Research Explainer: The chemistry across a “forest” of giant clams

On July 19, 2020July 20, 2020 By Dan KillamIn clams1 Comment

Another one of my PhD chapters is published in the journal G-cubed, resulting from work I did in the summer of 2016 in Israel and Jordan around the Red Sea. This is my first geochemistry article in a journal, so it is a big deal to me! I thought I’d write up a clamsplainer about what I was looking for and how we went about achieving the paper.

A slice of giant clam shell. You can see the difference between the inner and outer layers. The inner layer has visible annual growth lines.

I study the chemistry of giant clam shells. You might already be familiar with the concept of tree rings, a field called dendrochronology. It’s like reading the diary of a tree, where every “ring” is a page in the record of its life. The related field of sclerochronology looks at rings in the hard parts of shelled organisms. We can count those rings to figure out the ages of clams, or their health, and we can measure the chemistry of those rings to understand the temperature the clam grew at, and even what it ate.

A giant clam growing on the reef flat in Eilat

Giant clams are bivalves of unusually large size which achieve a very rapid growth rate through the help of symbiotic algae in their flesh. The clams are farmers, and their crop is inside their tissue! They grow their shells very quickly (sometimes up to 5 cm a year, equivalent to if a six foot tall man grew a foot every year from birth), and live a very long time, up to 100 years (their growth slows later in life). A whole bunch of talented researchers have measured the chemistry of giant clams all around the world to reconstruct past climate and even measure historic storms!

If we want to understand the ecology of a forest, we can’t measure just one tree!

But if you come back to the analogy of tree rings, we essentially have measured the rings and chemistry of individual “trees” in a bunch of different places, but don’t have as good an idea of how the chemistry varies within a “forest” of giant clams in a particular place. In our new study, we set out to describe exactly that, focusing on the Northern Red Sea.

A map of the Northern Red Sea. The right “toe” is the Gulf of Aqaba

Sites where we sampled shells along the northernmost tip of the Gulf of Aqaba

The Gulf of Aqaba represents the northernmost toe of the Red Sea, bordered by Egypt, Israel, Jordan and Saudi Arabia. It hosts some of the northernmost coral reefs in the world, aided by tropical temperatures and clear waters due to the lack of rainfall in the surrounding deserts. Here, we can find three species of giant clams including the small giant clam Tridacna maxima, the fluted giant clam T. squamosa and the very rare T. squamosina, which is found only in the Red Sea and nowhere else (as far as we know). In summer 2016, I went all around the Gulf of Aqaba collecting shells of clams from the beaches, fossil deposits, and even were able to work with shells confiscated from smugglers at the Israel-Egypt border. We cut these shells into slices and used tiny drill bits to sample powder from the cross section of their shells, which we could then conduct geochemistry with! We sampled large areas in bulk from the inner and outer portions of the shell (more on why later) using a Dremel tool, and also sampled more finely in sequential rows with a tiny dental drill bit (same brand your dentist uses!) to see how the measured temperatures varied through seasons. By “we”, I mean my coauthor and friend Ryan Thomas, who spent every Friday morning for several weeks milling out most of the powder we needed for this study. This data became part of his senior thesis at UCSC!

Two giant clams thriving on the shallow reef near Eilat, Israel

What kind of chemistry did we measure? The shells of clams are made of calcium carbonate, the same stuff Tums is made of. Calcium carbonate contains one calcium atom, one carbon atom, and three oxygen atoms. It turns out that all of those atoms come in “flavors” that we call isotopes, relating to the weight of those atoms. When you take the shell powder and put it into a machine called a mass spectrometer, you can figure out the proportions of isotopes of different elements present in the samples

The first isotope “flavors” we were interested were carbon-12 and carbon-13. The ratio of the two is thought to relate back mostly to the action of the algae inside (its symbionts) and outside the clam’s body (the floating algae the clam filters out of the water as an additional meal). This happens because as algae take carbon from the environment and bind it into sugars through photosynthesis, they naturally weight the dice in favor of carbon-12 making it into the sugars. So carbon-13 is left out in the water, and potentially in the clam’s shell. When photosynthesis is more active, it would leave the shell with proportionally more carbon-13. At least that’s what other researchers have confirmed happens in corals, and suspect happens in clams. In the world of isotope chemistry, this phenomenon is called “fractionation,” when a process causes isotopes to form fractions separated by mass. We wanted to test if that was true for giant clams, and could do so by comparing T. squamosina and T. maxima, which have more active photosynthesis, to the less photosynthetic T. squamosa.

Comparing carbon isotopes across different species and shell layers. The results are fairly flat all the way across.

It turns out that the more symbiotic species don’t have more carbon-13 in their shells. We set out several reasons that might be the case, including that the symbionts of these clams are actually more carbon-limited than many researchers might expect. Essentially, the algae lack an excess of carbon atoms to choose from, so they can’t be picky with which isotopes they use to make sugars. Therefore, the fractionation effect weakens and becomes possibly too subtle to manifest in the shell, even in the best-case scenario of three closely related species living the same area. This represents what I’d term a “null result.” We had a hypothesis and we demonstrated that hypothesis was not the case in our data. It was important to publish this result, because other researchers will know not to try the same thing. This means that when we try to search for evidence of symbiotic algae in fossil clams, we will likely need to use other types of chemistry to figure it out. But don’t worry, as finding such a “smoking gun” for algal symbiosis in fossil bivalves is part of my life’s work! I have a few projects in the works looking for exactly that kind of evidence! 😉

A look at how temperatures measured via oxygen isotopes vary through the lives of the animals. This is how scientists can use very old shells to figure out how temperatures varied through a year in prehistoric times!

But we had additional data we collected in addition to the carbon isotopes which actually turned out to provide some interesting results. This other type of measurement regarded the oxygen isotope ratio of the shells. Previous research has shown that the ratio of oxygen-18 to oxygen-16 in carbonate skeletons directly relates to temperature, a principle that has birthed a field known as paleothermometry. There are thousands of papers which use shells of corals, clams, cephalopods, microbes and more to reconstruct temperatures in ancient times. Giant clams have proven to be effective weather stations going all the way back to the Miocene epoch, millions of years ago! Because they grow so quickly (putting down a new layer every day), live for a long time, and don’t stop growing, they form very complete, high-resolution, and long records of past climate.

But no past studies had ever compared different species of giant clams from the same place. There would be interesting new lessons to draw from such a comparison, including seeing if one species preferred to grow at warmer parts of the reef. As complex, three dimensional structures, there are many remarkably different micro-environments throughout a reef, from the hot, sun-exposed reef flat and crest to the cooler, current-swept, deeper fore-reef. Do any of the species of giant clams show a consistently higher temperature than the others, and what would that mean if they did?

*T. squamosina* records higher temperatures than the other species. Outer shell layers also record higher temperatures than inner shell layers. More on that later in the post 😀

It turns out that the rare T. squamosina, only found in the Red Sea, does record a higher average temperature, almost 3 degrees C higher than the other two species. This is of interest because this species had been proposed by prior researchers to only live on the sun-drenched reef crest, at the shallowest part of the reef. We believe these results corroborate that observation. The previous research on the habitat of T. squamosina was limited to a single study which only was able to find 13 live animals along the coast of the Red Sea. But by independently confirming this life habit, we can ask further questions that may be borne out by further research.

An example of *T. squamosina* showing signs of possible bleaching (light parts at the center of its body).

Being restricted to the shallowest waters, is T. squamosina at greater risk of harvesting by humans along the shores of the region than its counterparts? Illegal poaching of giant clams along the Red Sea is believed to be a major stressor on their population size in the area. Could this explain why T. squamosina is so rare today, despite being proposed to have been more common in the past? In addition, being restricted to the top few feet of depth in the water could leave the species more vulnerable than the others to atmospheric warming. As with corals, when giant clams overheat they will “bleach”, expelling their symbiotic algae as a stress response. While the clams can recover, it is sometimes a fatal form of stress that leads to their death.

An excellent cartoon of the different shell layers in giant clams. From a peer of mine who also studies them, Michelle Gannon!

More research is needed to answer those questions. But the last aspect of this study relates to what is happening inside of the bodies and shells of the clams themselves. Giant clam shells have two layers. The outer layer grows forward away from the hinge, increasing clam’s length. The clam also makes an internal layer, growing inward to thicken the shell and add weight. We can read the growth lines of the clam’s diary within either layer, and different studies have used one or the other to make records of climate change. But very few studies have compared the two layers of the same individual. Do they record the same temperatures? Figuring it out would be important to determine how studies with just the inner layer or outer layer can be compared to each other across time and space.

A vividly blue example of the small giant clam, *T. maxima*. From user arthur_chapman on iNaturalist

In our studied clams, it turns out that the outer layer records warmer temperatures on average than the inner one! After ruling out other possible explanations behind this difference (the details are complicated and hard for even shell nerds to wrap our heads around), we settled on the idea that the outside of the clam is indeed warmer on average than the inside! This means that the outer layer, recording temperatures of the outer mantle, is indeed forming at a higher temperature than inside! Why is this?

Unlike us, clams are ectothermic. They generally stay the same temperature as their surrounding environment and don’t use their metabolism to generate internal heat. But that doesn’t mean that the clam doesn’t have hotter and cooler spots in its body. It makes sense that it would be hotter at the outer part of its body, facing the sun, as the solar rays hitting its outer mantle would then radiate out again as heat. The outer mantle is also darker in color than the inner mantle, allowing it to absorb more solar energy, much as you might feel hotter wearing a darker t-shirt in the sun than a white one. Photosynthesis itself produces a warming effect, a phenomenon known as non-photochemical quenching, and so the outer mantle, which contains the vast majority of the symbiotic algae, may be partially warmed by the activity of the symbionts!

More research is needed to confirm if this is true. As of yet, no researcher has ever stuck a temperature probe in multiple parts of a clam to see if the outside of it is indeed warmer than the inside. But until that day, it is interesting to think of how this would influence comparisons of diaries from the inner and outer layers of different bivalves. The effect is on the small side, so it doesn’t really mean one layer or the other should be preferred for future shell-based studies of climate change. But it could be an additional aspect to consider in the future as a way to record temperature differences within the body of an animal, and look into how those differences influence its overall level of stress.

Examples of juvenile smooth giant clams, *T. derasa*, that we’re growing at Biosphere 2. Photo by Katie Morgan.

So I hope this long explanation of my paper helps you to have a better idea of the work I did during my PhD thesis. There were other aspects to the paper that are too wonkish to get into here, particularly concerning the correlation we found between carbon and oxygen isotope ratios, but if you have questions or want a copy of the PDF, please message me! I have more clam papers in the pipeline, and my new postdoc at Biosphere 2 involves growing three species of giant clams in a controlled environment, where I hope to answer some of the physiological questions I mentioned above! But until then, stay hinged and happy as a clam (as much is possible in this chaotic time), and take comfort knowing there are colorful bivalves out there all at this very moment, harvesting sunlight for food and growing huge shells.

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!