by Helen Scales
In thousands of studies, conotoxins have aided researchers in understanding the fundamental processes of living things; they have advanced our understanding of how muscles contract, how blood pressure is regulated and how kidneys and retinas work. Conotoxins are showing just how many types of receptor there are and revealing the complexity of the human brain. Researchers are now figuring out the roles played by distinct receptors in neurological diseases like Parkinson’s, Alzheimer’s and alcoholism. And as well as helping to understand diseases, conotoxins are also helping to build a new arsenal of medicines to tackle them. For 50 million years cone snails have been evolving and perfecting the precision of their toxins; now biochemists are tapping into this immense repertoire and finding conotoxins that have specific therapeutic effects on the human nervous system. Dozens of conotoxin-inspired medicines are in development for treating an immense variety of disorders.
In the 1980s, Olivera’s team found a conotoxin extract from the Geography Cone Snail that induced a sleep-like state in laboratory mice. This ‘sleeper peptide’ is one of the soporific drugs that net-hunting cone snails waft into the water to sedate their prey. The active ingredient was later identified as conantokin-G, a conotoxin in the ‘nirvana cabal’ that blocks a specific type of ion channel receptor for the neurotransmitter glutamate, called the NMDA receptor. Clinical trials are currently underway to see if this conotoxin could help calm the hyperactive nerves of people suffering from intractable epilepsy. It could also stop the breakdown of nerves in patients with Alzheimer’s and Parkinson’s. Other conotoxins are being investigated as treatments for heart attacks, multiple sclerosis and ADHD. And for more than a decade now, people suffering from chronic pain have been deliberately injected with cone snail stings.
Ziconotide, marketed as Prialt (the ‘primary alternative’ to morphine), is an artificial version of a conotoxin originally found in the Magician’s Cone Snail. Prialt blocks calcium channels that transmit pain signals from nerves to the spinal cord and on up to the brain. It is a thousand times more potent than morphine, with much lower risks of addiction. The main drawback, though, is that it must be injected directly into the spinal fluid using a small pump inserted under the skin, an obviously intrusive procedure. A research team at the University of Queensland, where Bob Endean’s early studies of cone snails were carried out, are now working on conotoxin pills. To do so, David Craik and his colleagues are making and testing synthetic conotoxins that are looped around into rings, making them more stable and likely to survive passage through the human digestive tract.
And it’s not just conotoxins that are inspiring new drugs from cone snails. It turns out their weaponry is even more complex than previously thought. In 2015, a startling new finding emerged from a team at the University of Utah that included Baldomero Olivera. The study, led by Helena Safavi-Hemami, revealed that some cone snails send fish to sleep using sedatives laced with insulin. The peptide hormone elicits hypoglycaemic shock – a dangerous drop in blood sugar levels – making the fish pass out. Various forms of insulin are components of the ‘nirvana cabal’ of the Geography Cone and Tulip Cone, and they have evolved to be structurally more akin to fish hormones than molluscan varieties. These are the first known cases of weaponised insulin, and they open new avenues of research into how insulin works and, while it’s still a way off, there’s the potential for developing new drugs to treat diabetes.
Researchers have come a long way from watching sea snails master the unlikely skill of fish hunting. With so much knowledge generated and so many new ideas for medicines, the most astonishing thing of all is to contemplate what we still don’t know. There is so much left to discover inside cone snails and their tiny harpoons. So far, only around a hundred conotoxins have been studied in detail from six species, which surely means these masters of chemistry still have a thing or two left to teach us.
Sticking, gluing and digging
Down at the coast there are seashells that sit tight in some most improbable places. Mussels glue themselves to wet, slippery rocks despite the relentless crashing and sucking of waves around them, and for decades scientists have watched in envy, desperate to find out how they do it. Underwater superglue is another of the latest inspirations from molluscs.
Herbert Waite was one of the first people to begin unlocking the mussel’s sticky secret. As a graduate student at Harvard in the 1970s, he began gathering mussels from the northern shores of Long Island Sound in Connecticut, on the US Atlantic coast. Back in the lab, he scrutinised the byssus fibres that mussels use to anchor themselves, and Sardinian weavers use to spin golden threads. He broke the proteins down into their separate components, and among them he discovered a rare amino acid called L-dopa.
L-dopa had been known of for a while. It is present in various plants and animals and the human body makes it as a precursor to the neurotransmitter dopamine. For this reason it was developed as a treatment for Parkinson’s disease and other conditions. The 1990 movie Awakenings tells the true story of how neurologist Oliver Sacks used L-dopa to rouse patients from decades of catatonia.
Waite was the first to pinpoint the role of L-dopa in mussel glue. He worked out that this amino acid is key in allowing the liquid protein, secreted by the mussel’s byssal gland, to set hard in saltwater and stick the mussel in place. Since his discovery, many different glue proteins – referred to as Mussel Adhesive Proteins or MAPs – have been identified, and they all contain this molecule.
Currently researchers, including Herbert Waite and his lab now at University of California, Santa Barbara, are studying how MAPs work. The full picture has yet to be revealed but an important factor has been established. L-dopa molecules contain side-chains called catechols that interact directly with surfaces, be it a rock, a boat hull or whatever a mussel is trying to stick to, forming bonds that fix the mussels in place.
Synthetic glues laced with L-dopa or other catechol-containing compounds are being developed, with lots of potential applications. Most immediately, mussel-inspired glues are likely to be used inside the human body. A glue that works in blood vessels, with blood coursing through them, will be incredibly useful to surgeons. In particular, foetal membranes are very difficult to repair and bio-glues are being tested as a suture-free way of operating on unborn babies.
In addition, patients suffering from atherosclerosis and the build-up of plaque inside their blood vessels could be treated with a squirt of glue in their arteries, to help prevent heart attacks and strokes. Currently, stents or balloon angioplasties inserted into blood vessels to widen them are smeared in anti-inflammatory drugs, but around 95 per cent of the drug gets washed away in the blood flow. Bio-glue could see an end to this wastage.
Diabetes could also one day be treated with a dab of mussel-inspired glue. Instead of having to inject insulin, an alternative is for diabetics to have pancreatic cells from donors transplanted inside their bodies to produce insulin on their behalf. Currently, it’s possible to insert these cells inside the liver, but they only work for a few years. Using bio-glue, it might be possible to find somewhere else to stick these cells, on the outside of the liver, perhaps, without triggering inflammation and giving them a much longer lifespan.
One of the latest ideas to emerge from Herbert Waite’s lab is a synthetic polymer, covered in catechol-rich proteins, that can heal itself. Potential applications for this new material include the manufacture of hip and knee replacements that would require little or no surgery to maintain. It could also be used to reinforce hairline cracks in brittle bones. The mussel-inspired polymers could even one day be used to make self-repairing surfboards.
Beyond the human body, and wave riders, there is another, rather unexpected use of mollusc glues. MAPs could be used to stop molluscs from sticking. Fouling organisms are the weeds of the sea, growing in places they aren’t wanted. When molluscs and barnacles clamp on to boat hulls they increase drag in the water, pushing up fuel bills. And while many boats these days are metal or fibreglass, shi
pworms and their boring habits are still a threat to wooden jetties and pontoons.
Various treatments have been developed over the years to try to stop these nuisance creatures from getting a grip, but one in particular turned out to be grimly toxic. Tributyl tin, or TBT, was banned worldwide in 2008 after it was found to cause all sorts of ecological problems when used as anti-fouling paint (TBT compounds deter marine larvae from settling on treated surfaces). Alarms were raised when marine biologist Stephen Blaber found that female dog whelks around the British coasts were sprouting male genitalia. Far from being a minor inconvenience, a female whelk exposed to trace amounts of TBT will sprout a penis so large it blocks her oviducts, preventing eggs from being released and rendering her infertile (ecologists now routinely measure the length of wild female whelk penises as a gauge of environmental pollution). Maritime industries are still hunting for replacements for TBTs; one possibility is to use mussel glues to stick other, less harmful anti-fouling agents firmly to boats to keep their bottoms clean.
While mussels use chemistry to spend their lives stuck implausibly to rocks, another group of bivalves have become masters of physics and move in a way that at first seems impossible. Razor clams are long, narrow bivalves that spend much of their lives buried in sandy, muddy shores. They dig using a two-anchor system, opening their twinned shell slightly to hold it fast while pushing their muscly foot into the sediment. The clam then pumps blood into its foot, making it swell up and act as a second anchor while it pulls the shell downwards.
Based on the shape, size and strength of razor clams, calculations indicate that they should only be able to dig a short way before getting stuck by the pressure of mud and sand crushing down on them. Researchers have tested this by shoving model razor clams into a sandy beach. Test shells only penetrated a couple of centimetres (about an inch) beneath the surface. By contrast, the Atlantic Jackknife Clam, which measures around 20 centimetres (eight inches), can dig far deeper than its muscles and shell alone should allow. Razor clams have evolved a way of being so energy efficient they could burrow half a kilometre (a third of a mile) using just the power in a household AA battery. It turns out that the key to their digging skills lies in a little puddle of quicksand. Repeatedly opening and shutting their shells causes hard sediment around a razor clam to collapse, and water seeps inwards, creating a pocket of liquidised sand or mud. This reduces drag and cuts down the energy needed to burrow by around 10 times.
A robotic version of a razor clam is helping Amos Winter to explore this idea of quicksand digging. Along with his colleagues at the Global Engineering and Research Lab at MIT, Winter has spent a lot of time wading through mudflats in Gloucester, Massachusetts with RoboClam in tow.
Winter trained RoboClam to be as good at digging as possible by using an algorithm inspired by natural selection. This approach mimics biological evolution by generating hundreds of random tweaks in the design of little piston-powered clams, and testing which works best. The result is an imitation clam that can dig as effectively as a real one. Winter envisages that RoboClam will one day lead to small, low-power digging systems that could drastically cut the costs of anchoring boats. Even more excitingly, engineers have their eyes on international internet traffic, now that almost all this information passes over the seabed along submerged communication highways (a far cheaper method than using satellites). RoboClam’s descendants might one day be used to pin down the fibre-optic cables that reach between continents and wire up the Earth.
Their ability to stir up pools of quicksand is not the only thing that makes razor clams such expert diggers. They also depend on their shells not snapping in two. And like all molluscs, their shells are surprisingly strong.
Shiny on the inside
Being essentially made of chalk, seashells should be easily shattered. If you’ve ever snapped a stick of chalk in two you’ll know what I mean. And yet you can squeeze them, drop them, hit them with hammers, do what you like (within reason), and many mollusc shells will stay in one piece. This poses yet another conundrum that endlessly teases scientists and engineers. Why don’t seashells break all the time? Why don’t they snap as easily as a stick of chalk?
Hunting for answers to these questions has led researchers into the inner structure of seashells and the nano-world of the incredibly small. At this scale molluscs have evolved elegant solutions to the problems of attack, and found ways of not getting smashed. And it’s here we find inspiration for constructing protective shells of our own.
In the last few years there’s been a surge of interest among scientists and engineers in mother-of-pearl. This is the shiny layer on the inside of shells, also known as nacre, and the stuff that pearls are made of. It’s well known that pearls are the result of parasites or bits of grit that irritate a bivalve’s soft innards; the mollusc envelops these foreign bodies in layers of smooth nacre to protect itself (a fact that gem merchants rarely admit). Material scientists are busy learning lessons from nacre; not, though, on how to make things iridescent and pretty, but on how to make things incredibly strong.
Adult mollusc shells are commonly made of aragonite, a tough form of calcium carbonate that offers good protection against crab claws, fish jaws and even attacks from its own kind as they try to drill and stab their way in. However, the layer of calcium carbonate is prone to fractures. When cracks appear on the outside of a shell they spread, but they stop as soon as they reach the shiny inner layer of nacre. This happens because of nacre’s unique architecture.
Seen through an electron microscope, nacre is composed of diamond-shaped crystals, stacked like bricks in layers on top of each other, made of 95 per cent aragonite. At around 300 to 500 nanometres thick, these layers are just the right size to make waves of visible light bounce around between them, creating the structural colours that give mother-of-pearl its gleam. These nacreous bricks are mortared together with much thinner layers of chitin, the protein component of insect and crustacean exoskeletons. When cracks spread through nacre they are sent on a tortuous path between those microscopic bricks, which saps their energy and stops them in their tracks. The bricks slide over each other and the chitin protein stretches, helping to dampen the growing fracture.
Layers of nacre are also somewhat kinked, which seems to make them even more crack resistant. Mohammad Mirkhalaf and François Barthelat from McGill University demonstrated the importance of this waviness in nacre by mimicking its nano-scale structure. They used a laser to etch wavy lines into small glass plates and showed, counter-intuitively, that the glass became tougher by scratching it.
Over recent years several research teams have used a range of ingredients to make synthetic nacre. A team from Manchester and Leeds Universities used calcium carbonate mixed with polystyrene to make crack-resistant ceramics that could one day be used in building materials and bone replacements. Lorenz Bonderer and a team at ETH Zurich mimicked nacre using thin plates of aluminium oxide coated in chitosan (a material derived from shrimp and crab shells with added sodium hydroxide) to make a composite material that could be used to make aeroplanes and spacecraft. And in 2012, a group from Cambridge University were the first to make artificial nacre the way nature intended. Alex Finnemore and Ulli Steiner headed up a team that imitated the steps molluscs themselves take to produce nacre. They laid down layers of calcium carbonate and then sandwiched them between protein sheets pitted with pores. This artificial nacre looks, feels and behaves just like the real thing.
The next, eagerly awaited step is to start using mollusc-inspired materials for applications in the human world. In the pipeline are visors for motorcycle and space helmets, based on a mollusc named, appropriately enough, the Windowpane Oyster. These oysters, also known as capiz shells, were traditionally used in Asia to make windows, and more recently have been made into all sorts of decorations; a roaring export trade in lampshades, candle holders and Christmas lanterns called paról has wiped out stocks of these molluscs in the Philippines. The shells are not just decorative an
d see-through – they also happen to be incredibly strong.
Ling Li and Christine Ortiz from MIT worked out that Windowpane Oysters, which are 99 per cent calcite, have a similar nanostructure to nacre, with layers of elongated hexagonal crystals. In the lab, Li and Ortiz whacked chunks of these shells with a diamond-tipped hammer, then inspected the damage under an electron microscope. They saw the crystals behaving in various complicated ways including so-called nanocracking, visco-plastic stretching and nanograin formation; suffice to say that at a nano scale, the crystals put a halt to the spread of damage and set up a no-go buffer zone that cracks don’t cross. It means that unlike artificial ceramics, damaged shells stay largely intact and crystal clear. So, if their oyster-inspired helmets get cracked, astronauts will still be able to see out.
Composite ceramics, based on ideas borrowed from deep-sea snails, could one day show up in military body armour and vehicles. The Scaly-foot Snail was discovered on the Kairei hydrothermal vent field in the middle of the Indian Ocean, more than 2,000 metres (1.2 miles) beneath the waves. The scaly-foot gets its name from its strange covering that looks rather like the sclerites that covered the Cambrian creature Wiwaxia. That would seem odd enough for a modern mollusc, but even more bizarrely their scales are made of iron, and their shells are too. No other organisms are known to make skeletal structures clad in iron.
The Scaly-foot Snails make tri-layered shells that trump those of all the other molluscs. Sandwiched between hard layers of iron sulphide on the outside and calcium carbonate on the inside lies a spongy organic sheet. Back in Christine Ortiz’s lab at MIT, her research team tested the protective capabilities of these peculiar iron shells, again by bashing them with sharp probes, then bathing them in hot acid and programming computer simulations of predator attack. They worked out that each layer in the shell has its own distinctive role in protecting the squashable snail inside.
