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Seeing the Whole Elephant: Imaging Flow Cytometry Reveals Extensive Morphological Diversity within Blastocystis Isolates.

Yason JA, Tan KS - PLoS ONE (2015)

Bottom Line: The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans.Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate.We discuss the possible biological implications of these unusual forms.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

ABSTRACT
Blastocystis is a common protist isolated in humans and many animals. The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans. There are biological and molecular differences between Blastocystis subtypes although microscopy alone is unable to distinguish between these subtypes. Blastocystis isolates also display various morphological forms. Several of these forms, however, have not been properly evaluated on whether or not these play significant functions in the organism's biology. In this study, we used imaging flow cytometry to analyze morphological features of Blastocystis isolates representing 3 subtypes (ST1, ST4 and ST7). We also employed fluorescence dyes to discover new cellular features. The profiles from each of the subtypes exhibit considerable differences with the others in terms of shape, size and granularity. We confirmed that the classical vacuolar form comprises the majority in all three subtypes. We have also evaluated other morphotypes on whether these represent distinct life stages in the parasite. Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate. Granular forms were present as a continuum in both viable and non-viable populations, with non-viable forms displaying higher granularity. By analyzing the images, rare morphotypes such as multinucleated cells could be easily observed and quantified. These cells had low granularity and lower DNA content. Small structures containing nucleic acid were also identified. We discuss the possible biological implications of these unusual forms.

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Image gallery of rare Blastocystis multinucleated cells.The nuclei is visualized by Hoechst staining. The nuclei number more than three and some appear to be concentrated in the center and not along the edges (A). Some of these cells also appear to show nuclear condensation. (B) Image gallery showing small structures which contains DNA.
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pone.0143974.g008: Image gallery of rare Blastocystis multinucleated cells.The nuclei is visualized by Hoechst staining. The nuclei number more than three and some appear to be concentrated in the center and not along the edges (A). Some of these cells also appear to show nuclear condensation. (B) Image gallery showing small structures which contains DNA.

Mentions: We used Hoechst staining and the EDF setting of the imaging flow cytometer to analyze nuclear arrangement of viable Blastocystis cells. More than 80% of the population from the three subtypes studied featured 1–2 nuclei (Fig 7) located at the edge of the cells. ST4-WR1 isolates all feature one or two nuclei (and not more). This reflects the previous observation [31] whereby 98.4% of the cells have a similar arrangement. This study therefore supports the classical representation of Blastocystis. More infrequently, in the other STs (1–2% in ST1 and 2–8% in ST7), other nuclear features were observed. These cells either show more than 2 nuclei or that the nucleic acid stain covers a large area of the cell (Fig 8). The latter show an apparent nucleic acid condensation which may represent cells about to undergo binary fission. Surprisingly, we have observed multinucleation only in round cells in these two subtypes. We did not find evidence to support alternative modes of reproduction such as plasmotomy and budding in Blastocystis as suggested by others [16,18,22] since we did not observe multinucleation in irregularly-shaped viable cells. Some of the multinucleated cells show the nuclei located in the center of the cell with the size of the central vacuole diminished (as indicated by lower intensity of CFSE staining) (Fig 8A). If this is a reproductive stage, it may be an evidence of a multiple fission event in Blastocystis as suggested [33]. These, however, showed lower granularity (Fig 9). Cells with single or two nuclei also showed higher DNA content. This is consistent with the data above (Fig 6) and these suggests that multinucleated cells may not be in active reproductive stage as compared to the other cells. We have also observed small structures containing nucleic acid (Fig 8B). These structures are rare (2–4% of viable cells). They also do not appear to have vacuoles as indicated by low CFSE staining but consist mainly of nucleic acid material in its compartment. Our experiments did not determine whether these structures originate from bigger and multinucleated cells. It may also be possible that these represent starving cells or a state where Blastocystis sheds off its vacuole.


Seeing the Whole Elephant: Imaging Flow Cytometry Reveals Extensive Morphological Diversity within Blastocystis Isolates.

Yason JA, Tan KS - PLoS ONE (2015)

Image gallery of rare Blastocystis multinucleated cells.The nuclei is visualized by Hoechst staining. The nuclei number more than three and some appear to be concentrated in the center and not along the edges (A). Some of these cells also appear to show nuclear condensation. (B) Image gallery showing small structures which contains DNA.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4664278&req=5

pone.0143974.g008: Image gallery of rare Blastocystis multinucleated cells.The nuclei is visualized by Hoechst staining. The nuclei number more than three and some appear to be concentrated in the center and not along the edges (A). Some of these cells also appear to show nuclear condensation. (B) Image gallery showing small structures which contains DNA.
Mentions: We used Hoechst staining and the EDF setting of the imaging flow cytometer to analyze nuclear arrangement of viable Blastocystis cells. More than 80% of the population from the three subtypes studied featured 1–2 nuclei (Fig 7) located at the edge of the cells. ST4-WR1 isolates all feature one or two nuclei (and not more). This reflects the previous observation [31] whereby 98.4% of the cells have a similar arrangement. This study therefore supports the classical representation of Blastocystis. More infrequently, in the other STs (1–2% in ST1 and 2–8% in ST7), other nuclear features were observed. These cells either show more than 2 nuclei or that the nucleic acid stain covers a large area of the cell (Fig 8). The latter show an apparent nucleic acid condensation which may represent cells about to undergo binary fission. Surprisingly, we have observed multinucleation only in round cells in these two subtypes. We did not find evidence to support alternative modes of reproduction such as plasmotomy and budding in Blastocystis as suggested by others [16,18,22] since we did not observe multinucleation in irregularly-shaped viable cells. Some of the multinucleated cells show the nuclei located in the center of the cell with the size of the central vacuole diminished (as indicated by lower intensity of CFSE staining) (Fig 8A). If this is a reproductive stage, it may be an evidence of a multiple fission event in Blastocystis as suggested [33]. These, however, showed lower granularity (Fig 9). Cells with single or two nuclei also showed higher DNA content. This is consistent with the data above (Fig 6) and these suggests that multinucleated cells may not be in active reproductive stage as compared to the other cells. We have also observed small structures containing nucleic acid (Fig 8B). These structures are rare (2–4% of viable cells). They also do not appear to have vacuoles as indicated by low CFSE staining but consist mainly of nucleic acid material in its compartment. Our experiments did not determine whether these structures originate from bigger and multinucleated cells. It may also be possible that these represent starving cells or a state where Blastocystis sheds off its vacuole.

Bottom Line: The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans.Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate.We discuss the possible biological implications of these unusual forms.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.

ABSTRACT
Blastocystis is a common protist isolated in humans and many animals. The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans. There are biological and molecular differences between Blastocystis subtypes although microscopy alone is unable to distinguish between these subtypes. Blastocystis isolates also display various morphological forms. Several of these forms, however, have not been properly evaluated on whether or not these play significant functions in the organism's biology. In this study, we used imaging flow cytometry to analyze morphological features of Blastocystis isolates representing 3 subtypes (ST1, ST4 and ST7). We also employed fluorescence dyes to discover new cellular features. The profiles from each of the subtypes exhibit considerable differences with the others in terms of shape, size and granularity. We confirmed that the classical vacuolar form comprises the majority in all three subtypes. We have also evaluated other morphotypes on whether these represent distinct life stages in the parasite. Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate. Granular forms were present as a continuum in both viable and non-viable populations, with non-viable forms displaying higher granularity. By analyzing the images, rare morphotypes such as multinucleated cells could be easily observed and quantified. These cells had low granularity and lower DNA content. Small structures containing nucleic acid were also identified. We discuss the possible biological implications of these unusual forms.

Show MeSH
Related in: MedlinePlus