Analysis of X-ray irradiation effects on the mortality values and hemolymph immune cell composition of Apis mellifera and its parasite, Varroa destructor.

Varroa destructor is one of the most destructive enemies of the honey bee, Apis mellifera all around the world. Several control methods are known to control V. destructor, but the efficacy of several alternative control methods remains unexplored. Irradiation can be one of these unknown solutions but before practical application, the effectiveness, and the physiological effects of ionizing radiation on the host and the parasite are waiting to be tested. Therefore, the objective of our study was to investigate the effects of different doses (15, 50, 100, and 150 Gy) of high-energy X-ray irradiation through mortality rates and hemocyte composition changes in A. mellifera workers and record the mortality rates of the parasite. The mortality rate was recorded during short-term (12, 24, and 48 h) and long-term periods (3, 6, 12, 18, and 24d). The sensitivity of the host and the parasite in case of the higher doses of radiation tested (50, 100, and 150 Gy) been demonstrated by total mortality of the host and 90 % of its parasite has been observed on the 18th day after the irradiation. V. destructor showed higher sensitivity (1.52-times higher than the adult honey bee workers) at the lowest dose (15 Gy). A. mellifera hemocytes were influenced significantly by radiation dosage and the elapsed time after treatment. The higher radiation doses increased plasmatocyte numbers in parallel with the decrease in prohemocyte numbers. On the contrary, the numbers of granulocytes and oencoytes increased in the treated samples, but the putative effects of the different dosages on the recorded number of these hemocyte types could not be statistically proven. In summary, based on the outcome of our study X-ray irradiation can be deemed an effective tool for controlling phoretic V. destructor. However, further research is needed to understand the physiological response of the affected organisms.

Varroa destructor mite population dynamics in africanized honeybee (Apis mellifera) colonies in a semi-arid region.

Most published data on mite infestation rates in semi-arid regions have been collected over only 3 or 4 months during a specific period of the year. Therefore, the aim of this study was to observe parasite-host dynamics of hygienic and non-hygienic Africanized bee colonies considering environmental factors that may influence Varroa destructor mite infestation rates in a semi-arid region. To this end, the brood puncture method was applied to 37 colonies, forming two groups, namely G1, encompassing 16 hygienic colonies, and G2, comprising 21 non-hygienic colonies. After forming the groups, 300 worker bees from each colony were examined monthly for mite infestations and the data were correlated with climatological records. The monthly infestation average was considered low, below 10%, except in November, when it reached 12.19% ± 6.45. No statistically significant difference was observed for inter-group infestation rates (P > 0.05). When mite infestation rates were associated with climatic variables, they were linked to colony losses (32%) due to swarming. No significant correlations between hygienic behaviour and parasite infestation rates were noted. Nonetheless, these results support the idea that there is no need to apply acaricides for V. destructor control in Brazil.

The Effectiveness of Varroa destructor Infestation Classification Using an E-Nose Depending on the Time of Day.

Honey bees are subject to a number of stressors. In recent years, there has been a worldwide decline in the population of these insects. Losses raise a serious concern, because bees have an indispensable role in the food supply of humankind. This work is focused on the method of assessment of honey bee colony infestation by Varroa destructor. The approach allows to detect several categories of infestation: "Low", "Medium" and "High". The method of detection consists of two components: (1) the measurements of beehive air using a gas sensor array and (2) classification, which is based on the measurement data. In this work, we indicate the sensitivity of the bee colony infestation assessment to the timing of measurement data collection. It was observed that the semiconductor gas sensor responses to the atmosphere of a defined beehive, collected during 24 h, displayed temporal variation. We demonstrated that the success rate of the bee colony infestation assessment also altered depending on the time of day when the gas sensor array measurement was done. Moreover, it was found that different times of day were the most favorable to detect the particular infestation category. This result could indicate that the representation of the disease in the beehive air may be confounded during the day, due to some interferences. More studies are needed to explain this fact and determine the best measurement periods. The problem addressed in this work is very important for scheduling the beekeeping practices aimed at Varroa destructor infestation assessment, using the proposed method.

A long-term field study on the effects of dietary exposure of clothianidin to varroosis-weakened honey bee colonies.

Clothianidin is a commonly used systemic insecticide in seed treatments. Residues of clothianidin can occur in nectar and pollen as a result of within-plant-translocation. Foraging bees can collect contaminated nectar or pollen. Concerns have been brought forward that exposure to pesticide residues might affect colonies especially if they are weakened by varroosis. However, there are few scientific studies investigating such multiple-stressor scenarios in the context of the entire colony. To close this gapa field trial with 24 colonies was set up. The study design comprised four groups of six colonies each fed with uncontaminated sugar syrup ('C0'), or syrup spiked with 10 µg L-1 clothianidin ('C10'), 50 µg L-1 clothianidin ('C50') or 200 µg L-1 clothianidin ('C200'). C10 represented a residue concentration that may exceptionally occur and therefore a worst-case scenario, the higher dietary concentrations exceed and do not reflect fieldrealistic levels. A substantial load of 8 mites of Varroa destructor per ten gram bees in autumn was adjusted. The colonies were followed up for 328 days. The amount of brood and the strength of each colony were regularly assessed. Colony health, bee mortality, overwintering success, hive weights, and levels of in-hive residues were determined. Varroosis turned out to be the significant key factor for the endpoint colony strength. Clothianidin did not have a statistically significant impact on C0, C10 and C50 colonies. No statistical evidence was found for an interaction between varroosis andexposure to clothianidin.

Genetic variability and pyrethroid susceptibility of the parasitic honey bee mite Varroa destructor (Acari: Varroidae) in Iran.

The ectoparasitic honey bee mite Varroa destructor Anderson & Trueman (Acari: Varroidae) is one of the major concerns for worldwide beekeeping. The use of synthetic pyrethroids for controlling the mite was among the most popular treatments until resistance evolved in the mid 1990's. In Iran, beekeepers are dealing with the parasite and they also used pyrethroids for controlling the mite for a long time. After the evolution of resistance to pyrethroids, they based mite control mostly on treatments with amitraz, organic acids and several management practices. Here we conducted a comprehensive characterization of V. destructor populations parasitizing Apis mellifera in Iran. We determined the genetic variability of mites collected from 28 localities distributed throughout the country. The haplotype of V. destructor was determined by PCR-RFLP, analyzing a fragment of the mitochondrial cox1 gene. It was found that only the Korean haplotype was present in samples from all localities. DNA fragments from cox1, atp6, cox3 and cytb mitochondrial genes were sequenced and the results showed that all samples were identical to the K1-1 or the K1-2 V. destructor haplotypes. Moreover, as it has been reported that resistance to pyrethroids in V. destructor is associated with mutations at position 925 of the voltage-gated sodium channel, a TaqMan®-based allelic discrimination assay was conducted to genotype the mites collected. The results showed that all the mites tested were homozygous for the wild-type allele and, therefore, susceptible to treatment with pyrethroids.

Use of costic acid, a natural extract from Dittrichia viscosa, for the control of Varroa destructor, a parasite of the European honey bee.

Costic acid has been isolated from the plant Dittrichia viscosa and its efficacy against Varroa destructor, a parasite of Apis mellifera, the European honey bee, has been studied. Costic acid exhibited potent in vivo acaricidal activity against the parasite. Initial experiments showed that the compound is not toxic for human umbilical vein endothelial cells (HUVEC) at concentrations of up to 230 micromolar (µM), indicating that costic acid could be used as a safe, low-cost and efficient agent for controlling varroosis in honey bee colonies.

Prevalence of Varroa destructor in Honeybee (Apis mellifera) Farms and Varroosis Control Practices in Southern Italy.

The majority of honeybee farms in industrialized countries currently base their Varroa destructor control programs on the use of acaricides in conjunction with other management practices. However, the outcomes of these practices are often misunderstood and have only been studied to a limited extent. Better yields are guaranteed by having hives with low infection levels in the spring. Therefore, it is crucial to understand which beekeeping practices can result in increased control effectiveness. This study aimed to analyze the potential effects of environmental factors and beekeeping practices on the dynamics of V. destructor population. Experimental evidence was obtained by interpolating percentage infestation data from diagnoses conducted on several apiaries in the Calabria region (Southern Italy) with data acquired from a questionnaire on pest control strategies. Data on climatic temperature during the different study periods were also taken into account. The study was conducted over two years and involved 84 Apis mellifera farms. For each apiary, the diagnosis of infestation was made on a minimum of 10 hives. In total, 840 samples of adult honeybees were analyzed in the field to determine the level of infestation. In 2020, 54.7% of the inspected apiaries tested positive for V. destructor, and in 2021, 50% tested positive, according to a study of the field test findings (taking into account a threshold of 3% in July). A significant effect of the number of treatments on parasite prevalence was found. The results showed a significant reduction in the infestation rate in apiaries that received more than two treatments each year. Furthermore, it was shown that management practices, such as drone brood removal and frequent queen replacement, have a statistically significant impact on the infestation rate. The analysis of the questionnaires revealed some critical issues. In particular, only 50% of the interviewed beekeepers diagnosed infestation on samples of adult bees, and only 69% practiced drug rotation. In conclusion, it is only possible to maintain the infestation rate at an acceptable threshold by implementing integrated pest management (IPM) programs and using good beekeeping practices (GBPs).

Molecular Detection and Differentiation of Arthropod, Fungal, Protozoan, Bacterial and Viral Pathogens of Honeybees.

The honeybee Apis mellifera is highly appreciated worldwide because of its products, but also as it is a pollinator of crops and wild plants. The beehive is vulnerable to infections due to arthropods, fungi, protozoa, bacteria and/or viruses that manage to by-pass the individual and social immune mechanisms of bees. Due to the close proximity of bees in the beehive and their foraging habits, infections easily spread within and between beehives. Moreover, international trade of bees has caused the global spread of infections, several of which result in significant losses for apiculture. Only in a few cases can infections be diagnosed with the naked eye, by direct observation of the pathogen in the case of some arthropods, or by pathogen-associated distinctive traits. Development of molecular methods based on the amplification and analysis of one or more genes or genomic segments has brought significant progress to the study of bee pathogens, allowing for: (i) the precise and sensitive identification of the infectious agent; (ii) the analysis of co-infections; (iii) the description of novel species; (iv) associations between geno- and pheno-types and (v) population structure studies. Sequencing of bee pathogen genomes has allowed for the identification of new molecular targets and the development of specific genotypification strategies.

Residual Tau-Fluvalinate in Honey Bee Colonies Is Coupled with Evidence for Selection for Varroa destructor Resistance to Pyrethroids.

Varroa destructor is considered one of the most devastating parasites of the honey bee, Apis mellifera, and a major problem for the beekeeping industry. Currently, the main method to control Varroa mites is the application of drugs that contain different acaricides as active ingredients. The pyrethroid tau-fluvalinate is one of the acaricides most widely used in beekeeping due to its efficacy and low toxicity to bees. However, the intensive and repetitive application of this compound produces a selective pressure that, when maintained over time, contributes to the emergence of resistant mites in the honey bee colonies, compromising the acaricidal treatments efficacy. Here we studied the presence of tau-fluvalinate residues in hives and the evolution of genetic resistance to this acaricide in Varroa mites from honey bee colonies that received no pyrethroid treatment in the previous four years. Our data revealed the widespread and persistent tau-fluvalinate contamination of beeswax and beebread in hives, an overall increase of the pyrethroid resistance allele frequency and a generalized excess of resistant mites relative to Hardy-Weinberg equilibrium expectations. These results suggest that tau-fluvalinate contamination in the hives may seriously compromise the efficacy of pyrethroid-based mite control methods.

Study of Morphological Features in Pre-Imaginal Honey Bee Impaired by Varroa destructor by Means of Computer Tomography.

The honey bee (Apis mellifera L. 1778) is an essential element in maintaining the diversity of the biosphere and food production. One of its most important parasites is Varroa destructor, Anderson and Trueman, 2000, which plays a role in the vectoring of deformed wing virus (DWV) in honey bee colonies. Our aim was to measure the potential morphometric changes in the pre-imaginal stage of A. mellifera caused by varroosis by means of computed tomography, hence supplying evidence for the presumable role that V. destructor plays as a virus vector. Based on our results, the developmental disorders in honey bees that ensued during the pre-imaginal stages were evident. The total-body length and abdomen length of parasitized specimens were shorter than those of their intact companions. In addition, the calculated quotients of the total-body/abdomen, head/thorax, and head/abdomen in parasitized samples were significantly altered upon infestation. In our view, these phenotypical disorders can also be traced to viral infection mediated by parasitism, which was confirmed by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Capitalizing on a non-destructive method, our study reveals the deformation of the honey bee due to mite parasitism and the intermediary role this pest plays in viral infection, inside the brood cell.

Spatial and temporal analysis of varroosis from 2005 to 2018.

This paper describes the global distribution and temporal trend of Varroa spp. during 13 years (2005-2018) using the information retrieved from the OIE World Animal Health Information Database (WAHIS). During the period of study 53.4% of the countries reported the presence of the mite at least once. Countries were classified in five categories: 22% as Enzootic, 18% as Epizootic, 9% as Free, 12% and 26% respectively as disease Present or Absent at least once since 2005. Twelve percent of the countries were not able to provide any information on the presence of the disease. The average percentage of countries reporting the disease present was stable along the study, but it was observed a statistically significant increasing trend in the number of outbreaks reported per year. The number of outbreaks were different among the climate regions. Based on the seasonality analysis, the second semester of each year was the period characterized by the highest number of outbreaks reported.

Assessment of listing and categorisation of animal diseases within the framework of the Animal Health Law (Regulation (EU) No 2016/429): infestation with Varroa spp. (varroosis).

Infestation with Varroa spp. (varroosis) has been assessed according to the criteria of the Animal Health Law (AHL), in particular criteria of Article 7 on disease profile and impacts, Article 5 on the eligibility of varroosis to be listed, Article 9 for the categorisation of varroosis according to disease prevention and control rules as in Annex IV and Article 8 on the list of animal species related to varroosis. The assessment has been performed following a methodology composed of information collection and compilation, expert judgement on each criterion at individual and, if no consensus was reached before, also at collective level. The output is composed of the categorical answer, and for the questions where no consensus was reached, the different supporting views are reported. Details on the methodology used for this assessment are explained in a separate opinion. According to the assessment performed, it is inconclusive whether varroosis can be considered eligible to be listed for Union intervention as laid down in Article 5(3) of the AHL because there was no full consensus on the criterion 5 A(v). Consequently, the assessment on compliance of varroosis with the criteria as in Annex IV to the AHL, for the application of the disease prevention and control rules referred to in Article 9(1), and which animal species can be considered to be listed for varroosis according to Article 8(3) are also inconclusive.