The mushroom body development and learning ability of adult honeybees are influenced by cold exposure during their early pupal stage.

The honeybees are the most important pollinator in the production of crops and fresh produce. Temperature affects the survival of honeybees, and determines the quality of their development, which is of great significance for beekeeping production. Yet, little was known about how does low temperature stress during development stage cause bee death and any sub-lethal effect on subsequent. Early pupal stage is the most sensitive stage to low temperature in pupal stage. In this study, early pupal broods were exposed to 20°C for 12, 16, 24, and 48 h, followed by incubation at 35°C until emergence. We found that 48 h of low temperature duration cause 70% of individual bees to die. Although the mortality at 12 and 16 h seems not very high, the association learning ability of the surviving individuals was greatly affected. The brain slices of honeybees showed that low temperature treatment could cause the brain development of honeybees to almost stop. Gene expression profiles between low temperature treatment groups (T24, T48) and the control revealed that 1,267 and 1,174 genes were differentially expressed respectively. Functional enrichment analysis of differentially expressed genes showed that the differential expression of Map3k9, Dhrs4, and Sod-2 genes on MAPK and peroxisome signaling pathway caused oxidative damage to the honeybee head. On the FoxO signal pathway, InsR and FoxO were upregulated, and JNK, Akt, and Bsk were downregulated; and on the insect hormone synthesis signal pathway, Phm and Spo genes were downregulated. Therefore, we speculate that low temperature stress affects hormone regulation. It was detected that the pathways related to the nervous system were Cholinergic synapse, Dopaminergic synapse, GABAergic synapse, Glutamatergic synapse, Serotonergic synapse, Neurotrophin signaling pathway, and Synaptic vesicle cycle. This implies that the synaptic development of honeybees is quite possibly greatly affected by low temperature stress. Understanding how low temperature stress affects the physiology of bee brain development and how it affects bee behavior provide a theoretical foundation for a deeper comprehension of the temperature adaptation mechanism that underlies the "stenothermic" development of social insects, and help to improve honeybee management strategies to ensure the healthy of colony.

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°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.