The applicability of qPCR to quantify the activity of decomposers in managed and abandoned alpine soils

Julia Seeber

1 Introduction

The study of the function and behaviour of soil animals is mostly a difficult tasks due to the intransparency of the soil environment. Most research questions have to be investigated under controlled conditions in laboratories (e.g. Seeber et al. 2006, Pieper and Weigmann 2008, Eisenhauer et al. 2010) or in the field by e.g. using tracers such as labelled stable isotopes (e.g. Setälä and Aarnio 2002, Caner et al. 2004, Seeber et al. 2009). In the last decade molecular methods, for instance diagnostic PCR, have become a research tool which offers efficient ways to study soil animals (e.g. Boyer et al. 2011, Heidemann et al. 2011). However, most of these methods are qualitative ones. Here we propose a molecular technique, namely real-time PCR (qPCR), to quantify the presence of two important groups of soil macro-invertebrates in managed and abandoned alpine soils, namely decomposers (earthworms and millipedes) and agricultural pests (wireworms and larvae of cockchafers). Molecular methods have to be developed and tested in laboratories before using them in field studies, as many factors, e.g. climatic conditions, UV light, etc., have an impact on the detectability of DNA (Oehm et al. 2011). Therefore, the development and optimization of the qPCR approach in the lab will be an important step towards further taking molecular methods into the field.

Detritivoral soil animals play essential roles in humus and soil formation processes by fragmenting and mixing the organic matter into more accessible units for microbial attack and by incorporating it into the soil (Bardgett 2005). By doing this, decomposer animals leave behind apparent traces of their activities. Earthworms build burrows with their faeces and their mucus, faecal pellets of millipedes and insect larvae form an important part of humus layers. The soil animals thereby loose cells from their body containing detectable DNA. In a laboratory experiment, we were able to assign faecal pellets of millipedes to their invertebrate producers using diagnostic PCR (Seeber et al. 2010). Species-specific identification was possible for up to 5 days after defecation (no longer time intervals have been tested). In theory, it should also be possible to detect the DNA of decomposers in field samples. With insect pests this might be more difficult as they do not regularly defecate. In the proposed study we will therefore test and compare the qPCR approach for these two groups of soil animals to be able to evaluate its applicability in both decomposition and agricultural pest studies.

2 Aims and impact of the project

Aim of the project is to develop a reliable, rapid and non-destructive molecular tool which will allow us to quantify the presence of two important groups of soil animals (decomposers and pests). Up to now this had to be done by taking destructive soil samples, extracting the

soil animals by heat over a longer period of time, identifying them morphologically and counting their abundances. The development of the qPCR assay will allow to determine the animals’ DNA copy numbers in a known amount of humus or soil material. By normalizing and validating the assay it might be possible to relate the copy numbers directly to the animals’ biomass. The next step will then be the screening of field samples to test the applicability of diagnostic PCR and qPCR in field studies.

We aim at answering two central questions:

  • Is it generally possible to detect the producer’s DNA in a soil sample? It has already been proven for decomposers that their DNA can be ampflified from their own faeces, therefore it will most likely be possible from soil samples. However, as pest animals do not defecate or excrete mucus, it might be more difficult to detect their DNA.
  • If we succeed in detecting the producer’s DNA from soil samples, we will analyse the association between the copy numbers and the biomass of the animals. If there is a correlation, this will allow to draw conclusions on the presence of the invertebrates.

The results of this laboratory experiment will be a prerequisite (1) to test the applicability of this approach for field studies and, if this proves to be valid, (2) to be able to correctly interpret results from field samples. Furthermore, for soil pests it would be the first evidence that their presence might be analysed via DNA traces. Additionally, results might provide important cues for other molecular methods such as pyrosequencing, which is increasingly applied in studies on soil community composition (e.g. Hamilton et al. 2009, Wu et al. 2009).