Worker Honeybees Research Articles

Deciphering the CircRNA-Regulated Response of Western Honey Bee (Apis mellifera) Workers to Microsporidian Invasion.

Simple SummaryThe western honey bee (Apis mellifera) is commercially kept for crop pollination and production of bee products in China and many other countries. Currently, the circRNA-regulated response of A. mellifera to Vairimorpha ceranae infection is completely unknown. Here, characterization of circRNAs engaged in the response of A. mellifera workers to V. ceranae infection was conducted for the first time, followed by an in-depth investigation of the regulatory role of the hosts’ differentially expressed circRNAs (DEcircRNAs). Our findings not only revealed the underlying molecular mechanism of host response, but also offered novel insights into honey bee–microsporidian interaction.Vairimorpha ceranae is a widespread fungal parasite of adult honey bees that leads to a serious disease called nosemosis. Circular RNAs (circRNAs) are newly discovered non-coding RNAs (ncRNAs) that regulate biological processes such as immune defense and development. Here, 8199 and 8711 circRNAs were predicted from the midguts of Apis mellifera ligustica workers at 7 d (Am7T) and 10 d (Am10T) after inoculation (dpi) with V. ceranae spores. In combination with transcriptome data from corresponding uninoculated midguts (Am7CK and Am10CK), 4464 circRNAs were found to be shared by these four groups. Additionally, 16 circRNAs were highly conserved among A. m. ligustica, Apis cerana cerana, and Homo sapiens. In the Am7CK vs. Am7T (Am10CK vs. Am10T) comparison group, 168 (306) differentially expressed circRNAs (DEcircRNAs) were identified. RT-qPCR results showed that the expression trend of eight DEcircRNAs was consistent with that in the transcriptome datasets. The source genes of DEcircRNAs in Am7CK vs. Am7T (Am10CK vs. Am10T) were engaged in 27 (35) GO functional terms, including 1 (1) immunity-associated terms. Moreover, the aforementioned source genes were involved in three cellular immune-related pathways. Moreover, 86 (178) DEcircRNAs in workers’ midguts at 7 (10) dpi could interact with 75 (103) miRNAs, further targeting 215 (305) mRNAs. These targets were associated with cellular renewal, cellular structure, carbohydrate and energy metabolism, and cellular and humoral immunity. Findings in the present study unraveled the mechanism underlying circRNA-mediated immune responses of western honey bee workers to V. ceranae invasion, but also provided new insights into host–microsporidian interaction during nosemosis.

Impact of Imunit® insecticide on survival and detoxification enzymes activity of adult worker honey bee (Apis mellifera L.)

آلفاسایپرمترین+تفلوبنزورون (ایمونیت®) حشره‌کشی است که توسط سازمان حفظ نباتات ایران برای کنترل کرم میوه‌خوار (Helicoverpa armigera Hübner) در مزارع گوجه‌فرنگی توصیه می‌شود. در این پژوهش، تأثیر ایمونیت® روی نرخ بقای حشرات بالغ زنبورعسل کارگر (Apis mellifera L.) در شرایط آزمایشگاهی بررسی شد. زنبورهای تازه‌متولدشده (با سن کمتر از 24 ساعت) و چراگر (با سن بیشتر از 21 روز) به سه روش تماس با باقی‌مانده، گوارشی، و پاششی با دُز توصیه‌شدۀ مزرعه-ای این حشره‌کش تیمار شدند. همچنین، اثرات زیرکشندگی ایمونیت® روی فعالیت ویژه برخی از آنزیم‌های سم‌زُدا از قبیل آلفا-استراز، بتا-استراز، و گلوتاتیون اس-ترانسفراز مورد مطالعه قرار گرفت. آزمایش‌های زیست‌سنجی نشان داد که سمیت ایمونیت® با توجه به سن زنبور و روش استفاده از حشره‌کش فرق داشت. به استثنای روش پاششی، زنبورهای چراگر در مقایسه با زنبورهای تازه‌متولدشده از حساسیت بیشتری به این حشره‌کش برخوردار بودند. سمیت تماس با باقی‌مانده و پاششی ایمونیت® روی زنبورهای تازه‌متولدشده در مقایسه با سمیت گوارشی آن به طور معنی‌داری بیشتر بود، در حالی که زنبورهای چراگر بیشترین حساسیت را پس از تماس با باقی‌مانده این حشره‌کش نشان دادند. فعالیت آنزیم‌های سم‌زُدا نیز با توجه سن زنبورها و روش قرارگیری در معرض ایمونیت® تغییر پیدا کرد. به طور کلی، یافته‌های ما بیانگر پتانسیل بالای ایمونیت® برای ایجاد مسمومیت در زنبورعسل می‌باشد.

Immunohistochemical Distribution of Serotonin Transporter (SERT) in the Optic Lobe of the Honeybee, Apis mellifera.

Simple SummarySerotonin is ubiquitously expressed in vertebrates and invertebrates, where it regulates specific behavioural patterns. Though the specific effects of serotonin release in the optic lobe are not entirely known, increasing evidence associates the serotonergic system with optic lobe-mediated behaviours. In this study, the localization of serotonin transporter (SERT) was immunohistochemically analysed in the optic lobes of moderate, docile and aggressive worker honeybees. SERT-immunoreactive fibres were stratified in the optic lobe and distributed in the three visual neuropils: lamina, medulla and lobula. Interestingly, SERT immunoreactivity was inversely related to aggressive behaviour. The present study indicates that low levels of serotonin in the optic lobe are associated with aggressive behaviour.Visual information is processed in the optic lobes, which consist of three retinotopic neuropils. These are the lamina, the medulla and the lobula. Biogenic amines play a crucial role in the control of insect responsiveness, and serotonin is clearly related to aggressiveness in invertebrates. Previous studies suggest that serotonin modulates aggression-related behaviours, possibly via alterations in optic lobe activity. The aim of this investigation was to immunohistochemically localize the distribution of serotonin transporter (SERT) in the optic lobe of moderate, docile and aggressive worker honeybees. SERT-immunoreactive fibres showed a wide distribution in the lamina, medulla and lobula; interestingly, the highest percentage of SERT immunoreactivity was observed across all the visual neuropils of the docile group. Although future research is needed to determine the relationship between the distribution of serotonin fibres in the honeybee brain and aggressive behaviours, our immunohistochemical study provides an anatomical basis supporting the role of serotonin in aggressive behaviour in the honeybee.

Bees under interactive stressors: the novel insecticides flupyradifurone and sulfoxaflor along with the fungicide azoxystrobin disrupt the gut microbiota of honey bees and increase opportunistic bacterial pathogens

The gut microbiome plays an important role in bee health and disease. But it can be disrupted by pesticides and in-hive chemicals, putting honey bee health in danger. We used a controlled and fully crossed laboratory experimental design to test the effects of a 10-day period of chronic exposure to field-realistic sublethal concentrations of two nicotinic acetylcholine receptor agonist insecticides (nACHRs), namely flupyradifurone (FPF) and sulfoxaflor (Sulf), and a fungicide, azoxystrobin (Azoxy), individually and in combination, on the survival of individual honey bee workers and the composition of their gut microbiota (fungal and bacterial diversity). Metabarcoding was used to examine the gut microbiota on days 0, 5, and 10 of pesticide exposure to determine how the microbial response varies over time; to do so, the fungal ITS2 fragment and the V4 region of the bacterial 16S rRNA were targeted. We found that FPF has a negative impact on honey bee survival, but interactive (additive or synergistic) effects between either insecticide and the fungicide on honey bee survival were not statistically significant. Pesticide treatments significantly impacted the microbial community composition. The fungicide Azoxy substantially reduced the Shannon diversity of fungi after chronic exposure for 10 days. The relative abundance of the top 10 genera of the bee gut microbiota was also differentially affected by the fungicide, insecticides, and fungicide-insecticide combinations. Gut microbiota dysbiosis was associated with an increase in the relative abundance of opportunistic pathogens such as Serratia spp. (e.g. S. marcescens), which can have devastating consequences for host health such as increased susceptibility to infection and reduced lifespan. Our findings raise concerns about the long-term impact of novel nACHR insecticides, particularly FPF, on pollinator health and recommend a novel methodology for a refined risk assessment that includes the potential effects of agrochemicals on the gut microbiome of bees.

Infestation of <em>Apis mellifera</em> workers and larvae with <em>Varroa destructor</em> affects gut bacterial diversity

The present work aims to investigate the midgut bacteria of honey bee (Apis mellifera lamarckii) workers and larvae infested with Varroa destructor. To achieve this task, uninfested and infested honey bee larvae and workers with the parasitic mites were obtained from natural bees apiaries in Qaliubiya Governorate, Egypt. Aerobic bacteria were isolated from the midgut of uninfested and infested A. mellifera on nutrient agar medium and identified using a culture-dependent isolation along with 16S rRNA sequences. The results revealed that both uninfested and V. destructor infested workers and larvae harbored gut bacteria belonging to Gamma-proteobacteria strains. The uninfested workers contained Enterobacter cloacae and Serratia nematodiphila, while V. destructor infested workers harbored Enterobacter species. Uninfested larvae contained E. hormaechei and Klebsiella variicola. V. destructor infested larvae harbored K. oxytoca as a result of infection. We concluded that the honeybee microbiome composition is likely to be impacted through isolates variation by stage and diversity disruption of isolates by V. destructor infection.

Asymmetry in the ovarioles, mandibular glands and hypopharyngeal glands of honeybee (Apis mellifera) workers developing in queenright or queenless colonies

Asymmetry in the ovarioles, mandibular glands and hypopharyngeal glands of honeybee (Apis mellifera) workers developing in queenright or queenless colonies

Colonization dynamics of the gut flora in western honey bee workers within 7-day post-emergence

Colonization dynamics of the gut flora in western honey bee workers within 7-day post-emergence

Metabolomics-Guided Comparison of Pollen and Microalgae-Based Artificial Diets in Honey Bees.

Managed honey bee colonies used for crop pollination are fed artificial diets to offset nutritional deficiencies related to land-use intensification and climate change. In this study, we formulated novel microalgae diets using Chlorella vulgaris and Arthrospira platensis (spirulina) biomass and fed them to young adult honey bee workers. Diet-induced changes in bee metabolite profiles were studied relative to a natural pollen diet using liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) metabolomics. Untargeted analyses of pollen- and microalgae-fed bees revealed significant overlap, with 248 shared features determined by LC-MS and 87 shared features determined by GC-MS. Further metabolomic commonalities were evident upon subtraction of unique diet features. Twenty-five identified metabolites were influenced by diet, which included complex lipids, essential fatty acids, vitamins, and phytochemicals. The metabolomics results are useful to understand mechanisms underlying favorable growth performance as well as increased antioxidant and heat shock protein gene expression in bees fed the microalgae diets. We conclude that the tested microalgae have potential as sustainable feed additives and as a source of bee health-modulating natural products. Metabolomics-guided diet development could eventually help tailor feed interventions to achieve precision nutrition in honey bees and other livestock animals.

Pesticide risk assessment: honeybee workers are not all equal regarding the risk posed by exposure to pesticides.

Toxicological studies in honeybees have long shown that a single pesticide dose or concentration does not necessarily induce a single response. Inter-individual differences in pesticide sensitivity and/or the level of exposure (e.g., ingestion of pesticide-contaminated matrices) may explain this variability in risk posed by a pesticide. Therefore, to better inform pesticide risk assessment for honeybees, we studied the risk posed by pesticides to two behavioral castes, nurse, and forager bees, which are largely represented within colonies and which exhibit large differences in their physiological backgrounds. For that purpose, we determined the sensitivity of nurses and foragers to azoxystrobin (fungicide) and sulfoxaflor (insecticide) upon acute or chronic exposure. Azoxystrobin was found to be weakly toxic to both types of bees. However, foragers were more sensitive to sulfoxaflor than nurses upon acute and chronic exposure. This phenomenon was not explained by better sulfoxaflor metabolization in nurses, but rather by differences in body weight (nurses being 1.6 times heavier than foragers). Foragers consistently consumed more sugar syrup than nurses, and this increased consumption was even more pronounced with pesticide-contaminated syrup (at specific concentrations). Altogether, the stronger susceptibility and exposure of foragers to sulfoxaflor contributed to increases of 2 and tenfold for the acute and chronic risk quotients, respectively, compared to nurses. In conclusion, to increase the safety margin and avoid an under-estimation of the risk posed by insecticides to honeybees, we recommend systematically including forager bees in regulatory tests.

Differences in ASP1 expression and binding dynamics to queen mandibular pheromone HOB between Apis mellifera and Apis cerana workers reveal olfactory adaptation to colony organization

The eastern Apis cerana (Ac) and the western Apis mellifera (Am) are two closely related and most economically valuable honeybee species managed extensively worldwide. However, how worker bees of Ac and Am are adapted to their colony organization remains to be uncovered. Here, we found that the expression level of gene encoding antennae-specific proteins 1 (ASP1, a key regulator in recognizing queen mandibular pheromone) was positively correlated with the colony sizes in both bee species, and the expression level in Am was higher than that in Ac, suggesting that ASP1 may play an important role in maintaining colony homeostasis. Using competitive binding assay, molecular docking, and site-directed mutagenesis, we then confirmed the good binding affinities of both Ac-ASP1 and Am-ASP1 to methyl p-hydroxy benzoate (HOB), and Val115 was the key amino acid. However, the affinity of Am-ASP1 was stronger than that of Ac-ASP1. EAG analysis further demonstrated that antennae of Am worker bees had faster depolarization and repolarization in response to HOB stimulation. Taken together, these findings indicate that the differences in expression levels and binding dynamics allow ASP1 recognizing HOB to potentially serve as a specific regulator of colony organization in Ac and Am.

Butenolide Insecticide Flupyradifurone Affects Honey Bee Worker Antiviral Immunity and Survival.

Honey bees face many environmental stressors, including exposure to pesticides and pathogens. A novel butenolide pesticide, flupyradifurone, was recently introduced to the US and shown to have a bee-friendly toxicity profile. Like the much-scrutinized neonicotinoids that preceded it, flupyradifurone targets the insect nervous system. Some neonicotinoids have been shown to interfere with antiviral immunity, which raised the concern that similar effects may be observed with flupyradifurone. In this study, we investigated how flupyradifurone and a neonicotinoid, clothianidin, affect the ability of honey bee workers to combat an infection of Israeli acute paralysis virus (IAPV). We exposed workers to field-realistic doses of the pesticides either with or without co-exposure with the virus, and then tracked survival and changes in viral titers. We repeated the experiment in the spring and fall to look for any seasonal effects. We found that flupyradifurone caused elevated mortality in the fall, but it did not lead to increased virus-induced mortality. Flupyradifurone also appeared to affect virus clearance, as bees co-exposed to the pesticide and virus tended to have higher viral titers after 48 hours than those exposed to the virus alone. Clothianidin had no effect on viral titers, and it actually appeared to increase resistance to viral infection in spring bees.

Phosphoproteomics analysis of hypopharyngeal glands of the newly emerged honey bees (Apis mellifera ligustica)

The most important honey bee queen food royal jelly is produced by the exocrine hypopharyngeal glands (HGs) of the worker honey bees. The HGs exhibits diverse gene and protein that create age-related physiological adaptations. However, limited knowledge is available on how the phosphorylation process is responsible for physiological alterations across the development of HGs in newly emerged bees. This study measured the acinus of HGs and characterized its phosphoproteomics analysis between the newly emerged bees of royal jelly bees (RJBs) and Italian bees (ITBs). Phosphopeptides of HGs were enriched by Ti4+-IMAC reagents, followed by protein identification via Q-Exactive LC-MS/MS. Our findings indicated that the mean acinus size of HGs of newly emerged bees of RJBs was significantly larger (56.18 ± 1.72 µm) than ITBs (45.98 ± 1.62 µm). A total of 1576 phosphopeptides with 1800 phosphosites containing 525 phosphoproteins were identified in RJBs, while 746 phosphopeptides, of which 846 phosphosites correspond to 317 phosphoproteins were identified in ITBs. Most proteins were phosphorylated on 1 residue followed by 2 and 3 residues in newly emerged bees of both bee stocks. In addition, serine phosphorylation was most observed, followed by threonine and tyrosine in both bee stocks. In newly emerged bees of RJBs, the protein metabolic process, glycolytic process, and formation of translation preinitiation were uniquely enriched, while protein translation, peptide metabolic process and elongation were enriched in ITBs. This research shows detailed phosphorylation of HGs and provides helpful information for understanding the biological activities of HGs development in newly emerged bees from both bee stocks.

Longevity-enhancing Effects of Rosmarinic Acid Feeding on Honey bees (Apis mellifera L.) after Exposure to Some Pesticides Used in Strawberry Greenhouse

Bee pollination plays important roles in increasing the yield, weight, and quality of strawberries grown in greenhouses. Use of pesticides is often included in the pest management strategy to control insects, fungi and weeds. However, these pesticides can negatively affect bees, compromising greenhouse pollination programs. In this study, we investigate the effect of rosmarinic acid (RA) feeding on the longevity of honey bees after oral and contact exposure of bees to some selected 14 different pesticides and G-3KM (commercial product) was used as a positive control. Among the 14 pesticides tested, six were found to be highly toxic to honey bee workers as indicated by their 48h-LD50 (㎍/bee) values obtained from oral and spray bioassays; thiamethoxam (0.0002/0.0011), dinotefuran (0.005/0.018), emamectin benzoate (0.002/0.0002), spinetoram (0.018/0.001), sulfoxaflor (0.04/0.01), and cyantraniliprole (0.1/0.03), respectively. It was interesting to note that RA and G-3KM-supplemented feeding reduced honey bees mortality by 20-30% after they were exposed to high concentrations of the 6 highly toxic pesticides. The results indicate that RA could be used effectively in reducing honey bee mortality caused by pesticides.

Anadolu Arı Ekmeklerinin Fizikokimyasal ve Kimyasal Özelliklerine Dayalı Temel Bileşen Analizi ile Karakterizasyonu

Bee bread is a fermented bee product which is the mixture of pollen and honey. This substance is actually the main source of food for honey-bee workers. Due to its rich therapeutic properties, bee bread has gained an increasing interest for human consumption in recent years. The purpose of this study was to compare the physicochemical characteristics and chemical composition of bee bread samples collected from seven different regions in Anatolia. Various measurements such as pH, electrical conductivity, colour, colour intensity, and optical density were performed. Together with the mentioned, the content of moisture, ash, total protein, and free acidity were analyzed. Principal component analysis (PCA) was applied to analyze results in order to classify the bee breads from different regions. This study contributes to the chemical and physicochemical knowledge of this scarcely explored natural bee-product.

Effect of honeybee venom and Egyptian propolis on the honeybee (Apis mellifera L.) health in vivo

BackgroundHoneybees are one of the most important pollinators in the world, and their products are nowadays included in most anticancer, antiallergic, antimicrobial drugs and are included in cosmetic treatments. In the present study, honeybee venom and Egyptian ethanolic propolis extract (EP) were focused to test their effect on health and some genes for honeybee workers (defensin2, abaecin, hymenoptaecin, vitellogenin, and juvenile hormone esterase).ResultsHoneybee venom and Egyptian propolis extract (EP) were used as supplements in the nutrition with different concentrations in Varroa mites-infected colonies to measure the colonies' activities after treatment. The immune-related genes and antimicrobial peptides (AMPs) were evaluated by using qRT-PCR. Treated colonies with HBV and EP showed up-regulation of immune and immune-related genes' expressions and increased the life span, activities and their density of bee workers. The data illustrated that the highest gene expression fold of juvenile hormone esterase was detected in the treated colonies with Egyptian ethanolic propolis extract (EP), while the highest vitellogenin expression fold in treated colonies was with honeybee venom. The up-regulation of antimicrobial peptides occurred in colonies with both treatments.ConclusionThe findings suggest that honeybee venom and Egyptian ethanolic propolis extract (EP) could be used as potential supplements, even at the lowest concentration to develop the immunity of worker bees to increase their efficiency and prevent loss of honeybee colonies due to several diseases closely associated with Varroa mites' infections that cause sudden death.

Social microbiota and social gland gene expression of worker honey bees by age and climate

Winter forage dearth is a major contributor to honey bee colony loss and can influence disease susceptibility. Honey bees possess a secretory head gland that interfaces with the social environment on many levels. During winter or forage dearth, colonies produce a long-lived (diutinus) worker phenotype that survives until environmental conditions improve. We used a known-age worker cohort to investigate microbiome integrity and social gene expression of workers in early and late winter. We provide additional context by contrasting host-microbial interactions from warm outdoor and cold indoor environments. Our results provide novel evidence that social immune gene expression is associated with worker longevity, and highlight the midgut as a target of opportunistic disease during winter. Host microbial interactions suggest opportunistic disease progression and resistance in long-lived workers, but susceptibility to opportunistic disease in younger workers that emerged during the winter, including increases in Enterobacteriaceae, fungal load and non-core bacterial abundance. The results are consistent with increased social immunity, including host associations with the social microbiota, and a social immune response by long-lived workers to combat microbial opportunism. The cost/benefit ratio associated with limited expression of the diutinus phenotype may be a strong determinant of colony survival during winter forage dearth.

Correction: Queen cells acceptance rate and royal jelly production in worker honey bees of two Apis mellifera races

[This corrects the article DOI: 10.1371/journal.pone.0248593.].

Feeding probiotics and organic acids to honeybees enhances acinal surface area of their hypopharyngeal glands

Modern bee keeping demands more scientific and environment compatible methodologies to improve honeybee's health and efficiency. The current study was designed to explore the hidden potential of probiotics (Lactobacillus rhamnosus) and organic acids (lactic acid and acetic acid) on bee's growth especially hypopharyngeal gland (HPG). Large sized HPG has the ability to produce more royal jelly than smaller ones. For the purpose, the experimentation was carried out in 7 different treatment groups in which probiotics and organic acids were provided in different proportions. Significant increase in acinal surface area of bees in all the experimental groups was observed. Control bees which were fed with pollens and sugar syrup only for two weeks depicted the mean ± SE value of 0.011 ± 0.001 for acinal surface area. Similarly, worker bees of the experimental group 3 [pollens +50% (w/v) sucrose in 1.96% acetic acid], group 4 [pollens + L. rhamnosus in 50% (w/v) sucrose in distilled water], group 5 [pollens + L. rhamnosus in 50% (w/v) sucrose in 2.99% lactic acid], group 6 [pollens + L. rhamnosus in 50% (w/v) sucrose in 2.91% acetic acid] and group 7 [pollens + L. rhamnosus in 50% (w/v) sucrose in 1.96% acetic acid] showed mean ± SE values of 0.019 ± 0.001, 0.017 ± 0.001, 0.013 ± 0.001, 0.016 ± 0.001 and 0.014 ± 0.001 mm2, respectively. The use of acidifying agents and probiotics resulted in enhanced growth of HPG of Apis mellifera workers. Our findings of the present study will be helpful to obtain higher royal jelly yields.

Influence of sugary feeding with some vitamins on phenoloxidase and antioxidant activity in honeybee workers, Apis mellifera L.

Influence of sugary feeding with some vitamins on phenoloxidase and antioxidant activity in honeybee workers, Apis mellifera L.

Evaluation of acaricide toxicity to the parasitic mite (Varroa destructor), honeybee workers (Apis mellifera L.) and their residues in honey and beeswax.

Evaluation of acaricide toxicity to the parasitic mite (Varroa destructor), honeybee workers (Apis mellifera L.) and their residues in honey and beeswax.