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Biology Honors 718 (June 1, 2013)

Microbial Encystment in Euglena gracilis

JOHN AHMED DELLAS West Windsor Plainsboro High School South, Princeton Junction, New Jersey, U.S.A.

Abstract In this experiment I attempt to prove the possibility of Microbial Cyst formation (a process also known as Encystment) in certain micro-organisms. Microbial cysts are fundamental in the survival and life cycle of certain species, allowing them to endure harsh and unfavorable environmental conditions while economizing energy and reducing metabolic reactions to near zero activity. Giardia is a parasitic protozoan that latches itself on to the internal walls of the small intestine and encysts to protect itself from harsh stomach acids making it an incredibly resilient and serious threat to all animals (including human beings). The parasitic soybean cyst nematode is the primary cause of soybean crop failure around the world and owes its incredible propagative and resistant capabilities to the deep integration of cysts in their life cycles. Unraveling the micro-organic mysteries that are cysts would allow for the elimination of these common threats. The experimental procedure consists of several subcultures of Euglena gracilis placed on slides and allowed to individually undergo different harsh conditions in order to trigger encystment. These conditions include: harsh light (heat), darkness (starvation), and low moisture. After a set amount of time, whatever harsh circumstance was applied on the sub culture was returned to normal inducing the euglena to excyst and continue their regular trophozoite life stages (under the observation of a microscope). However, I was not able to induce the euglena to excyst because they never encysted in the first place or remained in a cyst throughout the experiment suggesting they died. In this experiment I attempted to prove the possibility for certain microorganisms to form protective cysts by examining Euglena gracilis. Surprisingly, I found it to be of a greater difficulty than I had originally expected; changing the scope of my experiment from detailed reports on cysts themselves to contributions in information for future methods on conducting cyst research.

Introduction The two current most problematic parasites that humanity faces today are Giardia Lamblia and the Soy Bean Cyst Nematode. Both of these parasites owe their resilience, reproductive capabilities and propagation to the non-trophozoite cystic stages of their life cycles. Giardia is a

parasitic protozoan that propagates through the feces of infected animals in bodies of water used as a drinking source for other animals, including human beings. It has no effective cure, and continues to pose a threat for wild life and human beings (Center for Disease Control and Prevention, 2011). The Soy Bean Cyst Nematode is the primary cause of Soy Bean agricultural failure world

wide, and causes an estimated 1.5 billion dollars of damage annually alone (S. Chen et al, 2001). Microbial cystic formation plays a key role in the survival, propagation and reproduction of certain microorganisms. This applies to many protists, and is incredibly common to parasitic protozoans. Parasites and other pathogenic microorganisms with encystment capabilities are a able to disperse across large distances over long periods of time awaiting to excyst at the encounter of a host (Hugo Aguilar-Daz et al, 2011). These temporary protective cysts can also last for a short amount of time as well. Another property of encystment utilized by several parasitic protozoans is that of reproductive cysts. Reproductive cysts allow for an individual microorganism to encyst, reproduce with in that cyst and have all of its off -spring mature safely awaiting ideal environmental conditions to excyst and begin their lives. This is the primary asset possessed by the Soy Bean Cyst Nematode, as it can maintain its resilience and parasitic presence in a soy bean field for generations. Reproductive cysts have the capability to contain up to 64 individual organisms, similar to the maximum number present in a Mucilagenous Palmella cyst (F. Hindak et al, 2000). Comprehending microbial cysts and forwarding more research on the topic would give the scientific community the ability to develop medical and chemical solutions to the modern problems posed by parasitic protozoans with cysts heavily integrated into their life cycles. Materials and Methods: Biological Requirements: The biological section of requirements for this experiment consists uniquely of a

culture of Euglena gracilis. Euglena were chosen as the microorganism the cyst formation trials would be conducted on as it has the capability to form reproductive cysts, palmella cysts and temporary protective cysts under a wide range of conditions including harsh exposure to light, starvation, thermal extremes and (primarily) low moisture conditions (, 2010); all which are easy to simulate. Euglena gracilis also require virtually nothing to survive over a long period of time other than an adequate source of light. Experimental Design: Small sub cultures of Euglena were abducted from the main culture, and each one was placed on a depression slide. Each individual sub culture underwent a different harsh environmental condition as an attempt to trigger encystment. Each individual simulation received 3 trials. Low Moisture Simulation: Euglena gracilis are freshwater microorganisms and their survival depends heavily on surrounding moisture conditions which tend to vary incredibly. As a response to this common problem, Euglena have evolutionarily developed a cyst forming mechanism triggered by these common low moisture conditions that allows them to out last unfavorably dry environmental conditions. This also renders this dry conditions a strong stimulation for encystment in Euglena; a concept which this trial attempts to exploit. Low moisture conditions were simulated by allowing the Euglena to dry out in open light without a cover slip over the sub culture. After ensuring that no water remained, they stayed in these dry conditions for a time period of 30 minutes. Once the time elapsed, the subculture was

rehydrated with natural spring water boiled prior to its appliance. To ensure that the sub culture would remain hydrated, a cover slip with petroleum jelly smeared on its edges was placed above the droplet. The hydrophobic properties of the petroleum jelly repelled the spring water and allowed it to stay within the microenvironment for a period of up to three weeks, simulating ideal conditions for the Euglena to excyst. After the microenvironment was successfully established, the Euglena sub culture was kept under the constant observation of a microscope for signs of any trophozoite activity. Harsh Light (Heat) Simulation: To ensure a stable microenvironment where all conditions are ideal except for the variable, the subculture was placed on a depression slide and ensured constant sufficient moisture by placing the petroleum jelly cover slip first. Following this, the Euglena were placed under direct sunlight and allowed to sit for a time period of 3 hours. When the time period elapsed, the Euglena were moved to a location with a more adequate level of light reestablishing original ideal conditions for excystation (if the euglena entered a state of suspended animation). Observation under a microscope followed to record results. Starvation Simulation: Since Euglena are photoautotrophic life forms, they depend on light as a primary food source. While they can also assume the role of a heterotroph, Euglena gracilis can merely feed of off bio molecules freely available in its environment and is not capable of actual predation (also known as chemo-organo-heterotrophy). Therefor, by

placing the euglena in complete darkness with no freely available bio molecules, it is possible to simulate starvation in the sub culture. Starvation is one of the primary reasons that protozoans (capable of encystation) encyst, considering that once in this state of suspended animation: metabolic activity and energy consumption are reduced to virtually zero. This allows for the euglena to travel and disperse within this cyst until a source of light or basic chemicals for chemoheterotrophy are found. Once in the presence of these requirements they excyst and continue their normal lives. This part of the experiment aims to simulate such conditions for encystment through starvation. Firstly, 1 ml of Euglena containing liquid was subtracted from the main parent culture (using a pipette) and moved to an separate test tube. Following this, the test tube was placed within a cardboard box and relocated to a closet to ensure maximum darkness. After a period of exactly 48 hours, the Euglena sub culture was removed from the box and a (once again using a pipette) a small amount of the subculture was extracted and placed on a depression slide for observation under a microscope. In the case that the observed Euglena were in a form of suspended animation and non-motile, the a grain of rice would be placed at the bottom of the test tube and the entire subculture would be relocated next to a strong, cool-blue fluorescent light to accelerate photosynthetic processes without over heating the Euglena to simulate ideal conditions for excystment. Results: Low Moisture Simulation: Even under the constant observation of a microscope for three days after rehydration, I was not able to observe excystation in any

of the trials for this simulation of the experiment. The Euglena sub culture remained in this condition of suspended animation caused by the original dehydration throughout the entire experiment and never returned to its normal trophozoite life stage. Harsh Light (Heat) simulation: The Euglena subculture was unaffected by the perpetual harsh light. It remained in its regular trophozoite life stage without ever encysting. Starvation simulation: Like the harsh light simulation, the sub culture was unaffected by the 48 hours it spent in the dark and none of the Euglena entered a state of suspended animation throughout all trials of this simulation.

Discussion: The results from the experiment were not at all what I had expected them to be, yet they still contributed to answering the general question of cyst formation in that they show what simulations and unfavorable conditions not to use in future research for encystment. The most surprising result was that of the Starvation simulation. The euglena some how managed to survive for two entire days without light or any basic bio molecules for chemo-heterotrophy without ever entering a state of suspended animation. This period of nutrient deprivation did not trigger encystment or even kill the cells. Upon further research, I speculate that the Euglena were able to survive due to their natural ability to store photosythate in the unusual form of Paramylon, a starch-like carbohydrate with incredibly diverse and

60 IndividualCellNumber 50 40 30 20 10 0 LowMoisture HarshLight(Heat) SubCulureSimulation Starvation Numberinsuspendedanimation Numberexcysted

Chart1:Abargraphrepresentingthe(average)numberofEuglenathathaveenteredsuspended animationandthenumberthathaveexcystedacrossthedifferentsimulationsthroughoutthe experiment.Asstatedintheresults,theonlysubculturestoentersuspendedanimationwerethose oftheLowMoistureSimulation.However,noneofthesubculturesweretriggeredintoexcystment.

Image1:Amicroscopicpictureofthelowmoisturesimulationsubculturetaken30minutesafter rehydration.ThiswastheclosestresulttosuccessfulencystmentIreceivedinmyentireexperiment. AsdepictedbyChart1,thissimulationssubculturesweretheonlyonesthatenteredsuspended animation.However,tothispointIamnotsureiftheEuglenainthispictureareincystsoriftheyare simplydead.

flexible methods of storage that allow Euglena to endure periods of starvation for incredibly long amounts of time (J. Z. Kiss et al, 1987). Upon learning this information, I know realize that I should have kept the Euglena starvation simulation subculture in the dark for a longer amount of time; ideally for a minimum time of two weeks. I had doubted that harsh light had the ability to trigger encystment on photo autotrophic life form from the beginning, yet I believed that the heat generated by this harsh light would bring about unfavorable conditions in the sub culture and eventually lead to encystment. Now I realize that it will

take a source of light far more powerful than the light on my windowsill (which I had strongly believed would suffice) to generate such conditions. I had based this simulation off of research found at, where evidently proof of euglena encystment in strong illumination had been achieved. The Low Moisture Simulation simply failed to trigger excystment of the Euglena after successful suspended animation achieved from the dehydration. Originally, I thought that I had managed to stimulate Euglena Gracilis into encysting and forming Mucilagenous Palmella Cysts. Palmella

cysts are cysts containing up to 64 individual euglena. They are formed by the Euglena excreting a gelatinous mucilage to form a "public" cyst, which any euglena can enter or leave (F. Hindak et al, 2000). Under a microscope, it resembles a group of cells together within a "bubble made up of ooze". I had observed something incredibly similar, and thought that I had successfully triggered excystation in my subcultures. Apparently what I had been observing was the cytoplasm being crushed out of the cells under the weight of the coverslip after all of the water supporting its weight had dried. This is the reason depression slides are used throughout my experiment instead of normal slides; the indentation in the middle prevents the euglena from being crushed by the weight of the cover slip. Euglena gracilis and Euglena viridis Mucilagenous Palmella cysts were successfully formed in a neustonic film

experiment by F. Hindak et. al in 2000. This work was incredibly useful to me in my experiment, providing me with the background information I needed to perform my experiment. However, even after reviewing their work my results remain inconsistent with Hindak's. Over all, my results did not manage to support my original hypothesis. Yet this was was merely because I was not able to replicate conditions necessary to trigger encystment; further research necessary to prove cyst formation must involve more time as well as greater background information on the topic. However, my data still relates and contributes to answering the original question in that if Euglena are capable of forming cysts, clearly the conditions used in this experiment are not conditions that will trigger encystment in future research (at least not to the extent applied in this experiment).

Image2:AmicroscopicpictureofagroupofEuglenatogetherinamucilaginousbubblewhichI originallythoughtwasaPalmellacyst.Ilaterfoundoutthatitwasactuallythecytoplasmbeing crushedoutofofthesecellsundertheweightofthecoverslip.

Conclusion In this experiment I attempted to prove the possibility for protozoans (in this case Euglena gracilis) to form microbial cysts under three different unfavorable environment simulations: Low Moisture, Harsh Light (heat) and Starvation. My original hypothesis was that cyst formation in Euglena is possible, and while my results did not directly disprove my hypothesis, they have changed the overall goal of my experiment from forming cysts to contributing to the overall effort of the scientific community to prove them caused by my surprising results. The results and data I acquired from my simulations were unexpected and did not support my hypothesis. For the low moisture simulation, all rehydrated Euglena remained in a state of suspended animation and did not excyst. For both the heat simulation and the starvation simulation, the euglena subcultures were unaffected by the trials and never entered a state of suspended animation; they remained in their regular trophozoite life stages throughout the experiment. Nonetheless, my results contributed to answering the general question in that if Euglena gracilis and other protozoans are capable of forming cysts, the micro-environmental unfavorable conditions simulated in this experiment are not the conditions to be used in future research. References: Center for Disease Control and Prevention. Gov., 1 Mar. 2011. Web. 5 June 2013. <>. Kiss, J. Z., et al. "X-Ray and Dissolution Studies of Paramylon Storage Granules from Euglena." PDF. 1987. Web. 1 June 2013. < ers/lreso/l118.pdf>. Chen, S., Assist Prof., D. H. MacDonald, Prof., and J. E. Kurle, Assist Prof. "The Soybean Cyst Nematode." SoyBeans UMN., 2001. Web. 1 June 2013. < 5.pdf>. "Reproduction in Euglena." N.p., 31 Aug. 2010. Web. 1 June 2013. <>. Diaz, Hugo Aguilar, Julio Cesar Carrero, and Raul Arguello Garcia. "Cyst and encystment in protozoan parasites: optimal targets for new life-cycle interrupting strategies?" Trends in Parisitology., 19 July 2011. Web. 1 June 2013. < stract/S1471-4922(11)00118-8?script=true>.