Friday, December 9, 2016

Beauveria bassiana: Pathogenic Pesticide

Pesticides. Toxic, unhealthy, chemicals. The word is increasingly becoming associated with concerns regarding chemical residues on produce as well as toxic runoff and groundwater contamination. Although chemical pest control has proven to be effective, it is clear that the side effects of widespread chemical treatments have negative impacts on both the environment and human health. Current legislation does not even require potential toxins to be listed on pesticide labels, and agricultural runoff is vastly unregulated (1). Acquired resistance of insects to these chemicals additionally undermines their applicability as sustainable pesticides. However, there are safer alternatives to chemical pesticides; one such option being the fungus Beauveria bassiana. That’s right. A fungus used as a pesticide, specifically called a microbial pesticide. Microbial pesticides are a type of biopesticide that utilize living organisms to control pests. Microbial pesticides present significant benefits over chemical pesticides as they are nontoxic to humans and wildlife and are specific to a particular group of insects (2).
Beauveria bassiana is a species of ubiquitous soil fungus and insect pathogen capable of growing in a variety of climates (3). Its multifaceted life cycle allows B. bassiana to transition between these lifestyles (3). One stage of this life cycle allows the fungi to produce spores capable of infecting insects, causing white muscardine disease (3). This disease is characterized by a white, spore-covered mold that grows from inside the body of the infected insect. B. bassiana is also found in a wide range of geographical locations, likely due to its high natural genetic variability between populations (4). It is thought that this variability also contributes to the diversity of insects that strains of B. bassiana are able to infect. The variety of potential species-specific isolates makes this fungus an attractive candidate to control a variety of pests.
B. bassiana’s mode of infection also contributes to its potential for large scale use as a biopesticide. Unlike common chemical pesticides, which must be directly consumed by insects, B. bassiana can infect insects simply by coming into contact with them. The ease of insect infection by B. bassiana presents a benefit of using the fungus as an alternate means of insect control. During the infectious stage of its life cycle, the fungus produces spores that adhere to and grow on the bodies of insects (3). Once mature, the fungi can enter the insect using a combination of mechanical pressure and secretion of specialized enzymes to degrade the outer layer of the insect (3). As previously mentioned, the enzymes secreted vary based on the species of insect infected, highlighting the versatility of B. bassiana as a microbial pesticide. Once inside the insect, B. bassiana switches its growth to single celled structures called blastospores (3). These blastospores are able to circulate through the body of the insect, utilize its nutrients, and secrete toxins that kill the host (3). Interestingly, the blastospores are able to shed components of their cell surfaces to evade detection by the insect’s immune system (8). The components shed include receptors which likely allow for recognition by the host immune system (8). After the death of the insect, its body is again ruptured so the fungus can release spores to initiate a new infection (3).
 B. bassiana has a host range of over 700 insect species including crop pests, ecologically hazardous pests, and insect disease vectors such as mosquitos and ticks (3, 5). A vector is an organism that spreads infection by transmitting pathogens between hosts. Although B. bassiana has a wide host range, the capability of the fungus to infect different hosts depends on the specific strain. To target a specific pest using B. bassiana as a microbial pesticide, it is possible to isolate a strain of the fungus capable of infecting that particular pest. The ability of strains of this fungus to infect such a wide variety of insects is likely due to host-specific gene expression. In fact, it has been found that approximately 2500 genes are differentially expressed by B. bassiana based on the environment in which it is grown (6). Recently, a group of researchers grew B. bassiana on samples of media containing extracts of different crop pests. They determined that the enzymes produced by B. bassiana differed depending on the media on which they were grown (6). These findings are important because they show that B. bassiana can regulate its gene expression to live in different environments and infect different hosts, making it a marketable microbial pesticide. Determining the genes involved in this differential expression can allow us to elucidate virulent strains of B. bassiana that are specific to particular species of insects. A virulent organism is one that is capable of causing disease in another organism. The more virulent the strain, the more effective it will be as a microbial pesticide.
The wide host range of B. bassiana strains also allows the fungus to infect human disease vectors, providing a potential solution to chemical pesticide-resistant insects. Over time, mutations in the DNA of the insects can result in individuals that become resistant to a particular chemical insecticide. These individuals survive the toxic chemicals, and are able to mate and expand the population of resistant insects. At this point, a new chemical has to be developed to inhibit the resistant populations of insects. Microbial pesticides offer an alternative to the multitude of chemicals to which insects are already resistant. It was determined that B. bassiana is capable of infecting and killing species of insecticide-resistant Anopheles arabienses mosquitos (7). A. arabienses are the mosquitos that infect humans with the bacteria that cause malaria. These mosquitos become difficult to control as they develop resistance to chemical insecticides. This finding eliminates the possibility that resistance to insecticides also confers resistance to fungal pathogens (7).
Progress is currently being made to increase the applicability of B. bassiana as a viable biocontrol agent. To use the fungus as an insecticide, labs develop and collect the infectious spores to incorporate into a formula to be applied to plants. Solid-state fermentation has been identified as the most efficient way to produce these spores. Using this method, the fungi are grown on media saturated with water and plant byproducts (9). Solid-state fermentation is advantageous over other methods in that it is relatively simple, cost effective, energy-efficient, and uses less water (9). Production methods that use less water yield spores with higher shelf lives (9). Ideally, streamlining the industrial production methods of B. bassiana as a microbial pesticide will help increase its appeal for use in large scale insect control.
Although microbial pesticides offer a nontoxic, species-specific approach to insect control, there are potential issues to using living organisms to control pests. The majority of these concerns involve manufacturing and storage procedures of the pesticides, which are currently being refined. Microbial pesticides are also typically more susceptible to heat and desiccation, and therefore proper timing and application procedures are especially important (2). Additionally, the species specificity of microbial pesticides often results in the need to utilize several different pesticides to control additional pests in the area (2). The insect selectivity of microbial pesticides also has the potential to limit the market for their use as biocontrol agents (2).
In all, the fungus Beauveria bassiana expresses many characteristics that make it an appealing alternative to potentially harmful chemical pesticides currently used to control insects. A variety of species-specific strains found in a multitude of climates make isolates of B. bassiana suitable candidates for use against both agricultural pests and human disease vectors. An efficient and sustainable method of microbial pesticide production has been determined. Genes associated with pathogenesis are being studied to identify particularly virulent strains of B. bassiana. The research presented indicates that the widespread success of this fungus as an insect pathogen can translate into its potential as an effective biopesticide.


Citations

1.     Joe Magner. “Is Water Getting Cleaner? An Introduction to Water Quality.” Department of Biosystems & Bioproducts Engineering. 2016. Web.

2.     Canan Usta. “Microorganisms in Biological Pest Control — A Review (Bacterial Toxin Application and Effect of Environmental Factors).” Current Progress in Biological Research. N.p., 2013. Web.

3.     Claudio A. Valero-Jimenez et al. “Genes Involved in Virulence of the Entomopathogenic Fungus Beauveria bassiana.” Journal of Invertebrate Pathology 133 (2016): 41–49. Web.

4.     P. Maurer et al. “Genetic Diversity of Beauveria bassiana and Relatedness to Host Insect Range.” Mycological Research 101.2 (1997): 159–164. Web.

5.     Sandhya Galidevara, Anette Reineke, and Uma Devi Koduru. “In Vivo Expression of Genes in the Entomopathogenic Fungus Beauveria Bassiana during Infection of Lepidopteran Larvae.” Journal of Invertebrate Pathology 136 (2016): 32–34. Web.

6.     Akbar Ali Pathan et al. “Analysis of Differential Gene Expression in the Generalist Entomopathogenic Fungus Beauveria Bassiana (Bals.) Vuillemin Grown on Different Insect Cuticular Extracts and Synthetic Medium through cDNA-AFLPs.” Fungal Genetics and Biology 44.12 (2007): 1231–1241. Web.

7.     Christophe K Kikankie et al. “The Infectivity of the Entomopathogenic Fungus Beauveria Bassiana to Insecticide-Resistant and Susceptible Anopheles Arabiensis Mosquitoes at Two Different Temperatures.” Malaria Journal 9.71 (2010): n. pag. Web.

8.     J. C. Pendland, S. Y. Hung, and D. G. Boucias. “Evasion of Host Defense by In Vivo-Produced Protoplast-Like Cells of the Insect Mycopathogen Beauveria Bassiana.” Journal of Bacteriology 175.18 (1993): 5962–5969. Web.

9.     Pham, Tuan Anh, Jeong Jun Kim, and Keun Kim. “Optimization of Solid-State Fermentation for Improved Conidia Production of Beauveria Bassiana as a Mycoinsecticide.” Mycobiology 38.2 (2010): 137–143. PMC. Web.

Thursday, December 8, 2016

ALGAE...IN SPACE!

by SH

A few billion years after the Earth first formed, algae started producing oxygen through a process known as photosynthesis. It would take a couple more billion years for enough oxygen to accumulate and contribute to the formation of the ozone layer. This layer of the atmosphere is what protects us from the sun’s lethal ionizing radiation. But how were the algae able to survive exposure to that deadly radiation before the atmosphere protected the Earth? Scientists may have partially answered that question by analyzing the results of experiments that involved sending algae into orbit. Amazingly, the researchers found that mutations in a gene that codes for proteins involved in photosynthesis allow the algae to survive, even with no atmosphere!
Figure 1: Structure of a chloroplast. Chlorophyll, the
pigment involved in photosynthesis, is contained in
the membrane of the thylakoids.
Before we get into how the algae grew in space, let’s do a quick review. Photosynthesis is a process by which plants are able to use electromagnetic energy from sunlight, carbon dioxide, and water to make glucose for chemical energy, with oxygen being released as a by-product (1). Plants and algae that use this process contain green organelles known as chloroplasts, which are shown in figure 1. The pigment chlorophyll, which is located in the membrane of the flat, sac-like structures called thylakoids in the chloroplast, absorbs blue and red light wavelengths of light (1). Green light is not absorbed but reflected, giving plants and algae their green color (1).
The chlorophyll pigments in the thylakoids are part of a photosystem, which captures and processes light photons (1). When photons of the blue and red wavelength come in contact with the chlorophyll, the energy created excites an electron, which transfers it to a high-energy state (1). This then energizes the chlorophyll and starts the process of photosynthesis in photosystem II (PSII), the first protein complex of the system (1). After a few more steps, a sugar is produced that the algae are able use for food.
Figure 2: Chlamydomonas reinhardtii. This is an
of the algae.
There are a few photosynthetic life forms that are considered model organisms for scientists to study. Model organisms are easily grown and maintained in a laboratory setting and are able to be experimented on. Chlamydomonas reinhardtii, as seen in the ECM image in figure 2, is a unicellular, photosynthetic alga that has been studied for many years. The photosystems in these algae are similar to those of vascular plants, and it has a cell wall, like plant cells, as seen in figure 3. The DNA contained in C. reinhardtii can be genetically modified, which makes it a good model for studying how the cell works. It is also easily grown and maintained on Tris-Acetate-Phosphate (TAP) medium in the lab (2). All of these things make C. reinhardtii an ideal organism to experiment on.
Figure 3Chlamydomonas reinhardtii. A
is colorized to show the different
parts of C. reinhardtii.
The PSII of C. reinhardtii has a reaction core that consists of two proteins, D1 and D2 (3). These proteins bind to chlorophyll to assist in the process of energy transfer in the photosystem (3). When exposed to harsh environments, it has been found that the D1 protein is damaged, which then causes damage to the PSII (3). An example of a harsh environment would be the exposure to ionizing radiation, like the type that the algae of early Earth were exposed to. Without the protection of the atmosphere, the membranes of cell organelles are damaged. As photosynthesis occurs in the thylakoid membrane, the radiation present in space affects how well the algae is able to perform photosynthesis (3).
Since the genome of C. reinhardtii was sequenced nine years ago, scientists are now able to make mutants to see how the absence of different proteins affects the algae. In 2007, a team of scientists from Germany and Italy collaborated on an experiment to see how PSII would be affected if C. reinhardtii colonies were exposed to the ionizing radiation of space without the protection of our atmosphere (3). The team made a C. reinhardtii strain that had a mutation in the psbA gene that encodes the D1 protein in the reaction center of the photosystem and exposed some of the cells to ultraviolet light (3). Keeping some of the cells on Earth for controls, colonies of C. reinhardtii were sent into space on a Foton pacecraft, as shown in figure 4, aboard a Soyuz capsule (3). The colonies were protected from the cold and vacuum of space, but were exposed to the ionizing radiation and weightless conditions (3). After fifteen days in space, the spacecraft was returned to Earth and the C. reinhardtii colonies were collected (3).
Figure 4: Foton spacecraft. C. reinhardtii colonies are housed in the red circular area on the magenta sphere.
The researchers found that even though the C. reinhardtii colonies were exposed to ultraviolet light that was five times higher than that on Earth and ionizing radiation that was not diffused by an atmosphere, many cells survived and were still able to perform photosynthesis (3). The cells that were exposed to ultraviolet light on Earth before the trip to space survived in better condition than cells that were not exposed (3). It appears that PSII is involved in capturing the energy from ionizing radiation and using it for photosynthesis, though more studies are needed to see if this hypothesis is true (3).
In 2013, a group of scientists from Germany, Italy, and the United States worked together on another experiment involving the PSII D1 protein in C. reinhardtii in the environment of space (4). In this experiment, Chlorophyll a, which produces fluorescence when energy is dispersed in the photosynthesis process, was monitored in real time while the algae were in space. This gave the scientists a better idea of how the algae reacted while in the harsh environment of space, rather than waiting to see if any algae survived after the spacecraft landed (4). The same mutation was made in the psbA gene that encodes the D1 protein in the reaction center of the photosystem as in the experiment described previously (4). Colonies of mutated and wild type C. reinhardtii were sent into space for fifteen days on a Foton spacecraft aboard a Soyuz capsule (4). After the spacecraft landed, the algae colonies were transferred to fresh TAP medium so that they could be analyzed (4).
The real time monitoring of C. reinhardtii colonies in space during the second experiment showed that the wild type strain’s photosynthetic efficiency dropped daily, though they were still able to perform the process (4). The mutated strain, however, was able to maintain a high level of PSII activity, showing a slight increase in fluorescence emission towards the final days of the spaceflight (4). When the colonies were returned to Earth, both the wild type and the mutated strains were able to grow in the lab, though the mutated strain grew better than the wild type strain (4). This showed that mutation to the psbA gene made the D1 protein less susceptible to damage from ionizing radiation (4).

Since science has not yet found a way to make a time machine, we are not able to definitively say what happened in Earth’s history. However, scientists are able to perform experiments that give us some idea of what the Earth was like billions of years ago. Both of these experiments have shown us that even without a protective atmosphere, Earth’s early algae colonies could survive. Thanks to the single cell algae and their by-product of oxygen, other life forms on this planet have since been able to grow and thrive.





References



1.    Freeman, S., Allison, L., Black, M., Podgorski, G., Quillin, K., Monroe, J., Taylor, E. (2014) Biological Sciences, custom edition for University of Minnesota. Pearson Learning Solutions, Boston, MA.


2.    Harris, E.H. (1989) The Chlamydomonas sourcebook: a comprehensive guide to biology and laboratory use. Academic Press, INC, San Diego, California.

3.    Bertalan, I., Esposito, D., Torzillo, G., Faraloni, C., Johanningmeier, U., Giardi, M.T. (2007) Photosystem II stress tolerance in the unicellular green alga Chlamydomonas reinhardtii under space conditions, Microgravity Science and Technology 19(5/6):122-127

4.    Giardi, M.T., Rea, G., Lambreva, M.D., Antonacci, A., Pastorelli, S., Bertalan, I., Johanningmeier, U., Mattoo, A.K. (2013) Mutations of photosystem II D1 protein that empower efficient phenotypes of Chlamydomonas reinhardtii under extreme environment in space, PLoS ONE 8(5):1-10, e64352

Another Health Issue You Can Get Without Ever Smoking

by JW

           My generation has grown up being told that smoking is bad for you and it causes countless ailments. One thing that no one thought to warn us about is microbes that can cause things such as acute respiratory distress syndrome, and the dimorphic fungus Blastomyces dermatitidis is one of those microbes. It was first found in Canada in 1910 but as also been found in Midwestern and southern United States as well as parts of Canada that are along bodies of water like the Great Lakes (1). Diagnosis and identification of this fungus is primarily done from a histological exam on sputum or bronchial washings where they are looking for a broad-based budding yeast with refractile cell walls (2). When asexually reproducing by budding, often the daughter cells are as large as the mother cell before detachment. Patients that come into a hospital with an infection of B. dermatitidis often aren’t diagnosed until it is too late. In a study from a rural area of northwestern Ontario, Canada, A 27-year old woman went to an emergency department with signs of labored breathing and decreased air entry into the left upper lung (2). The first few days of being in the hospital the patient was stable, but after five days the hospital staff requested that the patient be airlifted to an intensive care unit in another facility. After only being in the intensive care unit for a day the patient went into cardiac arrest, and the cause of death was determined to be from acute respiratory distress syndrome.
                        While some infections of B. dermatitidis result in hospitalization, most infected patients remain asymptomatic (3). A more common illness caused by B. dermatitidis is Blastomycosis and is caused by inhaling the fungus from the environment in its mold phase and it converts to a yeast phase in the lungs. Often patients that have symptoms of Blastomycosis aren’t correctly diagnosed because the illness can mimic those of bacterial pneumonia, acute respiratory distress syndrome, or tuberculosis. Symptoms of blastomycosis typically appear after two to six weeks of incubation of the yeast phase in the lungs. Most often it involves the lungs and approximately 50% of pulmonary infections do not require treatment, however blastomycosis can affect nearly every organ including the skin, bones, eyes, and others. Symptoms of the illness are only considered a confirmed illness when the symptoms persist even after multiple courses of antibacterial therapy. Patients with an acute case are often given antibacterial medications and experience improvement within two to three weeks. This improvement is actually from the self-limited nature of the infection and not from the treatment with antibacterial medications.  
            A survey in Wisconsin showed that the average annual incidence for blastomycosis was 40.4 per 100,000 persons in one county (3). However, mandatory public health reporting for this illness is not required everywhere. It is only required in six states in the United States and only two Canadian provinces, making the true rate of occurrence of this infection in humans unknown. However, while we don’t know the true infection rate in humans, we do know that B. dermatitidis also infects animals, and especially dogs. In the same areas of Wisconsin where they reported blastomycosis, they also reported canine cases to be high at the same rate. One report even showed that as many as a third of patients who had blastomycosis and owned a dog reported that their dog was diagnosed with blastomycosis, some times even before the patient themselves (3).
            While the fungus B. dermatitidis is in its mold phase in nature it produces conidia, which are spores that are produced from asexual reproduction and are placed at the end of branching filaments called hyphae. This structure is shown in the picture below. The conidia are aerosolized during activities where the soil or decaying wood are disturbed. There are occupational risk factors for the blastomycosis disease. People that live in densely wooded areas with moist soil, open bodies of fresh water, and those that spend a large amount of time in these outdoor environments are at a higher risk of inhaling the aerosolized conidia. There is a higher amount of cases reported in men than in women and that most likely has something to do with men having more of an exposure to the environment with jobs that require moving wood and farming.
http://thunderhouse4-yuri.blogspot.com/2012/12/blastomyces-dermatitidis.html
            The virulence of B. dermatitidis is difficult to evaluate given that the clinical course of blastomycosis is seen to be correlated with the amount of aerosolized conidia initially inhaled. Without consistency for the amount of inoculum inhaled in the cases reported, it is difficult to tell if the difference in the disease process is from the virulence of from the amount that was inhaled. This means that it is difficult to tell if acute respiratory distress syndrome is caused from a prolonged infection with B. dermatitidis conidia or if it the amount of conidia that the patient first encountered. Most cases of blastomycosis-induced acute respiratory distress syndrome follow weeks to months of symptoms for pneumonia before development as seen in the case study of the 27 year old woman from rural Ontario, Canada.
This map above shows the areas that B. dermatitidis is located at in blue. As shown from the multiple reports out of Wisconsin and the map above, the area of this state is surrounded by the Great Lakes along with having lakes in the state itself. The other factor of decaying wood being a source for conidia is also very common for Wisconsin as the state is 46% covered in trees. With the ecosystem like this in Wisconsin it’s no wonder that there is such a high reporting of blastomycosis (4). There are occupational risks for those working outside in the south and Midwestern regions of the United states and Canada because we still aren’t able to understand how the virulence of the fungus comes into play for how the infection of B. dermatitidis. It can either be a mild form of blastomycosis that is asymptomatic or a life threatening illness such as acute respiratory distress syndrome which leads to complications. While the dangers of smoking are still ever present, there are a few other things to watch out for that can cause lung problems, especially if you work outside in Wisconsin.


(1) Kroll, R. R., & Grossman, R. F. (2013). Pulmonary blastomycosis in a professional
diver: An occupational risk. Canadian Respiratory Journal : Journal of the Canadian Thoracic Society20(5), 340–342.
(2) Dalcin D, Rothstein A, Spinato J, Escott N, Kus JV. Blastomyces gilchristii as cause
of fatal acute respiratory distress syndrome. Emerg Infect Dis. 2016 Feb [11/5/16]. http://dx.doi.org/10.3201/eid2202.151183
(3) Castillo, C. G., Kauffman, C. A., & Miceli, M. H. (2016). Blastomycosis. Infectious
Disease Clinics of North America, 30(1), 247–64. doi:10.1016/j.idc.2015.10.002
(4) Natasha Kassulke, K. E. (2005). Fast forestry facts -- Wisconsin Natural Resources magazine -- December 2005. Retrieved from http://dnr.wi.gov/wnrmag/html/supps/2005/dec05/forest.htm