Post-transcriptional regulation of Dufour's gland reproductive signals in bumble bees.

Pheromone communication is a key mechanism by which the reproductive division of labor is maintained within insect communities. Understanding how pheromones evolved to regulate social behavior requires knowledge of the molecular regulation of their production. However, even in cases where pheromones were identified, our understanding of their biosynthesis and molecular regulation remains limited. Bumble bees provide a unique system to explore pheromone biosynthesis since workers produce ester sterility signals in their Dufour's gland that differ from gyne-specific esters and are not produced by queens. These esters are hypothesized to be produced in the exocrine gland where they are stored, and indeed queens, gynes and workers differ significantly in the expression of Dufour's gland genes coding to enzymes involved in the biosynthesis of esters. However, a previous transcriptome analysis revealed no gene expression differences in the Dufour's gland of workers despite differences in both ester production and ovarian activation, suggesting that ester production may be regulated lower down. Proteomics of the Dufour's gland of queens, gynes, and workers recovered over 2400 proteins and broadly matched the previous RNAseq data. However, more than 100 differentially expressed proteins were found between the worker groups, including key enzymes in fatty acid biosynthesis, indicating that the regulation of reproductive signal biosynthesis in workers is done post-transcription. Overall, our data provide evidence that pheromone biosynthesis in the Dufour's gland is caste specific, that gynes and workers are likely using different enzymes to make their respective wax esters, and that the regulation on pheromone production in queens, gynes and workers is likely done at different regulatory levels, with workers signals being subjected to regulation at the protein level.

Surviving high temperatures: The crucial role of vesicular inhibitory amino acid transporter in Asian honeybee, Apis cerana.

Asian honeybees (Apis cerana) play a crucial role as pollinators to service for the ecological stability. However, their proliferation and growth are significantly impacted by environmental temperature stress. This study delves into the function of the Apis cerana vesicular inhibitory amino acid transporter gene (AcVIAAT) in safeguarding Asian honeybees against high-temperature stress. The AcVIAAT gene exhibits positive responsiveness in honeybees subjected to varying thermal conditions by triggering the genes associated with oxidative stress. Molecular docking, co-immunoprecipitation, and ELISA verify the capacity of the AcVIAAT protein to interact with γ-aminobutyric acid (GABA), a key inhibitory neurotransmitter. Administering GABA to honeybees significantly improves their survival rate under high-temperature stress and also simultaneously upregulating oxidative stress-related genes. Therefore, these findings reveal that the AcVIAAT gene enhances the thermoregulatory capacity of honeybees by modulating oxidative stress-related genes through facilitating GABA transport. The characterization of six non-synonymous SNPs in the AcVIAAT gene among A.cerana populations distributed across both the northern and southern regions indicates a potential association between gene variation and environmental adaptation. Our results contribute to elucidating the molecular mechanisms underlying high-temperature tolerance in Asian honeybees and provide a promising genetic marker for enhancing heat tolerance through genetic improvement.

Systemic analysis of lipid metabolism from individuals to multi-organism systems.

Lipid metabolism is recognised as being central to growth, disease and health. Lipids, therefore, have an important place in current research on globally significant topics such as food security and biodiversity loss. However, answering questions in these important fields of research requires not only identification and measurement of lipids in a wider variety of sample types than ever before, but also hypothesis-driven analysis of the resulting 'big data'. We present a novel pipeline that can collect data from a wide range of biological sample types, taking 1 000 000 lipid measurements per 384 well plate, and analyse the data systemically. We provide evidence of the power of the tool through proof-of-principle studies using edible fish (mackerel, bream, seabass) and colonies of Bombus terrestris. Bee colonies were found to be more like mini-ecosystems and there was evidence for considerable changes in lipid metabolism in bees through key developmental stages. This is the first report of either high throughput LCMS lipidomics or systemic analysis in individuals, colonies and ecosystems. This novel approach provides new opportunities to analyse metabolic systems at different scales at a level of detail not previously feasible, to answer research questions about societally important topics.

The effects of queen mandibular pheromone on nurse-aged honey bee (Apis mellifera) hypopharyngeal gland size and lipid metabolism.

Queen honey bees (Apis mellifera) release Queen Mandibular Pheromone (QMP) to regulate traits in the caste of female helpers called workers. QMP signals the queen's presence and suppresses worker reproduction. In the absence of reproduction, young workers take care of the queen and her larvae (nurse tasks), while older workers forage. In nurses, QMP increases lipid stores in abdominal fat tissue (fat body) and protein content in hypopharyngeal glands (HPG). HPG are worker-specific head glands that can synthesize proteinaceous jelly used in colony nourishment. Larger HPG signifies ability to secrete proteinaceous jelly, while shrunken glands characterize foragers that do not make jelly. While it is known that QMP increases abdominal lipid stores, the mechanism is unclear: Does QMP make workers consume more pollen which provides lipids, or does QMP increase lipogenic capacity? Here, we measure abdominal lipogenic capacity as fatty acid synthase (FAS) activity while monitoring abdominal protein content and HPG size in caged workers. Cages allow us to rigorously control worker age, pheromone exposure, and diet. In our 2-factorial design, 3- vs. 8-day-old workers (age factor) were exposed to synthetic QMP or not (pheromone factor) while consuming a lipid deficient diet. We found that QMP did not influence abdominal FAS activity or protein content, but QMP still increased HPG size in the absence of dietary lipids. Our data revealed a positive correlation between abdominal protein content and HPG size. Our findings show that QMP is not a strong modulator of lipogenic capacity in caged worker bees. However, our data may reflect that QMP mobilizes abdominal protein for production of jelly, in line with previous findings on effects of honey bee Brood Pheromone. Overall, our study expands the understanding of how QMP can affect honey bee workers. Such insights are important beyond regulatory biology, as QMP is used in various aspects of beekeeping.

Acetylcholine and choline in honey bee (Apis mellifera) worker brood food are seasonal and age-dependent.

Nursing honeybees produce brood food with millimolar concentrations of acetylcholine (ACh), which is synthesized through head gland secretions mixed with honey stomach contents. While we previously demonstrated the necessity of ACh for proper larval development, the dynamics of ACh levels throughout ontogenesis and their seasonal variations have remained unclear until now. Our HPLC analysis reveals dependencies of choline and ACh levels on larval development days (LDDs), influenced by seasonal (April-September) variations. Median ACh concentrations peak on LDD 2, declining significantly toward cell capping, while choline levels are lowest during the initial LDDs, rising markedly toward cell capping. Seasonal patterns show peak ACh levels from April to June and a low in August, paralleling choline's peak in July and low in August. This seasonality holds consistently across multiple years (2020-2022) and colonies, despite potential variations in colony performance and environmental conditions. Our analysis found no correlation between temperature, sunshine, precipitation, or favourable foraging days and ACh/choline levels, suggesting the involvement of additional factors. These findings underscore the seasonal fluctuation of ACh levels and its potential implications for the genetic programs governing winter bee development.

Fat body lipogenic capacity in honey bee workers is affected by age, social role and dietary protein.

All organisms need to balance processes that consume energy against those that produce energy. With an increase in biological complexity over evolutionary time, regulation of this balance has become much more complex, resulting in specialization of metabolic tasks between organelles, cells, organs and, in the case of eusocial organisms, between the individuals that comprise the 'superorganism'. Exemplifying this, nurse honey bees maintain high abdominal lipids, while foragers have very low lipid stores, likely contributing to efficient performance of their social role, and thus to colony fitness. The proximate mechanisms responsible for these metabolic differences remain poorly understood. Here, we investigated the effects of age, worker class and dietary macronutrients on the abdominal activity of fatty acid synthase (FAS), the enzyme responsible for de novo synthesis of fatty acids, as well as the effects of age on lipase activity, enzymes responsible for the breakdown of stored lipids. We found that FAS but not lipase activity declines as bees age past peak nursing age. Feeding both nurses and foragers carbohydrates increased FAS activity compared with starved bees, but, whether fed or starved, nurses had much higher FAS activity than similarly treated foragers, implicating reduced lipid synthesis as one component of foragers' low lipid stores. Finally, we used artificial diets with different amounts of protein and fat to precociously induce low, forager-like FAS activity levels in nurse-age bees deprived of protein. We speculate that reduced protein appetite and consumption during the nurse-forager transition is responsible for suppressed lipid synthesis in foragers.

Age-dependent hypopharyngeal gland size and protein content of stingless bee workers, Tetragonula pagdeni.

Eusocial insects, such as stingless bees (Meliponini), depend on division of labour, overlapping generations, and collaborative brood care to ensure the functionality and success of their colony. Female workers transition through a range of age-specific tasks during their lifespan (i.e., age-polyethism) and play a central role in the success of a colony. These age-specific tasks (e.g., brood care or foraging) often closely coincide with key physiological changes necessary to ensure optimal performance. However, our understanding of how nutrition, age, and polyethism may affect the development of such physiological traits in stingless bees remains limited. Here we show that pollen consumption and age-polyethism govern hypopharyngeal gland (HPG) acini size and protein content in Tetragonula pagdeni. By conducting a controlled laboratory experiment we monitored the effect of pollen consumption on worker bee survival as well as assessed how a pollen diet and age affected their HPG acini width and protein content. Further, we sampled nurses and foragers from field colonies to measure the effect of age-polyethism on HPG acini width. We found that pollen consumption enhanced survival and led to increased HPG acini width and protein content and that HPG acini were as expected largest in nurse bees. Our findings highlight the beneficial effects of an adequate diet for physiological development and health in stingless bees and reveal that age-polyethism is the key factor governing HPG size in worker bees. As HPGs are imperative for collaborative brood care-an essential component of eusociality-the data provide a foundation for future studies to investigate the impact of potential environmental stressors on a critical physiological trait in stingless bees which may serve as a proxy to understand the effects at the colony level.

Brazilian stingless bee honey: A pioneer study on the in vitro bioaccessibility of phenolic compounds.

In this study, the effect of in vitro gastrointestinal digestion of phenolic compounds, the total phenolic content, and the antioxidant potential of stingless bee honey were investigated. Among the 33 phenolic compounds investigated, 25 were quantified, and only eight were not bioaccessible (p-aminobenzoic acid, sinapic acid, pinobanksin, isorhamnetin, quercetin-3-glucoside, syringaldehyde, coumarin, and coniferaldehyde). Benzoic acid was predominant in most undigested samples (21.3 to 2414 µg 100 g-1), but its bioaccessibility varied widely (2.5 to 534%). Rutin, a glycosylated flavonoid, was quantified in all samples and might have been deglycosylated during digestion, increasing the bioaccessibility of quercetin in a few samples. Overall, the concentration of phenolic compounds prior digestion and their bioaccessibility varied greatly among samples. Nevertheless, higher concentrations before digestion were not correlated to greater bioaccessibility. This study is the first to assess the in vitro bioaccessibility of phenolic compounds in SBH, providing novel insights into SBH research.

Identification of low-abundance proteins in the royal jelly using the Osborne classification method.

Royal jelly (RJ) is recognized as healthy food, with a high content of proteins. These proteins play important roles in honeybee caste and human health, but the proteomic analysis of low-abundance proteins in RJ has long been a challenge. Herein, we used the Osborne classification method to separate the RJ proteins of Xinjiang black bees into various fractions. The globulin, ethanol-soluble protein, and glutelin fractions were further separated by SDS-PAGE, and proteomic analysis was carried out by LC-MS/MS and searched against the UniProt database. A total of 23 secretory proteins were identified by proteomic analysis, in which 7 proteins were identified for the first time in RJ. The Osborne classification method combining one-dimensional gel electrophoresis-based proteomic analysis allows the identification of low-abundance proteins in the RJ and greatly extends the knowledge about the components and functions of RJ proteins. The raw data are available via ProteomeXchange with the identifier PXD023315. SIGNIFICANCE: This study makes an important contribution to the research of the components and functions of low-abundance royal jelly proteins for the following reasons.

The effect of pollen monodiets on fat body morphology parameters and energy substrate levels in the fat body and hemolymph of Apis mellifera L. workers.

Human activities associated with large-scale farms and the monocultures expose honey bees to one type of food. Moreover, there is an ongoing decline of plant species producing pollen and nectar in Europe. A poorly balanced diet affects a number of processes occurring in a bee's body. The fat body and hemolymph are the tissues that participate in all of them. Therefore, the aim of our study was to determine the effect of hazel, pine, rapeseed, buckwheat, phacelia and goldenrod pollen on the morphological parameters of fat body trophocytes, the diameters of cell nuclei in oenocytes and the concentrations of compounds involved in energy metabolism (glucose, glycogen, triglycerides and protein). In the cage tests, the bees were fed from the first day of life with sugar candy (control group) or candy with a 10% addition of one of the 6 pollen types. Hemolymph and fat body from various locations were collected from 1-, 7- and 14-day-old workers. Pollen produced by plant species such as hazel and pine increased glucose concentrations in the bee tissues, especially in the hemolymph. It can therefore be concluded that they are valuable sources of energy (in the form of simple carbohydrates) which are quickly used by bees. Pollen from plants blooming in the summer and autumn increased the concentrations of proteins, glycogen and triglycerides in the fat body, especially that from the third tergite. The accumulation of these compounds was associated with an increased the length and width of trophocytes as well as with enhanced metabolic activity, which was evidenced in the increasing diameter of oenocyte cell nuclei. It seems a balanced multi-pollen diet is more valuable for bees, but it is important to understand the effects of the particular pollen types in the context of a mono-diet. In the future, this will make it possible to produce mixtures that can ensure homeostasis in the apian body.

Evolution of Acquired Perfumes and Endogenous Lipid Secretions in Orchid Bees.

Male orchid bees are unique in the animal kingdom for making perfumes that function as sex pheromone. Males collect volatile chemicals from the environment in the neotropical forests, including floral and non-floral sources, creating complex but species-specific blends. Male orchid bees exhibit several adaptations to facilitate perfume collection and storage. When collecting volatile compounds, males apply lipid substances that they secrete from cephalic labial glands onto the fragrant substrate. These lipids help dissolve and retain the volatiles, similar to the process of 'enfleurage' in the traditional perfume industry. We investigated how the chemical composition of acquired perfume and labial gland secretions varied across the phylogeny of orchid bees, including 65 species in five genera from Central and South America. Perfumes showed rapid evolution as revealed by low overall phylogenetic signal, in agreement with the idea that perfume compounds diverge rapidly and substantially among closely related species due to their role in species recognition. A possible exception were perfumes in the genus Eulaema, clustering closely in chemospace, partly mediated by high proportions of carvone and trans-carvone oxide. Labial gland secretions, in contrast, showed a strong phylogenetic signal at the genus level, with secretions of Eufriesea and Exaerete dominated by fatty acids and Eulaema dominated by saturated acetates of chain lengths 12 to 16 C-atoms. Secretions of the majority of Euglossa were heavily dominated by one unsaturated long chain diacetate, (9Z)-Eicosen-1,20-diyldiacetate. However, we also identified few highly divergent species of Euglossa in four subclades (11 species) that appear to have secondarily replaced the diacetate with other compounds. In comparison with environment-derived perfumes, the evolution of labial gland secretion is much slower, likely constrained by the underlying biochemical pathways, but perhaps influenced by perfume-solvent chemical interactions.

Hydroxy Fatty Acid Synthesis-Related mRNA as the Biomarker for Detecting Mislabeling of Honey Entomological Origin.

The authentication of the entomological origin of honey is a widespread concern, necessitating the prompt establishment of an effective approach for distinguishing between Apis cerana cerana honey (ACH) and Apis mellifera ligustica honey (AMH). Hydroxy fatty acids (HFAs) found in honey are bee-derived components synthesized by the mandibular glands of worker bees. We previously discovered significant variations in the hydroxy fatty acid composition between ACH and AMH, suggesting their potential as indicators for identifying the authenticity of the entomological origin of honey. Herein, we identified differentially expressed genes associated with HFA synthesis by conducting transcriptome sequencing of the mandibular glands of AC and AM honeybees. Subsequently, we proposed a method for the relative quantitative analysis of bee-derived RNA components using real-time fluorescence quantitative polymerase chain reaction, which was supplemented by multivariate statistical analysis to further discern differences in HFA synthesis-related mRNA between ACH and AMH. The results showed that the mRNAs of FAXDC2 (fatty acid hydroxylase domain-containing protein 2) and FAS (fatty acid synthase) may serve as indicators to discern the entomological origin of honey. This study presents two novel biomarkers for detecting mislabeling of the entomological origin in ACH and AMH based on variations in bee-derived components.

From consumption to excretion: Lithium concentrations in honey bees (Apis mellifera) after lithium chloride application and time-dependent effects on Varroa destructor.

BACKGROUND: Owing to its systemic mode-of-action and ease of application, lithium chloride (LiCl) is an ideal varroacide for the control of Varroa destructor infestations in honey bee colonies. To better understand how LiCl functions within a colony, we screened different parts of honey bee anatomy for lithium accumulation. We wanted to elucidate the time-dependent effects of LiCl on V. destructor and its metabolism within honey bees when they were fed continuous LiCl treatments, as well as evaluate potential adverse effects such as accumulation in the hypopharyngeal glands of nurse bees, which could negatively impact queens and larvae. RESULTS: Cage experiments reveal rapid acaricidal onset, with >95% mite mortality within 48 h of treatment. Bee hemolymph analysis supports these observations, showing a rapid increase in lithium concentration within 12 h of treatment, followed by stabilization at a constant level. Lithium accumulates in the rectum of caged bees (≤475.5 mg kg-1 after 7 days of feeding 50 mm LiCl), reflecting the bees' metabolic and excretion process. Despite concerns about potential accumulation in hypopharyngeal glands, low lithium levels of only 0.52 mg kg-1 suggest minimal risk to the queen and 1st- and 2nd-instar larvae. Cessation of LiCl treatment results in a rapid decline in mite mortality in the first 5 days, which increases again thereafter, resulting in mite mortality of 77-90% after 10 days. CONCLUSION: These findings help optimize LiCl application in colonies to achieve high Varroa mortality without unwanted adverse effects and provide important baseline data for future registration. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Cuticular hydrocarbon profiles reveal geographic chemotypes in stingless bees (Hymenoptera: Meliponini).

Cuticular hydrocarbon (CHCs) variation has been detected in various insect taxa, but the potential contribution of cuticular compounds for analyzing intraspecific diversity at the population level has been little explored. Here we assess for the first time intraspecific variation in the CHC profile of stingless bees, using the species Melipona beecheii and Nannotrigona perilampoides. The objective is determining whether intraspecific variation can be useful for population identification. We found species-specific chemical patterns and extensive variation within each species. Notably, chemotypes were significantly associated to geographic origin in N. perilampoides but less so in M. beecheii and we discuss possible explanations for these patterns. Our results support the use of CHCs in conjunction with other methods in emerging problems such as undetected colony mobilization across regions. As CHCs are involved in several aspects of stingless bee recognition and interactions, it would be essential to unravel how these chemical signatures evolve across populations.

Caste-specific development of the dopaminergic system in bumble bees (Bombus ignitus).

The dopaminergic system is involved in caste-specific behaviors in eusocial bumble bees. However, little is known about how the caste differences in dopaminergic system are formed during pupal stages in the brains of bumble bees. Thus, we investigated the levels of dopamine-related substances and expression of genes encoding enzymes involved in dopamine synthesis and metabolism, dopamine receptors, and a dopamine transporter in the brain of female Bombus ignitus. The levels of dopamine and dopamine-related substances in the brain were significantly higher in gynes than in workers from the late pupal stage to emergence, but the dynamics were similar between the castes. The relative expression levels of genes encoding enzymes involved in dopamine synthesis (BigTh and BigDdc) and dopamine metabolism (BigNat) increased significantly from pupal stage to emergence, but there were no differences in the relative expression levels of these genes between castes. A similar pattern was seen in the relative expression levels of four dopamine receptor genes (BigDop1, BigDop2, BigDop3, and BigDopEcR) and a dopamine transporter gene (BigDat). Compared with the honey bee Apis mellifera, the caste-specific dopaminergic system in the bumble bee is less differentiated, which might reflect the degree of behavioral specialization in these two species.

Body-size-dependent effects of landscape-level resource energetics on pollinator abundance in woodland remnants.

Land use change alters floral resource availability, thereby contributing to declines in important pollinators. However, the severity of land use impact varies by species, influenced by factors such as dispersal ability and resource specialization, both of which can correlate with body size. Here. we test whether floral resource availability in the surrounding landscape (the 'matrix') influences bee species' abundance in isolated remnant woodlands, and whether this effect varies with body size. We sampled quantitative flower-visitation networks within woodland remnants and quantified floral energy resources (nectar and pollen calories) available to each bee species both within the woodland and the matrix. Bee abundance in woodland increased with floral energy resources in the surrounding matrix, with strongest effects on larger-bodied species. Our findings suggest important but size-dependent effects of declining matrix floral resources on the persistence of bees in remnant woodlands, highlighting the need to incorporate landscape-level floral resources in conservation planning for pollinators in threatened natural habitats.

Enhanced sensitivity of chimeric insect olfactory co-receptors for detecting odorant molecules.

Insect olfactory receptors (ORs) are seven-transmembrane domain ion channels that function by forming heteromeric complexes with olfactory receptor co-receptors (Orcos). In this study, we investigated the potential for enhancing sensitivity of odor detection and responsivity through genetic modification of Orcos, considering its wider application in odor sensing. First, we measured the intensity of response to 1-octen-3-ol for the mosquito Aedes aegypti OR (AaOR8) when complexed individually with an Orco from the same mosquito (AaOrco), the honeybee Apis mellifera (AmOrco), the silkworm Bombyx mori (BmOrco), or the fruit fly Drosophila melanogaster (DmOrco). Relative to the other Orcos, AmOrco demonstrated higher sensitivity and responsivity, with a 1.8 to 21-fold decrease in the half-maximal effective concentration (EC50) and a 1.6-8.8-fold increase in the maximal effect (Emax), respectively. Furthermore, AmOrco co-expressed with AaOR10, BmOR56, or DmOR47a showed higher sensitivity and responsivity than AaOrco, BmOrco, or DmOrco co-expressed with their respective ORs. To further increase sensitivity and responsivity, we engineered chimeric Orcos by fusing AmOrco with DmOrco, considering the domain characteristics of Orcos. The response to 1-octen-3-ol was evaluated for AaOR8 when complexed individually with AmOrco, as well as for a mutant that combines DmOrco from the N-terminal (NT) to the C-terminal region of the fourth transmembrane domain (TM4) with the region of AmOrco following TM4 (Dm[NT-TM4]AmOrco). When compared to AmOrco, Dm(NT-TM4)AmOrco showed higher sensitivity and responsivity, with a 1.4-fold decrease in the EC50 and a 1.4-fold increase in the Emax, respectively. In addition, Dm(NT-TM4)AmOrco co-expressed with either DmOR47a or BmOR56 demonstrated higher sensitivity and responsivity than AmOrco co-expressed with their respective ORs. These results suggest that AmOrco could be a relatively more sensitive Orco, and further enhancement of sensitivity and responsivity could be achieved through recombination with heterologous Orcos near the TM4 of AmOrco.

Being Stung Once or Twice by Bees (Apis mellifera L.) Slightly Disturbed the Serum Metabolome of SD Rats to a Similar Extent.

In most cases, the number of honeybee stings received by the body is generally small, but honeybee stings can still cause serious allergic reactions. This study fully simulated bee stings under natural conditions and used 1H Nuclear Magnetic Resonance (1H NMR) to analyze the changes in the serum metabolome of Sprague-Dawley (SD) rats stung once or twice by honeybees to verify the impact of this mild sting on the body and its underlying mechanism. The differentially abundant metabolites between the blank control rats and the rats stung by honeybees included four amino acids (aspartate, glutamate, glutamine, and valine) and four organic acids (ascorbic acid, lactate, malate, and pyruvate). There was no separation between the sting groups, indicating that the impact of stinging once or twice on the serum metabolome was similar. Using the Principal Component Discriminant Analysis ( PCA-DA) and Variable Importance in Projection (VIP) methods, glucose, lactate, and pyruvate were identified to help distinguish between sting groups and non-sting groups. Metabolic pathway analysis revealed that four metabolic pathways, namely, the tricarboxylic acid cycle, pyruvate metabolism, glutamate metabolism, and alanine, aspartate, and glutamate metabolism, were significantly affected by bee stings. The above results can provide a theoretical basis for future epidemiological studies of bee stings and medical treatment of patients stung by honeybees.

Ame-miR-1-3p of bee venom reduced cell viability through the AZIN1/OAZ1-ODC1-polyamines pathway and enhanced the defense ability of honeybee (Apis mellifera L.).

Bee venom serves as an essential defensive weapon for bees and also finds application as a medicinal drug. MicroRNAs (miRNAs) serve as critical regulators and have been demonstrated to perform a variety of biological functions. However, the presence of miRNAs in bee venom needs to be confirmed. Therefore, we conducted small RNA sequencing and identified 158 known miRNAs, 15 conserved miRNAs and 4 novel miRNAs. It is noteworthy that ame-miR-1-3p, the most abundant among them, accounted for over a quarter of all miRNA reads. To validate the function of ame-miR-1-3p, we screened 28 candidate target genes using transcriptome sequencing and three target gene prediction software (miRanda, PITA and TargetScan) for ame-miR-1-3p. Subsequently, we employed real-time quantitative reverse transcription PCR (qRT-PCR), Western blot and other technologies to confirm that ame-miR-1-3p inhibits the relative expression of antizyme inhibitor 1 (AZIN1) by targeting the 3' untranslated region (UTR) of AZIN1. This, in turn, caused ODC antizyme 1 (OAZ1) to bind to ornithine decarboxylase 1 (ODC1) and mark ODC1 for proteolytic destruction. The reduction in functional ODC1 ultimately resulted in a decrease in polyamine biosynthesis. Furthermore, we determined that ame-miR-1-3p accelerates cell death through the AZIN1/OAZ1-ODC1-polyamines pathway. Our studies demonstrate that ame-miR-1-3p diminishes cell viability and it may collaborate with sPLA2 to enhance the defence capabilities of honeybees (Apis mellifera L.). Collectively, these data further elucidate the defence mechanism of bee venom and expand the potential applications of bee venom in medical treatment.

Expression and function of the vitellogenin receptor in the hypopharyngeal glands of the honey bee Apis mellifera (Hymenoptera: Apidae) workers.

The vitellogenin receptor (VgR) is essential for the uptake and transport of the yolk precursor, vitellogenin (Vg). Vg is synthesized in the fat body, released in the hemolymph, and absorbed in the ovaries, via receptor-mediated endocytosis. Besides its important role in the reproductive pathway, Vg occurs in nonreproductive worker honey bee, suggesting its participation in other pathways. The objective was to verify if the VgR occurs in the hypopharyngeal glands of Apis mellifera workers and how Vg is internalized by these cells. VgR occurrence in the hypopharyngeal glands was evaluated by qPCR analyses of VgR and immunohistochemistry in workers with different tasks. The VgR gene is expressed in the hypopharyngeal glands of workers with higher transcript levels in nurse honey bees. VgR is more expressed in 11-day-old workers from queenright colonies, compared to orphan ones. Nurse workers with developed hypopharyngeal glands present higher VgR transcripts than those with poorly developed glands. The immunohistochemistry results showed the co-localization of Vg, VgR and clathrin (protein that plays a major role in the formation of coated vesicles in endocytosis) in the hypopharyngeal glands, suggesting receptor-mediated endocytosis. The results demonstrate that VgR performs the transport of Vg to the hypopharyngeal glands, supporting the Ovary Ground Plan Hypothesis and contributing to the understanding of the role of this gland in the social context of honey bees.