Fig. 3: Molecular mechanism of femoral morphogenesis for plant-mimicking camouflage.

A Photos of T2 and T3 legs across the developmental stage. Scale bar = 1 mm. B Gene family analysis showing that the Cuticle genes were significantly expanded in the genomes of H. coronatus and D. lobata. C Heatmaps of the differentially expressed genes in the leg tissues across the developmental stages. The red and blue colors indicate high and low expression levels, respectively. D Expression level analysis of the Arm gene at day 3 after two injections of Arm double-stranded RNA (dsArm). Ctrl, control group. Data represent the mean ± standard deviation of five replicates. *p = 0.02 by two-sided Student’s t tests. E Comparison of the size of ‘petal-like’ femoral lobes (the red dotted area) between the dsArm and control groups. The numbers indicate the areas of these expansions. F Gene Ontology (GO) enrichment of differentially expressed genes with FDR-corrected p value < 0.05 and |log2-fold-change | > 1 in the legs between the dsArm and control groups. The enriched GO terms with corrected p < 0.05 determined by Fisher’s exact test are presented. The size of the circles represents the number of differentially expressed genes for each GO term. The red and cyan colors represent the molecular function and biological process categories, respectively. G Bubble plot showing the expression levels of cuticle mRNAs between the dsArm and control groups. H Reconstruction of gene regulatory pathways related to femoral remodeling. The red and blue colors represent significantly up- and downregulated genes in the leg tissues of the fifth-instar larvae compared to the first-instar larvae. co, coxa. tr, trochanter. fe, femur. ti, tibia. ts, tarsus.