Identiﬁcation and Expression Analysis of Odorant-Binding and Chemosensory Protein Genes in Virus Vector Nephotettix cincticeps

Simple Summary: Nephotettix cincticeps , one of the destructive pests of rice plants, not only directly damages hosts by sucking, but also indirectly transmits plant viruses in the ﬁeld. However, the available resources of olfactory of N. cincticeps are little known, especially information regarding the odorant binding proteins (OBPs) and chemosensory proteins (CSPs) which are involved in the ﬁrst barriers for receiving and sensing chemical signals. In the present study, the N. cincticeps adults head and the rest of the body transcriptome were sequenced and analyzed using Illumina sequencing. Twenty putative OBP and 13 CSP genes were identiﬁed. A total of 12 OBP and 10 CSP genes were detected, and nine OBP and three CSP genes were highly expressed in N. cincticeps antennae with tissue expression levels analysis. This study will help us understand the molecular mechanism of N. cincticeps in the detection and recognition of rice volatiles. Abstract: The insect odorant binding proteins (OBPs) and chemosensory proteins (CSPs) are involved in the perception and discrimination of insects to host odor cues. Nephotettix cincticeps , one of the destructive pests of rice plants, not only directly damages hosts by sucking, but also indirectly transmits plant viruses in the ﬁeld. Previous study found that two rice volatiles (( E )- β -caryophyllene and 2-heptanol) induced by rice dwarf virus (RDV) mediated the olfactory behavior of N. cincticeps , which may promote virus dispersal. However, the OBPs and CSPs in N. cincticeps are still unknown. In this study, to identify the OBP and CSP genes in N. cincticeps , transcriptomic analyses were performed. In total, 46,623 unigenes were obtained. Twenty putative OBP and 13 CSP genes were discovered and identiﬁed. Phylogenetic analyses revealed that ﬁve putative OBPs belonged to the plus-C OBP family, and the other classic OBPs and CSPs were distributed among other orthologous groups. A total of 12 OBP and 10 CSP genes were detected, and nine OBP and three CSP genes were highly expressed in N. cincticeps antennae compared with other tissues. This study, for the ﬁrst time, provides a valuable resource to well understand the molecular mechanism of N. cincticeps in the perception and discrimination of the two volatiles induced by RDV infection.


Introduction
Insect chemoreception system plays a pivotal role in many aspects of insect behaviors, such as foraging, mate recognition, oviposition, and avoiding predators [1,2]. In the insect olfactory perception process, various olfactory proteins containing odorant binding proteins (OBPs), chemosensory proteins (CSPs), odorant receptors (OR), ionotropic receptors (IR) and odorant degrading enzymes are involved [3,4]. Among these olfactory proteins, OBPs and CSPs could Insects 2022, 13, 1024 3 of 16 abdomen, leg and wing were quickly separated and stored in liquid nitrogen until RNA extraction. There were four biological replicates for different tissues.

cDNA Library Construction and Transcriptome Sequencing
Total RNA was independently extracted from the female, male head, and other body parts of adult N. cincticeps using Trizol reagent (Invitrogen, Waltham, CA, USA) following the manufacturer's procedure. Quantity and purity of the RNA were analyzed with Bioanalyzer 2100 and RNA 1000 Nano LabChip Kit (Agilent, Santa Clara, CA, USA) (RIN number > 7.0). Poly(A) RNA is purified from total RNA using poly-T oligo-attached magnetic beads with two rounds of purification. After purification, the mRNA is fragmented into small pieces using divalent cations under elevated temperatures. Then the cleaved RNA fragments were reverse-transcribed to create the final cDNA library in accordance with the protocol for the mRNA-Seq sample preparation kit (Illumina, San Diego, CA, USA), the average insert size for the paired-end libraries was 300 bp (±50 bp). Then the paired-end sequencing on an IlluminaHiseq4000 at the (LC Sciences, Houston, TX, USA) was performed following the vendor's recommended protocol.

De Novo Assembly, Unigene Annotation, and Functional Classification
Clean short reads were obtained by removing those containing an adapter or poly-N (>5%) and of low quality (Q ≤ 20) from the raw reads [40]. De novo assembly of the transcriptome was performed with Trinity 2.4.0 [41].

Identification of OBP and CSP Genes
The head and other body transcriptome dataset of N. cincticeps was constructed in our laboratory using the Illumina platform. Putative NcinOBP and NcinCSP genes were identified by searching the keywords (odorant binding protein and chemosensory protein) in the annotated unigenes. In addition, we obtained the putative OBP and CSP genes from the genome dataset of N. cincticeps (provided by Professor Li Yi of Peking university). Then all the candidate OBP and CSP genes of N. cincticeps were confirmed by comparing the sequence against the NCBI non-redundant (nr) protein database using BLASTX. The conserved domains of these putative OBPs and CSPs were predicted by NCBI online tool (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi? (accessed on 28 January 2021). The open reading frame (ORF) of each candidate unigene was predicted using the ORF finder tool (https://www.ncbi.nlm.nih.gov/orffinder/ (accessed on 28 January 2021). The signal peptides of putative NcinOBP and NcinCSP proteins were predicted by the SignalP V 5.0 program (https://services.healthtech.dtu.dk/service.php?SignalP-5.0 (accessed on 28 January 2021).

Sequence Alignment and Phylogenetic Analysis
The amino acid sequences of candidate OBP and CSP of N. cincticeps (without signal peptide sequences) were aligned with their orthologs from other Hemipteran insect species by Clustal W [43]. A total of 161 OBP protein sequences from 9 Hemipteran species were used to construct the phylogenetic tree including 20 OBPs of N. cincticeps discovered in the present study, 44 Table S1). In addition, 103 CSP protein sequences from nine Hemipteran species were selected for the phylogenetic analysis including 13 CSPs of N. cincticeps identified in this study,  Table S2). These insect OBP and CSP sequences other than N. cincticeps were obtained from Zhao [44]. The OBP and CSP phylogenetic trees were constructed by MEGA 6.0 software (Mega Limited, Auckland, New Zealand) with the neighbor-joining method using the Poisson correction distance and 1000 bootstrap replications [45]. The final phylogenetic trees were optimized and visualized by an online tool, iTOL (https://itol.embl.de/ (accessed on 12 March 2021).

Tissue Expression Profile Analysis
The expression profiles for different tissues (female and male adults) of these candidate OBPs and CSPs were evaluated by qRT-PCR. The total RNA of different tissue of N. cincticeps including antennae, heads, thoraxes, abdomens, legs, and wings were extracted and reversely transcribed into cDNA as described previously [46]. The β-actin as a reference gene was used for normalizing target gene expression and to correct for sample-to-sample variation. The specific primers used for qRT-PCR analysis were designed with Primer 3.0 (http://bioinfo.ut.ee/primer3-0.4.0/ (accessed on 18 March 2021) (Table S3). cDNA templates and primer sets were mixed with SYBR ® Premix Ex Taq TM II (Tli RNaseH Plus; Takara, Shiga, Japan) and real-time PCR was performed on the CFX Connect TM Real-Time Detection System (Bio-Rad, Hercules, CA, USA). Negative controls were non-template reactions (replacing cDNA with sterile H 2 O). Four biological replicates were conducted for each experiment. The relative quantification was calculated using the comparative 2 −∆∆Ct method [47].

Statistical Analysis
All data were performed with SPSS 20.0 software(IBM, Armonk, NY, USA). The comparative analysis of each OBP and CSP genes among various tissues was determined using a one-way tested analysis of variance (ANOVA), followed by Tukey's multiple range test (p < 0.05). The values were presented as the mean ± standard error.

Overview of the Head and Rest of the Body Transcriptomes
Three mixed adult head and the rest of the body (10 females and 10 males) cDNA libraries were constructed and sequenced respectively using the IlluminaHiseq4000 platform. After filtering, a total of 55,049,478, 53,867,286, 54,310,996 (head samples) and 56,650,118,55,478,556,54,656,850 (rest of the body samples) clean data were generated with a Q20 scores (98.98%, 98.95%, 98.74% head samples; 98.89%, 98.98%, 98.50% rest of the body samples) (Table S4). Subsequently, all the clean reads were assembled together and obtained 46,623 unigenes with lengths ranging from 201 to 16,461 bp, with a mean length of 456 bp (Table S5).
Only 13,891 (29.79%) assembled unigenes were annotated in three different functional groups (biological process, cellular component, and molecular function) with the GO category analysis ( Figure S2). In the biological process category, the terms oxidationreduction process and biological process were the most abundant. In the cellular component category, the cytoplasm and nucleus were most represented. In the molecular function category, the protein and ATP binding were the most abundant.
In KEGG annotation, 12,142 assembled transcripts were divided into six classes including organismal systems, metabolism, human diseases, genetic information processing, environmental information processing, and cellular process ( Figure S3). In all pathways, several major ones in each class were identified containing transport and catabolism, signal transduction, translation, infectious diseases, carbohydrate metabolism, and the immune system.

Identification of Candidate OBP and CSP Genes in N. cincticeps
Based on the transcriptome and genome data, a total of 20 putative OBP genes were identified with the verification by BLASTX and BLASTN online tools ( Table 1). The size of these OBP genes ranged from 118 to 296 amino acids. Most of the annotated OBP genes contained a signal peptide at their N-terminal part (other than NcinOBP4, 6, 11, 12, 15, and 17), which is a signature of secretory proteins. Based on the numbers and locations of the conserved cysteines, the candidate NcinOBP1, OBP6, OBP8, OBP12, and OBP13 genes belonged to the plus-C OBP family, while the other OBP genes were divided into the classic OBP family ( Figure 1).  Similarly, 13 transcripts encoding putative CSPs were obtained in transcriptome and genome data ( Table 2). Most of the CSP sequences contained the complete ORFs with the size ranging from 109 to 207 amino acids (except for CSP2, 8,12), and all the sequences had a signal peptide. All the annotated CSP proteins had four conserved cysteines (Cys-X6-8-Cys-X18-19-Cys-X2-Cys) [48] (Figure 2).   Similarly, 13 transcripts encoding putative CSPs were obtained in transcriptome and genome data ( Table 2). Most of the CSP sequences contained the complete ORFs with the size ranging from 109 to 207 amino acids (except for CSP2, 8,12), and all the sequences had a signal peptide. All the annotated CSP proteins had four conserved cysteines (Cys-X6-8-Cys-X18-19-Cys-X2-Cys) [48] (Figure 2).

Phylogenetic Analysis of N. cincticeps OBP and CSP Genes
Due to E. onukii and N. cincticeps belonging to Cicadellidae, L. striatella, S. furcifera, N. lugens, and N. cincticeps having the same habitats, and the OBP/CSP of the other four insects being well annotated, thus we chose these species to construct the phylogenetic analysis. A phylogenetic tree containing of nine insect species of 161OBPs protein sequences in Hemiptera was constructed (Figure 3). These candidate OBP proteins were segmented into two classes of OBP sub-family (Plus-C and classic OBP family). NcinOBP1, OBP6, OBP8, OBP12, and OBP13 proteins were clustered into the Plus-C family, while the other OBP proteins were in the classic OBP family, indicating that these five genes have different evolutionary relationships with the other 15 OBP genes. The putative OBP proteins of N. cincticeps clustered into a different branch, but most sequences were gathered with the OBP proteins of E. onukii. In addition, NcinOBP4 was clustered with AlucOBP28 and

Phylogenetic Analysis of N. cincticeps OBP and CSP Genes
Due to E. onukii and N. cincticeps belonging to Cicadellidae, L. striatella, S. furcifera, N. lugens, and N. cincticeps having the same habitats, and the OBP/CSP of the other four insects being well annotated, thus we chose these species to construct the phylogenetic analysis. A phylogenetic tree containing of nine insect species of 161OBPs protein sequences in Hemiptera was constructed (Figure 3). These candidate OBP proteins were segmented into two classes of OBP sub-family (Plus-C and classic OBP family). NcinOBP1, OBP6, OBP8, OBP12, and OBP13 proteins were clustered into the Plus-C family, while the other OBP proteins were in the classic OBP family, indicating that these five genes have different evolutionary relationships with the other 15 OBP genes. The putative OBP proteins of N. cincticeps clustered into a different branch, but most sequences were gathered with the OBP proteins of E. onukii. In addition, NcinOBP4 was clustered with AlucOBP28 and AlinOBP10. NcinOBP9 was clustered with SfurOBP10 and LstrOBP1, while NcinOBP11 was clustered with BtabOBP1, implying that these genes may have similar functions.
The neighbor-joining tree of 103 CSPs with nine insect species in Hemiptera was established ( Figure 4). In this phylogenetic tree, all the putative CSPs could cluster with at least one hemipteran ortholog. Similarly, most of the NcinCSP proteins were clustered with the CSP proteins of E. onukii, because the two insects are members of Cicadellidae. Moreover, AgosCSP4 and NcinCSP3 were gathered with EonuCSP14, and Nc-inCSP11 was clustered with BtabCSP7, which suggests that these CSP genes maybe showed common functions.

Tissue Expression Levels of Candidate OBP and CSP Genes
The expression levels of these identified OBP and CSP genes in the antennas, heads, thoraxes, abdomens, legs, and wings of N. cincticeps were measured by qRT-PCR. Nine candidate OBP genes (NcinOBP1-9) were mostly expressed in antennas, with 53 to 1042 times higher than in wings ( Figure 5). While NcinOBP12 and NcinOBP14 exhibited a relative expression level of 493 and 116 times higher in heads than in wings respectively. The expression level of NcinOBP13 was higher in antennas and heads than in other tissues. The other eight putative OBP genes (NcinOBP10-11, [15][16][17][18][19][20] were not detected by qRT-RCR, maybe due to their expression levels being too low to detect.  The neighbor-joining tree of 103 CSPs with nine insect species in Hemiptera was established ( Figure 4). In this phylogenetic tree, all the putative CSPs could cluster with at least one hemipteran ortholog. Similarly, most of the NcinCSP proteins were clustered with the CSP proteins of E. onukii, because the two insects are members of Cicadellidae. Moreover, AgosCSP4 and NcinCSP3 were gathered with EonuCSP14, and NcinCSP11 was clustered with BtabCSP7, which suggests that these CSP genes maybe showed common functions.

Tissue Expression Levels of Candidate OBP and CSP Genes
The expression levels of these identified OBP and CSP genes in the antennas, heads, thoraxes, abdomens, legs, and wings of N. cincticeps were measured by qRT-PCR. Nine candidate OBP genes (NcinOBP1-9) were mostly expressed in antennas, with 53 to 1042 times higher than in wings ( Figure 5). While NcinOBP12 and NcinOBP14 exhibited a rel-  expression level of NcinOBP13 was higher in antennas and heads than in other tissues. The other eight putative OBP genes (NcinOBP10-11, [15][16][17][18][19][20] were not detected by qRT-RCR, maybe due to their expression levels being too low to detect. Figure 5. Relative expression levels of OBPs in different tissues of N. cincticeps analyzed by qRT-PCR. Four biological replicates were conducted for each experiment. The relative quantification was calculated using the comparative 2 −ΔΔCt method. The comparative analysis of each OBP genes among various tissues was determined using a one-way tested analysis of variance (ANOVA), followed by Tukey's multiple range test (p < 0.05). Different lowercase letters indicate significant differences between different tissues. The values were presented as the mean ± standard error. Four biological replicates were conducted for each experiment. The relative quantification was calculated using the comparative 2 −∆∆Ct method. The comparative analysis of each OBP genes among various tissues was determined using a one-way tested analysis of variance (ANOVA), followed by Tukey's multiple range test (p < 0.05). Different lowercase letters indicate significant differences between different tissues. The values were presented as the mean ± standard error.
For CSPs, four CSPs (NcinCSP2, 3, 4, 6) were specifically expressed in antennas, with 31 to 63 times higher than in wings ( Figure 6). NcinCSP1 and NcinCSP5 were comparatively highly expressed in the abdomen and leg respectively, compared with the expression levels of other tissues. The expression level of NcinCSP7 was higher in the wing than that in other tissues. NcinCSP8 was comparatively highly expressed in the head and abdomen than that in other tissues, while NcinCSP11 was highly expressed in antennae, head, and leg than that in the thorax, abdomen, and wing. NcinCSP9 was more highly expressed in the head than that in the thorax. Due to the undetectable expression levels (Ct value > 35), the other three CSP genes (NcinCSP10, 12,13) were not detected by qRT-RCR. that in other tissues. NcinCSP8 was comparatively highly expressed in the head and abdomen than that in other tissues, while NcinCSP11 was highly expressed in antennae, head, and leg than that in the thorax, abdomen, and wing. NcinCSP9 was more highly expressed in the head than that in the thorax. Due to the undetectable expression levels (Ct value > 35), the other three CSP genes (NcinCSP10, 12,13) were not detected by qRT-RCR. Figure 6. Relative expression levels of CSPs in different tissues of N. cincticeps analyzed by qRT-PCR. Four biological replicates were conducted for each experiment. The relative quantification was calculated using the comparative 2 −ΔΔCt method. The comparative analysis of each CSP genes among various tissues were determined using one-way tested analysis of variance (ANOVA), followed by Tukey's multiple range test (p < 0.05). Different lowercase letters indicate significant differences between different tissues. The values were presented as the mean ± standard error. Four biological replicates were conducted for each experiment. The relative quantification was calculated using the comparative 2 −∆∆Ct method. The comparative analysis of each CSP genes among various tissues were determined using one-way tested analysis of variance (ANOVA), followed by Tukey's multiple range test (p < 0.05). Different lowercase letters indicate significant differences between different tissues. The values were presented as the mean ± standard error.

Discussion
N. cincticeps is one of the serious insect pests affecting rice production in East Asia [36]. RDV transmitted by N. cincticeps threatens rice crop yield and leads to enormous economic losses [49]. Two rice plant volatiles ((E)-β-caryophyllene and 2-heptanol) induced by RDV infection influenced the olfactory behavior of N. cincticeps independently [38]. The relevant OBP and CSP gene information will show a promising starting point to understand the molecular mechanism of N. cincticeps in the detection and recognition of the two volatiles. According to the transcriptomic analyses, 20 OBP genes and 13 CSP genes were identified. As well, nine of the OBP genes and three of the CSP genes were highly expressed in N. cincticeps antennae compared with other tissues. This information could be helpful for developing new prevention and control strategies with the two volatiles.  [53] were reported. But it is significantly lower than in E. onukii (44 OBP genes) and in A. lucorum (38 OBP genes), respectively [44,54]. The number of N. cincticeps CSP genes, it is approximately close to L. striatella and S. furcifera [55], but it is markedly lower than in E. onukii (28 CSP genes) [44]. Different Hemipteran insect species have different numbers of OBP and CSP genes, which might be associated with insect habits or other environmental factors. For example, E. onukii had more OBP and CPS genes compared to other insects, which may be due to the wide range of host plants and complicated environment [56,57]. The internal mechanisms of different Hemipteran insect species with a different number of OBP and CSP genes need to be further investigated.
The evolutionary analysis will provide new insights into the evolution and function of N. cincticeps OBP and CSP genes with other Hemipteran species. In this study, most OBP and CSP genes of N. cincticeps were clustered into the OBP and CSP genes of E. onukii, indicating that the two insects had high homology relations. But the N. cincticeps OBPs are in different little branches, and the OBP sequences consistency is low, implying that the functions of these OBPs are differentiation. The OBP genes were divided into two classes of OBP sub-family (Plus-C and classic OBP family) ( Figure 3). The plus-C NcinOBP genes were subsequently clustered into two independent orthologous groups. NcinOBP6 was in one orthologous group, and NcinOBP1, OBP8, OBP12, and OBP13 were in another orthologous group, suggesting that the two plus-C OBP genes orthologous groups were perhaps derived from common ancestors and then diverged before speciation [58]. The NcinCSP genes were clustered with different hemipteran ortholog groups, indicating that the CSP genes have an earlier origin and are more conserved compared with the OBP genes [59,60]. L. striatella, S. furcifera, N. lugens, and N. cincticeps have the same habitats (feeding host plants of Poaceae, such as rice and maize). Previous studies showed that (E)-β-caryophyllene had attractions to S. furcifera and N. cincticeps [38,61]. The phylogenetic tree suggested that NcinOBP9 was clustered with SfurOBP10. Thus, we speculated that the two OBP genes might have similar functions.
Different locations of OBP and CSP genes in insect tissues might have different functions [62][63][64][65][66]. Therefore, investigation of the tissue expression patterns of OBP and CSP genes in N. cincticeps might help to predict their physiological functions. Herein, we found that most OBP genes (9 of 12) were significantly expressed in N. cincticeps antennae compared with other tissues, which is consistent with other Hemipteran species (e.g., [55,59,66]). Insect antennae are thought to be a mainly olfactory organs to perceive the host plant volatiles, alarm pheromones, and sex pheromones [67][68][69][70]. Thus, we speculate that the nine OBPs and three CSPs, which are highly expressed in N. cincticeps antennae, might be involved in the perception and discrimination of the two rice volatiles induced by RDV infection. In addition, two OBP (NcinOBP12, OBP14) and two CSP genes (NcinCSP1, CSP5, CSP7, CSP8) are markedly expressed in N. cincticeps head and leg compared with antennae respectively, which may play other functions, for instance, carriers of visual pigments, regeneration and development, anti-inflammatory action, nutrition or insecticide resistance [4,24,71]. The specific functions of these OBP and CSP genes will be studied in the next step.

Conclusions
In summary, a total of 20 putative OBP and 13 CSP genes are identified from the N. cincticeps transcriptome. Twelve OBP and 10 CSP genes are detected by qRT-PCR and, nine OBP and three CSP genes are highly expressed in N. cincticeps antennae compared with other tissues. Further study needs to analyze the effects of RDV infection on the expression of these OBP and CSP genes, which could be selected as the potential target proteins. Then the molecular mechanism of non-viruliferous and viruliferous N. cincticeps perception of the two volatiles induced by RDV would be analyzed respectively, which will