RNAi in Beetles and Leps – Why the Difference?

on. Image credit: www.biocomicals.com by Alper Uzun, PhD

Gene Silencing
Image credit: www.biocomicals.com by Alper Uzun, PhD

It is well established that the RNAi response of Coleoptera and Lepidoptera differ greatly but less is known about the basis for these differences.  Skukla et al (2016) systematically investigated this question and report their insights in a recent paper in RNA Biology.

The authors investigated the RNAi response of two Coleopteran cell lines and two Lepidopteran cell lines as well as whole insects.

Coleopteran cell lines were sensitive to gene silencing following incubation with specific dsRNAs (3 genes were used as targets).  Lepidoptera cell lines showed no significant gene silencing under identical conditions.  One possible reason might be differential uptake of dsRNA by cells.  Using fluorescein-labeled dsRNA (~250 bp, GFP) uptake could be detected after just 30 minutes into both Coleopteran and Lepidopteran cell lines.  Using pH sensitive labels the authors found that dsRNA taken up by Lepidopteran cells in vivo accumulates in endocytic compartments of the cells.  The authors performed experiments to show that fluorescent signals observed were inside cells and not attached to the outside of the cells.

Simplified Illustration of RNAi

Simplified Illustration of RNAi

Although Lepidopteran cells take up dsRNA, did they process the dsRNA into siRNAs.  The short answer is “no”, according to the authors and this conclusion is based on experiments in which dsRNA was labeled with P32 and after being taken up by cells total RNA was recovered and size fractionated by electrophoresis.  RNA from Coleopteran cells had evidence of P32 labeled siRNA based on size while labeled siRNA could not be found among the total RNAs recovered from Lepidopteran cells.

Colorado potato beetle.jpg

Leptinotarsa decimlineata. Image Credit: Scott Bauer, USDA ARS

Labelled dsRNA was also used to assess the kinetics of degradation following dsRNA injection into the hemocoel of Lepidoptera (Heliothis verescens) and Coleoptera (Leptinotarsa decimlineata).  dsRNA was degraded rapidly in the hemolymph of Lepidoptera and dsRNA that was taken up by other tissues before degradation was not processed into siRNAs.  This is quite different from what was observed in Coleoptera.  dsRNA degradation was observed in the hemolymph of Coleoptera but the rate of degradation was much slower.

Similar results were obtained after feeding dsRNA to H. verescens and L. decimlineata.

File:Heliothis virescens.jpg

Heliothis virescens. Image credit: R.J. Reynolds Tobacco Company Slide Set, R.J. Reynolds Tobacco Company, Bugwood.org

This is an interesting report that enlightens us about the differences in RNAi in Coleoptera and Lepidoptera.  In addition to dsRNA degradation, it seems that a major difference is in the fate of dsRNA once taken up by Lepidoptera and Coleoptera.  In the case of Coleoptera the dsRNA is processed into siRNA whereas this appears not to be the case in Lepidoptera.

 

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