In honey bees, reproductive division of labor is maintained by social suppression of worker fertility, yet queen loss can trigger ovary activation in workers. Here, we tested whether endogenous dopaminergic signaling is progressively remodeled across successive stages of ovarian activation and how these changes relate to key hormone pathways. Newly emerged Apis mellifera workers were introduced into queenright or queenless colonies, collected after 14 days, and classified as having inactive, partially activated, or fully activated ovaries. We quantified brain dopamine and measured expression levels of genes involved in dopamine synthesis, transport, metabolism, and reception in both brain and ovary tissues, together with transcriptional markers of juvenile hormone (JH) and 20-hydroxyecdysone (20E) signaling. Brain dopamine increased with ovary activation and peaked in fully activated workers, coincident with elevated transcripts of tyrosine hydroxylase, dopa decarboxylase, dopamine transporter, and arylalkylamine N-acetyltransferase in the brain. Dopamine receptor genes were stable in the brain but were remodeled in the ovary, with Amdop1 increasing and Amdop3 decreasing during activation. Markers of JH signaling and ovarian 20E pathway activity also rose with ovarian development, consistent with early endocrine priming following queen loss. Collectively, these results support an integrated neuroendocrine framework in which dopaminergic remodeling and hormone pathway activation jointly accompany worker reproductive activation under queenless conditions.

Temporal reproductive plasticity is a central life-history trait for most iteroparous animals, yet its underlying molecular mechanisms remain poorly understood. We utilize the reversible reproductive arrest experienced by honey bee queens during colony swarming to investigate how these reproductive specialists dynamically adapt their physiology. We demonstrate that pre-swarming nutritional restriction triggers a resource reallocation, whereby ovarian mass declines as flight muscle function is enhanced, providing empirical support for a flight-fecundity trade-off. This transition is accompanied by elevated juvenile hormone and suppressed ecdysteroid and vitellogenin levels, together with the engagement of three spatially distinct oogenesis checkpoints. These checkpoints engage programmed cell death pathways: autophagy predominates in germarial stem cells and follicle cells, while apoptosis is the primary mechanism in vitellarium oocytes and nurse cells, collectively orchestrating oogenesis arrest. Integrated whole-transcriptomic and metabolomic analyses revealed broad molecular remodeling, including changes in FoxO-, Notch-, Wnt-, and Hippo-related signatures that accompanied oogenesis suppression. Our results indicate a "nutrition-hormone-checkpoint-programmed cell death" model that links colony-level swarming to individual ovary suppression. This work provides a systematic framework for understanding the molecular regulation of the reversible reproductive plasticity of honey bee queens.

The ectoparasitic honeybee (Apis mellifera) mite Tropilaelaps mercedesae represents a serious threat to Asian apiculture and a growing concern for global beekeeping due to its high reproductive capacity and host adaptability. However, the regulatory mechanisms underlying its host adaptation across life stages remain poorly characterized. Here, we performed integrated transcriptomic, proteomic, and metabolomic analyses of female mites at 4 key postembryonic developmental stages: protonymphs, deutonymphs, mature adults, and reproductive adults. Our multi-omics approach reveals distinct stage-specific regulatory programs. Nymphal stages exhibit coordinated activation of chitin metabolism pathways for structural growth, neuroactive ligand-receptor interactions for neural maturation, and ATP-binding cassette (ABC) transporters for detoxification. Adult maturation involves enhanced membrane dynamics, improved energy metabolism, and increased antioxidant defenses to support reproductive capacity and environmental resilience. Reproductive adults prioritize lipid metabolism and vitellogenin synthesis to fuel embryonic development. Crucially, host adaptation emerges as a critical driver of this stage-specific regulation. Additionally, multi-omics integration highlights a developmental transition from transcriptional dominance in early stages to post-transcriptional regulation in later phases. These insights provide a systems-level understanding of the physiological orchestration enabling this parasite's ecological success, while identifying valuable molecular targets for formulating more effective strategies to control this formidable pest.

Cadmium (Cd), a pervasive heavy metal and potent environmental contaminant, threatens ecosystem integrity, yet its impacts on pollinating insects remain insufficiently characterized. In this study, we systematically investigated the consequences of larval-stage Cd exposure on honeybee (Apis mellifera) queens through integrated physiological, metabolomic, and proteomic analyses. Sublethal Cd exposure disrupted hormone balance, induced midgut epithelial degeneration, and impaired hemolymph lipid homeostasis, collectively retarding queen developmental progression and physiological maturation. Ovarian proteomic profiling further revealed the Cd-mediated suppression of key proteins involved in muscle contraction, tracheal function, neural conduction, and antioxidant defense, ultimately leading to diminished reproductive vitality. Critically, high-dose Cd exposure elicited transgenerational impairments: F1 workers showed reduced body size, lowered sucrose responsiveness, and impaired olfactory learning, while F1 queens developed smaller ovaries with fewer ovarioles. Together, these results demonstrate that Cd imposes substantial multigenerational fitness costs on honeybee colonies via both direct physiological impairment and transgenerational toxicity. Our findings underscore the urgency of mitigating environmental Cd pollution to protect pollinator health and ensure sustainable ecosystems.

Fenazaquin, a potent insecticide widely used to control phytophagous mites, has recently emerged as a potential solution for managing Varroa destructor mites in honeybees. However, the comprehensive impact of fenazaquin on honeybee health remains insufficiently understood. Our current study investigated the acute and chronic toxicity of fenazaquin to honeybee larvae, along with its influence on larval hemolymph metabolism and gut microbiota. Results showed that the acute median lethal dose (LD50) of fenazaquin for honeybee larvae was 1.786 μg/larva, and the chronic LD50 was 1.213 μg/larva. Although chronic exposure to low doses of fenazaquin exhibited no significant effect on larval development, increasing doses of fenazaquin resulted in significant increases in larval mortality, developmental time, and deformity rates. At the metabolic level, high doses of fenazaquin inhibited nucleotide, purine, and lipid metabolism pathways in the larval hemolymph, leading to energy metabolism disorders and physiological dysfunction. Furthermore, high doses of fenazaquin reduced gut microbial diversity and abundance, characterized by decreased relative abundance of functional gut bacterium Lactobacillus kunkeei and increased pathogenic bacterium Melissococcus plutonius. The disrupted gut microbiota, combined with the observed gut tissue damage, could potentially impair food digestion and nutrient absorption in the larvae. Our results provide valuable insights into the complex and diverse effects of fenazaquin on honeybee larvae, establishing an important theoretical basis for applying fenazaquin in beekeeping.

Ectoparasitic mites of the genera Varroa and Tropilaelaps have evolved to exclusively exploit honey bees as food sources during alternating dispersal and reproductive life history stages. Here we show that the primary food source utilized by Varroa destructor depends on the host life history stage. While feeding on adult bees, dispersing V. destructor feed on the abdominal membranes to access to the fat body as reported previously. However, when V. destructor feed on honey bee pupae during their reproductive stage, they primarily consume hemolymph, indicated by wound analysis, preferential transfer of biostains, and a proteomic comparison between parasite and host tissues. Biostaining and proteomic results were paralleled by corresponding findings in Tropilaelaps mercedesae , a mite that only feeds on brood and has a strongly reduced dispersal stage. Metabolomic profiling of V. destructor corroborates differences between the diet of the dispersing adults and reproductive foundresses. The proteome and metabolome differences between reproductive and dispersing V. destructor suggest that the hemolymph diet coincides with amino acid metabolism and protein synthesis in the foundresses while the metabolism of non-reproductive adults is tuned to lipid metabolism. Thus, we demonstrate within-host dietary specialization of ectoparasitic mites that coincides with life history of hosts and parasites.

The threat of neonicotinoids to insect pollinators, particularly honeybees (Apis mellifera), is a global concern, but the risk of chiral neonicotinoids to insect larvae remains poorly understood. In the current study, we evaluated the acute and chronic toxicity of dinotefuran enantiomers to honeybee larvae in vitro and explored the mechanism of toxicity. The results showed that the acute median lethal dose (LD50) of S-dinotefuran to honeybee larvae was 30.0 μg/larva after oral exposure for 72 h, which was more toxic than rac-dinotefuran (92.7 μg/larva) and R-dinotefuran (183.6 μg/larva). Although the acute toxicity of the three forms of dinotefuran to larvae was lower than that to adults, chronic exposure significantly reduced larval survival, larval weight, and weight of newly emerged adults. Analysis of gene expression and hormone titer indicated that dinotefuran affects larval growth and development by interfering with nutrient digestion and absorption and the molting system. Analysis of hemolymph metabolome further revealed that disturbances in the neuroactive ligand-receptor interaction pathway and energy metabolism are the key mechanisms of dinotefuran toxicity to bee larvae. In addition, melatonin and vitellogenin are used by larvae to cope with dinotefuran-induced oxidative stress. Our results contribute to a comprehensive understanding of dinotefuran damage to bees and provide new insights into the mechanism of enantioselective toxicity of insecticides to insect larvae.

Tropilaelaps mercedesae, an ectoparasitic mite of honeybees, is currently a severe health risk to Apis mellifera colonies in Asia and a potential threat to the global apiculture industry. However, our understanding of the physiological and developmental regulation of this pest remains significantly insufficient. Using ultra-high resolution mass spectrometry, we provide the first comprehensive proteomic profile of T. mercedesae spanning its entire post-embryonic ontogeny, including protonymphs, deutonymphs, mature adults, and reproductive mites. Consequently, a total of 4,422 T. mercedesae proteins were identified, of which 2,189 proteins were significantly differentially expressed (FDR < 0.05) throughout development and maturation. Our proteomic data provide an important resource for understanding the biology of T. mercedesae, and will contribute to further research and effective control of this devastating honeybee pest.

Aging, marked by a gradual decline in physiological functions, presents significant global health challenges. While royal jelly (RJ) is known for its biological activities, its anti-aging mechanisms require further investigation. Using a D-galactose (D-gal)-induced aging mouse model, this study applied untargeted serum metabolomics to explore RJ's effects. RJ administration significantly (P < 0.05) improved locomotor and cognitive abilities impaired by D-gal-induced aging. Metabolomic analysis showed that RJ modulates key pathways in amino acid, lipid, and nucleotide metabolism, which are essential for cellular function and energy supply. RJ enhanced the tricarboxylic acid cycle (P < 0.05), increased essential amino acids, and elevated nucleotide availability, supporting cognitive function and energy metabolism in aging mice. Notably, RJ had a stronger effect on male mice, particularly in improving lipid and nucleotide metabolism. These findings highlight RJ's potential as a promising anti-aging intervention, warranting further investigation for therapeutic applications in aging.

Environmental variation selects for the adaptive plasticity of maternal provisioning. Even though developing honeybees find themselves in a protected colony environment, their reproductively specialized queens actively adjust their maternal investment, even among worker-destined eggs. However, the potentially adaptive consequences of this flexible provisioning strategy and their mechanistic basis are unknown. Under natural conditions, we find that the body size of larvae hatching from small eggs in large colonies converges with that of initially larger larvae hatching from large eggs typically produced in small colonies. However, large eggs confer a persistent body size advantage when small and large eggs are cross-fostered in small and large colonies, respectively. We substantiate the increased maternal investment by identifying growth-promoting metabolomes and proteomes in large eggs compared to small eggs, which are primarily enriched in amino acid metabolism and cell maturation. Thus, our study provides a comprehensive adaptive explanation for the worker egg size plasticity of honeybees.

Egg size plasticity represents an adaptive reproductive strategy in numerous organisms, including the honey bee, Apis mellifera . However, the proximate causation of this plasticity and egg size in general is unknown. We show that honey bee queens predictably and reversibly adjust egg size in response to their colony size and that this plasticity is an active response to the queens’ perception of colony size instead of a consequence of egg laying rate. The egg size increase involves changes of 290 ovarian proteins, mostly related to increased energy metabolism, protein transport, and cytoskeleton functions. Spatio-temporal expression analysis of the small GTPase Rho1 indicates its central role in egg size regulation, which we confirm by RNAi-mediated gene knock-down and expression analyses. The molecular adjustments that promote maternal investment of honey bee queens in response to their social environment thus reveal a novel mechanism of egg size regulation.

10-Hydroxydec-2-enoic acid (10-HDA), an unsaturated hydroxyl fatty acid from the natural food royal jelly, can protect against cell and tissue damage, yet the underlying mechanisms are still unexplored. We hypothesized that the neutralization of the hydroxyl free radical (•OH), the most reactive oxygen species, is an important factor underlying the cytoprotective effect of 10-HDA. In this study, we found that the •OH scavenging rate by 10-HDA (2%, g/ml) was more than 20%, which was achieved through multiple-step oxidization of the –OH group and C=C bond of 10-HDA. Moreover, 10-HDA significantly enhanced the viability of vascular smooth muscle cells (VSMCs) damaged by •OH (P < 0.01), significantly attenuated •OH-derived malondialdehyde production that represents cellular lipid peroxidation (P < 0.05), and significantly increased the glutathione levels in •OH-stressed VSMCs (P < 0.05), indicating the role of 10-HDA in reducing •OH-induced cytotoxicity. Further proteomic analyses of VSMCs identified 195 proteins with decreased expression by •OH challenge that were upregulated by 10-HDA rescue and were primarily involved in protein synthesis (such as translation, protein transport, ribosome, and RNA binding) and energy metabolism (such as fatty acid degradation and glycolysis/gluconeogenesis). Taken together, these findings indicate that 10-HDA can effectively promote cell survival by antagonizing •OH-induced injury in VSMCs. To the best of our knowledge, our results provide the first concrete evidence that 10-HDA-scavenged •OH could be a potential pharmacological application for maintaining vascular health.

The eastern Apis cerana (Ac) and the western Apis mellifera (Am) are two closely related and most economically valuable honeybee species managed extensively worldwide. However, how worker bees of Ac and Am are adapted to their colony organization remains to be uncovered. Here, we found that the expression level of gene encoding antennae-specific proteins 1 (ASP1, a key regulator in recognizing queen mandibular pheromone) was positively correlated with the colony sizes in both bee species, and the expression level in Am was higher than that in Ac, suggesting that ASP1 may play an important role in maintaining colony homeostasis. Using competitive binding assay, molecular docking, and site-directed mutagenesis, we then confirmed the good binding affinities of both Ac-ASP1 and Am-ASP1 to methyl p-hydroxy benzoate (HOB), and Val115 was the key amino acid. However, the affinity of Am-ASP1 was stronger than that of Ac-ASP1. EAG analysis further demonstrated that antennae of Am worker bees had faster depolarization and repolarization in response to HOB stimulation. Taken together, these findings indicate that the differences in expression levels and binding dynamics allow ASP1 recognizing HOB to potentially serve as a specific regulator of colony organization in Ac and Am.

【Objective】The objective of this study is to investigate the proteome profile of the postcereberal gland (PGld) and thoracic gland (ThGld) between high royal jelly producing bees (Apis mellifera liguatica, RJBs) and Italian bees (Apis mellifera liguatica, ITBs) with the aim of revealing the molecular basis of salivary gland regulating royal jelly production, and to provide a basis for analyzing the high-yield mechanism of royal jelly.【Method】PGld and ThGld were dissected from nurse bees of RJBs and ITBs. After protein extraction and enzyme digestion, the peptide samples were analyzed by liquid chromatography coupled with tandem mass spectrometry. Furthermore, the mass spectral data were qualified and quantified by MaxQuant software, and the following bioinformatic analysis was conducted using Perseus software, prediction of secretory protein was achieved by SignalP database, biological process and KEGG pathway were enriched by Cluego software.【Result】Totally 2 335 proteins were identified in salivary glands of RJBs and ITBs nurse bees, including 1 823 proteins in PGld and 1 922 proteins in ThGld. The expression profiles of the core proteins in the PGld and ThGld of RJBs and ITBs were similar, mainly involved in RNA metabolism, nucleic acid metabolism, ATP metabolism, protein translation, translation regulation and catabolism. The principal component analysis (PCA) showed that the molecular basis of salivary gland of RJBs and ITBs had exerted varying extent of differentiation during the selective breeding. Quantitatively, the PGld of ITBs and RJBs expressed 254 and 333 up-regulated proteins, respectively, corresponding to the small molecule and carbohydrate metabolism pathway in ITBs, and the organic nitrogen compound synthesis, cell redox homeostasis, amino acid metabolism in RJBs. Those proved that the protein synthesis, amino acid metabolism and energy supply of salivary gland cells in RJBs were more active than ITBs. In the same way, the up-regulated expressions of 412 and 162 proteins were detected in the ThGld of ITBs and RJBs, respectively, which involved in the pathways of oxidative phosphorylation, translation regulation in ITBs, and oxidative phosphorylation, response to toxic substances in RJBs, indicating that the level of resistance of RJBs ThGld cells was increased. A total of 43 secretory proteins were identified in the salivary glands of RJBs and ITBs, in which 15 were detected in royal jelly. Major royal jelly proteins 1, 2, 3, 4, 5 and 7 were detected in PGld and ThGld, indicating that both of the glands were involved in the synthesis of royal jelly main protein. The identification of α-glucosidase related to nectar transformation and odorant binding proteins 3, 13, 17 and 21 involved in the synthesis and release of chemical pheromones in both PGld and ThGld indicated their basic function of nectar transformation and pheromone synthesis. The enhanced expression of major royal jelly proteins 1, 2, 3 and 7, hexamerin 70a and 110, odorant binding proteins 3, 13, 17 and 21, transferrin and apolipophorin-III-like protein in the salivary gland of RJBs demonstrated that the synthesis of pheromone and royal jelly protein was stronger than ITBs.【Conclusion】The core proteome with similar pattern in salivary glands of RJBs and ITBs ensures the synthesis and secretion of royal jelly protein, pheromone and invertase. Molecular variation between the salivary glands of RJBs and ITBs was developed after long-term selective breeding. Relative to ITBs, the salivary glands of RJBs enhance the ability of protein synthesis, amino acid metabolism, cell energy supply and stress resistance, and upregulate the expression of most secretory proteins, which benefits to a better protein synthesis system with promising efficiency and lasting, and contributes to the high producing of royal jelly.

(接2022年第3期) 3.2 不同地区和不同蜜粉源植物样品理化指标分析 蜂王浆的成分会因为地理、蜜粉源植物、蜂种、温度和降水量等因素的影响而存在一定差异[25,26].我们按不同地区(图2左侧)和不同蜜粉源植物(图2右侧)对所有样品的理化指标进行了分析.四个地区(图2A左)和不同蜜粉源植物(图2A右)蜂王浆的含水量之间没有显著差异(P＞0.05).从图2A左图的圆圈分布能看出,中西北部、中部和东南部地区样品的水分含量相对集中,而东北部样品的含水量差异较大.而不同蜜粉源植物种中,洋槐和椴树的含水量数值分布差异较大.

Reproduction involves the investment of resources into offspring. Although variation in reproductive effort often affects the number of offspring, adjustments of propagule size are also found in numerous species, including the Western honey bee, Apis mellifera . However, the proximate causes of these adjustments are insufficiently understood, especially in oviparous species with complex social organization in which adaptive evolution is shaped by kin selection. Here, we show in a series of experiments that queens predictably and reversibly increase egg size in small colonies and decrease egg size in large colonies, while their ovary size changes in the opposite direction. Additional results suggest that these effects cannot be solely explained by egg-laying rate and are due to the queens’ perception of colony size. Egg-size plasticity is associated with quantitative changes of 290 ovarian proteins, most of which relate to energy metabolism, protein transport, and cytoskeleton. Based on functional and network analyses, we further study the small GTPase Rho1 as a candidate regulator of egg size. Spatio-temporal expression analysis via RNAscope and qPCR supports an important role of Rho1 in egg-size determination, and subsequent RNAi-mediated gene knockdown confirmed that Rho1 has a major effect on egg size in honey bees. These results elucidate how the social environment of the honey bee colony may be translated into a specific cellular process to adjust maternal investment into eggs. It remains to be studied how widespread this mechanism is and whether it has consequences for population dynamics and epigenetic influences on offspring phenotype in honey bees and other species.

Behavioral specialization is key to the success of social insects and leads to division of labor among colony members. Response thresholds to task-specific stimuli are thought to proximally regulate behavioral specialization, but their neurobiological regulation is complex and not well understood. Here, we show that response thresholds to task-relevant stimuli correspond to the specialization of three behavioral phenotypes of honeybee workers in the well-studied and important Apis mellifera and Apis cerana. Quantitative neuropeptidome comparisons suggest two tachykinin-related peptides (TRP2 and TRP3) as candidates for the modification of these response thresholds. Based on our characterization of their receptor binding and downstream signaling, we confirm a functional role of tachykinin signaling in regulating specific responsiveness of honeybee workers: TRP2 injection and RNAi-mediated downregulation cause consistent, opposite effects on responsiveness to task-specific stimuli of each behaviorally specialized phenotype but not to stimuli that are unrelated to their tasks. Thus, our study demonstrates that TRP signaling regulates the degree of task-specific responsiveness of specialized honeybee workers and may control the context specificity of behavior in animals more generally.

Although the antimicrobial, nutritional, and health-promoting properties of royal jelly (RJ) have been widely confirmed, the effects of storage temperature and time on RJ quality remain to be further explored. Herein, the antimicrobial and proteomic dynamics of RJ stored under different conditions were comprehensively investigated to identify consistent and sensitive markers of RJ degradation. We confirmed the negative correlation between antimicrobial properties and increased the storage temperature and duration in RJ. Using surface plasmon resonance, we showed the protein degradation-induced conformation changes in RJ, which reflected the overall variation in RJ proteins caused by the storage conditions. Further proteomic and western blotting analyses demonstrated the sensitivity and reliability of major RJ protein 4 (MRJP4) as a measure of temperature- and time-dependent RJ changes. Based on these results, we developed a colloidal gold immunoassay strip for MRJP4 detection, providing a reliable, simple, and rapid method for the evaluation of RJ freshness.

本研究旨在利用昆虫细胞-杆状病毒表达系统表达蜂王浆主蛋白2(MRJP2),为后续MRJP2功能的深入研究提供材料.根据GenBank中意大利蜜蜂(Apis mellifera L.)MRJP2基因序列,经PCR扩增、克隆至真核表达载体pFastBac1,构建重组杆状病毒质粒MRJP2-Bacmid并转染至Sf9昆虫细胞,以P2代杆状病毒感染Sf9细胞进行诱导表达,利用层析柱和离子交换柱对表达产物进行纯化,并通过SDS-PAGE、Western blotting及四极杆静电场轨道阱高分辨质谱仪(Q Exactive)对目的蛋白进行分析验证.结果显示,本试验成功构建杆状病毒质粒MRJP2-Bacmid,转染至Sf9昆虫细胞并获得表达产物.SDS-PAGE和Western blotting验证结果表明,本研究成功利用昆虫细胞-杆状病毒表达系统表达出大小约为52 ku的MRJP2重组蛋白,且纯度较高;质谱分析结果显示,该重组蛋白匹配到蜜蜂蛋白质数据库中MRJP2的特异性肽段为39个,序列覆盖率为61％,且与MRJP2的匹配得分最高,进一步确认该重组蛋白为MRJP2.本研究利用昆虫细胞-杆状病毒表达系统成功表达出MRJP2,为后续该蛋白生物学功能的深入研究奠定了基础.