Fig. 1

Experimental design for dual insect and blood-meal mitochondrial DNA (mtDNA) sequencing using nanopore adaptive sampling (NAS). a Hematophagous insects having had recent blood meals are collected from natural populations. Whole genomic DNA is extracted from individual or pooled insects. b Genomic DNA is prepared for sequencing on an Oxford Nanopore Technologies device (MinION platform) with access to the NAS bioinformatic pipeline. NAS is performed on blood-feeding insect DNAs with a reference file containing mtDNA sequences of congeneric or conspecific insect species and a diverse selection of putative blood-meal hosts. Exact species matches are not needed for NAS references as the method will retain sequences sharing at least 75% identity, which are suitable for guided discovery of cryptic insects and blood-meal hosts. c Mitochondrial sequences from NAS vector and host matches are quality filtered, and species barcoding genes (i.e. those encoding COI, cyt-b, D-loop, etc.) are used for phylogenetic analyses and species identification. d Complete mitogenomes are recovered from the sampled insect, thus expanding molecular resources for vector species. Recovered mt sequences of blood-meal hosts elucidate potential disease transmission pathways. Collective results of NAS experiments directly inform One Health initiatives focused on hematophagous insect biology and vector-borne disease transmission. COI, Cytochrome c oxidase subunit 1; cyt-b, cytochrome b; mtDNA D-loop, mt displacement loop