[P007] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P04-487] Identification and cross-species comparison of low molecular-weight cuticular protein genes highly expressed during cuticle maturation in the Japanese rhinoceros beetle

○Minto Takahashi¹, Satoshi Murata¹, Kousuke Kataoka¹, Daivid Kisailus^2,3, Atsushi Arakaki¹ (1. Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology (Japan), 2. Department of Materials Science and Engineering, University of California Irvine (USA), 3. Materials and Manufacturing Technologies Program, University of California Irvine (USA))

Keywords:

Cuticular proteins (CPs),Beetle,Low molecular-weight proteins,Bio-inspired materials

[Purpose]Beetle elytra are lightweight yet strong structures, and their formation mechanisms have attracted attention in bio-inspired materials research. Although cuticular proteins (CPs) are known to play essential roles in cuticle formation, recent studies suggest that previously unclassified proteins may also contribute. Therefore, a broader re-evaluation of proteins involved in elytral maturation is needed. In this study, we used the Japanese rhinoceros beetle, Trypoxylus dichotomus, to identify genes associated with post-eclosion elytral maturation and to assess their lineage distribution through a cross-species comparison. [Method]Using a public T. dichotomus genome and existing RNA-seq data, we performed gene predictions with BRAKER3 (v3.0.8) and extracted highly expressed post-eclosion candidates based on transcript abundance quantified with Salmon (v1.10.3). Sequence modules were extracted from the candidate protein sequences, and were used to built profile hidden Markov models. Motif occurrences were evaluated across 18 arthropod datasets using hmmsearch (v3.4). We constructed a species phylogeny with IQ-TREE (v3.0.1) and mapped motif presence onto the tree. We also visualized the three-dimensional structures of the identified candidate proteins using AlphaFold3. [Results]Among the top 10 highly expressed protein-coding genes at 0 h and 24 h after eclosion, we identified multiple low molecular-weight protein genes (70–280 aa). Proteins highly expressed at 0 h tended to contain AAP-repeat motifs, whereas those highly expressed at 24 h had significant Gly-rich motifs. Cross-species comparison further showed that proteins containing AAP-repeat motifs are broadly distributed across Insecta, whereas proteins containing Gly-rich motifs are detected mainly in Coleoptera. AlphaFold3 predictions suggested that AAP-repeat proteins are largely disordered, while Gly-rich proteins contain β-sheet-forming regions. [Consideration]AAP-repeat motifs are enriched in proline residues, suggesting increased chain flexibility, while their elastin-like sequence and structural characteristics may also indicate a potential contribution to elasticity. The lineage-biased distribution of Gly-rich motifs containing proteins, along with their potential to form β-sheet structures, suggests that they may contribute to structural strength and thus be involved in forming rigid cuticle in Coleoptera. Moreover, the motif-level shift observed between 0 h and 24 h suggests stage-dependent functional differentiation of low molecular-weight proteins during post-eclosion cuticle maturation. [Conclusion]Our results identify candidate genes and sequence features linked to post-eclosion elytral maturation in T. dichotomus, and highlight candidate cuticular proteins that may contribute to rigid cuticle formation. Importantly, these findings provide molecular-level insights into mechanisms that could inform future materials designs.