Replicated Differential Expression Analysis in a Green‐Brown Polymorphic Grasshopper Reveals Role of Beta‐Carotene‐Binding Protein in Body Coloration

Orthoptera provide a well-documented case of transspecies colour polymorphism, with green and brown morphs coexisting in many species. This colour polymorphism is likely under long-term balancing selection, but the genetic and molecular mechanisms underlying the variation remain poorly understood. Here, we used transcriptome data alongside a novel chromosome-level assembly to perform differential gene expression analysis in the club-legged grasshopper Gomphocerus sibiricus (Caelifera: Acrididae: Gomphocerinae), aiming to identify the specific genes involved in the differentiation between green and brown morphs. Since differential expression analyses are prone to false positives, we replicated the analysis using an independent sample of individuals of the same species. We found six genes consistently upregulated in green individuals across both datasets, all annotated as beta-carotene-binding proteins (βCBPs). βCBPs are known to play a key role in the colour regulation in both the migratory locust Locusta migratoria and the desert locust Schistocerca gregaria, although their exact role may differ in the club-legged grasshopper. The gene tree and chromosomal positions of βCBP copies in G. sibiricus, L. migratoria and S. gregaria indicate both ancestral (pre-speciation) and lineage-specific duplications. Our screening of publicly available orthopteran genomes revealed that homologues of the βCBP genes are largely absent from non-Caelifera species when using conservative homology thresholds. This restricted distribution suggests that βCBP-mediated pigmentation may represent a Caelifera-specific mechanism that is involved in the production of green body coloration, while other orthopteran lineages likely rely on distinct genetic pathways. Together, our findings provide new insights and lay the groundwork for understanding the evolutionary diversification of pigmentation mechanisms in Orthoptera.