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Appendix S1. Experimental procedures
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Honey bee sample collection
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A. mellifera colonies were sampled from 62 apiaries in 44 among total 47 prefectures in
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Japan in collaboration with the Japan Beekeeping Association. 50 workers were
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collected and pooled from a single colony in an apiary (62 colonies in total). A total of
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35 A. c. japonica samples (each sample: 50 pooled workers from a single colony) were
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collected from 19 managed and 16 feral colonies. All of above were apparently healthy
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colonies and the workers were collected from brood nests inside the hives for managed
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colonies and at the hive entrances for feral colonies, respectively. In addition,
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approximately 50 dying workers (crawling on the ground in front of the hive entrance)
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were sampled from 28 and 9 collapsing A. mellifera and A. c. japonica colonies.
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RT-PCR detection of viruses
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Total RNA was isolated from 20 workers collected from single colonies by using Trizol
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reagent (Invitrogen). Total RNA (1 µg) was used for reverse transcription reaction by
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using ReverTra Ace reverse transcriptase (TOYOBO) and a random primer. The reverse
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transcription products were then used for PCR with KOD FX DNA polymerase
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(TOYOBO) and the primer sets for detecting ABPV (Acute bee paralysis virus), BQCV
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(Black queen cell virus), CBPV (Chronic bee paralysis virus), DWV (Deformed wing
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virus), IAPV (Israeli acute paralysis virus), KBV (Kashmire bee virus), and SBV
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(Sacbrood virus). The primer sequences are the same as those described in (Kojima et
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al.,
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5-CGGACGACGATTGGCGCTCA-3). Honey bee EF-1alpha was used as a positive
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control to verify the quality of RNA extraction as well as reverse transcription, and also
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serves as an internal loading standard. The primer set, 5'-TGCAAGAGGCTGTTCCTG
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GTGA-3' and 5'-CGAAACGCCCCAAAGGCGGA-3', was used. The thermal cycling
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conditions were as follows: one cycle of initial denaturation at 94°C for 2 min, 35
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cycles of denaturation at 98°C for 10 s, annealing at 55°C for 30 s, and extension at
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68°C for 30 s. A negative control lacking template DNA and a positive DNA control
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were performed for each PCR reaction. Positive identification was confirmed by
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sequencing the PCR products. The PCR product was analyzed by 2% agarose gel
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electrophoresis.
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PCR detection of Nosema ceranae and N. apis
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Total genomic DNA was isolated from 20 workers collected from single colonies by
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using DNAzol reagent (Invitrogen), and dissolved in 100 µL of 8 mM NaOH followed
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by neutralization by adding 1 µL of 1 M HEPES. Total DNA (0.1 µg) was used for PCR
2011a)
except
for
CBPV
(5-GACCCCCGTTGGAACGACGC-3
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and
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with
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5-CCATTGCCGGATAAGAGAGT-3 and 5-CCACCAAAAACTCCCAAGAG-3 for
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N. apis, and 5-CGGATAAAAGAGTCCGTTACC-3 and 5-TGAGCAGGGTTCTAGG
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GAT-3 for N. ceranae (Chen et al., 2009). As a control, a honey bee genomic DNA
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fragment encoding a part of AmHsTRPA (Kohno et al., 2010) was PCR amplified with
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the following primers: 5-CACGACATTCAAGGTTTAAGAAATCACG-3 and 5-TCA
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GTTATTCTTTTCCTTTGCCAGATTT-3. The thermal cycling conditions and the gel
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electrophoresis were the same as above. A negative control lacking template DNA and a
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positive DNA control were performed for each PCR reaction. Positive identification
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was confirmed by sequencing the PCR products.
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PCR detection of tracheal mite (A. woodi)
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Total genomic DNA prepared above was used for PCR detection of tracheal mite with
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the primer set (5'-TCTTCAATTTTAATTATACGT-3' and 5'-CAAAAATCAGAATAAA
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TGTTGAAATA-3') as described in (Kojima et al., 2011b). The thermal cycling
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conditions and the gel electrophoresis were the same as above. A negative control
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lacking template DNA and a positive DNA control were performed for each PCR
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reaction. Positive identification was confirmed by sequencing the PCR products.
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PCR detection of C. mellificae and A. bombi
KOD
FX
DNA
polymerase
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and
the
following
primer
sets:
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The reverse transcription products prepared above were used for PCR detection of C.
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mellificae with the primer sets (5'-TGAACGGCCACCGCATCCTG-3' and 5'-GGGCC
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AGGCAGTTGGTCGTG-3') based on the GAPDH gene sequence. The size of
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amplified product is 279-bp. Total genomic DNA prepared above was used for PCR
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detection of A. bombi as described in (Meeus et al., 2010) with the following primer set:
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5'-CCAGCATGGAATAACATGTAAGG-3' and 5'-GACAGCTTCCAATCTCTAGTCG
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-3'. The thermal cycling conditions and the gel electrophoresis were the same as above.
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A negative control lacking template DNA and a positive DNA control were performed
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for each PCR reaction. Positive identification was confirmed by sequencing the PCR
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products.
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Sequencing of SSU-rRNA, GAPDH, and Cyt b genes of C. mellificae
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To sequence 523-bp DNA fragments of SSU-rRNA gene of 14 Japanese C. mellificae
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isolates, genomic PCR products obtained with a primer set (5'- CGGGAGCGGGGGAT
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TAGGGTT-3' and 5'- AACAAAAGCCGAAACGGTAGCCT-3') were sequenced. To
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determine 450-bp GAPDH mRNA sequences, RT-PCR products obtained with a primer
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set (5'-CACGGCCGCCCGAAGTACAC-3' and 5'- CGGGATGATGTTCACCGAGCA
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-3') were sequenced. To determine 413-bp DNA sequences of Cyt b gene, genomic PCR
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products obtained with a primer set (5'-GT (A/T)TT(G/A)TTTTT(G/A)TG(G/A)GATT
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TTG-3', and 5'-CATAAACG(T/C)TCACAATAAAATGC-3') were sequenced. We also
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sequenced RT-PCR products of Cyt b mRNA, and found that their sequences were
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identical to the kinetoplast DNA sequences of two haplotypes, suggesting that RNA
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editing does not occur with C. mellificae Cyt b mRNA.
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Phylogenetic analysis of Cyt b sequences
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To construct phylogenetic tree of Cyt b sequences of two Japanese C. mellificae isolates
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and other related trypanosomatids, the appropriate Cyt B sequences were retrieved from
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a GenBank, and these are indicated by organism names with the accession numbers. All
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sequences were first aligned using MUSCLE program (Edgar, 2004), and then the
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Tamura 3-parameter with a discrete gamma distribution model (TN92+G) was selected
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as the best-fit substitution model. The condensed phylogenetic tree was then constructed
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using the maximum likelihood method and a bootstrap value of 1000 replicates with
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MEGA5 (Tamura et al., 2011).
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Supporting Information References
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Chen, Y.P., Evans, J.D., Zhou, L., Boncristiani, H., Kimura, K., Xiao, T., Litkowski,
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A.M., and Pettis, J.S. (2009) Asymmetrical coexistence of Nosema ceranae and Nosema
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apis in honey bees. J Invertebr Pathol 101: 204–209.
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Edgar, R.C. (2004) MUSCLE: multiple sequence alignment with high accuracy and
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high throughput. Nucleic Acids Res 32: 1792-1797.
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Kohno, K., Sokabe, T., Tominaga, M., and Kadowaki, T. (2010) Honey bee
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thermal/chemical sensor; AmHsTRPA; reveals neofunctionalization and loss of
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Transient Receptor Potential channel genes. J Neurosci 30: 12219-12229.
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Kojima, Y., Toki, T., Morimoto, T., Yoshiyama, M., Kimura K., and Kadowaki, T.
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(2011a) Infestation of Japanese native honey bees by tracheal mite and virus from
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non-native European honey bees in Japan. Microbial Ecol 62: 895-906.
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Kojima, Y., Yoshiyama, M., Kimura, K., and Kadowaki, T. (2011b) PCR-based
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detection of a tracheal mite of the honey bee Acarapis woodi. J Invertebr Pathol 108:
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135-137.
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Meeus, I., de Graaf, D.C., Jans, K., and Smagghe, G. (2010) Multiplex PCR detection
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of slowly-evolving trypanosomatids and neogregarines in bumblebees using
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broad-range primers. J Appl Microbiol 109: 107-115.
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Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., and Kumar, S. (2011)
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MEGA5: molecular evolutionary genetics analysis using maximum likelihood;
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evolutionary distance; and maximum parsimony methods. Mol Biol Evol 10:
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2731-2739.
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