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INTRODUCTION
Everything on Earth is subject to gravity. Since gravity on Earth is inaccurately considered a constant (1 g), its biological consequences are usually ignored. Human travel and colonization of space require knowledge of the effects of altered gravity on living systems as they transition from a nominal 1 g Earth environment to one of chronic altered (higher or lower) g. The clinostat is a ground-based instrument that attempts to simulate microgravity conditions (Albrecht-Buehler, 1992; Hader, 1999). Hyper g conditions can be simulated using a centrifuge designed to accommodate the organism under investigation (Benjaminson and Brown, 1988). In our lab, we have custom designed and fabricated instruments that simulate both g conditions for non-human model systems (Benjaminson et al., 2006).
The Dictyostelium discoideum, Enterobacter aerogenes predator-prey system has proved to the authors to be a valuable tool for the study of the D. discoideum developmental cycle. D. discoideum has previously been used to explore the effects of gravity on development in both ground-based experiments (Kawasaki et al., 1990) and in space flight (Takahashi et al., 2001). Our experiments have revealed evidence that the rate at which Dictyostelium completes its life cycle is sensitive to changes in gravity (Benjaminson et al., 2006; Benjaminson, 1989a; Benjaminson, 1989b). Gravity has been shown to influence stalk height growth as well as germination efficiency, cell differentiation, and spore formation (Kawasaki et al., 1990). Here we report that the developmental morphology of Dictyostelium is also influenced by the gravity environment.
The transition from single celled amoeba, through a sequence of morphogenic changes, to multicellular spore-bearing reproductive sorocarp stage is well known to biologists (Athanasius and Hogeweg, 2001; Breen et al., 1992; Loomis, 1975). Under standard laboratory conditions, Dictyostelium sorocarps grow upward, away from the agar surface and opposite to the gravity vector. We had also observed, however, that cultures grown on agar surfaces in the inverted position with sorocarp erection in the same direction as the gravity vector, skip the migratory slug stage and tend to be taller than the upward facing and growing culminants (Benjaminson, unpublished data). Insights into the governance of size through the allocation of energy are helping us to answer important questions about the growth of individuals and the growth of populations (Kempes et al., 2012; McNab, 1984). The prospect of combining advanced energetic techniques and biotechnology in the search for knowledge of the balance among forces determining the fate of living things is an attractive one. Aside from the important role played by energetics, diffusing factors under genetic control determine size in Dictyostelium through governance of the number of cells in the multicellular stages (Brock and Gomer, 1999; Breen et al., 1992; Kamboj et al., 1990; Kopachik, 1982; Okuwa et al., 2001). Therefore, the interplay among genes, gene products, and gravity appears to have prescribed the final height of the sorocarps observed.
MATERIALS AND METHODS
Recognizing the probable variation in local environments inherent in our laboratory and in order to reduce the number of environmental variables, each individual experimental environment (inverted, centrifuged, clinorotated) housed its own controls. This provided both experimental subjects and their controls with essentially the same conditions.
Dictyostelium Culture
The D. discoideum strain used in these studies was the CB Wild Type stock, routinely fed on Enterobacter aerogenes bacteria (Carolina Biological, Burlington, NC 27215-3398). The experimental inocula were 24 hour co-cultures of CB amoebae and E. aerogenes in 0.1% Lactose-Peptone Broth (LPB), 0.1% lactose, 0.1% peptone in deionized water. One standard loopful (10 μl) of the mixed inoculum was spotted in the center of a 35X10 mm Lactose-Peptone Agar (LPA) plate. The Petri dishes were supplied by Falcon (Becton Dickinson, Franklin Lakes, NJ USA 07417) and the medium consisted of 0.1% lactose, 0.1% peptone, and 2% agar (all media components from BBL) dissolved in deionized water. All media were sterilized in an autoclave (Wisconsin Aluminum Foundry Co. Inc., WI) at 15 psi for 15 min. All plates were sealed with labeling tape to prevent desiccation and maintained in the dark at 25 degrees C throughout the tests to eliminate the effects of light on development (Benjaminson, in preparation).
Inverted
The first treatment involved merely inverting the experimental group of Petri dishes, orienting them with their lids down so that at culmination, the base of the sorocarp faced upwards away from the surface of the Earth and the spore-bearing sorophore hung downward.
Hyper-g
The second experimental condition involved exposure to hyper-g. The experimental cultures were subjected to 10 g, the same g value as those cultures centrifuged previously in Dr. Brown’s facility (Benjaminson and Brown, 1988). The Model D1006 centrifuge used in the current experiments was designed and fabricated by Lehrer Engineering, Pompton Lakes, New Jersey. This centrifuge was designed to provide a maximum hyper-g environment of 10 g. Precise rotational speed control and, hence, precise g values are obtained using a speed control with a feedback loop. The controls for this experiment were stacked on the floor of the centrifuge chamber. The centrifuge is shown in Figure 1.
Figure 1.
Photograph of M1006 centrifuge showing housing and rotor.
Simulated Reduced-g
The reduced-g experimental group was rotated on a specially designed clinostat at 5.708 rpm, a speed calculated to simulate reduced-g (Benjaminson et al., 2006), Figure 2. The Model D1003 clinostat was designed and manufactured by Lehrer Engineering, Pompton Lakes, New Jersey. The reduced-g simulation is obtained by slowly rotating the clinostat disk holding the experimental cultures. Earth’s gravity vector with respect to the specimen is averaged out to approximately zero over each revolution of the disk. The rotational speed is sufficiently low (0.1 to 5.7 rpm) so that the effect of the centrifugal force induced by the rotation is essentially zero. The reduced gravity equals 0.0058 g at 5.7 rpm. The controls for this portion of the experiment were stowed in the clinostat’s upright U-shaped bracket alcove.
Figure 2.
The D1003 clinostat viewed from above showing the rotor assembly with culture and the upright U-shaped bracket alcove.
Measurements
The mature sorocarps were prepared for measurement by removing the tape seals and placing a plastic cover slip (Thomas Scientific, Swedesboro, NJ, USA 08085) on the exposed culture so that its weight forced the sorocarps into a supine position on the surface of the agar; see Figure 3. The length of the sorocarp from base-agar interface to distil terminus of the stalk where it joins the fruiting body was the dimension measured; see Figure 4.
Figure 3.
Photograph of supine sorocarps prepared for measurement.
Figure 4.
A rendition of a mature sorocarp showing the base, stalk, and fruiting body. The bracket delineates the measurement target.
Microscopy
The microscope used was an Olympus IMT-2 inverted microscope (Olympus Corporation, Center Valley, PA 18034-0610) using the 2X objective. The 10X oculars were used for sighting. The randomly selected sorocarp images were captured and saved using a QICAM monochrome video camera equipped with QICAM operating software. Northern Eclipse software was used to measure the height of the sorocarps (all Empix Imaging, Inc., Mississauga, ON, Canada, L5L 5M6), and Excel software was employed for sorting and analyzing the data. Images were captured with the exposure time set at 16.4 ms. The Northern Eclipse measurement software was calibrated using a stage micrometer and the Petri dish was manually scanned. Images were loaded into Northern Eclipse software. Measurements were made using the tracing tool on the sorocarp image longitudinally (the tracing tool keeps track of the length of the trace in millimeters); the collected data were automatically saved to an Excel spreadsheet.
RESULTS
The three experiments were analyzed separately comparing the experimental group to its control group in each case. The independent student’s t test was used to compare the mean heights. Results can be seen in Table 1.
Results.
GROUP Measurement
Inverted Experimental
Inverted Control
Hyper g Experimental
Hyper g Control
Simulated Reduced Gravity Experimental
Simulated Reduced Gravity Control
# of sorocarps (n)
678
642
698
435
1,121
1,353
Mean Height
1.84 mm
1.64 mm
1.13 mm
2.06 mm
2.12 mm
1.79 mm
+/- Standard Deviation
0.7 mm
0.66 mm
0.53 mm
0.75 mm
0.73 mm
0.68 mm
Range: High to Low
0.48 mm to 4.35 mm
0.44 mm to 3.89 mm
0.50 mm to 5.01 mm
0.42 mm to 3.14 mm
0.14 mm to 3.14 mm
0.37 mm to 5.07 mm
Inverted
In those Dictyostelium cultures that had simply been inverted, the sorocarps numbered 678. The average height of the sorocarps was 1.84 mm, ranging from 0.48 mm to 4.35 mm, a difference of 3.87 mm, with a standard deviation equal to 0.70 mm. The controls for this experiment were an average of 1.64 mm high and ranged in height from 0.44 mm to 3.89 mm, a difference of 3.45 mm, in the 642 measured, with a standard deviation equal to 0.66 mm.
For the inverted experiment, the calculated t = 10.42. For mean sorocarp height there is a significant difference between the inverted experimental group and the control group, p < 0.05.
Hyper-g
In the group centrifuged at 10 g, the number of sorocarps measured was 698, and they ranged in height from 0.50 mm to 5.01 mm, a difference of 4.51 mm, averaging 1.13 mm in length, with a standard deviation equal to 0.53 mm. The control for this group numbered 435 sorocarps. They ranged from 0.42 mm to 3.14 mm, a difference of 2.72 mm, with an average height of 2.06 mm, and a standard deviation equal to 0.75 mm.
For the hyper-g experiment, the calculated t = 31.16. For mean sorocarp height there is a significant difference between the hyper-g experimental group and the control group, p < 0.05.
Simulated Reduced-g
In the case of the clinorotated cultures, 1,121 sorocarps were measured. The average height of the experimental group was 2.12 mm, ranging from 0.14 mm to 3.14 mm, a difference of 3.0 mm, with a standard deviation equal to 0.73 mm. The number of controls was 1,353 sorocarps, whose average height was 1.79 mm, ranging from 0.37 mm to 5.07 mm, a difference of 4.7 mm, with a standard deviation equal to 0.68 mm.
For the simulated reduced-g experiment, the calculated t = 28.6. For mean sorocarp height there is a significant difference between the simulated reduced-g experimental group and the control group, p < 0.05.
DISCUSSION
A number of gravity sensitive parameters have been previously identified in the life cycle (developmental pathway) of Dictyostelium. These included the rate of development and the rate of induction of germination (Benjaminson, 1997). Development is known to be governed by an array of gene products whose activity drives the life cycle forward (Loomis, 1975). Ultimately, the development of Dictyostelium is determined by the constraints imposed by its genes’ range of activity and the environment in which it develops. In the current study, we demonstrate that the final height of the sorocarp is dependent partially on the gravitational environment.
All the Dictyostelium amoebae employed in these experiments were derived from the same culture, and they all fed on the same population of bacteria under the same environmental conditions. Thus the control and experimental populations of Dictyostelium we used theoretically had access to equivalent amounts of stored energy partitioned for differentiation, movement, and biosynthesis (Benjaminson, 1997). To our knowledge the only experimental variable in this study was the gravitational environment.
The results of the experimental treatments vs. control treatments demonstrated that different gravitational environments had significant effects on sorocarp height each of the experimental groups being statistically significantly different in height from its control group. The inverted and simulated reduced gravity (clinorotated) groups had taller sorocarps as compared to their respective 1 g controls. The hyper-g experimentals (centrifuged) had shorter sorocarps as compared to their controls
Inverted
In the “inverted” experiment, the control group was cultured with a typical orientation, i.e., the agar surface facing upward, resulting in Dictyostelium sorocarp assembly being vertical and perpendicular to the Earth’s surface (upright position). In this case, the gravity vector was in the opposite direction as compared to the sorocarp assembly direction. For the experimental group, Dictyostelium was cultured in the inverted position, i.e., agar surface facing down. For the experimental group the gravity vector was in the same direction as the sorocarp assembly direction. Based on the results of this experiment, it appears that the gravitational environment of the experimental group somehow favored sorocarp assembly (erection) resulting in experimental (inverted) sorocarps being significantly taller (p < 0.05) compared to controls. Based on our interpretation of graphs (Figures 5 and 6 in Kawasaki et al., 1990), similar results have been reported, in that the inverted fruiting bodies height (stalk height) was positively influenced (taller) by the inverted growing condition. We hypothesize that the inverted sorocarps received “help” from gravity for sorocarp assembly. This “help” may have been related to increased available energy consumption. Perhaps energy availability increased as a result of the elimination of the slug stage. Altered gene expression, epigenetic phenomena, other physical factors, and/or mechanical alterations may have played a role as well (Benjaminson, 1996).
Hyper-g
The hyper-g environment (10 g) produced shorter sorocarps when compared to 1 g controls. In the hyper-g experiment, the control group was housed on the centrifuge base in the upright position, both initiating and completing its life cycle in the 1 g environment. The 1 g gravity vector for the controls was in the opposite direction compared to the sorocarp assembly direction. For the experimental group, Dictyostelium was cultured in the upright position as well, i.e., agar surface facing upward. The gravity vector experienced by the experimental group was in the opposite direction as the sorocarp assembly direction, but exposed to 10 times (10 g) the force of Earth’s gravity (Benjaminson et al., 2006). Based on the results of this experiment, the gravitational environment of the experimental group negatively affected the sorocarp assembly resulting in experimental (centrifuged) sorocarps being significantly shorter (p < 0.05) compared to controls. This is in contrast to earlier experiments in hyper-g by Kawasaki et al. (1990) who reported that 3 g centrifugation throughout the entire Dictyostelium life cycle produced taller fruiting bodies (stalks) compared to 1 g controls. Our experiment used 10 g centrifugation on a different instrument platform and the number of stalks measured was different. In the measurement of the heights of fruiting bodies, Kawasaki reports that in the experiment, “thirty to 50 fruiting bodies were counted in each Petri dish, and three to 6 petri dishes measured.” That is, a maximum of 300 sorocarps were measured for height by Kawasaki’s group, compared to 678 (experimental) for a total of 1,121 in the experiments reported here. These differences may account for the conflicting results obtained.
We hypothesize that the hyper-g environment made sorocarp assembly more difficult, possibly requiring more energy expenditure and thereby resulting in shorter sorocarps than the 1 g controls.
Simulated Reduced-g
In the clinorotated experiment, the control group was housed on the clinostat chasis itself, completing its life cycle in the 1 g environment. The 1 g gravity vector for the controls was in the opposite direction compared to the sorocarp assembly direction. For the experimental group, the gravity vector theoretically approaches zero due to the Dictyostelium being attached to a substrate and going through 360 degrees of rotation during its entire life cycle, including sorocarp assembly. Theoretically, the clinostat speed set at 5.708 rpm partially cancels out the gravity vector through 360 degrees over time (Benjaminson et al., 2006). As with the inverted experimental group, the simulated reduced-g environment somehow favored sorocarp assembly, which resulted in experimental (clinorotated) sorocarps being significantly taller than their 1 g controls. This result is also in contrast to Kawasaki et al. (1990), who reported that chronic “artificial microgravity” produced by clinorotation, applied throughout the entire Dictyostelium life cycle, produced shorter fruiting bodies (stalks) compared to 1 g controls.
We hypothesize that the clinorotated sorocarps were taller due to the clinorotation reduced influence of a gravity vector acting in the opposite direction of sorocarp assembly and a reduced requirement for the expenditure of energy to counterbalance gravity’s effect on the process of development.
Hypothetical Mechanisms
The allocation of energy for morphogenesis is governed by mechanisms that are sensitive to chronic simulated gravitational environments (Vasiev and Cornelis, 2003; Benjaminson, 1996; Benjaminson, 1997; McNab, 1984). Cell allocation may also have influenced morphogenesis as was described by Maeda and Maeda (1974), and reported by Kawasaki et al. (1990). These may account for the ordering of average heights of the experimental groups, ranging from longest to shortest, clinorotated to inverted to centrifuged.
CONCLUSIONS
The three experimental gravitational alterations produced measurable differences in the experimental groups. Our work, which expanded on Kawasaki et al. (1990) experiments, supports the hypothesis that gravity has an influence on morphogenesis in D. discodium. Although our results differ from Kawasaki et al. (1990), the influence of gravity has been demonstrated in both experiments. How this influence is exerted and why results were different between our experiment and Kawasaki (Kawasaki et al., 1990) remains to be investigated.
FUTURE EXPERIMENTS
Based on the significant differences in Dictyostelium discoideum sorocarp heights observed in this study, we believe future investigations will confirm the results reported in this paper, and should extend to other model systems such as Drosophila and activated plant seeds. In addition, experiments designed to determine whether these effects are a universal phenomenon and to identify the underlying mechanisms of gravity’s influence on morphogenesis are planned.
