Effects of Bacterial Cell-Free Supernatant on Nutritional Parameters of Apis Mellifera and Their Toxicity Against Varroa Destructor

In their role as pollinators, honey bees are essential for agriculture, and therefore for the worldwide economy (Watson & Stallins, 2016), however colony loss syndrome, always on the increase, remains a serious issue (Lee et al., 2015; Seitz et al., 2015). The scientific community agrees that this scenario could be explained through a combination of such stressors (e. g. Neumann & Carreck, 2010) as pathogens (Bahreini & Currie, 2015), pesticides use (Kakumanu et al., 2016), monocultures (Maggi et al., 2016), climate change and invasive species (Nieto et al., 2014).

The ectoparasitic mite Varroa destructor has become the main pest of Apis mellifera. This mite feeds on the hemolymph of bees (e. g. De D’Aubeterre et al., 1999), although in recent studies Ramsey et al. (2018, 2019) has proposed that it feeds on bee fat body. However, the damages remain the same, a consequent reduction of protein content (Tewarson, 1983) and hemocytes (Amdam et al., 2004) leading to an imbalanced gut microbiota (Hubert et al., 2017) and decreased host's immune response (Gregory et al., 2005). In addition, Varroa mites act as a virus-vector (Antúnez et al., 2015). Scientists (e. g. Simion et al., 2011; Medici et al., 2015) have been alarmed by increasingly restricted parasite control and increasing acaricide resistance and residues in beehive products have resulting in several EU countries banning some of these compounds (European Commission, 2010) and seeking natural alternatives. The focus is on the mutualistic relationship between bees and their microbiota (e.g. Crotti et al., 2013; Alberoni et al., 2016). Commensal bacteria contribute to an increase in nutrient availability (Crotti et al., 2012) through the metabolism of toxic carbohydrates (Newton et al., 2013), the degradation of food components (Kwong & Moran, 2016; Kesinerova et al., 2017) as well as pesticides and antibiotics. Moreover, these supply part of the fatty acid, amino acid, metabolite and vitamin bee demands through microbiota secondary products (Brodschneider & Crailsheim, 2010; Crotti et al., 2013). Gut symbionts are involved in the stimulation of the host's immune system (Caccia et al., 2016) and contribute to the first line of bees’ defense of biofilm formation (Engel et al., 2012) by competing with microorganisms or by secreting antimicrobial compounds (Crotti et al., 2013; Alberoni et al., 2016). There is strong evidence that they induce an increase of antimicrobial peptide (AMPs) (Jefferson et al., 2013; Janashia & Alaux, 2016). In vitro trials have confirmed the ability of lactobacilli and bifidobacteria to inhibit honey bee pathogens (Audisio et al., 2011; Vásquez et al., 2012). Similarly, a reduction in N. ceranae intensity has been reported with the supply of Bacillus spp. (Sabaté et al., 2012) or their metabolic products (Porrini et al., 2010), a mix of bifidobacteria and lactobacilli (Baffoni et al., 2016) and Lactobacillus spp. (Maggi et al., 2013). Other evidence also supports a lower incidence of V. destructor in colonies supplied with lactobacilli (Audisio et al., 2015) or Bacillus subtilis subsp. subtilis Mori2 (Sabaté et al., 2012).

The balance of the beneficial microbiota can be disturbed by the combination of different stressors (Audisio, 2016; Kakumanu et al., 2016). Researchers have been joining efforts to determine the beneficial effects of symbionts and how to utilize them to improve bee health and thus colony performance (e.g. Alberoni et al., 2016; Audisio, 2016). In this scenario, bacterial metabolites appear as an ecological and environmentally-friendly alternative (Crotti et al., 2012; Moran, 2015). In the present study, L. johnsonii AJ5, E. faecium SM21 and B. subtilis subsp. subtilis Mori2 strains were selected based on their effects reported previously (Audisio & Benítez-Ahrendts, 2011; Sabaté et al., 2012). The purpose of this study was to determine the effect of bacterial cell-free supernatant on A. mellifera L., bee nutritional parameters, and their toxicity against V. destructor mite.

Bacterial gut symbionts and their secondary metabolites are increasingly being considered as a solution for gut microbial imbalance in bees due to their central role strengthening bees’ immune system (Crotti et al., 2012; Alberoni et al., 2016). While several studies have addressed the beneficial effects of using this bacterial strain on bee health (e. g. Porrini et al., 2010; Audisio et al., 2015), this study aimed to explore an alternative treatment determining the effect of their cell free supernatants.

Preliminary results of CFSs toxicity on bees suggest that no CFS is lethal after 72 h of consumption by bees. So, bees survival was higher than 0.75 in most CFS concentrations. Except for CFS 2 where all concentrations showed the lowest survival values, even the controlgroupsCFS1andCFS3yieldedthehighest survival in the lowermost concentrations. These results suggest that the different CFSs at low concentrations tend to enhance or maintain bee survival as compared to control groups. On the other hand, Ptaszyńska et al. (2016) after administering a prebiotic (inulin) product did not observe any difference in bee survival. However, even though bacteria strain administration was considered as a probiotic, the authors reported low survival of bees after feeding on Lactobacillus rhamnosus and a mix of this probiotic with inulin. Nonetheless, several studies on colony health and beehive performance parameters revealed positive effects with probiotics as L. johnsonii CRL 1647 (Audisio & Benítez-Ahrendts, 2011), B. subtilis subsp. subtilis Mori2 (Sabaté et al., 2012), and prebiotics L. johnsonii CRL 1647 metabolite (Maggi et al., 2013). Soluble proteins and fat bodies were analyzed in order to complement toxicity and survival information in an attempt to find CFSs concentration which significantly affected bee survival. Both parameters are associated with the nutritional status of individual bees (Ament et al., 2011; Nilsen et al., 2010). High nutrition standard is evidenced by the storage of nutrients in trophocytes and oenocytes that constitute the fat body (Nilsen et al., 2010), a major storage site of lipids and proteins (de Oliveira & da Cruz-Landim, 2003) and where such proteins as vitellogenin (Corona et al., 2007; Ament et al., 2011) and antimicrobial peptides (Wilson-Rich et al., 2008) are synthesized. Fifty percent of its dry weight are lipids, which is an indicator of bee health (Arrese & Soulages, 2010). In our study, no CFSs concentration led to an increase in lipid mass on bees’ abdomen, which suggests that the different CFS concentrations would maintain even bee nutritional status. These results vary with respect to those published by Maggi et al. (2013), who reported an increased fat body mass in bees of colonies supplemented with bacterial metabolites.

Two high values of abdominal soluble protein content of were obtained in CFS 1/syrup (40% v/v) and 30% v/v CFS 3/syrup treatments. This is the result of a lactic-acid rich supernatant, which along with short-chain fatty acids and acetic acids was described by Engel et al. (2012) as a bee symbiotic taxa product that could act as a supplement to the honeybee diet. Crotti et al. (2013) proposed that such bee microbiota products as acetic acid or amino acid play a role in regulating bees developmental rate, enhancing body size and improving energy metabolism. The parasitic relationship between Varroa and honey bees has been studied for years, but current studies are now centering on the relationship between mite and honey bee microbiota. Increasing evidence indicates that the degree of Varroa infestation and its chemical control affect the beneficial bacteria composition (Sandionigi et al., 2015; Hubert et al., 2017). In accordance with this matter, we tried to measure the effects of different CFSs produced by bacterias of the bees microbiota against the mite. Mite mortality at contact exposure below ten percent suggests that CFSs at those concentrations were not toxic for bees. All concentrations of CFSs administered to bees performed around 20% of efficiency and showed no significant differences with respect to control groups. However, CFS 1/syrup (40% v/v) showed 56 % of efficiency compared to control groups, which is in agreement with the high protein values obtained in bees. In some studies, where beneficial bacteria were administered to bees inside the hives, a reduction of Varroa incidence was detected. Márquez Gutiérrez et al. (2003) reported high death mite rate in hives after one application of a Bacillus thuringiensis product. Later, other studies described a low incidence of mites in colonies when lactobacilli (Audisio et al., 2015) and a B. subtilis subsp. subtilis Mori2 (Sabaté et al., 2012) product were administered. The metabolic pathway by which CFS 1 causes Varroa mortality through the bee remains unknown. Although our obtained values cannot be compared to other mite control products, it could be promising as a natural alternative considering its positive impact on soluble protein in bees’ abdomen. Our results support the potential use of symbiont-derived bee products to improve bee health. Commensal microbiota enhance immunity improving bee health through mechanisms that remain partially unknown (Crotti et al., 2013). Thus it is of paramount importance to address this lack of information in order to understand bee health (Caccia et al., 2016; Kwong et al., 2017). Consequently, there is a current need for more research in this area in order to explore the molecular mechanisms involved in this symbiotic relationship.