Hexokinase as a versatile molecular genetic marker for Microsporidia

Abstract

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.

Spore yield of the microsporidium Nosema pyrausta (Paillot) Weiser, 1961 is influenced the insect host rearing temperature and diet composition

“…The microsporidium used for the purposes of the present study showed 100% sequence identity to the respective Genbank record # MH669406 corresponding to N. pyrausta hexokinase (Tokarev et al, 2019). This verifies the diagnosis of the parasite as N. pyrausta.…”

“…This "natural" isolate was propagated in O. furnacalis laboratory culture using alimentary infection of 2 nd -3 rd instar larvae followed by spore isolation from fully-grown larvae and pupae (Grushevaya et al, 2018). Prior to further experiments, a spore aliquot was used for DNA extraction and PCR using NbHKfor1 and NpyrHKrev1 primers specific to N. pyrausta hexokinase (Tokarev et al, 2019) to confirm the species diagnosis. Total DNA was extracted using a simplified protocol of Sambrook et al (1989) without addition of phenol, with adjustments in the volumes of DNA washing agents .…”

Susceptibility of the Gypsy Moth Lymantria dispar (Lepidoptera: Erebidae) to Nosema pyrausta (Microsporidia: Nosematidae)

“…The PCR products were directly sequenced using an ABI Prism Genetic Analyzer 3500 (Applied Biosystems, Waltham, MA, USA). Alternatively, the N. pyrausta- specific NpyrHKfor1:NbHKrev1 primers were used for direct PCR detection of the pathogen [ 33 ].…”

Transovarial transmission of Nosema pyrausta in three generations of Ostrinia nubilalis in laboratory tests

“…For DNA analysis, genomic DNA was extracted routinely (Malysh et al., 2019) from spore samples, infected larvae, and experimental egg batches, followed by PCR and amplicon sequencing with 18f:1047r flanking a ca. 850 bp long region of microsporidia small subunit ribosomal RNA (SSU rRNA) gene (Weiss & Vossbrinck, 1999) and NbHKfor1:NpyrHKrev1 primers flanking a 709 bp long region of N. pyrausta hexokinase (Tokarev et al., 2019). For gene expression analysis, total RNA was extracted using ‘Trizol reagent’ and deoxyribonuclease I (both Thermo Fisher Scientific, Waltham, MA, USA), cDNA was synthesized using RevertAid reverse transcriptase and Oligo(dT)18 primer (Thermo Fisher), and PCR was run using primers NpyrHKfor1:NbHKrev1 flanking a 175 bp long region of N. pyrausta hexokinase (Tokarev et al., 2019).…”

“…850 bp long region of microsporidia small subunit ribosomal RNA (SSU rRNA) gene (Weiss & Vossbrinck, 1999) and NbHKfor1:NpyrHKrev1 primers flanking a 709 bp long region of N. pyrausta hexokinase (Tokarev et al., 2019). For gene expression analysis, total RNA was extracted using ‘Trizol reagent’ and deoxyribonuclease I (both Thermo Fisher Scientific, Waltham, MA, USA), cDNA was synthesized using RevertAid reverse transcriptase and Oligo(dT)18 primer (Thermo Fisher), and PCR was run using primers NpyrHKfor1:NbHKrev1 flanking a 175 bp long region of N. pyrausta hexokinase (Tokarev et al., 2019). Quantitative data of microsporidian prevalence levels were analyzed using Fisher’s exact test (Fisher, 1954).…”