Stable isotope ratios, carbon (δ¹³C) and nitrogen (δ¹⁵N), and fatty acids validated the trophic connection between farmed fish in a commercial nearshore fish farm and sea cucumbers in the Mediterranean Sea. This dual tracer approach evaluated organic matter transfer in integrated multi-trophic aquaculture (IMTA) and the ability of sea cucumbers to incorporate fish farm waste (fish faeces and uneaten artificial fish feed) into their tissue. Between October 2018 and September 2019, Holothuria (Roweothuria) poli Delle Chiaje, 1824, co-cultured at IMTA sites directly below one of the commercial fish cage , at 10 m and 25 m from the selected fish cage, and at two reference sites over 800 m from the fish farm. Sea cucumbers were sampled from each site in February, May and September, except at 0 m due to mass mortalities recorded here in the first month of study. Isotopic mixing models revealed that fish farm organic waste was the dominant dietary source for H. poli in IMTA at 10 m and 25 m from the cage. The contribution of marine plant-derived organic matter, Posidonia oceanica leaves and rhizomes, was least important. The isotopic signatures of sea cucumber tissues at reference sites were not explained by the sampled food resources. Importantly, fatty acid profiling revealed a high abundance of individual terrestrial plant fatty acids, such as oleic (18:1n-9), linoleic (18:2n-6) and eicosenoic (20:1n-9) acids in sea cucumber tissue at 10 m and 25 m from the fish cage, presumably linked to the terrestrial plant oil content of the fish feeds. At the reference sites, sea cucumber tissues were characterised by higher relative abundance of arachidonic acid (20:4n-6) acid, and the natural marine-based eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids. These analyses revealed important differences in the composition of H. poli between the IMTA and reference locations, driven by aquaculture-derived waste near fish cages. Moreover, this study revealed temporal variation in food availability and quality, and possible differences in the physiological responses of H. poli. Stable isotope analysis and fatty acid profiling provided complementary evidence for the important dietary preferences of H. poli and validated the potential of sea cucumbers to uptake aquaculture organic waste as part of inshore fish-sea cucumber IMTA. It reveals the important implications that an established trophic link has on the viability of using sea cucumbers for the development of IMTA and the sustainable expansion of aquaculture.

The shape of sagitta otoliths was used to compare individuals of little tunny (Euthynnus alleteratus) harvested on board commercial fishing vessels from the coastal areas along the Eastern Atlantic, including the Mediterranean Sea. Fish sampling and selection was designed to cover possible seasonal changes and tuna size. The research encompassed both morphometric and shape analyses of left sagittal otoliths extracted of 504 fish specimens. Four shape indices (Circularity, Roundness, Rectangularity, and Form-Factor) were significantly different between two groups, showing a statistical differentiation between two clear spatial units. The degree of divergence was even more pronounced along the rostrum, postrostrum, and excisura of the generated otolith outlines between these two groups. One group corresponds to the samples from the coastal areas in the Northeast Temperate Atlantic and Mediterranean Sea (NETAM Area) and a second group from the coastal areas off the Eastern Tropical Atlantic coast of Africa (ETA Area). This study is the first to use otolith shape to differentiate tunas from separate spatial units. These results could be used to re-classify previously collected samples and to correct time series of data collected.

The role of seagrasses in providing a complex habitat for marine organisms is globally documented; however, few studies have investigated the trophic incorporation of endemic Mediterranean Posidonia oceanica into marine food webs. Meadows of P. oceanica are declining due to climate change and anthropogenic pressures, emphasising the need to determine its contribution in local trophic dynamics. We investigated whether benthic marine invertebrate (BMI) and fish consumers assimilate carbon directly from P. oceanica seagrass or other sources along the seagrass meadow margins in Malta. We sampled and analysed the δ¹³C and δ¹⁵N isotope values of P. oceanica, particulate organic matter (POM), macroalgae, 14 invertebrate taxa, and 10 fishes at three locations marginal to P. oceanica seagrass meadows. Stable isotope ratios were significantly different between all taxa (F₂₆ = 17.37, R² = 0.68, p < 0.01) and locations (F₂ = 34.22, R² = 0.10, p < 0.01). The source, invertebrate, and fishes were enriched in both ¹³C and ¹⁵N at Baħar iċ-Ċagħaq relative to the other locations, L’Aħrax and Golden Bay, likely due to the increased effluent. Stable isotope mixing models were somewhat confounded as POM and macroalgae had similar δ¹³C and δ¹⁵N values at each site, hampering efforts to define the resource use of the sampled taxa. However, Posidonia oceanica made the lowest contribution for both consumer groups at all locations, consistent with the results of other Mediterranean studies, suggesting that P. oceanica does not contribute significantly to the diet of consumers at seagrass meadow margins within Maltese waters.