Figure 4. Ectopic AgOR2 protein reduces the transcript levels of AgOR isoforms.

(A) Volcano plot of differentially expressed isoforms. Using Wald tests, we evaluated whether the 13,224 isoforms present in 3 triplicates of control and 3 triplicates of ORCO > AgOR2 antennae (~200 antennae per sample) were differentially expressed. Non-A. gambiae OR and A. gambiae OR isoforms are shown as black or red dots, respectively. Only 0.63% of the transcriptome is differentially regulated in ORCO > AgOR2, in which 49% of those transcripts are AgORs (Figure S6; Data S2). log(q) is the level of significance of β₁, which, for each isoform, is defined as TPM_control −TPM_experimental. The 41 AgORs found to be significant from the Wald tests are labeled according to their gene annotation in the volcano plot. AgORCO, while not differentially expressed, is also indicated on the plot. All AgORs (with the exception of AgOR2) that are differentially expressed are downregulated in the experimental condition when compared to wild type. Interestingly, AgIR8a and AgIR75k (indicated on the volcano plot) are downregulated in ORCO > AgOR2. The remaining AgIRs and AgGRs are unaffected.

(B and C) Heatmap of the AgOR gene family. A Z score was computed for each cell in the heatmap by subtracting the mean isoform TPM from the TPM of the cell divided by the standard deviation (SD) of the isoform TPM. AgORs are sorted along the x axis according to their significance level (q value) from the Wald tests in (A) Darker orange is most significant and yellow is not significant. Note that ORCO > AgOR2 is listed as O > AgOR2. (C) Ectopic AgOR2 protein is required for the observed downregulation of native AgOR transcripts. Ternary plots were used to visualize the relative ratio of a genotype to the relative abundance level of an isoform using the formula (TPM_genotypex)/(mean TPM_control + mean TPM_ORCO>AgOR2 + mean TPM_{ORCO>mutAgOR2}) × 100. There are equal ratios of transcript abundance levels for non-AgOR genes among the 3 genotypes (left). However, the relative contribution to transcript abundance levels of control, ORCO > AgOR2, and ORCO > mutAgOR2 are skewed in the AgOR gene family such that AgOR gene levels in ORCO > mutAgOR2 and control are relatively similar and higher than ORCO > AgOR2 levels, with the exception of AgOR2 itself (whose abundance is similar between ORCO > AgOR2 and ORCO > mutAgOR2) (right).