Whitten et al. (2016) in a recent issue of the Proceedings of the Royal Society of London B use natural bacterial symbionts to silence their insect host’s genes. This ‘symbiont-mediated RNAi’ method could be an important advance for insect molecular biology research because RNAi is a major method for manipulating gene expression.
RNAi is used in basic insect research to investigate gene function and as a chemical-free insect control method.
In non-model insects a big challenge in using RNAi is the delivery of synthetic dsRNA or siRNA (the triggers for RNAi). Up until now there were two major delivery routes: injection directly into the haemolymph or feeding. These delivery methods are problematic for studies on long term phenotypic changes or insect control methods because knockdown of gene expression is transient. Furthermore, repeated injection or feeding of the insect is inefficient and can be harmful.
In contrast, the symbiont-mediated RNAi method described by Whitten et al. continually delivers dsRNA to the host, offering a new method for long term RNAi studies.
In order to use the authors’ RNAi method one must be able to obtain culturable bacterial symbionts from the insect. Whitten et al. designed dsRNA expression cassettes (for any target gene) to drive constitutive expression of the dsRNA. They used electroporation to introduce the cassette into Rhodococcus rhodii and BFo2, gut symbionts of Rhodnius prolixus and Frankliniella occidentalis, respectively. A crucial feature of the authors’ method was that the bacteria were modified to be deficient in RNaseIII. This modification prevented RNA degradation and permitted stable synthesis of dsRNA in the bacteria.
The authors resuspended the recombinant bacteria in a food appropriate for the host insect species and the insects were fed only once or twice. They showed that the recombinant bacteria established themselves in the insect’s gut and the knockdown of gene expression spread to tissues beyond the gut (systemic RNAi).
Whitten et al. demonstrated that when recombinant R. rhodnii colonised its insect host this had no fitness effect on the host and persisted in the host’s gut for over 250 days after delivery. When R. rhodnii expressed vitellogenin dsRNA it caused knockdown of vitellogenin in the insect host. These insects had a reduction in fertility.
Further, RNAi was horizontally transmitted between generations of R. prolixus because R. rhodnii is horizontally transmitted via contaminated faeces. R. prolixus’ long lifespan increases the chance that horizontal transmission occurs. Interestingly, this horizontal transmission means that if the recombinant R. rhodnii targets a gene that reduces R. prolixus fitness it might still efficiently spread in the R. prolixus population.
Whitten et al. also demonstrated that when BFo2 expressed alpha-tubulin dsRNA it caused knockdown of alpha-tubulin in the insect host. These insects had a high mortality rate.
Insect-Symbiont Systems to Investigate Symbiont-Mediated RNAi.
– Disease vector
|Frankliniella occidentalis (western flower thrips)
– Agricultural pest & disease vector
|Type of food (with symbiont added) insect ingests||Blood||Artificial feeding mixture (LB, sucrose, NaCl &
methylene blue to identify when insect has fed)
|Bacterial symbiont||Species||Rhodococcus rhodnii||BFo2 (species unknown)|
|Gram stain test||Positive||Negative|
|Type of symbiont||Obligate (necessary for normal insect development)||Facultative|
|Location in insect||Midgut||Hindgut & posterior midgut|
|dsRNA||Expression cassette||Stably integrated into symbiont’s chromosome||Plasmid|
Any insect with a culturable symbiont is a great candidate for symbiont-mediated RNAi so this RNAi method will be of interest to a large section of the insect research community.
Symbiont-mediated RNAi is a molecular biology method that has a double layer of specificity to an insect species. First, dsRNA is designed to target a sequence found only in that species. Second, each bacterial symbiont has a limited insect host range.
It is interesting to note that the symbiont-mediated RNAi method has an associated patent (WO2013117910 A1 ).
Whitten MMA, Facey PD, Del Sol R, Fernandez-Martınez LT, Evans MC, Mitchell JJ, Bodger OG, Dyson PJ. 2016 Symbiont-mediated RNA interference in insects. Proc. R. Soc. B 283: 20160042. http://dx.doi.org/10.1098/rspb.2016.0042