Periodic Reporting for period 1 - ChemoSense (Elucidating the Mechanisms of Insect’s Chemical Taste to Understand Specific Host-Plant Selection)
Okres sprawozdawczy: 2016-12-01 do 2018-11-30
Similar to humans and other animals, the sense of taste enables an insect to decide what it eats and drinks. One essential checkpoint for evaluating a plant as suitable food source are gustatory receptors (GRs) and their associated neurons. In concert, they decode the plant’s chemical composition by identifying different nonvolatile compounds that contribute to the insect’s behavioural distinction between edible and toxic. Despite their essential role in the insect’s overall food intake and survival, little is known about GRs and its neurons, especially in plant-feeding beetles. This is surprising given their importance as agricultural and forestry pests, their global distribution and huge species numbers. The EU-funded project ChemoSense, led by the Marie Skłodowska-Curie fellow Dr Stefan Pentzold, aimed at identifying and characterising those gustatory mechanisms that mediate host plant selection in a herbivorous pest, the poplar leaf beetle (Chrysomela populi). These beetles specifically feed on leaves of poplar trees and damage short-rotation plantations worldwide. By combining various state-of-the-art methods, ChemoSense has revealed novel insights in understanding taste sensation and host plant selection at different levels. The beetle’s molecular equipment of GR genes was identified and their spatiotemporal expression was exemplary visualised in different taste organs. Using electrophysiological, microscopic, chemo-analytical and behavioural assays, ChemoSense has also shown how C. populi taste their favourite food plant and how single compounds such as sugars and salicinoids affect the beetles’ feeding choice. The results may pave the way for developing sustainable pest management strategies, e.g. taste-based feeding traps with natural tastants.
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.
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.
ChemoSense has advanced our understanding of how, when and where GRs and associated neurons function as regulators of host plant selection in a specialist beetle. The new knowledge provides an intriguing example of the complexity between the herbivore’s gustatory repertoire and host’s chemical diversity. The outcomes such as the “RNA-i-FISH” approach improve optical imaging of chemosensory genes in pigmented insects, and the autofluorescence-based identification of antennal taste sensilla will be very helpful in insect taste research. The results of ChemoSense were widely disseminated throughout the course of the project, including international scientific conferences and seminars as well as public outreach, social media and supervision of high school, bachelor and master students. ChemoSense has also led to the establishment of an European collaboration through the secondment at CNRS-EGCE resulting in synergistic effects and exchange of knowledge and skills. The fellowship extended my scientific knowledge, skills and network finally acquiring a skill set of high professional maturity. I have learned many new cutting-edge biotechniques as well as transferable skills, which qualifies me for a future leading position. Finally, the outcomes of ChemoSense hold the potential for designing novel, sustainable and innovative crop protections strategies.