Monitoring ecosystems and studying biodiversity and food webs are important facets of ecological science. They are key to guiding conservation biology and management of increasingly threatened wilderness resources. The study of indicator species is one means of evaluating biodiversity and changes in ecological processes.
An indicator species is a dominant or keystone species that can be representative of a set of other species. This species might be sensitive to environmental changes. The population status of these species reflects the overall ecosystem health. Any decline may act as an early warning sign to adverse environmental events such as pollution, disease, climate change, and the loss of habitat or biological resources.
NGS-based diet analysis
Diet analysis is one aspect of an indicator species study that can help us better understand food web interactions. To replace labor-intensive or observational methods that lack scalability or taxonomical resolution, molecular approaches to diet analysis, including the application of next-generation sequencing (NGS), have been adopted.
Typical NGS diet analysis involves sampling scat, followed by amplification and sequencing of a genomic “barcode” region. DNA barcodes are short sections of DNA that contain sufficient sequence diversity to provide taxonomical identification. The best known genomic barcoded region for animals is the mitochondrial cytochrome c oxidase 1 gene (COI) used by the Barcode of Life project. To help boost the number of sequencing reads from food diet organisms, blocking probes are used to suppress detection of host DNA, and optimized PCR primers that amplify a barcode target region from a broad range of prey species with minimal amplification of predator DNA.
Use of the Ion PGM™ System for diet analysis
A number of publications have highlighted the adoption of the Ion PGM™ System for diet analysis research studies. Recently, an Australian study described amplification and sequencing of the 3´ end of the SSU (small subunit) rDNA region—with the use of blocking probe—from Adélie penguin scat. 
The Adélie penguin is a key Southern Ocean indicator species. They have a wide distribution in the Antarctic, a generalist diet, and host their breeding colonies on pack ice. Changes to the penguin diet could be due to such man-made changes as competition with fisheries operating in the Southern Ocean and environmental changes such as sea ice distribution fluctuations.
The Australian team adopted a sequencing approach, replacing previous diet estimate methods that included labor-intensive and invasive stomach flushing or stable isotope analysis from material such as feather or eggshell. These methods only provide a broad outline of diet without taxonomical identification. The new NGS method increased sample processing and decreased costs while providing new insight into the diet of the Adélie penguin: “The results indicate that this approach is well suited to Southern Ocean ecosystem monitoring and the broad applicability of the methods suggest that it would be valuable for dietary studies of any other bird or mammal indicator species” the authors wrote.
Other studies have used semiconductor-based sequencing to investigate prey consumption by a number of bat species. Researchers from Germany and the United Kingdom used molecular analysis of a target region of the COI gene combined with morphological analysis to help define prey consumption and the ecological niches that shape two bat species: Myotis dasycneme and M. daubentonii. Using a similar approach, three additional studies determined the dietary diversity of Daubenton’s bats, big brown bats, and the migrant bat Pipistrellus nathusii from fecal DNA extractions.
NGS and diet estimate quantification
Ecologists are becoming more interested in improving the quantification of diet estimates by NGS sequencing. Canadian and Australian researchers used captive harbor seals and food controls to improve the quantification of diet estimates. Amplification of a mitochondrial 16S gene fragment was used as the molecular barcode and sequenced on the Ion PGM™ System. Using matched controls, the authors generated tissue and digestion correction factors that reduced the estimated error and improved diet estimates, suggesting that accurate and quantitative data based on sequence reads is possible.
Another major concern of ecological scientists is that blocking probes might inhibit identification of prey species that are phylogenetically close to the predator. Authors of a recent study investigated the diet of a spider without the use of blocking probes. Amplification of the COI gene was performed and subsequent sequencing using the Ion PGM™ System demonstrated that sufficient sequence reads from prey species could be generated in an affordable manner.
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