Raising a Stink Over Sea Lions

If asked to describe the work of a marine biologist, the average person may envision a somewhat idyllic lifestyle: swimming with dolphins, diving on remote coral reefs, or piloting submersibles to uncharted depths. The reality, however, is that most marine biology is not this romantic. In the case of scientists studying the decline of Steller sea lion populations in Alaska, one important but less glamorous aspect of the work involves collecting and analyzing samples of scat, or fecal matter.

Arguably not a job for the weak-of-stomach, scat analysis has proven to be a useful tool for furthering our understanding of the sea lion diet. By removing and identifying the 'hard parts' from a scat sample, such as bones from different fish species, researchers can deduce the contents of a sea lion's recent meals. But what about soft-bodied prey such a squid, or fish with fragile bones that do not survive digestion? Because the analysis of 'hard parts' does not always paint a complete dietary picture, scientists have turned to DNA analysis to fill in the blanks.

In a study recently published in the journal Molecular Ecology, Bruce Deagle and Mark Hindell (University of Tasmania), Dominic Tollit and Andrew Trites (University of British Columbia), and Simon Jarman and Nick Gales (Australian Antarctic Division) conducted controlled feeding trials on two female Steller sea lions at the Vancouver Aquarium Marine Science Centre. The sea lions were placed on three diets consisting of varying amounts of herring, smelt, salmon, squid, capelin and pollock. Regular scat samples were collected, and DNA analysis was performed on both intact sub-samples and 'blended' samples.

Detection of DNA from the fish prey species fed during the feeding trials: (A) shows six DNA samples which have been extracted from fish and separated vertically on a gel. (each dark band corresponds to a single species). Sample 1 has a mix of DNA from all five fish prey species Samples 2-6 show DNA bands from individual species (Pacific herring, smelt, pollock, sockeye salmon and capelin respectively). (B) shows six samples of fish DNA obtained from sea lion scats. By comparison with bands from the potential prey species, we can determine that four of these samples contain salmon, smelt and herring DNA (samples 1, 2, 5, 6) and two samples contain only smelt and herring (samples 3, 4).

While much of the DNA obtained came from cells shed from the sea lions’ gut, the researchers also successfully detected DNA from each prey type. In fact, they detected DNA from relatively small portions of the diet (squid and sockeye salmon) just as successfully as that of the more abundant menu items (smelt and herring). Prey DNA was detected more consistently from the blended samples than from scat sub-samples, suggesting that prey DNA is not distributed evenly within a single scat – a finding that will likely influence sampling protocols in future studies.

One of the study's more significant findings was that the amounts of prey DNA in the scat samples were roughly proportional to the mass of the prey consumed. Based on this result, it may be possible to combine DNA analysis with other approaches to assess both the type and quantity of prey species in the Steller sea lion diet.

In order to fully address the sharp population decline of Alaska's Steller
sea lions, scientists must better understand their subjects from the inside
out. The field of scat analysis may not serve to inspire legions of future marine biologists, but it is lending considerable insight into what sea lions eat and how they metabolize different prey species. This information, in conjunction with ongoing research into sea lion energy requirements, could play an invaluable role in saving the species from extinction.

25 April 2005 Publication:
Quantitative analysis of prey DNA in pinniped faeces: potential to estimate diet composition? Deagle, B.E. and D.J. Tollit. 2007. Conservation Genetics 8:743-747. abstract Recent studies have shown prey DNA can be consistently recovered from faeces and effectively used to provide dietary information. We investigate the possibility of using the relative amounts of DNA recovered from different prey in faeces to obtain quantitative diet composition data. Faecal samples were obtained from captive Steller seas lions (Eumetopias jubatus) being fed a fish diet consisting of 50% Pacific herring (Clupea pallasii), 36% surf smelt (Hypomesus pretiosus) and 14% sockeye salmon (Oncorhynchus nerka) by mass. Quantitative real-time PCR was used to measure the amount of mtDNA from the three fish species in: (i) a blended tissue mix representative of the sea lion diet and (ii) the sea lion faecal samples. The percent composition of fish mtDNA extracted from the undigested tissue samples corresponded reasonably well to the mass of fish in the mixture. In the faecal samples (n = 23) the absolute amount of fish mtDNA recovered varied 100-fold, but the percent composition of the three fish was relatively consistent (57.5 ± 9.3% for herring, 19.3 ± 6.6% for smelt and 23.2 ± 12.2% for salmon). Differences between the mtDNA proportions in the tissue samples compared to the faecal samples indicate there are prey-specific biases in DNA survival during digestion. These biases may be less than those commonly observed in the conventional analysis of prey hard remains. Further investigation of this approach is warranted.
Estimating diet composition in sea lions: which technique to choose? Tollit, D.J., S.G. Heaslip, B.E. Deagle, S.J. Iverson, R. Joy, D.A.S. Rosen and A.W. Trites. 2006. In A.W. Trites, S. Atkinson, D.P. DeMaster, L.W. Fritz, T.S. Gelatt, L.D. Rea and K. Wynne (eds), Sea Lions of the World. Alaska Sea Grant College Program, University of Alaska, Fairbanks. pp. 293-307. abstract Accurate estimates of diets are vital to monitor impacts of sea lion populations on their ecosystems, their interactions with fisheries and to understand the role of food to animal nutrition and health. Approaches include using: (1) prey remnants in stomach contents, spews and scats, (2) prey DNA in scats (3) fatty acid signatures in blubber and (4) stable isotope ratios in predator's tissue. Each methodology has particular advantages and limitations, many of which can be assessed and improved through controlled captive feeding trials. Analysis of prey remnants from captive sea lion scats have shown significant variability in digestion between and within prey species, which coupled with preferential regurgitation and enumeration biases, can confound accurate diet quantification, but does not prevent spatial or temporal comparisons. Correction for partial digestion and use of additional structures besides otoliths can provide accurate prey size estimates. Prey DNA can be reliably isolated from soft remains in scats from captive sea lions and with further development this approach may allow quantification of diet. Genetic methods can be expensive and representative of only one to two days foraging (like prey remnant analysis), but may be less affected by differential digestion and can identify prey in scats that could not be identified through structural remnants. Validation of fatty acid signature analysis to quantify diet at longer temporal scales in sea lions is ongoing, but this new technique promises to be particularly useful to assess biases in traditional methods, identify the onset of weaning and to highlight what prey most contribute to lipid reserves. Stable isotope analysis of predator tissues gives only trophic level data, but can provide data on diet changes on many temporal scales. Remote video monitoring of foraging events and lavage/enema techniques can provide valuable diet information, but, like many newer techniques, animal capture is required. Ideally a suite of techniques should be used to study diet. While methods and correction factors developed for Steller sea lions can likely be applied to the other five sea lion species, they should be verified experimentally.
Molecular scatology as a tool to study diet: analysis of prey DNA in scats from captive Steller sea lions. Deagle, B.E., D.J. Tollit, S.N. Jarman, M.A. Hindell, A.W. Trites and N.J. Gales. 2005. Molecular Ecology 14:1831-1842. abstract The DNA of prey present in animal scats may provide a valuable source of information for dietary studies. We conducted a captive feeding trial to test whether prey DNA could be reliably detected in scat samples from Steller sea lions (Eumetopias jubatus). Two sea lions were fed a diet of fish (five species) and squid (one species), and DNA was extracted from the soft component of collected scats. Most of the DNA obtained came from the predator, but prey DNA could be amplified using prey-specific primers. The four prey species fed in consistent daily proportions throughout the trial were detected in more than 90% of the scat DNA extractions. Squid and sockeye salmon, which were fed as a relatively small percentage of the daily diet, were detected as reliably as the more abundant diet items. Prey detection was erratic in scats collected when the daily diet was fed in two meals that differed in prey composition, suggesting that prey DNA is passed in meal specific puls! es. Prey items that were removed from the diet following one day of feeding were only detected in scats collected within 48 hours of ingestion. Proportions of fish DNA present in eight scat samples (evaluated through the screening of clone libraries) was roughly proportional to the mass of prey items consumed, raising the possibility that DNA quantification methods could provide semi-quantitative diet composition data. This study should be of broad interest to researchers studying diet since it highlights an approach that can accurately identify prey species and is not dependent on prey hard parts surviving digestion.