IntroductionHeavy metal pollution poses a persistent global threat to pollinator health and biodiversity. Lead is environmentally stable and can induce multifaceted physiological disorders in pollinating insects. However, the comprehensive toxic impacts of lead on bumblebees, particularly the interconnections between host gene expression and gut microbiota, remain largely underexplored.MethodsIn this study, Bombus terrestris individuals were chronically exposed to three lead concentrations (0.95, 3.6, and 80 mg/L), selected based on acute toxicity data and field-relevant pollution levels. Following oral exposure via sugar water, we examined the expression profiles of genes associated with learning and memory, detoxification, and immune defense, as well as structural alterations in gut microbial communities.ResultsLead exposure significantly altered the transcription of DopR1, DopR2, NMDA, GST, PPO, defensin, and Hymen. Gut microbiota profiling further revealed distinct operational taxonomic unit (OTU) distributions across treatment groups, indicating that lead exposure reshaped the diversity and community structure of bumblebee gut microbes.DiscussionThese findings uncover the coordinated toxic responses of host genes and gut bacteria under lead stress, and provide critical insights for a more comprehensive assessment of the ecological risks that heavy metal pollution poses to bumblebee populations.

As the predominant native pollinator across Asia, Apis cerana is essential for the maintenance of biodiversity and agricultural productivity. The gut microbiota of honeybees plays a central role in host nutrition, detoxification, and immune function. p-Coumaric acid, a widespread phenolic acid enriched in pollen and nectar, has been reported to promote honeybee health by prolonging lifespan and increasing the expression of detoxification-related genes, hence improving tolerance to pesticides. Its influence on gut microbial communities, however, remains insufficiently characterized in A. cerana. This study evaluated the effects of dietary p-coumaric acid on survival, sucrose solution consumption, and gut microbiome composition in A. cerana workers using absolute quantification sequencing. Bees were provided sucrose solutions containing p-coumaric acid at concentrations of 41.0, 82.0, and 164.0 mg/L for durations of 5 and 10 days. The results indicated no effect on survival but revealed time-dependent changes in sucrose solution consumption. p-Coumaric acid exposure altered the abundance of non-core bacterial taxa, including Bombella and Apilactobacillus, whereas the core gut microbiota (Lactobacillus, Gilliamella, Snodgrassella, Apibacter, and Bifidobacterium) remained stable. These results suggest that p-coumaric acid modulates sucrose solution consumption and selectively influences non-core gut bacteria without disrupting survival or core microbiota stability, underscoring its role in regulating host-microbe interactions in honeybees.

Heavy metal pollution has emerged as a major challenge in global environmental science in the 21st century because of its extensive distribution, long-term environmental persistence, and non-degradability. As indispensable pollinators in ecosystems, honey bees have undergone marked population declines, a trend closely associated with multiple environmental stressors, including heavy metal contamination. Notably, lead (Pb), a widespread legacy toxic metal pollutant, is released into the environment through industrial emissions, vehicle exhaust, and other anthropogenic sources, contributing to long-term environmental contamination. Moreover, Pb can be transferred to honey bees through contaminated pollen and nectar. This exposure route may pose a risk to honey bee health, particularly under chronic or repeated exposure. This review synthesizes current evidence on Pb exposure routes, bioaccumulation, sublethal toxicity, and physiological responses in honey bees, while highlighting major uncertainties in colony-level risk assessment. Pb-associated physiological effects may impair individual bee health, but their long-term implications for colony health require further study.

Bumblebees are dominant pollinators threatened by environmental antibiotic residues. This study investigated sublethal chloramphenicol (12 and 120 μg/L) effects on Bombus terrestris after 15 days' exposure. The results showed that chloramphenicol exposure had no significant effect on the survival rate and cumulative food intake of bumblebees, confirming the sublethal property of the tested concentrations. However, chloramphenicol significantly dysregulated the expression of genes related to learning-memory (DopR2, Oamb, NMDA), immunity (abaecin, defensin) and detoxification (cyp9Q6) in bumblebees. High-dose chloramphenicol significantly increased carboxylesterase activity and reduced malondialdehyde content, while superoxide dismutase activity remained unchanged. In addition, chloramphenicol exposure significantly reshaped the gut microbiota structure of bumblebees, reduced the abundance of core beneficial symbiotic bacteria, and increased the proportion of drug-resistant bacteria. Our findings indicate that sublethal concentrations of chloramphenicol can impair bumblebee health through multiple pathways, including regulating gene expression, altering antioxidant enzyme activity and disrupting gut microbiota homeostasis. This study provides multi-dimensional toxicological data and a scientific basis for the ecological risk assessment of agricultural antibiotic residues to pollinator insects.

Heavy metal pollution is a growing global environmental concern, and its potential risks to pollinating insects are receiving increasing attention. Previous research has mainly focused on toxic hazards occurring during the adult developmental stage of bees. However, the toxic effects of lead (Pb) on honeybee larvae and their underlying mechanisms remain insufficiently understood. This study systematically evaluates the multidimensional impacts of Pb exposure on the growth and development, immune metabolism, and molecular responses of honeybee larvae (Apis mellifera). Following exposure to sublethal Pb concentrations of 1.7 and 8.5 mg/kg for 72 h, larval survival, growth and development were significantly reduced. It also leads to significant decreases in morphological indicators of newly emerged adult bees. Pb exposure induced pronounced oxidative stress, characterized by a decrease in antioxidant and immune enzyme activities and total antioxidant capacity, accompanied by the accumulation of lipid peroxidation products (MDA and 4-HNE) and DNA oxidative damage markers (8-OHDG). At the molecular level, high-concentration Pb exposure suppressed pathways related to cytochrome P450-mediated xenobiotic metabolism and amino acid metabolism. At the metabolic level, the arachidonic acid metabolism pathway was significantly activated, with key inflammatory mediators, such as leukotrienes and prostaglandins, showing marked accumulation, thereby exacerbating toxic damage through interactive inflammatory and oxidative pathways. This study provides insights into understanding the toxic effects of the heavy metal Pb on bee larvae. It emphasizes the importance of the larval developmental stage in colony health risk assessment and offers an important basis for deepening the understanding of sublethal effects and ecological risks of heavy metals on pollinators.

Bumblebees are efficient pollinators of fruits and vegetables in greenhouses and field crops. However, pesticide use in agricultural landscapes is causing a sharp decline in pollinating insect populations. The impact of pesticides on bumblebee health is a growing concern. Cyfluthrin, atrazine, and prothioconazole are 3 commonly used pesticides in agricultural production. Although the Food and Agriculture Organization of the United Nations has published the acute median lethal dose (LD50) data for these 3 pesticides on Apis mellifera honey bee, there is still a lack of LD50 data for non-Apis bees, such as Bombus terrestris (Linnaeus, 1758). Therefore, this study determined the oral median lethal dose (LD50) of 3 pesticides, cyfluthrin, atrazine, and prothioconazole, in European bumblebees (Bombus terrestris). The active ingredient of each pesticide was first dissolved in dimethyl sulfoxide and then diluted in a sucrose solution to prepare the pesticide-sucrose mixture for feeding. The oral LD50 values of cyfluthrin for worker bees were 4.27, 3.36, and 2.16 μg/bee at 24, 48, and 72 h, respectively. The 24-h LD50 for virgin queens was 13.49 μg/bee. For 24-h exposures in worker bees, the oral LD50 values of atrazine, prothioconazole, and their mixture were 355.3, 530.0, and 480.4 μg/bee, respectively. Pesticide-sucrose solution intake decreased as pesticide concentration increased. This study provides a preliminary evaluation of the toxicity of 3 pesticide types on bumblebees and offers insight for improving the conservation and sustainability of pollinators in agriculture. Additionally, the findings contribute to regulatory assessments by providing crucial data on pesticide effects on B. terrestris, supporting more comprehensive and effective pesticide regulations.

Across agricultural systems, crop production relies heavily on insect-mediated pollination. Both honey bees and bumble bees contribute substantially to crop productivity, with honey bee colonies providing large foraging workforces. On the other hand, bumble bees' specialized pollination behaviors offset the limitations of honey bees, making them especially well-suited for greenhouse production. However, pesticides may pose significant risks to bumble bee health. Here, we used 16S rRNA gene sequencing to examine the effects of treatment with cyfluthrin, atrazine, and prothioconazole on the gut bacterial community of the European bumble bee (Bombus terrestris Linnaeus, 1758). Only atrazine treatment elicited a concentration-dependent response in intake and mortality. In bumble bees that survived treatment with cyfluthrin and atrazine, beta diversity of the gut bacterial community was significantly altered. In contrast, prothioconazole significantly altered both alpha and beta diversity, suggesting a stronger impact on gut microbial structure. Cyfluthrin significantly increased the relative abundance of Proteobacteria while decreasing Firmicutes, atrazine significantly reduced Proteobacteria while increasing Firmicutes and Bacteroidota, and prothioconazole significantly reduced only Bacteroidota. At the genus level, cyfluthrin significantly decreased Lactobacillus and increased Pseudomonas and Brevundimonas, atrazine significantly increased Apibacter and Lactobacillus, and prothioconazole significantly decreased Apibacter and Bifidobacterium. Snodgrassella, Gilliamella, Apibacter, and Lactobacillus remained the dominant genera across all treatments. The results of this study clarify pesticide-specific effects on the bumble bee gut microbiome, providing exploratory evidence for potential modes of action and informing future risk-related studies.

Honeybees are vital pollinators that contribute substantially to global ecosystem stability and agricultural productivity. Camellia reticulata, a cross-pollinated crop species, depends on honeybees for successful reproduction. Apis cerana shows reluctance to pollinate C. reticulata, yet the molecular mechanisms underlying this phenomenon remain unexplored. In this study, we performed controlled feeding experiments in which adult worker A. cerana were supplied with stachyose, raffinose, and their combination. We assessed physiological traits including survival rate, sucrose solution consumption, and body weight gain, alongside histological changes in intestinal cell structures. We conducted RNA-seq of gut tissues as well as 16S rRNA sequencing and metabolomic profiling. Our findings revealed that the mixed oligosaccharide treatment significantly reduced the survival rate of workers, and three oligosaccharide treatments significantly reduced sucrose consumption in A. cerana. Both mixed and single-oligosaccharide treatments caused pronounced intestinal cell damage and disrupted the gut microbial community structure. Among the gut microbes, Gilliamella exhibited the most substantial decline in the stachyose group. Metabolomic analysis further demonstrated that oligosaccharide feeding significantly altered amino acid and galactose metabolism pathways, which may play critical roles in oligosaccharide utilization and directly influence honeybee survival. In summary, this study provides new insights into the molecular mechanisms underlying A. cerana mortality associated with C. reticulata pollination. These findings not only enhance our understanding of host-diet-microbiota interactions in honey bees but also offer a theoretical basis for the integrated management of A. cerana for C. reticulata pollination and the development of oligosaccharide-adapted bee diets.

The accumulation of cocoons within brood cells of old combs is a key factor causing a series of negative impacts on bee colonies. Previous studies did not sufficiently address this dynamic nature as the core microenvironment for preimaginal bee development. During this accumulation, the enrichment of potentially harmful microorganisms and chemical substances may pose a latent threat to colony health. This study combined microbiome and metabolomics analyses to systematically investigate the potential colony health risks posed by multi-generational accumulation of cocoons in Apis mellifera combs. The results demonstrated that with the growing number of brood rearing generations, the microbial diversity within the cocoons underwent significant shifts. For the bacterial community within multiple-generation cocoons, the Simpson index exhibited a significant increase, whereas indices including Sobs, Ace, and Chao showed significant decreases (p < 0.05). In the fungal community, the Shannon and Pielou_e indices significantly increased, while the Simpson and Faith_pd indices significantly declined (p < 0.05). Potential pathogens such as Melissococcus and the mycotoxin-producing fungus Wallemia became significantly enriched, reaching alarming relative abundances of 42.70% and 13.52%, respectively, in the multiple-generation cocoons. Metabolomic analysis further revealed the enrichment of 685 differential metabolites, including persistent exogenous pesticides such as cyanazine and pymetrozine, etc. Correlation analysis uncovered a significant positive relationship (r > 0.8) between these pesticide residues and pathogen abundance, indicating interactions between pollutants and pathogens that may exacerbate risks. This study reveals the aggravation of microecological imbalance and chemical pollution load within the cocoons of old combs and therefore provides strong scientific support for risk assessment of comb age in colony health management and offers practical guidance for the sustainable development of beekeeping.

At present, there is no clear consensus on the impact of carbohydrate feeds on bee colony health, and comprehensive research and evaluation in this context is lacking. To comprehensively and objectively examine the health status of honeybees after consuming those carbohydrates from multiple perspectives, experimental techniques, including high-throughput sequencing of the transcriptome, proboscis extension reflex (PER), and measuring bee growth parameters were employed. This study showed that compared with honey, feeding high fructose syrup (HFS) resulted in a decrease in the survival rate and body weight of bees, while sucrose decreased the learning and memory ability of bees. After feeding on honey, the main antimicrobial peptides including abaecin, apidaecin1, hymenoptin, and defensin in bees, are all upregulated in expression. The 14 DEGs significantly enriched in the axonal regeneration pathway were all downregulated in the sucrose group and HFS group. This study demonstrated that the expression of multiple genes involved in oxidative phosphorylation was downregulated in bees fed with HFS, moreover, HFS also affected the biosynthesis of unsaturated fatty acids. These effects may lead to energy and metabolic disorders (including fatty acids), thereby inhibiting the growth and development of bees. Sucrose can decrease the learning and memory ability of bees, which may be due to the downregulation of genes related to learning and memory in the axonal regeneration pathway. Honey can upregulate antimicrobial peptides and other immune-related proteins, activating the bee's immune system and boosting bees' immunity to pathogens.

Limosilactobacillus reuteri is recognized as a GRAS organism and has been extensively applied in food, pharmaceutical, and feed industries. This study systematically investigated the probiotic properties and safety of γ-aminobutyric acid (GABA)-producing L. reuteri LP4, a strain isolated from the intestinal tract of adult Apis cerana, through comprehensive phenotypic assays and whole-genome sequencing. In vitro experiments demonstrated that strain LP4 exhibits robust tolerance to digestive fluids, strong adhesion capacity, and notable antioxidant activity. The strain demonstrated significant antagonistic effects against common gastrointestinal pathogens, including Escherichia coli K88, Staphylococcus aureus, and Salmonella typhi. Additionally, GABA synthesized by LP4 could persistently accumulate in the extracellular environment. Critically, the strain displayed no hemolytic activity and lacked the ability to synthesize biogenic amines such as cadaverine, putrescine, or agmatine. Whole-genome sequencing revealed that the LP4 genome comprises a total length of 2,552,459 bp and encodes 2455 predicted genes. Genomic annotation identified multiple probiotic-associated genes involved in antienvironmental stress, adhesion, antibacterial activity, and biosynthesis of GABA and vitamins. Importantly, no definitive virulence or antibiotic resistance genes were detected. These findings align closely with the in vitro experimental results, highlighting the exceptional probiotic potential and safety profile of LP4, establishing a foundation for its application in the food industry and human health-related fields.

While the health benefits of lactic acid bacteria (LAB)-fermented feed on farmed animals are well-established, its potential benefits for honeybees, specifically Apis cerana cerana, remain largely unexplored. The present study aimed to optimize an enzymatic hydrolysis process for tea bee pollen, employing a complex enzyme comprising acid cellulase and pectinase, followed by fermentation with Limosilactobacillus reuteri LP4. A. c. cerana workers were subsequently fed tea bee pollen processed with this optimized method. Under the optimal processing condition of fermented tea bee pollen, the pH value was 4.41, the protein content was 27.75 %, and the viable count of LAB was 2.31×10⁹ CFU/g. No molds and yeasts as well as pathogens were detected. Compared to the unfermented pollen, honey bee workers administrated with fermented tea pollen with L. reuteri LP4 showed significantly increased survival rate by 24.34 % on day 15. Moreover, the relative abundances of Lactobacillus and Bifidobacterium were elevated, while those of Enterococcus and Bacteroides were diminished. Concurrently, the relative expression levels of immune-related genes including Abaecin, PPO, Defensin, and Vg were significantly upregulated. These findings provide a scientific foundation for application of fermented feeds to enhance the health of A. c. cerana populations and contribute to the sustainable development of apiculture in China.

ABSTRACTLong‐term positioning experiments have demonstrated significant benefits in agricultural production and environmental protection. Faba bean‐wheat intercropping with nitrogen fertiliser can effectively mitigate the occurrence of faba bean wilt disease. Identifying the optimal nitrogen application rate is essential for enhancing the disease control efficacy of intercropping. This study aimed to investigate the long‐term effects of varying nitrogen application levels on the physical, chemical, and biological changes in the rhizosphere soil of faba bean under intercropping conditions and to examine their relationship with the incidence of faba bean wilt disease. In a 9‐year field experiment, two treatments of faba bean‐wheat intercropping for 1 year (IF‐1) and 9 years (IF‐9) were established to investigate the incidence of faba bean wilt under four nitrogen levels (N0: 0 kg ha−1; N1: 45 kg ha−1; N2: 90 kg ha−1; N3: 135 kg ha−1). Rhizosphere soil from faba bean plants was collected to assess the corresponding physical, chemical, and biological indicators. Long‐term intercropping promoted the growth of faba bean plants and effectively controlled faba bean wilt disease by improving soil structure and fertility and soil quality (SQI). Under different nitrogen application levels, certain soil physical properties (moisture content, macroaggregate proportion, MWD, and GMD) and chemical properties (SOM, total carbon, SOC, total nutrients, and available nutrients) peaked under N2 (90 kg ha−1), with SQI showing a similar trend. Additionally, long‐term intercropping enhanced enzyme activity in the faba bean rhizosphere, reshaped microbial community composition, maximised the benefits of beneficial microbes, reduced the abundance of the pathogenic fungus Fusarium, and achieved optimal disease control under N2. Under long‐term intercropping with nitrogen fertiliser application at N2 (90 kg ha−1), the physical structure of the faba bean rhizosphere soil was significantly improved, soil quality and fertility were enhanced, and the abundance of plant pathogens was reduced by modifying the microbial community composition. This effectively alleviated faba bean disease, promoted healthy plant growth, and maintained soil function.

BACKGROUND:Bumblebees (Bombus spp.) are essential pollinators of agricultural crops, relying primarily on nectar and pollen for nutrition. However, these floral resources often contain pesticide residues that may exert sublethal effects on colony performance. While acute impacts are difficult to observe, chronic exposure can undermine colony development and increase the risk of collapse. The field-level effects of commonly used pesticides such as cyfluthrin, atrazine, and prothioconazole on the European bumblebee (Bombus terrestris) remain poorly understood. To address this gap, we evaluated colony development under natural conditions simulating long-term exposure to field-relevant concentrations of these pesticides in nectar. RESULTS:Developmental parameters included nest weight, pesticide-sucrose solution consumption, number of sealed pupae, number of virgin queens, and queen birth weight. Colonies exposed to cyfluthrin exhibited significantly lower nest weight, though other measures were unaffected. Atrazine exposure resulted in reduced nest weight, pesticide-sucrose consumption, and virgin queen birth weight, indicating impaired colony development. In contrast, prothioconazole exposure increased nest weight, and virgin queen birth weight, while pesticide-sucrose consumption, pupal and queen numbers were unaffected. CONCLUSION:These findings demonstrate the cyfluthrin exerts relatively mild effects, atrazine negatively impacts colony growth, and prothioconazole may unexpectedly enhance colony development. Current policy debates on pollinator safety largely emphasize neonicotinoids. However, our results highlight the need to consider the risks associated with other pesticide classes. Evaluating impacts on managed non-Apis species such as B. terrestris is particularly important for developing sustainable crop protection strategies that balance agricultural productivity with pollinator health. © 2025 Society of Chemical Industry.

Plastics and pesticides are commonly used and often coexist in the environment. As pollinating insects, honeybees are simultaneously exposed to both these toxins. However, there has been no study on the toxic effects of nano-polystyrene plastics (nanoPS) and cyfluthrin (Cy) on the Apis cerana cerana Fabricius until now. This study found that nanoPS did not significantly impact the mortality of Apis cerana cerana but could reduce cyfluthrin-induced mortality. Additionally, nanoPS caused damage to the honeybee gut and hindered the development of the hypopharyngeal glands, whereas cyfluthrin did not produce these pathological changes. Concerning the detoxification-related genes, the two toxins alone and in combination significantly promoted the expression of P450 9E2 and Cyp9Q3 genes, and the upregulation trend was found more significant for the combination. Regarding immune gene expression, exposure to a single toxin or both toxins significantly down-regulated the abaecin gene, but only exposure to nanoPS significantly decreased apidaecin expression. The changes in metabolites and metabolic pathways in honeybees after ingesting nanoPS were also studied. This study highlights the toxicity of nano-microplastics and Cy alone and in combination to Apis cerana cerana Fabricius and provides new insights into the potential ecological risks of nanoPS.

Lactobacillus helveticus is a probiotic bacterium widely used in the food industry. In this study, we evaluate the safety and probiotic properties of L. helveticus strain KM7, isolated from the gut of Apis cerana, through whole-genome sequencing and in vitro experiments. The complete genome consists of 2,164,024 bp, encoding 2192 genes with an average GC content of 36.82

Plant specialized metabolites are species-specific compounds that help plants adapt and survive in constantly changing ecological environments. Nectar contains various specialized metabolites, essential for maintaining nectar homeostasis. In this study, we employed high-performance liquid chromatography (HPLC) to compare the sugar composition between spoilage nectar and natural nectar, with further analysis of variations in color, odor, pH, and hydrogen peroxide (H₂O₂) content. Microbial strains in Camellia reticulata nectar were isolated and identified using the spread plate method coupled with DNA sequencing. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was implemented to characterize metabolite differences between spoilage and natural nectars. Subsequent in vitro experiments were conducted to validate the effects of screened nectar metabolites on the isolated microbial strains. The results showed that some C. reticulata nectar could spoil and deteriorate, which disrupted nectar homeostasis and significantly reduced the pollination efficiency by pollinators. Spoilage nectar had significant differences in color, odor, sugar composition, pH, and H2O2 content compared to natural nectar. The number of microbial species and quantity in spoilage nectar were much higher. The H2O2 content in natural nectar could reach (55.5 ± 1.80) μM, while it was undetectable in spoilage nectar. A total of 15 distinct microbial strains and 364 differential metabolites were isolated and identified from two types of nectar. In vitro experiments demonstrated that H2O2 could inhibit all the bacteria in C. reticulata nectar except Serratia liquefaciens. 12-Methyltetradecanoic Acid inhibited Bacillus subtilis, Curtobacterium flaccumfaciens, and Rothia terrae, and Myristic Acid only inhibited Rothia terrae. The nectar metabolites screened in this study had no effect on the nectar specialist yeast Metschnikowia reukaufii. In conclusion, the findings of this study revealed that C. reticulata nectar regulates the growth of microorganisms through its metabolites to maintain nectar homeostasis and prevent spoilage. This study improves the understanding of the physiological mechanisms of C. reticulata in maintaining nectar homeostasis and provides theoretical support for controlling nectar diseases and sustaining the reproductive fitness of C. reticulata. Future research could focus on further exploring the complex interactions between different metabolites in C. reticulata nectar and a wider range of microorganisms. Moreover, the development of practical applications based on these findings, such as the development of natural preservatives for nectar-related products or the optimization of pollination efficiency in C. reticulata cultivation, could be an important area for future exploration.

Honey bees transform nectar into honey through a combination of physical and chemical processes, with the physical process primarily involving the evaporation of excess water to concentrate the nectar. However, the factors affecting evaporation efficiency, such as evaporation duration, cell type, and bee species, remain incompletely understood. This study aimed to examine how these factors affect nectar evaporation efficiency during honey production. We measured the sucrose content in solutions subjected to combined active and passive evaporation, as well as passive evaporation alone. The results showed that eastern honey bee (EHB; Apis cerana) colonies were more efficient at concentrating sucrose solutions in worker cells than in drone cells under both combined active and passive evaporation conditions, as well as passive evaporation alone. Conversely, western honey bee (WHB; Apis mellifera) colonies exhibited greater efficiency in drone cells. Additionally, EHB colonies were more effective than WHB colonies in converting sucrose into fructose and glucose. Under passive evaporation, EHB colonies required at least 48 h to significantly concentrate the sucrose solution, while WHB colonies achieved similar concentrations in just 24 h. Sucrose content increased with the duration of passive evaporation. These findings provide insights into how honey bee colonies can efficiently produce mature honey during periods of abundant nectar flow.

Italian honey bees (IHBs, Apis mellifera ligustica) exhibit superior comb-building abilities compared with Chinese honey bees (CHBs, Apis cerana cerana), which often fail to fully utilize wax foundations, resulting in incomplete comb structures. The present study aimed to accelerate comb construction in CHB colonies using IHBs. In the experiment, IHB colonies, each with approximately 42,000 adult workers, required over four hours to construct a semi-drawn comb on CHB wax foundations. These semi-drawn combs were then transferred to experimental CHB colonies, where they were left to complete the comb-building process for an additional 24 h (4 + 24 h relay). In contrast, control CHB colonies were allowed to build combs on fresh CHB wax foundations for 28 h. The results showed that the combs built by CHBs in 28 h, those built by IHBs in 4 h, and the 4 + 24 h relay combs all shared a foundation size of 41.7 cm × 19.7 cm, with average thicknesses of 10.40 mm, 5.60 mm, and 12.20 mm, respectively. The average percentage of cells built in the 4 + 24 h relay combs was significantly higher than that in the 28 h combs. Additionally, cells in the relay combs were significantly larger and deeper than those in the combs built solely by CHBs. Although these findings prove that utilizing IHB colonies to form a base structure can enhance the efficiency of comb construction in CHB colonies, further research is needed to confirm the effects of 4 + 24 h operation on brood rearing and worker bee size.