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Novel life cycle stages of Colpodella sp. (Apicomplexa) identified using Sam-Yellowe’s trichrome stains and confocal and electron microscopy

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Colpodella spp. are free-living flagellates closely related to the apicomplexans. Human infections by Colpodella sp. have been reported. A biflagellated trophozoite and cyst stage comprise the known life cycle stages of Colpodella sp. However, the process of encystation and excystation within the life cycle is unclear. Life cycle stages initiating human infections are unknown. We performed a detailed investigation of the life cycle of Colpodella sp. (ATCC 50594) in culture using Sam-Yellowe’s trichrome stains and differential interference contrast (DIC) for light microscopy and fluorescence microscopy of Congo red-stained cells and investigated ultrastructure using transmission electron microscopy (TEM). We report previously undocumented stages of Colpodella sp. Asymmetric and asynchronous division was detected inside cysts by trichrome staining and by TEM. Odd-numbered juveniles and cysts containing more than four juvenile trophozoites were identified. Live imaging of active cultures captured the excystation and egress of juvenile trophozoites and confirmed the presence of multinucleate cysts. The ultrastructure of the multinucleate cyst is reminiscent of apicomplexan schizonts. Insights gained from the life cycle stages observed in culture allowed the construction of the life cycle of Colpodella sp. Knowledge of the life cycle will aid biochemical and molecular characterization of Colpodella sp. and help identify stages in human infections.

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We gratefully acknowledge the Cleveland Clinic Lerner Research Institute, Cleveland Clinic NIH that shared instrument grant for Orbitrap Elite LC-MS instrument; Cleveland Clinic NIH grant 1S100D023436-01 for Fusion Lumos instrument; and Cleveland Clinic Lerner Research Institute, Imaging Core, and MR4-BEI Resources for anti-EBA-175 antiserum.


The study was supported by funds from the Cleveland State University Undergraduate Summer Research Award 2019

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Correspondence to Tobili Y. Sam-Yellowe.

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Supplementary Fig. 1

Volocity movie of Colpodella sp. and B. caudatus in myzocytosis. The tubular tether bridging the two protists can be seen. The nucleus and kinetoplast in B. caudatus is DAPI stained (top). The nucleus of Colpodella sp. (below) also DAPI stained can be seen. DIC image shows morphology of both protists (AVI 52497 kb)

Supplementary Fig. 2

Volocity movie of Colpodella sp. (below) and B. caudatus (top) cysts. DAPI staining shows six nuclei of Colpodella sp. DIC image of cyst morphology is shown. (AVI 66691 kb)

Supplementary Fig. 3

Volocity movie of Colpodella sp. and B. caudatus cysts. DAPI staining shows four nuclei of Colpodella sp. DIC image of cyst morphology is shown. (AVI 12835 kb)

Supplementary Fig. 4

Normal rabbit serum, 1:100 was used as a negative control in IFA. There was no reactivity with proteins of Colpodella sp. and B. caudatus. The merged image shows two separate attachments of Colpodella sp. and B. caudatus trophozoites. B. DAPI staining shows the nucleus of Colpodella sp. and B. caudatus. C. Background reactivity was observed on cells with NRS. D. DIC images show the attachment of Colpodella sp. and the tubular tethers. (PNG 234 kb)

High resolution image (TIF 316 kb)

MP4 video of trophozoites egressing from Colpodella sp. cysts. Four trophozoites egress individually from a cyst (top, at 10-26 s) followed by egress of eight trophozoites (far right, at 34 s to 3 min 34 s). In the second cyst, the first four trophozoites egress individually and swim away followed by two pairs of trophozoites still joined at the anterior ends. Both pairs of trophozoites complete cytokinesis and separate outside the cyst. Bodo caudatus prey with multiple Colpodella sp. attachments can be seen in the video. The predator-prey connections in myzocytosis are not static but move within the culture. (MP4 61073 kb)

Supplementary Fig. 6

Sam-Yellowe’s Trichrome stained smears of Colpodella sp. life cycle stages. Sam-Yellowe’s Trichrome A stain was used to visualize life cycle stages of Colpodella sp. in culture. Black arrows identify demilune cysts of Colpodella sp., black arrowheads identify mature Colpodella sp. cysts and red arrows identify cysts of Bodo caudatus. (PNG 589 kb)

High resolution image (TIF 782 kb)


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Sam-Yellowe, T.Y., Getty, T.A., Addepalli, K. et al. Novel life cycle stages of Colpodella sp. (Apicomplexa) identified using Sam-Yellowe’s trichrome stains and confocal and electron microscopy. Int Microbiol 25, 669–678 (2022).

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