Molecular detection of small hive beetle Aethina tumida Murray (Coleoptera: Nitidulidae): DNA barcoding and development of a real-time PCR assay

Small hive beetle (SHB), Aethina tumida can feed on honey, pollen and brood in honey bee colonies. It was endemic to Africa, but since 1996 has been detected in a number of countries worldwide, including Australia, Brazil, Canada, Italy, Mexico, South Korea, Philippines and the USA where it has had economic effects on local apiculture. To improve SHB identification, we obtained the first reference sequences from the DNA barcoding 5′ COI gene region for SHB and some species of the family Nitidulidae associated with beehives. Phylogenetic analysis of SHB COI sequences (3′ COI) revealed two divergent lineages, with those from Australia and USA being genetically different from the recent detection in Italy. Many countries, including New Zealand, are currently free from SHB, and require a rapid detection method for biosecurity. Here we present the development and validation of a real-time PCR assay for detection of SHB. The assay showed high specificity and sensitivity for detecting SHB, with no cross-reaction observed with closely related species, such as A . concolor . The real-time PCR is sensitive, detecting the target sequences up to 100 copies/µL. This assay should prove a useful biosecurity tool for rapid detection of SHB worldwide.