Category Archives: Evolution
25th December 2018
Translated from the original article in Catalan.
We humans are destroying the planet Earth. Besides climate change (there are still ignorant people who do not believe it), the depletion of natural resources and the massive extinction of animal and plant species, one of the most visual effects is the coverage of the planet with rubbish. Since 71% of the surface is marine, most of the non-degrading waste finishes in the sea. In the oceans there are already large expansions covered by floating debris, especially plastics, called “plastic islands” (Figure 1). In the North Pacific area, where different sea currents come together, the “island” reaches 1500 km of radius, with plastics up to 200 meters deep, and continues to grow. There is more information of it, and also about the environmental consequences, in the Wikipedia article Great Pacific garbage patch.
Figure 1. Small portion of the Great Pacific Garbage Patch (From oceanandreserveconservationalliance.com)
Although there are many types of plastics, one of the most used and most abundant in waste and “plastic islands” is polyethylene terephthalate, known as PET or PETE (Figure 2). It is a type of thermoplastic polymer, vulgarly plastic, which belongs to the so-called polyesters, and is obtained by synthesis from petroleum. It is harmless, very resistant and lightweight and has multiple applications (Figure 3). Counting only bottles of PET for refreshing beverages, 1 million of them per minute are sold in the world. It is a recyclable material (see Pet bottle recycling in Wikipedia) but very resistant to biodegradation. In nature it can last some hundreds of years.
Figure 2. PET, polyethylene terephthalate.
Figure 3. Several applications of PET (From http://www.technologystudent.com).
PET is “eaten” by Ideonella sakaiensis
I. sakaiensis (Figure 4) are bacteria with rod shape, gram-negative, non esporulate aerobic heterotrophic, mobile with a flagellum, and catalase (+) and oxidase (+) (Tanasupawat et al 2016). They grow at neutral pH and are mesophilic, with optimum at 30-37°C. They belong to the phylogenetic group of betaproteobacteria, which include, besides many others, the known Neisseria (gonorrhoea and meningitis) and the nitrifying Nitrosomonas.
Figure 4. Scanning electron microscope images (false colour) of Ideonella sakaiensis cells grown on PET film for 60 h (From Yoshida et al 2016).
The 201-F6 strain, the first of the new species I. sakaiensis, was isolated from a landfill and identified in 2016 by a Japanese group of the Kyoto Institute of Technology that looked for bacteria using plastic as carbon source, from samples of remains of PET bottles (Yoshida et al 2016). They saw that these bacteria adhere to a low-grade PET film and can degrade it, by means of two enzymes characterized by these authors: a PETase and a MHETase, which produce terephthalic acid and ethylene glycol acid (Figure 5), which are benign environmental substances and that the bacteria can be metabolized. A colony of I. sakaiensis completely degraded a low-grade PET bottle in 6 weeks. High-grade PET products need to be heated to weaken them before the bacteria can degrade them. This is the first bacterium found as a PET degrader, and uses it as the only carbon source and energy source. Since PET has existed only for 70 years, these bacteria should have evolved in this short period until being able to degrade PET in a few weeks, instead of hundreds of years in nature (Sampedro 2016).
Figure 5. Predicted metabolic pathway of PET degradation by I. sakaiensis: extracellular PETase hydrolyses PET giving monohydroxyethyl terephthalic (MHET) and terephthalic acid (TPA). MHETase hydrolyses MHET to TPA and ethylene glycol (EG). The TPA is incorporated through a specific transporter (TPATP) and is catabolized to cyclohexadiene and this to protocatechuic acid (PCA) by the DCDDH. Finally, the PCA ring is cut by a PCA 3.4 dioxygenase with oxygen, as known for degradation of phenolic compounds and other xenobiotics. The numbers in parentheses are the ORF of the corresponding genes (From Yoshida et al 2016).
Previously, only some tropical microfungi (Fusarium solani) were known to degrade PET, and they also excreted esterases. In this case, Fusarium would be used to modify the polyester fabric, to achieve more hydrophilic and easier to work (Nimchua et al 2008). It is important to remember the structural similarity of synthetic PET fabrics (Figure 3) to those of natural fibre such as cotton, since these contain cutin, which is a polyester, a waxy polymer from the external parts of the plants. Therefore, the enzymes of Fusarium or Ideonella must be relatively similar to those that were already in nature long before the plastics were invented.
Recent genetic improvement of the enzyme PETase of Ideonella sakaiensis
In order to better understand the function and specificity of the PETase, a group of American and British researchers have recently characterized the structure of this enzyme (Austin et al 2018), mainly by high resolution X-ray crystallography, comparing it with a homologous cutinase obtained from actinobacteria Thermobifida fusca. The main differences between the two have been a greater polarization in the surface of the PETase (pI 9.6) than in the cutinase (pI 6.3), and on the other hand (Figure 6), a greater width of the active-site cleft in the case of PETase of I. sakaiensis. The cleft widening would be related with an easy accommodation of aromatic polyesters such as PET.
Figure 6. Compared structures (left) of the PETase of I. sakaiensis (above) and the cutinase of actinobacterium Thermobifida fusca (below), obtained by high resolution X-ray crystallography (0.92 Å). The active-site cleft is marked with a red dotted circle. Details (right) of the active site with different cleft widths in the PETase of I. sakaiensis (above) and the cutinase of T. fusca (below) are shown. (From Austin et al 2018).
Hypothesizing that the structure of the active site of the PETase would have resulted from a similar cutinase in an environment with PET, Austin et al (2018) proceeded to make mutations in the PETase active-site to make it more similar to cutinase and obtained a double mutant S238F/W159H which theoretically would make the entry of the active site closer (Figure 6). But their surprise was capital when they saw that the mutant degraded the PET better (an improvement of 20%), with an erosion of the PET film (Figure 7 C) even greater than the original PETase (Figure 7B). The explanation was that mutant changes in amino acid residues favoured PET intake in the active site, despite making a closest cleft (Austin et al 2018).
Figure 7. Scanning electronic microscopy images of a piece of PET without microorganisms (A), after incubating 96 h with PETase of the I. sakaiensis 201-F6 (B), and with PETase of the double-mutant S238F/W159H (C) (From Austin et al 2018).
In addition, these authors have shown that this PETase degrades also other similar semi aromatic polyesters, such as polyethylene-2,5-furonicarboxylate (PEF), and therefore this enzyme can be considered an aromatic polyesterase, but it does not degrade aliphatic ones.
The conclusion of their work is that protein engineering is feasible in order to improve the performance of PETase and that we must continue to deepen in the knowledge of their relationships between structure and activity for the biodegradation of synthetic polyesters (Austin et al 2018).
Other plastic-eating microbes ?
The discovery of I. sakaiensis has been very important for the possibility of establishing a rapid recycling process for PET, but it is not the first organism that has been found as plastic consumer. By the way, we can see the formulas of the main plastics derived from petroleum in Figure 8.
Figure 8. Formulas of the most common petroleum plastics: polyethylene (PE), polyvinyl chloride (PVC), polypropylene (PP), polystyrene (PS), polyethylene terephthalate (PET or PETE) and polyurethane (PU) (From Shah et al 2008).
Reviewing the bibliography, we see that many cases of plastic degrading microorganisms have been described (Shah et al 2008), especially polyethylene, polyurethane and PVC: various Pseudomonas, Rhodococcus and Comamonas among bacteria, and some Penicillium, Fusarium and Aspergillus between fungi.
Among the polyurethane consumers, mushrooms are highlighted (Howard 2002), and especially the plants endophyte Pestalotiopsis microspora, which can use polyurethane as the only source of carbon (Russell et al 2011).
On the other hand, the ability of the mealworms, the larval forma of the darkling beetle Tenebrio molitor, to chew and degrade the polystyrene foam is well known (Yang et al 2015). Fed only with the PS, these larvae degrade it completely in relatively short periods. As expected, the degradation of the PS is carried out by the intestinal bacteria of the animal (Figure 9). It has been demonstrated because degradation stops when administering antibiotics to the larva (Yang et al 2015). One of the isolated bacteria that has been shown to degrade PS is Exiguobacterium, from Bacillales group, but it is not the only one. In fact, when performing studies of metagenomics from gut of larvae eating PS, a large variety of bacteria have been found, and these vary depending on the kind of plastic, since the degradation of polyethylene has also been seen. Some of the bacteria with DNA found as predominant would be the enterobacteria Citrobacter and Kosakonia. It seems that the intestinal microbiota of Tenebrio is modified and adapted to the different ingested plastics (Brandon et al 2018).
Figure 9. Biodegradation of polystyrene by the intestinal bacteria of Tenebrio, the mealworm (Yang et al 2015).
Finally, as we see the microbial biodegradation of non-biodegradable or recalcitrant plastics should not surprise us, since on the one hand, there are natural “plastics” such as polyhydroxybutyrate or polylactic acid that are easily degradable (Shah et in 2008), and on the other hand the adaptive capacity of the microorganisms to be able to break the most recalcitrant chemical bonds is very large. Microbes evolve rapidly, and acquire better strategies to break the plastics made by humans (Patel 2018). We have seen in this case the degradation of PET, which in less than 70 years some microbes have already found a way to take advantage of it.
The problem is that we are generating too much plastic waste in no time and the microorganisms have not had time yet to degrade them. It is clear that we will have to help our microbial partners, not generating more degrading polymers, and recycling and degrading them, by using these same degrading microbes, among other ways.
Austin HP et al (2018) Characterization and engineering of a plastic-degrading aromatic polyesterase. Proc Nat Acad Sci 115, 19, E4350-E4357
Brandon AM et al (2018) Biodegradation of Polyethylene and Plastic Mixtures in Mealworms (Larvae of Tenebrio molitor) and Effects on the Gut Microbiome. Environ Sci Technol 52, 6526-6533
Howard GT (2002) Biodegradation of polyurethane: a review. Int Biodeterior Biodegrad 42, 213-220
Russell JR et al (2011) Biodegradation of polyester polyurethane by endophytic fungi. Appl Environ Microbiol 77, 17, 6076-6084
Sampedro J (2016 marzo 10) Descubierta una bacteria capaz de comerse un plástico muy común. El País
Shah AA et al (2008) Biological degradation of plastics: a comprehensive review. Biotechnol Adv 26, 246-265
Tanasupawat et al (2016) Ideonella sakaiensissp. nov., isolated from a microbial consortium that degrades poly(ethylene terephtalate). Int J Syst Evol Microbiol 66, 2813-2818
Yang et al (2015) Biodegradation and mineralization of polystyrene by plastic-eating mealworms: Part 2. Role of gut microorganisms. Environ Sci Technol 49, 12087-12093
Yoshida et al (2016) A bacterium that degrades and assimilates poly(ethylene terephthalate). Science 351,1196–1199
March 20th, 2016
The Arctic Ocean
Interestingly and coincidentally, “Arctic” comes from the Greek word αρκτος -arctos-, which means “bear” and is a reference to the constellation Ursa Minor, where is the North Star, which indicates the geographic North Pole .
The Arctic constitutes a unique ecosystem of the Earth, consisting of a large ice field, or ice-covered ocean, sometimes regarded as the northern part of the Atlantic Ocean, and it is surrounded by land, which is permafrost, with complete absence of trees. Life in the Arctic consists of organisms adapted to ice, including zooplankton and phytoplankton, fish, marine mammals, birds, land animals, plants and human societies fully adapted to the extreme conditions of the environment.
Due to global warming, isotherms are moving northward at a rate exceeding 50 km per decade over the past 30 years, so if we define the Arctic from a defined temperature or the tree line, its size is diminishing, being the reduction of sea ice the most visible effect.
Anthropogenic climate change: global warming, especially in the Arctic
Yes: climate change is here and it is generated by human activities, that is, it is anthropogenic. Previously there have been on Earth fluctuations in global temperature caused by natural phenomena, usually long-term and cyclical variations. For example, glaciations since about 2 million years are repeated every 100,000 years, and last ice age ended 15,000 years ago. So we are living now in an interglacial period and the next ice age could become not before 50,000 years. The cause of this cycle of glaciations seems to be orbital variations of the Earth, resulting in a lower insolation in high latitudes of the northern hemisphere during glacial periods.
Solar activity, like other stars, has cycles and roughly every 600 years there are periods of little activity (absence of very few solar spots and auroras), with lower energy output, which corresponds to cold periods in the Earth’s climate. The last minimal was in the period 1645-1715, and therefore from the middle of the eighteenth century we enjoy a maximum solar activity, with small cycles of minimum and maximum every 11 years.
Discounting these natural variations, it is clear that throughout the 20th century and especially since the 1960s there has been a steady increase in global average temperature (Figure 1), reaching almost 1ºC more than the beginning of the 20th century. In the early years of the current century the trend is worsening. The last 10 years have been the warmest since there are records, and the forecast is to continue increasing. Most experts agree that humans exert a direct impact on the heating process known as the greenhouse effect. The causes of this effect are some of gaseous components of the atmosphere, especially CO2, which has grown in parallel with rising temperatures, from about 300 ppm at the beginning of 20th century to nearly 400 ppm today. This CO2 and other gases as water vapour, methane and other exclusively anthropogenic absorb radiation and the result is that the atmosphere warms further.
Figure 1. Increase in average global temperature compared to the beginning of 20th century (from GISTEMP).
This global warming is particularly evident in the Arctic. The temperature increases are higher in northern latitudes, especially 60-70º N, where this past December 2015 (Figure 2) have raised to 9ºC above average in large areas of North America and Eurasia. This is called Polar Warming Amplification (PWA). The cause of this overheating in the Arctic respect of the rest of Earth is partly due to the loss of snow and ice (retroactive effect) because the largest area of land and water absorbs more solar energy than white ice (albedo effect), but also the PWA is partly due to the dynamic atmospheric transport, which transports heat energy from the clouds and subtropical regions to the north (Taylor et al 2013).
Figure 2. Thermal anomaly registered in December 2015 with respect to the average 1951-1980 (from GISTEMP).
Besides the consequences of this warming on the Arctic ice that we will comment below, another serious problem is the melting of permafrost, since then methane gas trapped under the frozen ground is released. This way, vast quantities of methane are released, and this greenhouse gas is contributing further to accelerate the global warming.
Less and less ice in the Arctic
Linear trends of sea ice extent and sea ice in the Arctic from 1979 to date are negative year after year, for any month is considered, but it is more clear by comparing Septembers, at the end of the summer when the ice is melting (Figure 3). Of the approximately 7 million km2 minimum in September (the maximum in March is about 16 million), about 100,000 km2 are melt per year, almost 9% every 10 years (Serreze et al 2007), so that there is now almost half ice than in 1979 (Figure 4).
Figure 3. Comparison of the extent of sea ice (in red): September 1979 and 2012 (from The Cryosphere Today).
Figure 4. Average monthly extension of Arctic sea ice since 1979 (Reeves et al 2013).
In addition to the reduction in surface ice, keep in mind the reduction in volume, representing now a third of what it was in September 1979.
There is a big difference between the different models for predicting the disappearance of Arctic sea ice. Half of them expect the total disappearance by September 2100. Predictions move since September 2040 the less optimistic until well past 2100 for the other (Serreze et al 2007).
Other problems resulting from the disappearance of sea ice are the ship traffic, which could shorten distances trips between the ports of northern countries, and on the other hand the exploitation of oilfields and other fossil fuels and minerals, since there is a large part of global reserves in the Arctic (Figure 5).
Figure 5. Left: forecast paths for open sea ships (blue) and for icebreakers (red) for 2040-2059. Right: Distribution of the potential major reserves of oil and gas (yellow) and licenses (red) and wells in operation or to operate (black). The dashed line indicates the limit of Conservation of Arctic Flora and Fauna (CAFF) declared by the Working Group of the Arctic Council (www.arctic-council.org). Figures from Reeves et al (2013).
Ecological consequences of the disappearance of the Arctic ice pack
There are many living beings linked to the ice. The polar bears roam on the Arctic ice, so we are feared for his fate. Many fish, seals and crustaceans (krill) form a food chain that starts from the algae that grow under the ice in a very consistent environment, rich in nutrients, especially favourable for marine life (Figure 6 A). Moreover, floating sea ice in summer is a good corridor for dispersion of terrestrial vertebrates (for instance arctic foxes) and plants.
The gradual disappearance of sea ice and warming in the Arctic coast involves a series of ecological imbalances (Figure 6 B). We see for example how walruses forced to remain grouped on the ground are more predisposed to disease transmission. The loss of sea ice diminishes dispersion by ice corridors and then the land populations are most isolated, thus gene flow is restricted. Polar bears and other predators that hunt on the sea ice have it much harder and their populations are at risk. Phytoplankton productivity decreases significantly, thereby reducing zooplankton, and then the whole food chain (fish, seals, etc.) is affected (Post et al 2013).
Figure 6. Ecological interactions influenced by sea ice. A: The distribution and seasonality of sea ice affects the abundance, distribution and interactions of the entire ecosystem in balance. B: The longest period without ice and less sea ice extent have disastrous consequences on the balance of the ecosystem (Post et al 2013).
The polar bear tries to survive
The polar bear (Ursus maritimus) is considered an endangered animal. There are only about 25,000 worldwide. The impact of climate change affects the exclusive habitat of polar regions and forecasts suggest that in a few years from now the ice of the Arctic will melt permanently and polar bears may become extinct because of warming area.
The polar bear is basically carnivorous, unlike others such as brown bears, and remains above the ice hunting seals. With the gradual disappearance of the ice it has more trouble finding preys, and some have begun to learn how to catch salmon rivers, as we see in the images (Figure 7).
Figure 7. White Bear dedicated to fishing salmons in order to survive (www.youtube.com/watch?v=9m_Q9Ojbcmw).
We have also seen groups of polar bears at sea fishing (see video) and dive emerging alternately as if they were dolphins or porpoises. Despite these small adaptations, the food is very low and it is clear that their populations are declining rapidly.
Orcas thrive north
The disappearance of the northern ice is a dramatic ecological change that is causing the disappearance of some species like the polar bear, but interestingly these imbalances benefit some other emerging species. This is the case of the killer whale (Orcinus orca), which is thriving more and more to the north (Figure 8).
Figure 8. Places (marked with numbers) of the Canadian Arctic where groups of orcas were repeatedly photographed between 2004 and 2009 (Young et al 2011).
Eskimo Inuit people live around the American Arctic (from Quebec to Alaska including Hudson Bay and adjacent islands) and the west coast of Greenland, and they are the first witnesses since the mid-twentieth century observing whales in their waters, unknown before. Moreover, in recent years scientists have made numerous orca’ sightings, they have been photographed individually (Young et al 2011), and their travels have been followed through bioacoustics (Ferguson et al 2010) and other techniques.
Figure 9. (Top): Narwhals with the characteristic great tusk, which gave rise to the myth of the unicorn. (Low): Group of orcas attacking narwhals cornered on the beach. Watch the video of PBS Nature.
For some years attacks by orcas on narwhals (as in Figure 9) have been observed repeatedly by Inuit Eskimos and studied in detail by several scientists. Laidre et al (2006) observed that before approaching whales, the narwhals tend to group, are more quiet and swim closer to the beach in shallow waters. During the attack, the narwhals disperse significantly but nevertheless mortality is very high. After predation, which can last several hours, oily stains are observed in sea surface, which come from fat of depredated narwhals (Figure 10).
Figure 10. Group of orcas surrounded by patches of oil on the sea surface from the fat of attacked narwhals (Laidre et al 2006).
Orcas’ attacks on narwhals are so common and effective that are beginning to affect the population. The effects are even worse in other cetaceans with smaller population such as whales of Greenland or bowhead (Balena mysticetus), which are now virtually extinct (Figure 11).
Figure 11. Scheme of preys’ proportions by a group of orcas from Hudson Bay (Ferguson et al 2010).
In conclusion, anthropogenic climate change is affecting the Arctic ecosystem severely (and all the other ecosystems), and although this problem is becoming known, effective policy measures to reduce emissions of CO2 and other greenhouse gases are so scarce that hardly will arrive in time. We are leading the planet Earth to a massive extinction of species and ecological changes ever seen in the history of humans.
The picture says it all: polar bear habitat is running out.
Arctic Council: http://www.arctic-council.org
Ferguson S.H., Higdon J.W. & Chmelnitsky E.G. (2010) The rise of killer whales as a major Arctic predator. In S.H. Ferguson, et al. (eds.): A little less Arctic: top predators in the world’s largest northern inland sea, Hudson Bay. Pp. 117–136. New York: Springer
GISTEMP, Goddard Institute for Space Studies Surface Temperature Analysis (NASA-GISS): http://data.giss.nasa.gov/gistemp/
Hawkings E (2014) nov 28: http://www.climate-lab-book.ac.uk/2014/hiatuses-in-the-rise-of-temperature/
Laidre KL, Heide-Jørgensen MP, Orr J (2006) Reactions of narwhals, Monodon monoceros, to killer whale, Orcinus orca, attacks in the Eastern Canadian Arctic. Can. Field Nat., 120, 457–465
Morell V (2012) Killer whale menu finally revealed. http://www.sciencemag.org/news/2012/01/killer-whale-menu-finally-revealed
Post et al. (2013) Ecological Consequences of Sea-Ice Decline. Science. DOI: 10.1126/science.1235225: http://www.carbonbrief.org/knock-on-effects-for-wildlife-as-the-arctic-loses-ice
Reeves RR et al (2014) Distribution of endemic cetaceans in relation to hydrocarbon development and commercial shipping in a warming arctic. Marine Policy 44, 375-389
Serreze MC, Holland MM, Stroeve J (2007) Perspectives on the Arctic’s shrinking sea-ice cover. Science 315, 5818, 1533–6.
Taylor PC, Cai M, Hu A, Meehl J, Washington W, Zhang GJ (2013) Decomposition of feedback contributions to Polar Warming Amplification. J Climate 26, 7023-43
The Cryosphere Today: http://arctic.atmos.uiuc.edu/cryosphere/
Young BG, Jeff W. Higdon JW, Steven H. Ferguson SH (2011) Killer whale (Orcinus orca) photo-identification in the eastern Canadian Arctic. Polar Research Vol 30
It seems to be so: the microbes in our gastrointestinal tract (GIT) influence our choice of food. No wonder: microbes, primarily bacteria, are present in significant amounts in GIT, more than 10 bacterial cells for each of our cells, a total of 1014 (The human body has about 1013 cells). This amounts to about 1-1.5 kg. And these bacteria have lived with us always, since all mammals have them. So, they have evolved with our ancestors and therefore they are well suited to our internal environment. Being our bodies their habitat, much the better if they can control what reaches the intestine. And how can they do? Then giving orders to the brain to eat such a thing or that other, appropriate for them, the microbes.
Well, gone seriously, there is some previous work in this direction. It seems there is a relationship between preferences for a particular diet and microbial composition of GIT (Norris et al 2013). In fact, it is a two-way interaction, one of the many aspects of symbiotic mutualism between us and our microbiota (Dethlefsen et al 2007).
There is much evidence that diet influences the microbiota. One of the most striking examples is that African children fed almost exclusively in sorghum have more cellulolytic microbes than other children (De Filippo et al 2010).
The brain can also indirectly influence the gut microbiota by changes in intestinal motility, secretion and permeability, or directly releasing specific molecules to the gut digestive lumen from the sub epithelial cells (neurons or from the immune system) (Rhee et al 2009).
The GIT is a complex ecosystem where different species of bacteria and other microorganisms must compete and cooperate among themselves and with the host cells. The food ingested by the host (human or other mammal) is an important factor in the continuous selection of these microbes and the nature of food is often determined by the preferences of the host. Those bacteria that are able to manipulate these preferences will have advantages over those that are not (Norris et al 2013).
Recently Alcock et al (2014) have reviewed the evidences of all this. Microbes can manipulate the feeding behaviour of the host in their own benefit through various possible strategies. We’ll see some examples in relation to the scheme of Figure 2.
Figure 2. As if microbes were puppeteers and we humans were the puppets, microbes can control what we eat by a number of marked mechanisms. Adapted from Alcock et al 2014.
People who have “desires” of chocolate have different microbial metabolites in urine from people indifferent to chocolate, despite having the same diet.
Dysphoria, id est, human discomfort until we eat food which improve microbial “welfare”, may be due to the expression of bacterial virulence genes and perception of pain by the host. This is because the production of toxins is often triggered by a low concentration of nutrients limiting growth. The detection of sugars and other nutrients regulates virulence and growth of various microbes. These directly injure the intestinal epithelium when nutrients are absent. According to this hypothesis, it has been shown that bacterial virulence proteins activate pain receptors. It has been shown that fasting in mice increases the perception of pain by a mechanism of vagal nerve.
Microbes can also alter food preferences of guests changing the expression of taste receptors on the host. In this sense, for instance germ-free mice prefer more sweet food and have a greater number of sweet receptors on the tongue and intestine that mice with a normal microbiota.
The feeding behaviour of the host can also be manipulated by microbes through the nervous system, through the vagus nerve, which connects the 100 million neurons of the enteric nervous system from the gut to the brain via the medulla. Enteric nerves have receptors that react to the presence of certain bacteria and bacterial metabolites such as short chain fatty acids. The vagus nerve regulates eating behaviour and body weight. It has been seen that the activity of the vagus nerve of rats stimulated with norepinephrine causes that they keep eating despite being satiated. This suggests that GIT microbes produce neurotransmitters that can contribute to overeating.
Neurotransmitters produced by microbes are analogue compounds to mammalian hormones related to mood and behaviour. More than 50% of dopamine and most of serotonin in the body have an intestinal origin. Many persistent and transient inhabitants of the gut, including E. coli, several Bacillus, Staphylococcus and Proteus secrete dopamine. In Table 1 we can see the various neurotransmitters produced by GIT microbes. At the same time, it is known that host enzymes such as amine oxidase can degrade neurotransmitters produced by microorganisms, which demonstrates the evolutionary interactions between microbes and hosts.
Table 1. Diversity of neurotransmitters isolated from several microbial species (Roschchina 2010)
|GABA (gamma-amino-butyric acid)||Lactobacillus, Bifidobacterium|
|Norepinephrine||Escherichia, Bacillus, Saccharomyces|
|Serotonin||Candida, Streptococcus, Escherichia, Enterococcus|
Some bacteria induce hosts to provide their favourite nutrients. For example, Bacteroides thetaiotaomicron inhabits the intestinal mucus, where it feeds on oligosaccharides secreted by goblet cells of the intestine, and this bacterium induces its host mammal to increase the secretion of these oligosaccharides. Instead, Faecalibacterium prausnitzii, a not degrading mucus, which is associated with B. thetaiotaomicron, inhibits the mucus production. Therefore, this is an ecosystem with multiple agents that interact with each other and with the host.
As microbiota is easily manipulated by prebiotics, probiotics, antibiotics, faecal transplants, and changes in diet, controlling and altering our microbiota provides a viable method to the otherwise insoluble problems of obesity and poor diet.
Alcock J, Maley CC, Aktipis CA (2014) Is eating behavior manipulated by the gastrointestinal microbiota? Evolutionary pressures and potential mechanisms. BioEssays 36, DOI: 10.1002/bies.201400071
De Filippo C, Cavalieri D, Di Paola M, Ramazzotti M, et al (2010) Impact of diet in shaping gut microbiota revealed by a comparative study in children from Europe and rural Africa. Proc Natl Acad Sci USA 107:14691–6
Dethlefsen L, McFall-Ngai M, Relman DA (2007) An ecological and evolutionary perspective on human-microbe mutualism and disease. Nature 449:811-818
Lyte M (2011) Probiotics function mechanistically as delivery for neuroactive compounds: Microbial endocrinology in teh design and use of probiotics. BioEssays 33:574-581
Norris V, Molina F, Gewirtz AT (2013) Hypothesis: bacteria control host appetites. J Bacteriol 195:411–416
Rhee SH, Pothoulakis C, Mayer EA (2009) Principles and clinical implications of the brain–gut–enteric microbiota axis. Nature Reviews Gastroenterology and Hepatology 6:306-314
Roschchina VV (2010) Evolutionary considerations of neurotransmitters in microbial, plant, and animal cells. In Lyte M, Freestone PPE, eds; Microbial Endocrinology: Interkingdom Signaling in Infectious Disease and Health. New York: Springer. pp. 17–52
It is really surprising, but it seems so: Italian and Austrian researchers have published a paper (Campisano et al. 2014) which shows that the bacterial species Propionibacterium acnes, related to human acne, can be found as obligate endophytes in bark tissues of Vitis vinifera, the grapevine.
Some bacterial pathogens of humans, such as Salmonella, are able to colonize plant tissues but temporarily and opportunistically (Tyler & Triplett 2008). In fact, there is a temporary mutual benefit between plants and bacteria, so some of these enterobacteria pathogenic to plants do not live endophytically and can be beneficial for them. These pathogens to humans, in its life cycle, use plants as alternative hosts to survive the environment, passing to the plants through contaminated irrigation water. Therefore, some bacteria are often temporary endophyte guests of plants.
But on the other hand, there are relatively rare cases of bacteria changing the host and adapting to the new host, finally being endophytes. This horizontal transfer happens mostly between evolutionarily close hosts, such as symbiotic bacteria of aphids (insects), which has proven to transfer to other species of aphids (Russell & Moran 2005). It has also been suggested the horizontal transfer of beneficial lactic acid bacteria (Lactobacillus reuteri) in the intestinal tract of vertebrates, since strains of this L. reuteri are similar in several species of mammals and birds.
Well, going beyond, the work of Campisano et al. subject of this review, concludes that bacteria associated with human acne should have passed on the vine, that is, the bacteria would have made a horizontal transfer interregnum, from plants to mammals.
Propionibacterium acnes type Zappae
Acne, as you know, is a common human skin disease, consisting of an excess secretion of the pilosebaceous glands caused by hormonal changes, especially teenagers. The glands become inflamed, the pores obstructed and scarring appears. The microorganism associated with these infections is the opportunistic commensal bacterium P. acnes, a gram-positive anaerobic aero tolerant rod, which fed fatty acids produced by the glands.
Young with acne (Wikimedia, public)
Propionibacterium acnes at the scanning electron microscope (left) and dyed with violet crystal (right). From Abate ME (2013) Student Pulse 5, 9, 1-4.
Interestingly, other species of the same genus Propionibacterium well known in microbial biotechnology industry are used for the production of propionic acid, vitamin B12, and the Swiss cheeses Gruyere or Emmental.
Campisano et al. have made a study of the vineyard endomicrobioma by the sequencing technique (Roche 454) amplifying the V5-V9 hyper variable region of the bacterial 16S rDNA present in the tissues of vine. In 54 of the 60 plants analyzed, between 0.5% and 5% of the found sequences correspond to the species Propionibacterium acnes. This observation has been confirmed by fluorescent in situ hybridization (FISH) with fluorochromes and specific probes of P. acnes.
Location of P. acnes (fluorescent blue spots) in the bark of a vine stem, seen with FISH microscopy with specific probes for this bacterium (Campisano et al 2004).
The authors of this work proposed for this bacterium the name of P. acnes Zappae, in memory of the eccentric musician and composer Frank Zappa, to emphasize the unexpected and unconventional habitat of this type of P. acnes.
Frank Zappa (1940-1993), the eccentric and satiric singer, musician and composer. Photo: Frank Zappa reviews.
And how did this human bacteria arrive into the vineyard?
To solve this riddle, Campisano et al. have taken the 16S rDNA sequences and from other genes (recA and tly) from these strains of P. acnes Zappae found in vine and have compared with those P. acnes of human origin in databases. Comparing phylogenies and clusters deducted from them, these researchers have concluded that P. a. Zappae has diversified evolutionarily recently. Studying in detail the recA gene sequences of P. a. Zappae, and taking into account the likely mutation rate and generation time (about 5 hours), they deduce that the diversification from other P. acnes occurred 6000-7000 years ago.
This date coincides with the known domestication of the vine by humans, which is believed to have occurred about 7000 years ago in the southern Caucasus, between the Black Sea and the Caspian Sea, the area of modern Turkey, Georgia, Armenia and Iran (Berkowitz 1996). The vineyard has its origins in a wild subspecies of Vitis that survived the Ice Age and was domesticated. This plant came out to three subspecies, and one of them, Vitis vinifera pontica, spread in the mentioned area and further south in Mesopotamia and then to all south Europe thanks to the Phoenicians.
Therefore, the conclusion is that P. acnes Zappae originated from human P. acnes 7000 years ago, by contact of human hands with grapes and other parts of the vineyard during the harvest and carrying them. As the authors say, this case would be the first evidence of horizontal transfer interregnum, from humans to plants, of a obligate symbiotic bacterium. This also makes more remarkable the adaptability of bacteria. Their ability to exploit new habitats can have unforeseen impacts on the evolution of host-symbiont relationship or even host-pathogen.
Harvesting by hand in Chile (Fine Wine and Good Spirits)
Berkowitz M (1996) World’s earliest wine. Archaeology 49, 5, Sept./Oct.
Campisano Aet al. (2014) Interkingdom transfer of the acne-causing agent, Propionibacterium acnes, from human to grapevine. Mol Biol Evol 31, 1059-1065.
Gruber K (4 march 2014) How grapevines got acne bacteria. Nature News 4 march 2014.
Russell JA, NA Moran (2005) Horizontal transfer of bacterial symbionts: heritability and fitness effects in a novel aphid host. Appl Environ Microbiol 71, 7987-7994.
Tyler HL, EW Triplett (2008) Plants as a habitat for beneficial and/or human pathogenic bacteria. Ann Rev Phytopathol 46, 53-73.
Walter J, RA Britton, S Roos (2011) PNAS 108, 4645-4652.
A few weeks ago I saw the following headline in “Recercat” (the electronic newsletter of research in Catalonia): “Científics descobreixen com es formen les molècules bàsiques de la vida“, that is, “Scientists discover how the basic molecules of life are made”. Oops what a heading ! I was very surprised, of course, because I have always been passionate for this subject of life’s origin. So, I quickly read it, and looked for the original article .
Well, once seen in detail, it is clear that the heading is very, very exaggerated, as supposed at first glance. Before discussing why I think it is exaggerated, I want to mention another detail of this headline that I think is superflous: Why the word “scientists” ? Whoelse might be working on the study of the formation of the basic molecules of life besides scientists ? Politicians perhaps? or economists or maybe the bishops ? It is clear that must be scientists and therefore it is no need to say it. The heading would be enough like this: “It has been discovered how…. ” . Or else, it could tell where scientists are from: “English scientists, or American, or Japanese, have discovered … “.
Well, going to the specific discovery, and as Recercat itself summarizes, some Americans and British researchers (Martins et al. 2013) have published in Nature Geoscience their laboratory work, where they simulated the impact of comets on the surface of a planet, shooting a projectile with a compressed air pistol at speeds of 7 km/s (25,000 km/h). They have seen that by the impact and the heat generated, amino acids are synthesized from water, CO2 and ammonia. This is what they call “shock synthesis”. Among others, the amino acids detected were glycine, D-alanine, L-alanine, amino isobutyric acid, isovaline, norvaline and other precursors of amino acids, both isomers D and L. The amounts detected were between nanograms and one microgram.
This synthesis process shows that a simple mechanism, such as the impact of comets on the surface of a rocky planet, can transform basic inorganic molecules to more complex organic molecules such as amino acids, which are the monomers of proteins, basic constituents of all living beings. And therefore, this shock synthesis could have been a step in the appearance of terrestrial life.
Now, is this the first work to demonstrate the possible formation of basic molecules of life ? Well, absolutely not ! This work has its value but does not deserve this headline so exaggerated.
So, let’s briefly review what is already known, since more than 50 years, many different researchers have worked on this topic and have been discovering aspects that reinforce the scientific hypotheses of abiogenesis. This, also known as biopoesis, is the natural process by which living organisms originated on earth from simple molecules about 3,700 million years ago.
Biopoesis, the process of emergence of living beings on Earth
This process of biopoesis necessarily involved several steps:
1) The formation and appearance of basic organic molecules of living beings, it is, the monomers such as amino acids, monosaccharides, fatty acids and nitrogenous bases.
2) From the above monomers, the formation of biogenic macromolecules, it is, polysaccharides, polypeptides, lipoids and others, by polymerization, probably on the surface of inorganic substrates such as clay or iron minerals.
3) And the formation of the protobionts, the precursors of first cells, from macromolecules. This key step, the most difficult to prove, was probably linked to the parallel acquisition of the three basic properties of living beings: i) a structure envelope (the membrane) of lipidic consistency; ii) some reactions transforming nutrients and energy (rudimentary metabolism); and iii) the ability of transfering characteristics to the offspring (hereditary mechanism) with an information-carrying molecule, probably RNA.
By the moment I will leave the steps 2 and 3, maybe for discussing in a future post, and I will focus on the first one, related to the mentioned article of Martins et al. (2013). This formation and appearance of compounds in the early Earth may have been by three mechanisms: a) in situ production, b) contributions from abroad: c) synthesis due to impacts.
These three categories of mechanisms were already raised as an inventory of the possible origins of life in 1992 in an article of Nature by Christopher Chyba and Carl Sagan, the famous pioneer of exobiology and science writer, very known by the extraordinary TV series Cosmos, and author of the phrase “We are stardust“. Coincidentally, he was the first husband of Lynn Margulis, who spread the endosymbiotic theory of the bacterial origin of mitochondria and chloroplasts.
Endogenous synthesis of organic compounds on the primitive Earth
Well, as I said, it has been demonstrating since many years the possibility of the formation of organic molecules in situ, id est, endogenous synthesis in the primitive Earth, without external inputs. The Oparin’s hypothesis that the reducing anaerobic conditions of the early atmosphere, along with solar energy, should favoured the synthesis of organic molecules forming the “prebiotic soup”, was demonstrated as possible by the known experiments of Miller and Urey:
In 1952, the graduate student Stanley Miller with his professor Harold Urey introduced a mixture of water, hydrogen, methane and ammonia in a cyclical container, where electrical sparks were applied. A week later, when analyzing the components, they found that 15% of the carbon coming from methane had been transformed in various organic compounds, including 5 amino acids, both D-and L-.
Schema of the experiments of Miller and Urey. From GCSE-Bitesize (BBC).
Recently (Parker et al 2011) the tubes with the extracts of original Miller- Urey experiments have been analyzed again with current sophisticated analytical techniques and equipment, and many more compounds that the found originally in the 1950s have been discovered, in particular 23 other amino acids.
The synthesis of these organic molecules in the early Earth was probably facilitated by energy sources of the atmospheric activity such as electrical discharges that were used by Miller and Urey experiments, but there were other possibilities, such as the sunlight, with more UV radiation than at present (there was no ozone layer since it was formed later, from oxygen), and also more volcanic activity and radioactivity in a younger Earth, and impacts of meteorites and comets, which is related to the mentioned work of Martins et al. (2013).
Another important contribution in the search for prebiotic organic synthesis was the demonstration made by Joan Oró (born in Lleida, Catalonia) working at NASA (Oró 1961) that adenine can be synthesized heating ammonium cyanide solutions. Similarly, recently it has been demonstrated the synthesis of pyrimidines (cytosine and uracil), adenine and triazines (other nitrogenous bases) from urea by freeze-thaw cycles and electric shocks (Menor-Salván et al. 2007).
As demonstrated by Joan Oró, adenine can be synthesized from 5 molecules of hydrogen cyanide. Adenine is a key molecule for life, since it is part of nucleic acids and ATP.
Contribution of organic molecules by extraterrestrial objects
In addition to the in situ synthesis of organic compounds in the primitive Earth, they could also have come from outside. The contribution of organic molecules by extraterrestrial objects, comets or meteorites or other, it is increasingly more evident scientifically. Recent studies suggest that the so-called massive bombing that took place 3.5 billion years ago provided an amount of organic compounds comparable to those produced in situ.
Simulation of meteor shower similar to this of the early Earth. From AZ-Revista de Educación y Cultura
It has been shown that organic compounds are relatively common in extraterrestrial space, especially in the outer solar system where volatile compounds are not evaporated by solar heat. Many comets have an outer layer of a material with appearance of tar, which contains organic compounds formed by reactions caused by radiation, especially UV. Apart from that long ago they had been detected by telescope spectrography, a few years ago it has been identified in situ the amino acid glycine in comet Wild -2 in samples taken by NASA’s Stardust spacecraft (Dolmetsch 2006) .
The Murchison meteorite, about 100 kg, fell in Australia in 1969 and broke in several fragments that have been well studied. This meteorite is of type carbonaceous chondrites, which are rich in carbon, and indeed contains amino acids, both common (glycine, alanine and glutamic acid) as the most unusual (isovaline, pseudoleucine), with concentrations up to 60 ppm (Kvenvolden et al. 1970). It also contains aromatic and aliphatic hydrocarbons, alcohols and other organic compounds such as carboxylic acids and fullerenes.
A fragment of the Murchison meteorite, fell in Australia in 1969, of the type carbonaceous chondrites, which contains amino acids and other organic compounds. Image from Wikipedia
The isotope ratio 12C/13C of uracil and other organic compounds in the Murchison indicates an origin no terrestrial (Martins et al. 2008). In fact, besides the Murchison, the analyses made with many other meteorites show that organic compounds can be formed in outer space.
Model studies done with computer suggest that prebiogenic organic compounds might be formed in the protoplanetary disk of dust that surrounded the sun before the formation of the Earth, and that the same process may happen around other stars (Moskowitz 2012).
Moreover, studies of infrared emission spectra (Kwok & Zhang 2011) of cosmic dust have concluded that complex organic molecules are produced in the supernova stars, and that these molecules are expelled to the interstar space by effect of the explosion of the star. Surprisingly, this organic dust is similar to compounds found in meteorites. Since meteorites are remnants of the early solar system, we can now suggest that organic compounds found in meteorites had formed in distant stars .
Infrared spectrum of organic compounds, superimposed on an image of the Orion Nebula where these complex organic compounds (with the formulas) have been found. Image taken from NASA (C.R. O’Dell and S.K. Wong, Rice University.
In recent years there has been a breakthrough in the detection of organic molecules in galactic space thanks to the radio telescopes such as the Green Bank (100 m diameter) in West Virginia (USA), and the ALMA (Atacama Large Millimeter Array, at 5000 m in the Atacama Desert, northern Chile) which is composed of 66 radio antennas of 12 m diameter connected to each other by optical fiber. These astronomical radio telescopes or interferometers capture wavelengths around mm.
With these telescopes, as commented by Pere Brunet last month in his post post “Som pols d’estrelles ?” (Are we stardust ?, of the blog Fractal Ara-Ciència), different compounds have been detected around other stars: propenal, cyclo-propenone, acetamide, and glycolaldehyde (CHO-CH2OH). The latter is quite significant, since it is the smallest sugar and it is necessary for the formation of RNA. It has been detected with ALMA, around a young binary sun-type star (IRAS 16293-2422 ), 400 light years from Earth, relatively close, within the Milky Way (Jørgensen et al. 2012) .
Synthesis of organic compounds due to impacts of extraterrestrial objects
Finally, we have this third mechanism related with the article object of this post (Martins et al. 2013). Well, as we said before, this possibility was already reviewed by Chyba & Sagan (1992), because experiments in this direction had been made years ago. Carl Sagan itself, with other authors (Bar-Nun et al. 1970) had shown that applying a thermal shock, simulating impacts of comets and micrometeorites, to a gas mixture similar to the primitive atmosphere, there appeared amino acids.
However, thinking about the impacts of extraterrestrial bodies the first idea is the opposite, that they are antagonists of life on Earth because we remember the impact which caused extinctions and catastrophes such as the asteroid 65 million years falling in the Chicxulub crater in Yucatan, or a much smaller scale, the meteorite that fell last year in Chelyabinsk, Russia, with the appearance of fireballs. However, although these collisions can cause adverse effects on living beings where they fall, at the same time the energy released by the shock can be a source of reactions that generate prebiogenic organic compounds, as evidenced by the work of Martins et al. (2013).
At the same time, it has been shown that biological compounds that were present in the meteorite can “survive” impacts. Indeed, it has been shown that these compounds may be captured in the carbonaceous pores, inside the molten material due to the temperature and pressure of impact, particularly in analyses made with material of Darwin Crater in Tasmania, coming from a meteorite that impacted 800,000 years ago (Howard et al. 2013).
Besides from Martins et al., other studies have also simulated the effects of impacts. Furukawa et al. (2009) simulated the impact of a meteorite chondrite-type in a primitive ocean. They used a propulsion gun to create a high-speed impact in a mixture of carbon, iron, nickel, nitrogen and water, and then they recovered several organic molecules, including fatty acids, amines and one amino acid.
Thus, these experiments suggest that the impact of frequent extraterrestrial bodies in the primitive Earth had resulted in a great contribution to the formation of many different organic compounds, and as I said, adding that to the contribution of the already previously synthesized in outer space and to in situ synthesis in the same Earth.
Bar-Nun A, Bar-Nun N, Bauer SH, Sagan C. 1970. Shock synthesis of amino acids in simulated primitive environments. Science 168, 470-472.
Chyba C, Sagan C. 1992. Endogenous production, exogenous delivery and impact-shock synthesis of organic molecules: an inventory for the origins of life. Nature 355, 125–32
Dolmetsch C. 2006. NASA Spacecraft Returns With Comet Samples After 2.9 Bln Miles. Bloomberg.com. 2006-01-15
Editorial. 2013. The upside of impacts. Nature Geoscience 6, 987.
Furukawa Y, Sekine T, Oba M, Kakegawa T, Nakazawa H. 2009. Biomolecule formation by oceanic impacts on early Earth. Nature Geoscience 2, 62–66
Generalitat de Catalunya. 2014. “Científics descobreixen com es formen les molècules bàsiques de la vida”. Recercat 94, gener 2014.
Howard KT et 12 al. 2013. Biomass preservation in impact melt ejecta. Nature Geoscience 6, 1018-1023.
Jørgensen JK, Favre C, Bisschop SE, Bourke TL, van Dishoeck EF, Schmalzl M. 2012. Detection of the simplest sugar, glycolaldehyde, in a solar-type protostar with ALMA. Astrophysical Journal Letters 757, L4, 1-13.
Kvenvolden KA, Lawless J, Pering K, Peterson E, Flores J, Ponnamperuma C, Kaplan IR, Moore C. 1970. Evidence for extraterrestrial amino-acids and hydrocarbons in the Murchison meteorite. Nature 228, 923–926
Kwok S, Zhang Y. 2011. Mixed aromatic–aliphatic organic nanoparticles as carriers of unidentified infrared emission features. Nature, DOI:10.1038/nature10542
Martins Z, Botta O, Fogel ML, Sephton MA, Glavin DP, Watson JS, Dworkin JP, Schwartz AW, Ehrenfreund P. 2008. Extraterrestrial nucleobases in the Murchison meteorite. Earth and Planetary Science Letters 270, 130–136
Martins Z, MC Price, N Goldman, MA Sephton, MJ Burchell. 2013. Shock synthesis of amino acids from impacting cometary and icy planet surface analogues. Nature Geoscience 6, 1045-1049.
Moskowitz C. 2012. Life’s Building Blocks May Have Formed in Dust Around Young Sun. Space.com
Menor-Salván C, Ruiz-Bermejo DM, Guzmán MI, Osuna-Esteban S, Veintemillas-Verdaguer S. 2007. Synthesis of pyrimidines and triazines in ice: implications for the prebiotic chemistry of nucleobases. Chemistry 15, 4411–8.
Oparin A. 1952. The origin of life. New York: Dover.
Oró J. 1961. Mechanism of synthesis of adenine from hydrogen cyanide under possible primitive Earth conditions. Nature 191, 1193–4.
Parker ET, Cleaves HJ, Dworkin JP et al. 2011. Primordial synthesis of amines and amino acids in a 1958 Miller H2S-rich spark discharge experiment. PNAS 108, 5526–31.
Wikipedia: http://en.wikipedia.org/wiki/Abiogenesis (very good review of life’s origin andthe different hypothesis)
As I mentioned in this blog a year and a half before (August 30th, 2012 “Is there life on Mars?”), the Curiosity rover began to walk by Mars, specifically inside the Gale crater, carrying its sophisticated instruments to analyze rocks, soil and atmosphere, that is, a well equipped geochemical laboratory analyzing the surface of another planet for the first time. You can see a video of 2 minutes of the 1st year of Curiosity here. As you know, one of the objectives of this analysis is to find evidence of whether there were favorable conditions for life on Mars, albeit in very bygone eras.
The Curiosity rover is walking inside Gale Crater, east of Syrtis Major, the Mars most prominence visible through a telescope. Image NASA/ESA/Hubble.
Mars is a planet with some similar features on Earth, such as the length of day, slightly more than 24 hours, but the greater distance from the Sun and very tenuous atmosphere make the average temperature to be -63 ° C, with daily oscillations from +20°C to -140°C. Having no oceans, the land surface of Mars is equivalent to the Earth, because Mars is smaller.
Comparison of Earth and Mars. The diameter of Mars is 3400 km, approx half of the Earth’s, but excluding oceans, the solid surface is similar on both planets. Image taken from T.E. Harrison, New Mexico State University.
Despite the difficulties to demonstrate the current existence of liquid water on Mars, from some years ago evidences have been found that there was water in the Martian surface, and it is being confirmed that during the first thousand million years there was plenty of water on Mars (Remind that both Mars and Earth are about 4.5 billion years).
Appearance of a region of Mars indicating the flow of water in the distant past. Image taken from T.E. Harrison, New Mexico State University.
Water history in Mars, in billions of years. Image from T.E. Harrison, New Mexico State University.
Curiosity shows an “habitable” lake 3800 million years ago
Thus, Curiosity rover has been taking samples in various ways (see in Figure below the 5 cm holes taking samples) and analyzing them. Studying the data, scientists have been able to write a series of articles that have been published in Science and other prestigious journals.
Two 5 cm holes made by Curiosity rover sampling in Yellowknife Bay of Gale Crater on Mars (NASA/JPL-Caltech/MSSS)
Among other articles, by last May was already published one (Williams et al 2013) with observations that the material extracted from the Yellowknife Bay of Gale Crater shows textures typical of fluvial sedimentary conglomerates, with rounded pebbles that indicate fluvial abrasion, and by their characteristics a water flow of nearly 1 meter per second has been deduced, with a depth of near 1 meter. Therefore, when this area was formed the climatic conditions, with abundant rivers, would have been very different from the current hyperarid and very cold environment.
A few weeks ago, on December 9th, Science Online has released a special edition dedicated to the latest findings of Curiosity, with 6 articles asserting that at Gale Crater there would have been a lake theoretically habitable for some organisms.
In one of the articles, maybe the most relevant one, Grotzinger et al (2013) describe the sedimentary rocks found by the rover and demonstrate that it corresponded to an aqueous environment with neutral pH, low salinity and different redox states of Fe and S. The presence of C, H, O, S, N and P (i.e., all biogenic elements) is also shown, and the authors estimate that this favorable environment for life could have lasted some few hundred thousand years, and therefore it demonstrates the biological feasibility of this fluvio-lacustrine environment of post-Noachian Mars, that is, about 3800 million years ago.
Therefore, these scientists deduced that this lake had fresh water, a water we could drink, and that around this lake various fluvial and lacustrine environments were present. This environment should be habitable for some organisms, and very similar to some current earth environments. The commented chemical compounds would be suitable for the survival of bacteria chemolithotrophic bacteria, id est, those which use inorganic compounds (such as Fe or S) as an energy source and use CO2 as a carbon source. On Earth we have many kind of these microbes, such as iron bacteria (Thiobacillus) or sulfur bacteria (including some sulfur thermophilic archaea) and the nitrifying ones. Although most of these chemolithotrophic bacteria on Earth are aerobic because they use atmospheric oxygen as electron acceptor, this metabolism is also known in some anaerobes, such as those that do the anoxic ammonium oxidation (process “Anammox”), for example Brocadia anammoxidans, or the same nitrifying bacteria in anaerobic conditions.
Therefore, with this habitable lake for some time, and with other similar environments, we can not reject the possibility that living beings had been sometimes in Mars.
View from Yellowknife Bay in Gale Crater, looking W-NW. This area of sedimentary deposits was the bottom of a freshwater lake. Photo: NASA/JPL-Caltech.
Estimated size and shape of the lake that was in the Gale crater. There must have been other similar. The arrows indicate the direction of the alluvial fan that flowed into the crater from its wall. Photo: NASA/JPL-Caltech/MSSS.
Another aspect studied in this extra issue of Science is the possible presence of methane in the Martian atmosphere (Webster et al 2013). Just like on Earth, methane is a potential sign of biological activity in the past. Previous observations made from Earth or from the Mars orbit speculated on the presence of some 10 ppb of methane, and its subsurface biological origin was also discussed. Nevertheless, in the measurements made in situ by laser spectrometry of Curiosity rover using a specific spectral methane pattern, it has not been detected since the values found are lower than 1 ppb. This reduces the probability of recent methanogenic microbial activity on Mars and it is limiting the possible contribution of recent geological and extraplanetary sources.
Finally, in another article (Hassler et al 2013) about measures of cosmic rays on the surface of Mars that Curiosity has made over a year, models of radiation are elaborated, for the time calculation of possible subsurface microbial survival, and also for the preservation of organic compounds of biological origin finding them billions of years later. Unfortunately, the conclusion is that the powerful effect of cosmic rays would cause very difficult any of these possibilities. Unlike the Earth, the magnetic field of Mars is very weak and the atmosphere too, things that help bombardment of cosmic rays and the solar wind on the Martian surface. However, there is evidence that in the past Mars had a magnetic field more effective, so hope is not lost …..
Achenbach J. 2013. NASA Curiosity rover discovers evidence of freshwater Mars lake. The Washington Post, Health & Science online Dec 9.
Anderson PS. 2013. Curiosity rover confirms ancient martian lake. Spaceflight Insider online Dec 11.
Grotzinger JP et 72 al. 2013. A Habitable Fluvio-Lacustrine Environment at Yellowknife Bay, Gale Crater, Mars. Science DOI: 10.1126/science.1242777 online Dec 9, 2013.
Harrison, T.E., New Mexico State University: http://astronomy.nmsu.edu/tharriso/ast105/Mars.html
Hassler DM et 23 al. 2013. Mars’ Surface Radiation Environment Measured with the Mars Science Laboratory’s Curiosity Rover. Science DOI: 10.1126/science.1244797 online Dec 9, 2013.
Kerr, RA. 2013. New Results Send Mars Rover on a Quest for Ancient Life. Science Now News, online Dec 9, 2013.
Science Special Collection Curiosity 2013. Curiosity at Yellowknife Bay, Gale Crater. Online: http://www.sciencemag.org/site/extra/curiosity/
Webster CR et al 2013. Low upper limit to methane abundance on Mars. Science 18 October 2013, Vol. 342 no. 6156 pp. 355-357
Williams RME et 38 al. 2013. Martian Fluvial Conglomerates at Gale Crater. Science 31 May 2013, Vol. 340 no. 6136 pp. 1068-1072
All we that studied “Bios” probably remember two known aspects of the symbiotic relationships of plant roots with microorganisms:
1) The bacterial Rhizobium nodules on the roots of legumes (Figure 1). These bacteria, with the nitrogenase complex, are among the few organisms capable of fixing atmospheric N2 transforming it into organic nitrogen, which is used by the plant, and symbiotically, the plant provides organic compounds to the bacteria. Thanks to these bacteria, plants such as legumes do not require nitrogen fertilizers.
Figure 1. Rhizobium nodules
2) The mycorrhizae, that is, the symbiotic relationships between fungi and plant roots. The most commonly known are the mushrooms always associated with some trees (Figure 2), such as the Lactarius sanguifluus associated with pines. In fact, mycorrhizae are present in most plants. Through this symbiosis, the fungi receive organic nutrients of the plant, and this can capture more easily water and mineral nutrients (especially P, Zn and Cu) by means of the fungus. In addition, mycorrhizae increase the resistance of plants to diseases coming from the soil and facilitate them inhabiting badlands.
Figure 2. Mycorrhizae of mushrooms with trees. Image from Shannon Wright
But these are only the best known of the symbiotic relationships between microorganisms and plant roots. Indeed, as the soil is full of microorganisms, many of these, including bacteria, fungi, algae, protozoa or viruses, are beneficial, symbiotic or otherwise, for the plants. And what is biotechnologically more interesting, more potential applications of these microorganisms to benefit crop plants are being found, which can be a good alternative to the use of fertilizers and pesticides.
Different microorganisms can have direct positive effects on plant nutrition as nitrogen fixation, mineralization of organic compounds, and solubilisation of elements not available to the plant (such as phosphates, K, Fe), but also indirectly positive effects, such as the production of hormones and growth factors, or protection against pathogens (García 2013).
Thus, there is a growing interest in the biological control of plant pathogens. It has been proven that some of these pathogens are inhibited by antibiotics produced by microorganisms in the rhizosphere (Raaijmakers et al 2002). Bacteria are being used (bacterization) for some years in soil or with seeds or other plant parts, with the aim of improving the growth and health of the plant.
Some of the best known and used bacteria in this sense have been Bacillus and Paenibacillus. Several species of these genera of aerobic spore bacteria are abundant in agricultural soils and can promote plant health in different ways, suppressing pathogens with antibiotic metabolites, stimulating plant defence, facilitating nutrient uptake by the plant, or promoting symbiosis with Rhizobium or with mycorrhizae (McSpadder 2004).
The genus Paenibacillus was reclassified from Bacillus in 1993, and includes P. polymyxa, a species N2 – fixing, which is used in agriculture and horticulture. This and other Paenibacillus species give complex and regular colonial forms in agar, even surprising (Figure 3), which vary according to environmental conditions. For this, a self-organizing and cooperative behaviour between individual bacterial cells is needed, using a system of chemical communication. This bacterial social behaviour would be an evolutive precursor of multicellular organisms.
Figure 3. Colonies of Paenibacillus dendritiformis, 6 cm diameter each, branched (left) and chiral (right) morphotypes. From Wikipedia Creative Commons.
The colonization of plant roots by these bacteria has been demonstrated, and also that they do it by forming biofilms (Figure 4). The inoculation of these bacteria to the roots promotes the growth, as shown in peppers (Figure 5). This appears to be due to the nitrogen fixing bacteria, which increases the formation of plant proteins and chlorophyll, thus increasing photosynthesis and physiological activities. And on the other hand, it has been shown that these bacteria produce siderophores, which facilitate Fe uptake by the plant (Lamsal et al 2012).
Figure 4. Colonization of Paenibacillus polymyxa and biofilm formation on roots of Arabidopsis thaliana. Adapted from Timmusk et al 2005.
Figure 5. Promoting growth effect of peppers (Capsicum annuum) by inoculation with Bacillus subtilis (AB17) and Paenibacillus polymyxa (AB15), respect the non-inoculated control. From Lamsal et al. 2012.
Moreover, bacteria such as Paenibacillus can be effective against plant pathogens. For example, it has been shown that a strain of P. lentimorbus (B-30488r) reduces the incidence of disease done by the fungus Alternaria solani in tomato. It has been tested (Figure 6) that after inoculating with Paenibacillus a plant infected with Alternaria, resistance to the fungus was induced in the plant. The bacteria degraded the cell walls of the fungus and also inhibited it by competition of nutrients. In addition, it was found that Paenibacillus has no negative effect on the microbial population in the rhizosphere of tomato (Khan et al 2012). These treatments are a good alternative to the use of fungicides, avoiding the environmental and health problems of these compounds.
Figure 6. Schema of the influence of Paenibacillus lentimorbus B-30488r in the interactions of tomato plant with Alternaria solani, a fungus pathogen (Khan et al 2012).
Finally, these Paenibacillus can also be useful to avoid the transmission of human pathogens such as Salmonella through the crop plants. Indeed, on the east coast of the USA a few years ago were detected outbreaks of Salmonella on tomatoes due to contamination of water. When they analyzed the microbiome present in the roots of tomatoes and these were compared with those of other places where there were no Salmonella contamination occurred, it was found that these tomatoes of the East Coast had no Paenibacillus, which were present in tomatoes of other places. With this, they decided to inoculate tomatoes with several Paenibacillus and found that Salmonella disappeared. Among the inoculated strains, one was selected as more effective, P. alvei TS -15 , for which a patent was obtained as a biocontrol agent of foodborne human pathogens (Brown et al. 2012) .
Thus, knowledge of the soil microbiota and the many forms of relationships between microorganisms and plants lead to find new strategies for using “good” microbes to prevent food safety problems of transmission of pathogens, while at the same time it can be a good ecological alternative to the massive use of pesticides.
Brown EW, Zheng J, Enurach A, The Government of USA (2012) Paenibacillus alvei strain TS-15 and its use in controlling pathogenic organisms. Patent WO2012166392, PCT/US2012/038584
Conniff R (2013) Super dirt. Scientific American 309, sept, 76-79.
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García, Sady (2013) Los microorganismos del suelo y su rol en la nutrición vegetal. Simposium Perú “Manejo nutricional de cultivos de exportación”. Slideshare.net
Khan N, Mishra A, Nautiyal CS (2012) Paenibacillus lentimorbus B-30488r controls early blight disease in tomato by inducing host resistance associated gene expression and inhibiting Alternaria solani. Biological Control 62, 65-74
Lamsal K, Kim SW, Kim YS, Lee YS (2012) Application of rhizobacteria for plant growth promotion effect and biocontrol of anthracnose caused by Colletotrichum acutatum on pepper. Mycobiology 40, 244-251.
McSpadden Gardener BB (2004) Ecology of Bacillus and Paenibacillus spp. in agricultural systems. Phytopathology 94, 1252-1258
Raaijmakers JM, Vlami M, De Souza JT (2002) Antibiotic production by bacterial biocontrol agents. Antonie van Leeuwenhoek 81, 537-547
Timmusk S, Grantcharova N, Wagner EGH (2005) Paenibacillus polymyxa invades plant roots and forms biofilms. Applied and Environmental Microbiology 71, 7292-7300
Tuberculosis and its agent
Tuberculosis is a common and often deadly infectious disease if left untreated, caused by mycobacteria, mainly Mycobacterium tuberculosis. Mycobacteria such as M. tuberculosis and M. leprae (the leprosy) are considered as gram-positive bacteria because they have glycopeptides, but they are not stained with crystal violet, because outside of glycopeptide they have a good layer of fat, called the mycolic acid (Figure 1). Precisely the prefix “myco” comes from the fungic appearance on the surface of liquid cultures of these mycobacteria.
Figure 1. Mycobacterium tuberculosis with his particular cell wall, and the more used antibiotics against this bacterium. (Figure from National Institute of Allergy and Infectious Diseases)
This mycobacterium was discovered as causing tuberculosis by the German Robert Koch (1882) and for this reason, it is also called Koch’s bacillus. It is a strict aerobic bacterium, since it needs high levels of oxygen, and therefore mostly infects the lungs. It has a generation time of about 15-20 hours, so it has a very slow growing compared to other bacteria (remember the 20 minutes in Escherichia coli). From a taxonomic point of view they are Actinobacteria, or gram-positive of high G+ C, as corynebacteria or Actinomycetales.
The virulence of M. tuberculosis is very complex and has many facets (Todar K.). Although apparently it produces no toxin, it has a large repertoire of physiological and structural properties that explain its virulence and pathology of tuberculosis. Once in the lungs, the bacteria are captured by alveolar macrophages, but they cannot be digested due to the structure of the bacterial cell wall and because bacteria neutralize reactive compounds from macrophages. The mycolic acid also makes the cell not permeable to lysozyme. M. tuberculosis can grow intracellularly without being affected by the immune system, and at the same time, it secretes several proteins involved in the pathogenesis. For instance, the antigen 85 complex, which binds fibronectin, facilitates the formation of tubercles in the lungs (Figure 2).
Figure 2. Progress of infection by M. tuberculosis in the lungs, with the formation of tubercles (From humanorgans.org/tuberculosis)
Tuberculosis usually attacks the lungs but can also affect many other organs. The classic symptoms of tuberculosis are a chronic cough with bloody sputum, fever and other symptoms. The diagnosis relies on radiology of the thorax, a tuberculin skin test and blood analysis, and a microscopic examination and microbiological culture of body fluids. The treatment of tuberculosis is difficult and requires long treatment with various antibiotics. The most commonly used are rifampicin and isoniazid (Figure 1). However, antibiotic resistance is a growing problem in some types of tuberculosis. Prevention is based mainly on vaccination, usually with the Bacillus Calmette-Guérin vaccine (BCG).
Tuberculosis is transmitted by air when infected people cough, sneeze or spit. One third of the world’s current population is infected with M. tuberculosis, and there is a new infected person every second. However, in most of these cases the disease is not fully developed, as the latent and asymptomatic infections are the most common. Approximately one in ten latent infections eventually it progress to active disease, which, if left untreated, kills more than half of the victims. In one year (2004), the global statistics included 14.6 million chronic active cases, 8.9 million new cases and 1.6 million deaths, mostly in developing countries. In addition, a growing number of people in the developed world are contracting tuberculosis because their immune systems are compromised by immunosuppressive drugs, substance abuse, or AIDS.
When the human tuberculosis originated ?
The origin of the population infectious diseases (such as plague, cholera, etc.), id est., those that spread easily where there are many humans together, has been associated with the Neolithic demographic transition, about 10,000 years ago, when the revolution of agriculture allowed the establishment of the first sedentary villages, with numbers of humans living together. Many of these diseases are associated with other animals, from which they are transmitted to humans. Until recently it was believed that tuberculosis was of this type, having also led the Neolithic, but in fact the majority of tuberculosis have no relationship with other animals.
However, a recent study (Comas et al. 2013) provides data in the sense that the disease originated long before, more than 70,000 years ago, going with the modern humans out of Africa and spreading to the world. Effectively, this work of 22 co-authors from 9 countries (and the first author of which is the young Valencian Iñaki Comas) have analyzed and compared the genomes of 259 strains of the complex M. tuberculosis to see their evolutionary history. In doing so, within the 4.4 Mb genome of this species the authors have identified 34,167 SNPs polymorphic sites, i.e., sites of DNA that have some different nucleotide in function of strains, and with which they have been able to reconstruct the phylogeny of this bacterium. In this way, they have confirmed seven lineages (groups of strains) that had been suggested by other techniques. The most interesting thing is that this phylogeny of genomes of M. tuberculosis is very similar to the human mitochondrial genome phylogeny, as shown in Figure 3. Therefore, this suggests that the evolution of tuberculosis bacteria goes parallel to that of modern humans.
Figure 3. The phylogeny of genomes from 220 strains of M. tuberculosis, with the different lineages (MTBC, left), is similar to that of mitochondrial genomes (right) of 4995 humans, with their main haplogroups (from Comas et al. 2013, Fig. 1 c, d).
Based on these phylogenies and on the frequencies of mutations observed in the genomes of M. tuberculosis, the origin of the different lineages of this bacterium has been established, calculating at what time of the human Palaeolithic the different branches were appearing, from approx. 73,000 to 42,000 years (Figure 4), which, having seen the parallel with mitochondrial DNA, coincide with the proposed dates of the modern humans expansion from Africa to Eurasia.
Figure 4. Expansion of complex M. tuberculosis going out of Africa, with the origin of different lineages and the approximate data (thousands years) of the evolutionary branches (from Comas et al. 2013, Fig. 2a).
Therefore, we can conclude that tuberculosis and its bacterial agent has been a constant companion of the modern humans during their evolution and their global spread, at least in the last 70,000 years, and also that the bacterium M. tuberculosis has been able to adapt to changes in human population. In addition, in this regard last years an increase in resistant strains to multiple antibiotics has been observed. The study of these mechanisms of bacterial adaptation may help to predict future patterns of disease and to design rational strategies to fight it.
Comas, Iñaki, et al. (2013) Out-of-Africa migration and Neolithic coexpansion of Mycobacterium tuberculosis with modern humans. Nature Genetics 45, 1176-1182.
Todar K., Todar’s Online Textbook of Bacteriology.
Mycobacterium tuberculosis in wikipedia: http://en.wikipedia.org/wiki/Mycobacterium_tuberculosis
Tuberculosis in viquipèdia (Catalan): http://ca.wikipedia.org/wiki/Tuberculosi.
It is the “pearl of Siberia”, so named for its beauty and nature. As shown on the map below, it is located in the south of central Siberia, in the Russian Federation, quite near of Mongolia and China. Historically, the chinese called it as the North Sea. The Russians do not began to explore the lake until the end of 17th century. Although today most of the population is of russian origin, the south of the lake is inhabited by buryats, of Mongolian origin. They are the largest ethnic minority group in Siberia, with their own language. They are about 400,000 and their principal city is Ulan-Ude.
The famous Trans-Siberian railway passes beside the lake, bordering it by the southwest corner, with stops in the cities of Irkutsk and Ulan-Ude. This section is spectacular, with 200 bridges and 33 tunnels. This area is “only” about 5000 km far from Moscow (3 days) and 4000 km of Vladivostok (3 days) in the Pacific.
As you can see in this map, the lake is shaped like a crescent. It is the largest freshwater lake by volume in the world (23,600 km3), it contains more water than the North American Great Lakes combined, although it is not the largest by surface (31,000 km2), due to because it is the deepest (1600 m maximum depth). It is aldo the world’s oldest (about 25-30 million years ago). The lake is in a rift, where tectonic plates are separating, and so it widens gradually.
It has an enormous biodiversity, with over 1700 species of plants and animals, 2/3 of which are endemic, and in 1996 UNESCO declared it a World Heritage Site. With minimum temperatures of -20°C most of the winter, lake Baikal is frozen for half the year.
The Baikal seal
The Baikal seal or “nerpa” is Pusa sibirica (formerly classified as Phoca sibirina), which is across all the lake, but nowhere else. The lake is 2400 km far from the Arctic ocean where the lake waters flow through the siberian river Yenissei.
The nerpa is one of the few species of freshwater seals. It is relatively small, measuring just over 1 meter and weighs about 60-70 kg. It eats several species of fish, but especially a so-called “golomyanka”, which is endemic of lake Baikal. This fish is very abundant in the lake, it is translucent, very fat (it is known as “oilfish”) and lives at depths of 200-500 meters. Seals dive down to eat them, they can resist up to 40 minutes underwater.
Well relaxed Baikal seals. Picture from Uryah. http://diertjevandedag.classy.be/zoogdieren/roofdieren/zeehond/baikalrob.htm
Well, and how this seal came to lake Baikal ?
Well, it is not very clear, it is almost a mystery, since the seal lives far from the open seas, where all other species of seals live. But anyway, as for everything, we can look for the more reliable scientific hypothesis. Let’s see what we can found ….
This species is most similar to the Arctic seal or ringed seal, Pusa hispida, which lives in the Arctic. Like this and other of the family of Phocids (such as the common seal, Phoca vitulina), it is earless, unlike other Pinnipedia (sea lions and fur seals). Another species quite similar is the Caspian seal, Pusa caspica, which is also a curious case, because the Caspian is also an isolated sea. But the Caspian is almost a real sea, with a salinity of 1.2% (the third of the others seas and oceans), and therefore the Caspian seal, such as the Arctic, is not a freshwater seal.
However, there are two subspecies of the arctic seal, with few numbers, which are freshwater, like that of Baikal lake: they are the seals of lakes Saimaa (Finland) and Ladoga (Russia, near Finland), which are relatively near of the Arctic ocean. These two subspecies probably came from the Arctic, after the last glaciation, and they remained confined to these lakes. Probably, to change from salt water to freshwater is a relatively easy adaptation for mammals like these.
Another common feature of the Baikal seal and these of the same genus Pusa (the arctic and the Caspian ones), and others, is that their pups have white fur, changing it shortly after to the grey fur typical of adults. This suggests the origin of the common ancestor in a icy place, the arctic or a related environment.
Therefore, the prevailing scientific theory by 1960  was that the origin of Baikal and Caspian seals from the Arctic would have been during one of the glacial periods of the Pleistocene, perhaps around 90,000 years ago, that is relatively recent in terms of evolution. As shown in the map below, during this period, north ice of the arctic covered part of Siberia (red line) and functioned as a barrier to all waters coming from the south, which currently drain to the Arctic ocean (by the rivers Ob and Yenissei, and others). Thus, a large lake was formed, which communicated probably with the Baikal and the Caspian, and all water flowed westward towards the Black Sea, as shown. Geologically, these connections of the Aral and Caspian to the Black Sea seem demonstrated [3, 4]. With this, the seals of the Arctic would have migrated south and led to the Caspian, and the Baikal perhaps.
Map collage of this from Mangerud  (left) with the one of freeworldmaps.net (right). The area in white until the red line was covered by north ice sheet, by 90,000 years ago.
But, as I mentioned, 90,000 years is a very short evolutionary time to explain the differences between these species, although they are closely related.
Therefore, it seems likely a previous common origin, and to prove it, molecular tools have been used the recent years. In this way, specific gene sequences of 12 mitochondrial proteins from different species have been compared. This study has been done including all pinnipeds .
Thus, in developing the corresponding dendrogram, as we see below, it can be observed that the evolutionary separation of the Baikal seal from the Arctic (ringed seal), and the Caspian, and the grey seal, occurred about 5 million years ago, at the beginning of the Pliocene. Therefore, these species of seals must have a common geographical origin, relating to these arctic lakes or internal seas. Molecular similarities have been also found between arctic amphipods (small crustaceans) and those of the Caspian Sea.
Furthermore, at the period when this seal speciation took place, and from the Oligocene (20 million years) to the Pliocene, the sea Paratethys (see diagram below) was extending from central Europe to this part Asia, over the Alps, Carpathians and other mountain ranges separating Paratethys from the Tethys sea. In fact, the current Black, Caspian and Aral seas, and other lakes in central Asia, are relics of what was the Paratethys. This great sea had connections with the Arctic during different periods.
Diagram from Woudloper: http://commons.wikimedia.org/wiki/User:Woudloper
Therefore, it is likely that the Caspian seal was originated from the Arctic ones or backwards, through these geographical connections of the Paratethys until the Pliocene. However, for our protagonist, the Baikal seal, there is not enough evidence to say that the Baikal was connected to the other seas of Paratethys because it is quite to the east. So, either Pusa sibirica came across this alleged connection with Paratethys, or maybe there was a separate settlement from the Arctic across the river Yenissei, also at this period. Nothing is discarded. Therefore, the Baikal seal will keep some mystery ……
 Arnason U. et al (2006) Pinniped phylogeny and a new hypothesis for their origin and dispersal. Molecular Phylogenetics and Evolution 41, 345-354
 McLaren I.A. (1960) On the origin of the Caspian and Baikal seals and the paleoclimatological implication. American Journal of Science 258, 47-64
 Mangerud J. et al. (2004) Ice-dammed lakes and rerouting of the drainage of northern Eurasia during the Last Glaciation. Quaternary Science Reviews 23, 1313-1332
 Mangerud J. et al. (2001) Huge ice-age lakes in Russia. Journal of Quaternary Science 16, 773-777
 ……. and Wikipedia, of course !!