Nosema Apis Infection in Worker and Queen Apis Mellifera Thomas Webster, Kirk Pomper, Greg Hunt, Etta Thacker, Snake Jones
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Nosema apis infection in worker and queen Apis mellifera Thomas Webster, Kirk Pomper, Greg Hunt, Etta Thacker, Snake Jones To cite this version: Thomas Webster, Kirk Pomper, Greg Hunt, Etta Thacker, Snake Jones. Nosema apis infec- tion in worker and queen Apis mellifera. Apidologie, Springer Verlag, 2004, 35 (1), pp.49-54. 10.1051/apido:2003063. hal-00891869 HAL Id: hal-00891869 https://hal.archives-ouvertes.fr/hal-00891869 Submitted on 1 Jan 2004 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Apidologie 35 (2004) 49–54 © INRA/DIB-AGIB/ EDP Sciences, 2004 49 DOI: 10.1051/apido:2003063 Original article Nosema apis infection in worker and queen Apis mellifera Thomas C. WEBSTERa*, Kirk W. POMPERa, Greg HUNTb, Etta M. THACKERa, Snake C. JONESa a Land Grant Program, Kentucky State University, Frankfort, Kentucky 40601, USA b Department of Entomology, Entomology Hall, Purdue University, West Lafayette, Indiana 47907-1158, USA (Received 6 December 2002; revised 15 June 2003; accepted 30 June 2003) Abstract – Worker and queen honey bees were fed individually with Nosema apis spores in sucrose solution and then returned to cages containing several hundred of their worker bee nestmates. After 3 to 7 days, the workers and queens that had been fed spores were sacrificed. Worker and queen ventriculi were removed and examined for spores by light microscopy, and DNA was extracted. The DNA was subjected to amplification with polymerase chain reaction, using primer sequences specific to N. apis DNA. The PCR analysis was more sensitive than examination for spores by light microscopy, in detecting N. apis infection. Worker bees and queen bees were infected at similar rates by the inoculation procedure. Apis mellifera / Nosema apis / PCR / queen / worker 1. INTRODUCTION disease by culling older comb and by treating their hives with fumagillin (Fries, 1997). The microsporidian Nosema apis Zander The effects of N. apis on queen bees are causes a destructive disease of honey bees, Apis particularly serious for practical beekeeping. mellifera L., worldwide (Bailey and Ball, 1991; Infected queens are often superseded (Farrar, Fries, 1997). By infecting the ventriculus of 1947; Furgala, 1962). Nurse worker bees, adult bees (White, 1919; Bailey, 1955) this which may feed their queen, suffer reduced organism shortens bee life span (Kleinschmidt and Furguson, 1989) and causes greater colony hypopharyngeal gland activity when infected mortality in winter (Nitschmann, 1957). Earlier (Wang and Moeller, 1969, 1971). observations that the disease is distributed Our present study was a test of the polymer- widely across the US by the mailing and truck- ase chain reaction for the amplification and ing of honey bees (Farrar, 1947, 1954; Jay, detection of N. apis DNA extracted from 1966) probably hold true today. honey bee ventriculi. We also wished to com- A bee becomes infected when it ingests pare worker bees to queen bees for sensitivity spores while cleaning feces from the comb, to this pathogen. Perhaps the queen is less sen- left by other infected bees (Fries, 1988). Con- sitive than the workers, an adaptation that taminated food and water may also be sources would minimize the overall effects of the dis- of spores. Beekeepers routinely control the ease on the colony. * Corresponding author: [email protected] 50 T.C. Webster et al. 2. METHODS the presence of spores at 400× by light microscopy using phase contrast optics (Cantwell, 1970). The tube containing the remainder of each macerated 2.1. Inoculum preparation ventriculus was then frozen at –70 °C until the DNA extraction procedure. Spores were collected from diseased bees by adding water to the abdomens, macerating the The microcentrifuge tubes were autoclaved before tissues in a blender, filtering through Whatman 4 use. To prevent cross contamination, the fine for- filter paper, and centrifuging the filtrate. The pellet ceps used for dissection were cleaned carefully was resuspended in sucrose solution, at a final before removing the ventriculus from a bee. No concentration of 50% sucrose and 3.7 × 106 spores ventriculi ruptured during the dissection procedure. per mL. One ml of this diet weighed 1.28 g, so that Each was rinsed with distilled water immediately each mg of diet contained 2.89 × 103 spores. before placing it in the tube. 2.2. Preparation of experimental hives 2.5. Primer for N. apis PCR analyses The sequences for the small subunit of the Thirteen small honey bee hives were established ribosomal RNA gene of a number of Nosema with mature queen cells on 22 and 23 August 2001. species and close relatives were obtained from The queen cells had been reared from larvae taken internet searches of Genbank and aligned with the from the same hive. Each colony was established in Clustal program. Most of the sequences were highly a box made to hold 5 deep Langstroth frames, and conserved but two polymorphic regions were found kept at an apiary site in Franklin Co., KY. that allowed us to design specific primers specific to N. apis (Fig. 1). Primers were designed so that the 2.3. Inoculation with Nosema apis spores 3’ terminus of each primer would end in this polymorphic region, as shown in the figure. Primers The queen and approximately 200 workers from were checked with Amplify Software version 1.2 each hive were caged, during 19–30 October 2001. (Engels, 1993) to predict whether they would At this time brood rearing had nearly ended and amplify one product from the gene. workers were relatively old. Shortly after collection The Nosema apis sequence was tested for simi- each cage was filled with CO2 for several seconds larity against the Genbank non-redundant database to immobilize the bees. The queen and 20 workers with a standard nucleotide BLAST. This database were then removed, restrained on paraffin blocks contains 1.77 million sequences. There were many with insect pins, and painted with distinctive colored significant “hits” with other microsporidia RNA ge- marks on their thoraces. When the pinned bees nes. However, none of the twenty most significant revived, they were each fed with a microcapillary alignments nor the sequences we compared in our ta- tube containing the above diet of N. apis spores, a ble showed a PCR product when tested with the Am- method similar to that of Furgala and Maunder plify Software. Therefore, there is no reason to (1961). The microcapillary tube was weighed before believe that we would get a product amplified from and after feeding to determine the weight of diet and these species with our standard PCR procedure. number of spores consumed. The worker bees consumed (73.1 + 3.5) × 103 spores (mean + S.E.). The queen bees consumed (66.8 + 10.9) × 103 spores 2.6. N. apis DNA extraction (mean + S.E.). After inoculation, each bee was returned to its cage through a hole at the bottom. The following procedure was used for the Nosema apis Each cage of bees was then provided with a feeder isolation and identification of DNA in honey bee tissues. Honey bee ventriculi known to be containing 50% sucrose in water, and kept in an infected or free of N. apis were collected and stored incubator at 25 °C for 3 to 7 days. at –70 °C until their use as positive and negative controls. Approximately 0.05 g of tissue was used 2.4. Examination of tissues for spores for DNA extraction using a modified protocol for the Wizard Genomic DNA Purification Kit (Promega After either 3 (2 cages), 4 (4 cages), 6 (2 cages) Corporation, Madison, WI). Tissue was ground in a or 7 days (5 cages) the bees in each cage were mortar and then transferred to a 1.5 mL microfuge immobilized with CO2. Surviving, marked bees tube. Nuclei Lysis Solution (NLS, 200 µL) was were removed for dissection. The ventriculus was added to each tube and it was then vortexed for 3 s removed from each bee, placed in a 1.5 mL plastic in order to wet the tissue. The sample was then microcentrifuge tube, and macerated with a small incubated at 65 °C for 60 min, after which 2 µL plastic pestle. A small drop of the preparation was RNAse (10 mg/mL; Sigma Chemical Co., Saint removed and examined on a microscope slide for Louis, MO) solution was added and mixed by Nosema apis in Apis mellifera 51 Figure 1. Host species and alignment of DNA sequence for small subunit RNA from Nosema species used for primer design. Genbank (http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers: N. ceranae, 857489; N. spp. from silk worm 1339944 (now revised to 1838930); N. vespula, 507913, Vairimorpha sp., from human, 954829; N. Oulemae from Oulema melanopus L. (Coleoptera), 849158; N. apis from Apis mellifera, 857487. The primer sequences are underlined. inverting, and the tube incubated at 37 °C for another Biotech, Cambridge, England). All samples were 15 min. The sample was then allowed to cool to stored at 4 °C until needed. room temperature for approximately 5 min prior to adding 67 µL protein precipitation solution. The sample was then vortexed vigorously for about 20 s 2.7. PCR amplification conditions and then centrifuged (15 800 g) for 5 min. The A 20 µL reaction volume was used that contai- supernatant was removed without disturbing the pellet and transferred to a new tube containing ned 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 250 µL isopropanol.