Med. Weter. 2013, 69 (12) 733
Praca oryginalna Original paper
Nosemosis is a parasitic disease affecting adult bees, which causes significant economic losses in apicultures worldwide. The disease is caused by two microspo-ridian species, Nosema apis and N. ceranae. Those highly specialized fungi (2, 15) develop inside host cells, existing outside the host only as metabolically inactive spores. Nosema apis infection is restricted to the midgut epithelium of adult bees, Nosema ceranae was shown to have also infected Malpighian tubules, fat body, hypopharyngeal glands, salivary glands (7).
N. apis has been well-known for a long time while N. ceranae was first identified as a disease of Apis mellifera in Europe in 2006, based on its genetic
sequence (9). Today N. ceranae is the predominant species known to shorten bee life spans. The parasite causes massive colony depopulations accompanied by a drop in honey production, as well as a decline in the number of pollinators and bee-pollinated plants, which can adversely affect natural ecosystems and agriculture (8).
Nosema spp. spores are routinely identified at the
species level (N. apis and N. ceranae) based on their morphological structure, but this method of species identification may be inaccurate due to minor dif-ferences in the size and shape of spores (4, 6). Both
species also have similar life cycles, which is why nowadays Nosema spp. species are identified by the PCR technique with the use of primers designed for the small subunit 16S rRNA (14). The presence of both microsporidian species has been confirmed in Poland (13).
Since apiculture is particularly well-developed in north-eastern Poland, and bee queens (genetic mate-rial) are imported from various parts of Europe, the objective of this study was to perform a phylogenetic analysis of a fragment of the small subunit 16S rRNA of N. apis and N. ceranae in honey bee (Apis mellifera) colonies from this region.
Material and methods
Sampling. The experimental material comprised worker
bees from 30 bee colonies infected with Nosema spp. spores (the presence of spores was confirmed by light microscopy at 400 × magnification), kept in 15 apiaries in north-eastern Poland (Bartoszyce, Braniewo, Ełk, Nidzica, Olsztyn, Suwałki). Each apiary comprised 60 to 100 bee colonies. The distance between apiaries was less than 80 km. Ten worker bees from frames of open broods, randomly selected from each colony, were put down and stored at a tempera-ture of –18°C until analysis. All collected samples were analyzed.
DNA isolation. For DNA isolation, 10 abdomens were
ground in a mortar with the addition of 10 ml of water. The
Phylogenetic analysis of Nosema apis and Nosema
ceranae small subunit 16S rRNA in honey bees
(Apis mellifera) from north-eastern Poland*
)
MARIA MICHALCZYK, RAJMUND SOKÓŁ, ANNA SZCZERBA-TUREK*
Department of Parasitology and Invasive Disease, *Department of Epizootiology, Faculty of Veterinary Medicine, University of Warmia and Mazury in Olsztyn, Oczapowskiego 13, 10-718 Olsztyn, Poland
*) This research project was funded by the National Science Center, grant
number 5914/B/PO1.2011/40.
Michalczyk M., Sokół R., Szczerba-Turek A.
Phylogenetic analysis of Nosema apis and Nosema ceranae small subunit 16S rRNA in honey bees (Apis mellifera) from north-eastern Poland
Summary
In order to perform a phylogenetic analysis of Nosema spp. spores, samples of 10 worker bees each were collected from 30 infected colonies (the presence of spores was confirmed by light microscopy) kept in 15 apiaries in north-eastern Poland. Both N. apis and N. ceranae are common in this region (mixed infection N. apis/ N. ceranae – 60%, N. ceranae – 37%, N. apis – 3%). The DNA samples of N. apis were 100% identical with the N. apis sequences deposited in the GenBank database in Queensland, Australia, Spain, New Zealand, Lithuania and Tasmania in Australia. The DNA samples of N. ceranae were found to be 99.5%-100% identical with the N. ceranae sequences previously published in Italy, Germany, Switzerland and Austria.
Med. Weter. 2013, 69 (12) 734
Fig. 1. Phylogenetic tree based on small subunit 16S rRNA. The evolutionary history was inferred using the UPGMA me-thod. The optimal tree with the sum of branch length = 4.58671062 is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method and are in the units of the number of base substitutions per site. The analysis involved 21 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. All posi-tions containing gaps and missing data were eliminated. There were a total of 172 posiposi-tions in the final dataset. Evolutionary analyses were conducted in MEGA5.
N.ceranae Turin Italy (HM859898) N.ceranae Poland (JX860434) N.ceranae Spain (DQ329034) N.ceranae France (DQ374655) N.ceranae Germany (DQ374656) N.ceranae Switzerland (DQ673615) N.ceranae Austria (EU045844) N.ceranae Mexico (HM581509)
N.ceranae Nova Scotia Canada (EU545141) N.ceranae Austria (EF458656)
N.ceranae Italy (HM859899) N.ceranae Thailand (GU045466)
N.ceranae Queensland Australia (FJ789797) N.ceranae Taiwan (DQ486027)
N.ceranae Taiwan (DQ078785) N.apis New Zealand (U97150) N.apis Spain (DQ235446)
N.apis Queensland Australia (FJ789793) N.apis Tasmania Australia (FJ789790) N.apis Lithuania (JQ639306) N.apis Poland (JX860435) 0.0 0.2 0.4 0.6 0.8
resulting suspension was filtered through muslin gauze and centrifuged at 800 g for six minutes. Genomic DNA was isolated using the Genomic DNA Mini Kit (A&A Biotech-nology, Gdynia, Poland). Purified DNA was stored in test- -tubes at a temperature of –20°C for further analysis.
PCR procedure. The multiplex PCR assay involved the
amplification of the small subunit 16S rRNA sequences of N. apis and N. ceranae. The sequence of DNA primers developed by the Institute of Biochemistry and Biophysics of the Polish Academy of Sciences was obtained from the OIE Terrestrial Manual (http://www.oie.int/fileadmin/ Home/eng/ Health_standards/tahm/2.02.04_NOSEMO-SIS.pdf). The following primer sequences were used: for N. cerana – MITOC FOR (5’-CGGCGACGATGT-GATATGAAAATATTAA-3’) and MITOC REV (5’-CCC-GGTCATTCTCAAACAAAAAACCG-3’), amplifying products with the length of 218 bp, and for N. apis – APIS FOR (5’-GGGGGCATGTCTTTGACGTACTATGTA-3’) and APIS REV (5’-GGGGGGCGTTTAAAATGTGAAA-CAACTATG-3’), amplifying products with the length of 321 bp. The multiplex PCR analysis was carried out using HotStarTaq Plus Polymerase (Qiagen). The reaction mixture of 20 µl comprised around 120 ng isolated DNA (from 1 to 3 µl), 10 µl HotStarTaq Plus Master Mix 2 ×, 2 µl CoralLoad Concentrate 10 ×, 0.1 µl of each primer (with a final concentration of 0.5 µM), supplemented with RNase-Free Water to 20 µl. The reaction was carried out in the Eppendorf Mastercycler thermocycler. PCR commenced with initial denaturation for 5 minutes at 95°C. The reaction mixture was then cycled 35 times, in the following steps: denaturation at 94°C for 45 seconds, primer annealing at 55°C for 45 seconds, and extension at 72°C for 1 minute. The last reaction was followed by final chain synthesis at 72°C for 10 minutes.
Separation and analysis of PCR products. The products
of the multiplex PCR reaction were separated by
electro-phoresis in 2% agarose gel in 1 × TAE at 5 V/cm. The size of the obtained products was evaluated by comparison with the GeneRulerTM 100 bp 36 Ladder Plus (Fermentas) mo-lecular size marker. Ethidium bromide was added to the gel at 0.5 µg/ml to visualize the resulting DNA fragments of Nosema spp. Electrophoresis results were archived using the GelDoc (Bio-Rad) gel documentation system.
Phylogenetic analysis. The amplicons were purified
using a clean-up kit (A&A Biotechnology, Poland) in ac-cordance with the manufacturer’s instructions. Purified amplicons were sequenced in the Genomed S.A., Warsaw (Poland). Sequence data from the specimens were compared with the nucleotide sequence 16S rRNA from NCBI using BLASTIN version 2.2.18. (1). A sequence analysis was performed using the BioEdit Sequence Alignment Editor and Clustal W software (11). The evolutionary history was inferred from phylogenetic analysis using the UPGMA
Med. Weter. 2013, 69 (12) 735 method (16). The evolutionary distances were computed
using the Maximum Composite Likelihood method (17), and phylogenetic analyses were conducted using the freeware Computational Evolutionary Biology package MEGA5 (18).
The sequence of our strains of N. apis and N. ceranae has been published GenBank and assigned the numbers (N. apis – JX860435) and (N. ceranae – JX860434).
Results and discussion
Nosema spp. spores were found in all 30 samples.
The presence of mixed infection (N. apis and N.
cera-nae) was confirmed in 18 samples (60%), N. ceranae
was identified in 11 samples (37%), and N. apis in one sample (3%). The sequences of all N. apis and
N. ceranae amplicons were analyzed.
The sequenced amplicons of N. apis were 100% identical with the N. apis sequences deposited in the GenBank databases in Queensland, Australia (Acc. No. FJ789793), Tasmania, Australia (Acc. No. FJ89790), New Zealand (Acc. No. U97150), Spain (Acc. No. DQ235446), and Lithuania (Acc. No. JQ639306). The sequenced amplicons of N. ceranae were found to be 99.5%-100% identical with the N. ceranae sequences previously published in Italy (Acc. No. HM859898), Germany (Acc. No. DQ374656), Switzerland (Acc. No. DQ673615), Austria (Acc. No. EU045844) and Iran (Acc. No.JF431546). The sequenced amplicons of N. ceranae from Poland were shown to be 97% identical with the N. ceranae sequences deposited in the GenBank database in Taiwan (Acc. No. DQ078785) and Thailand (Acc. No. GU045466) (Fig. 1).
The use of highly sensitive PCR methods has sup-ported the identification of two Nosema spp. species in honey bees (9, 10, 14). Medici et al. (12) docu-mented the presence of N. ceranae in honey bees from Argentina. Their DNA samples were 98% identical with the sequences deposited in the GenBank database (Canada, Austria, Switzerland, Germany, Spain, China) and 83% identical with the sequences of N. apis (Spain, New Zealand, Canada, Austria), while only 2.5% of the tested samples contained N. apis DNA.
In our experiment, the DNA sequences of N.
cera-nae (Acc. No. JX860434) were highly similar to the
sequences from Italy, Germany, Switzerland, Austria, Iran and the DNA sequences of N. apis (Acc. No. JX860435) were similar to the reference sequences from Australia (Acc. No. FJ89793), Lithuania (Acc. No. JQ639306). This was the first study of the type conducted in our country. We demonstrated a high level of genetic similarity between N. ceranae reported in Poland and other European countries.
According to Chaimanee et al. (3), a sequence analysis of the 16sRNA gene fragment, performed as part of a phylogenetic analysis of N. ceranae was not always sufficient to elucidate genetic relationships among N. ceranae strains isolated from four different host species. The cited authors concluded that the use
of the above gene as a marker was limited. Therefore, further in-depth analysis of the parasite’s genetic mate-rial obtained from bee colonies in Poland is required.
N. apis and N. ceranae infect Apis mellifera and share
similarities in sequences of rRNA gene. Phylogenetic analysis showed that N. apis is not the closest relative of N. ceranae (5).
References
1. Altschul S. F., Schäffer A. A., Zhang J., Zhang Z., Miller W., Lipman D. J.: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997, 25, 3389-3402.
2. Cavalier-Smith T.: A revised six – kingdom system of life. Biol. Rev. 1998, 73, 203-266.
3. Chaimanee V., Chen Y., Pettis J. S., Cornman R. S., Chantawannakul P.: Phylogenetic analysis of Nosema ceranae isolated from European and Asian honeybees in Northern Thailand. J. Invertebr. Pathol. 2011, 107, 229-233. 4. Chen Y. P., Evans J. D., Murphy C., Gutell R., Zuker M.,
Gundensen-Rindal D., Pettis J. S.: Morphological, molecular, and phylogenetic
charac-terization of Nosema ceranae, a microsporidian parasite isolated from the European honey bee, Apis mellifera. J. Eukaryot. Microbiol. 2009, 56, 142-147. 5. Chen Y. P., Huang Z. Y.: Nosema ceranae, a newly identified pathogen
of Apis mellifera in the USA and Asia. Apidologie 2010, 41, 364-374. 6. Fries I.: Nosema ceranae in European honey bees (Apis mellifera).
J. Invertebr. Pathol. 2010, 103, 73-79.
7. Gisder S., Möckel N., Linde A., Genersch E.: A cell culture model for Nosema ceranae and Nosema apis allows new insights into the life cycle of these important honey bee-pathogenic microsporidia. Environ. Microbiol. 2011, 13, 404-413.
8. Higes M., Garcia-Palencia P., Martin-Hernandez R., Meana A.: Experi- mental infection of Apis mellifera honeybees with Nosema ceranae (Micro- sporidia). J. Invertebr. Pathol. 2007, 94, 211-217.
9. Higes M., Martin-Hernández R., Meana A.: Nosema ceranae, a new micro- sporidian parasite in honeybees in Europe. J. Invertebr. Pathol. 2006, 92, 93-95. 10. Klee J., Beasana A. M., Genersch E., Gisder S., Nanetti A., Tam D. Q., Chinh
T. X., Puerta F., Ruz J. M., Kryger P., Message D., Hatjina F., Korpela S., Fries I., Paxton R. J.: Widespread dispersal of the microsporidian Nosema
ceranae an emergent pathogen of the western honey bee, Apis mellifera. J. Invertebr. Pathol. 2007, 96, 1-10.
11. Larkin M. A., Blackshields G., Brown N. P., Chenna R., McGettigan P. A.,
McWilliam H., Valentin F., Wallace I. M., Wilm A., Lopez R., Thompson J. D., Gibson T. J., Higgins D. G.: Clustal W and Clustal X version 2.0.
Bioinformatics. 2007, 23, 2947-2948.
12. Medici S. K., Sarlo E. G., Porrini M. P., Braunstein M., Eguaras M. J.: Genetic variation and widespread dispersal of Nosema ceranae in Apis mellifera apiaries from Argentina. Parasitol Res. 2012, 110, 859-864.
13. Michalczyk M., Sokół R., Szczerba-Turek A., Bancerz-Kisiel A.: A comparison of effectiveness of the microscopic and the multiplex PCR method in iden-tifying and discriminating the species of Nosema spp. spores in worker bees (Apis mellifera) from winter hive debris. Pol. J. Vet. Sci. 2011, 14, 385-391. 14. Paxton R., Klee J., Korpela S., Fries I.: Nosema ceranae has infected Apis
mellifera in Europe since at least 1998 and may be more virulent than Nosema apis. Apidologie 2007, 38, 558-565.
15. Sina M., Alastair G., Farmer M., Andersen R., Anderson O., Barta J.,
Bowser S., Brugerolle G., Fensome R., Fredericq S., James T., Karpov S., Kugrens P., Krug J., Lane C., Lewis L., Lodge J., Lynn D., Mann D., Maccourt R., Mendoza L., Moestrup O., Mozley S., Nerad T., Shearer C., Smirnov A., Spiegel F., Taylor M.: The New Higher level classification of
Eukaryotes with emphasis on the taxonomy of Protists. J. Eukar. Microbiol. 2005, 52, 399-451.
16. Sneath P. H. A., Sokal R. R.: Numerical taxonomy; the principles and practice of numerical classification. San Francisco, WH Freeman 1973, 230-234. 17. Tamura K., Nei M., Kumar S.: Prospects for inferring very large phylogenies
by using the neighbor-joining method. Proc. Natl. Acad. Sci. 2004, 101, 11030-11035.
18. Tamura K., Peterson D., Peterson N., Stecher G., Nei M., Kumar S.: MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol. Biol. Evol. 2011, 28, 2731-2739.
Corresponding author: M. Michalczyk, Oczapowskiego 13, 10-718 Olsztyn, Poland; e-mail: maria.michalczyk@uwm.edu.pl
