In the Netherlands several protected animals that live in surface waters are monitored to meet Dutch directives or laws. Current animal monitoring is mainly based on visual observations which can be time consuming and labor intensive. These methods are also not always effective for animal species that are rare, have a hidden lifestyle or have ambiguous morphological characteristics. Alternative monitoring methods were introduced for animal species that live (partly) in water based on detection of DNA - traces excreted into the environment (eDNA). Due to the fact that this method detects DNA, it is not required to catch or even observe the target organisms. eDNA can be detected and quantified using quantitative polymerase chain reaction (qPCR). This project aimed to develop and validate eDNA detection methods for protected weatherfish (Misgurnus fossilis) and great crested newt (Triturus cristatus) species, and for seven invasive freshwater crayfish species (Astacidae & Cambaridae). Target species are selected because they are rare (T. cristatus and M. fossilis) or have a hidden lifestyle (all). Besides, early detection of exotic and invasive species (Astacidae & Cambaridae) is essential for success of a control plan. Furthermore was examined under which circumstances eDNA detection method provide better results. Finally, two different sample processing procedures were compared for DNA yields and detection probability.
eDNA detection methods require robust qPCR-protocols for amplification and quantification of target species DNA. Specific primers were developed targeting COI, CytB and 16S genes present on mitochondrial DNA. Mitochondrial DNA (mtDNA) is known for its high copy number which increases detection chance. Besides, these DNA regions are variable between species but conserved within species. Both properties make mtDNA a suitable target for species specific detection methods. Species specific qPCR-protocols were proposed after extensive in silico and in vitro analysis. Primers and probes were analyzed for amplification efficiency of target- and reference species DNA. Specific M. fossilis and T. cristatus assays were successfully developed targeting a region in the COI gene. Assays for invasive Astacidae & Cambaridae species were not successful yet.
The sensitivity of the methods was successfully determined by analyzing samples taken from locations where target species were present. Developed eDNA methods were validated in a field study. Twelve locations were sampled in triplicate at several water bodies in the Alblasserwaard polder during early spring and late spring. Results were compared to conventional screening data from the same period. In general M. fossilis and T. cristatus DNA was detected in all samples taken from locations where species were observed. Moreover, DNA was amplified in more samples if collected during late spring than during early spring. This is most likely due to inactive M. fossilis species or absent T. cristatus species during colder periods. Finally, two different sampling methods and procedures were applied: 1 L filtration versus 15 mL precipitation. Sample filtration results in extraction of merely cellular DNA while sample precipitation results in extraction of both cellular- and extracellular DNA. Comparison of the results slightly indicated that precipitation of 15 mL volumes give higher DNA yields and filtration of 1 L volumes give higher detection probability. In conclusion, eDNA detection methods designed for Misgunrus fossilis and Triturus cristatus species show potential to be sensitive and specific tools in species monitoring but more research is needed before it can be applied.
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