An improved DNA method to unambiguously detect small hive beetle Aethina tumida, an invasive pest of honeybee colonies

The scavenger and invasive species Aethina tumida threatening the honey bee has been recently introduced in Europe. We present a new, reliable and rapid multiplex real‐time PCR for efficient diagnostics enabling surveillance programs. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

The small hive beetle (SHB) Aethina tumida Murray (Coleoptera: Nitidulidae) is a scavenger native to sub-Saharan Africa and is a pest of honey bees without provoking significant damage within its endemic range. 1,2 Since the first report in 1996 out of its native range 3 in North Carolina, USA, the beetle became an invasive species in Australia, and in central and North America. It was introduced in Europe in 2004 in Portugal where an eradication program was effective and then in Italy in 2014 where infestation is still ongoing 4 probably originating from an African population. 5 Its life cycle is intimately linked to the honey bee where it mates and reproduces inside the colony and where the larvae feed on beebread, honey and brood causing destruction. 1,6,7 Besides honey bees, it can also affect bumble bees and stingless bees (see review in reference 2 ). The economic damage to the beekeeping industry can therefore be substantial thereby explaining why the SHB is a statutory notifiable pest in the European Union (EU  8 where an amplification system targeting the cytochrome oxidase subunit I (COI) gene was proposed. However, the sequence of the reverse primer contains an important internal mismatch of three nucleotides with all currently published A. tumida COI sequences, rendering its application tedious and susceptible to false negative diagnostic. In this study, we propose a new SHB diagnostics using a multiplex PCR approach targeting COI gene and a common region of 18S ribosomal gene as internal control.
These results further confirm the validity of the proposed multiplex PCR system for rapid, reliable and specific diagnostics of the small hive beetle (SHB) that may be found in the hive and thus facilitate for example early detection programs.
The DNA extraction method was chosen according to a previous study comparing different extraction procedures of genomic DNA from ticks providing material allowing maximal recovery and good quality for a consistent amplification. 9 Briefly, insects stored in ethanol 70% (v/v) were air-dried prior to dissection into four quarters and then DNA was extracted using GeneJet Genomic Kit (Thermo Fisher, Waltham, MA, USA) following the manufacturer's instructions and eluted into a final volume of 100 L. Then, 4 L (0.8-10 ng) of insect genomic DNA were used for amplification. PCR was performed in a 20 L reaction volume containing 1x  wileyonlinelibrary.com/journal/ps After optimization of the concentration of the two probes, to further confirm the specificity of the primers the multiplex assay was tested on a total of 49 DNAs extracted from different insects which can be found in the vicinity of colony hives or are common in European fields. The DNA test set included 12 A. tumida individuals of different geographical origins and relatives from the Nitidulidae family. All the DNA extracted from A. tumida were positive for both the internal control 18S and COI gene amplification. Interestingly, threshold cycles (Cq) for the two systems never diverged for more than 6.4 cycles ( Table 2). All the other 37 insect samples analyzed were positive for 18S, with Cq ≤ 42.1, and negative for COI ( Table 2).
These observations prove the reliability of our multiplex PCR system, despite single nucleotide mismatches present in the COI reverse primer and at 5 ′ extremity of the COI probe with three known sequences of A. tumida (KT380625.1, KT380626.1, AF227647.1). These single nucleotide mismatches apparently did not influence COI amplification, nor its specificity.
These results further confirm the validity of the proposed multiplex PCR system for a rapid, reliable and specific diagnostics of SHB that may be found in the hive and thus facilitate for example early detection programs.