It’s the End of the Frogs as We Know it?

by BP

Figure 1:  Recorded distribution of Batrachochytrium dendrobatidis infections,

with red indicating recorded sites, white indicating negative sites, and blue

indicating negative or unknown sites. Image from BD-Maps.   

In recent history, worldwide amphibian populations have been on a sharp decline, in a trend that points towards extinction for many species. In some part, this is due to habitat loss as humans use up resources and rapidly change ecosystems, but the primary cause of this extinction points towards an emerging fungal parasite with a wide host range. In the early 1990’s, people found dead frogs infected with fungal zoospores across Australia. An experiment in 1999 identified this fungal species in the skin of infected amphibians, and named it Batrachochytrium dendrobatidis (Bd) [1]. Since then however, more deceased amphibians were being discovered across the Americas, Europe, Africa, and other regions of the world, all infected with the same kind of fungus [Figure 1]. Bd is so frightening to modern scientists because it is highly transmittable, and has been described as the single worst infectious disease among vertebrates based on the number of species affected and the rates of population decline [2]. These massive declines in amphibian populations are a major problem because most of the species affected serve important roles to their ecosystems. Because of this, scientists have been rushing to find the mechanism behind this disease and focus efforts on preventing its spread.

So what is this disturbing fungal parasite? Well, Bd is a chytrid fungus that infects the keratin-rich skin of amphibians, and causes the disease known as chytridiomycosis. Bd has a life cycle that involves two stages [3]. First, as a zoospore, Bd is mobile, and can detect and move towards various macromolecules required for life on the skin of a frog, in a process called chemotaxis. Once settled, it matures into a reproductive body called a zoosporangium, which releases more zoospores to either re-infect the host or infect nearby amphibians [Figure 2]. As zoospores accumulate in the host, symptoms start to appear. These symptoms include both physical and behavioral changes, as the frog’s skin becomes irritated and start to shed unnaturally, and frogs start to behave abnormally, losing their ability to right themselves, and failing to eat or seek shelter properly [4].

Figure 2:  Batrachochytrium dendrobatidis zoosporangium, which can open and

release zoospores for further infection. Image from University of New Hampshire.

All these symptoms can obviously leave hosts vulnerable to predators and the elements, but more often than not, amphibians die from the parasite because of how it disrupts homeostasis in the host’s skin. A study in 2009 by Voyles et. al looked at what the mechanism is by which Bd kills [5]. Their results showed that the physiological changes in amphibian skins, caused by Bd, are what kill the hosts. Most amphibians use their skins as a sort of gateway, which they can use to either “breathe” air in, or allow transport of water across to “drink” it. When their skins suffer from the symptoms of infection, it disrupts the ability of sodium and potassium pumps to maintain electrolyte balance. Without the function of these pumps in their skin to maintain homeostasis, they are effectively killed off either by cardiac arrest, as happens in tree frogs, or by suffocation, as seen in some other amphibian species [5].

Not all is hopeless for our amphibian friends, however, as some species have demonstrated resistance to infections by Bd. Originally, scientists suspected that some predisposed genetic trait may be the cause of this resistance. Some studies, however, have discovered ways that these species have shown resistance even in the presence of lethal amount of Bd cells. A recent study by Woodhams et. al looked at the survivability of four different amphibian species, and found two things [6]. First, they found that the number of certain innate immune system cells, rather than lymphocyte numbers, that differed between the susceptible and resistant members of one species. They also found that more members of species were resistant when there were more effective antimicrobial skin proteins. Taken together, these results suggest that the innate immune system in amphibians plays a strong role in resistance to Bd. Another study, by Ramsey et. al, came to a similar conclusion, finding that higher amounts of skin peptides and antibodies defended amphibians from Bd, showing that the resistant amphibian species likely have a stronger adaptive and innate immune [7]. These results suggest that the difference of amphibians in susceptibility to Bd comes from the strength of the immune response rather than a predisposed inability to be infected.

The big question circulating around concerned scientists right now is whether there is hope for amphibian populations to recover from these sharp population declines. The answer, unfortunately, seems to be uncertain at the moment. As previously stated, Bd is a highly transmittable fungus that has at this point already reached most corners of the globe. This makes it very difficult for uninfected populations to escape the spread of chytridiomycosis. Scientists are also concerned with resistant individuals carrying Bd to susceptible populations, furthering spread of the disease. The main hope is that the number of infected amphibians in a population doesn’t cross a threshold in which recovery is nigh impossible [8]. Even though the disease can infect about a third of amphibian species, the other hope is that Bd doesn’t evolve mechanisms to infect other amphibian species as well. Only time and further study can tell if there is hope for the future for amphibians. In the meantime efforts focus on how resistance can be attained for susceptible species, and preserving infected species from total extinction. The best thing that we can do, as people, is to try to prevent the spread of Bd ourselves when we are near wetland areas, where we can transfer chytrid fungi to the environments in which amphibians inhabit. Maybe with luck, nature will help us find a way to these vulnerable amphibians.


References:

1. Longcore, J. E., Pessier, A. P., & Nichols, D. K. (1999). Batrachochytrium dendrobatidis gen. et sp. nov., a chytrid pathogenic to amphibians. Mycologia, 219-227.

2. Gascon, C. (2007). Amphibian conservation action plan: proceedings IUCN/SSC Amphibian Conservation Summit 2005. IUCN.

3. Berger, L., Hyatt, A. D., Speare, R., & Longcore, J. E. (2005). Life cycle stages of the amphibian chytrid Batrachochytrium dendrobatidis. Diseases of aquatic organisms, 68, 51-63.

4. Padgett-Flohr, G.E. (2007). "Amphibian Chytridiomycosis: An Informational Brochure" (PDF). California Center for Amphibian Disease Control. Retrieved 12 November 2015.

5. Voyles, J., Young, S., Berger, L., Campbell, C., Voyles, W. F., Dinudom, A., ... & Speare, R. (2009). Pathogenesis of chytridiomycosis, a cause of catastrophic amphibian declines. Science, 326(5952), 582-585.

6. Woodhams, D. C., Ardipradja, K., Alford, R. A., Marantelli, G., Reinert, L. K., & Rollins‐Smith, L. A. (2007). Resistance to chytridiomycosis varies among amphibian species and is correlated with skin peptide defenses. Animal Conservation, 10(4), 409-417.

7. Ramsey, J. P., Reinert, L. K., Harper, L. K., Woodhams, D. C., & Rollins-Smith, L. A. (2010). Immune defenses against Batrachochytrium dendrobatidis, a fungus linked to global amphibian declines, in the South African clawed frog, Xenopus laevis. Infection and Immunity, 78(9), 3981-3992.

8. Vredenburg, V. T., Knapp, R. A., Tunstall, T. S., & Briggs, C. J. (2010). Dynamics of an emerging disease drive large-scale amphibian population extinctions. Proceedings of the National Academy of Sciences, 107(21), 9689-9694.

Living On Radiation, Fungal Adaptation in The Chernobyl Reactor

by MW

In 1991 Russian scientists published an article inspired by the observation of fungal growth within nuclear reactor no. 4 in Chernobyl, the site of the tragic meltdown in 1986. [1] Which irradiated the surrounding town making it unsuitable for human life. Since then, the site had been a hotbed of biological research into the effects of prolonged radiation on living organisms. The fungal growth that the researchers observed was within the reactor itself, the point with the highest level of radiation. The fungi appeared to be thriving in the unlikely environment; the radioactive water that was used to cool the reactor was blackened with growth. This rose questions into the radiodurability of these fungi, and how they were able to thrive is such a strange place. The authors of the article were forthcoming with a theory; that the fungi were using the radiation as an energy source, the same way some bacteria and all plants use sunlight. However, in this instance the authors hypothesized the fungi were accomplishing it with the pigment melanin as their chlorophyll. This was a grand theory, but was equally hard to prove. The effects of radiation on these fungi are still poorly understood.

Devastation at Reactor No. 4, Chernobyl.
Reactor where Fungi were observed.

The idea of radiotrophy is not so far-fetched, but is definitely odd by todays understanding of how most life works. The radiation from a nuclear reactor is extremely harmful to most organisms, given that it easily produces double-stranded breaks in DNA, which causes very damaging mutations. However, life on Earth has been exposed to various sources of radiation throughout history, sometimes for very extended periods of time. It’s not so crazy to believe that at some point, organisms evolved to not only cope with the danger, but also use it to their advantage.

First, let’s start with a quick review of what radioactive means. At its most basic level, radiation is the emission of some form of energy; this includes acoustic, electro-magnetic, and particle radiation. The types that come from things we think of as radioactive, like sites of nuclear reactor meltdown or (fission) nuclear weapons, are gamma and neuton radiation. The radiotrophic fungi are theorized to use the gamma radiation as an energy source, and seem resistant to lingering neutron radiation. Another name for gamma radiation is photon radiation, or light. However the type of gamma radiation coming from the reactor in Chernobyl isn’t visible to humans like the visible spectrum of light we are familiar with, its way more energetic and is closer to x-rays, or the cosmic radiation that bombards the atmosphere.

Normally, organisms use a variety of chlorophylls to harvest light and convert it to chemical energy. These compounds may blue, green, or several other colors, depending on the wavelengths, or energy level, of light they absorb. The process depends on the excitation of an electron in the pigment structure when a photon hits it, and the complex transfer of the energy to a chemical form. A similar process is theorized to take place with melanin, a dark pigment that appears black in high concentrations but in lower concentrations is what causes the appearance of a tan on human skin after some time is spent in intense sunlight. In most organisms, melanin is theorized to be a protective compound, helping to prevent damage to cells by UV light. However in the radiotrophic fungi, melanin is proposed to function a little like chlorophyll does for plants, as an antenna that gathers electromagnetic radiation allowing its conversion to chemical energy that the cell can use. Melanin is unique however in its ability to absorb much more energetic wavelengths than just visible and UV light.

a) Electron microscope image of C. neoformans.

b) C. neoformans stained with India Ink.

c) C. sphaerospermum. d) W. dermatitidis.

All Melanin-producing microscopic eukaryotes.

Three melanin-producing fungal organisms have been directly studied for radiotrophism in response to the Chernobyl observations, one of which was isolated directly from the reactor. Cladosporium cladosporioides, Penicillium roseopurpureum, and Penicillium hirsutum were exposed to gamma-radiation producing particles on a solid medium, and the direction of their growth evaluated. It was shown that even when alternative chemical energy sources were available in a different region of the medium, growth more often developed towards the radioactive source. [2] This however does not prove radiotrophism. The correlation between growth and radioactivity could be due to a number of causes, including any adaptive mechanism to protect the cells from the radiation. Could it be possible the organisms were creating a living shield? As an initial observation, the behavior is still very interesting. In a follow up study, melanized cells fared better than non-melanized cells when exposed to radiation and actually grew faster when exposed to radiation when compared to an identical culture grown without radiation. [2] However, this again does not prove that the melanized cells were deriving energy from gamma radiation. In the comparison between melanized and non-melanized cells exposed to radiation, the melanized cells would be expected to fare better due to the well known protective properties of the pigment. This however does not explain the increased growth of irradiated, melanized cells in comparison to non-irradiated, melanized cells.

To further explore the possible role of melanin in radiotrophy, researchers next inspected the properties of melanin as an energy-collecting molecule. Melanin is unique in its ability to quench free radicals in solution and absorb a wide spectrum of electromagnetic radiation, but its properties are still not fully understood. The researchers showed gamma radiation induced reductive potential in melanin, allowing it to reduce NAD+ to NADH, an electron shuttle used by every living organism that is central to energy metabolism. [3] This suggests a possible mode by which melanin may input energy in response to radiation. But still, a mechanism by which fungi can use melanin to collect energy has not been well characterized or even observed in a living system. The reductive potential of the compound alone is not sufficient to suggest a clear radiotrophy. The possibility of radiotrophism is still exciting, but needs significantly more research to confirm.

The current research is somewhat limited, but the implications of radiotrophy are exciting. Not only does it suggest an even wider range of conditions conducive to life, but it would add a powerful tool to bioengineering technology as well. Bioremediation of dangerous radioactive substances is only one of the useful innovations that might be made possible. Ultimately, radiotrophism may be the key to engineering organisms that can survive conditions that have since been inaccessible to bioengineering, such as the upper atmosphere, and the unshielded regions of orbiting space stations.

Articles Cited

(1) Zhdanova, N., Lashkom T., Vasiliveskaya, A., Bosisyuk, L., Sinyaveskaya O., Gavrilyuk, V., and Muzale, P. “Interaction of soil micromycetes with ‘hot’ particles in a model system.” Microbiologichny Zhurnal, 1991.

(2) Dadachova, E. and Casadevall, A. “Ionizing Radiation: how fungi cope, adapt, and exploit with the help of melanin.” Curr Opin Microbiol, 2008, DOI:10.1016/j.mib.2008.09.013

(3) Dadachova, E., Bryan, R.A., Huang, X., Moadel, T., Schweitzer, A.D., Aisen, P., Nosanchuk, J.D., and Casadevall, A. “Ionizing Radiation Changes the Electronic Properties of Melanin and Enhances the Growth of Melanized Fungi.” PLoS ONE, 2007, DOI:10.1371/journal.pone.0000457

Fusarium & trichothecene mycotoxins: Implications for use as biological weapons

by JS

The intentional use of infectious agents as weapons of war has been a heinous aspect of humanity’s past, with records dating as far back as 1155 AD (1). Even in the early history of the United States, the British used smallpox-infested blankets as biological weapons against the Native Americans, killing as many a 100,000 (1;2). Despite international treaties made in 1925 and 1972 that banned the use of biological weapons, advances in research and large-scale production of disease-causing agents exhibit daunting weapons for acts of terrorisms or use in warfare (3). One group of these biological warfare (BW) agents, trichothecene mycotoxins, pose a great threat to humans, agricultural crops, and, potentially, water supplies.

            Trichothecene mycotoxins have been found to be produced by five genera of fungi: Alternaria, Aspergillus, Claviceps, Penicillium, and Fusarium (4). Fusarium species have been identified to produce more than 40 trichothecene mycotoxins (5). The molecular structure of trichothecenes differ, but the presence of an epoxide group is responsible for their toxicity. Trichothecene toxins kill rapidly dividing cells by inhibiting protein synthesis after penetration of the skin (6). The toxin binds the ribosomal machinery used for translation and disrupts the activity of peptidyl transferase which normally forms bonds between amino acids to create proteins (6).

Although the CDC (8) does not have Fusarium, or the trichothecene mycotoxins they produce, listed as biological threats, there is contradicting evidence that the toxins have been used as weapons by the Vietnamese during the Korean War in 1981 (1). Analysis of “yellow rain” samples collected in Laos at this time revealed the presence of four trichothecene mycotoxins: T-2 toxin (T-2), nivalenol (NIV), 4-deoxynivalenol (DON) and diacetoxyscirpenol (DAS) (9). Skeptical scientists suggested that toxins present in the samples were produced by naturally occurring Fusarium, but Rosen and Rosen (9) countered this with the finding of synthetic material, possibly used as an emulsifier for the dispersal of toxins in water. Other reports indicate that trichothecenes have also been used as BW agents in Cambodia, Afghanistan, and Iraq (4).

            As a result of these biological threats, the U.S. Department of Agriculture began research on trichothecene toxins in 1984 (5). These toxins have been found to be the only BW toxin with cutaneous activity and manifestations, allowing for easier contraction of ailment and disease (10). Topical exposure of trichothecene mycotoxins causes blistering and death of skin (4). Studies of dermal exposure to trichothecenes often use solvents to enhance absorption (11), as was found in yellow rain samples from Laos (9). As far back as the 1800s, outbreaks of a human disease associated with consumption of Fusarium graminearum-contaminated grains occurred in Japan, China, Korea, and Russia (5). Ingestion of grains contaminated with F. sporotrichioides caused thousands of deaths in Russia during World War II (5). Ingestion causes alimentary toxic aleukia with symptoms including vomiting, diarrhea, inflammation, and hemorrhage (4;5;10). Creasia and colleagues (12) showed that inhalation is a more potent route of infection compared to ingestion. Inhalation of these toxins can cause death in humans within hours (10).

            Yet another difficult aspect of trichothecenes is their stability in water. In Cleveland, Ohio, fatal pulmonary hemorrhage in infants was thought to have been caused by trichothecene poisoning (10). The toxin was proposed to have come from sources growing in damp walls of insufficiently maintained homes. Trichothecenes have been shown to have stability in water at room temperature, specifically the T-2 toxin (4). Breakdown of the toxin does not occur rapidly enough to relieve health concerns over a seven day period. Although research has shown that cleanup is efficient, as removal from water using reverse osmosis is 99.9% efficient (4), the stability of trichothecene toxins still impose threats to persons and plants in exposed environments for an extended period of time.

Along with clinical symptoms in humans and animals and contamination of water, Fusarium species also have pathology in plants. Trichothecene-producing Fusarium can infect maize, wheat, rye, barley, and rice (13). However, the production of trichothecene toxins are not the sole cause of disease in plants. Fusarium oxysporum, for example, is found in soil throughout the world and pathogenic strains causes root rot in numerous plants, including agriculturally important crops (14). In 1973, the bioweapons program of the former Soviet Union was reportedly weaponizing Fusarium species to be used as anticrop agents (15). Through horizontal gene transfer, genetic information is exchanged between individual organisms allowing non-pathogenic Fusarium to become infectious (14). The easy transfer of pathogenicity poses a large issue for both intentionally released and naturally occurring Fusarium in our agricultural crops.

The threats to water, food, animal and human health make Fusarium and trichothecene mycotoxins potentially devastating weapons for use in warfare or terroristic crimes. Trichothecene mycotoxins have been shown to be deliverable as dusts, droplets, aerosols, or smoke (6), which greatly increases threats to to humans as topical exposure causes severe skin ailments and inhalation causes death. While warfare and acts of terrorism have become ongoing and often occurrences in our present world, the ability to protect ourselves, our food sources, and our water sources from BW attacks seems necessary.

References

1.     Frischknecht F. 2003. The history of biological warfare. Human experimentation, modern nightmares and lone madmen in the twentieth century. EMBO Rep 4 Spec No:S47–52.

2.     Brown T. 2006. Did the U.S. Army Distribute Smallpox Blankets to Indians? Fabrication and Falsification in Ward Churchill’s Genocide Rhetoric. Plagiary Cross-Disciplinary Stud Plagiarism, Fabr Falsif.

3.     Christopher GW, Cieslak TJ, Pavlin JA, Eitzen EM. 1997. Biological warfare. A historical perspective. JAMA 278:412–7.burrow

4.     Burrows WD, Renner SE. 1999. Biological warfare agents as threats to potable water. Environ Health Perspect 107:975–84.

5.     Desjardins AE. 2009. From yellow rain to green wheat: 25 years of trichothecene biosynthesis research. J Agric Food Chem 57:4478–84.

6.     Wannemacher RW Jr, Wiener SL 1997. Trichothecene mycotoxins. In: Medical Aspects of Chemical and Biological. Washington, DC:TMM Publications. 655-676.

7.     Cundliffe E, Davies JE. 1977. Inhibition of Initiation, Elongation, and Termination of Eukaryotic Protein Synthesis by Trichothecene Fungal Toxins. Antimicrob Agents Chemother 11:491–499.

8.     CDC. 2015. Bioterrorism Agents/Diseases: Emergency Preparedness & Response. Retrieved November 13, 2015, from http://emergency.cdc.gov/agent/agentlist.asp

9.     Rosen RT, Rosen JD. 1982. Presence of four Fusarium mycotoxins and synthetic material in “yellow rain”. Evidence for the use of chemical weapons in Laos. Biomed Mass Spectrom 9:443–50.

10.  McGovern TW, Christopher GW, Eitzen EM. 1999. Cutaneous Manifestations of Biological Warfare and Related Threat Agents. Arch Dermatol 135:311–322.

11.  Pang V. 1987. The toxicity of T-2 toxin in swine following topical application *1I. Clinical signs, pathology, and residue concentrations. Fundam Appl Toxicol 9:41–49.

12.  Creasia DA, Thurman JD, Jones LJ, Nealley ML, York CG, Wannemacher RW, Bunner DL. 1987. Acute Inhalation Toxicity of T-2 Mycotoxin in Mice. Toxicol Sci 8:230–235.

13.  Desjardins AE, Hohn TM, McCormick SP. 1993. Trichothecene biosynthesis in Fusarium species: chemistry, genetics, and significance. Microbiol Mol Biol Rev 57:595–604.

14.  van der Does HC, Lievens B, Claes L, Houterman PM, Cornelissen BJC, Rep M. 2008. The presence of a virulence locus discriminates Fusarium oxysporum isolates causing tomato wilt from other isolates. Environ Microbiol 10:1475–85.

15.  Wilson TM, Logan-Henfrey L, Weller R, and Kellman B. 2000. Agroterrorism, biological crimes, and Biological Warfare Targeting Animal Agriculture. In: Emerging diseases of animals. Washington, DC: ASM Press. 23-57.