بررسی روابط پلیوتروپیک ویتلوژنین در شبکه پاسخ به تنش زنبور عسل با استفاده از رویکرد تحلیل شبکه (PPI)

Introduction
The honey bee (Apis mellifera) is a cornerstone of global biodiversity and agricultural stability, serving as a primary pollinator for a vast array of crops. However, the survival and productivity of honey bee colonies are increasingly threatened by escalating environmental challenges, most notably thermal stress and oxidative pressure. These stressors trigger complex molecular responses that aim to maintain cellular homeostasis and prevent damage to proteins and membranes. Rather than acting through isolated, independent genes, the honey bee’s physiological resilience is orchestrated by an intricate, multi-layered network of interacting proteins. A key player in this molecular landscape is vitellogenin (Vg). Historically categorized primarily as a yolk precursor protein, Vg has recently been recognized as a highly pleiotropic molecule that regulates several critical physiological processes, including immunity, longevity, metabolic homeostasis, and redox balance. Given its widespread involvement in diverse biological functions, Vg is hypothesized to act as a central integrator within the stress response network. However, the precise network-level role of Vg and its interactions with key antioxidant and immune-related proteins have not been comprehensively mapped. This study was designed to bridge this knowledge gap by employing a systems biology approach, specifically protein-protein interaction (PPI) network analysis, to elucidate the structural and functional architecture of the honey bee stress response and to characterize the topological role of Vg as a potential network mediator.
Method
To construct a representative model of the honey bee stress response, a curated dataset of twenty-five candidate genes was selected based on an extensive review of existing literature, ensuring comprehensive coverage of four fundamental physiological axes: antioxidant defense (e.g., superoxide dismutases), heat-shock response (chaperones), innate immunity (antimicrobial peptides), and metabolic-hormonal regulation (signaling pathways). Subsequently, the protein-protein interaction (PPI) network was constructed using the STRING database (version 12.0), applying high-confidence interaction thresholds to minimize false-positive links and ensure the reliability of the derived interactome. The resulting network was then visualized and analyzed with Cytoscape (version 3.10.4), and to quantify the importance of specific nodes within this network, several topological metrics were computed using the CytoNCA plugin, including Degree Centrality for identifying highly connected hubs, Betweenness Centrality for pinpointing bottleneck nodes that control communication flow between functional modules, Closeness Centrality for assessing the efficiency of information propagation from a specific protein, and Eigenvector Centrality for evaluating a node’s influence based on its neighbors’ connectivity. Furthermore, functional modules, representing densely connected protein complexes, were identified within the network using the MCODE algorithm. Finally, to validate the biological relevance of the observed network topology, comprehensive functional enrichment analyses were performed utilizing Gene Ontology (GO) for both biological processes and molecular functions, in conjunction with pathway analyses via the KEGG and Reactome databases, with statistical significance for enrichment being defined at an FDR (False Discovery Rate) of < 0.05.
Results
The analysis revealed that the honey bee stress response network has a highly organized, modular topology. The network is not a collection of random interactions but is instead structured into four distinct, yet interrelated, functional modules: (1) the Antioxidant/Redox Module, containing key enzymes like Sod1, Sod2, Trx-2, Gtpx2, and GstD1; (2) the Heat-Shock/Proteostasis Module, involving chaperones such as Hsp90 and Hsc70-4; (3) the Innate Immunity Module, comprising antimicrobial peptides and signaling components like Def1 and ABAE-APIME; and (4) the Metabolic-Hormonal Module, featuring regulatory proteins such as Akt1, Tor, and Vg. Topological metrics identified several critical hub genes that maintain the structural integrity of the network. Specifically, Sod1, Trx-2, and Sod2 exhibited the highest degree centrality (16) and were identified as the principal network hubs. Their high connectivity underscores the paramount importance of antioxidant enzymes in the honey bee’s defense against oxidative stress. Crucially, the analysis identified vitellogenin (Vg) as the most significant regulatory bridge in the entire network. While it did not have the highest degree, Vg exhibited an exceptionally high Betweenness Centrality of 80.4931, identifying it as a key bottleneck node. This high betweenness indicates that Vg is strategically positioned to mediate communication between the disparate functional modules (antioxidant, immune, and metabolic). In the Vg-centered subnetwork analysis, Vg exhibited Degree Centrality of 11 and Closeness Centrality of 1.0000, confirming its role as a highly accessible and central coordinator. The Vg subnetwork demonstrated direct protein-protein interactions with 11 unique neighbors, representing a cross-section of all four functional axes, thereby confirming its pleiotropic role in integrating stress-related signals. Functional enrichment results provided strong biological validation for these topological findings. GO Biological Process analysis was significantly enriched for terms such as cellular response to oxidative stress, response to heat, and regulation of metabolic processes. GO Molecular Function analysis highlighted antioxidant enzyme activity and chaperone-related functions. Furthermore, KEGG and Reactome pathway analyses identified significant enrichment in the Insulin/TOR signaling pathway and redox homeostasis pathways, highlighting the critical connection between energy metabolism (via Akt1 and Tor) and the stress response (via Vg).
Conclusions
This study provides a comprehensive systems-level view of the honey bee stress response, demonstrating that it is a highly coordinated, multi-modular process. The results indicate that the antioxidant defense system, led by hubs such as Sod1 and Sod2, forms the structural core of the network. At the same time, vitellogenin (Vg) serves as the essential physiological bridge integrating antioxidant, immune, and metabolic regulatory systems. The identification of Vg as a high-betweenness node suggests that it is a master regulator capable of synchronizing different defensive modules to ensure colony-wide resilience. These findings have significant implications for honey bee biology and management; specifically, Sod1, Sod2, and Vg represent high-priority candidate molecular markers for breeding programs aimed at developing more resilient bee lineages. However, it is important to note that our results are derived from a prediction-based PPI analysis. While the topological evidence is robust, future research must prioritize in vivo experimental validation to confirm the specific biochemical interactions and functional consequences of these hubs under diverse environmental stress conditions.