Dermal Denticles: Up Close and Personal

After visiting the Smithsonian’s National Museum of Natural History in May to excise small pieces of skin from across the bodies of nearly 40 specimens, representing 36 species and 16 families of sharks (nearly half of all the extant families), and meticulously treating the skin with Clorox to separate the individual denticles from the underlying tissue, here is a sneak preview of the final product under a scanning electron microscope. The denticle crown definition is quite stunning, right?

Scanning electron microscope (SEM) images of shark dermal denticles from the three main shark families represented in our sediment samples. A blacknose shark (Carcharhinus acronotus) denticle is shown for family Carcharhinidae, a scalloped hammerhead (Sphyrna lewini) denticle for family Sphyrnidae, and a nurse shark (Ginglymostoma cirratum) denticle for family Ginglymostomatidae. All of the denticles were isolated from pieces of skin excised from preserved museum specimens. Scale bar = 100µm.

And for the viewing pleasure of those more artistically-minded folks, a colorful rendition edited by my research advisor is shown below. Enjoy!

To the Motherlode of Preserved Sharks

After a long series of flights over the course of an entire day, I found myself in Washington, D.C. It’s almost ironic how working abroad has actually brought me directly to the heart of American government itself and to a place where the institution I work for rings more bells in people’s minds. After spending the last five months in Panama, returning to the States and seeing the Stars and Stripes again sparked a resurgence of patriotism within me. Having never visited D.C. before, I wasn’t really sure what to expect, and frankly, now that I’m here I wish I had given myself more time to explore the city. Rather than meandering the streets and ogling historic buildings as a tourist, I’ve plunged right in to city life, joining the workers as they briskly walk their daily routes to and from the office, headphones in and consumed in their thoughts and the fast-paced lives they live. I love this routine and seem to fit right in. I’ve even been asked for directions. This city is a breath of fresh air. In some respects, I’d refer to it as the government’s college campus. Its layout, general air, momentous architecture, and work-oriented yet hip culture bring me back to my time at Stanford. For that alone, my experience here has been incredible.

But now, why am I here? I’ve come to the nation’s capital to sample the Smithsonian Institution National Museum of Natural History’s ichthyology collections and excise pieces of shark skin for inclusion in my dermal denticle reference collection. I’ve encountered a great diversity of denticle morphologies in my sediment samples. While the number of denticles I find per kilogram sediment provides insight into the relative abundance of sharks in the past vs. today, examining and categorizing denticle morphology will hopefully reveal whether shark communities have shifted over time. I’ve introduced the previously-documented denticle functional morphotypes in my last two blog posts, but now I intend to not only refine my denticle classifications but to take them one step further. My next challenge is to identify the denticles to taxonomic family. This is easier said than done, as denticles are known to vary across species and families as well as across the body of an individual shark. The key is to find common themes and patterns in denticle morphology amidst all this variation, which is where the reference collection comes into play.

Most studies of denticle morphology examine entire pieces of skin with the denticles still intact and overlapping. However, I encounter single, isolated denticles in my sediment samples. In order to compare apples to apples so to speak, I am constructing a reference collection of individual denticles from standardized locations across the body of sharks from families known to inhabit my study areas. By capturing this variation in the form of individual denticles, I can get closer to producing a set of criteria by which to group the denticles taxonomically. This secondary set of classifications will accompany and reinforce the picture painted by the functional morphotype data.

But first, this involves a little fishing, although not in the traditional sense. Isolating the denticles requires collecting skin samples, and to collect these skin samples, I need to locate the proper preserved sharks. These are stewing in massive tanks of ethanol in a pod within the Smithsonian’s Museum Support Center in Maryland. For those of you who read Dan Brown’s The Lost Symbol, this is the real Pod 5. Each metal tank contains between 10 and 40 sharks, ranging from around 30cm to over a meter in length, submerged in a semi-murky bath of 75% ethanol. This is where you must be prepared to “fish.” Donning thick, black rubber gloves that slid up to my armpits and a heavy apron, I would reach down into each tank to find the species or specific catalog number of shark that I needed for my reference collection. In most cases, I wanted to find the largest shark of each species on my list since denticles are also known to vary with ontogeny. This broadened my search since I wasn’t targeting specific catalog numbers. However, some sharks were more elusive than others. Sometimes the one I wanted would be right on top, but most of the time I ended up removing most of the sharks from a given tank before finding the one I needed. This literally left me with a pile of dead, preserved sharks balanced on a large plastic tray, ready to be re-submerged in ethanol, and my prized shark of interest sitting alone on a separate tray, ready to be sampled. While this was a time-consuming activity, I luckily had help. It was also fun to look at all of the different sharks in each tank as I pulled them out one by one… or maybe that was just the ethanol fumes getting to me.

Tank Room, Pod 5, Smithsonian Museum Support Center

Preserved sharks soaking in a bath of ethanol in a tank

Upon finding the shark that I wanted to sample, I transferred it to another large plastic tray, measured its total length, took a picture, and then covered it with ethanol-soaked cheesecloth to prevent it from drying out. Then, scalpel in hand, I would cut small squares of skin from the body and fins. The layer of skin was so thin on the fins that I found it easier to just “shave” off a small piece. I guess it was a bit like peeling a vegetable to an extent. Each piece of skin was placed with a corresponding label into a separate plastic bag called a Whirl-pak to be brought back to Panama for processing, analysis, and maintenance. After sampling each shark, I would return it to the tank of ethanol, making sure that it and all of its compatriots were fully submerged. This became a three-dimensional shark jigsaw puzzle when the ethanol level was low and there were a lot of specimens in the tank, as bits of the fins or head of the sharks would occasionally refuse to fit completely beneath the surface. In some cases, a lot of rearranging was required.

My sampling station

Carcharhinus limbatus, ready to be sampled

Over the course of four days at the facility, I sampled 40 individual sharks, representing 16 different families, for a grand total of nearly 160 skin samples. Most of the actual dissections went pretty quickly once the shark was located, with the exception of one family: the nurse sharks (Ginglymostomatidae).

Nurse sharks are bottom-dwelling reef sharks and are one of the most common sharks remaining in the Caribbean, as they are of low commercial and nutritional value, making them one of the last sharks to decline in the face of anthropogenic stressors. Due to their demersal nature, they have large, thick “abrasion strength-type” denticles that resemble little stones. This I had read in the scientific literature. What I wasn’t aware of was that sampling their skin would be like attempting to slice through armor attached to beef jerky. Good thing I brought extra scalpel blades with me because I only two I broke during my visit where while sampling this family. My surgery-level scissors also met their match and could hardly cut through these sharks’ sturdy exterior. In comparison, they were rarely needed to sample the other families. This striking contrast certainly highlighted the difference between denticles in demersal vs. faster predatory sharks in my mind.

Ginglymostoma cirratum denticles, 80x magnification

All in all, the trip ran really smoothly, and I was able to sample all of the sharks on my list with a little time to spare. Everyone I interacted with at the museum was incredibly welcoming and organized, making sampling a pleasant and efficient endeavor. While I successfully sampled all of the material I need for the time being, I hope to get the opportunity to work more with the Smithsonian in D.C. in the future. However, now I have my work cut out for me in terms of processing and photographing all of the skin samples I excised to actually build and publish the reference collection. But first, I think I’ve earned a day of rapid-fire sightseeing in D.C. before I return to Panama.

Form Fits Function

Denticles are known to vary greatly in form, both across the body of a shark and between shark families. Previous studies have organized them into five different functional groups: generalized functions, abrasion strength, defense, drag reduction, and denticles associated with bioluminescence. Generalized function denticles are a more ancestral form found in most sharks – particularly those living closer to the bottom – that play a variety of roles. The other four denticle types represent more derived, specialized forms. Abrasion strength denticles are thicker and are either ornamented or knob-like and smooth, acting to protect demersal sharks from the rocky and coralline substrates that they inhabit. They can also be found on abrasion-prone regions of other sharks, including the head and leading edges of fins. Defensive denticles are thorn-shaped with upward-pointing cusps, deterring the settlement of ectoparasites and epibionts in demersal and schooling sharks. Drag reduction denticles have narrowly-spaced ridges aligned with the direction of fluid flow that interfere with the boundary layer created by water moving past the shark as it swims to improve its hydrodynamic efficiency. Mesopelagic sharks carrying photophores have developed bioluminescent-type denticles that are either spine-shaped or square with concave facets that permit light to pass between them. Intermediate forms also exist.

Within this spectrum of functional morphotypes, one can place the different shark families based on their habitat preference and ecological niche. For example, drag reduction denticles almost completely cover the bodies of both near-shore and pelagic predatory sharks such as those from the families Sphyrnidae (hammerheads) and Carcharhinidae (requiem sharks). The distance between the ridges on the denticle crowns may even be used to differentiate between these two families due to differences in their swimming speeds, with faster sharks tending to have denticles characterized by more narrowly-spaced ridges. In contrast, demersal sharks such as the nurse shark (family Ginglymostomatidae) have been found to have a wider diversity of denticle morphotypes, ranging from thicker abrasion strength and generalized function denticles to delicate, ornamented defense-type denticles.

Denticle functional forms (Reif 1985, Ferron et al. 2014)

This variation in denticle form can also be observed in the fossil record. Thus, by assessing not only the number of denticles found in reef sediments but also the different morphotypes present, we can begin to reveal what a “pristine” shark community may have looked like, both in terms of shark abundance and taxonomic diversity. Preliminary data suggests that shark functional community composition may have shifted over time, particularly in terms of the relative abundance of demersal and predatory sharks, which play important and diverse roles in maintaining coral reef health and resiliency.

However, given the large amount of denticle diversity I’ve encountered thus far in my samples, identifying them has been challenging. While I’ve begun to refine my denticle groupings based on functional morphology, I also hope to take a step further and classify them to shark family. To do this, I will soon be visiting the Smithsonian ichthyology collections at the National Museum of Natural History in Washington, D.C. to sample skin from preserved modern sharks in order to build a reference collection of denticles. More on this in my next post!

It’s All in the Process

In science, there’s a long road between the beginning and the end of a project. In many cases, there really is no clear-cut finish, rather just a segue into the exploration of a new idea. Discovery happens in the process.

The development of the research project I’m working on in Panama has followed suite. From collecting samples to counting and identifying denticles, I’m on a journey, specifically one propelled forward by exciting day-to-day moments and epiphanies. These can be as simple as being mesmerized by the miniature explosions of sediment as acetic acid reacts with the carbonate in my samples or as intricate as re-reading a scientific paper and suddenly connecting new dots in my mind. I’ve found that new inspiration can hide around unexpected corners.

Lately, it has come in the form of finding a strikingly large number of denticles in one of my samples of reef sediment. We had originally hoped to find ~30 denticles in each ~8kg bag of sediment, best case scenario. While most have yielded between 16 and 30 denticles, I recently extracted a grand total of 75 denticles from one bag. As I picked out each denticle from the rest of the sediment particles, the look of disbelief on my face grew to grand proportions. I was absolutely shocked. Why was there such a significantly higher number of denticles accumulating on this particular reef? Was this just an outlier or would the remainder of the samples collected from this site yield an equally high abundance of denticles? I couldn’t wait to find out.

I’ve also begun to classify the denticles according to functional morphotype, prompting me to acknowledge the astounding diversity of denticle forms present in my samples. While the denticles I’ve extracted thus far can largely be placed into five functional groups and three different shark families, very few look exactly alike.

While taking pictures of the denticles under a microscope is an easy method of documentation, putting pencil to paper and actually drawing them is a much better way for me to familiarize myself with their subtle differences. By sketching all of the diverse denticle forms, I’ve begun to identify patterns and distinguishing characteristics to better make order from chaos. This is particularly exciting, as there is so much potential in this novel analysis. Notably, these groupings will help reveal whether the composition of shark communities changed as humans began to harvest them, in addition to how their abundance shifted over time.

Examples of denticle sketches (modern reef sediment, Bocas del Toro)

Who knew that drawing for work could actually be an acceptable, productive task? Science certainly can shape a person into a jack-of-all-trades. My daily routine is so varied that spending time in the lab never gets boring. One moment I’ll be drawing, another I’ll be diluting acid, and the next I’ll be filming videos, writing grants, fiddling around with seemingly random items I picked up at the local hardware store, or searching for denticles under a microscope. Every day is a winning game of roulette. Who knows what tomorrow will reveal.

“Buying paintbrushes? I see you’ve picked up a hobby here”… or not

As I peered down through the ocular of my microscope at the diverse array of particles illuminated by the bright light above, I spotted countless sponge spicules (miniscule glassy spears that compose their porous skeletons), fish teeth, bone fragments, otoliths (fish ear bones), and the occasional strand of organic material or remnant chunk of calcium carbonate. These stood out against the black gridded background of my metal picking dish, recounting the history of the menagerie of vertebrate and invertebrate creatures that left their mark on this particular patch of sediment before it was collected from the reef. Paintbrush in hand, I was ready to begin. However, I would be painting no masterpieces today.

I spread a small scoop – maybe equivalent to the size of a pinch of salt when cooking – of my sediment sample out over the area of the picking dish, trying to evenly distribute it and produce a single layer of particles. The sediment grains that I was looking at today were particularly small (less than 250µm, but greater than 63µm), so a little material went a long way. For reference, there are 25,400µm in an inch; an individual particle merely looks like a speck to the naked eye. And most of these specks are of no particular interest to me personally. So why spend hours upon hours systematically picking through every single one of them? In order to find something like this:

Isn’t she a beaut? Dermal denticles are rare – so rare that I may only find ten of them in a little over a kilogram of sediment. Consequently, I collected between 40 and 60 kilograms of sediment at each reef site, so hopefully this will give me enough material to work with. All in all, my teammates and I extracted nearly half a ton of sediment. Talk about a good workout.

Their elusiveness also means that I can go hours without spotting one, which frankly is just a tad discouraging. But plod on, I must. On the other hand, each time I find one, a little flurry of excitement wells up within me and I suddenly feel like I have the endurance to pick for ten more hours (as well as the inspiration to write a blog post). It’s a seemingly endless treasure hunt, but my tiny yet precious haul of denticles validates every minute of the search.

As I was picking through this particular size class of particles and not finding any denticles, I secretly hoped that perhaps I wouldn’t actually find any here anyway, which would limit the scope of my search. Essentially, it would mean that I would have less sediment to pick through in the future. This, of course, has its pros and cons. For example, if I found a denticle here, I would have motive to continue picking through the smaller size fractions for dermal denticles, providing me with a larger sample size – an incredibly promising prospect. On the downside, picking through sediment is a time-consuming and tedious process in which hours are spent recovering a handful of precious denticles. However, this thought was instantaneously quelled when I spotted a delicate semi-transparent figurine among the sea of particles. Not only was it a tiny dermal denticle, but it was a denticle of a morphology representing an entirely different family of sharks that I had not yet encountered in this sample. Instantaneously, I experienced a sequence of contradictory reactions. There I was, humming along with my music, when suddenly the denticle’s unique form caught my eye. Yay!, I thought, Another denticle, how fantastic!, and then milliseconds later, Crap… now I actually have a reason to finish sorting through this box of sediment. My work just doubled. Well, all I can say is that it’s a good thing that I’m returning in January to continue working on this project because at the end of the day, the more denticles the merrier.

The tools of the trade

Panning for… Denticles?

Like a gold miner crouched on the bank of a stream, shoveling scoopfuls of sediment into his sieve and hoping for a golden glint to catch his eye, I found myself standing at the edge of a giant metallic washing table, placing mounds of sediment onto my stack of sieves and hoping for the best. However, I had not one, but five sieves for the sediment to pass through, and the reward was not so immediate, as I’m interested in something far more minuscule. Shark dermal denticles generally range from 100µm to 1.2mm in size, requiring a microscope to spot in the samples. My collection of sieves helps narrow the search, though, given that they allow me to separate out the sediment by particle size. The large pieces of dead coral and shell remain on the top mesh screen, while the smaller particles fall through, continuing onward through the progression of increasingly finer mesh until they too are trapped. The smallest of particles – those less than 63µm – pass freely through the entire stack of sieves and into the sink, forming a trail of silt toward the drain.

With each scoop placed onto the uppermost sieve, my 10kg haul of sediment – filling up part of a formidably-large five gallon bucket – was gradually diminished. However, the sediment required extensive washing and vigorous shaking before the size fractions could be transferred onto separate trays to dry. Who needs a Shake Weight when you can just sieve sediments! This was quite the arm exercise, let me tell you, and it was a gratifying feeling when all of the water I added to wash the sediment particles finally passed through the final sieve, allowing me to empty each sieve and pour another scoopful of sediment over the top of the stack. Five hours later and I could see the bottom of the bucket. What a sight to behold after all that work. Yet the sample was not quite ready for analysis. I still needed to let it dry completely and digest away the carbonate before I could even begin to pick through it for my metaphorical “gold.” Then multiply this procedure by over forty samples; it was just the beginning of a long, but hopefully fruitful process.

My stack of sieves

The First Sample

Who knew that a bag of sand could help unravel the history of coral reefs ecosystems? Next time you’re at the beach, grab a handful of sediment and take a look. What you will see will likely not be uniform, for these sediments contain a menagerie of preserved evidence of past organisms mixed in with carbonate and other mineral and organic material. However, fossils don’t have to be large – like massive megalodon teeth or the dinosaur skeletons that tower over avid onlookers at natural history museums – to be momentous. Small shark teeth and dermal denticles that are barely visible with the naked eye can also paint a vivid picture of the historical abundances of these awe-inspiring organisms. But as with larger fossils, finding them can be a challenge. Dermal denticles (tooth-like scales on the skin of sharks) are rare, meaning that I will have to collect and pick through a lot of sediment to find them. The sediment samples I work with are composed largely of carbonate, so first I have to digest it all away. If you’ve ever put an egg in vinegar as part of a science class or cooking experiment, you know that the shell dissolves. This is the result of a simple gas-forming chemical reaction between the acetic acid in the vinegar and the carbonate of the eggshell. Instead of dissolving an egg, though, I was dissolving away part of the sediment. After multiple acid washes over the course of several days, I was left with a collection of sponge spicules, urchin spines, fish and shark teeth, otoliths, and *fingers crossed* my precious dermal denticles. It was the moment of truth – were there actually dermal denticles present in this sediment sample?

I started picking through a portion of the sample, unsure of whether I would find what I was looking for. I swept aside fish teeth, spicules, and the remaining bits of carbonate with my fine paintbrush until… I spotted a small black shark tooth amidst the sea of white particles. The first evidence of sharks! Soon after, I spotted a strange semi-translucent orange and white figurine with a shiny, tooth-like gloss. Except it definitely wasn’t a tooth. I couldn’t believe my eyes – it had to be a denticle. With my heart racing, I carefully transferred my miniscule treasure to a separate container. Victory! After reviewing pictures of dermal denticles in the scientific literature, there was not a doubt in my mind: I had found my first denticle.

Fossilized megalodon tooth from the Gatun Formation

Dermal denticle (potentially from a nurse shark given the denticle’s morphology), viewed under a dissecting microscope