A fatty acid-binding protein links lipid handling to chitin synthase-dependent cuticle formation and nanoscale organization

Background: The insect cuticle is a chitin-containing extracellular matrix whose formation requires precise coordination of chitin biosynthesis, fibril organization, and post-synthetic maturation. Although lipids are essential components of the cuticle, the molecular mechanisms linking lipid transport to chitin deposition remain poorly understood. Here we identify TcFabp8, a fatty acid-binding protein in Tribolium castaneum, as a functionally important regulator of chitin synthase 1 (Chs1)-dependent cuticle formation. Results: RNAi-mediated depletion of TcFABP8 caused molting defects, reduced cuticle thickness, loss of apical TcChs1 immunoreactivity, and reduced chitin deposition and organization. The finding that TcCHS1 transcript levels were unaffected by RNAi targeting TcFABP8 suggests that a post-transcriptional step, potentially involving vesicular TcChs1 transport, is affected, thereby impairing chitin synthesis and deposition. Synchrotron X-ray diffraction analysis of adult elytra revealed that TcFabp8 depletion preserved characteristic -chitin-associated scattering features, but altered chitin fibril accumulation, crystalline coherence, and the spatially ordered alignment of fibrils within the cuticular matrix. The TcFabp8 gene locus encodes splice variants yielding different N-terminal architectures: TcFabp8X1 carries a Sec/SPI signal peptide which is absent in TcFabp8X2 and TcFabp8X3. Immunocytochemistry using TcFabp8-specific antibodies detected intracellular signals in subapical cytoplasmic puncta and extracellular signals within the cuticle layers, which partially colocalized with Nile red fluorescence indicating lipid-rich environments. Conclusions: Together, our data demonstrate that TcFabp8 is a critical facilitator of TcChs1-dependent chitin deposition and extends functional connection between lipid transport and chitin biosynthesis in insects.