Alternative strategies based on transgenic Drosophila melanogaster for the functional characterization of insect Ionotropic Receptors

Authors: CRAVA, CRISTINA - Valencia University; Walker Iii, William; CATTANEO, ALBERTO - Swedish University Of Agricultural Sciences

Submitted to: Biological Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/28/2025
Publication Date: 6/9/2025
Citation: Crava, C.M., Walker Iii, W.B., Cattaneo, A.M. 2025. Alternative strategies based on transgenic Drosophila melanogaster for the functional characterization of insect Ionotropic Receptors. Biological Research. 58(36). https://doi.org/10.1186/s40659-025-00619-0.
DOI: https://doi.org/10.1186/s40659-025-00619-0

Interpretive Summary: The codling moth and spotted wing drosophila are prominent agricultural pest insects of several fruit crops around the world. A better understanding of the basic biology of these insects could lead to improved ways to control this pest by disrupting behaviors, such as host-finding or mate-seeking, that are regulated by the insect sense of smell. Detection of odors from the environment result in activation of behavioral responses. Odorant detection is mediated by multiple different families of proteins that function as olfactory receptors, including the sub-family known as “ionotropic receptors”. Within codling moth and spotted wing drosophila, very little is known about the odorant detection capabilities of the ionotropic receptors. Researchers at the USDA-ARS in Wapato, WA, the Swedish University of Agricultural Sciences, and the University of Valencia, studied the function of four odorant receptors identified from codling moth or spotted wing drosophila. They discovered that two of these receptors detect odorants not previously known to influence respective behaviors of either species. These results provide a first-time functional analysis of olfactory ionotropic receptors for codling moth and spotted wing drosophila and will allow the researchers to find ways to block these receptors and influence or manipulate insect behavior in the orchards as a way to enhance olfactory-based control of these important agricultural pest insects.

Technical Abstract: Background 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. Results 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. Conclusion 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.