In 2010, it seemed that biology textbooks would have to be rewritten. At the bottom of the Mediterranean Sea, in one of the most extreme environments on Earth, one research team found evidence of an animal able to live its entire life without oxygen.

Not one of the other million or so known animal species can do that. Oxygen, in some form, is often assumed to be vital for animal life. Yet the existence of these creatures seemed to blow a hole in this theory, with far-reaching implications for our understanding of life on Earth.

The tiny Mediterranean animals belong to a group called the loriciferans – an animal group so unusual that it was not discovered until the 1980s.

Because the mud at the bottom of the L’Atalante basin is completely devoid of oxygen, the team did not expect to find “higher lifeforms”

Loriciferans are about the size of a large amoeba. They live in muddy sediments at the bottom of the seas. But supposedly, that mud should contain some oxygen to allow the animals to breathe. The mud in the L’Atalante basin at the bottom of the Mediterranean does not.

Over a period of a decade, Roberto Danovaro at the Polytechnic University of Marche, Italy, and his colleagues trawled the depths of the L’Atalante basin. It lies 3.5km beneath the surface, about 200km (124 miles) off the western coast of Crete. The inner part of the basin is completely devoid of oxygen, because ancient salt deposits buried beneath the seafloor have dissolved into the ocean, causing the water to become extra salty and dense.

The dense water does not mix with the normal oxygen-rich seawater above, and becomes trapped in seafloor valleys. The oxygen-free water has been in place for over 50,000 years.

Because the mud at the bottom of the L’Atalante basin is completely devoid of oxygen, the team did not expect to find “higher lifeforms” – which basically means animals – living there. But in fact they found three new species of loriciferans, apparently thriving in the mud.

It is not just zero oxygen levels that the critters must contend with. Loriciferans are surrounded by poisonous sulphides, and live in such extreme salty water that normal cells would turn into dried out husks.

We took 10 years to confirm through experiments that the animals were really actually living without oxygen

“When we first saw them we couldn’t believe it,” says Danovaro. “Before this study only two [loriciferan] specimens had ever been found in the deep Mediterranean. There were more organisms in 10 square centimetres of anoxic basin than in the rest of the Mediterranean Sea put together!”

But the biggest surprise of all was the fact that the tiny animals seemed to survive without any oxygen at all.

“We knew that some animals, such as parasitic flatworm nematodes, can spend part of their lives without oxygen, living in the intestine,” says Danovaro. “However, they don’t spend their whole life cycle this way. Our discovery challenged all previous thoughts and assumptions about the metabolism of animals.”

He says this made their discovery difficult for other scientists to believe. “Indeed we didn’t believe it ourselves at first. We took 10 years to confirm through experiments that the animals were really actually living without oxygen.”

Those experiments were difficult to perform. The scientists could not bring the living animals up to the surface, because the journey would instantly kill them. What they could do was test the tiny animals for signs of life in the seafloor.

They showed that fluorescent molecules that are only taken up by living cells were incorporated into the loriciferans’ bodies. They also used a stain that reacts only to the presence of active enzymes. The stain reacted with loriciferans from the basin, but not from the obviously dead remains of other microscopic animals found in l’Atalante.

The closer the researchers’ samples came to the anoxic basin of water, the fewer living loriciferans they found

What’s more, some of the loriciferans appeared to have eggs in their bodies, suggesting that they were reproducing. Others loriciferans were found in the process of shedding their shell and moulting, a further indication that they were alive.

Finally, the loriciferans in l’Atalante were completely intact and not at all decomposed – unlike other microscopic animals the researchers found in the salty, oxygen-absent environment.

After this careful work Danovaro and his colleagues made their findings public: the loriciferans were, indeed, living in an environment completely devoid of oxygen. Their 2010 paper, published in the journal BMC Biology, was a scientific sensation.

Even so, some other researchers are not convinced. A second team visited the Mediterranean in 2011 to examine for themselves the loriciferans and their unusual environment. Their findings, which were published late in 2015, challenge the idea that the loriciferans really do live without oxygen.

Joan Bernhard at the Woods Hole Oceanographic Institution in Massachusetts led this second team. She and her colleagues collected mud and water samples from just above the anoxic pools of L’Atalante. Due to technical difficulties, the pools themselves were too dense for their remotely operated vehicle to penetrate.

If the tiny animals really were dead and inhabited by bacteria, this would have been obvious

The team found the same species of loriciferans discovered by Danovaro. But these loriciferans were living in environments with normal levels of oxygen, and in the upper layers of the sediment above the anoxic pools, which had low levels of oxygen.

The closer the researchers’ samples came to the anoxic basin of water, the fewer living loriciferans they found.

Bernhard argues that it is extremely unlikely that loriciferans would be adapted to live both in areas totally without oxygen and high in salt, and also in environments with plentiful oxygen and normal levels of salt.

Instead, her team argues that cadavers of dead loriciferans could have floated down into the muddy sediments of the L’Atalante basin, where they were inhabited by “body-snatching” bacteria. Many species of bacteria are known to be able to live without oxygen, and they could have incorporated the biomarkers into the loriciferans’ bodies, potentially fooling Danovaro and his colleagues into believing that the loriciferans were alive.

However, in June 2016 Danovaro and his team came back fighting against this alternative scenario. They say that, because Bernhard’s team did not collect mud samples from the areas of the basin that are permanently without oxygen, they cannot be sure that loriciferans do not live there.

All lifeforms on Earth must generate energy if they are to eat, reproduce, grow and move around

Danovaro’s team also points out that, if the tiny animals really were dead and inhabited by bacteria, this would have been obvious when the loriciferans were examined under a microscope. But, in fact, the loriciferans showed no sign of being decayed and decomposed by microbes. Additionally, no bacteria were seen living inside the loriciferans, and a dye used to stain living tissue stained all parts of the loriciferans’ bodies, not just the parts where bacteria would likely colonise a dead animal.

Finally, they say that the thick layers of ancient mud deposits further support their argument.

“We were able to prove that these animals were present in different layers within the mud,” says Danovaro. “Some of the layers are several thousand years old and so, if these animals were just dead and preserved, it’s a bit unbelievable that the animals in 3,000-year-old mud are just as maintained as those found at the surface. The most likely explanation is that the animals can penetrate sediments, and swim and push to go down.”

But why is there such a controversy over whether animals can survive without oxygen anyway? No one doubts that bacteria can survive without oxygen, for instance. Why does it seem so unlikely that animals can?

Answering this question requires an explanation for why animals like us breathe oxygen in the first place. All lifeforms on Earth must generate energy if they are to eat, reproduce, grow and move around. That energy comes in the form of electrons, the same negatively-charged particles that flow through electrical wires and power your laptop.

On primordial Earth the atmosphere was heavy with a smog of carbon dioxide, methane and ammonia

The challenge for all life on Earth is the same, whether it is a virus, bacterium or elephant: you have to find both a source of electrons and a place to dump them to complete the circuit.

Animals get their electrons from the sugar in the food they eat. In a series of chemical reactions that happen inside animal cells, these electrons are released and bind to oxygen. That flow of electrons is what powers animal bodies.

Earth’s atmosphere and oceans are full of oxygen, and the reactive nature of the element means that it is “eager” to steal electrons. For animals, oxygen is a natural choice for an electron dump.

However, oxygen was not always as plentiful as it is now. On primordial Earth the atmosphere was heavy with a smog of carbon dioxide, methane and ammonia. When the spark of life first ignited, there was little oxygen around. In fact, oxygen levels in the oceans were probably extremely low up until about 600 million years ago – about the same time that animals first appeared.

This means that older, more primitive lifeforms evolved to use other elements as their electron dumps.

Many of these lifeforms – such as bacteria and archaea – are still living happily without oxygen today. They thrive in places on Earth that have little oxygen, for example in mud banks and near geothermal vents. Instead of passing electrons to oxygen, some of these creatures can pass on their electrons to metals like iron, meaning that they effectively conduct electricity. Others can “breathe” sulphur or even hydrogen.

The theory is that the evolution of life exploded when oxygen became available in the atmosphere and ocean

The one thing that unites these oxygen-free lifeforms is their simplicity. They all consist of just one cell. Until the 2010 discovery of the loriciferans, no complex multicellular lifeforms had been found that can live entirely without oxygen. But why is that?

According to Danovaro, this stems from two fundamental points. First, breathing oxygen is far and away a better approach to generating energy. “Complexity and organisation requires oxygen, because this is more efficient for the production of energy,” he says.

When oxygen levels rose, hundreds of millions of years ago, it was as if a brake had been taken off evolution’s ambitions. A group of lifeforms called the eukaryotes – which includes animals – took advantage, adapting to harness the new substance in their metabolism and becoming far more complex as a consequence.

“The theory is that the evolution of life exploded when oxygen became available in the atmosphere and ocean,” says Danovaro.

But this is only part of the story. Some species of microbe also began to breathe oxygen but, unlike animals and some other eukaryotes, they did not become complex. Why not?

Danovaro says the key to understanding the mystery comes from looking at mitochondria, the tiny structures inside eukaryotic cells that act as the lifeform’s powerhouse. Inside these mitochondria, nutrients and oxygen are combined to generate a substance called ATP, the body’s universal energy currency.

It wouldn’t work if they were the size of an elephant

Mitochondria are found in almost all eukaryotes. But bacteria and archaea do not carry mitochondria, and this is a key difference.

“When mitochondria evolved, they made the process of making energy and ATP much more efficient, but they needed oxygen to do this,” says Danovaro.

In other words, animal life arose as a consequence of two points. First, the eukaryotes had gained mitochondria inside their cells. Then, when oxygen levels rose, these mitochondria allowed some of those eukaryotes to gain complexity and become animals.

So how come loriciferans can get by without oxygen when other animals cannot?

“They are very tiny, about the size of a large amoeba,” says Danovaro. “The small size helps. It wouldn’t work if they were the size of an elephant. As they are small their energy requirement is less.”

The loriciferans might differ from other animals in another important respect. They seem to lack the oxygen-using mitochondria found in all other animals. Instead, they may carry structures related to mitochondria called hydrogenosomes.

Some animals – like the loriciferans – may have stuck it out and lived without oxygen, remaining small as a consequence

These use protons instead of oxygen as their electron dump. Hydrogenosomes may even be one of many primitive types of mitochondria, which evolved in early eukaryotes to produce energy before atmospheric oxygen levels arose.

“I think the eukaryote common ancestor was a facultative anaerobe that could live with or without oxygen, much like E. coli, a well-known bacterium,” says William Martin, a professor of molecular evolution at the University of Dusseldorf, Germany.

This has important ramifications for understanding how and in what conditions complex life first appeared. The first eukaryotes probably evolved before oxygen was routinely freely available in the ocean, so the mitochondria-like structures inside their cells might have been adapted to both oxygen-present and oxygen-absent conditions. Then, as oxygen became more abundant, first in the atmosphere and then in the ocean, some eukaryotes adapted to their new oxygen-rich environments and became large and complex. They became animals.

But some animals – like the loriciferans – may have stuck it out and lived without oxygen, remaining small as a consequence.

For this scenario to work, the loriciferans must have retained their ability to live without oxygen from their ancient ancestors. But there is an alternative: the loriciferans might have gained their ability to do without oxygen very recently, perhaps by stealing genes from other species in a process known as horizontal gene transfer.

As soon as you put it under the microscope you kill it

“This could be evolution in action, as all previously-known species of loriciferans respire oxygen,” says Danovaro. “It is possible that this is an extreme adaptation to allow the loriciferans to live in an environment without competitors or predators.”

For now the scientific community waits with bated breath for more evidence confirming or disproving the original finding. “I think it is a stalemate at present,” says Martin. “What is needed are more samples for closer study.”

Final proof would be seeing the animals swimming around in the mud, but according to Danovaro, the small size of loriciferans and their difficult-to-reach environment makes it hard to make those sorts of observations.

“The animal is one-tenth of a millimetre so it requires a special system, because as soon as you put it under the microscope you kill it,” he says. “In principle you can extract its DNA, which is the next thing we are working on, but someone could still say, ‘well, that animal is dead’. It’s a very long track to get final confirmation but we are very optimistic.”

Source: BBC.com