Acrasis Rosea Research Articles

A contemporary evaluation of the acrasids (Acrasidae, Heterolobosea, Excavata)

Sorocarpic protists are organisms that individually aggregate and work together to form a fungus-like fruiting body (sorocarp). The amoeboid forms are often colloquially referred to as “cellular slime molds” or “acrasids”. We argue the latter term should be used only to refer to members of Acrasidae in Heterolobosea. Here we study the diversity of two Acrasidae genera, Acrasis and the closely similar Pocheina, using a combination of morphological characteristics and small subunit rRNA gene sequences. A total of eight isolates of Acrasis and an example of Pocheina were examined. Acrasis/Pocheina form a well-supported monophyletic group that is the highly supported sister to a clade containing Allovahlkampfia and several other amoebae. Four molecular lineages of Acrasis were resolved, each of which is characterized by a distinctive fruiting body morphology. Each lineage represents a species, two of which are novel, Acrasis kona n. sp. and Acrasis takarsan n. sp. An isolate identified as Pocheina rosea is nested within the clade containing isolates of the taxon Acrasis rosea, into which P. rosea is tentatively subsumed. One member of the tightly knit allovahlkampfid clade was induced to form a simple sorocarp, leading us to include this clade in Acrasidae.

A Morphologically Simple Species of Acrasis (Heterolobosea, Excavata), Acrasis helenhemmesae n. sp.

In the course of a large-scale global survey of mycetozoans, amoeboid organisms that form fruiting bodies, a new species of Acrasis was discovered from several subtropical locales in Hawaii, Australia, Bermuda, and South Africa. We isolated four strains from dead, still attached, plant material, and one strain from attached bark of a tree. Each isolate forms simple uniseriate multicellular fruiting bodies typically consisting of two bottle-shaped, basal stalk cells and a chain of <20 spores. The isolate from Bermuda often forms dichotomous simple branches, each consisting of <10 spores. Amoebae from these new isolates are limax with eruptive pseudopodial formation and display rapid locomotion-characters indicative of amoebae in the excavate taxon Heterolobosea. These isolates form simpler fruiting bodies than is typical of the well-known Acrasis rosea. Although in the original description, A. rosea is known to form uniseriate fruiting bodies similar to our isolates, A. rosea isolates typically form more complex fruiting structures along side simple ones, but never strictly simple ones. Nuclear-encoded 18S rRNA gene phylogenies demonstrate that our five isolates form a highly supported clade that is sister to A. rosea. Given the differences both in gene sequences and fruiting body morphology between our isolates and A. rosea, we propose the new species, Acrasis helenhemmesae n. sp.

Ultrastructure and Phylogenetic Placement within Heterolobosea of the Previously Unclassified, Extremely Halophilic Heterotrophic Flagellate Pleurostomum flabellatum (Ruinen 1938)

Although Pleurostomum was described almost a century ago, flagellates assigned to this taxon have been recorded only in very occasional faunistic studies of highly saline habitats, and their phylogenetic position has remained uncertain. We report the cultivation, ultrastructure, and phylogenetic relationships of Pleurostomum flabellatum isolated from a Korean saltern pond of 313 per thousand salinity. This isolate is biflagellated with a cytostomal groove, and is not distinguishable from previous accounts of P. flabellatum from saturated brines in India and Australia. Pleurostomum flabellatum shows ultrastructural features characteristic of many Heterolobosea: (1) a striated rhizoplast, (2) an absence of stacked Golgi bodies, (3) parallel basal bodies and flagella, and (4) a large number of peripheral microtubules supporting a rostrum. 18S rRNA gene phylogenies strongly confirm the affinities of P. flabellatum within Heterolobosea. Furthermore, the 18S rRNA gene of P. flabellatum has the heterolobosean-specific helix 17_1, and a group I intron in the same position as in Acrasis rosea. Within Heterolobosea, the 'amoeboflagellate' genera Naegleria and Willaertia were its closest relatives with high bootstrap support and posterior probability. P. flabellatum was observed only as a flagellate, and never as an amoeba. Since light microscopy and electron microscopy observations indicate that P. flabellatum flagellates are capable both of feeding and division, there might be no amoeba stage. Being morphologically distinct from its closest relatives and phylogenetically distant from other flagellate-only Heterolobosea, P. flabellatum cannot be moved into any previously described heterolobosean genus. Instead, we move Pleurostomum into Heterolobosea, and assign as the type species Pleurostomum salinum Namyslowski 1913, a species that closely resembles P. flabellatum. The optimal temperature for growth of P. flabellatum is 40 degrees C. Interestingly, P. flabellatum grows optimally at 300 per thousand salinity and fails to grow below 200 per thousand salinity, indicating that it is an 'extreme halophile'. The optimal salinity for growth is the highest for any eukaryote examined to date.

The chaperonin genes of jakobid and jakobid-like flagellates: implications for eukaryotic evolution.

The jakobids are free-living mitochondriate protists that share ultrastructural features with certain amitochondriate groups and possess the most bacterial-like mitochondrial genomes described thus far. Jakobids belong to a diverse group of mitochondriate and amitochondriate eukaryotes, the excavate taxa. The relationships among the various excavate taxa and their relationships to other putative deep-branching protist groups are largely unknown. With the hope of clarifying these issues, we have isolated the cytosolic chaperonin CCTalpha gene from the jakobid Reclinomonas americana (strains 50394 and 50283), the jakobid-like malawimonad Malawimonas jakobiformis, two heteroloboseans (Acrasis rosea and Naegleria gruberi), a euglenozoan (Trypanosoma brucei), and a parabasalid (Monocercomonas sp.). We also amplified the CCTdelta gene from M. jakobiformis. The Reclinomonas and Malawimonas sequences presented here are among the first nuclear protein-coding genes to be described from these organisms. Unlike other putative early diverging protist lineages, a high density of spliceosomal introns was found in the jakobid and malawimonad CCTs-similar to that observed in vertebrate protein-coding genes. An analysis of intron positions in CCT genes from protists, plants, animals, and fungi suggests that many of the intron-sparse or intron-lacking protist lineages may not be primitively so but have lost spliceosomal introns during their evolutionary history. In phylogenetic trees constructed from CCTalpha protein sequences, R. americana (but not M. jakobiformis) shows a weak but consistent affinity for the Heterolobosea and Euglenozoa.

Cell shape, motility and distribution of F-actin in amoebae of the mycetozoans Protostelium mycophaga and Acrasis rosea. A comparison with Dictyostelium discoideum

Summary We investigated, by a computer-assisted video analysis, by F-actin cytochemistry, and by scanning electron microscopy (SEM), the dynamic morphology, the locomotory behavior, and the actin cytoskeleton of trophic amoebae of the mycetozoans Protostelium mycophaga and Acrasis rosea, and compared them with amoebae of the cellular slime mold Dictyostelium discoideum. The three species represent protist taxa marked by different reproductive systems and by differences of amoeboid motility in the non-reproductive phase of the life cycle. Live observations and the numerical analysis of video recordings revealed characteristic traits of cell shape changes and motility in each species. The leading edge of Protostelium amoebae is dominated by a large lamellipodium that is studded with pseudodigits. We computed a mean cell size (outline area) of 498 μm2 and a mean velocity of 26.5 μm/min (centroid displacement) for this species (N = 30, as for the other organisms). Most amoebae followed a sinuous path during the observation period, which resulted in a mean standard deviation of vector direction (SDV) of 0.64, but some hardly moved at all. Amoebae of Acrasis are essentially monopodial, with lobose, smooth pseudopodia. Their mean cell size was 759 μm2 and their mean velocity 71.6 um/min. They are thus among the fastest “crawling” eucaryotic cells, which may be related to the fact that they contain no cytoplasmic microtubules. Most amoebae followed a straight path, but some made one or two abrupt turns during the observation period (SDV = 0.58). The corresponding values for Dictyostelium amoebae with filose or lobose pseudopodia were 220 μm2, 10.3 μm/min, and SDV = 0.80. In amoebae of all the three species F-actin was localized in the cell cortex, especially in hyaline pseudopodia. As seen by SEM, Protostelium amoebae had the largest area of contact with the substratum, whereas Acrasis amoebae adhered with a foot-like structure, the leading edge and the uropod being detached from the support.

Alpha-tubulin from early-diverging eukaryotic lineages and the evolution of the tubulin family.

The tubulin gene family, which includes alpha-,beta-, and gamma-tubulin subfamilies, is composed of highly conserved proteins which are the principle structural and functional components of eukaryotic microtubules. We are interested in (1) establishing when in eukaryotic evolution the duplications leading to paralogous alpha, beta, and gamma subfamilies occurred and (2) the possible utility of tubulin sequences in reconstructing organismal phylogeny. To broaden the taxonomic representation of alpha-tubulins so that it roughly equals that of beta-tubulins, alpha-tubulin genes from three Microsporidia (Encephalitozoon hellem, Nosema locustae, and Spraguea lophii), two Parabasalia (Monocercomonas sp. and Trichomitus batrachorum), and one Heterolobosean (Acrasis rosea) were sequenced. With these new genes, phylogenetic trees of alpha- and beta-tubulins were constructed and compared. Trees were congruent with each other, but incongruent with other molecular phylogenies. The agreement between alpha- and beta-tubulin trees could arise by the co-adaptation of one molecule to variants of the other as a result of their intimate steric association in microtubules. Thus, these trees may not be providing independent support for the phylogenetic results. However, one of these unexpected results, that microsporidia cluster with fungi, is supported by other circumstantial evidence, and may therefore reflect a real relationship despite the basal position usually assigned to microsporidia. Relationships between the three tubulins were also examined by constructing trees of all three types. These trees were found to be of limited value for determining the position of the root within each subfamily because of the great interfamily distances, but they do confirm the classification of all known genes into three monophyletic subfamilies. Divergent genes from Caenorhabditis elegans and Saccharomyces cerevisiae that have been proposed to represent the novel classes delta- and epsilon-tubulin were found to be specifically related to gamma-tubulins from animals and fungi respectively, and therefore are best seen as rapidly evolving orthologues of gamma-tubulin.

Evidence for the Heterolobosea from phylogenetic analysis of genes encoding glyceraldehyde-3-phosphate dehydrogenase.

The phylogenetic relationships between major slime mould groups and the identification of their unicellular relatives has been a subject of controversy for many years. Traditionally, it has been assumed that two slime mould groups, the acrasids and the dictyostelids were related by virtue of their cellular slime mould habit; a view still endorsed by at least one current classification scheme. However, a decade ago, on the basis of detailed ultrastructural resemblances it was proposed that acrasids of the family Acrasidae were not relatives of other slime moulds but instead related to a group of mostly free-living unicellular amoebae, the Schizopyrenida. The class Heterolobosea was created to contain these organisms and has since figured in many discussions of protist evolution. We sought to test the validity of Heterolobosea by characterizing homologs of the highly conserved glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from an acrasid, Acrasis rosea; a dictyostelid, Dictyostelium discoideum; and the schizopyrenid Naegleria andersoni. Phylogenetic analysis of these and other GAPDH sequences, using maximum parsimony, neighbour-joining distance and maximum likelihood methods strongly supports the Heterolobosea hypothesis and discredits the concept of a cellular slime mould grouping. Moreover, all of our analyses place Dictyostelium discoideum as a relatively recently originating lineage, most closely related to the Metazoa, similar to other recently published phylogenies of protein-coding genes. However, GAPDH phylogenies do not show robust branching orders for most of the relationships between major groups. We propose that several of the incongruencies observed between GAPDH and other molecular phylogenies are artifacts resulting from substitutional saturation of this enzyme.

A novel type of unorthodox mitosis in amoebae of the cellular slime mold (Mycetozoan) Acrasis rosea

Summary We investigated mitosis in trophic amoebae of Acrasis rosea (Olive and Stoianovitch) by video microscopy of live cells, by immunofluorescence with an antibody against tubulin, and by transmission electron microscopy. As interphase cells contain neither microtubules (MTs) nor microtubule-organizing centers (MTOCs) the mitotic spindle forms entirely de novo from presumptive diffuse intranuclear MTOCs. Mitosis is closed and devoid of any visible pole organelles during all its stages. The spindle axis is probably established by parallel alignment of MTs due to spatial constraints or lateral interaction. Chromosomes condense only when spindle formation is well advanced; their kinetochores presumably acquire MTs (kMTs) by capture. The two main elements of the nucleolus, viz. granular body and dense granules, disperse, but do not disintegrate. They later arrange in the center of the spindle along its axis and eventually segregate as two granular bodies and two groups of granules. Reconstruction of the nucleolus during telophase involves the compaction of the granular bodies and coaggregation of the dense granules. At metaphase the chromosomes are aligned at the equator of a spindle that tapers towards the blunt spindle poles. Chromosomes are small, but they have distinct layered kinetochores with two MTs each that terminate in their outer layer. Cytokinesis is accomplished ca. eight minutes after the first signs of prophase. Chromosome segregation during anaphase is effectuated almost solely by spindle elongation, which begins when chromosomes are still undivided and aligned at the equator. The velocity of spindle elongation and chromosome segregation was 6 μm/min. Chromosome congression and segregation are most likely driven by interactions, static or dynamic, between kMTs and non-kinetochore MTs (nkMTs), whereas the mechanism of spindle elongation is probably based on movements between staggeredly overlapping nkMTs. At the end of telophase the closure of the nuclear envelope around the daughter nuclei pinches off a membrane tube containing remnant nkMTs. Mitosis in A. rosea differs markedly from that in other cellular slime molds and has much in common with that in several protozoa.

COPROMYXELLA: A NEW GENUS OF ACRASIDAE

A new genus of cellular slime molds, Copromyxella, is described together with four species that comprise it. Since it resembles Zopf's Copromyxa more than any genus presently recognized, but differs from it in important characteristics such as the smaller dimensions of its cellular elements and the delicacy of its fructifications, the name Copromyxella is chosen for the new taxon. Four species are included: C. silvatica, C. filamentosa, C. spicata, and C. coralloides. All are characterized by small myxamoebae with lobose and potentially “explosive” pseudopodia and may assume limax forms in liquid media; all possess nuclei with centrally positioned nucleoli; all lack true contractile vacuoles (with a single possible exception); all aggregate without stream formation; and all form fructifications with no demarcation into stalks and sori. Taxonomically the genus belongs in the Acrasidae with Copromyxa, Guttulinopsis and Acrasis rosea rather than in the better known Dictyostelidae that includes Dictyostelium and Polysphondylium.

Copromyxella: A New Genus of Acrasidae

A new genus of cellular slime molds, Copromyxella, is described together with four species that comprise it. Since it resembles Zopf's Copromyxa more than any genus presently recognized, but differs from it in important characteristics such as the smaller dimensions of its cellular elements and the delicacy of its fructifications, the name Copromyxella is chosen for the new taxon. Four species are included: C. silvatica, C. filamentosa, C. spicata, and C. coralloides. All are characterized by small myxamoebae with lobose and potentially “explosive” pseudopodia and may assume limax forms in liquid media; all possess nuclei with centrally positioned nucleoli; all lack true contractile vacuoles (with a single possible exception); all aggregate without stream formation; and all form fructifications with no demarcation into stalks and sori. Taxonomically the genus belongs in the Acrasidae with Copromyxa, Guttulinopsis and Acrasis rosea rather than in the better known Dictyostelidae that includes Dictyostelium and Polysphondylium.

Acrasis rosea and the Possible Relationship between Acrasida and Schizopyrenida

Summary The Acrasida, a taxonomically isolated group of mycetozoans, and the Schizopyrenida (Vahlkampfiidae), an order of amoebae, show certain characters suggestive of a relationship. Acrasis rosea , the best-known acrasid, has a locomotive pattern identical with that of the vahlkampfiids but a mitotic pattern different from that of vahlkampfiids. Evidence reviewed from the literature also fails to confirm a relationship between Acrasis and vahlkampfiids. However, the hypothesis merits further investigation, and lines of inquiry are suggested.

Natural Variants of the Cellular Slime Mold Acrasis rosea

SYNOPSIS. Twenty different isolates of the cellular slime mold Acrasis rosea, obtained from diverse sources and geographic regions, were studied to determine similarities and differences in their development and structure in culture and their sensitivity or resistance to selected chemicals incorporated into the culture media. Six different classes of fruiting were defined based on the size, distribution, and type of sorocarps formed on the yeast, Rhodotorula, streaked on agar. In the course of these studies a significant mutant, NC‐18V (variant), developed spontaneously from the wild type, normal parent strain NC‐18N. The mutant differed considerably from all other Acrasis isolates, appeared several times in purified parental cultures, and represents the first laboratory derived variant of A. rosea to be described. Purified strains of the variant (V) and normal (N) cultures were obtained by single‐spore isolation. Normal and variant amebae were mixed in ratios of 10:1 and 100:1 (N:V) and spore and stalk cells were selected from different sorocarps in various regions of the culture plate for analysis. The results of these selection experiments clearly indicate that the individual variant amebae have increased migratory ability and that they develop smaller, morphologically different, and more numerous sorocarps that form at distances further from the food source than NC‐18N. Some isolates of Acrasis no longer were able to fruit and were classified as “non‐fruiters” and a few other isolates formed only a few, small sorocarps on rare occasions. These isolates were mixed together in various combinations of 2 and 3 to screen for cell interaction, but no synergism contributing to fruiting was found. Although fruiting of many A. rosea isolates was inhibited by exposure to continuous light or constant darkness, some “escape”fruiting was noted in certain isolates even when small inocula were used. Single spore isolates of these escape fruiters still fruited in continuous light or dark, but fruiting was always greatly enhanced by a routine 12 hr light : 12 hr dark incubation cycle. It was shown by biochemical studies that actidione, crystal violet, malachite green, ethyl violet, and 5‐fluorodeoxyuridine selectively killed some isolates and permitted a classification of isolates as either sensitive or resistant. In a further study of cell interaction between 2 different sets of Acrasis isolates with contrasting biochemical and morphologic markers the formation of neotypes or recombinants could not be demonstrated.The results of this study clearly indicate the existence of significant variation in A. rosea and the potential for application of these differences to developmental studies.

THE ACTION SPECTRUM OF LIGHT INDUCED AGGREGATION INPOLYSPHONDYLIUM PALLIDUM, AND A PROPOSED GENERAL MECHANISM FOR LIGHT RESPONSE IN THE CELLULAR SLIME MOLDS

In the cellular slime mold, Polysphondylium pallidum, aggregations are induced by a brief period of light coming after a long (5 hour) dark period. The action spectrum of this response was determined by illuminating small populations of amebae with a constant sub-saturation amount of light at each of several different wavelengths and measuring the number of aggregates induced in each case. Peaks in response occurred at 475 and 675 mµ. One of these peaks falls on the absorption maximum of hexane extracted carotenoids from another cellular slime mold, Acrasis rosea, suggesting that the receptor pigment may be a carotenoid.The light responses of the cellular slime molds are briefly reviewed. It is suggested that the events during light stimulation of aggregation and during other light induced responses may be similar to those which occur during light stimulation of the metazoan retina. In that case, the initial cellular even common to all light responses of the cellular slime molds may be the stimulation of cyclic-AMP production.

Developmental responses of Acrasis rosea to the visible light spectrum

Low energies of light in the blue region were highly stimulatory for inducing fruiting of the NC-18 isolate of the cellular slime mold Acrasis rosea. A comparison of energy input versus fruiting response at 420, 450, and 475 mμ indicated that 450 mμ (± 7 mμ) was the most efficient tested wavelength for induction of fruiting. Different wavelengths between 537 mμ and 790 mμ, at 2–3 times the energy input of the blue region, were weakly stimulatory or nonstimulatory for fruiting. At higher energies, broad-spectrum red light, but not far red, induced good fruiting responses. Cultures first exposed to white light and given a subsequent dark period of 6 hours were exposed to different wavelengths of light for 4 and 10 minutes and returned to darkness for 8 hours. Blue light irradiations were the only low energy light conditions which caused discernible inhibitions of fruiting. High energies of broad-spectrum red light, but not far red, also delayed fruiting. Fruiting of NC-18 amoebae, previously stimulated by white light, occurred in continuous far red. There was no evidence of any red- far red interaction similar to that reported in higher plants.

The Effects of Light on the Development of the Cellular Slime Mold Acrasis rosea

The Effects of Light on the Development of the Cellular Slime Mold Acrasis rosea

Silica gel as a preserving agent for the cellular slime mold Acrasis rosea.

SYNOPSIS. Modifications of the silica gel technic of Perkins and Ogata have been successfully used to preserve Acrasis rosea for 6–13 months and Dictyostelium purpureum for 31 months. The technic is simple, reliable and inexpensive and is recommended for its possible application for the general preservation of certain fungi and protozoa.

Evidence for Mixed Cytoplasm in Heterocaryons of Colletotrichum lagenarium

Spores of race 2 but not of race 1 of Colletotrichum lagenarium are ingested by myxamoebas of strain NE-30 of the slime mold Acrasis rosea. Sporulating colonies of three heterocaryons of C. lagenarium involving auxotrophic strains with different color markers of races 1 and 2 were inoculated with myxamoebas. Although spores of race 1 produced on homocaryons were not ingested by the myxamoebas, they were ingested when produced on heterocaryons involving race 2. Spores from sectors of the heterocaryons yielded colonies of race 1; spores from these colonies were not ingested by the myxamoebas.

Variation in the Cellular Slime Mold Acrasis rosea*

A number of isolates of the cellular slime mold, Acrasis rosea, differed from each other in the range of microorganisms on which they could grow and fruit. In a test for the possible occurrence of sexual reproduction and genetic recombination, two of the most widely divergent of these physiological races were studied further. Mixed colonies were obtained in agar plate cultures of Rhodotorula mucilaginosa, a yeast utilized by both. When spores from mature fruiting bodies or amoebae migrating out from the mixed colonies were isolated singly, regrown on Rhodotorula, and tested on a critical spectrum of food microorganisms, about 3‐4% proved to be distinct from the original two strains. Although some of these new types remained stable for several transfers, they eventually reverted to one or both of the parental types. The possibilities of explaining the neotypes on the basis of diploidy, heterokaryosis, or mixed cytoplasmic effects are discussed. The occurrence of cell anastomoses and occasional binucleate cells favors the latter two possibilities.