Worker Honeybees Research Articles

Vitellogenin expression in the ovaries of adult honeybee workers provides insights into the evolution of reproductive and social traits.

Social insects are notable for having two female castes that exhibit extreme differences in their reproductive capacity. The molecular basis of these differences is largely unknown. Vitellogenin (Vg) is a powerful antioxidant and insulin-signalling regulator used in oocyte development. Here we investigate how Royal Jelly (the major food of honeybee queens) and queen mandibular pheromone (a major regulator of worker fertility), affect the longevity and reproductive status of honey bee workers, the expression of Vg, its receptor VgR and associated regulatory proteins. We find that Vg is expressed in the ovaries of workers and that workers fed a queen diet of Royal Jelly have increased Vg expression in the ovaries. Surprisingly, we find that expression of Vg is not associated with ovary activation in workers, suggesting that this gene has potentially acquired non-reproductive functions. Therefore, Vg expression in the ovaries of honeybee workers provides further support for the Ovarian Ground Plan Hypothesis, which argues that genes implicated in the regulation of reproduction have been co-opted to regulate behavioural differences between queens and workers.

Consumption of field-realistic doses of a widely used mito-toxic fungicide reduces thorax mass but does not negatively impact flight capacities of the honey bee (Apis mellifera)

Commercial beekeepers in many locations are experiencing increased annual colony losses of honey bees (Apis mellifera), but the causes, including the role of agrochemicals in colony losses, remain unclear. In this study, we investigated the effects of chronic consumption of pollen containing a widely-used fungicide (Pristine®), known to inhibit bee mitochondria invitro, which has recently been shown to reduce honey bee worker lifespan when field-colonies are provided with pollen containing field-realistic levels of Pristine®. We fed field colonies pollen with a field-realistic concentration of Pristine® (2.3ppm) and a concentration two orders of magnitude higher (230ppm). To challenge flight behavior and elicit near-maximal metabolic rate, we measured flight quality and metabolic rates of bees in two lower-than-normal air densities. Chronic consumption of 230 but not 2.3ppm Pristine® reduced maximal flight performance and metabolic rates, suggesting that the observed decrease in lifespans of workers reared on field-realistic doses of Pristine®-laced pollen is not due to inhibition of flight muscle mitochondria. However, consumption of either the 230 or 2.3ppm dose reduced thorax mass (but not body mass), providing the first evidence of morphological effects of Pristine®, and supporting the hypothesis that Pristine® reduces forager longevity by negatively impacting digestive or nutritional processes.

Tissue-specific transcription patterns support the kinship theory of intragenomic conflict in honey bees (Apis mellifera).

Kin selection may act differently on genes inherited from parents (matrigenes and patrigenes), resulting in intragenomic conflict. This conflict can be observed as differential expression of matrigenes and patrigenes, or parent-specific gene expression (PSGE). In honey bees (Apis mellifera), intragenomic conflict is hypothesized to occur in multiple social contexts. Previously, we found that patrigene-biased expression in reproductive tissues was associated with increased reproductive potential in worker honey bees, consistent with the prediction that patrigenes are selected to promote selfish behaviour in this context. Here, we examined brain gene expression patterns to determine if PSGE is also found in other tissues. As before, the number of transcripts showing patrigene expression bias was significantly greater in the brains of reproductive vs. sterile workers, while the number of matrigene-biased transcripts was not significantly different. Twelve transcripts out of the 374 showing PSGE in either tissue showed PSGE in both brain and reproductive tissues; this overlap was significantly greater than expected by chance. However, the majority of transcripts show PSGE only in one tissue, suggesting the epigenetic mechanisms mediating PSGE exhibit plasticity between tissues. There was no significant overlap between transcripts that showed PSGE and transcripts that were significantly differentially expressed. Weighted gene correlation network analysis identified modules which were significantly enriched in both types of transcripts, suggesting that these genes may influence each other through gene networks. Our results provide further support for the kin selection theory of intragenomic conflict, and provide valuable insights into the mechanisms which may mediate this process.

Varroa destructor mite infestations in capped brood cells of honeybee workers affect emergence development and adult foraging ability.

Varroa destructor mite infestations in capped brood cells of honeybee workers affect emergence development and adult foraging ability.

Gut Microbiome of Two Different Honeybee Workers Subspecies In Saudi Arabia.

Honeybees play a vital role in the world’s food supply by acting as essential pollinators in the agricultural fields. Interestingly, more than one third of the world’s essential crops are honeybee’s dependant. The adult honeybeeworkers harbour a simple specific bacterial spectrum in their guts with vital role in bees’ health. Gut microbial diversity of adult honeybee workerswasstudied through targeting the V3 and V4 regions of the 16S rRNA geneviaIllumina MiSeq. The study identified four phyla of the gut microbiomesinadult workersof the two-honeybee subspecies A.m. jemeniticaandA.m. carnica. The most abundant phylum in microbiome of A.m. jemeniticawasFirmicutes (48%), while Protobacteria and Actinobacteriaphylawere less abundantat figures of31% and 10%, respectively. In microbiome of A.m. carnica,Firmicutes (57%) was also the most dominant phylum, while Protobacteria and Actinobacteria had lower prevalence at figures of 31% and 10%, respectively. At genus level, adult honeybee workers harboured a number ofLactobacillus spp.in their guts with relative abundance of 80% in A.m. jemeniticaworkers compared to52%forA.m. carnicaworkers.Up toour knowledge, this is the first study of its kind on gut microbiome diversity inhoneybee workersof different origins conducted in Saudi Arabia using high-throughput 16S rRNA gene sequencing technology. The results indicatedthat the variability inmonophyletic origin of host of honeybee workers affectedgut microbiota composition.

Seasonality in telomerase activity in relation to cell size, DNA replication, and nutrients in the fat body of Apis mellifera

In honeybees (Apis mellifera), the rate of aging is modulated through social interactions and according to caste differentiation and the seasonal (winter/summer) generation of workers. Winter generation workers, which hatch at the end of summer, have remarkably extended lifespans as an adaptation to the cold season when the resources required for the growth and reproduction of colonies are limited and the bees need to maintain the colony until the next spring. In contrast, the summer bees only live for several weeks. To better understand the lifespan differences between summer and winter bees, we studied the fat bodies of honeybee workers and identified several parameters that fluctuate in a season-dependent manner. In agreement with the assumption that winter workers possess greater fat body mass, our data showed gradual increases in fat body mass, the size of the fat body cells, and Vg production as the winter season proceeded, as well as contrasting gradual decreases in these parameters in the summer season. The differences in the fat bodies between winter and summer bees are accompanied by respective increases and decreases in telomerase activity and DNA replication in the fat bodies. These data show that although the fat bodies of winter bees differ significantly from those of summer bees, these differences are not a priori set when bees hatch at the end of summer or in early autumn but instead gradually evolve over the course of the season, depending on environmental factors.

Epizootic Situation on Honey Bees (Apis Mellifera) Varoosis in Selected Areas Within Chernivtsi Region (Ukraine)

Purpose. To describe the epizootic situation on honey bees (Apis mellifera Linnaeus, 1758) varroosis in four districts of Chernivtsi region. Methods. The material was collected in accordance with the existing "Rules for sampling of pathological material, blood, feed, water and their transfer for laboratory testing." The share of infested colonies, extensiveness of Varroa invasion of worker honey bees (EI, %) and the degree of brood damage were analysed using common methods. Results. The material was collected during the summer period (June – August) of 2020 from 203 bee colonies of 80 private apiaries within four administrative districts in Chernivtsi region (Ukraine). We found that the share of colonies with infested worker honey bees was 34.80 ± 14.97 % and with infested sealed brood 38.98 ± 9.51 % of the studied colonies. The studied areas were characterized by different ratios of degrees of extensiveness of Varroa infestation of worker honey bees. The total share of the colonies without mites or with a low degree of extensiveness of varroosis invasion was the highest (ranging between 95.3 % and 100 %) in all four districts. Two percent of the tested colonies from Khotyn and Storozhynets districts were characterized by a medium degree of damage and only 2.35 % of the colonies of Khotyn district had a high degree of extensiveness of Varroa invasion. According to the results of the analysis of the brood, 1.23 % of the surveyed colonies in Khotyn district were damaged to a high degree, and 2 % of the tested colonies in Storozhynets district to the extremely high degree. Conclusions. The results of our research indicate the need to perform annual monitoring of the epizootic situation on varroosis during summer period for the early detection of Varroa mites and improving methods for varroosis prevention and treatment.

Time-accuracy trade-off and task partitioning of hygienic behavior among honey bee (Apis mellifera) workers

Behavioral specialization and cooperation are fundamental in the organization and success of social groups. Honey bee workers display hygienic behavior, defined as the detection, uncapping, and removal of unhealthy brood. We present detailed analyses of behavioral specialization and task partitioning among hygienic worker bees, focusing on uncapping of brood cells and removal of the cells’ content in freeze-killed brood assays. We demonstrate specialization of hygienic workers on either uncapping or removal and task partitioning among multiple individuals. Speed of hygiene decreases with the number of behavioral instances, suggesting a time cost for cooperation of multiple individuals. Additional analyses of an individual agent-based simulation of hygienic removal of Varroa-mite-infested brood demonstrate that erroneous removal of healthy brood can be reduced by task partitioning due to collective decision-making. Combined, our results indicate a speed-accuracy trade-off in the collective performance of hygienic behavior: Hygienic behavior may take longer when many individuals contribute and specialize on different tasks, but this organization of work also can prevent costly mistakes. This trade-off may explain the observed combination of elite workers and numerous other workers that contribute only little. Honey bees and other social animals form successful groups that are characterized by individual specialization on different tasks. Hygienic behavior of honey bees removes unhealthy brood from the nest and is important for the health of honey bee colonies. Here, we observed that individual worker bees specialize on either of the two main tasks involved in hygienic behavior: uncapping cells or removing brood. We also found that a few individuals perform the majority of the work and that the speed of hygienic behavior is higher when one individual works continuously instead of multiple individuals. Additionally, we show in computer simulations that the number of erroneous brood removals is decreased when separate workers contribute to the different aspects of hygienic behavior. Thus, our study indicates that a speed-accuracy trade-off might drive the evolution of task partitioning in hygienic behavior.

Effect of E-field at frequency 50 Hz on protein, glucose, and triglycerides concentration in honeybee hemolymph

The development of technology contributes to the increase in the exposure of living organisms to the E-field. While searching for food in the environment, the honeybee is exposed to the influence of this factor. The aim of our research was to determine the E-field effect with a frequency of 50 Hz at various intensities and exposure times on the concentration of protein, glucose, and triglycerides in the honeybee hemolymph. The analysis was carried out on hemolymph taken from at the age of 2 days old worker honeybees. The research showed changes in the tested parameters. Change related to exposure time and field intensity was noticed. The lowest protein concentration in the hemolymph of worker honeybee was observed in all control groups (0.13 mg/mL). The highest in the experimental groups were 23.0 kV/m and 34.5 kV/m with 12 h exposure time (1.04 mg/mL and 1.25 mg/mL). In all experimental groups, statistically significant lower values of glucose concentration in the hemolymph of worker honeybee were observed than in the control groups (23.43–23.55 mmol/l). The highest concentration of triglycerides was observed in the control groups (1.55 mmol/l). Lower but not statistically significant values were observed in the 5.0 kV/m 1 h, 11.5 kV/m 1 and 3 h, and 23.0 kV/m 3 h groups, while the differences were statistically significant in the other groups. The results of the research confirm our earlier observations that the E-field can induce similar reactions in the body to the pathogen. Changes in these important indicators of the body’s nutritional status such as protein, glucose, and triglyceride levels can have long-term effects.

RNA m6A Modification Functions in Larval Development and Caste Differentiation in Honeybee (Apis mellifera)

Genetically identical female honeybee larvae with different diets develop into sterile workers or fertile queens. It remains unknown whether the reversible RNA N6-methyladenosine (m6A) mark functionally impact this "caste differentiation." Here, we profile the transcriptome-wide m6A methylome of honeybee queen and worker larvae at three instar stages and discover that m6A methylation dynamics are altered by differential feeding. Multiple methylome comparisons show an obvious increase in m6A marks during larval development and reveal a negative correlation between gene expression and m6A methylation. Notably, we find that worker larvae contain more hypermethylated m6A peaks than do queen larvae, and many caste-differentiation-related transcripts are differentially methylated. Chemical suppression of m6A methylation in worker larvae by 3-deazaadenosine (DAA) reduces overall m6A methylation levels and triggers worker larvae to develop queen caste features. Thus, our study demonstrates that m6A functionally impacts caste differentiation and larval development, yet it does not exclude potential contributions from other factors.

Gut digestive enzymes and bacterial and fungal diversity of Apis mellifera adansonii (Hymenoptera: Apidae) from three ecological zones of Nigeria

ABSTRACT Gut digestive enzymes and microbial diversity of worker honeybees,Apis mellifera adansoniiL. from rainforest, guinea savannah and derived savannah zones of Nigeria during the onset of rain, the wet season, and dry season was evaluated. The pour-plate method was used for microbial analysis while spectrophotometric method was used to determine activities of gut digestive enzymes. Gut activities of lipase and proteinase were not significantly different in the honeybees from the three ecological zones during the seasonal periods. However, amylase and α-glucosidase were significantly affected by seasonal periods and ecological zones. Bacterial count was higher on the body surface than in the gut of the honeybees. The lowest (gut and body surface) bacterial count was recorded during the dry season period. Fungal load counts on the body surface and gut were significantly different between the ecological zones during each of the seasonal periods. Bacterial species varied in the honeybees from the ecological zones and this was higher on the body surface than in their gut. Yeast isolate of the genus Candida was the only identified yeast on the body surface and in the gut of the honeybees. The gut of honeybees could selectively harbour microbes while performing digestive roles.

Workflow of Lotmaria passim isolation: Experimental infection with a low-passage strain causes higher honeybee mortality rates than the PRA-403 reference strain.

The impact of trypanosomatid parasites on honeybee health may represent a major threat to bee colonies worldwide. However, few axenic isolates have been generated to date and with no details on cell culture passages, a parameter that could influence parasite virulence. To address this question, a trypanosomatid isolation protocol was developed and a new strain was obtained, named L. passim C1. Using experimental infection of worker honeybees, we compared the virulence and mortality rates of the ATCC PRA-403 reference strain and C1 strain, the latter showing higher virulence from 10 days post-infection onward. This study highlights the impact of cell culture passages on the pathogenicity of L. passim in honeybees, providing new evidence of its negative effects on honeybee health.

Classifying the Biological Status of Honeybee Workers Using Gas Sensors.

Honeybee workers have a specific smell depending on the age of workers and the biological status of the colony. Laboratory tests were carried out at the Department of Apiculture at UWM Olsztyn, using gas sensors installed in two twin prototype multi-sensor detectors. The study aimed to compare the responses of sensors to the odor of old worker bees (3–6 weeks old), young ones (0–1 days old), and those from long-term queenless colonies. From the experimental colonies, 10 samples of 100 workers were taken for each group and placed successively in the research chambers for the duration of the study. Old workers came from outer nest combs, young workers from hatching out brood in an incubator, and laying worker bees from long-term queenless colonies from brood combs (with laying worker bee’s eggs, humped brood, and drones). Each probe was measured for 10 min, and then immediately for another 10 min ambient air was given to regenerate sensors. The results were analyzed using 10 different classifiers. Research has shown that the devices can distinguish between the biological status of bees. The effectiveness of distinguishing between classes, determined by the parameters of accuracy balanced and true positive rate, of 0.763 and 0.742 in the case of the best euclidean.1nn classifier, may be satisfactory in the context of practical beekeeping. Depending on the environment accompanying the tested objects (a type of insert in the test chamber), the introduction of other classifiers as well as baseline correction methods may be considered, while the selection of the appropriate classifier for the task may be of great importance for the effectiveness of the classification.

Effects of Synthetic Acaricides and Nosema ceranae (Microsporidia: Nosematidae) on Molecules Associated with Chemical Communication and Recognition in Honey Bees.

Acaricides and the gut parasite Nosema ceranae are commonly present in most productive hives. Those stressors could be affecting key semiochemicals, which act as homeostasis regulators in Apis mellifera colonies, such as cuticular hydrocarbons (CHC) involved in social recognition and ethyl oleate (EO) which plays a role as primer pheromone in honey bees. Here we test the effect of amitraz, coumaphos, tau-fluvalinate and flumethrin, commonly applied to treat varroosis, on honey bee survival time, rate of food consumption, CHC profiles and EO production on N. ceranae-infected and non-infected honey bees. Different sublethal concentrations of amitraz, coumaphos, tau-fluvalinate and flumethrin were administered chronically in a syrup-based diet. After treatment, purified hole-body extracts were analyzed by gas chromatography coupled to mass spectrometry. While N. ceranae infection was also shown to decrease EO production affecting survival rates, acaricides showed no significant effect on this pheromone. As for the CHC, we found no changes in relation to the health status or consumption of acaricides. This absence of alteration in EO or CHC as response to acaricides ingestion or in combination with N. ceranae, suggests that worker honey bees exposed to those highly ubiquitous drugs are hardly differentiated by nest-mates. Having determined a synergic effect on mortality in worker bees exposed to coumaphos and Nosema infection but also, alterations in EO production as a response to N. ceranae infection it is an interesting clue to deeper understand the effects of parasite-host-pesticide interaction on colony functioning.

MORPHOMETRIC CHARACTERISTICS OF HEMOCYTES IN WORKER BEES APIS MELLIFERA L.

Introduction. Hemocytes of bees perform many functions. Cellular immunity is one of the most important among them. This part of the immune system provides wound healing, protection against parasites and pathogens. Research of hemocytes composition is important for the study of the cellular component of bee immunity under the influence of biotic and abiotic factors.Aim. The aim of our research was to study the hemocytes composition of worker bees Apis mellifera L. The experiment was performed during the autumn-winter period on worker honey bees.Methods. Insects were selected from the colonies zoned in Chernivtsi region. Then hemolymph was selected, smears were made, fixed and stained, and microscopy was performed. Longitudinal and transverse diameters were measured in the identified hemolymph cells and their nuclei.Results. The detected prohemocytes are the cells of the smallest size, of round or oval shape, with a compact nucleus stained in dark purple. The cytoplasm surrounds the nucleus with a thin layer. Longitudinal and transverse diameters of prohemocyte nuclei are 5.41 ± 0.12 and 4.99 ± 0.10 μm. Some of the detected plasmatocytes are oval-shaped, others are spindle-shaped. The nuclei of plasma cells, located mainly in the center of the cells, are stained in dark pink or pink-purple. Chromatin is condensed in the form of grains. The cytoplasm is transparent. It contains light pink or light purple small granules, sometimes it contains vacuoles. The average values of the longitudinal and transverse diameters of oval plasmatocytes are 12.02 ± 0.15 and 7.03 ± 0.12 μm and for their nuclei they are 7.64 ± 0.13 and 4.09 ± 0.11 μm. The average values of the longitudinal and transverse diameters of spindle-shaped plasmatocytes are 15.30 ± 0.21; 5.21 ± 0.16 μm, and for their nuclei they are 8.42 ± 0.08; 4.26 ± 0.07. The granulocytes have a large round or oval pink-purple nucleus. The dimensions of the nucleus vary considerably: the longitudinal diameter is from 10.85 to 19.85 μm, and the transverse diameter is from 8.24 to 19.63 μm. Longitudinal and transverse diameters of the 1st type permeabilized cells nuclei are 7.64 ± 0.26 мкм and 6.75 ± 0.24 μm, permeabilized cells of the 2nd type have an amoeboid shape with the longitudinal and transverse nucleus diameters 7.31 ± 0.21 and 6.56 ± 0.23 μm. Transitional form of cells are morphologically different from differentiated cells.Conclusion. Prohemocytes, oval and spindle-shaped plasmatocytes, granulocytes, permeabilized cells and transitional cell forms were detected in hemolymph of worker honey bees during the autumn-winter period. Key words: Apis mellifera; hemocytes; morphometric characteristic

Queen pheromone modulates the expression of epigenetic modifier genes in the brain of honeybee workers.

Pheromones are used by many insects to mediate social interactions. In the highly eusocial honeybee (Apis mellifera), queen mandibular pheromone (QMP) is involved in the regulation of the reproductive and other behaviour of workers. The molecular mechanisms by which QMP acts are largely unknown. Here, we investigate how genes responsible for epigenetic modifications to DNA, RNA and histones respond to the presence of QMP in the environment. We show that several of these genes are upregulated in the honeybee brain when workers are exposed to artificial QMP. We propose that pheromonal communication systems, such as those used by social insects, evolved to respond to environmental signals by making use of existing epigenomic machineries.

Individual variations lead to universal and cross-species patterns of social behavior

The duration of interaction events in a society is a fundamental measure of its collective nature and potentially reflects variability in individual behavior. Here we performed a high-throughput measurement of trophallaxis and face-to-face event durations experienced by a colony of honeybees over their entire lifetimes. The interaction time distribution is heavy-tailed, as previously reported for human face-to-face interactions. We developed a theory of pair interactions that takes into account individual variability and predicts the scaling behavior for both bee and extant human datasets. The individual variability of worker honeybees was nonzero but less than that of humans, possibly reflecting their greater genetic relatedness. Our work shows how individual differences can lead to universal patterns of behavior that transcend species and specific mechanisms for social interactions.

A scientific note on the comparison of PCR based quantification methods of Melissococcus plutonius in honey bees

Melissococcus plutonius is the causative agent of European foulbrood of honey bee larvae (Apis mellifera). The early diagnosis of disease is necessary for its elimination. Previously, we found false-positive results for PCR detection of M. plutonius in honey bee workers by qPCR. Here we report the comparison of PCR methods for quantification of M. plutonius in the bacteria spiked samples of honey bee workers and detection limits to eliminate false-positive results. We compare available primers of qPCR and PCR detection. The obtained detection limit was 102 copies of M. plutonius. The results of this comparison show conventional PCR detection using EFB_primers as a suitable method to confirm visual observation of European foulbrood using samples of honey bee workers. Based on our estimation, the numbers of M. plutonius should be quantified by copies of the Naþ/H þ 50 antiporter marker (MP_primers) in honey bee worker samples

Long-term effects of antibiotic treatments on honeybee colony fitness: A modelling approach.

Gut microbiome disequilibrium is increasingly implicated in host fitness reductions, including for the economically important and disease‐challenged western honey bee Apis mellifera. In laboratory experiments, the antibiotic tetracycline, which is used to prevent American Foulbrood Disease in countries including the US, elevates honey bee mortality by disturbing the microbiome. It is unclear, however, how elevated individual mortality affects colony‐level fitness.We used an agent‐based model (BEEHAVE) and empirical data to assess colony‐level effects of antibiotic‐induced worker bee mortality, by measuring colony size. We investigated the relationship between the duration that the antibiotic‐induced mortality probability is imposed for and colony size.We found that when simulating antibiotic‐induced mortality of worker bees from just 60 days per year, up to a permanent effect, the colony is reduced such that tetracycline treatment would not meet the European Food Safety Authority's (EFSA) honey bee protection goals. When antibiotic mortality was imposed for the hypothetical minimal exposure time, which assumes that antibiotics only impact the bee's fitness during the recommended treatment period of 15 days in both spring and autumn, the colony fitness reduction was only marginally under the EFSA's threshold. Synthesis and Applications. Modelling colony‐level impacts of antibiotic treatment shows that individual honey bee worker mortality can lead to colony mortality. To assess the full impact, the persistence of antibiotic‐induced mortality in honey bees must be determined experimentally, in vivo. We caution that as the domestication of new insect species increases, maintaining healthy gut microbiomes is of paramount importance to insect health and commercial productivity. The recommendation from this work is to limit prophylactic use of antibiotics and to not exceed recommended treatment strategies for domesticated insects. This is especially important for highly social insects as excess antibiotic use will likely decrease colony growth and an increase in colony mortality.

Caste differentiation of spermatheca and organs related to sperm use and oviposition in the honeybee, Apis mellifera

Striking queen–worker dimorphism associated with each task is a key feature of highly eusocial insects, including Apis mellifera. The present study histologically examined the developmental process of the internal reproductive organs including spermatheca, valve-fold in the vagina, semi-circular muscle surrounding the common oviduct, and abdominal ganglia in honeybee queens and workers. During the pupal stage, queens showed an increased spermathecal reservoir, development of the tracheal network surrounding the spermathecal reservoir, and elongation of the spermathecal gland. Compared with queens, these developmental processes were never observed during the pupal stage in workers. Moreover, development of the valve-fold and semi-circular muscle was aborted, and they became rudimentary at the middle pupal stage in workers. Morphological caste differences in the abdominal ganglia were observed from the prepupal stages, showing that the most posterior ganglion was fused with the anterior ganglia in queens but not in workers.