Nuclear Features of the Heterotrich Ciliate Blepharisma americanum: Genomic Amplification, Life Cycle, and Nuclear Inclusion

Abstract

Blepharisma americanum, a member of the understudied ciliate class Heterotrichea, has a moniliform somatic macronucleus that resembles beads on a string. Blepharisma americanum is distinguishable by its pink coloration derived from the autofluorescent pigment blepharismin and tends to have a single somatic macronucleus with 3–6 nodes and multiple germline micronuclei. We used fluorescence confocal microscopy to explore the DNA content and amplification between the somatic and germline nuclei of B. americanum through its life cycle. We estimate that the DNA content of the macronucleus and micronucleus are 43 ± 8 Gbp and 83 ± 16 Mbp respectively. This correlates with an approximate DNA content difference of 500‐fold from micronucleus to macronucleus and a macronuclear ploidy of ~1,100 N as compared to the presumably diploid micronucleus. We also investigate a previously reported macronuclear inclusion, which is present sporadically across all life cycle stages; this inclusion looks as if it contains blepharismin based on its fluorescent properties, but its function remains unknown. We also provide additional detail to our understanding of life cycles changes in B. americanum by analyses of fluorescent images. Overall, the data analyzed here contribute to our understanding of the diversity of nuclear architecture in ciliates by providing details on the highly polyploid somatic macronucleus of B. americanum.

Twisted Tales: Insights into Genome Diversity of Ciliates Using Single-Cell ‘Omics

“…The stochasticity in chromosome copy number for Blepharisma may be a byproduct of the massive genome amplification that occurs during development ( Santangelo and Barone 1987 ), as the somatic nucleus is estimated to have >1,000× more DNA than the germline nucleus ( Ovchinnikova et al. 1965 ; Wancura et al. 2017 ).…”

“…Ciliates in the Heterotrichea are often very large (some species are > 1 mm in length) with correspondingly large somatic nuclei that contain from ∼1,000 to >13,000 times more DNA than their germline nuclei ( Ovchinnikova et al. 1965 ; Wancura et al. 2017 ).…”

Resting Cysts of the Pigmented Ciliate Blepharisma sinuosum Sawaya, 1940 (Ciliophora: Heterotrichea)

“…Distinctly, in B. lateritium Kahl, 1932(Larsen 1983) and B. seculum Isquith, 1965(Isquith et al 1965, the compact form of macronucleus in vegetative cells is maintained in the mature cysts. Recently, blepharismin was found in macronuclear inclusions in B. americanum, with unknown chemical identity and function (Wancura et al 2017). Here, it should be noticed that the accumulated pigmentation toward the plug in later mature cysts does not overlaps with the macronuclear region (compare Fig.…”

“…Recently, blepharismin was found in macronuclear inclusions in B. americanum, with unknown chemical identity and function (Wancura et al. ). Here, it should be noticed that the accumulated pigmentation toward the plug in later mature cysts does not overlaps with the macronuclear region (compare Fig.…”

Techniques and tools for species identification in ciliates: a review

“…Sodium thiosulphate is added to bleach the fixed sample and reduce darkness of the Lugol's fixed cells, which enhances the brightness of the fluorescence. Cells can also be fixed with either 8 % paraformaldehyde/0.2 % Triton-X 100 in a 1 : 5 ratio, or 20 % paraformaldehyde/50 % RNALAter/5 % Trizol in a 1 : 6 ratio for 15 min on microscopic slides and washed [60]. Further, 1 µg ml À1 DAPI solution is added to the sample and incubated for 3-4 min in the dark.…”