Detection of infective Nosema ceranae (Microsporidia) spores in corbicular pollen of forager honeybeesAcknowledgmentsReferencesShort CommunicationDetection of infective Nosema ceranae (Microsporidia) spores incorbicular pollen of forager honeybeesMariano Higesa,*, Raquel Martı´n-Herna´ndeza, Encarna Garrido-Bailo´na,Pilar Garcı´a-Palenciab, Ara´nzazu MeanacaRegional Apicultural Center, Direccio´n General de la Produccio´n Agropecuaria, Consejerı´ade Agricultura,Junta de Comunidades de Castilla-La Mancha, 19180 Marchamalo, Guadalajara, SpainbDepartment of Veterinary Medicine and Surgery, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, 28040 Madrid, SpaincDepartment of Animal Health, Faculty of Veterinary Medicine, Universidad Complutense de Madrid, 28040 Madrid, SpainReceived 12 March 2007; accepted 11 June 2007Available online 20 June 2007AbstractNosema ceranae is a Microsporidia recently described as a parasite in Apis mellifera honeybees in Europe. Due to the short time sinceits description, no epidemiological data are available.In this study, spore detection in both pollen baskets and pollen collected from commercial traps is described (PCM, TEM and PCRmethods). Spore infectivity is shown after artificial infection of Nosema-free adult bees.The epidemiological consequences of the presence of Nosema spores in corbicular pollen require more study and must be considered inbeekeeping practices. 2007 Published by Elsevier Inc.Keywords: Nosema ceranae; Apis mellifera; Corbicular pollen; PCR; EpidemiologyPollen is the only dietary source of protein, lipids, vita-mins and minerals for the nutrition of developing larvaeand adult bees. While overwintering, colonies rely on pol-len reserves stored during the previous foraging season toprovide nutrients, especially for brood rearing, beginningin early spring (Seeley, 1985). Forager bees gather loadsof pollen from flowers and pack it on the outer surfaceof their hind legs, in the pollen basket. This corbicular pol-len is deposited in a cell, usually just above or beside thebroodnest.Nosema ceranae is a Microsporidia recently described asa parasite in Apis mellifera honeybees in Europe (Higeset al., 2006) and Asia (Huang et al., 2007). Nosema ceranaehas been described as highly pathogenic for this new host(Higes et al., 2007), but no epidemiological data are avail-able due to the short time since its description. No data ofany kind on spore sources for bees and ways of infectiontransmission are available.In this study, spore detection in both pollen baskets andpollen collected from commerc ial traps is described. Sporeinfectivity after artificial infection of Nose ma-free adultbees is also shown.Forager bee samples were obtained from weak coloniesbelonging to a commercial beekeeper and from a naturallyinfected Nosema colony located in our experimental apiary.Thirty forager honeybees were taken from the previouslysealed hive entrance. Corbicular pollen was present in morethan 95% of the bees and was removed from the honeybees’corbicula by using sterile tweezers. This pollen was kept infrozen storage until its analysis. Another sample of com-mercial pollen obtained by using a trap was analysed atthe same time.The presence of microsporidia was first confirmed in beeand pollen samples. Samples were macerated in ddH2O andanalysed in phase-contrast microscopy (PCM) with 400·0022-2011/$ - see front matter  2007 Published by Elsevier Inc.doi:10.1016/j.jip.2007.06.002*Corresponding author. Fax: +34 949 250 176.E-mail address: [email protected] (M. Higes).www.elsevier.com/locate/yjipaAvailable online at www.sciencedirect.comJournal of Invertebrate Pathology 97 (2008) 76–78Journal ofINVERTEBRATEPATHOLOGYmagnification in corbicular pollen balls, bees and a 1 g oftrap pollen. Spores were identified by a previouslydescribed morphology (Fries et al., 1996) while ultrastruc-tural analyses of spores were done in recently obtained cor-bicular pollen that were isolated from a hive located in ourexperimental apiary. Pollen balls were prefixed in 2% glu-taraldehyde–2.5% paraformaldehyde and processed byTransmission Electron Microscopy (TEM) analyses (Higeset al., 2007). Nosema species was determined by PCR aspreviously described (Higes et al., 2006).Nosema spores were identified in all the samples byeither PCM or TEM (Fig. 1), and the species identifiedwas N. ceranae (PCR).To confirm corbicular spores infectivity, newl y emergedNosema-free honeybees were infected in a similar way aspreviously described (Higes et al., 2007). An aliquot of pre-viously diluted pollen samples (in ddH2O) was centrifuged(800g for 6 min.), resuspended three times again, eliminat-ing supernatant, and then counted to prepare infectiondoses.Three replicates of 20 newl y emerged workers werestarved for 5 h. Afterwards, two of them were collectivelydosed with 100,000 spores in 1 ml of sucrose (50% w/w inwater), and 2% of Promotor L (Calier lab.) was added asa dietary supplement for 24 h. The third cage acted as unin-fected control. Once the total infection dose was consumed,bees were fed ad libitum with the same control food. Beeswere checked and the food changed daily. Afterwards,infection was determined in one bee per group after 3, 7and 21 days p.i.Infected bees were positive to PCM spore detection bothafter 3 days p.i. and 7 days p.i. Quantification of sporesshowed a 10-fold increase in the number of spores fromday 3 to day 7. Around a million spores per infected beewere present a week after the infection with the corbicularpollen spores. After 20 da ys p.i. all the infected beesbecame visibly less active, with sluggish movements, orimmobile, not reacting to exter nal stimulus. The next dayall of them died. No similar signs were observed in controlgroups, with all bees alive except one by day 21 p.i. Nospores were detected in any of them, including the deadbee. Spore quantifications of dead infected bees showed amean of 21.5 million per bee in group 1 and 22.1 millionspores per bee in group 2. Spore identification by PCR con-firmed that they were N. ceranae.This is the first report about the detection of N. ceranaeinfective spores in corbicular pollen. Pollen store d in combcells of the hive has been reported as a bee pathogen reser-voir (Mehr et al., 1976; Moffet et al., 1978; Gilliam et al.,1988; Chen et al., 2006). Viable spores of Ascosphaera apishave been shown to survive in ‘‘bee bread’’ for at least12 months