**4. Discussion**

This project was initiated in an effort to identify the olfactory proteins of parasitoid wasps. In recent years, many aspects about the perception of pheromones and other odorants have been elucidated [36]. Olfaction is used by insects to recognize volatile cues that allow the detection of food, predators, and mates [37]. We identified 43 OBPs, 69 CSPs, 60 GRs, 689 ORs, 26 IRs, and 14 SNMPs in *D. longicaudata* (Table 2, Table S2) and some of them were differentially expressed when they were maintained with fruit flies fed with different fruits (Figure 4C, Table S4). This is the first time the chemosensory genes in *D. longicaudata* have been investigated and our findings provide a basis for elucidating the molecular mechanisms of the olfactory-related behaviors of parasitoid wasps.

OBPs are a group of proteins that specialize in the transport of lipids. In this study, we identified 43 transcripts encoding OBPs and this number is similar to the number of genes encoding OBPs in the *Drosophila* genome [38]. Addittionally, classic OBPs have been reported to contain six Cys in their sequences [38,39]. Because all the identified OBPs in this study were general OBPs (GOBPs) (Table S2), we found conserved Cys in their sequences (Figure S1), which increased the confidence of using the transcriptome to identify the OBPs in *D. longicaudata*. In detail, the *D. longicaudata* OBPs were grouped into four subfamilies: OBP-56, -69, -72, and -83 (Table S2). However, no studies have been demonstrated to distinguish their functions. In general, according to their names, they are called GOBPs because they bind general odorants that are likely to be represented by the same volatiles for most of the species [40]. We also showed the differential expression of nine transcripts encoding OBPs in parasitoid wasps in response to the fruit flies with different scents (Figure 4, Table S4). This might indicate that some specific OBPs are expressed to discriminate different scents.

Similar to OBPs, CSPs are another group of proteins that mediate the olfactory recognition in insects [41]. They are thought to be expressed during nearly the whole life circle of insects [42,43]. The number of CSP genes varies in species. For example, only 4~8 CSP genes in ants, flies, bees, wasps, and anopheline mosquitoes [44]; 19~20 in butterfly, moth, and beetle [45–47]; and 27 to 83 in *Culex* mosquito species [48]. We identified 69 CSP transcripts produced by 58 genes (Table S2), the number of which is similar to *Culex* mosquito species. CSPs function not only in the chemical communication between insects and the environment but also in some other cellular processes, such as lipid transport, general immunity, insecticide resistance, and xenobiotic degradation [45,49]. In ants, CSPs have been proposed to mediate the recognition of chemical signatures composed of cuticular lipids [50]. The differential expression of CSP transcripts identified in this study (Table S4) may support the ability to recognize different scents from their hosts.

In the present study, we also identified some other olfactory gene families, such as OR, IR, GR, and SNMP, which were differentially expressed in response to the fruit fly with different scents (Table S2, Table S4). OR is the name for all molecules that are expressed in the cell membranes of olfactory receptor neurons and are responsible for the detection of odorants. The ORs form a multigene family consisting of around 800 genes in humans and 1400 genes in mice [51]. We identified 689 OR transcripts derived from 637 genes (Table 2, Table S2). The diversity of ORs might help insects to discriminate as many different odors as possible. GRs are found be expressed exclusively in gustatory receptor neurons [52]. However, many GRs are not related to taste receptors but function in the detection of sugars, bitter compounds, and non-volatile pheromones [53]. Interestingly, GR28B represents a new class of thermosensor and is required for thermotaxis [54]. We found GR28B differentially expressed in C and M (Figure 4C, Table S2). This might be evidence of its new role in the detection of different scents. IR genes are expressed in coeloconic sensilla of the antenna and respond, among others, to water and amines [55]. IRs are not related to insect ORs but rather have evolved from ionotropic glutamate receptors (iGluRs), a conserved family of synaptic ligand-gated ion channels [56]. In this study, we identified 26 IR transcripts, and 18 of them encode IR25A (Table 2, Table S2). It is conceivable that the IR25A ancestor initially evolved as a sensory detector for external glutamate, analogous to the synaptic function of iGluRs, and that it only later acquired a co-receptor function after duplication and diversification of the IR repertoire [56]. We also identified 14 SNMP transcripts in the parasitoid wasps, including 11 encoding SNMP1 and 3 encoding SNMP2 (Table 2, Table S2). The SNMP1 has been shown to be antenna specific and play an important role in pheromone detection [57]. While SNMP2, which acts as a second lepidoperan and also associates with pheromone-sensitive sensilla, has been shown to be expressed in sensilla support cells rather than neurons [58,59]. The identification and differential expression of olfactory-related transcripts revealed the complex chemosensory system of *D. longicaudata* and supported a diverse function of olfactory genes in discriminating different chemical cues.

The limitations of this project may include the use of the tissue mixture of insects. It is said that some OBPs are expressed in insects with a sex preference. For example, MsepOBP5 exhibited female-biased expression in 0- and 5-day-old adults; MsepOBP22 displayed female-biased expression in 0- and 5-day-old adults but was male-biased in 3-day-old adults [60]. Due to the difficulty of the sample preparation, it is hard to ge<sup>t</sup> enough material for sequencing with the same sex. Additionally, it is difficult to determine the tissue-specific olfactory genes. However, our findings provide a basis of future studies about the olfactory system in *D. longicaudata*.
