In this article we will discuss about the somatic phase and reproductive phase in the life cycle of physarum with the help of suitable diagrams.
On the onset of conditions favourable for growth the spiny thick-walled resting meiospores germinates releasing usually 1 to 4 biflagellate swarm cells or myxamoebae which function as gametes. Copulation between two swarm cells or myxamoebae is followed by karyogamy. The resultant fusion cell is the zygote. It is amoeboid in form and has a single diploid nucleus.
The amoeboid zygote constantly puts forth slender processes, the pseudopodia and withdraws them. Thus, it has no definite shape. With the help of pseudopodia it creeps over the substratum engulfing food particles.
Assimilation of food results in the synthesis of more protoplasm resulting in growth which is irreversible increase in size. Growth in zygote is accompanied by repeated and successive meiotic divisions of the diploid parent nucleus.
There is thus repeated karyokinesis but no cytokinesis. The numerous nuclei come to lie in the cytoplasm of the same zygote. It becomes clear from the above account that as a result of growth, repeated karyokinesis but no cytokinesis, the amoeba-like zygote gradually changes into a multinucleate amoeboid mass of protoplasm with diploid nuclei. It is termed the plasmodium.
Plasmodium (Fig. 2.4):
The somatic phase of a true slime mold is thus a thallus consisting of a free living irregularly shaped mass of slimy protoplasm with several diploid nuclei embedded in it. Technically it is called the plasmodium. The Plasmodium is a cellular and is the product of syngamy, hence a diploid structure. It has neither any definite shape nor definite size. The plasmodium constitutes the assimilative phase in the life cycle.
It contains and secretes slime which protects it from dehydration. Probably correlated with the presence of slime in the growing Plasmodium is its inability to undergo cytokinesis. The Plasmodia are often very colourful.
The colour ranges from red, showing gross structure. (Diagrammatic) violet, orange, brown, yellowish, black, slightly greenish to even colourless. The plasmodium of P. polycephalum is bright yellow depending upon the species. However, the Plasmodium lacks chlorophyll.
In some slime molds, under favourable conditions, the Plasmodium increases in size, as it develops, by repeated synchronous (simultaneous) mitotic nuclear division of diploid nuclei and also by annexing zygotes and other plasmodia of the same species it comes across as it creeps over the substratum. Their nuclei, however, do not unite.
Consequently the Plasmodium, in some cases, may be organised into an extensive multinucleate amoeboid mass of protoplasm bounded by a plasma membrane (syncytium) attaining a size several square centimetres. It is known as the macroplasmodium or more appropriately the phaneroplasmodium. It is characteristic of Physarum polycephalum.
The mature Plasmodium is a massive structure which becomes differentiated into an anterior fan-shaped sheet of granular protoplasm with a network of veins and a posterior zone which consists of a complex reticulate network of thick branched veins or strands, also called the channels (Fig. 2.5.)
Each strand is a tubular structure of jellified protoplasm. It contains more fluid portion of the protoplasm (endoplasm) which circulates actively within the strands; carrying with it the numerous nuclei, vacuoles and other inclusions.
The enodplasm in the strands, as mentioned above, shows streaming movements. It is in a constant state of rotation. It circulates actively within the strands carrying with it the numerous nuclei, vacuoles and other inclusions first in one direction for about 40-60 seconds. It then slows down, finally stops momentarily and then streams in the reverse. This unending, oscillatory endoplasmic streaming is termed cyclosis.
The motive force which brings about cyclosis has been considered to be associated with metabolism. It is generated through the interaction of a contractile protein with ATP (Adenosin triphosphate). It has now been determined that actin and myosin are the two contractile proteins which occur in the Plasmodium.
Wholfarth (1962) showed that these are located in the cytoplasmic filaments and fibril structures. Daniel and Jarlfors (1972) opined that “location and structure of the micro-channel/cortex system strongly suggests it as the site for localisation of contractile function implicated in cyclosis and motility”.
Actin, actinin and myosin have now been extracted from the Plasmodium of Physarum. These contractile proteins, according to Hatano et al, (1980) organise to form the subcellular structures responsible for the generation of motive force involved in cyclosis. With the cyclic contraction of plasmodial gel (ectoderm) induced by contraction of actomyosin systems, F-actin filaments in the gel layer form bundles.
The inner sol (endoplasm) is squeezed out into the plasmodial strands. According to Kumiya and Kurdo (1958) this causes the flow of sol in the strands. On relaxation of the gel layer, the fibrils disappear.
Thus, actin which exists as cytoplasmic fibrils or actin filaments are not perpetual structures. These appear and disappear according to the contraction and relaxation of the gel layer. Actinin is considered to play a role in regulating sol-gel transformation.
Structure of Plasmodium:
(i) Gross structure:
Careful observation has revealed that the apparently naked Plasmodium is bounded by a thin flexible non-cellular slimy layer distinct from the protoplasm. It is very thin at the advancing front end where food particles are engulfed in an amoeboid manner. Within the slime layer or sheath, the plasmodial protoplasm at its air interface is differentrated into a distinct plasma membrane.
It is an integral part of the protoplasm and completely invests it. Within the plasma membrane, the plasmodial protoplasm is differentiated into two zones, the outer and inner. The outer zone is gelatinous. It consists of firmer cytoplasm of less liquid consistency.
It is devoid of nuclei and other cell organelles. The inner zone which is called the endoplasm contains cytoplasm of more fluid consistency. It is in the plasmosol state. It contains numerous tiny nuclei, small vacuoles, contractile vacuolas, pigment granules and other inclusions. In fact the endoplasm is coarsely granular. The two phases of the protoplasm blend gradually.
(ii) Fine structure (Fig. 2.6):
The plasmodial protoplasm at its air interface is differentiated into a distinct plasma membrane. In Physarum it undergoes extensive invaginations. Coating the plasma membrane is a non-cellular thin slime layer that contains micro-fibrils. Probably the slime sheath consists of mucopolysaccharide protein complex. Henney and Asgari (1975) reported that Slime is composed of protein consisting of 17 different amino acids.
The other component is a polysaccharide consisting of hexose-galactose. Within the plasma membrane, the plasmodial protoplasm is differentiated into two zones, the outer and the inner. The outer zone consists of finer cytoplasm of less liquid consistency. It is in the plasmogel state and is devoid of nuclei and other larger cell organelles and large vacuoles.
This layer or zone of cytoplasm is termed the ectoplasm or cortex. Occasionally the ectoplasm exhibits fine fibrils. The inner zone which is called the endoplasm contains cytoplasm of more fluid consistency which is in the plasmosol state.
The endoplasm shows a complex network of micro-channels and contains numerous discrete vacuoles. The obvious cell organelles which the endoplasm contains are the nuclei, mitochondria, endoplasmic reticulum and the numerous ribosomes.
These are fine tubular structures, approximately 1µ in diameter coursing through the endoplasm. They arise as invaginations of the plasma membrane from the exterior into the cytoplasm. Each microtubule contains a fibrous material similar to and contiguous with the slime layer coating the plasma membrane.
In the cytoplasm adjacent to each microtubule is a layer of fine filaments constituting the filament sheath? The latter surrounds each microchannel and in the area of invagination at the plasmodial surface it appears as a continuation of the ectoplasm. The filaments in the sheath run parallel to each other and to the microchannel.
These are membrane bound, spherical structures of varying sizes found dispersed in the endoplasm. They lack cytoplasm and are devoid of filament sheath.
The diploid nuclei have each a distinct nucleolus. The nuclear membrane has pores.
The mitochondria are round or elongate in form. The cristae form ribbon-like configurations.
Plasmodial movement is termed locomotion. Under non-growth conditions, it is rapid whereas under conditions of rapid growth it is very slow. The Plasmodium moves with the help of pseudopodia which are blunt finger-like processes or lobes of its body. It puts out one or more pseudopodia in one direction. The entire protoplast of the body then flows in the direction of the pseudopodium thus formed.
In this way the Plasmodium creeps slowly over the surface of the substratum. The contractile proteins which occur in the microchannel-cortex system form the mechanico-chemical basis for motility. Actinin is suggested to play a vital role in regulating sol- gel transformation. It moves towards moisture and shuns strong light during the somatic phase, which is thus found in the dark.
The decaying vegetable matter constitutes the chief food of the slime mold Plasmodium. It is absorbed saprophytically through the ectoplasm. The Plasmodium also feeds on small organisms such as bacteria, protozoa or fungi, other smaller micro-organisms and bits of non-living solid, nutritious decayed organic matter it comes in contact with. It is thus phagotrophic in its nutrition.
The food is engulfed by the Plasmodium flowing around it. The ingested food is digested within food vacuoles by the enzymes and is used to build new protoplasm. The waste matters are rejected and simply left behind with the slime trail as the plasmodium moves ahead.
The Plasmodium also contains contractile vacuoles which probably have an excretory function. The somatic or vegetative stage of slime molds thus resembles amoeba of the animal kingdom.
To sum up, the diploid plasmodium, which represents the acellular somatic phase in the life cycle has the capacity for engulfment of particulate matter, for migration over the substratum and for somatic fusion (plasmodial coalescence).
The duration of this phase in the life cycle depends on certain external conditions such as low light intensity, abundance of moisture and plenty of available food. These conditions prolong the growth of the somatic or assimilative phase of the slime mold plasmodium.
After a period of food intake and crawling movements the Plasmodium attains its maximum size and reaches maturity. In the normal course of events the reproductive phase ushers in at this stage and the Plasmodium forms fruit bodies. However, under conditions of stress and strains the phaneroplasmodium of Physarum becomes converted into a hard, irregularly shaped structure known as the sclerotium.
Sclerotia formation (Fig. 2.7):
Under certain conditions such as drought, starvation, cold and absence of light the phaneroplasmodium by differentiation and cleavage becomes transformed into an irregular hard structure consisting of thick-walled cellular units. It is the sclerotium. The sclerotium thus represents the only diploid cellular stage in the slime mold life cycle.
It remains dormant for a long period under conditions unfavourable for growth. With return of suitable conditions, the sclerotium grows into a Plasmodium again. During the early period of sclerotium development, there is condensation of the plasmodial protoplast through desiccation and extrusion of slime. This is followed by cleavage of the protoplast into polynucleate units or portions.
Finally a thick, two-layered wall is formed around each unit or portion. The sclerotium thus consists of small cells named macrocysts or more appropriately called spherules. The latter in a sclerotium remain grouped together in a berry-like form and are covered by a common coat.
The thick walls of the spherules separate them from the adjacent ones. The spherules in old sclerotia are lobed and not rounded. According to Jump (1954), the sclerotia are induced under gradual desiccation, low temperature (5°C), high osmotic solutions of certain heavy metals, starvation and low (2.0)pH.
Spherules (Fig. 2.7. A-B):
The manitol-induced spherules exist as separate units (A). They are round in form and vary in size from 6-12µ. The spherule has a thick hard wall. Within the cytoplasmic membrane which closely invests the cytoplasm (B), the latter contains a few diploid nuclei and vacuoles.
Each nucleus has a nucleolus. The vacuoles contain small dense hollow granules which vary in dia. from 0.1- 2 µ. The spherule germinates under normal conditions. The thick hard wall cracks. The polynucleate spherule protoplast emerges through the split. It feeds and grows to form the new diploid plasmodium.
The sclerotia and spherules primarily serve as means of perennation. Secondarily they constitute the vegetative methods of asexual reproduction in the life cycle and serve to prolong the diploid stage in the life cycle of Physarum. The prolonged diploid stage is unusual among the true fungi. Some myxomycetes including Physarum overwinter in the sclerotial stage.
Alexopoulos (1966) reported that the aphano-plasmodia of some myxomycetes (stemomtales) do not form sclerotia. Instead they form cysts directly from the plasmodial veins.
Normally when the phaneroplasmodium of Physarum attains its maximum size and reaches maturity, it passes into the reproductive phase. The Phaneroplasmodium at this stage contains about one hundred to many thousand diploid nuclei. During the reproductive phase, the entire Plasmodium is converted into one or more fruiting bodies which bear the spores.
The fruiting body is called the sporophore or sporangium. The spores are cleaved from the sporangial protoplast. This process is termed sporulation. Since both the somatic and reproductive phases do not coexist in the same Plasmodium, the slime molds are said to be holocarpic.
The conditions which promote fruit formation are not definitely known. Moisture, light, temperature, pH and exhaustion of food have been suggested to be related to fruiting by some workers. There are others who proved this claim to be untenable on the basis of their experimental results.
Under this heading we study:
(i) Development of sporangium
(ii) Its structure and
(i) Development of Sporangium (Fig. 2.8):
At the fruiting time which is influenced by conditions not definitely known, the phaneroplasmodium changes its behaviour. It comes out of darkness or diffuse light and moves to exposed sites which are dry. The amoeboid life ceases. The protoplast of the quiescent plasmodium then becomes concentrated in some places to form hemispherical mounds.
The slime layer of the plasmodium dries or simply remains as a sheath on the substratum to constitute the hypothallus. Owing to the upward pressure of the protoplast from within with a pulsating movement, each mound elongates into a column like structure known as the papilla (A).
As the papillae elongate their bases constrict to form the stalk of the sporangium and free ends swell up to form the body (B). A large number of nuclei along with cytoplasm stream into the swollen tip which is then separated from the stalk by a septum. The swollen multinucleate terminal body along with the stalk functions as the sporangium (C).
(ii) Structure of Sporangium:
The sporangia in slime molds may be stalked or sessile. They are beautifully coloured. White, purple, orange and brown are the usual tints. They vary in form and may be spherical, ovoid, elongated etc.
The stalk which is thin and slender also varies in length. P. polycephalum has stalked clustered sporangia. Each stalk may end in one or more dark- coloured more or less globular sporangia. In height the sporangium varies from 1-3 mm including the stalk.
The sporangium in Physarum is enclosed in a tough, slimy, non-cellular layer, or wall called the peridium. It is hard, brittle and has a rigid wrinkled texture. According to Kislev and Chet (1973), the peridium surface is marked by evenly distributed small holes or cavities about 5- 10µ in dia. and is covered with numerous head-like granules up to 2µ in dia.
Within the peridium, in a mature sporangium, are the numerous tiny, rounded spores which are close packed in between fine tube-like structures constituting the capillitium. The spores are, however, free from them when mature.
The cleavage furorows in the dividing proloplast of the young sporangium are filled with a fungus cellulose material in the form of an intricate network of fine hyaline threads. This network is called the capillitium (C). It appears to consist of a network of calcareous nodules connected by hyaline tubular threads which adhere to the peridial wall.
According to Kislev and Chet (1973), the capillitium in Physarum is an intricate network of fine tube-like structures of differing diameters. The capillitial tubes may be simple or branched, have rigid walls and open at the peridial surface (wall).
These capillitial openings look funnel-like cavities on the peridial surface. Fine particles or granules are found on the surface and bead-like granules within the tubes. These structures probably of calcium carbonate provide rigidity to the tubes.
Functions of capillitium:
(i) It serves as an endoskeleton providing mechanical support to the delicate, slime mold sporangium,
(ii) It serves as a conducting system through which excretory products and lime migrate outward to the peridium and become deposited thereon and
(iii) Perhaps assists in the despersal of spores.
It is a process whereby haploid spores (meiospores) are fashioned from the diploid multinucleate protoplast of the sporangium by meiosis. Meiosis is a special kind of nuclear division in which the diploid nuclei undergo two successive divisions but the chromosomes are replicated only once.
Location of Meiosis:
The numerous diploid nuclei in the sporangium protoplast undergo synchronous division. Some workers hold that this division is meiotic. The resultant protoplast with haploid nuclei undergoes cleavage into uninucleate tiny daughter Micro-body protoplasts. Each of these becomes Nucleolus rounded and secretes a thick wall around it to become a meiospore.
Thus, droplet according to this view, meiotic division in Physarum occurs in the precleavage sporangium.
The opposite view held by Stock et al. (1964), Aldrich (1967) and Pandall and Lynch (1974) advocates meiosis in P. polycephalum in the post cleavage spores. According to them, nuclear division in the precleavage sporangium is mitotic. Randall and Lynch (1974) observed nuclei in mature spores containing distinct synaptonemal complexes, a feature characteristic of meiotic prophase.
Their observation thus confirms the view that meiosis in this slime mold occurs after spore cleavage and not prior to it. In fact the young cleaved spores are uninucleate and diploid. The diploid nucleus later undergoes division in which synaptonemal complexes characteristic of meiosis become apparent.
Immediately the second division follows:
Each spore becomes quadrinucleate. The four nuclei represent the tetrad. Three of these disintegrate; consequently the spore becomes uninucleate and haploid. Technically the resultant unmucleate haploid spore is called a meiospore.
(iv) Dispersal of spores:
Wind, rain and mites play an important role as agents of spore dispersal in slime molds. The peridium disintegrates exposing the capillitium and mass of mature spores it supports. The hygroscopic capillitium twists in various ways as it absorbs and loses water These movements cause the spore mass to break and thus loosen and free the spores which are dispersed by air currents.
At the same time the capillitium expands carrying the spores high up with it from this position the dry, powdery spores are easily disseminated by wind. The wind disseminated spores is a plant characteristic. Only plants produce wind borne spores and sporangia. The fruiting stage of slime molds thus reminds one of a fungus.
(v) Spore ultra-structure (Fig. 2.9):
The purplish brown meiospores of P. polycephalum are resting spores. They are small, round, uninucleate structures not more than 10 µ in diameter with a spiny spore wall. The thick, beautiful sculptured spore wall is reported to contain cellulose which is typical of plant cells. McCormick, Blomquist and Rusch (1970) found it to contain mainly galactosamine and some protein and melanin.
The spore wall is differentiated into two layers, the outer and the inner. According to Randall and Lynch (1974), the outer layer, which is thicker, is electron opaque with a granular composition. Externally it bears randomly distributed cone- shaped spines. The inner layer of the spore wall which is comparatively thinner is electron transparent and has a fibrous appearance.
Within the spore wall is the spore protoplast which at its periphery is differentiated into a distinct unit membrane, the plasma membrane. The latter occasionally invaginates to form pocket-shaped vesicles which extend into the spore cytoplasm.
Gaither (1974) reported that the meiospores contain the usual complement of discrete organelles embedded in a dense cytoplasm. Numerous spheroidal mitochondria are dispersed throughout the cytoplasm.
Randall and Lynch (1974) reported the occurrence of electron-dense granules and occasional presence of electron-dense helical filaments within mitochondrial cristae. The single nucleus has a distinct nucleolus. The nuclear membrane has pores.
Near the periphery of mature spores in close proximity to vacuoles Randall and Lynch (1974) rarely observed dictyosomes consisting of stacks of 3 or 4 parallel cisternae. The other inclusions found in the cytoplasm are the lipid droplets, glycogen-like granules, vacuoles and microbodies.
(vi) Germination of Meiospores (Fig. 2.10):
The meiospores (A) remain viable for a considerable period. They are exceptionally resistant to prolonged periods of desiccation owing to the thick spore wall and physico- chemical structure of spore protoplasm (A).
They germinate on moist substrata such as dead leaves, logs, soil or substratum in favourable temperature and in rain water in nature. On germination the spore wall either cracks open (B) or a tiny pore appears in it. The contents usually escape in the form of spindle-shaped structures through it (C).
Each has two flagella inserted at its anterior end. One of them is shorter than the other but both are of whiplash type. The shorter flagellum is directed backward or appressed against the body. This flagellated structure is known as the swarm cell. The swarm cells greatly resemble colourless algal flagellates or protozoans (zooflagellates).
The number of swarm cells produced by each resting spore varies from one to four. The anteriorly biflagellate swarm cells are naked and amoeboid. Each contains a single nucleus toward the anterior end and contractile vacuoles at the posterior end (C).
Sometimes, instead of swarm cells, one to four amoeboid cells known as myxamoebae emerge when a spore germinates (D). Presence of moisture favours the flagellate form and drier conditions induce the amoeboid form. The swarm cells as such do not divide. The myxamoebae normally divide and increase in number.
(vii) Sexual Reproduction (Fig. 2.11):
The sexual stage intervenes between the resting meiospore stage and diploid Plasmodium. The swarm cells or myxamoebae which function as gametes are produced by the germination of meiospores or resting spores differentiated by meiosis from the diploid protoplast of the sporangium.
The sporangium in Physarum thus is an organ of sexual reproduction. The swarm cells (A) or myxamoebae (a) get their nutrition from the surrounding medium by absorption, and also by ingesting bacteria, fungal spores, yeast cells and small particles of organic matter at their sticky posterior end.
The sexual process consists of the following events:
Depending upon the species, fusion between gametes (swarm cells or myxamoebae) takes place in either of the following two ways:
(i) In P. polycephalum swarm cells directly function as planogametes (A). After liberation they swim about with a rotary movement and finally come in contact at their sticky posterior ends in pairs (B). Fusion thus starts at their posterior ends. The cytoplasm fuses with the cytoplasm and nucleus with the nucleus.
The young zygote thus formed is, at first, binucleate and flagellate (B2). It swims about for a while. Finally it retracts the flagella and changes into a myxamoeba (C). The two nuclei in it have fused to form a diploid nucleus. In P. polycephalum and P. flavicomum fusion takes place reproduction between swarm cells (gametes) of opposite mating strains.
They are thus heterothallic. Clark and Collins (1976) studied mating systems of eleven species of myxomycetes. They reported that P. cinereum is also heterothallic but P. compressum, P. gyrosum and P. pusillum are homothallic.
(ii) In some other species myxamoebae emerge from the resting spore’s one each (a). Under favourable conditions each myxamoeba may divide repeatedly to form a number of daughter cells (a). The latter function as gametes and copulate in pairs (b) to form a zygote (c).
(viii) Germination of Zygote:
The naked zygote (C) formed in either case has a single diploid nucleus. It creeps over the substratum feeding on bacteria and organic matter synthesising more protoplasm resulting in growth. The growth in size is accompanied by repeated and successive divisions of the diploid parent nucleus.
The divisions are mitotic. As a result of subsequent growth, repeated karyokinesis but no cytokinesis, the zygote gradually becomes changed into a large multinucleate amoeboid mass of protoplasm, called the macro Plasmodium (D). The numerous nuclei embedded in its protoplasm are diploid in nature. Favourable temperature, abundant moisture and food favour its growth, movement and reproduction.
In many cases, the young diploid plasmodia may combine with zygotes or other Plasmodia of the same species or a number of zygotes may coalesce to form a single larger plasmodium. In all these cases, the union involves the fusion of their cytoplasm only. There is no fusion between the nuclei.