Function Analysis Of PGRP-LB And PGRP-SB In Immunity Andmodulation Of Intestinal Microbiota In Bactrocera Dorsalis

The Oriental fruit fly, Bactrocera dorsalis(Hendel), is one of the most important agricultural pests because the larvae stay inside the fruits and can harm a wide range of fruits and vegetables. Gut microbes are closely related to the insect itself. They affect many physiological functions of the hosts such as metabolism, immunity, etc. Using RT-PCR and RACE, we have cloned genes PGRP-LB and PGRP-SB. We have performed analyses of PGRP-LB and PGRP-SB functions by infecting with Escherichia coli before or after RNAi. We also analyzed the changes of commensal bacteria flora in the gut after the genes PGRP-LB and PGRP-SB were silienced by using RNAi, in order to explore the role of these genes in maintaining the intestinal microflora homeostasis of B.dorsalis and provide a new target and strategy for control of B.dorsalis.1 Gene cloning and expression pattern of PGRP-LB and PGRP-SBThe length of the nucleic acid sequence of the ORF of the PGRP-LB we’ve got from NCBI was 738 bp. We have cloned PGRP-SB, the length of the nucleic acid sequence of the ORF was 558 bp. Sequence analyses indicated that the homology of each gene is 86% and 91% between B.dorsalis and Ceratitis capitata. The expression patterns indicated that the PGRP-LB highly expressed in the midgut of the adult; the PGRP-SB highly expressed in the third larva and fatbody of the adult.2 The immune responses of PGRP-LB and PGRP-SB to opportunistic pathogensThe immune responses of B. dorsalis after infection with E. coli showed that the expression levels of Diptericin, PGRP-LB and PGRP-SB raised significantly. The maximum up-regulation of Diptericin is 6.40 times at 6h after systemic infection and 4.52 times at 12 h after orval infection. The maximum up-regulation of PGRP-LB and PGRP-SB after systemic infection is 2.97 times at 6h and 1.61 times at 24 h. The maximum up-regulation of PGRP-LB and PGRP-SB after orval infection is 54.32% at 12h and 30.26% at 12 h. But the immune response for systemic infection was much stronger than oral infection. These results indictated that bacteria E. coli can leads to a systemic and local immune response. Besides the antibacterial peptide gene Diptericin and genes PGRP-LB and PGRP-SB play important roles in the process of intestinal defense.3 The effect of PGRP-LB and PGRP-SB on immune responses of B.dorsalisWe analysed the functions of PGRP-LB and PGRP-SB to immune responses of B. dorsalis by infection with E. coli after RNAi. We found that PGRP-LB and PGRP-SB-RNAi flies had stronger and more sustained immune responses than wild-type flies, as measured by the expression of the antibacterial peptide gene Diptericin. These results revealed that PGRP-LB and PGRP-SB could prevent immune activation of B.dorsalis, and acted as negative regulators of the Imd pathway.4 The effect of PGRP-LB and PGRP-SB on the intestinal microbial community composition and structure of B.dorsalisTo confirm the role of PGRP-LB and PGRP-SB in regμLation of commensal bacteria flora in the gut, we next detected the microflora variation in the gut between the control and PGRP-LB and PGRP-SB-RNAi flies. q RT-PCR showed that the expression levels of PGRP-LB and PGRP-SB reduced significantly within 5 days after RNAi and returned to normal at 15 days. Moreover, the quantity of gut bacteria has changed a lot. The number of total bacteria, γ-Proteobacteria, Actinobacteria and Firmicutes downregμ Lated 18.78%, 56.67%, 61.98% and 67.46% respectively at 5 days after silencing PGRP-LB gene. The number of total bacteria, α-Proteobacteria, γ-Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes down-reg μ Lated 31.73%, 40.41%, 44.64%, 50.26%, 52.19% and 0.18% respectively at 5 days after silencing PGRP-SB gene. The total bacteria, α-Proteobacteria, γ-Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes up-regμ Lated 2.16 times, 1.04 times, 1.65 times, 0.38 times, 2.70 times and 0.84 times respectively at 15 days after silencing PGRP-LB gene.The α-Proteobacteria, γ-Proteobacteria up-regμ Lated 53.77% and 60.48% and the Bacteroidetes and Firmicutes downed-regulated 14.89% and 63.60% at 15 days after silencing PGRP-SB gene. All bacteria have no significant difference from the control at 20 dyas after silencing PGRP-LB or PGRP-SB gene, except that the total bacteria and γ-Proteobacteria from down-regulated 45.96% and 62.06% after silencing PGRP-LB gene. These reluts confirmed that the silence of PGRP-LB and PGRP-SB lead to changes of the intestinal microflora. PGRP-LB and PGRP-SB play an indispensable role in maintaining the intestinal microflora homeostasis of B.dorsalis.