In the silkmoth, B. mori (Lepidoptera), no potent RNAi response is induced following injection or feeding of dsRNA (2). This observation prompted us to evaluate factors that could contribute to the (lack of) RNAi efficiency in the silkmoth, such as:
Expression pattern of basic intracellular RNAi factors
Expression studies suggested that the absence of R2D2 expression, an essential co-factor of Dicer-2 and Ago-2, may play a role in the refractoriness of the systemic RNAi response in Bombyx (3). However, functional studies indicate that the intracellular RNAi machinery can work efficiently in the absence of R2D2 in silkmoth-derived Bm5 cells (4).
Expression of dsRNA-degrading enzymes
It was demonstrated that a non-specific DNA/RNA nuclease (“dsRNase”) has a broad expression in many different tissues and is capable both to degrade dsRNA intracellularly and to interfere with dsRNA-mediated gene silencing (5).
DsRNA as (non-specific) “pathogen-activated molecular pattern” (PAMP)
It was observed that injection of dsRNA into the hemolymph induces the expression of genes of the RNAi machinery (Dicer-2, Ago-2) and dsRNase in the midgut, while the expression of the innate immune Toll9-1 receptor was inhibited (6). Ectopic expression of Toll9-1 receptor in Bm5 cells was observed to modulate the response against the PAMPs dsRNA and lipopolysaccharide (LPS) with respect to the expression of the RNAi machinery and innate immunity genes (7).
Persistent RNA virus infection
It is hypothesized that persistent virus infection can severely affect the function of the RNAi machinery according to several different molecular mechanisms (8). Next-generation sequencing was used to evaluate the transcriptome and small RNA response during infection of silkmoth larvae with cytoplasmic polyhedrosis virus (CPV), characterized with a segmented dsRNA genome (Cypovirus, Reoviridae). Analysis reveals a unique response to dsRNA virus infection in the silkmoth, with no overlap with the classical innate immune pathways triggered by bacteria or fungi (9).
1. Swevers, L. and Smagghe, G. (2012). Use of RNAi for control of insect crop pests. In:” Arthropod-Plant Interactions, Novel Insights and Approaches for IPM”, Progress in Biological Control, Volume 14. G. Smagghe & I. Diaz (Eds.), pp 177-197. Springer-Verlag, Dordrecht.
2. Terenius, O., Papanicolaou, A., Garbutt, J.S., Eleftherianos, I., Huvenne, H., Sriramana, K., Albrechtsen, M., An, C., Aymeric, J.-L., Barthel, A., Bebas, P., Bitra, K., Bravo, A., Chevalier, F., Collinge, D.P., Crava, C.M., de Maagd, R.A., Duvic, B., Erlandson, M., Faye, I., Felföldi, G., Fujiwara, H., Futahashi, R., Gandhe, A.S., Gatehouse, H.S., Gatehouse, L.N., Giebultowicz, J., Gómez, I., Grimmelikhuijzen, C.J., Groot, A.T., Hauser, F., Heckel, D.G., Hegedus, D.D., Hrycaj, S., Huang, L., Hull, J., Iatrou, K., Iga, M., Kanost, M.R., Kotwica, J., Li, C., Li, J., Liu, J., Lundmark, M., Matsumoto, S., Meyering-Vos, M., Millichap, P.J., Monteiro, A., Mrinal, N., Niimi, T., Nowara, D., Ohnishi, A., Oostra, V., Ozaki, K., Papakonstantinou, M., Popadic, A., Rajam, M.V., Saenko, S., Simpson, R.M., Soberón, M., Strand, M.R., Tomita, S., Toprak, U., Wang, P., Wee, C.W., Whyard, S., Zhang, W., Nagaraju, J., ffrench-Constant, R.H., Herrero, S., Gordon, K., Swevers, L., and Smagghe, G. (2011). RNA interference in Lepidoptera: an overview of successful and unsuccessful studies and implications for experimental design. J. Insect Physiol. 57, 231-245.
3. Swevers, L., Liu, J., Huvenne, H., and Smagghe, G. (2011). Search for limiting factors in the RNAi pathway in silkmoth tissues and Bm5 cells: the RNA-binding proteins R2D2 and Translin. PLoS ONE 6:e20250.
4. Liu, J., Swevers, L., Iatrou, K., Huvenne, H., and Smagghe, G. (2012). Bombyx mori DNA/RNA non-specific nuclease isoforms: expression in insect culture cells, subcellular localization and functional assays. J. Insect Physiol. 58, 1166-1176.
5. Kolliopoulou, A., and Swevers, L. (2013). Functional analysis of the RNAi response in ovary-derived silkmoth Bm5 cells. Insect Biochem. Mol. Biol. 42, 654-663.
6. Liu, J., Smagghe, G., and Swevers, L. (2013). Transcriptional response of BmToll9-1 and RNAi machinery genes to exogenous dsRNA in the midgut of Bombyx mori. J. Insect Physiol. 59, 646-654.
7. Liu, J., Kolliopoulou, A., Smagghe, G., and Swevers, L. (2013). Modulation of the transcriptional response of innate immune and RNAi genes upon exposure to dsRNA in silkmoth-derived Bm5 cells overexpressing BmToll9-1 receptor. Submitted for publication.
8. Swevers, L., Vanden Broeck, J., and Smagghe, G. (2013). The possible impact of persistent virus infection on the function of the RNAi machinery in insects: a hypothesis. Frontiers in Integrative Physiology (In Press).
9. Swevers, L., Van Nieuwerburgh, F., Kolliopoulou, A., Sun, J., and Smagghe, G. (2013). Transcriptome and small RNA analysis of larval midgut tissue persistently and acutely infected by cytoplasmic polyhedrosis virus (CPV) in the silkmoth Bombyx mori. 38th FEBS Congress, St. Petersburg, July 6-11. FEBS Journal 280 (Suppl. 1) 584-585.
