Introduction

1. Simulated Microgravity and its Effects on the Feeding Habits of the Giant Amoeba Chaos carolinensis Matt Bowen, Floyd Inman III, Leonard D. Holmes Sartorius-stedim Biotechnology Laboratory, Biotechnology Research and Training Center University of North Carolina at Pembroke, Pembroke, North Carolina 28372 Introduction Results of Simulated Microgravity Test Discussion Food Chain Preparation Chaos carolinensis is a giant amoeba, some measuring up to 5 mm. These amoebas are macroscopic, and can be seen with the naked eye if fully mature. C. carolinensis amoebas are heterotrophs, meaning they rely on another photosynthetic organism for their energy. C. carolinensis often feed on Paramecium, bacteria and other small organisms. C. carolinensis have only one nucleus, and may have many finger like projections called pseudopods. This study investigated the effects of simulated microgravity on Chaos carolinensis. Factors such as growth patterns, maturation time, feeding habits, and locomotion were observed and recorded. Control cultures were grown on bench top, to determine if simulated microgravity was the cause of change. To produce enough amoeba to run experiments, small food chains had to be created. In order to mature C. carolinensis into division phase, Paramecium had to be added to the cultures of amoebas. Paramecium served as the primary food source for the C. carolinensis . Cultures of Chilomonas were started in the hay infused media, to produce food for the Paramecium. After the Chilomonas population reached an appropriate level, Paramecium were added to begin feeding and multiplying. Once a culture of C. carolinensis reached a age of 7-8 days old, 10 amoebas were captured, and subjected to simulated micro gravity in the clinostat. Paramecium were also added to the clinostat as the primary food source for the C. carolinensis. 40-50 Paramecium were added to the clinostat per amoeba. The clinostat was allowed to spin for 24-48 hours and amoeba counts were performed using a dissecting scope. After 24 hours of being subjected to simulated microgravity, all C. carolinensis were removed from the clinostat, and counted. The amoebas moved across culture dishes the same as the controls, however no divisions were recorded. At 36 hours of subjected simulated microgravity, some amoebas were dead, but most were alive in the clinostat. Paramecium count remained the same, no divisions of C. carolinensis. After prolonged periods (more than 36 hours) of being subjected to microgravity, all amoebas were dead, but Paramecium were still alive. The Paramecium had no defined path of travel. Paths seemed uncontrollable, and very unpredictable. By testing C. carolinensis under simulated microgravity conditions, we can obtain results that will let us understand more about how microgravity effects the normal characteristics of small unicellular animal like protist. Under normal conditions, small food chains are relatively easy to maintain. Cultures of Chilomonas were grown in hay infused media, then Paramecium were added to feed upon the Chilomonas and multiply. After addition of C. carolinensis the food chain was complete, and the amoebas began to multiply. Under simulated microgravity conditions, C.caroinensis did not thrive. After 36 hours of exposure the population of Paramecium remained constant, and the population of C. carolinensis began to drop. The remaining amoebas looked small and starved. The Paramecium had a change in the regular defined path of travel. Under normal conditions the path of travel was very straight and direct, meanwhile under simulated microgravity the path was very unpredictable, and very different from the control. Simulated microgravity seemed to have an effect on the C. carolinensis ability to feed properly. All surviving amoebas seemed small and very thin, with several pseudopods projecting out from the body in search of food. Starvation could be in part to the change in the path of travel of the Paramecium. The turning of the clinostat could have caused complications for the amoeba to grasp, therefore making conditions unfavorable for feeding. No distinct differences were found with the locomotion of the amoebas. When viewing under a dissecting scope, they moved in a linear path in search of food. Figure 1: Photograph of Chaos carolinensis with 3 visible pseudopods. Materials and Equipment Figure 3: Photograph of two Chilomonas, the Paramecium food source Chaos carolinensis cultures were purchased from Carolina Biological. Media used was an amoeba growth media made from Timothy Hay and wheat kernels. A Synthecon High Aspect Ration Vessel (Figure 2) will be used to simulate microgravity. Feeding of Chaos Once ready to start a culture of C. carolinensis an appropriate amount of hay infused media was placed into a culture dish. 5-6 C. carolinensis amoebaswere captured using a dissecting scope and a pipet, then added to the plate. Paramecium were caught and added to the dishes, in approximate numbers of 40 Paramecium per amoeba. If amoebas looked small, and only a few Paramecium remained, more Paramecium were added to the culture. Acknowledgements Figure 5: Photograph of a well fed amoeba with few thickened pseudopods. Partial financial support was provided in part by the: North Carolina Biotechnology Center (grant # 2010-IDG-1008), UNC-Pembroke Department of Chemistry & Physics and Farm Bureau of Robeson County. Additionally, thanks is given to other undergraduate research students who provided laboratory assistance and to the following sponsors. Figure 4: Paramecium, the primary food source of C.carolinensis Figure 6: 2-3 day starvation of amoeba, notice all of the long thin projections from the body searching for food. Figure 2: High aspect ration vessel (50 mL ) used to simulate microgravity conditions