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.

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.

Varroa destructor is a major parasitic mite of the Western honey bee (Apis mellifera) responsible for severe colony losses. Control of Varroa relies heavily on synthetic acaricides such as tau-fluvalinate, a pyrethroid that acts on the voltage-gated sodium channel (VGSC) of mites. However, repeated use of tau-fluvalinate has led to the evolution of resistance, jeopardizing treatment efficacy. This study aimed to assess the relationship between an in vitro phenotypic bioassay for taufluvalinate susceptibility and a molecular assay targeting the L925V mutation in the VGSC, a known marker of pyrethroid resistance. The objective was to evaluate the reliability of the L925V mutation as a predictive indicator of phenotypic resistance across multiple French Varroa populations. Thirteen Varroa destructor populations were sampled from treated and untreated colonies in France. Susceptibility to taufluvalinate was assessed through an in vitro phenotypic assay quantifying mite mortality following exposure to the active substance (at the lethal concentration for 90% of susceptible mites). Following exposure, mites were classified as susceptible (dead or immobile) or resistant (surviving). In parallel, a TaqMan allelic discrimination assay was used to genotype individual mites for the L925V mutation in the VGSC gene. For each population, phenotypic mortality data were compared with genotype frequencies to evaluate the degree of concordance between phenotypic and molecular indicators of resistance. Additional parameters were also evaluated, including the distribution of the resistance within an apiary and its evolution in French apiaries over several years. The phenotypic and genotypic assessments showed a strong concordance, with a correlation coefficient of 0.89 between taufluvalinate mortality rates and the frequency of the VGSC L925V mutation. Phenotypically resistant mites had a 63% probability of carrying the L925V mutation, indicating moderate predictive power for detecting resistance. In contrast, phenotypically susceptible mites exhibited a 97% probability of not carrying the mutation, confirming its high reliability for identifying susceptibility. The study also suggested that sampling approximately 50% of beehives within an apiary could provide a representative estimate of the resistance distribution. Overall, the study demonstrated that the L925V mutation accounts for most taufluvalinate resistance observed in the tested populations and represents a valuable molecular marker for susceptibility screening. All of these findings highlight the usefulness of integrating phenotypic and genotypic approaches to improve monitoring strategies and support more effective resistance management in beekeeping practices.

Tropilaelaps mites (Laelapidae) are ectoparasites of honey bees native to Southeast Asia. They reproduce and feed within brood, causing colony weakness, mortality, and significant production losses. The European Union (EU) currently remains free of this regulated disease. However, global trade and climate change are facilitating the spread of Tropilaelaps beyond its native range, becoming an increasingly threat to EU beekeeping. Therefore, EU official laboratories must be prepared to reliably identify these mites and ensure early diagnosis. In 2022-2023, a Tropilaelaps collection campaign was organised in Thailand by the European Union Reference Laboratory (EURL) for Bee health in collaboration with Chiang Mai University. The study aimed to: i) describe infestation patterns in managed Apis mellifera and wild Apis dorsata colonies, and ii) characterise Tropilaelaps specimens using morphological and molecular approaches. A total 80 A. mellifera brood samples (from 8 colonies across 10 apiaries) and 11 A. dorsata brood samples (from 11 colonies) were collected in northern Thailand. Mites (Tropilaelaps spp. and Varroa spp.) were extracted using standard procedures recommended by the World Organisation for Animal Health (WOAH) Tropilaelaps specimens were selected from two colonies per A. mellifera apiary and from three A. dorsata colonies. Morphological identification followed the validated EURL method, also described in the WOAH Terrestrial Manual. Genus confirmation was performed by COI conventional PCR, using EURL and WOAH protocols, and species identification by real-time PCR with high-resolution melting (HRM) analysis. Conventional PCR amplicons were sequenced using the same primers, and phylogenetic analyses were conducted on the resulting 538 bp fragments. Haplotypes were determined by comparison with reference sequences from international databases. In A. mellifera apiaries, Tropilaelaps were detected in 98% (78/80) of sampled colonies, with apiary-level prevalence ranging from 88 to 100%. Co-infestation with Varroa spp. occurred in 70% (7/10) of apiaries and 35% (28/80) of colonies, with a prevalence of co-infested colonies within apiaries ranging from 0% to 88%. Tropilaelaps mercedesae was the only species identified in both A. mellifera (European honey bee) and A. dorsata colonies (giant honey bee, original host of T. mercedesae). Phylogenetic analyses showed that some sequences were closely related to sequences from Thailand, with no clear genetic differentiation between the two host species. These results provide well-characterised reference material for EU official laboratories and valuable epidemiological data to support surveillance and risk mitigation strategies against Tropilaelaps introduction into the EU.