Expression and functional analysis of two odorant binding proteins

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Agricultural and Environmental Chemistry

Expression and functional analysis of two odorant binding proteins from Bradysia odoriphaga (Diptera: Sciaridae) Bowen Tang, Shulei Tai, Wu Dai, and Chunni Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00568 • Publication Date (Web): 14 Mar 2019 Downloaded from http://pubs.acs.org on March 17, 2019

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Journal of Agricultural and Food Chemistry

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Expression and functional analysis of two odorant binding proteins from

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Bradysia odoriphaga (Diptera: Sciaridae)

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Bowen Tang, Shulei Tai, Wu Dai*, Chunni Zhang*

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Key Laboratory of Plant Protection Resources and Pest Integrated Management of the

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Ministry of Education, College of Plant Protection, Northwest A&F University,

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Yangling, Shaanxi, China

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*Corresponding

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Wu Dai, Tel. 86-13389220985; email: [email protected];

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Chunni

author.

Zhang,

Tel.

86-18729551958;

email:

1

ACS Paragon Plus Environment

[email protected]


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ABSTRACT

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Two OBP genes, BodoOBP1 and BodoOBP2 were cloned from Bradysia

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odoriphaga, a major agricultural pest of Chinese chives. The amino acid sequence

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alignment of both BodoOBPs showed high similarity. Fluorescence competitive

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binding assays revealed that both BodoOBPs have a moderate binding affinity to

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dipropyl trisulfide. Tissue expression profiles indicated that both BodoOBPs are

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antennae-specific and more abundant in the male antennae than in the female

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antennae. Developmental expression profile analysis indicated that expression levels

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of both BodoOBPs were higher in the male adult stage than in the other

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developmental stages. Both BodoOBPs also showed differential expression in pre-

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and post-mating adults. RNAi assays indicated that ability of dsOBPs-males to detect

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females was significantly reduced compared to controls. Attraction of plant volatile

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dipropyl trisulfide to dsOBPs-treated adults was also significantly lower than in the

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control. Our findings indicate that both BodoOBPs are involved in host-seeking

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behavior and in detecting sex pheromones.

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KEYWORDS: Bradysia odoriphaga, odorant binding protein, binding affinity,

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expression profile, RNA interference

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INTRODUCTION

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The olfactory system is essential for multiple behaviors of insects, including

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locating sexual partners and oviposition sites, host-seeking, and avoiding predators1-4.

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The insect olfactory system is mediated by eight main types of proteins: soluble

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binding proteins [odorant binding proteins (OBPs), chemosensory proteins (CSPs)

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and Niemann-Pick C2 protein (NPC2)]5, 6, membrane-bound receptors [odorant

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receptors (ORs), gustatory receptors (GRs), ionotropic receptors (IRs) and sensory

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neuron membrane proteins (SNMPs)] and odorant-degrading enzymes (ODEs)4.

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OBPs are small soluble proteins that are recognized as playing a central role in the

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initial phases of olfactory perception. OBPs are highly concentrated in the sensillum

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lymph of chemosensilla and are capable of binding and transporting hydrophobic

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odorant molecules via the chemosensilla lymph to corresponding chemosensory

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receptors1, 4, 7, 8.

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To date, a large number of OBP sequences have been identified from different

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insect orders including Hemiptera, Lepidoptera, Diptera, Hymenoptera, Coleoptera,

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Orthoptera, Isoptera, Anoplura, Blattaria and Neuroptera1, 9-15. Based on the number

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of cysteine residues they contain, OBPs are now classified as “Classic”, “Dimer”,

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“Minus - C”, “Plus - C”, and “Atypical” OBPs15. Most insect OBPs are characterized

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by six conserved cysteines (Classic OBPs) that form three disulphide bridges and play

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an important role in stabilizing the secondary and tertiary structure16, 17. The number

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of OBPs is highly variable even between closely related species. For example, there

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are 33 OBPs in Lygus lineolaris (Hemiptera: Miridae)18, 28 OBPs in Nysius ericae 3



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(Hemiptera: Lygaeidae)19, 49 OBPs in Episyrphus balteatus (Diptera: Syrphidae), 44

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OBPs in Eupeodes corollae (Diptera: Syrphidae)20. It has been demonstrated that

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many of the identified OBP genes are highly expressed in the antennae11,

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OBPs were also reportedly expressed in other chemosensory organs, including

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mouthpart palps, maxillae, labium, legs, and wings, suggesting that some OBPs might

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also be involved in recognition of taste compounds18,

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studies have demonstrated that several OBPs are specifically distributed in

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non-chemosensory organs, such as the male reproductive organs35, 36. For example, in

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Bactrocera dorsalis, OBP19c was abundantly expressed in the thorax and abdomen37.

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All of these studies revealed that OBPs play significant roles in insect olfactory

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systems, and may have different functions in non-sensory organs of the insect body,

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such as pheromone delivery, solubilisation of nutrients, development and insecticide

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resistance8.

26-34.

18, 21-25.

However, some recent

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The Chinese chive maggot, Bradysia odoriphaga Yang et Zhang (Diptera:

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Sciaridae) is a major agricultural pest that causes severe economic damage in

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liliaceous vegetable crops, edible mushrooms and humus38,

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(Allium tuberosum) suffers the most severe damage in Northern China, with yield loss

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of 30%–80% and economic losses of over 30%38, 40. B. odoriphaga larvae tend to

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aggregate in the roots, bulbs, and immature stems of Chinese chives, resulting in

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moisture loss and even death41. Since the larvae primarily damage the underground

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portions of plants, infestation is difficult to prevent or control. Management practices

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against B. odoriphaga still largely rely on the application of chemical insecticides. 4


39.

The Chinese chive

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However, insecticide use causes environmental pollution, may affect human health

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and may also lead to pesticide resistance. Because B. ororiphaga presumably locate

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their host plants by detecting volatile compounds released by the plants and also use

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pheromones in courtship42-44, identification and functional characterization of OBP

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and other olfactory genes are of potential importance for developing olfaction-based

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and environmentally friendly techniques to control the pest. At present, 49 OBP genes

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have been identified from B. odoriphaga transcriptomic data14, but their functions

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have not been addressed.

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In this study, in order to explore the possible olfactory roles of the two B.

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odoriphaga OBPs, quantitative real-time PCR was used to assess the expression

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profiles of the two OBPs genes in different tissues of both sexes, at different

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developmental stages, and before and after mating. In addition, the binding abilities of

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the two OBPs to host plant volatiles were measured using fluorescence competitive

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binding assays and the roles of two BodoOBPs in the detection of odorants were

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investigated by RNAi. Our systematic studies provide a better understanding of the

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molecular basis of B. odoriphaga olfaction.

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MATERIALS AND METHODS

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Insect rearing and sample collection. A laboratory colony of B. odoriphaga was

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established using specimens collected from a Chinese chive field in Shangcun Town,

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Zhouzhi County, Xi’an City, China in 2014. The insects were reared on fresh

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rhizomes of Chinese chive (1 cm in length) in petri dishes at 25 ± 1 °C, 70±5 %

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relative humidity with a photoperiod of 16 : 8 h (L : D). For gene expression pattern 5



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study, 1st to 4th instar larvae, pupae and adults were collected into RNase-free tubes

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and immediately immersed in liquid nitrogen. The antennae, head, thorax, abdomen,

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legs and wings were dissected under a microscope while soaking in Ringer’s Solution

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(6.5g/L NaCl, 0.2g/L NaHCO3, 0.14g/L KCl, 0.01g/L NaH2PO4, 0.12g/L CaCl2) and

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immediately transferred into RNase-free tubes and immersed into liquid nitrogen. The

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individual virgin adults were reared in a separate plastic box after emergence. After

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24h, 5 adults (male and female separately) were collected into RNase-free tubes;

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meanwhile other mature virgin males and females were placed into one plastic box.

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The mating behavioral assays were conducted at 25°C between 8:00 AM and 11:00

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AM. 5 post-mating adults (male and female separately) were collected into

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RNase-free tubes at 0h and 1h after the first successful mating. All samples were

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collected in three biological replicates and stored at -80 °C until further experiments.

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Total RNA isolation and cDNA synthesis. Total RNA for each sample was isolated

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by using the RNAiso plus Reagent (TaKaRa, Japan). The quality and concentration of

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total RNA were determined by agarose gel electrophoresis and spectrophotometer

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analysis (Infinite M200 PRO, Tecan Group Ltd., Mannedorf, Switzerland). For gene

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cloning, total RNA of adults was digested with DNase I to remove genomic DNA,

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and then was used to synthesize first-strand cDNA using a RevertAid First Strand

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cDNA Synthesis Kit (Thermo Scientific, USA). For quantitative real-time PCR

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analysis, 1μg of each RNA sample was employed as template to synthesize

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first-strand cDNA by PrimeScript™ RT reagent Kit with gDNA Eraser, Perfect Real

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Time (TaKaRa, Japan). All operations strictly followed the manufacturer’s handbook.

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The cDNA template was stored at -20 °C until use.

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Cloning of the full-length OBPs cDNA. The partial sequence of two candidate OBPs

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was identified based on the previously obtained transcriptome sequence. To obtain the

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full‐length cDNA, 5’ and 3’ RACE was performed. Specific primers for RACE were

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designed based on identified PCR fragment sequences (Table S1). The first strand

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cDNA for 5’ and 3’ RACE was synthesized from 1μg of total RNA using a

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SMARTer™ RACE cDNA Amplification Kit (TaKaRa, Japan). 5’RACE was carried

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out by nested PCR using OBP1-5-GSP1/ OBP2-5-GSP1 and the outer Primer from

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the primary PCR reactions and the second PCR reactions were carried out using

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OBP1-5-GSP2/ OBP2-5-GSP2 combined with the inner Primer. 3’ RACE was

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conducted by nested PCR using OBP1-3-GSP1/ OBP2-3-GSP1 and Long Primer for

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the primary PCR and the second PCR was carried out using

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OBP1-3-GSP2/OBP2-3-GSP2 combined with the Short Primer. For the second PCR

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amplification, a 50-fold dilution of the primary reaction mixture was used as template.

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All thermal cycling followed a standard program: 94 °C for 3 min; 35 cycles of 30 s at

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94 °C, 30 s at 54-58 °C and 60 s at 72 °C; 72 °C for 10 min. All amplified products

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were cloned in pMD18-T Vector (TaKaRa, Japan) and sequenced completely. Based

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on the sequences obtained from the 3′- and 5′-RACE, the putative full-length OBP

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genes were amplified with specific primers OBP-F and OBP-R. The amplified

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products were gel-purified, and cloned into pMD18-T vectors (TaKaRa, Japan) and

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sequenced completely. 7



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Sequence analysis. The ExPASy Proteomics Server

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(http://cn.expasy.org/tools/pi_tool.html) was used to compute the isoelectric points

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and molecular weights of deduced protein sequences. The signal peptide was

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predicted by SignalP 3 Serve (http://www.cbs.dtu.dk/service/SignalP/). Alignments of

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nucleotide and amino acid sequences were made using DNAMAN software. 3D

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structure analysis was performed with the program SWISS MODEL

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(https://swissmodel.expasy.org/).

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Expression and purification of recombinant BodoOBP1 and BodoOBP2 proteins.

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The coding sequences of mature BodoOBPs were amplified by PCR using specific

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primers carrying endonuclease restriction sites (Table S1). The purified PCR products

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of two OBPs were subcloned into pMD18-T vector (TaKaRa, Japan ), then the

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plasmids containing the correct sequence were digested by specific restriction

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enzymes. The digested fragments were purified and ligated into the expression vectors

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pET28a (+) (Novagen, Madison, WI) previously digested with the same enzymes

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(TaKaRa, Japan). These constructed vectors were confirmed by sequencing. The

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recombinant plasmids were transformed into Escherichia coli BL21(DE3) competent

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cells. The expression of recombinant proteins was induced at 28 °C for 8 h with a

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final concentration of 0.5 mM isopropyl β-d-1-thiogalactopyranoside (IPTG).

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Bacteria cells were collected by centrifugation at 6,000 g for 10 min, suspended in

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balance buffer (300 mM NaCl, 50 mM NaH2PO4, 10 mM imidazole, 10 mM Tris

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base, pH8.0) and sonicated in ice (15 s, 20 passes), then centrifuged again at 12,000 g

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for 20 min. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 8


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showed that the OBPs were expressed mainly in inclusion bodies.

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The inclusion bodies were washed by 50 mM Tris buffer (pH 6.8) with 0.2%

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Triton X-100 and dissolved in 5mL of 8M urea. The proteins were refolded according

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to the protocol of Prestwich45. Protein purification was accomplished by Ni-NTA

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Resin (TransGen Biotech, Beijing, China). Purified recombinant proteins were

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analyzed by SDS-PAGE and dialyzed overnight on ice with 10 mM

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phosphate-buffered saline (PBS) (pH 7.4).

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Fluorescence competitive binding assay. 10 typical chemical components of volatile

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oils from the Chinese chive46 were purchased from Aladdin (China Shanghai), HX-R

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(China, Chengdu), Sigma-Aldrich (USA) and TCI (Japan). The purity of all ligands

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and the fluorescent probe N-phenyl-1-naphthylamine(1-NPN) is higher than 95% and

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all of these were dissolved independently in GC grade methanol (Aladdin, China) to

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create 1mM stock solutions. Fluorescence competitive binding assays were performed

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at 25 °C on a Hitachi F-4500 spectrofluorimeter with a 1 cm light path fluorimeter

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quartz cuvette and 10-nm slits for both excitation and emission. The fluorescent probe

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was excited at 337 nm and emission spectra were recorded between 350 and 600 nm.

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The affinity of the fluorescent probe to each OBP was measured by titrating a 2 μM

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solution of the protein in 50 mM Tris-HCl (pH 6.8) with aliquots of 1 mM 1-NPN to

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final concentrations of 2-20 μM. In competitive binding assays, the ligands from 0 to

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50μM were added into each protein (2 μM) and 1-NPN (2 μM).

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The fluorescence intensities at the maximum fluorescence emission between 350 and 600 nm were plotted against the free ligand concentration to determine the 9



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binding constants. All data were analysed based on the assumption that the protein

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was 100% active and had a stoichiometry of 1 : 1 (protein : ligand) at saturation. The

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binding curves with 1-NPN were linearized using a Scatchard plot. The dissociation

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constants of the competitors were calculated from the corresponding IC50 values

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based on the following equation: Ki=[IC50]/(1+[1-NPN]/K1-NPN), where [1-NPN] is

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the free concentration of 1-NPN and K1-NPN is the dissociation constant of the

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complex protein/1-NPN47. If the IC50 value of the candidate competitive ligand

189

exceeded 30 μM, the further calculation of Ki was not considered.

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Quantitative real-time PCR. Quantitative real-time PCR (qRT-PCR) was

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performed using a Cycler IQ Real-Time PCR detection system (Bio-Rad, Hercules,

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CA, USA). 1μg of each RNA sample was employed as template to synthesize

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first-strand cDNA using a PrimeScript™ RT Reagent Kit with gDNA Eraser (Perfect

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Real Time) (TaKaRa, Japan). Each reaction included 1μL of template, 1 μL (0.6 μL

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for BodoOBP2) of each primer (10 mM), 10μL 2× UltraSYBR Mixture (KWBIO,

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Beijing, China) and added RNase-free water to a total volume of 20μL. Amplification

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conditions were: 95 °C for 10 min; followed by 40 cycles of 95 °C for 15 s, and 60 °C

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for 60 s. A melting curve analysis was conducted by a protocol that varied

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temperature from 55 to 95 °C with increments of 0.5 °C every 30 s with continuous

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fluorescence acquisition to ensure consistency and specificity of the amplified product.

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The amplification efficiency (E) of all primer pairs was validated by constructing a

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standard curve using a 10-fold serial dilution of cDNA template using the equation: E

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= (10[-1/slope]-1) ×100. No-template controls were included. Each experiment contained 10


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three biological replicates (three technical replications for each biological replication).

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Relative expression levels were determined using the 2−ΔΔCT method48. The 18S,

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RPS15 and RPS7 was used as a reference to normalize the expression of the target

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genes across different developmental stage. The 18S, RPS7 and Tubulin was used as a

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reference to normalize the expression of the target genes across various tissues.

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Results are shown as means ± SE. Statistical analyses of the gene expression data

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were performed using ANOVA in SPSS 20.0 (IBM Inc., Chicago, Illinois, USA).

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dsRNA synthesis and RNA interference assay. Primers of dsRNA fragments were

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designed according to the two OBP sequences, and the selected region for dsRNA

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synthesis was different from the region for qRT-PCR. These primers contained a T7

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polymerase binding sequence required for dsRNA synthesis. PCR conditions were

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94 °C for 3 min, followed by 35 cycles of 94 °C for 30 s, 60 °C for 30 s and 72 °C for

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60 s, with a final extension at 72 °C for 10 min. The amplified PCR products were

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separated on an agarose gel and purified by a Biospin Gel Extraction Kit (Bioer

218

Technology, Hangzhou, China). dsRNAs were synthesized using the T7

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RiboMAXTM Express RNAi System (Promega) following the manufacturer’s

220

instructions. dsRNA was purified and precipitated using 3M sodium acetate (pH 5.2)

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and isopropanol, then washed using 0.5ml of cold 70% ethanol. The purified dsRNA

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was eluted into diethyl pyrocarbonate (DEPC) -treated nuclease-free water and stored

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at -80 °C until use. As controls, double strand Green fluorescent protein (dsGFP,

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GenBank accession number: KF410615.1) was synthesized using the protocol

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described above. The dsRNA concentration was determined by a Nanodrop™ 1000 11



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(Thermoscientific, Lithuania). The primers used for dsRNA synthesis are shown in

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Table S1.

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30 nL dsRNA (6 μg/μL ) was injected into the abdomen of 1-day-old pupae

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using a Nanoject III (Drummond Scientific Company, USA) and each insect was

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injected only once. Pupae were injected with an equivalent amount of dsGFP as a

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control. The injected pupae were reared under the conditions described above and

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adults emerged on the third day of the pupal stage. 5 adults (male and female

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separately) were collected into RNase-free tubes and immersed into in liquid nitrogen

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at 72 h after dsRNA injection. RNA was extracted with RNAiso plus Reagent

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(TaKaRa, Japan) to measure the transcript levels of BodoOBP1 and BodoOBP2 by

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qRT-PCR as described above.

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Y-tube olfactometer assay. In order to study the role of OBPs in B. odoriphaga

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olfactory recognition behavior, a Y-tube olfactometer with diameter 2.5 cm (each arm

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with length of 15 cm and stem with length of 15 cm) was used in this study. The air

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was first filtered through active carbon and then humidified with doubly distilled

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water before entering the Y-tube. The air that travelled through both arms of the

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olfactometer was pumped in at a flow rate of 300 mL min-1. Each individual virgin

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adult was placed at the middle position of the olfactometer stem, and its choice was

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recorded when it passed the middle position of an arm and stayed for 30 s. Virgin

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adults that made no choice within 5 min were considered nonresponders. In these

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assays, 10 μL of the dipropyl trisulfide (10 μg/μL in methanol (HPLC)) applied to

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6mm diameter filter paper or virgin females (15 individuals, 24 hours after emergence) 12


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were placed in the incoming airstream of one of the arms of the Y-tube olfactometer.

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Each group included at least 30 individuals with 3 replicates. The number of adults

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that travelled upwind towards the odour sources in each arm was recorded. The time

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spent by individuals moving toward the odorant source was measured using a digital

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chronometer. The positions of the odour sources were exchanged after every 15 tests.

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The Y-tube olfactometer was cleaned with 75% ethanol and dried after every

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exchange. An attraction index was calculated from each replicate using the formula:

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(number of individuals in the treatment arm – number of individuals in the control

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arm)/total number of tested individuals49.

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RESULTS

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cDNA sequence analysis of BodoOBP1 and BodoOBP2

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The full length cDNA sequences of BodoOBP1 and BodoOBP2 were obtained

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by PCR and submitted to GenBank (accession numbers: KX099613 and KX099614).

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BodoOBP1 and BodoOBP2 are 622 bp and 690 bp in length, respectively, and both

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contain open reading frames (ORF) of 453 bp which encode a protein of 150 amino

263

acids. The calculated molecular masses of BodoOBP1 and BodoOBP2 proteins are

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17.3 kDa and 17.5 kDa, and the predicted isoelectric points are 5.67 and 6.03,

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respectively. Both proteins all have predicted signal peptides of 17 amino acids at the

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N-terminus.

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BodoOBP1 and BodoOBP2 proteins have the typical six cysteine signature

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(C1-X25−30-C2-X3-C3-X36−42-C4-X8−14-C5-X8-C6) and belong to the Classic OBP

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subfamily (Fig. 1). The amino acid sequence alignment revealed that both BodoOBPs 13



270

share very high similarities (identity >42%) with OBPs from other Dipteran insects

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(Fig. 1). The amino acid sequence of BodoOBP1 showed 86.7% identity with

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BodoOBP2. Both BodoOBPs have approximately 58% and 55% identities with

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AgamOBP1 (GenBank accession number: AAL84179.1), respectively (Fig. 2).

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AgamOBP1 was used as template to predict the 3D structure of BodoOBP1 and

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BodoOBP2 proteins according to SWISS MODEL (Fig. 3). 3D models of BodoOBP1

276

and BodoOBP2 proteins showed that both have the typical predicted structure of

277

OBPs, with six α-helices (labeled α1 to α6) surrounding a central hydrophobic cavity.

278

Four helices (α1, α4, α5 and α6) converge to form a binding pocket. The narrow end

279

of the binding pocket is open with the opposite end being capped by the α3 helix. The

280

binding pocket is delimited by the hydrophobic sides of these helices, and therefore

281

well suited to being a binding site for hydrophobic ligands. The 3D structures of

282

BodoOBP1 and BodoOBP2 are similar, suggesting that they may have similar

283

functions.

284

Bacterial expression and purification of BodoOBP1 and BodoOBP2

285

The genes encoding BodoOBP1 and BodoOBP2 were amplified from cDNA and

286

ligated into the expression vector pET-28a (+), as described in the Materials and

287

Methods section. The two OBPs were mainly present in inclusion bodies and highly

288

expressed, yielding approximately 30 mg/L culture. The denaturation, renaturation

289

and purification were performed as described in the Materials and Methods. The

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SDS-PAGE analysis showed that the molecular weights of the recombinant proteins

291

(contain His-Tag) were approximately 19 kDa (Fig. 4). 14


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Ligand-binding assays To investigate the ligand binding properties of BodoOBP1 and BodoOBP2 to

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different host volatiles, the fluorescence competitive binding assay was further

295

performed using 1-NPN as the fluorescence probe. The binding curves with 1-NPN

296

and Scatchard plots indicated that the bindings between the proteins and 1-NPN were

297

saturable with a single population of binding sites, without an apparent allosteric

298

effect (Fig. 5). The results showed that both recombinant BodoOBPs could well

299

interact with 1-NPN with dissociation constants of 4.03±0.46 μM and 6.91±0.61 μM.

300

The above results indicated that using 1-NPN as the fluorescence probe is suitable for

301

a fluorescence competitive binding assay.

302

Ten chemicals were selected based on previous reports (Table S2) and used in

303

the competitive binding assay. Competitive binding curves of these ligands to

304

BodoOBP1 and BodoOBP2 are shown in Fig. 6. When the concentration of dipropyl

305

trisulfide reached 15.46 μM and 12.06 μM, respectively, the fluorescence intensity of

306

the BodoOBP1-2 /1-NPN complex rapidly decreased to approximately 50%. These

307

results showed that the both BodoOBPs have moderate binding affinity to dipropyl

308

trisulfide, with Ki values of 12.39±0.83 μM and 10.53±0.36 μM, respectively (Table

309

S2). For other chemicals, the two BodoOBPs showed weak or no binding affinity

310

(Ki > 30 μM).

311

Tissue Expression Patterns of BodoOBPs

312 313

The quantitative real-time PCR (qRT-PCR) was used to measure transcript levels of BodoOBP1 and BodoOBP2 in different tissues. The results revealed that both 15



314

genes were significantly more highly expressed in the antennae of both sexes than in

315

other tissues (Fig. 7). Furthermore, expression levels of both genes were much higher

316

in male than in female antennae, approximately 3-fold higher for BodoOBP1 and

317

9-fold higher for BodoOBP2. The expressions of the two OBP genes were also

318

detected in the head of both sexes.

319

Expression patterns of BodoOBPs in different life stages

320

The expression patterns of BodoOBP1 and BodoOBP2 in different life stages

321

were measured by RT-qPCR. The transcript levels of both BodoOBPs were higher in

322

the male adult stage than in the other developmental stages (Fig. 8). Compared to

323

females, expression of both BodoOBPs was significantly higher in the adult and pupal

324

stage of males.

325

The results showed that expression of BodoOBP1 and BodoOBP2 in males was

326

significantly higher in the 24 h after eclosion than in the female (Fig. 9). Expression

327

of both BodoOBPs was down-regulated in 0h post-mating males but up-regulated in

328

1h post-mating males compared to virgin males. Compared to virgin females, the

329

expression of both BodoOBPs was up-regulated in 1h post-mating females. No

330

significant differences were detected in the expression of OBP genes between mature

331

virgin females and 0h post-mating females.

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Effect of dsRNA treatment on B. odoriphaga behavior

333

To test the effect of reducing transcript levels of BodoOBP1 and BodoOBP2

334

genes on B. odoriphaga behavior, we injected pupae with dsOBPs, and measured

16


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335

transcript levels of both BodoOBPs at 72 h post-injection. After treatment with

336

dsOBPs, BodoOBP expression was reduced by 85% or more in adults (Fig. 10).

337

To detect the role of BodoOBP1 and BodoOBP1 in response to sex pheromones,

338

we used virgin female adults as odour sources. The Y‐tube assay indicated that ability

339

of dsOBPs-males to detect females was significantly reduced when compared to

340

control insects (Fig. 11A). The attraction indices of males treated with dsOBP1 to

341

females were significantly lower than for those injected with dsOBP2. Virgin males

342

responded to virgin females with an attraction index of 53%, whereas the attraction

343

index was only 4% and 31%, respectively, for dsOBP1-treated and dsOBP2-treated

344

virgin males.

345

The attraction of plant volatile dipropyl trisulfide to dsGFP-treated male or

346

female adults was 32% and 31%, respectively, which was not significantly different

347

(Fig. 11B). However, the attraction of dipropyl trisulfide to dsOBP1-treated males or

348

dsOBP1-females was significantly lower than dsGFP-treated insects, with attraction

349

index of 9% and 7%, respectively, which was similar to results for attraction of

350

dipropyl trisulfide to dsOBP2-treated male or female adults (Fig. 11B).

351

The time spent by dsOBPs-treated virgin male adults moving toward females or

352

dipropyl trisulfide was significantly longer than for dsGFP-treated groups (Fig.12A,

353

B). The response time of dsOBPs-treated female individuals to dipropyl trisulfide

354

increased, but no significant difference was observed (Fig. 12C).

355

DISCUSSION

356

The olfactory recognition system plays a vital role in interactions of insects with 17



357

their environment, and is important for their survival and reproduction4. Our study

358

demonstrates that OBPs play a central role in the initial phases of olfactory perception

359

in B. odoriphaga. OBPs are responsible for transporting hydrophobic ligands to

360

specific ORs4. This suggests that OBPs could be useful for controlling insect

361

populations by blocking the first step of the olfactory recognition process. Although

362

the chemosensory genes of B. odoriphaga have been detected previously in antennal

363

and whole-body transcriptomes14, there was no previous information on expression

364

profiling of OBPs in B. odoriphaga across different developmental stages, which is

365

necessary to determine the role of these proteins on reproduction. Identification and

366

functional analysis of odorant-binding proteins is needed to provide new and more

367

environmentally friendly pest control strategies utilizing olfactory interference.

368

In this study, two OBP genes, BodoOBP1 and BodoOBP2 were cloned and

369

characterized from B. odoriphaga, and recombinant proteins were expressed in E.

370

coli. The amino acid sequence alignment revealed that both BodoOBPs are similar

371

(identity >42%) to OBPs from other dipteran insects. Both BodoOBPs have high

372

homology with Drosophila melanogaster OBPs OS-E and OS-F, which clustered

373

together with DmelOBP83a/83b14, an OBP group associated with the detection of

374

volatile pheromones in D. melanogaster27, 50. Binding assays showed that dipropyl

375

trisulfide, which has been isolated and identified from the B. odoriphaga host plant

376

Allium tuberosum, had the highest binding affinity of the tested ligands to BodoOBP1

377

and BodoOBP2. For other chemicals, these two BodoOBPs showed weak binding or

378

no binding affinity. Thus, we infer that both BodoOBPs are involved in host-seeking 18


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Page 19 of 48

379


behavior.

380

Previous work has reported that the transcripts of BodoOBP1 and BodoOBP2

381

were expressed specifically at very high levels in the female and male antennae14, as

382

also observed in our study, which implies that the two proteins are involved in

383

olfactory chemoreception. Interestingly, BodoOBP1 and BodoOBP2 were expressed

384

much more highly in male antennae than female antennae (Fig. 7), suggesting that

385

they could also play a role in male chemosensory behavior, such as sex pheromone

386

detection. This has been reported for the mosquito Aedes aegypti, in which OBP10

387

enriched in antennae may play a role in adult male chemosensory behavior51. The

388

results of qRT-PCR indicated that expression of BodoOBP1-2 differs during different

389

developmental stages of B. odoriphaga, with higher expression in male adults than in

390

other developmental stages (Fig. 8). Previous study of D. melanogaster showed that

391

Obp19a-d was expressed only in adults52. A similar result was observed in

392

Chrysomya megacephala with qPCR results showing that higher expression of two

393

OBP genes in adults than in larvae53. The male adult-biased expression observed for

394

BodoOBP1-2 suggests putative roles for these proteins in mate location.

395

For B. odoriphaga, we found a difference in expression levels between virgin

396

and post-mating adults for BodoOBP1. Both BodoOBPs were down-regulated in 0h

397

post-mating males but up-regulated in 1h post-mating males compared to virgin males

398

(Fig. 9). Similar results were observed in Anastrepha fraterculus post-mating males,

399

in which OBP56d and OBP57c were up-regulated in the 6h post-mating54. Higher

400

expression of OBPs in post-mating males would suggest that these genes could be 19



401

related to mate seeking including responding to the female pheromones55. Due to the

402

habit of multiple mating in B. odoriphaga male adults, we speculate that the higher

403

expression of both BodoOBPs in the 1h post-mating males could signify preparation

404

for re-mating. The expression of both BodoOBPs was up-regulated in 1h post-mating

405

females compared to virgin females (Fig. 9). Although in A. fraterculus females, most

406

OBP genes were down-regulated post-mating, in A. obliqua females the majority of

407

OBPs were up-regulated post-mating54, the same pattern observed in D. melanogaster

408

females52. Previous research indicated that behavioral changes in adults due to mating

409

were mainly related to responses of female moths to plant volatiles and of male moths

410

responding to sex pheromones56-59. Mature virgin females of the Mediterranean fruit

411

fly, Ceratitis capitata, have a strong preference for a male-produced pheromone,

412

whereas mated females are more attracted to host plant odors60, 61.

413

To test the effect of reducing transcript levels of BodoOBP1 and BodoOBP2

414

genes on B. odoriphaga behavior, we performed RNA interference of both

415

BodoOBPs. Our Y-tube olfactometer assay indicated that ability of dsOBP1-males to

416

detect females was significantly reduced when compared to control insects (Fig. 11).

417

Similar results were obtained for dsOBP2-males. Our findings are consistent with the

418

previous finding that in Rhodnius prolixus, RproOBP27 is significantly expressed in

419

male antennae33. After treatment with dsOBP27, ability of male adults to detect

420

females was reduced, and adult males spent significantly less time close to females as

421

compared to controls, indicating that RproOBP27 may be involved in the perception

422

of semiochemicals related to mate finding33. Additionally, our results indicated that 20


Page 20 of 48

Page 21 of 48


423

attraction of plant volatile dipropyl trisulfide to dsOBPs-treated adults was

424

significantly lower than dsGFP-treated insects (Fig. 11). Therefore, BodoOBP1 and

425

BodoOBP2 may be required for host plant recognition. In Holotrichia oblita,

426

dsOBP-treated female beetles showed significantly lower attraction to (E)-2-hexenol

427

and phenethyl alcohol than those treated with GFP-dsRNA, indicating that

428

HoblOBP13 and HoblOBP9 are essential for H. oblita perception of these volatile

429

compounds49. Taken together, the results presented here strongly suggest that both

430

BodoOBPs are likely involved in the perception of sex pheromone(s) and

431

host-seeking.

21



432

ACKNOWLEDGMENT

433

We thank Dr. Chris Dietrich (University of Illinois, IL, USA) and Dr. John Richard

434

Schrock (Emporia State University, Emporia, KS, USA) for their comments on an

435

earlier draft of this paper.

436

AUTHOR CONTRIBUTIONS

437

W.D. and C.N.Z. designed the experiment. B.W.T., S.L.T. and C.N.Z., performed the

438

experiments. B.W.T., W.D. and C.N.Z. prepared the manuscript. All authors

439

contributed to data analysis.

440

FUNDING

441

The research was supported by the National Natural Science Foundation of China

442

(31672037) and the Special Fund for Agro-scientific Research in the Public Interest

443

from the Ministry of Agriculture of China (201303027).

444

NOTES

445

The authors declare no competing financial interest.

446

SUPPORTING INFORMATION

447

The Supporting Information is available free of charge on the ACS Publications

448

website.

449

Table S1. Primers used for cloning of genes, qRT-PCR and RNAi. Table S2. Binding

450

affinities of selected host plant compounds to the recombinant proteins.

22


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Holotrichia oblita based on semiochemical-induced expression alteration and gene

652

silencing. Insect Biochemistry and Molecular Biology, 2019, 104, 11-19.

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FIGURE CAPTIONS

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Figure 1. Alignment of amino acid sequences of OBPs from dipteran insects. The six

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conserved cysteines are marked with a black star.

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Figure 2. Alignment of amino acid sequences of BodoOBP1, BodoOBP2 and

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AgamOPB1(AAL84179.1). Conserved amino acids in all OBPs are marked with

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black triangles. Predicted signal peptides are indicated by bold underline.

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Figure 3. 3D structure of BodoOBP1 and BodoOBP2. A: BodoOBP1. B: BodoOBP2.

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3D structure analysis was performed with the program SWISS MODEL using the

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structure of AgamOBP1 as a template.

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Figure 4. Expression and purification of recombinant BodoOBP1 and BodoOBP2. All

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proteins were expressed in E. coli BL21(DE3) using the vector pET28a(+) and

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purified by Ni-NTA Resin. Lane 1 and Lane 15: Molecular weight markers. Lane 2:

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supernatant of pET28a(+) (no insert). Lane 3: inclusion body of pET28a(+) (no

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insert). Lane 4: supernatant of BodoOBP1 without induction. Lane 5: inclusion body

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of BodoOBP1 without induction. Lane 6: supernatant of BodoOBP1after induction

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with 0.4mM IPTG. Lane 7: inclusion body of BodoOBP1 after induction with 0.4mM

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IPTG. Lane 8: purified BodoPBP1. Lane 9: supernatant of BodoOBP2 without

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induction. Lane 10: inclusion body of BodoOBP2 without induction. Lane 11:

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supernatant of BodoOBP2 after induction with 0.4mM IPTG. Lane 12: inclusion body

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of BodoOBP2 after induction with 0.4mM IPTG. Lane 13: purified BodoPBP2.

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Figure 5. Binding curves and Scatchard plots of 1-NPN to BodoOBP1 and 33



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BodoOBP2.

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Figure 6. Competitive binding curves of some ligands to BodoOBP1 and BodoOBP2.

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A and B: BodoOBP1. C and D: BodoOBP2.

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Figure 7. Expression patterns of BodoOBP1 and BodoOBP2 in different tissues. The

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data and error bars represent the means and standard errors of three biological

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replicates. Different letters above the bars indicate significant differences (P< 0.05,

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Student–Newman–Keuls (SNK) in one way ANOVA). “*” indicates significant

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differences, * p < 0.05; ** p < 0.01; *** p < 0.001 (Student's t test).

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Figure 8. Expression patterns of BodoOBP1 and BodoOBP2 (relative to the 1st) in

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different developmental stages. M/F-Pupae: Male or female pupae. M/F-Adults: Male

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or female adults. The data and error bars represent the means and standard errors of

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three biological replicates. Different letters above the bars indicate significant

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differences (P< 0.05, Student–Newman–Keuls (SNK) in one way ANOVA). “*”

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indicates significant differences, * p < 0.05; ** p < 0.01; *** p < 0.001 (Student's t

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test).

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Figure 9. Expression levels of BodoOBP1 and BodoOBP2 (relative to 24h after

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eclosion in female) in pre- and post-mating adults. The data and error bars represent

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the means and standard errors of three biological replicates. Different letters above the

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bars indicate significant differences (P< 0.05, Student–Newman–Keuls (SNK) in one

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way ANOVA).

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Figure 10. Effect of RNAi treatment on the transcript levels of BodoOBP1 and 34


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BodoOBP2. The data and error bars represent the means and standard errors of three

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biological replicates.

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Figure 11. The attraction indexes of B. odoriphaga (A) virgin female adults to male

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adults and (B) dipropyl trisulfide to adults. The data and error bars represent the

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means and standard errors of three biological replicates. “*” means significant

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difference between dsOBP1 or dsOBP2 treated group and dsGFP treated group, * p

Expression and functional analysis of two odorant binding proteins

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