Genetic polymorphism of natural isolates of Nosema Pyrausta (Microsporidia: Nosematidae)

Cover Page


Background. Microsporidia are ubiquitous parasites of animals, most abundant in arthropods and fishes. Many species of these parasites are important from standpoints of medicine, veterinary and agriculture. Microsporidium Nosema pyrausta is an important disease agent in corn borer populations causing adverse effect on host fitness. Genotyping of this parasite is necessary for proper species identification and intraspecific polymorphysm studies.

Materials and Methods. Microsporidia-infected larvae of corn borers of the genus Ostrinia were recovered from Krasnodar Territory in Russia and Gomel Region in Belarus. Small subunit ribosomal RNA (SSU rRNA) was amplified and sequenced directly, while intergenic spacer (IGS) was amplified, cloned and sequenced (1-5 clones per sample) for four isolates of microsporidia. Sequences were aligned and compared using standard bioinformatics tools (Clustal W and BLAST).

Results. SSU rRNA genotyping showed allocation of all four isolates to N. pyrausta with 100% identity to each other and 99.7% similarity to Nosema bombycis, the type species of the genus Nosema. High levels of IGS sequence variation (61-74%) is observed both between isolates of different species and populations of microsporidia as well as between molecular clones within parasite isolates from individual hosts.

Conclusion. N. pyrausta is widespread in corn borer populations and its genetic structure is complicated, as in other species of these parasites. Further studies of molecular markers are needed for genetic differentiation of geographic isolates of N. pyrausta.

Inna V Grushevaya

Author for correspondence.
All-Russian Scientific Research Institute of Plant Protection (VIZR)
Russian Federation, Pushkin, Saint Petersburg, Russia

Agronomist, Laboratory of agricultural entomology

Anastasia N Ignatieva
All-Russian Scientific Research Institute of Plant Protection (VIZR)
Russian Federation, Pushkin, Saint Petersburg, Russia

Junior Researcher. Laboratory of microbiological control

Julia M Malysh
All-Russian Scientific Research Institute of Plant Protection (VIZR)
Russian Federation, Pushkin, Saint Petersburg, Russia

Researcher, Laboratory of agricultural entomology

Lyudmila I Trepashko
Institute for Plant Protection
Russian Federation, Priluki, Republic of Belarus

Head, Laboratory of entomology

Yuri S Tokarev
All-Russian Scientific Research Institute of Plant Protection (VIZR)
Russian Federation, Pushkin, Saint Petersburg, Russia

Senior researcher, Laboratory of microbiological control

Andrei N Frolov
All-Russian Scientific Research Institute of Plant Protection (VIZR)
Russian Federation, Pushkin, Saint Petersburg, Russia

Head, Laboratory of agricultural entomology

  • Karpov SA, Mamkaeva MA, Aleoshin VV, et al. Morphology, phylogeny, and ecology of the aphelids (Aphelidea, Opisthokonta) and proposal for the new superphylum Opisthosporidia. Frontiers in Microbiology. 2014;28(5):112. doi: 10.3389/fmicb.2014.00112.
  • Исси И.В. Микроспоридии как тип паразитических простейших // Микроспоридии. Серия «Протозоология». – Л.: Наука, 1986. – Т. 10. – С. 6–136. [Issi IV. Microsporidia as a phylum of parasitic protozoa. In: Microsporidia. Ser. Protozoologiya. Leningrad: Nauka; 1986;10:6-135. (In Russ.)]
  • Becnel JJ, Andreadis TG. Microsporidia in insects. ASM Press, Washington D.C.: The microsporidia and microsporidiosis; 1999. P. 447-501.
  • Rode NO, Lievens EJ, Segard A, et al. Cryptic microsporidian parasites differentially affect invasive and native Artemia spp. Int J Parasitol. 2013;43(10):795-803. doi: 10.1016/j.ijpara.2013.04.009.
  • Bacela-Spychalska K, Rigaud T, Wattier RA. A co-invasive microsporidian parasite that reduces the predatory behaviour of its host Dikerogammarus villosus (Crustacea, Amphipoda). Parasitology. 2014;141(2):254-258. doi: 10.1017/S0031182013001510.
  • Gegner T, Otti O, Tragust S, Feldhaar H. Do microsporidia function as “biological weaponˮ for Harmonia axyridis under natural conditions? Insect Sci. 2015;22(3):353-359. doi: 10.1111/1744-7917.12224.
  • Vilcinskas A, Schmidtberg H, Estoup A, et al. Evolutionary ecology of microsporidia associated with the invasive ladybird Harmonia axyridis. Insect Sci. 2015;22(3):313-324. doi: 10.1111/1744-7917.12159.
  • Fries I. Nosema ceranae in European honey bees (Apis mellifera). J Invertebr Pathol. 2010;103(1): S73-S79. doi: 10.1016/j.jip.2009.06.017.
  • Paillot A. Sur deux protozoaires nouveaux parasites des chenilles de Pyrausta nubilalis Hubner. CR Acad Sci Paris. 1927;185:673-675.
  • Lipa JJ. Thelohania Ostriniae n. sp., a new microsporidian parasite of the European corn borer Ostrinia nubilalis Hnb. (Lepidoptera, Pyralidae). Acta Protozoologica. 1977;16(1):151-155.
  • Pezzutti R, Serini Bolchi G. Ostrinia nubilalis (Hb.) (Lepidoptera Pyralidae) e il suo parassita Perezia pyraustae Paillot (Sporozoa Microsporidia) in colture maidicole lombarde. Bolletino di Zoologia Agraria e di Bachicoltura. 1977;14:181-188.
  • Cagáň Ľ, Bokor P, Plačková A. Dissemination of the parasite Nosema pyrausta in populations of European corn borer (Ostrinia nubilalis) in Slovakia, the Czech Republic and Poland. Scientific journal for phytotechnics and zootechnics. Nitra: Slovenska polnohospodarska univerzita. 1998;9(3):81-85.
  • Малыш Ю.М., Токарев Ю.С., Ситникова Н.В., и др. Зараженность микроспоридиями стеблевых мотыльков рода Ostrinia (Lepidoptera: Crambidae) в Краснодарском крае // Паразитология. – 2011. – Т. 45. – Вып. 3. – С. 234–244. [Malysh YuM, Tokarev YuS, Sitnikova NV, et al. Incidence of microsporidian infection of stem borers of the genus Ostrinia (Lepidoptera: Crambidae) in the Krasnodar territory. Parazitologiya. 2011;45(3):234-244 (In Russ.)]
  • Caffrey DJ, Worthley LH. A progress report on the investigations of the European corn borer. In: USDA Agricultural Bulletin. 1927;1476:1-155.
  • Hill RE, Gary WJ. Effects of the microsporidium, Nosema pyrausta, on field populations of European corn borers in Nebraska. Environmental Entomology. 1979;8:91-95.
  • Siegel JP, Maddox JV, Ruesink WG. Seasonal progress of Nosema pyrausta in the European corn borer, Ostrinia nubilalis. J Invertebr Pathol. 1988;52(1): 130-136.
  • Lewis LC, Sumerford DV, Bing LA, Gunnarson RD. Dynamics of Nosema pyrausta in natural populations of the European corn borer, Ostrinia nubilalis: A six-year study. Biocontrol. 2006;51:627-642.
  • Lewis LC, Bruck DJ, Prasifka JR, Raun ES. Nosema pyrausta: Its biology, history, and potential role in a landscape of transgenic insecticidal crops. Biological Control. 2009;48:223-231.
  • Sambrook J, Fritsch E, Maniatis T. Molecular cloning: a laboratory manual. New York: Cold Spring Harbor Laboratory, Cold Spring Harbor; 1989.
  • Weiss LM, Vossbrinck CR. Molecular biology, molecular phylogeny, and molecular diagnostic approaches to the Microsporidia. The Microsporidia and Microsporidiosis. Washington, ASM Press; 1999. P. 129-171.
  • Huang WF, Tsai SJ, Lo CF, et al. The novel organization and complete sequence of the ribosomal RNA gene of Nosema bombycis. Fungal Genetics and Biology. 2004;41(5):473-481.
  • Vogelstein B, Gillespie D. Preparative and analytical purification of DNA from agarose. Proceedings of the National Academy of Sciences. USA. 1979;76:615-619.
  • Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium. 1999;41:95-98.
  • Liu H, Pan G, Luo B, et al. Intraspecific polymorphism of rDNA among five Nosema bombycis isolates from different geographic regions in China. J Invertebr Pathol. 2013;113(1):63-69. doi: 10.1016/j.jip.2013.01.008.
  • Fu Z, He X, Cai S et al. Quantitative PCR for detection of Nosema bombycis in single silkworm eggs and newly hatched larvae. J Microbiol Methods. 2016;120:72-78. doi: 10.1016/j.mimet.2015.12.003.
  • Tokarev YuS, Malysh JM, Kononchuk AG, et al. Redefinition of Nosema pyrausta (Perezia pyraustae Paillot 1927) basing upon ultrastructural and molecular phylogenetic studies. Parasitology Research. 2015;114(2):759-761. doi: 10.1007/s00436-014-4272-3.
  • Токарев Ю.С., Воронин В.Н., Сендерский И.В., Исси И.В. Микроспоридия Glugea gasterostei Voronin 1974 (Microsporidia: Marinosporidia) из трехиглой колюшки Gasterosteus aculeatus (Actinopterygii: Gasterosteiformes) как самостоятельный вид // Паразитология. – 2015. – Т. 49. – С. 81–92. [Tokarev YS, Voronin VN, Senderskiy IV, Issi IV. The microsporidium Glugea gasterostei Voronin 1974 (Microsporidia: Marinosporidia) from three-spined stickleback Gasterosteus aculeatus (Actinopterygii: Gasterosteiformes) as an independent species. Parazitologiya. 2015;49:81-92. (In Russ.)]
  • Franzen C, Fischer S, Schroeder J, et al. Morphological and molecular investigations of Tubulinosema ratisbonensis gen. nov., sp. nov. (Microsporidia: Tubulinosematidae fam. nov.), a parasite infecting a laboratory colony of Drosophila melanogaster (Diptera: Drosophilidae). J Eukaryot Microbiol. 2015;52:141-52.
  • Bjornson S, Le J, Saito T, Wang H. Ultrastructure and molecular characterization of a microsporidium, Tubulinosema hippodamiae, from the convergent lady beetle, Hippodamia convergens Guerin-Meneville. J Invertebr Pathol. 2011;206:280-288.
  • Malysh JM, Tokarev YS, Sitnicova NV, et al. Tubulinosema loxostegi sp. n. (Microsporidia: Tubulinosematidae) from the beet webworm Loxostege sticticalis L. (Lepidoptera: Crambidae) in Western Siberia. Acta Protozool. 2013;52:299-308.
  • Трепашко Л.И., Надточаева С.В., Головач В.В. Опасные вредители кукурузы // Защита и карантин растений. – 2012. – № 9. – С. 44–49. [Trepashko LI, Nadtochaeva SV, Golovach VV. Injurious pests of maize. Zaschita i karantin rasteniy. 2012;(9):44-49 (In Russ.)]
  • Ironside JE. Diversity and recombination of dispersed ribosomal DNA and protein coding genes in Microsporidia. PLoS ONE. 2013;8(2): e55878. doi: 10.1371/journal.pone.0055878.
  • Sagastume S, del Aguila C, Martín-Hernández R et al. Polymorphism and recombination for rDNA in the putatively asexual microsporidian Nosema ceranae, a pathogen of honeybees. Environ Microbiol. 2011;13(1):84-95. doi: 10.1111/j.1462-2920.2010.02311.x.

Supplementary files

Supplementary Files Action
1. Table 1. Levels of similarity and dissimilarity of SSU rRNA locus of microsporidia of the genus Nosema Download (42KB) Indexing metadata
2. Table2. Levels of similarity and dissimilarity of molecular clones of IGS in four isolates of microsporidium Nosema pyrausta Download (55KB) Indexing metadata


Abstract - 255

PDF (Russian) - 177

Copyright (c) 2017 Grushevaya I.V., Ignatieva A.N., Malysh J.M., Trepashko L.I., Tokarev Y.S., Frolov A.N.

Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.