**1. Introduction**

*Diachasmimoorpha longicaudata* (Ashmead, *D. longicaudata*) is a solitary species of parasitoid wasp of several fruit fly species and has been introduced to many countries as a biological control agent. Its host, *Bactrocera dorsalis* Hendel, can attack many fruit species and some other plants, such as Caricaceae, Moraceae, Myrtaceae, Rosaceae, and Solaneaceae [1]. It is said that the female Ashmead

insects can detect the fly larvae by sound in rotting fruit and that the attractant could be the fungal fermentation products rather than the chemical substances produced by the fly larvae [2,3]. Carrasco and colleagues reported that the presence of fly larvae was essential for the orientation of wasps [4]. Further, it was proposed that cues from the fruit can be used by the wasps directly and that the presence of the host can enhance the attraction towards a patch [5]. Interestingly, chemical compounds produced by the larvae can be detected by wasps to locate the host [6,7]. Once the female parasitoid is on the fruit, a specific chemical compound released by some Tephritidae species can be used to enhance the host search [7]. However, much is unknown about the chemosensory system of parasitoid wasps in response to their hosts.

Animals can use the chemosensory system to detect and discriminate chemical cues in the environment [8]. The chemical sensors of insects are mainly from the antennae system, which is a highly specific and extremely sensitive chemical detector, and olfactory proteins in the antennae can be used by the insects to detect very low-abundance odorants from thousands of odors and further to guide their behaviors, such as forage, mate hunting, host plant location, shelter, and selection of spawning sites [9]. Since the first odorant-binding protein (OBP) was identified in *Antheraea polyphemus* [10], research on insect OBP has become a hot spot in the field of entomology. However, OBP-related studies have mainly been demonstrated in some important pests [11], and very few have been reported in parasitoid wasps, which are very important natural enemies of the pests.

The chemoreception of insects involves three important events: (i) The uptake of signal molecules from the external environment; (ii) transport (di ffusion) through the sensory hair; and (iii) interaction with the chemoreceptor, which in turn activates the cascade of events leading to spike activity in sensory neurons [12]. Some important protein families have been reported to participate in these events, such as OBP, sensory neuron membrane protein (SNMP), chemosensory protein (CSP), odorant receptor (OR), gustatory receptor (GR), and odorant ionotropic receptor (IR) [12]. Insect OBPs are small globular proteins (~135 to 220 amino acids) and are characterized by a specific domain that comprises six α-helices joined by three disulphide bonds [12]. They can be categorized into two subgroups: Pheromone-binding proteins, which are mainly distributed in the male antenna, and general OBP (GOBP), which can be found in multiple tissues of male and female insects and function in the recognition of odorants from plants and other animals [13]. There are about 300 OBPs in the NCBI database and not many studies have been demonstrated to identify the OBPs in parasitoid wasps. Xu and colleagues used transcriptome sequencing and identified 1 CSP, 21 OBPs, 53 ORs, 29 IRs, and 4 SNMPs in *Bactrocers minax* [14], an oligophagous tephritid insect whose host selection, and oviposition behavior largely depend on the perception of chemical cues. Zhu et al. reported Sgua-OBP1 and Sgua-OBP2 in *Scleroderma guani* (Hymenoptera: Bethylidae) and NvitOBP in *Nasonia vitripennis* [15]. Zhang identified 10 OBPs in *Microplitis mediator* (Halidag) [16]. Zhao et al. identified 25 OBPs, 80 ORs, 10 IRs, 11 CSP, 1 SNMPs, and 17 GRs in adult male and female *Chouioia cunea* antennae [17]. However, little is known about the olfactory proteins in *D. longicaudata*.

Harbi and colleagues demonstrated a multistep assay (e.g., olfactory, laboratory, and semi-field trials) and reported the preference of medfly-infected fruits, including apple, orange, peach, and clementine mandarins [18]. This experiment supports that di fferent olfactory genes are expressed in response to di fferent fruit scents. In this study, we used transcriptome sequencing to study the olfactory genes in *D. longicaudata*. By similarity, we identified a number of OBPs, CSPs, ORs, IRs, SNMPs, and GRs expressed in the Ashmead insects. Our results also showed that di fferent olfactory genes were expressed in the search of their host with di fferent fruit scents. This is the first time to identify the gene and protein sequences for the olfactory products in this species. Our findings will provide a basis for future molecular studies and improve our understanding of the chemosensory system of parasitoid wasps.

#### **2. Materials and Methods**

## *2.1. Insect Rearing*

The Ashmead and fruit fly larvae were obtained from the Institute of Plant Protection (IPP), Hainan Academy of Agricultural Sciences and maintained in the experimental fields. The fruit flies were fed with guava (G), mango (M), and carambola (C) separately. A mixture of yeas<sup>t</sup> and sucrose (1:1) was used as supplementary nutrition for the fruit flies in the adult stage. Then, late-second and early-third instar fruit flies were used as hosts for the Ashmead insects. The Ashmead insects were fed with 15% honey water and clean water; the fifth, sixth and seventh generations of the Ashmead adults, which were parasitic to the fruit flies, maintained with G (G1~G3), M (M1~M3), and C (C1~C3), were used as biological replicates. The antenna, head, breast, abdomen, and feet tissues of the one male and two female wasps were mixed together for RNA extraction.

#### *2.2. RNA Isolation, Library Construction, and Deep Sequencing*

Total RNA was extracted from the insect tissues using the TRIzol reagent, as previously described [19,20]. The quality and quantity of total RNA were determined by multiple instruments, including a Nanodrop 2000 (Thermo Scientific, MA, USA), Qubit 4 Fluorometer (Invitrogen, CA, USA), and Agilent 2100 Bioanalyzer. Then, the total RNA (1 μg) of each sample was used to build the cDNA library using the TruSeq RNA Library Preparation Kit v2 protocol (Illumina, CA, USA), as described [20]. After the cDNA libraries were quality controlled by the Agilent 2100 Bioanalyzer and qRT-PCR, they were sequenced on the Illumina HiSeqXTEN platform with the paired-end 150 strategy. Raw sequencing reads of these samples can be accessed from the NCBI SRA platform under the accession numbers SRR10766480~SRR10766488.
