The plant’s chemical composition is highly diverse, but plant-feeding insects need to decode this complexity to identify their host. For close range assessment, GRs constitute the first checkpoint to convert a molecular information encoding a plant compound into a unique physiological signal that is relevant for the insect’s feeding choice. More details can be found in a viewpoint paper (doi:10.1039/c7np00002b) that I led as author.
In case of C. populi as model system for ChemoSense, it was hypothesised that salicin in poplar leaves is a major taste stimulant and enhances feeding activity. However, it was unknown if and how salicin, or, alternatively other taste stimulants from poplar are detected by specific GRs and how this interaction impacts on the beetle’s final feeding choice. To elucidate these mechanisms at different levels, the following work packages were carried out. (i.) Identification of GR genes: High-throughput transcriptome sequencing using RNA isolated from different developmental stages and organs of C. populi. Bioinformatic de novo assembling, annotation and construction of a transcriptomic library resulted in over 50 putative candidate genes encoding taste receptors. Most GRs were lowly expressed, even in typical external taste organs, e.g. such as tarsi, mouthparts and antennae. As validated by quantitative real-time PCR, some GRs were even expressed in inner organs such as brain and gut. (ii.) Visualisation of GR genes: Optical imaging turned out to be challenging, especially when using intact appendages. While such whole-mount samples retained the spatial information on the expression of GR genes better than e.g. ultra-thin sectioning, the pigmented cuticle constituted a serious barrier for microscopy. While chemical bleaching via e.g. hydrogen peroxide bleached the cuticle, it largely destroyed cellular RNA. Therefore, another approach was developed by taking advantage of characterised pigmentation genes such as laccase2. RNA interference (RNAi) based silencing of lac2 led to clearing of cuticular pigmentation and retained RNA level fully. This enabled the subsequent use of RNA fluorescence in situ hybridisation (FISH) and exemplary imaging of CpopGR1 in intact palps by confocal laser scanning microscopy. I was the lead author of this research methods paper (doi:10.1242/jeb.185710). (iii.) Functional analysis of single GRs: To revel if and how understand certain salicinoids from poplar can be tasted and contribute to the beetle’s feeding decision, insect cell expression and quantitative calcium imaging showed that a certain GR binds salicin and salicortin, but not tremulacin. Subsequent gene silencing showed that C. populi beetles prefer feeding on salicortin-rich poplar species (Populus trichocarpa) over salicortin-poor species (P. nigra). In contrast, RNAi-silencing of CpopGR1 led to an overall decrease in feeding intensity on P. trichocarpa in comparison to controls. (iv.) Identification of taste sensilla and their contribution to the beetle’s feeding choice: To close the gap between the molecular and behavioural findings from above experiments, taste responses at the physiological were analysed. Therefore, putative taste sensilla, hair-like cuticular microstructures, were identified using a novel approach based on cuticular autofluorescence scanning. The gustatory function of these antennal sensilla chaetica was validated by single taste sensillum recordings using salicin and sucrose. Both ligands were found to be higher concentrated in poplar compared to nonhost control leaves (willow), and may therefore contribute to the observed feeding preference for poplar over willow. This feeding choice resulted in higher weight gain when rearing beetles on poplar compared to willow leaves. I was the lead author of this research paper (doi:10.3389/fphys.2019.00343). The results from ChemoSense may provide the basis to develop taste-based feeding traps for poplar leaf beetles. Protection of poplar leaves is envisioned by designing mechanical traps containing an artificial diet soaked with salicin and sucrose. Such taste-based feeding traps using natural compounds are a sustainable alternative to transgenic poplar plants and synthetic insecticides and may inspire developing eco-friendly protection against other pest species.
