The boring giant clam is anything but.

On June 21, 2018June 24, 2018 By Dan KillamIn Biology, Chemistry, clams3 Comments

crocea2 — *Tridacna crocea*, bored into a coral head on a reef in Palau

There are many types of giant clam. Not all of them are giant; the boring giant clam, Tridacna crocea, only grows to 10 cm long or so. The boring giant clam is not so named because it’s dull; its main skill is its ability to bore into the coral of its coral reef home and live with its entire shell and body embedded in the living coral. They sit there with their colorful mantle edge exposed from a thin opening in the coral, harvesting energy from sunlight like the other giant clams. When disturbed by the shadow of a human or other such predator, they retract their mantle and close their shell, encased by an additional wall of coral skeleton. It’s a clever defensive strategy, and they are some of the most numerous giant clams in many reefs in the Eastern and Southern Equatorial Pacific.

But it’s always been a mystery of how they bore away at the coral so efficiently, and how they continue to enlarge their home as they grow their shell. There are other bivalves that are efficient borers, including the pholad clams (“piddocks”) which use sharp teeth on their hinge to carve their way into solid rock, and the shipworms, which have abandoned their protective shell and instead use their two valves as teeth to burrow into wood. Both of these methods of boring are pretty straightforward.

bivalve_pholas_dactylus_common_piddock_24-08-10_shells_1 — Piddocks in next to holes that they made in solid rock. Source: Aphotomarine

medium — Shipworm embedded in wood. Source: Michigan Science Art via Animal Diversity Web

But the boring giant clam has no such adaptation. It does not have large teeth on its hinge to carve at the coral. Such abrasion of the coral would also not explain how they widen the opening of their cubby-holes to allow their shell to grow wider. This mystery has long confounded giant clam researchers. I myself have wondered about it, and was surprised to find there was no good answer in the literature about it. But now, a team of scientists may have cracked the problem once and for all.

At the back of T. crocea‘s shell at the hinge, there is a large “byssal opening” with a fleshy foot which they can extend out of the opening to attach themselves to surfaces. Giant clams that don’t embed in coral (“epifaunal,” resting on the surface of the coral rather than “infaunal,” buried in the coral) lack this opening. The researchers suspected that the foot was the drilling instrument the clam used to create its home.

dscn0083 — Byssal opening of *T. crocea* with the foot retracted. Source: NickB on Southwest Florida Marine Aquarium Society

How could a soft fleshy foot drill into the solid calcium carbonate (CaCO3) skeleton of corals? I can confirm from experience that my own foot makes for a very ineffective drilling instrument in such a setting. But T. crocea has a secret weapon: the power of acid-base chemistry. CaCO3 can be dissolved by acids. You may well have taken advantage of this chemistry to settle your acid stomach by taking a Tums, which is made of CaCO3 and reacts with the excessive hydrochloric acid in your stomach, leaving your tummy with a more neutral pH. pH is a scale used to measure acidity, with low numbers indicating very acidic solutions like lemon juice, and high pH indicating a basic solution like bleach.

Scientists are well aware of the hazards corals face from decreasing pH (increasing acidity) in the oceans. All the CO2 we are emitting, in addition to being a greenhouse gas, dissolves in the ocean as carbonic acid and gets to work reacting and dissolving away the skeletons of corals and any other “calcifying” organisms that make shells. It makes it harder for corals to form their skeletons and is already worsening die-offs of corals in some areas. The researchers suspected that the clams use this phenomena to their advantage at a small scale, lowering the pH with their foot somehow to dissolve away the coral to make their borehole.

Using a wedge to keep open a *Tridacna* shell in my Red Sea work. We took a small blood sample with permission of local authorities. This caused no lasting effects to the clams.

But they needed to prove it, and that was a challenge. Giant clams can be unwilling research participants. I myself have observed this in trying to take samples of their body fluid for my own research. When they sense the presence of a predator, they immediately clam up in their protective shell. I used a small wedge to keep their shells open to allow me to take a sample of their body fluid, but the researchers working on T. crocea needed to convince the clam to place its foot on a piece of pH-sensitive foil, keep it there and do whatever acid-secreting magic allows it to burrow into coral. They would then be able to measure whether it indeed is making the water around its foot more acidic, and by how much.

In what I can only assume was an extended process of trial and error and negotiation with a somewhat unwilling research subject, the researchers found exactly the right angle needed to convince the clam that it was safe enough to try making a coral home. But it was not in coral, instead sitting in an aquarium, on top of a special type of foil that changes color when exposed to changing pH, like a piece of high-tech litmus paper. The researchers discovered that their suspicions were correct: the clams do make the area around their feet significantly more acidic than the surrounding seawater, as much as two to four pH units lower. Where seawater is around a pH of around 8, the clams were regularly reducing pH to as low as 6 (about the level of milk) and sometimes as low as 4.6 (about the pH of acid rain). Small differences in pH can make a big difference in the power of an acid because each pH unit corresponds to 10x more protons (hydrogen ions, H+) in the water. The protons are the agent that dissolves CaCO3. Each proton can take out one molecule of coral skeleton. The clams are dissolving away coral skeleton to make holes with only their feet!

Footage of the pH- sensitive foil, with darker areas corresponding to lower pH. The areas of low pH (high acidity) correspond exactly to the “footprint” of the clam!

But what in T. crocea‘s foot allows them to make acid? I know that my foot does not do this, though that would be a very entertaining and obscure superpower. The researchers found the enzymes called vacuolar-type H+-ATPase (VHA) present in great quantities in the outermost cells of the clam’s feet. These enzymes are found throughout the tree of life and are proton pumps that can quickly reduce pH through active effort. Other prior researchers like the influential Sir Maurice Yonge, a legendary British marine biologist who worked extensively with giant clams, had suspected that the clams had used acid but had never been able to detect a change in pH in the seawater around the clams’ feet through more conventional methods. It was only because of new technologies like the pH paper that this research team was able to finally solve this issue. And now, I suspect other groups will want to re-investigate the importance of VHA in their study organisms. Many branches of the tree of life may be utilizing acid-base chemistry to their advantage in ways we never had previously imagined.

You are Isotopes (Part III)

On August 30, 2017August 30, 2017 By Dan KillamIn Biology, ChemistryLeave a comment

This is the third part of a series about isotopes and why they’re useful and interesting to scientists.

Isotopes are the flavors of elements. And because our universe is made up of atoms of elements, every object can be thought of as a delicious smoothie of flavors. Scientists like me are trying to reverse engineer those mixtures and pick out individual tastes, in order to answer questions about our world.

For example, I work with giant clams. These guys build enormous shells made of a mineral called calcium carbonate: CaCO₃. That means that every molecule in a clam’s shell contains a calcium atom, a carbon and three oxygens. But as you might know from reading the previous entries in this series, not all of those atoms are the same. They are a mixture of different flavors. We have some carbon-12 and 13 in there (so named for their atomic weights), and some oxygen-16, 17 and 18. Here I’m focusing on the stable isotopes, which are not radioactive and are called “stable” because they’re not going to self-destruct. There are radioactive isotopes in there too, but I don’t use those nearly as often in my work.

calcium-carbonate-powder-1291396 — Officer, this is a pile of giant clam powder, I swear!

I am measuring stable isotopes of carbon and oxygen in my shell samples. To do this, I take a sample of powder, grind it up, weigh it, and put it into tiny little cups. We only need a very small sample: about 50 micrograms of shell material. A typical pill of tylenol contains over 300 mg of active ingredient, so about 6,000 of my samples will fit in a single tylenol regular strength pill, if you suddenly decided you needed a giant clam prescription.

deflect — Simplified representation of what’s happening in a mass spec. Source

This tiny sample is one of thirty that I can measure at a time. Those samples are reacted with acid and the CO2 gas that is released as a result of the reaction can be processed by a machine called a mass spectrometer. The mass spec, which is in the Stable Isotope Laboratory in my building, ionizes the molecules in that gas (gives them a bit of electric charge) and then those ions are flung through an electromagnetic field. That beam of charged gas is flung around a curve. That curve is where the magic of making a mass spectrum happens.

Think of the atoms in the CO2 gas from my sample as a bunch of racecars exiting the straightaway and starting around the curve on the racetrack. Only these racecars vary in weights. And the race organizers have greased the track at the curve so that they fling into the sides of the track when they try to turn. As the racecars fling into the sides of the track, they will separate according to their mass. The lighter cars will be able to make it further around the curve before they meet their demise because they have less inertia forcing them forward, whereas the SUVs in the race will barrel forward straight into the sides of the track. At the end, you have a spectrum of racecars poking out of the walls of the track, with SUVs first, then the coupes, then the compact cars and then the motorcycles, which almost made it around the bend, but not quite. Atoms in the mass spectrometer act the same way, and we measure how many collisions happen along each point of the bend in order to not only “weigh” the sample of gas, but also figure out how many molecules of each weight there are!

It turns out that it is quite difficult to measure the exact number of atoms of a particular isotope in gas, however. It is much more economical and feasible for the purposes of most researchers to simply compare our mass spectrum to the results from a standard. Much like there is a literal standard kilogram and standard meter in a lab somewhere in France which is used to keep track of how much mass is actually in a kilogram, there is a standard used by all researchers like me to describe our samples of carbonate.

6852716289_cca3c0a635_b — A collection of belemnite fossils from the Pee Dee formation, similar to the one used for the PDB standard. Source

The most common standard used is from a belemnite fossil from the Pee Dee formation in North Carolina. Belemnites are extinct squid-like creatures that formed an internal shell, and one of those internal shells was fossilized, unearthed by a researcher and ground up to become the reference for all other researchers following. Samples of the carbonate in its fossil had more carbon-13’s per unit mass than most other fossil specimens known. Almost everything you measure will be “lighter” in terms of carbon, because carbon-12 is naturally so common on our planet.

Scientists needed a convenient way to put a number on this, so a simple formula was developed which would allow us to quickly communicate to each other how isotopically “heavy” or “light” a particular sample is in comparison to the Pee Dee Belemnite. The formula isn’t that important for our purposes but the units of its output are in parts per thousand, or “per mil” for short (same idea of how we shorten parts per hundred to “percent”).

The symbol for per mil is a percent sign with an extra little loop at the end: ‰. To make the shorthand complete, we also need to note that this is how much the carbon-13 to carbon-12 isotope ratio of a sample differs from the Pee Dee Belemnite. We do so, we use the Greek delta symbol (δ), commonly used in science and math to represent “difference or change from.” So a sample that has a carbon-13 to carbon-12 ratio which is 20 parts per thousand less than that of the Pee Dee Belemnite is written -20 ‰ δ¹³C_PDB. There are other samples that can be used as well, including Standard Mean Ocean water (SMOW), and the Vienna Pee Dee Belemnite (VPDB). It’s important to note which you are using so that people know the scale of your measurement!

Phew, hopefully that didn’t confuse the hell out of you! Next time, I’ll talk about how different δ¹³C (and for oxygen isotopes, δ¹⁸O) can tell us different details about the life of an organism. Here’s a cute gif of a scallop as a chaser after all that science you read.

You are isotopes (Part II)

On August 14, 2017 By Dan KillamIn Chemistry2 Comments

This is the second part in a series how isotopes work and how they are scientifically fascinating. Part I here.

It turns out a horse is not just a horse, of course. The horse is a collection of atoms, and each of those atoms has a particular isotopic “flavor”, and the collection of isotope types in the horse tells a story. At the end of the day, scientists are simply interested in reading and telling stories about our world. The tail….er, tale of the horse is written by myriad interacting processes in the universe which influence the horse’s stable isotope ratios.

As I mentioned last time, carbon-12 is much, much more common than carbon-13 is on our planet, due to nuclear fusion of helium-4 in the sun. there are nearly 99 carbon-12’s on earth for every carbon-13. But that’s the base ratio if you took our whole planet, put it in a blender and mixed it all up. If you measured a particular object, such as a horse, it likely does not follow that measure exactly. It has become differentiated from the global average by numerous factors which have altered the isotope ratio.

In isotopic chemistry, fractionation is our name for any process which creates a preference for a certain isotope. If chemical reactions had no bias toward any particular isotope, that 99 to 1 ratio of carbon-12 to carbon-13 would be present in literally everything including you and me. But it turns out that the biochemical dice are loaded- to make the ratio even more biased!

The enormous Rubisco enzyme. No one said photosynthesis was simple. Source: Wikipedia

Photosynthesis is the process by which plants take carbon dioxide gas in the atmosphere and “fix” it to make sugars, which they then use for food. The core enzyme responsible for this carbon fixation is called Rubisco (short for Ribulose-1,5-bisphosphate carboxylase/oxygenase). This enormous molecule is likely the most abundant enzyme on earth. And it turns out that it has a favorite flavor when it comes to the carbon it fixes into sugar.

In fact, the entire plant is discriminating against carbon-13 in several of the processes of photosynthesis. Carbon dioxide molecules diffuse more quickly into the plant’s leaves if they include the lighter carbon-12 rather than carbon-13. “Light” CO₂ also dissolves more easily in the plant’s fluids. But the biggest fractionation happens when the Rubisco molecule gets hold of CO₂ and breaks it. At each of these steps, the light carbon-12 is more likely to be used by the plant than its heavier siblings. There are various thermodynamic reasons for why this is the case, but the plant is essentially a sieve removing more of those heavy carbons at every step. At the end of the process, the plant is left isotopically “lighter” than the CO₂ gas surrounding it that it breathes in.

Because you are what you eat, this means that you are suspiciously carbon-light, and there’s nothing you can do about it. Should have thought of that before you decided to be dependent on plants as the factory for your carbon-based molecules. Next time, we’ll talk about how we measure this, and the kinds of science that can happen once you have a nice consistent measurement to use to compare isotopic ratios between samples.

You are isotopes (Part I)

On July 31, 2017July 31, 2017 By Dan KillamIn Chemistry5 Comments

As you may well know, every element is defined by its number of protons contained the nuclei of its atoms. Hydrogen has one. Carbon has six. This is non-negotiable. But every element can be found in multiple “flavors” known as isotopes. This flavor depends on the isotope’s atomic mass, which is determined by the number of neutrons present in the nucleus of that atom. Neutrons are kind of like atomic ballast. Unlike protons, which have a positive charge, they are neutral, but they do have a mass. Different isotopes have different numbers of neutrons, determining their atomic mass but preserving its particular elemental identity (which would only change if you changed the number of protons present).

Let’s focus on carbon, an element which I think about daily, though every element has isotopes and I could pick many other examples. Hope you’re OK with that, but if not it’s my blog so deal with it. So carbon has been found or created in up to 15 flavors. A whopping 98.9% of all the carbon on Earth occurs as carbon-12 (written as ¹²C), which has six protons and six neutrons, adding up to an atomic mass of about 12 atomic mass units (amu). It’s the most common because it’s the product of three helium-4 isotopes fusing together, each weighing 4 amu + 4 amu + 4 amu adding to make a single carbon-12. This is a very common reaction in stars, and because you are stardust, it is also the most common flavor of carbon in you.

But we make other flavors by adding neutrons. You can make carbon-13 with six protons and seven neutrons. This is a rare flavor, accounting for almost all of the remaining 1.1% of carbon found on earth. It is also the only other stable form of carbon. I note that it’s stable because all the other 13 known flavors of carbon are unstable, and many are only known from the laboratory because they are too short-lived to be found in the environment.

It turns out that if an element’s atomic nucleus is too light, or too heavy, that element will become radioactive and decay with time, continuously firing off pieces of itself out of frustration. Carbon-14 is the most famous and common of these radioactive isotopes of carbon, and it still only makes up 1 in every million million atoms of carbon on earth. Carbon-14 fires off particles and decays into nitrogen-14 because it is more stable orientation for the protons and neutrons to be in, for physics reasons I won’t get into here.

Carbon-14 does this in a very predictable, methodical pattern. It’s difficult to predict when an individual carbon-14 atom will do this, but if you take any object you have just created, like a piece of pottery, for example, you can be pretty much certain that in 5,730 years, only 1/2 of the carbon-14’s will still be present. The rest decided they’d rather be nitrogen-14. This is non-negotiable and you’d best learn to accept it. But it means that we can sniff out the age of a lot of interesting mysterious objects if we know the amount of carbon-14 present in the environment (which we often do) and measure the amount present in the object today. You have some restrictions. For example, for objects that are too old, too little of the carbon-14 would be left for you to measure accurately.

Carbon-14 dating, often just called radiocarbon dating, is very useful in figuring out the ages of stuff, but I’m mostly interested in the stable isotopes of carbon. Next week I’ll talk about why that is, and what kind of questions I can answer by looking at amounts of different stable carbon isotopes in a sample. See you then!