Worker Honeybees Research Articles

Impact of odorants on perception of sweetness by honey bees.

Organic volatiles produced by fruits can result in overestimation of sweetness by humans, but it is unknown if a comparable phenomenon occurs in other species. Honey bees collect nectar of varying sweetness at different flowering plants. Bees discriminate sugar concentration and generally prefer higher concentrations; they encounter floral volatiles as they collect nectar, suggesting that they, like humans, could be susceptible to sweetness enhancement by odorant. In this study, limonene, linalool, geraniol, and 6-methyl-5-hepten-2-ol were tested for their ability to alter behaviors related to perception of sweetness by honey bees. Honey bees were tested in the laboratory using proboscis extension response-based assays and in the field using feeder-based assays. In the laboratory assays, 6-methyl-5-hepten-2-ol and geraniol, but neither linalool nor limonene, significantly increased responses to low concentrations of sucrose compared with no odorant conditions in 15-day and 25-day-old adult worker honey bees, but not in 35-day-old bees. Limonene reduced responding in 15-day-old bees, but not in the older bees. There was no odorant-based difference in performance in field assays comparing geraniol and limonene with a no odorant control. The interaction of the tested plant volatiles with sucrose concentration revealed in laboratory testing is therefore unlikely to be a major determinant of nectar choice by honey bees foraging under natural conditions. Because geraniol is a component of honey bee Nasonov gland pheromone as well as a floral volatile, its impact on responses in the laboratory may reflect conveyance of different information than the other odorants tested.

Biochemical Indicators and Mortality in Honey Bee (Apis mellifera) Workers after Oral Exposure to Plant Protection Products and Their Mixtures

The honey bee obtains food from bee forage, which comprises crops grown in multi-hectare agricultural fields where various types of plant protection products such as pesticides are used. Some of these negatively affect the honey bee organism. In our research, we aimed to evaluate the effects of three pesticide groups: fungicides (tebuconazole), insecticides (acetamiprid), herbicides (glyphosate), and their mixtures on the functioning of honey bee workers (A. mellifera carnica). Pesticides in various proportions and dilutions were added to sugar syrups and then fed to the bees. Mortality and food intake were recorded daily, while hemolymph analysis was performed after seven days of exposure. Food intake, mortality, and the results of various biochemical analyses differed between the experimental group and the control group receiving untreated sugar syrup. PPP’s mixture of glyphosate tebuconazole and acetamiprid is more toxic to bees than single pesticides. Certain protection products such as pesticides can disrupt the antioxidant and detoxification systems associated with immunity in honey bees. Consequently, honey bees experience weaker conditions and their proper functioning deteriorates. The results obtained from biochemical changes provide a basis for field studies.

Neuroplasticity in honey bee brains: An enhanced micro-computed tomography protocol for precise mushroom body volume measurement

BackgroundIn insect brains, mushroom bodies are associated with memory and learning behavior. It has been demonstrated that the volume of the mushroom bodies in the brain of a worker honey bee changes during the adult stage. Changes in mushroom body volume imply high neuroplasticity in the brains and may be related to the age polyethism of honey bees. A suitable volume measurement method is needed to understand the correlation between behavioral changes and mushroom body volume changes in honey bees. New methodWe developed a new protocol for insect micro-computed tomography by modifying a previously reported method. Permount™ mounting medium was used as the embedding medium for micro-computed tomography scanning. ResultsThis protocol can generate images with high contrast inside the brain and reduce the marked shape changes during specimen processing. From the resulting high-contrast images, we used freeware to generate a three-dimensional model and calculate the volumes of the mushroom bodies in honey bees. The measured volumes of the mushroom bodies were larger than the values reported in most previous studies. There was no significant difference between the left and right mushroom body volumes, but the volumes of honey bee mushroom bodies significantly increased with age. Comparison with existing methodsPrevious protocols for micro-computed tomography using dried samples would cause brain shrinkage; protocols using ethanol-preserved or resin-embedded samples generated images with lower contrast. ConclusionsThe embedding protocol for micro-computed tomography is suitable for calculating volume of the mushroom bodies in honey bee brains.

Effect of commercial bioinsecticides formulated from entomopathogenic fungi on Apis mellifera (Hymenoptera: Apidae)

Bees are essential for agricultural productivity, however, during foraging, they can be exposed to contamination with the residue of synthetic insecticides. Entomopathogenic fungi, an efficient and economically viable alternative to the use of these insecticides, are increasingly used in pest control. However, the viability of agricultural use of these fungi depends on formulations that can increase nontarget organism mortality. Thus, the objective of this study was to evaluate the effects of commercial bioinsecticides formulated from isolates of Beauveria bassiana (Boveril®) and Metarhizium anisopliae (Metarril®) on Apis mellifera workers under laboratory conditions and record the infectious process by scanning electron microscopy. To accomplish this, bees were exposed by direct contact to concentrations of 1.0 × 104,105,106 and 107 conidia.mL−1 applied by means of a continuous-flow manual sprayer. Evaluations were performed at 24-h intervals for five days. Among the different concentrations of bioinsecticides, pathogenicity did not significantly differ, but the effects reduced the mobility of A. mellifera and caused mortality between 55 and 58% at the highest concentration tested after 120h. Scanning electron microscopy analyses showed an accumulation of non-germinated conidia in pollen combs, indicating that grooming performed by workers can attenuate infection by entomopathogenic fungi. Results indicate a moderate lethal effect on bees in the laboratory, requiring further studies to determine the impact of bioinsecticides formulated from entomopathogenic fungi on colonies of A. mellifera under field conditions.

Effects of the combination of Lactobacillus helveticus and isomalto-oligosaccharide on survival, gut microbiota, and immune function in Apis cerana worker bees.

The adult worker bees were fed sucrose syrup or sucrose syrup supplemented with Lactobacillus helveticus KM7, prebiotic isomalto-oligosaccharide (IMO), or L. helveticus KM7 combined with IMO. Survival rate, gut microbiota, and gene expression of gut antimicrobial peptides in worker honey bees were determined. Administration of L. helveticus KM7 and IMO significantly increased the survival rate in worker bees relative to bees fed sucrose only. Then, higher concentration of both lactic acid bacteria and Bifidobacterium in the gut and lower counts of gut fungi, Enterococcus, and Bacteroides-Porphyromonas-Prevotella were observed in bees fed the combination of KM7 and IMO compared with control bees. The combination of L. helveticus KM7 with IMO showed a greater or comparable modulating effect on those bacteria relative to either KM7 or IMO alone. Furthermore, the combination treatment of L. helveticus KM7 and IMO enhanced mRNA expression of antimicrobial peptide genes, including Abaecin, Defensin, and the gene encoding prophenoloxidase (PPO) in the gut compared with both control bees and those either L. helveticus KM7 or IMO alone. These results suggest that the combination of L. helveticus KM7 and IMO synergistically modifies the gut microbiota and immunity and consequently improves the survival rate of Apis cerana adult workers.

Studying the Effect of Using Super Protein as a Food Substitute on Honey Bee Colony Activity

The research was conducted in a private apiary in Al-Buaitha region - Baghdad governorate to evaluate the effect of adding super protein to the diet of honey bee colonies, and to note its effect on the colonies’ activity, growth, development, productivity and some characteristics of the honey produced. It is clear from the results that the best acceptance rates were achieved by the super protein treatment, and according to the results of the statistical analysis, it was significantly superior to all other treatments in terms of measuring the honey area 2.631 cm2, the brood area 1.622 cm2, and the pollen area 0.378 cm2 It was also noted that there was a positive effect in terms of honey area, where the treated cults produced more honey than the comparison treatment, with an average of (2.631 cm, 1.997 cm), respectively. The results showed that feeding with super protein encouraged the worker honey bees to raise more brood and thus increase the area of the brood, as it outperformed the comparison treatment with an average of (1.622 cm2 1.024 cm2), respectively.

Novel Insights into the circRNA-Modulated Developmental Mechanism of Western Honey Bee Larval Guts.

Circular RNAs (circRNAs) are a class of novel non-coding RNAs (ncRNAs) that play essential roles in the development and growth of vertebrates through multiple manners. However, the mechanism by which circRNAs modulate the honey bee gut development is currently poorly understood. Utilizing the transcriptome data we obtained earlier, the highly expressed circRNAs in the Apis mellifera worker 4-, 5-, and 6-day-old larval guts were analyzed, which was followed by an in-depth investigation of the expression pattern of circRNAs during the process of larval guts development and the potential regulatory roles of differentially expressed circRNAs (DEcircRNAs). In total, 1728 expressed circRNAs were detected in the A. mellifera larval guts. Among the most highly expressed 10 circRNAs, seven (novel_circ_000069, novel_circ_000027, novel_circ_000438, etc.) were shared by the 4-, 5-, and 6-day-old larval guts. In addition, 21 (46) up-regulated and 22 (27) down-regulated circRNAs were, respectively, screened in the Am4 vs. Am5 (Am5 vs. Am6) comparison groups. Additionally, nine DEcircRNAs, such as novel_circ_000340, novel_circ_000758 and novel_circ_001116, were shared by these two comparison groups. These DEcircRNAs were predicted to be transcribed from 14 and 29 parental genes; these were respectively annotated to 15 and 22 GO terms such as biological regulation and catalytic activity as well as 16 and 21 KEGG pathways such as dorsoventral axis formation and apoptosis. Moreover, a complicated competing endogenous RNA (ceRNA) network was observed; novel_circ_000838 in the Am4 vs. Am5 comparison group potentially targeted ame-miR-6000a-3p, further targeting 518 mRNAs engaged in several developmental signaling pathways (e.g., TGF-beta, hedgehog, and wnt signaling pathway) and immune pathways (e.g., phagosome, lysosome, and MAPK signaling pathway). The results demonstrated that the novel_circ_000838-ame-miR-6000a-3p axis may plays a critical regulatory part in the larval gut development and immunity. Furthermore, back-splicing sites of six randomly selected DEcircRNAs were amplified and verified by PCR; an RT-qPCR assay of these six DEcircRNAs confirmed the reliability of the used high-throughput sequencing data. Our findings provide a novel insight into the honey bee gut development and pave a way for illustration of the circRNA-modulated developmental mechanisms underlying the A. mellifera worker larval guts.

Understanding the differential performance in hygienic behavior against dead brood of drones and workers of Apis mellifera: a chemical approach

Hygienic behavior is a social health mechanism displayed by honey bee workers of Apis mellifera by which dead or diseased brood is removed from the hive. Such behavior is known to be induced by olfactory cues. The efficiency of hygienic behavior toward drone brood cells is significantly lower than that of worker brood cells. A possible explanation for this difference lies in the cell wax cap of drone brood, which may act as a barrier to the diffusion of characteristic odors associated with diseased brood. The aim of this research was to explore the importance of drone cell wax cap as an interfering factor in the diffusion of chemical signals within honey bee colonies. To this end, brood removal percentages were compared among several types of brood: pin-killed worker, pin-killed drone, pin-killed worker with a healthy drone wax cap, healthy worker with a pin-killed drone wax cap, and healthy worker covered with a healthy drone wax cap. Results showed that the removal behavior did not differ between cells with pin-killed worker and pin-killed worker with a healthy drone wax cap, but was significantly higher towards healthy worker pupae covered with dead drone wax cap compared to the negative control (healthy worker pupa covered with a healthy drone cell wax cap). Present evidence suggests that chemical signals associated with the dead drone brood would be less volatile and retained to some extent in the wax cap, and partially explain the differential hygienic behavior between both types of brood.

Visitation of Apis mellifera L. in Runner Bean (Phaseolus coccineus L.) and Its Exposure to Seasonal Agrochemicals in Agroecosystems

Plant species and abiotic factors including season appear to be the most important variables influencing the frequency of visits by honeybees (Apis mellifera L.). In the present study, we evaluated the activity of honeybee workers visiting runner bean (Phaseolus coccineus L.) local cultivar ‘Piękny Jaś’. The runner beans are widely cultivated in south-eastern Poland, and are an important forage plant for honeybees in agroecosystems. We aimed at a comprehensive monitoring of the health of colonies and symptoms in A. mellifera in response to acute exposure to pesticides. The most numerous visits of A. mellifera were observed at the highest flower opening of the runner bean. A very weak positive correlation was observed between the number of honeybees on P. coccineus, the number of visited flowers, the time spent per flower and air temperature. The visitation rates of honeybees were more frequent at mid-day and decreased after 15:00. Signs of poisoning were detected in two out of seven apiaries monitored for acute pesticide exposure symptoms on runner bean plantations. The analysis of dead honeybee samples revealed the acute exposure of honeybees to the imidacloprid (neonicotinoid) and chlorpyrifos (organophosphorus) insecticides, which are highly toxic and banned in the European Union. Hazard quotient (HQ) screening showed an elevated burden of imidacloprid and chlorpyrifos corresponding to 7.1% and 10% of the LDD50, respectively, most likely indicating bee poisoning due to chronic exposure to these substances with contaminated food. Noteworthy was the presence of three fungicides that could pose a risk of poisoning in honeybees.

The Longevity Of Africanized Apis mellifera L. Influenced By Synthetic Phytosanitary Products Used In Soybean

Honey bees are the main pollinators, but they are in decline and synthetic phytosanitary products (SSPs) are suspected to be related. Therefore, this study aimed to evaluate the effect of three commercial SPPs, used to control the stink bug in soybean, formulated with Acetamiprid + Alpha-cypermethrin; Imidacloprid + Beta-Cyfluthrin and Fenitrothion + Esfenvalerate, on adult worker Africanized Apis mellifera. For this, worker honey bees was exposed to SPPs through three bioassays: 1) sprayed on the bees, 2) sprayed on Petri dishes in which the bees was subsequently placed, and 3) mixed with the Candy paste supplied to the bees. The honey bees in bioassay 3 had their midgut submitted to histopathological analysis. All bioassays was maintained in a climatized room (34 ± 2ºC, RH 60 ± 5%) and the workers longevity was verified at 1 up to 120 hours. In bioassay 1 at the time of 6 hours evaluation, 100% of the bees of all the SPPs was dead. In bioassay 2 all bees was dead after two hours of exposure of the three SPP. In bioassay 3 the SPP1 and SPP2 caused 100% mortality after 60 hours of exposure, to theSPP3 all bees was dead after 96 hours, while for the control, after 96 hours only 45% of the bees were dead. Histological analysis revealed the destruction of the midgut cells of all workers from the three SPPs treatments. The three evaluated SPPs showed no selectivity for A. mellifera workers, reducing bee longevity.

Transcriptome analysis of Apis mellifera antennae reveals molecular divergence underlying the division of labour in worker bees.

The olfactory system plays a fundamental role in mediating insect behaviour. Worker bees exhibit an age-dependent division of labour, performing discrete sets of behaviours throughout their lifespan. The behavioural states of bees rely on their sense of the environment and chemical communication via their olfactory system, the antennae. However, the olfactory adaptation mechanism of worker bees during their behavioural development remains unclear. In this study, we conducted a comprehensive and quantitative analysis of antennal gene expression in the Apis mellifera of newly emerged workers, nurses, foragers and defenders using RNA-seq. We found that the antenna tissues of honey bees continued developing after transformation from newly emerged workers to adults. Additionally, we identified differentially expressed genes associated with bee development and division of labour. We validated that major royal jelly protein genes are highly and specifically expressed in nurse honey bee workers. Furthermore, we identified and validated significant alternative splicing events correlated with the development and division of labour. These findings provide a comprehensive transcriptome profile and a new perspective on the molecular mechanisms that may underlie the worker honey bee division of labour.

Trypanosomatids in honey bee colonies in Spain: A new specific qPCR method for specific quantification of Lotmaria passim, Crithidia mellificae and Crithidia bombi

Bee trypanosomatids have not been widely studied due to the original belief that these organisms were not pathogenic to honey bees. However, trypanosomatids have been linked to increased winter mortality in honey bee colonies in recent years and it has been shown that these pathogens can shorten a honey bee worker’s lifespan in laboratory conditions. These studies found that this mortality corresponded to dose-dependent infection. Although Lotmaria passim is the most prevalent species worldwide, the natural load in colonies remains poorly investigated. Here we describe a new highly specific and sensitive qPCR method that allows the differentiation and quantification of the parasitic load of each of the three most common trypanosomatid species described to date in honey bee colonies: L. passim, Crithidia mellificae, and Crithidia bombi. We have used this new method to analyze honey bee colonies in central Spain and confirm that L. passim is the most common species and the one with higher parasitic loads in the colonies, which increased over the years, being higher in spring than in autumn. Crithidia mellificae was present along the study, with the highest prevalence in autumn 2019 and lately it was only found in non-quantifiable loads. Crithidia bombi was not detected in any of the colonies analyzed.

Evaluating the Impact of Neonicotinoid and Sulfoximine Pesticides on Honey Bee Workers, Apis mellifera L., under Laboratory Conditions

Evaluating the Impact of Neonicotinoid and Sulfoximine Pesticides on Honey Bee Workers, Apis mellifera L., under Laboratory Conditions

Toxicity and sublethal effects of lead (Pb) intake on honey bees (Apis mellifera)

Heavy metal pollution is becoming a worldwide problem affecting pollinators. The massive use of lead (Pb), the most harmful metal for the biosphere, in industries has increased the risk for honey bees. Pb exerts toxicity on living organisms inducing mainly oxidative stress. We assessed the toxicity and sublethal effects of Pb ingestion on protein content, catalase (CAT) activity, fat content and fatty acid (FA) profile of honey bee workers (Apis mellifera L.) under different nutritional conditions during chronic exposure tests. The LD50 was 15.13 ± 6.11 μg Pb2+/bee, similar to other reports. A single oral sublethal dose of 15 μg of Pb2+ affected the survival of bees fed with sugary food for ten days after Pb ingestion while supplementing the diet with bee bread improved Pb tolerance. The highest protein content was found in bees fed with the sugar paste and bee bread diet without Pb. CAT activity tended to decrease in bees of Pb groups independently of diet. Fat content was not affected by the diet type received by bees or Pb ingestion, but the FAs profile varied according to the nutritional quality of the diet. The results highlight that a single sublethal dose of Pb negatively affected the body proteins of bees despite the nutritional condition but did not disturb the FAs profile of workers. Nutrition plays an important role in preventing Pb-induced toxicity in honey bees.

The effect of forager loss on honeybee workers temporal polyethism and social network structure

The western honeybee Apis mellifera is perhaps the best-known example of an advanced eusocial species displaying temporal polyethism, a process in which workers perform different tasks in the colony as they age. Previous studies demonstrated that this temporal division of labour is not only regulated by the age of the bees but also by the proportion of workers performing the required tasks in the colony. As we progress through the Anthropocene, the Intergovernmental Panel on Climate Change predicted that a significant increase in both the frequency and intensity of severe weather events can be expected in the coming decades. Here, we performed a controlled interrupted time series experiment with the objective of quantifying the possible effects that these weather events might have in the honeybee colony dynamics. By simulating a significant loss of foragers in the colony, we observed that honeybee workers quickly replaced the missing foragers by accelerating their transition to a subsequent task and, in some cases, completely skipped several of the natural task transitions with respect to their age. In addition, we analysed how the colony social network structure is affected by the sudden loss of foragers. Indeed, our data show that honeybee colonies increased their network cohesion with workers having a higher number of interactions as well as becoming more closely connected to one another soon after the disturbance. Overall, our data shows that even when faced with a substantial perturbation, honeybees can respond swiftly in order to maintain colony homeostasis and likely increase their resilience against future perturbations.

Midgut health of Apis mellifera workers on oral administration of Lactobacillus rhamnosus and organic acids

A study about the provision of Lactobacillus rhamnosus NR_113332) and organic acids was carried out. 108 colony-forming units of probiotics were provided to the caged workers with and without the addition of 2.99% lactic acid, 2.91% acetic acid, and 1.96% acetic acid. After two weeks of oral administration, on termination, five bees were collected and dissected from each cage, and their midguts were cut into 8 μm thick sections by microtome. Subsequently, these histologically processed preparations were observed under camera fitted microscope. It was observed that the experimental workers were provided with diets: (pollen+ 50% w/v sucrose in 1.96% acetic acid, pollen+ L. rhamnosus in 50% w/v sucrose in distilled water, pollen+ L. rhamnosus in 50% w/v sucrose in 2.99% lactic acid, pollen+ L. rhamnosus in 50% w/v sucrose in 2.91% acetic acid, pollen+ L. rhamnosus in 50% w/v sucrose in 1.96% acetic acid) had a considerable quantity of peritrophic membranes. The mean midgut lumen diameters (µm2) were 373.33 ± 98.38, 296.67 ± 23.33, 243.33 ± 71.72, 426.67 ± 40.55, 576.67 ± 93.87 as compared to control’s 240.00 ± 30.55. The provision of probiotic and organic acids resulted in a slightly disturbed midgut epithelial wall, the presence of more quantity of peritrophic membranes, saved regeneration crypts, more gastric cells discharging, and some cytoplasmic vacuolization of caged worker bees. Our study suggests that probiotics and organic acids can be used as an environmentally friendly apipromotor, but verification by field studies is needed.

Physiological Impacts of Some Food Additives on Honeybee Workers (Apis mellifera L.)

Physiological Impacts of Some Food Additives on Honeybee Workers (Apis mellifera L.)

Differences in the suitability of published honey bee (Apis mellifera) reference genes between the African subspecies Apis mellifera scutellata and European derived Apis mellifera.

Quantitative real-time polymerase chain reaction (qPCR) is a method widely used to determine changes and differences in gene expression. As target gene expression is most often quantified relative to the expression of reference genes, the validation of suitable reference genes is of critical importance. In practice, however, such validation might not be thoroughly conducted if the same species and the same tissue or body parts are used for qPCR experiments. Here we show, that qPCR reference genes published for workers of European honey bee (Apis mellifera) subspecies fail to be stably expressed in workers of the African subspecies Apis mellifera scutellata. This is the case even when the sampled workers are in the same life stage, the same organ was dissected and the same reagents were used. Thus, reference genes need to be thoroughly re-tested before they can be used as suitable references even when the only thing that changes is the subspecies used.

Effects of the insecticide imidacloprid on the post-embryonic development of the honey bee Apis mellifera (Hymenoptera: Apidae)

The widespread use of pesticides in agriculture has been linked to declines in bee populations worldwide. Imidacloprid is a widely used systemic insecticide that can be found in the pollen and nectar of plants and has the potential to negatively impact the development of bee larvae. We investigated the effects of oral exposure to a realistic field concentration (20.5 ng g−1) of imidacloprid on the midgut and fat body of Apis mellifera worker larvae. Our results showed that larvae exposed to imidacloprid exhibited changes in the midgut epithelium, including disorganization of the brush border, nuclear chromatin condensation, cytoplasm vacuolization, and release of cell fragments indication cell death. Additionally, histochemical analysis revealed that the midgut brush border glycocalyx was disorganized in exposed larvae. The fat body cells of imidacloprid-exposed larvae had a decrease in the size of lipid droplets from 50 to 8 μm and increase of 100 % of protein content, suggesting possible responses to the stress caused by the insecticide. However, the expression of de cdc20 gene, which plays a role in cell proliferation, was not affected in the midgut and fat body of treated larvae. These results suggest that imidacloprid negatively affects non-target organs during the larval development of A. mellifera potentially impacting this important pollinator species.

Mandibular glands secrete 24-methylenecholesterol into honey bee (Apis mellifera) food jelly

Honey bee (Apis mellifera) workers feed their larvae with food jelly that is secreted by specialized glands in their heads – the hypopharyngeal and the mandibular glands. Food jelly contains all the nutrients the larvae need to develop into adult honey bees, including essential dietary sterols. The main sterol in food jelly, 24-methylenecholesterol (24MC), is pollen-derived and delivered in food jelly to the larvae in a complex with two proteins, major royal jelly protein 1 (MRJP1) and apisim. Whereas the proteins are synthesized in the hypopharyngeal glands, the sterol-secreting gland has not been identified. We here identified the mandibular glands as sterol-secreting gland for food jelly production by direct detection of the four main honey bee sterols (24MC, campesterol, β-sitosterol and isofucosterol). Furthermore, 24MC seems to be specifically enriched in the mandibular glands, thereby ensuring that food jelly contains the amounts of 24MC necessary for complex formation with MRJP1 and apisimin.