Expression analysis

Heatmap plots were generated for the binary logarithm of raw FPKM-plus 1 values. For each plot, the minimum value was set to the number type, with a value of zero, and displayed as yellow, the midpoint was set to percentile type, with a value of 100, and displayed as blue, and the maximum was set to the highest value type, and displayed as red. These plots were made and edited using ITOL tool.

Quantitative reverse transcription PCR was performed to validate the expression of candidate chemosensory receptors in A. gifuensis. The collection of antennae and body tissues without antennae of each sex were collected respectively (antennae: 400 of each sex; body tissues: 20 of each sex) and were frozen in liquid nitrogen. For the host aphid specific expression analysis, A. gifuensis reared on different aphids were collected (whole body and 20 of each sex) and frozen in liquid nitrogen. Total RNA of A. gifuensis was extracted using TRIzol reagent (Takara Bio, Tokyo, Japan), as per manufacturer’s instructions. The temple RNA was treated using Dnase I and incubated at 42 °C for 2 min to remove the genomic DNA. Next, the cDNA was synthesized from total RNA using Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Mannheim, Germany) according to the standard manufacturer’s protocol. Gene-specific primers were designed by Primer Premier 5 (PREMIER Biosoft International, Palo Alto, CA, USA), and are shown in Table ^S3. qPCR was conducted in 20 μl reactions containing 50 × SYBR Premix, Ex Taq (10 μL), primer (10 mM), sample cDNA (0.8 μL), and sterilized ultra-pure grade H₂O (7.6 μL). Cycling conditions were 95 °C for 30 s, 40 cycles of 95 °C for 5 s, and 55 °C for 30 s. Each sample had three technical replicates and three biological replicates. Relative quantification was performed using the Comparative 2^−ΔΔCT method. Transcription levels of these receptor genes were normalized by 18 S RNA, and the normalization of each gene was compared with the lowest expression level in different tissues⁶⁸. The expression data among the different tissues and host aphids of each sex were subjected to one-way analysis of variance (ANOVA); means were separated using Duncan’s test at P  < 0.05.