Eggs, nymphs, and pupae were collected from leaves of collard plants (Brassica oleracea L.) on which the isogenic MEAM1 colony was reared. Tissues were surface sterilized by submersion in a petri dish containing 70% ethanol. The eggs were gently separated from nymphs and pupae using a small paintbrush. Isolated nymph and pupa samples were rinsed with sterile water. Approximately 1500 adult whiteflies reared on broccoli (B. oleracea L. var. botrytis) at the USDA-ARS in Charleston, SC were transferred to either TYLCV-infected or uninfected tomato (Solanum lycopersicum cv. Moneymaker) cuttings and allowed to feed for 24, 48, or 72 h, respectively. For each treatment and time point, two compound leaves were collected from TYLCV-infected or uninfected plants and transferred to a flask filled with water, which was then sealed with Parafilm and placed in an insect-proof cage. Whiteflies were added to each cage and allowed to feed for 24, 48, or 72 h under controlled conditions at 28 ± 1 °C, a 14:10 (L:D) h photoperiod, and ~60% humidity. A total of 200–500 living whiteflies were collected at the end of each time point and stored at –80 °C until processing. Three biological replicates were performed for each sample. A similar experiment under the same environmental conditions was performed using adults from a MEAM1 colony maintained at the USDA-ARS in Salinas, California (CA), but these white flies were fed on ToCV-infected or uninfected tomato (cv. Moneymaker) plants.
For insecticide treatment experiments, adults of two MED populations, PyriR, which is susceptible to the insecticide Mospilan (acetamiprid), and 9-2103, which is resistant, were fed on cotton seedlings (Gossypium hirsutum L. cv. Acala) treated with the insecticide Mospilan at an LC30 dose (lethal concentration required to kill 30% of the population; 2 ppm for PyriR and 100 ppm for 9-2013) with the dipping method, as previously described [64 (link)]. Whiteflies fed on untreated cotton seedlings were used as controls. The experiments were conducted under standard rearing room conditions of 25 °C, 50% relative humidity, and a light regime of 10 h light and 14 h dark. Three to four biological replicates, each containing a pool of 200–500 adult whiteflies, were collected from each treatment. The insects were kept at –80 °C until use.
Total RNA was purified using the Ambion TRIzol Reagent (Thermo Fisher, USA) according to the manufacturer’s protocol. Strand-specific RNA-Seq libraries were constructed following the protocol described in Zhong et al. [65 (link)] and sequenced on the Illumina HiSeq 2500 system. Raw RNA-Seq reads were first processed to remove adapter and low-quality sequences using Trimmomatic [57 (link)]. Reads shorter than 40 bp after trimming were discarded. The resulting reads were then aligned to the ribosomal RNA database [66 (link)] and the three bacterial symbiont genomes using Bowtie [67 (link)], allowing up to three mismatches. The aligned reads were not used for further analysis. To assist gene prediction, the high-quality cleaned RNA-Seq reads were aligned to the assembled B. tabaci genome using TopHat [68 (link)], and the aligned reads were assembled into transcripts using Cufflinks [69 (link)]. For gene expression analysis, the RNA-Seq reads were aligned to the assembled B. tabaci genome using HISAT [70 (link)]. Raw counts for each B. tabaci predicted gene were derived from the read alignments and normalized to fragments per kilobase of exon model per million mapped fragments (FPKM). Differential expression analyses were performed using edgeR [71 (link)]. The resulting raw P values were adjusted for multiple testing using the false discovery rate (FDR) [72 ]. For each comparison, genes with FDR <0.05 and fold change no less than 1.5 were considered as differentially expressed genes.
For insecticide treatment experiments, adults of two MED populations, PyriR, which is susceptible to the insecticide Mospilan (acetamiprid), and 9-2103, which is resistant, were fed on cotton seedlings (Gossypium hirsutum L. cv. Acala) treated with the insecticide Mospilan at an LC30 dose (lethal concentration required to kill 30% of the population; 2 ppm for PyriR and 100 ppm for 9-2013) with the dipping method, as previously described [64 (link)]. Whiteflies fed on untreated cotton seedlings were used as controls. The experiments were conducted under standard rearing room conditions of 25 °C, 50% relative humidity, and a light regime of 10 h light and 14 h dark. Three to four biological replicates, each containing a pool of 200–500 adult whiteflies, were collected from each treatment. The insects were kept at –80 °C until use.
Total RNA was purified using the Ambion TRIzol Reagent (Thermo Fisher, USA) according to the manufacturer’s protocol. Strand-specific RNA-Seq libraries were constructed following the protocol described in Zhong et al. [65 (link)] and sequenced on the Illumina HiSeq 2500 system. Raw RNA-Seq reads were first processed to remove adapter and low-quality sequences using Trimmomatic [57 (link)]. Reads shorter than 40 bp after trimming were discarded. The resulting reads were then aligned to the ribosomal RNA database [66 (link)] and the three bacterial symbiont genomes using Bowtie [67 (link)], allowing up to three mismatches. The aligned reads were not used for further analysis. To assist gene prediction, the high-quality cleaned RNA-Seq reads were aligned to the assembled B. tabaci genome using TopHat [68 (link)], and the aligned reads were assembled into transcripts using Cufflinks [69 (link)]. For gene expression analysis, the RNA-Seq reads were aligned to the assembled B. tabaci genome using HISAT [70 (link)]. Raw counts for each B. tabaci predicted gene were derived from the read alignments and normalized to fragments per kilobase of exon model per million mapped fragments (FPKM). Differential expression analyses were performed using edgeR [71 (link)]. The resulting raw P values were adjusted for multiple testing using the false discovery rate (FDR) [72 ]. For each comparison, genes with FDR <0.05 and fold change no less than 1.5 were considered as differentially expressed genes.
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