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.