3.1. Problem for Pollinators
At this time there is a global trend of pollinators decreasing [64
]. Possible reason for this is the toxic pesticides effects [69
]. The active usage of pesticides in agriculture and the decline in the growth of colonies of pollinating insects can be interrelated processes. Recent studies have shown that pesticides can affect on bumblebee colonies—An important and annually decreasing group of pollinators [71
]. It has been shown that the effect of thiamethoxam, a neonicotinoid insecticide, on bumblebees reduces the proportion of incubating queens in the hive that lay eggs, and also increases the vulnerability of individuals to pathogenic microorganisms [72
]. Thus, neonicotinoids have a significant effect on the dynamics of the pollinators population. Current studies examine neonicotinoid target sites, as well as the metabolism of neonicotinoids, not only in pests, but also in beneficial insects such as bees [73
]. The obtained data contribute to the development of a new generation of pesticides targeted at nicotinic acetylcholine receptors of insects.
It is known that a honeybee has a special reproductive behavior that can change under the influence of stress factors. Widely used insecticide fipronil, as shown by studies, leads to a decrease in sperm concentration and their viability in combination with an increased rate of spermatogenesis, which has led to a deterioration in the fertility of male bees [74
]. The results of this study indicate the detrimental consequences of pesticides usage on the reproductive potential of bees. Pesticides can contribute to honeybee population reduce as a result of distorted fertility, an example of which is fipronil. Similarly, an experiment was conducted with another high-potent insecticide. Chronic consumption of neonicotinoid thiacloprid by bees influenced the behavior of insects in the field. The behavior, navigation, and social communication of bees were disrupted. It has been shown that thiacloprid applicable at low-dose at the feeding sites accumulates over time in the food [75
]. Thus, pesticides pose a significant risk to honey bees, disrupting learning and memory functions, and also lead to the losses of insects.
Currently, in addition to stress, bees are constantly exposed to a mixture of agrochemicals and at the same time suffer by parasites. Stressors do not act separately, the effect of pesticides can disrupt both detoxification mechanisms and immune responses, making bees more susceptible to parasites [76
]. Chronic exposure to several interacting stressors leads to the loss of honey bees colonies and a decrease in the abundance of wild pollinators. Analyzing the history of pollinators extinction, an assessment was made for extinction rate of bees in Britain from the middle of the 19th century to the present [78
]. The fastest extinction phase is associated with changes in agricultural policy that began in the 1920s and ends with the ill-considered application of farming methods.
One of the mechanisms of the toxic effect of small doses of pesticides may be their effect on the microbiological composition of insects. Next, data on the effect of pesticides on the microbiome of animals of various taxonomic groups will be considered.
3.2. Microbiome of Bumblebees and Honey Bees and the Impact of Pesticides
The study of intestinal microbiota is an important area of the research of the ecological and functional dynamics of the intestinal environment. Especially the study of the intestinal microbiota of honey bees and bumblebees, which are important pollinators. Due to the specific intestinal habitat, Apis mellifera
is a valuable model system. All these data together helps to research the microbiota and develop experiments in this field [79
Intestinal microbial communities play a crucial role in maintaining the health of many insect species. However, these bacteria are very sensitive and it is still unknown how the host and pathogen interact at anthropogenic changes in the environment. The bacterial composition of the microbiota of bumblebees Bombus terrestris
taken from forest and urban habitats was analyzed [80
]. The results showed a significant predominance of bee-specific main bacteria, such as Snodgrassella
in urban areas, and their small abundance in the forest zone. Conversely, the abundance of pathogens Crithidia bombi
and Nosema bombi
was greater in the forest zone compared to the urban one. This confirms the role of beneficial bacteria in protecting against pathogens in the body.
In addition, similar studies support this hypothesis. When analyzing the effect of the parasite Crithidia bombi
on the intestinal microbiota of Bombus terrestris
, differences were found: After microbiota transplantation, bumblebees with different resistance caused a different immune response [81
]. In studies related to the infection of bumblebees with Apicystis bombi
, a eukaryotic parasite, insects had a higher abundance of Arsenophonus
sp. and Phyllobacterium
sp., which are symbionts. These organisms are mainly present in the fat body, also a correlation has been found between bacteria in the intestine and the fat body [82
]. Studies conducted by Näpflin and Schmid-Hempel in Switzerland were aimed at researching the body’s properties that provide protection and resistance against pathogens, in particular Crithidia bombi
, since these mechanisms are still unknown [83
]. It turned out that the structure of the microbial community does not differ between infected and uninfected individuals. Although the microbial communities of the intestines and their functions in adult bumblebees are widely studied, data on the microbiota of the intestines of the larvae are still limited. It has been shown that the microbiota of adults and larvae differs significantly, in particular, typical main intestinal bacteria in adult bumblebees are absent in larval bumblebees, where Enterobacteriaceae and Lactobacillaceae predominate [85
]. The functions of this microbiota have yet to be determined.
As known, intestinal symbionts of insects inhibit the action of pathogens. The main symbiont bacteria, Snodgrassella alvi
] and Gilliamella
], were studied in the body of Apis mellifera
sp. It turned out that bees contain a greater number of bacterial strains than bumblebees, which confirms the hypothesis that the foundation of colonies by swarms of workers allows to keep a greater diversity than the foundation of colonies by one queen. Analysis of the intestinal isolates of the bumblebees Bombus pascuorum
, Bombus terrestris
, Bombus lucorum,
and Bombus lapidarius
showed the presence of several new bacterial taxa and species [88
], in particular, 4 new Gilliamella
bacteria species [89
]. The presence of accurately identified microbial isolates will contribute to a future assessment of the functional potential of the bumblebee intestinal microbiota.
As described above, it has been suggested that the use of pesticides has a negative effect on the microbiome of the honey bee. However, the general mechanisms of the toxic effect of insecticides on microbiomes are poorly understood and require detailed study to minimize this problem. Apis mellifera
honey bees were exposed to nitenpyram [90
] and imidacloprid [91
]. Decrease in the general condition, behavioral characteristics (food intake), and overall insect survival were observed. The result of sequencing of the 16S rRNA gene showed a change in the intestinal microbiota of bees, i.e., the reduction of microbial density that contribute to metabolic homeostasis and insect immunity in general.
Another study related to the effects of neonicotinoids on the body in general and intestinal microbiomes in particular, was an experiment with Bombus terrestris
]. A relationship was found between the effects of clothianidin on the microbial composition of the intestine, both at the individual level and at the colony level. A xenobiotic has been shown to reduce the size and weight of bumblebees, and reduce reproductive ability, but clotianidin does not cause a decrease in resistance to pathogenic microorganisms.
Intestinal microorganisms play a dominant role in the nutrition and other vital processes of the bee. There is a dependence of the intestinal microflora of the honeybee on the general health of the insect [93
]. This composition of the microbial community can be used as an indicator of the biological health of the insect. Diaz et al. found a negative effect of imidacloprid insecticide on the composition of the microbial community of the intestines of bees [94
]. Gram-positive and Gram-negative biomarkers were affected, whereas fungal biomarkers were unaffected. Both insecticides and generally accepted safe herbicides aimed at controlling weeds cause changes in the intestinal microbiome and increase the body’s sensitivity to pathogenic microflora, which leads to the death of insects [95
In this regard, research directions are currently being actively developed related to the study of microflora changes under the influence of various pollutants, including pesticides. Majority of chemical compounds, used as pesticides, has serious threat to the intestinal microflora [96
], and they also negatively affect to the endocrine and digestive systems. These substances play a significant role in the development of metabolic disorders, which leads to intestinal dysbiosis.
The most widely-used pesticides, such as amitraz, chlorpyrifos, dimethoate, were studied for acute or chronic toxicity in Apis mellifera
and Apis cerana
]. It was found that dimethoate most strongly affects the survival rate and diet consumptionof bees. Data on the highest susceptibility of A. mellifera
to chlorpyrophos and dimethoate were obtained, while A. cerana
was more sensitive to amitraz. All major intestinal bacterial types of honey bees were found, including Proteobacteria, Firmicutes, and Bacteroidetes. However, during the experiment, a significant difference in the species diversity of the intestinal microflora of insects appeared.
3.3. Pollinators Microbiome Healing
Today, the honey bee is under stress from a number of biotic and abiotic factors such as pesticides that reduce pollination processes and the overall productivity of the hive. Researches of intestinal microbiota are very important for understanding the general ecological mechanisms [79
]. The knowledge concerning the effect of the composition of the intestinal microbiota on the health of bees allows us to develop optimal concepts for increasing the productivity of apiaries and general natural populations of honey bees. Thus, the “probiotic concept” is actively transferred to beekeeping. A significant correlation was found between the increase in honey yield and the use of probiotics (Lactobacillus salivarius
) comparing with the control group which did not receive probiotic [99
]. Adding a probiotic to honeybee feed reduces yeast colonies [100
], which subsequently affects the overall immunity of insects [101
The main probiotic strains of the intestines of bees are Lactobacillus
. The beneficial effects of these microorganisms on the health and productivity of bee hives were indicate, especially if strains of bee origin are used [102
]. Lactic acid bacteria are widespread in nature due to their beneficial effects on the body and are actively used as probiotics. They are used in animal breeding, beekeeping, poultry farming to improve health and increase reproductive functions. Lactic acid bacteria isolated from the hive showed inhibition of twenty human and animal pathogens, some were resistant to antibiotics [103
]. The intestinal microflora of the honeybee has been classified as heteroenzymatic lactic acid bacteria [105
]. They also inhibited the growth of the main pathogen of the honey bee—Paenibacillus larvae
, which means that the studied lactobacilli have healthy probiotic properties. Lactobacilli are able to survive at acidic pH values, bile salts, and show vitality in the gastric juice [106
], which makes them a potential candidate for use in the nutraceutical and pharmaceutical industries. Tests with Lactobacillus
bacteria also revealed their probiotic effect [107
]. One of the results of their application was an increase in egg laying, an increase in the number of bees, and therefore a high yield of honey. Probiotics also reduced the incidence of honey bees by nosematosis and warrosis. The addition of a prebiotic preparation containing Lactobacillus johnsonii
CRL1647 to the hives, in general, favorably affected the activity of honey bees [108
]. The commercial probiotic Lactobacillus rhamnosus
and inulin as a prebiotic caused the survival of honey bees that were infected with the pathogen Nosema ceranae
]. However, the study pays great attention to the correct choice of doses of pro- and prebiotics used, since excessive saturation of the diet of honey bees with these food additives could not prevent the development of the disease from the pathogen, but rather disrupt the regulation of the immune system of bees and increase insect mortality.
In addition to lactobacilli, Brevibacillus laterosporus
was isolated from the digestive tract of the honey bee, which can stimulate the growth of bee colonies [111
]. This bacterium has a unique shape and morphological structure. The cyclic peptide (Leu-Pro) of the bacterium has biological activity against several pathogenic microorganisms, in addition, the total number of broods, production of bee pollen, honey is better with bees treated with a suspension of B. laterosporus
. Similar results were obtained in the analysis of Bacillus subtilis
subsp. subtilis Mori2, which increases the productivity of bees, stimulates egg laying, the growth of bee generations, therefore, causes an increase in honey, and also reduces the spread of bee diseases [112
Modulation of the intestinal microbiota of the honeybee is recognized as a successful practical approach. In European studies, the preparation of sugar syrup containing bifidobacteria and lactobacilli isolated from the intestines of the bees was sprayed onto a part of an apiary located in an open field [113
]. Analysis of the hives showed a significant increase in the brood population, as well as of pollen and collected honey. Analysis of intestinal microbiota showed an increase in the number of species of Acetobacteraceae
, which contribute to the normal metabolism of the insect.
The probiotic effects of seven isolated intestinal bacteria species from Apis mellifera jemenitica
to larvae of a bee infected with Paenibacillus
were studied. The following intestinal bacteria were used in the experiment: Fructobacillus fructosus
, Proteus mirabilis
, Bacillus licheniformis
, Lactobacillus kunkeei
, Bacillus subtilis
, and Enterobacter kobei
, Morganella morganii
]. It turned out that adding these probiotic microorganisms to the diet significantly reduces the mortality rate of honey bee larvae. The use of Bacillus licheniformis
and Lactobacillus kunkeei
showed the largest positive effect.
American foulbrood is a disease of honey bees associated with the bacterial pathogen Paenibacillus larvae
. This disease can be treated with antibiotics (e.g., tylosin), which is not desirable. There is interest to the search for an alternative medicine in the form of a probiotic [115
]. The antagonistic effect of lactic acid bacteria against the pathogen was most effective. During the experiment, it was shown that this probiotic under laboratory conditions affects the decrease in the number of pathogen, but in practice this experiment did not show a positive effect [115
]. The mechanisms governing the dynamics of bee microbiota are complex, and potential drugs identified in laboratory studies must be thoroughly tested in situ.
European foulbrood is a bacterial disease of honey bee larvae. Studies have shown that the intestinal bacteria of bees act as probiotic microorganisms and reduce the risk of developing this disease [117
]. The analysis showed that most isolates of intestinal bacteria belong to Bacillus
. Under the influence of pathogenic bacteria, the inhibitory effect of Bacillus
on these pathogens is shown.