from adults and larvae and indirectly from the diet of a mosquito consumer, the lesser horseshoe bat (Rhinolophus hipposideros) using a combination of DNA-based techniques including Sanger sequencing, DNA metabarcoding and real-time PCR. We discuss the development and application of each technique to different DNA sources and how the combination of these molecular techniques can be used to monitor mosquito distribution and diversity.