Validation of an Effective Protocol for Culicoides Latreille (Diptera: Ceratopogonidae) Detection Using eDNA Metabarcoding.

eDNA metabarcoding is an effective molecular-based identification method for the biosurveillance of flighted insects. An eDNA surveillance approach maintains specimens for secondary morphological identification useful for regulatory applications. This study identified Culicoides species using eDNA metabarcoding and compared these results to morphological identifications of trapped specimens. Insects were collected using ultraviolet (UV) lighted fan traps containing a saturated salt (NaCl) solution from two locations in Guelph, Ontario, Canada. There were forty-two Culicoides specimens collected in total. Molecular identification detected four species, C. biguttatus, C. stellifer, C. obsoletus, and C. mulrennani. Using morphological identification, two out of these four taxonomic ranks were confirmed at the species level (C. biguttatus and C. stellifer) and one was confirmed at the subgenus level (Avaritia [C. obsoletus]). No molecular detection of Culicoides species occurred in traps with an abundance of less than three individuals per taxon. The inconsistency in identifying Culicoides specimens to the species level punctuates the need for curated DNA reference libraries for Culicoides. In conclusion, the saturated salt (NaCl) solution preserved the Culicoides' morphological characteristics and the eDNA.