Cooperation of two opposite flagella allows high-speed swimming and active turning in zoospores

Phytophthora species cause diseases in a large variety of plants and represent a serious agricultural threat, leading, every year, to multibillion dollar losses. Infection occurs when these biflagellated zoospores move across the soil at their characteristic high speed and reach the roots of a host plant. Despite the relevance of zoospore spreading in the epidemics of plant diseases, it is not known how these zoospores swim and steer with two opposite beating flagella. Here, combining experiments and modeling, we show how these two flagella contribute to generate thrust when beating together, and identify the mastigonemes-attached anterior flagellum as the main source of thrust. Furthermore, we find that steering involves a complex active process, in which the posterior flagellum is stopped, while the anterior flagellum keeps on beating, as the zoospore reorients its body. Our study is a fundamental step towards a better understanding of the spreading of plant pathogens’ motile forms, and shows that the motility pattern of these biflagellated zoospores represents a distinct eukaryotic version of the celebrated “run-and-tumble” motility class exhibited by peritrichous bacteria.

2-APB-potentiated channels amplify CatSper-induced Ca2+ signals in human sperm

Ca2+i signalling is pivotal to sperm function. Progesterone, the best-characterized agonist of human sperm Ca2+i signalling, stimulates a biphasic [Ca2+]i rise, comprising a transient and subsequent sustained phase. In accordance with recent reports that progesterone directly activates CatSper, the [Ca2+]i transient was detectable in the anterior flagellum (where CatSper is expressed) 1–2 s before responses in the head and neck. Pre-treatment with 5 μM 2-APB (2-aminoethoxydiphenyl borate), which enhances activity of store-operated channel proteins (Orai) by facilitating interaction with their activator [STIM (stromal interaction molecule)] ‘amplified’ progesterone-induced [Ca2+]i transients at the sperm neck/midpiece without modifying kinetics. The flagellar [Ca2+]i response was unchanged. 2-APB (5 μM) also enhanced the sustained response in the midpiece, possibly reflecting mitochondrial Ca2+ accumulation downstream of the potentiated [Ca2+]i transient. Pre-treatment with 50–100 μM 2-APB failed to potentiate the transient and suppressed sustained [Ca2+]i elevation. When applied during the [Ca2+]i plateau, 50–100 μM 2-APB caused a transient fall in [Ca2+]i, which then recovered despite the continued presence of 2-APB. Loperamide (a chemically different store-operated channel agonist) enhanced the progesterone-induced [Ca2+]i signal and potentiated progesterone-induced hyperactivated motility. Neither 2-APB nor loperamide raised pHi (which would activate CatSper) and both compounds inhibited CatSper currents. STIM and Orai were detected and localized primarily to the neck/midpiece and acrosome where Ca2+ stores are present and the effects of 2-APB are focussed, but store-operated currents could not be detected in human sperm. We propose that 2-APB-sensitive channels amplify [Ca2+]i elevation induced by progesterone (and other CatSper agonists), amplifying, propagating and providing spatio-temporal complexity in [Ca2+]i signals of human sperm.

The zoospore of Schizochytrium aggregatum

The zoospore ultrastructure of the marine protist Schizochytrium aggregatum was examined in detail. The zoospores of S. aggregatum differed considerably from the zoospores of the presumably closely related marine protist Thraustochytrium. The flagella of S. aggregatum emerged from an erumpent area and followed along shallow grooves on the spore body. The mastigonemes of the anterior flagellum are localized along one-third the circumference of the flagellar shaft. There is a concertina-like structure just distal to the kinetosome terminal plate and osmiophilic granules are present within the kinetosome lumen. Microtubular bundles are associated with the kinetosomes and form a "backbone root" system. Serial reconstructions show that each zoospore contains a single tubular mitochondrion. Closely associated with the mitochondrion is a single microbody which in turn is appressed to the nuclear membrane. The ultrastructural data suggest that Schizochytrium aggregatum can be accommodated among the Fungi.

Renaming of Salpingoeca Sensu Grøntved

Salpingoeca natans Grentved and 5. spinifera Throndsen have been removed from Salpingoeca James-Clark and made the nomenclatural types of Calliacantha gen.nov., and Bicosta gen.nov., respectively.James-Clark (1876) erected the genus Salpingoeca J.-Clk, 1867 for solitary choanoflagellates with colourless, transparent, thecae.* As for most choanoflagellates the protoplasts possessed a single anterior flagellum surrounded by a hyaline collar which, when viewed with the light microscope, appeared membranous.

External morphology of the choanoflagellate Salpingoeca gracilis James-Clark

In his search for evidence illustrating the botanical/zoological affinities of the sponges (Porifera), James-Clark (1867) studied a number of colourless flagellates. The genus Salpingoeca (Craspedophyceae, Christensen, 1962; cf. Chadefaud, 1960) was thus erected to comprise certain solitary, lorica-dwelling organisms, characterized by a single anterior flagellum encircled by a ‘filmy, membranous, colourless collar’ (James-Clark, 1867).About fifty species (approximately twenty marine) are at present allocated to this genus, provided it is defined as e.g. by Norris (1965) and Bourrelly (1968) (i.e. including Lagenoeca Kent and Pachysoeca Ellis).

THE STRUCTURE, ORIGIN, ISOLATION, AND COMPOSITION OF THE TUBULAR MASTIGONEMES OF THE OCHROMONAS FLAGELLUM

The structure, assembly, and composition of the extracellular hairs (mastigonemes) of Ochromonas are detailed in this report. These mastigonemes form two lateral unbalanced rows, each row on opposite sides of the long anterior flagellum. Each mastigoneme consists of lateral filaments of two distinct sizes attached to a tubular shaft. The shaft is further differentiated into a basal region at one end and a group of from one to three terminal filaments at the free end. Mastigoneme ontogeny as revealed especially in deflagellated and regenerating cells appears to begin by assembly of the basal region and shaft within the perinuclear continuum. However, addition of lateral filaments to the shaft and extrusion of the mastigonemes to the cell surface is mediated by the Golgi complex. The ultimate distribution of mastigonemes on the flagellar surface seems to be the result of extrusion of mastigonemes near the base of the flagellum, and it is suggested that mastigonemes are then pulled up the flagellum as the axoneme elongates. Efforts to characterize mastigonemes biochemically after isolation and purification on cesium chloride (CsCl) followed by electrophoresis on acrylamide gels have demonstrated what appear to be a single major polypeptide and several differentially migrating carbohydrates. The polypeptide is not homologous with microtuble protein. The functionally anomalous role of mastigonemes in reversing flagellar thrust is discussed in relation to their distribution relative to flagellar anatomy and to the plane of flagellar undulations.

EXTRACELLULAR MICROTUBULES

Mastigonemes (Flimmer) from the sperm of Ascophyllum and Fucus were found to consist of a tripartite structure—a ca. 2000-A tapered basal region, a closed microtubular shaft, and a group of terminal filaments. Each of these regions appears to be constructed of globular subunits with a center-to-center distance of about 45 A. The mastigoneme microtubule is of smaller diameter (170–190 A) than cytoplasmic microtubules in these or other plant cells. During the initial stages of flagellar ontogeny, structures similar to mastigonemes (presumptive mastigonemes) are found within membrane-limited sacs in the cytoplasm or within the perinuclear space. Mastigonemes at this time are generally not found on the flagellar surface. Later, when the anterior flagellum acquires mastigonemes, the presumptive mastigonemes are absent from the cytoplasm. The regularity of attachment of mastigonemes to the flagellar surface suggests that specific attachment sites are constructed on the plasma membrane during flagellar ontogeny. No evidence for penetration of the mastigoneme through the plasma membrane was obtained. The origin and structure of mastigonemes are discussed in relation to reports of the origin and structure of other microtubular systems.

THE FLAGELLATION, MOVEMENT, AND ENCYSTMENT OF SOME PHYCOMYCETOUS ZOOSPORES

The anterior flagellum of the zoospores of Phytophthora fragariae, P. megasperma, P. cambivora, Saprolegnia parasitica, Achlya americana, and Pythium aphanidermatum projects straight in front of the zoospore and never moves except during encystment whereas the posterior flagellum is active during the swimming period. In the secondary zoospore the anterior and posterior flagella are attached a short distance apart in the center of the depression on the concave side and respectively pass forward and backward through a groove and form a central axis about which the zoospore rotates. Hyaline vesicles which also have been called beads or paddles form at the base of the flagella at the beginning of encystment and glide part or all the way down the flagella. Movement of flagella after they are released from the zoospore is reported for the first time. Encystment may result from contact stimulus except in the case of Allomyces anomalus. A filament on which vesicles may occur may be secreted or retracted by the Allomyces zoospore.

Investigations of the Biology of Peranema trichophorum (Euglenineae)

The feeding apparatus of Peranema trichophorum, consisting of cytostome and rodorgan, is independent of the reservoir system; the latter is the same in structure and function as that of other Euglenineae. There are two flagella, one directed forward, the other backward and adherent to the ventral body surface. The anterior flagellum is longer and thicker than the adherent one. Both flagella are composed of a central core and an outer sheath. Electron micrographs suggest that the core consists of many longitudinal fibrils, and the sheath of many short fibrils radiating from the core, giving the whole flagellum the appearance of a test-tubebrush. Treatment with certain proteindispersing agents cause the unfixed anterior flagellum todissociate into three fibrils. Peranema multiplies freely on a diet of living yeast-cells; dead yeast is not suitable. Euglena viridis, E. gracilis, and certain other unicellular algae can also serve as food. Egg-yolk, and especially milk, can be used to maintain bacteria-free pure cultures. Casein is suitable in combination with soil-extract or beef-extract, but never as good as milk. With the latter the individuals are larger and tnore numerous than with yeast as food, although the cultures decline earlier. Clear liquid media of many various kinds did not support growth: paniculate food seems to be essential. Peranema is capable of ingesting a great variety of living organisms provided these are motionless. Small organisms are swallowed whole; larger ones are either engulfed or cut open by the rod-organ and their contents sucked out. The rod-organ can be protruded out of thecytostome and used in holding on to, and cutting, the periplast of the prey. Starch-grains, oil-droplets, and protein-particles are engulfed and digested. The main food reserves are paramylon-granules and oil-droplets. H+ ions, decomposition products of proteins, and other substances diffusingout of living, and particularly dead, organisms attract gliding Peranema. Chemotaxis plays an important role in leading Peranema to its prey. Both gliding and swimming have been observed in normal individuals, although the latter is less frequent. While there can be no doubt that swimming results from the action of the anterior flagellum, it does not seem to play an appreciable part in gliding. Nothing is known about the function of the adherent flagellum.