Fish consuming (piscivore) bird species such as the Common Kingfisher (Alcedo atthis) or the Bald Eagle (Haliaeetus leucocephalus) or the Eurasian otter (Lutra lutra) serve as ecosystem indicators and even flagship species for conservation. In Europe, such species the ecosystems they live in are protected under the Habitats Directive and the Bird Directive of the European Union. Piscivore species occurring in high population densities on the other hand are seen as competitors to humans. Good examples are cormorants and herons. The problem is that we still don't have sufficient information on both groups' impact on local fish fauna but on the other hand any management of piscivores aiming at either protecting or regulating their predators requires a much better understanding of their feeding ecology.
DNA-based prey identification of stomach contents and quite a few methods have been developed over the last decade, DNA barcoding being one of them and certainly a very successful one.
Whilst sequence-based methods such as next-generation sequencing provide information on the prey range at high taxonomic resolution, they are time-consuming and expensive, especially when dealing with high sample numbers. Diagnostic multiplex PCR provides a valuable alternative to sequence-based approaches when a defined set of prey organisms is to be detected: multiplexing of taxon-specific primers allows the identification of several prey taxa within one reaction, based on differences in amplicon size. Depending on the information needed, the taxonomic level of prey identification can be selected and through balancing primer concentrations, equal sensitivity can be reached across the targeted taxa.
Colleagues from Austria have developed a two-step multiplex PCR system, allowing for the detection of fish DNA in dietary samples of piscivore predators. This system encompasses fish and lamprey species native to Central European freshwaters and enables the identification of 31 species, six genera, two families, two orders and two fish family clusters. The multiplex PCR is based on barcodes and 16S rRNA primers. The authors conducted feeding trials with Eurasian otters at the zoo and collected feces samples. In addition they used field-collected dietary samples from kingfishers (feces) and cormorants (feces and pellets).
The two-step multiplex PCR approach presented here provides an efficient, easy to use and cost-effective tool to examine the diet of piscivores in great detail. Although the system has been developed for Central Europe, it will be applicable to other regions where the targeted fish species occur; however, we strongly recommend evaluating specificity a priori. Furthermore, the application of the multiplex PCR system is not restricted to prey identification, but the assays or single primer pairs will be useful to any approach where fish DNA needs to be identified with high specificity and sensitivity such as environmental monitoring, studies on environmental DNA or species-specific identification of fish eggs, larvae and carcasses.