SLIME MOLDS

Slime molds are intriguing organisms who have been traditionally studied by mycologists for years. Many laboratories around the world are engaged in using slime molds to study fundamental biological processes such as cell movement, cell differentiation and the mechanics of nuclear division. Even though their distinctiveness from true fungi have been recognized in the recent years, slime molds remain under the study of mycologists.

Three main characters distinguish slime molds from true fungi.
1. Slime molds lack cell walls but may contain a slime sheath around its protoplasm.
2. Propagate by spores that germinate to form amoeboid or flagellate cells. Spores of true fungi are mostly non motile.
3. Exhibit phagocytic mode of nutrition instead of absorptive form of nutrition as true fungi.

Organisms that are considered as slime molds are classified in two kingdoms and belong to 5 different phyla. The classification is shown below.

Kingdom Protoctista

Phylum Myxomycota
Phylum Plasmodiophoromucota
Phylum Dictyosteliomycota
Phylum Acrasiomycota

Kingdom Stramenopila

Phylum Labyrinthulomycota

The slime molds that belong into these 5 phyla can be divided into three categories:

True slime molds : Phylum Myxomycota (plasmodial or acellular slime molds) - free living
Phylum Plasmodiophoromycota - endoparasitic
These organisms are regarded as true slime mold because they form a true plasmodium ( a naked multinucleate mass of protoplasm that moves and feeds in amoeboid fashion)

Cellular slime molds : Phylum Dictyosteliomycota
Phylum Acrasiomycota
These slime molds differ from true slime molds in that their stalks consist of walled cells and that their plasmodial stage is consisted of a pseudoplasmodium where component cells remain as individual cells but function as a group.

Net Slime molds : Phylum Labyrinthulomycota
These slime molds differ form the other two types in that these organisms have a thallus consisting of branched tubes within which amoeboid cells crawl.

The focus of today's lab is to observe different stages of slime molds that belong to the above mentioned phyla and try to categorize the available samples to those phyla using a dichotomous key.

Phylum Myxomycota
Members of this phylum make haploid spores that germinate to give rise to myxamoebae or swarm cells depending on the environmental conditions. These myxamoebae and swarm cells function as gametes and unite in pairs (swarm+swarm, myxamoeba+myxamoeba or swarm + myxamoeba) to give rise to a zygote thus initiating the diploid phase of the life cycle. As the zygote grows, its nucleus undergoes synchronous mitotoc divisions without cytokinesis resulting in a multinucleate, amoeboid structure, the plasmodium. The plamodium can increase in size by uniting with other plasmodia or with zygotes of the same strain. The plasmodium does not have a definite shape or size and is ever changing and ever flowing. It creeps over the surface of the substrate and engulfs particles of food within its path. At this stage, protoplasm streaming is visible while the plasmodium extends itself in different directions. Under unfavorable conditions, the plasmodium becomes converted into irregular, hardened mass known as the sclerotium. Sclerotia can remain dormant for a long period of time and will grow into a plasmodium on the return of favorable conditions.
Reproductive phase of the myxomycetes is marked by the conversion of the entire plasmodium into one or more sporophores. Myxomycetes produce four general types of sporophores: sporangia, aethalia, pseudoaethalia, plasmodiocarps. (see below for definitions) Meiosis occurs in the young spores resulting in haploid uninucleate spores that are liberated from their sporophores by wind, water and activities of animals.
The life cycle of a typical myxomycete is shown in fig: 1.

Lab Work
1. Observe the structures that are defined below
Peridium : The outside covering or wall of a fructification
Sporangia : sac like structure containig protoplasm which is converted to spores
Plasmodiocarp: a curved or branched, vein like fruiting structiure of some myxomycetes
Capillitium: sterile, thread like structures present among spores in the fruiting bodies
Columella: sterile structure within a sporangium or other fructification: extension of a stalk
Aethalium: Large, sometimes massive cushion shaped fructification
Learn how to use the dichotomous key to identify the specimans provided. Draw everything you see; describe the characteristics of the specimen and write down the order, family, genus and species of the specimen.

2. Observe protoplasmic streaming
Protoplasmic streaming is a characteristic of many cells but the streaming observed in myxomycete plasmidia is on an unusually massive scale. Obtain a growing plasmodium of Physarium polycephalum. Using a dissecting microscope and without opening the plate, focus on one big vein and obeserve the movement of protoplasm. Some questions to help you in your observations:
Is the streaming unidirectional?
Is the rate in which streaming occur constant or variable?
Does it ever stop?
Does streaming occur in all of the veins at the same time, rate and direction?
Does streaming continue even if smaller veins join together?

3. "Pet" Physarium polycephalum
Transfer a small agar block containing a growing plasmodium onto a new oat flake agar plate. This will be your `pet' Physarium. Using your creativity, you can do whatever you like with it to answer your own question. A `class pet' will serve as a control. This Physarium will be grown under optimal conditions, i.e. fed with oatflakes as needed, kept in the dark at optimum temperature of 25 C. Next week, we eill compile all your observations to help us better understand the growth and life cycle of this myxomycete.



Phylum Plasmodiophoromycota
Members of this phylum are obligate endoparasites of vascular plants, algae and other fungi. These slime molds produce a true plasmodium but are different from plasmodium of a myxomycete due to the absence of translocational movement. Furthermore, they lack the ability to phagotocize food material and exist wholly within the cells or hyphae of their hosts. A typical lifecycle of a plasmodiophoromycota is shown in fig. 2.
Resting spores of Plamodiophorids (borne in a sporosorus) exist in soil or water as a result of disintegration of tissues of infected hosts. Each resting spore germinates to form a primary zoospore (thick walled) which attaches to a susceptible host and encysts. Once the host is punctured, the protoplast of the zoospore enters the host cell and is carried around inside the host cell. Cruciform mitotoc divisions occur in the protoplast resulting in the formation of the primary or the sporangial plasmodium. Once the plasmodium reaches a certain size, the it cleaves into segments that develop into zoosporangia. Secondary zoospores (thin walled) are cleaved within and are released directly into other host cells or to the outside of the root. The secondary zoospores can enter a host cell as primary zoospores and form a secondary or sporogenic plasmoduim. As secondary plasmodium establishes itself in the host cells, the host cells undergo extensive hypertrophy and hyperplasia resulting in distortion of the root shape. Eventually, these plasmodia undergo cleavage followed by meiosis to form resting spores which are typically produced in masses termed sporosori (except in the P. brassicae where they are produced free or in loose associations). The resting spores are released into the environment after the death and disintegration of the host cells. These spores can exist dormant for long periods of time before germinating to form primary zoospores.

The lifecycle of Plasmodiophora brassicae is shown in fig.3 as an example of theses slime molds.

Lab work
1. Observe the prepared specimen of P. brassicae to identify sporangia or sori and resting spores of these plant pathogen.


Phylum Dictyosteliomycota
The basic unit of structure of members of this phylum is a uninucleate, haploid amoeba that feeds by engulfing bacteria. These amoebae have filose pseudopodia in contrast to lobose pseudopodia made by members of phylum acrasiomycota. Under certain conditions the amoeba encysts forming a microcyst which act as a resting structure. Once the environmental conditions are favorable, the microcyst germinates producing an amoeba.
Each amoeba can divide mitotically resulting in two uninucleate amoebae. This process can occur repeatedly until the population reaches a minimum number of cells when amoebae stop feeding and aggregate to a certain aggregation center in streams. Aggregation center is formed by one or more cells that secrete a chemical (cAMP for some dictyostelids) and other amoebae are attracted to this along the chemical gradient. Aggregation results in the formation of a pseudoplasmodium in which the amoeboid cells do not fuse but exist as intimately associated uninucleate cells. The cells of the pseudoplasmodium become specialized at an early stage. The cells at the anterior 1/3 of the slug consists of prestalk cells and the posterior portion of the slug consist of prespore cells. The pseudoplasmodium migrates on the substrate for a while before becoming globose, flattened at the base and developing a papilla of prestalk cells. This is in preparation for the development of the sorocarp. This preparation is known as culmination. Prestalk cells elongate, produce cellulose to form a stalk tube and push down on the cell mass while the prespore cells rise to the top of the fully developed stalk. These prespore cells are transformed into spores.
Two uninucleate, haploid amoebae can at times fuse with each other giving rise to a diploid zygote. Other uninucleate amoebae surround the zygote and secrete a primary wall around the zygote and themselves. The zygote feeds on these trapped amoebae and lays down other wall material within the primary wall. This is known as a macrocyst and meiosis takes place within. Uninucleate amoebae divide mitotically to produce large number of nuclei. After several weeks the macrocyst cytoplasm cleaves to produce uninucleate haploid amoebae. These amoebae will be released through the cyst wall and can give rise to a pseudoplasmodium.

The lifecycle of Dictyostelium discoideum is shown in fig.4

Lab Work

Try to observe the moving slug, the pseudoplasmodium and a mature sorocarp for the plates inoculated with D. discoideum.




Phylum Acrasiomycota
Phylum Acrasiomycota differs from Dictyosteliomycota in a number of morphological and life cycle details.

1.Amoebae have lobose pseudopodia.
2. Amoebae of acrasid slime molds aggregate singly or in groups rather than in streams.
3. Amoebae of acrasid slime molds do not respond to cAMP as the aggregation chemical.
4. Pseudoplasmodium of members of the phylum acrasiomycota does not have a migration phase. Once aggregation occurs and pseudoplasmodium is formed, sorocarp development begins.
5. Stalk cells of the sorocarps do not form cellulose to form a stalk tube.
6. No distinct sori and sorospores in the sorocarps. All cells of the sorocarp may
germinate to produce amoebae.

The lifecycle of Acrasis rosea (member of Acrasiomycota) is shown in fig.5

We do not have specimen for this phylum. Know how this phylum differ from other phyla of the slime molds.

Phylum Labyrinthulomycota
There is not much known about different species of this phylum. The classification of these organisms is speculative. Historically, the most important character of the organisms that belong to this group is the presence of an ectoplasmic network of branched, anastomosing, wall less filaments produced by cells with a specialized cel surface organelle known as a bothrosome.
These slime molds have a thallus consisting of branched tubes within which amoeboid cells crawl. They have biflagellated spores with both whiplash and tinsel type flagella. One important species that belong to this group is Labyrinthula macrocystis, a pathogen of eel grass.

There are no lab specimen belonging to this group.