Colpodella sp. (ATCC 50594) Life Cycle: Myzocytosis and Possible Links to the Origin of Intracellular Parasitism

Colpodella species are free living bi-flagellated protists that prey on algae and bodonids in a process known as myzocytosis. Colpodella species are phylogenetically related to Apicomplexa. We investigated the life cycle of Colpodella sp. (ATCC 50594) to understand the timing, duration and the transition stages of Colpodella sp. (ATCC 50594). Sam-Yellowe’s trichrome stains for light microscopy, confocal and differential interference contrast (DIC) microscopy was performed to identify cell morphology and determine cross reactivity of Plasmodium species and Toxoplasma gondii specific antibodies against Colpodella sp. (ATCC 50594) proteins. The ultrastructure of Colpodella sp. (ATCC 50594) was investigated by transmission electron microscopy (TEM). The duration of Colpodella sp. (ATCC 50594) life cycle is thirty-six hours. Colpodella sp. (ATCC 50594) were most active between 20–28 h. Myzocytosis is initiated by attachment of the Colpodella sp. (ATCC 50594) pseudo-conoid to the cell surface of Parabodo caudatus, followed by an expansion of microtubules at the attachment site and aspiration of the prey’s cytoplasmic contents. A pre-cyst formed at the conclusion of feeding differentiates into a transient or resting cyst. Both DIC and TEM microscopy identified asynchronous and asymmetric mitosis in Colpodella sp. (ATCC 50594) cysts. Knowledge of the life cycle and stages of Colpodella sp. (ATCC 50594) will provide insights into the development of intracellular parasitism among the apicomplexa.


Introduction
Colpodella species are free-living terrestrial, fresh water or marine predators that feed on protists and algae. Trophozoite and cyst stages have been described in the life cycles of Colpodella species [1]. Trophozoites have a cone shaped microtubular structure which forms the pseudo-conoid contained in the rostrum, used for feeding [1,2]. Colpodella species possess a pellicle and apical complex organelles, like pathogenic apicomplexans, that include rhoptries, micronemes, pseudo-conoid, polar rings and microtubules, which facilitate predation. Trophozoites possess hetero-dynamic flagella that originate from separate flagella pockets and possess transversal plates in the transitional zone. A thin wall cylinder lies over the transversal plate. [2,3]. Some Colpodella species possess tricho-cysts which are organelles that are ejected in response to stimuli [3].
The mechanisms of encystation, excystation and transformation of life cycle stages is unknown among colpodellids and has not been described in culture or in the environment. The life cycles of Colpodella vorax, C. unguis, C. turpis and C. pugnax have been reported [1,3]. However, its duration and the timing of stage transformations were not described in these studies [1,3]. Studies aimed at investigating the biology of Colpodella species would benefit

Time Course Studies of Colpodella sp. (ATCC 50594) Development in Culture
In order to identify life cycle stages of Colpodella sp. (ATCC 50594) in Hay medium culture, a time course analysis of Colpodella sp. (ATCC 50594) development was performed. Subculture of 500 µL resting cyst stages of Colpodella sp. (ATCC 50594) and P. caudatus was performed in 10 mL of Hay medium in each T25 flask (nine flasks), to initiate the time course. The initial subculture of cells was collected at T = 0 and then cells were collected every four hours for 36 h (T = 1, T = 2, T = 3, T = 4, T = 5, T = 6, T = 7, T = 8, and T = 9). The first time course experiment was performed in four replicates. Each replicate was performed for 36 h using slide culture chambers for the first two replicates and tissue culture flasks for the second two. The four replicates were performed to determine the reproducibility of the life cycle and stage transitions. Slide culture chambers contained 2 mL cultures in duplicate per slide. Encysted cultures were also collected 5 and 7 days after encystation to identify the morphology of the resting cyst stages. In order to observe cells in the most active period of the life cycle, an additional time course experiment was performed for 40 h. Cells were collected for fixation and staining every four hours up until twenty-two hours. At twenty-two hours cells were collected every hour until 30 h. Then cells were collected for fixation and staining every two hours until 40 h. A final time course experiment was performed to identify the predominant cyst stage of Colpodella sp. (ATCC 50594) in resting cultures. Cells were collected every 24 h for eight days and formalin-fixed for staining with Sam-Yellowe's trichrome staining [18]. Days five, seven and eight cultures were fixed, stained and counted. One hundred cysts on duplicate slides were counted to obtain the percentage of each Colpodella sp. (ATCC 50594) life cycle stage present in resting cultures.

Immunofluorescence and Differential Interference Contrast Microscopy
Immunofluorescence assay (IFA) was performed on cells from the di-protist culture fixed using 5% formalin. Formalin fixed cells were permeabilized with 0.1% Triton X-100 then blocked with 3% bovine serum albumin (BSA). After blocking, incubation occurred with primary antibodies specific for antigen. This incubation was followed by three washes with 1× dPBS and then the smears were incubated with species specific secondary antibody conjugated to an Alexa fluorophore. Alexa fluorophores used include Alexa 488 and Alexa 647. Antibodies specific to apical complex organelle proteins were used to identify Colpodella sp. (ATCC 50594) proteins as described [17]. Antiserum 686 [21] specific for the rhoptry protein RhopH3 was used for IFAs. Other antibodies used were anti-Py235 [22], Anti-plasmepsin II [23], anti-EBA175 [24], anti-AMA1 [25], anti-IMC3, anti-IMC3 FLR, anti-IMC7 [26][27][28] and anti-RhopH3 full length (FL) [29]. In some experiments, cells were stained with Actin green 488 to detect actin and then reacted with antibodies for immunofluorescence. IFA slides were examined at the imaging core (Learner Research Institute, Cleveland Clinic). Confocal, fluorescent and differential interference contrast (DIC) images were collected using a Leica SP8 True Scanning Confocal (TCS) DM18 inverted microscope (Leica Microsystems, GmbH, Wetzlar, Germany). Stained and confocal images were adjusted to 300 dpi using CYMK color mode, RGB color mode, auto color and auto contrast on Adobe Photoshop (CC). 3D reconstructions of confocal z-stacks were performed using Volocity v.6.3.0 software (Quorum Technologies Inc., Puslinch, ON, Canada).

Transmission Electron Microscopy
An aliquot of Colpodella sp. (ATCC 50594) in culture medium was added to an equal volume of 8% paraformaldehyde in 0.1 M cacodylate buffer and spun down for 10 min at 3500 rpm. The cell pellet was fixed with 2.5% glutaraldehyde, 2% paraformaldehyde in 0.1 M cacodylate buffer. The fixation continued in the same fixative solution for a total of 2 h at room temperature. The pellets were thoroughly rinsed in 0.1 M cacodylate buffer, and then postfixed for 2 h in an unbuffered 1:1 mixture of 2% osmium tetroxide and 3% potassium ferrocyanide. After rinsing with distilled water, the specimens were soaked overnight in an acidified solution of 0.25% uranyl acetate. After another rinse in distilled water, they were dehydrated in ascending concentrations of ethanol, passed through propylene oxide, and embedded in an EMbed 812 embedding media (Electron Microscopy Sciences, Hatfield, PA, USA). Thin sections (70 nm) were cut on a RMC MT6000-XL ultramicrotome. These were mounted on T-300 mesh nickel grids (Electron Microscopy Sciences, Hatfield, PA, USA) and then sequentially stained with acidified methanolic uranyl acetate and stable lead staining solution. They were then coated on a Denton DV-401 carbon coater (Denton Vacuum LLC, Moorestown, NJ, USA), and observed in a FEI Tecnai Spirit (T12) transmission electron microscope with a Gatan US4000 4k × 4k CCD.

General Staining
Sam-Yellowe's trichrome staining protocols were used to stain formalin-fixed cells of Colpodella sp. (ATCC 50594) from di-protist cultures. Two major life cycle stages occur in Colpodella sp. (ATCC 50594) and the staining protocol distinguishes these. The stages are trophozoites and cysts of predator and prey. prey at a time for feeding in the process of myzocytosis. Figure 2 shows as many as seven predators attached to one prey ( Figure 2A, blue arrows, red arrowhead indicates prey) and six predators attached to one prey in Figure 2B (blue arrows, red arrow indicates prey). Egressed trophozoites from Colpodella sp. (ATCC 50594) cysts were identified, still attached at the anterior ends ( Figure 2C,D). the cyst, separating the cysts from bacteria. Mature cysts of Colpodella sp. (ATCC 50594) are identified by black arrowheads (panels B, D and G) and P. caudatus cysts are identified by the red arrowhead (panel A). Predator (yellow arrow)-prey (red arrow) in myzocytosis with tubular tethers of varying lengths (open black arrows), used for attachment, are shown in panels E and F. A large posterior food vacuole (Fv) is formed in the predator as cytoplasmic contents are aspirated from the prey. Multiple predators can attach to one prey at a time for feeding in the process of myzocytosis. Figure 2 shows as many as seven predators attached to one prey ( Figure 2A, blue arrows, red arrowhead indicates prey) and six predators attached to one prey in Figure 2B (blue arrows, red arrow indicates prey). Egressed trophozoites from Colpodella sp. (ATCC 50594) cysts were identified, still attached at the anterior ends ( Figure 2C,D).
.     Figure 3A-Z and Table S2. Parabodo caudatus trophozoites and Colpodella sp. (ATCC 50594) in myzocytosis were identified at 20 h (T = 5, Figure 3A,B), Colpodella sp. (ATCC 50594) pre-cysts and cysts were identified (panels C and D), with the active feeding by myzocytosis observed from 22 (T = 6) to 30 h (T = 13) ( Figure 3E-T). Cells were found to be most active during the 20 h to 28 h time points. Multiple myzocytosis attachments were observed. Early and mature cysts were also seen as Colpodella sp. (ATCC 50594) encysted. Pre-cysts of Colpodella sp. (ATCC 50594) were observed (panels 3C and 3J). In cells undergoing myzocytosis, young and mature cysts were observed up to 30 h (T = 13). By 32 h, more Colpodella sp. (ATCC 50594) cysts were observed and young trophozoites excysting from cysts were observed (panels 3U to 3Y). By 36 h, cyst stages of both Colpodella sp. (ATCC 50594) and P. caudatus were present in the culture and remained predominant up to 40 h (T-18). Clear zones separating Colpodella sp. (ATCC 50594) cysts from bacteria were observed (panels 3D and 3U). during the 20 h to 28 h time points. Multiple myzocytosis attachments were observed. Early and mature cysts were also seen as Colpodella sp. (ATCC 50594) encysted. Pre-cysts of Colpodella sp. (ATCC 50594) were observed (panels 3C and 3J). In cells undergoing myzocytosis, young and mature cysts were observed up to 30 h (T = 13). By 32 h, more Colpodella sp. (ATCC 50594) cysts were observed and young trophozoites excysting from cysts were observed (panels 3U to 3Y). By 36 h, cyst stages of both Colpodella sp. (ATCC 50594) and P. caudatus were present in the culture and remained predominant up to 40 h (T-18). Clear zones separating Colpodella sp. (ATCC 50594) cysts from bacteria were observed (panels 3D and 3U).  A third time course was performed to identify the predominant Colpodella sp. (ATCC 50594) cyst stage in a resting culture. Cells were formalin-fixed for staining every 24 h for eight days. Additionally, cysts from day five and cysts from day seven resting cultures were each pooled and formalin-fixed for trichrome staining. The results show that the predominant Colpodella sp. (ATCC 50594) life cycle stage in resting cultures for up to eight days were mature cysts with a single nucleus. Cysts on days seven and eight were the same stage. A few mature cysts were observed with two or more nuclei and some young demilune cysts were also observed ( Figure 3A'-H'). In order to determine the percentage of different cyst stages present in the resting culture, cysts from pooled day five and seven cultures were counted. One hundred cysts were counted on duplicate slides from cysts stained from day 5 and 7 cultures. Of the cysts, 85% were mature cysts on day 5 and 93% on day 7 (Table 1). A few Colpodella sp. (ATCC 50594) and P. caudatus trophozoites excysted during these time points.

Differential Interference Microscopy (DIC)
We performed DIC microscopy and DAPI staining to identify the morphology of life cycle stages and the number of nuclei present in trophozoites and cysts, respectively. Single or double Colpodella sp. (ATCC 50594) (yellow arrows) feeding on P. caudatus (red arrow) were identified. DAPI staining identified the kinetoplast and nucleus of P. caudatus and the central nucleus of Colpodella sp. (ATCC 50594) trophozoites (white arrow). DIC microscopy and DAPI staining are shown in Figure 4A,C and DAPI staining alone shown in panels Figure 4B,D. A Colpodella sp. (ATCC 50594) trophozoite (yellow arrow) feeding on P. caudatus (red arrow) in myzocytosis is shown by DIC and DAPI staining in Figure 4E.  Figure S1). DIC microscopy and DAPI stained pre-cysts of Colpodella sp. (ATCC 50594) are shown in Figure 5A,C,E. The yellow arrow shows the frayed and disintegrated anterior end of the trophozoite and the forming cyst. Panels B, D and F show DAPI stained nucleus and aspirated cytoplasmic contents of the prey, respectively. DIC microscopy and DAPI staining show a four-nuclei cyst ( Figure 5G) and a five-nuclei cyst ( Figure 5I). The nuclei in each cyst were identified by the blue DAPI staining. A young trophozoite identified by DIC microscopy and DAPI staining shows a central nucleus ( Figure 5K,L). are shown in Figure 5A,C,E. The yellow arrow shows the frayed and disintegrated anterior end of the trophozoite and the forming cyst. Panels B, D and F show DAPI stained nucleus and aspirated cytoplasmic contents of the prey, respectively. DIC microscopy and DAPI staining show a four-nuclei cyst ( Figure 5G) and a five-nuclei cyst ( Figure 5I). The nuclei in each cyst were identified by the blue DAPI staining. A young trophozoite identified by DIC microscopy and DAPI staining shows a central nucleus ( Figure 5K,L).

Transmission Electron. Microscopy
Cells from di-protist cultures were prepared for TEM to investigate the ultrastructure of Colpodella sp. (ATCC 50594) life cycle stages. Colpodella sp. (ATCC 50594) trophozoites were identified in Figure 6A,B ( Figure S6A). Organelles indicated by black arrows are rhoptries with the bodies extending into the cytoplasm. Colpodella sp. (ATCC 50594) trophozoites (yellow arrows) and P. caudatus (red arrows) in myzocytosis were also identified in Figure 6C (enlarged in Figure 6D and Figure S6B), in Figure 6E (enlarged in Figure 6F) and in Figure 6G. Initial contact is made by the pseudo-conoid of Colpodella sp. (ATCC 50594) to the plasma membrane of P. caudatus. The point of attachment is indicated by blue arrows. Microtubular organization in the cytoskeleton in areas of close proximity to the point of attachment is seen in panels 6 D to G ( Figures S6B and S7A,B). The flow of cytoplasmic contents from the prey (open white arrows) into the predator was identified in Figure 6G (enlarged in Figure S7B). The flow of cytoplasmic contents from the prey into Colpodella sp. (ATCC 50594) (white arrow) was observed ( Figure S6C). The plasma membrane of the prey pulled into the predator is indicated by black arrows. The initial attachment of the pseudo-conoid with bands of microtubules organized at the point of attachment ( Figure S7A) and extension of the plasma membrane of Colpodella sp. (ATCC 50594) (blue arrows) with foci of microtubules is shown ( Figures S6C and S7B) in a two-step process for myzocytosis. The plasma membrane of the prey pulled into the cytoplasm of the predator is broken down to allow for aspiration of cytoplasmic contents of the prey (Figure S7B). Bacteria (B) taken up by P. caudatus were observed in the cytoplasm.

Transmission Electron Microscopy
Cells from di-protist cultures were prepared for TEM to investigate the ultrastructure of Colpodella sp. (ATCC 50594) life cycle stages. Colpodella sp. (ATCC 50594) trophozoites were identified in Figure 6A,B ( Figure S6A). Organelles indicated by black arrows are rhoptries with the bodies extending into the cytoplasm. Colpodella sp. (ATCC 50594) trophozoites (yellow arrows) and P. caudatus (red arrows) in myzocytosis were also identified in Figure 6C (enlarged in Figure 6D and Figure S6B), in Figure 6E (enlarged in Figure 6F) and in Figure 6G. Initial contact is made by the pseudo-conoid of Colpodella sp. (ATCC 50594) to the plasma membrane of P. caudatus. The point of attachment is indicated by blue arrows. Microtubular organization in the cytoskeleton in areas of close proximity to the point of attachment is seen in panels 6 D to G ( Figures S6B and S7A,B). The flow of cytoplasmic contents from the prey (open white arrows) into the predator was identified in Figure 6G (enlarged in Figure S7B). The flow of cytoplasmic contents from the prey into Colpodella sp. (ATCC 50594) (white arrow) was observed ( Figure S6C). The plasma membrane of the prey pulled into the predator is indicated by black arrows. The initial attachment of the pseudo-conoid with bands of microtubules organized at the point of attachment ( Figure S7A) and extension of the plasma membrane of Colpodella sp. (ATCC 50594) (blue arrows) with foci of microtubules is shown ( Figures S6C and S7B) in a two-step process for myzocytosis. The plasma membrane of the prey pulled into the cytoplasm of the predator is broken down to allow for aspiration of cytoplasmic contents of the prey ( Figure S7B). Bacteria (B) taken up by P. caudatus were observed in the cytoplasm.
Formation of the large posterior food vacuole (Fv) in Colpodella sp. (ATCC 50594) (yellow arrow) is seen in Figure 6H. A high magnification of the tubular tether holding predator and prey together shows the plasma membrane and cytoplasm of P. caudatus being pulled into the predator surrounded by the plasma membrane of the predator ( Figure 6I, large black arrowheads). The direction of flow of cytoplasmic contents, including mitochondria (m) and other organelles, aspirated from the prey into the cytoplasm of the prey is shown (blue arrows, Figure 6I, enlarged in Figure S6C) and Figure S7B.
Following myzocytosis, the anterior end of the Colpodella sp. (ATCC 50594) trophozoite disintegrates resulting in loss of the flagella and organelles to form the pre-cyst formed from the posterior food vacuole (Fv), remnant cytoplasm and nucleus ( Figure 6J and enlarged in Figure 6K). The young cyst stages of Colpodella sp. (ATCC 50594) with developing trophozoites (DT) are shown in Figure 6L,M with the residual food vacuole (Fv) and a thin cyst wall (black arrow). Cysts containing two developing trophozoites ( Figure 6N-P), three ( Figure 6Q), four ( Figure 6R) and seven ( Figure 6S) developing trophozoites are shown. Colpodella sp. (ATCC 50594) cysts can have both asymmetric and symmetric division as both odd and even-numbered juvenile trophozoites were observed within mature cysts. In Figure 6O,P,R,S, juvenile trophozoites at different stages of maturity were observed. Asynchronous development was observed where one trophozoite already had a developed pseudo-conoid. The developed pseudo-conoid in the cysts indicated by yellow arrows was identified along with flagella (anterior and posterior).
The thin cyst wall is indicated by black arrows. A trophozoite of P. caudatus is shown in Figure 6T

Immunofluorescence of Colpodella sp. (ATCC 50594) in Diprotist Culture Using Anti-RhopH3 Antibodies
In order to determine if proteins associated with host cell invasion in pathogenic apicomplexans are shared by the free-living Colpodella sp. (ATCC 50594), we performed immunofluorescence assay (IFA) using Plasmodium sp. and Toxoplasma gondii specific antibodies ( Table 2). We investigated cross-reactivity of the antibodies to proteins in different life cycle stages of Colpodella sp. (ATCC 50594). DAPI staining identified the round central nucleus of Colpodella sp. (ATCC 50594) trophozoites (red arrow) and the kinetoplast (gold arrowhead) and nucleus (grey arrowhead) of prey P. caudatus ( Figure 7A). Antiserum 686 against the RhopH3 rhoptry protein of P. falciparum (green) and antiserum FL against the RhopH3 protein of P. berghei (red) reacted with structures in the cytoplasm of Colpodella sp. (ATCC 50594) trophozoites. Antibody reactivity was also observed in the tubular tether with what appears to be a spherical structure in the tube. Anti-RhopH3 antibody reactivity was also observed in the cytoplasm of the prey ( Figure 7B,C). Antibody/DAPI and Antibody/DAPI/ DIC merged images identified colocalization of the two RhopH3 specific antibodies, the morphology of the predator and prey, the long tubular tether and the enlarged posterior food vacuole of the predator ( Figure 7D,E). Volocity videos generated from z-stack acquisitions of immunofluorescence, DAPI and DIC images show the predator and prey in myzocytosis ( Figure S2). Antibody 686 and 676 reactive with P. falciparum RhopH3 and rhoptries, respectively, reacted with discrete structures at the apical end of the attached Colpodella sp. (ATCC 50594) trophozoites and within the cytoplasm of the trophozoite (Figures S3 and S4). Open arrows show the point of attachment ( Figure S3). Volocity videos generated from z-stack acquisitions of immunofluorescence, DAPI and DIC images show the predator and prey in myzocytosis ( Figure S4). Still images of Figure S4 are shown in Figure S5 and identify the prey's destruction upon aspiration by the predator. Cytoplasmic contents of Colpodella sp. (ATCC 50594) identified by antibody reactivity as particulate circular structures could be seen ( Figures S4 and S5).
arrows show the point of attachment ( Figure S3). Volocity videos generated from z-stack acquisitions of immunofluorescence, DAPI and DIC images show the predator and prey in myzocytosis ( Figure S4). Still images of Figure S4 are shown in Figure S5 and identify the prey's destruction upon aspiration by the predator. Cytoplasmic contents of Colpodella sp. (ATCC 50594) identified by antibody reactivity as particulate circular structures could be seen (Figures S4 and S5).

Immunofluorescence of Colpodella sp.(ATCC 50594) with Antibodies against IMC3, IMC7, Py235, EBA175, AMA1 and Plasmepsin II Proteins
Different antibodies against known apical and non-apical proteins of P. falciparum and Toxoplasma gondii were used in IFA. We wanted to know if antigens recognized by these antibodies could be localized in Colpodella sp. (ATCC 50594). Antisera were diluted at 1:50, 1:100, 1:200, 1:500, and 1:1000. Anti-IMC3 FLR which recognizes the full-length antigen was reactive with proteins in the cysts and trophozoites of Colpodella sp. ( Figure  8A-P) and weakly in P. caudatus. Colpodella sp. (ATCC 50594) two-way cysts were reactive with anti-IMC3FLR (yellow arrows). Red arrows show weak to no reactivity in P. caudatus. Colpodella sp. (ATCC 50594) trophozoites were reactive with the antibody (yellow arrowhead, Figure 8H,P)). No reactivity was obtained with P. caudatus trophozoites (red arrowheads, Figure 8H,P). A young Colpodella sp. (ATCC 50594) trophozoite reacted with anti-IMC3 FLR. Both flagella are shown (double yellow arrow, Figure 8M,N). IMC3 is an inner membrane complex protein found in apicomplexans and was identified in Colpodella sp. (ATCC 50594). IMC7 is another inner membrane complex protein identified in apicomplexan parasites. There was no reactivity with anti-IMC7 antibody as the reactivity was observed as background reactivity (Figure 9 A-D). There was no cross reactivity observed Different antibodies against known apical and non-apical proteins of P. falciparum and Toxoplasma gondii were used in IFA. We wanted to know if antigens recognized by these antibodies could be localized in Colpodella sp. (ATCC 50594). Antisera were diluted at 1:50, 1:100, 1:200, 1:500, and 1:1000. Anti-IMC3 FLR which recognizes the full-length antigen was reactive with proteins in the cysts and trophozoites of Colpodella sp. (Figure 8A-P) and weakly in P. caudatus. Colpodella sp. (ATCC 50594) two-way cysts were reactive with anti-IMC3FLR (yellow arrows). Red arrows show weak to no reactivity in P. caudatus. Colpodella sp. (ATCC 50594) trophozoites were reactive with the antibody (yellow arrowhead, Figure 8H,P)). No reactivity was obtained with P. caudatus trophozoites (red arrowheads, Figure 8H,P). A young Colpodella sp. (ATCC 50594) trophozoite reacted with anti-IMC3 FLR. Both flagella are shown (double yellow arrow, Figure 8M,N). IMC3 is an inner membrane complex protein found in apicomplexans and was identified in Colpodella sp. (ATCC 50594). IMC7 is another inner membrane complex protein identified in apicomplexan parasites. There was no reactivity with anti-IMC7 antibody as the reactivity was observed as background reactivity ( Figure 9A-D). There was no cross reactivity observed with Py235 antisera with proteins of Colpodella sp. (ATCC 50594) ( Figure 9E-H). The antibody is specific for the 235 kDa rhoptry protein of P. yoelii, which is a rodent parasite. Only faint background reactivity was observed. The black arrow identifies the tubular tether formed between predator (yellow arrow) and prey (red arrow) Figure 9E,H. DAPI stained cytoplasmic contents aspirated from the prey were identified (black arrowhead, Figure 9E). AMA1 is a microneme protein used in cell invasion among parasitic apicomplexans. Intense cross reactivity was obtained with the antibodies on proteins of Colpodella sp. trophozoites and weakly on P. caudatus trophozoites ( Figure 10A-H).   The cross reactivity with anti-AMA1 antibody in Colpodella sp. (ATCC 50594) trophozoites was observed towards the apical end ( Figure 10B,D,F,H, yellow arrowhead). The central nucleus of Colpodella sp. (ATCC 50594) trophozoites was identified by white arrows and the trophozoites showing strong anti-AMA1 reactivity are identified by yellow arrows. Parabodo caudatus trophozoites (red arrows) showed weak to no reactivity with the antibody. DAPI stained cytoplasmic contents aspirated from the prey were identified ( Figure 10A,D, grey arrowhead). EBA175 is a microneme protein that functions in P. falciparum merozoite invasion. It is used to initiate invasion of red blood cells. Antibody cross reactivity was identified in Colpodella sp. (ATCC 50594) at the apical end of the cell and weakly in P. caudatus ( Figure 11A-H). The DAPI stained nucleus (n) and kinetoplast (k) of P. caudatus and the nucleus of Colpodella sp. (ATCC 50594) trophozoite (white arrow) is shown in Figure 11A. Anti-EBA175 reactivity was observed in the anterior of Colpodella sp. (ATCC 50594) trophozoites ( Figure 11B,D, yellow arrow). Parabodo caudatus with weak reactivity was identified (red arrows) and a group of four Colpodella sp. (ATCC 50594) (indicated by yellow arrowhead) feeding on a single prey (red arrow) were identified. Antibody reactivity was strongest at the apical ends of Colpodella sp. (ATCC 50594) trophozoites ( Figure 11F,H). Plasmepsin II is an aspartic protease located in the food vacuole of P. falciparum where it degrades hemoglobin. Cross reactivity was observed between the antisera and antigen in Colpodella sp. (ATCC 50594) pre-cyst stages ( Figure 12A-H, grey arrowheads). The posterior food vacuole (Fv) in the pre-cyst stages of Colpodella sp. (ATCC 50594) reacted with anti-plasmepsin II ( Figure 12D,H). Weak to no antibody reactivity was observed in unattached P. caudatus trophozoites ( Figure 12A, red arrowheads). The nucleus (n) of the pre-cyst was identified ( Figure 12A,F) and flagella (yellow arrowhead) still   The cross reactivity with anti-AMA1 antibody in Colpodella sp. (ATCC 50594) trophozoites was observed towards the apical end ( Figure 10B,D,F,H, yellow arrowhead). The central nucleus of Colpodella sp. (ATCC 50594) trophozoites was identified by white arrows and the trophozoites showing strong anti-AMA1 reactivity are identified by yellow arrows. Parabodo caudatus trophozoites (red arrows) showed weak to no reactivity with the antibody. DAPI stained cytoplasmic contents aspirated from the prey were identified ( Figure 10A,D, grey arrowhead). EBA175 is a microneme protein that functions in P. falciparum merozoite invasion. It is used to initiate invasion of red blood cells. Antibody cross reactivity was identified in Colpodella sp. (ATCC 50594) at the apical end of the cell and weakly in P. caudatus ( Figure 11A-H). The DAPI stained nucleus (n) and kinetoplast (k) of P. caudatus and the nucleus of Colpodella sp. (ATCC 50594) trophozoite (white arrow) is shown in Figure 11A. Anti-EBA175 reactivity was observed in the anterior of Colpodella sp. (ATCC 50594) trophozoites ( Figure 11B,D, yellow arrow). Parabodo caudatus with weak reactivity was identified (red arrows) and a group of four Colpodella sp. (ATCC 50594) (indicated by yellow arrowhead) feeding on a single prey (red arrow) were identified. Antibody reactivity was strongest at the apical ends of Colpodella sp. (ATCC 50594) trophozoites ( Figure 11F,H). Plasmepsin II is an aspartic protease located in the food vacuole of P. falciparum where it degrades hemoglobin. Cross reactivity was observed between the antisera and antigen in Colpodella sp. (ATCC 50594) pre-cyst stages ( Figure 12A-H, grey arrowheads). The posterior food vacuole (Fv) in the pre-cyst stages of Colpodella sp. (ATCC 50594) reacted with anti-plasmepsin II ( Figure 12D,H). Weak to no antibody reactivity was observed in unattached P. caudatus trophozoites ( Figure 12A, red arrowheads). The nucleus (n) of the pre-cyst was identified ( Figure 12A,F) and flagella (yellow arrowhead) still present in the pre-cyst ( Figure 12G,H) were observed. present in the pre-cyst ( Figure  12G,H) were observed.   There was no reactivity observed with normal mouse (Figure 13C,G) and rabbit serum ( Figure 13E-H) used as negative controls. Aspiration of DAPI stained contents (grey arrowhead, Figure 13A There was no reactivity observed with normal mouse (Figure 13C,G) and rabbit serum ( Figure 13E-H) used as negative controls. Aspiration of DAPI stained contents (grey arrowhead, Figure 13A    There was no reactivity observed with normal mouse (Figure 13C,G) and rabbit serum ( Figure 13E-H) used as negative controls. Aspiration of DAPI stained contents (grey arrowhead, Figure 13A,B) from the prey was detected in the posterior food vacuole of a Colpodella sp. (ATCC 50594) trophozoite (yellow arrow) attached to P. caudatus prey (red arrow) ( Figure 13A,E). The nucleus (n) and kinetoplast (k) of P. caudatus and the central nucleus of Colpodella sp. (ATCC 50594) trophozoite (white arrow) were identified by DAPI staining. .

Discussion
In this study, Colpodella sp. (ATCC 50594) was grown in tissue culture flasks in Hay medium [19,20] and used in studies aimed at identifying the specific time points and stage transitions for each life cycle stage. Three time course experiments with four replicates of experiment one were performed. Cells were formalin-fixed and stained with Sam-Yellowe's trichrome [5,18] stains to view the cells. We used the first four time course replicates to demonstrate duration and reproducibility of the life cycle stages and timing of stage transitions in Colpodella sp. (ATCC 50594) cultures. These four replicates revealed that P. caudatus excysts much earlier than the predator approximately four hours after subculturing and dominates in the culture until about 20 h when majority of Colpodella sp. (ATCC 50594) trophozoites begin to egress from their cysts. Young trophozoites of Colpodella sp. (ATCC 50594) emerge and begin myzocytosis lasting between 20 and 30 h in culture. At 28 h, P. caudatus trophozoites begin to encyst with Colpodella sp. (ATCC 50594) trophozoites beginning to encyst at 30 h. By 36 h the culture is mostly quiet with only a few predator and prey trophozoites remaining. Clear zones surrounding Colpdella sp. (ATCC 50594) cysts, separating them from bacteria were observed. It is unclear if these zones represent anti-bacterial activity or artifacts of fixation. Additional investigations will be required to understand the identity and significance of these structures. These type of experiments have not been performed previously for any Colpodella species. Recent investigations in our lab identified previously undocumented life cycle stages in the di-protist culture using Sam-Yellowe's trichrome stains, confocal and DIC microscopy and TEM [5]. The appearance of the early cyst stage of Colpodella sp. (ATCC 50594) shows an irregular dual-colored, dark blue-purple and white colored cyst which we designated a demilune cyst [5,18]. The stage before the demilune cyst when the food vacuole and nucleus are still visible and the anterior end of the trophozoite is disintegrated, we have designated as the pre-cyst stage. Mature Colpodella sp. (ATCC 50594) cysts stained dark-red blue and contained two or more juveniles [5,18]. These designations allow for stage transitions to be identified. In the current study, timing and transitions of these newly described stages were identified.
The second and third time course experiments focused on the most active time period of the life cycle and identification of the predominant cyst stage present in resting cultures, respectively. Predator and prey remain encysted until they are sub-cultured and are viable up to 14 days. The cells must be sub-cultured by 14 days or the cysts start to deteriorate, and the cell yield is low. Smears from di-protist cultures stained from pooled day five and seven cultures were counted to determine the percentage of each cyst stage present in the resting culture. Eighty to ninety percent of the Colpodella sp. (ATCC 50594) cysts were single nuclei mature cysts. Not all Colpodella sp. (ATCC 50594) cysts were mature with a single nucleus, and some had multiple asymmetric nuclei or symmetric nuclei with two or more juvenile trophozoites. Early Colpodella sp. (ATCC 50594) demilune cysts were also counted but there only seemed to be a relatively small number. Even though these cultures were primarily resting, free swimming predator and prey trophozoites, encysting and excysting during these time points, were also detected. The most active part of the life cycle was between 20 and 30 h, with peak activity observed between 20 and 28 h. The cultures looked the same from 36 to 40 h. Knowledge of a general life cycle reflecting transition times in culture will aid future investigations of Colpodella sp. (ATCC 50594) and help in the identification of infective stages in opportunistic human infections caused by Colpodella species. Furthermore, Colpodella species in ticks that feed on animals, with potential for zoonotic infections in humans, can be identified.
Single predator prey attachments were the most common pairings observed, similar to reports in other Colpodella species [1,3,5]. Two to three predators on one prey can also occur. In the most active stage of the life cycle observed in the present study, five to seven predators feeding on one prey were observed. Multiple attachments and different lengths of the tubular tethers have not been reported previously. One of the reasons for so many Colpodella sp. attaching to one prey may be connected to the smaller number of prey available when the majority of Colpodella sp. (ATCC 50594) trophozoites egress from cysts. Colpodella sp. (ATCC 50594) trophozoites exceed the number of P. caudatus trophozoites because of early prey encystation. This has only been observed in culture and may be different in the natural environment and in different culture conditions as summarized in Table S3. The description of the life cycle for C. vorax shows one to two trophozoites feeding on P. caudatus resulting in cysts that contain two to four juveniles [3]. Both C. turpis and C. pugnax were shown to have conjugation in their life cycles along with the formation of cysts containing two or four juveniles [2]. In C. unguis, oblique-transverse fission was identified in the life cycle [2,14]. In the current study, we did not observe fission or conjugation in the life cycle of Colpodella sp. (ATCC 50594). Knowledge of the timing of life cycle stage transitions will also facilitate investigations of organelles by isolating them. Colpodella species from natural habitats and their life cycle timing and stages can be investigated.
In the current study, transmission electron microscopy was performed to investigate the ultrastructure of Colpodella sp. (ATCC 50594) life cycle stages. Asymmetric and asynchronous multinucleate cyst stages were identified and Colpodella sp. (ATCC 50594) trophozoites in myzocytosis with P. caudatus prey were identified. Different stages of predator-prey attachment were observed, from initial attachment of the pseudo-conoid to the prey's plasma membrane and reorganization of microtubules and apical complex organelles at the point of attachment, after which the membrane of the prey is engulfed, pulled into the predator, degraded, and cytoplasmic contents of the prey flow into the predator's cytoplasm toward the posterior food vacuole. The extended plasma membrane of the predator with foci of microtubular organization was observed. Myzocytosis in Colpodella vorax also showed microtubules at the point of attachment of predator and prey [3]. In the present study, the anterior portion of the predator was observed to disintegrate after feeding, leading to loss of the flagella and cytoplasmic organelles and the rounding of the posterior food vacuole, along with the nucleus, to form the cyst.
Electron microscopy images identified the ultrastructure of Colpodella sp. (ATCC 50594) and P. caudatus cysts. The cysts of Colpodella sp. (ATCC 50594) have a thin cyst wall and a remnant food vacuole associated with undifferentiated trophozoites. The electron microscopy images confirmed that cysts of Colpodella sp. (ATCC 50594) can have asymmetric division as shown previously [5]. Results also showed that mitosis in mature    Not much is known about the process of cell division inside the cysts of Colpodella species. Closed mitosis is characteristic of apicomplexans where the nuclear envelope remains intact and the mitotic spindle is intranuclear [30][31][32][33]. Within apicomplexan life cycles, the plastid, flagella and asexual division have been the focus of investigations in order to gain insights into the origins of intracellular parasitism [30,33]. The mechanism of myzocytosis would also be instructive in the understanding of zoite invasion into host cells, since apical complex proteins are used in both processes. Genes encoding proteins for flagella and photosynthesis were lost in some apicomplexan lineages that are obligate intracellular parasites. Conversely, genes encoding secretory proteins required for host cell invasion and intracellular survival were gained [30]. In apicomplexa, asexual division results in the release of merozoites (zoites) that invade host cells. Both intracellular apicomplexan pathogens and their free-living relatives possess an apical complex with the presence of the secretory organelles, rhoptries and micronemes whose proteins initiate host cell invasion and maintain the trophozoite within the host cell [30,33]. In the current study, transmission electron microscopy showed asynchronous budding and development of Colpodella sp. (ATCC 50594) trophozoites within the cyst, with immature and mature juveniles present within the same cyst. Furthermore, flagella were also identified within the cyst. Mitosis in C. vorax was described as semi-open resulting in two and four trophozoites [3]. Cysts of Chromera velia also produce more than four juveniles [34], with the development of the juveniles shown to be closely associated with flagella and apical organelle complex formation [34].
In order to identify cross reactivity of antibodies specific for invasion and food vacuole proteins of P. falciparum in Colpodella sp. (ATCC 50594) life cycle stages, we performed immunofluorescence and confocal microscopy. Antibodies specific for apical and nonapical complex proteins of P. falciparum and non-apical complex proteins of Toxoplasma gondii was used in IFA with DAPI used to stain the nuclei of the predator and prey. The morphology of each protist was identified by DIC microscopy. Antiserum 686 specific for the RhopH3 rhoptry protein reacted with Colpodella sp. (ATCC 50594) trophozoites in free swimming trophozoites and in trophozoites attached to P. caudatus in myzocytosis, similar to previous reports [18]. The spherical structures reactive with rhoptry and RhopH3 specific antibodies were recognized within the cytoplasm of Colpodella sp. (ATCC 50594), at the points of attachment of predator and prey and within prey attached to Colpodella sp. (ATCC 50594). The microneme specific antibodies against AMA-1 [25] and EBA175 [24] reacted with the apical end of Colpodella sp. (ATCC 50594) trophozoites. The IFA data showing cross reactivity of apical complex protein specific antibodies is suggestive of a similar use of apical complex proteins for myzocytosis as reported for zoite invasion into host cells in parasitic apicomplexa. Additional investigation using Colpodella sp. (ATCC 50594) specific antibodies and molecular characterizations will determine if the predator-prey interactions observed use similar mechanisms to the zoite-host cell interactions recognized in pathogenic apicomplexan where intracellular host cell infections are dominant. The use of microneme and plasmepsin II specific antibodies in immunofluorescent assay studies to characterize Colpodella species proteins has not been performed previously. The specific structures containing the cross-reactive proteins are unknown. However, the cross reactivity of antibodies against apical complex proteins with Colpodella sp. (ATCC 50594) proteins suggests that events of myzocytosis may have preceded events that led to zoite internalization within host cells in intracellular parasitism. Discrete spherical and particulate structures were identified with RhopH3 specific antibodies in Colpodella sp. (ATCC 50594). There are no antibodies against apical complex or food vacuole proteins of Colpodella species. Antibodies against IMC3 cross reacted with proteins on Colpodella sp. (ATCC 50594) with a more diffuse staining pattern on the cells. There was no reactivity with antibodies against IMC7 and Py235. Interestingly, the antibodies against plasmepsin II, an aspartate protease found in the food vacuole of P. falciparum, reacted with proteins in the pre-cyst stages of Colpodella sp. (ATCC 50594) suggestive of a similar localization to the posterior food vacuole of Colpodella sp. (ATCC 50594) [23]. In a previous study anti-EBA175 was shown to react weakly with the cysts of Colpodella sp. (ATCC 50594) [5].
The type of genes activated and what proteins are expressed during each stage of the life cycle of Colpodella sp. (ATCC 50594) are unknown. Identifying genes conserved in Colpodella sp. (ATCC 50594) that have been identified among the parasitic apicomplexans can help with phylogenetic studies. Reactivity of antibodies against the microneme proteins, EBA175 and AMA1 used for merozoite invasion in P. falciparum will need to be confirmed by identifying the genes encoding these proteins in Colpodella sp. (ATCC 50594). The gene encoding AMA1 is highly conserved among the parasitic apicomplexans [35][36][37]. A proteomic study of the dinoflagellate Perkinsus marinus, identified liver stage antigen 3 and merozoite surface protein 3 of P. falciparum and Py235 rhoptry protein of P. yoelii [38]. In previous studies and in the current study, antibodies specific for rhoptry proteins and whole rhoptries of Plasmodium sp. cross-reacted with apical proteins in Colpodella sp. (ATCC 50594) and Voromonas pontica [17,39,40]. These results suggest that the dinoflagellates and colpodellids with synapomorphies of the apical complex containing rhoptries and micronemes may possess conserved genes encoding rhoptry and microneme proteins. Understanding the role played by the apical complex proteins may provide additional insights to help understand the origins of intracellular parasitism.