Identification of new viruses specific to the honey bee mite Varroa destructor

Abstract

Large-scale colony losses among managed Western honey bees have become a serious threat to the beekeeping industry in the last decade. There are multiple factors contributing to these losses but the impact of Varroa destructor parasitism is by far the most important, along with the contribution of some pathogenic viruses vectored by the mite. So far, more than 20 viruses have been identified infecting the honey bee, most of them RNA viruses. They may be maintained either as covert infections or causing severe symptomatic infections, compromising the viability of the colony. In silico analysis of available transcriptomic data obtained from mites collected in the USA and Europe as well as additional investigation with new samples collected locally allowed the description of three novel RNA viruses. Our results showed that these viruses were widespread among samples and that they were present in the mites and in the bees but with differences in the relative abundance and prevalence. However, we have obtained strong evidence showing that these three viruses were able to replicate in the mite, but not in the bee, suggesting that they are selectively infecting the mite. To our knowledge, this is the first demonstration of Varroa-specific viruses, which open the door to future applications that might help controlling the mite through biological control approaches.

A toolkit for studyingVarroagenomics and transcriptomics: Preservation, extraction, and sequencing library preparation

“…RNA sequence data were analyzed using Bowtie2 and Samtools, and then taxonomically classified using Kraken [14]. We compared this protocol with previously published RNA-seq libraries deposited on NCBI, which were mapped to the reference genome in a similar way (SRR8867385 [24], SRR5109825 & SRR5109827 [8], SRR533974 [25], SRR5760830 & SRR5760851 [26], SRR8100122 & SRR8100123, SRR5377267 & SRR5337268, and SRR8864012 [27]).…”

“…Raw sequence data are available under DDJB/NCBI BioProject PRJDB10252. To compare our results using the following sequences obtained from sequence read archive NCBI: SRR8867385 [24]: RNA-seq of pooled V. destructor adults https://www.ncbi.nlm.nih.gov/sra/?term=SRR8867385 SRR5109825 & SRR5109827 [8]: Small RNA of Varroa destructor : South Africa https://www.ncbi.nlm.nih.gov/sra/?term=SRR5109825 SRR533974 [25]: RNA-seq of Varroa destructor https://www.ncbi.nlm.nih.gov/sra/?term=SRR533974 SRR5760830 & SRR5760851 [26]: GSM2686390: R245-9; Varroa destructor; RNA-Seq https://www.ncbi.nlm.nih.gov/sra/?term=SRR5760830 SRR8100122 & SRR8100123: RNA-seq of Varroa destructor https://www.ncbi.nlm.nih.gov/sra/?term=SRR8100122 SRR5377267 & SRR5337268: VD_FemaleFound1 https://www.ncbi.nlm.nih.gov/sra/?term=SRR5377267 SRR8864012 [27]: RNA-seq of Varroa destructor adult female https://www.ncbi.nlm.nih.gov/sra/?term=SRR8864012 …”

“…SRR8864012 [27]: RNA-seq of Varroa destructor adult female https://www.ncbi.nlm.nih.gov/sra/?term=SRR8864012…”

Covert RNA viruses in medflies differ in their mode of transmission and tissue tropism

“…Then, the levels of the 13 covert RNA viruses previously described in the medfly, as well as the expression of the medfly gene encoding the ribosomal protein L23a (Genbank acc: XM004518966), were assessed through RT-qPCR (CFX96 Touch Real-Time PCR Detection System, BioRad) using specific primers (Hernández-Pelegrín et al, 2022;Llopis-Giménez et al, 2017) (Table S1). The normalized viral RNA levels refer to the levels of viral molecules in relation to the levels of the reference gene L23a, and were calculated by comparing Ct values of RNA viruses and L23a, after adjusting for primer efficiency (Herrero et al, 2019).…”

“…Three microliter of the cDNA was used for the PCR reaction with DreamTaq DNA polymerase (Thermo Fisher Scientific), using a forward primer annealing to the overhang of the primer used to make the cDNA and a reverse primer specific for each virus (Table S1). The PCR reaction was performed as follows: one step of 95 °C for five minutes, 35 cycles of annealing at 52 °C for 30 s, and elongation at 72 °C for one minute (Herrero et al, 2019). PCR results were visualized in 1% agarose gels.…”