WGCNA based identification of hub genes associated with cold response and development in Apis mellifera metamorphic pupae

Honeybee is a crucial pollinator in nature, and plays an indispensable role in both agricultural production and scientific research. In recent decades, honeybee was challenged with health problems by biotic and abiotic stresses. As a key ecological factor, temperature has been proved to have an impact on the survival and production efficiency of honeybees. Previous studies have demonstrated that low temperature stress can affect honeybee pupation and shorten adult longevity. However, the molecular mechanism underlying the effects of low temperatures on honeybee growth and development during their developmental period remain poorly understood. In this paper, the weighted gene co-expression analysis (WGCNA) was employed to explore the molecular mechanisms underpinnings of honeybees' respond to low temperatures (20 degrees C) during four distinct developmental stages: large-larvae, prepupae, early-pupae and mid-pupae. Through an extensive transcriptome analysis, thirteen gene co-expression modules were identified and analyzed in relation to honeybee development and stress responses. The darkorange module was found to be associated with low temperature stress, with its genes primarily involved in autophagy-animal, endocytosis and MAPK signaling pathways. Four hub genes were identified within this module, namely, loc726497, loc409791, loc410923, and loc550857, which may contribute to honeybee resistance to low temperature and provide insight into the underlying mechanism. The gene expression patterns of grey60 and black modules were found to correspond to the developmental stages of prepupae and early-pupae, respectively, with the hub genes loc409494, loc725756, loc552457, loc726158, Ip3k and Lcch3 in grey60 module likely involved in brain development, and the hub genes loc410555 in black module potentially related to exoskeleton development. The brown module genes exhibited a distinct pattern of overexpression in mid-pupae specimens, with genes primarily enriched in oxidative phosphorylation, citrate cycle and other pathways, which may be related to the formation of bee flying muscle. No related gene expression module was found for mature larvae stage. These findings provide valuable insights into the developmental process of honeybees at molecular level during the capped brood stage.