Ripidomyxa

1. Ripidomyxa australiensis Chakraborty & Pussard, 1985 (ref. ID; 7744 original paper)

Ripidomyxa australiensis Chakraborty & Pussard, 1985 (ref. ID; 7744 original paper)

Diagnosis

Descriptions

• Trophozoite morphology: Trophozoites of this amoeba exhibit two distinctly different forms, a branched flattened form and a tubular limax form. Majority of the trophozoites in both agar and aqueous media produce the branched flattened form. On agar, they may appear as irregular masses whose outlines are determined by the crowding of individuals. More commonly, however, they are fan-shaped with broad convex or truncate advancing margins. They can also be branched, somewhat dichotomously and quite extended. Although crowding in common, especially in young cultures, trophozoites have never been seen to anastomose so as to give a reticulate appearance. Trophozoites range from 80 to 150 um in length and 28 to 95 um in breadth. The cell cytoplasm is mostly composed of a granular endoplasm except in the advancing margins, where a hyaline ectoplasmic layer is present. Trophozoites in microcultures may form eruptive layers of hyaline ectoplasm in regions other than the advancing margin if the illumination is too intense. The endoplasm is finely granular and usually contains many vacuoles up to 16 um in diameter. The nucleus is readily visible in live trophozoites. They measure 6-10 um in diameter and contain an elongated to oval nucleolus 3-5 um in diameter. Trophozoites are predominantly uninucleate, although up to 6 nuclei have been observed in some. In microcultures, trophozoites sometimes do not undergo cytokinesis following mitosis and this may result in the formation of multinucleate plasmodia; up to 14 nuclei have been seen in such forms. However, of the 200 or so trophozoites examined so far, only less than approx. 5% of the trophozoites have more than one nucleus. Small in determinate pseudopodia of the lobosa are formed from the advancing margin and this gives an irregular outline to the trophozoite. Lobopodia are formed by short eruptive action of the ectoplasm into which the granular endoplasm may flow. Often in the branched forms, each branch produces a tuft of terminal pseudopodia. Two or more branches may advance in different directions, resembling a polyaxial type of locomotion. A true polyaxial locomotion, for prolonged periods however, has not been observed. Unlike in the limax form, locomotion in the branched form is rarely unidirectional. The rate of locomotion can be as rapid as 5 um sec-1 in the branched form and this is even faster in limax trophozoites. As the trophozoite advances, the posterior margin forms a variable number of fine filiform structures with somewhat pointed ends and in trophozoites with a broad posterior region, such structures may be observed along the entire retracting margin. Similar filiform structures are also formed when a lobose pseudopod is retraced. Although morphologically somewhat similar, these structures differ from filopodia in not being actively produced by the trophozoite. The limax form is less common and is mainly observed in aqueous media which are disturbed when this form occurs at the interface between the suspension and the air. It appears that the limax form only exists for a short time and trophozoites soon settle down to the bottom to produce the stable form with characteristic flattened morphology. This change between the two forms can be readily observed in fresh hanging drop preparations. Limax forms are narrower at the posterior end than at the anterior end and measure 50 to 180 um in length and 20 to 40 um in width. Cell cytoplasm is divided into a granular endoplasm and a hyaline ectoplasmic zone which is present as a cap at the anterior margin and can be undulated, smooth or rounded in shape. Apart from the single nucleus, which is very distinct in this form, the only other cytological structure visible by light microscopy are the vacuoles. These vacuoles are usually present at the posterior end of the trophozoite and can be from one to many in number. (ref. ID; 7744)
  [Cyst morphology]: Cysts begin to appear in cultures after about 3-4 days of starting a new culture. They are usually spherical to oval in shape, but this can vary if individuals are crowded, or if cysts are formed within the confines of lysed fungal cells. For instance, cysts often form within empty sporangia of Phytophthora cinnamomi Rands. Cysts range from 15 to 40 um in diameter. Although the trophozoite morphology somewhat resembles that of Leptomyxa flabellata Goodey, 1915 (Pussard and Pons 1976), the cyst morphology of R. australiesis is quite distinct and all the cyts examined so far have a single visible wall which is smooth and does not have any visible lamellations or ornamentations. No endocysts, like those in L. reticulata Goodey, 1915 or L. flabellata Goodey, 1915 (Pussard and Pons 1976) have been observed in cultures. Majority of the cysts are uninucleate and only rarely up to 3 nuclei have been seen in some cysts. Excystment can be easily observed by placing cysts in sterile amoeba saline or on PJA bearing the food bacterium. Excystment commences with the appearance of initially small and then large vacuoles and protoplasmic streaming. A single trophozoite invariably emerges from the cyst. After excystment, the empty cyst wall looks largely intact and in the absence of visible germ-pores, it is not clear how the trophozoite overcomes the cyst-wall barrier. (ref. ID; 7744)
• Mitosis: Nuclear division in R. australiensis is of mesomitotic type according to the revised of definition of Pussard (1973). The onset of mitosis is marked by the cessation of locomotion, withdrawal of pseudopodia and the gradual formation of a more or less spherical outline by the trophozoite. Cytoplasmic streaming and the formation and bursting of vacuoles continue but no pseudopod is formed. The interphase nucleus is characterised by its enlarged size, is often spherical in shape and the nucleolus appears less-dark in colour. Disappearance of the nucleolus commences in prophase and the nucleolar outline appears somewhat irregular. The nucleolus may either fragment into 2-10 small sections or the entire nucleolus may gradually become faint and disappear; in the later case, often the nucleolar material forms a ring-like structure before disappearing. The amoeba is very sensitive at this stage and if illumination is too intense the nucleolus reappears quickly and the amoeba may resume locomotion. However, it is possible to observe this disappearance and reappearance of the nucleolus for as many as 5 times by alternately decreasing and increasing the illumination. The other form of disturbance is due to any slight movement of the microscope objective, especially in the oil immersion types, as a result of camera shutter operation during photomicrography. With the disappearance of the nucleolus, protoplasmic streaming slows down and almost stops during the major part of mitosis; the formation and bursting of vacuoles, however, can be seen throughout the entire process. Metaphase exists for a short time and a very clear metaphase plate is never observed. Chromosomes are arranged in an irregular band at the equatorial plane. As chromosomes start moving towards opposite poles, the general spherical shape of the nucleus is lost and it gradually becomes elongated. Towards the end of anaphase, chromosomes appear in two distinct groups at each pole and the nuclear membrane is drawn out into a extended form. As the two poles move further apart, the middle portion gradually becomes narrower and extends into a fine thread-like structure which finally snaps. The nuclear membrane persists throughout anaphase. After the separation, the two daughter nuclei are not discernible inside the granular endoplasm and they are difficult to detect till the end of cytokinesis. At the end of anaphase the cytoplasm starts streaming, pseudopodia are gradually formed and the general spherical shape of the trophozoite is lost. The amoeba becomes more active and the parent trophozoite separated into two halves which gradually draw away from each other finally dividing into two trophozoites. The whole process, starting from the diappearance of the nucleolus to the end of cytokinesis takes about 15-25 minutes. (ref. ID; 7744)

Notes

• (1) Number of nuclei - As emphasised earlier, trophozoites of R. australiensis are predominantly uninucleate and more than one nucleus has been observed only rarely. Trophozoites of L. flabellata, on the other hand, are usually multinucleate, although the number of nuclei per trophozoites is less than 20 (Pussard and Pons 1976).
• (2) Mitosis - In describing the mitosis of L. flabellata, Pussard and Pons (1976) considerd that the process was essentially similar to that of L. reticulata. In L. reticulata, the nucleolus does not disappear and can be seen alongside the chromosomes during metaphase and anaphase. This is not the case in R. australiensis, where the nucleolus either gradually becomes faint and disappears or fragments into smaller pieces before disappearing and no nucleolus is visible from the end of prophase till the end of telophase when the nucleoli are reconstructed in the daughter nuclei. Also, during cytokinesis in L. flabellata, Pussard and Pons (1976) have reported the frequent occurrence of a trifoliate form of the trophozoite which leads to the formation of two multinulceate individuals. In R. australiensis, no trifoliate stage was seen. Cytokinesis is always binary resulting in two uninucleate trophozoites; only in exceptional cases was a multinucleate plasmodium formation observed in microculture.
• (3) Cysts - Both Goodey (1915) and Pussard and Pons (1976) have described the cyst morphology of L. flabellata in some detail. The cyst wall always has two envelopes and an amorphous layer. The external layer may further bear one of the four types of ornamentations (Pussard and Pons 1976). Cysts of R. australiensis, on the other hand, always have one single layer in their wall which is smooth and without any ornamentations. The other important difference is the occurrence of endocysts in L. flabellata and not in R. australiensis.

Prey organisms

Observed habitat