Spotlight: DNA barcoding to discover chick diets

by Deborah Buehler originally published in Wader Study 131(3)

You can learn a lot from poo.

For example, how can one determine what birds eat across the vast stretches of the Arctic tundra? The Arctic is a place where it is hard enough to find birds, and much harder to see what goes into their beaks. However, understanding the diet of birds that breed in the Arctic is paramount for understanding their habitat needs in a rapidly changing climate. Of particular interest is the possibility of mismatch between the peaks of breeding and food sources. Such mismatches could be caused by shifts in the timing and intensity of the seasons in the Arctic region, where climatic changes are most pronounced. To test for such mismatches, researchers need to understand both the peak abundance of various prey species (usually arthropods) and what growing chicks of various species actually eat after hatching. In this issue of Wader Study, Mikhail Zhemchuzhnikov and colleagues present the first use of DNA metabarcoding to identify multiple prey species in faecal samples from chicks of several shorebird species, breeding in Greenland¹.

The researchers collected the data between 6 and 25 July 2022 in the high Arctic tundra at Zackenberg in northeast Greenland (74°28’N, 20°34’W). They captured shorebird chicks from six species that breed regularly in the area: Dunlin Calidris alpina, Red Knot Calidris canutus, Red-necked Phalarope Phalaropus lobatus, Red Phalarope Phalaropus fulicarius, Ringed Plover Charadrius hiaticula and Ruddy Turnstone Arenaria interpres. The researchers captured the chicks by hand during the pre-fledging period (before the chicks were old enough to fly). To collect the faecal samples, they put the chicks in a disinfected and sectioned plexiglass box. Each section contained one chick and was lined with two layers of fresh tissue paper. To minimize any negative impacts, chicks were released after 10 minutes if no defecation occurred. If a chick did leave a sample, it was scraped from the tissue using a clean disposable plastic spoon and stored in 96% ethanol at -20°C until laboratory analysis.

How exactly can DNA barcoding a poo sample determine what a shorebird chick has eaten? DNA barcoding can identify taxonomic groups (e.g., species, family, or class) using short, standardized segments of DNA²; the DNA of what a bird has eaten remains at least somewhat intact within its droppings. The segment of DNA commonly used for Metazoans is usually a section of the gene known as “Cytochrome c oxidase subunit I”, located in the mitochondria of cells. This gene has a mutation rate roughly three times that of other forms of DNA². That means it is more likely to harbour genetic differences, even between closely related organisms. Primers specific to the gene are used in a polymerase chain reaction (PCR) to make many copies of this piece of DNA. These copies are then sequenced to determine their exact code. Every species has a unique sequence (“barcode”), just as every person has a unique fingerprint or each item for sale in a store or shop has a barcode. These DNA barcodes are then compared with a reference library to identify taxonomic groups. In DNA metabarcoding, a sample contains sequences of many potential organisms. For example, different arthropod groups have been found in a sample of shorebird poo. The proportion of copies for one particular sequence variation divided by the total number of copies (reads) is called Relative Read Abundance (RRA).

How could the researchers be sure that what turned up after barcoding the samples truly reflected everything the birds had eaten? Additionally, how could they be sure that digestion did not degrade the DNA of some species more than others? These important questions were addressed in an earlier study on cavity-nesting birds that allowed the filming of parents feeding chicks³. Data from the camera footage validated that DNA metabarcoding of faeces accurately represented the ingested arthropod groups. This also validated that digestion did not bias the results for any particular group. Perhaps most exciting was that the study reported a high correlation between the relative read abundance of different variations and the consumed biomass of arthropod groups seen on the camera footage. If this is generally the case, then it means that read numbers retrieved from DNA metabarcoding can also be used to quantify the relative biomass of arthropod groups in bird diets.

Following the DNA metabarcoding method used by Verkuil and colleagues³, Zhemchuzhnikov and colleagues showed that two-winged flies (Diptera) are a core component of Arctic shorebird chick diets: they were present as a majority in all droppings, except one Ringed Plover sample. Within Diptera, the dominant groups were Culicidae (mosquitoes), Chironomidae (non-biting midges), Muscidae (house flies), and Empididae (dagger or dance flies).

Interestingly, the Ringed Plover sample showed a different arthropod composition. There were many reads from Lepidoptera (mainly geometer moths) and Hymenoptera (mainly parasitic wasps) and fewer from Diptera. The researchers speculated the parasitic wasps might represent secondary prey species (prey of the prey eaten by the chick). Blattellidae (cockroaches) were also found in this sample (and four others) but probably represent contamination during sample handling.

The authors acknowledge that they sampled only a short time of the year and had only a small number of faecal samples per shorebird species. The next steps might include a longer sampling period to capture diet shifts within species during the breeding season. Additionally, the practice of using read numbers to approximate the relative contributions of prey taxa is quite new and has only been validated for an insectivorous songbird³.

Despite the study’s limitations, Zhemchuzhnikov and colleagues have expanded the use of DNA metabarcoding to a new location in Northeast Greenland. They have provided new insights into the needs of Arctic breeding birds at a time when this region is warming and changing rapidly. This study is part of a rapidly increasing pool of knowledge generated by DNA barcoding, showing how this method can be used to better understand ecological diversity in various environments and how these environments support the harboured biodiversity. To quote Ben Panko in Smithsonian Magazine⁴ “The key to protecting life on earth may be barcoding it”.

 

¹ Zhemchuzhnikov, M.K., T.S L. Versluijs, R.A. Bom, R. Blok, J.D.L. van Bleijswijk, J. Reneerkens & J.A.van Gils. 2024. A DNA-based dietary study of shorebird chicks in Greenland. Wader Study 131: 178–179.

 ² International Barcode of Life. 2024. What is DNA barcoding? International Barcode of Life. Accessed 29 Nov 2024 at https://ibol.org/about/dna-barcoding/

³ Verkuil, Y.I., M. Nicolaus, R. Ubels, M.W. Dietz, J.M. Samplonius, A. Galema, K. Kiekebos, P. de Knijff & C. Both. 2022. DNA metabarcoding quantifies the relative biomass of arthropod taxa in songbird diets: Validation with camera‐recorded diets. Ecology & Evolution 12: e8881.

⁴ Pando, B. 2017. The key to protecting life on Earth may be barcoding it. Smithsonian.com. Accessed 29 Nov 2024 at https://www.smithsonianmag.com/science-nature/how-dna-barcoding-opens-new-doors-conservation-180963431/

 

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Featured image: Incubating sanderling. Zackenberg, Greenland.