Abstract
Nosema apis and Nosema ceranae are microsporidia which present resistant spores for the transmission stage (environmental spores) that play an important role for epidemiology and for laboratory studies of honey bee microsporidiosis. In this study, the long-term longevity of N. apis and N. ceranae spores exposed to 4 °C, room temperature (mean 25 °C) and 35 °C for 6-month long and to −20 °C for 10-month long has been assessed by flow cytometry. Storage temperature and the length of storage duration had adverse effects on spore viability of both Nosema spores, with significant differences between the two species. The greatest increase in spore mortality was observed in N. apis spores stored at 33 °C (64, 89 %) and in N. ceranae spores at −20 °C (53.55 %) and at 33 °C (51.97 %). For N. ceranae spores at −20 °C, the loss in viability was very quick, getting an increase over 20 % just after 6 days of exposure. Results on viability were confirmed by the infectivity tests where the lowest infectivity for N. ceranae was observed with spores stored for 10 months at −20 °C (79 %; P < 0.05) and for N. apis with spores stored at 33 °C (71 %; P < 0.05). For both Nosema species, the best storage temperatures were 25 and 4 °C, especially for N. apis that was almost unaffected at those temperatures.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Adl SM, Simpson AGB, Farmer MA, Anderesen RA, Anderson OR, Barta JR, Bowser SS, Brugerolle G, Fensome RA, Fredericq S, James TY, Karpov S, Kugrens P, Krug J, Lane CE, Lewis LA, Lodge J, Lynn DH, Mann DG, McCourt RM, Mendoza L, Moestrup Mozley-Standridge SE, Nerad TA, Shearer CA, Smirnov AV, Spiegel FW, Taylor MFJR (2005) The new higher level classification of eukaryotes with emphasis on the taxonomy of protist. J Eukaryot Microbiol 52:399–451
Amigó JM, Gracia MP, Comas J, Salvadó H, Vivarés CP (1994) Comparative study of micrsporidian spores by flow cytometric analysis. J Eukaryot Microbiol 41:210–214
Amigó JM, Gracia MP, Ruis M, Salvadó H, Maillo PA, Vivarés CP (1996) Longevity and effects of temperature on the viability and polar-tube extrusion of spores of Glugea stephani, a microsporidian parasite of commercial flatfish. Parasitol Res 82:211–214
Bailey L (1954) The control of Nosema disease. Bee World 35:111–113
Cali A, Takvorian PM (1999) Developmental morphology and life cycles of the microsporidia. In: Wittner M, Weiss LM (eds) The Microsporidia and microsporidiosis. Am Soc Microbiol, Washington
Cornejo LG, Rosi CO (1975) Enfermedades de las abejas: su profilaxis y prevención. Ed. Hemisferio Sur, Buenos Aires
Fenoy S, Rueda C, Higes M, Martín-Hernández R, del Águila C (2009) High-level resistance of Nosema ceranae, a parasite of the honeybee, to temperature and desiccation. Appl Environ Microbiol 75(21):6886–6889
Fries I (2010) Nosema ceranae in European honey bees (Apis mellifera). J Invertebr Pathol 103:73–79
Fries I, Feng F, da Silva A, Slemenda SB, Pieniazek NJ (1996) Nosema ceranae n. sp. (Microspora, Nosematidae), morphologycal and molecular characterization of a Microsporidian parasite of the Asian honey bee Apis cerana (Hymenoptera, Apidae). Eurp J Protistol 32:356–365
Gisder S, Hedtke K, Möckel N, Frielitz MC, Linde A, Genersch E (2010) Five-year cohort study of Nosema spp. in Germany: does climate shape virulence and assertiveness of Nosema ceranae? Appl Environ Microbiol 76(9):3032–3038
Higes M, García-Palencia P, Martín-Hernández R, Meana A (2007) Experimental infection of Apis mellifera with Nosema ceranae (Microsporidia). J Invertebr Pathol 94:211–217
Higes M, Martín-Hernández R, Garrido-Bailón E, García-Palencia P, Meana A (2008a) Detection of infective Nosema ceranae (microsporidia) spores in corbicular pollen of forager honeybees. J Invertebr Pathol 97(1):76–78
Higes M, Martín-Hernández R, Garrido-Bailón E, Botías C, García-Palencia P, Meana A (2008b) Regurgitated pellets of Merops apiaster as fomites of infective Nosema ceranae (Microsporidia) spores. Environ Microbiol 10(5):1374–1379
Higes M, Martín-Hernández R, García-Palencia P, Marín P, Meana A (2009) Horizontal transmission of Nosema ceranae (Microsporidia) from worker honey bees to queens (Apis mellifera). Environ Microbiol Rep 1(6):495–498
Higes M, Martín-Hernández R, Meana A (2010) Nosema ceranae in Europe: the background and an update of emergent nosemosis type C. Apidologie 41(3):375–392
Higes M, Meana A, Bartolomé C, Botías C, Martín-Hernández R (2013) Nosema ceranae (Microsporidia), a controversial 21st century honey bee pathogen. Environ Microbiol Rep 5(1):17–29
Kramer JP (1970) Longevity of microsporidian spores with special reference to Octosporea muscadomesticae Flu. Acta Protozool 8:217–224
Kucerova Z, Moura H, Leitch GJ, Sriram R, Bern C, Kawai V, Vargas D, Gilman RH, Ticona E, Vivar A, Visvesvara G (2004) Purification of Enterocytozoon bieneusi spores from stool specimens by gradient and cell sorting techniques. J Clin Microbiol 42(7):3256–3261
Leiro J, Cano E, Ubeira FM, Orallo F, Sanmartín ML (2004) In vitro effects of resveratrol on the viability and infectivity of the microsporidian Encephalitozoon cuniculi. Antimicrob Agents Chemother 48:2497–2501
Malone LA, Gatehouse HS, Tregida EL (2001) Effects of time, temperature and honey on Nosema apis, a parasite of the honey bee (Apis mellifera). J Invertebr Pathol 77:258–268
Martín-Hernández R, Meana A, García-Palencia P, Marín P, Botías C, Garrido-Bailón E, Barrios L, Higes M (2009) Effect of temperature on the biotic potential of honeybee microsporidia. Appl Environ Microbiol 75(8):2554–2557
Martín-Hernández R, Botías C, Barrios L, Martínez-Salvador A, Meana A, Mayack C, Higes M (2011a) Comparison of the energetic stress associated with experimental Nosema ceranae and Nosema apis infection of honey bees (Apis mellifera). Parasitol Res 109:605–612
Martín-Hernández R, Botías C, Garrido Bailón E, Martínez-Salvador A, Prieto L, Meana A, Higes M (2011b) Microsporidia infecting Apis mellifera: coexistence or competition. Is Nosema ceranae replacing Nosema apis? Environ Microbiol 14(8):2127–2138
McGowan J (2012) Viability Assessment and cryopreservation of the honey bee (Apis mellifera) parasite, Nosema ceranae. Thesis presented at the University of Guelph, Ontario, Canada. Available at: https://dspace.lib.uoguelph.ca/xmlui/bitstream/handle/10214/3801/Janine%20McGowan%20Thesis%20Defense%20Copy%20July%202012.pdf?sequence=3. Accessed on 15 Jul 2013
Moffet JO, Wilson WT (1971) The viability and infectivity of frozen Nosema spores. Am Bee J 111:55–56
Moss D, Croppo GP, Wallace S, Visvesvara G (1999) Flow cytometric analysis of microsporidia belonging to the genus Encephalitozoon. J Clin Microbiol 37(2):371–375
Revell IL (1960) Longevity of refrigerated Nosema spores—Nosema apis a parasite of honey bees. J Econ Entomol 53:1132–1133
Smith ML (2012) The honey bee parasite Nosema ceranae: transmissible via food exchange? PLoS ONE 7(8):e43319. doi:10.1371/journal.pone.0043319
Van der Zee R (2010) Colony losses in the Netherlands. J Apic Res 49:121–123
Zander E (1909) Tierische Parasiten als Krankenheitserreger bei der Biene. Münchener Bienenzeitung 31:196–204
Acknowledgments
The authors wish to thank the Culture Unit at the CAI, the Faculty of Medicine and Biology of the University of Alcalá de Henares (Spain) and especially Isabel Trabado for her help developing the flow cytometry analysis. We also like thank J. Almagro, J. García, V. Albendea and T. Corrales of the honey bee pathology laboratory for their technical support. JGSC granted by INIA. Financial support for this work was provided by INIA-FEDER (RTA-2009-000105-C02-01).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Sánchez Collado, J.G., Higes, M., Barrio, L. et al. Flow cytometry analysis of Nosema species to assess spore viability and longevity. Parasitol Res 113, 1695–1701 (2014). https://doi.org/10.1007/s00436-014-3814-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00436-014-3814-z