Animals have developed numerous strategies to contend with environmental pressures. We observed that the same adaptation strategy may be used repeatedly by one species in response to a certain environmental challenge. The ladybird Harmonia axyridis displays thermal phenotypic plasticity at different developmental stages. It is unknown whether these superficially similar temperature-induced specializations share similar physiological mechanisms. We performed various experiments to clarify the differences and similarities between these processes. We examined changes in the numbers and sizes of melanic spots in pupae and adults, and confirmed similar patterns for both. The dopamine pathway controls pigmentation levels at both developmental stages of H. axyridis. However, the aspartate-β-alanine pathway controls spot size and number only in the pupae. An upstream regulation analysis revealed the roles of Hox genes and elytral veins in pupal and adult spot formation. Both the pupae and the adults exhibited similar morphological responses to temperatures. However, they occurred in different body parts and were regulated by different pathways. These phenotypic adaptations are indicative of an effective thermoregulatory system in H. axyridis and explains how insects contend with certain environmental pressure based on various control mechanisms.

The ladybird Harmonia axyridis is an insect that exhibits pupal attachment to plants, which facilitates development and environmental adaptation. The cremaster is highly specialized for this behavior. However, the underlying molecular regulation of the cremaster remains unclear; therefore, we performed experiments to investigate the transcriptional regulation of cremaster development. First, we examined the morphological structure of the cremaster to reveal its function in pupal attachment of H. axyridis. Next, we analyzed the Hox gene Ha-Abd-B using RNA interference (RNAi) to determine its function in regulating cremaster formation; Ha-Abd-B up-regulation promoted effective pupal attachment, whereas successful RNAi caused severe down-regulation of this gene, and pupae were unable to attach. Furthermore, successful RNAi and subsequent Ha-Abd-B down-regulation caused phenotypic changes in cremaster structure, including its complete disappearance from some individuals. Finally, we observed unique development of the cremaster and dynamic expression of Ha-Abd-B during pre-pupal development; consequently, we hypothesized that there was specific pre-pupal development of the cremaster. Overall, based on these results, the specialized cremasteric structure located on the posterior side of H. axyridis was determined to be a key organ for pupal attachment. Cremaster identification in H. axyridis is regulated by Ha-Abd-B and exhibits preferential development. Pupal attachment of H. axyridis reveals an environmental adaptation of this species; thus, this study and future molecular studies will help determine the role of Hox genes in regulation of insect attachment and further our understanding of the multiple functions of Hox genes.