Glucose concentration can influence the pattern of cytoplasmic streaming in the true slime mold Physarum polycephalum.

Windel, N. and Alexander, R. Department of Biology, University of the Ozarks, Clarksville, Arkansas 72830.

Abstract:

Organisms have ways of sensing and responding to chemical signals, and knowing how an organism senses and responds to its chemical environment is important to biological science because it provides us with an understanding of the organism as well as information about the evolution of chemical recognition. To test the effects of glucose concentration on the growth of Physarum polycephalum, the slime mold was cultured in three separate petri dishes each with 0.06 M and 0.60 M glucose saturated paper discs, and plasmodia growth was observed and recorded for four days. Over the observation period, the organism demonstrated positive chemotaxis to the 0.06 M glucose concentration and negative chemotaxis to the 0.60 M concentration. The growth of the organism in each dish had reached the 0.06 M glucose concentration by the end of the experimental period.

****Note from Dr. Coleman conclusion might be better as last sentence.****

Introduction:

An ability to sense and respond to the environment is an essential mechanism for all life on earth. Many organisms have developed means to transport themselves from one location to another to exploit new resources or to avoid an unfavorable environment. In addition, sometimes making a choice between two environments may lead to more success for the organism: a hungry nocturnal animal may come out to eat during the day if it is starving and experience greater chances of survival as a result (Alcock 1998). Likewise, on a cellular level it is important for organisms to distinguish small differences or changes in the microscopic environment. Organisms have evolved many ways to sense these change, including the ability to sense and respond to chemical differences or changes. Known as chemotaxis, the ability of an organism to move in response to a chemical stimulus can be important for the success and survival of an organism; this movement can be positive, movement favoring an environment, or negative, movement avoiding an environment (Bozzone 2001;Widman 1996). Many organisms use chemical messages to find food, avoid inhospitable environments, and reproduce. Chemical pathways transfer these chemical messages from the outside of the cell to the inside and then into a cellular response (Campbell 2002).

Differences in chemical concentration gradients can significantly influence the behavior of organisms because some cellular regulatory mechanisms depend on specific ranges of osmotic pressure. Osmotic effects of chemical concentration gradients at the cellular level can mean life or death for cells. Hypotonic and hypertonic environments can significantly influence cellular function and sometimes even cause cells to die (Campbell 2002). Because of this, it is reasonable that a cell be able to differentiate between chemical concentration gradients and effectively respond to changes in them. One such organism is Physarum polycephalum, an extensively used scientific model species and the subject of this investigation.

P. polycephalum is a true slime mold that is responsive to both light and chemical stimulus. Because it is only one cell, the organism lends itself to studies of cellular function with macroscopic results; in its plasmodial stage it can be observed forming a network of veins, or streams, that branch out to exploit new resources (Wong 2003). In previous experiments, the organism has been observed moving away from light and towards various food resources, including glucose. This behavior leads to many interesting questions about how P. polycephalum interacts with its environment, especially concerning the organism’s preferred, or optimal, environmental conditions. Previous experiments with P. polycephalum have indicated that it responds not only to different substances, but also to varying concentrations of those substances (Bozzone 2001). The number of dissolved particles in solution influences P. polycephalum, like any other cell, these osmotic effects are essential for cell function but can be dangerous.

The purpose of this experiment was to determine the effect of 0.06 M and 0.60 M glucose concentrations on the directional growth of P. polycephalum. Our hypothesis was that P. polycephalum can detect differences in glucose concentrations and will travel toward a higher concentration of glucose.

The direction and distance of growth to 0.06 M and 0.60 M glucose concentrations of P. polycephalum was measured over four days to determine the effect of glucose concentration on cellular response. The organism exhibited a positive chemotactic response to the 0.06 M glucose solution and a negative chemotactic response to the 0.60 M glucose solution.

Materials and Methods:

Three 100x15mm petri dishes were prepared with 2% agar. Each dish was prepared with a spot of the organism placed in the center of the dish and two glucose saturated paper discs positioned to each side of the organism (Figure 1). One sterile paper disc was saturated with 0.06 M glucose solution and the other with 0.60 M glucose solution. The glucose discs were placed on either side of the organism 21.0-28.0mm away. An additional drop of solution was added to each disc on the morning of the second day. The petri dishes were sealed and covered with aluminum foil to eliminate any sunlight from reaching the organism. Measurements of the distance of cytoplasmic streaming were made three times a day from March 10-13, 2003. Measurements were made from the initial location of the organism to the edge of the growth toward the glucose concentrations. Initial distance from organism to glucose disc was measured and distance traveled was calculated as a percentage of the initial distance from the glucose disc. A chi square was calculated to determine the probability of the results.

Results:

There was no observable growth toward either glucose concentration during the first eight hours of the experimental period, and there was no initial chemotactic response to the 0.60 M glucose in any of the trials. The plasmodium had a positive chemotactic response to 0.06 M glucose and a negative chemotactic response to 0.60 M glucose. The organism grew to the 0.06 M glucose in all three trials and grew past the 0.06 M glucose disc in two of the three trials (Table 1). In one trial, this indirect growth resulted in a final distance that was closer to the 0.60 M glucose disc than the initial distance. The average percent distance traveled to 0.06 M was 100%; the average percent distance traveled to the 0.60 M glucose disc was 7% (Table 2). Statistical analysis of the data indicates a high probability that these differences were not obtained through random sampling error alone (Chi square=65.0;p=0.95)(Table 3).

Discussion:

These results support the hypothesis that glucose concentration does effect the direction of cytoplasmic streaming in P. polycephalum, but do not support the hypothesis that P. polycephalum growth would favor the 0.60 M glucose concentration. Experimental data suggests the opposite—that this organism actually selects a lower glucose concentration over a higher glucose concentration. These results actually make sense when chemical concentration gradients are considered. Many organisms often rely on an ability to sense and respond to changes in osmotic pressure. It is reasonable that 0.60 M glucose is hypotonic to P. polycephalum and it prefers a less concentrated environment.

In two of the three trials the plasmodium grew past the 0.06 M glucose and around the edge of the petri dishes. Interestingly, in one of these trials the final distance of the plasmodium from the 0.60 M glucose was closer than the initial distance. However, it cannot be determined from these results if the organism was actually growing toward the 0.60 M glucose because it never reached that paper disc and only traveled an average of 7% of the total distance to the 0.60 M glucose. It is important to emphasize that in all three trials the plasmodium grew toward and contacted the 0.06 M glucose relatively quickly considering that it never contacted the 0.60 M glucose.

The results of this experimental trial are promising but there are some weaknesses that should be addressed. There were only three experimental replications, which is far fewer than is needed to draw any suitable conclusions about the effect of glucose concentration on P. polycephalum growth. However, this experiment could be very easily replicated and manipulated any number of times and more definitive conclusions could be formed. In addition, the consistency of directional growth to the 0.06 M glucose in all three trials does allow some confidence in concluding from these results that glucose concentration does affect the pattern of cytoplasmic streaming in P. polycephalum.

Future study is important for determining the extent of the influence of glucose concentration on P. polycephalum growth. The effects on P. polycephalum growth when its only source of food is 0.60 M glucose should be considered in order to determine if this is a survivable environment for P. polycephalum. 0.60 M glucose may not be lethal to this organism but it could be sub-optimal relative to 0.06 M glucose, which could cause it to favor the lesser concentration. It would also be interesting to determine the response of P. polycephalum to different but higher concentrations of glucose (i.e. response to 0.60 M and 1.2 M). These concentrations could be lethal, but it is possible that one is closer to optimal conditions and therefore favored. If given a choice between these two concentrations, P. polycephalum might prefer an environment that it might otherwise avoid.

Literature Cited:

Alcock, J. 1998. Animal Behavior: An Evolutionary Approach. Sinauer Associates:

Sunderland, MA.

Bozzone, D. M. 2001. Cells with “Personality”: Physarum polycephalum. Carolina Tips.

http://www.carolina.com

Campbell, N. A., and J. B. Reece. 2002. Biology. Benjamin Cummings: San Francisco.

Introduction to the “Slime Molds.”

http://www.ucmp.berkeley.edu/protista/slimemolds.html

Wong, G. Myxomycota. 2003.

http://www.botany.hawaii.edu/faculty/wong/Bot201/Myxomycota/

Widman, M. 1996. What is chemotaxis?

http://www.egr.msu.edu/widmanm1/chemotax.html

Figure 1: Diagram of experimental setup with petri dishes, P. polycephalum, and 0.06 M and 0.60 M glucose saturated paper discs.

Table 1: Initial, final, and change in distance between organism and glucose saturated paper discs for trials A, B, and C.
	Distance to 0.06 M			Distance to 0.60 M
Dish	Initial	Final	Change	Initial	Final	Change
A	22.0	0.0	22.0	28.0	28.0	0.0
B	22.0	0.0	22.0	21.0	21.0	0.0
C	24.0	0.0	24.0	25.0	20.0	5.0

Table 2: Average distance traveled to 0.06 M and 0.60 M glucose concentration as a percent of the initial distance.
	M	% Traveled
	0.06 M	100.0
	0.60 M	7.0

Table 3: Observed and expected change in distances (mm) to 0.60 M glucose solutions by P. polycephalum and Chi square values
Distance to 0.60 M
Observed	Expected
0.0	28.0	28.0
0.0	21.0	21.0
5.0	25.0	16.0
	Chi square	65.0

****Note from Dr. Coleman Figure Legends and notes should go after the table or figure.****