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

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.

Mitochondrial genome sequencing, mapping, and assembly benchmarking for Culicoides species (Diptera: Ceratopogonidae)

“…Consequently, additional efforts to validate the identity of the generated mitochondrial genomes are needed [9]. The mitogenome of the host Bos taurus was recovered from C. biguttatus DNA reads, confirming the potential of metagenomics for studying vectors and their hosts simultaneously, as well as potentially vectored pathogens [35][36][37]. Notably, all C. biguttatus specimens used for mitogenome reconstruction were sampled in a dairy barn.…”

“…The low mtDNA concentrations are inevitably linked to the limited amount of starting material, which presents a challenge regardless of the applied mitochondrial isolation methods/kits and can only be increased by pooling multiple individuals prior to DNA extraction. Pooling individuals was not considered in the present experimental design to avoid further complications during mitogenome reconstruction, given the possibility of the presence of different haplotypes and since the mix of genetic variants is a recognized challenge during insect genome assembly and annotation [ 36 ]. The low number of generated mtDNA reads might also be due to the lower depth of coverage used when sequencing these libraries (up to tenfold less than that for whole-genome sequencing libraries), which should be increased in future attempts.…”

“…7 ). All C. biguttatus specimens were collected with miniature Centre for Disease Control (CDC) light traps with UV lights (Bioquip, CA, USA) at the Ontario Veterinary College Dairy Barn, University of Guelph, Guelph, ON Canada [ 36 ]. C. sonorensis specimens belonged to a colony of specimens that originated in Idaho (Owyhee County).…”

“…1 ). The integrity of DNA extracts was further validated by amplifying the COI barcode gene with the COI-specific primers and PCR conditions described in Milián-García et al 2020 and 2021 [ 36 , 45 ] during the first steps of library preparation for Illumina MiSeq sequencing.…”

“…De novo assemblers SPAdes, MEGAHIT, and MitoZ are recommended in the lack of a species-specific reference genome for Culicoides to avoid mapping bias. We plan to sequence the mitogenomes of other Culicoides species, including the 38 Culicoides species currently described for Ontario, and eventually reach the 57 species found in Canada [36,39]. The establishment of a complete molecular database containing all genomes of all viral serotypes transmitted by Culicoides will be beneficial for the detection of viral DNA in Culicoides HTS datasets.…”