35℃ is considered the optimal temperature for honeybee development, and our previous studies have shown through tissue sections that brain development of new pupae stagnates after exposure to cold stress, and learning and memory are impaired in adults [20]. However, the physiological processes and molecular mechanisms behind this phenomenon of cold-stress effect have not been thoroughly investigated. With this aim in mind, we analyzed the trends of DEGs in the pupae heads of white-eyed pupae with or without cold treatment, suggesting that ER stress-associated cell apoptosis may be the key factor contributing to brain developmental defects during cold exposure. At the same time, we found that cold stress may inhibit the synthesis of ecdysteroids in the pupae head and interfere with epigenetic regulation associated with brain development, which are also possible factors for the inhibition of pupae brain development. We also found that DEGs associated with neuronal differentiation and neuronal damage repair showed an up-regulation trend during cold stress, suggesting a potential repair mechanism for pupae in response to cold stress. This study also lays the groundwork for the subsequent functional validation of genes related to brain development.
Accumulation of misfolded proteins exacerbates endoplasmic reticulum (ER) stress-induced cell apoptosis
RNF5 and SVIP, which were identified in Profile 1 and annotated in protein processing in endoplasmic reticulum pathways, are involved in endoplasmic reticulum-associated protein degradation (ERAD). The ubiquitin/protease system is responsible for clearing misfolded proteins during protein synthesis through various physiological steps catalyzed by E3 ubiquitin ligase[24]. RNF5 encodes E3 ubiquitin-protein ligase RNF5 and is involved in degrading misfolded proteins within the endoplasmic reticulum [25, 26]. SVIP, on the other hand, encodes the Small VCP/p97 interacting protein (SVIP) in Drosophila, serving as an interaction cofactor that regulates protein quality control and responds to cellular stress [27]. The highly conserved VCP/p97 in eukaryotic cells binds to SVIP, recognizing ubiquitinated proteins and transferring them from the endoplasmic reticulum to the cytosol for degradation [28–30]. The expression levels of RNF5 and SVIP downregulated in response to cold stress, indicating that the degradation of misfolded proteins in the endoplasmic reticulum of head tissue cells was inhibited, leading to the accumulation of misfolded proteins (Figure 6).
The downregulation of FBXO32 in Profile 1 could play a crucial role in regulating cell apoptosis induced by endoplasmic reticulum stress (ER stress). The build-up of misfolded proteins in the endoplasmic reticulum results in endoplasmic reticulum stress, which triggers unfolded protein responses (UPR) [31, 32]. Adverse environmental stress can induce endoplasmic reticulum stress, which triggers unfolded protein responses (UPR) [33]. This activation of the IRE1 pathway in Apis mellifera worker cells leads to the splicing of XBP1u mRNA, turning it into XBP1s mRNA. As a result, XBP1s are produced and enter the cell nucleus to facilitate the generation of chaperones and other folding-promoting factors [34]. If the stress intensity exceeds a certain threshold or persists for an extended period, ER stress will ultimately result in cell apoptosis [35]. Additionally, XBP1s has been shown to enhance the synthesis of the CHOP transcription factor [36–38], thereby exacerbating honeybee cell apoptosis [39]. FBXO32 encodes a ubiquitin-E3 ligase that plays a role in regulating cellular autophagy and maintaining endoplasmic reticulum homeostasis [40]. Mutations in FBXO32 result in increased CHOP expression and worsen cell apoptosis induced by ER stress [41]. In this study, FBXO32 were downregulated in Profile 1, annotated in the FoxO signalling pathway. It is hypothesized that cold exposure will induce ER stress in the brain cells of new pupae, resulting in elevated apoptosis and hindrance to normal pupal brain development (Figure 6).
The downregulation of the UBL5, annotated in the thermogenesis pathway, could be another factor that intensifies ER-stress-associated brain cell apoptosis. UBL5 is known to play a crucial role in the organisms’ stress response [42]. UBL5 encode Ubiquitin-like protein 5 (UBL5) is not only involved in the response to unfolded protein reactions in cell mitochondria [43] but it has also been implicated in ER stress-induced cell apoptosis. The PERK pathway plays a role in the cellular response to unfolded protein responses, as demonstrated in a series of studies [44–46]. Additionally, this pathway is a crucial UPR regulatory pathway present in insects [47, 48]. When unfolded protein reactions take place in the endoplasmic reticulum, the cellular PERK pathway plays a crucial role in degrading the UBL5 protein, ultimately resulting in heightened cell apoptosis [49]. The study indicated a downward trend in the expression of UBL5, indicating that, alongside the IRE1 pathway, cold stress can also trigger the activation of the PERK pathway, leading to increased apoptosis in brain cells of honeybee pupae, and may potentially inhibit brain development (Figure 6).
PET191 encodes a protein that plays a role in the assembly of cytochrome oxidase [50, 51], an enzyme involved in the mitochondrial electron transport chain. Cytochrome oxidase is important for neuronal metabolic activity and is considered a marker of neuronal function and size [52]. Inhibition of cytochrome oxidase activity in neuronal mitochondria can exacerbate endoplasmic reticulum stress-induced neuronal apoptosis [53]. PET191 were downregulated in Profile 1, annotated in the thermogenesis pathway, suggesting that it could be a crucial factor in the regulation of apoptosis in honeybee pupae brain cells (Figure 6).
Inhibition of ecdysteroids synthesis in pupae head
Multiple members of the cytochrome P450 gene family play a role in regulating ecdysteroid production, and their presence in Profile 1 implies that cold stress could potentially impede the synthesis of ecdysteroids. Ecdysone and 20-hydroxyecdysone are important hormones in insect moulting, collectively known as ecdysteroids. The brain is the main site for ecdysone synthesis in honeybee workers, with the conversion to 20-hydroxyecdysone occurring mainly in the brain and fat body tissue [54]. The CYP306A1 enzyme is crucial for ecdysone biosynthesis, as it catalyzes the conversion of ketodiol to ketotriol [55]. Insects' development process is hindered by the reduction in CYP306A1 expression level. Silencing the CYP306A1 gene expression in third instar larvae of Chilo suppressalis resulted in a significant decrease in pupation rate and pupal weight [56]. The CYP307B1 enzyme is also important for ecdysone biosynthesis [57, 58], catalyzing the conversion of 2-deoxydecanone to 3-deoxydecanone [59]. In this study, the downregulated genes CYP306A1 (phm) and CYP307B1 (spookiest) were enriched in the KEGG signalling pathway of insect hormone biosynthesis (P = 0.027, Q = 0.484). It is suggested that cold stress reduces the activity of CYP306A1 and CYP307B1 enzymes, leading to inhibition of ecdysteroid synthesis in the honeybee pupal head.
Epigenetic regulation in brain development
The gene LSD1, which is downregulated in Profile 1, is believed to play a crucial role in regulating the development of the worker pupal brain. LSD1 is involved in epigenetic regulation and acts as a lysine-specific demethylase targeting histones H3K4 and H3K9, as well as non-histone substrates, to either inhibit or activate transcription and regulate gene expression [60]. Studies have shown that LSD1 is important for various aspects of brain development [61] and that mutations in LSD1 can lead to paralysis in adult mice, extensive neuronal death in the hippocampus and cortex, and learning and memory disorders [62]. In this study, the expression of LSD1 decreased with prolonged exposure to cold stress. Our team plans to further investigate the role of LSD1 in honeybee pupal brain development by using RNAi methods to confirm the relationship between LSD1 downregulation and inhibition of brain development.
Neurodevelopment & neuronal repair
The DEGs identified in Profile 6 were found to be significantly enriched in three KEGG signalling pathways: apoptosis, axon regeneration, and signalling pathways regulating pluripotency of stem cells (Q < 0.05). The upregulated gene EIP93F, involved in cell apoptosis, has been shown to regulate the development of the Drosophila nervous system, promote autophagy of mushroom body neurocytes, and facilitate the remodelling process of the mushroom body during abnormal development [63]. Additionally, the upregulated gene EPHB1, present in the axon regeneration pathway, enhances the expression of damaged motor neurons to initiate neuronal repair [64]. Previous research by our team revealed that 70% of bee individuals subjected to 48 h of cold treatment died before completing eclosion [20]. It is speculated that the head tissue development of honeybee worker bees continues to progress even under cold stress. There seems to be a mechanism in place for repairing neural tissue damage, which helps counteract the abnormal apoptosis of head tissue cells caused by the stress. Profile 6 cluster shows an upregulate trend initially, then followed by a plateau phase suggesting a limited ability for damage repair. This aligns with the observation that only 70% of pupae successfully emerge after undergoing 48 hours of cold treatment during the pupal stage.