Varroa destructor and Varroa-transmitted viruses: impact on environmentally monitored apiaries.

INTRODUCTION World-wide Honey bee population declines and colony collapse is caused by many factors including diseases and climate change serving as the most important (Goulson et al., 2015. Science 347: 1255957;). Acute Bee Paralysis Virus (ABPV), Deformed Wing Virus A and B (DWV-A and DWV-B) are among six RNA viruses with highest adverse effects on European honey bee (Apis mellifera) health (Schurr, et al., 2019. J. Virol. Methods 270, 70–78;). While persistence of these pathogens within colonies is primarily maintained through vertical transmission in honey bees, it is furthermore enhanced via horizontal transmission mainly by ubiquitous A. mellifera ectoparasite Varroa destructor (V. destructor) (Chen et al., 2006. Appl. Environ. Microbiol. 72, 606-611;). Since such virus are able to develop infections with no visible clinical symptoms; monitoring their viral load titer by accurate (precise and specific) RT-qPCR is critical in the control and prevention of honey bees’ death (Amiri et al., 2015. PLOS ONE 10, e0140272). In this study, we conducted a molecular survey by investigating the prevalence of V. destructor-borne viral diseases on a chronological basis to find possible associations between clinical signs’ demonstrations, V.destructor prevalence and anti-parasitic treatments. MATERIALS AND METHODS Honey bees and V. destructor mites were collected from 40 colonies of two separate apiaries in the province of Cuneo (Piedmont region, Italy.) between March and October 2021. 140 honey bee (~200 μg of homogenate per each hive) and 104 V. destructor samples (pools varying in mite number per each hive) were used for total RNA extraction by Tri Reagent® (Sigma-Aldrich; Merck KGaA) and blackPREP Tick DNA/RNA Kit (Analytik Jena, Germany) respectively. cDNA synthesis was performed using QuantiTect® Reverse Transcription Kit (Qiagen, Hilden, Germany) in a final volume of 25 μl from 1 μg and 15ng of every honey bee and V. destructor mite RNA samples respectively. In order to investigate the presence and prevalence of ABPV, DWV-A and DWV-B viruses, qualitative PCR and Two-Step quantitative PCR were performed using specific primer and probes on both bee and mite samples. RESULTS Until now, more than 200 honey bee samples are investigated for DWV-A and ABPV viruses; among them, 11 samples from different sampling periods were tested positive for DWV-A while only one sample was positive for ABPV suggesting a prevalence in line with results reported in other studies. COCLUSION Our results while demonstrating the presence of both DWV-A and ABPV viruses are also suggestive for an association between viral prevalence and anti-parasitic treatments efficacy aiming at V. destructor elimination. Moreover, increased positive results in samples collected following anti-parasitic treatment may be informative of probable resistance to such agents in V. destructor mites.