Mosquito Identification From Bulk Samples Using DNA Metabarcoding: a Protocol to Support Mosquito-Borne Disease Surveillance in Canada

Abstract

Approximately 80 species of mosquitoes (Diptera: Culicidae) have been documented in Canada. Exotic species such as Aedes albopictus (Skuse) (Diptera: Culicidae) are becoming established. Recently occurring endemic mosquito-borne diseases (MBD) in Canada including West-Nile virus (WNV) and Eastern Equine Encephalitis (EEE) are having significant public health impacts. Here we explore the use of DNA metabarcoding to identify mosquitoes from CDC light-trap collections from two locations in eastern Canada. Two primer pairs (BF2-BR2 and F230) were used to amplify regions of the cytochrome c oxidase subunit I (CO1) gene. High throughput sequencing was conducted using an Illumina MiSeq platform and GenBank-based species identification was applied using a QIIME 1.9 bioinformatics pipeline. From a site in southeastern Ontario, Canada, 26 CDC light trap collections of 72 to >300 individual mosquitoes were used to explore the capacity of DNA metabarcoding to identify and quantify captured mosquitoes. The DNA metabarcoding method identified 33 species overall while 24 species were identified by key. Using replicates from each trap, the dried biomass needed to identify the majority of species was determined to be 76 mg (equivalent to approximately 72 mosquitoes), and at least two replicates from the dried biomass would be needed to reliably detect the majority of species in collections of 144–215 mosquitoes and three replicates would be advised for collections with >215 mosquitoes. This study supports the use of DNA metabarcoding as a mosquito surveillance tool in Canada which can help identify the emergence of new mosquito-borne disease potential threats.

Analysis of trapped mosquito excreta as a noninvasive method to reveal biodiversity and arbovirus circulation

“…Taxonomic identification is limited with our method to the genetic diversity that can be captured and amplify over a short mitochondrial sequence. Here, taxonomic identifications are biased toward arthropods because we used a degenerated primer set specifically designed to amplify arthropods (Mechai et al, 2021 ). The use of primers targeting the mitochondrial DNA of vertebrates on the same target might further increase our ability to identify the diversity of vertebrate species on which mosquitoes fed prior to being trapped (Reeves et al, 2018 ).…”

“…Virus detection, sequencing and metabarcoding approaches were performed on the same eluates to facilitate the procedure. A ~460 bp mitochondrial DNA section corresponding to a subfragment of the classical Folmer cytochrome c oxidase subunit I (COI) fragments (Folmer et al, 1994 ), was amplified with universal primers BF2/BR2 (Table S1 ) that are routinely used for macroinvertebrate monitoring (Hajibabaei et al, 2019 ; Mechai et al, 2021 ). Illumina Nextera universal tails sequences were added to the 5′ end of each of these primers to facilitate library preparation, as described above for the WNV tiling amplicon‐based sequencing procedure.…”

“…Taxonomic identification is limited with our method to the genetic diversity that can be captured and amplify over a short mitochondrial sequence. Here, taxonomic identifications are biased toward arthropods because we used a degenerated primer set specifically designed to amplify arthropods (Mechai et al, 2021).…”

Mosquito excreta reveals circulation of West Nile virus and its underlying ecosystem

“…Taxonomic identification is limited with our method to the genetic diversity that can be captured and amplify over a short mitochondrial sequence. Here, taxonomic identifications are biased toward arthropods because we used a degenerated primer set specifically designed to amplify arthropods 38, 39 . The use of primers targeting vertebrates’ mitochondrial DNA on the same target might further increase our ability to identify the diversity of vertebrate species on which mosquitoes fed prior to be trapped 40 .…”

“…Virus detection, sequencing and metagenomic approaches were performed on the same eluates to facilitate the procedure. A ∼ 460 bp mitochondrial DNA section corresponding to a sub fragment of the classical Folmer cytochrome c oxidase subunit I (COI) fragments 15 , was amplified with universal primers BF2/BR2 68 (Supplementary table 1) that are routinely used for macroinvertebrate monitoring 38, 39 . Illumina Nextera® universal tails sequences were added to the 5’ end of each of these primers to facilitate library preparation, as described above for the WNV tiling amplicon-based sequencing procedure.…”