Methodological advances
Assets of RESIST include various methodological advances in experimental design, bioanalytical tools, data storing and data analysis, a few of which are highlighted here.
Heating module for the ExStream system
Phase I was based to a large extent on experiments with the ExStream outdoor mesocosm system. For these experiments, up to 128 circular mesocosms are fed with stream water and multiple stressors can be simultaneously applied in a full factorial design with eight replicates per stressor combination each. While ExStream systems have been used for several years, the system still faced some technical difficulties in the application of stressors. This is particularly relevant for temperature manipulations, as the control of water temperature for constantly supplied stream water poses major challenges. In Phase I, we developed a heating module that enables an automated differential temperature regulation between two or more warming treatments (see Madge Pimentel et al., 2023). We successfully applied the new system in our two main mesocosm experiments involving warming as a stressor.
Simultaneous application of several (bio-)analytical tools
We applied indoor flumes as a second experimental system (AquaFlow) to test for effects of stressor increase and reduction under highly controlled conditions. Here we simultaneously used non-target screening, microeukaryotic and prokaryotic DNA metabarcoding as effective approaches for detecting environmental impacts on freshwater ecosystems. We specified differences in their detection capabilities and their temporal resolution. Microeukaryotic DNA metabarcoding consistently showed exceptional detection capabilities, particularly for short- and mid-term effects (see Sieber et al., 2023).
Use of stable isotopes for dispersal research
Isotopic enrichment with 15N is a non-invasive method with the potential of labeling large quantities of macroinvertebrates but has rarely been applied systematically. We tested and compared isotopic mass enrichment across 12 macroinvertebrate taxa and feeding types. Macroinvertebrate density estimates on the stream bottom averaged a total of ~3.4 million labeled individuals. Hence, isotopic enrichment can greatly assist the analysis of macroinvertebrate dispersal through mark-and-recapture experiments, as it allows to measure the movement at the level of individual specimens (see Enss et al., 2024).
Reverse stable isotope labelling (RSIL)
We applied and refined a new method to measure microbial activity in river sediments and water. Microcosms were set up with a bicarbonate buffer containing 10 atom percent of labeled H13CO3-. Microbial degradation of organic material leads to the release of carbon dioxide containing a natural abundance of 13C (1.01%), which dilutes the label in the bicarbonate buffer. This change in the isotopic composition of the bicarbonate buffer due to microbial activity can be very precisely measured with stable isotope analyses for CO2. We observed that RSIL successfully determined microbial activity under temperature, salinity and combined temperature and salinity stress as well as in control experiments (Baikova et al., in prep.).
New laboratory protocols for DNA/RNA extraction and amplification
For the analysis of different sample types (macroinvertebrate bulk samples, sediment, plant material, bacteria), several new protocols were developed and published in an open-source repository (link to repository) to facilitate and upscale the processing of samples.
Introducing a data reuse identifier (DRI)
Not all publicly deposited data are associated with a respective publication, which can be the result of many different circumstances including the early release of data by sequencing centers. In the past, unjust re-use of public data has infrequently created tension and issues in the community which at times led to retraction of publications. We tackle this issue by suggesting the introduction of a data reuse identifier (DRI) to label data that is currently being worked on. This DRI tag carries ORCIDs of the data creators which allows for discussing the data reuse or harnessing more metadata for richer data mining publications.