Development and Application of Molecular Techniques to Assess Feeding Interactions in a Forest Invaded by the Asian Earthworm Amynthas agrestis

Straube, Daniela

ABSTRACT: The invasion of alien species into new habitats is an on-going global phenomenon and alien species can affect the invaded habitat at different levels. For instance, invasive earthworms are known to change soil properties and community composition, but little is known about their influence on the diet of native predators, and even less on whether native predators utilize the alien species as prey. The invasive Asian earthworm Amynthas agrestis is known to have invaded the Great Smoky Mountains National Park (GSMNP, U.S.) as recently as the 1990s. The park is rich in species and provides a variety of generalist predators for investigations of the effects of A. agrestis on their diet. In this thesis non-invasive sampling methods for dietary analyses were developed for carabid beetles and salamanders. They are known to generally prey on earthworms, and are very abundant in the GSMNP. Non-invasive sampling methods are necessary to investigate protected and rare species, and to minimize the effect on community composition due to sampling. We investigated if and how long prey DNA is detectable in these predators by conducting feeding experiments. Furthermore, the prey spectrum of carabid beetles was examined to answer the question if A. agrestis is utilized as prey and indirectly changes the prey spectrum of carabid beetles due to its pronounced effects on faunal composition. Different preservation and extraction methods for DNA samples were evaluated to detect differences in DNA amplification success of different target genes, and to find an efficient way to ship all samples from the U.S. to Austria. Feeding experiments with carabid beetles and salamanders were conducted to examine how long prey DNA is detectable in regurgitates and faecal pellets, and if additional prey and species of predator influences the DNA detection success. Carabid beetles were either fed with Eisenia spp. and Tenebrio sp. consecutively or with one prey only and regurgitates were then analysed using a molecular approach. Salamanders were fed with three different prey species (Eisenia spp., crickets, and termites) and additional Tenebrio sp. was offered. Salamander faecal pellets were then molecularly analysed to detect prey DNA. Carabid beetles were collected from study sites in the GSMNP to investigate their regurgitates to evaluate whether they utilize A. agrestis as prey, and if the prey spectrum as well as the detection frequency of different prey species changes. Therefore, a multiplex PCR system targeting different prey species was designed. For this study, preservation by freeze drying samples in combination with peqGold™ extraction are the preferred methods for long distance shipping, and was used for DNA samples generated in the studies. The feeding experiment with carabid beetles showed that the influence of additional prey on the detection of a target prey was low, but significant in one instance; feeding on Eisenia spp. prior to feeding on Tenebrio sp. does reduce the 50% detection probability for Tenebrio sp. DNA. The species identity of the predators had no significant influence on the detection success of the two prey species.