Grin and Water Bear It

Biology concepts – tardigrade, cryptobiosis, anhydrobiosis, eutely, hyperplasia, hypertrophy, dry vaccine, dormancy

The original toughman contests were supposed to show us the

toughest of the general population. I’m not so sure how tough

it is to have a nickname like “Butterbean,” but maybe that

proves his toughness. On the other hand, do you get a cereal

box cover and a mohawk because your tough, or do you get

tough because you have a mohawk.

In the early 1980’s, a forerunner to the mixed martial arts craze was temporarily popular in the United States. Called the “ToughMan” contests, non-boxers would enter the ring for fights against other nonprofessional fighters. The rules were supposedly the same as in boxing, although many contests were run without proper supervision, and the tournaments sometimes required a participant to fight several times in one evening. Think legalized bar fights.

Famous participants in the toughman contests included “Butterbean” Esch – a 350 lb. boxer with one punch and an iron jaw, and of course, who can forget Mr. T. The contests are still held in many states, but MMA now has the majority of the fan base, even more than traditional boxing between trained fighters.

These guys were tough, but were they the toughest? Humans as a rule are weak for their size, scared of more things than they should be, and less inclined to fight to the death for a morsel of food or potential mate – well most are. So…..

Questions of the Day: What is the world’s toughest animal?

Ask a hundred people and you may get a hundred different answers. The bull elephant can fight off an entire pride of lions and can lift five tons. But can you really give the prize to an animals that is scared of a mouse?! Maybe they aren’t afraid of mice, but they will avoid them if possible, according to one of the more scientifically consistent episodes of Mythbusters.

A good second choice might be the honey badger. It supposedly knows no fear, and proves it by depriving lions of the prey they just killed. In one case, three honey badgers stole a entire carcass from seven lions! The South Africa National defense calls their armored personnel vehicles ratels, the afrikaans word for honey badger.

This is the toughest animal on Earth, although it may not

look it. The water bear looks more like a teddy bear,

although the claws might do some damage if you are a

bacterium or a protist. The mouth has stylets to puncture

plant cells and suck out the liquid nutrition.

But I will try to convince you that a type of bear is the toughest animal on the face of the Earth, a water bear to be specific. This animal has long claws on each foot and a mouth that takes up a good portion of its head. On the other hand, it's less than 1 mm long!

The water bear is more scientifically known as a tardigrade (latin for slow walker), a phylum that falls somewhere near arthropods and nematode worms. There are two classes (eutardigradia and heterotardigradia) and more than 900 species, but there may be some overlap in those descriptions.

The adult tardigrade will have 40,000 cells, and will never have more. What is more, every species of tardigrade is matures with a specific number. This is called eutely (eu = good, and telos = end). Many lower organisms may be eutelic; their cells have a limited number of divisions, so they grow to that number and then stop.

It isn’t just whole organisms that might be eutelic, organs can be as well. For example, the nematode Ascaris ALWAYS has 162 neurons. The research model nematode C. elegans has exactly 959 somatic cells, although a 2011 study has shown that C. elegans can lose critical cell nuclei as they age – tell me about it. Other nematodes, rotifers, and gastrotrichs have also been shown to have cell constancy at the body and/or organ level.

Tardigrades do grow after they reach adulthood, just not by adding cells. Growth by additional cells is called hyperplasia (excess formation), while growth by existing cells becoming larger is called hypertrophy (excess nourishment). 

The gingiva around the teeth can overgrow in response

to some developmental disorders, but more often it is a

result of drugs given for epilepsy or other diseases. The

point here is that whether it is from hypertrophy

(increased cell size) or hyperplasia (increased cell

number), it looks the same. These are histologic

determinations and don’t really matter for clinical evaluation.

Prostate enlargement is often due to an increase in cells, hence the name benign prostatic hyperplasia, but hyperplastic growth doesn’t have to be pathologic. When you lose part of your liver, some can grow back by through hyperplasia. Likewise, hypertrophy is great when it is your muscles getting bigger, but not necessarily so good when your heart’s ventricles overgrow (ventricular hypertrophy).

Different tardigrade species are adapted to nearly every environment on Earth. They live in the Arctic and the Antarctic, in the mountains and the oceans, in the deserts and the jungles. All are found near water, some marine and some limnal (freshwater), some in the water and some just next to the water held in mosses or lichens.

But wherever you find them, you’ll find them in great numbers. The density of tardigrades can approach two million per square meter. Yellow crazy ants (Anoplolepis gracilipes) form supercolonies of incredible density, yet they can only muster about 2000 individuals per square meter. Haven’t heard of crazy ants? You will – look them up.

Tardigrade toughness doesn’t come from their pursuit of prey or their ability to fend off predators, but their willingness to live in conditions that would kill anything else, and I mean anything, else.

Cold, not a problem. Tardigrades can have liquid nitrogen (-346˚F/-210˚C) poured on them and they’re just fine. Heat – boil them for a couple of hours and then watch them lay eggs and go back to eating. Radiation isn’t a problem either; they can take 5700 grays of ionizing radiation without blinking.... well, they could if they had eyes. Humans curl up in a ball and die when exposed to 5 gray.

It is important to know how much radiation is absorbed

by the body, not just how much is in the air. The Gray,

named for Harold Louis Gray, is equal to 1 joule of energy

absorbed per kilogram of matter. Harold Gray is considered

the Father of Radiobiology. The old dose name was the

rad, and 1 Gray is equal to 100 rads. A chest X-ray is

typically about 0.0006 Grays.

Some tardigrades live in black smokers at the bottom of the ocean, yet most of them can take 6000x normal pressure in stride. To sum it all up, in 2007 the Russians fired tardigrades into space for 12 days (near absolute zero, total vacuum, cosmic radiation). They came back and starting having babies. Now that’s tough.

How do they manage these amazing feats? Basically – they die and then come back to life. Technically, it’s called cryptobiosis (hidden life), but death and self-resurrection is not a bad description. During cryptobiosis, metabolism is reduced by 1000x fold or even more, down to the level where there is NO detectable chemical activity.

There are five recognized types of cryptobiosis, based on the noxious environmental condition that triggers it – anhydrobiosis (without water), chemobiosis (chemicals), cryobiosis (cold), anoxybiosis (lack of oxygen), and osmobiosis (change in osmotic potential).

The primary form for tardigrades is anhydrobiosis. They drop their claws, retract their legs and roll up into a ball called a tun. 99% loss of water, roll up into “tun” this is important because it regulates the rate of evaporative water loss. At this level, they don't hold enough water for damaging reactions to take place or even enough water to form ice crystals. The water is replaced by a sugar called trehalose.

Trehalose production is similar to the way many organisms can protect their structures and biochemistry from environmental damage, but apparently scientists have just touched the surface of how tardigrades react to uncomfortable environments. A 2013 study indicates that there are many unidentified organic molecules present in the tuns of tardigrades that are not present in the organisms under normal physiologic situations.

The tun of a tardigrade is a very regulated structure.

When the claws are dropped and the legs retracted, the

tardigrade coils into almost a ball. In this situation, the

loss of water can be carefully controlled. The plates on

the back also form a protective armor for non-

environmental assaults – the spikes look imposing.

Amazingly, tardigrades are the only animal that can undergo all five types of cryptobiosis.  It's really like dying and coming back to life. Reviving from cryptobiosis can take a little while, usually the longer they have been in anhydrosis, the longer it takes to recover. They can’t survive this way forever either.

Earlier reports had professed that 120 year old tardigrades were revived from dried lichen and moss samples in the British Museum, and that decades old samples were just fine. But Dr. James Garey of the University of South Florida tells me that many of these reports have been called into question and cannot be repeated.

Dr. Garey’s estimate is that tardigrades can survive 1-5 years as a tun, with decreasing viability upon hydration after that. Still, could any other animal you know of be dead for five years, with no air, no water, high radiation, liquid nitrogen, and taunts about their size and lineage – and then come right back to life when the opportunity is right?

Cryptobiosis is quite different than dormancy. Dormancy doesn’t bring a huge change in physiology – like 99% dessication. Also, dormancy is preemptive while cryptobiosis is reactive. However, a very good 2011 review of tardigrade reactions shows that they can undergo both dormancy and crytobiosis – sometime simultaneously!

The question is – how do they survive the bad conditions WHILE they are forming the tun? It takes about 20 minutes for tun formation to occur, so it appears that many of the conditions they can endure require them to already be in the cryptobiologic state. They can survive the radiation when they are dessicated, they can survive boiling when they are dessicated. I don’t think it makes them any less amazing.

In early 2013, researchers from King’s College in England

developed a silicon mold with dissolvable sugar micro-

needles that can deliver a dry vaccine powder.  The system

would induce immunity through activation of skin immune

cells, would require almost no training to deliver, and no

refrigeration. The anhydrobiotic live vaccine is based on

tardigrade cryptobiotic features.

Tardigrades aren’t even considered extremophiles, since they are not designed to live in extreme environments. But this is precisely why I think they are so tough, because few of them are adapted to extreme conditions, but they can survive deadly situations anyway.

Can the exploits of this microanimal help humanity? You betcha. Tardigrades' ability to undergo anhydrobiosis has begun to influence the design of medicines. In third world countries, a lack of reliable refrigeration requires vaccines and medicines don’t need refrigeration, and can be reactivated upon ingestion.

Dry vaccines are a current goal, so the National Institutes of Health recently put out a call for proposals for research into more thermostable and reactivateable preparations. A late 2012 paper has identified a tablet form for deliver of some protein drugs, with reactivation of the molecules with saliva. This would be much better than the current reliance on hypodermics and refrigeration. So tardigrades are tough for themselves, and may fight for us as well.

Next week, just how do you describe the climate of your hometown? Biomes are scientific entities, but it seems we can't agree on what each looks like or is called.



Borde, A., Ekman, A., Holmgren, J., & Larsson, A. (2012). Effect of protein release rates from tablet formulations on the immune response after sublingual immunization European Journal of Pharmaceutical Sciences, 47 (4), 695-700 DOI: 10.1016/j.ejps.2012.08.014
 
McGee, M., Weber, D., Day, N., Vitelli, C., Crippen, D., Herndon, L., Hall, D., & Melov, S. (2011). Loss of intestinal nuclei and intestinal integrity in aging C. elegans Aging Cell, 10 (4), 699-710 DOI: 10.1111/j.1474-9726.2011.00713.x  

Guidetti, R., Altiero, T., & Rebecchi, L. (2011). On dormancy strategies in tardigrades Journal of Insect Physiology, 57 (5), 567-576 DOI: 10.1016/j.jinsphys.2011.03.003

 

It’s An Airtight Case

Biology concepts – respiration, aerobe, anaerobe, CAM plants, plastron respiration, cutaneous respiration

Question of the Day – what living thing can hold its breath the longest?

It may seem like an exaggeration, but people whose
tissues are low on oxygen (hypoxic) can have a bluish

hue (cyanosis). Blood that is oxygenated is redder

than blood that is deoxygenated. In animals with more

hemoglobin than humans, like whales, the blood can

actually turn almost purple. Blue Man Group will turn

you blue - from laughter, not disease.

The world’s record for holding one’s breath (voluntarily) was set in May, 2012 by Denmark’s Stig Severinsen. Even though it wasn’t technically cheating, he did hyperventilate with almost pure oxygen for 19 minutes before he then held his breath for an amazing 22 minutes and 0 seconds!

Hyperventilation works by reducing the CO₂:O₂ ratio in your blood. CO₂ concentration is measured by sensors in your large blood vessels. Signals are sent to the brain to increase the breathing rate if there is too much CO₂ in the blood or to reduce the breathing rate if the pCO₂ is too low. By breathing fast and breathing pure O₂, there will be less CO₂ in the blood and your brain will tell you to stop breathing until the pCO₂ increases to normal levels.

Stig said he used two mental tricks to increase the time he can hold his breath. One is scientifically valid; biofeedback allows Stig to slow his own body activity. By concentrating on his heartbeat, Stig has learned to stimulate neural pathways to reduce his cardiac output and his overall metabolism rate -lower metabolism and heart rate - less need for oxygen. His second technique? He thinks about dolphins.

Twenty-two minutes seems like a long time; indeed, this feat required arduous training by Stig. In truth, 22 minutes isn’t very long at all. In fact, humans are weenies when it comes to going without oxygen. Lots of animals can hold their breath longer than we can.

Sperm whales and elephant seals are the banner carriers for the marine mammals in our contest. Sperm whales can submerge for more than two hours, while elephant seals may stay under water for an hour or more. Being mammals, they have physiology very similar to humans in some respects, but they do have modifications that allow for the long submersions.

Elephant seals have a large proboscis; hence the

elephant name. There are two species, Northern and

Southern, with the Southern species being slightly

larger. Males can weigh over 5 tons and they fight for

females. The females are more like 1500-2000 pounds,

making this the largest relative difference in weights

of two sexes for any mammal.

Whales and seals have more blood than humans do – duh! Even compared pound for pound, they have 3x as much blood. More blood means more oxygen carrying capability. They also have increased oxygen carrying proteins in their blood and tissues. In addition, they can reduce their metabolism in all but the most necessary organs, and they can divert their blood to just these organs.

New research shows that marine mammals also have oxygen carrying proteins in their brains, called neuroglobin and cytoglobin. So, while blood levels of oxygen may plummet when diving, the brain remains oxygenated.

Sea turtles and crocodilians are examples of reptiles that can hold their breath for amazing lengths of time. Aligators and crocodiles can stay submerged for a couple of hours, while a Galapagos sea turtle can easily stay underwater for 4-7 hours, depending on its level of activity. Maybe they think about Stig in order to stay submerged longer.

Sea turtle hibernation is controversial, but some freshwater turtles do just that. They can stay submerged for weeks or perhaps months at a time! But many turtles cheat at our contest, they have a bimodal respiratory system, through their lungs and through their skin. Cutaneous gas exchange is apparent in all freshwater turtles to some degree, but it is much more efficient in some soft shell turtles, according to a 2001 study.

Let’s look at a different type of reptile. The Belcher’s sea snake (Hydrophis belcheri) is the most venomous snake on the face of the Earth, or under it, as the case may be. It is a sea snake that can remain submerged for 7-8 hours. Sea snakes like H. belcheri have a single lung that runs almost the entire length of their body, and their trachea can also transfer oxygen to the blood. This reduces the “dead space” in their respiratory system and allows them to absorb more of the oxygen they inhale.

This diagram gives you an idea of how little respiratory

space humans use for gas exchange. The only places

that move oxygen in to the blood are the pinkish

alveoli at the ends of each airway. Sea snakes use

their available respiratory space to exchange gasses.

Humans, as a comparison, only absorb about 15% of the oxygen in each breath, partly because the gas exchange takes place only in the alveoli (terminal air sacs). Oxygen in the nose, pharynx, trachea, bronchi, and bronchioles is just exhaled without any chance to be used by the body.

However, sea snakes are cheaters as well. Their bodies have been streamlined to help them move through the water. One adaptation in this direction is the complete loss of scales. As a result, these snakes have evolved the ability to exchange some oxygen and carbon dioxide with the water through their skin. So they aren’t really holding their breath when submerged.

Almost all amphibians are cutaneous (skin surface) breathers as well. In air, most amphibians can survive exclusively by exchanging gasses through their skin, and in water, adult gills or rudimentary lungs are supplemented by exchange of gas from the water. Cold water and turbulent water contains more oxygen, so in these environments amphibians can survive indefinitely by garnering oxygen from water.

Indeed, the largest family of salamanders (the plethodontidae), don’t have any lungs at all. They exchange gasses only through their skin and the mucosa surfaces of their mouths. And many of these salamanders are primarily aquatic, they don’t take a breath in their entire lives – but they aren’t holding their breath either.

But none of these animals are the champion breath holders. There are organisms that laugh at holding their breath for a couple of hours. But let’s limit our discussion to those organisms that require oxygen. It’s no fun watching an anaerobic bacterium hold its breath; it doesn’t need oxygen! In many cases, air kills them!

Cockroaches, ticks, and ants do last a long time underwater, just try flushing one. But they can’t win our contest either. They seem to trap a bubble of air as they submerge. They have long hairs on their abdomens that trap air via the surface the surface tension and cohesion of water.

A plastron is the bottom portion of the turtle or tortoise shell,

made up of flat pieces. On the right is the plastron of a tick.

In some ticks there can be gas exchange from water to bug

through the plastron. This is called plastron respiration.

Surrounded by the bubble, they can oygenate their tissues via the breathing holes on the sides of their bodies (spiracles). This allows them to be underwater for nearly an hour and still be breathing. New research in ticks shows that the plastron (flat portion under the abdomen) is capable of some gas exchange itself via the air trapped by the hydrophobic hairs on the abdomen.

We make a big deal about how plants take in carbon dioxide and give off oxygen, and they do during photosynthesis in their chloroplasts. But that’s only half the story. They also have mitochondria that produce ATP from photosynthesis products via oxidative phosphorylation, just like we do.

For plants that grow in hot, dry environments, loss of water is a serious threat. To minimize water loss, some can close the pores in their leaves (stomata), but this also prevents gas exchange, including taking up carbon dioxide and oxygen. The stomata will open only at night, when the temperatures are cooler and water loss would be lost. This is the only time they exchange CO₂ and O₂ with the environment as well.

CAM (crassulacean acid metabolism) plants can store the carbon dioxide they take in at night in the form of malate. They then can perform photosynthesis even though their stomata are closed. CAM physiology also reduces the amount of O₂ bound by RuBisCo enzyme instead of CO₂. RuBisCo + O₂ leads to inefficient carbon fixation, so waiting until night time when CO₂ is relatively more abundant and more soluble will increase photosynthesis productivity. As a result, CAM plants hold their breath for 8-15 hours every day!

CAM plants close their stomata during the hot day, but

exchange gasses during the cooler night. They convert

carbon dioxide to malate as a temporary fixation, which

they store in the central vacuole. During the day, they

convert the malate to carbon dioxide and then to

carbohydrate in the chloroplast using normal

photosynthesis pathways. CAM plants include the

prickly pear, as shown on the extreme right and left.

But the winners of our lack of oxygen survival contest – bacteria, of course! Bacteria come in many flavors, including those that don’t need oxygen for respiration (chemosynthesizers and anaerobes), those that can take or leave oxygen (facultative bacteria), and those that must have oxygen in order to make ATP (obligate aerobes).

Mycobacterium tuberculosis is an example of an obligate aerobe. I talked to Martin Gengenbacher at the Max Planck Institute in Berlin about M. tuberculosis and its survival time without oxygen. He has recently published a great review of M.tuberculosis biology. In a series of experiments that resulted in the development of something called the Wayne model, M. tuberculosis was sealed in a vessel in which they consumed all the available oxygen over time.

However, even after the oxygen was gone, the organisms remained viable for 25 days! They do seem to go dormant, but this dormancy is not the same as ceasing activity completely. It seems that some metabolism and respiration is maintained in the complete absence of oxygen – even though we know that M. tuberculosis absolutely requires oxygen to survive.

These 25 days make M. tuberculosis better than any of our other example organisms at living without gas exchange, though there may be other obligate aerobes that can perform similar feats. But there’s more to the skills of the tuberculin bacterium. In tuberculosis, the body has a difficult time killing off the organism, so it does the next best thing – it walls off the bacteria and traps them in a prison cell of immune cells. These whirls of cells are called granulomas and are very complex structures.

On the left shows a tuberculin granuloma forming and breaking

down. You can see in the middle a formed granuloma, with

macrophages surrounded by a fibrous cuff and lymphocytes.

When immunosuppression sets in, the granuloma breaks down

and the organisms is released to cause disease. On the right is a

photomicrograph of granulomas. In the right corner is a

tuberculosis bacterium before granuloma formation.

Granulomas are extremely hypoxic (oxygen poor), and M. tuberculosis does undergo dormancy in these structures. But again, Dr. Gengenbacher states that this is a metabolically active dormancy, which would by definition require ATP, and therefore require cellular respiration.

The patient still has TB, but no symptomology. This remains the case until the patient undergoes some form of immunosuppression, some disease or condition that prevents the immune cells from keeping the organism in prison. There have been cases where TB has reactivated some 50 years after the original infection. So – M. tuberculosis can hold its breath for half a century! We have a winner.

Next week, another question to ponder. Just how many species call Earth home? 



Gengenbacher, M., & Kaufmann, S. (2012). Mycobacterium tuberculosis: success through dormancy FEMS Microbiology Reviews, 36 (3), 514-532 DOI: 10.1111/j.1574-6976.2012.00331.x  

Fielden, L., Knolhoff, L., Villarreal, S., & Ryan, P. (2011). Underwater survival in the dog tick Dermacentor variabilis (Acari:Ixodidae) Journal of Insect Physiology, 57 (1), 21-26 DOI: 10.1016/j.jinsphys.2010.08.009  

Williams, T., Zavanelli, M., Miller, M., Goldbeck, R., Morledge, M., Casper, D., Pabst, D., McLellan, W., Cantin, L., & Kliger, D. (2008). Running, swimming and diving modifies neuroprotecting globins in the mammalian brain Proceedings of the Royal Society B: Biological Sciences, 275 (1636), 751-758 DOI: 10.1098/rspb.2007.1484