Skip to main content Accessibility help

Hexokinase as a versatile molecular genetic marker for Microsporidia

  • Yuri S. Tokarev (a1), Sergei A. Timofeev (a1), Julia M. Malysh (a1), Alexander A. Tsarev (a1), Anastasia N. Ignatieva (a1), Oksana G. Tomilova (a2) and Vyacheslav V. Dolgikh (a1)...


Hexokinase (HK) is a core glycolytic enzyme of Microsporidia which regulates host cell metabolic processes. The goal of the present study was to test for the utility of HK for molecular phylogenetics, species identification and molecular detection of microsporidia in infected insects. HK sequence-based reconstructions were essentially similar to those based upon largest subunit RNA polymerase (RPB1) gene sequences, as well as previously published rRNA gene and genome-based trees. Comparing HK sequences allowed clear differentiation of closely related taxa, such as Nosema bombycis and Nosema pyrausta. In Nosema ceranae, unique SNPs were found for an isolate from wild colonies of the Burzyan dark honey bee as compared with the isolates from domesticated European honey bee. Similarly, in Encephalitozoon cuniculi, HK was as effective as RPB1 for discrimination of isolates belonging to different ITS genotypes. Amplification using species-specific primers flanking short fragments at the 3′-end of HK gene showed the presence of infection in insect tissues infected with N. pyrausta, Nosema ceranae and Paranosema (Antonospora) locustae. For the latter parasite species, HK expression was also demonstrated at early stages of infection using total mRNA extracts of locust larvae. These results indicate the suitability of HK as a novel tool for molecular genetic studies of Microsporidia.


Corresponding author

Author for correspondence: Julia M. Malysh, E-mail:


Hide All
Bass, D, Stentiford, GD, Littlewood, DTJ and Hartikainen, H (2015) Diverse applications of environmental DNA methods in parasitology. Trends in Parasitology 31, 499513.
Cornman, RS, Chen, YP, Schatz, MC, Street, C, Zhao, Y, Desany, B, Egholm, M, Hutchison, S, Pettis, JS, Lipkin, WI and Evans, JD (2009) Genomic analyses of the Microsporidian Nosema ceranae, an emergent pathogen of honey bees. PLOS Pathogens 5, e1000466.
Cuomo, CA, Desjardins, CA, Bakowski, MA, Goldberg, J, Ma, AT, Becnel, JJ, Didier, ES, Fan, L, Heiman, DI, Levin, JZ, Young, S, Zeng, Q and Troemel, ER (2012) Microsporidian genome analysis reveals evolutionary strategies for obligate intracellular growth. Genome Research 22, 24782488.
Deng, L, Li, W, Zhong, Z, Gong, C, Cao, X, Song, Y, Wang, W, Huang, X, Liu, X, Hu, Y, Fu, H, He, M, Wang, Y, Zhang, Y, Wu, K and Peng, G (2017) Multi-locus genotypes of Enterocytozoon bieneusi in captive Asiatic black bears in southwestern China: high genetic diversity, broad host range, and zoonotic potential. PLoS ONE 12, e0171772.
Dolgikh, VV, Senderskiy, IV, Pavlova, OA, Naumov, AM and Beznoussenko, GV (2011) Immunolocalization of an alternative respiratory chain in Antonospora (paranosema) locustae spores: mitosomes retain their role in microsporidial energy metabolism. Eukaryotic Cell 10, 588593.
Freeland, J (2011) Molecular markers in ecology. In Freeland, J, Petersen, S and Kirk, H (eds), Molecular Ecology. Oxford, Hoboken, NJ: Wiley–Blackwell, pp. 3162.
Grushevaya, IV, Ignatieva, AN, Malysh, SM, Senderskiy, IV, Zubarev, IV and Kononchuk, AG (2018) Spore dimorphism in Nosema pyrausta (Microsporidia, Nosematidae): from morphological evidence to molecular genetic verification. Acta Protozoologica 57, 4952.
Hall, TA (1999) Bioedit: a user–friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium 41, 9598.
Henry, JE (2017) The path to registration of a microbial pesticide. Protistology 11, 175182.
Ironside, JE (2013) Diversity and recombination of dispersed ribosomal DNA and protein coding genes in Microsporidia. PLoS ONE 8, e55878.
Jones, DT, Taylor, WR and Thornton, JM (1992) The rapid generation of mutation data matrices from protein sequences. Computer Applications in the Biosciences 8, 275282.
Kaushik, S, Saha, R, Das, S, Ramachandran, V and Goel, A (2017) Pragmatic combination of available diagnostic tools for optimal detection of intestinal Microsporidia. In Cohen, IR, Lajtha, A, Lambris, JD, Paoletti, R and Rezaei, N (eds), Advances in Experimental Medicine and Biology. Boston, MA: Springer, pp. 110.
Ndikumana, S, Pelin, A, Williot, A, Sanders, JL, Kent, M and Corradi, N (2017) Genome analysis of Pseudoloma neurophilia: a Microsporidian Parasite of Zebrafish (Danio rerio). Journal of Eukaryotic Microbiology 64, 1830.
Nikolenko, AG, Ilyasov, RA, Poskryakov, AV and Petukhov, AV (2015) Genetic differentiation of local population of dark European bees Apis mellifera mellifera L. in Ural. Russian Journal of Genetics 51, 792798.
Reinke, A, Balla, KM, Bennett, EJ and Troemel, ER (2017) Identification of microsporidia host-exposed proteins reveals a repertoire of rapidly evolving proteins. Nature Communications 8, 14023.
Sambrook, J, Fritsch, E and Maniatis, T (1989) Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory.
Senderskiy, IV, Timofeev, SA, Seliverstova, EV, Pavlova, OA and Dolgikh, VV (2014) Secretion of Antonospora (paranosema) locustae proteins into infected cells suggests an active role of microsporidia in the control of host programs and metabolic processes. PLoS ONE 9, e93585.
Silvestro, D and Michalak, I (2012) raxmlGUI: a graphical front-end for RAxML. Organisms Diversity and Evolution 12, 335337.
Timi, JT, Luque, JL and Poulin, R (2010) Host ontogeny and the temporal decay of similarity in parasite communities of marine fish. International Journal for Parasitology 40, 963968.
Timofeev, SA, Senderskiy, IV, Tsarev, AA, Tokarev, YS and Dolgikh, VV (2017) Heterologous expression of Paranosema (antonospora) locustae hexokinase in lepidopteran, Sf9, cells is followed by accumulation of the microsporidian protein in insect cell nuclei. Journal of Invertebrate Pathology 143, 104107.
Tokarev, Y, Malysh, JM, Kononchuk, AG, Seliverstova, EV, Frolov, AN and Issi, IV (2015) Redefinition of Nosema pyrausta (Perezia pyraustae Paillot 1927) basing upon ultrastructural and molecular phylogenetic studies. Parasitology Research 114, 759761.
Tokarev, YS, Simakova, AV, Timofeev, SA, Malysh, JM, Sokolova, OI and Issi, IV (2016) Host specificity in Microsporidia. Parazitologiia 50, 446459 (in Russian).
Vogelstein, B and Gillespie, D (1979) Preparative and analytical purification of DNA from agarose. Proceedings of the National Academy of Sciences, USA 76, 615619.
Vossbrinck, CR and Debrunner-Vossbrinck, BA (2005) Molecular phylogeny of the Microsporidia: ecological, ultrastructural and taxonomic considerations. Folia Parasitologica 52, 131142.
Vossbrinck, CR, Debrunner-Vossbrinck, BA and Weiss, LM (2014) Molecular phylogeny of the Microsporidia. In Weiss, LM and Becnel, JJ (eds), Microsporidia: Pathogens of Opportunity. Oxford: Wiley–Blackwell, pp. 203220.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

  • ISSN: 0031-1820
  • EISSN: 1469-8161
  • URL: /core/journals/parasitology
Please enter your name
Please enter a valid email address
Who would you like to send this to? *



Altmetric attention score

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed