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Phylogenetic analysis of Nosema ceranae isolated from European and Asian honeybees in Northern Thailand
Journal of Invertebrate Pathology 107 (2011) 229–233
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Phylogenetic analysis of Nosema ceranae isolated from European and Asian honeybees in Northern Thailand Veeranan Chaimanee a, Yanping Chen b, Jeffery S. Pettis b, R. Scott Cornman b, Panuwan Chantawannakul a,c,⇑ a
Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand USDA-ARS, Bee Research Laboratory, Beltsville, MD 20705, USA c Materials Science Research Center, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand b
a r t i c l e
i n f o
Article history: Received 16 January 2011 Accepted 3 May 2011 Available online 10 May 2011 Keywords: Nosema ceranae Apis mellifera Apis cerana Apis dorsata Apis florea Polar tube protein (PTP 1) gene Phylogenetic relationship
a b s t r a c t Nosema ceranae was found to infect four different host species including the European honeybee (A. mellifera) and the Asian honeybees (Apis florea, A. cerana and Apis dorsata) collected from apiaries and forests in Northern Thailand. Significant sequence variation in the polar tube protein (PTP1) gene of N. ceranae was observed with N. ceranae isolates from A. mellifera and A. cerana, they clustered into the same phylogenetic lineage. N. ceranae isolates from A. dorsata and A. florea were grouped into two other distinct clades. This study provides the first elucidation of a genetic relationship among N. ceranae strains isolated from different host species and demonstrates that the N. ceranae PTP gene was shown to be a suitable and reliable marker in revealing genetic relationships within species. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Nosema genus is a parasitic microsporidia commonly infecting Lepidoptera (Tsai et al., 2003) and Hymenoptera (Fries et al., 1996; Higes et al., 2006) but has also been found in Orthoptera (Henry, 1971) and Amphipoda (Terry et al., 1999). Nosema apis and Nosema ceranae are the two microsporidian species that are reported as important honeybee pathogens. N. apis has long been known as a pathogen that infects the European honeybee, Apis mellifera (Zander, 1909). However recent studies have shown that N. ceranae, a species that was first known to infect the Asian honeybee, Apis cerana (Fries et al., 1996) has become the dominant species infecting honeybees worldwide (Fries et al., 2006; Higes et al., 2006; Chauzat et al., 2007; Cox-Foster et al., 2007; Huang et al., 2007; Klee et al., 2007; Paxton et al., 2007; Chen et al., 2008; Williams et al., 2008; Chaimanee et al., 2010). N. ceranae infection has impacts at both the individual honeybee (Paxton et al., 2007; Higes et al., 2009; Martín-Hernández et al., 2007) and colony level, and has been associated with honeybee colony losses in Spain (Higes et al., 2008, 2009). The early classification of Nosema species was based on morphological characters (eg. spore size, shape and ultrastructure), life ⇑ Corresponding author at: Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. Fax: +66 05 3892259. E-mail address: [email protected] (P. Chantawannakul). 0022-2011/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2011.05.012
cycle and pathological symptoms within the host range. However, it is difficult to differentiate the closely related species using morphological characters (Weiss and Vossbrinck, 1999). The genes coding for the rRNAs of the small and large subunit of the ribosomes have been used in the taxonomic determination of microsporidia and in the differentiation of Nosema species (Ku et al., 2007; Huang et al., 2008; Williams et al., 2008; Chen et al., 2009). The rRNA genes have been widely used for identification, molecular characterization and as a molecular marker for phylogenetic analysis among eukaryotes. The multiple copies of rRNA are a common phenomenon of microsporidia (Tay et al., 2005; O’Mahony et al., 2007), then the rRNA genes may not be ideal choices for the phylogenetic analysis of microsporidia. Therefore, the study of phylogenetic relationships among species or strains of Nosema need to be based on non-repeated loci (Sagastume et al., 2011). Recently, Chaimanee et al. (2010) reported that N. ceranae had been detected not only in the European honeybee (A. mellifera), but also in the cavity nesting Asian honeybee (A. cerana), the dwarf Asian honeybee (Apis florea) and the giant Asian honeybee (Apis dorsata) in Northern Thailand. However, the genetic relationship of possible N. ceranae isolates from different host species has not been determined. In this study, we conducted phylogenetic studies using sequences of the 16S rRNA gene and of the gene coding for the polar tube protein 1 (PTP 1) of N. ceranae independently to determine the genetic relationship of N. ceranae strains that were isolated from four different honeybee species.
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2. Materials and Methods 2.1. Samples The samples of Nosema infected A. mellifera (N = 10), A. cerana (N = 8), A. florea (N = 10) and A. dorsata (N = 10), were collected from Northern Thailand in 2008–2009 and stored at 20 °C prior to molecular analysis.
closely linked to that of E. cuniculi. Phylogenetic trees were constructed with the Mega 4 program (Tamura et al., 2007) using the maximum parsimony method (MP) and the close-neighborinterchange algorithm with search level one in which the initial trees were obtained with the random addition of sequences. One thousand bootstrap replicates were assessed to test the robustness of the tree. 3. Results
2.2. DNA extraction 3.1. Detection of N. ceranae in different honeybee species Genomic DNA was extracted from individual bees, homogenized with a pestle and the crude homogenate suspended in 400 ll of CTAB buffer (100 mM Tris–HCl, pH8.0; 20 mM EDTA, pH 8.0; 1.4 M sodium chloride; 2% (w/v) cetyltrimethylammonium bromide; 2% 2-mercaptoethanol). The 0.15 g of glass beads (425– 600 lm, Sigma-Aldrich, St.Louis, MO) were added in the spore suspensions and the mixture shaken at the maximum speed for 4– 5 min using a FastPrep cell Disrupter (Qbiogene, Carlsbad, CA). The mixture was incubated with proteinase K (200 lg/ml) at 55 °C overnight. DNA was extracted from suspensions using a DNA purification Kit, DNAzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The concentration of total DNA was determined by using the NanoDrop spectrophotometer (Thermo Scientific, USA). 2.3. PCR amplification and DNA sequencing A region of the 16S rRNA gene was amplified using the primer set described by Chen et al. (2008). Two primer sets, N. apisforward/N. apis-reverse (50 -CCATTGCCGGATAAGAGAGT-30 /50 -CA CGCATTGCTGCATCATTGAC-30 ) and N. ceranae-forward/N. ceranaereverse (50 -CGGATAAAAGAGTCCGTTACC-30 /50 -TGAGCAGGGTT CTAGGGAT-30 ), were individually used to amplify a 269 bp fragment of N. apis and a 250 bp product of N. ceranae, respectively. A pair of primers based on the hypothetic PTP gene of N. ceranae isolated from A. mellifera (XM_002995447) were designed to amplify a 838 bp product. The sequences of the primers used for PCR were as follows: NcORF 1664-forward: 50 -GACAACAAGGAAGA CCTGGAAGTG-30 and NcORF-1664 reverse: 50 -TGT GAATAAGAGGGTGATCCTGTTGAG-30 . PCR amplification was carried out in a 25 ll reaction mixture containing 1 high fidelity PCR buffer, 0.1 mM each dNTP, 2 mM MgSO4, 0.2 lM of each primer, 1U of Platinum Taq High Fidelity polymerase (Invitrogen; Carlsbad, CA) and 500 ng of total genomic DNA. The amplication conditions were 95 °C for 2 min, 40 cycles of denaturation at 94 °C for 15 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s with a final extension at 72 °C for 10 min on a thermal cycler. The PCR product from each reaction was electrophosed on 1.0% low melting point agarose gel (Invitrogen). The specificity of amplified PCR products was verified by purifying PCR fragments using Wizard PCR Prep DNA Purification System (Promega, Medison, WI) according to the manufacturer’s instruction and sequenced using both forward and reverse primers by the Center for Biosystems Research, University of Maryland Biotechnology Institute. 2.4. Phylogenetic analysis The DNA sequences of PTP obtained from this study were aligned using the ClustalX version 1.83 program. After alignment the unaligned sequences at both ends were truncated. Encephalitozoon cuniculi (GenBank Accession No. NM_001041403.1), a mammalian microsporidian species, was used as an outgroup to root the phylogenetic tree since the gene encoding this protein is
When we amplified the partial 16S rRNA gene of Nosema using the primer set N. apis-forward/N. apis-reverse and N.ceranaeforward/N.ceranae-reverse, a 250 bp fragment specific for N. ceranae was amplified from all samples. No N. apis specific PCR products were genereated from any samples examined. This is consistant with our previous findings that N. ceranae but not N. apis could be isolated from four different honeybee species (Chaimanee et al., 2010). The primer set of NcORF-1664 forward/NcORF-1664 reverse amplifying a 808 bp-fragment of the PTP 1 gene yielded a PCR-product of the expected size (Fig. 1A). Sequence analysis further confirmed the specificity of PCR amplification by PTP primers. 3.2. Phylogenetic analysis Based on the partial polar tube protein (PTP 1) sequences of N. ceranae isolates, no difference has been found amongst N. ceranae isolates from A. mellifera and A. cerana. We observed 22 and 7 nucleotide differences amongst the N. ceranae isolates from A. florea and A. dorsata and share 97% and 99% similarity, respectively with isolates from A. mellifera and A. cerana. No differences N. ceranae strains found within host species. Deletion occurrence in N. ceranae sequences that found in Thailand at 325th bp to 354th bp when compared to the N. ceranae sequence (XM_002995447) from USA (Fig. 2). We analyzed the phylogenetic relationship amongst N.ceranae isolates found in A. mellifera, A. cerana, A. florea and A. dorsata based on the partial sequences of 16S rRNA and PTP genes separately. The phylogenetic tree based on the partial rRNA gene sequences resulted in N. ceranae from the four different honeybee species forming one clade. However, the phylogenetic tree based on the partial protein coding region (PTP 1) gene sequences showed three distinct clades. N. ceranae isolated from A. mellifera grouped into the same clade as N. ceranae isolated from A. cerana supported by a high bootstrap value (97%). However, N. ceranae isolated from A. dorsata and from A. florea formed district clades with bootstrap value 95% and 99%, respectively (Fig. 1B). 4. Discussion Recently, it has been shown that N. ceranae is capable of infecting multiple host species including European honey bees, Asian honey bees, and bumble bees (Chaimanee et al., 2010; Plischuk et al., 2009). The wide host range of this parasite is of significant epidemiological concern. However, the host-parasite interactions that determine host susceptibility to the parasite remain poorly understood and the genetic relationship of different N. ceranae isolates from different host species had not been previously explored. While the phylogeny of the hosts should play an important role in host-parasite interactions and the establishment of successful infection, the phylogeny of the parasites may also aid in our understanding of host range expansion. In this study, we analyzed the phylogenetic relationships between N. ceranae isolated from four different honeybee species.
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Fig. 1. PCR amplification and phylogenetic analysis of N. ceranae isolated from four different honeybee species (A. mellifera, A. cerana, A. dorsata and A. florea). The partial PTP 1 gene sequences were amplified with a primer set NcORF-1664 forward/NcORF-1664 reverse (A). Phylogenetic analysis of N. ceranae isolates based on the PTP 1 gene. The maximum parsimony tree was generated by MEGA 4 software using the close-neighbor-interchange algorithm with search level one in which the initial trees were obtained with the random addition of sequences (10 replicates). E. cuniculi was used as an outgroup for polar tube protein phylogenetic tree. The tree used 1000 bootstrap replicates (B).
One of the major prerequisites of phylogenetic studies is the identification of useful genetic markers. The 16S rRNA genes contain highly conserved regions among all living organisms. This trait makes rRNAs the most widely used molecular genetic marker for microbial studies. However, the use of rRNA markers in genetic studies of microsporidia has some limitations. The presence of multiple non-homologous copies of rRNA is a common feature of microsporidia because the large demand for ribosomes for driving protein synthesis can only be met by multiple copies of rRNA. A previous study reported the presence of variants of the small subunit gene in the same isolate of N. ceranae (Sagastume et al., 2011). The presence of multiple copies of ribosomal RNA genes in the same cell was also found in N. bombi (Tay et al., 2005; O’Mahony et al., 2007). Thus, Fries (2010) pointed out that the ribosomal RNA sequences are probably not appropriate for phylogenetic approaches in Nosema species. Sagastume et al. (2011) also suggested that rRNA gene sequences are not suitable for a reliable differentiation of N. ceranae strains and the study of phylogenetic relationships among Nosema species needs to be established on the basis of non-repeated loci. In agreement with previous conclusions, our present study based on the partial 16S ribosomal RNA gene
showed that sequence variation of rRNA genes of N. ceranae was insufficient for elucidating the genetic relationship of N. ceranae isolates. The first phylogenetic relationship analysis of microsporidia based on the protein-coding genes was conducted using sequences of the largest subunit of RNA polymerase II (RPB1) (Cheney et al., 2001) and focused on resolving the phylogenetic relationships of polyporous species. They reported that a RPB1 tree of 14 mainly polyporous species gave a better resolution than 16S rRNA gene tree. More recently, Ku et al. (2007) suggested that the partial sequences of the less conserved gene (alpha-tubulin, beta-tubulin and RPB1 genes) will be better candidates than rRNA genes for Nosema species identification. Similarly, Lee et al. (2008) reconstructed the phylogenetic tree between Brachiola algerae and Antonospora locustae based on the alpha- and beta-tubulin sequences. They revealed closely related relationships between B. algerae and A. locustae that were previously unclear from SSU rRNA sequences. Since the polar tube proteins (PTP 1, PTP 2 and PTP 3) play an important role in the host invasion process, N. ceranae may have adapted the polar tube component to be more host specific as the host and parasite co-evolve. Moreover, the
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Fig. 2. Sequence alignment of the partial PTP 1 gene of N. ceranae isolated from A. mellifera, A. cerana, A. florea and A.dorsata in Thailand and N. ceranae isolated from A. mellifera in USA (XM_002995447). The asterisk () indicates the location of one hundred percent identical aligned nucleotide. Single nucleotide differences are shaded in dark grey and columns with less than 80% conservation are boxed. Gaps introduced for optical alignment are indicated by a dash.
O-mannosylated moiety of PTP 1 can interact with a host cell mannose receptor (Xu and Weiss, 2005). This interaction could be an important part of the infection eventually leading to the cell invasion (Texier et al., 2010). However, it was recently shown that cell invasion of N. ceranae and N. apis obviously does not depend on prior interaction of the polar tube with the cell surface (Gisder et al., 2011). In the current study, we designed the primer set for amplification of the partial PTP 1 gene. When we reconstructed the phylogenetic tree based on the partial sequences of the PTP1 gene, it easily distinguished the N. ceranae isolates found in the four honeybee species, demonstrating PTP is a useful genetic marker for studying the relationships of N. ceranae at the strain level. Both A. mellifera and A. cerana are the cavity-nesting honeybees and therefore more closely related than other single comb nesting, A. dorsata and A. florea species (Alexander, 1991). The formation of a single clade for N. ceranae isolates from the cavity-nesting species reflects the genetic lineage of A. mellifera and A. cerana and suggests ongoing co-evolution of this pathogen and its hosts. Acknowledgments We would like to thank the Thailand Commission on Higher Education for grant support funding under the program of Strategic Scholarships for Frontier Research Network for the Joint Ph.D. Program Thai Doctoral degree and National Research University Project under Thailand’s Office of the Higher Education Commission. We also thank the Thailand Research Fund (RSA 5280010) and the USDA-ARS Bee Research Laboratory for financial support. We thank Michele Hamilton for her excellent technical help with the project. References Alexander, B., 1991. A cladistic analysis of the genus Apis. In: Smith, D.R. (Ed.), Diversity in the Genus Apis. Westview Press, Boulder, pp. 1–28. Chaimanee, V., Warrit, N., Chantawannakul, P., 2010. Infections of Nosema ceranae in four different honeybee species. J. Invertebr. Pathol. 105, 207–210.
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V. Chaimanee et al. / Journal of Invertebrate Pathology 107 (2011) 229–233 Ku, C.T., Wang, C.Y., Tsai, Y.C., Tzeng, C.C., Wang, C.H., 2007. Phylogenetic analysis of two putative Nosema isolates from Cruciferous Lepidopteran pests in Taiwan. J. Invertebr. Pathol. 95, 71–76. Lee, R.C.H., Williams, B.A.P., Brown, A.M.V., Adamson, M.L., Keeling, P.J., Fast, N.M., 2008. F- and b- tubulin phylogenies support a close relationship between the microspoidia Brachiola algerae and Antonospora locustae. J. Eukaryote. Microbiol. 55, 388–392. Martín-Hernández, R., Meana, A., Prieto, L., Martínez Salvador, A., Garrido-Bailón, E., Higes, M., 2007. Outcome of colonization of Apis mellifera by Nosema ceranae. Appl. Environ. Microbiol. 73, 6331–6338. O’Mahony, E.M., Tay, W.T., Paxton, R.J., 2007. Multiple rRNA variants in a single spore of the microsporidian Nosema bombi. J. Eukaryot. Microbiol. 54, 103–109. Paxton, R., Klee, J., Korpela, S., Fries, I., 2007. Nosema ceranae has infected Apis mellifera Europe in at least 1998 and may be more virulent than Nosema apis. Apidologie 38, 558–565. Plischuk, S., Martín-Hernández, R., Lucía, M., Prieto, L., Botías, C., Meana, A., Abrahamovich, A.H., Lange, C., Higes, M., 2009. South American native bumblebees (Hymenoptera: Apidae) infected by Nosema ceranae (Microsporidia), an emerging pathogen of honeybees (Apis mellifera). Environ. Microbio. Reports 1, 131–135. Sagastume, S., DelÁguila, C., Martín-Hernández, R., Higes, M., Henriques-Gil, N., 2011. Polymorphism and recombination for rDNA in the putatively asexual microsporidian Nosema ceranae, a pathogen of honeybees. Environ. Microbiol. 13, 84–95. Tamura, K., Dudley, J., Nei, M., Kumar, S., 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0.. Mol. Biol. Evol. 24, 1596– 1599.
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Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Phylogenetic analysis of Nosema ceranae isolated from European and Asian honeybees in Northern Thailand Veeranan Chaimanee a, Yanping Chen b, Jeffery S. Pettis b, R. Scott Cornman b, Panuwan Chantawannakul a,c,⇑ a
Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand USDA-ARS, Bee Research Laboratory, Beltsville, MD 20705, USA c Materials Science Research Center, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand b
a r t i c l e
i n f o
Article history: Received 16 January 2011 Accepted 3 May 2011 Available online 10 May 2011 Keywords: Nosema ceranae Apis mellifera Apis cerana Apis dorsata Apis florea Polar tube protein (PTP 1) gene Phylogenetic relationship
a b s t r a c t Nosema ceranae was found to infect four different host species including the European honeybee (A. mellifera) and the Asian honeybees (Apis florea, A. cerana and Apis dorsata) collected from apiaries and forests in Northern Thailand. Significant sequence variation in the polar tube protein (PTP1) gene of N. ceranae was observed with N. ceranae isolates from A. mellifera and A. cerana, they clustered into the same phylogenetic lineage. N. ceranae isolates from A. dorsata and A. florea were grouped into two other distinct clades. This study provides the first elucidation of a genetic relationship among N. ceranae strains isolated from different host species and demonstrates that the N. ceranae PTP gene was shown to be a suitable and reliable marker in revealing genetic relationships within species. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction Nosema genus is a parasitic microsporidia commonly infecting Lepidoptera (Tsai et al., 2003) and Hymenoptera (Fries et al., 1996; Higes et al., 2006) but has also been found in Orthoptera (Henry, 1971) and Amphipoda (Terry et al., 1999). Nosema apis and Nosema ceranae are the two microsporidian species that are reported as important honeybee pathogens. N. apis has long been known as a pathogen that infects the European honeybee, Apis mellifera (Zander, 1909). However recent studies have shown that N. ceranae, a species that was first known to infect the Asian honeybee, Apis cerana (Fries et al., 1996) has become the dominant species infecting honeybees worldwide (Fries et al., 2006; Higes et al., 2006; Chauzat et al., 2007; Cox-Foster et al., 2007; Huang et al., 2007; Klee et al., 2007; Paxton et al., 2007; Chen et al., 2008; Williams et al., 2008; Chaimanee et al., 2010). N. ceranae infection has impacts at both the individual honeybee (Paxton et al., 2007; Higes et al., 2009; Martín-Hernández et al., 2007) and colony level, and has been associated with honeybee colony losses in Spain (Higes et al., 2008, 2009). The early classification of Nosema species was based on morphological characters (eg. spore size, shape and ultrastructure), life ⇑ Corresponding author at: Department of Biology, Faculty of Science, Chiang Mai University, Chiang Mai 50200, Thailand. Fax: +66 05 3892259. E-mail address: [email protected] (P. Chantawannakul). 0022-2011/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2011.05.012
cycle and pathological symptoms within the host range. However, it is difficult to differentiate the closely related species using morphological characters (Weiss and Vossbrinck, 1999). The genes coding for the rRNAs of the small and large subunit of the ribosomes have been used in the taxonomic determination of microsporidia and in the differentiation of Nosema species (Ku et al., 2007; Huang et al., 2008; Williams et al., 2008; Chen et al., 2009). The rRNA genes have been widely used for identification, molecular characterization and as a molecular marker for phylogenetic analysis among eukaryotes. The multiple copies of rRNA are a common phenomenon of microsporidia (Tay et al., 2005; O’Mahony et al., 2007), then the rRNA genes may not be ideal choices for the phylogenetic analysis of microsporidia. Therefore, the study of phylogenetic relationships among species or strains of Nosema need to be based on non-repeated loci (Sagastume et al., 2011). Recently, Chaimanee et al. (2010) reported that N. ceranae had been detected not only in the European honeybee (A. mellifera), but also in the cavity nesting Asian honeybee (A. cerana), the dwarf Asian honeybee (Apis florea) and the giant Asian honeybee (Apis dorsata) in Northern Thailand. However, the genetic relationship of possible N. ceranae isolates from different host species has not been determined. In this study, we conducted phylogenetic studies using sequences of the 16S rRNA gene and of the gene coding for the polar tube protein 1 (PTP 1) of N. ceranae independently to determine the genetic relationship of N. ceranae strains that were isolated from four different honeybee species.
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2. Materials and Methods 2.1. Samples The samples of Nosema infected A. mellifera (N = 10), A. cerana (N = 8), A. florea (N = 10) and A. dorsata (N = 10), were collected from Northern Thailand in 2008–2009 and stored at 20 °C prior to molecular analysis.
closely linked to that of E. cuniculi. Phylogenetic trees were constructed with the Mega 4 program (Tamura et al., 2007) using the maximum parsimony method (MP) and the close-neighborinterchange algorithm with search level one in which the initial trees were obtained with the random addition of sequences. One thousand bootstrap replicates were assessed to test the robustness of the tree. 3. Results
2.2. DNA extraction 3.1. Detection of N. ceranae in different honeybee species Genomic DNA was extracted from individual bees, homogenized with a pestle and the crude homogenate suspended in 400 ll of CTAB buffer (100 mM Tris–HCl, pH8.0; 20 mM EDTA, pH 8.0; 1.4 M sodium chloride; 2% (w/v) cetyltrimethylammonium bromide; 2% 2-mercaptoethanol). The 0.15 g of glass beads (425– 600 lm, Sigma-Aldrich, St.Louis, MO) were added in the spore suspensions and the mixture shaken at the maximum speed for 4– 5 min using a FastPrep cell Disrupter (Qbiogene, Carlsbad, CA). The mixture was incubated with proteinase K (200 lg/ml) at 55 °C overnight. DNA was extracted from suspensions using a DNA purification Kit, DNAzol (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The concentration of total DNA was determined by using the NanoDrop spectrophotometer (Thermo Scientific, USA). 2.3. PCR amplification and DNA sequencing A region of the 16S rRNA gene was amplified using the primer set described by Chen et al. (2008). Two primer sets, N. apisforward/N. apis-reverse (50 -CCATTGCCGGATAAGAGAGT-30 /50 -CA CGCATTGCTGCATCATTGAC-30 ) and N. ceranae-forward/N. ceranaereverse (50 -CGGATAAAAGAGTCCGTTACC-30 /50 -TGAGCAGGGTT CTAGGGAT-30 ), were individually used to amplify a 269 bp fragment of N. apis and a 250 bp product of N. ceranae, respectively. A pair of primers based on the hypothetic PTP gene of N. ceranae isolated from A. mellifera (XM_002995447) were designed to amplify a 838 bp product. The sequences of the primers used for PCR were as follows: NcORF 1664-forward: 50 -GACAACAAGGAAGA CCTGGAAGTG-30 and NcORF-1664 reverse: 50 -TGT GAATAAGAGGGTGATCCTGTTGAG-30 . PCR amplification was carried out in a 25 ll reaction mixture containing 1 high fidelity PCR buffer, 0.1 mM each dNTP, 2 mM MgSO4, 0.2 lM of each primer, 1U of Platinum Taq High Fidelity polymerase (Invitrogen; Carlsbad, CA) and 500 ng of total genomic DNA. The amplication conditions were 95 °C for 2 min, 40 cycles of denaturation at 94 °C for 15 s, annealing at 55 °C for 30 s and extension at 72 °C for 30 s with a final extension at 72 °C for 10 min on a thermal cycler. The PCR product from each reaction was electrophosed on 1.0% low melting point agarose gel (Invitrogen). The specificity of amplified PCR products was verified by purifying PCR fragments using Wizard PCR Prep DNA Purification System (Promega, Medison, WI) according to the manufacturer’s instruction and sequenced using both forward and reverse primers by the Center for Biosystems Research, University of Maryland Biotechnology Institute. 2.4. Phylogenetic analysis The DNA sequences of PTP obtained from this study were aligned using the ClustalX version 1.83 program. After alignment the unaligned sequences at both ends were truncated. Encephalitozoon cuniculi (GenBank Accession No. NM_001041403.1), a mammalian microsporidian species, was used as an outgroup to root the phylogenetic tree since the gene encoding this protein is
When we amplified the partial 16S rRNA gene of Nosema using the primer set N. apis-forward/N. apis-reverse and N.ceranaeforward/N.ceranae-reverse, a 250 bp fragment specific for N. ceranae was amplified from all samples. No N. apis specific PCR products were genereated from any samples examined. This is consistant with our previous findings that N. ceranae but not N. apis could be isolated from four different honeybee species (Chaimanee et al., 2010). The primer set of NcORF-1664 forward/NcORF-1664 reverse amplifying a 808 bp-fragment of the PTP 1 gene yielded a PCR-product of the expected size (Fig. 1A). Sequence analysis further confirmed the specificity of PCR amplification by PTP primers. 3.2. Phylogenetic analysis Based on the partial polar tube protein (PTP 1) sequences of N. ceranae isolates, no difference has been found amongst N. ceranae isolates from A. mellifera and A. cerana. We observed 22 and 7 nucleotide differences amongst the N. ceranae isolates from A. florea and A. dorsata and share 97% and 99% similarity, respectively with isolates from A. mellifera and A. cerana. No differences N. ceranae strains found within host species. Deletion occurrence in N. ceranae sequences that found in Thailand at 325th bp to 354th bp when compared to the N. ceranae sequence (XM_002995447) from USA (Fig. 2). We analyzed the phylogenetic relationship amongst N.ceranae isolates found in A. mellifera, A. cerana, A. florea and A. dorsata based on the partial sequences of 16S rRNA and PTP genes separately. The phylogenetic tree based on the partial rRNA gene sequences resulted in N. ceranae from the four different honeybee species forming one clade. However, the phylogenetic tree based on the partial protein coding region (PTP 1) gene sequences showed three distinct clades. N. ceranae isolated from A. mellifera grouped into the same clade as N. ceranae isolated from A. cerana supported by a high bootstrap value (97%). However, N. ceranae isolated from A. dorsata and from A. florea formed district clades with bootstrap value 95% and 99%, respectively (Fig. 1B). 4. Discussion Recently, it has been shown that N. ceranae is capable of infecting multiple host species including European honey bees, Asian honey bees, and bumble bees (Chaimanee et al., 2010; Plischuk et al., 2009). The wide host range of this parasite is of significant epidemiological concern. However, the host-parasite interactions that determine host susceptibility to the parasite remain poorly understood and the genetic relationship of different N. ceranae isolates from different host species had not been previously explored. While the phylogeny of the hosts should play an important role in host-parasite interactions and the establishment of successful infection, the phylogeny of the parasites may also aid in our understanding of host range expansion. In this study, we analyzed the phylogenetic relationships between N. ceranae isolated from four different honeybee species.
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Fig. 1. PCR amplification and phylogenetic analysis of N. ceranae isolated from four different honeybee species (A. mellifera, A. cerana, A. dorsata and A. florea). The partial PTP 1 gene sequences were amplified with a primer set NcORF-1664 forward/NcORF-1664 reverse (A). Phylogenetic analysis of N. ceranae isolates based on the PTP 1 gene. The maximum parsimony tree was generated by MEGA 4 software using the close-neighbor-interchange algorithm with search level one in which the initial trees were obtained with the random addition of sequences (10 replicates). E. cuniculi was used as an outgroup for polar tube protein phylogenetic tree. The tree used 1000 bootstrap replicates (B).
One of the major prerequisites of phylogenetic studies is the identification of useful genetic markers. The 16S rRNA genes contain highly conserved regions among all living organisms. This trait makes rRNAs the most widely used molecular genetic marker for microbial studies. However, the use of rRNA markers in genetic studies of microsporidia has some limitations. The presence of multiple non-homologous copies of rRNA is a common feature of microsporidia because the large demand for ribosomes for driving protein synthesis can only be met by multiple copies of rRNA. A previous study reported the presence of variants of the small subunit gene in the same isolate of N. ceranae (Sagastume et al., 2011). The presence of multiple copies of ribosomal RNA genes in the same cell was also found in N. bombi (Tay et al., 2005; O’Mahony et al., 2007). Thus, Fries (2010) pointed out that the ribosomal RNA sequences are probably not appropriate for phylogenetic approaches in Nosema species. Sagastume et al. (2011) also suggested that rRNA gene sequences are not suitable for a reliable differentiation of N. ceranae strains and the study of phylogenetic relationships among Nosema species needs to be established on the basis of non-repeated loci. In agreement with previous conclusions, our present study based on the partial 16S ribosomal RNA gene
showed that sequence variation of rRNA genes of N. ceranae was insufficient for elucidating the genetic relationship of N. ceranae isolates. The first phylogenetic relationship analysis of microsporidia based on the protein-coding genes was conducted using sequences of the largest subunit of RNA polymerase II (RPB1) (Cheney et al., 2001) and focused on resolving the phylogenetic relationships of polyporous species. They reported that a RPB1 tree of 14 mainly polyporous species gave a better resolution than 16S rRNA gene tree. More recently, Ku et al. (2007) suggested that the partial sequences of the less conserved gene (alpha-tubulin, beta-tubulin and RPB1 genes) will be better candidates than rRNA genes for Nosema species identification. Similarly, Lee et al. (2008) reconstructed the phylogenetic tree between Brachiola algerae and Antonospora locustae based on the alpha- and beta-tubulin sequences. They revealed closely related relationships between B. algerae and A. locustae that were previously unclear from SSU rRNA sequences. Since the polar tube proteins (PTP 1, PTP 2 and PTP 3) play an important role in the host invasion process, N. ceranae may have adapted the polar tube component to be more host specific as the host and parasite co-evolve. Moreover, the
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Fig. 2. Sequence alignment of the partial PTP 1 gene of N. ceranae isolated from A. mellifera, A. cerana, A. florea and A.dorsata in Thailand and N. ceranae isolated from A. mellifera in USA (XM_002995447). The asterisk () indicates the location of one hundred percent identical aligned nucleotide. Single nucleotide differences are shaded in dark grey and columns with less than 80% conservation are boxed. Gaps introduced for optical alignment are indicated by a dash.
O-mannosylated moiety of PTP 1 can interact with a host cell mannose receptor (Xu and Weiss, 2005). This interaction could be an important part of the infection eventually leading to the cell invasion (Texier et al., 2010). However, it was recently shown that cell invasion of N. ceranae and N. apis obviously does not depend on prior interaction of the polar tube with the cell surface (Gisder et al., 2011). In the current study, we designed the primer set for amplification of the partial PTP 1 gene. When we reconstructed the phylogenetic tree based on the partial sequences of the PTP1 gene, it easily distinguished the N. ceranae isolates found in the four honeybee species, demonstrating PTP is a useful genetic marker for studying the relationships of N. ceranae at the strain level. Both A. mellifera and A. cerana are the cavity-nesting honeybees and therefore more closely related than other single comb nesting, A. dorsata and A. florea species (Alexander, 1991). The formation of a single clade for N. ceranae isolates from the cavity-nesting species reflects the genetic lineage of A. mellifera and A. cerana and suggests ongoing co-evolution of this pathogen and its hosts. Acknowledgments We would like to thank the Thailand Commission on Higher Education for grant support funding under the program of Strategic Scholarships for Frontier Research Network for the Joint Ph.D. Program Thai Doctoral degree and National Research University Project under Thailand’s Office of the Higher Education Commission. We also thank the Thailand Research Fund (RSA 5280010) and the USDA-ARS Bee Research Laboratory for financial support. We thank Michele Hamilton for her excellent technical help with the project. References Alexander, B., 1991. A cladistic analysis of the genus Apis. In: Smith, D.R. (Ed.), Diversity in the Genus Apis. Westview Press, Boulder, pp. 1–28. Chaimanee, V., Warrit, N., Chantawannakul, P., 2010. Infections of Nosema ceranae in four different honeybee species. J. Invertebr. Pathol. 105, 207–210.
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