# Comparative Assessment of Environmental DNA and Bulk-Sample Metabarcoding in Biosecurity Surveillance for Detecting Biting Midges (Ceratopogonidae)

## TL;DR

This study compares DNA metabarcoding and eDNA methods for detecting biting midges in biosecurity surveillance, finding that bulk-sample DNA metabarcoding is more accurate and efficient.

## Contribution

The study introduces DNA metabarcoding of bulk insect samples as a more accurate and efficient alternative to traditional microscopy and eDNA methods for biosecurity surveillance.

## Key findings

- DNA metabarcoding of bulk insect samples had over 81% accuracy in detecting biting midges compared to morphology.
- eDNA from trap fluids had lower accuracy (55.26–68.42%) due to extraction issues or low species abundance.
- Both methods provided similar insect community data, suggesting potential for biodiversity monitoring.

## Abstract

Culicoides biting midges are vectors that can transmit pathogens, causing many diseases of veterinary concern, including bluetongue virus, which significantly affects primary production animals. New Zealand is free of these midges, and national surveillance programmes are in place to detect them and their vector-borne diseases early. Traditionally, insects from these trap samples are identified under the microscope, which is slow and requires specialized taxonomic expertise. This study compared two molecular approaches, DNA metabarcoding using insect bulk samples from traps and environmental DNA (eDNA) from trap liquids, considering morphological outcomes for detecting biting midges and Culicoides identification. The DNA metabarcoding of bulk insect samples was more accurate (over 81%) compared to eDNA samples from trap fluids (55.26–68.42%) in matching morphological outcomes. Both methods provided similar information about insect communities in traps, suggesting non-destructive eDNA metabarcoding could be useful for biosecurity surveillance and biodiversity monitoring. Overall, DNA metabarcoding with insect bulk samples could make the diagnostic process and surveillance strategy more efficient, reducing workload and screening time.

Biting midges, Culicoides spp. (Diptera: Ceratopogonidae), are significant vectors capable of transmitting arboviruses, such as bluetongue virus, to livestock. New Zealand is free of Culicoides, and a national surveillance programme is in place for the early detection of an incursion. Traditionally, insect trap samples from the surveillance programme are analyzed using morphology-based diagnostics under microscopes, which is time-consuming and relies on specialized taxonomic expertise. Here, we assessed the effectiveness of DNA metabarcoding using insect bulk samples and environmental DNA (eDNA) from liquid samples collected in surveillance traps. Two Cytochrome oxidase I (COI) barcoding primer sets were employed to study biodiversity and detect exotic species. The results indicated that DNA metabarcoding with homogenized insect bulk samples had a higher overall detection accuracy rate (over 81% for both primer pairs) compared to ethanol fluid-derived eDNA samples from traps (68.42% and 55.26% for the primer sets LCO1490/HCO2198 and mlCOIintF/jgHCO2198, respectively) based on congruence with morphological identification. Detection failures were likely due to eDNA extraction issues or low target species abundance. Both approaches showed similar insect community composition and diversity in the surveillance trap samples, suggesting the potential of DNA metabarcoding for biosecurity surveillance and biodiversity assessments. Overall, DNA metabarcoding using bulk insect samples could enhance the efficiency of Culicoides surveillance, reducing workload and screening time.