Timing of chitin/chitosan matrix assembly in insects

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The N-acetyl-glucosamine polysaccharide chitin and its deacetylated derivative chitosan are major extracellular scaffolds in the cuticle of insects. In different body parts and stages, specific sets of cuticle proteins interact with chitin/chitosan forming a crystalline, amorphic or composite structures that define the region-specific material and physical properties of the cuticle. The assembly of the matrix composed of chitin/chitosan and the associated proteins in time and space including its impact on the mechanical and biological function is unexplored. In line with the CodeXi preamble, we reckon that the tissue-specific ratio between chitin and chitosan (FA) and pattern of deacetylation (PA) are interpreted by cuticle proteins depending on the position in the body. In this joint project, we address these problems using the leg and optionally the wing of the fruit fly Drosophila melanogaster as a model cuticle system. At the genetic and molecular level, we explore the functions of the two chitin/chitosan-binding proteins Pro-Resilin and Cpr56F focusing on the trochanter (needed for locomotion) and the tarsus (needed for adhesion). To begin with, we will monitor the temporal and spatial distribution of fluorescently tagged versions of Pro-Resilin and Cpr56F that we have already generated applying the gene editing protocol of Crispr/Cas9. In parallel, we will examine the expression of tagged versions of the chitin deacetylases Vermiform (Verm) and Serpentine (Serp) as well as the C-type lectin Schlaff (Slf) that is needed for correct Pro-Resilin localization. Using fungal chitosanases and chitosan-binding proteins, we map chitosan distribution in the leg segments. Gene editing will be applied to swap respective promotors and chitin/chitosan-binding domains in order to refine the study of temporal and spatial impact of Pro-Resilin and Cpr56F on cuticle function. The cuticle properties of the genetically modified flies are subsequently characterised in extended histological, biomechanical and behavioural essays, and eventually linked to the specific and local chitin/chitosan code in the insect cuticle. This work will serve as an integral example for the molecular mechanisms of generation, interpretation, and use of a chitin/chitosan matrix in the living world.