Year 11 | 12 November 2019 | firstname.lastname@example.org
Researchers from the Government’s National Bee Unit and Aberdeen University have worked out how to "silence" natural functions in the mites genes to make them self destruct
The parasitic mite Varroa destructor is considered the major pest of the European honey bee (Apis mellifera) and responsible for declines in honey bee populations worldwide.
The mite causes damage by feeding on the haemolymph of both the developing bee within brood cells and the adult bee. Mites also transmit a variety of pathogens, most notably deformed wing virus. In terms of both the number of enterprises affected and the impact of global food production, varroasis is arguably the most serious disease of livestock in any species. Previous control of V. destructor by chemical treatment is increasingly ineffective due to the development of widespread resistance in mites to the limited available acaricides
There is an urgent need to harness modern molecular techniques for research into the biology and, ultimately, the control of this non-model organism, V. destructor. RNA interference (RNAi) is a gene silencing technique that is becoming an ever more powerful tool in investigating the functional role of specific genes that may be potential targets for chemotherapeutic intervention. The RNAi mechanism involves the in vivo production of small interfering RNA molecules (siRNAs) from larger introduced double-stranded RNA (dsRNA). siRNA molecules target and destroy specific mRNA, silencing the target gene at the post-transcriptional stage.
Various less invasive techniques of introducing dsRNA into V. destructor were investigated that would improve speed, ease of operation, increase throughput, could be performed without expensive equipment and, most importantly, increase survivability of the treated groups. Topically applied dsRNA to immobilized mites for several hours failed to induce gene knockdown, presumably because the dsRNA was unable to cross the cuticle. We tried the addition of the detergent Triton X-100 to break the surface tension of the water allowing the dsRNA solution to become more in contact with the cuticle rather than held off it by the fine hair layer. However, Triton X-100 caused rapid death of the mites. Next we assessed total immersion of mites as a possible route for dsRNA administration as was previously successfully performed with salmon lice in seawater . Mites immersed in 0.2% Triton X-100 died rapidly and, surprisingly, mites in distilled water died within a few hours. We postulated that the hypo-osmolarity of the distilled water caused an osmotic influx of water into the mite tissues and this was exacerbated in the presence of Triton X-100 which may have allowed water to enter through the spiracles more rapidly with the reduced water tension.
Increasing the osmotic concentration of the immersion solution by using 0.9% NaCl allowed the mites to remain under solution overnight at 4°C with 75-80% survival and achieved excellent gene silencing. It is not known what is the site of dsRNA entry into the mite, but this is of considerable interest and importance. We presumed entry to be either by ingestion or though the tracheal system via the spiracles. Adult female V. destructor enter the prepupal cells 24 - 48 h before the cell is capped and they remain hidden from vigilant nurse bees by submerging themselves in the liquid brood food with the peritremes surrounding the spiracles protruding from the liquid enabling the mites to breathe. It may be possible to improve survivability and, possibly increase gene knockdown efficacy, if the osmolality of the immersion solution were increased to that of liquid brood food that is presumed to be greater than that of physiological saline (0.9% NaCl). Indeed, further optimisation of this approach could be attempted, if thought necessary, by altering immersion temperature and time, but the method presented here is a good starting point.
In conclusion, scientists have established dsRNA knockdown in the critically important bee parasite, V. destructor, using a mu-class GST as the candidate gene. A simple, inexpensive, high throughput method of dsRNA administration was established with low mortality rates for V. destructor.
The dsRNAi approach will allow researchers to make full use of the V. destructor gene sequences becoming available to investigate biological questions in this important parasite, notably to develop control strategies in the era when current suitable acaricides are no longer effective due to widespread resistance. Peculiarly to Varroa, because of the relatively close relatedness between the parasite and host (Varroa and bee) it is especially difficult to develop pesticides effective against the Varroa, but innocuous to the bee. In this respect, the specificity of dsRNAi may allow targeting of V. destructor genes as a control strategy, as suggested for other insect pests.
Dr Giles Budge from National Bee Unit, part of the Food and Environment Research Agency (Fera), said:
“This cutting edge treatment is environmentally-friendly and poses no threat to the bees. With appropriate support from industry and a rigorous approval process, chemical-free medicines could be available in five to ten years.”
Not only was gene knockdown achieved at the transcriptional step (mRNA) but this was also apparent at the translational step (protein) with decreased GST catalytic activity.
by Graziano Alderighi
03 january 2011, Technical Area > Science News