CCAP receptor 2, rather than CCAPR1, functions in pupal ecdysis in the 28-spotted larger potato ladybird

Crustacean cardioactive peptide receptor (CCAPR), a G protein-coupled receptor, is critical for pupal ecdysis in Drosophila melanogaster and Acyrthosiphon pisum, both of which possess a single ccapr gene. In this study, we identified two ccapr genes in Henosepilachna vigintioctomaculata, a potato-defoliating ladybird widely distributed in Asia, and found that the two receptors have diverged functionally in their requirement for pupal ecdysis. Bayesian phylogenetic analysis recovered well-supported within-lineage relationships and placed HvCCAPR1 and HvCCAPR2 as a sister pair within the coleopteran clade. Spatiotemporal expression analysis showed that Hvccapr2 was upregulated immediately before the onset of pupal ecdysis and exhibited a stronger bias toward neural and muscular tissues. Injection of in vitro-synthesized dsRNA or oral administration of bacterially expressed dsRNA targeting the common region shared by both genes, as well as dsccapr2 but not dsccapr1, impaired larval-pupal transition. Moreover, knockdown of Hvccapr1 + Hvccapr2 or Hvccapr2, but not Hvccapr1, markedly prolonged the durations of ecdysis and postecdysis behavioral sequences. As a consequence, most Hvccapr1 + Hvccapr2 and Hvccapr2 RNAi prepupae failed to shed the old larval cuticle. In contrast, pupal cuticles beneath the retained larval exoskeleton became pigmented in developmentally arrested Hvccapr1 + Hvccapr2 and Hvccapr2 RNAi beetles. Together, these findings demonstrate that CCAPR2, rather than CCAPR1, plays the predominant role in coordinating pupal ecdysis behavior in H. vigintioctomaculata.