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Identification and Expression Analysis of an Olfactory Receptor Gene Family in the Yemma signatus Hsiao (Hemiptera: Berytidae)

PJZ_52_5_1911-1919

 

 

Identification and Expression Analysis of an Olfactory Receptor Gene Family in the Yemma signatus Hsiao (Hemiptera: Berytidae)

Yue Qin Song, Hui Zhong Sun* and Jun Feng Dong

Forestry College, Henan University of Science and Technology, Luoyang 471000, China

ABSTRACT

Olfactory receptors (ORs) in the dendritic membrane of olfactory cells are the key elements in the molecular recognition and discrimination of odorants. On the basis of female and male antennal transcriptomes of Yemma signatus adults, a total of 66 candidate Y. signatus olfactory receptor genes (YsigORs), including one olfactory co-receptor (Orco), were identified in this study. All the sequences were further validated by cloning and sequencing. Tissue expression profiles of all YsigOR genes in the antennae of females and males were analyzed using real-time quantitative PCR (RT-qPCR). The result showed that some YsigOR genes displayed significant differences in the expression levels between sexes. YsigOrco had the highest expression level in all YsigOR genes; however, the expression level in males was twice as that of females. Our study provides valuable biological information for studying the olfactory communication system of Y. signatus.


Article Information

Received 16 June 2018

Revised 01 March 2019

Accepted 23 April 2019

Available online 19 May 2020

Authors’ Contribution

SYQ and SHZ designed and performed the research, analyzed the data, and wrote the paper. All authors were involved in writing and revisions of the manuscript.

Key words

Yemma signatus, Antennal transcriptome, Olfactory receptor family, Expression patterns.

DOI: https://dx.doi.org/10.17582/journal.pjz/20180616040644

* Corresponding author: [email protected]

0030-9923/2020/0005-1911 $ 9.00/0

Copyright 2020 Zoological Society of Pakistan



Introduction

The olfactory system plays a crucial role in most insects in the detection and discrimination between small, volatile compounds in the environment. The ability to sensitively and specifically recognize odors is crucial for their survival as these chemical signals are important for avoiding predators and can provide essential information about the sources of food, mating, and oviposition (Field et al., 2000; Zhang et al., 2015). Receptor proteins in the dendritic membrane of olfactory cells are the key elements in molecular recognition of odorants. These receptor proteins include three large, distinct families: olfactory receptors (ORs), gustatory receptors (GRs), and ionotropic receptors (IRs) (Clyne et al., 1999, 2000; Benton et al., 2009).

Insect OR genes were the first chemoreceptor family to be found in the Drosophila melanogaster genome (Gao et al., 1999). Unlike vertebrate ORs, which are G-protein coupled receptors (GPCRs), these ORs are seven transmembrane domain (TMD) receptors with an inverted membrane topology containing an intracellular N-terminus and an extracellular C-terminus (Benton et al., 2006; Lundin et al., 2007; Smart et al., 2008). These proteins are specifically expressed in the olfactory sensory neurons (OSNs) of the antennae and maxillary palps of the insect, where they are concentrated in the sensory dendrites of the cells. Subsequently, the OR genes of other insects have also been sequenced and identified using bioinformatics methods. A specific ligand-binding OR type that forms a heteromer with a second common co-receptor from the OR gene family has been reported (Benton et al., 2006). Formerly, it was named Or83b in Drosophila and OR2 or OR7 in other insects. According to the most recent nomenclature, this protein is referred to as Orco. The number of OR genes in different insect species varies greatly, from 10 in Pediculus humanus (Kirkness et al., 2010) to 400 in Pogonomyrmex barbatus (Smith et al., 2011). It is speculated that these variations in the number of OR genes among species reflects evolutionary adaption to certain ecological and physiological demands in the search for food or the major importance of odorants in social communication between insects living in colonies. In recent years, many Lepidopteran OR genes were explored using the Xenopus oocyte expression system (Mitsuno et al., 2008). However, to date, the exact functions of insect OR genes are largely unknown.

Yemma signatus (Hsiao) (Hemiptera: Berytidae) is an identified omnivorous insect that feeds on plants and small insects. It was recorded in China as a pest that sucks juices out of Paulownia tree leaves (Yang, 1982). Interestingly, Y. signatus is also considered beneficial as it feeds on small insects, such as Cicadellidae insects, that damage fruit trees (Liang et al., 1992). Currently, little molecular information is available on Y. signatus. In this study, we sought to identify and annotate olfactory receptor genes in Y. signatus antennae using de novo transcriptome sequencing and assembly. Next, tissue expression profiles of all YsigOR genes in the antennae of females and males were analyzed by real-time quantitative PCR (RT-qPCR). The present study provides bases for the functional study of ORs of Y. signatus.

 

Materials and methods

Insects and sample collection

The laboratory strain of Y. signatus was collected from cotton in Luoyang, Henan, China (112-26´E, 34-43´N) in 2014 and reared on cotton plants in a greenhouse at 27 ± 3°C with a 14 h:10 h light/dark cycle and 60%–80% relative humidity. Approximately 500 female and 500 male adult antennae were respectively dissected from Y. signatus (3–4 days old), immediately frozen in liquid nitrogen, and stored at −80°C until RNA isolation.

cDNA library construction and sequencing

Total RNA from the antennal tissue was extracted using RNAiso Plus kit (TaKaRa, Dalian, China) and treated with DNase I (TaKaRa, Dalian, China) following the manufacturer’s instructions. The concentration, quality, quantity, and integrity of the RNA sample were detected using agarose gel electrophoresis, Nanodrop (Thermo Scientific, USA), Qubit 2.0 (Life Technologies, USA), and Agilent 2100 (Agilent, USA). The antennal RNAs from female and male adults were mixed in a 1:1 ratio to conduct transcriptome sequencing.

Following the TruSeq RNA Sample Preparation Guide v2 (Illumina), mRNA was enriched using magnetic beads crosslinked with oligo (dT) and was fragmented into small pieces using the fragmentation buffer. First-strand cDNA was synthesized using small mRNA fragments, random primers, and reverse transcriptase, and second-strand cDNA synthesis was conducted by adding dNTPs, DNA polymerase I, and RNase H. Next, the double-stranded cDNA was purified with AMPure XP beads (Beckman Coulter, USA) and then treated for end-repairing, poly-A tailing, and sequencing adapter linking processes. The size of the fragment was chosen using AMPure XP beads, and the cDNA library was constructed by PCR amplification (Veriti® 96-Well Thermal Cycle, Applied Biosystems, USA). The concentration and insert size of cDNA library were detected using Qubit 2.0 and Agilent 2100, respectively, and the DNA was quantified with q-PCR (CFX-96, Bio-Rad, USA).

Finally, sequencing was performed in Illumina HiSeqTM 2500 platform to generate 125-bp pair-end reads. Sequencing analysis was performed by the Genomics Services Lab of the Beijing Novogene Technologies Co., Ltd. (Beijing, China). Raw data processing and base calling were performed using the Illumina instrument software.

De novo contig assembly and unigene annotation

Clean reads were obtained by removing short or low quality and adaptor sequences. The transcriptome was assembled using Trinity (version: trinityrnaseq_r20131110) using default settings, except for setting min_kmer_cov to 2 (Grabherr et al., 2011). Unigene function was annotated based on searches against seven databases: NCBI non-redundant protein sequences (Nr, NCBI blast 2.2.28+, e-value = 1e-5), NCBI nucleotide sequences (Nt, NCBI blast 2.2.28+, e-value = 1e-5), protein family (Pfam, HMMER 3.0 package, hmmscan, e-value = 0.01), euKaryotic Ortholog Groups (KOG, NCBI blast 2.2.28+, e-value = 1e-3), Swiss-Prot (NCBI blast 2.2.28+, e-value = 1e-5), Kyoto Encyclopedia of Genes and Genomes (KEGG, KAAS, KEGG Automatic Annotation Server, e-value = 1e-10), and Gene Ontology (GO, Blast2GO v2.5, e-value = 1e-6). Coding sequences (CDS) were predicted by aligning transcriptome sequences to the Nr and Swiss-Prot databases or using ESTScan 3.0.3 (Iseli et al., 1999). The read count of each gene was obtained by mapping clean reads back onto the assembled transcriptome using RSEM software (bowtie2 parameters: mismatch 0). Lastly, the read count was calculated as fragments per kilobase of transcript per million fragments mapped (FPKM).

Bioinformatics analyses

Similarity searches were performed with the NCBI Blast network server (http://blast.ncbi.nlm.nih.gov/). The transmembrane domains (TMDs) of ORs were predicted using TMHMM v. 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0/). The amino acid sequence alignments of the candidate ORs were aligned using MAFFT (http://mafft.cbrc.jp/alignment/server/clustering.html), and phylogenetic trees were constructed using PhyML in Seaview v.4 based on the Jones–Taylor–Thomton (JTT) model with 1000-fold bootstrap replication in neighbor-joining method.

Tissue-specific expression of ORs

All total RNA samples were extracted using the RNAiso Plus kit (TaKaRa, Dalian, China), and the isolated RNA was transcribed to first-strand cDNA by PrimeScriptTM RT reagent Kit with gDNA Eraser (TaKaRa, Dalian, China) following the manufacturer’s instructions. The nucleotide sequences of all 66 YsigORs were confirmed by cloning and sequencing. Real-time quantitative PCRs (RT-qPCRs) were performed with SYBR® Premix Ex TaqTM II (TaKaRa, Dalian, China). The Y. signatus β-actin gene was used as endogenous control correct for sample-to-sample variation. A 200-ng/l concentration cDNA sample was used for different tissues. Primers for RT-qPCR were designed using Primer Premier 5.0 software and are listed in Supplementary Table I. The RT-qPCR reactions were conducted in 20-μL reaction mixtures containing 10-μL SYBR Premix Ex Taq II, 20-ng cDNA templates, 0.2-μM of each primer, and nuclease-free water. The cycling conditions were as follows: one cycle of 95°C for 5 min, followed by 40 cycles of 95°C for 5 s and 55°C for 30 s. Melting curve conditions were 95°C for 10 s and 65°C for 30 s. A no-template control (NTC) was also included to detect for possible contamination. Three biological replicates were analyzed and relative expression levels of OR genes across the samples were measured by the 2-∆∆CT method. Expression levels were calculated relative to the expression level in male antennae of YsigOR56, which was arbitrarily set at 1. The differences in the expression of YsigOR genes between tissues of females and males were compared by a one-way nested analysis of variance (ANOVA), followed by Tukey’s honestly significance difference (HSD) test using SPSS software (SPSS Institute 17.0, IBM, Chicago, IL, USA).

 

Results

Analysis of Y. signatus antennae transcriptome

To identify candidate OR genes from Y. signatus, the transcriptomes of the antennae of males and females were generated using the HiSeq 2500 platform. A total of 62530320 raw reads were produced from the female and male antennae mixture sample, and after filtering, 61080938 clean reads were assembled into 115491 (mean length, 578 bp) unigenes. All sequences from Y. signatus antennal transcriptome were registered in the NCBI database (GenBank: SRR3348966). The assembly of all clean reads together led to the generation of 148736 transcripts with a mean length of 687 bp (Supplementary Table II). BLASTx and BLASTn homology searches of all 115491 unigenes with an E-value < 1.0E-5 showed that 32842 unigenes (28.43%) had BLASTx hits in the Nr databases and 14986 (12.97%) had BLASTn hits in the Nt databases (Supplementary Table III).

GO assignments were used to functionally classify the predicted proteins. Of all the unigenes, 32216 (27.89%) could be classified into three functional categories: molecular function, biological process, and cellular component (Fig. 1). In molecular function category, the genes expressed in the antennae were mostly linked to binding (16903/52.47% unigenes) and catalytic activity (14742/45.76% unigenes). In terms of the biological process, the most represented biological processes were cellular processes (17,197/21.23% unigenes), metabolic processes (17756/22.98% unigenes), and the single-organism process (13552/17.54% unigenes). Among the cellular component terms, cell (9238/19.77% unigenes) and cell part (9237/19.76% unigenes) constituted the most abundant categories.

Identification of Y. signatus ORs

A total of 66 different sequences that encode candidate OR genes were identified, and six of the 66 analyzed candidate genes were partial sequences of genes. They were named YsigOR1 to YsigOR65, and one of the genes was named YsigOrco. The 58 full-length ORs had ORFs measuring about 1200 bp with 4–8 predicted transmembrane domains. We searched for homology of the OR sequences using BLASTx and found that the


 

amino acid sequences of candidate ORs had high sequence conservation with ORs from Halyomorpha halys, followed by Cimex lectularius. The Orco sequence of Y. signatus had very high nucleotide identity (92%) with H. halys Orco, which is often the only one conserved olfactory co-receptor in most insect species (Table I).

Phylogenetic analysis of YsigOR sequences

To better understand the relationship between the different OR genes that were identified in the transcriptome, we conducted a phylogenetic analysis of the 66 candidate YsigOR genes along with OR sequences from Hemipteran insects, Apolygus lucorum, H. halys, Acyrthosiphon pisum, Nilaparvata lugens, and C. lectularius. In the phylogenetic tree, OR genes were extremely divergent and formed various clades, indicating their distinct function in responding to different odors. By contrast, YsigOrco clustered with Orco sequences of different insect species and formed a clear orthologous lineage due to high sequence similarity (Fig. 2).


 

Table I.- Sequences information of ORs in Yemma signatus.

Gene

name

Accesion No.

ORF (aa)

BLASTx best hit (Reference/Name/Species)

E-value identity

Iden-tity

Full length

TM (No.)

Olfactory co-receptor

YsigOrco

MG2046701

474

ref|XP_014279419.1| odorant receptor coreceptor isoformX1 [Halyomorpha halys]

0.0

92%

Yes

7

Other olfactory receptors

YsigOR1

MG204636

413

ref|XP_014271039.1| odorant receptor 83a-like [Halyomorpha halys]

8e-30

25%

Yes

6

YsigOR2

MG204637

410

ref|XP_014287492.1| odorant receptor 4-like [Halyomorpha halys]

8e-63

33%

Yes

6

YsigOR3

MG204638

434

ref|XP_014289672.1| odorant receptor 49a-like [Halyomorpha halys]

7e-68

35%

Yes

6

YsigOR4

MG204639

380

ref|XP_014288704.1| odorant receptor 67c-like isoform X1 [Halyomorpha halys]

8e-32

30%

Yes

6

YsigOR5

MG204640

376

ref|XP_014286385.1| putative odorant receptor 92a

[Halyomorpha halys]

1e-55

33%

Yes

4

YsigOR6

MG204641

381

gb|XP_014257038| odorant receptor Or2-like isoform X2

[Cimex lectularius]

2e-47

30%

Yes

6

YsigOR7

MG204642

354

gb|KPJ01705.1| Putative odorant receptor 30a [Papilio xuthus]

2e-08

26%

Yes

4

YsigOR8

MG204643

437

ref|XP_014289672.1| odorant receptor 49a-like [Halyomorpha halys]

2e-60

35%

Yes

6

YsigOR9

MG204644

434

ref|XP_014289672.1| odorant receptor 49a-like [Halyomorpha halys]

1e-58

34%

Yes

6

YsigOR10

MG204645

415

ref|XP_014271039.1| odorant receptor 83a-like [Halyomorpha halys]

1e-64

32%

Yes

6

YsigOR11

MG204646

417

ref|XP_014274444.1| odorant receptor 47a-like [Halyomorpha halys]

2e-19

25%

Yes

7

YsigOR12

MG204647

429

ref|XP_014271039.1| odorant receptor 83a-like [Halyomorpha halys]

3e-53

31%

Yes

7

YsigOR13

MG204648

392

ref|XP_014273330.1| odorant receptor 85b-like [Halyomorpha halys]

4e-118

47%

Yes

6

YsigOR14

MG204649

418

ref|XP_014271039.1| odorant receptor 83a-like [Halyomorpha halys]

6e-38

26%

Yes

5

YsigOR15

MG204650

230

ref|XP_014289672.1| odorant receptor49a-like [Halyomorpha halys]

1e-46

38%

No (5′ lose)

3

YsigOR16

MG204651

379

ref|XP_014249919.1|putative odorant receptor 69a, isoform A

[Cimex lectularius]

2e-17

26%

Yes

4

YsigOR17

MG204652

416

ref|XP_014287492.1| odorant receptor 4-like [Halyomorpha halys]

2e-73

35%

Yes

6

YsigOR18

MG204653

385

ref|XP_014274900.1| odorant receptor 30a-like [Halyomorpha halys]

5e-39

27%

Yes

6

YsigOR19

MG204654

364

ref|XP_014261210.1| odorant receptor 45b-like [Cimex lectularius]

1e-85

41%

Yes

3

YsigOR20

MG204655

391

ref|XP_014287040.1| odorant receptor 4-like [Halyomorpha halys]

7e-81

36%

Yes

6

YsigOR21

MG204656

404

ref|XP_014242040.1| odorant receptor 85b-like [Cimex lectularius]

2e-67

35%

No (3′ lose)

4

YsigOR22

MG204657

398

ref|XP_014273330.1| odorant receptor 85b-like [Halyomorpha halys]

3e-32

27%

Yes

5

YsigOR23

MG204658

407

ref|XP_014275988.1| odorant receptor 22c-like [Halyomorpha halys]

1e-37

26%

Yes

5

YsigOR24

MG204659

447

ref|XP_014289672.1| odorant receptor 49a-like [Halyomorpha halys]

2e-59

32%

Yes

6

YsigOR25

MG204660

379

ref|XP_014282544.1| odorant receptor 4-like [Halyomorpha halys]

4e-72

36%

Yes

6

YsigOR26

MG204661

231

ref|XP_014276741.1| odorantreceptor94a-like [Halyomorpha halys]

5e-31

36%

No (5´ lose)

2

YsigOR27

MG204662

225

gb|AIG51873.1| odorant receptor [Helicoverpa armigera]

4e-10

27%

No (5´ lose)

2

YsigOR28

MG204663

426

ref|XP_014271039.1| odorant receptor 83a-like [Halyomorpha halys]

1e-30

27%

Yes

6

YsigOR29

MG204664

383

ref|XP_014294439.1| odorant receptor Or1-like isoform X1 [Halyomorpha halys]

1e-52

30%

Yes

4

YsigOR30

MG204665

373

ref|XP_014289672.1| odorant receptor 49a-like [Halyomorpha halys]

4e-59

33%

Yes

5

YsigOR31

MG204666

422

ref|XP_014294765.1| odorant receptor 4-like [Halyomorpha halys]

9e-47

28%

Yes

5

YsigOR32

MG204667

431

ref|XP_014242937.1| odorant receptor Or2-like [Cimex lectularius]

3e-41

27%

Yes

7

YsigOR33

MG204668

437

ref|XP_014276985.1| odorant receptor 22c-like [Halyomorpha halys]

5e-59

32%

Yes

7

YsigOR34

MG204669

414

ref|XP_014281005.1| odorant receptor 4-like [Halyomorpha halys]

5e-59

33%

Yes

6

YsigOR35

MG204670

397

ref|XP_014292083.1| odorant receptor 85b-like [Halyomorpha halys]

3e-66

32%

Yes

6

Gene

name

Accesion No.

ORF (aa)

BLASTx best hit (Reference/Name/Species)

E-value identity

Identity

Full

length

TM (No.)

YsigOR36

MG204671

427

ref|XP_014274444.1| odorant receptor 47a-like [Halyomorpha halys]

5e-121

43%

Yes

5

YsigOR37

MG204672

402

ref|XP_014276741.1| odorant receptor 94a-like [Halyomorpha halys]

2e-42

29%

Yes

5

YsigOR38

MG204673

401

ref|XP_014281005.1| odorant receptor 4-like [Halyomorpha halys]

3e-24

26%

Yes

6

YsigOR39

MG204674

227

ref|XP_014257038.1| odorant receptor Or2-like isoform X2 [Cimex lectularius]

1e-19

27%

No (5′ lose)

3

YsigOR40

MG204675

282

ref|XP_014289672.1| odorantreceptor49a-like [Halyomorpha halys]

6e-09

26%

Yes

4

YsigOR41

MG204676

404

ref|XP_014292083.1| odorant receptor 85b-like [Halyomorpha halys]

9e-150

53%

Yes

5

YsigOR42

MG204677

427

ref|XP_014271039.1| odorant receptor 83a-like [Halyomorpha halys]

6e-34

27%

Yes

5

YsigOR43

MG204678

224

ref|XP_014282386.1| odorant receptor 49a-like isoform X1 [Halyomorpha halys]

1e-15

30%

No (5′ lose)

3

YsigOR44

MG204679

388

ref|XP_014294439.1| odorant receptor Or1-like isoform X1 [Halyomorpha halys]

2e-87

40%

Yes

6

YsigOR45

MG204680

444

ref|XP_014282386.1| odorant receptor 49a-like isoform X1 [Halyomorpha halys]

7e-08

27%

Yes

6

YsigOR46

MG204681

402

ref|XP_014275988.1| odorant receptor 22c-like [Halyomorpha halys]

3e-68

34%

Yes

8

YsigOR47

MG204682

392

ref|XP_014257038.1| odorant receptor Or2-likeisoformX2

[Cimex lectularius]

6e-24

24%

Yes

6

YsigOR48

MG204683

380

ref|XP_014287367.1| odorant receptor 85b-like [Halyomorpha halys]

1e-53

33%

Yes

4

YsigOR49

MG204684

375

ref|XP_014282544.1| odorant receptor 4-like [Halyomorpha halys]

3e-99

46%

Yes

6

YsigOR50

MG204685

396

ref|XP_014274900.1| odorant receptor 30a-like [Halyomorpha halys]

4e-84

37%

Yes

6

YsigOR51

MG204686

401

ref|XP_014281005.1| odorant receptor 4-like [Halyomorpha halys]

5e-39

28%

Yes

5

YsigOR52

MG204687

363

ref|XP_014287367.1| odorant receptor 85b-like [Halyomorpha halys]

4e-51

33%

Yes

4

YsigOR53

MG204688

437

ref|XP_014289672.1| odorant receptor 49a-like [Halyomorpha halys]

6e-61

32%

Yes

4

YsigOR54

MG204689

435

ref|XP_014242937.1| odorant receptor Or2-like [Cimex lectularius]

4e-46

28%

Yes

6

YsigOR55

MG204690

373

ref|XP_014294439.1| odorant receptor Or1-like isoform X1 [Halyomorpha halys]

6e-67

33%

Yes

4

YsigOR56

MG204691

157

ref|XP_014282386.1| odorant receptor 49a-like isoform X1 [Halyomorpha halys]

2e-14

29%

No (5′ lose)

2

YsigOR57

MG204692

381

ref|XP_014274900.1| odorant receptor 30a-like [Halyomorpha halys]

2e-32

25%

Yes

8

YsigOR58

MG204693

392

ref|XP_014257038.1| odorant receptor Or2-like isoform X2 [Cimex lectularius]

4e-28

25%

Yes

6

YsigOR59

MG204694

435

ref|XP_014289672.1| odorant receptor 49a-like [Halyomorpha halys]

3e-67

33%

Yes

6

YsigOR60

MG204695

415

ref|XP_014249551.1| putative odorant receptor 92a

[Cimex lectularius]

2e-100

37%

Yes

5

YsigOR61

MG204696

394

ref|XP_014274900.1| odorant receptor 30a-like [Halyomorpha halys]

2e-129

47%

Yes

7

YsigOR62

MG204697

387

ref|XP_014273330.1| odorant receptor 85b-like [Halyomorpha halys]

1e-53

31%

Yes

6

YsigOR63

MG204698

372

ref|XP_014287367.1| odorant receptor 85b-like [Halyomorpha halys]

1e-12

25%

Yes

6

YsigOR64

MG204699

425

ref|XP_014261210.1| odorant receptor 45b-like [Cimex lectularius]

1e-112

43%

Yes

5

YsigOR65

MG204700

396

ref|XP_014257038.1| odorant receptor Or2-like isoform X2

[Cimex lectularius]

3e-33

29%

Yes

6

 

Transcript expressions of YsigOR genes

The expression profiles of 66 YsigORs in the antennae of females and males were evaluated using RT-qPCR. The result showed that YsigOrco had the highest expression level among all YsigOR genes; however, the expression level of males was twice as that of females. The expression levels of 11 YsigOR genes (YsigOR2, YsigOR8, YsigOR21, YsigOR31, YsigOR35, YsigOR42, YsigOR48, YsigOR49, YsigOR50, YsigOR59, and YsigOR62) were significantly higher in the antennae of males than in those of females, while the expression levels of YsigOR19 was significantly higher in the latter than in the former. Additionally, other YsigOR genes showed comparable expression levels in the antennae of both sexes (Fig. 3).


 

Discussion

In the present study, we generated a transcriptome of the Y. signatus antennae and identified candidate chemosensory genes encoding 66 ORs. To our knowledge, this is the first comprehensive study of olfactory genes in the Berytidae family. Subsequently, all the sequences were further validated by cloning and sequencing.

Previously, it was reported that the number of identified OR genes ranged from 10 in P. humanus (Kirkness et al., 2010) to 400 in P. barbatus (Smith et al., 2011). In this study, 66 ORs were identified in the antennae of Y. signatus. This number is much lower than that for other Hemipteran insects, such as 83 ORs in Nysius ericae (Zhang et al., 2016), 110 ORs in A. lucorum (An et al., 2016), and 88 ORs in Adelphocoris lineolatus (Xiao et al., 2017); however, it is higher than 63 ORs in Sogatella furcifera (He et al., 2015) and 45 ORs in Aphis gossypii (Cao et al., 2014). This may be caused by the adaptation of distinct species to their hosts during evolution. In different insect species, OR genes are extremely divergent and formed different clades in our OR phylogenetic tree. This may be a result of adaptation of distinct species to their hosts during evolution (Sanchez-Gracia et al., 2009). We also found a co-receptor YsigOrco gene, which had characteristics common with those of other insect species’ Orco genes, such as seven transmembrane domains, a high degree of similarity with Orco genes of other insects, and high expression levels (Smart et al., 2008; Dong et al., 2016). Unlike divergent ORs, Orcos from different insect species could be easily assigned and formed a clear orthologous lineage.

For a better understanding of the function of these YsigORs, the expressions of the antennae of females and males were evaluated using RT-qPCR methods. The results showed that 11 YsigOR genes (YsigOR2, YsigOR8, YsigOR21, YsigOR31, YsigOR35, YsigOR42, YsigOR48, YsigOR49, YsigOR50, YsigOR59, and YsigOR62) were expressed more in antennae of males than of females, whereas YsigOR19 was expressed more in the latter, indicating sex-specific functions of these genes. The male-dominant expression of ORs may be involved in the recognition of the sex pheromone or in other male-specific behaviors, while female-dominant expression of ORs may be related to oviposition site selection or male-produced courtship pheromone detection (Anderson et al., 2009; Zhang et al., 2014, 2015b). The sex-specific functions of these ORs need further investigation.

In conclusion, we successfully constructed the first antennal transcriptome of Y. signatus, and identified 66 candidate YsigOR genes. Simultaneously, the phylogenetic relationships between YsigORs and other Hemipteran ORs were also analyzed. For a better understanding of their functions, the expression patterns of these YsigOR genes in the antennae of females and males were executed using RT-qPCR. We successfully identified 11 male antennae-specific genes and one female antenna-specific YsigOR gene. Our results will aid better understanding of the mechanisms of hemipteran chemosensory system.

 

Acknowledgments

This research was supported by Natural Science Foundation of China (31701788) and the Startup Project of Doctor scientific research (4026-13480047) of Henan University of Science and Technology.

 

Supplementary material

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20180616040644

 

Statement of conflict of interest

The authors declare no conflict of interest.

 

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