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Article Cite This: ACS Omega 2019, 4, 3800−3811
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Responses to Host Plant Volatiles and Identification of Odorant Binding Protein and Chemosensory Protein Genes in Bradysia odoriphaga Yuxin Zhang, Yanping Ren, Xiaolan Wang, Yong Liu, and Ningxin Wang* Department of Entomology, College of Plant Protection, Shandong Agricultural University, Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests, Taian 271000, Shandong, China
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S Supporting Information *
ABSTRACT: The chive maggot Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae) is a devastating agricultural pest that feeds on liliaceous vegetables and edible mushrooms. The shared host plant volatiles and chemosensory genes of B. odoriphaga may together play crucial roles for insects to identify their host. However, the responses of B. odoriphaga to host volatiles remain unclear. Electroantennography (EAG) and behavioral bioassays were performed on 12 volatiles shared in Allium and Pleurotus. Hexanal evoked in both male and female adults extremely significant EAG responses. In behavioral assays, 3rd-instar larvae and female adults can be significantly repelled by methyl propyl disulfide, 1-octen-3-ol, or hexanal at the concentration of 100 mg/mL. Third-instar larvae and female adults were significantly attracted by limonene with a concentration of 10 and 100 mg/mL, respectively. In addition, 57 chemosensory genes, including 51 odorant binding proteins (OBPs) and 6 chemosensory proteins (CSPs), were identified based on transcriptomes of larvae and pupae. Compared with previous adult transcriptomes, 11 BodoOBPs were specifically expressed in adults, and 6 BodoOBPs were specifically expressed in larvae and pupae. BodoCSP2 and BodoCSP3 were exclusively expressed in the adult stage. Our results provided the potential substances in new ecofriendly pest management and the targets for further study of chemosensory gene functions.
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host plants of insects.9 B. odoriphaga feeds on plants of both Allium and Pleurotus. Although the characteristic odors of Allium spp. are distinct from that of Pleurotus, there are still some common volatiles in between them.10−15 The effects of shared host odors in Allium spp. and Pleurotus spp. to B. odoriphaga are still unclear. The discovery of an attractive or repellent substance that can “pull” pests into traps or “push” them away from crop areas will greatly facilitate the application of this method to the control of certain pests.16 Electroantennography (EAG) is often used to analyze changes in the antennae electrical signals of insects to a certain odorant and further to compare the differences in responses of males and females.17 Significant EAG responses were demonstrated in B. odoriphaga to trans-2-hexenal, which is an effective insect repellent and can be fumigated to kill pests at a relatively low concentration.18 Another study suggested that female adults of B. odoriphaga are attracted by body surface volatiles of 4th-instar larvae, and the main components of these volatiles include dipropyl disulfide and 2,2-dimethyl1,3-dithiane.19 The olfactory system of insects, which serves as the major perception of surrounding environment, plays a
INTRODUCTION Bradysia odoriphaga Yang et Zhang (Diptera: Sciaridae), the Chinese chive root maggot, is a serious agricultural pest in China and can damage host roots and cause crop failures. B. odoriphaga feeds on plants from seven families, including Liliaceae, Cucurbitaceae, and Compositae, and is also a potential pest of mushrooms.1 A recent study demonstrated that B. odoriphaga exhibits better life parameters when they are reared on Chinese chive and oyster mushroom.1 Third-instar larvae of B. odoriphaga, the most serious stage of damage, can cause 50% yield reduction in Chinese chives.2,3 At present, the control of chive root maggot relies mainly on chemical pesticides, and only a handful of four pesticides have been approved for use in China.4 Pesticides are not the most effective treatment because of widespread drug resistance and slow toxicity and their association with many problems, such as pesticide residues and food safety.4−6 As an alternative to chemical insecticides, non-pesticide control methods are urgently needed for application as an efficient and environment-friendly control strategy. Push−pull strategy is considered an effective pest control and has been applied to a variety of pest control.7 Chemical signals of the host volatiles are essential for insects in feeding and oviposition host selection.8 It has been reported that the shared host odors may contribute to the search for © 2019 American Chemical Society
Received: December 19, 2018 Accepted: January 24, 2019 Published: February 21, 2019 3800
DOI: 10.1021/acsomega.8b03486 ACS Omega 2019, 4, 3800−3811
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Table 1. List of Host Volatiles for EAG and Behavioral Experiments
critical role in insect life activities, including food seeking, mating, host finding, and many additional behaviors.20,21 The first step of olfactory recognition is that odor molecules dissolve and penetrate into the sensilla lymph from the external environment. Odor molecules are then transferred to olfactory sensory neurons by odorant binding proteins (OBPs) and chemosensory proteins (CSPs).22,23 OBPs, which act as carriers in signaling and behavior regulation,24,25 are small soluble proteins and generally contain six conserved cysteine residues forming three disulfide bridges.26 According to the number of conserved cysteine residues, OBPs can be divided into Classic OBPs, Minus-C OBPs, Plus-C OBPs, and atypical OBPs (existing and evolving only in Anopheles and its relatives).27,28 CSPs, small soluble proteins that are abundantly expressed in the sensilla lymph, are also binding proteins of chemical substances with four cysteine residues.23,29 To date, the mechanism of odor recognition of B. odoriphaga has not been elucidated, and the characterization of OBPs and CSPs of B. odoriphaga based on antennae and body transcriptomes of adults was reported in a recent study.5 The attraction of host volatiles and specific recognition of odor molecules by chemoreceptors together play crucial roles in host recognition of pests. In order to reveal the olfactory
recognition process of insects, it is necessary to study both aspects. In this study, some host volatiles were used to stimulate B. odoriphaga, and their effects on this pest were determined, including electrophysiological and behavioral responses. OBP and CSP families were identified based on the transcriptomes of pupae and 3rd- and 4th-instar larvae, which were most harmful stages to plants.30 Compared with the adult transcriptomes, some genes were specifically expressed in different stages.
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MATERIALS AND METHODS
Insects Rearing and Tissue Collection. A laboratory population of B. odoriphaga was raised at 25 ± 1 °C with a relative humidity (RH) of 70 ± 5% and a photoperiod of 14 h light/10 h dark. B. odoriphaga was reared on fresh Chinese chive bulbs in culture dishes that were maintained in an artificial climate incubator to maintain proper conditions. Samples of diverse tissues (approximately 1500 antennae, 500 heads, 80 thoraxes, 50 abdomens, 1000 legs, and 1200 wings) were separately collected from newly hatched adults without mating. All collected tissues were frozen in RNAhold (TransGen Biotech, Beijing, China) and stored at −80 °C until use.
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Electroantennograms. Twelve host volatiles from Allium and Pleurotus were selected to stimulate the antennae of male and female adults.10−15,31−35 All test host volatiles from Allium and Pleurotus are listed in Table 1. All chemicals were purchased from J&K Company (Beijing, China) and were ≥95% pure. Before the EAG experiments, each compound was dissolved in paraffin oil at concentrations of 10, 100, and 500 mg/mL. Fresh antennas were cut from the base and immediately attached to the fork electrode with an electrode gel (Parker Laboratories, INC., USA). Filter paper strips (0.5 cm × 2.0 cm) loaded with 10 μL each solutions were inserted into a glass Pasteur pipette. An air stimulus controller (CS-55; Syntech, Hilversum, Netherlands) was used for gas delivery. Only one antenna was stimulated (1 s) per adult, and each antenna was stimulated thrice at an interval of 1 min. In both females and males, 10 antennae were tested at each concentration of each compound. The EAG response values were calculated using the following formula: the EAG response = R1 − R2 (R1 is the EAG response for a certain compound, and R2 is the EAG response for paraffin oil before the stimulus). The EAG responses of females and males to tested volatiles were compared using a t-test. Statistical analysis was conducted using SPSS 23.0 (SPSS Inc., Chicago, IL, USA). Behavioral Assays. A 2.4 cm diameter glass Y tube, consisting of a 14 cm stem and two 12 cm arms (angled at 75°), was used for behavioral bioassays. Twelve volatile compounds dissolved in paraffin oil were tested with concentrations of 10 and 100 mg/mL, respectively. 10 μL of each test volatile solution was added to filter paper strips (0.5 cm × 2.0 cm), which were placed in glass jars. 10 μL of solvent paraffin oil was used as a control. A stream of clean, moist, and charcoal filtered air was pumped through jars containing odor source and into the arms of glass Y tube. The flow rate was maintained at 1 L/min with a flowmeter, and a continuous gas flow was maintained for 10 min before experiments to stabilize the gas flow within the apparatus. The newly hatched females without mating and 3rd-instar larvae were tested, and insects were individually placed into the stem of the glass tube and observed for no more than 10 min until 20 individuals made choice. When 3rd-instar larvae crossed the arm over 1 cm or female adult exceeded 1/2 of the arm length and stayed for more than 10 s, it was considered to make a choice. For each treatment, three biological replicates were performed. For each test of 10 individuals, a new odor source was replaced, and Y-tube was washed with ethanol and the position of two arms was adjusted. Statistical analysis was performed by t-test using SPSS 23.0 software (SPSS Inc., Chicago, IL, USA). Acquisition of Putative Chemosensory Genes. We downloaded the previously reported total seven transcriptome sequencing results with accession numbers of SRX744222, SRX1459738, SRX1473238, and SRX1473242,30,36 including three developmental stages (3rd- and 4th-instar larvae and pupae), from the NCBI SRA database (https://www.ncbi.nlm. nih.gov/sra). The assembly was accomplished using Trinity.37 The assembly sequences were considered as unigenes, and the following steps were performed. Putative OBP and CSP genes from unigenes were screened using the tBLASTn program at NCBI (https://www.ncbi.nlm.nih.gov/). To verify the authenticity of these candidate genes, all putative chemosensory genes were confirmed by BLASTx analysis based on the nonredundant protein sequence database and via the CD-search
program (https://www.ncbi.nlm.nih.gov/Structure/cdd/ wrpsb.cgi). RNA Isolation and cDNA Synthesis. To explore the expression levels of the chemosensory genes, RNA was isolated from six tissues using Trizol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. The quantity and purity of isolated RNA were determined by spectrophotometric analyses, and the integrity of RNA was analyzed by 1% agarose gel. Then, cDNA templates were synthesized using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix, which eliminated genomic DNA contamination, according to the manufacturer’s instructions. Previously, RNA concentrations from all tissues were set to the same standard, and cDNA synthesis was performed in 20 μL volume containing 100 ng RNA. Sequence and Phylogenetic Analysis. The open reading frames (ORFs) of chemosensory genes were predicted by using ORF Finder (https://www.ncbi.nlm.nih.gov/ orffinder/). The putative N-terminal signal peptides were predicted using the SignalP V4.1 Server (http://www.cbs.dtu. dk/services/SignalP/).38 The molecular weight and isoelectric points of OBPs and CSPs were computed using an online tool provided by ExPASy (https://web.expasy.org/compute_pi/). We utilized Clustal X 2.0 to align the sequences of putative OBPs and CSPs39 and generated the diagrams of sequence alignments by DNAMAN (LynnonBiosoft, USA). All phylogenetic trees were constructed using MEGA7.0 software based on chemosensory genes of B. odoriphaga and several other species,40 and then trees were visualized by EvolView.41 Neighbor-joining trees of the putative olfactory gene sequences with complete ORFs were constructed using default settings, and the bootstrap values at nodes were based on 1000 replicates.42 All the sequences of OBPs and CSPs used for evolutionary analysis are listed in Table S1. 3D Structure Prediction of BodoOBPs and BodoCSPs. The three-dimensional structures of BodoOBPs and BodoCSPs were predicted using an online tool Phyre2 with normal modeling mode.43 All amino acid sequences of BodoOBPs and BodoCSPs used for structural prediction are provided in Table S2. The top-ranking templates of proteins were utilized to predict protein models. The authenticity analysis of the protein models were conducted using PROCHECK,44 Verify 3D,45 and ERRAT methods.46 Quantitative Real-Time PCR. Analysis of quantitative real-time polymerase chain reaction (qRT-PCR) included 13 new BodoOBPs and 3 new BodoCSPs. The previously reported β-actin gene of B. odoriphaga was considered as housekeeping gene, and we utilized the same primer pairs for the β-actin gene reported in Gao’s research.30 Other specific primers of chemosensory genes were designed by primer premier 5.0 and Beacon designer 8.13 (Palo Alto, CA, USA). All primers used for qRT-PCR are provided in Table S3. qRTPCR was conducted using the CFX96 Touch Real-Time PCR Detection System (Bio-Rad, USA) with a 20 μL system containing 1 μL of cDNA of diverse tissues, 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), 7 μL of RNase-free ddH2O, and 10 μL of 2× TransStart Top Green qPCR SuperMix (TransGen Biotech, Beijing, China). The qRT-PCR reaction was performed as follows: 95 °C for 3 min, 45 cycles of 95 °C for 10 s, and 60 °C for 30 s, and a melting curve analysis from 65 to 95 °C. The expression levels of OBPs and CSPs in six tissues were calculated based on Ct-values using Bio-Rad CFX Manager 3.1 software. To ensure the 3802
DOI: 10.1021/acsomega.8b03486 ACS Omega 2019, 4, 3800−3811
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Figure 1. EAG responses of male and female adults to 12 host volatiles. The concentrations of tested compounds were 10, 100, and 500 mg/mL in paraffin oil. The names of compounds numbered 1−12 are listed on the right. *, **, and *** indicate significant differences between males and females at P < 0.05, 0.01, and 0.001 (t-test), respectively.
Figure 2. Behavior response of 3rd-instar larvae of B. odoriphaga to 12 host volatiles in Y-tube olfactometer. The concentrations of tested compounds were 10 and 100 mg/mL in paraffin oil. The names of compounds numbered 1−12 are listed on the right. Bars signify percentages of selecting test substances, and negative responses represent repellent effect. Asterisk indicates significant differences between choosing test compound and choosing control at P < 0.05 (t-test).
1-Octen-3-ol and linoleic acid are widely found in Pleurotus hosts. In addition, 1-octen-3-ol is a component of the odor of cattle and human sweat49,50 that could elicit significant EAG responses in Anopheles gambiae, Anopheles funestus, and Aedes albopictus.49,51−53 Our EAG results revealed that adults had significant electrophysiological responses to 1-octen-3-ol. Linoleic acid, a precursor of 1-octen-3-ol, which is extremely common in living organisms, can induce only a weak EAG responses in both males and females.54 Compounds numbered 7−12 are derived from the volatiles present in both Allium and Pleurotus and could be identified in a wide variety of organisms and essential oils. Hexanal was the most active compound that could trigger EAG response in our study, and the EAG amplitude of males to 500 mg/mL hexanal was approximately 2.5 mV. Hexanal is a common component of the three plant hosts of A. gambiae and is also found in human breath and skin emanations.55,56 Because of the strong attraction of hexanal to A. gambiae, hexanal could be used as bait for mosquitoes.55,56 Benzaldehyde only induced relatively weak EAG responses in B. odoriphaga adults, and there were no significant differences in EAG amplitudes between male and female insects. In a study of Drosophila melanogaster, benzaldehyde was the most repulsive among 110 tested odors.57 Moreover, 1-octen-3-ol was also repellent to D. melanogaster, linoleic acid had little effect, and hexanal and furfural were attractants.57 EAG experiments provided the electrophysiological responses of pests to host volatiles.
credibility of the experiments, three technical replications and three biological replications were constructed for each sample.
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RESULTS AND DISCUSSION Effects of Host Plant Volatiles on EAG Responses of B. odoriphaga. Adults exhibit significant differences to certain odors at different concentrations (Figure 1). The EAG responses of males to five substances (compounds numbered 2, 3, 5, 9, and 10) at each test concentration were significantly stronger compared with females. Compounds numbered 1−4 were thioether compounds of Allium spp. and were also components in certain essential oils that were commercially formulated for pest management based on the repellent properties.47 Recent studies demonstrated that trisulfides and disulfides were repellents of Diaphorina citri and Cacopsylla chinensis. Among these compounds, trisulfides were more toxic, and the monosulfide had minimal effects on pests.47,48 EAG responses also exhibited increased amplitudes to disulfides (compounds numbered 1−3) compared with the monosulfide (compound numbered 4) in our study. Propyl disulfide, a main volatile of 4th-instar larvae of B. odoriphaga and some host plants in Allium, had a significant attractant effect on females compared with the control and could evoke a significant EAG reaction in our research.10,11,19 Surprisingly, the EAG responses to propyl disulfide and methyl propyl disulfide of males were significantly increased compared with females even though propyl disulfide was proven to be attractive to females.19 3803
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Figure 3. Behavior response of female adults of B. odoriphaga to 12 host volatiles in Y-tube olfactometer. The concentrations of tested compounds were 10 and 100 mg/mL in paraffin oil. The names of compounds numbered 1−12 are listed on the right. Bars signify percentages of selecting test substances, and negative responses represent repellent effect. Asterisk indicates significant differences between choosing test compound and choosing control at P < 0.05 (t-test).
Behavioral Bioassay of B. odoriphaga to Host Volatiles. The behavior responses of 3rd-instar larvae and female adults to chemical substances at 10 and 100 mg/mL were tested. For 3rd-instar larvae, limonene and hexanal at a concentration of 10 mg/mL were significantly attractive to pests, whereas methyl propyl disulfide, 1-octen-3-ol, and hexanal at a concentration of 100 mg/mL were significantly repellent (Figure 2). The host vicinity, such as bulbs of Chinese chives, is considered to be the preferred spawning site for females, and host volatiles may have more obvious effects on female adults.19 For female adults, behavioral analysis demonstrated that 1-octen-3-ol and hexanal had significant repellent effects at both 10 and 100 mg/mL concentrations (Figure 3). For both 3rd-instar larvae and female adults, methyl propyl disulfide, 1-octen-3-ol, and hexanal at a concentration of 100 mg/mL were significant repellents. Propyl disulfide at a concentration of 100 mg/mL exhibited significant attraction (average approximately 61.7%) to female adults, which was reported to be an attractant present in host and worm volatiles,19 so it was supposed to have an effect on B. odoriphaga oviposition choice. Methyl propyl disulfide at 100 mg/mL was a significant repellent, especially for female adults, with a 73.3% repellent rate. 1-Octen-3-ol is an effective mosquito trap when combined with carbon dioxide;58 however, it acts as an insect repellent for fungus gnats (Bradysia spp.).59 In our study, this major volatile component in mushrooms was not as attractive as mushroom, probably because of the concentration. Limonene could be an effective attractant for 3rd-instar larvae and female adults, with a selectivity of more than 65%. Limonene could cause a weak response of Maruca vitrata in electroantennographic detection, and it had a strong attractive ability in field-trapping experiments.8 Similarly, limonene elicited a moderate EAG response in our experiments, which was the most attractive of all tested substances to the chive maggots. BioUD, a repellent registered in 2007, has a repellent effect on mosquitoes and can be used as an alternative to DEET.60 The active ingredient of BioUD is 7.75% 2undecanone derived from wild tomato plants.60 2-Undecanone could elicit EAG responses in B. odoriphaga, and 100 mg/mL of it could significantly repel the females. In our study, hexanal could cause very strong EAG responses in adults and significant behavioral responses (attraction or repellent), which have been shown to have different effects on pests at different concentrations of the same substance.
Our results provided several potential attractants and repellents for 3rd-instar larvae and female adults. The discovery of a suitable insect attractant or repellent will make it possible to apply the “push−pull strategy” for the control of B. odoriphaga,7 a devastating agricultural pest, to improve the current situation of excessive use of chemical pesticides. Identification of Putative Chemosensory Genes. Sequence similarity analyses were performed between the assembled unigenes and previously reported chemosensory genes of other insects. A total of 57 chemosensory gene candidates, including 51 OBPs and 6 CSPs, had been confirmed by BLASTx and exhibited a conserved C-pattern in our study (Table 2). Compared with the previously identified 49 OBPs and 5 CSPs based on adult transcriptomes of B. odoriphaga, 13 new OBPs and 3 new CSPs were named BodoOBP50-62 and BodoCSP6-8, respectively (Table 2). To date, a total of 62 OBPs and 8 CSPs of B. odoriphaga were identified based on analysis of the transcriptome sequencing, and the amounts are similar to the average of 54 OBPs and 8 CSPs in Diptera.5 All 51 predicted OBPs (except BodoOBP32) had complete ORFs and encoded 125−444 amino acids (Table 2). According to the number of conserved cysteines, BodoOBPs were divided into Classic OBPs (=6), Minus-C OBPs (6). With the exception of BodoOBP22, 32, 53, and 59, the other OBPs have signal peptide sequences at the N-terminal domain. The molecular weight of BodoOBPs is between 13.6 and 52.0 kDa, and the isoelectric point is between 4.35 and 8.97. With the exception of individual BodoOBPs, most BodoOBPs have molecular weights between 10 and 30 kDa, which is consistent with previous reports of some other species.61 For CSPs, only BodoCSP7 lacked complete ORFs. The molecular weight of BodoCSPs is between 13.4 and 27.2 kDa, and the isoelectric point is between 7.64 and 9.57. Sequence and Phylogenetic Analysis. Sequence alignments were performed based on a total of 8 BodoCSPs and 62 BodoOBPs, including 43 Classic OBPs, 15 Minus-C OBPs, and 4 Plus-C OBPs. Amino acid sequence alignments of different OBP subfamilies and CSPs of B. odoriphaga are presented in Figure S1 and S2. The sequence alignments of BodoOBPs demonstrated that OBP family of Diptera has conserved C-patterns, whereas members of each subfamily also exhibit relatively large distinctions. 3804
DOI: 10.1021/acsomega.8b03486 ACS Omega 2019, 4, 3800−3811
3805
Minus-C
Classic
Minus-C
BodoOBP32
BodoOBP33
Classic
BodoOBP28
BodoOBP31
Classic
BodoOBP27
Classic
Minus-C
BodoOBP26
BodoOBP29
Classic
Classic
BodoOBP17
BodoOBP24
Classic
BodoOBP16
Minus-C
Classic
BodoOBP15
BodoOBP23
Minus-C
BodoOBP14
Classic
Classic
BodoOBP13
BodoOBP22
Classic
BodoOBP12
Classic
Classic
BodoOBP9
BodoOBP21
Classic
BodoOBP8
Plus-C
Classic
BodoOBP7
BodoOBP19
Classic
BodoOBP6
Classic
Classic
BodoOBP1
BodoOBP18
subfamily
gene name
152
222
141
239
145
154
153
144
141
147
144
252
138
127
144
149
143
148
143
148
144
147
132
150
ORF (aa)
Y
N
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
intact ORF
17
15
18
18
19
18
18
16
ND
22
17
20
18
18
24
16
24
19
18
17
22
20
17
signal peptide (aa)
17644.51
-
15750.74
27885.61
16649.41
16828.51
17940.86
15016.36
16174.51
16813.95
15599.94
29210.93
15369.82
14304.47
15373.68
16085.53
16481.84
17097.09
16257.96
16215.71
16556.06
16458.15
14799.29
17307.05
molecular weight (Da)
7.70
-
4.56
5.71
5.64
5.55
5.92
5.23
5.53
8.12
4.35
5.95
5.33
5.04
5.01
4.69
6.19
8.97
8.82
4.97
5.78
5.92
6.14
5.67
isoelectric point
100 100 100 100 100
3 × 10−91 4 × 10−104 2 × 10−102 2 × 10−102 2 × 10−102
100 100
7 × 10−101 6 × 10−86
100 100 100 100 100 100 100
2 × 10−103 6 × 10−100 1 × 10−98 4 × 10−110 1 × 10−108 8 × 10−102 1 × 10−180
100 100
1 × 10−164 5 × 10−109
3 × 10
100
100
4 × 10−99
−99
100
0.0
100
100
1 × 10−101
3 × 10−98
100
2 × 10−103
5 × 10 100
100
2 × 10−108
−107
identity (%)
E value
AWC08444.1
AWC08443.1
AWC08442.1
AWC08440.1
AWC08439.1
AWC08438.1
AWC08437.1
AWC08435.1
AWC08434.1
AWC08433.1
AWC08432.1
AWC08430.1
AWC08429.1
AWC08428.1
AWC08427.1
AWC08426.1
AWC08425.1
AWC08424.1
AWC08423.1
AWC08420.1
AWC08419.1
AWC08418.1
AWC08417.1
ANA52575.1
best BLASTx match ass. no.
Table 2. Characterizations and BLASTx Matches of B. odoriphaga Putative OBP and CSP Genesa
odorant binding protein 1 [B. odoriphaga] odorant-binding protein 6 [B. odoriphaga] odorant-binding protein 7 [B. odoriphaga] odorant-binding protein 8 [B. odoriphaga] odorant-binding protein 9 [B. odoriphaga] odorant-binding protein 12 [B. odoriphaga] odorant-binding protein 13 [B. odoriphaga] odorant-binding protein 14 [B. odoriphaga] odorant-binding protein 15 [B. odoriphaga] odorant-binding protein 16 [B. odoriphaga] odorant-binding protein 17 [B. odoriphaga] odorant-binding protein 18 [B. odoriphaga] odorant-binding protein 19 [B. odoriphaga] odorant-binding protein 21 [B. odoriphaga] odorant-binding protein 22 [B. odoriphaga] odorant-binding protein 23 [B. odoriphaga] odorant-binding protein 24 [B. odoriphaga] odorant-binding protein 26 [B. odoriphaga] odorant-binding protein 27 [B. odoriphaga] odorant-binding protein 28 [B. odoriphaga] odorant-binding protein 29 [B. odoriphaga] odorant-binding protein 31 [B. odoriphaga] odorant-binding protein 32 [B. odoriphaga] odorant-binding protein 33 [B. odoriphaga]
best BLASTx match
ND
Ceratitis capitata
Drosophila yakuba
Scirpophaga excerptalis
A. darlingi
Lucilia cuprina
ND
S. mosellana
Drosophila busckii
A. gambiae
Drosophila takahashii
B. dorsalis
Sitodiplosis mosellana
Delia platura
T. castaneum
Zeugodacus cucurbitae
Drosophila navojoa
A. gambiae
Bactrocera dorsalis
C. quinquefasciatus
Drosophila hydei
A. mellifera
Aedes aegypti
Anopheles darlingi
best BLASTx match species except B. odoriphaga
ACS Omega Article
DOI: 10.1021/acsomega.8b03486 ACS Omega 2019, 4, 3800−3811
3806
Minus-C
Minus-C
Plus-C
BodoOBP58
BodoOBP59
Classic
BodoOBP55
BodoOBP57
Minus-C
BodoOBP54
Classic
Minus-C
BodoOBP53
BodoOBP56
Classic
Classic
BodoOBP46
BodoOBP52
Classic
BodoOBP45
Classic
Minus-C
BodoOBP44
BodoOBP51
Minus-C
BodoOBP42
Classic
Classic
BodoOBP40
BodoOBP50
Classic
BodoOBP39
Classic
Classic
BodoOBP38
BodoOBP49
Classic
BodoOBP37
Classic
Classic
BodoOBP36
BodoOBP48
Classic
BodoOBP35
Classic
Plus-C
BodoOBP34
BodoOBP47
subfamily
gene name
Table 2. continued
228
140
143
125
144
444
221
140
144
146
144
149
125
138
140
131
144
142
137
180
132
239
149
189
ORF (aa)
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
intact ORF
ND
16
16
19
18
22
ND
22
18
18
21
21
18
20
19
17
18
24
19
22
19
20
24
17
signal peptide (aa)
26135.36
16017.44
16485.89
13618.69
14796.11
51987.86
25601.17
15895.33
15095.56
17013.71
15732.35
16794.56
14247.31
15357.83
15398.79
14804.03
16539.10
15835.59
15364.20
19879.09
14900.61
28065.14
16936.54
20375.82
molecular weight (Da)
6.22
5.54
6.29
4.80
5.17
6.01
6.43
4.88
5.14
5.63
5.20
5.15
5.22
5.03
5.56
4.58
5.87
6.37
6.35
7.58
8.62
8.48
7.56
5.54
isoelectric point
100 100 100 100 100
4 × 10−107 2 × 10−178 2 × 10−89 1 × 10−129 1 × 10−93
78 76 53 64 64 75 63
2 × 10−80 2 × 10−77 2 × 10−43 3 × 10−46 3 × 10−45 3 × 10−75 2 × 10−44
73 90
4 × 10−66 3 × 10−134
2 × 10
88
100
8 × 10−98
−89
100
100
4 × 10−98
9 × 10−103
100
9 × 10−96
100
100
4 × 10−91
8 × 10−86
100
1 × 10−103
1 × 10 100
100
8 × 10−135
−97
identity (%)
E value
AWC08430.1
AWC08434.1
AWC08425.1
AWC08417.1
AWC08435.1
XP_001849139.1
XP_001849139.1
AWC08451.1
AWC08435.1
AWC08439.1
AWC08460.1
AWC08459.1
AWC08458.1
AWC08457.1
AWC08456.1
AWC08455.1
AWC08453.1
AWC08451.1
AWC08450.1
AWC08449.1
AWC08448.1
AWC08447.1
AWC08446.1
AWC08445.1
best BLASTx match ass. no. odorant-binding protein 34 [B. odoriphaga] odorant-binding protein 35 [B. odoriphaga] odorant-binding protein 36 [B. odoriphaga] odorant-binding protein 37 [B. odoriphaga] odorant-binding protein 38 [B. odoriphaga] odorant-binding protein 39 [B. odoriphaga] odorant-binding protein 40 [B. odoriphaga] odorant-binding protein 42 [B. odoriphaga] odorant-binding protein 44 [B. odoriphaga] odorant-binding protein 45 [B. odoriphaga] odorant-binding protein 46 [B. odoriphaga] odorant-binding protein 47 [B. odoriphaga] odorant-binding protein 48 [B. odoriphaga] odorant-binding protein 49 [B. odoriphaga] odorant-binding protein 28 [B. odoriphaga] odorant-binding protein 24 [B. odoriphaga] odorant-binding protein 40 [B. odoriphaga] conserved hypothetical protein [C. quinquefasciatus] conserved hypothetical protein [C. quinquefasciatus] odorant-binding protein 24 [B. odoriphaga] odorant-binding protein 6 [B. odoriphaga] odorant-binding protein 14 [B. odoriphaga] odorant-binding protein 23 [B. odoriphaga] odorant-binding protein 19 [B. odoriphaga]
best BLASTx match
A. gambiae
Drosophila persimilis
Drosophila suzukii
C. quinquefasciatus
Clunio marinus
C. quinquefasciatus
C. quinquefasciatus
A. sinensis
Anopheles sinensis
A. darlingi
Adelphocoris lineolatus
S. mosellana
D. navojoa
S. mosellana
C. capitata
Glossina morsitans morsitans
Drosophila bipectinata
S. mosellana
A. darlingi
Rhyzopertha dominica
Musca domestica
C. capitata
S. mosellana
C. quinquefasciatus
best BLASTx match species except B. odoriphaga
ACS Omega Article
DOI: 10.1021/acsomega.8b03486 ACS Omega 2019, 4, 3800−3811
Tyrophagus putrescentiae Ostrinia furnacalis O. furnacalis T. putrescentiae T. putrescentiae Diachasma alloeum
Drosophila mojavensis
Drosophila erecta
A. gambiae
To investigate evolutionary relationships of these species, a total of 185 OBPs of five species (B. odoriphaga, Bombyx mori, Tribolium castaneum, Apis mellifera, and Chrysopa pallens) from Diptera, Lepidoptera, Coleoptera, Hymenoptera, and Neuroptera had been used to construct a phylogenetic tree (Figure 4). With the exception of few OBPs, other OBPs from three
ANA52574.1 AWC08463.1 AWC08465.1 ANA52574.1 ANA52574.1 AWC08464.1 100 100 100 94 82 72
Figure 4. Phylogenetic tree of OBPs from B. odoriphaga and other four species. Classic, Minus-C, and Plus-C OBPs are represented by red, green, and blue colors at the outermost, respectively. The five colors inside represent OBPs from B. odoriphaga (Bodo, red), B. mori (Bmor, orange), T. castaneum (Tcas, blue), A. mellifera (Amel, green), and C. pallens (Cpal, yellow). Five BodoOBPs with a homology to BmorOBPs are labeled with an asterisk.
subfamilies are clustered into diverse groups. A high degree of consistency may not exist between OBPs of the same species, and similar motif patterns of certain subfamilies are dominant in different species. Genes with closer relationships may be derived from gene duplication,62 and we hypothesized that the gene pairs that exhibited high bootstrap values in diverse species may play a semblable role in physiological activities of insects. In addition to five asterisk-labeled OBPs, all BodoOBPs harbor homologous genes in B. odoriphaga. The asterisk-labeled OBPs (BodoOBP7, 34, 38, 42, and 49) constituted paralogous gene pairs with BmorOBP41, 5, 38, 29, and 37, separately, indicating that B. odoriphaga has a closer relationship with B. mori. Compared with other three species, Diptera and Lepidoptera are separated from Mecopteria, which is consistent with the results of OBP gene evolution analysis. The OBP family has undergone evolution and a series of gene duplications perhaps to recognize more diverse odors from the environment.63 By comparing the OBP families of many insect species, it is possible to understand the evolutionary differences of OBPs and chemosensory mechanisms derived from adaptation to host and environment.63 A total of 78 CSPs with full ORFs from seven species were used to construct phylogenetic trees (Figure S3). BodoCSP1 and BodoCSP4 are homologous pairs to BodoCSP6 and BodoCSP8 respectively, and the other 3 BodoCSPs (BodoCSP2, 3, and 5) were homologous with CSPs from other Dipteran species. The portion marked by a gray dotted line indicated that BodoCSPs were more consistent with the CSPs of the other two Dipteran insects, and these Dipteran species exhibited more closely related relationships.
9.57 13474.77 18
ND: not detected; NC: not classified; “-”: not analyzed because of incomplete ORFs. a
14758.93 13583.77 27238.78 14833.00
NC NC NC NC NC NC BodoCSP1 BodoCSP4 BodoCSP5 BodoCSP6 BodoCSP7 BodoCSP8
129 118 235 129 63 116
Y Y Y Y N Y
18 18 20 18
7.64 9.42 8.52 7.64
2 1 3 4 4 3
× × × × × ×
10−92 10−96 10−174 10−76 10−22 10−65
AWC08454.1 76 1 × 10−68 Minus-C BodoOBP62
139
Y
16
15960.05
5.13
58 Minus-C BodoOBP61
136
Y
15
15594.91
5.91
4 × 10−50
AWC08452.1
odorant-binding protein 19 [B. odoriphaga] odorant-binding protein 41 [B. odoriphaga] odorant-binding protein 43 [B. odoriphaga] CSP [B. odoriphaga] CSP 3 [B. odoriphaga] CSP 5 [B. odoriphaga] CSP [B. odoriphaga] CSP [B. odoriphaga] CSP 4 [B. odoriphaga] AWC08430.1 84 8 × 10−135 Plus-C BodoOBP60
258
Y
17
29761.39
5.80
identity (%) isoelectric point molecular weight (Da) signal peptide (aa) intact ORF ORF (aa) subfamily gene name
Table 2. continued
Article
E value
best BLASTx match ass. no.
best BLASTx match
best BLASTx match species except B. odoriphaga
ACS Omega
3807
DOI: 10.1021/acsomega.8b03486 ACS Omega 2019, 4, 3800−3811
ACS Omega
Article
Figure 5. Expression levels of new BodoOBPs and BodoCSPs in adult tissues. qRT-PCR was performed to explore the expression levels of 16 new olfactory proteins in different adult tissues. Adult tissues include A, antennae; H, heads; T, thoraxes; Ab, abdomens; L, legs; and W, wings. Six BodoOBPs (BodoOBP51, 52, 55, 57, 60, and 61) were not detected in adults. The expression levels of OBPs and CSPs in six tissues were calculated based on Ct-values using Bio-Rad CFX Manager 3.1 software.
Predicted 3D Structure of BodoOBPs and BodoCSPs. All predicted protein models and authenticity verification results are presented in Table S4 and S5. The confidence levels of all protein models predicted using Phyre2 were greater than 94.9%. Ramachandran plot results also revealed that for all established models, greater than 94.9% of the residues were in the allowed region, indicating that the predicted results were reliable. Most predicted models of BodoOBPs had six α-helical domains, folding to form an empty cavity. In contrast, BodoCSPs structures were more conservative with all four αhelical domains except BodoCSP7, which does not have a full ORF. The model prediction results also conformed to the previous research results on the three-dimensional structure of insect olfactory proteins.26 In past studies, OBPs and CSPs exhibited additional functions besides chemosignal recognition, such as releasing chemical pheromones, regeneration, and development.29 These proteins with similar structures may play vastly different roles, and more research on the function of chemosensory genes is needed in the future. When studying and verifying protein functions, the acquisition of the three-dimensional structure of proteins is necessary, which has been proved in many studies.64,65 Our predicted 3D models might help to explore the combining ability and function of these proteins. Expression of Putative OBPs and CSPs. qRT-PCR was performed to determine the expression patterns of 13 new OBPs and 3 new CSPs in six tissues of adults, including antennae, heads, thoraxes, abdomens, legs, and wings. Seven BodoOBPs (BodoOBP50, 53, 54, 56, 58, 59, and 62) were detected in adult tissues, and five BodoOBPs (BodoOBP50, 53, 54, 56, and 62) were enriched in the antennae, the primary olfactory organ of insect, implying that they may play an important role in olfactory recognition.66 BodoOBP58 is primarily expressed in the antennae and abdomen, suggesting that BodoOBP58 may be associated with the binding and release of sex pheromones.5 BodoOBP59 is expressed in the antennae, heads, abdomens, and legs, with the highest expression in antennae and almost no expression in thoraxes (Figure 5). For BodoCSPs, three new BodoCSPs (BodoCSP68) have semblable expression profiles to BodoCSP2,5 which are enriched in antennae and heads, and exhibit increased
expression levels in heads. The 3D model predictions also revealed that these 4 BodoCSPs may exhibit very similar structures (Table S5); therefore, we hypothesized that they play similar roles in life activities of B. odoriphaga. The expression profiles of chemosensory genes in adults and other three stages (3rd- and 4th-instar larvae and pupae) were compared (Figure 6). Eleven BodoOBPs (BodoOBP2, 3, 4, 5,
Figure 6. Comparison of numbers of BodoOBPs (A) and BodoCSPs (B) expressed in different developmental stages. Yellow region indicates the olfactory proteins present in adults, and blue region indicates the olfactory proteins present in 3rd- and 4th-instar larvae and pupae.
10, 11, 20, 25, 30, 41, and 43) that have been identified in previous research were not identified in our transcriptome sequencing analysis using tBLASTn method, indicating that these 11 OBPs are specifically expressed in adults.5 Of note, 8 of them (except for BodoOBP3, BodoOBP25, and BodoOBP30) are mainly expressed in the antennae and minimally expressed in other tissues,5 and these eight BodoOBPs may be closely related to the odor recognition at adult stages of B. odoriphaga. Three BodoOBPs (BodoOBP2, 4, and 20) specifically expressed in male antennae may be involved in the binding and transport of sex pheromones, but further research is needed to prove this notion. Six new BodoOBPs (BodoOBP51, 52, 55, 57, 60, and 61) were not detected in all adult tissues by qRT-PCR, and we have not identified them in the unigenes derived from adult transcriptomes. This part of OBPs may play roles in life activities of the 3rd- and 4th-instar 3808
DOI: 10.1021/acsomega.8b03486 ACS Omega 2019, 4, 3800−3811
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Article
larva or pupa stage, such as the recognition and transfer of a part of host volatiles. All eight BodoCSPs were identified in adults, whereas BodoCSP2 and BodoCSP3 were not detected in our larvae and pupa transcriptome analyses, suggesting that they may be specifically expressed in adults.5 Exploring the molecular mechanisms of CSPs and interfering with the olfactory recognition of insects are feasible methods to control pests. By silencing CquiOBP1 of Culex quinquefasciatus, RNAi-treated mosquitoes exhibited a significantly weakened electrophysiological response to the mosquito oviposition pheromone (MOP).67 When OBP lush was absent, Drosophila exhibited no behavioral response to pheromone 11cis vaccenyl acetate, suggesting that OBP lush is necessary to recognize this substance.68 Silencing LmigOBP1 of Locusta migratoria could significantly reduce food consumption and EAG response levels to some volatiles. The absence of certain OBPs may inhibit the recognition of insect preferred odors, thus reducing the amount of plants pests consume.69 New nonpesticide control measures will be conducive to significantly improving the current management strategies of B. odoriphaga that are extremely dependent on chemical pesticides. In summary, responses to chemical signals and chemosensory genes are two crucial aspects of olfactory recognition in insects. Li et al. had combined EAG and the identification of chemosensory genes to reveal the recognition of semiochemicals in Carpomya vesuviana.70 In this study, we investigated the electrophysiological and behavioral responses of B. odoriphaga to different host volatiles and identified its chemosensory genes, which represent two critical factors of olfactory recognition. Hexanal could elicit extremely significant EAG responses and has significant impacts on the behavior of B. odoriphaga (attract or repel at different concentrations). In behavioral bioassays, methyl propyl disulfide, 1-octen-3-ol, and hexanal at the concentration of 100 mg/mL can remarkably repulse 3rd-instar larvae and female adults, whereas limonene could appreciably attract 3rd-instar larvae and female adults. Moreover, 11 BodoOBPs and 6 BodoOBPs were specifically expressed in adults and the other three stages (3rd- and 4thinstar larvae and pupae). Expression profiles of new genes in adult tissues and the enrichment differences of chemosensory genes between larvae and adults provided targets for further research on the binding ability to host volatiles. In short, our results provided a foundation for the application of new ecofriendly pest management for B. odoriphaga in the future.
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Table S5: predicted protein models and authenticity verification results of CSPs of B. odoriphaga (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Ningxin Wang: 0000-0002-7058-4959 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We are grateful to Jianning Liu for his help in transcriptomic data analysis. This work was supported by the Shandong Province Postdoctoral Innovation Project (201702044), the China Postdoctoral Science Foundation (2017M612312), a Project of Shandong Province Higher Educational Science and Technology Program (J17KA149), as well as the Provincial Key Research and Development Program of Shandong (2017CXGC0207). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article.
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ABBREVIATIONS Bodo, Bradysia odoriphaga; CSP, chemosensory protein; EAG, electroantennography; OBP, odorant binding protein; ORF, open reading frame; qRT-PCR, quantitative real-time PCR; RH, relative humidity
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REFERENCES
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsomega.8b03486. Figure S1: alignment of three subfamily OBPs of B. odoriphaga. Figure S2: alignment of CSPs of B. odoriphaga. Figure S3: phylogenetic tree of CSPs from B. odoriphaga and other six species. Table S1: amino acid sequences of OBPs and CSPs of B. odoriphaga and other insects used in phylogenetic tree construction. Table S2: amino acid sequences of OBPs and CSPs of B. odoriphaga used in 3D model prediction. Table S3: primers for qRT-PCR of new OBPs and CSPs of B. odoriphaga. Table S4: predicted protein models and authenticity verification results of OBPs of B. odoriphaga. 3809
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DOI: 10.1021/acsomega.8b03486 ACS Omega 2019, 4, 3800−3811
