Development of metabarcoding for tracking changes of soil fauna community under stress by application of ash

Ash is a waste product from combustion of bio-fuel in power plants. Application of ash on soil ensures nutrient recycling, but detrimental ecotoxicological consequences may arise since ash is a complex mixture that may contain compounds affecting soil invertebrates and their food and habitat condition. Here, we study the effects of ash on the abundance and composition of the soil fauna community. Over time, we will compare control plots with plots receiving three different concentrations of ash. Targeting soil fauna community includes protozoa, nematodes, enchytraeids, collembolans, mites and earthworms. For collembolans and mites, a morphospecies approach combined with DNA barcoding will be performed to quantify the species abundance. Traditional morphological methods of identifying arthropods are (1) time consuming, (2) require trained taxonomists and (3) hampered by cryptic life stages and species. DNA metabarcoding, which couples the principle of DNA barcoding with next generation sequencing technology, has the potential to simplify community diversity monitoring. However, sampling and DNA extraction methods for the purpose of soil microarthropod metabarcoding have not been yet fully developed. We therefore will start out by comparing the morphospecies approach with three molecular approaches, differing in the types of DNA extracted from soil samples: total soil DNA, extracellular DNA and DNA gained from animals collected from the soil. In order to target all faunal groups, we use a set of three mini-barcodes based on the mitochondrial cytochrome oxidase I encoding gene (COI), histone 3 protein encoding gene (H3) and 18S regions, respectively. Community amplicon sequencing will be performed on the MiSeq Illumina platform. The extraction method with highest quality and efficiency will be used for long term monitoring of changes in the soil mesofaunal biodiversity under the stress of ash. Hopefully, the long term monitoring data could help maintain the soil ecology and improve the management of ash.