*3.5. Phylogenetic Analysis*

We next compared the sequences of olfactory proteins identified in Ashmead and some other homology organisms and analyzed their phylogenetic relationship. First, we constructed the phylogenetic tree for the top 5 OBPs (Table 3) and 28 other OBP sequences obtained from NCBI. Detailed information, including accession numbers and species, can be accessed in Table S3. The phylogenetic tree of OBPs (Figure 3A) showed high similarity (68.9% to 96.5%) between Ashmead OBPs and other species, such as *Aethina tumida* (Atumi), *Aphidius gifuensis* (Agifu), *Cephus cinctus* (Ccinc), *Cotesia chilonis* (Cchil). *Diachasma alloeum* (Dallo), *Fopius arisanus* (Faris), *Megachile rotundata* (Mrotu), *Meteorus pulchricornis* (Mpulc), *Microplitis demolitor* (Mdemo), and *Microplitis mediator* (Mmedi). We next performed the phylogenetic analysis for CSPs. The top 5 highly expressed CSPs (Table 3) were compared with 22 CSPs from other species, like *Vespa velutina* (Vvelu), Ccinc, *Sclerodermus*, Mpulc, and *Yemma signatus*(Ysign). Detailed accession numbers of these CSPs can be accessed in Table S3. It is clear that the Ashmead CSPs can be clustered with other known CSPs. The phylogenetic analysis supported the identification and characterization of OBP and CSP transcripts in this study.

#### *3.6. Di*ff*erential Expression Analysis*

Another important goal of this study was to identify genes in the insects in response to their parasitic hosts, which had difference fruit scents. Using edgeR, we identified a total of 2650 transcripts differentially expressed in the Ashmead insects in response to the fruit flies with different scents (Table S4), and the number of differentially expressed transcripts can be seen in Figure 4A. Compared to G, there were 1466 upregulated and 53 downregulated transcripts identified in both C and M (Figure 4B). Some transcripts were specifically expressed in the insects when they were parasitic to the fruit flies with one fruit scent (Figure 4B). We next analyzed the differentially expressed transcripts encoding olfactory proteins in the Ashmead insects in response to the three fruit flies. A total of 58 transcripts encoding olfactory proteins were found, including 9 OBPs, 13 CSPs, 3 GRs, 4IRs, 25 ORs, and 4 SNMPs (Figure 4C). This evidence further supports the existence of multiple pathways of Ashmead insects in response to different fruits scents. In addition to olfactory proteins, some other protein families were differentially expressed in the wasps maintained with fruit flies supplied with different fruits, such as 2 LOC107047718 (Putative 7 transmembrane sweet-taste receptor of 3 gcpr), 88 ribosomal proteins, 109 transcription factors, 99 histones, 11 heat shock proteins, and 43 G-protein coupled receptors/regulators (Table S4). The di fferential expression of transcripts from di fferent families indicated the complicated regulation mechanisms of parasitoid wasps in response to their hosts with di fferent fruit scents. More experiments are required to explore their functions in this process.


**Table 3.** Top five transcripts encoding olfactory proteins in Ashmead insects.

Normalized expression, TMM.

a

**Figure 3.** Phylogenetic trees for OBPs ( **A**) and CSPs (**B**). Ashmead proteins are highlighted in red, the percentage represents the bootstrap value, and the scale bar represents the evolutionary distance.

**Figure 4.** Differentially expressed transcripts in the parasitoid wasps of fruit flies fed with different fruits. (**A**) Number of differentially expressed transcripts in the insects in response to two fruit scents. (**B**) Venn diagram of up- (upper panel) and downregulated (lower panel) transcripts identified in C and M, compared to G. (**C**) A heat map showed the differential expression of 9 OBPs, 13 CSPs, 3 GRs, 4IRs, 25 ORs, and 4 SNMPs in the parasitoid wasps of fruit flies fed with different fruits.
