Effects of Deformed Wing Virus-Targeting dsRNA on Viral Loads in Bees Parasitised and Non-Parasitised by Varroa destructor

Abstract

The Varroa destructor mite is a devastating parasite of honey bees; however the negative effects of varroa parasitism are exacerbated by its role as an efficient vector of the honey bee pathogen, Deformed wing virus (DWV). While no direct treatment for DWV infection is available for beekeepers to use on their hives, RNA interference (RNAi) has been widely explored as a possible biopesticide approach for a range of pests and pathogens. This study tested the effectiveness of three DWV-specific dsRNA sequences to lower DWV loads and symptoms in honey bees reared from larvae in laboratory mini-hives containing bees and varroa. The effects of DWV-dsRNA treatment on bees parasitised and non-parasitised by varroa mites during development were investigated. Additionally, the impact of DWV-dsRNA on viral loads and gene expression in brood-parasitising mites was assessed using RNA-sequencing. Bees parasitised during development had significantly higher DWV levels compared to non-parasitised bees. However, DWV-dsRNA did not significantly reduce DWV loads or symptoms in mini-hive reared bees, possibly due to sequence divergence between the DWV variants present in bees and varroa and the specific DWV-dsRNA sequences used. Varroa mites from DWV-dsRNA treated mini-hives did not show evidence of an elevated RNAi response or significant difference in DWV levels. Overall, our findings show that RNAi is not always successful, and multiple factors including pathogen diversity and transmission route may impact its efficiency.

Keywords: RNA interference; RNA-seq; biopesticide; deformed wing virus; double-stranded RNA; varroa destructor.

Figure 1

A mini-frame and mini-hive used in experiments. (a) Two mini-frames adhered together to fit as a single frame inside a hive for acquiring brood. Mini-frames were separated for one mini-frame of larvae to be introduced to the mini-hive. (b) A mini-hive with two plexiglass viewing windows in the front, and two chambers separated by a plexiglass partition to fit a mini-frame in the left chamber and the treatment pouch in the right foraging chamber.

Figure 2

Relative DWV levels of uncapped bees from sugar, non-specific dsRNA and DWV-dsRNA treated mini-hives. Coloured boxes show different parasitism phenotypes: non-parasitised (pink), parasitised with wing deformities (orange) and parasitised with normal wings (green). Raw data points (i.e., Relative DWV level of individual bees) for each box plot are overlayed. Upper and lower hinges of the boxes show 75% and 25% quantiles, respectively, separated by black lines showing the median. Upper and lower whiskers extend to 1.5* interquartile range.

Figure 3

Effect of treatment on the proportion of uncapped bees with wing deformities from each mini-hive. Bars show mean proportion of uncapped bees with wing deformities for sugar water (n = 4), non-specific dsRNA (n = 4), and DWV-dsRNA (n = 5) treated mini-hives with error bars showing standard deviation. No significant difference in proportion of bees with deformed wings was found between treatments (df = 2, chi-squared = 1.4251, p = 0.49).

Figure 4

Multidimensional scaling (MDS) plot of expression profile (TMM normalised gene counts) of candidate RNAi-associated genes for each mite sample. Each triangle denotes an individual mite sample with colours indicating which treatment group the sample belongs to: Non-specific dsRNA (orange), sugar control (green) or DWV-dsRNA (purple). Dimensions one and two are shown and account for 92% of the variation in the data.

Figure 5

Relative abundance of deformed wing virus loads represented as transcripts per million (TPM) in varroa mite RNA-seq samples from sugar water, non-specific dsRNA and DWV-dsRNA-treated mini-hives. Upper and lower hinges of the boxes show 75% and 25% quantiles, respectively. Whiskers extend to 1.5* interquartile range. Raw data is overlaid to show TPM values for each sample (black points). ANOVA results showed no significant difference in DWV loads in varroa mites between treatment groups (df = 2, F = 2.883, p = 0.09).

Figure 6

Unrooted Bayesian phylogenetic tree of DWV-A genome sequences, labelled with NCBI accession IDs. Branch labels show posterior probabilities and are coloured by country of isolate sample origin. The DWV contig identified in varroa samples from our study is shown in the green box.