Transcriptome-based screening of regulator genes for sexual dimorphism in pupae of Callipogon relictus

Sexual dimorphism in insects arises from morphological, physiological, and behavioral differences that often reflect reproductive strategies. However, its molecular basis remains poorly understood in non-model and endangered species.

To elucidate the molecular mechanisms underlying sexual dimorphism in Callipogon relictus-a long-horned beetle of high conservation importance.

We conducted a transcriptome-wide analysis of sex-biased gene expression in pupal tissues of C. relictus. RNA sequencing was performed on six pupal tissues (mandible, maxillary palpus, foreleg, midleg, elytra, and hindwing) from both sexes. Differentially expressed genes were defined using an absolute log₂ fold change > 1 and raw p-value < 0.05. For enrichment analyses, Benjamini-Hochberg false discovery rate (FDR) correction was applied. Throughout the manuscript, p-value refers to unadjusted values, and FDR indicates corrected values. Functional annotation and Gene Ontology enrichment were performed using Drosophila melanogaster orthologs, and conserved sex-biased genes were identified.

Overall, 2,913 sex-biased genes were detected in these tissues. Male tissues were enriched in cuticle formation, chitin metabolism, immune defense, and cell cycle regulation, consistent with the development of enlarged mandibles and reinforced cuticles. Female tissues were enriched in metabolic processes, hormone regulation, and muscle development, reflecting reproductive and flight-related investments. A conserved set of 33 male-biased and 14 female-biased genes was shared across tissues. The key male-biased genes were linked to juvenile hormone biosynthesis, chemosensory signaling, and cuticle reinforcement. The key female-biased genes are associated with oogenesis and hormonal metabolism.

These findings reveal the tissue-specific and conserved regulators of sexual dimorphism in C. relictus; they provide a foundation for functional studies and support conservation strategies, including captive rearing and sustainable management, for this endangered beetle.