Liu Qun, Liu Wei, Zeng Baosheng, Wang Guirong, Hao Dejun, Huang Yongping
Co-Innovation Center for the Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China; College of Forestry, Nanjing Forestry University, Nanjing 210037, China; Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200032, China.
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China.
Insect Biochem Mol Biol. 2017 Jul;86:58-67. doi: 10.1016/j.ibmb.2017.05.007. Epub 2017 May 31.
Olfaction plays an essential role in many important insect behaviors such as feeding and reproduction. To detect olfactory stimuli, an odorant receptor co-receptor (Orco) is required. In this study, we deleted the Orco gene in the Lepidopteran model insect, Bombyx mori, using a binary transgene-based clustered regulatory interspaced short palindromic repeats (CRISPR)/Cas9 system. We initially generated somatic mutations in two targeted sites, from which we obtained homozygous mutants with deletion of a 866 base pair sequence. Because of the flight inability of B. mori, we developed a novel method to examine the adult mating behavior. Considering the specialization in larval feeding, we examined food selection behavior in Orco somatic mutants by the walking trail analysis of silkworm position over time. Single sensillum recordings indicated that the antenna of the homozygous mutant was unable to respond to either of the two sex pheromones, bombykol or bombykal. An adult mating behavior assay revealed that the Orco mutant displayed a significantly impaired mating selection behavior in response to natural pheromone released by a wild-type female moth as well as an 11:1 mixture of bombykol/bombykal. The mutants also exhibited a decreased response to bombykol and, similar to wild-type moths, they displayed no response to bombykal. A larval feeding behavior assay revealed that the Orco mutant displayed defective selection for mulberry leaves and different concentrations of the volatile compound cis-jasmone found in mulberry leaves. Deletion of BmOrco severely disrupts the olfactory system, suggesting that BmOrco is indispensable in the olfactory pathway. The approach used for generating somatic and homozygous mutations also highlights a novel method for mutagenesis. This study on BmOrco function provides insights into the insect olfactory system and also provides a paradigm for agroforestry pest control.
嗅觉在许多重要的昆虫行为中起着至关重要的作用，如取食和繁殖。为了检测嗅觉刺激，需要一种气味受体共受体（Orco）。在本研究中，我们使用基于二元转基因的成簇规律间隔短回文重复序列（CRISPR）/Cas9系统，在鳞翅目模式昆虫家蚕中删除了Orco基因。我们最初在两个靶向位点产生了体细胞突变，并从中获得了缺失866个碱基对序列的纯合突变体。由于家蚕无法飞行，我们开发了一种新方法来检测成虫的交配行为。考虑到幼虫取食的特异性，我们通过对家蚕位置随时间的行走轨迹分析，研究了Orco体细胞突变体的食物选择行为。单感器记录表明，纯合突变体的触角对两种性信息素——蚕蛾醇或蚕蛾醛中的任何一种都无反应。成虫交配行为试验表明，Orco突变体对野生型雌蛾释放的天然信息素以及蚕蛾醇/蚕蛾醛11:1混合物的交配选择行为明显受损。突变体对蚕蛾醇的反应也有所降低，并且与野生型蛾类相似，它们对蚕蛾醛无反应。幼虫取食行为试验表明，Orco突变体对桑叶以及桑叶中发现的不同浓度挥发性化合物顺式茉莉酮的选择存在缺陷。BmOrco的缺失严重破坏了嗅觉系统，表明BmOrco在嗅觉通路中不可或缺。用于产生体细胞和纯合突变的方法也突出了一种新的诱变方法。这项关于BmOrco功能的研究为昆虫嗅觉系统提供了见解，也为农林业害虫防治提供了一个范例。
