85 ISJ 19: 85-90, 2022 ISSN 1824-307X SHORT COMUNICATION Stimulation effect of probiotic bacteria Bacillus spp. and inactivated yeast on the honey bees Apis mellifera physiology and honey productivity E Sokolova1,2,3*, S Mager2, E Grizanova1,3, G Kalmykova2, N Akulova2, I Dubovskiy1,2,3* 1Novosibirsk State Agrarian University, Laboratory of Biological Plant Protection and Biotechnology 2Siberian Federal Scientific Centre of Agro-BioTechnologies of the Russian Academy of Sciences 3Tomsk State University, Tomsk, Russia This is an open access article published under the CC BY license Accepted May 11, 2022 Abstract In order to find effective and safe ways to prevent the weakening and death of honey bee colonies from various stress factors, it is necessary to focus on the stimulation of physiological processes in the bee’s body, activating their own mechanisms of resistance. Bacteria Bacillus subtilis and Bacillus licheniformis produce important digestive enzymes, which have antimicrobial and detoxification effects, and also stimulate metabolic processes in the organism. The inactivated yeast Saccaromyces cerevisiae was used as a protein and vitamin component of the supplement. It was found that the addition of the studied supplement to the bees' feeding increased the activity of proteases 3.32-fold in the gut, non-specific esterases - 2.16-fold in the fat body, glutathione-S-transferases - 2.64-fold in the gut and 1.69-fold in the fat body in the Apis mellifera. Application of the supplement in the field has shown that the honey productivity per family increases 1.5-fold compared to the control. Key Words: probiotics; Apis mellifera; Bacillus; Saccaromyces cerevisiae; supplement; detoxification system Introduction Over the last twenty years, both technical and scientific progress and the growth of beekeeping have led to a significant increase in honey production, with an average annual growth of 35,000 tonnes since 2000, amounting to a total of 1.8 Mt of honey produced in 2016 worldwide (Pippinato et al., 2020). However, in some years, the world production of honey has reduced compared to the previous one (for example, in 2007, 2009, 2018, 2019) (FAOSTAT). High mortality in honey bee colonies has been reported worldwide in recent decades without definitive identification of the causes (Benaets et al., 2017). Nevertheless, recent investigations have established some of the most important factors contributing to honey bee losses, in particular, pests and diseases, bee management, including bee keeping practices and breeding, the change in climatic conditions (Potts et al., 2010), agricultural practices, and the use of pesticides (Gill et al., 2012; Hristov et al., 2020). The decline in honey bee populations causes serious damage not only to the production of honey, but also to pollination _________________________________________ Corresponding authors: Elina Sokolova Ivan Dubovsky Novosibirsk State Agrarian University Laboratory of biological plant protection and biotechnology st. Dobrolyubova, 154, 630039 Novosibirsk, Russia E-mails: elinq.98@mail.ru; dubovskiy2000@yahoo.com of plants affecting the functioning of natural and agricultural ecosystems (Klein et al., 2007; van Engelsdorp et al., 2008), therefore the attention of the international community is focused on this phenomenon. In the early spring period, with a meager flow, as well as during wintering, when environmental conditions become unfavorable for the vital activity of bees, they are most susceptible to the influence of various pathogenic factors (Becsi et al., 2021). To provide bees with the necessary amount of nutrients and to activate metabolic processes during these periods, beekeepers use various feedings. They can activate the defense systems of bees and increase the amount of obtained honey (Brodschneider and Crailsheim, 2010). To prevent and protect the honey bees from infections and parasitosis (such as varroatosis, acaripidosis), beekeepers generally use a variety of antibiotics and insecticides, which imposes restrictions on the production of organic beekeeping products (Ruoff and Bogdanov, 2004; Luttikholt, 2007) and their contamination may carry serious human health hazards (Al-Waili et al., 2012). Most importantly, it leads to the accumulation of a stockpile of resistance capabilities in the microbiota of a healthy gut, providing a source of resistance genes for pathogens themselves (Tian et al., 2012). Therefore, in order to find effective and safe ways to prevent the weakening and death of honey bee 86 colonies, it is necessary to focus on the stimulation of natural physiological processes in the body of bees, activating their own mechanisms of resistance. Bacteria Bacillus subtilis and Bacillus licheniformis are related species of gram-positive bacteria. As antagonists of pathogenic and opportunistic microorganisms (Staphylococcus sp, Salmonella sp, Shigella sp) (Moore, 2013), they stimulate the growth of normal intestinal microbiota (Mazkour et al., 2019). Reproducing in the intestinal lumen, these bacteria produce all the main digestive enzymes (proteases, amylases, lipases, pectinases, cellulases), stimulate metabolic processes in the microorganism (Suva et al., 2016). In addition, there are more than two dozen known antibiotics produced by B. subtilis (Kudriashova et al., 2005; Stein, 2005). It should be noted that multi-strain mixture of these microorganisms is able to mutually reinforce and complement each other's biological activity (Cutting, 2011). Probiotic bacteria are not only antagonists of opportunistic microbiota in the bee’s gut, but also a constant source of microbial protein. This is especially important during intensive brood growth - the sensitivity of adult honey bees to pesticides directly depends on the amount and quality of protein consumed in the first 10 days after hatching (Wahl and Ulm, 1983). Because bees fed a high protein diet were better able to survive insult with interacting stressors (Archer et al., 2014) the studied feeding included the inactivated yeast Saccaromyces cerevicaea as a protein and vitamin component, since it contains all the essential amino acids (Abbas, 2006) and is easily digested by honey bees (Abbasian and Ebadi, 2002). Honey bees and other insect pollinators utilize detoxification enzymes such as carboxylesterases and glutathione-S-transferases (GSTs) to mitigate the toxic effects of xenobiotics such as plant defense compounds and pesticides (Panini et al., 2016). Glutathione-S-transferases (GST) are the principal Phase II (conjugation of products of Phase I) enzymes, although they can also function in Phase I during which the toxin structure alters enzymatically (Berenbaum and Johnson, 2015). Indeed, esterase enzyme activity positively correlates with pesticide tolerance in many insect species, including bees of all stages of development (Milone et al., 2020). Total protease activity in honey bee midgut is also an important parameter related to protein digestion (Li et al., 2012). A decrease in its activity is not only associated with a low level of protein in the diet, it may also be related to inhibitory effects of various infections and parasitosis. The inhibition of the activity of the host's proteolytic enzymes by the parasite often occurs during endoparasitosis (Zółtowska et al., 2005). Beekeeping is still done mainly to produce honey (Crane, 2009). It was found that honey production is governed by the interaction of three primary factors: average daily brood production, length of worker life and individual productivity of workers (Woyke, 1984). The health and, consequently, the productivity of honey bee colony depends on abiotic factors such as pesticides, management, weather conditions (Abou-Shaara et al., 2017), biotic factors such as mites, viruses, bacteria and fungi as well as on the nutrition profile (Steinhauer et al., 2018). The aim of this study was to examine the effect of bacteria B. subtilis and B. licheniformis and inactivated yeast culture on the detoxifying and digestive enzymes of honey bees Apis mellifera, as well as on their honey productivity in the field. Material and methods Experimental design Adult worker bees of the Middle Russian race were collected from one hive of a medium-strong family (Novosibirsk region, 54.759912, 82.633827). The bees were taken from the surface of the combs and transported to the laboratory of Novosibirsk state agrarian university. The bees were kept under laboratory conditions in shaded cages at a temperature of 28 - 30 °C and a relative humidity of 60 – 65 % with approximately 200 bees in each variant. Experimental diet was performed with 60 % sugar syrup with the addition of bacteria with yeast additive (5 g/L of a mixture of Bacillus subtillis and Bacillus licheniformis and 30 g/L of inactivated yeast). In the control variant, no components were added to the sugar syrup. The syrup was prepared and replaced daily. Enzymes activity in the fat body and midgut 10 days after the start of the feeding with experimental diet, the honey bees (n = 30) were placed on ice and their fat body and gut were dissected at 4 °C (Carreck et al., 2013). Each organ was homogenized by ultrasound in 100 µl of 0,1 М Na-phosphate buffer pH 7.2 (PBS). The homogenates were centrifuged for 15 min, 10,000 g at 4 °C. The supernatant was used for the analysis of enzyme activity. The activity of glutathione-S-transferases was determined spectrophotometrically at 340 nm, calculating the rate of increase in the concentration of 5- (2,4-dinitrophenyl)-glutathione, which is a reaction product of dinitrobenzene and reduced glutathione (Habig et al., 1974). Incubation was carried out at a temperature of 28° C for 5 min with the following composition of the reaction mixture: 205 μL of PBS with the addition of 150 mM NaCl, 1 mM glutathione, 1 mM o-Dinitrobenzene, and 5 μL of the supernatant of the studied tissue (Grizanova et al., 2018). Nonspecific esterase activity was estimated by spectrophotometric analysis of the p- nitrophenylacetate hydrolysis rate (Prabhakaran and Kamble, 1993). Five microliters of the supernatant were incubated with 200 μL phosphate buffer with the addition of 0.54 mM 1-naphthyl acetate in darkness for 5 min at 28 °C, and then the transmission density was measured at 410 nm. The method for determining the total proteolytic activity was to measure the rate of hydrolysis of 0,3 % azocasein (Sigma) by bee intestinal proteinases with some modifications (Alarcón et al., 2002). 30 μL of intestinal supernatant was added to 210 μL of 0.3 % azocasein in PB, after which the reaction mixture was incubated at 37 °C for 1. The reaction 87 was stopped by adding 200 μL of a 30 % TCA solution and subsequent incubation at -18 °C for 30 min. Then the resulting mixture was centrifuged for 10 min, 10000 g, and 150 μL of the supernatant was taken from it. After adding 70 μL of 1M NaOH to the resulting mixture, the optical density was measured at 440 nm. Enzyme’s activity was measured in units of optical density (ΔA) of incubation mixture per 1 min and 1 mg of protein. The total protein concentration in all of the samples was determined according to Bradford (Bradford, 1976). Standard curves to estimate protein concentration in the samples were prepared using bovine serum albumin (BSA). Microscopic analysis of the fat body Fat bodies of 30 bees from the control and treated groups were assessed according to the developmental index (a scale from one to five, with five being the best developed structure) proposed by Maurizio (1954), examining the inner surfaces of tergites using a binocular microscope (Fliszkiewicz et al., 2012). Honey productivity Three honey bee colonies with one year-old sister queens were selected per variant (one of these colonies was used in laboratory tests of bees enzymes activity in the fatbody and midgut), feeding was carried out three times (every three weeks) during the spring of 2021. Bee colonies of the control variant received sugar syrup without additives, the experimental group of bees received the supplement with the studied additives - 10 g of inactivated yeast and 2 g of a mixture of B. subtilis and B. licheniformis per colony. The honey was pumped out twice - in July and in September. Colony honey production was determined by weighing the honey supers before and after extraction, after which the mass of honey per one bee family was calculated (Nelson and Gary, 1983). Statistic The data were analyzed using GraphPad Prism® ver. 8 (GraphPad Software, USA). The results are reported as the mean values ± SD. A Kolmogorov-Smirnov normality test was used to check the normal distribution of the data. The data of visual scoring of the fat body of bees was analyzed by a Mann Whitney test. Two-way ANOVA (with Dunnett’s multiple comparison test) was used to assess differences between activities of ferments in the insect midguts and fat bodies. Results and discussion It was shown that adding bacteria B. subtilis and B. licheniformis with inactivated yeast to the honey bees' diet significantly increased the activity of GST both in the gut (p < 0.001) and in the fat body (p