Organism: Entamoeba histolytica

by GP

Entamoeba histolytica is an anaerobic protozoan parasite that primarily infects digestive tract in humans and primates.  This pathogenic microorganism is the fourth leading cause of deaths and third leading cause of morbidity due to protozoan infections worldwide (1).  Transmission of E. histolytica occurs when a person ingests food or water that is contaminated with infected feces or during sexual intercourse (2).  This sort of transmission is more prevalent in developing countries with poor sanitary conditions.  The infection of E. histolytica is usually called amebiasis or amoebiasis (1,2).  Amebiasis occurs in the large intestines, which causes an internal inflammation. The internal inflammation is caused by trophozoites, which are infected cells that enter the intestines. The trophozoites come into contact with human cells, which induce a rapid influx of calcium in the cell.  This causes all membrane movement to stop.  The internal workings of the cell are disrupted, organelles lyse and, cells die. Consequently, the amoeba vacuums up the dead cell.

Figure 1. The Life Cycle

of E. histolytica

The course of infection for this parasite starts with mature cysts entering the human body, by ingesting mature cysts in fecally contaminated food or water.  Once inside the body, trophozoites develop in the large intestine and produce cysts. In some cases, the trophozotic cysts could remain undetected in the intestinal lumen and pass through by feces.  The cysts that exit the body by feces in cyst form can survive outside the body for several weeks.  In other cases, the trophozoites can pass the mucosal barrier or enter the bloodstream that causes the infection to occur (2, Figure 1). The symptoms of the disease include diarrhea and abdominal pain for mild cases.  For severe cases of the disease the symptoms may include stomach pain, blood and mucus in feces, and high fever (2).

Figure 2. Entamoeba histolytica with four nuclei

Often E. histolytica is misdiagnosed or mistaken for other similar infections.  When diagnosing amebiasis, stool samples are usually taken and it is difficult to distinguish if the infection is caused by E. histolytica due to the similarity to other infections and its symptoms as well.  However, a way to determine the presence of the parasite is to examine the number of nuclei (Figure 2). For example, the mature cysts have four nuclei, which is a telltale sign of Entamoeba histolytica cysts. Other than stool samples, enzyme-linked immunosorbent assay (ELISA) can be used to evaluate the sensitivity level of the parasite in cells.  ELISA is a useful tool in medicine to measure the interaction between antibodies and antigens from foreign microorganisms. ELISA is used to examine the trophozoites because at this stage in the cell cycle of Entamoeba histolytica, they are in the intestinal tract and can be easily extracted than cysts from stool samples.  The trophozoites are treated with different detection kits for ELISA and the level of expression of each kit are further analyzed in PCR, and this helps to know which substrate and substance identify the pathogenic parasite.  Thus, knowing which enzyme substrate and serum substances to use for identifying Entamoeba histolytica, the diagnosis of the disease, amebiasis, cannot be confused by other pathogens that share similar characteristics.  Stool samples examined under microscopy only show that parasites are present, but cannot tell what type of parasite it is.  Treating amebiasis with proper diagnosis and antibiotics, such as auranofin, can save lives and inform the public about how the parasite spreads. Diagnostic steps can be taken, like using stool samples and ELISA, to ensure the right diagnosis is made.

After diagnosing the patient with amebiasis, there are several treatments for Entamoeba histolytic, but the rise of antibiotic resistance is a major concern.  The treatment of amebiasis involved metronidazole, which E. histolytica is now resistant to (1) (Oh NO!).  Have no fear; new drugs are being made to target specific sites of amebiasis.  Drug screen tests have identified auranofin as an effective drug against the parasite.  This drug is originally used for treating rheumatoid arthritis.  To understand how the drug is targeting E. histolytica, transcriptional profiling and thioredoxin reductase assays show the drug targets the parasite’s thioredoxin reductase.   Thioredoxin is a protein that acts like an antioxidant and helps reduce other proteins by cysteine thiol-disulfide exchange. This is an important biological process because preventing thioredoxin reduction in E. histolytica means disulfide bonds in cells are not reduced.  Since the disulfide bonds are not reduced, sensitivity to reactive oxygen species (ROS) is increased in which cell structures can be damaged (1).

Along with disrupting thioredoxin reductase, the role of adherence to mucosal membrane is another target site for drugs (3).  As mentioned above, the course of infection mainly pertains to the large intestines, thus the parasite seems to adhere and degrade the mucosal barriers.  Research by Ravdin et al. found that the adherence of E. histolytica to target cells requires microfilament function, which lyse cells, and has a specific amebic receptor that is attracted to N-acetyl-d-galactosamine (GALNAc) (3).  The adherence of amebas to a functional group, CHO, on cells show carbohydrate specificity and it is a key component of the contact-mediated killing of target cells.  GALNAc inhibits the amebic cytolysis of target cells, so cells cannot be killed.  Since the cells cannot be killed, the parasite cannot adhere to the mucosal barriers and cannot enter the bloodstream (3,4).  To target the adherence site and adherence receptors on Entamoeba histolytica and cell surface, respectively, drug therapies can be made to specifically target that region, like using GALNAc.

All the aspects of Entamoeba histolytica is known in terms of being the first human amoeba to have its genome sequenced and analyzed.  The Sanger Institute is the first to sequence the genome and with their help, further insight of the amoeba parasite can provide evolutionary DNA markers and mechanisms of infection.  The human amoeba parasite, Entamoeba histolytica, infects 50 million people around world and 70,000 deaths occur.  All hands are on deck to fight Entamoeba histolytica and prevent misdiagnosis.

Citations

1. Sharon L Reed, et al. "A High-Throughput Drug Screen For Entamoeba Histolytica Identifies A New Lead And Target." Nature Medicine 18.6 (2012): 956-960. Academic Search Premier. Web. 15 Nov. 2012.
2. Centers for Disease Control and Prevention. Centers for Disease Control and Prevention, 02 Nov. 2010. Web. 15 Nov. 2012. 
3. Ravdin, Jonathan I., and Richard L. Guerrant. "Role of Adherence in Cytopathogenic Mechanisms of Entamoeba Histolytica." Journal of Clinical Investigation 68.5 (1981): 1305-313. Print.
4. Barbara J. Mann, et al. "Identification Of Entamoeba Histolytica Thiol-Specific Antioxidant As A Galnac Lectin-Associated Protein." Molecular & Biochemical Parasitology 127.2 (2003): 113. Academic Search Premier. Web. 15 Nov. 2012.
5. Mirelman, David. "Comparison of Use of Enzyme-linked Immunosorbent Assay-based Kits and PCR Amplification of RRNA Genes for Simultaneous Detection of Entameba Histolytic and E. Dispart." Journal of Clinical Microbiology 35.9 (1997): 2405-407. Print.

But it was just a dip in the lake: a look into the parasitic disease Schistosomiasis

by KS#1

You know the feeling you get after a Thanksgiving meal when your stomach is so full it makes your abdomen appear to protrude with a “food baby?”  Now imagine that same feeling, without being full.  In fact, imagine it while feeling starved, malnourished, and dehydrated.  These are a few of the symptoms of schistosomiasis; a disease caused by eukaryotic microbes from the Schistosoma genus. Why should we care?  Let’s imagine for a minute that we are 8 years old again. The summer temperature is scorching and we are craving nothing more than a dip in the lake out back.  Now, here in the US and in other highly developed countries, that would be fine.  However, in some regions of Africa, our 8 year old selves have just subjected our bodies to possible infection and subsequent schistosomiasis. Schistosomiasis is one of the most infectious parasitic diseases in the world, second only to malaria (1). Approximately 240 million people are infected each year (2), most commonly through unsanitary conditions and contaminated fresh water sources.  Unfortunately, that includes most lakes and rivers in southern and sub-Saharan Africa. Even the Nile River has been known to play host to the infectious larvae of schistosomes (1).  It should come as no surprise then, that children are the most commonly infected age group, as they are the most likely to play in contaminated waters. 

Figure 1: The life cycle of Schistosomes.  The human is the location of

 the adult blood fluke.  The fluke then lays eggs which are excreted into the

 environment to hatch into miracidium.  These miracidium can then  infect

 snails, where transformation from miracidium into sporocytes can occur.

  The sporocytes give rise to cercariae (the larval stage) which can swim

 freely in fresh water to infect humans through skin penetration.

Of clinical isolates, the two most commonly isolated schistosomes are Schistosoma haematobium and Schistosoma mansoni, both found in warmer climates with most infectious cases deriving from African populations (1).  The life cycle of these blood flukes is fairly simple, rotating between the human – where the adult fluke is found – and the snail, the intermediate host (3). The blood fluke miracida (the pre-larval hatched form of Schistosomas) infect snails where the fluke transforms into sporocysts.  These give rise to cercaria (infectious larvae) which can then survive in fresh water to infect humans through skin penetration.  Now, our bodies are well equipped to battle most pathogens, and if the initial burrow site causes any inflammation, the cercaria are destroyed by various immunological pathways.  Unfortunately, should our bodies not react to the initial infection, the cercaria make their way to the lungs or liver, where they develop into the adult form.  It is from there that they migrate into the intestinal (S. masoni) or urinary (S. haematobium ) tracts to lay their eggs.  The eggs are then excreted back into the environment to hatch into miracida and the cycle begins again (1, Figure 1).   

Going back to our example, simple childhood tendencies have given rise to a blood fluke navigating its way through our bodies. What does this mean as a patient? We can expect to develop a rash at the site of infection which is typically mild and often goes untreated.  In 1-2 months the symptoms have progressed and we begin to experience fever, chills, cough and muscle aches - common influenza symptoms.   Since viral infections pass, no drastic action is taken and only the symptoms are treated. A year or so later, we now experience abdominal pain, difficulty passing urine, and blood in the stool or urine.  These symptoms clearly indicate something is wrong and a trip to the doctor is arranged. The doctor sends urine and stool samples to the lab to check for the presence of eggs and sends a blood sample for serological analysis.  The urine and blood tests come back positive for infection with S. haematobium.  In some rare cases, eggs can be deposited in the brain or nervous tissue which can lead to severe migraine-like headaches, sporadic paraphasia, and brain lesions.  In such a case, diagnosis may need to occur through surgical excision of the lesions and biopsy (4).  Luckily, this time the fluke appears to have settled solely in the urinary tract. The doctor prescribes Praziquantel, the only anti-helmintic medicine available to treat helminthes infections.  Symptoms subside and diagnostic tests reveal we are no longer infected with the parasite. 

Great! Right…? Well, yes, but there are drawbacks to this route of treatment.  Unfortunately, Praziquantel is only a treatment, not prevention.  Subsequent infections may still occur upon exposure to more contaminated water.  Currently, there is ongoing research to find a suitable candidate for vaccine production against these parasites in hopes of combating this truly unfortunate disease.  Most recently, Lochmatter et al. discovered that immunization of mice with the first extracellular domain of SmTOR (S. mansoni tetraspanning orphan receptor) led to a decrease in adult worm burden.  Immunization also led to the production of anti-rSMTORRed1 (the first domain name) antibodies which may act as a memory response to any further inoculation with the parasite (5).  Lochmatter et al.’s paper is set to be put to print in December 2012 so currently no data on re-inoculation is available. 

So what can be done in the meantime?  Prevention is most successful through avoiding any interaction with contaminated waters; just a splash can cause infection if the skin is weak or already injured.  If contaminated waters are the only water source in the area, small amounts should be boiled for at least 1 minute and cooled before consumption or bathing. Control of the snail populations can also be an effective measure of prevention. However, any chemical treatments of contaminated waters may harm other species in the area, and if not continued, could allow re-inhabitation of the snails (1).  While we wait for a better solution, the best that community education can do is inform the public about these parasites and the subsequent disease, and hope that parents don’t let their children go for that dip in the lake.

References

1.     Global Health – Division of Parasitic Diseases and Malaria. Parasites – Shistosomiasis. Centers for Disease Control and Prevention.  Last updated 7 November 2012.  Accessed 14 November 2012.

2.     World Health Organization. Schistosomiasis. 2012. Accessed 14 November 2012.

3.     Mitreva, Maedonka.  The genome of a blood flue associated with human cancer. Nature Genetis. 27 January 2012. 44:116-118.

4.     Imai, Kentaro; T. Koibuchi, T. Kumagai, T Maeda, Y. Osada, N. ohta, M. Koga, H. Nakamura, T. Miura, A. Iwamoto, and T. Fujii.  Cerebral schistosomiasis due to Schistosoma haematobium confirmed by PCR analysis of the brain specimen.  Clinical Journal of Microbiology. October, 2011. 49(10):3703-3706. 

5.     Lochmatter, C.; C.L. Schneider, K. Ingram, J. Keiser, and J.A. Schifferli. Schistosoma masoni tetraspanning orphan receptor (SmTOR): a new vaccine candidate against schistosomiasis. Clinical & Experimental Immunology. 1 November 2012 (online publication, set for release in print December 2012).  170(3):342-357.

Heteromita globosa: The Antarctic Sewer Scrubber

Heteromita globosa and its flagella

by AB

Antarctica is home to icebergs, penguins, and…soil flagellates? With average temperatures as low as -80°F and no trees, flowering plants or nutrients, it is difficult to understand how any microorganism could survive in such a cold environment.  Studies show, however, that Antarctica is home to a range of terrestrial microbiota called psychrophiles that can operate at extremely cold temperatures.  These microorganisms not only survive the cold temperature fluctuations, but they fulfill important functions such as maintaining other microbial populations and preventing biofilm build-up. According to H.G. Smith, the microbiota that exists in this environment consists of those that can adapt physiologically in order to survive, grow, and reproduce in unfavorable climates (1).

The “small (5-20 Ixm), nonpigmented bacteriophagous heterotroph” Heteromita globosa is the most prevalent protozoan soil flagellate in the fell-fields of Antarctica (See Figure 1) (2). H. globosa implements a unique form of feeding and locomotion in which its flagella produces currents to bring in essential nutrients and swim from place to place. While these psychrophiles perform optimally at temperatures as low as 1.5°C, cold temperature-induced encystment, or enclosure in a cyst form, occurs below 1.5°C and excystment, or breaking out of the cyst, occurs slightly above 1.5°C (See Figure 2). Due to the extreme temperature fluctuations and thermal periods in Antarctic terrestrial environments, H. globosa can readily adapt, allowing them to survive the freeze and thaw cycles (2).

Heteromita globosa in a cyst

The combined effort of this organism’s psychrophilic adaptations for excystment and encystment, ability to feed off nutrients brought in by the flagella, and mobility make it successful.  Compared to other microorganisms of the same environment, H. globosa confers “greater mobility, growth rates and synchrony of temperature-induced excystement at low sub-optimal temperatures”, or temperatures below 1.5°C (2). From a practical perspective, H. globosa is a huge consumer of bacteria in biofilm and microbial communities.  This trait makes it an important component of biogeochemical cycling in the ground waters of Antarctica as well as a source of carbon transfer and nutrient regeneration (3).

In order to conserve this cycle of encystment and excystment, H. globosa implements specific mechanisms to maintain the fastest growth while competing with similar microorganisms (3). In a study, H.G. Smith compared the population growth rates of H. globosa with the ciliated Protozoa Colpoda cucullus.  While C. cucullus has not been isolated in the Antarctic zone, like H. globosa, it can survive in extremely dry and cold temperature-fluctuating conditions.  When growing H. globosa at sub-optimal temperatures, it was found that the growth rate was slower in conditions favorable to other soil flagellates. Comparing these low temperatures to C. cucullus showed that this organism was not able to withstand the same temperature fluctuations as H. globosa. In fact, C. cucullus could not grow in temperatures below 5°C while H. globosa grew in temperatures slightly lower than 1.5°C. The fact that H. globosa was able to withstand these cold temperature fluctuations demonstrates its versatility as a microorganism. Compared to other microorganisms that are found in similar environments, H. globosa has the mechanisms to best adapt to the Antarctic environments, making it a microbial force to be reckoned with.

In addition to housing the mechanisms needed to survive the frigid seasons in Antarctica, H. globosa is a significant biofilm reducer in aquifers at cold temperatures.  As previously stated, protozoan soil flagellates are the primary consumers of bacteria not only in Antarctica but in several environments. Bacteria clog the tiny pores spaces in aquifers inhibiting sewage disposal, “microbe-enhanced oil recovery, groundwater recharge, and in situ bioremediation” (4). They form biofilms in these pores by expelling exopolymer slime, creating insoluble biogas, and gathering into a biomass.  H. globosa grows in contaminated aquifers along with bacteria, producing nutrients that “stimulate bacterial metabolism” in order to cause biodegradation (4). H. globosa reverses the effects of bacterial biofilms by grazing on these biofilms and increasing porosity and permeability in these aquifers. In doing this, they induce bioremediation, or removal of pollutants, inhibiting the biofilm-forming bacteria from clogging the pores.

These findings have prompted environmental researchers to consider H. globosa as an important mediator of biofilm reduction.  By studying the relationship between bacteria and protozoan soil flagellates like H. globosa, new trategies that increase the effectiveness of these aquifers in cold regions like Antarctica can be improved.  It has been found that the bacterivorous protozoa like H. globosa “coexist with bacteria [by] thriving on organism contaminants on the subsurface” (4). This method of feeding allows H. globosa to remineralize components that bacteria generate, causing biodegradation and destruction of biofilms produced.  This is a huge advantage to bioremediation projects including maintaining hydrolytic conductivity in aquifers that are normally clogged with bacterial biofilms.  By inducing aquifers with H. globosa as well as other protozoan soil flagellates, bacterial biofilms will be reduced, maintaining hydrolytic power and proper sewage disposal.

While it is difficult to understand why anything would prefer to inhabit extremely cold, temperature-fluctuating regions and live in sewage systems, evidence shows that H. globosa contains the microbial machinery to thrive in this type of environment. Its unique flagellar feeding mechanisms, psychrophilic behavior, and ability to degrade bacterial biofilms enable it to be the most prevalent protozoan soil flagellate in Antarctica. So while most Antarcticans will spend the winter season in front of the fireplace, H. globosa will be scrubbing the sewers.

References:

1. H.G. SMITH . 1996. Diversity of Antarctic terrestrial protozoa. Biodiversity and Conservation (5). [cited 2012 Nov 15] 1379-1394. Available from: http://www.springerlink.com/content/t2xr52876rl78j67/fulltext.pdf?MUD=MP
2. H. G. Smith a1, J. Hughes a1 and S. J. Moore a1 . 2004. Growth of Antarctic and temperate terrestrial Protozoa under fluctuating temperature regimes. Antarctic Science. [cited 2012 November 15] Volume 2 / Issue 04 /pp 313-320. Available from: http://journals.cambridge.org/action/displayAbstract?fromPage=online&aid=222941
3. Denis V. Tikhonenkov 1 and Yuri A. Mazei 2 . 2006. Heterotrophic flagellates from freshwater biotopes ofMatveev and Dolgii Islands (the Pechora Sea). Protistology (4) [Internet]. [**Last Updated**, cited 2012 November 15] (4), 327337. Available from: http://www.egf-penza.ru/kaf/zoo/mazei/pdf/32.pdf
4. Richard G. Mattison, Hironori Taki, and Shigeaki Harayama. 2002. The Bacterivorous Soil Flagellate Heteromita globosa Reduces Bacterial Clogging under Denitrifying Conditions in Sand-Filled Aquifer Columns. Applied and Environmental Microbiology. [cited 2012 November 15] 68(9): 4539–4545. Available from: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC124123/

Euglena gracilis: A Closer Look

by Tina Soltani

Take a deep breath. As you inhale, oxygen enters into your lungs and carbon dioxide is then exhaled. Organisms like us require this oxygen for our everyday function. Most of us are generally aware that oxygen comes from trees and plants. But what many people are probably not aware of is that many microbes are also responsible for the oxygen we breathe. Through photosynthesis, phytoplanktons, such as diatoms and dinoflagellates, and euglenoids, like Euglena gracilis, contribute to the oxygen supply found on Earth. E. gracilis’s ability to photosynthesize allows it to consume carbon dioxide and release oxygen, which we then breathe. With carbon dioxide levels rising every year and contributing to global warming, microbes, such as E. gracilis, are essential in combating this issue. A species as versatile as this deserves a more in-depth analysis. Now take another breath and read on. 

Euglena gracilis in its free-living flagellated state (8)    

Euglena gracilis are free-living flagellated protists and contain chloroplasts; they are not known pathogens. They are part of one of the most primitive eukaryotic groups, the euglenoids. They are primarily found in freshwater habitats, but they can also inhabit marine and soil environments. E. gracilis tends to favor more alkaline environments; neutral or acidic environments are not conducive for nutrition or reproduction, although they can withstand such conditions (1). An interesting fact about E. gracilis: it is both chemoheterotrophic and photoautotrophic, meaning that it can not only consume organic molecules around it but it can produce its own food source as well. As a chemoheterotroph, E. gracilis is able to utilize a number of organic molecules such as ethanol, lactate, glucose, lactose and malate as carbon sources. As a photoautotroph, it utilizes carbon dioxide as its carbon source when undergoing photosynthesis (2,3). One of the most fascinating attributes of E. gracilis is its reaction to light beyond food production. E. gracilis is well known for its ability to lose chloroplasts when grown under constant darkness conditions, but then regain this ability when exposed to light again. When grown in the dark, they lose their chlorophylls and the chloroplasts regress to form proplastids; upon exposure to light they can re-differentiate chloroplasts (3).

There is no known sexual reproduction in E. gracilis. They usually reproduce by longitudinal binary fission in either a free-swimming or encysted state. If conditions are favorable, reproduction usually occurs in the free-swimming state, whereas if the environment is too hot or lacks water or the proper nutrients, E. gracilis resorts to an encysted state and remains that way until conditions become favorable again (1). The genome of E. gracilis has not been fully mapped yet. There has been difficulty conclusively classifying E. gracilis; they share similarities to protists, fungi animals and plants. Santos Ferreira et al were able to determine using expressed sequence tags (ESTs) of E. gracilis cDNAs that about 61% of these ESTs had similarities with proteins of known function. A breakdown of the known 61% of ESTs revealed that these proteins have similarities to different group classifications – 36% protist, 21% plant, 2% animal, 1% fungi and 1% prokaryote (4). E. gracilis is thought to have an endosymbiotic relationship with green algae, possibly due to the consumption of a eukaryotic alga. E. gracilis is well suited for the study of endosymbiosis and endosymbiotic gene transfer (EGT) because its plastid was acquired by secondary endosymbiosis, but no remains of the endosymbiotic nucleus is present (5). Endosymbiosis (or primary endosymbiosis) is when an organism lives inside another organism or its cells. Secondary endosymbiosis, then, occurs when an organism engulfs another organism that has underdone primary endosymbiosis. This loss of nucleus from the algae suggests that E. gracilis has transferred key genetic material from these algae, and a separate nucleus within E. gracilis is no longer required. The occurrence of EGT makes it difficult to map out a phylogenetic tree, and is certainly the case for E. gracilis. Phylogenetic trees are designed to reflect scenarios of vertical evolution with descent from a single common ancestor; E. gracilis has two (5).

Current levels of atmospheric CO2 levels as

of October 2012 (9)

E. gracilis is also a key component in biological carbon dioxide fixation. Because of its high tolerance of carbon dioxide, E. gracilis is one of the many favorable microalgae considered for projects centered on carbon dioxide sequestration (6). Chae et al used E. gracilis as a model by which to study the effects of elevated carbon dioxide and how it affects the ability to photosynthesize. They found that E. gracilis could withstand an environment containing up to 40% carbon dioxide. E. gracilis can also convert carbon dioxide to oxygen at a much more rapid rate than some other photosynthetic microbes (2,7). This suggests E. gracilis as one of the possible solutions to global warming attributed by the increase in carbon dioxide emissions.

Experiments by Santos Ferreira et al and Chae et al are just the beginning in understanding the complexity of E. gracilis and how, despite extensive research, there is still much unknown regarding Euglena species. Further research in their genomic sequence can give scientists further understanding in how their genetic makeup has evolved over time and why it shares so many similarities to other types of organisms found in other kingdoms. It can also highlight at what point in time E. gracilis began its endosymbiotic relationship with green algae. Additional investigations in its ability to withstand such high amounts of carbon dioxide and still be able to function relatively well can offer a better understanding of a possible evolutionary adaptation to climate change. Because current carbon dioxide levels are progressively increasing, photosynthetic organisms like Euglena are crucial for carbon fixation. No matter what the future holds, thanks to Euglena gracilis, we can all breathe a little easier.

References:

1. Tannreuther, George W., 1922. Nutrition and Reproduction in Euglena. Zoological Library, University of Missouri. Mit 52 Textabbildungen, 367-383.

2. Euglena and Euglena gracilis. <http://en.wikipedia.org/wiki/Euglena> and <http://microbewiki.kenyon.edu/index.php/Euglena_gracilis>

3. Regnault, Annie, Francoise Piton and Regis Calvayrac, 1990. Growth, Proteins and Chlorophyll in Euglena Adapted to Various C/N Balances. Phytochemistry, Vol 29(12): 3711-3715.

4. dos Santos Ferreira, Veronica, Iara Rocchetta, Visitacion Conforti, Shellie Bench, Robert Feldman and Mariano J. Levin, 2007. Gene expression patterns in Euglena gracilis: Insights into the cellular response to environmental stress. Gene 389:136-145.

5. Ahmadinejad, Nahal, Tal Dagan and William Martin, 2007. Genome history in the symbiotic hybrid Euglena gracilis. Gene 402:35-39.

6. Ono, Eiichi and Joel L. Cuello. Selection of optimal microalgae species for CO2 sequestration. <http://www.netl.doe.gov/publications/proceedings/03/carbon-seq/PDFs/158.pdf>

7. Chae, S.R., E.J. Hwang and H.S. Shin, 2006. Single cell protein production of Euglena gracilis and carbon dioxide fixation in an innovative photo-bioreactor. Bioresource Technology 97:322-329.

8. Figure 1 http://www.britannica.com/EBchecked/media/5411/Euglena-gracilis

9. Figure 2 http://www.co2now.org