Worker Honeybees Research Articles

Reproductive Cessation and Post-Reproductive Lifespan in Honeybee Workers

The post-reproductive lifespan is an evolutionary enigma because the cessation of reproduction in animals seems contrary to the maximization of Darwinian fitness. Several theories aim to explain the evolution of menopause, one of which suggests that females of a certain age receive more fitness benefits via indirect selection (kin selection) than they would directly from continuing reproduction. Post-reproductive lifespans are not very common in nature but have been described in humans, nonhuman primates, a few species of toothed whales, guppies, and in some insect societies consisting of clonal colony members, such as aphid and ant societies. Here, we provide evidence that menopause also exists in honeybee societies. Our study shows that workers with a short life expectancy (older and/or injured workers) invest fewer resources and less time in their own reproduction than workers with a long life expectancy (younger and/or uninjured workers), even if their colony is hopelessly queenless. These results are consistent with the kin selection explanation for the evolution of menopause and help us understand the net effects of relatedness and social cooperation in animals.

Increasing temperature and time in glasshouses increases honey bee activity and affects internal brood conditions

Honey bees are globally important pollinators, key to many aspects of ecosystem function and agricultural production. However they are facing an increasing array of stress factors. These stressors include exposure to pathogens and pesticides, agricultural intensification, and changes in climate, and likely contribute to colony dysfunction and colony losses. Here we use temperature-controlled glasshouse experiments to investigate the impact of a field-realistic temperature-range on honey bee colonies, including temperatures based on projections for near-future local conditions. We show that increased temperatures have a significant impact on honey bee worker activity, with increased worker movement in and out of colonies, particularly over 30 °C. In addition, increased glasshouse temperatures led to significantly higher brood (egg, larval and pupal cells) humidity. Finally, temperature had a more severe impact at the later end of the experiment than at the start (on worker movement and brood conditions), suggesting that colonies under stress (either due to exposure to thermal stress or glasshouse confinement) have more difficulty in manging thermoregulation. These results indicate the potential impact of higher temperatures on the healthy functioning of these important pollinators.

Analysis of X-ray irradiation effects on the mortality values and hemolymph immune cell composition of Apis mellifera and its parasite, Varroa destructor

Varroa destructor is one of the most destructive enemies of the honey bee, Apis mellifera all around the world. Several control methods are known to control V. destructor, but the efficacy of several alternative control methods remains unexplored. Irradiation can be one of these unknown solutions but before practical application, the effectiveness, and the physiological effects of ionizing radiation on the host and the parasite are waiting to be tested. Therefore, the objective of our study was to investigate the effects of different doses (15, 50, 100, and 150 Gy) of high-energy X-ray irradiation through mortality rates and hemocyte composition changes in A. mellifera workers and record the mortality rates of the parasite. The mortality rate was recorded during short-term (12, 24, and 48 h) and long-term periods (3, 6, 12, 18, and 24d).The sensitivity of the host and the parasite in case of the higher doses of radiation tested (50, 100, and 150 Gy) been demonstrated by total mortality of the host and 90 % of its parasite has been observed on the 18th day after the irradiation. V. destructor showed higher sensitivity (1.52-times higher than the adult honey bee workers) at the lowest dose (15 Gy). A. mellifera hemocytes were influenced significantly by radiation dosage and the elapsed time after treatment. The higher radiation doses increased plasmatocyte numbers in parallel with the decrease in prohemocyte numbers. On the contrary, the numbers of granulocytes and oencoytes increased in the treated samples, but the putative effects of the different dosages on the recorded number of these hemocyte types could not be statistically proven. In summary, based on the outcome of our study X-ray irradiation can be deemed an effective tool for controlling phoretic V. destructor. However, further research is needed to understand the physiological response of the affected organisms.

Quantifying Abdominal Coloration of Worker Honey Bees.

The main drawback in using coloration to identify honey bee subspecies is the lack of knowledge regarding genetic background, subjectivity of coloration grading, and the effect of the environment. The aim of our study was to evaluate the effect of environmental temperature on the abdominal coloration of honey bee workers and to develop a tool for quantifying abdominal coloration. We obtained four frames of honey bee brood from two colonies and incubated them at two different temperatures (30 and 34 °C). One colony had workers exhibiting yellow marks on the abdomen, while the other did not. We collected hatched workers and photographed abdomens. Images were analyzed using custom-written R script to obtain vectors that summarize the coloration over the abdomen length in a single value-coloration index. We used UMAP to reduce the dimensions of the vectors and to develop a classification procedure with the support vector machine method. We tested the effect of brood origin and temperature on coloration index with ANOVA. UMAP did not distinguish individual abdomens according to experimental group. The trained classifier sufficiently separated abdomens incubated at different temperatures. We improved the performance by preprocessing data with UMAP. The differences among the mean coloration index values were not significant between the gray groups incubated at different temperatures nor between the yellow groups. However, the differences between the gray and yellow groups were significant, permitting options for application of our tool and the newly developed coloration index. Our results indicate that the environmental temperature in the selected range during development does not seem to impact honey bee coloration significantly. The developed color-recording protocol and statistical analysis provide useful tools for quantifying abdominal coloration in honey bees.

The role of temperature on the development of circadian rhythms in honey bee workers.

Circadian rhythms in honey bees are involved in various processes that impact colony survival. For example, young nurses take care of the brood constantly throughout the day and lack circadian rhythms. At the same time, foragers use the circadian clock to remember and predict food availability in subsequent days. Previous studies exploring the ontogeny of circadian rhythms of workers showed that the onset of rhythms is faster in the colony environment (~2 days) than if workers were immediately isolated after eclosion (7-9 days). However, which specific environmental factors influenced the early development of worker circadian rhythms remained unknown. We hypothesized that brood nest temperature plays a key role in the development of circadian rhythmicity in young workers. Our results show that young workers kept at brood nest-like temperatures (33-35 °C) in the laboratory develop circadian rhythms faster and in greater proportion than bees kept at lower temperatures (24-26 °C). In addition, we examined if the effect of colony temperature during the first 48 h after emergence is sufficient to increase the rate and proportion of development of circadian rhythmicity. We observed that twice as many individuals exposed to 35 °C during the first 48 h developed circadian rhythms compared to individuals kept at 25 °C, suggesting a critical developmental period where brood nest temperatures are important for the development of the circadian system. Together, our findings show that temperature, which is socially regulated inside the hive, is a key factor that influences the ontogeny of circadian rhythmicity of workers.

Sex-specific element accumulation in honey bees (Apis mellifera).

Honey bees are social insects that show division of labor and sexual dimorphism. Female honey bees differentiate in two different castes, queens or worker bees, while males are called drones. Worker bees have different tasks in the hive including collection of food, its processing, caring for brood, protecting the hive, or producing wax. The drones' only role is to mate with a virgin queen. Many studies have dealt with differences in physiology, behavior, and morphology of workers and drones. This is the first study that demonstrates differences in element accumulation and composition between workers and drones honey bees. Using inductively coupled plasma mass spectrometry, we found that worker honey bees have higher concentrations of most elements analyzed. Drones had higher concentrations of elements essential to bees, Na, P, S, Zn, Cu, and especially Se (2.2 × higher), which is known to be important for sperm quality and fertility in many animals. Until now higher Se content was not observed in male insects. These differences can be attributed to different environmental exposure, reproductive role of drones, but mostly to the food workers and drones consume. Worker bees feed on bee bread, which is rich in minerals. Drones are fed food pre-processed by worker bees.

Histological Changes Of Bee Worker (Apis mellifera L.) By Bee Venom And Amino Acids Feeding

The effect of feeding honey bee colonies on a sugar solution to which ‎bee ‎venom has been added at a concentration of 5‎‏0‏‎ mg/liter sugar solution ‎and on amino ‎acids extracted from clover 4 cm amino acids/liter sugar ‎solution on the amount of ‎food consumed by honey bee workers as well as ‎the effect on gland tissues ‎Hypopharyngeal and maxillary glands and ‎histological changes that occur in them. ‎Nine honeybee colonies were tested in an apiary of the Bee Research ‎Department, Plant Protection ‎Research Institute, Agricultural Research Center, ‎Ministry of Agriculture, ‎Giza, Egypt, during 2020-2021. Statistically, there are no significant differences in the amount of food consumed by honeybee workers, whether the bee venom solution or the amino acid solution, ‎compared to ‎the control colonies that fed on the sugar solution only. No histological changes were observed in the ‎hypopharyngeal (HPGs) and mandibular‎ ‎glands (MGs) in workers fed sugary solution (control colonies). Feeding ‎with ‎amino acids resulted in histological ‎changes in the HPGs and MGs, ‎ as most of the ‎acini were atrophic and smaller ‎in size than usual, with the loss of vesicles in the ‎cytoplasm. As for the bees ‎that fed on a solution of bee venom, histological changes ‎ in the HPGs and ‎MGs were observed, where severe atrophy and contraction of the ‎acini with ‎an irregular outline were noted. Nuclear thickening of the ‎‎epithelial cell lining was observed. The histological structure of the lower glands ‎showed ‎atrophy in some individual cells, which led to damage to the ‎development of both the ‎HPGs and MGs.‎

A Hitchhiker's Ride: The Honey Bee Louse Braula Coeca (Diptera: Braulidae) Selects its Host by Eavesdropping.

The bee louse Braula spp. had until recently a distribution coincident with its host the honey bee. The adult fly usually attaches to a worker honey bee and steals food from its mouth. However, not all worker bees carry Braula spp. and the mechanism used by Braula spp. to select hosts is not well understood. Using choice remounting bioassays and chemical analyses, we determined host selection and the cues used by B. coeca, a species associated with the African honey bee Apis mellifera scutellata. Braula coeca successfully remounted bees from which they were initially removed and preferred their mandibular gland pheromones (MDG) over those of bees not carrying them. The bee lice did not show any preference for the cuticular hydrocarbons of both types of workers. Chemical analyses of the MDG extracts, revealed quantitative differences between the two categories of workers, with workers carrying B. coeca having more of the queen substance (9-oxo-2(E)-decenoic acid) and worker substance (10-hydroxy-2(E)-decenoic). Braula coeca showed a dose response to the queen substance, indicating its ability to use host derived kairomones as cues that allowed it to benefit from trophallactic dominance by individuals that have a higher probability of being fed by other workers.

Farnesol, a component of plant-derived honeybee-collected resins, shows JH-like effects in Apis mellifera workers

Farnesol, a sesquiterpene found in all eukaryotes, precursor of juvenile hormone (JH) in insects, is involved in signalling, communication, and antimicrobial defence. Farnesol is a compound of floral volatiles, suggesting its importance in pollination and foraging behaviour. Farnesol is found in the resin of Baccharis dracunculifolia, from which honeybees elaborate the most worldwide marketable propolis. Bees use propolis to seal cracks in the walls, reinforce the wax combs, and as protection against bacteria and fungi. The introduction within a honeybee hive of a compound with potential hormonal activity can be a challenge to the colony survival, mainly because the transition from within-hive to outside activities of workers is controlled by JH. Here, we tested the hypothesis that exogenous farnesol alters the pacing of developing workers. The first assays showed that low doses of the JH precursor (0.1 and 0.01 µg) accelerate pharate-adult development, with high doses being toxic. The second assay was conducted in adult workers and demonstrated bees that received 0.2 µg farnesol showed more agitated behaviour than the control bees. If farnesol was used by corpora allata (CA) cells as a precursor of JH and this hormone was responsible for the observed behavioural alterations, these glands were expected to be larger after the treatment. Our results on CA measurements after 72 h of treatment showed bees that received farnesol had glands doubled in size compared to the control bees (p < 0.05). Additionally, we expected the expression of JH synthesis, JH degradation, and JH-response genes would be upregulated in the treated bees. Our results showed that indeed, the mean transcript levels of these genes were higher in the treated bees (significant for methyl farnesoate epoxidase and juvenile hormone esterase, p < 0.05). These results suggest farnesol is used in honeybees as a precursor of JH, leading to increasing JH titres, and thus modulating the pacing of workers development. This finding has behavioural and ecological implications, since alterations in the dynamics of the physiological changes associated to aging in young honeybees may significantly impact colony balance in nature.

Seasonal Changes in Hemolymph Protein Level and Hypopharyngeal Gland Size Depending on Age and In-Nest Location of Honeybee Workers

A honeybee colony, as a super-organism, is regulated through age-polyethism. A honeybee worker’s age is considered by means of a chronological and biological approach. The biological age is estimated with physiologically related biological markers, e.g., total hemolymph protein content (THP) and hypopharyngeal gland size (HGs), which also vary seasonally. Contemporary insights into the age-related spatial workers’ distribution within the hive nest space regarding biological age are insufficiently clarified. This study aimed to monitor changes in selected physiological markers during the entire season in relation to worker age and their spatial position in the hive nest. THP content and HG size analysis was performed in nine colonies for the entire season to compare the physiological markers within and among the groups of the workers whose ages were known and sampled in different hive parts. Seasonal impact on the biomarkers’ development was confirmed in known-age workers. In the case of HGs, this impact was the most apparent in 4- and 5-week-old workers. For THP, the seasonal impact was the most obvious in 2-week-old workers. The highest THP was found in 1- and 2-week-old workers during the entire season. Biologically younger workers of the same age were located predominantly in upper hive parts consistently throughout the year and vice versa. These workers showed significantly higher THP in comparison with those sampled below. Regarding the chronological age, the downwards, spatially shifting mechanism of workers within the hive nest while they aged was characterized. We recommend storage of diluted hemolymph samples up to one month before performing an assay if necessary. The physiological context, relation to division of labor and benefits for beekeeping practices are discussed.

Fatty acid profile of Apis mellifera workers in the face of an immune challenge

Fatty acid profile of Apis mellifera workers in the face of an immune challenge

Developmental exposure to hormone-mimicking insect growth disruptors alters expression of endocrine-related genes in worker honey bee (Hymenoptera: Apidae) brains and hypopharyngeal glands.

Division of labor within a honey bee colony creates a codependence between bees performing different tasks. The most obvious example of this is between the reproductive queen and worker bees. Queen bees lay 1,000 or more eggs a day, while young worker bees tend and feed queens. Young workers and queens can be exposed to pesticides when foragers return to the hive with contaminated resources. Previous research has found negative effects of larval exposure to insect-grown disruptors (IGD) methoxyfenozide and pyriproxyfen, on adult responsiveness to artificial queen pheromone. The present work investigates potential physiological and molecular mechanisms underpinning this behavioral change by examining the development of hypopharyngeal glands and ovaries as well as the expression of genes related to reproduction and worker endocrine signaling in the brain and hypopharyngeal gland tissues. Though hypopharyngeal gland and ovary development were not altered by developmental exposure to IGDs, gene expression differed. Specifically, in the brain tissue, ilp1 was downregulated in bees exposed to pyriproxyfen during development, and Kr-h1 was downregulated in both methoxyfenozide- and pyriproxyfen-exposed bees. In the hypopharyngeal glands, Kr-h1, EcR-A, EcR-B, and E75 were upregulated in honey bees exposed to methoxyfenozide compared to those in the pyriproxyfen or control treatments. Here we discuss these results and their potential implications for the health and performance of honey bee colonies.

Profiling the neuroproteomics of honeybee brain: A clue for understanding the role of neuropeptides in the modulation of aggressivity

The aggressivity is modulated in honeybee brain through a series of actions in cascade mode, with the participation of the neuropeptides AmAST A (59–76) and AmTRP (254–262). The aggressivity of honeybees was stimulated by injecting both neuropeptides in the hemocoel of the worker honeybees, which were submitted to behavioral assays of aggression. The brain of stinger individuals were removed by dissection and submitted to proteomic analysis; shotgun proteomic approach of honeybee brain revealed that both neuropeptides activate a series of biochemical processes responsible by production of energy, neuronal plasticity and cell protection. In addition to this, AmTRP (254–262) elicited the expression of proteins related to the processing of the potential of action and lipid metabolism; meanwhile AmAST A (59–76) elicited the metabolism of steroids and Juvenile hormone-related metabolism, amongst others. Apparently, the most complex biochemical process seems to be the regulation of ATP production, which occurs at two levels: i) by a subgroup of proteins common to the three experimental groups, which are over−/under-regulated through glycolysis, pyruvate pathway, Krebbs cycle and oxidative phosphorylation; ii) by a subgroup of proteins unique to the each experimental group, which seems to be regulated through Protein-Protein Interactions, where the protein network regulated by AmTRP (254–262) seems to be more complex than the other two experimental groups. SignificanceRecently we reported the effect of the neuropeptides AmAST A (59–76) and AmTRP (254–262) in the modulation of the aggressive behavior of the worker honeybees. Up to now it is known that the simple presence of the allatostatin and tachykinin-related-peptide in bee brain, is enough for inducing the aggressive behavior. However, nothing was known about how these neuropeptides perform their action, inducing the aggressive behavior. The results of the present study elucidated some of the metabolic pathways that were activated or inhibited to support the complex defensive behavior, which includes the aggressivity. These results certainly will impact the behavioral research of honeybees, since we are paving the way for understanding the molecular base of regulation, of individual /nest defense of honeybees.

Memory consolidation in honey bees is enhanced by down-regulation of Down syndrome cell adhesion molecule and changes its alternative splicing.

Down syndrome cell adhesion molecule (Dscam) gene encodes a cell adhesion molecule required for neuronal wiring. A remarkable feature of arthropod Dscam is massive alternative splicing generating thousands of different isoforms from three variable clusters of alternative exons. Dscam expression and diversity arising from alternative splicing have been studied during development, but whether they exert functions in adult brains has not been determined. Here, using honey bees, we find that Dscam expression is critically linked to memory retention as reducing expression by RNAi enhances memory after reward learning in adult worker honey bees. Moreover, alternative splicing of Dscam is altered in all three variable clusters after learning. Since identical Dscam isoforms engage in homophilic interactions, these results suggest a mechanism to alter inclusion of variable exons during memory consolidation to modify neuronal connections for memory retention.

Simultaneous PCR detection of Paenibacillus larvae targeting insertion sequence IS256 and Melissococcus plutonius targeting pMP1 plasmid from hive specimens

Paenibacillus larvae and Melissococcus plutonius represent the most threatening bacterial diseases of honeybee (Apis mellifera)—American and European foulbrood, respectively. For efficient control of those diseases, rapid and accurate detection of the pathogens is crucial. Therefore, we developed a novel multiplex PCR method simultaneously detecting both pathogens. To design and optimize multiplex PCR reaction, four strains of P. larvae representing four ERIC genotypes I–IV (strain DSM 7030—ERIC I, DSM 25430—ERIC II, LMG 16252—ERIC III, DSM 3615—ERIC IV) were selected. Those strains were fully sequenced using long-read sequencing (Sequel I, Pacific Biosciences). For P. larvae, the multicopy insertion sequence IS256 identified in all genotypes of P. larvae was selected to provide high sensitivity. M. plutonius was detected by plasmid pMP1 sequence and the virulence verified by following detection of ETX/MTX2 toxin responsible for pore formation in the cell membrane. As an internal control, a gene encoding for major royal jelly protein 1 specific for honeybees was selected. The method was validated on 36 clinical specimens collected from the colonies suffering from American and European foulbrood in the Czech Republic. Based on the results, sensitivity of PCR was calculated to 93.75% and specificity to 100% for P. larvae diagnosed from hive debris and 100% sensitivity and specificity for honeybee workers and larval scales as well as for diseased brood infected by M. plutonius.

Some essential oils as potential control agents for varroa mite (Varroa destructor) in infected honey bees (Apis mellifera).

Ecto-parasite, varroa mite, (Varroa destructor), is the primary pest affecting the apiculture sector globally in various regions. This study examined the toxicity of nine essential oils to Apis mellifera L. and the acaricidal impact of those oils against V. destructor. The acaricidal effects of nine essential oils, extracted from plant materials were used. In the screening experiment, 10 mg of the active ingredients of the plant material extracts were prepared in an alcohol solution with concentrations of 5%, 10%, and 15%. For each type of plant extract, five female V. destructor were transferred to a Petri dish with five worker bees incubated at 70% humidity and 33°-34° for 2 days, for each treatment four replicates were used compared to the control. Forty-eight hours following treatment, the number of dead and live mites was counted to determine the mortality rate. In the second assay experiment, the best five essential oils of the previous experiment were selected to re-assess their effectiveness on varroa mites and honeybee workers by using a concentration of 15%. Five females of V. destructor were transferred to a Petri dish with 10 adult bees and treated with the solution of the selected oils. Five replicates and control treatments were taken for each sample simultaneously. Dead and live bees were counted for each replicate at 48 hours after treatment. There were no significant differences between the concentrations used of each oil on the rate of death of mites, and its effectiveness ranged between 70.0% and 53.3% compared to the control groups. In addition, the best oil used was bitter melon, with a death rate of 80% at a concentration of 15%, while peppermint oil showed the lowest death rate of 45% at a concentration of (5%). However, all these treatments were statistically highly significant compared with the natural death rate in control (2%). In the second test, the results of the statistical analysis indicated that there were highly significant differences (P0.05 <0.0001) in the average numbers of dead varroa mites compared to the control when using a 15% concentration of five selected oils. On the other hand, there was no statistically significant difference in the honey bee workers' mortality rate between the treatment and control groups (P0.05 <0.3390), and it was relatively low for all treatments except the basil oil, where the bee mortality rate was 16% compared to the control (10%). It is clear from this experiment that bitter melon oil can be used to control varroa mites and it can be considered safe for honey bees as well as for the environment.

A Comparative Study on Using Simple Pollen Substitutes to Improve Brood Rearing Activity and Characteristics of Honey Bee Workers

A Comparative Study on Using Simple Pollen Substitutes to Improve Brood Rearing Activity and Characteristics of Honey Bee Workers

Studying The Spread of Varroa on Honey Bee Workers and Incubators and Assessing The Health of Selected Strains

Studying The Spread of Varroa on Honey Bee Workers and Incubators and Assessing The Health of Selected Strains

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.