INVERTEBRATE PATHOLOGY

Journal of Invertebrate Pathology 95 (2007) 8486 www.elsevier.com/locate/yjipa

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Inuence of carbon dioxide on Nosema apis infection of honeybees (Apis mellifera)

Krystyna Czekonska
*
Department of Pomology and Apiculture, Agricultural University, Al. 29 Listopada 52, 31-425 Krakow, Poland Received 5 May 2006; accepted 6 February 2007 Available online 22 February 2007

Abstract Young workers of the honeybee Apis mellifera carnica were individually inoculated with Nosema apis spores subjected to carbon dioxide (CO2) treatment. The spores were kept in a CO2 atmosphere for 30, 35 and 40 h. The course of the infection was evaluated on the basis of the survival rate of bee workers and the number of N. apis spores in their digestive tracts. CO2 treatment of N. apis spores resulted in faster proliferation of the parasite as well as higher mortality among workers infected with spores kept in CO2 for 30 and 35 h. 2007 Published by Elsevier Inc.
Keywords: Nosema apis; Nosema disease; Honey bee; Apis mellifera; Carbon dioxide

1. Introduction The protozoan Nosema apis, traditionally classied as Mikrosporidia but now recognized by some researchers as a highly derived fungus (Hirt et al., 1999; Arisue et al., 2002), is one of the most common honeybee parasites, causing nosema disease (Nosemosis apium) (Bailey and Ball, 1991; Matheson, 1996; Fleche, 1997; Fries, 1997). The most intensive damage from this disease occurs in apiaries in temperate climatic zones (Fries, 1997). The presence of the parasite in honeybees leads to major losses in apiary productivity and may also cause the loss of entire bee colonies (Farrar, 1947; Woyke, 1984; Fries, 1988a, 1997). Prevention of the disease involves, above all, hygienic and sanitary procedures to control the development and spread of the parasites (Shimanuki and Knox, 1997). These procedures include disinfection of honeycombs by fumigation with chemical agents such as acetic acid or ethylene oxide. Carbon dioxide (CO2) is also used in fumigation, but it has been recommended chiey to combat the most

harmful pest of honeycombs, the greater wax moth (Galleria mellonella L.) (Cantwell et al., 1972; Williams, 1997; Shimanuki and Knox, 1997). In the literature there are no reports on how this gas aects the spores of N. apis: whether it is neutral, perhaps stimulates, or rather inhibits their development. The aim of this study was to examine the course of the disease in honeybee workers inoculated with N. apis spores previously subjected to high CO2 concentrations. 2. Materials and methods Young workers of Apis mellifera carnica were obtained from combs with emerging workers, and placed in an incubator. The bee workers were inoculated with a sugar solution containing N. apis spores earlier treated with CO2. An inoculating solution was prepared and divided into four doses of 5 ml each. Three doses were placed in anaerostates and exposed to CO2 (100% concentration) for 30, 35 and 40 h. The fourth sample, used as a positive control, was left at the same temperature as the other samples but without exposure to CO2. Workers emerging during 20 h were individually fed doses of 10 ll 54% sugar solution containing 9.08 103

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spores of N. apis, applied from a micro-pipette. Each of the four doses was used to inoculate 120 workers. Thereafter the workers were placed in cages (4 6.5 9 cm) equipped with small combs and a feeding tube. A fth group was formed of bee workers not inoculated with N. apis spores. In each of the ve groups there were three cages. Each cage contained 40 workers. The groups were given the designations G0, G30, G35, G40 and GC, corresponding with the duration of exposure of the inoculated spores to CO2, and the negative control group of non-inoculated workers. The cages with bee workers were kept in an incubator at 30 C. Every day the dead workers were removed from the cages and the food was replenished. The course of invasion by parasites was tested every third day by counting the number of N. apis spores in the digestive tracts of living workers. For this purpose, ve workers were taken from each cage. The workers removed from the cages were sacriced by applying CO2 and kept at 18 C until laboratory examination. Immediately prior to the laboratory tests, the insects were thawed, the ventriculus was dissected and macerated in 1 cm3 distilled water, and the spores were counted in a haemocytometer in 0.025 mm3 suspension. If less than 10 spores were found in the sample they were recounted in 0.2 mm3 suspension. The results are given as the number of spores per 1 cm3, treated as the number of spores per bee. To assess the signicance of dierences in numbers of infected workers at various days post-inoculation the Chi square test was used; for number of N. apis spores in infected workers, nested ANOVA analysis; and for survival rates of workers, the G test (Sokal and Rohlf, 1981). 3. Results On the 6th day after inoculation, in groups G0, G30, G35, G40 and GC spores of N. apis were found in 53.3%, 100%, 100%, 66.7% and 0% of the workers, respectively. There were signicant dierences between groups G0 and G35 (Chi square test: v2 = 6.71, df = 2, p = 0.009) as well as between groups G0 and G30 (v2 = 6.25, df = 2, p = 0.012). On the 9th day, 100% of the workers in G0 and G40 were infected. Only the workers from the GC group were free of parasites at all intervals of the experiment. There were signicant dierences in number of spores between groups inoculated with N. apis spores (nested ANOVA: F = 19.665, p < 0.001) and between days of experiment (F = 1641.853, p < 0.001) but not between cages (F = 1.197, p = 0.304) (Fig. 1). The highest numbers of spores on successive days were found in workers from the G40 group, and the lowest number in workers from group G0 (Fig. 1). Between groups there were signicant dierences in the survival rates of workers (G test: Gadj = 54.762, p < 0.001) (Fig. 2). At the end of the experiment, lasting 18 days, in the G0, G30, G35, G40 and GC groups, the respective proportions of surviving workers were 20.8%, 4.2%, 0%, 30.0% and 23.3%.

100

Log10 no. of spores x 10 per bee

10

40 h 35 h 30 h 0h

0.1 3 6 9 12 15 18 Days post-inoculation

Fig. 1. Rates of infection among workers inoculated with N. apis spores exposed to CO2 for 0, 30, 35 and 40 h.

100 80 Survival (%) 60 40 20 0 0 2 4 6 8 10 12 14 16 18 Days post-inoculation

40 h 35 h 30 h 0h Negative control

Fig. 2. Mortality rates in workers inoculated with N. apis spores exposed to CO2 for 0, 30, 35 and 40 h, and in the negative control group containing non-inoculated workers.

4. Discussion The presented study showed that extended exposure of the inoculated spores to CO2 accelerated the development and proliferation of N. apis spores, observed from the 6th day after inoculation. The epithelium of the middle section of the intestine becomes entirely infected by parasites in about 2 weeks (Fries, 1988b, 1989). After that time, an average of 3050 106 spores of N. apis are present there (Lotmar, 1943; Fries, 1988b, 1997; Bailey and Ball, 1991). In this study, such a high rate of parasite invasion was found as early as on the 9th and 12th days after inoculation in workers inoculated with doses of spores exposed to CO2 for 30, 35 and 40 h. Slow proliferation of N. apis spores was found only for the inoculum not treated with carbon dioxide. It is known that the invasion by the N. apis parasite depends on the temperature at which the infection occurs (Woyciechowski and Czekonska, 1999), as well as on the temperature at which development of N. apis spores takes place (Karmo and Morgenthaler, 1939; Lotmar, 1943). The present study suggests that the course of invasion may also depend on whether the spores are exposed to CO2. In the experiment, exposure to carbon dioxide stimulated the spores to develop. Similar eects of CO2 exposure, although applied at lower concentrations not exceeding

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K. Czekonska / Journal of Invertebrate Pathology 95 (2007) 8486 De Graaf, D.C., Masschelein, G., Vandergeynst, F., Brabander, H.F., Jacobs, F.J., 1993. In vitro germination of Nosema apis (Microspora: Nosematidae) spores and its eect on their aa-trehalose/D-glucose ratio. J. Invertebr. Pathol. 62, 220225. Farrar, C.L., 1947. Nosema losses in package bees as related to queen supersedure and honey yields. J. Econ. Entomol. 40, 333338. Fleche, C., 1997. Risks of spreading bee diseases through the international movement of bees and bee products. Rev. Sci. Tech. 16, 177186. Fries, I., 1988a. Contribution to the study of Nosema disease (Nosema apis Z.) in honey bee (Apis mellifera L.) colonies. Rapport 166. University of Agricultural Sciences, Sweden. Fries, I., 1988b. Infectivity and multiplication of Nosema apis Z. in the ventriculus of the honey bee (Apis mellifera L.). Apidologie 19, 319 328. Fries, I., 1989. Observations on the development and transmission of Nosema apis Z. in the ventriculus of the honey bee. J. Apicult. Res. 28, 107117. Fries, I., 1997. Protozoa. In: Morse, R.A., Flottum, K. (Eds.), Honey Bee Pests, Predators, and Diseases. A.I. Root Company, Medina, Ohio, USA, pp. 5776. Heath, L.A.F., Gaze, B.M., 1987. Carbon dioxide activation of spores of the chalkbrood fungus Ascosphaera apis. J. Apicult. Res. 26, 243246. Hirt, R.P., Logsdon, J.M., Healy, B., Dorey, M.W., Doolittle, W.F., Embley, T.M., 1999. Microsporidia are related to Fungi: Evidence from the largest subunit of RNA polymerase II and other proteins. Prac. Natl. Acad. Sci. USA 96, 580585. Karmo, E., Morgenthaler, O., 1939. The development of Nosema apis at various temperatures. Bee World 20, 5758. Lotmar, R., 1943. Uber den Einuss der Temperatur auf den Parasiten Nosema apis. Beih. Schweiz. Bienenztg. 1, 261284. Matheson, A., 1996. World bee health update 1996. Bee World 77, 4551. Nerun, K., Buelens, H., 1997. Hypoxia-controlled winter metabolism in honeybees (Apis mellifera). Comp. Biochem. Physiol. 117A, 445455. Seeley, T.D., 1974. Atmospheric carbon dioxide regulation in honey-bee (Apis mellifera) colonies. J. Insect Physiol. 20, 23012305. Shimanuki, H., Knox, D.A., 1997. Summary of control methods. In: Morse, R.A., Flottum, K. (Eds.), Honey Bee Pests, Predators, and Diseases. A.I. Root Company, Medina, Ohio, USA, pp. 493512. Sokal, R.R., Rohlf, F.J., 1981. Biometry. W.H. Freeman, New York. Williams, J.L., 1997. Lepidoptera (Moths). In: Morse, R.A., Flottum, K. (Eds.), Honey Bee Pests, Predators, and Diseases. A.I. Root Company, Medina, Ohio, USA, pp. 119141. Woyciechowski, M., Czekonska, K., 1999. The eect of temperature on Nosema apis Zander (Microsporida, Nosematidae) infection in honey bees (Apis mellifera). Parasite 6, 185187. Woyke, J., 1984. Increases in life span, unit honey productivity and honey surplus with fumagillin treatment of honey bee. J. Apicult. Res. 23, 209212.

12.5% by volume of air, were found for spores of Ascosphaera apis, a fungus causing a dangerous disease in honeybees called chalk brood (Heath and Gaze, 1987). Possibly what stimulated the N. apis spores to develop were bicarbonate ions, as suggested by de Graaf et al. (1993). This could be conrmed by an in vitro experiment. The presence of N. apis spores in honeybee workers shortens their lives (Fries, 1997). Although in this study the mortality among workers did not increase linearly with the time of CO2 exposure of the inocula, it was nevertheless highest in workers inoculated with spores exposed to CO2 for 3035 h. It cannot be excluded that the intensity of invasion might also aect the mortality among the workers (Lotmar, 1943; Fries, 1988b). It is not clear why workers mortality was relatively low in those inoculated with spores exposed to CO2 for 40 h. The results suggest that applying CO2 to fumigate honeycombs may increase invasion by the N. apis parasite in bee colonies. Moreover the results can explain why after winter when the concentration of CO2 in beehive air is higher (Chauvin, 1968; Seeley, 1974; Nerun and Buelens, 1997), the intensity of N. apis infection of bees is greater (Bailey and Ball, 1991). Acknowledgments I thank Adam Tolski for helpful comments on an earlier version of this paper. This work was funded by the Polish Ministry of Science and Higher Education, Grant No. 2 P06Z 031 29. References
Arisue, N., Hashimoto, T., Hasegawa, M., 2002. Early evolution of eukaryotes inferred from genome data. Int. Congr. Ser. 1246, 209215. Bailey, L., Ball, B.V., 1991. Honey Bee Pathology. Academic Press, London. Cantwell, G.E., Jay, E.G., Pearman, G.C., Thompson, J.V., 1972. Control of the greater wax moth Galleria mellonella (L.) in comb honey with carbon dioxide. Am. Bee J. 112, 302303. Chauvin, R., 1968. Energetique (calorimetrie des abeilles). In: Chauvin, R. (Ed.), Traite de biologie de Labeille. I. Biologie et physiologie generales. Masson et Cie Paris, pp. 253255.