Knowledge of zooplankton in situ diet is critical for accurate assessment of marine ecosystem function and structure, but due to methodological constraints, there is still a limited understanding of ecological networks in marine ecosystems. Here, we used DNA-metabarcoding to study trophic interactions, with the aim to unveil the natural diet of zooplankton species under temporal variation of food resources. Several target consumers, including copepods and cladocerans, were investigated by sequencing 16S rRNA and 18S rRNA genes to identify prokaryote and eukaryote potential prey present in their guts. During the spring phytoplankton bloom, we found a dominance of diatom and dinoflagellate trophic links to copepods. During the summer period, zooplankton including cladocerans showed a more diverse diet dominated by cyanobacteria and heterotrophic prey. Our study suggests that copepods present trophic plasticity, changing their natural diet over seasons, and adapting their feeding strategies to the available prey spectrum, with some species being more selective. We did not find a large overlap of prey consumed by copepods and cladocerans, based on prey diversity found in their guts, suggesting that they occupy different roles in the trophic web. This study represents the first molecular approach to investigate several zooplankton-prey associations under seasonal variation, and highlights how, unlike other techniques, the diversity coverage is high when using DNA, allowing the possibility to detect a wide range of trophic interactions in plankton communities.

Alternative pathways of energy transfer guarantee the functionality and productivity in marine food webs that experience strong seasonality. Nevertheless, the complexity of zooplankton interactions is rarely considered in trophic studies because of the lack of detailed information about feeding interactions in nature. In this study, we used DNA metabarcoding to highlight the diversity of trophic niches in a wide range of micro- and mesozooplankton, including ciliates, rotifers, cladocerans, copepods and their prey, by sequencing 16- and 18S rRNA genes. Our study demonstrates that the zooplankton trophic niche partitioning goes beyond both phylogeny and size and reinforces the importance of diversity in resource use for stabilizing food web efficiency by allowing for several different pathways of energy transfer. We further highlight that small, rarely studied zooplankton (rotifers and ciliates) fill an important role in the Baltic Sea pelagic primary production pathways and the potential of ciliates, rotifers and crustaceans in the utilization of filamentous and picocyanobacteria within the pelagic food web. The approach used in this study is a suitable entry point to ecosystem-wide food web modelling considering species-specific resource use of key consumers.

Marine communities undergo rapid changes because of human-induced ecosystem pressures. The Baltic Sea pelagic food web has experienced several regime-shifts during the past century, resulting in a system where competition between planktivorous mesopredators is assumed to be high. While the two clupeids sprat and herring reveal signs of competition, the stickleback population has increased drastically during the past decades. Here, we investigate diet overlap between the three dominating planktivorous fish in the Baltic Sea, utilizing DNA metabarcoding, targeted qPCR, and microscopy. Our results show a niche differentiation between clupeids and stickleback and that rotifers play an important function in niche partitioning of stickleback, as a resource that is not being used, neither by the clupeids nor by other zooplankton. This study suggests that rotifers and other understudied soft-bodied prey may have an important function in the pelagic food web and that the growing population of pelagic stickleback is supported by the unutilized feeding niche offered by the rotifers.

Estimating energy fluxes, or weight of trophic interactions, in food webs allows linking food web structure to properties of ecosystem functioning. Due to limitations in traditional methods, resolved food-web models are often binary or based on body size. Species-specific feeding traits and weights of interactions are typically not included, particularly at the base of the pelagic food web. Consequently, models are prone to underestimate the trophic diversity of species interactions.Here, we focus on trophic pathways of primary production in the Baltic Sea using a bioenergetic model that includes several trophic levels from primary producers to fish. For the first time, dietary DNA metabarcoding data of zooplankton are combined with accumulated biomass data from long-term pelagic monitoring and metabolic theory in a network model to describe energy pathways, including the diversity of planktonic organisms.We show that picocyanobacteria and filamentous cyanobacteria are the main contributors to secondary production in the Baltic Sea and that the latter experience high predation pressure from zooplankton. In contrast, the combination of high biomasses and low predation pressure on dinoflagellates and diatoms suggests that a significant fraction of the spring bloom is not utilized in the pelagic food web, explaining the high export of biological material to the sea seafloor.This study can be used to address ecosystem management objectives in the Baltic Sea under changing environmental conditions. Furthermore, the novel framework presented in this study, integrating DNA metabarcoding and biomonitoring data to assess energy fluxes, can be extended to dynamic food web modeling in other ecosystems.