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

SIMPLE SUMMARY: All organisms shed genetic material into the environment, which is known as environmental DNA. Current molecular technologies allow for sequencing molecular markers in complex environmental samples. The use of these methods permits an effective identification and monitoring of flighted insects such as Culicoides species. These biting midges are agricultural pests of significant economic concern. This study identified Culicoides species using a novel molecular-based approach for this group and compared these results to morphological identifications of the specimens collected. There were forty-two Culicoides specimens collected in total, using a saturated salt solution as a collection fluid. Molecular identification detected four species. Using morphological identification, we identified two out of these four taxonomic ranks at the species level and one at the subgenus level. The inconsistency in identifying Culicoides specimens to the species level indicates the need for curated DNA reference libraries for molecular-based identification. The saturated salt solution used in the traps preserved the morphological characteristics and the organisms’ environmental DNA, which is an essential contribution of this study. ABSTRACT: 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.