Functional identification of the sex pheromone receptor MsepOR6 in the natural population of <i>Mythimna separata</i> (Walker)

doi:10.3785/j.issn.1008-9209.2022.04.261

Journal of Zhejiang University (Agriculture and Life Sciences)

2023, Vol. 49

Issue (2): 149-158 DOI: 10.3785/j.issn.1008-9209.2022.04.261

Special Topic: Insect Physiology and Biochemistry & Pest Biological Control

Functional identification of the sex pheromone receptor MsepOR6 in the natural population of Mythimna separata (Walker)

Yongle ZHANG¹(

),Jianjun CHENG¹,Jingjun ZHENG²,Yongjun DU¹(

)

^1.Institute of Pesticide and Environmental Toxicology, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, Zhejiang, China
^2.Agricultural Pest Precaution and Management Center of Guangdong Province, Guangzhou 510500, Guangdong, China

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Abstract

In order to further understand the calling behavior of Mythimna separata (Walker) (Lepidoptera, Noctuidae) and explore the olfactory molecular recognition mechanism of male moths to intra- and inter-specific sex pheromones, the male moths collected by sex pheromone trapping with the mixture of Z11-16：Ac and Z11-16：OH in the field were used in this study. According to the reported fragment, we amplified the full-length sequence of MsepOR6 by rapid amplification of cDNA ends (RACE) technology. The responses of MsepOR6 cRNA to pheromone compounds were identified by two-electrode voltage clamp. The behavioral responses of male moths to active compounds were further verified by sex pheromone trapping experiment in the field. Electrophysiological assay results showed that MsepOR6 is one of the sex pheromone receptor genes, which was sensitive to two intra-specific sex pheromones of Z11-16：Ald and 16：Ald, and four inter-specific sex pheromones of Z9-14：Ac, Z7-12：Ac, Z9-16：Ac, and Z9-14：OH. Field bioassay results showed that Z9-14：Ac was antagonist and Z7-12：Ac was synergist in sex pheromone trapping of male moths. This study identified the function of MsepOR6 in the natural population of male moths, which is helpful to understand the regulatory mechanism of calling behavior and provides theoretical support for further development of sex pheromone application technology.

Key words： Mythimna separata (Walker) olfactory receptor two-electrode voltage clamp sex pheromone inter-specific chemical communication

Received: 15 March 2022 Published: 27 April 2023

CLC:

S433.4

Corresponding Authors: Yongjun DU E-mail: 2908085596@qq.com;yongjundu@zju.edu.cn

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Cite this article:

Yongle ZHANG,Jianjun CHENG,Jingjun ZHENG,Yongjun DU. Functional identification of the sex pheromone receptor MsepOR6 in the natural population of Mythimna separata (Walker). Journal of Zhejiang University (Agriculture and Life Sciences), 2023, 49(2): 149-158.

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https://www.zjujournals.com/agr/10.3785/j.issn.1008-9209.2022.04.261 OR https://www.zjujournals.com/agr/Y2023/V49/I2/149

东方黏虫［Mythimna separata (Walker)］田间自然种群的性信息素受体MsepOR6功能鉴定

为进一步理解东方黏虫[Mythimna separata (Walker)]的求偶行为，探明雄蛾对种内和种间性信息素的嗅觉分子识别机制，本研究以顺11-十六碳烯乙酸酯（Z11-16：Ac）和顺11-十六碳烯醇（Z11-16：OH）为性信息素进行田间诱捕到的东方黏虫作为研究对象，根据文献报道的MsepOR6基因片段序列，采用cDNA末端快速扩增（rapid amplification of cDNA ends, RACE）技术扩增出其全长，采用双电极电压钳技术鉴定MsepOR6对东方黏虫及其同作物生境害虫的性信息素的电流反应，并通过田间诱捕试验验证雄性东方黏虫对其中的活性化合物的行为反应。电生理测定结果表明：MsepOR6是东方黏虫雄蛾的性信息素受体基因，其cRNA对种内性信息素顺11-十六碳烯醛（Z11-16：Ald）、十六醛（16：Ald），以及种间性信息素顺9-十四碳烯乙酸酯（Z9-14：Ac）、顺7-十二碳烯乙酸酯（Z7-12：Ac）、顺9-十六碳烯乙酸酯（Z9-16：Ac）、顺9-十四碳烯醇（Z9-14：OH）有显著的电生理反应。田间生物测定结果表明，Z9-14：Ac可显著抑制东方黏虫性信息素的引诱活性，Z7-12：Ac则显著提高东方黏虫性信息素对雄蛾的引诱效果。本研究有助于理解东方黏虫的性信息素行为调控机制，为开发应用性信息素技术提供了理论支撑。

关键词： 东方黏虫, 嗅觉受体, 双电极电压钳, 性信息素, 种间化学通信

序号 No.	基因 Gene	引物序列（5 $′$ →3 $′$ ） Primer sequence (5 $′$ →3 $′$ )	用途 Usage
1	MsepOR6	F: TACTCTGATTACGCAATGGA	qPCR
1	MsepOR6	R: AGTTGACCACATACGCATT	qPCR
2	RPS3	F: AATGAGTTCTTGACCAGGGAG	qPCR
2	RPS3	R: GTGTCCTCGTCGCCATAAT	qPCR
3	Actin	F: ATCAAGGAGAAGCTCTGCTACG	qPCR
3	Actin	R: ATCGTTCGTTTCCGATGGTG	qPCR
4	MsepOR6	F: ACTTTGGCAGATCTGATAgccaccATGCAAAGATTGAAAAGAAAGTT	同源重组 Homologous recombination
4	MsepOR6	R: CCGGGCCCACTAGTGATCTACTTGCTTCGAAGGAAAGT	同源重组 Homologous recombination
5	MsepORco	F: GGCAGATCTGATATCgccaccATGATGACCAAAGTGAAGGCCCA	同源重组 Homologous recombination
5	MsepORco	R: ACCGGGCCCACTAGTTTACTTGAGTTGCACCAACACCA	同源重组 Homologous recombination

Table 1 Primers used in this experiment

Table 2 Compounds used in the electrophysiological test

Fig. 1 Multiple sequence alignment result of MsepOR6

Fig. 2 Phylogenetic tree of MsepOR6andMsepORco in Mythimna separataHvir: Heliothis virescens; Harm: Helicoverpa armigera; Hass: Helicoverpa assulta; Aseg: Agrotis segetum; Msep: Mythimna separata; Slitu: Spodoptera litura; Psau: Peridroma saucia; Sfru: Spodoptera frugiperda; Bmor: Bombyx mori.

Fig. 3 Specific expression of MsepOR6 in tissues of Mythimna separataAn: Antenna; He: Head; Th: Thorax; Ab: Abdomen; Le: Leg; Wi: Wing. Data are expressed as means±standard errors. Different lowercase letters above bars indicate significant differences at the 0.05 probability level; n=6.

Fig. 4 Responses of MsepOR6/MsepORco co-expressed in Xenopus oocytes to the compoundsA-B. Current responses of MsepOR6/MsepORco to 1.0×10^－4 mol/L intra- and inter-specific sex pheromones (n=6); C. Dose-response of MsepOR6/MsepORco to intra-specific sex pheromone of Z11-16：Ald (n=4); D. Dose-response of MsepOR6/MsepORco to inter-specific sex pheromone of Z9-14：Ac (n=4).

Fig. 5 Comparison of the attracting abilities of different proportions of sex pheromones against male Mythimna separataA. Relative trapping moth amount after adding different proportions of Z9-14：Ac; B. Relative trapping moth amount after adding different proportions of Z7-12：Ac. Data are expressed as means±standard errors. Different lowercase letters above bars indicate significant differences at the 0.05 probability level.


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