Hexokinase as a versatile molecular genetic marker for Microsporidia

AbstractHexokinase (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.

Download Full-text

Sutterella stercoricanis sp. nov., isolated from canine faeces

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.63098-0 ◽

2004 ◽

Vol 54 (5) ◽

pp. 1581-1584 ◽

Cited By ~ 20

Author(s):

Hazel L. Greetham ◽

Matthew D. Collins ◽

Glenn R. Gibson ◽

Catriona Giffard ◽

Enevold Falsen ◽

...

Keyword(s):

Type Strain ◽

Novel Species ◽

Molecular Genetic ◽

Gene Sequencing ◽

Sequence Divergence ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Anaerobic Conditions ◽

Genetic Studies ◽

Rrna Gene Sequencing

Morphological, biochemical and molecular genetic studies were carried out on an unknown non-spore-forming, Gram-negative, rod-shaped bacterium which was isolated from dog faeces. The bacterium grew under anaerobic conditions, was asaccharolytic, resistant to 20 % (v/v) bile and was oxidase- and urease-negative. Phylogenetic analysis based on comparative 16S rRNA gene sequencing showed that the unidentified bacterium clustered with Sutterella wadsworthensis, although a sequence divergence of >5 % indicated that the bacterium from dog faeces represented a previously unrecognized subline within the genus. On the basis of the presented findings, a novel species, Sutterella stercoricanis sp. nov., is described. The type strain of Sutterella stercoricanis is 5BAC4T (=CCUG 47620T=CIP 108024T).

Download Full-text

Polymorphism of 16s rRNA Gene: Any Effect on the Biomolecular Quantitation of the Honey Bee (Apis mellifera L., 1758) Pathogen Nosema ceranae?

Applied Sciences ◽

10.3390/app12010422 ◽

2022 ◽

Vol 12 (1) ◽

pp. 422

Author(s):

Giovanni Cilia ◽

Giacomo Luchetti ◽

Antonio Nanetti

Keyword(s):

Apis Mellifera ◽

16S Rrna ◽

Honey Bee ◽

Single Copy ◽

Nosema Ceranae ◽

Rrna Genes ◽

Rrna Gene ◽

Treatment Regimens ◽

Polymorphic Gene ◽

Type C

The microsporidian Nosema ceranae is a severe threat to the western honey bee Apis mellifera, as it is responsible for nosemosis type C, which leads the colonies to dwindle and collapse. Infection quantification is essential to clinical and research aims. Assessment is made often with molecular assays based on rRNA genes, which are present in the N. ceranae genome as multiple and polymorphic copies. This study aims to compare two different methods of Real-Time PCR (qPCR), respectively relying on the 16S rRNA and Hsp70 genes, the first of which is described as a multiple and polymorphic gene. Young worker bees, hatched in the laboratory and artificially inoculated with N. ceranae spores, were incubated at 33 °C and subject to different treatment regimens. Samples were taken post-infection and analyzed with both qPCR methods. Compared to Hsp70, the 16S rRNA method systematically detected higher abundance. Straightforward conversion between the two methods is made impossible by erratic 16s rRNA/Hsp70 ratios. The 16s rRNA polymorphism showed an increase around the inoculated dose, where a higher prevalence of ungerminated spores was expected due to the treatment effects. The possible genetic background of that irregular distribution is discussed in detail. The polymorphic nature of 16S rRNA showed to be a limit in the infection quantification. More reliably, the N. ceranae abundance can be assessed in honey bee samples with methods based on the single-copy gene Hsp70.

Download Full-text

Slackia faecicanis sp. nov., isolated from canine faeces

INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY ◽

10.1099/ijs.0.63531-0 ◽

2005 ◽

Vol 55 (3) ◽

pp. 1243-1246 ◽

Cited By ~ 14

Author(s):

Paul A. Lawson ◽

Hazel L. Greetham ◽

Glenn R. Gibson ◽

Catriona Giffard ◽

Enevold Falsen ◽

...

Keyword(s):

Novel Species ◽

Molecular Genetic ◽

Gene Sequencing ◽

16S Rrna Gene Sequencing ◽

Rrna Gene ◽

Strictly Anaerobic ◽

Anaerobic Conditions ◽

Genetic Studies ◽

The Family ◽

Rrna Gene Sequencing

Morphological, biochemical and molecular genetic studies were carried out on an unknown non-spore-forming, Gram-positive, rod-shaped bacterium that was isolated from dog faeces. The bacterium grew under strictly anaerobic conditions, was asaccharolytic, and possessed a relatively high G+C content of 61 mol%. Phylogenetic analysis based on comparative 16S rRNA gene sequencing showed that the unidentified bacterium was a member of the family Coriobacteriaceae and represents a hitherto unknown subline within the genus Slackia. Based on the presented findings, a novel species, Slackia faecicanis sp. nov., is described. The type strain of Slackia faecicanis is 5WC12T (=CCUG 48399T=CIP 108281T).

Download Full-text

First Identification of Nosema Ceranae (Microsporidia) Infecting Apis Mellifera in Venezuela

Journal of Apicultural Science ◽

10.1515/jas-2017-0010 ◽

2017 ◽

Vol 61 (1) ◽

pp. 149-152

Author(s):

Leonardo P. Porrini ◽

Martin P. Porrini ◽

Paula M. Garrido ◽

Judith Principal ◽

Carlos J. Barrios Suarez ◽

...

Keyword(s):

Apis Mellifera ◽

Honey Bee ◽

Pcr Primers ◽

Nosema Ceranae ◽

Rrna Gene ◽

Parasitic Fungi ◽

Multiplex Pcr Assay ◽

Etiologic Agents ◽

Microsporidian Infection ◽

The 16S Rrna Gene

Abstract Nosema ceranae is a pathogen of Apis mellifera, which seems to have jumped from its original host Asiatic honey bee Apis ceranae. Nosemosis which affects the honey bee Apis mellifera is caused by two parasitic fungi described as etiologic agents of the disease. Nosema apis was the only microsporidian infection identified in A. mellifera until N. ceranae in Taiwan and Europe. Nosema spp. positive samples of adult worker bees from the Venezuelean state of Lara were determined through light microscopy of spores. Samples were then tested to determine Nosema species (N.apis/N.ceranae) using previously reported PCR primers for the 16S rRNA gene. A multiplex PCR assay was used to differentiate both N. apis and N. ceranae species. Only N. ceranae was found in the analyzed samples and the percentage of infected foragers fluctuated between 18% and 60%.

Download Full-text

Simulating a base population in honey bee for molecular genetic studies

Genetics Selection Evolution ◽

10.1186/1297-9686-44-14 ◽

2012 ◽

Vol 44 (1) ◽

Cited By ~ 3

Author(s):

Pooja Gupta ◽

Tim Conrad ◽

Andreas Spötter ◽

Norbert Reinsch ◽

Kaspar Bienefeld

Keyword(s):

Honey Bee ◽

Molecular Genetic ◽

Base Population ◽

Genetic Studies

Download Full-text

Vairimorpha (Nosema) ceranae Infection Alters Honey Bee Microbiota Composition and Sustains the Survival of Adult Honey Bees

Biology ◽

10.3390/biology10090905 ◽

2021 ◽

Vol 10 (9) ◽

pp. 905

Author(s):

Yakun Zhang ◽

Meiling Su ◽

Long Wang ◽

Shaokang Huang ◽

Songkun Su ◽

...

Keyword(s):

Gut Microbiota ◽

Honey Bee ◽

Honey Bees ◽

Disease Transmission ◽

16S Rrna Gene Sequencing ◽

Nosema Ceranae ◽

Rrna Gene ◽

Gut Microbe ◽

Rrna Gene Sequencing ◽

Nosema Disease

Vairimorpha (Nosema) ceranae is the most common eukaryotic gut pathogen in honey bees. Infection is typically chronic but may result in mortality. Gut microbiota is a factor that was recently noted for gut infectious disease development. Interestingly, studies identified positive, instead of negative, associations between core bacteria of honey bee microbiota and V. ceranae infection. To investigate the effects of the positive associations, we added isomaltooligosaccharide (IMO), a prebiotic sugar also found in honey, to enhance the positive associations, and we then investigated the infection and the gut microbiota alterations using qPCR and 16S rRNA gene sequencing. We found that infected bees fed IMO had significantly higher V. ceranae spore counts but lower mortalities. In microbiota comparisons, V. ceranae infections alone significantly enhanced the overall microbiota population in the honey bee hindgut and feces; all monitored core bacteria significantly increased in the quantities but not all in the population ratios. The microbiota alterations caused by the infection were enhanced with IMO, and these alterations were similar to the differences found in bees that naturally have longer lifespans. Although our results did not clarify the causations of the positive associations between the infections and microbiota, the associations seemed to sustain the host survival and benefit the pathogen. Enhancing indigenous gut microbe to control nosema disease may result in an increment of bee populations but not the control of the pathogen. This interaction between the pathogen and microbiota potentially enhances disease transmission and avoids the social immune responses that diseased bees die prematurely to curb the disease from spreading within colonies.

Download Full-text