Common Soil Bacteria Negatively Impacts the Growth of Physarum Polycephalum

Common Soil Bacteria Negatively Impacts the Growth of Physarum Polycephalum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Common Soil Bacteria Negatively Impacts the Growth of Physarum Polycephalum aidan gao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5672670/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Physarum polycephalum, known as slime mold, is a multinucleate protist that develops networks to search for food. To understand the effects of bacteria and competition on P. polycephalum growth, this experiment investigated the effects of introducing Bacillus cereus , Micrococcus luteus , and Clostridium sporgenes into P. polycephalum in LB and pure agar media and measured the growth of P. polycephalum squares after four days. The introduction of bacteria to LB agar resulted in a decrease in growth of 86%, from an average of 14.6 to 1.67 squares covered with a P value < 0.001, whereas pure plates yielded no deviation from the control. These results suggest that the introduction of bacteria interferes with P. polycephalum growth, likely because bacterial clustering reduces predation ability. Figures Figure 1 Figure 2 Introduction Physarum Polycephalum is a species of slime mold, a protist primarily characterized by a multinucleate plasmodium that forms a network of tubes[ 1 ]. Owing to their pseudocognitive ability, slime molds, especially P. polycephalum, are typically used as model organisms for motility and chemotaxis[ 2 ]. As a model organism, it usually feeds on oats but also ingests bacteria, fungal spores, and other smaller protists through phagocytosis[ 3 ]. Bacillus cereus is an anaerobic and gram-positive bacteria commonly known for contaminating food and resulting in intestinal illness[ 4 ]. It is a common soil bacteria with a motile and spore-forming nature that grows optimally at temperatures ranging from 30–40 degrees Celsius and a neutral pH[ 5 ]. Micrococcus luteus is also a gram-positive bacterium but is nonmotile and aerobic. While unable to form spores, it can be dormant as an oligotroph and can survive in harsh conditions through a minimal metabolic state[ 6 ]. It is commonly found in soil and air and grows optimally at temperatures ranging from 25–37 degrees Celsius and a neutral pH[ 7 ]. Clostridium sporogenes is another gram-positive and anaerobic bacteria that produces endospores and is commonly found in soil[ 8 ]. Like B. cereus , it exhibits motility, and optimal growth occurs at approximately 37 degrees Celsius and a neutral pH[ 9 ]. In the natural environment of P. polycephalum soil, competition for nutrition and growth is abundant. Previous research has revealed the effects of competition among different species of slime mold and has demonstrated that often, only a single species thrives in the environment[ 10 ]. As a predator of bacteria, P. polycephalum feeds on bacteria in nature, much like other species of slime mold do, with some species even cultivating bacteria for later use[ 11 ]. Additionally, in previous research, the introduction of bacteria to cellular slime molds in forest soil environments was shown to increase slime mold growth[ 12 ]. While prior research has investigated the relationship between slime molds and bacteria in nature, little work has investigated their relationship when nutritional constraints are removed. By isolating both bacteria and slime molds and eliminating food concerns, this study aims to better understand predation and competition between slime molds and bacteria, with the three bacterial species above chosen due to being soil bacteria common around P. polycephalum. Owing to past research and with respect to P. polycephalum predation by bacteria, it can be hypothesized that all three bacterial species increase P. polycephalum growth by providing additional incentives. Materials and Methods Preliminary testing ensured that P. polycephalum would grow with a specific oat arrangement and that bacteria would grow alongside P. polycephalum, affecting its growth. P. polycephalum was subcultured from a five-day-old pure population, and both LB and pure agar were tested with and without bacteria added during preliminary testing, with growth measured after four days. The results indicated that placing oats in an X pattern with the initial P. polycephalum sample in the middle resulted in more noticeable growth differences and confirmed that bacteria affected the development of P. Polycephalum within the four days. Final testing utilized another five-day-old pure subculture, with four runs of 2% pure LB agar and four runs of 2% LB agar, each with three trials. Both groups of runs consisted of three runs, adding B. cereus , M. luteus , and C. sporogenes, with a negative control run to establish a baseline for the results. During each trial, a small square of P. Polycephalum was subcultured and placed upside down on the center of a square agar plate with either pure agar or LB agar, and the oats were then placed in an X pattern, with an oat on each square, as shown in Fig. 1 . If not a control run, bacteria were subcultured and streaked in a square pattern between the first and second layers of the X pattern, as shown in Fig. 1 . The agar plate was then sealed with Parafilm and placed in the dark to grow for four days. Figure 1 : A final trial of LB M. luteus, showcasing experimental setup. Oats are placed in an X pattern with one on each square, and the square of white dots is M. luteus streaked in the square pattern. The bottom two central squares would be counted as DV. P. Polyephalum growth was measured by counting squares with noticeable growth, with Fig. 1 counting the two bottom central squares. A T-test was employed to assess the statistical significance of the collected data since there is no expected value for the growth of Physarum in the null hypothesis. The chosen T-test was two-tailed and type two to determine any difference regardless of the direction and because datasets originate from different populations. The control variables kept constant during the experiment were the period of growth due to additional time resulting in growth differences, and the amount and position of oats as oats provide the incentive that dictates P. polycephalum movement is the streaking position of bacteria since it dictates P. polycephalum growth before interaction, and the sterilization of the plates as contamination results in external factors. To control these variables, the growth time was kept constant; one oat was placed on each square in the X pattern, all bacteria were streaked in a square pattern between the first and second oats in the X pattern, and all plates and tools were kept sterile. Results Figure 1 presents the key findings of this experiment: the introduction of bacteria to the LB plates significantly hindered the growth of P. Polycephalum, with a P value < 0.001, confirming an 86.4% decrease between the two statistical groups, A and B. The two control runs averaged 13.33 and 12.33 squares with %AADs of 11.6% and 3.6%, respectively. Compared with the two control runs, the pure runs of all three bacteria showed no significant difference in growth, with average growth values of 14, 10.33, and 10.67, corresponding to left to right on the graph, and %AADs of 9.5%, 10.7%, and 14.5%, respectively. The LB runs showed average growth rates of 2.33, 1.33, and 1.33, respectively, with %AADs of 19%, 33%, and 33%, respectively. The LB group yielded an average 86.0% decrease, with P < 0.001 between the two groups. Discussion The results of this study contradict the hypothesis that bacteria increase P. polycephalum growth, with extremely low P values (< 10 3 ), confirming that this difference is statistically significant. While the %AAD was high for most runs, and only three trials were conducted, leading to increased susceptibility to changes. While the differences between the LB and pure groups could have resulted from the difference between the agars, the significant difference, with a P value of 0.46 between the control runs, means that the difference is likely due to the environment on the pure agar being restrictive for bacterial growth, which is supported by the fact that all three bacteria are not autotrophs and thus cannot produce their own nutrients[13,14,15]. The inability of P. Polycelphalum to bypass streaked bacteria can be interpreted in two ways, the first being the development of bacterial colonies that are too large to bypass. As shown in previous studies, bacteria can cluster in response to predators[16]. Additionally, owing to streaking from the subculture of bacteria directly, the density of the bacteria placed on the agar plate is likely high, meaning that their ability to cluster is more pronounced. Research has shown that motile bacteria, especially B. cereus and C. sporogeneus, willingly exhibit clustering behavior and form microcolonies, which are more pronounced on soft surfaces such as agar[ 17 ]. Since P. Polycephalum feeds through phagocytosis, completely engulfing the organism, the ability of the bacteria tested to form colonies could mean that the microcolonies formed by all three bacteria are too large for the P. Polycephalum to ingest, forming P. Polycephalum to be confined within the square of bacteria or travel over it[ 18 ]. Thus, owing to the grouping behavior of the bacteria, it is difficult for the P. Polycephalum to predate on the colonies, leading to stagnation of growth for P. Polycephalum as it cannot reach food sources and naturally cannot obtain nutrients from LB agar, suggested by the lack of change between the LB and pure agar controls. The second interpretation of the stopped growth is the use of bacterial defenses to prevent predation from P. Polycephalum. While grouping is a defense mechanism in itself, the use of more active defenses like toxins could provide another explanation. All three bacteria have the ability to become pathogens, and B. cereus and C. sporogenes produce toxins and acids, respectively[19,20]. These toxins can deter predation by P. Physarum, leading to restricted growth and nutrient acquisition. However, with past research indicating that slime molds have some resistance to toxins and are able to withstand close to 1000 mg/L insecticides, this interpretation is less likely to be true[21]. The main source of uncertainty within this experiment was the age of the bacteria relative to the age of P. Polycephalum. Since the bacteria used in the experiment had variable time spans to incubate and multiply, their inconsistency themselves and P. Polycephalum could have artificially lowered P. Polycephalum’s ability to predate. Another source of uncertainty was the method of measurement. While counting squares is a good indicator of development, it is subject to visual bias and not a definitive indicator of growth, such as biomass. The final source of uncertainty was the sample size. Owing to only having three trials per run, slight changes would cause an extremely high %AAD. To counteract these uncertainties, simultaneously subculturing all the organisms would ensure that the age is constant, and counting by weighing the biomass with more trials per run would better confirm the results. Additionally, to confirm which of the interpretations is correct, additional testing with bacteria that do not produce toxins and bacteria that do not exhibit clustering behavior could single out possible explanations. Conclusion This study revealed that the presence of various common soil bacteria negatively affected the growth of Physarum Polycephalum, with a P value < 0.001. While there is uncertainty with the small sample size, high %AAD, and varying ages, the extremely small P values suggest that this relationship is not coincidental and is likely due to clustering behavior, which could decrease predation and increase competitiveness, as indicated by the results. Limitations This study has several limitations that warrant consideration when interpreting the results. The limited sample size of three trials per run may not capture the full variability in P. polycephalum growth and bacterial interactions, potentially affecting the reliability and generalizability of the findings. Additionally, the measurement method, which involves counting the number of squares covered by P. polycephalum, is prone to visual bias and may not accurately reflect the actual biomass or health of the slime mold. More precise measurement techniques, such as biomass weighing, could yield more accurate results. Declarations Author Contribution A.G. Conducted experimentation, data collection, and wrote manuscript text. All authors reviewed the manuscript Acknowledgments I would like to thank Milton Academy for giving me this opportunity, and my teacher, Mr. Carvalho, for offering advice and guiding me through the P. Polycephalum growing and sterilization process. References (N.d.). Geneseo.edu. Retrieved June 2, 2024, from https://milnepublishing.geneseo.edu/botany/chapter/physarum/ American Association for the Advancement of (2010, January 22). Slime design mimics Tokyo’s rail system: Efficient methods of a slime mold could inform human engineers. Science Daily . https://www.sciencedaily.com/releases/2010/01/100121141051.htm Utah State (n.d.). The blob . Usu.edu. Retrieved June 2, 2024, from https://www.usu.edu/herbarium/education/fun-facts-about-fungi/slime-molds McDowell, H., Sands, E. M., & Friedman, H. (2023). Bacillus Cereus . StatPearls Publishing. Drobniewski, A. (1993). Bacillus cereus and related species. Clinical Microbiology Reviews , 6 (4), 324–338. https://doi.org/10.1128/cmr.6.4.324 Micrococcus luteus Fact Sheet - Wickham Micro . (n.d.). co.uk. Retrieved June 3, 2024, from https://wickhammicro.co.uk/knowledge-and-education/micrococcus-luteus Kundrat, (2015, November 11). Environmental Isolate Case Files: Micrococcus luteus . Microbiologics Blog. https://blog.microbiologics.com/environmental-isolate-case-files-micrococcus-luteus/ Clostridium sporogenes - (n.d.). Kenyon.edu. Retrieved June 3, 2024, from https://microbewiki.kenyon.edu/index.php/Clostridium_sporogenes Clostridium sporogenes . (n.d.). Org.uk. Retrieved June 3, 2024, from https://www.culturecollections.org.uk/nop/product/clostridium-sporogenes-3 Horn, G. (1971). Food competition among the cellular slime molds (Acrasieae). Ecology , 52 (3), 475–484. https://doi.org/10.2307/1937630 Solomon, S. (2011, January 19). Slime molds can farm their own bacteria when food is short . Slate. https://slate.com/technology/2011/01/slime-molds-can-farm-their-own-bacteria-when-food-is-short.html Kuserk, T. (1980). The relationship between cellular slime molds and bacteria in forest soil. Ecology , 61 (6), 1474–1485. https://doi.org/10.2307/1939055 Bacillus cereus- nutrition . (n.d.). edu. Retrieved June 2, 2024, from http://bioweb.uwlax.edu/bio203/s2013/salaba_jaco/nutrition.htm Clostridium - What is it? Morphology, Classification, Characteristics . (n.d.). Retrieved June 2, 2024, from https://www.microscopemaster.com/clostridium.html Greenblatt, L., Baum, J., Klein, B. Y., Nachshon, S., Koltunov, V., & Cano, R. J. (n.d.). Micrococcus luteus - Survival in Amber . Calpoly.edu. Retrieved June 2, 2024, from https://digitalcommons.calpoly.edu/cgi/viewcontent.cgi?article=1363&context=bio_fac Salcher, M., Pernthaler, J., Psenner, R., & Posch, T. (2005). Succession of bacterial grazing defense mechanisms against protistan predators in an experimental microbial community. Aquatic Microbial Ecology: International Journal , 38 (3), 215–229. https://doi.org/10.3354/ame038215 Vourc’h, , Léopoldès, J., & Peerhossaini, H. (2020). Clustering of bacteria with heterogeneous motility. Physical Review. E , 101 (2). https://doi.org/10.1103/physreve.101.022612 Anderson, R. (1993). Fine structure observations of phagotrophic activity by plasmodia of Physarum polycephalum . The Journal of Eukaryotic Microbiology , 40 (1), 67–71. https://doi.org/10.1111/j.1550- 7408.1993.tb04884.x Drobniewski, A. (1993). Bacillus cereus and related species. Clinical Microbiology Reviews , 6 (4), 324–338. https://doi.org/10.1128/cmr.6.4.324 Wikoff, W. R., Anfora, T., Liu, J., Schultz, P. G., Lesley, S. A., Peters, E. C., & Siuzdak, G. (2009). Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites. Proceedings of the National Academy of Sciences of the United States of America , 106 (10), 3698–3703. https://doi.org/10.1073/pnas.0812874106 Terayama, , Honma, H., & Kawarabayashi, T. (1978). Toxicity of heavy metals and insecticides on slime mold Physarum polycerhalum. The Journal of Toxicological Sciences , 3 (4), 293–303. https://doi.org/10.2131/jts.3.293 Additional Declarations The authors declare no competing interests. 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Oats are placed in an X pattern with one on each square, and the square of white dots is M. luteus streaked in the square pattern. The bottom two central squares would be counted as DV.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-5672670/v1/d7a3b25b72e2f33ab07cc997.jpeg"},{"id":72306655,"identity":"c1b0dcbc-8a65-4438-a62e-8e67b2549c4d","added_by":"auto","created_at":"2024-12-25 04:24:19","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":28410,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure 1: \u003c/strong\u003eThe amount of P. polycephum growth measured through the number of squares with noticeable P. polycephalum growth for three species of bacteria across regular and LB agar plates. Error bars represent the absolute AAD, and statistical letters are derived from p values calculated between groups. T tests comparing the LB groups averaged P \u0026lt; 0.001, and t tests within the two groups returned 0.1 and 1 in B. T tests within the pure group all returned P \u0026gt; 0.05.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5672670/v1/8e85e5924f29884da9c5b5d9.png"},{"id":72306754,"identity":"5590a461-11d2-4d93-bff4-10562a466c02","added_by":"auto","created_at":"2024-12-25 04:32:23","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":349288,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5672670/v1/5253cb16-4c8e-4b18-8d24-d9653ef18799.pdf"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"Common Soil Bacteria Negatively Impacts the Growth of Physarum Polycephalum","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003ePhysarum Polycephalum is a species of slime mold, a protist primarily characterized by a multinucleate plasmodium that forms a network of tubes[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Owing to their pseudocognitive ability, slime molds, especially P. polycephalum, are typically used as model organisms for motility and chemotaxis[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. As a model organism, it usually feeds on oats but also ingests bacteria, fungal spores, and other smaller protists through phagocytosis[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eBacillus cereus\u003c/em\u003e is an anaerobic and gram-positive bacteria commonly known for contaminating food and resulting in intestinal illness[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. It is a common soil bacteria with a motile and spore-forming nature that grows optimally at temperatures ranging from 30\u0026ndash;40 degrees Celsius and a neutral pH[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. \u003cem\u003eMicrococcus luteus\u003c/em\u003e is also a gram-positive bacterium but is nonmotile and aerobic. While unable to form spores, it can be dormant as an oligotroph and can survive in harsh conditions through a minimal metabolic state[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. It is commonly found in soil and air and grows optimally at temperatures ranging from 25\u0026ndash;37 degrees Celsius and a neutral pH[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Clostridium sporogenes is another gram-positive and anaerobic bacteria that produces endospores and is commonly found in soil[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Like \u003cem\u003eB. cereus\u003c/em\u003e, it exhibits motility, and optimal growth occurs at approximately 37 degrees Celsius and a neutral pH[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the natural environment of P. polycephalum soil, competition for nutrition and growth is abundant.\u003c/p\u003e \u003cp\u003ePrevious research has revealed the effects of competition among different species of slime mold and has demonstrated that often, only a single species thrives in the environment[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. As a predator of bacteria, P. polycephalum feeds on bacteria in nature, much like other species of slime mold do, with some species even cultivating bacteria for later use[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, in previous research, the introduction of bacteria to cellular slime molds in forest soil environments was shown to increase slime mold growth[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. While prior research has investigated the relationship between slime molds and bacteria in nature, little work has investigated their relationship when nutritional constraints are removed. By isolating both bacteria and slime molds and eliminating food concerns, this study aims to better understand predation and competition between slime molds and bacteria, with the three bacterial species above chosen due to being soil bacteria common around P. polycephalum. Owing to past research and with respect to P. polycephalum predation by bacteria, it can be hypothesized that all three bacterial species increase P. polycephalum growth by providing additional incentives.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003ePreliminary testing ensured that P. polycephalum would grow with a specific oat arrangement and that bacteria would grow alongside P. polycephalum, affecting its growth. P. polycephalum was subcultured from a five-day-old pure population, and both LB and pure agar were tested with and without bacteria added during preliminary testing, with growth measured after four days. The results indicated that placing oats in an X pattern with the initial P. polycephalum sample in the middle resulted in more noticeable growth differences and confirmed that bacteria affected the development of P. Polycephalum within the four days.\u003c/p\u003e\n\u003cp\u003eFinal testing utilized another five-day-old pure subculture, with four runs of 2% pure LB agar and four runs of 2% LB agar, each with three trials. Both groups of runs consisted of three runs, adding \u003cem\u003eB. cereus\u003c/em\u003e, \u003cem\u003eM. luteus\u003c/em\u003e, and C. sporogenes, with a negative control run to establish a baseline for the results. During each trial, a small square of P. Polycephalum was subcultured and placed upside down on the center of a square agar plate with either pure agar or LB agar, and the oats were then placed in an X pattern, with an oat on each square, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. If not a control run, bacteria were subcultured and streaked in a square pattern between the first and second layers of the X pattern, as shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The agar plate was then sealed with Parafilm and placed in the dark to grow for four days.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e: A final trial of LB M. luteus, showcasing experimental setup. Oats are placed in an X pattern with one on each square, and the square of white dots is M. luteus streaked in the square pattern. The bottom two central squares would be counted as DV.\u003c/p\u003e\n\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eP. Polyephalum growth was measured by counting squares with noticeable growth, with Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e counting the two bottom central squares.\u003c/p\u003e\n\u003cp\u003eA T-test was employed to assess the statistical significance of the collected data since there is no expected value for the growth of Physarum in the null hypothesis. The chosen T-test was two-tailed and type two to determine any difference regardless of the direction and because datasets originate from different populations.\u003c/p\u003e\n\u003cp\u003eThe control variables kept constant during the experiment were the period of growth due to additional time resulting in growth differences, and the amount and position of oats as oats provide the incentive that dictates\u003c/p\u003e\n\u003cp\u003eP. polycephalum movement is the streaking position of bacteria since it dictates P. polycephalum growth before interaction, and the sterilization of the plates as contamination results in external factors. To control these variables, the growth time was kept constant; one oat was placed on each square in the X pattern, all bacteria were streaked in a square pattern between the first and second oats in the X pattern, and all plates and tools were kept sterile.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv class=\"BlockQuote\"\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e presents the key findings of this experiment: the introduction of bacteria to the LB plates significantly hindered the growth of P. Polycephalum, with a P value\u0026thinsp;\u0026lt;\u0026thinsp;0.001, confirming an 86.4% decrease between the two statistical groups, A and B. The two control runs averaged 13.33 and 12.33 squares with %AADs of 11.6% and 3.6%, respectively. Compared with the two control runs, the pure runs of all three bacteria showed no significant difference in growth, with average growth values of 14, 10.33, and 10.67, corresponding to left to right on the graph, and %AADs of 9.5%, 10.7%, and 14.5%, respectively. The LB runs showed average growth rates of 2.33, 1.33, and 1.33, respectively, with %AADs of 19%, 33%, and 33%, respectively. The LB group yielded an average 86.0% decrease, with P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 between the two groups.\u003c/p\u003e\n\u003c/div\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThe results of this study contradict the hypothesis that bacteria increase P. polycephalum growth, with extremely low P values (\u0026lt;\u0026thinsp;10\u003csup\u003e3\u003c/sup\u003e), confirming that this difference is statistically significant. While the\u003c/p\u003e \u003cp\u003e%AAD was high for most runs, and only three trials were conducted, leading to increased susceptibility to changes. While the differences between the LB and pure groups could have resulted from the difference between the agars, the significant difference, with a P value of 0.46 between the control runs, means that the difference is likely due to the environment on the pure agar being restrictive for bacterial growth, which is supported by the fact that all three bacteria are not autotrophs and thus cannot produce their own nutrients[13,14,15]. The inability of P. Polycelphalum to bypass streaked bacteria can be interpreted in two ways, the first being the development of bacterial colonies that are too large to bypass.\u003c/p\u003e \u003cp\u003eAs shown in previous studies, bacteria can cluster in response to predators[16]. Additionally, owing to streaking from the subculture of bacteria directly, the density of the bacteria placed on the agar plate is likely high, meaning that their ability to cluster is more pronounced. Research has shown that motile bacteria, especially \u003cem\u003eB. cereus\u003c/em\u003e and C. sporogeneus, willingly exhibit clustering behavior and form microcolonies, which are more pronounced on soft surfaces such as agar[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Since P. Polycephalum feeds through phagocytosis, completely engulfing the organism, the ability of the bacteria tested to form colonies could mean that the microcolonies formed by all three bacteria are too large for the P. Polycephalum to ingest, forming P. Polycephalum to be confined within the square of bacteria or travel over it[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Thus, owing to the grouping behavior of the bacteria, it is difficult for the P. Polycephalum to predate on the colonies, leading to stagnation of growth for P. Polycephalum as it cannot reach food sources and naturally cannot obtain nutrients from LB agar, suggested by the lack of change between the LB and pure agar controls.\u003c/p\u003e \u003cp\u003eThe second interpretation of the stopped growth is the use of bacterial defenses to prevent predation from P. Polycephalum. While grouping is a defense mechanism in itself, the use of more active defenses like toxins could provide another explanation. All three bacteria have the ability to become pathogens, and \u003cem\u003eB. cereus\u003c/em\u003e and C. sporogenes produce toxins and acids, respectively[19,20]. These toxins can deter predation by P. Physarum, leading to restricted growth and nutrient acquisition. However, with past research indicating that slime molds have some resistance to toxins and are able to withstand close to 1000 mg/L insecticides, this interpretation is less likely to be true[21].\u003c/p\u003e \u003cp\u003eThe main source of uncertainty within this experiment was the age of the bacteria relative to the age of P. Polycephalum. Since the bacteria used in the experiment had variable time spans to incubate and multiply, their inconsistency themselves and P. Polycephalum could have artificially lowered P. Polycephalum\u0026rsquo;s ability to predate. Another source of uncertainty was the method of measurement. While counting squares is a good indicator of development, it is subject to visual bias and not a definitive indicator of growth, such as biomass. The final source of uncertainty was the sample size. Owing to only having three trials per run, slight changes would cause an extremely high %AAD. To counteract these uncertainties, simultaneously subculturing all the organisms would ensure that the age is constant, and counting by weighing the biomass with more trials per run would better confirm the results. Additionally, to confirm which of the interpretations is correct, additional testing with bacteria that do not produce toxins and bacteria that do not exhibit clustering behavior could single out possible explanations.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThis study revealed that the presence of various common soil bacteria negatively affected the growth of Physarum Polycephalum, with a P value\u0026thinsp;\u0026lt;\u0026thinsp;0.001. While there is uncertainty with the small sample size, high %AAD, and varying ages, the extremely small P values suggest that this relationship is not coincidental and is likely due to clustering behavior, which could decrease predation and increase competitiveness, as indicated by the results.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Limitations","content":"\u003cp\u003e \u003cdiv class=\"BlockQuote\"\u003e \u003cp\u003eThis study has several limitations that warrant consideration when interpreting the results. The limited sample size of three trials per run may not capture the full variability in P. polycephalum growth and bacterial interactions, potentially affecting the reliability and generalizability of the findings. Additionally, the measurement method, which involves counting the number of squares covered by P. polycephalum, is prone to visual bias and may not accurately reflect the actual biomass or health of the slime mold. More precise measurement techniques, such as biomass weighing, could yield more accurate results.\u003c/p\u003e \u003c/div\u003e \u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eA.G. Conducted experimentation, data collection, and wrote manuscript text. All authors reviewed the manuscript\u003c/p\u003e\u003ch2\u003eAcknowledgments\u003c/h2\u003e \u003cp\u003eI would like to thank Milton Academy for giving me this opportunity, and my teacher, Mr. Carvalho, for offering advice and guiding me through the P. Polycephalum growing and sterilization process.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003e(N.d.). Geneseo.edu. Retrieved June 2, 2024, from https://milnepublishing.geneseo.edu/botany/chapter/physarum/\u003c/li\u003e\n\u003cli\u003eAmerican Association for the Advancement of (2010, January 22). Slime design mimics Tokyo\u0026rsquo;s rail system: Efficient methods of a slime mold could inform human engineers. \u003cem\u003eScience Daily\u003c/em\u003e. https://www.sciencedaily.com/releases/2010/01/100121141051.htm\u003c/li\u003e\n\u003cli\u003eUtah State (n.d.). \u003cem\u003eThe blob\u003c/em\u003e. Usu.edu. Retrieved June 2, 2024, from https://www.usu.edu/herbarium/education/fun-facts-about-fungi/slime-molds\u003c/li\u003e\n\u003cli\u003eMcDowell, H., Sands, E. M., \u0026amp; Friedman, H. (2023). \u003cem\u003eBacillus Cereus\u003c/em\u003e. StatPearls Publishing.\u003c/li\u003e\n\u003cli\u003eDrobniewski, A. (1993). Bacillus cereus and related species. \u003cem\u003eClinical Microbiology Reviews\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(4), 324\u0026ndash;338.\u0026nbsp;https://doi.org/10.1128/cmr.6.4.324\u003c/li\u003e\n\u003cli\u003e\u003cem\u003eMicrococcus luteus Fact Sheet - Wickham Micro\u003c/em\u003e. (n.d.). co.uk. Retrieved June 3, 2024, from https://wickhammicro.co.uk/knowledge-and-education/micrococcus-luteus\u003c/li\u003e\n\u003cli\u003eKundrat, (2015, November 11). \u003cem\u003eEnvironmental Isolate Case Files: Micrococcus luteus\u003c/em\u003e. Microbiologics Blog. https://blog.microbiologics.com/environmental-isolate-case-files-micrococcus-luteus/\u003c/li\u003e\n\u003cli\u003eClostridium sporogenes - (n.d.). Kenyon.edu. Retrieved June 3, 2024, from https://microbewiki.kenyon.edu/index.php/Clostridium_sporogenes\u003c/li\u003e\n\u003cli\u003e\u003cem\u003eClostridium sporogenes\u003c/em\u003e. (n.d.). Org.uk. Retrieved June 3, 2024, from https://www.culturecollections.org.uk/nop/product/clostridium-sporogenes-3\u003c/li\u003e\n\u003cli\u003eHorn, G. (1971). Food competition among the cellular slime molds (Acrasieae). \u003cem\u003eEcology\u003c/em\u003e, \u003cem\u003e52\u003c/em\u003e(3), 475\u0026ndash;484. https://doi.org/10.2307/1937630\u003c/li\u003e\n\u003cli\u003eSolomon, S. (2011, January 19). \u003cem\u003eSlime molds can farm their own bacteria when food is short\u003c/em\u003e. Slate. https://slate.com/technology/2011/01/slime-molds-can-farm-their-own-bacteria-when-food-is-short.html\u003c/li\u003e\n\u003cli\u003eKuserk, T. (1980). The relationship between cellular slime molds and bacteria in forest soil. \u003cem\u003eEcology\u003c/em\u003e, \u003cem\u003e61\u003c/em\u003e(6), 1474\u0026ndash;1485. https://doi.org/10.2307/1939055\u003c/li\u003e\n\u003cli\u003e\u003cem\u003eBacillus cereus- nutrition\u003c/em\u003e. (n.d.). edu. Retrieved June 2, 2024, from http://bioweb.uwlax.edu/bio203/s2013/salaba_jaco/nutrition.htm\u003c/li\u003e\n\u003cli\u003e\u003cem\u003eClostridium - What is it? Morphology, Classification, Characteristics\u003c/em\u003e. (n.d.). Retrieved June 2, 2024, from https://www.microscopemaster.com/clostridium.html\u003c/li\u003e\n\u003cli\u003eGreenblatt, L., Baum, J., Klein, B. Y., Nachshon, S., Koltunov, V., \u0026amp; Cano, R. J. (n.d.). \u003cem\u003eMicrococcus luteus - Survival in Amber\u003c/em\u003e. Calpoly.edu. Retrieved June 2, 2024, from https://digitalcommons.calpoly.edu/cgi/viewcontent.cgi?article=1363\u0026amp;context=bio_fac\u003c/li\u003e\n\u003cli\u003eSalcher, M., Pernthaler, J., Psenner, R., \u0026amp; Posch, T. (2005). Succession of bacterial grazing defense mechanisms against protistan predators in an experimental microbial community. \u003cem\u003eAquatic Microbial Ecology: International Journal\u003c/em\u003e, \u003cem\u003e38\u003c/em\u003e(3), 215\u0026ndash;229. https://doi.org/10.3354/ame038215\u003c/li\u003e\n\u003cli\u003eVourc\u0026rsquo;h, , L\u0026eacute;opold\u0026egrave;s, J., \u0026amp; Peerhossaini, H. (2020). Clustering of bacteria with heterogeneous motility.\u0026nbsp;\u003cem\u003ePhysical Review. E\u003c/em\u003e, \u003cem\u003e101\u003c/em\u003e(2). https://doi.org/10.1103/physreve.101.022612\u003c/li\u003e\n\u003cli\u003eAnderson, R. (1993). Fine structure observations of phagotrophic activity by plasmodia of \u003cem\u003ePhysarum polycephalum\u003c/em\u003e. \u003cem\u003eThe Journal of Eukaryotic Microbiology\u003c/em\u003e, \u003cem\u003e40\u003c/em\u003e(1), 67\u0026ndash;71. https://doi.org/10.1111/j.1550- 7408.1993.tb04884.x\u003c/li\u003e\n\u003cli\u003eDrobniewski, A. (1993). Bacillus cereus and related species. \u003cem\u003eClinical Microbiology Reviews\u003c/em\u003e, \u003cem\u003e6\u003c/em\u003e(4), 324\u0026ndash;338.\u0026nbsp;https://doi.org/10.1128/cmr.6.4.324\u003c/li\u003e\n\u003cli\u003eWikoff, W. R., Anfora, T., Liu, J., Schultz, P. G., Lesley, S. A., Peters, E. C., \u0026amp; Siuzdak, G. (2009). Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites. \u003cem\u003eProceedings of the National Academy of Sciences of the United States of America\u003c/em\u003e, \u003cem\u003e106\u003c/em\u003e(10), 3698\u0026ndash;3703. https://doi.org/10.1073/pnas.0812874106\u003c/li\u003e\n\u003cli\u003eTerayama, , Honma, H., \u0026amp; Kawarabayashi, T. (1978). Toxicity of heavy metals and insecticides on slime mold Physarum polycerhalum. \u003cem\u003eThe Journal of Toxicological Sciences\u003c/em\u003e, \u003cem\u003e3\u003c/em\u003e(4), 293\u0026ndash;303. https://doi.org/10.2131/jts.3.293\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"milton academy","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5672670/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5672670/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003ePhysarum polycephalum, known as slime mold, is a multinucleate protist that develops networks to search for food. To understand the effects of bacteria and competition on P. polycephalum growth, this experiment investigated the effects of introducing \u003cem\u003eBacillus cereus\u003c/em\u003e, \u003cem\u003eMicrococcus luteus\u003c/em\u003e, and Clostridium sporgenes into P. polycephalum in LB and pure agar media and measured the growth of P. polycephalum squares after four days. The introduction of bacteria to LB agar resulted in a decrease in growth of 86%, from an average of 14.6 to 1.67 squares covered with a P value\u0026thinsp;\u0026lt;\u0026thinsp;0.001, whereas pure plates yielded no deviation from the control. These results suggest that the introduction of bacteria interferes with P. polycephalum growth, likely because bacterial clustering reduces predation ability.\u003c/p\u003e","manuscriptTitle":"Common Soil Bacteria Negatively Impacts the Growth of Physarum Polycephalum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-25 04:16:14","doi":"10.21203/rs.3.rs-5672670/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"56f4d790-3393-47e9-af9b-3eb6262fb922","owner":[],"postedDate":"December 25th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-25T04:16:14+00:00","versionOfRecord":[],"versionCreatedAt":"2024-12-25 04:16:14","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5672670","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5672670","identity":"rs-5672670","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}