*3.2. Transcriptome Annotation*

We first annotated the assembled Ashmead transcriptome on the transcript level. All the transcripts were aligned to public databases for full annotation and Figure 1B showed 74,264, 68,185, 54,536, 58,109, 61,794, 58,113, and 22,546 transcripts were aligned to the NR, NT, UniProt/SwissProt, KEGG pathway, KOG, Pfam, and GO databases, respectively. Tthee NR mapping results (Figure 1C) showed the top 10 species aligned by the assembled Ashmead transcripts and the majority of the transcripts were aligned to *Diachasma alloeum* (45,569 transcripts), *Fopius arisanus* (5217 transcripts), and *Rhinolophus sinicus* (4920 transcripts). Unsurprisingly, the top two species together with Ashmead were all from the Braconidae family. GO annotation revealed that 11,131, 7722, and 9078 transcripts were involved in "binding", "membrane", and "cellular process", respectively (Figure 1C). Then, we categorized the KEGG pathway annotation (Figure 1E) into six groups: Cellular progresses, environmental information processing, genetic information processing, human diseases, metabolism, and organism systems. Among them, "signal transduction" is the most significant pathway, which involved 9231 transcripts. KOG annotation also revealed that 14,221 transcripts were involved in the signal transduction mechanisms (Figure 1F).

**Figure 1.** Overview of the assembled Ashmead transcriptome and annotation. (**A**) Length distribution of the assembled Ashmead transcriptome. (**B**) Number of transcripts and likely proteins aligned to different databases. (**C**) Number of the transcripts aligned to other species in the NR mapping results. (**D**) GO annotation for the assembled Ashmead transcriptome. (**E**) KEGG pathway annotation for the assembled Ashmead transcriptome. (**F**) KOG annotation for the assembled transcriptome.

Next, TranDecoder predicted 88,215 likely proteins encoded by the assembled Ashmead transcriptome and we annotated them using the Trinotate pipeline. It was shown that 59,887, 26,402, 53,725, and 52,572 were aligned to the UniProt/SwissProt, KOG, Pfam, and GO databases, respectively (Figure 1B). In addition, 6642 and 14,672 likely proteins were predicted to contain signal peptides and transmembrane helices (Figure 1B).

In addition, we predicted 74,963 of the unannotated transcripts using CPC2 had low coding probability. Interestingly, 2128 of the unannotated transcripts were predicted to be possible coding transcripts, which might be specific to the Ashmead and require further experiments to be verified. Next, we predicted 87 transcripts have the potential of producing miRNAs (Table S1). Notably, 84 of them were probably derived from the intron regions of coding genes while 1 and 3 derived from the Ashmead specific coding gene and long noncoding genes, respectively.

## *3.3. Olfactory Genes*

We next identified genes encoding the olfactory gene from five families, including OBP, CSP, OR, GR, IR, and SNMP. In the Ashmead transcriptome, we found 43 transcripts encoding OBPs (Table 2, Table S2) by similarity and 35 of them were predicted to have complete ORFs by TransDecoder. The Ashmead OBPs were categorized into four sub-families: OBP-56, -69, -72, and -83 (Table S2). Multiple sequence alignment (Figure S1) showed these OBPs have 6 conserved cysteine residues (Cys) and SignalP predicted that 26 of these OBPs have the signal peptides located in the first 23 amino acids (aa) and 2 located in the first 37 aa. We identified that 69 transcripts had the potential of encoding CSPs in the Ashmead transcriptome by aligning the likely proteins to the known CSPs (Table 2, Table S2) and 63 of them had intact ORFs. SignalP identified 58 CSPs with the signal peptides in the first 28 aa while transmembrane domains were found in 13 CSPs. Surprisingly, 689 Ashmead transcripts had the capacity of encoding ORs and 115 transcripts were found to encode OR-13 (Table 2, Table S2). We also identified nine transcripts encoding OR coreceptors (OR-co). Out of the 347 OR transcripts that had intact ORFs, 276 were predicted to have transmembrane domains (Table 2). In the Ashmead transcriptome, there were 26 transcripts encoding IRs, including 3 IR-21, 18 IR-25, 4 IR-68, and 2 IR-93 (Table 2). Two thirds of the IR transcripts that had intact ORFs were predicted to have transmembrane domains. In addition, we identified 60 GR and 14 SNMP transcripts (Table 2). GR-28 and SNMP-1 were the largest group, which corresponded 19 and 11 transcripts, respectively. Further, we found 55 transcripts that had either the 7tm chemosensory receptor (PF02949) or GOBP (PF01395) PFAM domain in their protein sequences (Table 2, Table S2).



a Transcripts containing the chemosensory receptor or the GOBP domain, by PFAM annotation. \* Products are not specified.

#### *3.4. Gene Expression Profile*

We next profiled the gene expression in the Ashmead insects maintained with the fruit flies fed with the three kinds of fruits. After lowly expressed genes (count < 5) were filtered, RSEM identified a total of 63,627 transcripts in the Ashmead animals, of which 46,607, 49,253, and 53,558 transcripts were distributed in G, M, and C, respectively (Table S3). The Venn diagram (Figure 2A) revealed 38,009 transcripts commonly expressed in all samples while 3115, 4571, and 8159 were specifically detected in G, M, and C, respectively. Interestingly, not all the olfactory genes were expressed in the insects and we found 39 OBPs, 65 CSPs, 29 GRs, 382 ORs, 15 IRs, and 13 SNMPs. Figure 2B,C showed the expression levels of these olfactory transcripts in the insects, and revealed that di fferent olfactory genes of Ashmead insects are responsible for the fruit flies with di fferent fruits. According to the average expression levels, we showed the top five highly expressed olfactory transcripts identified in this study (Table 3) and it was revealed that the identities of highly expressed olfactory transcripts were shared by the parasitoid wasps of the fruit flies fed with di fferent fruits. Notably, OBP56 and OBP69 were highly expressed in the insects; IR25a and SNMP1 were the only highly expressed transcript for the IR and SNMP groups, respectively.

**Figure 2.** Expression levels of olfactory transcripts in the Ashmead insects. (**A**) Number of transcripts identified in the insects stimulated by three fruits. (**B**) Heat maps of the expression levels of OBP, CSP, and OR transcripts. (**C**) Heat maps showing the expression levels of transcripts encoding GRs, IRs, and SNMPs in the Ashmead insects.
