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J. Biosci., Vol. 20, Number 1, January 1995, pp 1–6. © Printed in India.
Why does slug length correlate with speed during
migration in Dictyostelium discoideum?
J Τ BONNER
Department of Ecology and Evolutionary Biology. Princeton University, Princeton. New
Jersey 08544, USA
MS received 15 July 1994; revised 30 September 1994
Abstract. Taking advantage of the fact that static electricity in plastic Petri dishes will
produce very long, thin migrating slugs of Dictyostelium discoideum, it was shown that
these slugs moved particularly rapidly. This is consistent with the demonstration of Inouye
and Takeuchi that speed varies with length for slugs migrating on agar. Based on these
observations it is suggested that slug speed is controlled by both the resistance at the tip
and some factor that correlates With slug size, such as the concentration of endogenously
produced ammonia.
Keywords. Slime mould; Dictyostelium; migration speed.
1. Introduction
It was first observed by Francis (1959) and later put on a firm basis by Inouye
and Takeuchi (1979) that the speed of a migrating slug of Dictyostelium discoideum
correlates significantly with its length, and not its volume or surface area. Ever
since the advent of plastic Petri dishes many workers have observed that occasionally
slugs become very thin and long, and move straight up to reach the underside of
the lid. As will be shown here, this phenomenon is caused by static electricity,
and these especially long and thin slugs follow the length ∝-speed rule, and from
this fact it is possible to draw some inferences as to how migration speed is
governed in cellular slime moulds.
2. Methods
The amoebae of Dictyostelium discoideum NC-4 were grown in plastic Petri dishes
(100 × 15 mm) on 2%, non-nutrient agar at room temperature. Loopfuls of Escherichia
coli B/r were placed at three points on the agar surface roughly 1 cm from the
edge and each was inoculated with D. discoideum spores. The meniscus of the
agar on the side of the dish was cut out near each of the points of inoculation
to make viewing from the side possible. When migrating slugs appeared the lids
of the Petri dishes were rubbed with either paper tissues, or a piece of synthetic
velvet. That this does generate a considerable amount of static electricity is made
evident by the fact that if there are fruiting bodies on the plate, the spore masses
will fly lose from their stalks and splash on the underside of the Petri dish lid.
The charged spores are nolt held in by a sheath, as has been clearly demonstrated
recently by Sameshima (1993), and are pulled loose by the electrostatic forces.
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To obtain a side view of the rising slugs they were looked at from above,
through a prism complex put at the edge of the Petri dish nearest the slug. Both
the dish and the prism rested on a sheet of plastic to avoid grounding by the
metal of the microscope. This arrangement was placed under a microscope with a
50 mm lens and recorded with a time lapse video recorder (Panasonic video camera,
WW-1850; recorder, AG-6720A). The speeds of the migrating slugs were measured
over a period of 10 to 30 min; ten slugs were measured in all.
To examine the shape of the cells within slugs, they were knocked over, put
onto a microscope slide, and immediately submerged in a few drops of macerating
fluid ( 1: 1: 13 glycerol ; glacial acetic acid : water). This treatment partially separates
the cells and each retains its original shape.
3. Results
3.1 Description of slugs
If one takes two Petri dishes containing migrating slugs and rubs the upper surface
of the lid of one and not the other, the one in which static electricity has been
generated will, after an interval of time, have more slugs pointing straight upwards
towards the lid. Not all the slugs will show this response, but only those young
ones that happen to have their tips raised.
An attempt was made to see if one could distinguish prestalk and prespore zones
in these slugs by staining them with the vital dye neutral red, but they appeared
uniformly stained, which is characteristic of younger slugs (Bonner et al 1990).
It proved to be difficult to obtain good side views of these rising slugs for they
are very delicate and are affected by the slightest change in their environment,
including anything that might change the static electricity, distribution on the Petri
dish. Even the atmospheric conditions affected the experiments; cool, and especially
dry days, were clearly desirable.
The slugs attracted to the Petri dish lid by static electricity are not only thin
and long, but their posterior end rises on their condensed slime sheath so that they
are held up by the finest stiff, straight pedestal—as thin as a small piece of spider's
silk (figure 1a). To illustrate the fact that this bit of raised slime sheath is strong,
not infrequently the slug will not reach the lid, but change to the fruiting, or
culmination mode, in mid air. The slug's tip will reverse its forward motion to
form the beginning of a small fruiting body perched on the top of the slime sheath
thread, and then will proceed to culminate, the basal disc of the stalk beginning
in mid air (figure 1b). The slugs used for the culmination data are not included
in the migration data because it was not possible to measure both for one slug—the
technical problems were too great.
When the long thin slugs were placed in a macerating solution it could be seen
that the orientation of their cells was the same as one finds in normal slugs (Smith
1983; Feit 1994). The cells in and near the tip are isodiametric, while the cells
in the bulk of the slug are more or less elliptical in shape and oriented parallel
to the main axis of the slug.
3.2 Speed ∝—length
As a background to this study, Inouye and Takeuchi (1979) have measured the
Why does slug length correlate with Dlctyostelium discoideum
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Figure 1. (a) A photograph take from the video screen of a slug rising to the lid charged
with static electricity. (The slug is approximately 2 mm long). (b) A similar slug that has
changed into the fruiting mode in mid air and has formed a fruiting body standing on a
thread of congealed slime sheath. (The arrow indicates the junction between the basal disc
and the rigid slime sheath column. Note that the stalk is unusually thick, an anomaly that
occurs occasionally under normal conditions. Both photographs are at the same magnification).
length and the speed of a number of slugs, and I have plotted their data as solid
dots on a log-log scale (figure 2). The data obtained here for slugs attracted by
static electricity have been superimposed on the graph as open circles.
For comparison I have measured the length of a number of slugs, and then later
measured the rate at which they moved upward as they fruited. Clearly these
culmination speeds also vary with the length of the slugs, and although culmination
is a much slower process than migration, the slope of its length-speed relation is
parallel to that of migration (figure 2).
4. Discussion
There are some differences between slugs which lie flat on the agar and those rise
from the agar due to static electricity, but the interesting thing is that despite these
differences the speed-length relationship holds. These observations led to the obvious
hypothesis that speed is severely affected by the resistance at the tip of the migrating
slug. The static electricity reduces that resistance by attracting the slug tip, with
the result that the slugs become longer and thinner, and move more rapidly (no
slug on plastic or glass will be as long as one under the influence of static
electricity). It has been known for many years (Bonner 1994), and observed by
numerous workers, that when the tip of the slug rises into the air it becomes
relatively elongate and moves faster, and when it drops back onto the agar, it
slows and becomes broader. Francis (1959) showed that at low temperatures slugs
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J Τ Bonner
Figure 2. A log-log plot showing the relation of speed to slug length. The solid dots are
the data of Inouye and Takeuchi (1979) for slugs migrating on agar. The open circles are
the long, thin slugs under the influence of static electricity. The open squares are for the
speeds of culmination whose prior slug lengths had been measured while they were migrating
on agar.
not only moved more slowly, but became short and thick. All of these facts lead
to the conclusion that one of the reasons slug length correlates with its speed is
that a reduction of resistance at the tip produces both a thinning of the slug and
more rapid speed.
One question that must be asked here is why is culmination in D. discoideum
a slower process than migration. Before making any hypothetical suggestion as to
why this might be, let me remind the reader that while this is true for this species
(Bonner and Eldredge 1945, and this study), Cox et al (1988) showed that the
rate of culmination is the same regardless of size in Polysphondylium pallidum.
Polysphondylium differs from Dictyostelium in that it does not have prespore and
prestalk zones—all the cells in the 'slug' appear to be similar and as the cell mass
Why does slug length correlate with DIctyostelium discoideum
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rises, the stalk is continuously produced at the tip, and at the posterior of the mass
groups of cells are pinched off to form a whorl at quite regular intervals. On the
other hand in Dictyostelium, as it rises the posterior prespore cells turn into mature
spores fairly early in culmination. For this reason it is generally thought that the
motive force for the culmination movement comes entirely from the anterior prestalk
cells. Therefore, one of the reasons culmination might be slower than migration is
that during culmination the relative length of the prestalk zone is much shorter
than that of a migrating slug. This cannot be the only reason, for if one takes
into account the culminating prestalk zone length, it still moves more slowly than
the estimated speed of migrating slugs of similar length. Perhaps the speed is also
reduced during culmination because the process of forming the stalk at the tip in
itself provides extra resistance to the upward movement. (These explanations still
leave the problem of Polysphondylium where size does not seem to affect speed,
a matter that is clearly in need of further investigation).
The isodiametric shape of the cells in the tip region compared to the cells in
the rest of the slug, even in long thin slugs, could reflect this tip resistance.
Furthermore, it is well known that during culmination, where, as we have seen,
there is an even greater reduction in speed, the prestalk cells are flattened and
have their long axes perpendicular to the direction of movement.
The other known factors that affect speed are size, age, and the concentration
of ammonia (review: Bonner 1994). The reasons for the decline with age, which
was established by Inouye and Takeuchi (1979), could be a simple depletion of
energy substrates within the cells. In the case of NH 3 it has long been known that
it is given off by the slugs, and that the speed of slug movement is accelerated
by an appropriate increase in NH3 concentration.
By far the most significant factor is size. Previously we made the suggestion
that in larger slugs the concentration of internally generated NH3 would be higher
than in small slugs, because of the increase volume to surface ratio, which would
mean that the NH 3 diffuses away less effectively in large slugs (Bonner et al
1989). However this argument would not apply for long, thin slugs, where the
escape of NH3 would be facilitated. A good hypothesis to account for this apparent
contradiction would be that the speed of slugs is limited by both the internal
concentration of NH 3 dictated by the surface/volume ratio, and by the resistance
at the tip.
Acknowledgements
I would like to thank Jordan Poler for his helpful advice on how to deal with
static electricity, and for the time he took to devise and construct a special Petri
dish with which he could induce static electricity with high voltages. I also thank
the following individuals for helpful comments and criticisms of earlier versions
of the paper: Ε C Cox, Κ Inouye, V Nanjundiah, and Ε Palsson.
References
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J T Bonner
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