Isotopic and biochemical trophic markers reveal the complexity of interactions at the base of pelagic food webs (Mediterranean sea).

To gain insight into the impact of bottom-up changes in the plankton community on planktivorous fish in the context of the decline of small pelagic fisheries in the Northwestern Mediterranean Sea, we have conducted an extensive year-long study. The investigation combined biochemical analyses (proteins, carbohydrates, and lipids) with C and N stable isotope analyses (SIA) to simultaneously study phytoplankton, zooplankton, and eight planktivorous fish species (Engraulis encrasicolus, Sardina pilchardus, Sardinella aurita, Sprattus sprattus, Cepola macrophthalma, Chromis chromis, Boops boops, and Spicara maena). This study is the first to analyze both stable isotope and biochemical compositions in coastal particulate organic matter (POM) size classes (0.7-2.7 µm, 2.7-20 µm, and 20-200 µm), zooplankton size classes (200-300 µm, 300-500 µm, 500-1000 µm, 1000-2000 µm, and >2000 µm), and taxonomic groups. We demonstrated that: (1) POM stable isotope compositions varied based on its spatial origin, the taxonomic composition of its biota, and its biochemical content; (2) δ15N values increased with zooplankton size classes and groups, indicating different trophic levels; (3) Phytoplankton exhibited a lipid-rich composition (∼55%), while zooplankton and fish muscles were protein-rich (∼61% and ∼66%, respectively). Bayesian stable isotope mixing models revealed that, on average: (1) POM from oceanic waters contributed the most to the POM in the bay (>51%), with a dominance of pico-POM (∼43%); (2) The 200-1000 µm zooplankton primarily consumed nano-POM, the 1000-2000 µm zooplankton mostly consumed micro-POM (∼64%), and the >2000 µm zooplankton also mostly consumed micro-POM; (3) Mesozooplankton (200-2000 µm) constituted the main portion (∼42%) of the diet for planktivorous fish species, while macrozooplankton organisms (>2000 µm) were the primary food resource (∼43%) for both B. boops and S. sprattus. Our study underscores the complexity of the pelagic food web and highlights the bottom-up transfer of organic matter from the smallest phytoplankton size fractions to planktivorous fish.

Spatial variations of biochemical content and stable isotope ratios of size-fractionated plankton in the Mediterranean Sea (MERITE-HIPPOCAMPE campaign).

Plankton represents the main source of carbon in marine ecosystems and is consequently an important gateway for contaminants into the marine food webs. During the MERITE- HIPPOCAMPE campaign in the Mediterranean Sea (April-May 2019), plankton was sampled from pumping and net tows at 10 stations from the French coast to the Gulf of Gabès (Tunisia) to obtain different size fractions in contrasted regions. This study combines various approaches, including biochemical analyses, analyses of stable isotope ratios (δ13C, δ15N), cytometry analyses and mixing models (MixSiar) on size-fractions of phyto- and zooplankton from 0.7 to >2000 µm. Pico- and nanoplankton represented a large energetic resource at the base of pelagic food webs. Proteins, lipids, and stable isotope ratios increased with size in zooplankton and were higher than in phytoplankton. Stable isotope ratios suggest different sources of carbon and nutrients at the base of the planktonic food webs depending on the coast and the offshore area. In addition, a link between productivity and trophic pathways was shown, with high trophic levels and low zooplankton biomass recorded in the offshore area. The results of our study highlight spatial variations of the trophic structure within the plankton size-fractions and will contribute to assess the role of the plankton as a biological pump of contaminants.

Multi-trophic markers illuminate the understanding of the functioning of a remote, low coral cover Marquesan coral reef food web.

We studied the food web structure and functioning of a coral reef ecosystem in the Marquesas Islands, French Polynesia, characterized by low coral cover, high sea surface temperature and meso- to eutrophic waters. The Marquesas constitute a relevant ecosystem to understand the functioning of low diversity reefs that are also subject to global change. A multi-tracer assessment of organic matter pathways was run to delineate ecosystem functioning, using analysis of fatty acids, bulk and compound specific stable isotope analysis and stable isotopes mixing models. Macroalgae and phytoplankton were the two major food sources fueling this food web with, however, some marked seasonal variations. Specifically, zooplankton relied on phytoplankton-derived organic matter and herbivorous fishes on macroalgae-derived organic matter to a much higher extent in summer than in winter (~ 75% vs. ~ 15%, and ~ 70 to 75% vs. ~ 5 to 15%, respectively) . Despite remarkably high δ15N values for all trophic compartments, likely due to local dynamics in the nitrogen stock, trophic levels of consumers were similar to those of other coral reef ecosystems. These findings shed light on the functioning of low coral cover systems, which are expected to expand worldwide under global change.

Sources partitioning in the diet of the shipworm Bankia carinata (J.E. Gray, 1827): An experimental study based on stable isotopes.

Adaptations that allow teredinids to maintain and thrive on wood, a nutritionally unbalanced food, make these marine bivalves remarkable. Capable of filter-feeding, shipworms house endosymbiotic bacteria synthesizing cellulolytic enzymes for digestion of wood carbohydrates and providing nitrogen to their host through nitrogen fixation. To what extent each of these nutrition modes contributes to the shipworm's metabolism remains an open question. In this experimental study, we estimated source partitioning through the determination of δ13C and δ15N values in original biological samples. For this purpose, pieces of common alder (Alnus glutinosa) were immersed at a coastal station of the north-western Mediterranean Sea. The shipworm Bankia carinata infected wood logs and stable isotope mixing models suggested it got most of the carbon and nitrogen it needs from separate sources. From 71 to 77% of the carbon was derived from the digestion of wood carbohydrates, whereas between 42 and 82% of the nitrogen originated from N2 fixation. These first semi-quantitative estimations suggest that the contribution of N2 fixers to nitrogen requirements of this shipworm species is far from incidental.

Dietary composition of black drum Pogonias cromis in a hypersaline estuary reflects water quality and prey availability.

The Baffin Bay estuary is a hypersaline system in the Gulf of Mexico that supports an important recreational and commercial fishery for black drum Pogonias cromis, a benthic predator. Seasonal measurements of water quality variables, benthic macrofauna densities and biomass, and determination of P. cromis food sources using stomach-content and stable-isotope analyses were carried out to determine how P. cromis food sources change with water quality and how this may affect P. cromis diet. Gut-content analysis indicated P. cromis selectively consumed bivalves Mulinia lateralis and Anomalocardia auberiana. Isotope compositions demonstrated that P. cromis relied on these benthic food resources produced in the Baffin Bay estuary year-round. Biomass and densities of these bivalves were influenced by changes in water quality variables, particularly salinity and dissolved oxygen. Thus, this paper demonstrates the relationship between water quality variables, benthic macrofauna, and their use as food resources by a carnivorous fish species, and emphasizes the need for integrated assessments when studying the effects of water quality on ecosystem function. Holistic approaches such as these can provide important information for management and conservation of fishery resources and can improve predictions of ecosystem response to climate variability.

Mercury in blue shark (Prionace glauca) and shortfin mako (Isurus oxyrinchus) from north-eastern Atlantic: Implication for fishery management.

Pelagic sharks (blue shark Prionace glauca and shortfin mako Isurus oxyrinchus) caught by long-line Spanish and Portuguese fleets in the NE Atlantic, were sampled at Vigo fish market (Spain) for total mercury (Hg) analysis. Hg concentration in white muscle increased with size and weight in both species, but at a higher rate in shortfin mako than in the blue shark. No difference was found with sex, year and season. Spatial variation was observed in the blue shark with higher Hg values in the North of the Azorean archipelago, but not in the shortfin mako. These high-level predators are particularly susceptible to bioaccumulate contaminants (Hg) in their tissues (muscle). However, a significant positive relationship between Hg concentration and trophic level (δ15N) of individuals was observed only in the shortfin mako. Most sharks landed were juveniles which presented Hg concentration lower than the maximum limit allowed by the European Union (1mgkg-1 wet weight) for marketing. However, concentrations above this threshold were most recorded in blue sharks larger than 250cm total length (TL) and in shortfin makos larger than 190cm TL, raising the question of the commercialization of large-sized individuals.

Ecological structure and function in a restored versus natural salt marsh.

Habitat reconstruction is commonly employed to restore degraded estuarine habitats and lost ecological functions. In this study, we use a combination of stable isotope analyses and macrofauna community analysis to compare the ecological structure and function between a recently constructed Spartina alterniflora salt marsh and a natural reference habitat over a 2-year period. The restored marsh was successful in providing habitat for economically and ecologically important macrofauna taxa; supporting similar or greater density, biomass, and species richness to the natural reference during all but one sampling period. Stable isotope analyses revealed that communities from the natural and the restored marshes relied on a similar diversity of food resources and that decapods had similar trophic levels. However, some generalist consumers (Palaemonetes spp. and Penaeus aztecus) were more 13C-enriched in the natural marsh, indicating a greater use of macrophyte derived organic matter relative to restored marsh counterparts. This difference was attributed to the higher quantities of macrophyte detritus and organic carbon in natural marsh sediments. Reduced marsh flooding frequency was associated with a reduction in macrofaunal biomass and decapod trophic levels. The restored marsh edge occurred at lower elevations than natural marsh edge, apparently due to reduced fetch and wind-wave exposure provided by the protective berm structures. The lower elevation of the restored marsh edge mitigated negative impacts in sampling periods with low tidal elevations that affected the natural marsh. The results of this study highlight the importance of considering sediment characteristics and elevation in salt marsh constructions.

Does reef structure affect oyster food resources? A stable isotope assessment.

As ecosystem engineers, oysters create and maintain structured habitat and can influence trophodynamics and benthic-pelagic coupling in the surrounding landscape. The physical reef structure and associated biotic parameters can affect the availability of food resources for oysters. Oysters and potential composite food sources - suspended particulate organic matter (SPOM) and surface sediment organic matter (SSOM) - were assessed using a dual stable isotope (δ13C, δ15N) approach at three reef types (natural, restored, and unconsolidated) seasonally for two years to determine if changes in physical and/or biotic parameters affected the relative availability and/or use of food resources by oysters. SPOM was more depleted in 13C (-24.2 ± 0.6‰, mean ± SD) than SSOM (-21.2 ± 0.8‰). SPOM composition is likely dominated by autochthonous phytoplankton production, while SSOM includes trapped phytoplankton and benthic microalgae. SSOM was used by oysters in increasing proportions relative to SPOM over time at all reef types. This temporal trend is likely due to increased oyster biomass over time, promoting enhanced microphytobenthos growth through feedback effects related to oyster biodeposits. Structural differences between reef types observed in this study had no effect on food resource availability and use by oysters, indicating strong bentho-pelagic coupling likely due to shallow depths as well as strong and consistent winds. This study provides insights for restoration of oyster reefs as it highlights that food resources used by oysters remain similar among reef types despite changes in abiotic and biotic parameters among habitats and over time.

Seasonal Variation of Harbor Seal's Diet from the Wadden Sea in Relation to Prey Availability.

The Wadden Sea has an important role for marine mammals in terms of resting, nursing and foraging. Harbor seal is the most abundant marine mammal species in this area. The use of the food resources of the Wadden Sea by seals is not clear, and previous studies showed that this species can travel kilometers away from their haul-outs to forage in the North Sea. In this study, we analyzed the stable isotopes of vibrissae from 23 dead harbor seals found on the island of Sylt to investigate their diet. The predator´s carbon and nitrogen isotope compositions were compared to the compositions of different potential prey items from the Sylt-Rømø Bight and from the North Sea in order to study seasonal pattern in the diet and in the foraging location. In parallel, seasonal variation of abundance and biomass of the potential prey items from the Sylt-Rømø Bight were studied and compare to their contribution to the seal´s diet. The results revealed a change in the seal´s diet from pelagic sources in spring to a benthic based diet in summer, and an increasing use of the North Sea resources in fall and winter in accordance with the seasonal variation of the availability of prey in the Sylt-Rømø Bight.

Carbon Stable Isotope Analysis of Methylmercury Toxin in Biological Materials by Gas Chromatography Isotope Ratio Mass Spectrometry.

A critical component of the biogeochemical cycle of mercury (Hg) is the transformation of inorganic Hg to neurotoxic monomethylmercury (CH3Hg). Humans are exposed to CH3Hg by consuming marine fish, yet the origin of CH3Hg in fish is a topic of debate. The carbon stable isotopic composition (δ(13)C) embedded in the methyl group of CH3Hg remains unexplored. This new isotopic information at the molecular level is thought to represent a new proxy to trace the carbon source at the origin of CH3Hg. Here, we present a compound-specific stable isotope analysis (CSIA) technique for the determination of the δ(13)C value of CH3Hg in biological samples by gas chromatography combustion isotope ratio mass spectrometry analysis (GC-C-IRMS). The method consists first of calibrating a CH3Hg standard solution for δ(13)C CSIA. This was achieved by comparing three independent approaches consisting of the derivatization and halogenation of the CH3Hg standard solution. The determination of δ(13)C(CH3Hg) values on natural biological samples was performed by combining a CH3Hg selective extraction, purification, and halogenation followed by GC-C-IRMS analysis. Reference δ(13)C values were established for a tuna fish certified material (ERM-CE464) originating from the Adriatic Sea (δ(13)C(CH3Hg) = -22.1 ± 1.5‰, ± 2 SD). This value is similar to the δ(13)C value of marine algal-derived particulate organic carbon (δ(13)CPOC = -21‰).

Methods of lipid-normalization for multi-tissue stable isotope analyses in tropical tuna.

RATIONALE: The bias associated with lipid contents in fish tissues is a recalcitrant topic for trophic studies using stable isotopes. Lipids are depleted in the heavy carbon isotope ((13)C) and the lipid content varies considerably among species, tissues and in both time and space. We have applied and assessed different correction methods for tropical tuna tissues. METHODS: We tested two types of normalization methods to deal with variable lipid content in liver, gonads, and white and red muscles of yellowfin, bigeye and skipjack tuna: a chemical extraction using dichloromethane and a mathematical correction based on three modeling approaches (linear, non-linear and mass balance models). We measured isotopic ratios of bulk and lipid-free tissues and assessed the predictive ability of the correction models with the lipid-free measurements. The parameters of the models were estimated from our dataset and from results from published studies on other species. RESULTS: Comparison between bulk, lipid-free and lipid-corrected isotopic ratios demonstrated that (1) chemical extraction using dichloromethane did not affect δ(15)N values; (2) the change in δ(13)C values after extraction was tissue-specific; (3) lipid-normalization models using published parameter estimates failed to predict lipid-corrected δ(13)C values; and (4) linear and non-linear models using parameters estimated for each tissue from our dataset provided accurate δ(13)C predictions for all tissues, and mass balance model for white muscle only. CONCLUSIONS: Models using published estimates for parameters from other species cannot be used. Based on a range of lipid content that do not exceed 45%, we recommend the linear model to correct the bulk δ(13)C values in the investigated tissues but the parameters have to be estimated from a proportion of the original data for which chemical extraction is required and the isotopic values of bulk and lipid-free tissues are measured.

Retrospective monitoring of mercury in fish from selected European freshwater and estuary sites.

Levels and trends of total mercury (THg) and methylmercury (MeHg) compounds in bream (Abramis brama) from different European sites were compared. Bream were collected between 2007 and 2013 in the estuaries of the rivers Scheldt (Netherlands), Rhône (France), Göta älv (Sweden), Tees (United Kingdom), and Mersey (UK), and in Lake Belau (Germany). A direct mercury analyzer was used to determine THg concentrations while MeHg was measured by gas chromatography/inductively coupled plasma-mass spectrometry applying stable isotope dilution. THg and MeHg in annual pool samples of bream ranged between 15.9 and 251 µg kg(-1) wet weight (ww) with lowest concentrations found at the reference site Lake Belau and highest in samples from the river Rhône. The EU environmental quality standard (EQS) of 20 µg kg(-1) ww was exceeded at all sites and in all years except at Lake Belau in 2012. Significantly decreasing trends over time were observed only in bream from the Rhône, while THg increased in bream from the Western Scheldt. The MeHg fractions of THg were always >80% and a significant difference between sites was detected only in one case (Rhône vs. Lake Belau).