Functional Analysis Of PGRP-SC In Intestinal Immunity Of Musca Domestica

The entire life of Musca domestica has a closely connection with complex microbial environment, and its innate immunity can effectively protect the housefly from pathogens infection. Meanwhile, the microorganism also plays a crucial part in the development of housefly larvae, so that inappropriate immune responses may cause damages to the harmless symbiont, or to the host tissue. Therefore, a delicate balance of protecting microbiota and defending against pathogens should be kept by strictly control the strength of immune responses.As an pattern recognition receptor, peptidoglycan recognition proteins(PGRPs) play important roles in the regulations of responses to microbial infection. Our earlier study has cloned an M. domestica PGRP-SC which mainly expressed in gut, so we speculated that it may involve in the regulation of intestinal immune. To verify this speculation, we interfered the transcriptional expression of PGRP-SC in housefly larvae by RNAi, then investigated the functions of this PGRP in the intestinal immune of housefly. The main contents are as follows:(1) We attempted to knock-down the PGRP-SC in housefly larvae by method of injection and feeding. In the first place, the double-strand RNA targeting to PGRP-SC was transcribed in vitro and injected into 1st housefly larvae. The expression profiles of PGRP-SC in each groups were monitored by q PCR. The relative expression level of PGRP-SC showed highly significantly decreased in the interference group after 48 hours, and the maximum interference efficiency was 70.94%. In the next moment, we fed the 1st housefly larvae with Escherichia coli expressing ds RNA and then monitored the PGRP-SC expression profiles. The q PCR results showed, the relative expression level of PGRP-SC showed significantly decreased in the interference group at 24 h and the maximum interference efficiency was shown at 48 h which is 53.95%.(2) The tempo-spatial dynamics of the GFP-expressing bacteria ingested by housefly can be observed under fluorescent microscope. So we fed the treated larvae with GFP-expressing E. coli respectively in the negative control group and the interference group, and then a rapid fadeout of fluorescence in the interference group was observed compared with the negative control group after 30 min, which means the ingested bacteria was eliminated by the larvae.(3) The hydrogen peroxide contents in intestine of housefly larvae were tested by colorimetry in each group. The results shows no significant difference among each group, so we speculated that the synthesis of hydrogen peroxide may not involve in the elimination process mentioned before.(4) The expression profiles of four antimicrobial peptide genes(attacin, diptericin, muscin and cecropin) were tested by q PCR in the gut of housefly larvae. The results show that all of these four AMP genes were up-regulated in varying degrees after RNAi, which indicated that the PGRP-SC exhibits a regulatory effect in the sensitivity of intestinal immunity. Interfering the expression of PGRP-SC may result to the activation of IMD pathway, which will stimulate the synthesis of AMPs and consequently eliminate the ingested E. coli in gut.