Worker Honeybees Research Articles

Cuticle melanization and the expression of immune-related genes in the honeybee Apis mellifera (Hymenoptera: Apidae) adult workers.

The global decline of bee populations has several factors, including pathogens, which need overcome the insect defenses such as the physical barriers, the body cuticle and peritrophic matrix (primary defenses), as well as the secondary defenses with antimicrobial peptides (AMPs) and the enzyme lysozyme. The regulation of immune defenses according to the infection risks raises questions about the immunity of social bees due to their exposition to different pathogens pressures during the adult lifespan and tasks performed. This study evaluated the primary (body cuticle melanization, peritrophic matrix and cpr14 expression) and secondary (AMPs and lysozyme expression) defenses of the honeybee Apis mellifera workers according to the age and tasks. The expression of malvolio was used to detect precocious forage tasks outside the colony. Forager workers have higher amount of cuticular melanization in the body cuticle than nurse, but not when the age effect is retired, indicating the gradual acquisition of this compound in the integument of adult bees. The relative value of chitin in the peritrophic matrix and cpr14 mRNA are similar in all bees evaluated, suggesting that these components of primary defenses do not change according to the task and age. Differential expression of genes for AMPs in workers performing different tasks, within the same age group, indicates that the behavior stimulates expression of genes related to secondary immune defense. The expression of malvolio gene, accelerating the change in workers behavior, and those related to immune defense suggest the investment in secondary defense mechanisms when the primary defense of the body cuticle is not yet completed.

Effect of supplementary feeding on morphometry of worker honeybees in Western Himalayan region

Efficient management of bee colonies requires the beekeepers to provide artificial food to the bees when they need it. The need to feed artificial diet depends a lot on the amount of brood in the hive, the amount of stored pollen and nectar, current as well as future nectar and pollen conditions and demands to be placed on the hives. If hives are critically short of pollen and either have a large brood nest or are expanding their brood nest, then the beekeepers need to consider various options available to them for e.g. supplementary feeding, migration of bee colonies to the region rich in flora etc. to avoid a reduction in bee population. Feeding of artificially diet may have positive effect on colony attributes including morphometric characters. It was observed that feeding of diet led to significant increase in some of the morphometric characters like tongue length.

Morphological variations in Nigerian Apis mellifera Linnaeus, 1758 populations in Guinea savannah agro-ecological zone

Apis mellifera Linnaeus, 1758 is one of the most economically valuable insects which plays significant role in human medicine, nutrition and crop pollination. The morphometric variations of honeybee from different locations of the southern guinea and northern guinea savannah ecological zone of Nigeria were studied. Fifteen morphological traits were measured for variation on six hundred (600) worker honeybee samples purposively collected from ten different locations within the ecological zone. Data collected were subjected to one-way Analysis of Variance (ANOVA), SNK test (α=0.05) and Pearson correlation between morphometric characters were determined. At the same time a dendrogram of morphological proximity based on the fifteen morphological features was constructed. Results showed that morphometry variation (p<0.05) existed within the honeybee population in the guinea savannah agro-ecological zone of Nigeria, high morphological similarities were observed in the tibia length of the hind leg and the thorax length. The honeybee samples were classified into two distinct morphoclusters (A and B). Honeybee samples within morphocluster A were closely related in terms of the examined morphometric features and geographic distance (CV=1.65). In contrast, within cluster B, honeybee samples were closely related despite the vast geographical distance (CV=3.30, p<0.05). The body length was significantly positively correlated with the leg size, while hind wing length was positively associated with the proboscis, abdominal, thoracic, body length and hooks. Morphometric variations found in A. mellifera of guinea agro-ecological zone could significantly impact conservation and future bee breeding programmes of Nigeria.

Secondary effect of in-hive Metronidazole application on some biological, histological and biochemical activities of Honeybee workers (Apis mellifera L.)

Secondary effect of in-hive Metronidazole application on some biological, histological and biochemical activities of Honeybee workers (Apis mellifera L.)

Remarkable Sensitivity of Young Honey Bee Workers to Multiple Non-photic, Non-thermal, Forager Cues That Synchronize Their Daily Activity Rhythms

Honey bees live in colonies containing tens of thousands of workers that coordinate their activities to produce efficient colony-level behavior. In free-foraging colonies, nest bees are entrained to the forager daily phase of activity even when experiencing conflicting light-dark illumination regime, but little is known on the cues mediating this potent social synchronization. We monitored locomotor activity in an array of individually caged bees in which we manipulated the contact with neighbour bees. We used circular statistics and coupling function analyses to estimate the degree of social synchronization. We found that young bees in cages connected to cages housing foragers showed stronger rhythms, better synchronization with each other, higher coupling strength, and a phase more similar to that of the foragers compared to similar bees in unconnected cages. These findings suggest that close distance contacts are sufficient for social synchronization or that cage connection facilitated the propagation of time-giving social cues. Coupling strength was higher for bees placed on the same tray compared with bees at a similar distance but on a different tray, consistent with the hypothesis that substrate borne vibrations mediate phase synchronization. Additional manipulation of the contact between cages showed that social synchronization is better among bees in cages connected with tube with a single mesh partition compared to sealed tubes consistent with the notion that volatile cues act additively to substrate borne vibrations. These findings are consistent with self-organization models for social synchronization of activity rhythms and suggest that the circadian system of honey bees evolved remarkable sensitivity to non-photic, non-thermal, time giving entraining cues enabling them to tightly coordinate their behavior in the dark and constant physical environment of their nests.

No increase in alcohol dehydrogenase levels following repeated ethanol exposure in young honeybee workers

AbstractSome workers of the honeybee show high alcohol dehydrogenase (ADH) levels and high resistance to the sedative effects of alcohol, yet it is unknown whether these two issues are directly related. Here we looked for a link between ADH levels and sedation latency in response to alcohol exposure. We used molecular and immunoblotting methods to investigate a possible induction of ADH production in young, ADH‐lacking workers in response to repeated ethanol vapour presentation. Although we found increased sedation latency in individuals after several ethanol encounters, this was not accompanied by detectibly increased ADH levels in such workers. The lack of ethanol‐induced ADH production indicated that it was not needed for increased coping with alcohol inebriation. In this work, we expanded current knowledge about the effects of alcohol on honeybee workers and related it to the existing literature on the subject.

Reproductive plasticity and oogenesis in the queen honey bee (Apis mellifera)

In the honey bee (Apis mellifera), queen and worker castes originate from identical genetic templates but develop into different phenotypes. Queens lay up to 2000 eggs daily whereas workers are sterile in the queen’s presence. Periodically queens stop laying: during swarming, when resources are scarce in winter, and when they are confined to a cage by beekeepers. We used confocal microscopy and gene expression assays to investigate the control of oogenesis in the ovaries of honey bee queens that were caged inside and outside the colony. We find evidence that queens use a different combination of ‘checkpoints’ to regulate oogenesis compared to honey bee workers and other insect species. However, both queen and worker castes likely use the same programmed cell death pathways to terminate oocyte development at their caste-specific checkpoints. Our results also suggest that a key factor driving the termination of oogenesis in queens is nutritional stress. Thus, queens may regulate oogenesis via the same regulatory pathways that were utilised by ancestral solitary species but likely have adjusted physiological checkpoints to suit their highly-derived life history.

The Comparison of Juvenile Hormone and Transcriptional Changes between Three Different Juvenile Hormone Analogs Insecticides on Honey Bee Worker Larval’s Development

Juvenile hormones (JHs) play a crucial role in the development of honey bee (Apis mellifera) worker larvae. Juvenile hormone analogs (JHAs), insecticides widely used in pest control, have been reported to affect the health and survival of honey bee worker larvae. However, the molecular mechanisms of JHAs in the honey bee remain unclear. In this study, we treated honey bee worker larvae with pyriproxyfen, fenoxycarb, and methoprene, three different JHAs. We monitored the changes in the transcription of genes encoding major JH response enzymes (CYP15A1, CYP6AS5, JHAMT, and CHT1) using RT-qPCR and analyzed the transcriptome changes in worker larvae under JHA stress using RNA-seq. We found that the enrichment pathways differed among the treatment groups, but the classification of each pathway was generally the same, and fenoxycarb affected more genes and more pathways than did the other two JHAs. Notably, treatment with different JHAs in the honey bee changed the JH titers in the insect to various extents. These results represent the first assessment of the effects of three different JHAs on honey bee larvae and provide a new perspective and molecular basis for the research of JH regulation and JHA toxicity in the honey bee.

Antibacterial, Antifungal and Antihelminthic Properties of Ethanolic, Methanolic and Water Extracts of Pollen

Microorganisms and helminthes can cause serious diseases in humans as well as in animals. The use of antimicrobial and antihelminthic drugs have created selective pressure and caused resistance to antibiotics used against them, thus it necessitates the use of honey bee’s derived natural products. One such bee derived product is pollen, collected by worker honey bees from the flowering plants and modify it by adding its salivary secretions. The present study embodies use of pollen as antimicrobial and antihelminthic substance. Among microorganisms 4 Gram (+ve) bacteria; (Bacillus subtilis, Staphylococcus epidermidis, Staphylococcus aureus, Streptococcus pneumoniae) and 3 Gram (-ve) bacteria; (Escherichia Coli, Pseudomonas aeruginosa, Salmonella enteric) and 2 yeasts (Candida albicans and Saccharomyces cerevisiae) were used and the methodology used disc diffusion assay and broth dilution method. The antihelminthic effect was observed among amphistomes via bioassay method under in vitro conditions. For observations three types of pollen extracts (ethanolic, methanolic and water extract) were prepared and positive controls used were; Ampicillin for antibacterial, Amphotericin B for antifungal and Albendazole for anti-helminthes. The antimicrobial activities were determined by measuring the zones of inhibition diameters in millimeters after 24 hours of incubation at optimum temperature for each microbe and also by broth dilution method. Results obtained showed that the water extract of pollen was found to be most effective against bacteria used in the present study where; Gram (+ve) bacteria were more susceptible as compared to the Gram (-ve) bacteria. It was also observed that among yeasts; Saccharomyces cerevisiae was more susceptible towards ethanolic extract of pollen while Candida albicans showed more inhibitions towards water extract of pollen. Results also demonstrated that none of the extracts of pollen was found to be effective against Helminthes (amphistomes) used in the present study.

Searching beyond the streetlight: Neonicotinoid exposure alters the neurogenomic state of worker honey bees.

Neonicotinoid insecticides have been implicated in honey bee declines, with many studies showing that sublethal exposure impacts bee behaviors such as foraging, learning, and memory. Despite the large number of ecotoxicological studies carried out to date, most focus on a handful of worker phenotypes leading to a “streetlight effect” where the a priori choice of phenotypes to measure may influence the results and conclusions arising from the studies. This bias can be overcome with the use of toxicological transcriptomics, where changes in gene expression can provide a more objective view of how pesticides alter animal traits. Here, we used RNA sequencing to examine the changes in neurogenomic states of nurse and forager honey bees that were naturally exposed to neonicotinoids in the field and artificially exposed to neonicotinoids in a controlled experiment. We found that neonicotinoid exposure influenced the neurogenomic state of foragers and nurses in different ways; foragers experienced shifts in expression of genes involved in cognition and development, while nurses experienced shifts in expression of genes involved in metabolism. Our study suggests that neonicotinoids influence nurse and forager bees in a different manner. We also found no to minimal overlap in the differentially expressed genes in our study and in previously published studies, which might help reconcile the seemingly contradictory results often reported in the neonicotinoid literature.

Antioxidant Activities of Bingöl Royal Jelly on SH-SY5Y Cells

Royal Jelly is a bee product with high protein content is a unique nutrient for the queen honeybee. It leads to a substantial elongation of the lifetime of the queen in comparison to the worker honeybees via anti-inflammatory, anti-oxidant, anti-cancer and anti-microbial properties. Flavonoids naturally have reported to have anticancer activities thanks to their potent antioxidant activity. The antioxidant activity of RJ is attributed to its rich flavonoid content however anti-oxidant activities of the Bingöl RJ has yet to be explored in detail. The goal of the current study was to investigate the antioxidant activities of Bingöl RJ on SH-SY5Y neuroblastoma cells. The results of our study revealed that Bingöl RJ at a concentration of 0.3 mg/ml significantly augmented ROS level in SH-SY5Y cells while 0.5 mg/ml of RJ had almost no effect on ROS levels. The levels of malondialdehyde in SH-SY5Y cells considerably increased in the presence of 0.3 mg/ml RJ while 0.5 mg/ml RJ had no significant impact on MDA levels in SH-SY5Y cells. The results showed that RJ treatment 0.3 mg/ml) significantly lowered the activities of SOD and CAT activity while 0.5 mg/ml of RJ had negligible effect indicating that RJ could protect the cell membranes from radical mediated cell injuries.

Identification of circular RNAs and corresponding regulatory networks reveals potential roles in the brains of honey bee workers exposed to dinotefuran

Honey bees are important and highly efficient pollinators of agricultural crops and have been negatively affected by insecticides in recent years. Circular RNA (circRNA) plays an important role in the regulation of multiple biological and pathological processes; however, its role in the honey bee brain after exposure to dinotefuran is not well understood. Here, the expression profiles and potential modulation networks of circRNAs in the brains of workers (Apis mellifera) were comprehensively investigated using RNA sequencing and bioinformatics. In total, 33, 144, and 211 differentially expressed (DE) circRNAs were identified on the 1st, 5th and 10th days after exposure to dinotefuran, respectively. Enrichment analyses revealed that the host genes of DE circRNAs were enriched in the Hippo signaling pathway-fly, Wnt signaling pathway, and neuroactive ligand-receptor interaction. circ_0002266, circ_0005080, circ_0010239 and circ_0005415 were found to have translational potential due to the presence of an internal ribosome entry site (IRES). An integrated analysis of the DE circRNA-miRNA-mRNA networks suggest that circ_0008898 and circ_0001829 may participate in the immune response to dinotefuran exposure by acting as miRNA sponges. Our results provide invaluable basic data on A. mellifera brain circRNA patterns and a molecular basis for further study of the biological function of circRNAs in the development and immune response of honey bees.

Evaluating the Efficacy of 30 Different Essential Oils against Varroa destructor and Honey Bee Workers (Apis mellifera).

Simple SummaryWorldwide, mass losses of honey bee colonies are being observed more frequently due to Varroa mite infestation. Therefore, varroosis is considered a major problem in beekeeping participating to a large extent in colony collapse disorder. Except for direct damage of bees and suppressing their immune system caused by parasitism, Varroa mites transfer viral particles straight to bee hemolymph which can have a fatal impact. To control the mite population, several acaricidal treatments are used. Commonly used treatments are synthetic acaricides with a high risk of developing Varroa resistance population and contamination of bee products by acaricidal residues. Other commonly used treatments are organic acids, which are increasingly associated with damage of brood, adult bees, and premature deaths of queens. Therefore, in this study, we evaluated the varroacidal effect of 30 individual essential oils. The toxicity of the most effective oils selected by screening was subsequently tested on Varroa mites and adult honey bee workers simultaneously. In addition, the main components of these essential oils were specified. Several essential oils were proven to be effective against the adult female of Varroa mites and at the same dose safe for adult honey bee workers under laboratory conditions, especially manuka, peppermint, oregano, litsea, and cinnamon.Essential oils and their components are generally known for their acaricidal effects and are used as an alternative to control the population of the Varroa destructor instead of synthetic acaricides. However, for many essential oils, the exact acaricidal effect against Varroa mites, as well as the effect against honey bees, is not known. In this study, 30 different essential oils were screened by using a glass-vial residual bioassay. Essential oils showing varroacidal efficacy > 70% were tested by the complete exposure assay. A total of five bees and five mites were placed in the Petri dishes in five replications for each concentration of essential oil. Mite and bee mortality rates were assessed after 4, 24, 48, and 72 h. The LC50 values and selectivity ratio (SR) were calculated. For essential oils with the best selectivity ratio, their main components were detected and quantified by GC-MS/MS. The results suggest that the most suitable oils are peppermint and manuka (SR > 9), followed by oregano, litsea (SR > 5), carrot, and cinnamon (SR > 4). Additionally, these oils showed a trend of the increased value of selective ratio over time. All these oils seem to be better than thymol (SR < 3.2), which is commonly used in beekeeping practice. However, the possible use of these essential oils has yet to be verified in beekeeping practice.

Cosmetic Applications of Bee Venom.

Bee venom (BV) is a typical toxin secreted by stingers of honeybee workers. BV and BV therapy have long been attractive to different cultures, with extensive studies during recent decades. Nowadays, BV is applied to combat several skin diseases, such as atopic dermatitis, acne vulgaris, alopecia, vitiligo, and psoriasis. BV is used extensively in topical preparations as cosmetics and used as dressing for wound healing, as well as in facemasks. Nevertheless, the safety of BV as a therapeutic choice has always been a concern due to the immune system reaction in some people due to BV use. The documented unfavorable impact is explained by the fact that the skin reactions to BV might expand to excessive immunological responses, including anaphylaxis, that typically resolve over numerous days. This review aims to address bee venom therapeutic uses in skin cosmetics.

The effect of a lack of uncapped brood on social interactions between honey bee workers and the queen

Vibro-acoustic communication is used by honey bees in many different social contexts. Our previous research showed that workers interact with their queen outside of the swarming period by means of wing-beating behaviour. Therefore, the aim of this study was to verify the hypothesis that the wing-beating behaviour of workers attending the queen stimulates her to lay eggs. The behaviour of workers and the queen was recorded using a high-speed camera, at first in the presence of uncapped brood in the nest and then without one. None of the queens performed wing-beating behaviour. On the other hand, the workers attending the queen demonstrated this behaviour two times per minute, on average, even in the presence of uncapped brood in the nest. After removing the combs with the uncapped brood, the incidence of wing-beating behaviour increased significantly to an average of four times per minute. Wing-beating behaviour did not differ significantly in its characteristics when uncapped brood was present or absent in the nest. During 3 days after removing the combs with the uncapped brood, there was no significant increase in the rate of egg lying by the queen. Therefore, the results presented here do not convincingly confirm that the wing-beating behaviour of workers affects the rate of queen's egg-lying. This negative result can be related to colony disturbance and longer time required by the queen to increase egg production.

Bee year: Basic physiological strategies to cope with seasonality

Worker honey bees are subject to biochemical and physiological changes throughout the year. This study aimed to provide the reasons behind these fluctuations. The markers analysed included lipid, carbohydrate, and protein levels in the haemolymph; the activity of digestive enzymes in the midgut; the levels of adipokinetic hormone (AKH) in the bee central nervous system; the levels of vitellogenins in the bee venom and haemolymph; and the levels of melittin in the venom. The levels of all the main nutrients in the haemolymph peaked mostly within the period of maximal bee activity, whereas the activity of digestive enzymes mostly showed a two-peak course. Furthermore, the levels of AKHs fluctuated throughout the year, with modest but significant variations. These data suggest that the role of AKHs in bee energy metabolism is somewhat limited, and that bees rely more on available food and less on body deposits. Interestingly, the non-metabolic characteristics also fluctuated over the year. The vitellogenin peak reached its maximum in the haemolymph in winter, which is probably associated with the immunoprotection of long-lived winter bees. The analysis of bee venom showed the maximal levels of vitellogenin in autumn; however, it is not entirely clear why this is the case. Finally, melittin levels showed strong fluctuations, suggesting that seasonal control was unlikely.

Glucosinolate Bioactivation by Apis mellifera Workers and Its Impact on Nosema ceranae Infection at the Colony Level.

The microsporidian fungus Nosema ceranae represents one of the primary bee infection threats worldwide and the antibiotic fumagillin is the only registered product for nosemosis disease control, while few alternatives are, at present, available. Natural bioactive compounds deriving from the glucosinolate–myrosinase system (GSL–MYR) in Brassicaceae plants, mainly isothiocyanates (ITCs), are known for their antimicrobial activity against numerous pathogens and for their health-protective effects in humans. This work explored the use of Brassica nigra and Eruca sativa defatted seed meal (DSM) GSL-containing diets against natural Nosema infection in Apis mellifera colonies. DSM patties from each plant species were obtained by adding DSMs to sugar candy at the concentration of 4% (w/w). The feeding was administered in May to mildly N. ceranae-infected honey bee colonies for four weeks at the dose of 250 g/week. In the treated groups, no significant effects on colony development and bee mortality were observed compared to the negative controls. The N. ceranae abundance showed a slight but significant decrease. Furthermore, the GSL metabolism in bees was investigated, and MYR hydrolytic activity was qualitatively searched in isolated bee midgut and hindgut. Interestingly, MYR activity was detected both in the bees fed DSMs and in the control group where the bees did not receive DSMs. In parallel, ITCs were found in gut tissues from the bees treated with DSMs, corroborating the presence of a MYR-like enzyme capable of hydrolyzing ingested GSLs. On the other hand, GSLs and other GSL hydrolysis products other than ITCs, such as nitriles, were found in honey produced by the treated bees, potentially increasing the health value of the final product for human consumption. The results are indicative of a specific effect on the N. ceranae infection in managed honey bee colonies depending on the GSL activation within the target organ.

Humoral and Cellular Defense Mechanisms in Rebel Workers of Apis mellifera.

Simple SummaryImmune mechanisms in insects include cellular and humoral defenses. One of these cellular defense mechanisms is phagocytosis. The humoral defense components are produced in the fat body cells, the development of which depends on the juvenile hormone (JH) titers and vitellogenin (Vg) concentration. These mechanisms also regulate caste formation. In a colony, a queen, a worker, or a rebel can develop from eggs of the same genome. Rebels are reproductive workers that can lay eggs while maintaining tasks inside and outside the colony the same as sterile workers. The aim of our studies was to determine the phagocytic index, JH titer, Vg concentration, the number of oenocytes (fat body cells), and their size in the rebels and to compare them to those in normal workers. The rebels were characterized by high phagocytic indices, JH and Vg levels, and increased numbers and sizes of oenocytes in the fat body cells in comparison to the normal workers. These characteristics can be viewed as an evolutionary adaptation of these reproductive workers to life in a eusocial society.The physiological state of an insect depends on efficiently functioning immune mechanisms such as cellular and humoral defenses. However, compounds participating in these mechanisms also regulate reproductive caste formation and are responsible for reproductive division of labor as well as for labor division in sterile workers. Divergent reaction of the same genotype yielding reproductive queens and worker castes led to shaping of the physiological and behavioral plasticity of sterile or reproductive workers. Rebels that can lay eggs while maintaining tasks inside and outside the colony exhibit both queen and worker traits. So, we expected that the phagocytic index, JH3 titer, and Vg concentration would be higher in rebels than in normal workers and would increase with their age. We also assumed that the numbers of oenocytes and their sizes would be greater in rebels than in normal workers. The rebels and the normal workers were collected at the age of 1, 7, 14, and 21 days, respectively. Hemolymph and fat bodies were collected for biochemical and morphological analyses. The high levels of JH, Vg, and the phagocytic index, as well as increased numbers and sizes of oenocytes in the fat body cells demonstrate the physiological and phenotypic adaptation of rebels to the eusocial life of honeybees.

Pollen Treated with a Combination of Agrochemicals Commonly Applied During Almond Bloom Reduces the Emergence Rate and Longevity of Honey Bee (Hymenoptera: Apidae) Queens.

Honey bee (Apis mellifera L.) colonies that pollinate California’s almond orchards are often exposed to mixtures of agrochemicals. Although agrochemicals applied during almond bloom are typically considered bee-safe when applied alone, their combined effects to honey bees are largely untested. In recent years, beekeepers providing pollination services to California’s almond orchards have reported reductions in queen quality during and immediately after bloom, raising concerns that pesticide exposure may be involved. Previous research identified a synergistic effect between the insecticide active ingredient chlorantraniliprole and the fungicide active ingredient propiconazole to lab-reared worker brood, but their effects to developing queens are unknown. To test the individual and combined effects of these pesticides on the survival and emergence of developing queens, we fed worker honey bees in closed queen rearing boxes with pollen artificially contaminated with formulated pesticides containing these active ingredients as well as the spray adjuvant Dyne-Amic, which contains both organosilicone and alkyphenol ethoxylate. The translocation of pesticides from pesticide-treated pollen into the royal jelly secretions of nurse bees was also measured. Despite consistently low levels of all pesticide active ingredients in royal jelly, the survival of queens from pupation to 7 d post-emergence were reduced in queens reared by worker bees fed pollen containing a combination of formulated chlorantraniliprole (Altacor), propiconazole (Tilt), and Dyne-Amic, as well as the toxic standard, diflubenzuron (Dimilin 2L), applied in isolation. These results support recommendations to protect honey bee health by avoiding application of pesticide tank-mixes containing insecticides and adjuvants during almond bloom.

Coexistence between selected pathogens in honey bee workers

The aim of the study was to determine the occurrence of Nosema apis, N. ceranae, Lotmaria passim, Crithidia mellificae and Varroa destructor pathogens in bee colonies located in areas with different floral sources of pollen, and to identify the correlations between these pathogens. The study was conducted in three Polish regions: Warmia and Mazury (A), Mazowsze (B) and Wielkopolska (C). Three apiaries were analyzed in each region: A1, A2 and A3 in region A; B1, B2 and B3 in region B; and C1, C2 and C3 in region C. Ten bee colonies without any symptoms of disease were randomly selected from each apiary. Pathogens were identified by PCR in pooled samples of 30 worker bees each. Samples from 90 (100%) honey bee colonies from 9 apiaries were analyzed. N. ceranae was detected in 68 samples (76.75%), and N. apis was identified in 19 samples (21.11%) but never alone and always in co-infection with N. ceranae. Lotmaria passim was present in 49 samples (54.44%). The presence of V. destructor females was determined in 44 samples (48.89%). Coexistence between L. passim, N. apis and N. ceranae were observed in selected regions.