Journal of Agricultural Science and Technology A 1 (2011) 818-825 Earlier title: Journal of Agricultural Science and Technology, ISSN 1939-1250

Stingless Bee Propolis Effects on Experimental Infection of Apis florea with Nosema ceranae G. Suwannapong1, S. Maksong2 and M. E. Benbow3 1. Department of Biology, Faculty of Science, Burapha University, Chon Buri 20131, Thailand 2. Biological Science Program, Faculty of Science, Burapha University, Chon Buri 20131, Thailand 3. Department of Biology, University of Dayton, College Park, Ohio 45469-2320, USA Received: January 25, 2011 / Published: October 20, 2011. Abstract: Propolis collected by stingless bees from various types of plants has been used as an antimicrobial agent in several previous studies. We assessed the effect of propolis produced by a stingless bee, Trigona apicalis, on Apis florea experimentally infected with Nosema ceranae, a parasite of honeybees. For parasite inoculation each Nosema free-bee was fed 2 µL of 50% (w/v) sucrose solution containing N. ceranae spores at 40,000 spores/bee and 0 as a negative control (CO). Treated bees were provided with 0%, 10%, 20% and 50% propolis (w/v) in water, defined as 0P, 10P, 20P and 50P, respectively. We assessed the effects of propolis 14 days post inoculation. All propolis-treated bees had significantly higher survival than untreated bees. However, survival of Nosema-inoculated bees was lower than that of control bees. Bees treated with the highest propolis concentration (50P) had the highest survival ratio. No control bees became infected over the course of the study. However, N. ceranae infection rates of bees treated with 0P, 10P, 20P and 50P were 75 ± 1.4%, 72 ± 5.6%, 69 ± 4.2% and 47 ± 1.4%, respectively. In addition, propolis-treated bees had hypopharyngeal gland protein content that was significantly higher than 0P and CO bees. Overall, propolis treatment significantly reduced N. ceranae infection rate and bee mortality and was associated with increased hypopharyngeal gland protein concentration. Key words: Apis florea, hypopharyngeal glands, Nosema ceranae, ventricular cells.

1. Introduction Nosema infection is one of the most economically damaging honeybee diseases. It infects ventricular cells of adults after spores are ingested, especially through trophallaxis [1, 2]. N. apis infecting Apis mellifera has been reported worldwide and was initially described more than one hundred years ago by Zander [3, 4]. The microsporidian parasite may also infect the Asiatic honeybee, A. cerana [5, 6]. Recently, this parasite species also was found in cultivated A. mellifera colonies [7, 8]. It has become more widely distributed in the past decade by cross

Corresponding author: G. Suwannapong, assistant professor, Ph.D., research field: honeybee health and communication. E-mail: [email protected]; [email protected].

infection from A. cerana to European honeybees [9]. N. ceranae can negatively affect honeybee production capacity. Although infected bees do not exhibit obvious external disease symptoms, infection by N. ceranae causes digestive disorders, shortened life spans, lower colony population sizes [10] and reduction of honey production and crop products that rely on bees for pollination [11, 12]. Recently, Bromeshenk et al. (2010) demonstrated a significant association between honey bee colony collapse disorder and double infection with N. ceranae and a virus. Thus, Nosema infection is correlated with significant declines in the honey bee population. Propolis is a resinous hive product collected from various plant species by honeybee workers. Its chemical composition includes flavonoids, aromatic

Stingless Bee Propolis Effects on Experimental Infection of Apis florea with Nosema ceranae

acids, esters, aldehydes, ketones, fatty acids, terpenes, steroids, amino acids, polysaccharides, hydrocarbons, alcohols, hydroxybenzene, and several other compounds [13-15]; the composition varies according to the plant species in a specific geographic region. The flavonoids (mainly pinocembrin) are considered to be responsible for inhibitory effects on bacteria and fungus, but only traces of these compounds have been found in propolis of South American origin [16, 17]. More specifically, sinapic, isoferulic, and caffeic acids inhibited the growth of Staphylococcus aureus [17]. The ethanol extracts from propolis had a “marked synergistic effect” on the anti-Staphylococcus activity of two antibiotics, streptomycin and cloxacillin [18]. Further, two types of propolis (green propolis and Scaptotrigona sp. propolis) were efficient against Escherichia coli [18] and propolis of Melipona quadrifasciata was better than green propolis and Scaptotrigona propolis in tests of efficacy against Pseudomonas aeruginosa [18]. Although there have been reports of N. ceranae infecting two honeybee species of Thailand, A. florea and A. cerana [19-21], no published reports examine the efficacy of stingless bee propolis against N. ceranae in honeybees. We therefore tested the efficacy of a stingless bee propolis obtained from Trigona apicalis nests as an anti-N. ceranae treatment in the honey bee, A. florea. In our experiment, A. florea workers were infected with N. ceranae obtained from heavily infected A. florea workers. Survival, percentage of infected cells, and hypopharyngeal gland protein concentration were measured for honeybees treated with several concentrations of the propolis extract and control bees that received no propolis.

2. Materials and Methods 2.1 Spore Preparation Nosema ceranae spores were isolated from heavily infected A. florea workers from a colony near Samut Songkram Province, Thailand. Honeybee midguts

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were removed and transferred to 1.5 mL microcentrifuge tubes containing 200 µL sterile distilled water, and then homogenized and centrifuged at 6,000 g for 10 min. Supernatant was discarded. To determine each infection dosage, the pellet was diluted and spores were counted with a hemocytometer under a light microscope. 2.2 Propolis Extraction Propolis extract was obtained from propolis structures taken from three colonies of Trigona apicalis in an apiary located in Chanta Buri Province, Thailand. The propolis was dried in a hot air oven at 80 °C for 72 h and then extracted by 70% (w/v) ethanol; 60 g propolis in 100 mL 70% ethanol, yielding a crude ethanol extract that we define (for the purpose of our experiment) as a 100% propolis. Three concentrations of 0%, 10%, 20% and 50% propolis in distilled water (v/v) were prepared for the experiment. 2.3 Effect of Propolis on Experimental Inoculation: Infection Rate Frames of sealed brood were obtained from three Nosema-free colonies of A. florea located at Burapha University, Chon Buri Province, Thailand. They were kept in an incubator at 34 ± 2 °C with relative humidity between 50%-55% to provide newly emerged Nosema-free honeybee workers for the experiments. The emergent bees were carefully removed, confined to cages in groups of 50, and kept in the incubator for two days. Two days after eclosion, the bees were fed with 2 µL of 50% sucrose solution (w/w) in water containing N. ceranae spores at dosages of 40,000 spores/bee for five treatment groups (Table 1). Three replicated cages of 50 bees each were fitted with 2 gravity feeders, one with water and the other with sugar syrup (50% w/w sucrose solution) that was replenished during the experiment. Each cage was checked daily to remove and count any dead bees.

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Stingless Bee Propolis Effects on Experimental Infection of Apis florea with Nosema ceranae

Table 1 Propolis treated and untreated Apis florea workers infected with N. ceranae spores originally isolated from naturally infected A. florea workers. Bee group CO 0P 10P 20P 50P

Nosema treatment (spore/bee) 0 40,000 40,000 40,000 40,000

Propolis treatment (% v/v) 0 0 10 20 50

Food provided Pollen mix (17 g pollen mixed with 50% sucrose solution (w/w) in 35% ethanol) Pollen mix (17 g pollen mixed with 50% sucrose solution (w/w) in 35% ethanol) Pollen mix (17 g pollen mixed with 50% sucrose solution (w/w) in 10% propolis) Pollen mix (17 g pollen mixed with 50% sucrose solution (w/w) in 20% propolis) Pollen mix (17 g pollen mixed with 50% sucrose solution (w/w) in 50% propolis)

2.4 Infection Ratio or Parasitic Ratio 2.4.1 Light Microscopy Ventriculi of dead bees were individually checked to verify the presence of N. ceranae spores. Three bees from each cage were randomly collected at 14 days post inoculation (p.i.), and their ventriculi were processed for microscopic examination. The midgut was removed and fixed with Bouin’s fluid solution for 24 h, washed three times in 70% ethanol or until the solution became colorless, dehydrated with an alcohol series and embedded in melted paraplast. 6 µm sections were cut with a rotary microtome (Leica, Germany), stained with PAS and counterstained with light green, and examined under light microscope. The infection ratio was calculated as the proportion of infected cells to non-infected cells of representative tissue areas over one hundred counted cells. Bees from the control group were sacrificed and analyzed to confirm the absence of spores in ventriculi using the same methods. 2.4.2 Transmission Electron Microscopy For examination of ultrastructural change, midguts were placed in insect saline (NaCl 75 g/L, Na2HPO4 2.38 g/L, KH2PO4 2.72 g/L) and prefixed with modified Karnovsky fixative (4% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2) for one hour at 4 °C. Tissues were post-fixed in 2% osmium tetroxide in cacodylate buffer for one hour at room temperature. They were then washed three times in the same buffer for 10 min each. Specimens were contrasted for 12 h in 1% phospotungstic acid dissolved in 50% ethanol, dehydrated in a standard alcohol series (50%-100%), cleared in propylene oxide, and embedded in Epon

812-Aradite 502 resins. Tissue sections of 500 μm and 90 nm were cut using an LKB ultramicrotome. The sections were contrasted with 10% uranyl acetate and lead citrate dissolved in 50% ethanol for 15 min and examined under the TEM (JEOL CX 200). 2.5 Survival Analysis Daily for 30 days, the number of dead bees per cage was recorded and bodies removed. To evaluate mortality among the treatments, Kaplan-Meier survival curves were generated by plotting the number of surviving bees against days from the initiation of the experiment [22]. 2.6 Hypopharyngeal Gland Protein Contents Three bees were removed from each cage on days 6, 10, and 14 p.i. and stored frozen until analyzed. Bees were then thawed, decapitated and the hypopharyngeal glands removed. The dissected glands were stored in 50 µL phosphate buffer (pH. 7.8) in 1.5 mL microcentrifuge tubes, homogenized and then centrifuged at 1,000 rpm for 2 min. Supernatant from each tube was used for analysis by Bradford protein assay [23]. Standard curves were prepared using bovine serum albumin (BSA). Protein absorbance was measured at 595 nm against a blank reagent using a Shimadzu uv-visible spectrophotometer (UV-1610). Concentrations of protein (BSA) were plotted against the corresponding absorbance values to generate a linear regression standard curve which was used to predict

protein

concentration

(mg/bee)

from

absorbance. Data were analyzed using one-way ANOVA and Duncan’s Multiple Range Post-tests.

Stingless Bee Propolis Effects on Experimental Infection of Apis florea with Nosema ceranae

3. Results 3.1 Effect of Propolis on Infection Rates (Infectivity) Control bees (CO) contained no N. ceranae spores. However, all infected bees contained spores, to different degrees. The infection rates of bees in 0P, 10P, 20P and 50P were 75 ± 1.4%, 72 ± 5.6%, 69 ± 4.2%, 47 ± 1.4% and 49 ± 4.2% respectively. Thus, although 0P treated bees were treated with ethanol (the solvent used to prepare the propolis solution), ethanol did not inhibit Nosema growth. There was no significant difference between the infection ratio between the positive control and bees treated with 10% propolis were not statistically different. However, the infectivity of these groups were significantly lower from those of bees treated with 20P and 50P (F4,5 = 24.69, P < 0.0017). The lowest infection ratio was found in bees treated with 20% and 50% propolis which were not significantly different from each other (Fig. 1). 3.2 Infection Ratio After 14 d p.i. the highest mean infection ratio (89 ± 8.5% cells) was found in positive control bees that were not treated with propolis (Fig. 2). The mean infection ratio for inoculated bees treated with 50P was 28.5 ± 3.5% cells, while that for 20P bees was 37.5 ± 2.1% cells. There were significant differences

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between 0P, 10P and 50P (F3,4 = 47.70, P < 0.0014), but 20P bees were not statistically different from 10P or 50P (Fig. 2). 3.3 Light Microscopy and Transmission Electron Microscopy Nosema spores were detected in the ventricular cells of untreated infected bees (0P) while control bees (CO) were negative throughout the study (Fig. 3a). Each epithelial cell of positive control bees (0P) showed a high number of mature spores distributed throughout the cell cytoplasm, but particularly at the apical part of the cell (Fig. 3b). Mature spores also were clearly observed at the bottom of the cells near the basement membrane, in addition to notable degeneration of epithelial cells (Fig. 3c). The ventricular cells of bees treated with 10P and 20P had a lower infection ratio (Fig. 2) compared to that of 0P bees (Fig. 3d). In addition, electron micrographs of longitudinal sections of N. ceranae spores appeared abnormal in propolis treated bees (10P, 20P and 50P); the spores were irregular in shaped particularly where the anchoring disk became narrow (Fig. 3e). This was different compared to spores found in the cell cytoplasm of untreated bees (0P). The disorganized cellular organelles also were found in the ventricular cell cytoplasm of propolis treated bees (Fig. 3f).

% Infection rate (mean ± SD)

90 80

a

a

70 60

b b

50 40 30 20 10 0 0P

10P

Doses

20P

50P

Fig. 1 Mean (SD) N. ceranae infection rate of bees inoculated with 40,000 spores/bee treated with propolis at dosages of 0% (0P), 10% (10P), 20% (20P), 50% (50P). Vertical bars with different letters represent significant differences among groups (ANOVA- Duncan’s multiple range test, F4,5 = 24.69; P < 0.0017).

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Stingless Bee Propolis Effects on Experimental Infection of Apis florea with Nosema ceranae

% Infection ratio (mean ± SD)

100

a

90 80 70 60

b

50

bc

40

c

30 20 10 0 0P

10P

Doses

20P

50P

Fig. 2 Mean (SD) of N. ceranae spore infection ratio at 14 d p.i. of positive control bees (OP) and bees treated with 10% (10P), 20% (20P) and 50% (50P) propolis (v/v) in water. Vertical bars with different letters represent significant difference among groups (ANOVA- Duncan’s multiple range test, F3,4 = 47.70, P < 0.0014).

Fig. 3 (a) A. florea worker ventricular cells of a control bee (CO) (200 ×). (b) Ventricular cells of a bee inoculated with 40,000 spores per bee (0P) (100 ×). The swollen and broken apical parts of epithelial cell of the midgut were observed in cells infected with N. ceranae spores. (c) Cross section of ventricular cells of a bee treated with 10% propolis on day 14 p.i (toluidine blue, 100 ×). (d) Electron micrograph of a longitudinal section of a N. ceranae spore inside the ventricular cell of a 20P bee on day 14 p.i. (e) Electron micrograph of a longitudinal section of a N. ceranae spore inside the ventricular cell of a 20P bee on day 14 p.i showing abnormal shape at the anchoring end. It was surrounded by clear zone. (f) Electron micrograph of a longitudinal section of a N. ceranae spore inside the ventricular cell of a 50P bee. Abbreviations: bm, basement membrane; cz, clear zone; ex, exospores; en, endospore; ep, empty spore; lu, lumen of the mid gut; mv, microvilli; n, nucleus of ventricular cell; pf, polar filament; RER, rough endoplasmic reticulum; sp, spore.

Stingless Bee Propolis Effects on Experimental Infection of Apis florea with Nosema ceranae

3.4 Honeybee Survival Rate All propolis-treated bees had significantly higher survival than the untreated bees. There was no significant difference in survival among bees treated with the different concentrations of propolis. Kaplan-Meier curves showed that untreated bees infected with N. ceranae spores had the lowest survival (highest mortality rate), followed by bees treated with 0%, 10%, 20% and 50% (v/v) propolis, respectively. Bees treated with 50% (v/v) propolis had the highest survival, with significantly more bees alive on day 30 p.i. compared to the other treatments (Fig. 4).

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the protein content of bees without propolis treatment (0P) and uninfected bees (CO, Fig. 5).

4. Discussion This is the first report on the effect of propolis from the stingless bee, Trigona apicalis, on the experimental infection of the red dwarf honeybee, A. florea, with the microsporidium N. ceranae isolated from A. florea workers. Propolis treatement significantly reduced the proliferation of N. ceranae in infected bees, exhibiting a dose-response effect such that the highest dose of propolis provided the greatest inhibition of Nosema proliferation. This result corresponds to the study of

3.5 Propolis Effects on Hypopharyngeal Gland Protein Content

[24].where it was shown the potential of propolis to

The mean total protein contents of hypopharyngeal glands of bees inoculated with 40,000 spore per bee and treated with 0%, 10%, 20% and 50% propolis in water (v/v), taken on days 6, 10 and 14 p.i. are shown in Fig. 5. Bees treated with 50% (v/v) propolis had the highest

mellifera [24]. It is also related to a study of propolis from a stingless bee that evaluated its antifungal

protein concentration (272.11 ± 16.50 μg/μL) (F14,30 = 7.09; P < 0.0001). Bees treated with 0% (v/v) propolis

addition, propolis rescued the protein production of the

(0P) had lower protein concentration (242.39 ± 11.93 µg/µL) than those of others. Protein content of infected bees treated with propolis was significantly higher than

Nosema. Protein is vital for feeding larvae and thus for

control Nosema infection in the European honeybee, A.

properties [17, 19]. Although we used ethanol to prepare our propolis solution, diluted ethanol alone (0P treatment) was not effective against Nosema. In hypopharyngeal glands of worker bees infected with overall colony health. Finally, propolis treatment significantly enhanced the survival of treated bees.

Percentage of serviving

120 100 0P

80

10P 20P

60

50P

40

CO

20 0 1

3

5

7

9 11 13 15 17 19 21 23 25 27 29 Number of days

Fig. 4 Survival of A. florea after infection by spores of N. ceranae isolated from A. florea workers for 30 days after inoculation with 40,000 spores per bee and treated with 0% (0P), 10% (10P), 20% (20P), and 50% (50P) propolis, in addition to a negative (CO).

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Stingless Bee Propolis Effects on Experimental Infection of Apis florea with Nosema ceranae

Hypopharyngeal gland protein content (m g/bee)

16

a

14

a

b

12

a

c

a

a

a

b ab

bc

ab

a

a

a

CO

10

0P

8

10P

6

20P 50P

4 2 0 6

10

14

Days Fig. 5 Mean± SD protein content (mg/bee) of hypopharyngeal glands of A. florea at 6, 10 and 14 days after inoculation with 40,000 spores per bee and then treated with 0% (0P), 10% (10P), 20% (20P) and 50% (50P) in addition to a negative (CO). Vertical bars with different letters represent significant differences among groups (ANOVA-Duncan’s multiple range test, F14,30 = 7.09; P < 0.0001).

There are multiple ways in which propolis may counteract Nosema infection. Propolis could inhibit

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spore growth and differentiation. Immature stages of Nosema were found in the ventricular cells of 50P—treated bees, however, only mature and empty (final stage) spores, no meronts, were found in bees untreated

with

propolis.

However,

[2] [3]

direct

dose-response assays of propolis on N. ceranae spores

[4]

are needed to explore this in greater detail. These results suggest a new method of treating Nosema that relies upon extracts of a naturally obtained compound, propolis. This should be of significant interest to organic apiculturalists as well as to biologists

[5] [6]

interested in the treatment of microsporidian diseases. [7]

Acknowledgments The authors would like to thank James Nieh for his

[8]

comments on this manuscript. This study was supported by Thailand Research Fund. Our special thanks also go to Faculty of Science, Burapha University, Thailand for research facility and financial support. The University of Dayton Research Council and Department of Biology provided support to MEB.

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