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.

Updates on the Mobile Divider and Its Use in Calabria Region to Monitor and Control Aethina tumida Infestation

The small hive beetle (SHB), Aethina tumida was first detected in the Calabria and Sicily region (Southern Italy) in 2014. In this regard, a more effective and faster inspection method than the Official one (Ministry of Health) was tested to detect the beetle in the hive. In collaboration with Calabria beekeepers’ association, a “mobile divider”-based method was tested, in order to facilitate the detection of A. tumida and save time during hive inspection. In this short communication, we provide an update on the mobile divider technique and its related inspection procedure, which was first proposed and used in Calabria (Southern Italy) from 2014 to 2016. We report preliminary data concerning the number of detected SHBs and the time spent for their detection, based on the inspection of two apiaries in Calabria region, using both methods (official method and mobile divider method). The preliminary data presented here show that, on average, the mobile divider method seems to be able to recover a slightly higher number of beetles (0.9 adults) per inspected hive, in a shorter time (25 s).

New Matrixes to Diagnose Aethina Tumida Presence at Apiary Level

Aethina tumida (Coleoptera: Nitidulidae - Small Hive Beetle - SHB), is a parasite of honey bee colonies that causes the notifiable disease called aethinosis. In 2014, SHB was detected in Southern Italy, where it is still present (Calabria region). As part of surveillance activities, official diagnosis of the disease is performed by veterinary services via visual inspection of single hives in the apiaries. New outbreaks can be eradicated and the spread of SHBs limited by early detection of new introductions. We report an alternative protocol for the diagnosis of SHB through swabs and hive debris analysis by PCR. This was tested in three apiaries through the evaluation of different SHB infestation levels with a hive inspection method. This approach for sampling, followed by biomolecular methods, was fast and useful in unfavorable conditions (bad weather, aggressiveness, robbing), could be integrated in the official diagnosis procedures and may act as pre-clinic indicator.

Definition of a Protocol to Manage and Officially Confirm SHB Presence in Sentinel Honeybee Colonies

Given the consolidated circulation of Aethina tumida (SHB) in Reggio Calabria and Vibo Valentia provinces of Calabria region (Southern Italy), the need for a more effective and less time-consuming approach to SHB surveillance emerged. Accordingly, honeybee sentinel colonies were established in the infested areas under the supervision and management of the Veterinary Services of the Local Health Unit. In this short communication, we present the protocol adopted in the Calabria region to manage the SHB positive sentinel honeybee colonies. The procedures for safely packing and transport the SHB infested sentinel honeybee colonies from the field to the official laboratory and the subsequent procedure for their careful inspection in the laboratory are illustrated.

Cuticular Hydrocarbon Profile of Parasitic Beetles, Aethina tumida (Coleoptera: Nitidulidae)

Cuticular hydrocarbons (CHCs) cover insects’ bodies and play important roles in chemical communication, including nestmate recognition, for social insects. To enter colonies of a social host species, parasites may acquire host-specific CHCs or covertly maintain their own CHC profile by lowering its quantity. However, the chemical profile of small hive beetles (SHBs), Aethina tumida, which are parasites of honey bee, Apis mellifera, colonies, and other bee nests, is currently unknown. Here, adults of SHB and honey bee host workers were collected from the same field colonies and their CHC profiles were analysed using GC-MS. The chemical profiles of field-sampled SHBs were also compared with those of host-naive beetles reared in the laboratory. Laboratory-reared SHBs differed in their CHC profiles from field-sampled ones, which showed a more similar, but ten-fold lower, generic host CHC profile compared to host workers. While the data confirm colony-specific CHCs of honey bee workers, the profile of field-collected SHBs was not colony-specific. Adult SHBs often commute between different host colonies, thereby possibly preventing the acquisition of a colony-specific CHC profiles. An ester was exclusive to both groups of SHBs and might constitute an intraspecific recognition cue. Our data suggest that SHBs do not use any finely tuned chemical strategy to conceal their presence inside host colonies and instead probably rely on their hard exoskeleton and defence behaviours.

Identification and Expression Profile of Chemosensory Genes in the Small Hive Beetle Aethina tumida

Aethina tumida is a parasite and predator of honeybee causing severe loss to the bee industry. No effective and environmentally friendly methods are available to control this pest at present. Chemosensory genes play key roles in insect behavior which can potentially be used as targets for developing environmentally friendly pest control agents. In this study, the putative chemosensory genes in antennae and forelegs of A. tumida involved in olfaction or contact chemical communication of adults were investigated using RNA transcriptome sequencing and PCR methods. Based on transcriptomic data, unigenes encoding 38 odorant receptors (ORs), 24 ionotropic receptors (IRs), 14 gustatory receptors (GRs), 3 sensory neuron membrane proteins (SNMPs), 29 odorant binding proteins (OBPs), and 22 chemosensory proteins (CSPs) were identified. The analyses of tissue expression profiles revealed that genes encoding 38 ORs, 13 antennal IRs, 11 GRs, 1 SNMP, 24 OBPs, and 12 CSPs were predominately expressed in antennae. No significant differences in expression levels of these genes were found between males and females. Genes encoding 5 non-NMDA iGluRs, 3 GRs, 2 SNMPs, 5 OBPs, and 12 CSPs were predominately expressed in forelegs. RT-PCR assays for SNMPs, OBPs and CSPs further revealed that 3 OBPs (AtumOBP3, 26 and 28) and 3 CSPs (AtumCSP7, 8 and 21) were highly expressed in antennae. Our results enrich the gene inventory of A. tumida and facilitate the discovery of potential novel targets for developing new pest control measures.

First Report on the Mechanisms of Insecticide Resistance in Field Populations of the Small Hive Beetle in Florida

The small hive beetle (Aethina tumida Murray) is a serious threat to beekeeping and crops that rely on honeybees for pollination. The small hive beetle not only causes significant damage to honeybees by feeding on pollen and honey, attacking bee brood and causing stored honey to ferment, but also might serve as a vector of diseases. In addition, the small hive beetle has developed resistance to the pyrethroid and organophosphate insecticides registered for control of honeybee pests in the United States. The development of resistance in small hive beetle populations is a great concern to the beekeeping industry; thus, there is an urgent need for strategies to manage that resistance. Therefore, we used synergist probes to determine the mechanisms of resistance in a small hive beetle population to these insecticides. Our studies on the toxicity of insecticides alone or with the synergists piperonyl butoxide (PBO) and S,S,S,-tributyl phosphorotrithionate (DEF) suggested that mixed-function oxidases and esterases were the major resistance factors to these insecticides in a studied population of the small hive beetle. In contrast, there was no synergism with diethyl maleate (DEM), triphenyl phosphate (TPP) and formamidine. Therefore, glutathione-S-transferase, carboxylesterase and target site were not involved in insecticide resistance in the small hive beetle. Rotation of classes of insecticides (with different modes of action) and metabolic synergists were suggested for the development of successful resistance management programs. To the best of our knowledge, this is the first study of the mechanisms of resistance in small hive beetle populations in Florida and suggests an urgent need for alternative control strategies for these serious pests of honeybee colonies.