Kit v2

DNA was isolated using the DNeasy^® PowerLyzer^® PowerSoil^® kit (Qiagen, Hilden, Germany) as recommended by the manufacturer’s Quick-Start Protocol (Qiagen, Germantown, MD, USA). Approximately 0.300 g of sediment material was used for each of the 10 sampling points (in the relevant seasons), each in triplicate. Metabarcoding of 16S rRNA was performed within its V3–V4 region [17 (link)]. 341f and 785r primers were applied both for the amplification of the mentioned region and for the preparation of the library [17 (link),18 (link)]. The PCR was carried out as described by Wolińska, et al. [17 (link)] with the use of Q5 Hot Start-High Fidelity 2X Master Mix (New England Biolabs INC., MA, USA). When a positive effect of PCR was obtained, triplicate DNA isolates of one sample were pooled, which was in agreement with the recommendation of Kuźniar, et al. [19 (link)]. Next-Generation Sequencing (NGS) was performed by Genomed S.A. on a MiSeq sequencer, paired-end (PE) technology, 2 × 300 nt, using Illumina kit v2.

The high-throughput sequencing platform HiSeq 2000 was used to sequence libraries that were constructed from treated fat bodies using an Illumina kit v2. For the following bioinformatics analysis, a flux-capacitor was used to determine the reads count (FPKM) of the transcripts, and the DEGseq package in R language was employed to identify DEGs (52 (link)). The reference genome used in this experiment was downloaded from the VectorBase website (https://vectorbase.org/vectorbase/app/downloads/release-47/AaegyptiLVP_AGWG/fasta/data/). Genes were considered differentially expressed when the P value was <0.05. Cross comparison of each treated sample was normalized by its FPKM. The iEGFP sample was treated as the background when the fold change rate was calculated. The transcriptome data were deposited to the NCBI SRA (bioproject PRJNA744689). qRT-PCR was performed using SuperReal PreMix (Tiangen) on StepOnePlus (Thermo Fisher Scientific). Template concentrations were normalized to the internal reference RPS7 gene. The primers used in this experiment are listed in Table S5.