Sharks & Stable Isotopes: Making the most of mortality
During our dissections, we occasionally found really interesting critters in the stomachs of our sharks. Here you can see what we IDed tentatively as a margined snake eel (Ophichthus cruentifer), one of eight individuals removed from the stomach of a single Cuban dogfish.
Hi everyone!
I'm pleased to share another collaborative project that your donations helped make possible! Released this summer in the Journal of Experimental Marine Biology and Ecology, this publication was led by Ollie Shipley, now a PhD student at Stony Brook University, during his Master's thesis work. The effort involved researchers at Newcastle University, Banyan Tree Marine Lab, University of Exeter, University of Windsor, and our team from the Florida State University Coastal & Marine Lab and Cape Eleuthera Institute.
Background
Unfortunately, our research on the post-release mortality (PRM) of deep-sea sharks led to mortality for some of the individuals that we caught and monitored for 24 hours after longline capture (we estimated a mortality rate of ~50% for the Cuban dogfish, for instance - full results coming soon). In those situations, we gleaned all of the scientific value out of each mortality event that we possibly could. Animals were examined for gut contents and reproductive state, genetic samples were taken for species confirmation and population connectivity analysis, jaws were cleaned for educational use, and muscle samples were taken for two purposes- 1) to provide baseline measurements for comparison with samples from sharks exposed to the Deepwater Horizon oil spill in the Gulf of Mexico, and 2) to provide stable isotope data for Ollie's work, described below.
The study: Polar compounds preclude mathematical lipid correction of carbon stable isotopes in deep-water sharks
This one is a bit complicated for those who aren't already familiar with stable isotopes, so I'll do my best to break it down. First, a reminder from chemistry - the nucleus of every atom contains protons (positively charged sub-atomic particles) and neutrons (sub-atomic particles with no net charge). The number of protons for each atom defines that element (like carbon, for instance), and, when added to the number of neutrons for that element, gives you the atomic mass. So, using carbon as an example, you would typically see 6 protons and 6 neutrons in its nucleus giving you an atomic mass of 12 (written as 12C, the most common form of natural carbon in our world). But sometimes, carbon can have 6 protons and 7 neutrons, or one more neutron than normal. This would be referred to as 13C since we've added that one neutron to the atomic mass, and is an example of a heavy stable isotope of carbon. Being "stable" just means that it won't decay into other elements and being an "isotope" just means that it has a different number of neutrons than normal - changing the atomic mass but not the chemical properties of that element.
These heavy stable isotopes are preferentially retained in the tissues of organisms (like our sharks) during normal metabolic processes. Stable carbon isotopes, for instance, are retained during respiration. Essentially, being heavier keeps them in place more than the lighter versions of themselves, which can travel a bit easier. Examining the ratios of these heavy isotopes to the lighter, more common isotopes (this ratio is written shorthand as δ13C) in an animal's tissue can shed light on its habitat use and diet.
During dissections, we sometimes discovered pups inside of our sharks. Here, you can see a developing gulper shark and its egg yolk, the source of its energy during development, behind. We always measured and took genetic samples from these embryos to better understand the species' reproductive biology. They also confirmed that the mother was fully mature.
An interesting example of how stable isotopes can be used in this way comes from comparing hair samples from people around the world. In the US, human hair samples generally contain high levels of 13C because Americans eat a largely corn-based diet and corn is high in 13C. People who eat a primarily rice-based diet show far less 13C as rice is lower in 13C than corn. While it might seem like we don't eat that much corn here in the U.S, typically around 65% of our nutrition comes from corn agriculture in some form or another. Anyways, back to sharks...
When examining stable isotopic signatures in sharks, we're mostly interested in carbon and nitrogen. The δ13C values can help us understand the primary production source (i.e. where is sunlight converted into carbohydrates?) in the marine system where the shark is feeding. For nitrogen, we're interested in the ratio of 15N (the heavy stable isotope) to 14N (the standard form of nitrogen). The ratio between these, δ15N, can tell us a bit about the position on the food web where a species is feeding - essentially, it tells us whether that species is feeding low on the food web or, for instance, is a top-level predator. Basically, because 15N is heavy, it is transferred from prey species to predators as they are consumed. This results in higher level predators having more 15N in their tissues.
So... stable isotopes are valuable to scientists! Unfortunately, being able to accurately measure them in sharks can sometimes be difficult. Sharks retain certain compounds in their tissue, like lipids (e.g. fats) and urea (regularly found in urine), that make the interpretation of stable isotope values tricky. High concentrations of these compounds in shark tissue result in lower δ13C and δ15N values than would be expected for an animal with less lipids / urea in their tissues. Without correcting for these differences during analysis, scientists run the risk of misinterpreting values and might conclude that a species is lower on the food chain than it really is (among other things).
Because of this, it is important to know how to correctly prepare muscle samples from sharks before stable isotope analysis. Further, it is important to know how the type of shark species might influence this sample preparation. In this case, the team was interested specifically in how tissue samples from sharks living in the deep-sea need to be prepared to account for their unique concentrations of lipids/urea in their tissues.
With that, I'll leave you with the abstract of the paper itself to present the highlights:
Lipids affect stable isotope values generated for marine fishes, however these effects remain poorly described for many extant shark taxa, especially deep-sea species. Here, we report the effects of lipid extraction (LE) on δ13C, δ15N, and C:N values of seven deep-sea sharks, generate novel mathematical normalizations for δ13C based on the relationship between bulk and lipid extracted values (δ13CBulk and δ13CLE) and examine whether common normalized correction models provide a robust method for addressing lipid-biasing effects in two species, the Cuban dogfish (Squalus cubensis; n =20), and Greenland shark (Somniosus microcephalus; n=24). LE generally resulted in enrichment of 13C and 15N, but produced variable effects on C:N across all species. Novel mathematical normalizations for δ13C were derived from the pooled shark community, and a single species specific correction was generated for the Cuban dogfish, but could not be determined for the Greenland shark. Four common lipid correction models used for teleosts, failed to accurately predict δ13C values statistically similar to δ13CLE, in both Cuban dogfish and Greenland sharks, likely due to the confounding effects of lipids and urea on C:N. These observations suggest that chemical lipid extraction should be a mandatory procedure prior to interpreting stable isotope data for deep-sea sharks, at least for those species where lipid effects are large.
The Bottom Line
So what does all this mean? Well, as a result of this work, scientists now have a way to mathematically correct stable isotope values from Cuban dogfish tissue to account for their unique concentrations of lipids/urea. And, on top of that, it is recommended that lipids be removed through a chemical extraction process prior to interpreting stable isotope values for deep-sea sharks in general.
A Cuban dogfish, one of the two study species in this study (the other is the Greenland shark, the longest living vertebrate on Earth), awaits dissection.
Great work and a big thanks to Ollie for making it happen!
Read the full paper here!