Evolutionary trees are of central importance in biology to develop explanatory frameworks for many kinds of studies, including studies of behaviour, ecology, and conservation. Since the last major estimate of evolutionary relationships among many bumblebee species two decades ago, there have been revisions to their taxonomy, descriptions of new species, and sequencing of many rarer species. By combining: (1) earlier data mostly from slow-evolving nuclear genes as a backbone tree; with (2) new data from fast-evolving mitochondrial COI barcodes to resolve more of the twigs on the tree, including sequences obtained from rare old specimens with ancient DNA techniques; as well as (3) the results from broader genomic data for resolving deep relationships, we make a Bayesian estimate of evolutionary relationships with BEAST 2 among all 289 published and accepted extant bumblebee species, an increase of more than 29% of the species on the previous largest tree. The new tree will serve as a framework for future comparative studies that should enable broader insights into the evolution and ecology of all bumblebees. We illustrate this with an analysis of the evolution of some male morphological characters related to male mate-searching behaviour. We also present a novel map of spatial variation in net diversification rates among bumblebee species world-wide, which indicates an especially rapid net diversification within the more recent Mesoamerican and South American faunas.

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.

The mandibular glands and the tergal glands have been suggested to play a crucial role in queen pheromone production. However, very few studies have linked changes in mandibular and tergal gland morphology to variations in their pheromone secretions. In this study, we aimed to reveal the age-related morphological development of these glands based on the morphological images and traits. We provided detailed information about how the mandibles, the mandibular glands, and the tergal glands changed in Apis cerana virgin queens aged 0 to 10 days. We detected only slight variations in mandible size (length and width of bilateral mandibles) across different age groups. However, the age of virgin queens had a significant effect on the morphological development of their mandibular and tergal glands, as reflected by the mandibular gland area and the morphology of tergal gland cell nuclei. The mandibular gland area of the 7-day-old virgin queens was significantly larger than that of the other age groups. Concurrently, the development of tergal glands reached a stable plateau in queens aged 5 to 7 days. In addition, significant differences were also detected in the surface area of tergal gland cells and the cell nucleus, and the cell nucleus diameter among tergites III, IV, and V. Compared to tergites IV and V, the cell nucleus surface area and the nucleus/cytoplasm index in tergite III exhibited greater variations across different age groups. These results are valuable for expanding our understanding of exocrine gland development in A. cerana queens.

Pollen is one of the main food sources for honeybees. The honeybee gut microbiota plays a crucial role in maintaining digestive function and host health during long-term coevolution. While the consumption and utilization of pollen have been extensively studied, there is limited information about the effects of pollen on the gut microbiota of Apis cerana. In this study, we used 16S rRNA amplicon sequencing to evaluate the effects of four natural pollens (oilseed rape pollen, camellia pollen, lotus pollen and buckwheat pollen) and two pollen substitutes (Diet 1 and Diet 2) on the hindgut microbiota of newly emerged A. cerana worker bees, following feeding periods of 5, 10 and 15 days. The results showed that Firmicutes and Proteobacteria are dominant in the gut microbiota of A. cerana. A. cerana workers fed with pollen diets had a higher diversity of gut microbiota than those fed with pollen substitutes. There have been significant differences in the gut microbiota structure and relative abundance of the core microbial community among A. cerana workers supplied with different diets. Our results confirm that gut bacterial communities of A. cerana can be influenced by pollen diets and may play an important role in host adaptation.

Targeting odorant-binding proteins (OBPs) is a promising strategy for developing sustainable, behavior-modifying pest control agents. The unfamiliarly highly expressed OBP7 from Acyrthosiphon pisum (ApisOBP7) is a known binder of aphid alarm pheromone (E)-β-farnesene. Here, indole was identified as a moderate binder of ApisOBP7 (Ki = 28.47 μM) with repellent activity against A. pisum (repellent rate = 32.67%). Using this scaffold, 15 novel indole-2-carboxylate derivatives were designed and synthesized to discover selective ligands for ApisOBP7. Compound B02 exhibited an excellent affinity (Ki = 2.73 μM) to ApisOBP7. Molecular dynamics simulations suggested its high affinity originated from strong hydrophobic interactions and key hydrogen bonds. This strong binding conferred efficient repellency for B02 with 57.33% repellency rate at a 5 μg dose. Critically, RNAi study on ApisOBP7 significantly reduced repellent activity of indole (from 32.67% to 10%) and B02 (from 57.33% to 20%). This work validates ApisOBP7 as a valuable target for novel aphid repellent design.

The Qinghai-Tibet Plateau (QTP) region is the greatest hotspot of social bumblebee diversity worldwide. However, how the high diversity of bumblebees in the QTP evolved and contributed to their diversification in adjacent and far-reaching regions remains unclear. Here, we explored this question using a widespread bumblebee species, Bombus pyrosoma. Phylogenomic analysis revealed a history of dispersal out of the QTP and distinct lineage divergence along elevations within the species. Four major chromosomal inver-sion variants were identified, and shifts in their frequency with altitude were consistent with patterns of lineage divergence. The highland bumblebees showed stronger metabolic robustness and better flight performance under cold environments, whereas lowland ones excelled under warm conditions. Forty-one percent of the positively selected genes were located in the inversion regions and were mostly associated with fatty acid metabolism and information processing. Mutation with a strong candidate gene, elovl6, possibly modulated long-chain fatty acid elongation, which facilitated bumblebee flight under thermal stress. Finally, genomic inversion analyses across 22 bumblebee species-17 native to the QTP-corroborate the widespread involvement of inversions in the elevational diversification of bumblebees. Overall, our findings revealed that chromosomal inversions play a pivotal role in facilitating local adaptation and dispersal out of the QTP in a bumblebee, providing insights into the genomic underpinnings of bumblebees' diversification.

Bumblebees of the subgenus Sibiricobombus Vogt, 1911 of the genus Bombus Latreille, 1802 are associated with dry grasslands in the montane regions of Mongolia, Central Asia, the Qinghai-Tibetan Plateau and surrounding mountains, to as far west as Greece. Many of the taxa in this group were described initially from their colour patterns and have at different times been regarded as separate species or have been put into different combinations that were variously regarded as species. We examine integrated evidence for species as evolutionarily independent lineages, comparing evidence from species’ gene coalescents, based on fast-evolving barcode-like DNA sequences, and from discontinuous variation in skeletal morphology and in colour patterns. Many sequences of Sibiricobombus taxa appear from their AT bias at the third codon position to be recent low-divergence numts. Higher frequencies of these numts may be associated with more substitutions within the barcode-primer-binding regions of the mitogenome since the divergence of bumblebees from the lepidopteran source of the primers. Nonetheless, in this particular case, all but one of the 12 species’ gene coalescents support candidate species that are corroborated by morphological diagnoses. For the 11 corroborated species, the status of three species is revised and one new replacement name is proposed: Bombus sibiricus (Fabricius, 1781), B. semenovi Morawitz, 1887 stat. rev., B. oberti Morawitz, 1883, B. morawitzi Radoszkowski, 1876, B. sulfureus Friese, 1905, B. niveatus Kriechbaumer, 1870, B. obtusus Richards, 1951, B. tescorum Williams nom. nov. et stat. rev., B. longiceps Smith, 1878 stat. rev., B. falsificus Richards, 1930 stat. rev., and B. asiaticus Morawitz, 1875. Nine new synonyms are recognised. Estimates are provided for (1) an evolutionary tree for species and (2) for the ancestral species’ distributions.

Drones are the males in the honey bee population that provide high quantity and quality sperm to mate with the queen and ensure colony reproduction. To understand spermatogenesis in the drone testis at the molecular level, reverse transcription quantitative PCR (RT-qPCR) is a standard and useful means to investigate the target genes. However, a lack of appropriate reference genes hinders the accurate normalization of target genes. To identify stable reference genes in the drone testis during the meiosis stages of honey bee, we assessed eight candidate genes using BestKeeper, delta-CT, GeNorm, NormFinder, and RefFinder tools. Moreover, the expression pattern of stat92e and dicer1 was used to validate the stability of the selected reference genes. The results showed that the most stable reference gene in the testis during meiosis stages varied according to different algorithms, and gapdh was the most stable reference gene in the drone testis during meiosis stages when integrating these different algorithms. In addition, gapdh, rps18, and the combination of gapdh & rps18 were optimal reference genes to normalize the expression of the target genes in testis during meiosis stages, as validated by the expression of stat92e and dicer1. Our findings provide a conducive foundation for the accurate quantification of gene expression levels in the testis during the drone meiosis stages of the honey bee.

After the introduction of various Apis mellifera subspecies into China, prolonged breeding and selection have substantially altered the genetic structure and diversity of commercial populations. Currently, A. mellifera has become the most widely managed and numerically dominant honey bee species in China, providing important agricultural pollination services and substantial economic benefits. Nevertheless, the genetic diversity and progenitor of commercially managed A. mellifera populations within different regions in China remain poorly understood. To assess the maternal lineage diversity of A. mellifera populations and to support regional breeding strategies, we examined DNA sequence diversity in the cytochrome C oxidase I (COI) and tRNAleu-COII gene segment of the mitochondrial genome in 178 samples of A. mellifera drones from 12 sites in the urban districts of Beijing. Three haplotypes were found for the COI gene segment, and eight haplotypes were discovered for the tRNAleu-COII gene segment. Mitochondrial sequencing revealed that the frequency of C evolutionary lineage dominates in A. mellifera populations in Beijing. In addition, one unexpected haplotype from the Z lineage was detected. The subspecies Apis mellifera ligustica, representing the C1 haplotype, demonstrates the widest distribution and highest frequency. Our results also showed that significant genetic differentiation exists among commercial populations of A. mellifera in the urban districts of Beijing.

Chitinases are crucial enzymes in insect growth and development processes and have thus become potential targets for the control of insect pests. Rhodanine (5-alkenylthiothiazolidinone) is an important fragment with various biological activities in agriculture. Therefore, 24 novel 5-alkenylthiothiazolidinone derivatives were designed and synthesized by introducing a hydrophobic hydrocarbon chain and a benzylformamide group on the basis of the conserved binding cavity of three chitinases, OfChtI, OfChtII and OfChi-h. These 5-alkenylthiothiazolidinone analogs were found for the first time to have inhibitory effects on OfChtII, except for their ability to inhibit the other two known chitinases, OfChtI and OfChi-h. The inhibitory activities of the target compounds against OfChtI and OfChi-h ranged from 40 % to 95 % at a concentration of 100 mu M. In particular, some compounds were also found to have an inhibitory rate of 50 % against OfChtII. Among them, the Ki values of the most potent compound 5e-9 were 5.82, 27.5 and 6.77 mu M against OfChtI, OfChtII and OfChi-h, respectively. The inhibition mechanism studies revealed that both pi stacking interactions and hydrophobic interactions were vital for the binding of Compound 5e-9 with the three chitinases. A lethal and sublethal bioassay study revealed that Compound 5e-9 could affect the growth and development of Asian corn borers. This work suggested that 5-alkenylthiothiazolidinone could be a novel inhibitor scaffold against multichitinase for the development of insect growth regulators.

The development of novel insecticides with low bee toxicity has become increasingly urgent as many high bee-toxicity neonicotinoids have been progressively restricted. In this study, novel halogenated phenyl-substituted α-butenolide compounds were designed, optimized and synthesized through a progressive strategy based on insect nicotinic acetylcholine receptor and the calculated oil-water partition coefficient (ClogP) of compounds. Among these, the difluorophenyl-substituted compound 3cj (lethal medium concentration [LC50] = 40.96 µg/mL) exhibited the highest insecticidal activity against Myzus persicae and a relatively low ClogP of 4.25. The molecular docking study revealed that compound 3cj mainly interacted with two key residues TRP147 and TYR188 of Aplysia californica-acetylcholine-binding protein. The bee-toxicity bioassay and molecular mechanism study indicated that compound 3cj exhibited low acute contact toxicity against Apis mellifera, probably due to its reduced binding energy with the honeybee chimeric receptor AmeIα8/ratβ2. This study provides rational guidance for novel α-butenolide compounds as low bee-toxicity insecticidal candidates based on their target receptors and molecular physicochemical properties.

Due to the weak self-pollination of blueberries, pollinators are essential in greenhouse blueberry production. In this study, we examined foraging activity and pollen deposition after the flowers were visited by three pollinators (Bombus terrestris Linnaeus, Apis mellifera Linnaeus and Apis cerana Fabricius) in "Bluesouth (M7)" plants grown in greenhouses. Single visit duration of A. mellifera (18.55 +/- 0.44 s) was significantly longer than those of A. cerana (12.66 +/- 0.17 s) and B. terrestris (9.31 +/- 0.41 s). Evaluating pollen deposition on stigmas is an important method for comparing pollination efficiency. We found that B. terrestris deposits more pollen grains than A. cerana and A. mellifera on blueberry stigmas, both during a single visit and during a whole day of open pollination. Based on these findings, we concluded that B. terrestris is the most effective pollinator for "Bluesouth (M7)" blueberry grown in greenhouses in Northern China.

In recent years, honey bees have been stressed by multiple factors, with malnutrition posing a significant threat to the healthy development of honey bee colonies. To keep a colony healthy and productive, beekeepers supply their colonies with supplementary pollen or commercial pollen substitutes during periods of pollen dearth or insufficient pollen quantity or quality. In this study, we evaluated the effects of four natural pollen types (oilseed rape pollen, camellia pollen, lotus pollen and buckwheat pollen) and two commercial pollen substitutes (Diet 1 and Diet 2) against a control group (sucrose solution) on Apis cerana through cage experiments. The food consumption, live body weight, longevity, hypopharyngeal gland development and midgut proteolytic enzyme activity of caged workers were measured. The food consumption rates of oilseed rape pollen and buckwheat pollen were greater than the other diets. Oilseed rape pollen and camellia pollen were recognized as excellent-quality diets for hypopharyngeal gland development and midgut proteolytic enzyme activity. Over the entire experimental period, the caged workers fed with lotus pollen had a similar diet consumption and body weight to those fed with pollen substitutes, and these bees had a significantly higher survival rate than those fed with other diets. The results indicated that the commercial pollen substitutes appeared to be less beneficial to caged A. cerana workers than the natural pollen resources.

Pesticides represent a major threat to pollinators' health. Bumblebees, as essential pollinators, are one of the bees severely impacted by pesticide exposure. The lack of sensitivity data across diverse species hinders the breeding and application of pesticide-tolerance pollinators trains. This study assessed the acute oral toxicity of dimethoate, an organophosphorus insecticide and a positive control in environmental risk assessments, in five bumblebee species: the commercial European bumblebee species Bombus terrestris and four wild Chinese native bumblebee species (B. ignitus, B. ganjsuensis, B. pyrosoma, and B. lantschouensis). The 48-h median lethal doses (LD50) were determined as follows: 1.64 μg/bee for B. terrestris, 0.98 μg/bee for B. ignitus, 0.91 μg/bee for B. ganjsuensis, 0.71 μg/bee for B. pyrosoma and 0.70 μg/bee for B. lantschouensis. Morphological traits including body wight and forewing length were measured, revealing a positive correlation between them and a negative relationship between body weight and sensitivity to dimethoate. These results indicate that easily measurable morphological traits like body weight could serve as potential indicators to predicting dimethoate tolerance in bumblebees. Our findings are of importance for bumblebee toxicology, addressing the knowledge gap in non-commercial bumblebees of pesticide sensitivity. By expanding acute toxicity data to include underrepresented wild species, this study provided valuable insights for selecting and breeding pollinator strains with enhanced insecticide tolerance, highlighting the need for a broader species range in toxicity tests.

Background/Objectives: Apis cerana development is described as comprising four stages: embryo, larva, pupa, and adult. There are significant differences between workers and drones in terms of physiological functions and social roles, and the formation of the organ primordia occurs during the embryonic stage. Therefore, the objective of this study is to investigate the differential expression of and alternative splicing of genes in worker and drone embryos and to explain their unique developmental patterns. Methods: Long-read sequencing (PacBio Iso-Seq) and short-read sequencing (Illumina RNA-Seq) were used to investigate worker and drone embryo gene expression differences in A. cerana across five developmental points (12, 24, 36, 48, and 60 h). Results: The study identified 59,254 common isoforms, with 5744 and 5106 isoforms specific to worker and drone embryos, respectively. Additionally, a new transcript of the csd gene was identified. The number of differentially expressed genes (3391) and differential splicing events (470 genes) peaked at the 24-h embryonic stage. Differential splicing events of csd, dsx, and Y-y were observed in the worker and drone embryos. Conclusions: The gene expression results indicated that the 24-h embryonic point is a critical period for the expression of genes related to developmental and behavioral differences between workers and drones. The findings provide a theoretical basis for future research on the developmental differences between workers and drones.

Pesticides are considered a major factor in the decline of bee populations. Flupyradifurone, a novel insecticide, is believed to be relatively ‘bee-safe’. This study aims to evaluate the acute and chronic toxicity of flupyradifurone and assess its risks to both commercial bumblebee Bombus terrestris and the Asian native species B. lantschouensis. Oral toxicity tests demonstrated species-specific sensitivity, with B. lantschouensis exhibiting 5.4-fold higher acute toxicity (72-h LD50: 5.1 μg/bee vs. 28 μg/bee) and 3-fold lower chronic toxicity (No Observed Adverse Effect Concentration, NOAEC: 20 μg/mL vs. 60 μg/mL) compared to B. terrestris. Risk assessments indicated low Hazard Quotients (HQ) of 4 for B. terrestris and 20 for B. lantschouensis. However, the Exposure Toxicity Ratio (ETR) values from both screening and first-tier assessments exceeded the trigger levels, necessitating further testing. This study provides crucial data on the acute and chronic toxic effects of flupyradifurone and highlights the need for more comprehensive insecticide risk assessments, particularly for non-Apis pollinators, to better protect these vital species.

The application of forchlorfenuron (CPPU) and bee pollination are common artificial pollination practices in the protected cultivation of melon. Compared with the CPPU treatment, bee pollination significantly increases fruit quality. In this study, the effects of these two pollination methods on melon fruit growth dynamics and quality were compared. High-performance liquid chromatography (HPLC) and headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) were employed to analyse the sugar content and volatile organic compounds (VOCs) in mature fruits. The results showed that bumblebee-pollinated melons initially grew faster (0-9 days after anthesis) but were subsequently surpassed by CPPU-treated fruits until maturity. At maturity, the CPPU-treated melons had slightly greater fruit weight and flesh thickness, whereas the bumblebee-pollinated melons displayed a higher fruit set rate, a more uniform fruit shape, a greater seed count and denser rind netting. Compared with CPPU-treated fruits, bumblebee-pollinated melons contained higher sucrose levels but lower fructose and glucose levels. VOC profiling revealed that alcohols, aldehydes, and esters dominated the volatile profiles in both groups, but the total VOC content in bumblebee-pollinated melon flesh was 1.9 times greater than that in CPPU-treated melons. Nonmetric multidimensional scaling (nMDS) analysis indicated significant differences in VOC composition between the two pollination methods. Orthogonal partial least squares-discriminant analysis (OPLS-DA) identified five discriminant VOCs: 2-ethyl-1-hexanol, 2-nonanol, 1-nonanol, 6-nonen-1-ol, and 3,6-(E, Z)-nonadien-1-ol. These findings demonstrate that bumblebee pollination significantly improves the fruit appearance, flesh sweetness, and aroma of protected-cultivation melons and should be actively promoted as a sustainable alternative to CPPU application.

Polyandry is widespread among eusocial Hymenoptera, and the honey bee is a typical representative of this. It has been widely shown that polyandry can confer benefits to queens and their offspring, including enhanced productivity and fitness, stronger resistance to pathogens, and resilient division of labor, which promotes colony-level homeostasis. A previous study conducted in Australia demonstrated that 33.8% commercial Apis mellifera queens produced in autumn were not adequately mated. Beekeepers of Apis cerana in China also claimed that the queens reared in autumn are inferior to those reared in spring. To confirm whether the quality difference of queens produced in different periods is related to their mating frequency, we estimated the observed mating frequency (k) and the effective mating frequency (me) of A. cerana queens produced at the beginning and end of the queen production season in Liaoning Province. We found that all the queens were suitably mated and there was no significant difference in the mating frequency between early spring queens and late summer queens. In addition, our study indicated that the queens and their offspring workers owned a high level of heterozygosity and their inbreeding coefficients were universally low. Further studies on the queens’ performance and health are required to verify the statement that the queens reared in autumn are not as good as those reared in spring.

Developing novel-structured nAChR modulators is urgent due to the high bee toxicity of current neonicotinoids. In this work, a pharmacophore model of an nAChR modulator was generated, and a series of novel furan β-butenolide compounds were designed and synthesized. The bioassay study demonstrated that the insecticidal activity of compound 8c was the best (LC50 = 54.68 μg/mL) against Myzus persicae, possibly due to its binding mode being more similar to that of flupyradifurone, as well as its interactions with specific key residues (ARG55, TRP143, and TYR89) within Ls-AChBP. Notably, compound 8c also exhibited low acute contact toxicity to Apis mellifera. An innovative mutated AChBP model simulating bee nAChR was generated first in silico, and the low bee toxicity of compound 8c was attributed to the decrease of its binding energy with the mutated bee-like AChBP. This study presents an innovative approach to design and verify β-butenolide compounds as low-bee-toxicity insecticidal candidates.

The honeybee Apis cerana as an important pollinator contributes significantly to ecological diversity. In recent years, it has been used as a common pollinator in greenhouses, but it is highly susceptible to heat stress, which affects its behavior, physiology, survival, and gene expression. Here, we conducted transcriptomic analysis to identify differentially expressed genes (DEGs) and reveal the associated biological processes in the queen head and ovary of honeybee A. cerana under different temperatures. Differential expression analysis revealed 116 DEGs (72 upregulated, 44 downregulated) in the head and 106 DEGs (78 upregulated, 28 downregulated) in the ovary after 24 h of heat stress. At 96 h, 29 DEGs (17 upregulated, 12 downregulated) were identified in the head, and 44 DEGs (34 upregulated, 10 downregulated) in the ovary. After 168 h, the number of DEGs increased significantly: 846 DEGs (567 upregulated, 279 downregulated) in the head, 479 DEGs (296 upregulated, 183 downregulated) in the ovary, and 582 DEGs (338 upregulated, 244 downregulated) in the thorax. DEGs associated with metabolic processes, signaling, and transport pathways were significantly altered under heat stress, potentially contributing to the reduced reproductive and growth capacity of bees. Additionally, genes related to antioxidant activity, nutrient metabolism, heat shock proteins, zinc finger proteins, and serine/threonine-protein kinases were differentially expressed across treatments. Overall, the head and ovaries of honeybee queens show a significant response to heat shock, and these responses are related to antioxidant genes, heat shock proteins, and metabolic regulation, our findings provide genetic information for the breeding of heat-resistant bee strains.