The expression patterns of cwh genes of C. montrouzieri, H. axyridis, and P. japonica in different life stages and tissues were explored. The level of expression in 4th instar larva, pupa, and male adult stages, as well as in the head, gut, and gut-removed abdomen of male adults, was quantified by quantitative PCR (qPCR).

The expression patterns of cwh genes in response to infection by E. coli and B. subtilis were explored in C. montrouzieri, H. axyridis, P. japonica, and H. vigintioctopunctata. The bacterial cells were suspended at approximately 109 CFU/mL using phosphate-buffered saline (PBS). We injected 2 mL of the suspension into the species with small body sizes (C. montrouzieri and P. japonica) and 5 mL of the suspension into the species with large body sizes (H. axyridis and H. vigintioctopunctata) in the abdomens of male adults. Insects injected with the same volume of PBS were set as controls. Total RNA was extracted 24 h after infection for further qPCR.

The primers for cwh and the reference genes (Additional file 1: Table S2) were designed using NCBI primer-BLAST based on the transcriptome data (see the following methods for transcriptome sequencing) or referenced from previous studies [6668]. qPCR was performed on cDNA samples using a SYBR Green-based kit (Invitrogen, USA) according to the manufacturer’s protocol. Expression levels for cwh were normalized to those of two reference genes. Analyses of cwh expression included four technical replicates for each biological replicate and five biological replicates for each treatment. The Ct data of qPCR can be found in Additional file 2. Relative gene expression of each cwh in comparison to the average level of that in head and larva was analyzed by the 2−ΔΔCt method to calculate the fold changes [69]. To test for significant differences in the expression levels of each cwh gene under different temporal, spatial, and bacterium-infected conditions, we applied one-way ANOVAs followed by Tukey’s post hoc tests using SPSS 17.0 (SPSS Inc).

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