Hemolymph microbiota collection, 16S rRNA sequencing, and bioinformatics analysis

We prepared four larval groups, namely sixth-instar larvae at 24 h PE (feeding) and 96 h PE (wandering), as well as larvae at 144 h post-dsHaCTL3 or -dsGFP treatment. Each group contained three biological replicates, and each replicate contained pooled hemolymph samples from six larvae. Prior to dissection, larvae were surface-sterilized. Hemolymph was collected from each group by cutting the abdominal feet of larvae without touching guts and diluted threefold in sterile anticoagulant buffer. After centrifugation at 1,000 g for 10 min, the precipitation (containing hemocytes and microbiota) was harvested and used for extraction of total metagenomic DNA. Subsequently, the V3 and V4 hypervariable regions of the 16S rRNA gene pool were amplified with the primers 338F and 806R (S6 Table). PCR amplicon libraries were then generated and subjected to high-throughput sequencing on an Illumina MiSeq PE300 platform (Illumina, San Diego, CA, USA) at Majorbio Bio-Pharm Technology Co., Ltd. (Shanghai, China).

Raw sequences of each sample were processed to obtain clean data according to the method described previously [65]. OTU generation and clustering were performed with a cut-off of 97% similarity using USEARCH [66]. Phylogenetic affiliation of each 16S rRNA gene sequence was analyzed against the SILVA 16S rRNA database (http://www.arb-silva.de) with a confidence threshold of 70%. Differential analyses of the relative abundance of hemolymph microbiota between groups were performed at the family level.