Reliability of Morphological and PCR Methods for the Official Diagnosis of Aethina tumida (Coleoptera: Nitidulidae): A European Inter-Laboratory Comparison

SIMPLE SUMMARY: Aethina tumida, also called the Small Hive Beetle, is an insect that multiplies primarily in honeybee hives, causing honey losses and weakening colonies. It is native to sub-Saharan Africa and was introduced into different countries and continents over the last 20 years, posing a threat to beekeeping internationally. In case of introduction into a new area, officially approved laboratories (certified by government services) carry out analyses to confirm the outbreak. The reliability of the results is essential in the implementation of management measures. Therefore, a study was organised at the European level to compare the results between official laboratories for two types of methods, used routinely for the identification of A. tumida: morphological examination (form and structure) and DNA testing (genetics). The 22 participants analysed in a blinded way a panel of 12 samples (positive and negative samples). The results were very satisfactory, with the exception of one participant who encountered several anomalies for negative samples and especially for DNA tests, probably related to his inexperience with the method. This study proved the ability of laboratories and analytical methods to identify A. tumida, which is a key element in monitoring and managing this risk. ABSTRACT: The Small Hive Beetle (Aethina tumida Murray, 1867) is an invasive scavenger of honeybees. Originally endemic in sub-Saharan Africa, it is regulated internationally in order to preserve the areas still free from this species. To ensure the reliability of official diagnoses in case of introduction, an inter-laboratory comparison was organised on the identification of A. tumida by morphology and real-time PCR. Twenty-two National Reference Laboratories in Europe participated in the study and analysed 12 samples with adult coleopterans and insect larvae. The performance of the laboratories was evaluated in terms of sensitivity and specificity. Sensitivity was satisfactory for all the participants and both types of methods, thus fully meeting the diagnostic challenge of confirming all truly positive cases as positive. Two participants encountered specificity problems. For one, the anomaly was minor whereas, for the other, the issues concerned a larger number of results, especially real-time PCR, which probably were related to inexperience with this technique. The comparison demonstrated the reliability of official diagnosis, including the entire analytical process of A. tumida identification: from the first step of the analysis to the expression of opinions. The performed diagnostic tools, in parallel with field surveillance, are essential to managing A. tumida introduction.