Figure 6. The DmMesh-mediated DmDuox regulation in response to commensal bacteria in Drosophila.

(a–d) Oral introduction of A. thailandicus (A. tha) induced the DmMesh expression and DmDuox-mediated ROS in the gut of aseptic Drosophila. A. thailandicus was orally introduced into the germ-free DmMesh RNAi flies by food ingestion. Measurement of the average bacterial number introduced into each fly gut was presented by Supplementary Figure 18. The mRNA expression levels of DmMesh (a) and DmDuox (c) were determined by SYBR Green qPCR. (b) The protein level of DmMesh was determined by Western-blot with an anti-AaMesh murine antibody. (d) Detection of the ROS activity in the Drosophila midguts.

(e–f) The DmMesh-mediated DmDuox regulatory pathway in response to A. thailandicus. The DmMesh RNAi Drosophila strain was generated using a GAL4 line driven by a NP3084 promoter. The germ-free NP3084/GFP-RNAi flies that fed on the same bacteria served as negative controls. 6×10⁶ A. thailandicus was orally introduced into the germ-free DmMesh RNAi flies by food ingestion. (e) Measurement of DmDuox expression and (f) the ROS activity in the Drosophila midguts.

(g) Schematic representation of the AaMesh-mediated Duox regulatory pathway in response to symbiotic gut microbes. The expression of Mesh can be upregulated by the proliferation of the symbiotic microbiome in insect guts (1). Because Mesh is a positive regulator of Arrestin expression, induction of Mesh results in a higher abundance of Arrestins (2), and subsequently accelerates the phosphorylation of the ERK and JNK MAPK proteins (3). The activation of ERK and JNK leads to the downstream Duox transcription (4). Thus, higher DUOX-mediated ROS are generated in response to the gut microbial proliferation, thereby moderating the proliferation of symbiotic microbes in gut-microbe homeostasis (5).

(a, c, e) The Duox expression was assessed by SYBR Green qPCR and normalized against Drosophila actin. The qPCR primers are described in Supplementary Table 6.

(d, f) The ROS activity was detected by a H₂O₂ assay.

(a, c–f) The data are presented as the mean ± S.E.M. All results were repeated by 3 independent experiments. The data were analyzed using the non-parametric Mann-Whitney test.