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Praca oryginalna Original paper
Intestinal microflora is a very important part of the digestive system in all animals. It profoundly affects the anatomical, physiological and immunological de-velopment of the host, and through bacterial and fungal antagonism, inhibits intestinal colonization by exoge- nous pathogens. Furthermore, intestinal microflora benefits to the host is the supply of energy from the fermentation of undigested carbohydrates and the sub-sequent absorption of short chain fatty acids. Intestinal bacteria also play a role in synthesizing vitamins, e.g. vitamine B, which is essential to a proper development of animals, including bees and in metabolizing many toxic chemical compounds, e.g. xenobiotics.
The indigenous microflora of the gastrointestinal tract is generally stood in terms of species specific to a given insect and is largely independent of its habi-tat and diet (23). The intestinal flora of healthy bees is dominated by Gram-negative bacteria, mainly from the Enterobacteriaceae family (Citrobacter, Erwinia,
Enterobacter, Escherichia coli, Flavobacterium, Klebsiella, Proteus), as well as from Alcaligenaceae (Achromobacter) and Pseudomonadaceae (Pseudo- monas), which represent more than 70% of all micro-organisms. About 27% of intestinal microflora consists of Gram-positive bacteria, primarily from the genus Bacillus and 1% are yeasts and other fungi (3, 11, 12, 22).
Yeasts are prevalent organisms in every environ-ment in which bees conduct their life cycle and can be isolated, for example, from honey and nectar. Honey microflora is composed of Gram-positive bacteria and yeasts, such as Saccharomyces rouxi, S. mellis, S. bisporus, S. roesi, S. bailli, S. heterogenicus, Pichia (Hansenula) anomala. The pollen reserves flora, which is dominated by bacteria from the genera Pseudomonas and Lactobacillus and fungi from the genera Saccharomyces, Candida and Cryptococcus is far outnumbered than microflora of the honey (4, 7, 24).
Impact of nosemosis on the intestinal yeast flora
of honey bees
GRZEGORZ BORSUK#, ANETA A. PTASZYŃSKA*#,
KRZYSZTOF OLSZEWSKI, JERZY PALEOLOG
Department of Biological Basis of Animal Production, Faculty of Biology and Animal Breeding, University of Life Sciences, Akademicka 13, 20-950 Lublin, Poland
*Department of Botany and Mycology, Institute of Biology and Biochemistry, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Akademicka 19, 20-033 Lublin, Poland
#These authors equally contributed to this work.
Borsuk G., Ptaszyńska A. A., Olszewski K., Paleolog J. Impact of nosemosis on the intestinal yeast flora of honey bees
Summary
Intestinal microflora is a very important part of the digestive system in every animal, and plays a role in the synthesis of vitamins and the metabolism of many toxic chemical compounds. The indigenous intestinal flora of bees changes even as a result of changing their diet from natural to artificial or placing them in cages. Such factors have an impact on the health of bees and on the strength of whole colonies.
In our study, intestinal fungi isolated from healthy bees and from bees infected with Nosema spp. belonged to two genera: Candida and Saccharomyces. The approximate numbers of yeast CFUs (colony forming units) obtained from healthy Apis mellifera carnica and Buckfast bees were, respectively, 2880-5180 and 1056-4120. Apis mellifera carnica and Buckfast bees were similarly sensitive to slight Nosema spp. infections, but heavy infestations had a greater impact on the intestinal microflora of A. m. carnica.
The degree of Nosema spp. infestation had an impact on the quantitative composition of the intestinal microflora of bees. Slightly infected bees of Apis mellifera carnica had up to 44 915 yeast CFUs per bee, and Buckfast bees up to 28 705 yeast CFUs per bee. Surprisingly, a heavy infestation reduced the number of yeast CFUs to no more than 120 in A. m. carnica bees and to no more than 164 in Buckfast bees. Therefore, in studies in which the number of yeast CFUs is used as the main indicator of stress in bees, the potential presence and the degree of Nosema spp. infestation needs to be taken into account.
Med. Weter. 2013, 69 (12) 727 Surprisingly, as mentioned above, the gut flora of
healthy, free-flying bees contains only few yeasts if any. Even the placing of bees in cages changes the ratio of yeasts present in the gut microflora. Also diseases, malnutrition, antibiotics and insecticides cause an in-crease in the number of yeasts. Therefore, an inin-creased number of yeast colonies isolated from bees’ guts may be considered as a stress indicator (6, 9, 10, 14).
Therefore, it was interesting to investigate how yeasts belonging to intestinal microbiota respond to nosemosis. Nosema spp. complete their life cycle in the intestines of bees and cause many changes in their organism due to malnutrition. Nosema spp. belong to fungi (Microsporidia) and in fungal communities strong antagonisms are observed among species com-peting for the same substrate. The present study aimed to examine the relationships between fungi present in bees’ intestines under nosemosis infection.
Material and methods
Bees were collected from an apiary in the Lublin region during two years (2011, 2012). Each year, three independent cage experiments were conducted, during June (experi-ment 1) and July (experi(experi-ments 2) on Apis mellifera carnica and Buckfast bees. Each experiment was conducted during 12 days.
Combs with brood in the 20th day of a development were
put in an air-conditioned chamber and kept at a constant temperature of 35°C and a relative humidity of 60%. Bees emerged on the 21st day. Healthy bees of the same age
were placed in wooden cages (12 × 12 × 4 cm) with glass front screens, as well as ventilating and feeding slots. The following experimental groups were formed from two- -day-old bees:
1. Control – consisting of 6 cages with 40 A. m. carnica per cage and 6 cages with 40 Buckfast per cage. The bees were “inoculated” with sterile water.
2. Bees slightly infected with Nosema spp. – bees were in-oculated with suspension of approximately 50 000 Nosema spp. spores. On the 10th day of the experiment the Nosema
spp. spores were counted for bees collected from every cage, and cages settled by slightly infected bees, with the spore number of up to 10 million spores per bee, were selected for this group. Ultimately, this group consisted of 6 cages with A. m. carnica and 6 cages with Buckfast bees.
3. Bees heavily infected with Nosema spp. – bees were inoculated with suspension of approximately 1 000 000
Nosema spp. spores. On the 10th day of the experiment the Nosema spp. spores were counted for bees collected from
every cage, and cages settled by heavily infected bees, with the spore number of over 40 million spores per bee, were selected to this group. Ultimately, this group consisted of 6 cages with A. m. carnica and 6 cages with Buckfast bees.
Spore suspensions were obtained by crushing bees infected with Nosema spp. in sterile water, filtering the suspension through sterile meshes and centrifuging it for 3 minutes at 2000 × g. The control group was fed once with 10 µl of sterile water. Bees from the second and third groups were feed once individually with 10 µl of fresh Nosema spp.
spores inoculum on the first day of each experiment. After inoculation, bees were fed with 1:1 sugar-water syrup.
After the groups had been formed, the cages were trans-ferred to an air-conditioned chamber. The chamber was kept at a constant temperature of 26°C and a relative humidity of 65%.
Then, for every group, the numbers of Nosema spp. spores were estimated by grounding 20 whole bees in 20 ml of sterile distilled water and preparing microscope samples for examination (19). Three independent samples (per 20 bees) for each experimental group were prepared. The es-timation of the number of Nosema spp. spores per bee was accomplished by using Olympus BX 61 light microscopy and a haemocytometer (2, 16). For each spore suspension, the averages of two estimates of intensity were used.
To estimate the number of yeasts, intestinal tracts of healthy bees and those of bees infected with Nosema spp. were aseptically removed. From each group three pooled samples of 10 intestinal tracts were ground in sterile 0.85% NaCl solution (13). The intestinal tracts were weighed and an appropriate amount of NaCl solution was added to estimate the 1:10 ratio of the suspension. Then, 50 µl of such suspension was spread in duplicate on Petri dishes containing Sabouraud dextrose agar with chloramphenicol and gentamycin, and incubated for 5 days at 30°C. After that time, the number of colony forming units (CFU) per one bee was counted. The addition of antibiotics inhibited the growth of bacteria and improved the cultivation of fungi and yeasts. For every Petri dish, the number of CFUs per one bee was counted.
The API® strips-Yeasts (bio Mérieux Clinical
Diagnos-tics) were used to differentiate fungi isolated from bees’ intestinal tracts.
The results were analysed statistically with the SAS software by using ANOVA (a group effect was the experi-mental factor) and the HSD (honestly significant difference) test (20).
Results
During the first year of the experiment, the degree of Nosema spp. infestation of bees was obtained on lower range than during the second year. Differences between the results of experiments conducted in dif-ferent months (June and July) of each year were not statistically significant. Similarly, the results for CFUs were different for each year. Therefore, the results ob-tained during 2011 and 2012 are presented separately (Tab. 1).
Intestinal fungi isolated from healthy bees and from bees infected with Nosema spp. belonged to two gen-era: Candida and Saccharomyces. The approximate numbers of yeast CFUs obtained from healthy Apis mellifera carnica and Buckfast bees were, respectively, 2880-5180 and 1056-4120 (Tab. 1).
A weak degree of Nosema spp. infestation, in a range of up to 10 million spores per bee, caused an increase in the number of yeast CFUs isolated. Surprisingly, when bees where Nosema spp. infected in the highest obtained degree, in a range of up to 85 million spores
Med. Weter. 2013, 69 (12) 728
per bee (Tab. 1), it resulted in a significant decrease in the number of yeast CFUs.
Apis mellifera carnica slightly infected with Nosema spp. had higher CFU numbers than Buckfast, but after a heavy infection the results were reversed: the number of CFUs isolated from Buckfast was higher.
Discussion
Results obtained in this study for healthy bees were similar to data presented in earlier publications (7, 8). In the spring, approximately 2500 CFUs per bee were isolated from free-flying colonies, and 3500 CFU per bee from caged bees. In the fall, no intestinal yeasts were isolated from free-flying colonies and an average number of 44 CFUs per bee was obtained from caged bees.
Bees kept in cages, without a bee queen or the possibility of flying, and fed artificial diet are under stress conditions. Consequently, after 12 days of the experiment, yeasts were prevalent in the bees’ intes-tinal microflora. Nosemosis increased the stress on bees because, like all parasites, Nosema spp. depend on their hosts for energy. Furthermore, bees expend energy for mounting an immunological response to infection, and it is an energetically expensive process (21). Accordingly, the number of yeast CFUs isolated from bees’ intestines should be higher for bees infected with Nosema spp. than for healthy bees. This was true for a slight Nosema spp. infection, but when the level of infection increased, the number of CFUs decreased. The development of nosemosis probably inhibited the colonization of the bees’ intestinal tracts by yeasts through fungal antagonism and through impeding the access of yeasts to nutrients by degenerative changes in the gut. Therefore, in studies in which the number of yeast CFUs is used as the main indicator of stress in bees, the potential presence and the degree of Nosema spp. infestation needs to be taken into account.
Apis mellifera carnica and Buckfast bees were sim-ilarly sensitive to a slight Nosema spp. infection, but a heavy infestation probably had a greater impact on A. m. carnica. Studies conducted on Buckfast bees (5,
15, 17, 18) demonstrated that they are more resistant to nosemosis than other bee breeds. In our study, the number of yeast CFUs was high for slightly infected A. m. carnica and for heavily infected Buckfast bees. This may indicate that a strong infestation did not have as much impact on Buckfast bees as it did on A. m. carnica because nutrients left in the intestines of Buckfast bees were sufficient for the development of yeasts.
Proper development of bees is favoured by adequate nutrition (1). The indigenous intestinal flora of bees changes even as a result of changing their diet from natural to artificial or placing them in cages. Such factors have an impact on the health of bees and on the strength of whole colonies. In the complex microbial community of bees, saprophytic yeasts probably play a role in the regulation of homeostasis. The elucidation of the mechanisms that occur in the intestinal flora during Nosema spp. infection is therefore important for understanding the course of the disease and for the selection of potential drugs. Possibly crucial in treating nosemosis is to strengthen the natural intestinal flora of bees. The role of intestinal yeasts for the host and the interplay between yeasts and other members of the intestinal flora of bees should be further studied.
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Tab. 1. Average numbers of Nosema spp. and yeast CFUs (colony forming units) obtained during the two-year study
Bees Group
Number of Nosema spp. spores
per one bee (mln) Number of yeast CFUs per one bee Year
2011 2012 2011 2012
Apis mellifera carnica
Control – uninfected bees 0a 0a 2 880b 5 180b
Bees slightly infected with Nosema spp. 2.65b 4.54b 26 030c 44 915c
Bees heavily infected with Nosema spp. 53.06c 85.38c 640a 120a
Buckfast
Control – uninfected bees 0a 0a 1 056b 4 120b
Bees slightly infected with Nosema spp. 1.15b 6.40b 15 770c 28 705c
Bees heavily infected with Nosema spp. 40.01c 71.81c 960a 164a Explanations: a, b, c – differences in groups are significant at p ≤ 0.05 (ANOVA; Tukey’s test, F = 14,45)
Med. Weter. 2013, 69 (12) 729
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Corresponding author: Aneta A. Ptaszyńska PhD, Akademicka 19, 20-033 Lublin; e-mail: aneta.ptaszynska@poczta.umcs.lublin.pl
