Relationships between probiotic bacteria and honeybee diseases

The gut microbial composition of honey bees (Apis mellifera L.) is simple, unique and highly specialized; these microorganisms participate in various processes such as food digestion, detoxification from harmful molecules, supply of essential nutrients, participation in the host defense system and protection from pathogens and parasites. An imbalance of this microbiota (dysbiosis) can contributing to the phenomenon of Colony Collapse Disorder (CCD). The modulation of the honey bee gut microbiota, by supplementation of selected lactobacilli (LABs), has aroused special attention since it represents a strategy to improve the health status of colonies, in terms of productivity, as well as boosting the presence of beneficial microorganisms within the gut of new‐generation bees. Moreover, appropriate probiotics could be exploited to actively counteract bee pathogens and parasites, or to enhance immunity, and thus indirectly, increase the protection of the honey bees’ health. Preliminary studies (conducted at the Department of Agricultural, Environmental and Food Sciences of the University of Molise) showed that Apilactobacillus kunkeei strains could be functional for use in “probiotic syrup”, useful to restore the symbiotic communities of the intestine in case of dysbiosis and to exert a prophylactic action against Ascosphaera apis, an important pathogenic fungus of honeybee larvae. Selected Al. kunkeei strains were able to inhibit A. apis and highlighted their important properties, such as ability to form biofilms, high auto-aggregation, and hydrophobicity, all prerequisites for candidacy as probiotic microorganisms in a honeybee diet. In addition, the selected Al. kunkeei strains showed high osmotic tolerance, a functional requirement for the probiotication of sugar syrups to restore or strengthen the symbiotic communities in honeybee guts in case of microbial dysbiosis. The Lactiplantibacillus plantarum strains used in our first experiments have been shown to possess substances biologically active against A. apis. These results confirm the potentially antagonistic role of Lp. plantarum against pathogenic microorganisms that use the digestive channels of honeybees as the sites of infection. Five strains of Lp. plantarum are able to produce Extracellular Polymeric Substances (EPSs) and form biofilms, which are prerequisites for potential candidates to be used as probiotics in the honeybee diet. In addition, the antioxidant properties of the tested bacterial strains can help to increase the tolerance of these insects to endogenous and exogenous oxidative stress. These first results suggest the possibility to set up new strategies to improve honeybee health through nutritional approaches or the modulation of the gut microbiota using beneficial microbes and open up a new horizon for controlling honeybee pathogens. The study of the LAB community in the honey bee gut showed that Al. kunkeei and Lp. plantarum were the most numerically representative species. Al. kunkeei is a highly versatile bacterium and can be found in fructose-rich niches, including honey, beebread, flowers and the gastrointestinal tract of honey bees. Lp. plantarum is also a LAB usually isolated from the honey bee gut and it is sometimes numerically very representative. This confirms the extreme adaptability of this species to different environmental niches, including those rich in fructose. In further investigation, focused on European Foulbrood (EFB) and American Foulbrood (AFB), the eight LAB strains tested showed inhibitory activity against the vegetative growth of their causative agents, Paenibacillus larvae and Melissococcus plutonius, respectively. Results show the absence of inhibition in the control test, suggesting that the antagonistic action is not related to organic acids but to the presence of other metabolites capable of inhibiting the two pathogens. The analysis of enzyme activity profiles, determined using the API-ZYM test, showed that the strains of Lp. plantarum and Al. kunkeei possess glycosidase activity. Several carbohydrates present in the honey bee diet are toxic because these insects do not possess functional enzymes to metabolize them. The results obtained in the carbohydrate assimilation test showed that the four Lp. plantarum strains are capable of metabolizing the monosaccharides L-arabinose, galactose, mannose, and the oligosaccharides melibiose, lactose, melezitose and raffinose. The four Al. kunkeei assimilated L-arabinose, galactose, mannose, melibiose, lactose and melezitose. Lp. plantarum LP 179 and Al. kunkeei ALK 268 were also able to metabolize rhamnose and Lp. plantarum LP 31 was the only bacterium able to assimilate L-xylose. All of the above-mentioned carbohydrates are considered to be potentially toxic to the honey bee and may be contained in traces in the natural nectar derived from the hydrolysis of pectin, or synthesized as melezitose. This sugar can be produced by aphids and is the main carbohydrate contained in honeydew. The use and the role of the selected LAB strains as probiotics, thanks to their specific enzymatic activities, can contribute to the breakdown of complex polysaccharides and metabolize toxic sugars and consequently improve the dietary tolerance of honey bees. The ability of probiotics to adhere to the intestinal epithelium is an important pre-requisite for biofilm formation in the host intestinal tract. This ability, along with their antimicrobial activity are important features that can hinder the colonization of undesirable microorganisms. The gut adherence ability of probiotic bacteria involves different types of surface properties, including hydrophobicity and auto-aggregation. Auto-aggregation mechanisms generally involve various molecules including cell surface proteins, exopolysaccharides, carbohydrates, glycoproteins, teichoic and lipoteichoic acids. The eight LAB strains were observed to have a high level of autoaggregation, which is a recommended characteristic for a good probiotic strain. The adherence capacity is a strain-specific property due to several interactions between hydrophobic and hydrophilic components of the cell bacterial surface. In our tests, the hydrophobicity was evaluated by the BATH method using xylene and toluene as hydrocarbons. The results showed that the LAB strains had high values of hydrophobicity and were in line with previous investigations. Thanks to their cell surface properties, these LABs used as probiotic supplements in honey bees’ diets can persist in the intestinal tract where, especially during foraging, there is an intense flow of water and nectar. The virulent action of P. larvae and M. plutonius is based on a few key steps: growth dynamics; attachment to host cells by the production of biologically active compounds, such as adhesins; and the production of enzymes degrading the peritrophic matrix, enabling the pathogens to directly attack the epithelial cells. Probiotic bacteria, by binding to receptors in the intestinal mucosa, can inhibit the adherence of pathogenic microorganisms that are subsequently eliminated from the intestine. However, the evaluation of the functional properties of eight strains of Al. kunkeei and Lp. plantarum was carried out in vitro and, as a consequence, the good probiotic potential highlighted does not axiomatically result in health benefits for the honey bee colonies. Therefore, it is necessary to evaluate the contribution that these bacteria can make to the biocontrol strategy against EFB and AFB diseases. Finally, a comparative characterization of plant sources, microbial community, and phenolic compounds present in bee pollen (BP) and bee bread (BB) samples from two different apiaries (preparatory to the use of LABs in the Western honeybee diet), revealed that the microbial communities of BP and BB samples differed significantly according to the geographical location of the apiaries and the plant sources available. In the last year of the PhD course, also bees and honey as bioindicators of environmental contamination by polycyclic aromatic hydrocarbons, heavy metals and plasticizers residues were used. In addition, also the chemical composition and markers in the mediterranean orange blossom honey by UHPLC-HRMS metabolomics study integrating melissopalynological analysis, GC-MS and HPLC-PDA-ESI/MS were studied. In light of these studies, whether or not the bee gut microbial flora is influenced by different pollutants and whether or not the microbial flora influences the chemical composition of honey should be investigated. Future research activities will involve also the investigation of the nature of the antifungal compounds and evaluate the effects of these Lp. plantarum strains on honeybee health and productivity, and their efficacy in bee diseases biocontrol in vivo/in situ. Furthermore, it could be appropriate to characterize the microbiota of local population, also of the different subspecies, to verify possible symbiosis between microbes and subspecies/populations. In addition, it would be interesting to study the microbiota of wild colonies that have demonstrated to survive for several years in the natural environment. The overall results obtained in these studies could constitute an important basis for the development of biotechnological approaches able to improve the health of honey bees or to enhance the quality features of their products.