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This data set is composed to assess the accumulation of persistent organic pollutants (POPs) such as polychlorinated biphenyls (PCBs) dioxin like (DL) and not dioxin like (NDL), organochlorine pesticides (POCs) and polycyclic aromatic hydrocarbons (PAHs) in Thunnus thynnus and to elucidate the suitability of this species as a bioindicator for monitoring contaminations of these compounds in the marine ecosystems of the Straits of Messina.This investigation was conducted on liver samples of 14 T. thynnus collected during April 2015. Quantitative determination of PCBs (DL and NDL), POCs and PAHs in the examined samples has been carried out by HRGC-MS/MS.Among the PCBs, high prevalence of DL was found while, generally, the values detected for PCBs–NDL were lower than the legal limits. Tuna samples analyzed for PAHs residues revealed that all the samples were contaminated with acenaphthalene, fluorene, phenanthrene and anthracene. Moreover, generally the residual levels of DDT and DDT metabolites were low.The total content of PCB–DL, in almost all the samples, showed higher concentration than the legal limit. Keywords: Fish, Thunnus thynnus, POPs, PCBs, PAHs, HRGC-MS/MS

One of the most important bottleneck in the farming of Atlantic bluefin tuna (ABT) is the growth and survival during the larval rearing phase, mainly related with the diet quality. For the last ten years the Spanish Institute of Oceanography (IEO) in Mazarrón (Murcia, SE Spain) has developed a technique for ABT larval rearing and juvenile production. Taurine and selenium are essential water soluble compounds in live preys and inert diets for ABT larvae and juveniles. To know the importance of dietary taurine and selenium on growth and survival of ABT larvae, two experiments have been carried out with different taurine (Exp1) and selenium (Selplex® ) (Exp2) doses added to rotifers enriched with ALGAMAC 3050® . Both experiments were finished at 14 days post hatching. ABT fertilized eggs were collected from captive breeders spawning spontaneously in floating cages in the area. The eggs, transported to the IEO facilities, were quantified, cleaned, selected by buoyancy and distributed randomly in 1400 L fiber cylindrical glass tanks at a density of 10 ABT eggs per liter, whereas prey density was maintained at 5 rotifers per mL. Temperature ranges were: 24.0±0.5ºC and 26.0±0.5ºC for Exp1 and Exp2, respectively. Fig 1 shows the growth results in size for both experiments at 14 dph. Survival in both cases was close to 10% with no significant differences due to the different treatments. The lowest growth in size was observed in larvae fed dose 0 in both experiments Standard length average (mm ± confidence intervals) in Exp1 and Exp2 at 14 dph. Letters indicate statistically significant differences (95%)

Notochord flexion increases the swimming capacity of fish larvae, aids in the capture of mobile prey, and coincides with the timing of when the physiological capacities of larvae begin to develop significantly, allowing an early shift to piscivory. Therefore, reaching the flexion stage as soon as possible can be considered beneficial for the growth and survival of the larvae. Individual growth differences of larvae from the same cohort are very common before reaching flexion and the potential explanation is still unknown. In this study, we examined if the nutritional status of the larvae, measured by the RNA:DNA and DNA:DW ratios, explains the differences in the development of notochord flexion in laboratory-reared Atlantic bluefin tuna (ABFT; Thunnus thynnus) and Atlantic bonito (AB; Sarda sarda) larvae. Moreover, we described the ontogeny of the different stages and condition indices. The daily average condition of ABFT estimated by the RNA:DNA and DNA:DW ratios increased with larval age and developmental stage, whereas in AB it was not. In both species, bigger larvae at a specific age had higher nutritional condition than smaller larvae regardless of developmental stage. However, in ABFT the DNA:DW ratio indicated lower condition for the biggest larvae within developmental stages F0 (pre-flexion) and F3 (post-flexion). After removing the size effect, larger individuals of ABFT within flexion stages F1 and F2 had higher condition than any other larval stages, whereas in AB an indistinct trend within stages was found. Our results confirm that in ABFT the RNA:DNA ratio is correlated with faster development while the DNA:DW ratio is negatively correlated with the time to flexion. The trends found in the DNA:DW ratio points at cell growth mechanisms being a better indicator of larval condition that the RNA:DNA ratio. Our findings suggest that under culture, ad libitum conditions, larval nutritional conditions measured by nucleic acid ratios, might not be enough to explain developmental differences in fast growing species., SI

La presente comunicación muestra las últimas técnicas desarrolladas por el equipo de cultivo de túnidos del Instituto Español de Oceanografía (IEO) para la captura, la manipulación, el transporte y la adaptación a instalaciones en tierra, de juveniles de atún rojo (Thunnus thynnus). La técnica utilizada para la captura fue usando anzuelos sin muerte. El uso de bolsas de plástico llenas de agua de mar resultó muy eficaz al eliminar prácticamente los daños por abrasión, y permitiendo medir, pesar, marcar/identificar con pit-tags, y obtener muestras para caracterización genética, de forma no traumática. The present communication shows the last techniques developed by the tunid aquaculture team belonging to Spanish Institute of Oceanography (IEO), for capturing, handling, transporting and adapting to the land based facilities of bluefin tuna juveniles (Thunnus thynnus). The technique for capturing was using barbless hooks. The use of soft plastic bags filled with sea water was very effective avoiding practically the injures due to the abrasion, allowing to obtain the size and weight, the tagging with pit-tags and the samples collection for genetic characterization, in a non traumatic way.

Elongation of very long-chain fatty acid 4 (Elovl4) proteins are involved in the biosynthesis of very long-chain (> C24) fatty acids and in many teleost fish species they are key enzymes in the pathway for the production of docosahexaenoic acid (DHA; 22:6n-3) from eicosapentaenoic acid (EPA; 20:5n-3). Therefore, Elovl4 may be particularly important in Atlantic bluefin tuna (ABT; Thunnus thynnus) characterised by having high DHA to EPA ratios. The present study cloned and characterised both the function and expression of an elovl4 cDNA from ABT. The Elovl4 had an open reading frame of 915 base pairs encoding a putative protein of 304 amino acids. Alignment and phylogenetic analyses indicated that the Elovl4 isoform identified in the present study was an Elovl4b. Functional characterisation demonstrated that the Elovl4b enzyme had elongase activity towards all the polyunsaturated fatty acid (PUFA) substrates assayed. The ABT Elovl4b contributed to DHA biosynthesis by elongation of EPA and DPA to 24:5n-3, the latter being desaturated to 24:6n-3 by the action of fads2 (Δ6 desaturase). Additionally, the ABT Elovl4b has a role in the biosynthesis of very long-chain PUFA up to C34, compounds of key structural roles in neural tissues such as eye and brain, which had high levels of elovl4b transcripts. Surprisingly, while the relative expression of fads2, required for the production of DHA from EPA, was increased in liver of ABT fed a diet with reduced levels of EPA and DHA, expression of elovl4b was reduced. Results indicated that ABT has enzymes necessary for endogenous production of DHA from EPA and demonstrate that Elovl4b can effectively compensate for absence of Elovl2., SI

The present study aims to solve the problem related to the difficulty in measuring the bluefin tuna (Thunnus thynnus L.) larval length when the larvae curl for several reasons (freezing, fixing, handling or in notochord flexion phase). To do that, the relationship between the horizontal eye diameter and the notochord length has been studied in 1143 bluefin tuna larvae from the eye pigmentation up to the full notochord flexion (between 2 and 15 days post hatching) reared in the facilities of the Marine Aquaculture Plant in Mazarrón (Murcia, SE Spain) belonging to the Spanish Institute of Oceanography (IEO). Our results shown that the horizontal eye diameter (ED) could be used to estimate the notochord length (NL) of bluefin tuna larvae up to complete notochord flexion (around 15 days old), using the equation: NL= 1.81953 + 6.5031 ED (r2 = 89.79%, p, El presente estudio intenta resolver la problemática asociada a la dificultad de medir la longitud de larvas de atún rojo (Thunnus thynnus L.) cuando éstas se curvan debido a varios factores (al congelarlas, fijarlas, debido a la manipulación o en la fase de flexión de la notocorda). Para ello, se ha estudiado la relación existente entre el diámetro ocular (que usualmente no se ve afectada por los citados factores) y la longitud estándar en 1143 larvas de atún rojo desde la pigmentación del ojo hasta la completa flexión de la notocorda (entre dos y quince días después de la eclosión) cultivadas en las instalaciones de la Planta de Cultivos Marinos de Mazarrón (Murcia), del Instituto Español de Oceanografía (IEO). Nuestros resultados muestran que la medida horizontal del diámetro ocular (LO) puede ser utilizada para la estimación de la longitud de la notocorda (LN) de larvas de atún rojo que han completado la flexión de la notocorda (alrededor de 15 días de vida) usando la ecuación LN= 1,81953 + 6,5031 LO (r2 = 89,79%, p

En esta comunicación se presentan los resultados de crecimiento de alrededor de 80 juveniles de atún rojo criados entre el 15 de febrero de 2018 y el 22 de enero de 2019 (300 días) en un tanque de 22 m de diámetro y 10 m de profundidad de la Instalación del IEO para el Control de la Reproducción del Atún rojo (ICRA). Los datos de crecimiento en talla y en peso se obtuvieron de forma no invasiva mediante un sistema de estéreo-video cámaras. El peso medio inicial fue de 2,90 kg (CV=14,2%) y el final 14,1 kg (CV=23%) con una tasa específica diaria de crecimiento (SGR%) de 0,73. El índice de conversión FIFO estuvo en torno a 14 con una tasa de conversión de alrededor de 4 (considerando 70% de agua en la carnada). The growth of around 80 bluefin tuna juveniles, reared between 15th February 2018 and 22nd January 2019 (300 days) in a 22m diameter and 10m depth tank held in the IEO bluefin tuna reproduction facility (ICRA) are shown in the present study. The growth data in size and weight have been obtained in a non invasive way by means of a stereo-video camera system. The initial and final mean weight were 2.90 kg (VC=14.2%) and 14.1 kg (VC=23%) with a specific daily growth rate of 0.73, a FIFO index about 14 and a conversion rate close to 4 (considering 70% water in the bait)

Among the many fish species commercially exploited since prehistoric times, Atlantic bluefin tuna (Thunnus thynnus) is one of the most economically significant, having left an indelible imprint on several civilizations including the Phoenicians, Greeks, and Romans. Here, we describe our efforts to identify tuna specimens among the remains of 345 fish vertebrae and bones in several large collections from the Atlantic Ocean, Mediterranean Sea, and Black Sea, dating from the Late Iron Age (2nd century BCE) to the early 20th century (1911-1927). Unfortunately, ancient fish specimens are often mislabelled, which can cause a great deal of confusion among zoologists. Protocols were developed and optimized to overcome the unique challenges related to the compromised integrity of genetic material preserved in ancient bones. Three DNA isolation protocols were compared to maximize yields, and as reported for other faunal remains, a silica spin column-based method was proven most effective. Endogenous DNA was successfully extracted from the majority of bones and amplified using polymerase chain reactions (PCRs) and an assortment of four primer pairs targeting nuclear (internal transcribed spacer) and mitochondrial sequences (cytochrome oxidase subunit 1 and control region). Protocols targeting mitochondrial markers were more successful than those focused on nuclear targets. Due to the restricted length of the extracted DNA molecules, character-based keys containing diagnostic nucleotide substitutions were defined and used to identify 231 samples to genera, of which 171 were identified to species level. The success rate of assignment of specimens to species level varied between location and collection, reflecting variation in DNA preservation between different sites and environments. The methods detailed herein can be used to identify other ancient fish specimens and provide information about historical human diets, trade, species distribution, and biodiversity. The same tools can be applied to the analysis of processed food items with highly damaged DNA.

The spawning grounds of the Atlantic Thunnus thynnus are traditionally considered to be the Gulf of Mexico (GOM) and the Mediterranean Sea (MED). However, for the western Atlantic, unequivocal evidence of T. thynnus spawning outside the GOM has been presented. In this study, we show evidence for T. thynnus reproduction outside the MED in the eastern Atlantic. Using opportunistic ichthyoplankton samplings, 9 T. thynnus larvae were collected during July 2010 and 2015, in the Southern Bay of Biscay (SBB). The microscopic identification of Atlantic bluefin larvae, based on morphologic and meristic characters and pigmentation patterns was confirmed by genetic analysis. As for Sarda sarda, the spawning grounds for this species in the eastern Atlantic are located in the neritic regions of the tropical and subtropical Atlantic and of the MED. However, 6 larvae of this species were collected in the SBB since 2001. Therefore, the SBB should be evaluated as a spawning ground for T. thynnus, since its larvae appeared in 33% of the stations sampled during July 2010 in the slope-oceanic region, where this species reproduces.

dentification of the first annual increments in otoliths of Atlantic bluefin tuna juveniles is difficult due to the frequent appearance of numerous sub-annual bands which can be easily misinterpreted. Instead, the pattern of deposition of the annuli on the first dorsal fin rays (spines) is very distinct during the first years. Furthermore, the bias vector for correcting otoliths age, and developed from paired otolith-spine samples, allowed a better tracking of cohorts in the 2017 assessment. With this background, 262 paired structures with identical age estimates were used to revise reading criteria for annual increments of juvenile bluefin otoliths and to create a template. A table with first annuli measurements was built to serve as a reference and help identifying the first otolith annual bands. For this, it was necessary to establish the location of origin of the measurements (anchor point) and define them. In addition, otoliths from this set of paired structures can be used as a collection that will serve to test the new reading criteria and template.

[ES] Se obtuvieron valores de potencia del blanco o Target Strength (TS) de ecoregistros de ecosondas de 200 kHz en un tanque de crecimiento de doradas (Sparus aurata) y ecoregistros de ecosondas de 200 kHz y 333 kHz en tanques de crecimiento de atunes (Thunnus thynnus), con el fin de relacionar los valores de TS obtenidos con el desarrollo larvario. Para esto se realizaban dos experimentos en tiempos distintos, en un tanque de 1.6m de alto y 2m de diámetro, se llevaron a cabo medidas diarias de longitud total (mm) y diámetro (mm) de la vejiga natatoria si estaba presente. Las medidas fueron realizadas en las instalaciones del Centro de Cultivo de Mazarrón, en el tanque G4, con un transductor situado en el fondo del tanque, además se realizaron dos cierres en el flujo de aire uno nocturno y otro diurno, con el fin de obtener valores de TS control sin el efecto de las burbujas de aire. Los análisis de los valores de TS evidenciaron dos grupos a lo largo del tiempo, correspondiente a las larvas con y sin la vejiga natatoria desarrollada. Los resultados de la distribución del TS muestran una relación directa con la longitud y con el diámetro de la vejiga natatoria de las larvas., [EN] Target Strength (TS) values of 200 kHz echo sounder ecoregisters were obtained in a gilthead growth tank (Sparus aurata) and 200 kHz and 333 kHz echo sounder ecoregisters in tuna growth tanks (Thunnus thynnus), with the in order to relate the values of TS obtained with larval development. For this, two different experiments were performed at different times, in a 1.6m high tank and 2m in diameter, daily measurements of total length (mm) and diameter (mm) of the swim bladder were performed if present. The measurements were made at the facilities of the Mazarrón Cultivation Center, in the G4 tank, with the transducer located at the bottom of the tank, two closures were made in the air flow one night and one day, in order to obtain values of TS control without the effect of air bubbles. When analyzing the values of TS, two groups are observed over time corresponding to the larvae with and without the developed swim bladder. The results of the distribution of the TS show a direct relationship with the length and with the diameter of the swim bladder of the larvae.

Otolith reading bias for juvenile Atlantic bluefin tuna was detected at its 2017 assessment. To try to reduce this bias, the current standardized reading protocol was revised. The new protocol uses age estimates from the first dorsal fin ray (spine) to identify the growth increments in the otoliths removed from the same young specimen. An exchange involving 14 experienced otolith readers was conducted to verify if the new reading procedure minimized the difference between otolith and spine readings. The results showed that there is a good agreement in the first five years, but from age 6, otoliths ages tended to be higher than spine age. Some readers had difficulty following the new protocol despite being experts, which indicates that it is necessary to improve it. The use of annual band measurements has shown to be a good tool for the control of the quality of age estimates. The results indicate that we are progressing in the recognition of the deposition pattern of the first annuli. It is recommended to carry out an ageing workshop to achieve the tasks identified as necessary.

Taurine appears to be a crucial nutrient for teleosts, especially top predator species such as Atlantic bluefin tuna (Thunnus thynnus, L.; ABT). While dietary taurine supplementation has been highly recommended, there is a lack of studies on taurine assimilation and biosynthesis for this iconic species. The present study aims to provide insight into the molecular mechanisms involved in taurine biosynthesis and transport in ABT by studying tissue distribution and ontogenetic development of expression of cysteine dioxygenase (cdo), cysteine sulfinic acid decarboxylase (csad), 2-aminoethanethiol dioxygenase (ado) and taurine transporter (tauT) in response to graded levels of dietary taurine supplementation. The full open reading frame (ORF) for cdo and partial sequences for csad, ado and tauT were obtained, with the translated polypeptides being 202, 176, 166 and 324 amino acids, respectively. All three showed characteristics such as cupin motifs in Cdo and predicted N-glycosylation sites in Taut that are common to these genes in other species. Phylogenetic analysis showed that the ABT sequences clustered with sequences of other teleosts, and separately from mammals and molluscs. Tissue distribution varied, with adipose tissue, kidney, white muscle and testis/brain showing highest expression of cdo, csad, ado and tauT, respectively. Whole larvae expression of csad peaked at 15 dah, whereas the other genes generally increased throughout development to show highest expression at 25 dah. The nutritional trial was carried out by feeding ABT larvae from mouth opening to 14 days after hatching (dah) with rotifers (Brachionus rotundiformis) enriched with 4 different levels of taurine: 0.0 (tau0), 0.5 (tau0.5), 1.0 (tau1), and 2.0 g taurine per 106 rotifers (tau2). Rotifers effectively accumulated taurine with ABT larvae fed on treatment tau2 attaining the highest concentration of taurine. However, ABT larvae fed tau1 displayed higher growth and survival, and flexion index at 14 dah, than larvae fed the other taurine levels. Larvae fed tau1 also showed generally higher expression of tauT and cdo and digestive and antioxidant enzyme genes. While this study showed that larval ABT express taurine metabolism genes, suggesting possible synthesis that could contribute to the taurine pool in the fish, larval performance was enhanced by a level of dietary taurine (3.7 mg taurine g−1 rotifer) supplied by enrichment of rotifers at 1 g taurine per 106 rotifers., SI

Mass mortality is still one of the main constraints in larval rearing of Atlantic bluefin tuna (Thunnus thynnus L.; ABT). Early data related to the feeding sequence of ABT larvae suggested that mortality observed during the first stages of life could be partly due to nutritional deficiencies. Previous studies demonstrated that copepods ap- peared to be a superior live prey compared to rotifers during the first 2 weeks of life. Our overarching aim was to evaluate different feeding strategies during first feeding of ABT larvae from a performance, compositional and molecular perspective. In order to do so, two groups of ABT larvae were fed with either copepod (Acartia tonsa; C) nauplii or rotifers (Brachionus rotundiformis; R) enriched with Algamac 3050® from mouth opening to 13 days after hatching (dah). After this, the group C-larvae was fed either Artemia enriched with Algamac 3050® (CA), Acartia nauplii and copepodites (CC) or sea bream (Sparus aurata) yolk-sac larvae (CY), while the R group were fed on Artemia enriched with Algamac 3050® (RA) up to 18 dah. At 13 dah, larvae fed copepods (C) had grown better than those fed enriched rotifers (R) although there were no significant differences in survival. ABT larvae fed R accumulated highest eicosapentaenoate (EPA) but lowest docosahexaenoate (DHA) and total n-3 long- chain polyunsaturated fatty acids (LC-PUFA) than C-fed larvae, reflecting the dietary contents. There was no activation in the expression of the enzymes involved in EPA and DHA biosynthesis. However, the different live prey showed regulation of transcription factor, digestive enzyme, lipid metabolism and oxidative stress genes. At 18 dah, larvae fed CY and CA treatments were largest in size, with larvae fed RA displaying the lowest growth, with no significant differences in survival among the dietary treatments. The highest DHA contents were found in ABT larvae fed CC and CY, whereas the lowest contents were found in RA-fed larvae. Indeed, larvae fed RA showed the highest level of the intermediate product n-3 docosapentaenoate, which could reflect increased activity of the biosynthetic pathway although this was not supported by gene expression data., SI

This article shows the most relevant results obtained between August 2017 and October 2018 with two batches of 0+ tunas: cultured tunas (CT) born in the IEO facilities in June 2017 and wild tunas (WT) captured in Mazarron Bay in September 2017. Fertilized ABFT eggs were collected from cages owned by PisciAlba and obtained larvae were cultured in IEO facilities. At the end of July 2017, 500 fingerlings (CT, 2 g average weight) were move to ICRA facilities on 29th July and placed in a 1000 m3 tank. At the end of September 2017 mean weight were 350 gr. In that moment, a total of 68 0+ ABFT fingerlings (WT, 414 g mean weight), were captured from the Mazarron Bay and placed in a 3500 m3 tank in ICRA. From this moment temperature ranged between 18 and 27ºC, and feeding consisted in bait, mainly Sardinella aurita and Scomber scombrus, but also Scomber japonicus, Engraulis encrasicolus and Clupea harengus. During November 2017, mortality increased in CT without any symptoms of infectious disease. 80 healthier CT were then moved to a greater tank (2500 m3 ) and in March 2018, when completely recovered, they were placed together with WT and both batches were cultured in the same tank up to now. During the 13 months period mortality observed in CT was high (close to 85%) while mortality in WT was lower than 15%. Growth was also slightly faster in WT. Feeding intake ranged during the whole period between 3 and 26%, and feeding conversion rate estimated was close to 12. At the end of October 2018 mean weight of population was 11 kg.

[EN] In this work, acoustic and computer vision techniques are combined to develop an automatic procedure for biomass estimation of tuna during transfers. A side scan sonar working at 200¿kHz and a stereo camera, positioned facing towards the surface to record the ventral aspect of fish, are set as acquisition equipment. Moreover, a floating structure has been devised to place the sensors between cages in transfers, creating a transfer canal that allows data acquisition while fish swim from donor to receiving cage. Biomass assessment is computed by counting transferred tuna and sizing a representative sample of the stock. The number of transferred tuna is automatically deduced from acoustic echograms by means of image processing techniques, whereas tuna size is computed from the stereo videos using our automatic computer vision procedure based on a deformable model of the fish ventral silhouette. The results show that the system achieves automatic tuna counting with error below 10%, achieving around 1% error in the best configuration, and automatic tuna sizing of more than 20% of the stock, with highly accurate Snout Fork Length estimation when compared to true data from harvests. These results fulfil the requirements imposed by International Commission for the Conservation of Atlantic Tunas for compliant transfer operations., This work was supported by funding from the BIACOP project, ES13/41, from the EU Fisheries Control Programme through the Secretaría General de Pesca, Ministerio de Agricultura, Alimentación y Medio Ambiente (MAGRAMA), and by ACUSTUNA project ref. CTM2015-70446-R (MINECO/ERDF, EU). The BIACOP project has been possible thanks to the collaboration of the Spanish company Grup Balfegó S.L. that also supplied boats and divers to acquire underwater video in the Mediterranean Sea. Special personal acknowledgements to Manuel Guijarro and Antonio López, and to David Baizán from IMA – Acuicultura Integrada Multitrófica S.L.U., for their assistance with the design and construction of the floating structures.

Fish processing facilities generate and discharge substantial amounts of waste that contains considerable amount of lipid rich with long-chain omega-3 polyunsaturated (PUFA) fatty acids. Thus these by-products (heads, gut content, liver) could be converted into value-added products such as fish oil that has many applications as animal feed, cosmetics, and human nutrition. The potential utilization of fish by-products from different species (farmed bluefin tuna, sardine and farmed seabass and seabream) and different origin (wild and farmed) for oil production was investigated. The yield, physicochemical quality, fatty acid profile and nutritive value of the oils were determined and compared to the control oil. Among oils from different by-products, the yield ranged from 4.13 to 33.38 %, being the highest in oil from tuna liver. In terms of oxidative stability, tuna and farmed fish by-product oils had the lowest free fatty acid, peroxide, TOTOX and anisidine value. The crude oil stability at 80 and 100°C was related to the α-tocopherol content and naturally present α-tocopherol in fish oils (obtained through fish diet) had better oxidative protection than when it was added to the oil post-extraction. The predominant fatty acids in investigated oils were palmitic (C16:0 ; 12.16-22.14%), oleic (C18:1n-9c ; 8.48-18.37%), palmitoleic (C16:1 ; 3.55-8.81), eicosapentaenoic (EPA ; C20:5n-3 ; 3.33-14.20%) and docosahexaenoic acid (DHA ; C22:6 ; 8.26-21.29%). The n-3/n-6 ratio of the oils showed value ranged from 0.66 in farmed fish by-product oil to 11.04 in sardine oil. Fish by-products may be valuable source of quality fish oil and represent a good strategy to add economic value to the wastes since this latter is a problem related to food security, the environment and also it directly impact the profitability of the fish industries.

The development of formulated diets and feeds is essential to increase production of farmed tuna species. There is limited knowledge of this topic, mainly on Pacific Bluefin tuna (Thunnus orientalis) in Japan, whereas no major attempts have been made with Atlantic Bluefin tuna (Thunnus thynnus; ABT). In the present study, two trials were performed using inert formulated diets as on-growing feeds for weaned ABT juvenile in order to establish adequate dietary levels of both lipid and omega-3 long-chain polyunsaturated fatty acids (LC-PUFA). In a first trial, ABT (initial weight = 2.9±0.9g) were fed for 10 days with either a commercial (Magokoro®, MGK) or two experimental feeds with two different lipid levels (15 or 20%) using krill oil (KO) as the single lipid source in order to estimate the suitable lipid content. Fish fed MGK displayed the highest growth, followed by 15KO, with no differences in fish survival. Thus, a lipid content of 15% was considered better than 20% for ABT juveniles. In the second trial, fish (initial weight = 3.3 ± 0.6g) were fed either MGK, 15KO or a feed containing 15% lipid with a combination (1:1, v/v) KO and rapeseed oil (RO) (15KORO). Fish fed 15KO and 15KORO showed the highest growth in terms of weight and fork length (including weight gain and SGR). Increasing dietary lipid level or adding RO to the feeds did not increase liver lipid content. The liver fatty acid profile largely reflected dietary intake confirming very limited LC-PUFA biosynthetic activity for this teleost species. In this respect, liver of fish fed 15KO and 20KO displayed the highest contents of docosahexaenoic acid (DHA). The hepatic expression of genes of lipid and fatty acid metabolism, transcription factors, and antioxidant enzymes was investigated with many of the genes showing regulation by both dietary lipid and LC-PUFA contents. The present study showed promising results that suggested ABT juveniles can be on grown on inert dry feeds that supported good fish growth and the accumulation of the health-promoting fatty acid DHA. Further studies are required in order to fully elucidate lipid and fatty acid requirements of this iconic species regarding dietary sources and production costs.

There are still many issues that require to be solved in larval rearing of Atlantic bluefin tuna (Thunnus thynnus; ABT) to prevent “mass-mortality” during this developmental stage. Initial data related to the feeding sequence of ABT larvae suggested that mortality observed during the first stages of life could be due partly to nutritional deficiencies. Previous studies demonstrated that copepods appeared to be a superior live prey compared to rotifers during the first two weeks of life. Our overarching aim was to evaluate different feeding strategies during first feeding of ABT larvae from a performance, compositional and molecular perspective. In order to do so, two groups of ABT larvae were fed with either copepod (Acartia tonsa; C) nauplii or rotifers (Brachionus rotundiformis; R) enriched with Algamac 3050® from mouth opening to 13 days after hatching (dah). After this, the group C-larvae was fed either Artemia enriched with Algamac 3050® (CA), Acartia nauplii and copepodites (CC) or sea bream (Sparus aurata) yolk-sac larvae (CY), while the R group passed on to being fed on Artermia enriched with Algamac 3050® (RA) up to 18 dah. After 13 dah, larvae fed C grew more than those fed R although there were no differences in survival. ABT larvae fed R accumulated highest eicosapentaenoate (EPA) but lowest docosahexaenoate (DHA) and total n-3 long-chain polyunsaturated fatty acids (LC-PUFA) than C-fed larvae, reflecting dietary contents. Indeed, there was no activation in the expression of the enzymes involved in LC-PUFA biosynthesis. However, the different live prey elicited regulation of transcription factor, digestive enzyme, lipid metabolism and oxidative stress genes. At 18 dah larvae fed CY and CA were the largest size with larvae fed RA displaying the lowest growth with no differences in survival among the dietary treatments. The highest DHA contents were found in ABT larvae fed CC and CY, whereas the lowest contents were found in RA-fed larvae. Indeed, RA-fed larvae showed the highest level of the intermediate product n-3 docosapentaenoate, which could be reflecting up-regulation in the biosynthetic pathway although this was not supported by gene expression data.

Este estágio foi realizado no Grupo AquaBioTech (ABT Group) em Malta para completar o Mestrado em Aquacultura da Escola de Turismo e Tecnologias do Mar, (ESTM) do Instituto Politécnico de Leiria (IPL). O Grupo AquaBioTech, é uma empresa que está envolvida em estudos relacionados com biotecnologia marinha desenvolvidos no setor de aquacultura e pescas focando-se na sustentabilidade ambiental. Esta empresa opera de uma forma sustentável utilizando nos seus estudos Aquacultura em Sistema de Recirculação (RAS), fazendo também colaborações com várias empresas e instituições, apoiando o desenvolvimento de importantes projetos internacionais. O estágio teve uma duração de seis meses e foi dividido em duas vertentes diferentes: (i) a primeira ocorreu nas instalações da empresa, onde procedimentos básicos e avançados de aquacultura foram executados; (ii) participação no Projecto Atum em parceria com o The International Commission for the Conservation of Atlantic Tunas (ICCAT). A participação neste projecto, inicialmente a bordo de navios-fábrica e posteriormente no laboratório, possibilitou o estudo "Análise das relações de biométricas e dos otólitos asteriscus e lapillus em atum rabilho do Atlântico, Thunnus thynnus". Este estágio permitiu aprofundar os conhecimentos teóricos adquiridos durante o percurso académico e desenvolver capacidades pessoais e profissionais. O estudo "Análise das relações de biométricase dos otólitos asteriscus e lapillus em atum rabilho do atlântico, Thunnus thynnus", demonstrou que (i) é necessária uma atualização das equações biométricas utilizadas pela Standing Committee on Research and Statistics (SCRS), (ii) a diferenças de tamanhos e pesos entre machos e fêmeas é significativa, pode resultar da alimentação e/ou do seu processamento e (iii) a identificação do sexo de T. thynnus não é possivel ser verificada através da análise dos otólitos.

[EN] This study presents various models based on formulae relating weight and dimensions (length, height and width) of Bluefin tuna, Thunnus thynnus (L.), fattened in captivity. The main aim of establishing these expressions is to design tools for indirectly predicting the weight of a Bluefin tuna from measurements of one or more dimensions obtained using non-invasive methods such as stereoscopic cameras. Measurements of maximum length, height and width following slaughter were taken of fish fattened in captivity (n = 2078). Different relationships drawn from the dimensions of the tuna against their weight are fitted with part of the data collection and later checked against a reserved sample set. The resulting formulae are compared with the formulae most commonly used in the case of wild tuna. The results of this study confirm that, for tuna fattened in cages, the availability of more than one dimension to estimate weight improves the predictive power of the model and reduces error in the estimate., This work was supported by funding from ACUSTUNA project ref. CTM2015-70446-R (MINECO/ERDF, EU). The authors are grateful for the support provided by Grup Balfegó.

Atlantic bluefin tuna (Thunnus thynnus) is a high- value product and Croatia is one of the primary Mediterranean producers. The study aimed to examine the microbial community naturally associated with adult Atlantic bluefin tuna in the Adriatic, as a continuation of our earlier work. Microbiological analysis was conducted at commercial tuna farm in the Middle Adriatic. Samples of the swabs from the gills and skin were diluted in sterile PBS. Dilutions of swab samples were then plated onto non-selective TSA supplemented with 1% NaCl, and plates were incubated at 22 °C. To determine Vibrio count, samples were inoculated onto selective TCBS Agar and incubated at 35 °C. The isolates were characterised phenotypically using the API kits (bioMerieux, France): API 20 NE, API 20 E, API Staph, API Coryne and API 50 CHL. API results were interpreted with the aid of the APIWEB platform. The bacterial community associated with tuna in our study comprised at least 6 genera, most of which were Gram-negative. The dominant bacteria were Pasteurella and Moraxella, whereas Staphylococcus was the dominant Gram-positive genera. The proportion of isolates that was Gram- positive in our study was significantly higher on the skin (49.0%) than on the gills (6.6%). We characterised bacteria that are naturally associated with healthy farmed Atlantic bluefin tuna. These results will provide a valuable baseline reference for future studies of how farming conditions affect the health of farmed tuna, risk of food spoilage and the safety of raw and processed tuna as food for humans.

Life-history traits such as spawning migrations and timing of reproduction are adaptations to specific environmental constraints and seasonal cycles in many organisms’ annual routines. In this study we analyse how offspring fitness constrains spawning phenology in a large migratory apex predator, the Atlantic bluefin tuna. The reproductive schedule of Atlantic bluefin tuna varies between spawning sites, suggesting plasticity to local environ- mental conditions. Generally, temperature is considered to be the main constraint on tuna spawning phenology. We combine evidence from long- term field data, temperature-controlled rearing experiments on eggs and larvae, and a model of egg fitness, and show that Atlantic bluefin tuna do not spawn to optimize egg and larval temperature exposure. The timing of spawning leads to temperature exposure considerably lower than optimal at all spawning grounds across the Atlantic Ocean. The early spawning is constrained by thermal inhibition of egg hatching and larval growth rates, but some other factors must prevent later spawning. Matching offspring with ocean productivity and the prey peak might be an important driver for bluefin tuna spawning phenology. This finding is important for predictions of reproductive timing in future climate warming scenarios for bluefin tuna., SI

It has been demonstrated at least in the Murcia area, that the captive ABFT (Thunnus thynnus) for fattening activities reproduce actively in the farming cages in the natural spawning season (early June – middle July). Tens of millions of fertilized eggs coming from these cages had been collected in the last years and cultured in the facilities of the Spanish Institute of Oceanography (IEO) in Mazarrón (Murcia, SE Spain) and grown up to juveniles, demonstrating as well their viability. Taking into account that an ABFT female could spawn roughly hundred thousands of eggs per kg during all the spawning season and thousands of tones of ABFT adults have been farmed in the Murcia coast during the last years, the total fertilized eggs could reach hundreds of billions every year. Obviously the conditions in the Murcia coast are different of the natural spawning areas not only regarding the feeding availability but also the massive presence of many egg and larvae predators. It would be recommendable to carry out prospecting surveys in the aim to shed some light on this possible effect of farmed ABFT on the recruitment at least in Murcia.

In this manuscript, we test how an understanding of geographical variation in larval fitness in relation to temperature and habitat use could be a useful method to improve our understanding of recruitment and develop better indices of annual recruitment. On the basis of the assumption that growth and survival of tuna larvae are influenced by temperature, we have developed a potential larval survival index for Atlantic bluefin tuna (Thunnus thynnus) by combining empirical data from egg and larval rearing experiments with temperature data from hydrodynamic models. The experiments were designed to test the full range of temperature variability that bluefin larvae would experience in the field and provide a mechanistic understanding of the processes driving egg and larval survival. We then developed a biological model using the temperature-related growth expressions and a size-dependent survival function for the larvae. The biological model was applied to a time-series of spatially explicit temperature data for the western Mediterranean from the Strait of Gibraltar to 6 E, which includes the major recognized bluefin tuna eastern stock spawning area, the Balearic Sea. Our results show that areas with high probabilities of larval survival coincide with those that would be considered as optimal based on other data sources (ichthyoplankton surveys, spawning female locations from commercial fisheries data, and adult tracking data). However, evidence of spawning has been found in areas with suboptimal thermal habitats, as predicted by the model, which we discuss regarding sampling effort and salinity fronts. There was a good match between the survival index and recruitment indices from standardized CPUE fisheries data. These results have implications for our understanding of the recruitment process of the eastern stock of Atlantic bluefin tuna, since they suggest that the combined effects of temporal and spatial variability of the environment drive recruitment success, which has important implications for the management of the species., SI

The development of formulated diets and feeds is essential to increase production of farmed tuna species. There is limited knowledge of this topic, mainly on Pacific Bluefin tuna (Thunnus orientalis) in Japan, whereas no major attempts have been made with Atlantic Bluefin tuna (Thunnus thynnus; ABT). In the present study, two trials were performed using inert formulated diets as on-growing feeds for weaned ABT juvenile in order to establish adequate dietary levels of both lipid and omega-3 long-chain polyunsaturated fatty acids (LC-PUFA). In a first trial, ABT (initial weight = 2.9±0.9g) were fed for 10 days with either a commercial (Magokoro®, MGK) or two experimental feeds with two different lipid levels (15 or 20%) using krill oil (KO) as the single lipid source in order to estimate the suitable lipid content. Fish fed MGK displayed the highest growth, followed by 15KO, with no differences in fish survival. Thus, a lipid content of 15% was considered better than 20% for ABT juveniles. In the second trial, fish (initial weight = 3.3 ± 0.6g) were fed either MGK, 15KO or a feed containing 15% lipid with a combination (1:1, v/v) KO and rapeseed oil (RO) (15KORO). Fish fed 15KO and 15KORO showed the highest growth in terms of weight and fork length (including weight gain and SGR). Increasing dietary lipid level or adding RO to the feeds did not increase liver lipid content. The liver fatty acid profile largely reflected dietary intake confirming very limited LC-PUFA biosynthetic activity for this teleost species. In this respect, liver of fish fed 15KO and 20KO displayed the highest contents of docosahexaenoic acid (DHA). The hepatic expression of genes of lipid and fatty acid metabolism, transcription factors, and antioxidant enzymes was investigated with many of the genes showing regulation by both dietary lipid and LC-PUFA contents. The present study showed promising results that suggested ABT juveniles can be on grown on inert dry feeds that supported good fish growth and the accumulation of the health-promoting fatty acid DHA. Further studies are required in order to fully elucidate lipid and fatty acid requirements of this iconic species regarding dietary sources and production costs., SI

Being bigger is not always the best option to survive and mortality rates do not always decline with increasing larval size. Smaller size at a given age could under certain condition and at certain stages of development confer a survival advantage on individual members of a larval cohort. References

The Atlantic Bluefin tuna is an emblematic species which is feeding the Mediterranean human populations for centuries. In the last two decades its wild stocks have been severely overfished having been established important capture limits with the consequent reduction of the production. For satisfying the high market, it is essential the increase the bluefin tuna production coming from the self-sustained aquaculture. The Spanish Institute of Oceanography (IEO) has recently built a land-based large scale facility (ICRA) for the control of the reproduction of this species, capable of hosting big size breeders. This facility is near the already existing IEO Aquaculture facility in Mazarrón, (Murcia, SE Spain), devoted to the research on Atlantic Bluefin tuna larval rearing and juveniles production. Both facilities are a model of how research centres of excellence can enhance cooperation to generate new knowledge on performance that can be transferred directly to operators and provide the technical basis needed to take the sector forward. Co-funded by European Regional Development Funds (FEDER) and Spanish Institute of Oceanography (IEO)

Hasta el momento actual, el cultivo integral de atún rojo se inicia con la recogida de puestas de huevos embrionados procedentes de reproductores ubicados en jaulas flotantes en el mar. Uno de los graves problemas que plantea esta técnica es que los huevos vienen acompañados de huevos de otras especies que también se están reproduciendo en el área. Algunas de estas especies tienen fases larvarias piscívoras y alcanzan dicha fase antes que el propio atún, y por tanto depredan sobre él. La presencia de algunas de estas especies en los cultivos larvarios de atún rojo supone una mortalidad por depredación que reduce en gran medida la producción de juveniles de esta especie. En este trabajo se ha testado la posibilidad de eliminar estos huevos separándolos por flotabilidad ya que en la mayoría de los casos es menor que la de los huevos de atún rojo, ya que estos tienen una flotabilidad especialmente elevada. Justificación, Until present time, Atlantic bluefin tuna full life cycle aquaculture in Europe has started with the collection of fertilized eggs from breeders located in floating cages in the sea. One of the most relevant issues of that technique is the presence of eggs of not desired fish species with concomitant reproduction season mixed with tuna, which due to their fast development can be able to predate bluefin tuna larvae at their earlier stages. This fact seriously threatens the survival of bluefin tuna larvae and juveniles production. In the present study, the possibility of doing away with these undesirable eggs by using a differential buoyancy technique has been tested, which in most cases it has been proved to be inferior to the buoyancy of bluefin tuna eggs.

El conocimiento de la dieta y los hábitos alimentarios del atún rojo (Thunnus thynnus, L. 1758, ABFT) es un aspecto básico en la comprensión de su biología y mejora nuestra percepción e interpretación de su estructura y dinámica poblacional. Esta información es útil para lograr una adecuada gestión y explotación sostenible de este importante recurso. En la presente tesis doctoral se estudia la biología trófica del atún rojo en diferentes etapas de su ciclo vital, juveniles y adultos, en el Mediterráneo occidental y el Estrecho de Gibraltar (SoG) utilizando dos metodologías complementarias: análisis de contenidos estomacales (SCA) y de isótopos estables (SIA). En general, los juveniles de edad 0 analizados en el Mediterráneo Occidental se alimentaron principalmente de pequeños pelágicos (Engraulis encrasicolus, clupeidos) aunque los invertebrados (cefalópodos y pequeños crustáceos) contribuyeron significativamente a la dieta. La dieta de los juveniles varió entre años y zonas, por ejemplo, en Valencia los ejemplares se alimentaron de una dieta más diversa que en Cambrils, Carboneras y Fuengirola. Por otra parte, el SCA de subadultos y adultos capturados en el SoG mostró que los crustáceos mesopelágicos (Sergia robusta, Acanthephyra pelagica, Pasiphaea spp.) fueron la principal fuente de alimento, seguida de peces mesopelágicos (Stomiidae y Myctophidae) y clupeidos, mientras que la composición de la dieta estimada a partir de modelos de mezcla isotópicos estuvo formada principalmente por calamares. Los valores isotópicos del carbono y nitrógeno medidos en los atunes sugieren que existe una importante variabilidad espacial y temporal en la composición isotópica de los organismos que constituyen la línea base del ecosistema. En general, los valores de δ13C medidos en el tejido muscular y hepático de los juveniles fueron similares, lo cual podría indicar que los ABFT estudiados aquí estaban en equilibrio isotópico con las presas y, por tanto, se podría deducir que se alimentaron en la misma zona durante un período prolongado de tiempo. En general, pese a que los valores de la PT (posición trófica) estimados a partir de la pareja de aminoácidos, ácido glutámico-fenilalanina, fueron del orden de media unidad inferiores a los obtenidos con los datos de SCA y del orden de una unidad inferior a los valores de la PTSIA, los valores de la PT del atún de edad 0 estimados fueron próximos a 4, lo cual indica que el atún rojo juvenil se sitúa en la parte alta de la cadena trófica. Por otra parte, los atunes residentes en el SoG se comportan como mesodepredadores en lugar de superdepredadores, y probablemente compiten con otros depredadores pelágicos por los mismos recursos tróficos en la zona., Ministerio de Economía y Competitividad (BES-2012-058472)(MICINN, CTM2011-29525-C04-01).

Se ha realizado un video divulgativo del proyecto que puede visualizarse en: https://www.youtube.com/watch?v=X_fd8‐K1Xf8&t=13s Objetivos del proyecto BIACOP Mediante la utilización de sistemas de ecosondas y visión estereoscópica, el proyecto BIACOP propone dos objetivos principales: Objetivo 1: Desarrollo de un sistema totalmente automático para estimar la distribución de tallas de los atunes en jaulas flotante Objetivo 2: Desarrollo de una herramienta con ecosondas y sistemas estereoscópico para la de estimación de número de individuos y biomasa total de atún rojo durante el proceso de transferencia entre jaulas de transporte y de engrase, desde una perspectiva ventral. Los objetivos del proyecto han sido cumplidos en su totalidad y se han alcanzado resultados altamente prometedores, combinando tecnología de sensores acústicos y ópticos, que muestran que las técnicas desarrolladas posibilitan el conteo de los atunes en transferencias y su tallaje tanto en jaulas como en transferencias. Nuestros desarrollos permiten el tallaje y conteo con resultados independientes de fatigas y condiciones humanas.