Skip to main content
SpringerLink
Log in

Molecular characterization and phylogenetic relationships among microsporidian isolates infecting silkworm, Bombyx mori using small subunit rRNA (SSU-rRNA) gene sequence analysis

  • Published:
Acta Parasitologica Aims and scope Submit manuscript

Abstract

The life cycle, spore morphology, pathogenicity, tissue specificity, mode of transmission and small subunit rRNA (SSU-rRNA) gene sequence analysis of the five new microsporidian isolates viz., NIWB-11bp, NIWB-12n, NIWB-13md, NIWB-14b and NIWB-15mb identified from the silkworm, Bombyx mori have been studied along with type species, NIK-1s_mys. The life cycle of the microsporidians identified exhibited the sequential developmental cycles that are similar to the general developmental cycle of the genus, Nosema. The spores showed considerable variations in their shape, length and width. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIWB-15mb was found to be more virulent than other isolates. All of the microsporidians were found to infect most of the tissues examined and showed gonadal infection and transovarial transmission in the infected silkworms. SSU-rRNA sequence based phylogenetic tree placed NIWB-14b, NIWB-12n and NIWB-11bp in a separate branch along with other Nosema species and Nosema bombycis; while NIWB-15mb and NIWB-13md together formed another cluster along with other Nosema species. NIK-1s_mys revealed a signature sequence similar to standard type species, N. bombycis, indicating that NIK-1s_mys is similar to N. bombycis. Based on phylogenetic relationships, branch length information based on genetic distance and nucleotide differences, we conclude that the microsporidian isolates identified are distinctly different from the other known species and belonging to the genus, Nosema. This SSU-rRNA gene sequence analysis method is found to be more useful approach in detecting different and closely related microsporidians of this economically important domestic insect.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Altschul S.F., Gish W., Miller W., Myers E.W., Lipman D.J. 1990. Basic local alignment search tool. Journal of Molecular Biology, 215, 403–410.

    PubMed  CAS  Google Scholar 

  • Andreadis T.G. 1983. Life cycle and epizootiology of Amblyospora sp. (Microspora: Amblyosporidae) in the mosquito, Aedes cantator. Journal of Protozoology, 30, 509–518.

    Google Scholar 

  • Baker M.D., Vossbrinck C.R., Didier E.S., Maddox J.V., Shadduck J.A. 1995. Small subunit ribosomal DNA phylogeny of various microsporidia with emphasis on AIDS related forms. Journal of Eukaryotic Microbiology, 42, 564–570. DOI: 10.1111/j.1550-7408.1995. tb05906.x.

    Article  PubMed  CAS  Google Scholar 

  • Baker M.D., Vossbrinck C.R., Maddox J.V., Undeen A.H. 1994. Phylogenetic relationships among Vairimorpha and Nosema species (Microspora) based on ribosomal RNA sequence data. Journal of Invertebrate Pathology, 64, 100–106. DOI: 10.1006/jipa.1994.1077.

    Article  PubMed  CAS  Google Scholar 

  • Ball S.J., Pittilo R.M., Joyner L.P., Norton C.C. 1981. Scanning and transmission electron microscopy of Eimeria maxima microgametogenesis. Parasitology, 82, 131–135. DOI: 10.1017/S0031182000041937.

    Article  PubMed  CAS  Google Scholar 

  • Becnel J.J., Andreadis T.G. 1999. In: (Eds. W. Murray and M.W. Louis) The Microsporidia and microsporidiosis. Microsporidia in insects. American Society of Microbiology, Washington, DC, USA, 447–501.

    Google Scholar 

  • Becnel J.J., Sprague V., Fukuda T., Hazard E.I. 1989. Development of Edhazardia aedes (Kudo, 1930) N.G.N. Comb (Microsporidia: Amblyosporidae) in the mosquito Aedes aegypti (L.) (Diptera: Culicidae). Journal of Protozoology, 36, 119–130.

    PubMed  CAS  Google Scholar 

  • Brooks W.M., Cranford J.D. 1972. Microsporidoses of the hymenopterous parasites, Campoletis sonorensis and Cardiochiles nigriceps, larval parasites of Heliothis species. Journal of Invertebrate Pathology, 20, 77–94.

    Article  Google Scholar 

  • Burges H.D., Canning E.U., Hulls I.K. 1974. Ultrastructure of Nosema oryzaephili and the taxonomic value of the polar filament. Journal of Invertebrate Pathology, 23, 135–139.

    Article  PubMed  CAS  Google Scholar 

  • Canning E.U., Baker R.J., Page A.M., Nicholas J.P. 1985. Transmission of microsporidia especially Orthosoma operophterae (Canning, 1960) between generations of winter moth Operophtera brumata (L.) (Lepidoptera: Geometridae). Parasitology, 90, 11–19.

    Article  Google Scholar 

  • Canning E.U., Curry A., Cheney S., Lafranchi-Tristem N.J., Haque M.A. 1999b. Vairimorpha imperfecta n. sp., a microsporidian exhibiting an abortive octosporous sporogony in Plutella xylostella L. (Lepidoptera: Yponomeutidae). Parasitology, 119, 273–286.

    Article  PubMed  Google Scholar 

  • Canning E.U., Curry A., Cheney S.A., Lafranchi-Tristem N.J., Kawakami Y., Hatakeyama Y., Iwano H., Ishihara R. 1999a. Nosema tyriae n. sp. and Nosema sp., microsporidian parasites of cinnabar moth Tyria jacobaeae. Journal of Invertebrate Pathology, 74, 29–38. DOI: 10.1006/jipa.1999.4861.

    Article  PubMed  CAS  Google Scholar 

  • Didier E.S., Vossbrinck C.R., Baker M.D., Rogers L.B., Bertucci D.C., Shadduck J.A. 1995. Identification and characterization of three Encephalitozoon cuniculi strains. Parasitology, 111, 411–421.

    Article  PubMed  CAS  Google Scholar 

  • Dunn A.M., Terry R.S., Smith J.E. 2001. Transovarial transmission in the microsporidia. Advances in Parasitology, 48, 57–100.

    Article  PubMed  CAS  Google Scholar 

  • Dunn A.M., Terry R.S., Taneyhill D.E. 1998. Within host transmission strategies of transovarial, feminizing parasites of Gammarus duebeni. Parasitology, 117, 21–30.

    Article  PubMed  Google Scholar 

  • Fedorko D.P, Nelson N.A., Cartwright C.P. 1995. Identification of microsporidia in stool specimens by using PCR and restriction endonucleases. Journal of Clinical Microbiology, 33, 1739–1741.

    PubMed  CAS  Google Scholar 

  • Finney D.J. 1971. Probit analysis. 3rd edition, Cambridge University Press, London and New York, 333 pp.

    Google Scholar 

  • Fujiwara T. 1979. Infectivity and pathogenicity of Nosema bombycis to larvae of the silkworm. Journal of Sericultural Science Japan, 48, 376–380.

    Google Scholar 

  • Fujiwara T. 1980. Three microsporidians (Nosema spp.) from the silkworm, Bombyx mori. Journal of Sericultural Science Japan, 53, 398–402.

    Google Scholar 

  • Hall T.A. 1999. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98NT. Nucleic Acids Symposium Series, 41, 95–98.

    CAS  Google Scholar 

  • Hartskeerl R.A., van Gool T., Schuitema A.R., Didier E.S., Terpstra W.J. 1995. Genetic and immunological characterization of the microsporidian Septata intestinalis Cali, Kotler and Orenstein, 1993: reclassification to Encephalitozoon intestinalis. Parasitology, 110, 277–285. DOI: 10.1017/S0031182000080860.

    Article  PubMed  CAS  Google Scholar 

  • Hatakeyama Y., Bansal A.K., Iwano H., Kawakami Y., Ishihara R. 2000. Characterization of SSU-rRNA sequence of a new microsporidium Nosema sp. (Nosematidae: Microsporidia), isolated from Antheraea mylitta Drury (Lepidoptera: Saturniidae) in India. Indian Journal of Sericulture, 39, 131–134.

    Google Scholar 

  • Hatakeyama Y., Kawakami Y., Iwano H., Inoue T., Ishihara R. 1997. Analysis and taxonomic inferences of small subunit ribosomal RNA sequence of five microsporidia pathogenic to the silkworm, Bombyx mori. Journal of Sericultural Science Japan, 66, 242–252.

    CAS  Google Scholar 

  • Hazard E.I., Oldacre S.W. 1975. Revision of microsporidia (Protozoa) close to Thelohania with descriptions of one new family, eight new genera and thirteen new species. United States Department Agriculture Technical Bulletin, No. 1530, 104.

  • Henry J.E. 1971. Nosema cuneatum sp. n. (Microsporidia: Nosematidae) in Grasshoppers (Orthoptera: Acrididae). Journal of Invertebrate Pathology, 17, 164–171.

    Article  PubMed  CAS  Google Scholar 

  • Huang W.F., Tsai S.J., Lo C.F., Soichi Y., Wang C.H. 2004. The novel organization and complete sequence of the ribosomal RNA gene of Nosema bombycis. Fungal Genetics and Biology, 41, 473–481. DOI: 10.1016/fgb.2003.12.005.

    Article  PubMed  CAS  Google Scholar 

  • Hung H.W., Lo C.F., Tseng C.C., Peng S.E., Chou C.M., Kou G.H. 1998. The small subunit ribosomal RNA gene sequence of Pleistophora anguillarum and the use of PCR primers for diagnostic detection of the parasite. Journal of Eukaryotic Microbiology, 45, 556–560. DOI: 10.1111/j.1550-7408.1998.tb05116.x.

    Article  PubMed  CAS  Google Scholar 

  • Ishihara R. 1969. The life cycle of Nosema bombycis as revealed in tissue culture cells of Bombyx mori. Journal of Invertebrate Pathology, 14, 316–320. DOI: 10.1016/0022-2011(69)901 57-8

    Article  PubMed  CAS  Google Scholar 

  • Ishihara R., Fujiwara T. 1965. The spread of pebrine within a colony of silkworm, Bombyx mori (Linnaeus). Journal of Invertebrate Pathology, 7, 126–131.

    Article  PubMed  CAS  Google Scholar 

  • Iwano H., Ishihara R. 1991. Dimorphic development of Nosema bombycis spores in gut epithelium of larvae of the silkworm, Bombyx mori. Journal of Sericultural Science Japan, 60, 249–256.

    Google Scholar 

  • Kawarabata T. 2003. Biology of microsporidians infecting the silkworm, Bombyx mori, in Japan. Journal of Insect Biotechnology and Sericology, 72, 1–32.

    Google Scholar 

  • Kawakami Y., Inoue T., Kikuchi M., Takayanagi M., Sunairi M., Ando T., Ishihara R. 1992. Primary and secondary structures of 5S ribosomal RNA of Nosema bombycis (Nosematidae: Microsporidia). Journal of Sericultural Science Japan, 61, 321–327.

    CAS  Google Scholar 

  • Liu H., Pan G., Li T., Huang W., Luo B., Zhou Z. 2011. Ultrastructure, chromosomal karyotype, and molecular phylogeny of a new isolate of microsporidian Vairimorpha sp. BM (Microsporidia: Nosematidae) from Bombyx mori in China. Parasitology Research, Published on line: 03 June 2011. DOI: 10.1007,/s00436-011-2470-9.

  • Maddox J.V., Sprenkel R.K. 1978. Some enigmatic microsporidia of the genus Nosema. Miscellaneous Publications of the Entomological Society of America, 11, 65–84.

    Google Scholar 

  • Malone L.A., McIvor C.A. 1996. Use of nucleotide sequence data to identify a microsporidian pathogen of Pieris rapae (Lepidoptera: Pieridae). Journal of Invertebrate Pathology, 65, 269–273. DOI: 10.1006/jipa.1995.1041.

    Article  Google Scholar 

  • Mathis A., Michel M., Kuster H., Muller C., Weber R., Deplazes P. 1997. Two Encephalitozoon cuniculi strains of human origin are infectious to rabbits. Parasitology, 114, 29–35.

    Article  PubMed  Google Scholar 

  • Mercer C.F., Wigley P.J. 1987. A microsporidian pathogen of the poroporo stem borer, Sceliodes cordalis (Dbld) (Lepidoptera: Pyralidae): I. Description and identification. Journal of Invertebrate Pathology, 49, 93–101. DOI: 10.1016/0022-2011(87)90130-3.

    Article  Google Scholar 

  • Muller A., Stellermann K., Hartmann P., Schrappe M., Fatkenheuer G., Salzberger B., Diehl V., Franzen C. 1999. A powerful DNA extraction method and PCR for detection of microsporidia in clinical stool specimens. Clinical and Diagnostic Laboratory Immunology, 6, 243–246.

    PubMed  CAS  Google Scholar 

  • Nageli A. 1857. Ueber die neve krankheit der seidenraupe and verwandte organismen. Botanische Zeitung, 15, 760–761.

    Google Scholar 

  • Rao S.N., Muthulakshmi M., Kanginakudru S., Nagaraju J. 2004. Phylogenetic relationships of three new microsporidian isolates from the silkworm, Bombyx mori. Journal of Invertebrate Pathology, 86, 87–95. DOI: 10.1016/j.jip.2004.05.004.

    Article  Google Scholar 

  • Rao S.N., Nath B.S., Bhuvaneswari G., Urs S.R. 2007. Genetic diversity and phylogenetic relationships among microsporidia infecting the silkworm, Bombyx mori, using random amplification of polymorphic DNA: Morphological and ultrastructural characterization. Journal of Invertebrate Pathology, 96, 193–204. DOI: 10.1016/j.jip.2007.05.001.

    Article  PubMed  CAS  Google Scholar 

  • Rao S.N., Nath B.S., Saratchandra B. 2005. Characterization and phylogenetic relationships among microsporidia infecting silkworm, Bombyx mori, using inter simple sequence repeat (ISSR) and small subunit rRNA (SSU-rRNA) sequence analysis. Genome, 48, 355–366. DOI: 10.1139/g04-109.

    Article  PubMed  CAS  Google Scholar 

  • Raynaud L., Delbac F., Broussolle V., Rabodonirina M., Girault V., Wallon M., Cozon G., Vivares C.P., Peyron F. 1998. Identification of Encephalitozoon intestinalis in travelers with chronic diarrhea by specific PCR amplification. Journal of Clinical Microbiology, 36, 37–40.

    PubMed  CAS  Google Scholar 

  • Sato R., Kobayashi M., Watanabe H. 1982. Internal ultrastructure of spores of microsporidians isolated from the silkworm, Bombyx mori. Journal of Invertebrate Pathology, 40, 260–265. DOI: 10.1016/0022-2011(82)90124.0.

    Article  Google Scholar 

  • Solter S.L. 2006. Transmission as a predictor of ecological host specificity with a focus on vertical transmission of microsporidian. Journal of Invertebrate Pathology, 92, 132–140.

    Article  PubMed  Google Scholar 

  • Sprague V. 1977. In: (Eds. L.A. Bulla and T.C. Cheng) Classification and phylogeny of the microsporidia. Comparative Pathobiology. Plenum, New York, Vol. 2, 1–30.

    Google Scholar 

  • Sprague V. 1981. In: (Ed. S.P. Parker) Microspora: Synopsis and classification of living organisms. McGraw-Hill, New York, 589–594.

    Google Scholar 

  • Sprague V., Becnel J.J, Hazard E.I. 1992. Taxonomy of phylum microspora. Critical Reviews in Microbiology, 18, 285–395. DOI: 10.3109/10408419209113519.

    Article  PubMed  CAS  Google Scholar 

  • Swofford D.L. 2001. PAUP* Phylogenetic analysis using parsimony (*and other methods). Sinauer Associates Inc., Sunderland, MA, USA.

    Google Scholar 

  • Takizawa H., Vivier E., Petitprez A. 1975. Recherches cytochimiques sur la microsporidia Nosema bombycis au cours de son development chez le ver a soie (Bombyx mori). Journal of Protozoology, 22, 359–368.

    Google Scholar 

  • Tamura K., Dudley J., Nei M., Kumar S. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA), software version 4.0. Molecular Biology and Evolution, 24, 1596–1599. DOI: 10.1093/molbev/msm092.

    Article  PubMed  CAS  Google Scholar 

  • Terry R.S., Dunn A.M., Smith J.E. 1997. Cellular distribution of a feminizing microsporidian parasite: a strategy for transovarial transmission. Parasitology, 115, 157–163.

    Article  PubMed  Google Scholar 

  • Terry R.S., Smith J.E., Sharpe R.G., Rigaud T., Littlewood D.T.J., Ironside J.E., Rollinson D., Bouchon D., MacNeil C., Dick J.T.A., Dunn A.M. 2004. Widespread vertical transmission and associated host sex-ratio distortion within the eukaryotic phylum Microspora. Proceedings of the Royal Society of London, B, 271, 1783–1789.

    Article  Google Scholar 

  • Thompson J.D., Gibson T.J., Plewniak F., Jeanmougin F., Higgins D.G. 1997. The Clustal_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research, 25, 4876–4882. DOI: 10.1093/nar/25.24.4876.

    Article  PubMed  CAS  Google Scholar 

  • Tsai S.J., Lo C.F., Soichi Y., Wang C.H. 2003. The characterization of microsporidian isolates (Nosematidae: Nosema) from five important lepidopteran pests in Taiwan. Journal of Invertebrate Pathology, 83, 51–59. DOI: 10.1016/S0022-2011(03) 00035-1.

    Article  PubMed  CAS  Google Scholar 

  • Undeen A.H., Alger N.E. 1971. A density gradient method for fractionating microsporidian spores. Journal of Invertebrate Pathology, 18, 419–420. DOI: 10.1016/0022-2011(71)900 48-6.

    Article  Google Scholar 

  • Undeen A.H., Cockburn A.F. 1989. The extraction of DNA from microsporidia spores. Journal of Invertebrate Pathology, 54, 132–133. DOI: 10.1016/0022-2011(89)90151-1.

    Article  Google Scholar 

  • Undeen A.H., Vavra J. 1997. In: (Ed. L. A. Lacey) Research methods for entomopathogenic protozoa. Manual of Techniques in Insect Pathology. Academic Press, San Diego USA, 117–151.

    Google Scholar 

  • Vossbrinck C.R., Baker M.D., Didier E.S., Debrunner-Vossbrinck B.A., Shadduck J.A. 1993. Ribosomal DNA sequences of Encephalitozoon hellem and Encephalitozoon cuniculi: species identification and phylogenetic construction. Journal of Eukaryotic Microbiology, 40, 354–362. DOI: 10.1111/j.1550-7408.1993.tb04928.x.

    Article  PubMed  CAS  Google Scholar 

  • Wang L.L., Chen K.P., Zhang Z., Yao Q., Gao G.T., Zhao Y. 2006. Phylogenetic analysis of Nosema antheraeae (Microsporidia) isolated from Chinese oak silkworm, Antheraea pernyi. Journal of Eukaryotic Microbiology, 53, 330–313. DOI: 10.1111/j.1550-7408.2006.00106.x.

    Article  Google Scholar 

  • Wittner M., Weiss L.M. 1999. The microsporidia and microsporidiosis. ASM Press, Washington DC, 1–553 pp.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Molecular Pathology Division, Seribiotech Research Laboratory, Central Silk Board, CSB Campus, Carmelram Post, Kodathi, Bangalore, 560035, Karnataka, India

    B. Surendra Nath

  2. Silkworm Pathology Department, Central Sericultural Research and Training Institute, Central Silk Board, Berhampore, 742101, West Bengal, India

    S. K. Gupta & A. K. Bajpai

Authors
  1. B. Surendra Nath
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. S. K. Gupta
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. K. Bajpai
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to B. Surendra Nath.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Nath, B.S., Gupta, S.K. & Bajpai, A.K. Molecular characterization and phylogenetic relationships among microsporidian isolates infecting silkworm, Bombyx mori using small subunit rRNA (SSU-rRNA) gene sequence analysis. Acta Parasit. 57, 342–353 (2012). https://doi.org/10.2478/s11686-012-0051-4

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11686-012-0051-4

Keywords

Search

Navigation