Introduction
According to Guarner and Álvarez (2020) reports, the microorganism which colonized plants and animals are a constituent, functional and non dispensable part of the host organism. An unbalanced microbiota can cause a disease, affect the body development, alter the regulation of the immune system and the metabolism, and even, negatively influenced on behavior.
The microbiota of the digestive tract of bees is very varied and it is associated to the type of feeding applied in the beehive, to the production system, the seasonal nature and the state of health (Marche et al. 2019). Other authors refers that melliferous bees has a high specificity of the intestinal microbial community. They are constituted, approximately, by eight phylotypes (Moran et al. 2012 and Khan et al. 2020) and there are three groups in the intestinal community: Snodgrassella alvi, Gilliamella apicola and Lactobacillus spp. (Cariveau et al. 2014 and Rodríguez et al. 2021). Other studies show the presence of 174 species (Hernández et al. 2020).
At the international level, is working on the incorporation into animal production of zootechnical additives that have probiotic activity, since these constitute an alternative, due to their ability to modulate the immune system and the intestinal microbiota; in addition to having an antagonistic function against pathogens. In the last decades is stated that the probiotics and plant extracts are the most used to improve the productive and health indicators in bees, so in Cuba is working to get their standardization (Hernández et al. 2020). The objective of this study was to show a compilation about the history of apiculture, the main diseases of bees and the ways for their control and the use of microbial additives in apiculture.
Apiculture history
The first records of melliferous bees’ domestication are date back to the Egyptian tows, where hieroglyphics from the year 2400 before Christ (BC) were founded with the bee image and where the use of horizontal beehive of sun backed clay (figure 1) is observed. In the qualification of the Egypt king in the year 3100 (BC) the bee figure appears, so they had been important even before their domestication. The Persians, Syrians, Greeks and Romans towns has evidence of the apiculture practice. The honey, beeswax and propoli were used in food production, as in medicines, cosmetics and even, in the religious field. The honey was used as natural sweetener or for adding to alcoholic drinks. It was also used in holy rituals, in gods offering and in the embalming process of Egyptians nobles. In this first stage, the men begin to take out the beehives from its natural habitat and were transported. The beehives were made of different materials: clay, hollow trunk of trees, straw or wicker, and every time the honey were extracted, the bees were killing and the beehives were loss (Añón 2018).
Nowadays, the apiculture or bee culture is a livestock activity directed to bee breeding (Apis mellifera L.). Among the positive impacts that this activity generates the important function the melliferous bees plays in the maintenance in almost all life in the earth is highlighted, because they are the main pollinates agents of commercial crops and wild flora (Arredondo 2015 and Wu et al. 2021). In addition, bees take part in the production of a great amount of industrial products: honey, beeswax, royal jelly, propolis, among others (Jacovi 2019 and SEFC 2019). However, despite their importance, currently in the world there is a decrease in the number of beehive.
The decrease of the number of beehives and the increase of bee mortality is due to many stress factors, which varied according to the geographic area, local characteristics or climatic conditions. Among the factors is the dangerous impact of the invader exotic species as the mite Varroa (Varroa destructor), the little beehive beetle (Aethina tumida), the Asiatic wasp (Vespa velutina), Paenibacillus larvae (causal agent of American foulbrood), among others (Burnham 2019). There also considered the effect of certain active substances presents in the phytosanitary products and other biocides, the climatic change, the environmental degradation, the habitats degeneration and the progressive disappearance of angiosperms (Manzano 2018).
Main bee diseases and the control ways
Due to the colonial way of life of bees, they can be infected with a great variety of pathogens, among them are the mites, bacteria, fungus and virus (DeGrandi and Chen 2015). Among the most significant diseases that these microorganisms cause are the varroosis, American Foulbrood and nosemosis, caused by the microsporidian Nosema ceranae and Nosema apis (Pérez-Piñeiro 2018 and Burnham 2019).
The varroosis is a parasitic disease of great significance for the apicole sector. It is considered a world disease (Sanabria et al. 2015). Its importance lies in the many losses of colonies that annually caused and in the affectations on honey production. That is why it should regularly controlled (Pérez-Piñeiro 2018).
In Spain and the rest of the European Union have an endemic character, being the only bee disease that required a systematic treatment of the colonies to keep the parasitism rate under harmful values (Ministerio Agricultura, Pesca y Alimentación 2023). It is similar the performance in the Americas region, where it represents the main bees enemy, and constitutes the main risk for apiculture (Masaquiza et al. 2019).
Cuba is not immune to these circumstances. The current practice of Cuban apiculture was transformed after the presence of Varroa destructor mite on the island was diagnosed in 1996. This parasite induced significant changes in the beehives management, which jointly to the economic problems that Cuba has to face, affect negatively the apiculture. Specifically, in the beehives park, of a total of 60, 000 families, approximately 40, 000 were recovered (Pérez-Piñeiro 2018).
V.destructor is a parasite mite that cause damages in the bee, weakened, when depressed their immune system and favors the infection by other pathogens, is deadly if it is not adequately treated (Rosenkranz et al. 2010). It is all over the country, so it is needed to apply mite killer products systematically to avoid the beehives loss.
The presence of V.destructor in the beehives it is not only a problem itself, so it increase their virulence due to their association with different ARN virus. The virus of severe paralysis, chronic paralysis, black queen cell, sacbrood and the deformed wing are transmitted by these contaminant agents (Antúnez et al. 2013).
This mite feeds exclusively on the hemolymph (blood) of bees. It reproduces in the brood of worker bee and the drones, with a preference for the latter. V. destructor enters a cell shortly before percolation and lays eggs (first male egg, followed by a female egg) that develop inside the capped cell. The male can fertilize females that reach maturity, normally one or two within a worker bee cell, and three or four within a drone cell (Mendoza et al. 2008).
American Foulbrood (AFB) is produced by the pathogen Paenibacillus larvae, and is the most destructive disease affecting bee brood. P. larvae. is a bacillus Gram-positive, catalase-negative, and facultative anaerobic, with low percentage of GC (Guanine and Cytosine) and endospore-forming (Genersch et al. 2006).
The larvae of worker bee, queen bee and drone become infected by ingesting food contaminated with P. larvae spores, when fed by nurse bees (Hornitzky et al. 1989). Once ingested, the spores reach the lumen of the larval intestine, where they germinate, and giving rise to vegetative cells. They proliferate and move towards the epithelium and destroy cell-cell interactions and invade the intercellular space, reaching the host's hemolymph very quickly. Subsequently, the larva dies, which is accompanied by sporulation of vegetative cells. The spores are spread within the beehive when cleaning bees contaminate their mouthparts by removing dead larvae and subsequently transmit them to the larvae when feeding (Yue et al. 2008).
N. ceranae is a fungus formed by microsporidia, which are obligate intracellular parasites of insects such as bees. Its infection takes place after ingesting mature spores that germinate in the intestine and form a tube that extrudes and injects the sporoplasm into the cytoplasm of epithelial cells (Hernández et al. 2018).
The symptoms that are noticed in the beehive affected by N. ceranae are dissimilar: restlessness in the bees, malnutrition, short abdomen, decreased activity and weakness. Many bees are also seen crawling in the background and on the frames. When the roof is removed, outside the beehive, it is observed that the infected bees hardly manage to fly a few meters without landing. Other times they crawl on the ground or on the leaves. The abdomen is often stretched by fecal matter and will appear shiny and greasy (Li et al. 2018).
In Cuba, when large infestations by V. destructor, N. ceranae and Paenibacillus larvae occur, no drugs, antibiotics or chemical substances are applied to treat beehive pathologies, only integrated management is practiced, which consists of cleaning the beehives, harvesting in the apiary to avoid transporting honey or infected combs, changing queen bees and, if necessary, the beehives are sacrificed if there are outbreaks of serious infectious diseases, which leads to a decrease in the populations of these insects (Pérez-Piñeiro 2018).
In livestock production, the use of probiotics is known, but very little is its use in apiculture. Probiotics are live microorganisms that, when intake in adequate amounts, confer benefits to the host health (Milián et al. 2021). Obtaining probiotic biopreparations from the isolation and selection of beneficial microorganisms from the digestive tract of animals is the starting point for their application in animal production. These zootechnical additives are supplied with the purpose of being used as animal growth promoter, since they improve the composition of the gastrointestinal microbiota and the efficiency in the use of food, stimulate the immune system and inhibit pathogenic microorganisms without the use of antibiotics (Rondón et al. 2020 and Cabaña et al. 2021).
Recent studies by Khoury (2018) show that strains of Saccharomyces cereviseae var. bourlardii CNCM I-1079 and Pediococcus acidilactici CNCM MA 18/5M are effective in protecting bees against fungal infections known as nosemosis (table 1). This author hypothesizes that the mechanism of action might involve an enhanced immune system and tissue repair processes to protect the host from the damage caused by the parasite. However, it could be inferred that this result can be associated with the action of probiotics, since it is one of its main mechanisms. Specifically, the biopreparations obtained from Bacillus strains activate the immune system, since they are considered immunomodulators par excellence (Milián et al. 2022).
Survival of bees treated with probiotic microorganisms | ||
---|---|---|
Strains | Treatments | |
Sugar syrup (%) | Curatives fallowed by a nosemosis challenge (%) | |
|
28.8 | 23 |
|
29.9 | 41 |
Source: Khoury (2018)
Antunez et al. (2019) managed to reduce the infestation by V. destructor, by enhancing the effect of the synthetic acaricide and, at the same time, reducing the infection by N. ceranae. Probiotics can decrease the mortality rate of this infection in bees by up to 40 %.
According to Derome (2018), when bees are subjected to stress, the microorganism can evade their immune system, causing an infection that can affect feeding ability, stops larval care, alter bee orientation, and increase mortality. Hence, the researcher decided to evaluate the effectiveness of two probiotics (Bactocell® and Levucell®), these products are marketed in pig, chicken, shrimp and salmon farms. In addition, he evaluated two cultures of strains isolated from the intestinal microbiota of healthy bees, obtained under laboratory conditions, all were administered to the bees by mixing them with sugar syrups. After two weeks of testing, the mortality rate of infected bees was from 20 to 40 % lower in those receiving the probiotics and cultures than in the control group.
In recent years, the Bacillus spp. genus is recognized as one of the species most used as probiotics (Florencia et al. 2018). The Bacillus is Gram-positive bacteria with rod-shaped, spore-forming, aerobic or facultative anaerobic (Alou et al. 2015).
Due to their potential, Bacillus can synthesize metabolites with antifungal and antibacterial activity. These antimicrobial substances are biopeptides with different chemical structures, which are used as therapeutic agents against pathogenic bacteria and fungi, able of acting on microorganisms of diverse etiology. The biocontrol effect exerted by Bacillus spp. is the result of various mechanisms, including antibiosis, which occurs due to the production of peptides, lipopeptides and phospholipids (Kadaikunnan et al. 2015, Pedraza et al. 2018 and Milián et al. 2021).
Probiotics in apiculture
Researches on the use of probiotic biopreparations from Bacillus genus to be used in apiculture are few. Researchers carried out by Florencia et al. (2018), showed the probiotic potential of Bacillus 4A, 230P and 86B strains isolated from honey and pollen from native stingless bees. They showed a marked probiotic effect on the control of pathogens such as: Listeria innocua 6a, L. innocua 7, L. monocytogenes ATCC 7644, Pseudomonas aureuginosa ATCC 27853, Escherichia coli ATCC 25922 and Enterococcus faecalis ATCC 29212.
Audisio (2017) found beneficial results when evaluating the strain B. subtilis subsp. The spore counts of Nosema spp. and Varroa spp. in the treated hivebees were lower than those of the control group. These results in the experimental apiaries show that B. subtilis subsp. Mori2 favored the bees’ performance. In the first place, because the microorganism stimulated the laying of the queen's eggs, which resulted in a greater number of bees and, consequently, more honey. Second, because it reduced the prevalence of two important bee diseases worldwide: nosemiasis and varroosis.
Another study was reported by Hernández et al. (2021). In a laboratory test, species from Bacillus ssp. and Brevibacillus ssp. were used, associated with melliferous bees, as natural alternative for the AFB control of and plaster brood. In this experiment favorable results are reported, when observing inhibition faced pathogens. This constitutes the first study of associations between the presence of genes related with the synthesis of antimicrobial peptides and their antagonism faced P. larvae and A. apis.
One of the affectations that currently are in the beehives is the incidence by ants, considered common visitors in the beehives. Studies performed by Ruíz et al. (2021), showed the antifungal effect of the Bacillus subtilis strain against the fungus Aspergillus nigri and Penicillium serie crhysogenum isolated from the ants cuticle established in Apis mellifera L. beehives. activity that makes it possible to control the populations of ants that affect the beehives and an increase in the production of bees.
The studies carried out by Yépez (2019) showed the effect of Bacillus cereus sensu lato, when been related with the microbiota of bee honey. This author perfomed a microbilogical analysis to 38 samples of honey coming from Mejía canton, Ecuador, and determined factors associated to the presence of the group Bacillus cereus sensu lato. This group was in 53 % (20/38) of the collected samples. A total of three species were identified: Bacillus cereus, Bacillus mycoides and Bacillus thuringiensis. Its distribution in the 20 positive samples was: 1 (5 %) Bacillus cereus, Bacillus thuringiensis and Bacillus spp; 2 (10 %) Bacillus cereus and Bacillus mycoides; 3 (10 %) Bacillus cereus and Bacillus thuringiensis; 4 (20 %) Bacillus mycoides and Bacillus thuringiensis; 5 (25 %) Bacillus cereus and 6 (30 %) Bacillus cereus and Bacillus spp. This confirms the ability of sporulated Gram-positive bacilli to survive in honey.
At present, the study of the bee microbiota function continue, due to their contribution to health and to the productivity of this specie and by the influence the probiotics can have on their stabilization and on the stimulation of the immune system of bees (Al-Ghamdi et al. 2020).
In the Centro de Estudios Biotecnológicos, belongs to Facultad de Ciencias Agropecuarias de la Universidad de Matanzas, Cuba, is working on the isolation, selection and identification of the strains of acid lactic bacteria Bacillus spp. and yeasts with probiotic potential, from the digestive tract and the honey of Melipona beecheii B. The antimicrobial activity of Apis mellifera and Melipona beecheii honeys against pathogenic microorganisms is also evaluated.
Álvarez (2020) studied a total of eight pathogenic strains, four reference strains from Centro Provincial de Higiene y Epidemiología (Staphylococcus aureus ATCC 25923, Escherichia coli. ATCC 25922, Streptococcus sp. and E. coli) and four belonging to isolations of cows with mastitis (C7, C8, C9 and C15), which were identified by traditional methods (two Staphylococcus aureus and two Bacillus sp).The evaluation of the inhibitory effect was quantitatively performed, by numeric measurement of the inhibition halos, and qualitatively, taking as reference the pattern proposed by Duraffourd et al. (1987). The results of the inhibitory effect of M. beecheii and A. mellifera honeys against pathogenic microorganisms, are shown in table 2. It is observed that the Apis mellifera and Melipona beecheii honeys have inhibitory action in the most of the studied pathogenic microorganisms, so it constitutes an alternative for the treatment of cattle mastitis, as well as for some of the apiaries diseases.
Strains | TREATMENTS | ||||||
---|---|---|---|---|---|---|---|
MM100% | MM50% | MM30% | AB | MA100% | MA50% | MA30% | |
C9 ( |
22.6±2.5a (+++) | 21.3±2. 0a (+++) | 18.0±2.0a (+++) | 17.6±0.5a | 16.6±0. 5a (++) | 9.6±1.2b | 4.0±1.7c |
C7 |
20.3±1.5a (+++) | 19±2.0a (++) | 12.3±3.2b (+) | 23.6±0.5a | 11.0±0.3b (+) | 8.6±2.5b (+) | 1.2±0.2c (-) |
|
16.4±0.5a (++) | 15.2±0.7 a, b (++) | 13.5±0.7b (+) | 22.1±0.2c | 8.0±1.0d (+) | 6.0±1.0d, e (-) | 4.5±0.5e (-) |
|
19.6±0.6a (++) | 18.0±1.0a (++) | 15.3±0.6b (++) | 27.3±0.6c | 11.3±0.6d (+) | 1.2±0.2 e (-) | 0.8±0.01e (-) |
C8 ( |
6.6±0.6a (-) | 5.6±1.5a (-) | 0.7±0.2b (-) | 25.0±1.0c | 0.7±2.0b (-) | 0.4±0.15b (-) | 0.2±0.09b (-) |
C15( |
8.7±1.5 | 0.7±0.2b (-) | 0.6±0.2b (-) | 25.0±0.6c | 0.6±0.2b (-) | 0.4±0.1b (-) | 0.2±0.05b (-) |
|
5.1±2.0a (-) | 3.7±1.1a, b (-) | 0.7±0.1b (-) | 31.3±1.5c | 4.0±1.0a, b (-) | 1.0±0.1b (-) | 0.9±0.2b (-) |
5.0±1.0a (-) | 0.8±0.05b (-) | 0.6±0.05b (-) | 31.0±1.0c | 0.7±0.05b (-) | 0.6±0.05b (-) | 0.2±0.05b (-) |
Different letters in the same row show significant differences between means with P<0.05.
Duraffourd scale: (-) Null, (+) sensitive, (++) very sensitive, (+++) extremely sensitive.
M. beecheii (MM) and it is assigned the corresponding percentage (MM100 %, MM50 %, MM30 %) and for A. mellifera honey (MA100 %, MA50 %, MA30 %).
Conclusions
The use of probiotic bioprepartions from Bacillus sp. strains and other microbial genera reaffirm the real possibility of their use, since they represent economic advantages for any farmer, not only due to loss reduction, so because these products show for bees an impact on physiological and productive responses in the apiaries, which are superior to what its cost of production might be.