The Dictyostelids

The Dictyostelids

by Kenneth Bryan Raper

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Overview

The Dictyostelids by Kenneth Bryan Raper

Kenneth Raper tells how dictyostelids are isolated, cultivated, and conserved in the laboratory; how myxamoebae aggregate to form multicellular pseudoplasmodia; how fructifications arise by transformation of amoeboid cells into stalk cells and spores; and how similar cells can, under certain conditions, enter a sexual phase. For each known dictyostelid Professor Raper includes a complete description and photographic illustrations; one new species is described.

Originally published in 1984.

The Princeton Legacy Library uses the latest print-on-demand technology to again make available previously out-of-print books from the distinguished backlist of Princeton University Press. These editions preserve the original texts of these important books while presenting them in durable paperback and hardcover editions. The goal of the Princeton Legacy Library is to vastly increase access to the rich scholarly heritage found in the thousands of books published by Princeton University Press since its founding in 1905.

Product Details

ISBN-13: 9780691612553
Publisher: Princeton University Press
Publication date: 07/14/2014
Series: Princeton Legacy Library Series
Pages: 466
Product dimensions: 9.10(w) x 6.10(h) x 0.90(d)

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The Dictyostelids


By Kenneth B. Raper

PRINCETON UNIVERSITY PRESS

Copyright © 1984 Princeton University Press
All rights reserved.
ISBN: 978-0-691-08345-2



CHAPTER 1

Historical Background


Slime molds of the dictyostelid type have been known since Oskar Brefeld isolated and described Dictyostelium mucoroides in 1869. While examining the fungus flora of horse dung he observed spores of the same form but much smaller than those of Mucor mucedo. Unable at first to trace their origin, he fortunately, and soon thereafter, obtained on rabbit dung in relatively clean culture abundant fruiting structures bearing similar spores. Superficially these structures resembled the sporangia and sporangiophores of a Mucor, hence the specific name. Several unique features, however, refuted any probable relationship: the "sporangia" had no enveloping walls and the spores were suspended freely in droplets of thin slime; no vegetative hyphae were connected with the bases of the fructifications; the spores upon germination produced not hyphae but small amoeboid cells; the myxamoebae, following a period of growth and substantial multiplication, aggregated to certain points from which fructifications arose directly; and finally the stalks that supported the pseudosporangia consisted not of hyphae, as in the true fungi, but of closely packed parenchyma-like cells. The generic name Dictyostelium was chosen to indicate their net-like appearance (Fig. 1-1).

Using cooked horse dung as a solid substrate, and a decoction of the same for slide cultures, Brefeld succeeded in following the entire life cycle of his slime mold; and, except for a few misinterpretations, he presented a generally accurate picture of its growth and development as this is still known. He recognized the free-living myxamoebae as constituting the vegetative phase and noted a parallel between these and an early stage in the life cycle of the Myxomycetes with which he concluded Dictyostelium was related. He described the presence of a thin, open-ended sheath surrounding the cellular stalk and noted the cellulosic character of this and of the walls of spores and stalk cells as well. He may have sensed but did not elaborate upon a possible role for the open-ended tube in stalk construction. His major misinterpretation, and one that troubled him later, was his assumption that the myxamoebae upon aggregation fused to form a true Plasmodium, albeit a somewhat transitory one. This error was corrected in a second and more elaborate paper several years later (1884), but not before van Tieghem (1880) had demonstrated that the aggregated myxamoebae never actually fused.

It is to Ph. van Tieghem that we owe our first clear insight into the true and unique nature of the so-called cellular slime molds, including the dictyostelids. In two brief and regrettably unillustrated papers (1880, 1884) he showed that the myxamoebae formed their characteristic fructifications through the orderly assemblage, coordination, and subsequent differentiation of large populations of separate, collaborating cells. In the first of his brief communications (1880) he described a new genus and species, Acrasis granulata, two new species of Cienkowski's Guttulina (1873), and two new species of Dictyostelium, D. roseum and D. lacteum, in addition to including observations on strains of Brefeld's D. mucoroides that he had isolated. Whereas Acrasis granulata was the slime mold in which the absence of a true Plasmodium was first established, a fact reflected in the generic name selected for it, he clearly demonstrated that nonfusion of aggregated myxamoebae was equally characteristic of the other genera included in his "Plasmode Agrégé," or Acrasiées. In the second paper (1884) van Tieghem described a new and more complex genus and species, Coenonia denticulata, and in a few simple experiments demonstrated that the position of a myxamoeba within the frucitifying mass determined whether it would form a stalk cell or a spore. Unfortunately, neither Coenonia nor Acrasis granulata has been rediscovered.

In the same year (1884), but without reference to van Tieghem's earlier work (1880), Brefeld published a long and comprehensive paper that included further observations on Dictyostelium mucoroides together with a description and superb illustrations of an additional new genus and species, Polysphondylium violaceum (Figs. 1-2 and 1-3). He had by this time recognized the unique character of the aggregated cell associations in these genera and introduced the term Scheinplasmodium, or pseudoplasmodium, which was appropriately descriptive and has since been generally accepted.

Three additional reports appeared prior to 1900: one by E. Marchal (1885) wherein a fourth species of Dictyostelium, D. sphaerocephalum, was described; one by von M. Grimm (1895) on the structure and development of D. mucoroides that in the main confirmed Brefeld's account of 1884 with some additional information on nuclear division; and a third by G. A. Nadson (1899) in which, for the first time, a dictyostelid, D. mucoroides, was reported to be growing with a known species of bacteria, Bacillus fluorescens liquefaciens. He concluded that the two organisms were symbionts and that the bacteria favored the slime mold by creating an alkaline reaction in the culture medium. The myxamoebae, he believed, fed solely upon nutrients in solution.

Early in the next century very important papers were published by E. W. Olive in the United States (1901, 1902) and George Potts in Germany (1902), while works of lesser scope were reported in France by P. Vuillemin (1903) and E. Pinoy (1903, 1907).

In the first of his papers, entitled "A Preliminary Enumeration of the Sorophoreae" (1901), Olive listed, with brief notations, all the known slime molds that he believed were assignable to Zopfs "Gruppe I" of the Eumycetozoa (1885), together with a new genus, Guttulinopsis (with three species), three new species of Dictyostelium, and two new species of Polysphondylium. This was followed a year later (1902) by his much longer "Monograph of the Acrasieae" (Fig. 4). For this he used an Anglicized version of van Tieghem's more restrictive "Acrasiees," and excluded Barker's Diplophrys (1868) and Cienkowski's Labyrinthula (1867) because they lacked cell associations of true aggregative origin. Based upon his studies at Harvard University under the watchful eyes of Roland Thaxter, Olive attempted to present a comprehensive picture of what he and earlier investigators had learned about the growth, development, and possible relationships of the genera and species then known. His slime molds were primarily of coprophilous origin, and, like his predecessors, he relied heavily upon horse dung and dung decoctions, either liquid or "stiffened with agar," as culture substrates. Media containing peptone and potato decoction were used also, but concentrations were not given. Unidentified bacteria were constantly present. Their possible role in the nutrition of myxamoebae was noted but not explored.

Olive (1902) did emphasize the temporal separation of the vegetative (myxamoebal growth and division) and fruiting (cell aggregation and fructification) stages, and he surmised that the myxamoebae aggregated in response to chemotropic stimuli since Dictyostelium mucoroides and D. purpureum fruited separately when cells of the two species were intermixed. He included some further information concerning stalk and spore formation and, consistent with the times, dwelt at some length upon the cytology and nuclear states of the myxamoebae and of differentiating cells. His major contribution, however, related to the systematics of the group. For over three-quarters of a century his monograph has remained the one inclusive taxonomic work on the Acrasieae despite the publication of five new genera and many new species since that time.

Although differing in subject matter, and less frequently cited, Potts's paper "Zur Physiologie des Dictyostelium mucoroides" (1902) was no less significant. He cultivated Dictyostelium mucoroides upon sterilized dung and dung infusion, upon infusion agar made from the stems of Vicia faba, and upon an extract of corn (maize) grains which he employed either as a liquid or solid medium. Employing corn gelatin, which he found to be especially favorable, he observed that Dictyostelium developed only in the presence of bacteria, and he succeeded in isolating the slime mold with a single bacterial species which he described as Bacterium fimbriatum. He further studied the relationship between these organisms by cultivating them upon a large number of synthetic media, each of which contained some inorganic or organic source of nitrogen together with a sugar, organic acid, or other source of carbon. Although he obviously misinterpreted the manner in which the slime mold fed upon the accompanying bacteria, believing they were digested extracellularly, he made no mistake in his conclusion that the Dictyostelium was dependent upon bacteria for nutriment. Furthermore, he was not unmindful of the influence the bacteria might exert through altering the culture medium, and he reported that D. mucoroides grew well in media of slightly acid to mildly alkaline reaction. Potts succeeded in growing the myxamoebae with pure cultures of three additional bacteria, Bacillus subtilis, B. megatherium, and B. fluorescens liquefaciens, and noted that the list could probably be extended. He refuted Nadson's claim (1899) of symbiosis between the last of these bacteria and D. mucoroides. Exhaustion of the nutrient was believed to stimulate cell aggregation, for Potts succeeded in keeping cells in a vegetative stage for 3 ½ months by transferring them frequently to fresh substrata. Concerning cell aggregation, he concluded that the ray-like arrangement of the inflowing arms of myxamoebae around the midpoint appeared "almost to point to a chemotactic influence." Normal fruiting was reported to be strictly aerobic and strongly influenced by humidity, which in turn seemingly controlled transpiration of the developing fructifications, a process that Potts regarded as being of prime importance.

As the title "Une Acrasioe Bacteriophage" suggests, Vuillemin (1903) clearly perceived the relationship between Dictyostelium mucoroides and Bacillus fluorescens nonliquefaciens, the bacteria with which his slime mold was isolated and cultivated. The Dictyostelium grew only in the presence of bacteria, and he states unequivocally that the bacteria were ingested and digested by the myxamoebae. He appreciated also the interrelationship between the bacteria, the substrate, and the presence or absence of slime mold growth, for he concluded that the slime mold failed to grow on maltose-peptone medium with Bacillus pyocyaneus, a more proteolytic species, because of excess alkalinity.

Vuillemin's studies were extended in several ways by E. Pinoy (1903, 1907). He confirmed the engulfment and digestion of bacteria and succeeded in isolating from myxamoebae of D. mucoroides a preparation that liquefied gelatin and dissolved bacterial cells (B. coli communis) that had been killed by chloroform. To the active substance, or enzyme contained in this extract, he applied the name "acrasidiastase," and noted that it was similar in its action to a substance, or enzyme, earlier isolated by Mouton (1902) from a soil amoeba and termed an "amibodiastase." Dismissing symbiosis as a possibility, Pinoy (1907) considered the slime mold as living parasitically upon the colonies of associated bacteria. He cultivated the myxamoebae successfully with several Gram-negative bacteria including Bacterium coli, Bacillus kieli, Bacillus violaceus, and Vibrio cholorae, and noted that Gram-positive species such as Bacillus subtilis, Bacillus megatherium, and Bacillus anthracis were not consumed. Of the various substrates investigated, an agar medium based upon cooked flax seed proved most useful.

Pinoy made some comparisons between Dictyostelium mucoroides and D. purpureum and Polysphondylium violaceum, having obtained the latter cultures from Professor Thaxter. Sorus color in D. mucoroides, but not in D. purpureum or P. violaceum, was reported to vary with the identity of the bacterial associate and the composition of the underlying substrate. Since the fluorescent pigment of B. fluorescens liquefaciens could account for a yellow sorus in D. mucoroides, he surmised that van Tieghem's D. roseum (1880) might have been the same species in the presence of a redpigmented bacterium such as Bacillus kieli.

A decade later F. X. Skupienski initiated studies on sexuality among the slime molds (1917, 1918). Although concerned primarily with the true Myxomycetes, such as Didymium nigripes, he published on Dictyostelium mucoroides briefly in 1918, and at greater length in his "Recherches sur le Cycle Évolutif de Certain Myxomycètes," published privately in Paris in 1920. A sexual cycle was reported where (+) and (-) myxamoebagametes fused to form zygotes, and these then merged to produce a Plasmodium of short duration. This in turn was transformed into a single sporangium, rarely two. The spores and cells of the stalk(s) were thought to be derived by progressive division of the whole plasmodial mass as in the fruiting process of Didymium nigripes. This interpretation could not be substantiated by other investigators, and the discovery of a true sexual stage in Dictyostelium was delayed for a half-century until the origin, structure, and function of the macrocysts were revealed (Erdos, Nickerson, and Raper, 1972 and 1973; Clark, Francis, and Eisenberg, 1973; Erdos, Raper, and Vogen, 1973). Skupienski's cultural studies were informative and generally correct, except he concluded, like Nadson (1899) before him, that D. mucoroides and Pseudomonasfluorescens liquefaciens existed in a symbiotic relationship.

At about the same time (1922) R. Oehler in Wurzburg reported that Dictyostelium mucoroides could be isolated from compost and forest soil in addition to dung, and that it represented an ideal subject for laboratory demonstrations of spore germination and the nutrition of amoeboid organisms growing in association with living bacteria. Of such bacteria Gram-negative species were most suitable and "xerose-bacteria" (Corynebacterium xerosis?) were less so, while Gram-positive bacilli were not recommended.

Using cultures obtained from Oehler, W. von Schukmann took up the study of D. mucoroides and in 1924 and 1925 published papers on its biology and morphogenesis. In the first of these he reported that substrains which had ceased to produce pseudosporangia, or even pseudoplasmodia, could be restored to normal development by implanting myxamoebae on sterilized horse dung together with an indigenous dung bacterium. Once the ability to produce pseudosporangia had been restored, the revitalized slime mold could be cultivated successfully on agar media with B. coli. In the second paper he, like Potts (1902), recognized nutrient exhaustion as a primary stimulus for fructification, but claimed that negative hydrotropism also played a significant role. Enhanced fruiting on roughened agar surfaces created by the incorporation of inert objects such as glass wool and glass beads was cited as evidence. In still other experiments he attempted to immunize rabbits by injecting them with myxamoebae of D. mucoroides. Although not dramatic by current standards, his results were nonetheless interesting. Spores of D. mucoroides were not influenced by the immune serum.

Except for the pioneering investigations of E. W. Olive, already cited, the Dictyosteliaceae attracted little or no attention in America prior to the mid-twenties, when R. A. Harper of Columbia University began publishing his analytical studies of sorocarp construction in Dictyostelium mucoroides and Polysphondylium violaceum. Having previously studied processes of morphogenesis in coenobic algae such as Pediastrum and Hydrodictyon (1918), he was drawn to the Acrasieae because of their capacity to build from previously independent myxamoebae erect, multicellular fructifications of considerable symmetry. In the first of three papers, "Morphogenesis in Dictyostelium" (1926), he introduced the very useful term sorocarp for the completed fructification, and in great detail analyzed steps in its formation. Much emphasis was placed upon photographs of living material, and although not very clear they still supported his thesis of intercellular communication and regulation leading to fairly constant levels of proportionality in completed sorocarps and in the cells that comprised them. Recognizing that many other factors influenced the construction and pattern of sorocarps, Harper believed that cellular contacts and pressures, combined with stimuli of maximal weight resistance and diminishing load, were of prime importance in effecting the readjustments in cellular form and position required during fructification. When one considers the culture techniques employed (primarily hanging drop cultures in dung extract), it is remarkable that he achieved so much.


(Continues...)

Excerpted from The Dictyostelids by Kenneth B. Raper. Copyright © 1984 Princeton University Press. Excerpted by permission of PRINCETON UNIVERSITY PRESS.
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Table of Contents

  • FrontMatter, pg. i
  • CONTENTS, pg. v
  • PREFACE, pg. vii
  • BIOGRAPHICAL NOTE, pg. xi
  • CHAPTER 1. Historical Background, pg. 3
  • CHAPTER 2. Occurrence and Isolation, pg. 17
  • CHAPTER 3. Ecology, pg. 30
  • CHAPTER 4. Cultivation, pg. 48
  • CHAPTER 5. Culture Maintenance, pg. 77
  • CHAPTER 6. Vegetative Stage, pg. 87
  • CHAPTER 7. Cell Aggregation, pg. 104
  • CHAPTER 8. Fructification, pg. 134
  • CHAPTER 9. Macrocysts, pg. 178
  • CHAPTER 10. Acrasiomycetes, pg. 225
  • CHAPTER 11. Dictyostelidae, pg. 242
  • CHAPTER 12. Dictyosteliaceae: Dictyostelium, pg. 246
  • CHAPTER 13. Dictyosteliaceae: Polysphondylium, pg. 368
  • CHAPTER 14. Acytosteliaceae: Acytostelium, pg. 393
  • CHAPTER 15. Coenonia, pg. 408
  • EPILOGUE, pg. 413
  • BIBLIOGRAPHY, pg. 417
  • INDEX, pg. 443



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