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Department of Ecology and Evolutionary Biology, Princeton University, Princeton, New Jersey 08544
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMIGRATING SLUGSCONCLUSIONLITERATURE CITED |
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By comparing two species of cellular slime molds that have stalkless migration stages it is possible to gain interesting insights into how the cells move. In contrast to the familiar behavior of Dictyostelium discoideum, Dictyostelium polycephalum slugs can travel greater distances through soil and even can migrate through agar. In addition to the interest in the differences, these differences shed light on the mechanism of slug movement. Unlike D. discoideum, D. polycephalum does not have prestalk and prespore zones and severed sections of any part of these slugs move at a rate proportional to their length. This leads to the hypothesis that longer slugs move faster because the amoebae aligned along the inside of the slime sheath each contribute a forward push and the more extended the amoebae line is the faster the slug moves.
Key words: cellular slime mold, slug movement, soil ecology
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMIGRATING SLUGSCONCLUSIONLITERATURE CITED |
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Dictyostelium polycephalum is an unusual species and except for its original description by Raper (1956)
, which was extensive and detailed, almost no work has been done on its development and behavior. My current study builds on the work of Raper and includes comparisons with D. discoideum, a much studied species for which we have accumulated a wealth of physiological knowledge. The reason that the comparison is so interesting is that both species have stalkless migrations, leading cells from the site of aggregation to the site of fruiting. Yet as we shall see many significant differences in their migrations are not only of interest in themselves but shed light on the mechanism of slug movement.
The aggregation of D. polycephalum is a much less well organized than that of D. discoideum or any other of the larger Dictyostelium or Polysphondylium species. The formation of well defined streams is not always evident and the aggregation centers are broad mounds that develop papillae only secondarily; one mound may have 1–5 or more mounds and each becomes a migrating slug. As we shall see in detail those slugs differ from those of D. discoideum by being much thinner and longer, almost like strands of spaghetti. In the laboratory it is difficult to induce fruiting; without the right conditions they will migrate indefinitely, but with the help of activated charcoal or a pile of soil they can be persuaded to fruit. After migration the slug again forms a mound and 2–5 or more papillae appear on its surface. They rise to fruit in essentially the same manner as a fruiting body of D. discoideum with the difference being that the stalks of these individuals coalesce in a bundle part way up as they rise and they then separate like flowers sticking out of the top of a slender vase, to form a whorl of spore groups or sori. These are the basic facts of their life history, established by Raper (1956) (FIG. 1).
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MIGRATING SLUGS |
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TOPABSTRACTINTRODUCTIONMIGRATING SLUGSCONCLUSIONLITERATURE CITED |
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No zones in slugs.— –
No equivalent was found in D. polycephalum to the prestalk and prespore zones in D. discoideum when stained with the vital dye neutral red. D. polycephalum slugs are uniformly red along their axes. They resemble D. discoideum slugs that form right after aggregation and before they become two-toned (Bonner and Slifkin 1949, Bonner et al 1990).
These cells were seen clearly when the slug was immersed in mineral oil and examined under a microscope (40x). By following the slug with time-lapse video, the movement of the amoebae was observed in detail (FIG. 2). What one sees is that all the amoebae are churning about as they move as a group down the tube formed by the slime sheath. Furthermore one clearly can see no difference in the intensity of amoeba movement near the tip or farther back. This phenomenon is a contrast to what was found in D. discoideum where the anterior prestalk amoebae moved significantly faster than the posterior prespore amoebae (Francis 1959, 1962; Siegert and Weijer 1992; Bonner 1998).
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discovered. I conducted preliminary tests to determine the existence of a temperature-gradient orientation and found none. In these respects D. polycephalum differs from D. discoideum, which has strong photo- and thermotaxis. However, like D. discoideum and all other species that have been tested, D. polycephalum showed clear and sensitive gas orientation, presumably to minute gradients of ammonia and oxygen. Bonner and Lamont (2005) provided evidence that gas orientation is the major factor in guiding slugs in the soil. Migration through soil.— – D. polycephalum slugs are champions when it comes to migration. They can move greater distances through the soil than other species and they can migrate even through agar.
In a previous study (Bonner and Lamont 2005) D. discoideum and Polysphondylium pallidum were added to mounds of E. coli on nonnutrient agar and covered with forest soil of different depths and discovered that both could reach the surface only when the soil was 1 cm or less deep. These experiments were repeated with D. polycephalum slugs; they can migrate through as many as 7 cm of soil—the limit tested in the current study—and fruit on the surface. This was done in glass crystallizing dishes (5 x 9 cm) for the shorter distances and then in test tubes (2 x 15 cm) for the larger ones. In both cases a layer on nonnutrient agar (ca. 4 mm thick) was placed at the bottom and inoculated with 1–3 mounds of E. coli and D. polycephalum amoebae. These were covered with unsterilized forest soil and incubated at room temperature (23 C).
Migration under agar.— –
Even more remarkable was the spontaneous appearance of slugs penetrating the agar. When Petri dishes containing 1.5% agar and 0.1% peptone and lactose were inoculated with E. coli and D. polycephalum they produced abundant amoebae and migrating slugs. In some of the areas with dense growth small clusters of amoebae often appeared just below the agar surface and slugs occasionally formed from them and migrated into the agar. It was possible to follow their formation and their progress. Amoebae were recruited constantly at the posterior end of slugs as they became bigger and moved forward. They also occasionally appeared on 1.5% nonnutrient agar plates when the bacterium inocula and the amoebae were plunged deep into the agar (FIG. 3
). They moved far more slowly (at ca. < 1/10 the speed) than slugs on the agar surface. This ability might let D. polycephalum penetrate dense soil more effectively; it clearly is capable of migrating great distances below ground.
Beaded slugs.— –
I made another interesting observation, this time on the soil surface. Some slugs had a conspicuous beaded appearance (FIG. 4). When followed with the time-lapse camera it was evident that these beads did not move. Instead the amoebae within worked their way forward and at the tip they inflated a new tip as though they were blowing a bubble. This phenomenon indicated that the sheath is a firm membrane that can retain shape as the amoebae pass through. It took roughly 15 min to produce a bead. A less pronounced form of his phenomenon was reported by Francis (1962) for D. discoideum, but in neither case was there any understanding of the cause or the mechanism of the phenomenon.
Speed-length relationships.— –
It is well known through the work of a number of authors that in the case of D. discoideum slugs the longer the slugs are the faster they move and speed correlates with length far more strongly than with volume. (review: Bonner 1994). This turned out to be true for D. polycephalum as well (FIG. 5).
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. On the other hand when a D. polycephalum slug was cut each severed portion moved at speeds commensurate with their lengths. This phenomenon was measured by the movement of the posterior end of the slug, marking the distance and time on time-lapse video. I have two reasons for this kind of measurement: (i) the posterior end always was fixed to the agar surface, while the anterior end often wandered into the air and therefore was difficult to measure; (ii) in the case of posterior segments anterior ends rounded up and took time to recuperate from the trauma, while their posterior ends kept moving, immediately slowing to a speed commensurate to their initial lengths. Both slug fragments and whole slugs fell in line and the slope was almost parallel to the slope determined by Inouye and Takeuchi (1979) for D. discoideum (FIG. 5). It was not clear why the points for D. polycephalum were more scattered although there are a number of possible explanations; nevertheless the regression was highly significant. Because of the scatter it was impossible to say whether length, surface area or volume correlated best with its speed.
The polarity of amoebae movement within a slug is explained by the chemoattractant being pulsed (or in a gradient) from its high point at the tip, which orients the amoebae down the axis of the slug. It was pointed out by Odell and Bonner (1986) that each amoeba pushes against its neighbors but this behavior alone would not produce forward movement; movement can be achieved only by the peripheral amoebae pushing against the rigid inner wall of the slime sheath. However, as Kei Inouye had pointed out (pers comm), this required rigidity could come from the amoebae.
Results presented here on D. polycephalum shed some light on the question of why longer slugs are faster. This species does not have the anterior prestalk zone of D. discoideum which simplifies the problem: all parts of a D. polycephalum slug are similar and are doing the same thing. Therefore one could argue that, because each amoeba pushes, the longer these amoebae columns are the greater the combined push and the faster the overall speed. So when a slug is severed into two, those segments immediately move slower at speeds corresponding to their lengths.
Here are facts relevant to this hypothesis. In an unpublished study by Macko (1971) the speed of movement of haploid and diploid strains of D. discoideum for both separate amoebae and slugs was measured. The cell size of the 2n strain was roughly twice that of the 1n strain, and Macko found that the speed of movement of isolated larger amoebae was considerably faster than that of the smaller ones. (In a cAMP gradient, haploid amoebae average 0.28 mm/h ± 0.09 SD; diploid 0.43 mm/h ± 0.12 SD, n = 12). Yet when the speed of slugs of the same size of the two strains was compared they moved at the same rate. The 2n slugs presumably have approximately half the number of cells than 1n slugs, but their amoebae move much faster than the amoebae in a 1n slug. This helps explain why haploid and diploid slugs of the same size move at the same speed. The implication is that the critical element is the total mass of cytoplasm regardless of the size of the units in which it is packaged. The total amount of contractile protein correlates with speed; the longer the column of that contracting motor is the faster it moves. This lesson from D. discoideum presumably applies directly to what is found in D. polycephalum where the speed was proportional to the length of the column of amoebae.
One other set of experiments is of interest. When a D. polycephalum slug is measured and then punctured with a series of stabs (with an eyelash held in a glass needle) its speed immediately after wounding remained roughly the same as it was before. It could be that enough of the sheath and the internal rigidity remain so that traction is retained. However when the slug was savaged down its length, it took more than 1 h before it resumed its former speed, at which time it formed a new, slender anterior end that emerged from the rubble.
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CONCLUSION |
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TOPABSTRACTINTRODUCTIONMIGRATING SLUGSCONCLUSIONLITERATURE CITED |
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, Inouye and Takeuchi 1980) suggests that this is essentially true for D. discoideum too because isolated posterior portions, under the right circumstances, are known to continue their forward motion without the help of the more active zone at the tip. However, were it not for the lesson I learned from D. polycephalum, I still might be under the impression that an active anterior end is the key factor in making slug movement possible. As I pointed out at the beginning, there is no gainsaying the power of model organisms (e.g. D. discoideum) to teach us about biology and to help us understand some fundamental living processes. However by using a comparative approach it is clear that relatives of the models can tell us things as well.
Two other matters concerning D. polycephalum should be mentioned. First S.L. Baldauf, P. Schaap and colleagues (pers comm) recently constructed a molecular phylogeny of various Dictyostelids and they found that D. polycephalum is ancestral to and far removed from the Dictyostelium species. So perhaps it is not surprising that it behaves differently. Second is that fact that many Dictyostelids species are different morphologically, yet can co-exist in a patch of soil; this phenomena supports the argument that those differences may not be adaptive but phenotypically neutral (Bonner and Lamont 2005). D. polycephalum might be one of the exceptions and is specially adapted for conditions in the soil that require extended migration over considerable distances and possibly under difficult conditions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 E-mail: jtbonner{at}princeton.edu
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMIGRATING SLUGSCONCLUSIONLITERATURE CITED |
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———. 1994. The migration stage of Dictyostelium: behavior without muscles or nerves. (minireview). FEMS Microbiol Lett 120:1–8.[CrossRef]
———. 1998. A way of following individual cells in the migrating slugs of Dictyostelium discoideum. Proc Nat. Acad Sci USA 95:9355–9359.[CrossRef]
———, Feit IN, Selasse AK, Suthers HB. 1990. Timing of the formation of the prestalk and prespore zones in Dictyostelium discoideum. Dev Genet 11:439–441.
Francis DW. 1959. Pseudoplasmodial movement in Dictyostelium discoideum [Master’s thesis]. University of Wisconsin. Madison, Wisconsin.
———. 1962. The movement of pseudoplasmodia of Dictyostelium discoideum [Doctoral thesis]. Madison Wisconsin: University of Wisconsin.
Inouye K, Takeuchi I. 1979. Analytical studies on migrating, movement of the pseudoplasmodium of Dictyostelium discoideum. Protoplasma 99:289–304.[CrossRef]
———. 1980. Motive force of the migrating pseudoplasmodium of the cellular slime mould Dictyostelium discoideum. J Cell Sci 41:53–64.
Odell GM, Bonner JT. 1986. How the Dictyostelium discoideum grex crawls. Phil Trans R Soc B 312:487–525.[CrossRef]
Raper KB. 1956. Dictyostelium polycephalum n.sp.: a new cellular slime mould with coremiform fructifications. J Gen Microbiol 14:716–732.[Medline]
Siegert F, Weijer CJ. 1992. Three-dimensional scroll waves organize Dictyostelium slugs. Proc Natl Acad Sci USA 89:6433–6437.
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