The prophylaxis of Nosema disease in bees by laboratory methods - microscopic testing of the beehive products

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1 Romanian Biotechnological Letters Vol. x, No. x, 018 Copyright 018 University of Bucharest Printed in Romania. All rights reserved ORIGINAL PAPER The prophylaxis of Nosema disease in bees by laboratory methods - microscopic testing of the beehive products Received for publication, April, 0, 016 Accepted, January, 1, 018 BODESCU DAN 1, SAVU VASILICĂ, AGRIPINA SAPCALIU *, RĂDOI ION, MĂRGĂRIT GABRIELA, TĂNASE PETRUŢ 4, VICTOR CALIN 4, PÎRVULET CRISTINA ŞTEFANIA 1 University of Agronomical Sciences and Veterinary Medicine Iasi, Romania Beekeeping Research and Development Institute Bucharest, Romania, Bucharest, Romania University of Agronomical Sciences and Veterinary Medicine Bucharest, Romania 4 Spiru Haret University Bucharest, Romania Agricultural and Forestry Academy of Sciences Ghe. Ionescu Sisesti Bucharest, Romania *Corresponding author: Beekeeping Research and Development Institute Bucharest, Romania, 4 Ficusului Blvd., Bucharest, Romania, +40/0060, address: sapcaliuagripina@yahoo.com Abstract The purpose of this study was to monitor the load of Nosemaspp. spores in honey and pollen and to evaluate the pertinence of the method in the prophylaxis of Nosema spp. infestation by eliminating contaminated sources used in bee fed. We investigated 6 apiaries for a period of years at the end of the active bee season, the collected consisting in honey (8 ), pollen (8 ) and reserve honeycombs (80 ). The were processed and tested for spores of Nosema spp. through the method OIE/008, adapted for of honey, honeycomb and pollen. In the beginning and at the end of each active bee season, the infestation degree and the evolution of the disease amongst monitored bees were investigated. The originated from apiaries suspected of nosema disease. Within the lot with pollen (8 ), a number of 0 pollen belonging to the 4 dominant floral categories in Romania (fruit trees, rape, sun-flower, linden) were chosen in order to determine the infestation level with Nosema spp. spores. Most of the Nosema spp. spores positive were represented by the rape and sunflower pollen. Tests evidenced the presence of Nosema spp. spores in 0 of honey, pollen and honeycombs of the total of 0 examined during the monitoring process. The tests made on bee collected at the end of the beekeeping season, by comparison to the ones collected in the beginning of the following season, demonstrated a significant reduction in the infestation degree (7%) in bees by eliminating from consumption the sources of infestation (honey and pollen) in the winter season. Testing before the inactive season for Nosema spp. spores in the reserve honey and pollen represents an important prophylactic method against Nosema spp. infestation in bees. Keywords: microscopic testing, honey and pollen, prophylaxis, Nosema spp., bees, honeycombs 1. Introduction Nosema disease is severe honeybee diseases producing important economic losses and a high mortality rate within the honeybee populations. Nosemosis (Nosema disease) is a parasitic disease produced by the spores of two intestinal protozoa species: Nosema apis and Nosema ceranae, which especially affect the adult worker bees (age over 1 days) and this correlated with important losses within the colonies, up to depopulation []. The bees are infected as a result of ingesting faecis full of spores during the activity of hive cleaning or by ingesting food or water contaminated with spores of Nosema spp. Nosema apis produces the so called type A nosemosis, which specially occurs during the cold season when the occurrence of the watery brown diarrhea syndrome is to be found both in the hive and outside

2 the hive, the bees are no longer able to fly, with an apparent paralysis which finally produces a high mortality rate []. Nosema ceranae produces the type C nosemosis (Nosema disease) an infestation which highly increases during summer time (hot season), with a suppression of the digestive and immune functions, followed by the quick death of the bees far away from the apiary [1,] without noticing the diarrhea syndrome. Nosema ceranae is involved (is one of the incriminated factors) in the occurrence of CCD (colony collapse disorder) [1,]. The bees are infected by ingesting faecis full of spores during the activity of cleaning or by ingesting food or water contaminated with spores of Nosema spp. For an efficient prophylaxis of these diseases, the main causes leading to their occurence should be removed, one of these causes being the contaminated food resources which are consumed by the bees []. The purpose of this study was to monitor the load of Nosema spp. spores in honey and pollen and to evaluate the pertinence of the method in the prophylaxis of Nosema spp. infestation by eliminating contaminated sources used in bee fed.. Material and method During a year period a number of 6 apiaries were investigated at the end of the active beekeeping season. 0 representing honey (8 ), bee pollen (8 ) and reserve foundation combs (80 ) were collected (Table 1) originating from apiaries suspected of Nosema disease. At the beginning and the end of the active season the infestation level with Nosema spp. spores was investigated. The evolution of the disease within the monitored and studied honeybee colonies was also investigated. Table 1. Total number of investigated apiaries and Number of examined Number of honey Number of bee pollen Number of reserve apiaries foundation combs The collected were processed and analyzed to check the occurrence (presence) of the Nosema spp. spores using the OIE/008 [4] method that was adapted for the honey, bee pollen and foundation combs. At the beginning and the end of each active season the level of infestation with Nosema spp. spores and the evolution of the disease within the monitored honeybee s colonies. Within the lot with pollen (8 ), a number of 0 pollen belonging to the 4 dominant floral categories in Romania (fruit trees, rape, sun-flower, linden) were chosen in order to determine the infestation level with Nosema spp. spores. The potential variation of the infestation level correlated with the melliferous floral source was also studied (Table ) Table. Pollen from various floral melliferous species in Romania examined pollen rape pollen sunflower pollen linden (Tillia from fruit trees spp.) pollen Results and discussion The direct microscopic examination revealed the presence of the Nosema spp., spores in 0 of honey, bee pollen and reserve foundation combs of the total number of 0 analyzed (table and figure 1).

3 Table. The analyzed for the presence of Nosema spp. spores Sample type Number of examined Honey 8 Bee pollen 8 Reserve honey combs Total number of examined 80 0 (100%) Total number of positive 71 (8.%) 74 (87.1%) 7 (9.7%) 0 (88%) Total number of negative 14 (16.%) 11 (1.9%) (6.%) 0 (1%) The number of Nosema spp. spores positive is shown in Figure 1. Fig. 1. Graphic representation of the Nosema spp. spores positive Looking at the above table it was concluded that out of 8 honey 71 were positive (8.%) for the presence of Nosema spp., out of 8 bee pollen 74 were positive (87.1%) while out of the total number of 80 reserve honey combs, 7 were positive for Nosema spp., spores (9.7%). Analyzing the results of the study it can be said that within the studied lots a high level of contamination was noted for categories of hive products which are used as food by the honeybees (honey, pollen, combs). The results made it possible to classify in categories the level of Nosema spp. spores load: weak infestation (1- spores/microscopic field), medium (average) infestation (- spores/microscopic field) and massive infestation ((> spores/field). (Table 4 and Fig.,,4). Table 4. The infestation level of the honey, bee pollen and reserve honey combs Sample type Honey Bee pollen Reserve honey combs positive 71 (8.%) 74 (87.1%) 7 (9.7%) Weak (low) infestation (1- spores of Nosema spp./field) 64 (90.1%) 6 (7.6%) 8 (77.%) Infestation level Medium infestation (- spores of Nosema spp./field) (7.1%) 1 (17.6) 6 (8,0%) Massive (high) infestation (> spores of Nosema spp./field) (,8%) (6.8%) 11 (14.7%) negative sample 14 (16.%) 11 (1.9%) (6.%)

4 Fig.. Nosema spp. spores in honey (x400) Fig.. Nosema spp. spores in bee pollen (x400) Fig. 4. Nosema spp. spores in reserve honey combs (x400) Following the direct microscopic examination of the pollen, the results made it possible to clasify the categories for the Nosema spp. spores load: weak infestation (1- spores/microscopic field, medium (average) infestation (- spores/field) and massive (high) infestation > spores/field) (Table ). Table. Nosema spp. spores infestation level for the 0 pollen in correlation with the melliferous source examined Nosema spp. spores/field No./percent of infestated Fruit trees pollen Rape pollen Sunflower pollen Linden pollen > Neg > Neg > Neg > Neg. 1 8% % % % % 1 8% 6 46% 4 % 4 % 1 8% 7 8% According to the data shown in Table, from the total number of 1 pollen from fruit trees, 4 (1%) were positive for Nosema spp. spores. In the rest of the lot the positive values were as follows: 7 (4%) for the rape pollen, 8 (67%) for 4

5 the sunflower pollen, (4%) for linden pollen (Fig. ). Most of the Nosema spp. spores positive were represented by the rape and sunflower pollen. Fig.. Number of positive and negative pollen for the presence of Nosema spp. spores. The microscopic examinations of 0 of honeybees collected from 6 apiaries wereanalyzed during a year interval. The that were collected at the end of the beekeeping season (autumn), as compared to that were collected at the beginning of the next season (early spring), proved a significant decrease of the Nosema spp. infestation level in the examined bees (the percentage was 7%) as a result of removing from consumption the main infestation sources during the inactive season, the infested honey and bee pollen that were given as wintering food. The level of infestation with Nosema spp. spores at the end of the active beekeeping season (autumn) and at the beginning of the next active season (early spring) as well as the decrease of the infestation level is shown in table 6. Table 6. The Nosema spp. infestation level at the end and the beginning of the beekeeping season honeybee (6 examined colonies / years) 10 Level of infestation with Nosema spp. spores at the end of the active beekeeping season 119 (9%) Level of infestation with Nosema spp. at the beginning of the next active season 6 (0%) Decrease of the of infestation level with Nosema spp. spores 7% Removing the main contamination sources with Nosema spp., spores from the food of the honeybees during the inactive period (the wintering period) contaminated honey and bee bread is an undeniable proof of the significant decrease of the infestation level of the bees during the wintering season, an element that should be highly considered if the survival of the honeybees passing wintering period is wanted. 4. Conclusions Out of a total number of 0 examined of honey, bee pollen and reserve honey combs, a number of 0 (88%) were positive for the presence of Nosema spp. Spores. The infestation level with Nosema spp. spores of the examined ranged from 1- spores/microscopic field to more than spores/microscopic field.

6 Most of the Nosema spp. spores positive were represented by the rape and sunflower pollen. The microscopic tests carried out on honeybees at the end of the beekeeping season (autumn) as compared to the honeybees that were checked at the beginning of the next beekeeping season (early spring), proved a significant decrease for the infestation level, 7% respectively, following the removal of the contaminated food. The microscopic examination of honey, bee pollen and reserve honey combs for the presence of the Nosema spp. spores is an important prophylactic step against the infestation of the honeybees during the wintering period. The examination of the presence of Nosema spp. spores in the food reserves before wintering makes it possible to remove (eliminate) from consumption these infestation sources, a fact which significantly decreases the morbidity and the mortality rate among honeybees.. Acknowledgements This work was supported by a grant of the Romanian National Authority for Scientific Research, CNDI UEFISCDI, project number PN 108/01: Studies on the preparation and testing of an apiphytotherapeutical product for veterinary use NOSE-API for the treatment and prophylaxis of Nosema disease in bee families. References 1. M.E. COLIN, M. TOURNAIRE, L. GAUTHIER, On the epidemiology of Nosema ceranae in France. Apimondia 41st congress, Scientific programme, Oral presentation abstracts, Apimodia 41st Congress, Montpellier, , (009).. M. HIGES, R. MARTÍN-HERNÁNDEZ, C. BOTÍAS, E. GARRIDO-BAILÓN, A.V. GONZÁLEZ-PORTO, L BARRIOS, ET AL. How natural infection by Nosema ceranae causes honeybee colony collapse. Environ Microbiol 10: , (008).. HUANG, H.. Effects of Nosema on honey bee behavior and physiology. CAP Updates: 19. extension, (011). 4. OIE (World Organisation for Animal Health). Nosemosis of honey bees. In: Manual of Diagnostic Tests and Vaccines for Terestrial Animals (mammals, birds and bees), vol.1, 6 ed.: , (008).. V. SAVU, A. ŞAPCALIU. Patologia albinelor Editura Fundaţiei România de Mâine. Bucureşti. ISBN , pag 14-1 (01). 6