Transcriptional response of BmToll9-1 and RNAi machinery genes to exogenous dsRNA in the midgut of Bombyx mori

• Dominant expression of BmToll9-1 mRNA in the larval and pupal midgut tissues.
• High breakdown of dsRNA integrity in the insect midgut.
• Injection of dsRNA suppresses expression of BmToll9-1 mRNA in the larval midgut.
• Induction of BmDcr2 and BmAgo2 transcription in the larval midgut by dsRNA.

Injection of dsRNA is widely applied to silence endogenous genes and study gene function in insects. However, it is not yet clear to what extent it can also exert non-specific effects, for instance by interference with the innate immune response. In this study, we report on the transcriptional response of BmToll9-1 to lipopolysaccharide (LPS) and dsRNA in the silkmoth, Bombyx mori. BmToll9-1 encodes a Toll receptor highly expressed in midgut tissue and that shows limited similarity to the mammalian TLR3 endolysosome receptor for dsRNA; while Dcr2 and Ago2 encode two key components of the RNAi machinery.An expression pattern study of all 14 Toll receptors in B. mori showed that BmToll9-1 was expressed in different larval and pupal tissues with the highest expression level detected in the midgut, indicating a possible function in immunity against pathogens taken up by the food. In order to investigate the response of BmToll9-1, different ways to deliver dsRNA, specific for GFP (dsGFP), and LPS were applied in Bombyx 5th instar larvae. The feeding experiments suggested that dsGFP did not suppress the expression of BmToll9-1 significantly, while LPS could suppress the expression of BmToll9-1 after 3 h of feeding. On the other hand, the injection experiments showed that dsGFP, as well as LPS, could significantly inhibit the expression of BmToll9-1 in 3 h. Bacteria that constantly expressed dsGFP could also down-regulate the expression of BmToll9-1 to a greater extent than bacteria that do not express dsGFP. The failure of dsGFP by feeding to affect the expression of BmToll9-1 was correlated with the rapid degradation of dsGFP by dsRNase in the midgut juice. Expression of the RNAi machinery genes Dcr2 and Ago2, as well as dsRNase, was also affected by injection of dsRNA and not by feeding, but in these cases an induction was observed instead of a down-regulation.Because LPS is a well-known pathogen-associated molecular pattern (PAMP), it suggested that the decrease in BmToll9-1 expression is a consequence of the activation of the innate immune response by LPS. The similar response of BmToll9-1 between the two triggers, LPS and dsRNA, suggests that dsRNA can also act as a PAMP in the midgut of Bombyx. Furthermore, induction of the genes Dcr2, Ago2 and dsRNase may also constitute a defense mechanism against invading dsRNA.

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