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.

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.

Figure 2:  Batrachochytrium dendrobatidis
zoosporangium, which can open and
release zoospores for further infection.

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].
        

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.

From the Albert Einstein College of Medicine.

b) C. neoformans stained with India Ink. From

doctorfungus.org. c) C. sphaerospermum.

From discoverlife.org. d) W. dermatitidis.

From mold.ph. 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

GIARDIA: From Structure to Disease

by ET

Giardia lamblia is a flagellate protozoan, an intestinal parasite that commonly causes diarrheal disease throughout the world. The protozoan exhibit pear-shaped cells that are approximately 12-15 um wide. It is the common agent that causes most waterborne outbreaks of diarrhea in the United States, although it’s occasionally seen as a cause of food-borne diarrhea (1).
Infection is initiated through ingestion of Giardia lamblia cysts and numerous different strains may be found in one host during the course of the infection. About 10-25 cysts are capable of causing a disease with minor symptoms, however, ingestion of more than 25 cysts results in a 100% infection rate (3,4).

Giardia lamblia, cyst form   

The cyst morphology of this protozoan organism is smooth-walled, oval in shape and contains four nuclei. The cyst is relatively stable and can survive in a variety of environmental conditions for a prolonged time. In addition, it also survives acidic environments within the stomach and then initiates replication in the small intestine, where it causes symptoms of diarrhea and malabsorption. Upon excitation, a mature cyst releases 2 trophozoites. When the host is infected, thropozoites may appear in the duodenum within a few minutes. The major component of the cyst wall is N-galactosamine, which is produced by the enzymatic pathway that is induced during encystation (1,2).

The anatomical structure of this organism is quite unique. The ventral disk is an important component of Giardia lamblia cytoskeleton and is crucial in the survival of the organism in the intestine of the host. It is used by the organism for sucking, adhesion and holding abilities within the host. Giardia lamblia exhibits concave structure and covers the entire surface of the organism. The disk contains contractile proteins such as actin, myosin, and tropomyosin (2,3).

Also, the presence of the lectins on the surface of the organism ensures the attachment of the Giardia to the enterocyte within the host. Attachment to the host depends on the host’s active metabolism and is inhibited by temperature at 37C, high oxygen levels, and/or reduced cysteine concentrations. It is important to note that Giardia has nucleus and nuclear membrane, cytoskeleton and endomembrane system but it lacks other organelles such as nucleoli and peroxisomes that are nearly universal to eukaryotic organisms. Also Giardia are characterized by their lack of mitochondria and cytochrome mediated oxidative phosphorylation. They rely mostly on fermentation metabolism for energy conservation. Thus, glucose promotes growth of this microorganism but its not absolutely essential (1,2).

The development of good molecular classification tools and better understanding host specificity makes it reasonable to address the possibility of coevolution of different species or genotypes of this organism with their host. Several studies suggest that some genotypes of Giardia lamblia have a wide-ranging host specificity that includes humans while others appear to be more restricted and may not pose a risk of zoonotic transmission (1,2).

The mechanism by which Giardia causes symptoms of diarrhea and malabsorption are not yet fully understood. However, some recent research suggests that the damage of the endothelial brush border and endotoxins alter secretion and gut motility within the stomach. The damage of the endothelial wall occurs when Giardia lamblia releases substances that damage the intestinal epithelium. Also, lectins on the surface of the Giardia may contribute to the epithelial damage (4).

Giardia lamblia life cycle   

The life cycle of the Giardia lamblia is composed of two stages: the thropozite stage that freely exists within the human small intestine and the cyst stage, which is passed into the environment. No intermediate host is required within the life cycle. During the second stage excystation occurs in the stomach and the duodenum in the presence of acid and pancreatic enzymes. The thropozites pass into the bowel where they rapidly multiply within 9-12 hours. As they pass into the large bowel, encystation occurs in the presence of the neutral pH and bile salts. Then cysts are passed into the environment and the cycle is repeated again (1,2).

There are several types of transmission: person-to-person, water-borne and venereal transmission. Person-to-person transmission is mostly associated with poor hygiene and sanitation. Water-borne transmission is commonly associated with the ingestion of an unfiltered water surface. Venereal transmission types occur through fecal-oral contamination.

Giardia affects people of all ages. The infection is rare in newly-born infants, however, children that are older than 6 months have a higher susceptibility rate. High-risk groups for the infection include travelers who visit highly endemic areas, homosexual men and immuno-compromised individuals. Giardiasis is slightly more common in males than females. High incidence rates of the giardiasis are reported in daycare centers, institutions, and Native American Reservations.  The highest prevalence of the infection was documented in Western Nepal, Bangladesh, and Ethiopia.

The prognosis for the infected patients is generally excellent. Most patients are asymptomatic and most developing infections are self-limited. Also weight loss, disaccharide deficiency, and growth retardation are possible complications. This infection is rarely associated with mortality except in rare cases when extreme dehydration in infants or malnourished children occurs. Untreated giardiasis can last for weeks. The parasite persists in stool and re-infection is possible as well. Several antibiotic agents are available with good efficiency rates to shorten the course of the disease. However, drug resistance also has been observed in clinical experiments.

To prevent giardiasis, travellers should be educated regarding proper hygiene methods and symptoms of infection. Careful washing should be emphasized in order to prevent infection. Personal hygiene education to minimize person-to-person transmission in high-risk settings must be introduced.

References:

1.     Daly ER, Roy SJ, Blaney DD, et al. Outbreak of giardiasis associated with a community drinking-water source. Epidemiol Infect. Apr 2010;138(4):491-500.

2.     Adam, R. D. "Biology of Giardia Lamblia." Clinical Microbiology 14.3 (2001): 447-75.

3.     Farthing MJ. Giardiasis. Gastroenterol Clin North Am. Sep 1996;25(3):493-515

4.     Buret AG. Mechanisms of epithelial dysfunction in giardiasis. Gut. Mar 2007;56(3):316-7.