Floral handling can be energetically costly for bees, yet these costs are rarely measured. We provide the first direct quantification of the metabolic cost of floral buzzing in bumblebees and evaluate its ecological significance. Using flow-through respirometry synchronized with laser vibrometry, we measured carbon dioxide production during floral buzzing by Bombus terrestris and compared it with flight take-off, which is powered by the same thoracic muscles. Floral buzzing required high muscular effort compared with other behaviours, with approximately 0.10 J per event and mass-specific power approximately 293 W/kg. Overall costs were comparable to take-off because floral buzzing bouts are longer despite a lower metabolic rate. Metabolic rate scaled with body mass, whereas intertegular span did not, implying that transient load rather than structural size better explains energetic demand in short, high-intensity behaviours. Metabolic traits were highly repeatable within individuals, but variable between individuals, and colony identity also explained additional variance. Converting costs to nectar equivalents showed that floral buzzing required slightly more nectar than take-off and that requirements rose as nectar sugar concentration declined. We conclude that floral buzzing is a major, previously unquantified component of bee energy budgets that is likely to shape nectar demands, flower visitation patterns and generally plant-pollinator interactions.

The regulation of reproductive division of labor in eusocial insects is pivotal for the evolution of social behavior and the maintenance of eusociality. Primitively eusocial bumblebee workers retain reproductive totipotency, with dominant workers capable of ovarian activation and egg-laying. Here, we investigated the cellular and molecular basis of reproductive hierarchy in Bombus terrestris by constructing a single-nucleus transcriptomic atlas of the ovary in queenless bumblebee groups. Using single-nucleus RNA sequencing, we profiled ovarian cell types and revealed that α-worker bees possess more mature follicle cells, which are essential for ovarian development. Differential maturation of follicle cells, particularly at the vitellogenic stage, emerges as a key regulatory node in this process. More mature follicle cells promote the production of growth factors that activate PDK1. This activation subsequently induces AKT phosphorylation and downstream signaling. As a result, the levels of 20-hydroxyecdysone are elevated in dominant α-workers. By demonstrating how follicle cell maturation and signaling drive reproductive activation, our findings link cellular physiology to social organization and provide new insight into the molecular mechanisms underlying the evolution of eusociality.

Fenitrothion is a widely used insecticide, yet its effects on the health of bumble bees (Bombus terrestris) have not been reported until now. We carried out experiments to investigate the impact of field-realistic concentrations of fenitrothion on food consumption, body weight, survival probability, gut structure, gut detoxification enzyme activity, gut microbial diversity, gut transcriptional and gut metabolic profiles of Bombus terrestris. Exposure to 500 μg kg-1 fenitrothion significantly reduced survival probability, induced gut damage as evidenced by hematoxylin and eosin staining, decreased cytochrome P450 (P450) activity while increasing glutathione S-transferase (GST) activity, and down-regulated AChE1 expression in gut tissue. Furthermore, 16S rRNA sequencing revealed a decline in gut microbial diversity, while transcriptomic and metabolomic analyses indicated disruptions in gut gene and metabolic expression. Molecular docking revealed that fenitrothion binds within a hydrophobic pocket formed by residues Trp148, Ser186, Tyr185, and Gly182 of the AChE1 protein. These multiple sublethal alterations in bee behavior and physiology indicate that fenitrothion is likely to exert complex yet detrimental effects on Bombus terrestris health under field conditions. © 2026 Society of Chemical Industry.

Role of the expanded Bombus terrestris CYP6AQ cytochrome P450s subfamily in thiacloprid metabolism and their interaction with SBI fungicides

Bombus terrestris (Linnaeus, 1758) is a crucial pollinator for global greenhouse cultivation and is mass-reared for commercial pollination services in China. Diapause is an indispensable phase in the commercial production of B. terrestris, yet the optimal duration for maximizing colony output remains undefined. We conducted a large-scale study within a Chinese industrial rearing pipeline, assigning 2,455 B. terrestris queens to one of eight diapause durations (0, 4, 8, 12, 16, 20, 24, and 28 wk). Compared to non-diapause controls, diapause significantly improved reproductive performance and shortened colony development cycles. Reproductive success peaked at 12 wk, with 98% egg-laying success, 80% colony foundation success (ie colonies reaching ∼10 workers), and 64% medium colony success (ie colonies reaching ∼60 workers). Queen survival decreased significantly after 16 wk of diapause. Using orthogonal partial least-squares discriminant analysis, we identified seven reproductive performance indicators that contributed most to discriminating among diapause durations (eg time to first male emergence, colony foundation success). Post-diapause physiological status correlated with subsequent colony performance; in this dataset, queens with higher reproductive success tended to exhibit free water content around 55%, glycogen levels between 24 to 37 mg/g, and protein levels between 113 and 134 mg/g at diapause termination. Our findings identify a 12-wk diapause as associated with the highest reproductive performance in this commercial B. terrestris rearing system, offering a practical reference for similar operations.

Determination of the effects of bumblebee (Bombus terrestris), hand, and natural pollination on fruit set, yield, and quality in pitaya (Hylocereus spp.) cultivation under subtropical conditions.

Cotton bollworm, Helicoverpa armigera, is a major global pest of cotton. Delivery of double-stranded RNA (dsRNA) shows promise as a biopesticide for controlling this insect pest. However, employing RNA interference (RNAi) technology in the field requires enhancing its stability and demonstrating biosafety. Two genes, chitin synthase II and cytochrome p450 protein 6b6, were simultaneously targeted to disrupt the gut physiology of the cotton bollworm. Long dsRNA molecules (≈ 400 bp) encapsulated with a cationic polymer (chitosan) and a cross-linker (tripolyphosphate, TPP) were synthesized to produce nanoparticles ranging from 150 to 300 nm in size, with a positive charge ~40 mV. Encapsulation resulted in a 100% increase in the knockdown effect on larvae that were fed with a diet containing dsRNA-nanoparticle complex compared to those fed with naked dsRNA. Additionally, nanoparticles produced substantially higher mortality than naked dsRNA, even at 100-fold lower concentration. Overall, our findings suggest that chitosan/TPP/dsRNA nanoparticles at low doses (0.1-1 μg g-1) effectively enhance the RNAi effect in H. armigera larvae via oral delivery. In addition, acute oral toxicity tests were conducted on two relevant beneficial bees, Scaptotrigona postica and Bombus terrestris. Consistent with predictions from bioinformatic analyses, no significant lethality or signs of toxicity were observed in these non-target organisms. Delivery of dsRNA molecules via nanoparticles was demonstrated to be a promising approach to enhance RNAi activity against H. armigera. Thus, this technology represents an efficient and safe strategy for managing lepidopteran pests. However, further research to optimize these molecules for field application is crucial. © 2026 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Abstract The invasive bumblebee Bombus terrestris has established widespread populations across northern Japan since its introduction in 1991 and has become dominant in some agricultural regions. This dominance has raised concerns about ecological impacts on native pollinators; however, the consequences of its removal for crop pollination and yield remain poorly understood. We conducted a field‐based case study in squash ( Cucurbita maxima ) fields to examine differences in pollination dynamics between a year without queen removal (2022) and a year with intensive queen removal (2024). We quantified floral visitation, stigmatic pollen deposition, fruit set, fruit weight and seed production. Queen abundance was also monitored in 2025 to document population reappearance following the removal period. The abundance of B. terrestris workers during flowering was markedly lower in 2024 than in 2022. Although native bumblebees and alternative visitors such as Vespula were observed, their visitation did not compensate for pollen transfer and fruit and seed production were reduced relative to the non‐removal year. In 2025, B. terrestris queens were again recorded, indicating recolonisation from surrounding areas. These results indicate that B. terrestris currently plays a dominant functional role in pollination at the study site and that its removal may be associated with reduced pollination services and crop production. Management of invasive pollinators in agroecosystems should therefore consider potential trade‐offs between biodiversity conservation and pollination services and integrate control efforts with measures that support native pollinator communities.

Titanium dioxide nanoparticles (TiO2 NPs) are widely used in industrial, agricultural, and consumer products and are increasingly detected in terrestrial and floral environments that are critical for pollinators. However, the mechanistic and ecological consequences of TiO2 NPs exposure on pollinator health remain poorly understood. Here, we present a multi-level ecotoxicological assessment of dietary TiO2 NPs exposure in the bumblebee Bombus terrestris L. (Hymenoptera: Apidae), integrating colony performance, gut microbiota composition, and host transcriptomic responses. Using both environmentally relevant and worst-case concentrations, we conducted short-term (3-d) and chronic (15-d) exposures to evaluate physiological disruption, microbial dynamics, and potential compensatory or recovery processes. TiO2 NPs ingestion reduced worker survival and syrup consumption, delayed oviposition, and shortened egg development, indicating impaired colony performance. Gut microbiota exhibited pronounced dysbiosis after 3 d, followed by partial converged toward control-like profiles after 15 d, suggesting microbial potential resilience and compensatory restructuring. Transcriptomic analyses revealed dose-dependent activation of immune, oxidative stress, calcium signaling, and programmed cell death pathways. Correlative analyses identified associations between specific microbial taxa, including Apibacter and Klebsiella, and host gene expression linked to immunity, detoxification, and metabolism, highlighting coordinated microbe-host responses under TiO2 NPs stress. This study provides novel mechanistic insight into how engineered nanoparticles affect pollinators across biological scales, from colony physiology and microbial ecology to molecular pathways, and emphasizes the practical importance of safeguarding pollinator health for both wild plant reproduction and crop pollination. The findings advance understanding of nanoparticle ecotoxicology and support evidence-based strategies for sustainable nanomaterial use and pollinator protection.

Rapid learning of aversive stimuli is adaptive, but the persistence of the avoidance response in the absence of further reinforcement might depend on the severity of the adverse experience. For example, an experience involving injury would be expected to lead to more durable memory than the mere exposure to an unpleasant tastant, especially when new experiences indicate that the aversive stimulus is no longer present. We investigated how bumblebees (Bombus terrestris) learn and retain associations between flower colours and two types of aversive stimuli: electric shock and saturated salt (NaCl) solution. Using a conditioning paradigm, we examined how these stimuli influence avoidance learning across foraging bouts and tracked the process of extinction learning, the formation of new memory in response to the absence of the reinforcement, over two weeks. Our results show that bees rapidly learn to avoid both stimuli, and reach >90% accuracy of avoidance after six foraging bouts. We then examined how bees modified their avoidance behaviour in the absence of further aversive stimulation. Testing extinction learning on days 1, 3, 5, 7 and 14, we found that electric shock as a nociceptive stimulus induces a more persistent avoidance response, whereas exposure to the salt by engaging gustatory aversion pathways leads to a three times faster extinction rate. This suggests that although the initial training leads to equal levels of avoidance for both stimuli, bumblebees might display greater behavioural flexibility when updating the association between a colour and an unpleasant taste in comparison to a potentially injurious stimulus.

Male bumblebees (Bombus terrestris) are more active and behaviourally flexible than workers.

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.

Invasive species may exhibit shifts in their gut microbiome in response to novel environments and diet, but this may differ across host species and their time since colonisation. We investigate if site environmental variables and foraged pollen resources differentially shape the gut microbiomes of two bee species with contrasting introduction histories: The European honeybee, Apis mellifera (introduced 1831), and the recently invasive bumblebee, Bombus terrestris (invaded 1992). Using landscape-scale metabarcoding across the island state of Tasmania in Australia, we characterised gut bacteria (16S rRNA) and corbicular pollen diversity (ITS2) for each species. Gut bacterial composition was significantly associated with mean annual temperature for A. mellifera and with mean annual precipitation and percentage of pasture for B. terrestris. In B. terrestris, the core and facultative gut microbial diversity and richness showed associations with precipitation, foraged pollen diversity, wind velocity and temperature. Foraged pollen diversity of native plants more strongly predicted the facultative gut microbiome across species. Overall, the gut microbiome of B. terrestris showed a stronger response to abiotic and biotic predictors compared to A. mellifera. Our findings advance understanding of how environmental and dietary factors shape pollinator gut microbiomes at landscape scales, with implications for pollinator health and survival.

Intra-individual variation in pollen availability: An experimental analysis of its impact on plant-pollinator interactions.

Effects of ubiquitous plasticizers on olfactory perception in the buff-tailed bumble bee.

Insect pollinators, such as bees, provide essential ecosystem services yet face increasing environmental challenges, including pathogens, which can negatively impact host fitness. Central to host defences are immune genes and their products but for many bee species, our understanding of the conservation of the immune gene repertoire, as well as mechanisms that allow for functional diversity, is restricted to a few species. Here, we perform a pan-clade examination of the canonical immune genes and associated functional gene groups found across 70 bee species, representing six of the seven extant families. We show a high level of conservation of immune genes with all major immune gene groups represented, with elevated copy number variation found in CLIP-domain serine proteases, serpins, and small RNA regulatory proteins across bee species. Using the buff-tailed bumblebee Bombus terrestris, we further show that increases in immune-related gene group size are generally associated with increased nucleotide diversity, and transcriptional divergence among group members suggestive that group size, through mechanisms such as gene duplication, may allow for structural and functional diversity across immune components. However, we find that this pattern is non-linear indicating that gene group expansion is constrained. We also find lineage-specific losses of antimicrobial peptides highlighting that certain immune components may be dispensable or compensated by other elements. Our analyses show that the genetic components of bee immunity are largely conserved, with duplication and loss highlighted as mechanisms that shape immune diversity, which, collectively, has implications for understanding resilience of bee species to increasing pathogenic threats.

Antibiotic resistance gene profiles in the gut microbiomes of Apis cerana, Apis mellifera, and Bombus terrestris.

Understanding species-dependent siRNA length generation provides both fundamental and applied insights. At the basic level, it highlights an underappreciated dimension of RNAi diversity in insects, emphasizing that antiviral immunity cannot be fully understood from Drosophila melanogaster alone. At the applied level, these findings have direct implications for the design of dsRNA-based pest management strategies, where tailoring siRNA production to the target insect order could enhance both efficacy and specificity. Previously studies demonstrated that Dicer-2-generated siRNAs exhibit distinct, species-dependent length distributions: dipterans (D. melanogaster) and coleopterans (Tribolium castaneum) mainly produce 21-nt siRNAs; and hymenopterans (Bombus terrestris) and orthopterans (Locusta migratoria) generate siRNAs enriched at 22 nt; whereas lepidopterans such as Spodoptera exigua and Trichoplusia ni predominantly produce 20-nt siRNAs. The central biological question motivating this study was whether structural divergence in Dicer-2 may explain these lineage-specific differences in siRNA length profiles and antiviral RNAi efficiency. To address this, we interpreted observed structural variations in the context of the "molecular ruler" hypothesis and integrated them with previous experimental data on siRNA length variation across insect taxa. Future studies that combine high-resolution structural biology, comparative genomics, and functional assays will be essential to experimentally test whether the structural correlations proposed here determine Dicer-2 cleavage length in vivo and in vitro, and to leverage this knowledge for both agricultural and biomedical applications.

When making decisions, animals often rely on multiple cues simultaneously. These provide complementary sources of information, which can increase the accuracy of decisions. In this study, we tested the decision and learning strategies of insects faced with multiple cues in a foraging task, using the bumblebee Bombus terrestris. We presented combinations of visual-only features, as found in natural flowers: colors of varying discriminability, paired with shapes or patterns. We found that the bees relied exclusively on colors when these were easy to discriminate, and did not learn pattern or shape features simultaneously. With harder to discriminate colors, the bees learned both color and shape or pattern features. Our results demonstrate that bumblebees flexibly adjust their learning strategies when presented with visual features of varying discriminability, to balance the investment in learning time and accuracy during multicue learning and decision making. A difference in learning dynamics suggests that blocking could serve as a mechanism to implement this strategy switch.

Exposure to pesticides partly depends on the foraging behavior of bees, which may exhibit indifference, deterrence, or attraction to contaminated food. In the present study, we conducted laboratory experiments to test the foraging avoidance of Bombus terrestris for honey syrup contaminated with field-realistic concentrations of the neonicotinoid acetamiprid (ACE), herbicide glyphosate (GLY), and fungicide metalaxyl-M (MET). Tests were also conducted with the recommended field concentration of a biopesticide, the sweet orange essential oil (EOE). Bees’ behavior, and lethal and sublethal effects of the pesticides on bumble bees were assessed at the individual (isolated foragers) and colony (colony exposure) levels. Bees did not display any avoidance for contaminated or uncontaminated food at the individual or colony levels in the ACE, GLY, and MET treatments. However, the EOE treatment reduced the consumption of honey syrup in relation to non-treated bees, resulting in lower survival of individual bees. At the individual level, no behavioral differences were observed between non-treated and treated bees. At the colony level, however, pesticide treatments modified bees’ walking behavior. In general, pesticides increased resting time and meandering, while reducing speed, movement, distance walked, and time spent moving fast. Our results demonstrate that bumble bees did not reduce food consumption of syrup contaminated with realistic concentrations of different common pesticides, resulting in detrimental effects on their behavior. Furthermore, bumble bees treated with the biopesticide field concentration reduced food consumption both at individual and colony levels. This treatment caused negative behavioral changes on bees and decreased the survival of isolated individuals.