Fluorescent Nanoparticle Uptake by Myzocytosis and Endocytosis in Colpodella sp. ATCC 50594

Colpodella sp. (ATCC 50594) is a free-living biflagellate predator closely related to pathogenic Apicomplexa such as Plasmodium, Cryptosporidium and Toxoplasma gondii. Colpodella sp. (ATCC 50594) obtain nutrients by preying on Parabodo caudatus using myzocytosis. The organization of the myzocytic apparatus and the mechanism of nutrient uptake into the posterior food vacuole of Colpodella species is unknown. In this study, we investigated myzocytosis using light and transmission electron microscopy. We investigated the uptake of 40 nm and 100 nm fluorescent nanoparticles and E. coli BioParticles by Colpodella sp. (ATCC 50594) in a diprotist culture. Transmission electron microscopy was used to investigate the morphology of the tubular tether formed during myzocytosis. E. coli BioParticles were taken up by P. caudatus but not by Colpodella sp. (ATCC 50594). Both protists took up the 100 nm and 40 nm beads, which were observed distributed in the cytoplasm of free unattached Colpodella sp. (ATCC 50594) trophozoites, and also in feeding Colpodella sp. (ATCC 50594) trophozoites and in the pre-cysts. Fragments of the nucleus and kinetoplast of P. caudatus and the nanoparticles were identified in the tubular tether being aspirated into the posterior food vacuole of Colpodella sp. (ATCC 50594). Unattached Colpodella sp. (ATCC 50594) endocytose nutrients from the culture medium independently from myzocytosis. The mechanisms of myzocytosis and endocytosis among Colpodella species may provide important insights into nutrient uptake among the pathogenic apicomplexans.


Introduction
Colpodella species are free-living alveolate predators closely related phylogenetically to the pathogenic Apicomplexa [1,2]. Among the alveolates, Colpodella species, along with Voromonas pontica and the dinoflagellates Perkinsus and Psammosa species, possess two heterodynamic flagella and apical complex organelles, and are known as myzozoans [3]. The apical complex organelles, including the rhoptries, micronemes, polar rings and conoid participate in host cell invasion and nutrient uptake among the apicomplexans [4][5][6]. The apicomplexans and apicomplexan-like organisms are a polyphyletic group, thought to have evolved independently, at least three times [2]. Colpodella species possess an open conoid, known as a pseudoconoid [3]. Contents of the rhoptries are also used for nutrient acquisition in Plasmodium species and among the gregarines [7,8]. Colpodella species feed using a process known as myzocytosis, a characteristic of the myzozoans, where they attach to their flagellate and algae prey and aspirate the cytoplasmic contents into a posterior food vacuole. At the conclusion of feeding, most Colpodella species encyst. Variations exist in different Colpodella species regarding cyst formation. Resting cysts are found in the life cycle of Colpodella pseudoedax, in addition to division by longitudinal fission, C. unguis divides by Different sizes of the prey targeted by Colpodella sp. (ATCC 50594) trophozoites are shown in both panels. We reported in previous studies the presence of DAPI-stained contents in the food vacuole of Colpodella sp. (ATCC 50594) trophozoites [23]. In active cultures, free unattached Colpodella sp. (ATCC 50594) trophozoites were identified with DAPI-stained contents in the cytoplasm as shown in Figure 1C. Colpodella sp. (ATCC 50594) trophozoites in active culture were observed to attach to and detach from prey after making contact unlike the irreversible attachments of zoites during host cell invasion in pathogenic Apicomplexans such as Plasmodium species. We performed nutrient uptake experiments using fluorescent particles of 4 µm, 2 µm, 100 nm and 40 nm and E. coli BioParticles in order to determine the size of nutrients taken up in culture by Colpodella sp. (ATCC 50594) and its prey. The 4 and 2 µm particles were not taken up by Colpodella sp. (ATCC 50594). Fluorescent nanoparticles of 100 nm (Figure 2A-C) and 40 nm ( Figure 3A-H) were observed in the cytoplasm of Colpodella sp. (ATCC 50594) trophozoites, distributed within the cytoplasm. Colpodella sp. (ATCC 50594) trophozoites did not take up E. coli BioParticles. Parabodo caudatus trophozoites did not take up 4 µm or 2 µm particles but took up 100 nm, 40 nm and E. coli BioParticles from the culture. E. coli BioParticles were observed in the cytoplasm of P. caudatus ( Figure 2D, grey arrows). Different sizes of the prey targeted by Colpodella sp. (ATCC 50594) trophozoites are shown in both panels. We reported in previous studies the presence of DAPI-stained contents in the food vacuole of Colpodella sp. (ATCC 50594) trophozoites [23]. In active cultures, free unattached Colpodella sp. (ATCC 50594) trophozoites were identified with DAPI-stained contents in the cytoplasm as shown in Figure 1C. Colpodella sp. (ATCC 50594) trophozoites in active culture were observed to attach to and detach from prey after making contact unlike the irreversible attachments of zoites during host cell invasion in pathogenic Apicomplexans such as Plasmodium species. We performed nutrient uptake experiments using fluorescent particles of 4 µm, 2 µm, 100 nm and 40 nm and E. coli BioParticles in order to determine the size of nutrients taken up in culture by Colpodella sp. (ATCC 50594) and its prey. The 4 and 2 µm particles were not taken up by Colpodella sp. (ATCC 50594). Fluorescent nanoparticles of 100 nm (Figure 2A-C) and 40 nm ( Figure 3A-H) were observed in the cytoplasm of Colpodella sp. (ATCC 50594) trophozoites, distributed within the cytoplasm. Colpodella sp. (ATCC 50594) trophozoites did not take up E. coli BioParticles. Parabodo caudatus trophozoites did not take up 4 µm or 2 µm particles but took up 100 nm, 40 nm and E. coli BioParticles from the culture. E. coli BioParticles were observed in the cytoplasm of P. caudatus ( Figure 2D, grey arrows).
The nucleus and kinetoplast of P. caudatus were observed in the cytoplasm of an unattached Colpodella sp. (ATCC 50594) trophozoite below the nucleus (n) ( Figure 3H). In Figure 4, aspirated DAPI-stained nutrients were identified in the cytoplasm and food vacuole of two trophozoites (yellow arrows) attached to P. caudatus (red arrow), and in the tubular tether, along with 100 nm nanoparticles (red) also distributed in the cytoplasm of predator and prey ( Figure 4B, green arrows show DAPI-stained nutrients and yellow arrow head indicates aspirated nutrients in the tubular tether). Following uptake of nanoparticles and fixation of cells, fluorescent CellMask actin was applied to the smear and the distribution of actin relative to the distribution of nanoparticles was observed. In differential interference contrast (DIC) images showing DAPI staining ( Figure 5A), DAPIstained nucleus and kinetoplast from the prey was identified in the forming posterior food vacuole. Forty nanometer particles (green) as well as DAPI-stained nucleus and kinetoplast was observed in the cytoplasm of Colpodella sp. (ATCC 50594) trophozoites and in the forming posterior food vacuole ( Figure 5B,C,F). CellMask red actin label was observed in the cytoskeleton of predator and prey and in the tubular tether ( Figure 5D,E,G-J).   The nucleus and kinetoplast of P. caudatus were observed in the cytoplasm of an unattached Colpodella sp. (ATCC 50594) trophozoite below the nucleus (n) ( Figure 3H). In Figure 4, aspirated DAPI-stained nutrients were identified in the cytoplasm and food vacuole of two trophozoites (yellow arrows) attached to P. caudatus (red arrow), and in the tubular tether, along with 100 nm nanoparticles (red) also distributed in the cyto- was observed in the cytoskeleton of predator and prey and in the tubular tether ( Figure  5D,E,G-J).  In the pre-cyst of Colpodella sp. (ATCC 50594) the food vacuole contained the nucleus and kinetoplast of P. caudatus ( Figure 6A) and the green 40 nm nanoparticles ( Figure 6B,G). The CellMask actin distribution in the pre-cyst was observed in the cytoskeleton of the pre-cyst and in the remnants of the anterior region of the Colpodella sp. (ATCC 50594) trophozoite ( Figure 6D-F,H). We previously reported that initial contact is made by Colpodella sp. (ATCC 50594) trophozoites using a myzocytic aperture located posterior to the pseudoconoid followed by uptake of the prey's membrane, dissolution of the membrane and aspiration of the prey's cytoplasmic contents [24]. The process of myzocytosis is a sequential process and not a direct piercing of the prey's plasma membrane by the predator. Microorganisms 2023, 11, x FOR PEER REVIEW 5 of 9 In the pre-cyst of Colpodella sp. (ATCC 50594) the food vacuole contained the nucleus and kinetoplast of P. caudatus ( Figure 6A) and the green 40 nm nanoparticles ( Figure  6B,G). The CellMask actin distribution in the pre-cyst was observed in the cytoskeleton of the pre-cyst and in the remnants of the anterior region of the Colpodella sp. (ATCC 50594) trophozoite ( Figure 6D-F,H). We previously reported that initial contact is made by Colpodella sp. (ATCC 50594) trophozoites using a myzocytic aperture located posterior to the pseudoconoid followed by uptake of the prey's membrane, dissolution of the membrane and aspiration of the prey's cytoplasmic contents [24]. The process of myzocytosis is a sequential process and not a direct piercing of the prey's plasma membrane by the predator.  At the point of contact between predator and prey, there is a reorganization of microtubules ( Figure 7A,B) followed by uptake of the prey's plasma cell membrane ( Figure 8A-D, white arrows). The tip of the pseudoconoid containing the rostrum in the young trophozoite of Colpodella sp. (ATCC 50594) shown in the TEM micrograph is pointed away from the contact site (brown arrow), while the attachment site (black arrow) is in direct contact with the prey's membrane. From this contact site, aspiration of the cytoplasmic contents of the prey occurs following dissolution of the prey's plasma membrane. The prey's (red arrows) membrane is pulled in further into the predator's (yellow arrows) cytoplasm, as shown in Figure 9A and enlarged in 9B and 9C (white arrows). Extended membrane rims of Colpodella sp. (ATC C 50594) are seen surrounding the prey's plasma membrane ( Figure 9B,C, black arrows), forming the tubular tether.

Discussion
Colpodella species feed on their prey using myzocytosis. Juvenile Colpodella sp. (ATCC 50594) trophizoites have been observed feeding on mature P. caudatus trophozoites, and mature Colpodella sp. (ATCC 50594) trophozoites in culture feed on juvenile P. caudatus trophozoites. In culture, Colpodella sp. (ATCC 50594) trophozoites can be identified without prey attachments and trophozoites have been observed to detach from their prey after initial attachment and persist in culture. In this study, we investigated the hypotheses that unattached trophozoite stages would take up nutrients through means other than myzocytosis and that aspirated contents from the prey would be degraded before uptake and incorporation into the posterior food vacuole. We also wanted to identify the point of contact between predator and prey at the initial stages of myzocytosis. Fluorescent particles of 4 µm, 2 µm 100 nm and 40 nm were used to investigate the size of aspirated particles taken up during myzocytosis. Free, unattached, active young trophozoites of Colpodella sp. (ATCC 50594) and mature trophozoites were observed in culture. The size of aspirated nutrients has not been reported for Colpodella species, and in our previous light microscopy and ultrastructural studies, particulate nutrients were not observed in the pre-cysts and cysts of Colpodella sp. (ATCC 50594) [23][24][25].
In culture, young trophozoites of Colpodella sp. (ATCC 50594) can feed on different stages of P. caudatus trophozoites, forming tubular tethers that are used for aspirating cytoplasmic contents. In unattached Colpodella sp. (ATCC 50594) trophozoites, DAPIstained contents were identified in the cytoplasm, suggesting that nutrient uptake can take place independently of myzocytosis. Results of nutrient uptake experiments showing uptake of 40 nm and 100 nm beads into unattached trophozoites of Colpodella sp. (ATCC 50594) confirmed uptake by endocytosis. Although not considered bacteriotrophic, bacteria from culture were observed in the cytoplasm of Colpodella sp. (ATCC 50594) trophozoites. However, fluorescent E. coli BioParticles were not taken up. Aspirated cytoplasmic contents of P. caudatus were observed in the tubular tether between predator and prey during myzocytosis. Intact kinetoplast and nuclei of P. caudatus were observed in the posterior food vacuole of Colpodella sp. (ATCC 50594) in the trophozoite and pre-cyst stages of the life cycle (Figures 5 and 6). These data suggest that the kinetoplast and nuclei can be squeezed through the tubular tether during aspiration, further indicating that aspirated contents may not have to be digested before aspiration. Nuclei reportedly deform when cells squeeze through tight or constricted spaces [26]. Based on light and transmission electron microscopy, it appears the diameter of the tether may vary, just as the lengths of the tether vary [24], thus allowing nutrient content of varying sizes to be aspirated.
Among the Apicomplexa, trophozoites of blastogregarines and archigregarines attach to their host cells using the mucron, through which, nutrients are obtained by myzocytosis [16,17]. The apical complex organelles and the mucron are present throughout the life cycle. In pathogenic Apicomplexa, contents of apical complex organelles are secreted and participate in junction formation, entry into the host cell and parasitophorous vacuole (PV) formation in organisms that form the PV [6,27]. Colpodella species do not invade their prey, and so differ from Selendium species where the trophozoites obtain nutrients by myzocytosis from their host cells [17,27]. Cryptosporidium species interact with their host cells by forming a feeder organelle, composed of actin at the site of interaction and obtain nutrients in a manner similar to the archigregarines and Colpodella species [27]. The epimerite plasma membrane in gregarines forms a border with the invaginated host cell plasma membrane [16,17] similar to the tight interaction observed between Colpodella sp. (ATCC 50594) and the prey's membrane in [24] and the current study. The sequential progression of myzocytosis involving attachment to the prey, the uptake of the prey's membrane into the cytoplasm of the predator and dissolution of the membrane to permit aspiration of the prey's cytoplasmic contents into the posterior food vacuole argue against phagocytosis and trogocytosis [24,25]. Moreover, the presence of a "myzocytotic apparatus" posterior to the tip of the pseudoconoid demonstrates that attachment is not followed by the piercing of the prey's plasma membrane by the pseudoconoid. Rather, the uptake of the prey's membrane followed by its dissolution to create a channel through which cytoplasmic contents are aspirated is in agreement with the myzocytotic process described for Selenidium orientale where the cytostome is used and can re-open for intermittent feeding [28]. It is unclear if the "myzocytic aperture" in Colpodella sp. (ATCC 50594) is within the opening of the pseudoconoid or a separate structure such as the cytostome or micropore. Myzocytosis has been described as a type of endocytosis [29] and as a form of "apical phagotrophy" [17]. This form of nutrient acquisition prevalent among free-living predatory alveolates, archigregarines and possibly Cryptosporidium species may be the mode of feeding used by the common ancestor of the Apicomplexans [17]. Myzocytosis for predation as described for Colpodella species and V. pontica [3] represents the ancestral form of feeding among the apicomplexans, with Cryptosporidium species serving as the link between predatory feeding, extracellular parasitism and intracellular parasitism following host cell invasion [28].
The IMC is described as a barrier to endocytosis in Apicomplexa, with the micropore serving the role of an endocytotic apparatus, and the cytostome consists of proteins that are associated with endocytosis [12]. The observation that Colpodella sp. (ATCC 50594) trophozoites perform endocytosis, with the uptake of 100 nm and 40 nm beads (Figures 1-3), suggests that endocytosis may occur at the apical end where regions of the plasma membrane can attach with the membrane of the prey without obstruction by the IMC. This same area may be used for nutrient acquisition during myzocytosis. The size and shape of the 4 µm and 2 µm particles may have hindered uptake by Colpodella sp. (ATCC 50594) and P. caudatus, since the larger particles were not observed in the cytoplasm of the protists. The rhoptries and micronemes in Colpodella species are emptied during myzocyosis and may participate in the digestion of the prey's membrane and the uptake and digestion of aspirated nutrients. The unexpected observation in this study of intact nuclei and kinetoplast of P. caudatus in the cytoplasm of free unattached trophozoites and in food vacuoles of Colpodella sp. (ATCC 50594) in pre-cyst stages demonstrates that undigested particulate, as well as digested soluble nutrients, can be taken up by Colpodella sp. (ATCC 50594) trophozoites in diprotist culture, contrary to our hypothesis. A major challenge to performing nutrient uptake studies in diprotist culture with nanoparticles is the presence in the culture of more P. caudatus than Colpodella sp. (ATCC 50594) and the presence of large amounts of bacteria. We are currently investigating synchronization protocols to enrich for each protist and eliminate bacteria. This will facilitate quantitating particle uptake at each stage of the life cycle for each protist in future experiments, as well as performing endocytosis inhibition studies. The synchronization of life cycle stages will also aid the identification of proteins involved in endocytosis. The cytoskeleton participates in tubular tether formation, as we showed previously that cytochalasin D treatment distorts the tubular tether [24]. CellMask actin red distributes over the tether (Figure 5), in agreement with actin involvement. Additional studies will be required to identify proteins associated with endocytosis and to map their distribution in free trophozoites, trophozoites engaged in myzocytosis and in the pre-cysts and cyst stages of the life cycle. Due to the phylogenetic closeness of Colpodella species to the pathogenic Apicomplexa [1,2] and the recent data showing the association of endocytosis with artemisinin resistance in Plasmodium falciparum [12,30], the events of nutrient acquisition seen in the free-living alveolate relatives of pathogens such as Plasmodium species may provide clarity for understanding nutrient acquisition and the role of the food vacuole during intracellular infection.

Conclusions
Our data show for the first time that Colpodella sp. (ATCC 50594) use myzocytosis and endocytosis for nutrient acquisition during growth in diprotist culture. Unattached Colpodella sp. (ATCC 50594) trophozoites take up nutrients in culture independently of myzocytosis and may attach and feed intermittently before detaching to seek other prey. The presence of a "myzocytotic aperture", most likely a structure similar to a cytostome, may permit intermittent feeding. During myzocytosis, Colpodella sp. (ATCC 50594) trophozoites do not pierce the plasma membrane of its prey to obtain nutrients. Following attachment, the plasma membrane of the prey is taken up into the cytoplasm of Colpodella sp. (ATCC 50594), followed by dissolution of the membrane, to form a channel through which the cytoplasmic contents of the prey are aspirated into the posterior food vacuole. Nutrients as large as intact nuclei and kinetoplasts were identified in the posterior food vacuole of Colpodella sp. (ATCC 50594), suggesting that the organelles can be squeezed through the tubular tether with the cytoplasmic aspirate and deposited in the posterior food vacuole. Fluorescent nanoparticles of 40 nm and 100 nm were taken up by Colpodella sp. (ATCC 50594) and P. caudatus. Nanoparticles taken up by P. caudatus were aspirated by Colpodella sp. (ATCC 50594) during myzocytosis. Fluorescent E. coli BioParticles were only taken up by P. caudatus. In future studies, endocytosis inhibition will be performed to gain insights regarding the cytoskeletal components involved in nutrient uptake and proteins involved in endocytosis will be identified. The use of synchronized life cycle stages of predator and prey in future nutrient uptake studies will permit the quantitation of beads taken up by both protists. Myzocytosis for predation may be the ancestral mode of nutrient acquisition among the Apicomplexa.