Adductomics studies are used for the detection and characterization of various chemical modifications (adducts) of nucleic acids and proteins. The advancements in liquid chromatography coupled with high-resolution tandem mass spectrometry (HRMS/MS) have resulted in efficient methods for qualitative and quantitative adductomics. We developed an HRMS-based method for the simultaneous analysis of RNA and DNA adducts in a single run and demonstrated its application using Baltic amphipods, useful sentinels of environmental disturbances, as test organisms. The novelty of this method is screening for RNA and DNA adducts by a single injection on an Orbitrap HRMS instrument using full scan and data-independent acquisition. The MS raw files were processed with an open-source program, nLossFinder, to identify and distinguish RNA and DNA adducts based on the characteristic neutral loss of ribonucleosides and 2'-deoxyribonucleosides, respectively. In the amphipods, in addition to the nearly 150 putative DNA adducts characterized earlier, we detected 60 putative RNA adducts. For the structural identification of the detected RNA adducts, the MODOMICS database was used. The identified RNA adducts included simple mono- and dimethylation and other larger functional groups on different ribonucleosides and deaminated product inosine. However, 54 of these RNA adducts are not yet structurally identified, and further work on their characterization may uncover new layers of information related to the transcriptome and help understand their biological significance. Considering the susceptibility of nucleic acids to environmental factors, including pollutants, the developed multi-adductomics methodology with further advancement has the potential to provide biomarkers for diagnostics of pollution effects in biota.

Detritivores are essential to nutrient cycling, but are often neglected in trophic networks, due to difficulties with determining their diet. DNA analysis of gut contents shows promise of trophic link discrimination, but many unknown factors limit its usefulness. For example, DNA can be rapidly broken down, especially by digestion processes, and DNA provides only a snapshot of the gut contents at a specific time. Few studies have been performed on the length of time that prey DNA can be detected in consumer guts, and none so far using benthic detritivores. Eutrophication, along with climate change, is altering the phytoplankton communities in aquatic ecosystems, on which benthic detritivores in aphotic soft sediments depend. Nutrient-poor cyanobacteria blooms are increasing in frequency, duration, and magnitude in many water bodies, while nutrient-rich diatom spring blooms are shrinking in duration and magnitude, creating potential changes in diet of benthic detritivores. We performed an experiment to identify the taxonomy and quantify the abundance of phytoplankton DNA fragments on bivalve gut contents, and how long these fragments can be detected after consumption in the Baltic Sea clam Macoma balthica. Two common species of phytoplankton (the cyanobacteria Nodularia spumigena or the diatom Skeletonema marinoi) were fed to M. balthica from two regions (from the northern and southern Stockholm archipelago). After removing the food source, M. balthica gut contents were sampled every 24 hours for seven days to determine the number of 23S rRNA phytoplankton DNA copies and when the phytoplankton DNA could no longer be detected by quantitative PCR. We found no differences in diatom 18S rRNA gene fragments of the clams by region, but the southern clams showed significantly more cyanobacteria 16S rRNA gene fragments in their guts than the northern clams. Interestingly, the cyanobacteria and diatom DNA fragments were still detectable by qPCR in the guts of M. balthica one week after removal from its food source. However, DNA metabarcoding of the 23S rRNA phytoplankton gene found in the clam guts showed that added food (i.e. N. spumigena and S. marinoi) did not make up a majority of the detected diet. Our results suggest that these detritivorous clams therefore do not react as quickly as previously thought to fresh organic matter inputs, with other phytoplankton than large diatoms and cyanobacteria constituting the majority of their diet. This experiment demonstrates the viability of using molecular methods to determine feeding of detritivores, but further studies investigating how prey DNA signals can change over time in benthic detritivores will be needed before this method can be widely applicable to both models of ecological functions and conservation policy.