Illumina HiSeq 2000 sequencing; Receptors are affected by selection with each Bacillus thuringiensis israelensis Cry toxin but not with the full Bti mixture in Aedes aegypti
Receptors are affected by selection with each Bacillus thuringiensis israelensis Cry toxin but not with the full Bti mixture in Aedes aegypti
A total of five strains of Aedes aegyptiwere used in the present study. The Ae. aegypti laboratory Bora-Bora strain, susceptible to all insecticides, was used for selection using Cry4Aa, Cry4Ba and Cry11Aa toxins during 33 generations to obtain the LR4A, LR4B and LR11 strains resistant 1018 fold to Cry4Aa, 226 fold to Cry4Baa and 70 fold to Cry11Aa, respectively (Stalinski et al., 2016). These three strains had different crossresistance patterns; LR4A showed little cross-resistance (b4 fold crossresistance to Cry4Ba and Cry11Aa) while LR4B and LR11 had higher level of cross resistance (14–35 fold) to the two other Bti Cry toxins (Stalinski et al., 2016). A composite strain (LR3) was created by mixing together an equal amount of adults from each LR strain (30% each) at the generation 18 and 10% of adults from the susceptible Bora-Bora strain. This composite strain was then selected during 14 generations with unformulated Bti to obtain the LR3Bti strain exhibiting low levels of resistance to Bti Cry toxins (5–14 fold) (Stalinski et al., 2014). All strains shared similar low level of resistance to Bti (1.2–1.7 fold), (Stalinski et al., 2016). Mosquito breeding and selection were performed in standard insectary conditions (27 °C, 14/10 h light/dark period, 80% relative humidity) as previously described (Tetreau et al., 2012). The Tophat algorithm release 2.1.12 (https://ccb.jhu.edu/software/tophat) was run with default parameters to align all reads on the Ae. aegypti reference genome AaegL3 (available at http://aaegypti.vectorbase.org) by taking into account both already known and novel ab initio splice exon-exon junctions. Aligned reads were filtered on mapping quality (alignment score ≥ 97 and removal of non-primary multiple mapped reads) using the software Genespring NGS version 12.5 (Agilent). The Ae. aegypti genome was reannotated based on read distribution and RPKM were calculated for each known or new putative transcript using default parameters. Transcripts with RPKM N0.5 in at least one strain were retained for differential transcription analysis. Differential transcription level was tested for each transcript between each condition (unexposed vs exposed) using a Mann–Whitney U test. Genes with P b 0.05 and a 2 fold over- or under-transcription as compared to the parental Bora-Bora strain were considered differentially transcribed. For each strain, total RNA was extracted from three biological replicates, consisting of 40 larvae, using “absolutely RNA” miniprep Kit (Agilent) followingmanufacturer's instructions. RNA quantity and quality were assessed using an Agilent 2100 Bioanalyzer (Agilent). cDNA libraries were prepared from total RNA using the Illumina TruSEQ RNASeq kit (Illumina) and sequenced on an Illumina HiSEQ 2000 at Beckman Coulter Genomics facilities.