Worker Honeybees Research Articles

Expression of Concern: Morphometric study of Yemeni (Apis mellifera jemenitica) and Carniolan (A. m. carnica) honeybee workers in Saudi Arabia.

Expression of Concern: Morphometric study of Yemeni (Apis mellifera jemenitica) and Carniolan (A. m. carnica) honeybee workers in Saudi Arabia.

Workers of Apis mellifera Reared in Small-Cell Combs Show Higher Activity of the Proteolytic System in Hemolymph than Workers Reared in Standard-Cell Combs in Laboratory Cage Tests.

The aim of the study was to compare the activities of proteases and their inhibitors in the hemolymph of honeybee workers reared in small-cell combs (SMC) and standard-cell combs (STC) in laboratory cage tests. The analyses conducted in laboratory conditions facilitated assessment of the impact of the comb cell width (small vs. standard) along with eliminating the influence of many environmental factors on the results. The width of the comb cells in which the workers were reared had a significant effect on the protein concentrations and proteolytic system activities in the hemolymph. Irrespective of the age of the workers, higher protein concentrations were found in the hemolymph of the SMC workers. In turn, the activities of proteases and their inhibitors in the hemolymph of 1-day-old bees were higher in the STC workers. In older bees, aged 7-21 days, activity was higher in the SMC workers. The role of the considerable cell width variability in natural combs that were built without the use of an artificially produced wax foundation is worth investigating. It is highly probable that the impact of the comb cell width on the features of workers reared in these combs modifies the age polyethism in the worker caste as well. The investigation results of one-season studies of honeybees could be seriously affected by random factors. To reduce the risk of these effects, it is advisable to continue experiments over a few consecutive years.

Group Size Buffers against Energetic Stress in Honeybee Workers (Apis mellifera)

Individuals of the Western honeybee species, Apis mellifera, live in large groups of thousands of worker bees, a queen, and a few drones. Workers interact frequently with related individuals while performing various tasks. Although it is well understood why and under which conditions sociality can evolve, the consequences for individuals living in permanent groups are less well understood. As individuals of solitary species become stressed when kept at high density, it might be the opposite in obligate social species. Here, I use an experimental laboratory set-up to study the effect of varying group sizes on the magnitude and within-group variance of stress responses towards energetic and heat stress. While only a weak difference was found in the magnitude of an energetic stress response as a function of group size, the within-group variance showed a statistically significant positive relationship with group size for the glucose/trehalose ratio, a marker for energetic stress. The heat stress marker, hsp70AB gene expression, did not show any relationship to group size. Individuals of obligate social species seem to benefit from adaptations to permanent group living, e.g., buffering against stress, especially at a higher density of individuals. The consequences of infections and immune system activation in isolated individuals are discussed.

The diverging epigenomic landscapes of honeybee queens and workers revealed by multiomic sequencing

The role of the epigenome in phenotypic plasticity is unclear presently. Here we used a multiomics approach to explore the nature of the epigenome in developing honey bee (Apis mellifera) workers and queens. Our data clearly showed distinct queen and worker epigenomic landscapes during the developmental process. Differences in gene expression between workers and queens become more extensive and more layered during the process of development. Genes known to be important for caste differentiation were more likely to be regulated by multiple epigenomic systems than other differentially expressed genes. We confirmed the importance of two candidate genes for caste differentiation by using RNAi to manipulate the expression of two genes that differed in expression between workers and queens were regulated by multiple epigenomic systems. For both genes the RNAi manipulation resulted in a decrease in weight and fewer ovarioles of newly emerged queens compared to controls. Our data show that the distinct epigenomic landscapes of worker and queen bees differentiate during the course of larval development.

Accelerated abdominal lipid depletion from pesticide treatment alters honey bee pollen foraging strategy, but not onset, in worker honey bees.

Honey bee abdominal lipids decline with age, a change thought to be associated with the onset of foraging behavior. Stressors, such as pesticides, may accelerate this decline by mobilizing internal lipid to facilitate the stress response. Whether bees with stressor-induced accelerated lipid loss vary from controls in both the onset of foraging and nutritional quality of collected pollen is not fully understood. We asked whether stressors affect foraging behavior through the depletion of abdominal lipid, and whether stress-induced lipid depletion causes bees to forage earlier and for fattier pollen. We tested this by treating newly emerged bees with one of two pesticides, pyriproxyfen (a juvenile hormone analog) and spirodiclofen (a fatty acid synthesis disruptor), that may affect energy homeostasis in non-target insects. Bees fed these pesticides were returned to hives to observe the onset of foraging behavior. We also sampled foraging bees to assay both abdominal lipids and dietary lipid content of their corbicular pollen. Initially, spirodiclofen-treated bees had significantly more abdominal lipids, but these declined faster compared with controls. These bees also collected less, yet more lipid-rich, pollen. Our results suggest that bees with accelerated lipid decline rely on dietary lipid content and must collect fattier pollen to compensate. Pyriproxyfen treatment reduced the age at first forage but did not affect abdominal or collected pollen lipid levels, suggesting that accelerated fat body depletion is not a prerequisite for precocious foraging.

Artificial feeding of honey bee colonies by adding nutritional supplements to pollen substitutes and its effect on the development of the hypopharyngeal gland stages of honeybee workers Apis mellifera L.

Artificial feeding of honey bee colonies by adding nutritional supplements to pollen substitutes and its effect on the development of the hypopharyngeal gland stages of honeybee workers Apis mellifera L.

Vital responses of caged honeybee (Apis mellifera) workers fed on new substitute diets in the laboratory

Vital responses of caged honeybee (Apis mellifera) workers fed on new substitute diets in the laboratory

Evaluating the Impact of Commonly Used Pesticides on Honeybees (Apis mellifera) in North Gonder of Amhara Region, Ethiopia.

Global honeybee losses and colony decline are becoming continuous threat to the apicultural industry, as well as, for food security and environmental stability. Although the putative causes are still unclear, extensive exposure of bees to pesticides could be the possible factor for worldwide colony losses. This study was aimed at evaluating the impact of nine commonly used pesticide incidents on adult worker honeybees (A. mellifera) under the laboratory condition, in North Gonder of Amhara region, Ethiopia. Feeding test, contact test, and fumigation tests were carried out for each pesticide following the standard procedures, and each pesticide toxicity was compared to the standard toxic chemical, dimethoate 40% EC (positive control), and to 50% honey solution (negative control). The results revealed that all the tested pesticides caused significant deaths of the experimental bees (P < 0.05) in all the tests when compared to the negative control. Diazinon 60% EC, endosulfan 35% EC, and malathion 50% EC were appeared highly toxic causing 100% mortality of bees, while chlorsulfuron 75% WG killed 90% of the experimental bees as tested via feeding. On the other hand, agro-2, 4-D and its mixture with glycel 41% EC are moderately toxic, and mancozeb 80% WP and glycel 41% EC were slightly toxic to honeybees as compared to the positive control (dimethoate 40% EC). Suddenly, diazinon 60% EC and malathion 50% EC triggered 100% mortality of bees, while endosulfan 35% EC and chlorsulfuron 75% WG caused 63.63% and 90.82% of bee mortality, respectively, when evaluated via contact test. The fumigation test also showed that chlorsulfuron 75% WG, diazinon 60% EC, and endosulfan 35% EC caused 100%, 86.7%, and 65.6% mortality rate of bees. Our result also highlighted that tested LD50 of all pesticide incidents were significantly lower than the manufacturer-based LD50. This shows that local honeybees A. m. jemenetica are extremely sensitive to commonly used agricultural pesticides, which may affect the colony level due to the intensive application of these pesticides in Ethiopia.

Apis mellifera Worker Bees Selected for Varroa-sensitive Hygiene Show Higher Specific Sensitivity and Perception Speed Towards Low Concentrations of Chemical Cues Emitted by the Brood

Varroa-sensitive hygiene (VSH) is highly influenced by the worker bee’s olfactory ability. Workers bred for VSH and non-selected control line workers were tested for differences in their speed and perception ability when presented with highly diluted stimuli. Four different substances (citral – dilution 1:1300, linalool dilution 1:1300, Varroa-parasitized brood extract, isopropanol) were used as tactile stimuli for differential conditioning with the proboscis extension response (PER). Discrimination ability and generalization were assessed. In a second set of conditioning experiments differences in sensitivity to the highly diluted citral and the Varroa-parasitized brood extract as reinforced stimuli (Cs +) were explored between workers from both lines. The worker bees were classified into three groups (Time points) depending on how long before they started correctly extending their proboscis to the Cs + , and results were examined separately for each of the two stimuli and group. While the VSH-selected line exhibited a significantly higher perception ability for the parasitized brood extract than the non-selected line, the two lines showed no differences when conditioned with the floral stimulus citral as Cs + . Furthermore, the VSH-selected line displayed a significantly higher number of worker bees that perceived the complex bouquet of the Varroa-parasitized brood extract at the earliest time grouping (Time point 1). The odds of perception at the earliest possible time point were 2.6-times higher for the VSH-selected line. Although no comparison was made between healthy and parasitized brood, the results indicate an enhanced specific sensitivity in VSH-selected workers towards chemical cues emitted by the brood, which might play a role in the detection of Varroa destructor.

Susceptibility Comparison of Bumblebee and Honeybee to Acetamiprid and Imidacloprid

midacloprid and acetamiprid are neonicotinoid insecticides introduced in the market for the control of insect pests of agricultural crops, but unfortunately these insecticides are causing harmful effect on non-target organisms including honeybees and bumblebees. Insecticides of this group effect acetylcholine nicotinic receptors. Apis mellifera, the European honeybee and Bombus haemorrhoidalis are important pollinators for wild and managed crops in Pakistan, but unfortunately their population is declining day by day at alarming rate. This study was design to find out the susceptibility level and insecticidal toxicity comparison on A. mellifera and B. haemorrhoidalis to acetamiprid and imidacloprid under controlled laboratory conditions by using contact and oral exposure methods. The results of oral method by using sugar solution showed that honeybee is more susceptible as compared to bumblebee on both insecticides. Maximum mortality i.e. 20%, 35%, 66%, in honeybee and 16%, 29%, 45% in bumblebee workers at acetamiprid and at imidacloprid 46%, 67%, 83% in honeybee and 34%, 56%, 72% in bumblebee were observed respectively after the exposure of 3hrs. 6hrs. and 24hrs. Similar susceptibly results were found on pollen and contact method. Overall results demonstrate that these both neonicotinoid insecticides are toxic for bumblebees and honeybees, and honeybee workers are more susceptible as compared to bumblebee workers at company recommended field realistic dose.

Seasonal changes in ultrastructure and gene expression in the fat body of worker honey bees

The anatomical, physiological, and behavioral characteristics of honey bees are affected by the season as well as division of labor. In this study, we examined the structure, ultrastructure, and gene expression of fat body cells in both long-lived winter and short-lived summer worker bees (the youngest stage of hive bees and forager bees). In contrast to hive bees, foragers and winter bees have a higher metabolism due to intensive muscle activity during their flight (foragers) or endothermic heat production (winter bees). These workers differ from hive bees in the biology of their mitochondria, peroxisomes, and lysosomes as well as in the expression of the genes involved in lipid, carbohydrate, amino acid metabolism, insulin, and TGF- β signaling. Additionally, the expression of genes related to phospholipid metabolism was higher in the hive bees. However, we found no differences between workers in the expression of genes controlling cell organelles, such as the Golgi apparatus, endoplasmic reticulum, ribosomes, nucleus, and vacuoles, as well as genes for DNA replication, cell cycle control, and autophagy. Furthermore, lysosomes, autophagic processes and lipofuscin particles were more frequently observed in winter bees using electron microscopy.

Effect of Rearing in Small-Cell Combs on Activities of Catalase and Superoxide Dismutase and Total Antioxidant Capacity in the Hemolymph of Apis mellifera Workers

Honeybee nests constructed without man-made wax foundation have significantly more variability of cell widths/sizes than those in commercially-kept colonies. The effects of this natural variability in comb cell widths on individual and colony traits have not been explained to date. The investigation of this problem can lead to new findings about the biology, physiology, and possibly, the evolution of the honeybee. The aim of the study was to compare the catalase and superoxide dismutase activities and the total antioxidant capacity levels in the hemolymph of honeybee workers reared in small-cell combs and standard-cell combs in colonies kept simultaneously on standard- and small-cell combs. The ratio of the small-cell combs to the standard-cell combs in the nest was 1:1. The workers reared in small-cell combs were characterized by higher antioxidant activities in the hemolymph than those reared in standard-cell combs. Consequently, their hemolymph had a greater antioxidant capacity, which indicates that they may be better predisposed to be foragers than workers reared in standard-cell combs. To describe the physiological differences between worker bees reared in small- and standard-cell combs in the same colony, the role of the considerable variation in the cell width in natural combs built without the use of artificially produced wax foundation is worth elucidating. The comparison of the apiary and cage experiments indicated that changes in antioxidant activities predominantly result from worker activities, especially those requiring the intensification of metabolism, rather than the age of the worker bees. To reduce the impact on the results of random environmental factors potentially present in one-season studies of honeybee research, investigations should preferably be carried out over a few consecutive years.

Phenotypic dimorphism between honeybee queen and worker is regulated by complicated epigenetic modifications

Phenotypic dimorphism between queens and workers is an important biological characteristic of honeybees that has been the subject of intensive research. The enormous differences in morphology, lifespan, physiology, and behavior between queens and workers are caused by a complicated set of factors. Epigenetic modifications are considered to play an important role in this process. In this study, we analyzed the differences in chromosome interactions and H3K27ac andH3K4me1 modifications between the queens and workers using high-throughput chromosome conformation capture (Hi-C) and Chromatin immunoprecipitation followed by sequencing (ChIP-Seq) technologies. We found that the queens contain more chromosome interactions and more unique H3K27ac modifications than workers; in contrast, workers have more H3K4me1 modifications than queens. Moreover, we identified Map3k15 as a potential caste geneinqueen-worker differentiation. Our results suggest that chromosomal conformation and H3K27ac and H3K4me1 modifications are involved in regulating queen-worker differentiation, which reveals that the queen-worker phenotypic dimorphism is regulated by multiple epigenetic modifications.

Linking Histone Methylation States and hsp Transcriptional Regulation in Thermo-Tolerant and Thermo-Susceptible A. mellifera L. Subspecies in Response to Heat Stress.

Genetic and epigenetic responses to environmental cues of worker honeybees mediate hsp synthesis, a key mechanism to tolerate high ambient temperatures in Apis mellifera. In this study, the chromatin immunoprecipitation assay followed by qPCR were used to determine alterations in histone methylation states (H3K27me2, H3K27me3, H3K4me2, and H3K4me3) associated with hsp/hsc/trx in A. m. jemenetica (thermo-tolerant subspecies) and A. m. carnica (thermo-susceptible subspecies) after heat treatment. The results revealed significant changes in enrichment folds of histone methylation states associated with hsp/hsc/trx. Indeed, the enrichment of H3K27me2 decreased strongly in response to heat stress. Changes in histone methylation states were significantly higher in A. m. carnica samples compared to A. m. jemenitica samples. Our study provides a new perception on linking histone post-translational methylation as an epigenetic mechanism of gene regulation with hsp/hsc/trx in A. mellifera subspecies exposed to heat stress.

Targeted and Suspect Fatty Acid Profiling of Royal Jelly by Liquid Chromatography-High Resolution Mass Spectrometry.

Royal jelly (RJ) is a bee product produced by the mandibular and hypopharyngeal glands of worker honeybees which has attracted special attention because of its numerous pharmacological activities and its applications to dermatology and cosmetics. In 2020, we demonstrated a liquid chromatography-high resolution mass spectrometry (LC-HRMS) method for the determination of seven medium-chain FFAs in RJ samples. The aim of the present work was to extend our studies on FA profiling of RJ, exploring the presence of common long-chain saturated, mono-unsaturated and poly-unsaturated free FAs in RJ samples using this LC-HRMS method. Among twenty common FAs studied by a targeted approach, palmitic acid, stearic acid and oleic acid were found at concentrations higher than the rest of the FAs (the concentrations of these three acids ranged from 37.4 to 48.0, from 17.7 to 24.0 and from 9.4 to 11.1 mg/100 g of fresh RJ, respectively). The high mass accuracy of LC-HRMS allowed the application of a suspect approach, which enabled the exploration of various C9 and C11 FAs, as well as hydroxylated C12 FAs. Nonenoic acid was indicated as the most abundant among these acids. In addition, for the first time, the presence of a variety of regio-isomers of hydroxymyristic, hydroxypalmitic and hydroxystearic acids was demonstrated in RJ samples.

Whole-Genome Sequences of a Lactobacillus melliventris Strain and Its Myovirus Temperate Phage, phIBH004, Isolated from the Digestive Tract of Apis mellifera in Switzerland.

The complete genome sequence of Lactobacillus melliventris strain IBH004, isolated from the gut of a honeybee worker (Apis mellifera) and containing two plasmids and a temperate phage, was determined using hybrid assembly of Oxford Nanopore and Illumina reads. Phage-sequence relationships were identified from the coding sequences, and a proteomic tree was constructed.

Detailed chemical analysis of honey bee (Apis mellifera) worker brood volatile profile from egg to emergence.

Chemical communication is a widely used mode of communication for social insects and has been demonstrated to be involved in many behaviours and physiological processes such as reproduction, nutrition or the fight against parasites and pathogens. In the honey bee, Apis mellifera, the release of chemical compounds by the brood plays a role in worker behaviour, physiology, and foraging activities and colony health as a whole. Several compounds have already been described as brood pheromones, such as components of the brood ester pheromone and (E)-β-ocimene. Several other compounds originating from diseased or varroa-infested brood cells have been described as triggering the hygienic behaviour of workers. So far, studies of brood emissions have focused on specific stages of development and little is known about the emission of volatile organic compounds by the brood. In this study, we investigate the semiochemical profile of worker honey bee brood during its whole developmental cycle, from egg to emergence, with a specific focus on volatile organic compounds. We describe variation in emissions of thirty-two volatile organic compounds between brood stages. We highlight candidate compounds that are particularly abundant in specific stages and discuss their potential biological significance.

Genome-wide analysis of the chromatin sites targeted by HEX 70a storage protein in the honeybee brain and fat body.

Hexamerins, the proteins massively stored in the larval haemolymph of insects, are gradually used throughout metamorphosis as a source of raw material and energy for the development of adult tissues. Such behaviour defined hexamerins as storage proteins. Immunofluorescence experiments coupled with confocal microscopy show a hexamerin, HEX 70a, in the nucleus of the brain and fat body cells from honeybee workers, an unexpected localization for a storage protein. HEX 70a colocalizes with fibrillarin, a nucleolar-specific protein and H3 histone, thus suggesting a potential role as a chromatin-binding protein. This was investigated through chromatin immunoprecipitation and high-throughput DNA sequencing (ChIP-seq). The significant HEX 70a-DNA binding sites were mainly localized at the intergenic, promoter and intronic regions. HEX 70a targeted DNA stretches mapped to the genomic regions encompassing genes with relevant functional attributes. Several HEX 70a targeted genes were associated with H3K27ac or/and H3K27me3, known as active and repressive histone marks. Brain and fat body tissues shared a fraction of the HEX 70 targeted genes, and tissue-specific targets were also detected. The presence of overrepresented DNA motifs in the binding sites is consistent with specific HEX 70a-chromatin association. In addition, a search for HEX 70a targets in RNA-seq public libraries of fat bodies from nurses and foragers revealed differentially expressed targets displaying hex 70a-correlated developmental expression, thus supporting a regulatory activity for HEX 70a. Our results support the premise that HEX 70a is a moonlighting protein that binds chromatin and has roles in the brain and fat body cell nuclei, apart from its canonical role as a storage protein.

Development of a TaqMan qPCR assay for trypanosomatid multi-species detection and quantification in insects

BackgroundTrypanosomatid parasites are widely distributed in nature and can have a monoxenous or dixenous life-cycle. These parasites thrive in a wide number of insect orders, some of which have an important economic and environmental value, such as bees. The objective of this study was to develop a robust and sensitive real-time quantitative PCR (qPCR) assay for detecting trypanosomatid parasites in any type of parasitized insect sample.MethodsA TaqMan qPCR assay based on a trypanosomatid-conserved region of the α-tubulin gene was standardized and evaluated. The limits of detection, sensitivity and versatility of the α-tubulin TaqMan assay were tested and validated using field samples of honeybee workers, wild bees, bumblebees and grasshoppers, as well as in the human infective trypanosomatid Leishmania major.ResultsThe assay showed a detection limit of 1 parasite equivalent/µl and successfully detected trypanosomatids in 10 different hosts belonging to the insect orders Hymenoptera and Orthoptera. The methodology was also tested using honeybee samples from four apiaries (n = 224 worker honeybees) located in the Alpujarra region (Granada, Spain). Trypanosomatids were detected in 2.7% of the honeybees, with an intra-colony prevalence of 0% to 13%. Parasite loads in the four different classes of insects ranged from 40.6 up to 1.1 × 108 cell equivalents per host.ConclusionsThese results show that the α-tubulin TaqMan qPCR assay described here is a versatile diagnostic tool for the accurate detection and quantification of trypanosomatids in a wide range of environmental settings.Graphical

Respiration levels of honey bee castes as a possible indicator in Varroa mite attraction

It has been demonstrated that fatty acids esters play an important role in the caste selection Varroa mites. However, there may be another mechanism of attraction in the preferred host location. We hypothesize that it may be due to differential respiration rates between larvae, but no investigations have been reported. This research was developed to determine if there is a measurable level and significant difference in honey bee caste respiration that may be exploited by Varroa mites in choice selection. In this study, we measured the respiration of all stages of the Western honey bee Apis mellifera, L. castes to determine the rate of continuous respiration. An assay was conducted using a portable photosynthesis system for gas exchange and chlorophyll fluorescence measurement (Li-Core LI-6400XT) to measure the respiration rate. In this study, a comparative analysis resulted in respiration levels that indicated a significant difference in the CO2 levels of adult worker (777.0 ± 0.002 μg CO2 g insect-1 min-1) and drone (375.5 µg CO2) honey bees. The comparison of worker (5.59 ± 0.002 μg CO2 g insect-1 min-1) and drone (7.20 ± 0.002 μg CO2 g insect-1 min-1) pupae showed no significant difference in the respiration levels; however, there was a noticeable difference in respiration levels of the worker (6.33 ± 0.002 μg CO2 g insect-1 min-1) and drone (10.07 ± 0.002 μg CO2 g insect-1 min-1) larvae. Future research will utilize the knowledge obtained to examine the mite’s attraction to the honey bee castes in host location. This information is a crucial step for the advancement of research studies and the development of management protocols for Varroa.