Spotlight: Molecular Scatology – PCR on poo reveals wader diets

My school-aged kids and their friends are obsessed with poo. They talk about it, point it out on hikes, and try to decipher what the animal that made the deposit had eaten. They are little scatologists. Turning our attention to poo can tell us a lot about an animal, especially when one applies modern technology to the science of scatology.

The fusion of animal droppings with molecular lab techniques is called molecular scatology, or polymerase chain reaction (PCR) on poo. In this issue of Wader Study, Novic, Veit, Mizrahi and Symondson apply molecular scatology to shorebirds on stopover during spring migration in Delaware Bay. In their article¹, they describe how they collected fecal samples from shorebirds and performed amphipod specific PCR on these samples to figure out whether amphipods form part of the shorebird diet in Delaware Bay.

Delaware Bay is known as a critical stopover site for shorebirds on spring migration and it is well known that the eggs of spawning Horseshoe Crabs Limulus polyphemus provide an essential food source. An earlier study², conducted by the same researchers, focused on detecting Horseshoe Crab DNA in bird feces and confirmed the importance of Horseshoe Crab eggs for migrating shorebirds. But the eggs seemed more important for some species than others, making the researchers wonder about other invertebrate food sources for shorebirds in Delaware Bay. The amphipod Corophium volutator is a valuable prey item for shorebirds in the Bay of Fundy during fall stopover. Over 40 species of amphipod crustaceans have been reported in Delaware Bay, and the researchers wondered whether shorebirds were eating amphipods in the Bay.

Understanding how shorebirds use all food resources at this critical stopover site will support more effective conservation and management.

But why the fancy techniques, why not just look at the poo? Often shorebird biologists can figure out what the birds have been eating by looking closely at fecal samples. This is because some shorebirds, in some parts of the world, eat hard-shelled prey (like clams and mussels) whose shell fragments can still be identified in the feces. But in Delaware Bay, shorebirds are eating softer prey. Neither digested Horseshoe Crab eggs nor invertebrates like amphipods are easy to see in feces. Other methods, like flushing the stomach contents, are much more invasive, and small and soft prey is hard to see even then because shorebirds rapidly digest food. This is why molecular scatology is so useful.

To perform molecular scatology, the researchers first needed to ensure that the PCR primers reliably detected the amphipods they were targeting. To do this – and also to examine seasonal change in abundance of invertebrates throughout the stopover season – they took benthic core samples within foraging areas on the New Jersey side of Delaware Bay. They found that their primers did indeed detect amphipod species. They also found that densities of amphipods in Delaware Bay were considerably lower than those of C. volutator in the Bay of Fundy, and that the maximum densities of amphipods in the study sites were substantially lower than the densities of Limulus eggs. But were the birds eating the amphipods (even though the eggs were more abundant)? And were the amphipods a more important food source for some species but not others?

To find out, they needed to try their amphipod primers on poo. They caught shorebirds by mist-netting throughout May in both 2011 and 2012 and obtained fecal samples from individual birds by placing the birds in foil-lined boxes and collecting their droppings once they were deposited. As was the case with the benthic core samples, the primers successfully amplified amphipod DNA from the fecal samples. Amphipod DNA was present in samples from all four species tested: Dunlins, Least Sandpipers, Semipalmated Sandpipers and Short-billed Dowitchers. These species are eating amphipods, in addition to Horseshoe Crab eggs, during spring stopover in Delaware Bay.

In the 2012 data, they also found significant differences in occurrence of amphipods in feces of different species, with Least Sandpipers using this food sources more than Dunlins, Semipalmated Sandpipers and Short-billed Dowitchers. This is consistent with the Least Sandpiper tendency to forage in vegetated areas of tidal marshes where spawning crabs are uncommon. It is also consistent with the earlier finding that Limulus eggs were found in a smaller percentage of Least Sandpiper samples compared with other shorebird species². It seems that Least Sandpipers rely on amphipods, and probably other benthic invertebrates, rather than Horseshoe Crab eggs during spring stopover in Delaware Bay. The researchers also note that amphipods may also be an important food source for other shorebird species before crab eggs become available in early spring.

By blending old and new methods, the researchers gained a better understanding of how shorebirds use available resources at this critical stopover area. Such information is necessary for the effective conservation and management of both shorebirds and their foraging habitats. In Delaware Bay, we are learning that that tidal mudflats and marshes, where birds feed on various benthic invertebrates, are also important foraging habitats for shorebirds. Hence, new conservation strategies regarding these habitats should be considered in the future.

This study is one of many in which researchers have incorporated new technologies into the study of birds. Other innovations include the use geolocators for everything from discovering the flight paths of long distance migrants to studying their breeding biology in remote areas (as highlighted by a [paper] by Lisovski and colleagues, also in this issue of Wader Study³. It is truly and exciting time for technology in the study of waders and other birds⁴.

1. Novcic, I., R.R. Veit, D.S. Mizrahi & W.O.C. Symondson. 2016. Molecular analysis of amphipods in the diets of migrating shorebirds. Wader Study 123(3): 195-201.
2. Novcic, I., D.S Mizrahi, R.R. Veit & W.O.C. Symondson. 2015. Molecular analysis of the value of Horseshoe Crab eggs to migrating shorebirds. Avian Biology Research 8: 210–220.
3. Lisovski, S., K. Gosbell, C. Hassell & C. Minton. 2016. Tracking the full annual-cycle of Great Knot, Calidris tenuirostris, a long distance migratory shorebird of the East-Asian Australasian Flyway. Wader Study 123(3): 177-189.
4. Altshuler, D.L., K. L. Cockle, & W. A. Boyle. 2013 North American ornithology in transition. Biol Lett 9: 20120876. https://rsbl.royalsocietypublishing.org/content/9/1/20120876

PDF version can be downloaded here.