Simultaneous RNA and DNA Adductomics Using Single Data-Independent Acquisition Mass Spectrometry Analysis.

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.

Molecular diet analysis enables detection of diatom and cyanobacteria DNA in the gut of Macoma balthica.

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.

Phytoplankton settling quality has a subtle but significant effect on sediment microeukaryotic and bacterial communities.

In coastal aphotic sediments, organic matter (OM) input from phytoplankton is the primary food resource for benthic organisms. Current observations from temperate ecosystems like the Baltic Sea report a decline in spring bloom diatoms, while summer cyanobacteria blooms are becoming more frequent and intense. These climate-driven changes in phytoplankton communities may in turn have important consequences for benthic biodiversity and ecosystem functions, but such questions are not yet sufficiently explored experimentally. Here, in a 4-week experiment, we investigated the response of microeukaryotic and bacterial communities to different types of OM inputs comprising five ratios of two common phytoplankton species in the Baltic Sea, the diatom Skeletonema marinoi and filamentous cyanobacterium Nodularia spumigena. Metabarcoding analyses on 16S and 18S ribosomal RNA (rRNA) at the experiment termination revealed subtle but significant changes in diversity and community composition of microeukaryotes in response to settling OM quality. Sediment bacteria were less affected, although we observed a clear effect on denitrification gene expression (nirS and nosZ), which was positively correlated with increasing proportions of cyanobacteria. Altogether, these results suggest that future changes in OM input to the seafloor may have important effects on both the composition and function of microbenthic communities.

Mercury-methylating bacteria are associated with copepods: A proof-of-principle survey in the Baltic Sea.

Methylmercury (MeHg) is a potent neurotoxin that biomagnifies in marine food webs. Inorganic mercury (Hg) methylation is conducted by heterotrophic bacteria inhabiting sediment or settling detritus, but endogenous methylation by the gut microbiome of animals in the lower food webs is another possible source. We examined the occurrence of the bacterial gene (hgcA), required for Hg methylation, in the guts of dominant zooplankters in the Northern Baltic Sea. A qPCR assay targeting the hgcA sequence in three main clades (Deltaproteobacteria, Firmicutes and Archaea) was used in the field-collected specimens of copepods (Acartia bifilosa, Eurytemora affinis, Pseudocalanus acuspes and Limnocalanus macrurus) and cladocerans (Bosmina coregoni maritima and Cercopagis pengoi). All copepods were found to carry hgcA genes in their gut microbiome, whereas no amplification was recorded in the cladocerans. In the copepods, hgcA genes belonging to only Deltaproteobacteria and Firmicutes were detected. These findings suggest a possibility that endogenous Hg methylation occurs in zooplankton and may contribute to seasonal, spatial and vertical MeHg variability in the water column and food webs. Additional molecular and metagenomics studies are needed to identify bacteria carrying hgcA genes and improve their quantification in microbiota.

DNA epigenetic marks are linked to embryo aberrations in amphipods.

Linking exposure to environmental stress factors with diseases is crucial for proposing preventive and regulatory actions. Upon exposure to anthropogenic chemicals, covalent modifications on the genome can drive developmental and reproductive disorders in wild populations, with subsequent effects on the population persistence. Hence, screening of chemical modifications on DNA can be used to provide information on the probability of such disorders in populations of concern. Using a high-resolution mass spectrometry methodology, we identified DNA nucleoside adducts in gravid females of the Baltic amphipods Monoporeia affinis, and linked the adduct profiles to the frequency of embryo malformations in the broods. Twenty-three putative nucleoside adducts were detected in the females and their embryos, and eight modifications were structurally identified using high-resolution accurate mass data. To identify which adducts were significantly associated with embryo malformations, partial least squares regression (PLSR) modelling was applied. The PLSR model yielded three adducts as the key predictors: methylation at two different positions of the DNA (5-methyl-2'-deoxycytidine and N6-methyl-2'-deoxyadenosine) representing epigenetic marks, and a structurally unidentified nucleoside adduct. These adducts predicted the elevated frequency of the malformations with a high classification accuracy (84%). To the best of our knowledge, this is the first application of DNA adductomics for identification of contaminant-induced malformations in field-collected animals. The method can be adapted for a broad range of species and evolve as a new omics tool in environmental health assessment.

Denitrification responses to increasing cadmium exposure in Baltic Sea sediments.

Benthic ecosystems have come under intense pressure, due to eutrophication-driven oxygen decline and industrial metal contamination. One of the most toxic metals is Cadmium (Cd), which is lethal to many aquatic organisms already at low concentrations. Denitrification by facultative anaerobic microorganisms is an essential process to transform, but also to remove, excess nitrate in eutrophied systems. Cd has been shown to decrease denitrification and sequester free sulfide, which is available when oxygen is scarce and generally inhibits complete denitrification (i.e. N2O to N2). In polluted sediments, an interaction between oxygen and Cd may influence denitrification and this relationship has not been studied. For example, in the Baltic Sea some sediments are double exposed to both Cd and hypoxia. In this study, we examined how the double exposure of Cd and fluctuations in oxygen affects denitrification in Baltic Sea sediment. Results show that oxygen largely regulated N2O and N2 production after 21 days of exposure to Cd (ranging from 0 to 500 µg/L, 5 different treatments, measured by the isotope pairing technique (IPT)). In the high Cd treatment (500 µg/L) the variation in N2 production increased compared to the other treatments. Increases in N2 production are suggested to be an effect of 1) enhanced nitrification that increases NO3- availability thus stimulating denitrification, and 2) Cd successfully sequestrating sulfide (yielding CdS), which allows for full denitrification to N2. The in situ field sediment contained initially high Cd concentrations in the pore water (∼10 µg/L) and microbial communities might already have been adapted to metal stress, making the effect of low Cd levels negligible. Here we show that high levels of cadmium pollution might increase N2 production and influence nitrogen cycling in marine sediments.

Nitrogen fixation by cyanobacteria stimulates production in Baltic food webs.

Filamentous, nitrogen-fixing cyanobacteria form extensive summer blooms in the Baltic Sea. Their ability to fix dissolved N2 allows cyanobacteria to circumvent the general summer nitrogen limitation, while also generating a supply of novel bioavailable nitrogen for the food web. However, the fate of the nitrogen fixed by cyanobacteria remains unresolved, as does its importance for secondary production in the Baltic Sea. Here, we synthesize recent experimental and field studies providing strong empirical evidence that cyanobacterial nitrogen is efficiently assimilated and transferred in Baltic food webs via two major pathways: directly by grazing on fresh or decaying cyanobacteria and indirectly through the uptake by other phytoplankton and microbes of bioavailable nitrogen exuded from cyanobacterial cells. This information is an essential step toward guiding nutrient management to minimize noxious blooms without overly reducing secondary production, and ultimately most probably fish production in the Baltic Sea.

Bloom-forming cyanobacteria support copepod reproduction and development in the Baltic Sea.

It is commonly accepted that summer cyanobacterial blooms cannot be efficiently utilized by grazers due to low nutritional quality and production of toxins; however the evidence for such effects in situ is often contradictory. Using field and experimental observations on Baltic copepods and bloom-forming diazotrophic filamentous cyanobacteria, we show that cyanobacteria may in fact support zooplankton production during summer. To highlight this side of zooplankton-cyanobacteria interactions, we conducted: (1) a field survey investigating linkages between cyanobacteria, reproduction and growth indices in the copepod Acartia tonsa; (2) an experiment testing relationships between ingestion of the cyanobacterium Nodularia spumigena (measured by molecular diet analysis) and organismal responses (oxidative balance, reproduction and development) in the copepod A. bifilosa; and (3) an analysis of long term (1999-2009) data testing relationships between cyanobacteria and growth indices in nauplii of the copepods, Acartia spp. and Eurytemora affinis, in a coastal area of the northern Baltic proper. In the field survey, N. spumigena had positive effects on copepod egg production and egg viability, effectively increasing their viable egg production. By contrast, Aphanizomenon sp. showed a negative relationship with egg viability yet no significant effect on the viable egg production. In the experiment, ingestion of N. spumigena mixed with green algae Brachiomonas submarina had significant positive effects on copepod oxidative balance, egg viability and development of early nauplial stages, whereas egg production was negatively affected. Finally, the long term data analysis identified cyanobacteria as a significant positive predictor for the nauplial growth in Acartia spp. and E. affinis. Taken together, these results suggest that bloom forming diazotrophic cyanobacteria contribute to feeding and reproduction of zooplankton during summer and create a favorable growth environment for the copepod nauplii.

Mesozooplankton grazing on picocyanobacteria in the Baltic Sea as inferred from molecular diet analysis.

Our current knowledge on the microbial component of zooplankton diet is limited, and it is generally assumed that bacteria-sized prey is not directly consumed by most mesozooplankton grazers in the marine food webs. We questioned this assumption and conducted field and laboratory studies to examine picocyanobacteria contribution to the diets of Baltic Sea zooplankton, including copepods. First, qPCR targeting ITS-1 rDNA sequence of the picocyanobacteria Synechococcus spp. was used to examine picocyanobacterial DNA occurrence in the guts of Baltic zooplankton (copepods, cladocerans and rotifers). All field-collected zooplankton were found to consume picocyanobacteria in substantial quantities. In terms of Synechococcus quantity, the individual gut content was highest in cladocerans, whereas biomass-specific gut content was highest in rotifers and copepod nauplii. Moreover, the gut content in copepods was positively related to the picocyanobacteria abundance and negatively to the total phytoplankton abundance in the water column at the time of sampling. This indicates that increased availability of picocyanobacteria resulted in the increased intake of this prey and that copepods may rely more on picoplankton when food in the preferred size range declines. Second, a feeding experiments with a laboratory reared copepod Acartia tonsa fed a mixture of the picocyanobacterium Synechococcus bacillaris and microalga Rhodomonas salina confirmed that copepods ingested Synechococcus, even when the alternative food was plentiful. Finally, palatability of the picocyanobacteria for A. tonsa was demonstrated using uptake of (13)C by the copepods as a proxy for carbon uptake in feeding experiment with (13)C-labeled S. bacillaris. These findings suggest that, if abundant, picoplankton may become an important component of mesozooplankton diet, which needs to be accounted for in food web models and productivity assessments.

Trade-offs between predation risk and growth benefits in the copepod Eurytemora affinis with contrasting pigmentation.

Intraspecific variation in body pigmentation is an ecologically and evolutionary important trait; however, the pigmentation related trade-offs in marine zooplankton are poorly understood. We tested the effects of intrapopulation phenotypic variation in the pigmentation of the copepod Eurytemora affinis on predation risk, foraging, growth, metabolic activity and antioxidant capacity. Using pigmented and unpigmented specimens, we compared (1) predation and selectivity by the invertebrate predator Cercopagis pengoi, (2) feeding activity of the copepods measured as grazing rate in experiments and gut fluorescence in situ, (3) metabolic activity assayed as RNA:DNA ratio in both experimental and field-collected copepods, (4) reproductive output estimated as egg ratio in the population, and (5) total antioxidant capacity. Moreover, mitochondrial DNA (mtDNA) COI gene variation was analysed. The pigmented individuals were at higher predation risk as evidenced by significantly higher predation rate by C. pengoi on pigmented individuals and positive selection by the predator fed pigmented and unpigmented copepods in a mixture. However, the antioxidant capacity, RNA:DNA and egg ratio values were significantly higher in the pigmented copepods, whereas neither feeding rate nor gut fluorescence differed between the pigmented and unpigmented copepods. The phenotypic variation in pigmentation was not associated with any specific mtDNA genotype. Together, these results support the metabolic stimulation hypothesis to explain variation in E. affinis pigmentation, which translates into beneficial increase in growth via enhanced metabolism and antioxidant protective capacity, together with disadvantageous increase in predation risk. We also suggest an alternative mechanism for the metabolic stimulation via elevated antioxidant levels as a primary means of increasing metabolism without the increase in heat absorbance. The observed trade-offs are relevant to evolutionary mechanisms underlying plasticity and adaptation and have the capacity to modify strength of complex trophic interactions.