Varroa destructor resistance to tau-fluvalinate: relationship between in vitro phenotypic test and VGSC L925V mutation.

BACKGROUND: Varroa destructor is a parasitic mite of the honey bee, Apis mellifera. Its presence in colonies can lead to a collapse within a few years. The use of acaricides has become essential to manage the hive infestation. However, the repeated and possibly incorrect use of acaricide treatments, as tau-fluvalinate, has led to the development of resistance. The in vitro phenotypic test allows the proportion of susceptible or resistant individuals to be known following an exposure to an active substance. In Varroa mites, resistance to tau-fluvalinate is associated with the presence of mutations at the position 925 of the voltage-gated sodium channel (VGSC). RESULTS: Here, we compared the results obtained with an in vitro phenotypic test against tau-fluvalinate and those obtained with an allelic discrimination assay on 13 treated and untreated Varroa populations in France. The correlation between the phenotype and the genetic profile rate is found to be 0.89 Varroa mites having resistant phenotypic profile have a probability of 63% to present the L925V mutation (resistance detection reliability). However, 97% of the Varroa mites having the susceptible phenotype do not present the L925V mutation (susceptible detection reliability). CONCLUSION: The L925V mutation explains most of the resistance to tau-fluvalinate in V. destructor in the populations tested. However, other mutations or types of resistance may also be involved to explain the survival of Varroa mites in the phenotypic test. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

An international inter-laboratory study on Nosema spp. spore detection and quantification through microscopic examination of crushed honey bee abdomens.

Nosemosis is a microsporidian disease causing mortality and weakening of honey bee colonies, especially in the event of co-exposure to other sources of stress. As a result, the disease is regulated in some countries. Reliable and harmonised diagnosis is crucial to ensure the quality of surveillance and research results. For this reason, the first European Interlaboratory Comparison (ILC) was organised in 2017 in order to assess both the methods and the results obtained by National Reference Laboratories (NRLs) in counting Nosema spp. spores by microscopy. Implementing their own routine conditions of analysis, the 23 participants were asked to perform an assay on a panel of ten positive and negative samples of crushed honey bee abdomens. They were asked to report results from a qualitative and quantitative standpoint. The assessment covered specificity, sensitivity, trueness and precision. Quantitative results were analysed in compliance with international standards NF ISO 13528 (2015) and NF ISO 5725-2 (1994). Three results showed a lack of precision and five a lack of trueness. However, overall results indicated a global specificity of 98% and a global sensitivity of 100%, thus demonstrating the advanced performance of the microscopic methods applied to Nosema spores by the NRLs. Therefore, the study concluded that using microscopy to detect and quantify spores of Nosema spp. was reliable and valid.

Rapid Identification of Tropilaelaps Mite (Mesostigmata: Laelapidae) Species Using a COI Barcode-HRM.

Tropilaelaps mite (Mesostigmata: Laelapidae) is an ectoparasite of bees present, to date, only on the Asian continent. In the context of the threat of Tropilaelaps's introduction into new regions, accurate, rapid, and sensitive detection of the Tropilaelaps spp. is essential. In the present study, we developed a novel molecular method for bee mite's identification, which consists of a new real-time PCR method. A high-resolution melting analysis (HRM) was then performed on the amplified products to differentiate the species. PCR amplification was applied on the cytochrome c oxidase subunit I gene (580 bp). Short fragments from the most variable regions of this gene were identified in silico to amplify and discriminate among the four Tropilaelaps species. Four reference plasmids were synthesized to characterize species by well-distinguished melting curves. The method was then validated in terms of its specificity and sensitivity using a panel of 12 specimens. The results showed that an HRM method can be applied for the intended objective: for rapid and simultaneous identification of Tropilaelaps species. To our knowledge, this study reports the first direct HRM assay developed for the genome of a bee mite, specific for Tropilaelaps species. This COI barcode-HRM technique could be a promising tool for mite species identification.

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

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.