You cannot exactly ask a shark how old it is. And unlike a tree, you cannot simply count rings without getting rather more… invasive. For decades, shark researchers have relied heavily on growth bands in vertebrae, which can provide useful age estimates but generally require the animal to be killed (and has also been found to become less reliable in older sharks). So, what is one to do? Well, what about instead of looking inside a shark’s vertebrae, you could look at its blood, instead?
That is the premise behind a new study of zebra sharks (Stegostoma tigrinum), which has developed some of the first highly accurate epigenetic clocks for an elasmobranch. The researchers found that patterns of DNA methylation in blood can predict a zebra shark’s chronological age with a median error of roughly one to two years. Even more surprisingly, the models could still estimate age pretty well using information from just 10 spots in the genome.
But what exactly is an epigenetic clock?
Our DNA sequence is largely fixed, but the way that DNA is regulated changes throughout our lives. One important mechanism is “DNA methylation,” in which small chemical groups called methyl groups are added to DNA, most commonly at sites where a cytosine is followed by a guanine, known as CpG sites. These chemical tags can influence whether nearby genes are more or less active. As animals age, methylation patterns change across the genome (i.e., some regions tend to lose methylation while others gain it). Because these changes occur in relatively predictable ways, researchers can use them to build statistical models that estimate an individual’s chronological age. This idea has already transformed aging research in humans and other mammals — the first widely used human epigenetic clock (developed in 2013!) could estimate chronological age to within a few years using 353 DNA methylation sites. Since then, researchers have developed clocks for dozens of mammals as well as birds, reptiles, amphibians and bony fishes.
Sharks, however, have been something of a blind spot. Which is kind of ironic, because elasmobranchs are an extraordinary group for studying aging. Sharks, skates and rays diverged from the lineage leading to most other vertebrates more than 400 million years ago and the group as a whole display a remarkable variation in lifespan. Some sharks may live little more than a decade while Greenland sharks can apparently survive for centuries! Zebra sharks, the focus of this study, can live for more than 28 years. With this in mind, the researchers had one question they wanted sharks and their relatives to answer: exactly how much of the biology of aging is ancient?
To investigate, researchers collected blood samples from 70 zebra sharks ranging from less than a year old to approximately 30 years old. Of these, 51 were aquarium-bred sharks with known birth dates, giving researchers exactly what they needed to build and test an age predictor (aka sharks whose ages they knew for certain). They then used whole-genome enzymatic methyl-sequencing to examine more than 14 million CpG sites. Rather than simply looking for a handful of promising locations, this approach allowed the researchers to examine where age-associated methylation changes occurred throughout the zebra shark genome.
The results revealed that overall methylation declined slightly with age, with more individual sites losing methylation than gaining it. But the locations of those changes were not random; sites that gained methylation tended to occur in CpG-rich regions near genes and promoters, including regions associated with Polycomb repressive complexes. Sites that lost methylation? Well, they were more commonly found in CpG-poor regions and within gene bodies. Those resulting patterns were quite familiar to the researchers and left them a little bit stumped… because they resemble observations made in mammals. In other words, although a zebra shark and a human have been evolving along very different paths for hundreds of millions of years, some of the molecular geography of aging appears to look remarkably similar! Now, that does not mean sharks age like humans. Far from it! As the scientists pointed out, the study cannot yet tell us whether these methylation changes actually cause aspects of aging or simply reflect other biological processes occurring as an animal gets older. But those similarities suggest that some of the biological processes involved in aging may have been conserved across vertebrates for a very, very long time.
There was another interesting twist: methylation didn’t simply change in the same direction throughout a shark’s life. Many sites changed substantially between young and middle-aged animals, then changed direction later. The most common trajectory involved methylation decreasing early in life before increasing again later. Other sites showed a consistent decline. Overall, epigenetic changes were more pronounced during the earlier stages of life than during later adulthood. This raises an intriguing question about where “aging” actually begins. Are the epigenetic changes occurring during rapid growth and development truly part of aging, or are they something biologically distinct that later influences the aging process? Researchers are still debating that question across species. It also makes the “clock” itself more complicated. The simplest epigenetic clocks work particularly well when methylation changes predictably with age. Non-linear changes can make older individuals harder to estimate accurately. That was even seen with the study animal in question, where the zebra shark clock performed exceptionally well overall but became less precise in sharks older than 20 years.
Still… the potential conservation application is perhaps the most exciting part.
Zebra sharks are listed as Endangered, and many other elasmobranchs face serious population declines. Knowing the ages of animals in a population can tell scientists a great deal about whether that population is growing, stable or struggling to replace older individuals, yet obtaining those data is particularly difficult for threatened species when traditional age estimates require lethal sampling. A blood-based epigenetic clock could change all of that! Researchers could now potentially estimate the ages of sharks captured during routine monitoring, tagging or veterinary examinations without killing them. With enough individuals, scientists could begin constructing age structures for wild populations and use those data to improve population models and conservation decisions.
There is one important catch, however. The zebra shark clocks were trained primarily using aquarium-bred animals and captive conditions can differ dramatically from life in the wild. Diet, stress, growth rates, disease exposure and environmental conditions can all influence an animal’s physiology, potentially affecting its epigenome as well. Which means that means an aquarium-born shark is not necessarily the perfect biological equivalent of a wild shark. The researchers’ tests on wild-caught individuals produced encouraging but less precise results, highlighting exactly why more calibration work is needed. They say that future studies will need larger datasets containing known-age wild animals where possible, along with longitudinal sampling that follows the same individuals over time.
But for conservation scientists, this opens up the door to a flood of exciting (and currently unanswered) questions. If similar molecular signatures of aging continue to appear across mammals, birds, reptiles, fishes and elasmobranchs, what does that tell us about the evolutionary history of aging itself? Are some of these mechanisms fundamental features of vertebrate biology? Could environmental conditions accelerate or slow epigenetic aging in wild animals? And might an animal’s “epigenetic age” eventually tell us something more meaningful than its chronological age, such as whether it has experienced unusually high levels of environmental stress?
Age is one of the most important pieces of information we can have about a population, yet it remains surprisingly difficult to obtain for many of the animals we are trying hardest to protect. The zebra shark may be giving us a new way to read this sort of “hidden” history… and that’s exciting, even if it’s just at the beginning.
