Crava Cristina M, Walker William B, Cattaneo Alberto Maria
University Institute of Biotechnology and Biomedicine, University of Valencia, 46100, Burjassot, Spain.
USDA-ARS Temperate Tree Fruit and Vegetable Research Unit, 5230 Konnowac Pass Road, Wapato, WA, 98951, USA.
Biol Res. 2025 Jun 9;58(1):36. doi: 10.1186/s40659-025-00619-0.
Insect Ionotropic Receptors (IRs) are a relatively uncharted territory. Some studies have documented IR activation by recording neuronal activity in situ, others by their heterologous expression in Xenopus oocytes or mis-expressing IRs from Drosophila melanogaster or from the related D. sechellia into the D. melanogaster "ionotropic receptor decoder" neuron, which lacks the endogenous tuning receptor subunit but expresses IR-coreceptors.
In this study, we first made use of Drosophila olfactory sensory neurons (OSNs) different from the "ionotropic receptor decoder", demonstrating that by replacing or introducing IRs alongside the native D. melanogaster ones, functional heteromeric complexes can be formed. IR41a1 from the lepidopteran Cydia pomonella exhibits binding to polyamines and the IR75d from the dipteran Drosophila suzukii binds hexanoic acid. Secondly, expressing D. suzukii's putative acid sensor IR64a into the "ionotropic receptor decoder" of D. melanogaster inhibits the response to the main activators of neighboring neurons from the same sensillum, despite that IR64a does not respond to acids. In situ hybridization on the antennae of D. suzukii unveils wide expression of IR64a in neurons proximal to the sacculus. Structural modeling analysis does not explain its absence of binding to acids; conversely, this approach identifies key amino acids features explaining the binding of hexanoic acid by IR75d. Finally, we have also explored alternative methods to heterologously express IRs based on Human Embryonic Kidney cells (HEK293). Despite observing correct expression of IRs in transfected cells through immunohistochemistry experiments, this approach did not achieve successful deorphanization of these receptors.
Our findings highlight the potential use of Drosophila OSNs as a valuable tool for functional characterization of IRs from different insect species: for the first time, we have provided evidence of IR-functionalities within alternative OSNs from the Drosophila's "ionotropic receptor decoder" neuron to functionally characterize and deorphanize IRs from lineages that are evolutionarily distant from the D. melanogaster subgroup, contributing to the understanding of chemosensory modalities in D. suzukii and C. pomonella, two globally significant agricultural pests. Additionally, the unsuccessful deorphanization in HEK cells highlights the complex requirements for IR functionality, supporting the use of Drosophila OSNs as a more suitable expression system.
昆虫离子型受体（IRs）是一个相对未知的领域。一些研究通过记录原位神经元活动来证明IR的激活，另一些研究则通过在非洲爪蟾卵母细胞中进行异源表达，或将黑腹果蝇或相关的塞舌尔果蝇的IRs错误表达于缺乏内源性调谐受体亚基但表达IR共受体的黑腹果蝇“离子型受体解码器”神经元中来证明。
在本研究中，我们首先利用了不同于“离子型受体解码器”的黑腹果蝇嗅觉感觉神经元（OSNs），证明通过替换或引入与天然黑腹果蝇IRs一起的IRs，可以形成功能性异源复合物。来自鳞翅目苹果蠹蛾的IR41a1表现出与多胺的结合，而来自双翅目铃木果蝇的IR75d与己酸结合。其次，将铃木果蝇假定的酸传感器IR64a表达于黑腹果蝇的“离子型受体解码器”中，抑制了来自同一感器的相邻神经元对主要激活剂的反应，尽管IR64a对酸没有反应。对铃木果蝇触角进行原位杂交揭示了IR64a在球囊附近神经元中的广泛表达。结构建模分析无法解释其与酸不结合的原因；相反，该方法确定了解释IR75d与己酸结合的关键氨基酸特征。最后，我们还探索了基于人胚肾细胞（HEK293）异源表达IRs的替代方法。尽管通过免疫组织化学实验观察到转染细胞中IRs的正确表达，但该方法并未成功使这些受体去孤儿化。
我们的研究结果突出了黑腹果蝇OSNs作为一种有价值工具用于不同昆虫物种IRs功能表征的潜力：我们首次提供了证据，证明在黑腹果蝇“离子型受体解码器”神经元的替代OSNs中存在IR功能，以对与黑腹果蝇亚群进化距离较远的谱系中的IRs进行功能表征和去孤儿化，有助于理解铃木果蝇和苹果蠹蛾这两种全球重要农业害虫的化学感应方式。此外，在HEK细胞中去孤儿化未成功突出了IR功能的复杂要求，支持将黑腹果蝇OSNs用作更合适的表达系统。
