Figure 1.
Motility and budding of Ceratomyxa puntazzi in the bile of Diplodus puntazzo.
A–D: LM, E–G: SEM, H–K: CLSM (DAPI and Phalloidin stained). A) Small ellipsoidal stage. B) Pyriform stage with a wide hyaline area and refractive granules at rounded, anterior end of parasite. C) Pyriform stage showing large filopodia and abundant refractive bodies at rounded end and a large, rigid cytoplasm extension at posterior end. D) Pyriform stage with abundant vacuoles present in almost the whole body. Refractive bodies were concentrated at anterior end. E–G) Exogenous budding with several stages dividing by plasmotomy. Arrows indicate cytoplasm constrictions. Some filopodia and blebs can be seen on the surface of the stages. H) Three stages, a small ellipsoidal stage with 4 nuclei and two larger stages with 10 and 12 nuclei. I) Pyriform stage with abundant filopodia at round side, where F-actin (green stain) is accumulated, and rigid cytoplasmic extension at the posterior end. Four nuclei are visible. J) Several stages with a clear pattern of accumulation of F-actin in the hyaline area at the anterior end of the parasites where the filopodia are located. Upper parasite: exogenous budding of a round stage with three nuclei (arrow head) and an F-actin rich surface at opposite end from the “mother” parasite it is emerging from. K) Two stages showing exogenous budding with still attached buds moving in opposite directions. Abbreviations: HA: hyaline area; CE: cytoplasmic extension; FP: filopodia; RG: refractive granules; V: vacuole; B: bleb; Nu: Nuclei. Scale Bar: A = 3 µm; B–D = 10 µm; E–K = 4 µm.
Figure 2.
Schematic drawing of the locomotive action of an active pyriform stage.
Projection of filopodia from the anterior, median part radially to most posterior part of the hyaline area, allowing active parasite movement.
Figure 3.
End of motility and the lipid droplets of Ceratomyxa puntazzi from the bile of Diplodus puntazzo.
A: LM, B–H: CLSM. B: phalloidin stain, C–E: DAPI and phalloidin stains, F–H: Nile Red stain. A) Pseudoplasmodia with two almost mature spores. Hyaline area visible at rounded side of parasites and one cytoplasmic extension at posterior end. B) A early sporogonic stage, still pyriform, with a F-actin rich hyaline area and filopodia. Notice two polar capsules (arrow head) of the forming spores. C) Late pseudoplasmodium with two spores, where all nuclei, including the valvogenic ones are visible. D) Mature stage with two spores, where F-actin accumulation can be noted. Valvogenic nuclei have disintegrated. E) Mature spore liberated into the bile with two capsulogenic nuclei and two sporoplasm nuclei. Valve nuclei are absent. F) Pyriform stage with two early sporoblasts, abundant lipid droplets, filopodia and one larger posterior cytoplasmic extension. G) Almost mature stage showing abundant filopodia at rounded side (arrow) and abundant lipid droplets. Two polar capsules can be estimated (arrow head). H) Two mature stages harbouring two spores each, presenting smaller and randomly distributed lipid droplets than in early pseudoplasmodia. Abbreviations: HA: hyaline area; CE: cytoplasmic extension; VNu: Valvogenic nuclei; Ac: F-actin; SpNu: Sporoplasmogenic nuclei; CNu: Capsulogenic nuclei; FP: filopodia; SB: sporoblast; LD: Lipid droplets. Scale bar: A = 10 µm; B = 8 µm; C–H = 4 µm.
Figure 4.
Pre-sporogonic proliferative development of Ceratomyxa puntazzi from the bile of Diplodus puntazzo (TEM).
A) Early stage consisting of a P cell with two P cell nuclei. Notice the presence of vacuoles, mitochondria and absence of lipid droplets. B) Stage consisting of a P cell and a S cell with their respective nuclei. In the P cell, vacuoles are well defined and large mitochondria. In the S cell, small mitochondria are visible. C) Stage consisting in a P cell with two S cells, one of them with a forming tertiary cell (S-T doublet). Large mitochondria are present in the P cell and in the S cells. One of the S cell nuclei has an eccentric nucleoli. Notice the presence of small lipid droplets in the P cell. D) Detail of a stage showing two S cells and their nuclei, one of them with two eccentric nucleoli. Notice junction of the S cells (arrow head). Electron-dense lipid droplets where observed in the cytoplasm of the P cell and abundant mitochondria in the S cells. E) Large stage with several S cells and S-T doublet. Abundant electron-dense lipid droplets and mitochondria in the P cell. F) Detail of S-T doublet shown in Figure 4E, composed of an S cell and two T cells. Notice abundant rough endoplasmic reticulum in S cell cytoplasm. G) A stage with three S cells in a P cell. Notice cell junction of two S cells (arrow head), where partial engulfment was detected. H) Liberated cell doublet was observed, with a high electron dense cytoplasm, probably a S cell with a T cell in its cytoplasm. Note remnants of the P cell membrane (arrows). Abbreviations: P: primary cell; PNu: primary cell nucleus; M: mitochondria; S: secondary cell; SNu: secondary cell nucleus; V: vacuole; LD: lipid droplet; N: Nucleoli; T: tertiary cell; TNu: tertiary nucleus; rER: rough endoplasmic reticulum. Scale Bar: A–C = 1 µm; D–E = 2 µm; F = 1 µm; G = 2 µm; H = 1 µm.
Figure 5.
Pre-sporogonic proliferative development of Ceratomyxa puntazzi from the bile of Diplodus puntazzo (TEM).
A) Two large mitochondria in the P cell with numerous well-developed cristae. B) Detail of electron-dense lipid droplet in a P cell. Notice the lack of a delimiting membrane. C) Detail of non electron-dense vacuole with a single membrane (arrow head) filled with granular material. D) Pyriform stage with filopodia at rounded side and a large cytoplasmic extension at posterior end. Notice vacuoles with granular material. E) Stage with blebs on the surface. Notice electron-dense cytoplasm of an S cell densely packed with ribosomes. F) Large stage close to the epithelium of the gall bladder, with a large bleb filled with the same material as the cytoplasm of the P cell. G) Stages with blebs. Notice the presence of material inside, similar to the P cytoplasm content. H) Detail of a bleb. Abbreviations: P: primary cell; PNu: primary cell nucleus; M: mitochondria; S: secondary cell; SNu: secondary cell nucleus; V: vacuole; FP: filopodia; CE: cytoplasmic extension; B: bleb; H: host epithelium. Scale Bar: A = 1 µm; B–C = 0.5 µm; D–E = 1 µm; F–G = 2 µm; H = 1 µm.
Figure 6.
Sporogenesis of Ceratomyxa puntazzi from the bile of Diplodus puntazzo (TEM).
A) Initial sporoblast with two capsulogenic cells developing the external tube. Both capsulogenic cells are enveloped by a sporoplasmogenic cell harbouring two sporoplasmic nuclei. Laterally, a valvogenic cell and its nucleus. Lipid droplets and vacuoles are abundant in the cytoplasm of the P cell. B) Sporoblast with two capsulogenic cells, a sporoplasmogenic cell with two nuclei and two valvogenic cells. The nucleus of the valvogenic cell is connected by a cytoplasmic bridge (*). Notice formation of suture (arrows). C) Detail of a sporoblast, showing the binucleate sporoplasm, with two eccentric nucleoli. Abundant rough endoplasmic reticulum is present in the cytoplasm of the sporoplasmogenic cell. D) Capsulogenic cell with a prominent external tube and a capsular primordium. Note vesicular body associated to the membrane of the capsulogenic cell. E) Detail of the vesicular body of Figure 6D. F) Detail of a sporoblast with vesicular body between the membranes of the two capsulogenic cells and the sporoplasmogenic cell. Suture forming between the two valvogenic cells (arrows). G) Detail of vesicular body shown in Figure 6F. Abbreviations: P: primary cell; PNu: primary nucleus; CC: capsulogenic cell; CNu: capsulogenic nucleus; SP: sporoplasmogenic cell; SpNu: sporoplasmogenic nuclei; VC: valvogenic cell; VNu: Valvogenic nucleus; ET: external tube; rER: rough endoplasmic reticulum; LD: lipid droplets; M: mitochondria; V: vacuole; Cp: capsular primordium; N: nucleoli; VB: vesicular body. Scale Bar: A = 2 µm; B = 5 µm; C = 2 µm; D = 1 µm; E = 0.2 µm; F–G = 1 µm.
Figure 7.
Sporogenesis of Ceratomyxa puntazzi from the bile of Diplodus puntazzo (TEM).
A) Pseudoplasmodium harbouring two sporoblasts, with capsulogenic cells in different stages of maturation. One capsulogenic cell has a mature polar capsule, where sections of the coiled, twisted polar filament can be observed. The other three capsulogenic cells show an external tube and capsular primordium in their cytoplasm. Note abundant rough endoplasmic reticulum in the cytoplasm of the sporoplasmogenic cell. Formation of suture line between valvogenic cells can be observed (arrows). B) Two mature spores inside a P cell. Abundant lipid droplets are present in the degenerated P cell. C) Mature spore with two polar capsules, the opening for the extrusion of the polar filament, the suture (arrow head), one sporoplasm nucleus and sporoplasmosomes present in the sporoplasm cytoplasm. D) Detail of a mature polar capsule with twisted appearance of polar filament and capsular plug; polar capsule with an outer electron-dense layer and an inner electron-lucent layer. Apical junctions of the mature spore, surrounded by fibrous material between the capsulogenic cell and the valve cell (arrows). Suture between valves (arrow head). E) Detail of a mature spore: desmosome-like junction of the suture (arrow head) and sporoplasmosomes in sporoplasm cytoplasm. Abbreviations: CC: capsulogenic cell; CNu: capsulogenic nucleus; SP: sporoplasmogenic/sporoplasm cell; SpNu: sporoplasmogenic/sporoplasm nucleus; VC: valvogenic cell; PC: polar capsule; Cp: capsular primordium; ET: external tube; M: mitochondria; rER: rough endoplasmic reticulum; LD: lipid droplets; Spl: sporoplasmosomes; Op: opening for the extrusion of the polar filament; PF: polar filament; Pl: capsular plug; IL: inner electron-lucent layer; OL: outer electron-dense layer; GM: granular matrix. Scale Bar: A = 2 µm; B = 5 µm; C = 2 µm; D = 1 µm; E = 0.5 µm.
Figure 8.
Diagrams of some representative developmental stages of Ceratomyxa puntazzi in the bile of Diplodus puntazzo.
A) Early, active ellipsoidal stage with a few filopodia at anterior part, showing a primary cell with two primary nuclei and two secondary cells with a nucleus each, and refractive granules. B) Budding of a small stage from the “mother” parasite. Both stages show accumulation of F-actin in opposite poles allowing separation. “Daughter” parasite shows our suggestion of a secondary-tertiary cell doublets as proliferative stages. C) Active, pyriform stage, with many filopodia, at anterior part, some of them ramified. This stage possesses a primary cell with a primary nucleus, three single secondary cells and a secondary cell with two tertiary cells, abundant lipid droplets and refractive granules. D) Sporogonic stage with two forming spores, still showing motility and abundant lipid droplets. E) Sporogonic stage close to the end of spore development, with loss of motility and reduced size and number of lipid droplets. F) Mature spore with a binucleate sporoplasm, showing two capsulogenic cells with their nuclei and two polar capsules.