Are commercial probiotics and prebiotics effective in the treatment and prevention of honeybee nosemosis C?
The study was conducted to investigate the effect of Lactobacillus rhamnosus (a commercial probiotic) and inulin (a prebiotic) on the survival rates of honeybees infected and uninfected with Nosema ceranae, the level of phenoloxidase (PO) activity, the course of nosemosis, and the effect on the prevention of nosemosis development in bees. The cells of L. rhamnosus exhibited a high rate of survival in 56.56 % sugar syrup, which was used to feed the honeybees. Surprisingly, honeybees fed with sugar syrup supplemented with a commercial probiotic and a probiotic + prebiotic were more susceptible to N. ceranae infection, and their lifespan was much shorter. The number of microsporidian spores in the honeybees fed for 9 days prior to N. ceranae infection with a sugar syrup supplemented with a commercial probiotic was 25 times higher (970 million spores per one honeybee) than in a control group fed with pure sucrose syrup (38 million spores per one honeybee). PO activity reached its highest level in the hemolymph of this honeybee control group uninfected with N. ceranae. The addition of probiotics or both probiotics and prebiotics to the food of uninfected bees led to the ~2-fold decrease in the PO activity. The infection of honeybees with N. ceranae accompanied an almost 20-fold decrease in the PO level. The inulin supplemented solely at a concentration of 2 μg/mL was the only administrated factor which did not significantly affect honeybees’ survival, the PO activity, or the nosemosis infection level. In conclusion, the supplementation of honeybees’ diet with improperly selected probiotics or both probiotics and prebiotics does not prevent nosemosis development, can de-regulate insect immune systems, and may significantly increase bee mortality.
KeywordsApis mellifera survival Nosema ceranae Phenoloxidase activity Probiotic Prebiotic Lactobacillus rhamnosus, inulin
All members of the Animalia kingdom, including humans, have helpful symbiotic microbiota which are extremely important for the proper functioning of the gastrointestinal tract. These symbiotic microorganisms are responsible for the fermentation of carbohydrates as well as the production of some vitamins and amino acids that their hosts need. Furthermore, gut microbiota, through the “barrier effect,” prevent pathogenic microorganisms from colonizing the gastrointestinal tract. In particular, lactic acid bacteria (LAB) prove to be important inhabitants of animal and human intestinal tracts as they have a multifaceted, antimicrobial potential, mainly because of their ability to synthesize lactic acid, short-chain, volatile fatty-acid, and bacteriocin-like molecules (Jack et al. 1995; Wilson et al. 2005; Audisio et al. 2011). Lactic acid bacteria are usually considered probiotics, i.e., viable microorganisms that provide health benefits to their hosts (Schlundt 2012). Probiotics are helpful in the treatment of several human illnesses, including diarrhea, allergies, obesity, lactose intolerance, inflammation, Helicobacter pylori infections, necrotizing enterocolitis (NEC), eczema, and many others. Successful marketing strategies and the popularization of probiotics have led to these products being commonly used as dietary supplements. Also, prebiotics which are non-digestible fiber compounds cause specific changes, both to the composition and/or activity of gastrointestinal microflora, and confer benefits upon their hosts’ well-being and health (Roberfroid 2007). One such prebiotic is inulin, a linear chain of (2-1)-linked β-d-fructosyl units, which selectively promotes the growth and activity of bacteria from the genus Bifidobacterium that are beneficial for human and animal health (Cummings et al. 2001; Urías-Silvas et al. 2008).
Probiotics and prebiotics are recommended to be added not only to the human diet but also into the forage of different vertebrates as well as invertebrates (e.g., Weese and Arroyo 2003; Patterson and Burkholder 2003; Ötleş 2013; Verlinden et al. 2006; Bagheri et al. 2008; Talpur et al. 2012). Certainly, the most beneficial effect is observed when organisms are provided with probiotics that had been previously isolated from themselves. However, LAB isolated from humans were found to have been used with positive results in the husbandry of terrestrial animals and for agricultural health management; e.g., Lactobacillus rhamnosus and Lactobacillus bulgaricus were indicated to be protective against opportunistic pathogens in fish farming (Nikoskelainen et al. 2001; Ötleş 2013). Also, in beekeeping management, there are commercial diet supplements which contain probiotics and/or prebiotics. One such supplement recommended for the feeding of honeybees and other animals contains bacteria such as Lactobacillus casei, Lactobacillus plantarum, Rhodopseudomonas palustris, and yeast Saccharomyces cerevisiae. A further example, in addition to lactic acid bacteria (Lactobacillus acidophilus or L. casei) and Bifidobacterium lactis, also comprises prebiotics (Pătruică and Mot 2012; Pătruică and Hutu 2013; Andrearczyk et al. 2014).
In honeybee guts and crops, several symbiotic bacteria were reported (Engel et al. 2012; Corby-Harris et al. 2014). They mainly belong to the Lactobacillus and Bifidobacterium genera and to the Acetobacteraceae family. Additionally, two other probiotic bacterial species, i.e., Gilliamella apicola and Snodgrasella alvi, were identified in honeybee alimentary tracts (Engel et al. 2012; Corby-Harris et al. 2014).
Nosema ceranae, the causative agent of nosemosis C, is an obligate, intercellular pathogen which completes its life cycle in honeybee intestines (Wittner and Weiss 1999; Ptaszyńska et al. 2014; Roberts et al. 2015). N. ceranae suppresses immune responses in honeybees (Antúnez et al. 2009; Chaimanee et al. 2012), causing a degeneration of gut epithelial cells (Higes et al. 2007; Dussaubat et al. 2012), a shortening of bee lifespans (Paxton et al. 2007; Higes et al. 2007; Dussaubat et al. 2012), and finally leading to a depletion of honeybee colonies. Insects defend themselves against pathogen infections by cellular immunity and humoral immune responses. These processes such as phagocytosis and encapsulation, in connection with melanization, play an important role in the cellular response. Phenoloxidase (PO) lysozyme and antimicrobial peptides such as abaecin, apidaecin, defensin, and hymeoptaecin are humoral factors essential for the antimicrobial defense of honeybees (Schmid-Hempel 2003; Evans et al. 2006; Cerenius et al. 2008).
Honeybees are very important pollinators which strongly influence the genomic diversity of the plant community; hence, their role in shaping the ecosystem can hardly be overestimated (Bradbear 2009). Currently, there are only a few articles concerning the effect of commercial probiotics and prebiotics on honeybee health. Some data have shown that commercial probiotics increase honeybee mortality, whereas others suggest that the administration of probiotics and prebiotics has an excellent effect on the growth of bee colonies and increases honey production (Pătruică and Mot 2012; Pătruică and Hutu 2013; Andrearczyk et al. 2014). Therefore, we decided to study the effect on honeybee health of L. rhamnosus, which plays a predominant role in the probiotics market (Douillard et al. 2013), and of inulin, a well-known prebiotic, (Slavin 2013), by analyzing PO activity, as well as the role of these supplements on the treatment and the prevention of the nosemosis in honeybees.
Material and methods
Animals, culture conditions and N. ceranae infection
Honeybees, Apis mellifera carnica, were maintained with standard beekeeping management methods in the university apiary (University of Life Sciences in Lublin, Poland). Honeybees were collected between the end of May 2014 and August of the same year. Although no permission is needed to administer experiments on insects, our research was planned in a way that reduced the number of honeybees to the minimum necessary for the proper conduction of these experiments. To obtain 1-day-old healthy honeybees, combs with brood originating from one queen bee were transferred, on the 20th day of bee development, to an air-conditioned chamber and kept at a constant temperature of 35 °C and at a humidity of 60 %. After emerging, honeybees were kept under laboratory conditions, in complete darkness (30 °C; H = 65 %) in wooden cages, occupied by 40 specimens.
In all experiments, honeybees were fed with a daily prepared 56.6 % sugar-water syrup (1:1; w/v) supplemented with commercial probiotics and/or prebiotics. The control honeybees were fed with a pure sugar-water syrup. Doses of the commercial probiotics and prebiotics used in experiments, i.e., 3750 CFU/syrup mL (group L2) and 2 μg/syrup mL (group In), respectively, were estimated on the basis of the manufacturer’s advice concerning a daily dosage of these supplements, taking 160 mg as an average honeybee weight. The average weight of honeybees was established after weighing 50 randomly chosen specimens of those being used in the experiments and was estimated at 157.6 mg.
To induce nosemosis, the honeybees were inoculated with a fresh solution containing 4 million N. ceranae spores/mL, in the amount of 8 μL per honeybee, according to the methodology described by Forsgren and Fries (2010). The spore inoculums were prepared from the ventriculi of naturally infected honeybees directly before experiments (Fries et al. 2013).
Uninfected and N. ceranae-infected honeybees from variants A, B, and C were divided into six feeding groups, i.e., (1) SS; (2) L1; (3) L2; (4) In; (5) L1 + In; and (6) L2 + In. Concentrations of commercial probiotic and/or prebiotic among these groups were as follows: SS (control, pure sucrose syrup), L1 (1250 of Lactobacillus CFU/syrup mL, Biomed-Lublin, Poland), L2 (3750 of Lactobacillus CFU/syrup mL, Biomed-Lublin, Poland), In (inulin 2 μg/syrup mL, Frutafit® IQ, Orafti, Belgium).
In all experiments, dead bees were counted every day, and the volume of eaten sugar syrup was estimated. Additionally, at the end of the experiments, the number of N. ceranae spores was counted and hemolymph PO activity was estimated.
Estimation of the nosemosis level
Samples were prepared from every group in two repeats to count N. ceranae spores. For one sample, ten honeybee abdomens were grounded in 10 mL of sterile, distilled water, and the number of Nosema spores was counted according to Fries et al. (2013) and Hornitzky (2008) using a hemocytometer and Olympus BX61 light microscope. Furthermore, each sample was observed under bright field and differential interference contrast (DIC) to a proper differentiation of N. ceranae spores from other remains present in honeybee homogenates.
Isolation of total DNA from honeybees and molecular detection of N. ceranae
Total DNA from uninfected and N. ceranae-infected A. mellifera carnica was isolated using the DNeasy Blood and Tissue Kit (Qiagen) according to the manufacturer’s instruction. To identify N. ceranae, DNA in the investigated samples using duplex PCR was conducted with 321-APIS and 218-MITOC primers (Martín-Hernández et al. 2007) in a 25-μL reaction mixture of the Qiagen Taq PCR Core Kit (Qiagen Inc.) containing 2.5 μL PCR buffer with 5 μL Q solution, 0.1 mM dNTP mixture, 0.7 U Taq DNA polymerase, 0.2 μM of each forward and reverse primers, approximately 0.15 μg of DNA template, and ddH2O to a final reaction volume of 25 μL. For DNA amplification, the following PCR cycling conditions were used: 1 min at 94 °C, 1 min at 61.8 °C, and 1 min at 72 °C, repeated for 30 cycles, and 10 min at 72 °C.
The survival of L. rhamnosus (a commercial probiotic) in sugar syrup
The bacteria of the genus Lactobacillus used as the commercial probiotic were added to the number of 1250 and 3750 bacterial cells to 1 mL of 56.6 % sugar syrup. Resulting bacterial suspensions were left at 30 °C and at a humidity of 60 % to check the bacteria survival during their administration to the honeybees. After 1 min, and subsequently after 2, 3, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 38, 40, 48, and 96 h, the titer of the bacteria was determined by plating them on an MRS agar medium and incubating them for 24–48 h, at 37 °C, in anaerobic conditions. Ten colonies were then randomly selected to verify the taxonomic position of the cultured bacteria, on the basis of API® CH50 strips (bioMérieux Clinical Diagnostics).
Honeybee hemolymph collection
Hemolymph from ten individuals was collected in each experimental group in sterile-chilled Eppendorf tubes. The hemolymph was used to measure PO activity after the removal of hemocytes (Phenoloxidase (PO) activity assay section). For this purpose, first, the hemolymph was centrifuged at 4 °C at 200×g for 5 min, and next, the supernatant was centrifuged at 20,000×g for 15 min. After centrifugation, pooled supernatants were stored at −20 °C until used for PO activity measurement.
PO activity assay
PO activity was determined in pooled hemolymph samples, according to a modified method, previously described by Park et al. 2005; Zdybicka-Barabas and Cytryńska 2010; Andrejko et al. 2014; Zdybicka-Barabas et al. 2014. Two microliters of the hemolymph, twice diluted in tris-buffered saline (TBS) (50 mM Tris–HCl pH 6.8, 1 mM NaCl), was combined with 18 μL of TBS, containing 5 mM CaCl2 in the wells of a 96-well plate (to a final sample volume of 20 μL). After 20 min of incubation at room temperature, 180 μL of 2 mM L-dihydroxyphenylalanine (L-DOPA) in 50 mM sodium phosphate, pH 6.5, was added. PO activity was determined spectrophotometrically, on the basis of the amount of melanin formed (absorbance at 490 nm) over 60 min, at 2-min intervals, using a microtiter plate reader (Bio-Rad Laboratories, Hercules, CA, USA). The PO activity was determined in three independent experiments, in triplicate, for each hemolymph sample.
The SAS software (2002–2003) employing the ANOVA (a group and a variant effects were the experimental factors) and the Tukey’s honestly significant difference (HSD) test (SAS Institute 2002–2003) were used to prepare statistical analysis of the data obtained.
Results and discussion
The survival of honeybees depends on their successful defense against different microbial parasites. Indigenous gut bacterial flora with the dominant role of lactic acid bacteria plays an important role in the protection of bees and other insects against colonization by pathogens and in the control of the growth of undesirable microorganisms (Jack et al. 1995; Wilson et al. 2005; Audisio et al. 2011).
The research was conducted to investigate the effect of L. rhamnosus, an important commercial probiotic, and of inulin, a widely known prebiotic, on the survival rate of honeybees, infected and uninfected with N. ceranae, to investigate the level of PO activity in the hemolymph of insects, and, furthermore, to analyze the role of the commonly used probiotics and prebiotics in the protection of bees against nosemosis C (Fig. 1).
Supplementation of honeybee diet with the probiotic and the probiotic + prebiotic (feeding groups: L1, L2, L1 + In, L2 + In) for 9 days before Nosema infection (Fig. 6) had the largest impact on honeybee mortality. Inulin present in food together with L. rhamnosus promoted the mortality of honeybees associated with the probiotic. However, this prebiotic alone had no visible effect on honeybee death rate (Figs. 5 and 6). An especially high increase in bee mortality was found between the second and the fourth day after microsporidian infection (11th–13th days of the experiment) and reached up to seven specimens per cage (Fig. 6). Over the next few days until the end of the experiment, honeybee mortality was established as being at a constant level, i.e., 2.02 (±0.67) specimens per day per cage (Fig. 6). Generally, we conclude that feeding honeybees with commercial probiotics and probiotic + prebiotic not only does not prevent nosemosis development in bees but may even increase insect vulnerability to infection with N. ceranae.
Generally, the infection of honeybees with N. ceranae significantly reduced the level of PO activity in the hemolymph. Still, the lowest PO activity was noted when bees were fed for 9 days before infection with a sugar syrup supplemented with L. rhamnosus or L. rhamnosus together with inulin and reached ~0.14 and ~0.11, respectively (Fig. 7, variants C, groups: L1, L2 and L1 + In, L2 + In). These results clearly indicated the strong inhibition of the honeybees’ PO, not only by microsporidian infection, but also by feeding honeybees with the commercial probiotic and with probiotic in combination with prebiotic. This data suggests that the supplementation of honeybee diets with probiotic or both probiotic and prebiotic is not beneficial for the functioning of honeybee defense systems (Fig. 7).
We supposed that colonization of honeybees’ intestinal tracts by probiotic microorganisms ought to have inhibited the development of nosemosis, through the competition for binding sites and nutrients, as well as by positive modulation of the immune system. Surprisingly, honeybees fed with a sugar syrup supplemented with commercial probiotic (L. rhamnosus) were more susceptible to N. ceranae infection and nosemosis development, and the number of microsporidian spores in such bees was very high (Fig. 8). It is possible that lactic acid, produced by multiplying L. rhamnosus, could have increased acidity in the bee intestine and/or could have been the cause of degeneration of the gut, and through this, could have initiated favorable conditions for the germination of microsporidian spores and accelerated the infection of epithelial cells with N. ceranae (de Graaf et al. 1993; Bradley 2008; Feigenbaum and Naug 2010; Ptaszyńska et al. 2013). Consequently, mortality rates increased among honeybees fed with commercial probiotic containing L. rhamnosus. Therefore, preparations containing bacteria identified as probiotics for mammals should not be considered as probiotics for honeybees and possibly for other invertebrates.
Microorganisms selected as commercial probiotics are highly resistant and have a great ability to survive, even in unsuitable environments. Therefore, they can easily proliferate in honeybee intestines and, hence, may exclude natural symbiotic microorganisms. The elimination of honeybees’ natural microbiota can reduce the absorption of nutrients and can lead to the malnutrition of bees. Furthermore, the intensive development of microorganisms, which are non-natural for honeybees, can lead to the degeneration of the peritrophic membranes of the bee intestines which, together with exoskeleton cuticule, are the first lines of insects’ defense against various pathogens. That can be the reason of the increase in the mortality of foragers, as observed in our experiments. In earlier studies (Vásquez and Olofsson 2009; Martinson et al. 2011; Tajabadi et al. 2011, 2013a, b; Pattabhiramaiah et al. 2012; Vásquez et al. 2012; Audisio et al. 2015), different bacterial strains of the genus Lactobacillus were isolated from honeybee intestines, meaning these lactobacilli can probably be considered as probiotics, for these ecologically and economically crucial insects.
The supplementation of honeybee diet with improper probiotics or probiotics and prebiotics can disturb the natural microbiota composition, which is important in maintaining metabolic homeostasis in bee intestines. Furthermore, it can deregulate the immune system and, in consequence, may promote pathogen infections and increase honeybee mortality.
This research was supported by the Individual Research Grant of Vice-rector for Research and International Relations of UMCS (Lublin, Poland) and with the use of equipment purchased thanks to the financial support of 374 the European Regional Development Fund under the project: “Increase in R & D potential of 375 Departments of Chemistry, and Biology and Earth Sciences UMCS in Lublin” (POPW 376 .01.03.00-06-017/09-00).
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