Bumblebees are excellent pollinators sustaining ecosystems, however, their populations are declining due to land-use changes, lack of food resources, and pathogen-induced diseases. The gut microbiota is central to animal health, influencing nutrition and defense. We examined how pollen diet affects colony development, pathogen presence, and gut microbiota in two native South American bumblebee species, Bombus pauloensis and B. bellicosus. In B. pauloensis, monofloral pollen from Eucalyptus grandis increased worker body mass and showed a slight reduction in developmental time compared to polyfloral pollen. In B. bellicosus, no significant diet effects on body mass or development time were observed. Diet also altered gut microbiota structure. Based on diversity analyses, significant diet-associated shifts were observed in B. pauloensis queens and B. bellicosus workers. In B. pauloensis queens, monofloral pollen increased bacterial richness and favored Snodgrassella spp., whereas in B. bellicosus workers, monofloral pollen also increased diversity, but enriched ASVs were mainly environmental taxa. The microsporidian pathogen Nosema ceranae was detected in a single B. pauloensis colony, limiting conclusions about prevalence or diet. These findings demonstrate that nutritional quality of dietary pollen and/or pollen diversity modulates gut microbiota and colony development in native bumblebees, with species and caste-specific responses. Our study suggests a potential role of nutrition in host-microbiota interactions and indicates that agricultural intensification and the loss of floral diversity may affect bee health. By linking pollen nutritional quality/diversity, microbiota structure, and colony performance, these findings may help inform pollinator-friendly landscape management and could contribute to improving the resilience of South American agroecosystems.
Bumblebees are key pollinators in agricultural and natural ecosystems; however, climate change is driving shifts in population size, body size, and diversity. Accelerating global warming affects bumblebee body size, thereby influencing the strength of sexual selection and overall population fitness. Thus, this study aimed to investigate the effects of body mass and temperature on sexual selection in Bombus terrestris reared under warm conditions, with mating conducted between equal sex ratios at both optimal (23 °C) and elevated (32 °C) temperatures. Mating success was lower at 32 °C than at 23 °C, regardless of body mass; however, larger queens and males consistently exhibited higher mating success at both temperatures than their smaller counterparts. Mate-choice patterns were similar across temperatures: large queens predominantly mated large males, whereas small ones mainly mated small males. However, small queens tended to mate large males at 32 °C than at 23 °C. Meanwhile, matings between small queens and small males occurred relatively later at both temperatures. Although small males exhibited longer mating durations than large ones, the amount of sperm transferred to the spermathecae of the queens was positively associated with the body size of both males and queens. Our findings indicate that, at equal sex ratios, mate choice in bumblebees is primarily determined by body mass at all temperatures. Thus, smaller bumblebees associated with higher temperatures had the lowest mating success.
Facial expressions provide a critical window into the internal states of animals, allowing the identification of affective processes and their underlying neural mechanisms. In mammals, postconsumption orofacial reactions-such as tongue protrusions to sweet tastes-are among the most robust and widely accepted behavioral markers of positive hedonic impact (liking), distinct from motivation (wanting). However, no equivalent markers have been established in insects, leaving their capacity for affective evaluation unresolved. Here, we show that bumblebees exhibit discrete orofacial reactions that are functionally analogous to those described in mammals. Bees displayed postconsumption glossa protrusions to rewarding solutions and head-shaking and mouth-wiping to aversive tastes. These reactions were not tied to any specific taste identity. Behavioral and pharmacological experiments demonstrated that glossa protrusions did not automatically occur due to consumption, were independent of ongoing feeding mechanisms and dopamine-mediated motivation, and were enhanced by the endocannabinoid anandamide. Our findings support postconsumption glossa protrusions as a fast, reliable, and evolutionarily comparable candidate behavioral marker of affective evaluation in an invertebrate. By providing evidence that a behavior which tracks liking can be dissociated from motivational (wanting) responsiveness in bumblebees, this work establishes an experimental framework for investigating the neural basis and evolutionary origins of affective processing. More broadly, these results open the door to rigorous cross-phyletic approaches to emotion.
Insecticides and herbicides are utilized worldwide in agricultural practices, and these hazardous materials retained in the environment impose potential threats to pollinators, including bumblebees. However, whether and how short-term exposure to sublethal concentrations of insecticide/herbicide induces lasting toxicity effects in newly emerged gynes remain uncharacterized. In this study, we comprehensively investigated the long-term consequences of short-term exposure to sublethal imidacloprid (IMI) and glyphosate (GLY) (singly or combined) on bumblebee (Bombus terrestris) gynes under different exposure regimens through integrated physiological and transcriptomic analyses. Short-term exposure of gynes to IMI alone and IMI + GLY mixtures not only impacted survival, tissue development, nutrient reserves, mating, diapause energy metabolism, and reproduction of queens but also caused transgenerational toxicity affecting offspring development and performance. In contrast, GLY exposure alone adversely affected tissue development, nutrient reserves, diapause energy metabolism, and offspring development of queens. Notably, compared to direct exposure, starvation exacerbated the adverse effects of IMI exposure on queens' diapause survival and energy metabolism and aggravated the impacts of GLY exposure on gynes' ovarian development and lipid reserves. For combined IMI + GLY exposure, starvation mainly amplified physiological disturbances related to queens' diapause survival, energy metabolism, and offspring development and performance. The observed defects in short-term IMI- and IMI + GLY-exposed gynes may result from altered nutrient metabolic pathways. Collectively, our research unveils the lasting toxicity of short-term mixed insecticide/herbicide exposure on gynes/queens and their offspring from physiological and molecular perspectives, underscoring the urgent need for regulatory consideration of combined pesticide risks in bumblebee conservation.
Attention in vertebrates helps prioritise the processing of important sensory information and filter out irrelevant signals. The capture of attention by sudden or salient stimuli is typically called bottom-up attention. Little is known about similar attentional processes in insects, although they should be advantageous for insects as well. We therefore adapted two paradigms used to investigate bottom-up attention in primates to investigate it in bumblebees: a target detection task and a target discrimination task. For both tasks, we trained bees to choose between two locations on each side of a computer screen and collect a reward below a full contrast target displayed on the screen. During detection task tests, the contrast of the target was varied, and it could be preceded by a cue flashed on the side of the target, the opposite side of the screen or not flashed at all. The discrimination task tests were similar but with a full contrast target on one side and a variable contrast distractor on the opposite side of the screen. We tested if the presence of the flash influenced the orientation and choices of the bees as well as their contrast sensitivity as has been seen in primates. We analysed bee choices using both direct observation of their behaviours at the trained locations and a trajectory analysis of high-speed videos. Our results show no effect of the prior cue, suggesting that other paradigms might prove more useful to test these processes in insects. The online version contains supplementary material available at 10.1007/s10905-026-09908-2.
Nectar robbery is common in hummingbird-pollinated plants and is often assumed to reduce plant reproductive success by depleting floral rewards and disrupting pollination. However, its quantitative effects on plant fitness remain poorly resolved, particularly in systems where native and invasive nectar robbers coexist. We evaluated nectar robbery by the native bumblebee Bombus dahlbomii and the invasive B. terrestris in the hummingbird-pollinated shrub Desfontainia fulgens in southern Chile. Flowering and nectar robbery were strongly synchronized, with robbery frequency peaking during maximum flower availability. Primary nectar robbing substantially reduced floral rewards: nectar standing crop in pierced flowers was approximately nine times lower than in intact flowers, whereas nectar sugar concentration showed only minor and statistically non-significant changes. Small ants and flies were found inside the flowers, suggesting that these insects may act primarily as nectar thieves rather than effective pollinators. Their abundance was not significantly associated with corolla perforation. Floral visitation intensity differed among visitor species and foraging strategies, with bumblebees visiting more flowers per plant during nectar robbing than during legitimate pollination. However, these observations describe foraging modes rather than demonstrating that robbing itself caused a behavioral shift. The hummingbird Sephanoides sephaniodes was observed exclusively as a legitimate pollinator. Experimental manipulations showed no negative effects of nectar robbery on seed production when pollinators were present. Flowers exposed to pollinators produced substantially more seeds than flowers from which pollinators were excluded, confirming strong pollinator dependence. In contrast, both the probability of seed production and the number of seeds produced per flower were similar between flowers exposed to pollinators with and without nectar robbers. These results indicate that nectar robbery can markedly erode floral rewards without translating into reduced female reproductive output.
Heavy 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. In 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. Lead 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. These 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.
Male bees navigate complex tradeoffs between energy acquisition and reproductive signaling, yet their movement strategies remain understudied. Unlike workers that optimize foraging to support the colony, male bumblebees (Bombus terrestris) forage independently to collect nectar and deposit sex pheromones on selected plants. Using high-resolution 3D tracking in an indoor flight cage, we investigated how the spatial arrangement of nectar and scent-marking sites, along with nectar availability, influence male movement patterns. We manipulated the distribution of feeders (artificial flowers) and scent-marking locations (branches), and varied nectar delivery rates, to assess effects on foraging, scent-marking, and patrolling. Males responded strongly to spatial structure: in clumped arrays with evenly spaced resources, movements between consecutive visits were shorter and more localized, while in dispersed arrays with irregular spacing, transitions were longer and more variable. The combination of dispersed spacing and low nectar availability imposed the highest foraging demands, resulting in fewer feeding events and reduced total feeding time. Despite these increased costs, males maintained consistent investment in reproductive behaviors, suggesting a prioritization of mate-seeking over energy gain. Rather than reducing signaling, males adjusted their foraging strategy-favoring fewer but prolonged feeding bouts when nectar availability allowed. These findings reveal a unidirectional behavioral adjustment, in which foraging is modulated to sustain reproductive effort, and show how spatial resource structure and nectar availability together shape movement decisions in male pollinators.
Judgement bias tasks are increasingly used to assess affective states in animals, yet the extent to which they might reflect transient states or stable traits remains unclear. Here, we tested bumblebees (Bombus terrestris) in an active choice task across three repeated sessions to assess individual consistency in the absence of any manipulation. Bees were trained to associate each of two colours with either a high or a low reward, presented in separate chambers. During testing, they were presented with ambiguous colours. Bees were more likely to choose the high-reward chamber and to choose more quickly in response to colours closer to the positive colour. The latency to choose the cues showed significant and moderate repeatability across sessions, suggesting a stable, trait-like underlying component. In contrast, the repeatability of the chamber choices was negligible, indicating that such responses might be largely state-dependent and influenced by situational factors. These findings suggest that judgement biases, particularly as assessed through an active choice task, reflect states affected by external factors. Active choice tasks may help disentangle stable behavioural traits from transient affective states in invertebrates.
Pollinator decline is one of the most urgent environmental challenges of our time, and pesticide use is considered a major contributing factor. In this study, we investigated whether exposure to volatilized prallethrin, a widely used insecticide, impairs the homing ability of bumblebees. Using a consumer-grade vaporizing device, we exposed foraging bees to field-realistic doses of prallethrin and then tested their ability to return to their colony in a rural area where they were already familiar with the surroundings. Our results show that longer exposure durations reduced the bees' return rates, indicating disrupted navigational ability. However, among those that did return, homing time was not affected by the treatment. While our study only focuses on one key behavioural trait, the findings provide clear evidence of a sublethal effect of a commonly used household insecticide on an essential pollinator species.
[This corrects the article DOI: 10.1111/eva.70234.].
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.
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.
Bumblebees rely on diverse sensory information to locate flowers while foraging. The majority of research exploring the relationship between visual and olfactory floral cues is performed at local spatial scales and is applicable to understanding floral selection. Floral-cue use during search remains underexplored. This study investigated how the bumblebee Bombus impatiens uses visual versus olfactory information from flowers across behavioral states and spatial scales. At local spatial scales, non-flying animals in an associative learning paradigm will generalize to either unimodal attribute of a learned color+odor cue with equal likelihood. However, bumblebees flying in a wind tunnel shift cue-use strategy depending on the spatiotemporal scale of cue encounter. When both color and odor cues mimic local/within-patch spatial scale, bumblebees weigh color information of a learned floral-cue more heavily. When cues mimic an intermediate/between-patch spatial scale, bumblebees weigh color and odor information equally, and show the highest response to fully intact multimodal cues. Thus the spatiotemporal scale of sensory information influences how bumblebees utilize multimodal floral cues.
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.
Global climate change is disrupting key ecological processes and species interactions. In particular, the frequency and severity of heat waves have increased dramatically over the last decade. Bumblebees are key pollinators in natural and agricultural systems, representing great economic and biodiversity value. However, these insects are particularly vulnerable to heat stress, because they are exceptionally well adapted to cold environments. Previous studies showed that heat stress has negatively impacted bumblebee foraging in terms of flight performance and foraging success, but its effect on chemosensory orientation is still unknown. In this study, we experimentally investigated if heat wave-treated bumblebees have difficulties in sensing or locating the source of a synthetic floral blend. We found that the proportion of individuals that initiated foraging was significantly lower in the heat wave-treated group than in the control group. Moreover, heat wave-treated bees started foraging later and approached scent sources randomly, although they reached the first scent source with a latency comparable to that of control individuals. Contrary to our initial expectations, the heat wave treatment influenced the antennal response of bumblebees only in a body size-dependent manner. Our findings provide evidence that heat waves can reduce foraging activity and impair directional movements toward floral scents in buff-tailed bumblebees, and support the idea that climate change may be one of the most harmful anthropogenic factors affecting the foraging performance of this pollinator species.
Biological invasions can disrupt plant-pollinator interactions by altering pollinator behaviour and pollen transfer dynamics, yet the mechanisms and timing of these effects remain poorly understood. Most studies rely on observational comparisons or removal experiments in long-established invasions, but little is known about changes in pollination function at the onset of invasion. We investigated the pollination of Stachys sylvatica by combining field comparisons between pristine and invaded sites with an experimental introduction of Impatiens glandulifera into a previously uninvaded site. We quantified pollinator visitation, pollen loads carried by bumblebees, and conspecific and invasive pollen deposition on S. sylvatica stigmas. Across multiple field sites, stigmas of S. sylvatica in pristine habitats received approximately three times more conspecific pollen than those in invaded sites. Bumblebees dominated the pollinator assemblage across all sites, and in invaded habitats, they carried pollen loads strongly dominated by I. glandulifera. During the experimental introduction, bumblebees rapidly incorporated I. glandulifera into their foraging, while conspecific pollen deposition on S. sylvatica stigmas declined sharply, with 81.5% reduction within 4 days. Our results demonstrate that invasion by I. glandulifera can rapidly impair pollination function of a co-flowering native species through changes in pollen transport and transfer efficiency, even before strong shifts in visitation patterns become apparent. By capturing early invasion dynamics through experimental introduction, this study highlights the importance of direct pollen-based metrics for understanding how plant invasions disrupt pollination processes.
We developed a laboratory-based setup to perform behavioral tests of the effect of the neonicotinoid insecticide Thiacloprid in the CALYPSO® formulation on bumblebees. This setup simulates essential components of navigation and pollination under natural conditions. The behavioral components are exploration, exploratory learning, learning of a rewarded local cue in the context of a specific panorama, and retrieving the memory for this association. The walking bumblebees navigated under their own motivation between a fully functional colony and a training/test arena. They explored the arena and learned the association of a rewarded local cue in the context of a panorama. The rule of association was that the local cue was bound to a particular part of the panorama irrespective of where it appeared in its spatial relation to the entrance gate through which the animal came from the colony. Extinction tests were performed for two conditions, match and mismatch. The match condition resembled the training condition. In the mismatch condition the local cue appeared in a different part of the panorama. Solving this task requires the learning and remembering of a rule under variable conditions, mimicking the cognitive requirements faced by bumblebees under natural conditions. The control animals solved this task, whereas animals treated with Thiacloprid 400 ng CALYPSO® diluted in 4 µL per animal were significantly compromised, as shown by several parameters of the walking trajectories under the match and mismatch conditions. No dose-response functions were established, but a volume of 800 ng CALYPSO® diluted in 8 µL per animal did not show any significant differences from a volume of 4 µL CALYPSO®. The setup and the experimental paradigm are suitable for routine quantitative tests on the effects of insecticides on the cognitive faculties of insects during navigation and pollination.
The gut microbiome of eusocial corbiculate bees, which include honeybees, bumblebees, and stingless bees, consists of anciently associated, host-specific bacteria that play crucial role in nutrition, pathogen defense and host fitness. While the core microbiota of honeybees and bumblebees is well characterized, the composition, spatial organization, and evolutionary dynamics of the microbiota of stingless bees remain poorly understood. This gap is particularly evident in the diverse genus Melipona, where Snodgrassella and Gilliamella, ubiquitous symbionts of honeybees and bumblebees, appear rare or absent, indicating a shift in microbiota composition in these stingless bees. Here, we address this gap by characterizing the microbiota of multiple Melipona species using 16S rRNA amplicon sequencing of newly collected and previously published data from field-collected samples. We also mapped the spatial localization of the dominant microbiota members within the gut regions of Melipona quadrifasciata anthidioides through targeted dissection. The Melipona microbiota is dominated by members of the genera Bifidobacterium, Lactobacillus, Apilactobacillus, Floricoccus, and Bombella, with striking regional structure. Apilactobacillus and Bombella dominate in the crop, whereas Apilactobacillus and other members of the Lactobacillaceae are most abundant in the ventriculus. The ileum lacks Snodgrassella and Gilliamella but contains a putative new symbiont closely related to Floricoccus, as well as strains of Bifidobacterium, Lactobacillaceae (including Apilactobacillus), and Bombella. The rectum is dominated by Bifidobacterium and Lactobacillus. These findings reveal a distinct microbiota architecture in Melipona that differs from other corniculate bees yet retains compartment-specific specialization, suggesting an alternative symbiotic strategy that may reflect unique dietary ecology and evolutionary history. Understanding these patterns advances our knowledge of host-microbe symbiosis and provides a baseline for microbiome conservation in declining stingless bee populations.
To accommodate daily environmental changes, insects exhibit species-specific circadian rhythms in behavior and physiology. Compared to non-social insects, honeybees display robust, socially regulated circadian rhythms aligned with colony tasks, while bumblebees show more flexible rhythms linked to simpler social structures. The gut microbiota has been proposed to influence circadian rhythms in mammals through microbial metabolites, hormone regulation, and gene expression. However, its role in honeybee and bumblebee circadian rhythms remains unclear. In this study, we generated gnotobiotic honeybees and bumblebees to explore microbiota-driven circadian modulation through behavioral and transcriptional analyses. Behavioral assays showed that gut microbiota had no significant effect on bumblebee locomotor activity, rhythmicity strength, or free-running period under the tested conditions. In contrast, microbiota colonization in honeybees led to increased daily activity, enhanced rhythmicity strength, and stronger circadian oscillations without affecting the intrinsic free-running period. Notably, qPCR analysis revealed that, at T5, colonized bees exhibited higher per expression together with reduced cwo and serotonin receptor 5-ht1 expression in the honeybee brain. These transcriptional changes indicate potential links between the gut microbiota and honeybee circadian rhythms via the gut-brain axis, possibly involving altered serotonergic signaling. Overall, our results indicate an association between gut microbiota and circadian rhythmicity in honeybees, whereas bumblebees showed little responsiveness, suggesting potential species-specific interactions between microbial signals and host circadian systems. This study advances understanding of microbiota-driven behavioral regulation in eusocial insects and lays the groundwork for future mechanistic investigations.