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.

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.

Nitrogen-fixing cyanobacteria (NFC) are important primary producers in many freshwater and marine systems, including the Baltic Sea. In this system, NFC circumvent summer nitrogen limitation, while also generating a supply of novel combined nitrogen and thus supporting food webs. Using field observations on zooplankton and phytoplankton development during a growth season in the northern Baltic Proper, we show that cyanobacterial nitrogen is assimilated and transferred to zooplankton via both direct grazing on NFC and indirectly through grazing on picoplankton, such as picocyanobacteria. The key findings supporting these conclusions are: (1) all zooplankton grazers were found to ingest NFC (Nodularia spumigena) and picocyanobacteria (Synechococcus spp.); (2) ingestion of both NFC and picocyanobacteria measured by quantitative polymerase chain reaction analysis was highly correlated with ambient stocks of the respective cyanobacteria; (3) consumption of NFC and picocyanobacteria translated into decreased delta N-15 signature of zooplankton indicative of diazotrophic nitrogen input; (4) growth and reproduction indices in zooplankters were significantly positively related to NFC and picocyanobacteria; and (5) zooplankton biomass was positively related to the increasing nitrogen content of particulate organic matter (POM<10 mu m) and was highest at low POM delta N-15 values; the latter reflected overlap in zooplankton production and diazotroph seasonal dynamics. These findings provide empirical evidence that both NFC and picoplankton are readily ingested and assimilated by zooplankton, albeit with differential effects on growth and recruitment.

The aim of the study was to measure copepod reproduction, mortality and sex ratio in the field before, during and after a cyanobacteria bloom during the summer in the western Gulf of Finland. Environment and zooplankton samples were collected every fortnight, and the copepod Acartia spp. was incubated in the laboratory for reproductive output, i.e. egg production and egg hatching success. Other responses monitored were female: male ratio, mortality and body condition. In addition, molecular analyses of the nodularin-producing cyanobacterium Nodularia in Acartia gut contents (GCs) were assessed. Egg production and body condition decreased with increasing Nodularia GCs. During the bloom, hatching decreased as a response to Nodularia in the copepod gut. Although not related to cyanobacteria variables, male mortality was higher than female mortality, resulting in a female-biased sex ratio over most of the summer. The study demonstrates that Acartia reproductive output is constrained by cyanobacteria blooms in the Baltic Sea, and more generally that copepod population dynamics may be negatively affected by such blooms. This is especially significant considering that toxin-producing blooms are predicted to increase due to warming.

Filamentous, nitrogen-fixing cyanobacteria form extensive summer blooms in the Baltic Sea. Their ability to fix dissolved N-2 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.

Blooms of nitrogen fixing cyanobacteria (NFC) occur in many freshwater and marine systems, including the Baltic Sea. By fixing dissolved nitrogen, they circumvent general summer nitrogen limitation, while also generating a supply of novel bioavailable nitrogen for non-diazotrophic primary producers and ultimately supporting secondary production. Elucidating trophic links between primary consumers and NFC is essential for understanding role of these blooms for secondary production. However, until recently, there was no reliable method to quantify individual prey species for zooplankter feeding in situ. The development of PCR-based methods to detect prey-specific DNA in the diet of consumers, including microscopic animals, allows identification and quantification of trophic linkages in the field.

Using molecular diet analysis in combination with egg production measurements, biochemical markers of growth and condition; and stable isotope approach, we explored a possibility to determine (1) whether cyanobacteria are grazed and assimilated by mesozooplankters (Papers I and II), (2) which species/groups are particularly efficient consumers of cyanobacteria (Papers II and III), and (3) how feeding on cyanobacteria affects zooplankton growth and development (Paper I and III). Taken together, these laboratory and field observations, provided evidence that NFC contribute to feeding and reproduction of zooplankton during summer and create a favorable growth environment for the copepod nauplii (Paper I). The favorable growth conditions for juvenile copepods observed during NFC blooms were hypothesized to be mediated by picoplankton that take up bioavailable nitrogen exuded from cyanobacterial cells. This hypothesis found support in Paper II that provided quantitative estimates for the direct picocyanobacteria → mesozooplankton pathway, with highest weight-specific consumption observed in nauplii. Further, using field observations on zooplankton and phytoplankton development during a growth season in the northern Baltic proper, we found that NFC nitrogen is assimilated and transferred to zooplankton via both direct grazing and indirectly through grazing on small-sized phyto- and bacterioplankton (Paper III). Finally, these and other findings emphasizing the importance of NFC for Baltic Sea secondary production during growth season were synthesized to show that diazotrophic nitrogen enters food webs already at bloom initiation (Paper III) and is transferred via multiple pathways to pelagic and benthic food webs and, ultimately, to fish (Paper IV).

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.

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 C-13 by the copepods as a proxy for carbon uptake in feeding experiment with C-13-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.

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.