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

International audienceTropilaelaps 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

Phylogenetic analysis of <i>Aethina tumida</i> Murray (Coleoptera: nitidulidae) from Reunion Island

The small hive beetle (SHB), Aethina tumida Murray, native to sub-Saharan Africa, is a parasite of Apis mellifera causing significant damage to honey bee colonies. For the past decades, it has spread to many countries worldwide and once established, eradication is difficult or not feasible. In July 2022, an outbreak was reported for the first time in a French department in the Indian Ocean, Reunion Island. The origin and the pathway of the introduction were not identified. The molecular characterisation of SHB specimens collected in the southern part of the island, where the beetle has infested several apiaries, was investigated in order to provide elements on the invasive source. The sequencing of the partial Cytochrome oxidase I gene from two specimens showed that they belonged to the same new haplotype. Phylogenetic analysis suggests an introduction either from an unidentified African source or from the Asian continent since the haplotype is similar to those characterized from China and Philippines.</p

Reliability of Morphological and PCR Methods for the Official Diagnosis of <i>Aethina tumida</i> (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

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

International audienceThe 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, the European Reference Laboratory for Bee Health organised an inter-laboratory comparison on the identification of A. tumida by morphology and real-time PCR, two types of methods used routinely to confirm outbreaks. 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 present comparison is, to our knowledge, the first organised at the international level on the official diagnosis of A. tumida. It 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 in countries where its presence has not yet been detected and where early detection is crucial

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

International audienceThe 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