昆虫拥有一套高度灵敏的嗅觉系统，来识别环境中纷繁复杂的气味物质，如种内或种间信息素、植物挥发物等，借此高效完成定位寄主、寻找配偶和躲避天敌等一系列重要行为。触角是昆虫最重要的嗅觉器官，表面密布嗅觉感器；嗅觉感器中感受神经元树突膜上的气味受体（Odorant receptor，OR）负责将化学信号转换成电信号，因而在嗅觉感受的灵敏性和特异性中起关键作用。草地贪夜蛾和斜纹夜蛾均是灰翅夜蛾属重要害虫，但二者的寄主范围有所差异，草地贪夜蛾偏好玉米、水稻、牧草等禾本科植物，而斜纹夜蛾偏好烟草、大豆、棉花等双子叶植物。不同的寄主偏好性暗示两种夜蛾可能在植物挥发物的感受上存在分化。研究并明确这种分化及其分子机制，有助于深入理解近源种寄主分化的机制，并对研发基于嗅觉的行为调控技术具有重要意义。为此，本研究以两种夜蛾为对象进行比较研究，首先利用触角电位（Electroantennography，EAG）、Y型嗅觉仪和产卵选择等技术，测定并比较了二者对5种常见植物挥发物（壬醛、苯乙酮、苯甲醇、水杨酸甲酯和顺-3-己烯基乙酸酯）的电生理和行为反应的异同；然后通过系统发育分析获得两种夜蛾感受这些植物挥发物的候选OR基因，并利用qPCR技术明确了这些OR基因在雌雄触角中的表达水平；最后利用爪蟾卵母细胞-双极电压钳系统测定了这些候选OR基因的功能，探讨了两种夜蛾同源OR基因在感受常见寄主植物挥发物中的差异。主要结果如下：
1、草地贪夜蛾和斜纹夜蛾对5种寄主植物挥发物的电生理和行为反应
利用触角电位技术测定了两种昆虫雌雄蛾对5种植物挥发物的EAG反应，发现两种昆虫对于4种挥发物（壬醛、苯乙酮、苯甲醇和水杨酸甲酯）均可产生显著的EAG反应，且具有剂量依赖效应；但两种夜蛾对于顺-3-己烯基乙酸酯的EAG反应有差异，斜纹夜蛾雌雄蛾均可产生显著反应，而草地贪夜蛾只有雌虫可产生显著的反应。利用Y型嗅觉仪进行双向选择实验，发现两种昆虫均对壬醛、苯乙酮、顺-3-己烯基乙酸酯有显著的趋向行为，此外草地贪夜蛾还对水杨酸甲酯有显著的趋向行为。进一步利用产卵选择实验，测定了两种雌蛾对5种挥发物的产卵选择行为，结果壬醛、苯乙酮和水杨酸甲酯均能显著吸引两种雌蛾产卵，而顺-3-己烯基乙酸酯仅能吸引斜纹夜蛾产卵，对草地贪夜蛾没有显著影响。可见，两种夜蛾对5种植物挥发物无论在电生理还是在行为反应上均存在一定的差异。
2、两种夜蛾感受5种植物挥发物的OR同源基因的序列和性别表达差异分析
通过构建草地贪夜蛾、斜纹夜蛾、海灰翅夜蛾、棉铃虫、烟青虫和家蚕的OR系统发育树，根据后四种昆虫中OR的文献报道，将感受壬醛等5种植物挥发物的OR分支中对应的草地贪夜蛾和斜纹夜蛾的OR基因作为候选OR基因。序列分析表明，两种昆虫共5对候选OR同源基因所编码蛋白间的氨基酸相似性均很高：SfruOR47和SlituOR9为98.24%，SfruOR24和SlituOR55为89.66%，SfruOR25和SlituOR56为96.15%，SfruOR28和SlituOR69为95.36%，SfruOR58和SlituOR22为88.86%。利用qPCR对候选OR基因在雌雄触角中的表达差异分析发现，5对同源基因的性别表达差异完全相同，其中SfruOR47和SlituOR9均为雌蛾显著高于雄蛾，其余候选OR基因在雌雄蛾触角间的表达量没有显著差异。然而，两种夜蛾间5对同源基因在触角中的表达量存在明显不同，雌蛾触角中各候选OR基因的表达水平均为斜纹夜蛾显著高于草地贪夜蛾，雄蛾触角中除SfruOR58和SlituOR22的表达量无显著差异外，其他4对OR基因也表现为斜纹夜蛾显著高于草地贪夜蛾。
3、两种夜蛾感受5种植物挥发物的OR同源基因的功能分析
利用爪蟾卵母细胞-双极电压钳系统测定了5对候选OR基因对70种化合物的电生理反应。结果发现，同源基因SfruOR47和SlituOR9均对壬醛、癸醛、壬醇、癸醇4种化合物有电生理反应，并以壬醛的电生理反应最强，EC50值也基本相同；SfruOR25和SlituOR56都对苯乙酮等15种化合物有电生理反应，且都对苯乙酮的电生理反应最强，但两者的敏感性明显不同，前者的EC50值是后者的2倍左右；SfruOR24和SlituOR55都对苯甲醇、苯乙醇等6种化合物有电生理反应，且都对苯甲醇的电生理反应最强，前者的EC50值是后者的3倍左右；SfruOR58和SlituOR22都对水杨酸甲酯有特异性反应，但前者的EC50值仅是后者的1/6左右；SfruOR28和SlituOR69都对顺-3-己烯基乙酸酯、壬醛、癸醛、顺-3-己烯醇4种植物挥发物有电生理反应，且都对顺-3-己烯基乙酸酯的电生理反应最强，但前者的EC50值仅是后者的1/5左右。可见两种夜蛾5对同源OR的配体反应谱基本相同，但对最适配体的敏感性存在明显差异。
4、SfruOR47和SlituOR9与壬醛的相互作用
通过构建SfruOR47和SlituOR9的3D模型和分子对接确定了壬醛与两个OR结合的关键氨基酸位点。两个OR都存在His57和Glu61两个氨基酸位点与壬醛形成氢键，是壬醛与两个OR结合的主要作用力。进一步通过点突变，将His或Glu突变为Ala，或者同时将His和Glu突变为Ala，然后进行爪蟾卵母细胞-双电极电压钳系统检测点突变后的SfruOR47和SlituOR9对壬醛的电生理反应。结果显示，His57点突变后（SfruOR47-His57Ala和SlituOR9-His57Ala），两个OR对壬醛的电生理反应和敏感性都显著降低，EC50分别升高了103倍和388倍；Glu61点突变后（SfruOR47-Glu61Ala和SlituOR9-Glu61Ala），两个OR对壬醛的电生理反应降低，EC50分别升高了3倍和18倍；His57和Glu61同时突变后（SfruOR47-His57Ala-Glu61Ala和SlituOR9-His57Ala-Glu61Ala），两个OR对壬醛的电生理反应和敏感性都基本消失。点突变结果验证了His57和Glu61是两个OR结合壬醛所必需的氨基酸位点。
综上，本研究在触角电生理、行为学和OR表达量及配体反应等方面，揭示了草地贪夜蛾和斜纹夜蛾对5种常见植物挥发物的嗅觉分化。在电生理和行为反应上，两种夜蛾对顺-3-己烯基乙酸酯、水杨酸甲酯存在一定差异；两种夜蛾间5对同源OR的氨基酸序列相似性均很高（> 88%），但在触角中的表达量斜纹夜蛾普遍高于草地贪夜蛾；同源OR基因编码的蛋白的配体反应谱基本相同，但对最适配体的敏感性存在明显差异；His57和Glu61是两种夜蛾感受壬醛的受体（SfruOR47和SlituOR9）所必需的氨基酸位点。研究结果为深入解析两种昆虫主要寄主植物分化的嗅觉机制提供了数据，同时为开发更加经济高效、环境友好的行为防治技术奠定了基础。

Insect has a highly sensitive olfactory system to identify complex odorants in the environment, such as inter- and intra-specific pheromones, plant volatiles, which allow them a high efficiency in many important behaviors such as locating hosts, searching for mates, and avoiding predators. Antennae are the major olfactory organs, on which thousands of olfactory sensilla are located. The olfactory receptors on the dendritic membranes of the sensory neurons in the olfactory sensilla are responsible for converting chemical signals into electrical signals, and thus play a key role in the sensitivity and specificity of olfactory perception. The two important pests Spodoptera frugiperda and S. litura are congeneric, but differentiate in the host plants. S. frugiperda prefers to eat gramineous plants such as corn, rice, and forage plants, while S. litura prefers to dicotyledons such as tobacco, soyabean, and cotton. This difference in host preferences suggests a divergence in the perception of plant odors. Studying and clarifying this divergence and its molecular mechanisms can help us to deepen the understanding of the mechanism of host divergence among closely related species, and have important implications for developing behavioral technologies based on olfactory perception. Therefore, a comparative study between the two Spodoptera species was conducted. First, by using electroantennography (EAG), Y-tube olfactometer, and egg-laying selection equipment, the electrophysiological and behavioral responses of moths to five common plant volatiles (nonanal, acetophenone, benzyl alcohol, methyl salicylate, and cis-3-hexenyl acetate) were measured. Second, by means of construction of evolutionary tree of ORs, the homologous OR genes that were reported to tune for the five plant volatiles were obtained as candidate OR genes of the two Spodoptera species, and in addition, expression levels of these candidate OR genes in the male and female antennae were determined by qPCR. Finally, the ligand responses of these OR genes were clarified by the Xenopus oocyte-two electrode voltage clamp system. The main results are as follows:
1. The electrophysiological and behavioral responses to five host plant volatiles by adults of the two Spodoptera species
Both S. frugiperda and S. litura showed significant EAG responses to nonanal, acetophenone, benzyl alcohol and methyl salicylate, in a dose-dependent manner. However, significant difference was observed to cis-3-hexenyl acetate. Both male and female S. litura responded to cis-3-hexenyl acetate, while only female S. frugiperda showed a significant response. In a two-choice experiment using Y-tube olfactometer, females of both species showed a significant preference for nonanal, acetophenone and cis-3-hexenyl acetate, and S. frugiperda was also attracted by methyl salicylate. In further egg-laying assay, both species preferred laying eggs on soybean plants treated with nonanal, acetophenone, and methyl salicylate; in addition, S. litura also preferred laying eggs on plants with cis-3-hexenyl acetate.
2. The amino acid sequence and sex expression profile of the homologous OR genes between the two Spodoptera species
To obtain the candidate OR genes tuned the 5 host plant volatiles in the two species, a phylogenetic tree of ORs was constructed with 388 ORs from six moth species was built. Five OR clads were clearly showed, with each clad containing at least one OR for one of the 5 plant volatiles previously reported in S. littoralis, H. armigera, H. assulta or Bombyx mori. The 5 pairs of OR orthologous genes of S. frugiperda and S. litura in the 5 clads were considered to be candidate OR genes for the plant volatiles. Sequence analysis showed that homologous genes between the two insects shared with high amino acid identity of 98.24% between SfruOR47 and SlituOR9, 89.66% between SfruOR24 and SlituOR55, 96.15% between SfruOR25 and SlituOR56, 95.36% between SfruOR28 and SlituOR69, and 88.86% between SfruOR58 and SlituOR22. The qPCR results showed that the sex expression patterns of these candidate OR genes was exactly the same between the two species. Both SfruOR47 and SlituOR9 were significantly female biased, while other four pairs of candidate OR genes were all unbiased between sex. However，the expression levels of candidate OR genes in female and male antennae of S. litura were generally higher than those in S. frugiperda.
3. Response profiles of homologous OR genes to the 5 plant volatiles in the two Spodoptera species
The functions of candidate OR genes to 70 compounds were measured by Xenopus oocyte-two electrode voltage clamp system. The results showed that SfruOR47 and SlituOR9 had similar electrophysiology responses to four compounds (nonanal, decanal, nonanol, and decanol), with the strongest response to nonanal. SfruOR25 and SlituOR56 had similar electrophysiology responses to 15 compounds, with the strongest response to acetophenone, but for this compound, the EC50 value of SfruOR25 was about 2 times that of SlituOR56. SfruOR24 and SlituOR55 also had similar electrophysiological responses to 6 compounds including benzyl alcohol and Phenethyl alcohol, and both ORs had the strongest response to benzyl alcohol; but EC50 value of the former is about 3 times higher than that of the latter. SfruOR58 and SlituOR22 both specifically responded to methyl salicylate, but the EC50 value of the former was only about 1/6 of that of the latter. SfruOR28 and SlituOR69 had electrophysiological responses to 4 plant volatiles (cis-3-hexenyl acetate, nonanal, decanal, and cis-3-hexenol), and both had the strongest electrophysiological response to cis-3-hexenyl acetate. However, the EC50 value of the former is significantly lower than that of the latter, only about 1/5 of the latter. 
4. Interaction of attractant nonanal with SfruOR47 and SlituOR9
The key amino acid sites of SfruOR47 and SlituOR9 for binding nonanal were identified by constructing 3D models of the two ORs and molecular docking. Two amino acid sites, His57 and Glu61, are present in both ORs to form hydrogen bonds with nonanal, which is the main force for binding nonanal. Further site-directed mutagenesis results showed single mutant of His57Ala or Glu61Ala significantly reduced the binding affinity of SfruOR47 to nonanal, increasing the EC50 value to 1.986×10-3 M and 6.519×10-5 M (2.811×10-6 M for intact OR), respectively, and the double mutant further increased the EC50 value to 3.472×10-2M. Similarly, for SlituOR9, the EC50 value for single mutation of His57Ala or Glu61Ala increased to 2.11×10-3 and 9.829×10-5 M (2.792×10-6 M for intact SlituOR9), respectively; and the double mutant resulted in loss of the binding ability to nonanal. These results revealed the His57 and Glu61 residues in both SfruOR47 and SlituOR9 are essential for nonanal binding.
In summary, this study revealed the olfactory differentiation between S. frugiperda and S. litura to five common host-plant volatiles, in terms of antennal electrophysiology, behavioral response, and OR expression and ligand response profile. Differences were observed in EAG and behavioral response to cis-3-hexenyl acetate and methyl salicylate; the 5 pairs of homologous ORs shared high amino acid identity (> 88%), but the expression levels were generally higher in S. litura than in S. frugiperda; furthermore, homologous ORs between the two species shared with similar ligand specificity, but they showed significant differences in the sensitivity to the optimal ligand. By 3D structure prediction, molecular docking and site-directed mutagenesis, we revealed that the His57 and Glu61 residues in both SfruOR47 and SlituOR9 are essential for nonanal binding. This study provides data for further clarification of the mechanism underlying the host plants difference between the two congeneric species, and lays a foundation for developing more economically efficient and environmentally friendly behavioral control techniques for these two pests.

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