Fig 1.
Comparison of T. vaginalis (FMV1 strain) following CPD and HMDS drying procedures for SEM.
(A) Schematic representation of sample preparation procedures for SEM. (B–C) Representative SEM images illustrating morphological differences between parasites processed by CPD (B) and HMDS drying (C). In CPD-prepared samples (B), uropod-like structures (U) are observed protruding from the posterior pole, while microvesicle-like structures and few small tubular projections (white arrows) are visible on the parasites’ cell body; no projections are observed in the region where the anterior (AF) and recurrent (RF) flagella emerge. In contrast, HMDS-prepared samples (C) display a greater abundance of surface projections, including uropods (U) and numerous protrusions extending from the entire cell surface (white arrows). Cytoneme-like structures are observed emerging from the cell body (*), flagellar base region (white arrowheads), posterior pole (green arrowheads), and axostyle (Ax; yellow arrowheads). Filopodia (orange arrowheads) and pseudopods (P) are also seen. (D) Quantification of surface area in CPD- and HMDS-dried parasites. Bars represent the mean ± standard deviation from three independent experiments. SEM images of 50 randomly selected parasites per sample were analyzed using ImageJ software (area parameter). Individual points represent the measured surface area of each parasite. HMDS-dried parasites exhibit a significantly larger surface area compared to CPD-dried parasites. **p < 0.01, Mann–Whitney U test.
Fig 2.
Comparison of T. vaginalis (CDC1132 and G3 strains) following CPD and HMDS drying procedures for SEM.
(A, B) Representative SEM images of general and detailed views of the T. vaginalis CDC1132 strain processed by CPD and HMDS drying. (A) A myriad of surface projections is seen in parasites processed by both CPD or HMDS (white arrows). Cytonemes protruding from cell body and flagellar base region are indicated by asterisks and white arrowheads, respectively. Cytoneme-like projections emerging from the posterior pole and axostyle (Ax) are indicated by green and yellow arrowheads, respectively. In detail, the CPD-dried parasite displays only a few short tubular structures at the posterior pole, whereas the HMDS-dried parasite exhibits longer and more abundant posterior and axostylar cytonemes. Pseudopodia (P) and filopodia (blue arrowheads) are also seen. (B) In CPD-dried sample, projections such as pseudopods (P), uropods (U) and filopodia (Fi) in contact with adjacent parasites are seen ruptured (blue arrows). In contrast, these structures remain intact in HMDS-dried samples. Insets show well-preserved cytonemes in contact with the parasite body (B) and a region of contact between the tips of two cytonemes from adjacent parasites (yellow arrow). Flagellar cytonemes are indicated by white arrowheads. (C) Representative SEM images of general and detailed views of the T. vaginalis G3 strain processed by CPD and HMDS drying. In both conditions, parasites display well-preserved, typical piriform or ellipsoid morphology with a slightly irregular surface; no protrusions are observed. A parasite with a wrinkled surface in the CPD-dried sample is indicated by a white arrow. (D) Quantification of the percentage of parasites from FMV1, CDC1132, and G3 strains exhibiting filopodia and/or cytonemes on their cell surface following CPD and HMDS drying. Bars represent the mean ± standard deviation from three independent experiments performed in duplicate. 500 parasites were randomly counted per sample using SEM. **p < 0.01, Mann–Whitney U test. AF, anterior flagella; RF, recurrent flagellum.
Fig 3.
SEM of cytonemes and filopodia in HMDS-dried T. vaginalis FMV1 strain during parasite-to-parasite interactions in axenic culture.
(A) General and detailed views of a parasite clump. Cytonemes (Cy) and filopodia (Fi) emerging from anterior flagella (AF) and cell body (P) are observed connecting adjacent cells within the clump of parasites. Points of contact between cytonemes and the cell body, axostyle (Ax), and filopodia are indicated by yellow, orange, and dark blue arrows, respectively. The asterisk indicates a filopodium, which bifurcates. (B) Cytonemes (Cy) are seen contacting the surface of the recurrent flagellum (RF) of an adjacent parasite (light blue arrows). (C) Long cytonemes (Cy) are observed extending toward (upper image) and establishing contact with (lower image) a neighboring parasite (P). (D) General and detailed views of a cytoneme (Cy) connecting two parasites (P). The distal end of the projection is bifurcated (*) and is contacting the cell body of the adjacent parasite (yellow arrows). Microvesicle-like structures are seen on the proximal region of the cytoneme (white arrows). (E) A bifurcated filopodium (Fi) protruding from the parasite cell body (P) is shown. Inset: microvesicles are seen emerging near the base of the filopodium (white arrows). (F) A filopodium (Fi) and a cytoneme (Cy) emerge from a common bifurcation (*) on the parasite surface (P). (G) General and detailed views of multiple filopodia (Fi) protruding from a pseudopod-like structure (★). Flagellar cytonemes and posterior projections are indicated by white and green arrowheads, respectively. (H) General and detailed views showing the interaction between the distal ends of two filopodia (Fi) from adjacent parasites (P), forming a handshake-like contact structure (green arrow).
Fig 4.
Comparative analysis of CPD and HMDS drying reveals similar efficiency in preserving microvesicle-like structures on the surface of T. vaginalis FMV1 strain.
(A) Representative SEM images of general and detailed views of the T. vaginalis processed by CPD and HMDS drying. Numerous microvesicles (*) are seen protruding from the cell body of parasites processed by both drying methods (white arrows). Cytonemes and filopodia are observed in HMDS-dried parasites (arrowheads). (B) Quantification of the percentage of parasites exhibiting microvesicles on their cell surface upon CPD and HMDS drying. Bars represent the mean ± standard deviation from three independent experiments performed in duplicate. 500 parasites were randomly counted per sample using SEM. (C) SEM of microvesicles shedding from the anterior flagella (AF) of the parasites processed by CPD and HMDS drying. Cytonemes are observed in HMDS-dried parasites (arrowheads). RF, recurrent flagellum; Ax, axostyle; U, uropod-like projection.
Fig 5.
HMDS provides superior ultrastructural preservation of the amoeboid T. vaginalis surface (FMV1 strain) following adhesion to fibronectin-coated coverslips.
(A) Representative SEM images showing general and detailed views of parasites processed by CPD and HMDS drying. In CPD-dried sample, lamellipodia and pseudopods are retracted (white arrows), and disrupted filopodia and cytonemes are evident (orange arrowheads). An intact surface projection is indicated by a white arrowhead. No cytonemes are observed connecting adjacent parasites. In contrast, HMDS-dried sample display well-preserved, long cytonemes forming a network between adjacent parasites (white arrowheads), with few ruptured projections (orange arrowheads). Large, flattened lamellipodia and pseudopods are observed spreading across the substrate (*). (B) Quantification of the percentage of parasites displaying filopodia and/or cytonemes (top graph) and the percentage exhibiting disrupted surface projections (bottom graph) following CPD and HMDS drying. Bars represent mean ± standard deviation from three independent experiments performed in duplicate. 500 parasites were randomly counted per sample using SEM. *p < 0.05, Mann–Whitney U test. (C) Detailed SEM images highlighting differences in surface preservation between CPD- and HMDS-dried parasites. In CPD samples, numerous fragments resembling ruptured filopodia, cytonemes (orange arrowheads), lamellipodia, and blebs (★) are seen around a parasite. In HMDS, intact filopodia and cytonemes (white arrowheads) are seen extending from pseudopods and lamellipodia (*) of a parasite. Filopodia are also observed connecting lamellipodia (*) to large blebs (★), forming network-like arrangements (yellow arrowheads). Filopodia protruding from blebs are indicated by blue arrowheads. A cluster of projections at the posterior region of the parasites is indicated by a green arrow. Minor surface ruptures are indicated by an orange arrow. Ax, axostyle; U, uropod-like projection.
Fig 6.
HMDS enhances ultrastructural preservation of T. vaginalis during interaction with BPH1 prostate epithelial cells.
Representative SEM images showing general and detailed views of parasites (P) in contact with hosts cells (H) following by CPD and HMDS drying. (A) FMV1 strain. In CPD-dried sample (top row), no filopodia or cytonemes are observed on the surface of the parasites after contact with BPH1 cells. A cluster of small surface protuberances is observed on one parasite (*, inset), and a uropod-like projection (U) is seen contacting a host cell. In contrast, HMDS-dried samples (middle and bottom rows) reveal long cytonemes (white arrows) in contact with neighboring parasites, host cells, and a bacterium (Ba). Flagellar and bifurcated cytonemes are indicated by blue and yellow arrows, respectively. Posterior and axostylar cytonemes extending toward host cells are indicated by green arrows. Clusters of protrusions at the posterior region of parasites are indicated by asterisks (insets), and the letter (F) denotes filopodium. (B) CDC1132 strain. In both CPD- and HMDS-dried samples, filopodia and cytonemes are observed emerging from the flagellar base (blue arrows) and cell body (orange arrows) of parasites interacting with BPH1 cells. A cluster of projections at the posterior region is indicated by an asterisk. Elongated posterior and axostylar cytonemes are seen in HMDS-dried sample (green arrow). AF, anterior flagella; Ax, axostyle; RF, recurrent flagellum.