Western honey bees (Apis mellifera) are key to ecosystem services and are widely managed by beekeepers for pollination and honey production. Colonies of western honey bees (sometimes called European honey bees) have experienced a global decline, a major cause of which is the ectoparasitic mite Varroa destructor, against which the bees have limited innate defences. V. destructor feeds on both drones and workers, leading to high infestation levels, and the species is a viral reservoir transmitting pathogens like deformed wing virus. If untreated, colonies can collapse within a year. Because of the importance of pollination by this species to global food production, the infestation of Apis mellifera by these mites has been said to be the most serious disease of livestock of any species.
Traditional synthetic acaricides have previously been effective in controlling Varroa destructor levels, although resistance is now widespread. Furthermore, these chemical pesticides are harmful to both the bees and beekeepers. Because of this, beekeepers are increasingly shifting towards non-hard chemical controls to treat hives, which is great to see, as this aligns with target 1 of the European Commission’s ‘Farm to Fork’ strategy which aims to reduce synthetic pesticide use by 50% by 2030. RNA interference technology (RNAi) has been proposed as an alternate to chemical controls, as the dsRNAs utilised in this method have a limited impact on environmental and human health. RNAi is an intracellular mechanism of sequence-specific gene silencing; by delivering sequence-specific dsRNA that is complementary to mRNA within the target organism, the expression of gene transcripts encoding proteins important for survival and reproduction can be suppressed. Lab studies have shown for some time that RNAi has the potential to reduce mite infestations in honey bees, however studies on their use in the field are lacking.
In their recent article in Parasites & Vectors, Bortolin and colleagues incorporated dsRNA into western honey bees’ diet to investigate whether this method could reduce parasite load in the naturally unpredictable open field environment. The project was developed alongside beekeepers, who administered the dsRNA and whose feedback would be used to determine the feasibility of this potential method of parasite control in this highly managed species.
The team employed a multi-target approach, creating three dsRNA’s to target three genes important for mite survival. The A. mellifera genome was compared with that of V. destructor to ensure no off-target silencing of bee genes.
- VdACC-dsRNA: Targeting Acetyl-CoA-carboxylase (ACC), which is important for the biosynthesis of lipids.
- VdATPase-dsRNA: Targeting Na+/K+ ATPase, an enzyme which regulates membrane permeability and ion transport. This enzyme is also a natural target of some plant defence compounds which are toxic to mites.
- VdChit-dsRNA: Targeting chitinases which are involved in chitin degradation and reconstruction during moulting, inhibiting this gene can prevent the mite’s normal growth and development.
In the lab, Bortolin et al soaked mites in a solution with each of the dsRNAs to test the efficacy of this method in causing knockdown of their target genes. This method was shown to be suitable for gene silencing back in 2010, when Campbell and colleagues reported the first successful gene knockdown in any mite species. Bortolin and colleagues saw statistically significant gene silencing of targets 1 and 2, but not for the endochitinase gene.
The team aimed to test the dsRNAs in 50 colonies across five apiaries in northern Italy, in hives with a similar level of infestation. In the end, two colonies naturally collapsed, others were affected by nearby mosquito treatment, and one showed an anomaly in infestation dynamics, leaving 37 hives to analyse. Honey and pollen reserves, the number of adult bees, and brood size were measured at the start and end of the experiment to show that dsRNA did not affect the strength of the colonies. In Autumn 2022, bees were fed daily with a mixture of the three dsRNAs in a sucrose solution for 16 days, with a GFP-dsRNA and a 60% sucrose solution used as control groups. The effectiveness of RNAi technology was evaluated based on the variation in the level of phoretic infestation between days 1 and 37 (16 days after the end of administration).
The results of the field tests were promising, with most hives showing no signs of mite-transmitted disease. Furthermore, the mean phoretic infestation levels in the treated group were significantly lower than the GFP-dsRNA by 43%, and the 60% sucrose control by 33%. They also observed that the dsRNAs spread further through the hive by horizontal transfer due to trophallaxis, and that they were transferred across generations via royal jelly. This latter mechanism means the dsRNA could reach non-phoretic mites that are inside brood cells, which traditional chemicals typically cannot, showing an additional benefit to the use of RNAi technology for control of Varroa destructor. The team express the importance of a multi-target approach that inhibits genes involved in a variety of physiological processes for effective RNAi based mite control.
Aside from the measurable efficacy of using RNAi technology to control infestation of Apis mellifera, the techniques need to be transferable across all apiaries and must not be disruptive to beekeepers’ daily duties. It is important that projects on managed species like the western honey bee can be undertaken by non-experts and are maintainable long term. Bortolin and colleagues' choice to involve beekeepers in this field study adds to value of their research. Beekeepers confirmed that administering the dsRNA was manageable and did not disrupt their regular production activities. Furthermore, no PPE is required to administer the dsRNA, and RNA biopeptides have been classified as safe for use by the European Food Safety Authority. Bortolin et al have shown that RNAi technology is effective in the field as well as under laboratory conditions, and is a promising alternative to conventional chemical pesticides, which will benefit this declining bee species, as well as human and environmental health.