Beekeeping plays an important role in the development of agriculture in terms of the valuable bee products produced by beekeepers as well as its influence on the quantity and quality of crops. Unfortunately, bees are increasingly dying off mainly in the United States and Europe. Bober et al. (2016) report that the bee colony mortality rate reached 32.4% in European countries in the years 2012–2014, while in Poland the rate was 16%, resulting in average production losses of 4.5%. The situation has not improved in the years since then (Semkiw, 2020).
The causes of the decline in the number of pollinators are complex and often highly controversial (Di Noi et al., 2021). However, the indisputable overall weakness and increased mortality of honey bee colonies is due to the synergistic effect of multiple stressors – biological, climatic, nutritional and chemical. Cuesta-Maté et al. (2021) suggest four main areas of human impact responsible for the decline in bee populations: food stress caused by habitat loss and degradation, reduced genetic diversity in honey bees, antibiotics used in beekeeping mainly to control Nosema disease and pesticide use. All of these factors alter the defence mechanisms of the bee immune system, including physical barriers and the general cellular and humoral response, which causes bees to lose their innate ability to resist changes associated with civilization (Iorizzo et al., 2022). These factors, especially pesticides and medications used in beekeeping, have also been established to affect honey bees’ intestinal microbiota, which is of fundamental importance for their growth and development and supports their resistance and vigour (Iorizzo et al., 2022). The role of the intestinal microbiota in the health of bees is crucial, as it influences metabolism, development, immune function and thus protection against pathogens (Raymann & Moran, 2018). Dosch et al. (2021) experimentally both confirmed the positive role of the intestinal microbiota in the condition of honey bees infected with various viruses and showed that environmental stressors altering the composition of the intestinal microbiota of bees, including chemotherapeutics and pesticides, can make them more susceptible to infectious agents.
Laboratory studies confirm that such herbicides and insecticides as glyphosate or highly toxic neonicotinoids, including imidacloprid and thiamethoxam, disturb the population size of dominant members of the bacterial community and make honey bees more susceptible to pathogens (Motta et al., 2018; Blot et al., 2019; Rouzé et al., 2019). The magnitude of disturbances in the microbiota is determined by the concentrations of pesticides, the duration of exposure, the time of year and co-existing stressors (Hotchkiss et al., 2022).
Most studies on the honey bee microbiome concern changes caused by exposure to pesticides (Hotchkiss et al., 2022). However, there is a lack of information on how acaricides, including amitraz which is used to control
In addition, significant changes in the biology of
In Poland, many medications of varied compositions are authorized for controlling mites and varroosis (Strachecka et al., 2013). The most effective agents, acaricides, control the mites through direct contact during the development of several parasite generations. These include third-generation acaricides which are based on synthetic pyrethroids and agents whose active substance is amitraz. Although this substance has been used to control
The aim of the study was to analyse the intestinal microbiota of honey bees (
The study was conducted on six honey bee colonies of similar strengths and structures inhabiting Dadant polystyrene hives. In all colonies, the queens were sisters. Each colony occupied eight combs of the nest, while the free space outside the nest was occupied by two frames filled with polystyrene foam. The microbial composition of the bees’ intestines was determined. In addition, the hygiene conditions of the hive environment were assessed through analysis of the microbiological purity of the air and surfaces. Samples were taken twice in the second half of June, the first before the harvest of linden honey and the second seven days afterwards. This means that before amitraz application (2 strips 1000 mg amitraz per colony; 1st sampling) and seven days afterwards (2nd sampling), so that the first samples were taken in colonies in which there had been no treatment to control
Homogenate of intestines collected from ten bees was combined to form one sample and then tested for the total content of bacteria, fungi, lactic acid bacteria, enriched agar for total bacterial count - incubation for 24–48 h at 37°C, Sabouraud agar for total fungal count - incubation for 5–7 days at 25°C, mFC for MRS for total count of lactic acid bacteria of the genus BSM for bacteria of the genus TSC for total
Following incubation, the colonies were counted and their concentration was expressed as colony-forming units per g of intestines [cfu/g].
Assessment of the microbiological purity of the air in the honey bee colony involved determination of the levels of bacterial and fungal contamination. Air samples were collected by aspiration using a GilAir 5 sampling pump (Sensdidyne, Inc., Clearwater, USA). The total bacterial and fungal counts were determined by dilution plating on appropriate media:
tryptone soy agar (TSA) for total bacterial count - incubation for seven days at 37°C (1 day), 22°C (3 days) and 4°C (3 days), Sabouraud agar with chloramphenicol for total fungal count - incubation at 30°C (4 days) and 25°C (3 days).
Following incubation, the colonies were counted and their concentration was expressed as colony-forming units per m3 of air [cfu/m3].
Microbiological assessment of the inner surface of the top bar, brood cappings on the colony’s penultimate honeycomb, and the hive walls involved analysis of the total content of bacteria, including enriched agar for total bacterial count - incubation for 24–48 h at 37°C, Sabouraud agar total fungal count - incubation for 5–7 days at 25°C, mFC for
After the colonies were counted, the number of microbes per 100 cm2 of test surface [cfu/100 cm2] was determined.
The obtained research results were analysed statistically. The normality of the distribution was assessed by the Shapiro–Wilk test. If the distribution was normal, one-way analysis of variance (ANOVA) was performed. Statistical analysis of the results was performed by one-way analysis of variance (ANOVA). Differences were considered significant at p≤0.05. All statistical data were calculated using STATISTICA 13.1 software (StatSoft, Krakow, Poland).
In the present study, the microbiological profile of the honey bee intestines showed minor changes in the microbiota following the application of amitraz (Tab. 1). Comparison of the numbers of bacteria and fungi revealed a positive downward trend in the number of fungi. The number of bacteria decreased as well, including
Total microbial counts in intestinal homogenates [cfu/g]
Bacteria | 1.0×108 | 1.3×107 | 1.1×108 | 6.2×107 | 0.837 |
E. coli | 2.2×108 | 3.3×107 | 6.3×107 | 5.3×106 | 0.314 |
7.9×103 | 1.1×102 | 0.0 | 0.0 | 0.143 | |
Lactic acid bacteria | 2.2×108 | 4.9×107 | 1.9×108 | 1.0×107 | 0.887 |
1.7×107 | 1.3×107 | 4.4×106 | 4.2×105 | 0.069 | |
Fungi | 2.9×107 | 5.6×107 | 2.0×103 | 1.8×102 | 0.268 |
Total microbial count in air samples [cfu/m3]
Bacteria | 7.0×101 | 3.3×101 | 2.8×102 | 4.7×101 | 0.0001* |
Fungi | 7.4×101 | 4.3×101 | 2.9×102 | 1.1×102 | 0.003* |
values differ statistically
No statistically significant differences (p>0.05) were observed in the level of contamination of the hive’s surfaces (Tab. 3), which may be due to the thin layer of propolis protecting them against pathogenic bacteria and fungi growth.
Total microbial count in samples from swabs [cfu/100 cm2]
Top bar | |||||
Bacteria | 1.2×103 | 1.9×102 | 2.3×101 | 1.7×101 | 0.212 |
9.8×101 | 2.1×102 | 0.0 | 0.0 | 0.313 | |
Total fungi | 1.0×103 | 1.1×102 | 3.3 | 4.7 | 0.064 |
Yeasts | 1.6 | 3.7 | 0.0 | 0.0 | 0.341 |
Honeycomb | |||||
Bacteria | 6.7 | 7.5 | 6.7 | 1.1×101 | 1.000 |
0.0 | 0.0 | 0.0 | 0.0 | - | |
Total fungi | 1.7 | 3.7 | 1.1×102 | 2.4×101 | 0.340 |
Yeasts | 0.0 | 0.0 | 6.8×101 | 1.5×101 | 0.341 |
Hive wall | |||||
Bacteria | 1.2×101 | 2.2×101 | 5.0 | 5.0 | 0.522 |
0.0 | 0.0 | 0.0 | 0.0 | - | |
Total fungi | 5.0 | 1.1×101 | 0.0 | 0.0 | 0.341 |
Yeasts | 0.0 | 0.0 | 0.0 | 0.0 | - |
The intestinal bacteria of the honey bee come from their surrounding environment and food, so the microbiota of mature worker bees may differ somewhat depending on the food source, age of the bee, time of year and geographic location, although some microbe species are common everywhere. The cluster of
The predominant microaerophilic conditions, a temperature of 35°C and the presence of sugars from nectar in the digestive tract of the honey bee are ideal conditions for the development of lactic acid bacteria (Iorizzo et al., 2020). According to Dong et al. (2020), these bacteria colonize the intestines of worker bees up to three days after emergence. The importance of their role in the host organism is evidenced by their involvement in such functions, as inhibiting expansion of pathogens in the intestines during competition for nutrients, fighting pathogens with the products of their metabolism, producing bacteriocins, and significantly influencing immune modulation in the host. In addition, bacteria of the genus
Hotchkiss et al. (2022) emphasize that changes occur in the abundance of taxa of bees intestinal microbiota during exposure to insecticides, most frequently involving a decline in the populations of
A balanced microbiota in bees not only supports their defensive strength but are also a significant indicator of the health of bee colonies. Hygiene in the apiary and the hive itself plays an important role, as
The thin layer of propolis with which bees cover and seal the surface of the hive has antibacterial, antifungal and antiviral properties, and it simultaneously sterilizes the interior of the hive (Anderson et al., 2011). Homeostasis of the nest environment is also influenced through the maintenance of a constant temperature in the hive (33–36°C) and ventilation of the nest, which determines the direction of microbiological changes occurring in bee products, spoilage prevention and removal of harmful substances from the hive together with moist air (Peters et al., 2019).
This study is an introduction to more extensive research. However, the initial observations suggest that the use of amitraz to control