Impact of Co-Delivery of EGCG and Tuna Oil within a Broccoli Matrix on Human Gut Microbiota, Phenolic Metabolites and Short Chain Fatty Acids In Vitro.

(-)-Epigallocatechin gallate (EGCG) and tuna oil (TO) are beneficial bioactive compounds. EGCG, TO or a combination of, delivered by broccoli by-products (BBP), were added to an in vitro anaerobic fermentation system containing human fecal inocula to examine their ability to generate short-chain fatty acids (SCFA), metabolize EGCG and change the gut microbiota population (assessed by 16 S gene sequencing). Following 24 h fermentation, EGCG was hydrolyzed to (-)-epigallocatechin and gallic acid. EGCG significantly inhibited the production of SCFA (p < 0.05). Total SCFA in facal slurries with BBP or TO-BBP (48-49 µmol/mL) were significantly higher (p < 0.05) than the negative control with cellulose (21 µmol/mL). EGCG-BBP and TO-EGCG-BBP treatment increased the relative abundance of Gluconacetobacter, Klebsiella and Trabulsiella. BBP and TO-BBP showed the greatest potential for improving gut health with the growth promotion of high butyrate producers, including Collinsella aerofaciens, Bacillus coagulans and Lactobacillus reuteri.

Onset of nutrient consumption during early life stage digestive system development of two tuna species (Thunnus orientalis and Thunnus albacares).

To specify the timing of exogenous nutrient consumption in the larvae of two commercially important tuna species, the Pacific bluefin tuna (PBF) Thunnus orientalis and the yellowfin tuna (YFT) Thunnus albacares, the gene expressions of peptide transporter 1 (PEPT1) were examined. The mRNA expressions of PEPT1 first occurred at 2 days post hatching (dph) in PBF larvae and 3 dph for the YFT, and PEPT1 was found to only be expressed in the intestinal tract. The histological changes of the digestive tract of the YFT larvae were observed and compared to PBF larvae from a previous study. The intestines were developed at the hatching day for both species. It was found that the developmental timing of internal organs differed between the species, with the YFT showing an approximately one-day delay. The major organs such as liver, pancreas and gall bladder that excrete digestive enzymes appeared at 1 dph for PBF and 2 dph for YFT. The development of external morphological features was similar to organ development timings, with mouth-opening and first feeding starting at 2 dph for PBF, and 3 dph for YFT. Growth during the first month is rapid and variable for both species, ranging from 1.06 to 1.56 mm/d. Our findings provide new information about the early onset of feeding and larval development for the two species which would contribute to future aquaculture.

De novo transcriptome assembly, functional annotation and characterization of the Atlantic bluefin tuna (Thunnus thynnus) larval stage.

In the present work, we assembled and characterized a de novo larval transcriptome of the Atlantic bluefin tuna Thunnus thynnus by taking advantage of publicly available databases with the goal of better understanding its larval development. The assembled transcriptome comprised 37,117 protein-coding transcripts, of which 13,633 full-length (>80% coverage), with an Ex90N50 of 3061 bp and 76% of complete and single-copy core vertebrate genes orthologues. Of these transcripts, 34,980 had a hit against the EggNOG database and 14,983 with the KEGG database. Codon usage bias was identified in processes such as translation and muscle development. By comparing our data with a set of representative fish species, 87.1% of tuna transcripts were included in orthogroups with other species and 5.1% in assembly-specific orthogroups, which were enriched in terms related to muscle and bone development, visual system and ion transport. Following this comparative approach, protein families related to myosin, extracellular matrix and immune system resulted significantly expanded in the Atlantic bluefin tuna. Altogether, these results provide a glimpse of how the Atlantic bluefin tuna might have achieved early physical advantages over competing species in the pelagic environment. The information generated lays the foundation for future research on the more detailed exploration of physiological responses at the molecular level in different larval stages and paves the way to evolutionary studies on the Atlantic bluefin tuna.

Combining genetic markers with stable isotopes in otoliths reveals complexity in the stock structure of Atlantic bluefin tuna (Thunnus thynnus).

Atlantic bluefin tuna (Thunnus thynnus) from the two main spawning populations in the Mediterranean and Gulf of Mexico occur together in the western, central and eastern Atlantic. Stock composition of catches from mixing areas is uncertain, presenting a major challenge to the sustainable management of the fisheries. This study combines genetic and chemical markers to develop an integrated method of population assignment. Stable isotope signatures (δ13C and δ18O) in the otolith core of adults from the two main spawning populations (adult baselines) showed less overlap than those of yearlings (12-18 months old) from western and eastern nursery areas suggesting that some exchange occurs towards the end of the yearling phase. The integrated model combined δ18O with four genetic markers (SNPs) to distinguish the adult baselines with greater accuracy than chemical or genetic markers alone. When used to assign individuals from the mixing areas to their population of origin, the integrated model resolved some (but not all) discrepancies between the chemistry and genetic methods. Some individuals in the mixing area had otolith δ18O values and genetic profiles which when taken together, were not representative of either population. These fish may originate from another Atlantic spawning area or may represent population contingents that move away from the main spawning areas during the first year of life. This complexity in stock structure is not captured by the current two-stock model.

Age and growth of slender tuna (Allothunnus fallai) in an unexploited temperate population.

Many large predatory fishes are in decline and tuna sustainability is high on the global agenda. Slender tuna (SLT), Allothunnus fallai, is data-poor and a rare contemporary example of a globally unexploited temperate tuna. This study analysed 214 otoliths for age and growth of fish collected in the South Atlantic. Observed ages varied between 9 and 42 years for a size range of 68-90 cm fork length. We reveal important life history data for SLT before exploitation and underline the relevance of data-poor stocks in understanding wider questions for exploited tuna.

Growth-related trophic changes of Thunnus thynnus as evidenced by stable nitrogen isotopic values in the first dorsal spine.

The bluefin tuna, Thunnus thynnus, is a highly migratory and long-living fish at the top of the pelagic food web. As top predator, it plays a key role in the stability of marine food webs by exerting top-down control on its prey. The diet composition of bluefin tuna varies in relation to its growth, seasons and migratory patterns, making it difficult to evaluate spatial and temporal effects. This latter aspect is further complicated to be determined during the first months of life, when T. thynnus specimens have a rapid growth rate leading to changes in the trophic status. In this study, the potential collagen-related effects on δ15N and δ13C values were evaluated on the whole spine of adult tuna specimens collected in the central Mediterranean Sea. Obtained results showed non-significant differences between extracted and non-extracted collagen samples for δ15N in whole spine, allowing adopting the isotopic analysis both for annuli in the spine section of adults and for younger specimens, whose spine size does not permit the collagen extraction. Specifically, isotopic analysis of whole spine of the young of the year specimens, showed a rapid change in δ15N values with length, following an exponential model. For older specimens, δ15N values were higher and varied around a plateau, likely due to a higher specificity in the choice of prey and/or to change in the geographical location. Such variability was also mirrored in annuli of spines sections of adult tunas. As far as δ13C values are concerned, a strong collagen-related effect was evidenced, likely highlighting the influence of lipids. Consequently, δ13C analysis may be used only on adult specimens where collagen extraction is possible. This research also showed how isotopic analysis of both whole sample and sequence of annuli in the cross-section of dorsal spine might produce isotopic profiles useful to detect specific trophic dynamics along the bluefin tuna growth.

Mercury concentrations in reared Atlantic bluefin tuna and risk assessment for the consumers: To eat or not to eat?

This study determines the levels of total mercury in muscle tissue from 268 reared Atlantic bluefin tuna in the Mediterranean Sea, detects variations on mercury concentrations in relation to biological parameters and rearing period and evaluates the health risk posed by fish consumption taking into consideration the size, age and rearing period. A total of 30 pooled samples were analyzed. The total Hg concentrations ranged from 0.49 to 1.60 mg/kg w.w. and showed a significant positive relationship with the size and age. It was found that the longer the rearing period, the lower the mercury concentration. The total Hg exceeded the maximum level defined by the European Commission Decision in a proportion of 40.0%. Since the value of Target Hazard Quotient was higher than 1 in all different groups (medium, large, 6 months and 18 months reared specimens), the maximum possible safe weekly consumption (400 g) is suggested.

Ontogenetic change in the amount and position of slow-oxidative myotomal muscle in relationship to regional endothermy in juvenile yellowfin tuna Thunnus albacares.

Myotomal slow-oxidative muscle (SM) powers continuous swimming and generates heat needed to maintain elevated locomotor muscle temperatures (regional endothermy) in tunas. This study describes how the amount and distribution of myotomal SM increases with fish size and age in juvenile yellowfin tuna Thunnus albacares in relationship to the development of regional endothermy. In T. albacares juveniles 40-74 mm fork length (LF ; n = 23) raised from fertilised eggs at the Inter-American Tropical Tuna Commission Achotines Laboratory in Panama and larger juveniles (118-344 mm LF ; n = 5) collected by hook and line off of Oahu, Hawaii, USA, SM was identified by histochemical staining for the mitochondrial enzyme succinic dehydrogenase or by colour (in the two largest individuals). The cross-sectional area of myotomal SM at 60% LF , a position with maximal percentage of SM in larger T. albacares, increased exponentially with LF . The percentage of total cross-sectional area composed of SM at 60% LF increased significantly with both LF and age, suggesting that SM growth occurs throughout the size range of T. albacares juveniles studied. In addition, the percentage of SM at 60% LF that is medial increased asymptotically with LF . The increases in amount of SM and medial SM, along with the development of the counter-current heat-exchanger blood vessels that retain heat, allow larger tuna juveniles to maintain elevated and relatively stable SM temperatures, facilitating range expansion into cooler waters.

Effects of environmental change and early-life stochasticity on Pacific bluefin tuna population growth.

Species conservation and fisheries management require approaches that relate environmental conditions to population-level dynamics, especially because environmental conditions shift due to climate change. We combined an individual-level physiological model and a conceptually simple matrix population model to develop a novel tool that relates environmental change to population dynamics, and used this tool to analyze effects of environmental changes and early-life stochasticity on Pacific bluefin tuna (PBT) population growth. We found that (i) currently, PBT population experiences a positive growth rate, (ii) somewhat surprisingly, stochasticity in early life survival increases this growth rate, (iii) sexual maturation age strongly depends on food and temperature, (iv) current fishing pressure, though high, is tolerable as long as the environment is such that PBT mature in less than 9 years of age (maturation age of up to 10 is possible in some environments), (v) PBT population growth rate is much more susceptible to changes in juvenile survival than changes in total reproductive output or adult survival. These results suggest that, to be effective, fishing regulations need to (i) focus on smaller tuna (i.e., juveniles and young adults), and (ii) mitigate adverse effects of climate change by taking into the account how future environments may affect the population growth.

Early development of primordial germ cells in Pacific bluefin tuna Thunnus orientalis.

Bluefin tuna is one of the most important aquaculture species in several countries; however, information regarding the primordial germ cell (PGC) development and migration in this species is scarce. This information is vital for application in reproductive biotechnology, for example, induced sterility through targeted cell ablation or PGC manipulation. Teleost PGC can be visualized by injecting an RNA transcribed from the fusion product of a fluorescent protein gene attached to the 3' untranslated region (3'UTR) of zebrafish nanos3 (zf-nos3) into eggs. In this study, we identified the PGC and its migratory pathway during early embryogenesis and larvae development by injecting the GFP-zf-nos3 3'UTR mRNA into the Pacific bluefin tuna (PBT, Thunnus orientalis). PBT PGCs were initially found around the marginal and dorsal regions of the blastodisc at 50%-epiboly stage. The PGCs were aligned as two elongated lines at the posterior part of the embryonic body during the early segmentation period, and eventually formed a single tight cluster underneath somites 10 to 15 of the embryonic body until the late segmentation period. Although the aggregated PGCs stayed at the same position during hatching, they started migrating anteriorly and were split into two populations at 3 days after hatching (DAH). Until 15 DAH, these PGCs settled in two bilateral lines at the apex of the peritoneal cavity. Histological analysis of PBT larvae revealed that at 3 and 5 DAH, the PGCs were not enclosed by the somatic cells, whereas at 15 DAH, they were entirely covered by the somatic cells, indicating the development of the primordial gonads. These results are essential for future experiments in germ line control technologies for bluefin tuna.

Performance, feed utilization, and hepatic metabolic response of weaned juvenile Atlantic bluefin tuna (Thunnus thynnus L.): effects of dietary lipid level and source.

Two trials were performed using extruded diets as on-growing feeds for weaned Atlantic bluefin tuna (Thunnus thynnus; ABT) to establish adequate dietary levels of both lipid and omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), and impacts on lipid metabolism via liver gene expression. In trial A, ABT were fed with either a commercial feed (Magokoro®; MGK) as a reference diet or two experimental feeds differing in lipid levels (15 or 20%) using krill oil (KO) as the single lipid source in order to estimate suitable lipid content. Fish fed MGK displayed the highest growth, followed by 15KO, and therefore a dietary lipid content of 15% was considered preferable to 20% at this stage. In trial B, fish were fed MGK, 15KO, or a feed containing 15% lipid with a blend of KO and rapeseed oil (RO) (1:1, v/v; 15KORO). Fish fed 15KO and 15KORO showed no difference in weight gain, specific growth rate, and fork length. Increasing dietary lipid level or including vegetable oil, RO, in the feeds did not increase liver lipid content. Liver fatty acid compositions largely reflected dietary profiles confirming very limited endogenous LC-PUFA biosynthesis. Liver of ABT fed 15KO and 20KO displayed the highest contents of docosahexaenoic acid (DHA). The hepatic expression of genes encoding enzymes and transcription factors involved in lipid and fatty acid metabolism, as well as genes encoding antioxidant enzymes, showed that many of these genes were regulated by dietary lipid and LC-PUFA content. Results suggested that ABT juveniles can be on-grown on inert dry feeds that support good fish growth and the accumulation of DHA.

A Simplified Gas Chromatographic Fatty-Acid Analysis by the Direct Saponification/Methylation Procedure and Its Application on Wild Tuna Larvae.

A method for the direct preparation of fatty-acid methyl esters (FAME) was simplified for fatty-acid analysis of a single fish larva using gas chromatography (GC). The method included the isolation of a larval trunk and drying in a glass vial, followed by saponification of all the contents without prior lipid extraction. Thereafter, the fatty acids released were methylated by trimethylsilyldiazomethane. This method has advantages over another method, direct acid-catalyzed transesterification, because both the saponification and methylation at room temperature can reduce loss of unsaturated fatty acids and formation of artifacts unavoidable in acidic reaction at high temperature. GC of the products showed that the simplified method can yield methyl esters without artifacts interfering analysis. More than 50 fatty acids were determined, which are twice as many as those previously analyzed using high-performance liquid chromatography. Observation of consistent small impurities in GC of blank tests allowed the accurate determination of fatty acids by correcting the peak areas. Dry matter weights (<3 mg) and the total fatty-acid contents displayed a linear relationship. Fatty-acid analysis of wild larvae of bluefin tuna, yellowfin tuna, and skipjack tuna collected from the waters around Japan (n = 100) revealed that the eicosapentaenoic acid (EPA) level in bluefin tuna collected from the Japan Sea was significantly higher than that in the three species collected from Nansei Islands. The simplified direct saponification/methylation method will be a powerful tool for investigating growth and survival of individual larval tuna and other fish species.

Accounting for environmental and observer effects in estimating abundance of southern bluefin tuna from aerial survey data.

Southern bluefin tuna (SBT) is a valuable species that has been subject to high exploitation rates since the 1950s. In 2011, the spawning stock biomass was estimated to be at a historically low level, at only 5% of pre-fished biomass. A key component for managing and rebuilding the stock is having reliable, fishery-independent estimates of juvenile abundance. This paper describes how such estimates have been constructed from aerial surveys of juvenile (age 2-4) SBT conducted annually in the Great Australian Bight from 1993-2000 and 2005-2009. During these surveys, observers flew along pre-set transect lines searching for surface schools of SBT. Data were collected on the location and biomass of SBT sightings, and on the environmental conditions present during the survey. Sea surface temperature (SST) was found to correlate with the size (biomass) of schools, and several environmental variables, SST and wind speed in particular, were found to correlate with the number of sightings (presumably by affecting the ability of observers to see surface schools as well as whether fish were present at the surface). In addition, observers changed over time and differed in their aptitude for spotting tuna. Thus, generalized linear mixed models (GLMMs) were used to standardize the sightings and biomass data to a common set of observers and environmental conditions in order to produce an annual time series of relative abundance estimates. These estimates, which form one of two key inputs to the management procedure used by the international Commission for the Conservation of Southern Bluefin Tuna to set the global catch quota, suggest juvenile abundance was highest in the first years of the survey (1993-1996), after which it declined and fluctuated around a level about four times lower.

Robust estimates of a high Ne/N ratio in a top marine predator, southern bluefin tuna.

Genetic studies of several marine species with high fecundity have produced "tiny" estimates (≤10-3) of the ratio of effective population size (Ne) to adult census size (N), suggesting that even very large populations might be at genetic risk. A recent study using close-kin mark-recapture methods estimated adult abundance at N ≈ 2 × 106 for southern bluefin tuna (SBT), a highly fecund top predator that supports a lucrative (~$1 billion/year) fishery. We used the same genetic and life history data (almost 13,000 fish collected over 5 years) to generate genetic and demographic estimates of Ne per generation and Nb (effective number of breeders) per year and the Ne/N ratio. Demographic estimates, which accounted for age-specific vital rates, skip breeding, variation in fecundity at age, and persistent individual differences in reproductive success, suggest that Ne/N is >0.1 and perhaps about 0.5. The genetic estimates supported this conclusion. Simulations using true Ne = 5 × 105 (Ne/N = 0.25) produced results statistically consistent with the empirical genetic estimates, whereas simulations using Ne = 2 × 104 (Ne/N = 0.01) did not. Our results show that robust estimates of Ne and Ne/N can be obtained for large populations, provided sufficiently large numbers of individuals and genetic markers are used and temporal replication (here, 5 years of adult and juvenile samples) is sufficient to provide a distribution of estimates. The high estimated Ne/N ratio in SBT is encouraging and suggests that the species will not be compromised by a lack of genetic diversity in responding to environmental change and harvest.

Effects of age on growth in Atlantic bluefin tuna (Thunnus thynnus).

Atlantic Bluefin Tuna Thunnus thynnus (ABFT) is considered one of the most important socio-economic species but there is a lack of information on the physiological and molecular processes regulating its growth and metabolism. In the present study, we focused on key molecules involved in growth process. The aim of the present study was to associate molecular markers related to growth with canonical procedures like morphological measurements such as curved fork length (CFL) and round weight (RWT). The ABFT specimens (n = 41) were organized into three different groups A, B and C according to their age. The molecular analysis of liver samples revealed that igf1, igf1r and mTOR genes, involved in growth process, were differentially expressed in relation to the age of the fish. In addition, during the analyzed period, faster growth was evident from 5 to 8 years of age, after that, the growth rate decreased in terms of length yet increased in terms of adipose tissue storage, as supported by the higher fat content in the liver. These results are useful in expanding basic knowledge about the metabolic system of ABFT and provide new knowledge for the aquaculture industry.

Spillover of the Atlantic bluefin tuna offspring from cages in the Adriatic Sea: A multidisciplinary approach and assessment.

During routine monitoring of commercial purse seine catches in 2011, 87 fingerling specimens of scombrids were collected in the southern Adriatic Sea. Sequencing of the mitochondrial DNA control region locus inferred that specimens belonged to the Atlantic bluefin tuna, Thunnus thynnus (Linnaeus, 1758) (N = 29), bullet tuna, Auxis rochei (Risso, 1810) (N = 30) and little tunny, Euthynnus alletteratus, Rafinesque, 1810 (N = 28). According to previously published growth parameters, the age of the collected specimens was estimated at approximately 30-40 days, suggesting they might have been spawned in the Adriatic Sea, contrary to the current knowledge. A coupled modelling system with hydrodynamic (ROMS) and individual based model (IBM-Ichthyop) was set up to determine the location of the spawning event. Numerical simulations with the IBM model, both backward and forward in time, indicate commercial tuna cages in the middle Adriatic coastal area as possible spawning location. The two other non-commercial species likely opportunistically use the positive environmental (abiotic and biotic) conditions to spawn in the same area.

Adaptive responses of tropical tuna purse-seiners under temporal regulations.

The failure to achieve fisheries management objectives has been broadly discussed in international meetings. Measuring the effects of fishery regulations is difficult due to the lack of detailed information. The yellowfin tuna fishery in the eastern Pacific Ocean offers an opportunity to evaluate the fishers' responses to temporal regulations. We used data from observers on-board Mexican purse-seine fleet, which is the main fleet fishing on dolphin-associated tuna schools. In 2002, the Inter-American Tropical Tuna Commission implemented a closed season to reduce fishing effort for this fishery. For the period 1992-2008, we analysed three fishery indicators using generalized estimating equations to evaluate the fishers' response to the closure. We found that purse-seiners decreased their time spent in port, increased their fishing sets, and maintained their proportion of successful fishing sets. Our results highlight the relevance of accounting for the fisher behaviour to understand fisheries dynamics when establishing management regulations.

Reproductive Biology of Albacore Tuna (Thunnus alalunga) in the Western Indian Ocean.

The reproductive biology of albacore tuna, Thunnus alalunga, in the western Indian Ocean was examined through analysis of the sex ratio, spawning season, length-at-maturity (L50), spawning frequency and fecundity. From 2013 to 2015, a total of 923 female and 867 male albacore were sampled. A bias in sex ratio was found in favor of females with fork length (LF) < 100 cm. Using histological analyses and gonadosomatic index, spawning was found to occur between 10°S and 30°S, mainly to the east of Madagascar from October to January. Large females contributed more to reproduction through their longer spawning period compared to small individuals. The L50 (mean ± standard error) of female albacore was estimated at 85.3 ± 0.7 cm LF. Albacore spawn on average every 2.2 days within the spawning region and spawning months, from November to January. Batch fecundity ranged between 0.26 and 2.09 million oocytes and the relative batch fecundity (mean ± standard deviation) was estimated at 53.4 ± 23.2 oocytes g-1 of somatic-gutted weight. The study provides new information on the reproductive development and classification of albacore in the western Indian Ocean. The reproductive parameters will reduce uncertainty in current stock assessment models which will eventually assist the fishery to be sustainable for future generations.

Fisher-Level Decision Making to Participate in Fisheries Improvement Projects (FIPs) for Yellowfin Tuna in the Philippines.

This study identifies the capabilities needed by small-scale fishers to participate in Fishery Improvement Projects (FIPs) for yellowfin tuna in the Philippines. The current literature provides little empirical evidence on how different models, or types of FIPs, influence the participation of fishers in their programs and the degree which FIPs are able to foster improvements in fishing practices. To address this literature gap, two different FIPs are empirically analysed, each with different approaches for fostering improvement. The first is the non-governmental organisation-led Partnership Programme Towards Sustainable Tuna, which adopts a bottom-up or development oriented FIP model. The second is the private-led Artesmar FIP, which adopts a top-down or market-oriented FIP approach. The data were obtained from 350 fishers surveyed and were analysed using two separate models run in succession, taking into consideration full, partial, and non-participation in the two FIPs. The results demonstrate that different types of capabilities are required in order to participate in different FIP models. Individual firm capabilities are more important for fishers participation in market-oriented FIPs, which use direct economic incentives to encourage improvements in fisher practices. Collective capabilities are more important for fishers to participate in development-oriented FIPs, which drive improvement by supporting fishers, fisher associations, and governments to move towards market requirements.

Organochlorine pesticides in canned tuna and sardines on the Serbian market.

The aim of this study was to determine the level of organochlorine (OC) pesticides in 57 samples of canned tuna and 31 samples of canned sardines in vegetable oil, collected from supermarkets in Serbia. OC pesticides α-HCH, ß-HCH, δ-HCH, dichlorodiphenyltrichloroethane (DDT), DDE, DDD, dielderin, endosulfane I, endosulfane II, endosulan sulfate, endrin, endrin ketone, heptachlor, heptachlor epoxide, lindane, aldrin, metoxichlor, cis-chlordane and trans-chlordane were determined using a GS-MS method. The highest concentrations (µg kg-1, arithmetic means) in canned tuna were for δ-HCH (60.6 ± 97.0) and p, p´-DDT (55.0 ± 25.1), while the corresponding values in canned sardines were for δ-HCH (90.7 ± 102.7) and endosulfane II (78.0 ± 145.9). Mean level for the sum of endosulfans was above the maximum limit in canned sardines (85.0 µg kg-1). Also, dieldrin (39.7 µg kg-1) was measured above the ML.