Increasing marine trophic web knowledge through DNA analyses of fish stomach content: a step towards an ecosystem-based approach to fisheries research.

Multispecies and ecosystem models, which are key for the implementation of ecosystem-based approaches to fisheries management, require extensive data on the trophic interactions between marine organisms, including changes over time. DNA metabarcoding, by allowing the simultaneous taxonomic identification of the community present in hundreds of samples, could be used for speeding up large-scale stomach content data collection. Yet, for DNA metabarcoding to be routinely implemented, technical challenges should be addressed, such as the potentially complicated sampling logistics, the detection of a high proportion of predator DNA, and the inability to provide reliable abundance estimations. Here, we present a DNA metabarcoding assay developed to examine the diet of five commercially important fish, which can be feasibly incorporated into routinary samplings. The method is devised to speed up the analysis process by avoiding the stomach dissection and content extraction steps, while preventing the amplification of predator DNA by using blocking primers. Tested in mock samples and in real stomach samples, the method has proven effective and shows great effectiveness discerning diet variations due to predator ecology or prey availability. Additionally, by applying our protocol to mackerel stomachs previously analyzed by visual inspection, we showcase how DNA metabarcoding could complement visually based data by detecting overlooked prey by the visual approach. We finally discuss how DNA metabarcoding-based data can contribute to trophic data collection. Our work reinforces the potential of DNA metabarcoding for the study and monitoring of fish trophic interactions and provides a basis for its incorporation into routine monitoring programs, which will be critical for the implementation of ecosystem-based approaches to fisheries management.

Trophic niche overlap between round sardinella (Sardinella aurita) and sympatric pelagic fish species in the Western Mediterranean.

The northward expansion of round sardinella (Sardinella aurita) in the Mediterranean Sea, together with declines and fluctuations in biomass and landings of European sardine (Sardina pilchardus) and anchovy (Engraulis encrasicolus) observed in recent decades, may suggest potential inter-specific competition in the pelagic domain. The coexistence of sympatric zooplanktivorous fish species might therefore be exposed in part to trophic niche overlap and competition for food. Combining visual diet characterization under the microscope with DNA metabarcoding from stomach contents of fish collected in spring results show that predation on relatively large krill is equally important for sardinella than for the other two niche overlapping species. Furthermore, an important overlap is found in their isotopic niche, especially with anchovy, using nitrogen (δ15N) and carbon (δ13C) stable isotopes in muscle tissue. In fact, the three fish species are able to feed effectively in the whole prey size spectrum available during the sampled season, from the smallest diatoms and copepods to the larger prey (i.e., decapods and euphausiids), including fish larvae. Moreover, effective predation upon other large prey like siphonophores, which is observed only when multi-proxy analyses in stomach contents are applied, might also be relevant in the diet of sardinella. The overlapping diet composition in spring, together with the effective use of food resource by sardinella, can be of special interest in potential future scenarios with warmer water temperature leading to lower zooplankton and/or higher jellyfish availability, where sardinella may take advantage over other species due to its feeding plasticity.

Ingestion of microplastics and occurrence of parasite association in Mediterranean anchovy and sardine.

We quantified the incidence of microplastics in the gut contents of the European sardine (Sardina pilchardus) and anchovy (Engraulis encrasicolus) in the Northwestern Mediterranean Sea and tested which variables influence this abundance, including the prevalence of parasites (i.e., trematoda larvae and nematodes). We detected a 58% occurrence of microplastics ingestion in sardines and a 60% in anchovies. With respect to sardines, the individuals with lower body conditions were found to have the highest microplastics ingestion probabilities, whereas in anchovies such probabilities were observed in individuals with higher gonadosomatic indices and smaller size. The areas with the highest microplastics ingestion probabilities were the Gulf of Alicante for sardines and the Gulf of Lion - Ebro Delta for anchovies. Both species showed a positive relationship between parasites and microplastics ingestion. These results highlight that both parasitism and ingestion of microplastics are concerns for the health of marine stocks and human consumers.

A trophic latitudinal gradient revealed in anchovy and sardine from the Western Mediterranean Sea using a multi-proxy approach.

This work combines state-of-the-art methods (DNA metabarcoding) with classic approaches (visual stomach content characterization and stable isotope analyses of nitrogen (δ15N) and carbon (δ13C)) to investigate the trophic ecology of anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus) at high taxonomic and spatial resolution in the Western Mediterranean Sea. Gut contents observed are in accordance with the dietary plasticity generally described for anchovy and sardine, suggesting a diet related to the opportunistic ingestion of available prey in a certain area and/or time. Genetic tools also showed modest inter-specific differences regarding ingested species. However, inter-specific and intra-specific differences in ingested prey frequencies and prey biomass reflected a latitudinal signal that could indicate a more effective predation on large prey like krill by anchovy versus sardine, as well as a generalized higher large prey ingestion by both species southwards. In fact, both species presented lower δ15N in the northernmost area. This latitudinal gradient indicates changes in the trophic ecology of anchovy and sardine that coincide with previously described better biological conditions for fish in the southern part of the study area as well as higher landings of both species in recent years.

Bioenergetics modeling of the annual consumption of zooplankton by pelagic fish feeding in the Northeast Atlantic.

The present study uses bioenergetics modeling to estimate the annual consumption of the main zooplankton groups by some of the most commercially important planktivorous fish stocks in the Northeast Atlantic, namely Norwegian spring-spawning (NSS) herring (Clupea harengus), blue whiting (Micromesistius poutassou) and NEA mackerel (Scomber scombrus). The data was obtained from scientific surveys in the main feeding area (Norwegian Sea) in the period 2005-2010. By incorporating novel information about ambient temperature, seasonal growth and changes in the diet from stomach content analyses, annual consumption of the different zooplankton groups by pelagic fish is estimated. The present study estimates higher consumption estimates than previous studies for the three species and suggests that fish might have a greater impact on the zooplankton community as foragers. This way, NEA mackerel, showing the highest daily consumption rates, and NSS herring, annually consume around 10 times their total biomass, whereas blue whiting consume about 6 times their biomass in zooplankton. The three species were estimated to consume an average of 135 million (M) tonnes of zooplankton each year, consisting of 53-85 M tonnes of copepods, 20-32 M tonnes of krill, 8-42 M tonnes of appendicularians and 0.2-1.2 M tonnes of fish, depending on the year. For NSS herring and NEA mackerel the main prey groups are calanoids and appendicularians, showing a peak in consumption during June and June-July, respectively, and suggesting high potential for inter-specific feeding competition between these species. In contrast, blue whiting maintain a low consumption rate from April to September, consuming mainly larger euphausiids. Our results suggest that the three species can coexist regardless of their high abundance, zooplankton consumption rates and overlapping diet. Accordingly, the species might have niche segregation, as they are species specific, showing annual and inter-annual variability in total consumption of the different prey species. These estimates and their inter-annual and inter-specific variation are fundamental for understanding fundamental pelagic predator-prey interactions as well as to inform advanced multispecies ecosystem models.

Feeding Ecology of Northeast Atlantic Mackerel, Norwegian Spring-Spawning Herring and Blue Whiting in the Norwegian Sea.

The Norwegian spring-spawning (NSS) herring (Clupea harengus), blue whiting (Micromesistius poutassou) and Northeast Atlantic (NEA) mackerel (Scomber scombrus) are extremely abundant pelagic planktivores that feed in the Norwegian Sea (NS) during spring and summer. This study investigated the feeding ecology and diet composition of these commercially important fish stocks on the basis of biological data, including an extensive set of stomach samples in combination with hydrographical data, zooplankton samples and acoustic abundance data from 12 stock monitoring surveys carried out in 2005-2010. Mackerel were absent during the spring, but had generally high feeding overlap with herring in the summer, with a diet mainly based on calanoid copepods, especially Calanus finmarchicus, as well as a similar diet width. Stomach fullness in herring diminished from spring to summer and feeding incidence was lower than that of mackerel in summer. However, stomach fullness did not differ between the two species, indicating that herring maintain an equally efficient pattern of feeding as mackerel in summer, but on a diet that is less dominated by copepods and is more reliant on larger prey. Blue whiting tended to have a low dietary overlap with mackerel and herring, with larger prey such as euphausiids and amphipods dominating, and stomach fullness and feeding incidence increasing with length. For all the species, feeding incidence increased with decreasing temperature, and for mackerel so did stomach fullness, indicating that feeding activity is highest in areas associated with colder water masses. Significant annual effects on diet composition and feeding-related variables suggested that the three species are able to adapt to different food and environmental conditions. These annual effects are likely to have an important impact on the predation pressure on different plankton groups and the carrying capacity of individual systems, and emphasise the importance of regular monitoring of pelagic fish diets.