Trophic ecology of Atlantic bluefin tuna Thunnus thynnus larvae

Feeding intensity, diet composition, selectivity, energy ingestion and dietary niche breadth of larval Atlantic bluefin tuna Thunnus thynnus were studied on the eastern (Mediterranean) spawning grounds of the species. Larval T. thynnus were collected in the Balearic Archipelago (north-west Mediterranean Sea) during 2004 and 2005 using surveys specific for larval scombrids. Larvae between 2·6 and 8·7 mm standard length (L(S) ) are diurnal feeders, and 94% of the guts collected during daylight hours were full. The mean ±s.d. number of prey per gut was 7·1 ± 5·7, with mean ±s.d. ranging from 3·0 ± 1·6 in the smallest T. thynnus larvae to 11·1 ± 5·8 in 5·0-6·0 mm L(S) larvae. Up to 21 prey were found in a single larval gut (5·0-6·0 mm L(S) ) at the end of the day. Larvae progressively selected larger prey and exhibited increased carbon content concurrent with preflexion development of feeding and locomotory structures. Larvae of 5·0-6·0 mm L(S) exhibited positive selection of cladocerans over other prey (Chesson's index), whereas copepod nauplii dominated the diets of earlier stages. The dietary niche breadth measured increased initially but decreased at c. 5·5 mm L(S) . Appendicularians were found in the diet of larger larval sizes, but no piscivory was observed. Results are discussed in light of the sparse existing data for larval T. thynnus and other larval tuna species.     

Comparative analysis of male germ cell proliferation and apoptosis in wild and captive Atlantic bluefin tuna (Thunnus thynnus L.)

The most commonly observed reproductive dysfunction in male fishes reared in captivity is reduction in sperm volume and quality. The Atlantic bluefin tuna Thunnus thynnus (Osteichthyes: Scombridae) is one of the few large pelagic and migratory marine fishes maintained in captivity with the purpose of establishing breeding populations to support an aquaculture industry. The objectives of the present study were to compare male germ cell proliferation and apoptosis between wild and captive individuals at two different phases of the spermatogenetic cycle, and to evaluate sperm motility characteristics of captive individuals. Histological observations were performed to analyze testicular activity, and germ cell proliferation and apoptosis were evaluated through the immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and the terminal deoxynucleotidyl transferasemediated d'UTP nick end labeling (TUNEL) method, respectively. Computer‐assisted sperm analysis (CASA) was used to evaluate sperm motility. Results showed that germ cell proliferation was delayed and germ cell apoptosis increased in captive animals relative to wild individuals. Sperm motility of samples obtained from captive individuals was anomalous, both in terms of motility duration and swimming efficiency. Thus it appears that rearing in captivity impairs male reproductive function through, at least, changes in germ cell proliferation and apoptosis.     

Isolation and characterization of seven tetranucleotide microsatellite loci from Atlantic northern bluefin tuna Thunnus thynnus thynnus

Microsatellite‐enriched genomic libraries were obtained from the Atlantic northern bluefin tuna, Thunnus thynnus thynnus and seven tetranucleotide markers were successfully isolated and characterized from this library. These markers were found to have between 1 and 17 alleles in Atlantic northern bluefin tuna and heterozygosity ranged from 0 to 0.85. No deviations from the expectation of Hardy‐Weinburg equilibrium were found for any marker. Several of these markers amplify reliably in other tuna species.     

Microsatellite and mitochondrial DNA analyses of Atlantic bluefin tuna (Thunnus thynnus thynnus) population structure in the Mediterranean Sea

Genetic variation was surveyed at nine microsatellite loci and the mitochondrial control region (868 bp) to test for the presence of genetic stock structure in young-of-the-year Atlantic bluefin tuna (Thunnus thynnus thynnus) from the Mediterranean Sea. Bluefin tuna were sampled over a period of 5 years from the Balearic and Tyrrhenian seas in the western basin of the Mediterranean Sea, and from the southern Ionian Sea in the eastern basin of the Mediterranean Sea. Analyses of multilocus microsatellite genotypes and mitochondrial control region sequences revealed no significant heterogeneity among collections taken from the same location in different years; however, significant spatial genetic heterogeneity was observed across all samples for both microsatellite markers and mitochondrial control region sequences (FST=0.0023, P=0.038 and PhiST=0.0233, P=0.000, respectively). Significant genetic differentiation between the Tyrrhenian and Ionian collections was found for both microsatellite and mitochondrial markers (FST=0.0087, P=0.015 and PhiST=0.0367, P=0.030, respectively). These results suggest the possibility of a genetically discrete population in the eastern basin of the Mediterranean Sea.     

Comparative study of liver vitellogenin gene expression and oocyte yolk accumulation in wild and captive Atlantic bluefin tuna (Thunnus thynnus L.)

The sequence of vitellogenin A (VgA) and vitellogenin B (VgB) cDNAs in Atlantic bluefin tuna (Thunnus thynnus L.) were determined, and vitellogenin expression levels in the liver and oocyte yolk accumulation were compared in wild and captive-reared individuals. Liver and ovary samples were taken from 31 individuals reared experimentally in three commercial Atlantic bluefin tuna fattening sites in the Mediterranean Sea and from 33 wild individuals caught by commercial traps during the fish's migration towards their Mediterranean spawning grounds. The total length of VgA cDNA was 5585 nucleotides and that of VgB was 5267 nucleotides. The identity and similarity between deduced amino acid sequences of VgA and VgB were 60% and 78%, respectively. The Atlantic bluefin tuna VgA and VgB amino acid sequences have high similarities with those of other teleost fishes. Relative levels of VgA and VgB mRNAs were low in April, increased significantly during the reproductive period in May and June, and declined in July. There was a trend towards higher relative levels of VgA and VgB mRNAs in captive fish compared to wild individuals during the reproductive period. The surface occupied by eosinophilic yolk granules in fully vitellogenic oocytes, as well as the frequency of oocytes in late vitellogenesis, was significantly higher in captive compared to wild individuals. The study suggests that the experimental conditions under which Atlantic bluefin tuna individuals were reared allowed the occurrence of normal vitellogenesis, based on gene expression of VgA and VgB in the liver and yolk accumulation in the oocytes. The higher yolk accumulation and frequency of vitellogenic oocytes observed in the ovaries of captive fish suggest that improvements in feeding practices may result in an improved vitellogenic process.     

The muscle twitch and the maximum swimming speed of giant bluefin tuna, Thunnus thynnus L.

Sustained swimming of bluefin tuna was analysed from video recordings made of a captive patrolling fish school [lengths (L) 1.7–3.3 m, body mass (M) 54–433 kg]. Speeds ranged from 0.6 to 1.2 L s⁻¹ (86–260 km day⁻¹) while stride length during steady speed swimming varied between 0.54 and 0.93 L. Maximum swimming speed was estimated by measuring twitch contraction of the anaerobic swimming muscle in pithed fish 5 min after death. Muscle contraction time increased from the shortest just behind the head (30–50 ms at 20% L) to the longest at the tail peduncle (80–90 ms at 80% L) (all at 28°C). A fish (L = 2.26 m) with a muscle contraction time of 50 ms at 25% L can have a maximum tail beat frequency of 10 Hz and maximum swimming speed of 15m s⁻¹ (54km h⁻¹) with a stride length of 0.65L. With a stride length of 1 L a speed of 22.6 m s⁻¹ (81.4 km h⁻¹) is possible. Power used at maximum speed was estimated for this fish at between 10 and 40 kW, with corresponding values for the drag coefficient at a Reynolds number of 4.43 × 10⁷ of 0.0007 and 0.0027.     

Ultrastructure of oogenesis in the bluefin tuna, Thunnus thynnus

Ovarian ultrastructure of the Atlantic bluefin tuna (Thunnus thynnus) was investigated during the reproductive season with the aim of improving our understanding of the reproductive biology in this species. The bluefin, like the other tunas, has an asynchronous mode of ovarian development; therefore, all developmental stages of the oocyte can be found in mature ovaries. The process of oocyte development can be divided into five distinct stages (formation of oocytes from oogonia, primary growth, lipid stage, vitellogenesis, and maturation). Although histological and ultrastructural features of most these stages are similar among all studied teleosts, the transitional period between primary growth and vitellogenesis exhibits interspecific morphological differences that depend on the egg physiology. Although the most remarkable feature of this stage in many teleosts is the occurrence of cortical alveoli, in the bluefin tuna, as is common in marine fishes, the predominant cytoplasmic inclusions are lipid droplets. Nests of early meiotic oocytes derive from the germinal epithelium that borders the ovarian lumen. Each oocyte in the nest becomes surrounded by extensions of prefollicle cells derived from somatic epithelial cells and these form the follicle that is located in the stromal tissue. The primary growth stage is characterized by intense RNA synthesis and the differentiation of the vitelline envelope. Secondary growth commences with the accumulation of lipid droplets in the oocyte cytoplasm (lipid stage), which is then followed by massive uptake and processing of proteins into yolk platelets (vitellogenic stage). During the maturation stage the lipid inclusions coalesce into a single oil droplet, and hydrolysis of the yolk platelets leads to the formation of a homogeneous mass of fluid yolk in mature eggs.     

Microsatellite DNA markers for population‐genetic studies of Atlantic bluefin tuna (Thunnus thynnus thynnus) and other species of genus Thunnus

Twenty‐five microsatellites from Atlantic bluefin tuna (Thunnus thynnus thynnus) were characterized. All 25 microsatellites were polymorphic; the number of alleles among up to 56 individuals surveyed ranged from two to 23. Atlantic bluefin tuna are highly exploited and major questions remain as to stock structure and abundance in the eastern and western North Atlantic. The microsatellites will be useful in testing stock‐structure hypotheses and in generating estimates of effective population size. The polymerase chain reaction primer sets developed also amplified identifiable alleles in three other species of genus Thunnus: T. albacares (yellowfin tuna), T. alalunga (albacore tuna) and T. obesus (bigeye tuna).     

Digestive activity and stomach temperature in farmed bluefin tuna Thunnus thynnus measured by acoustic tag

Eight farmed Atlantic bluefin tuna Thunnus thynnus were tagged with temperature and depth transmitters inserted in chub mackerels Scomber colias to characterize their digestive activity, feeding physiology and behaviour in captivity. Results obtained in the experiment can be used to optimize daily T. thynnus feeding strategy in farms, reducing the early regurgitation of food and thus the environmental effects of inappropriate feeding practices.     

Age and growth of Atlantic bluefin tuna, Thunnus thynnus (Osteichthyes: Thunnidae), in the Mediterranean Sea

The objective of the study was to describe the biometry of Mediterranean bluefin tuna, Thunnus thynnus, the biology of which is not yet well understood. A total of 504 specimens was collected from 1998 to 2005 in the central part of the Mediterranean basin. They were sexed and measured; fork lengths (FL) ranged from 51.0 to 255.0 cm while body weights (W) ranged from 2.6 to 247.0 kg. The first spiniform ray (spine) of the first dorsal fin was removed and cross‐sectioned near the condyle base in order to count annuli for age estimation. The regression coefficient (b) of the female FL–W relationship was significantly higher than that of the male, and both sexes displayed a negatively allometric growth (b < 3); male regression equation: ln W = ?2.942 + 2.730 ln FL; female regression equation: ln W = ?3.660 + 2.878 ln FL. Based on counts of the translucent zones in the sections of the first ray of the first dorsal fin, estimated ages ranged from 1 to 15 years for males and 1 to 14 years for females. The correlation between the spine ray (R) and FL fit the allometric model best; the R–FL regression equations of the two sexes did not differ significantly and the overall equation was: ln FL = 3.721 + 0.851 ln R. Due to the R–FL allometric correlation, estimates of fork lengths at previous ages, FL_i, were back‐calculated with a body proportional hypothesis. Von Bertalanffy growth equations were derived from both observed and back‐calculated FLs‐at‐age, which did not differ significantly. Moreover, no significant difference was found between the growth equations of the two sexes; the overall equation was FL_t = 373.08 [1?e^{?0.07(t + 1.76)}]. Weight‐at‐age values were derived from the von Bertalanffy predicted FLs‐at‐age by the FL–W correlation equations for males and females. The paper represents the first comprehensive study on the biometry, including age and growth, of bluefin tuna captured in the Mediterranean Sea.     

Oogenesis in the bluefin tuna,Thunnus thynnus L.: a histological and histochemical study

Histology and histochemistry are useful tools to study reproductive mechanisms in fish and they have been applied in this study. In the bluefin tuna, Thunnus thymus L., oocyte development can be divided into 4 principal phases based on the morphological features of developing oocytes and follicles. The primary growth phase includes oogonia and basophilic or previtellogenic oocytes classified as chromatin-nucleolus and perinucleolus stages. The secondary growth phase is represented by vitellogenic oocytes at early (lipid globule and yolk granule 1), mid (yolk granule 2) and late (yolk granule 3) vitellogenesis stages. The maturation phase involves postvitellogenic oocytes undergoing maturation process. During the spawning period, both postovulatory follicles, which indicate spawning, and atretic follicles can be distinguished in the ovary. Carbohydrates, lipids, proteins and specially those rich in tyrosine, tryptophan, cystine, arginine, lysine and cysteine, as well phospholipids and/or glycolipids and neutral glycoproteins were detected in yolk granules. Moreover, affinity for different lectins (ConA, WGA, DBA and UEA) was detected in vitellogenic oocytes (yolk granules, cortical alveoli, follicular layer and zona radiata), indicating the presence of glycoconjugates with different sugar residues (Mannose- Man- and/or Glucose -Glc-; N-acetyl-D-glucosamine- GlcNAc- and/or sialic acid- NANA-; N-acetyl-D-galactosamine- GalNAc-; L-Fucose -Fuc-). Histochemical techniques also demonstrated the presence of neutral lipids in globules (vacuoles in paraffin sections) and neutral and carboxylated mucosubstances in cortical alveoli. By using anti-vitellogenin (VTG) serum, immunohistochemical positive results were demonstrated in yolk granules, granular cytoplasm and follicular cells of vitellogenic oocytes. Calcium was also detected in yolk granules and weakly in follicular envelope. In females, the gonadosomatic index (GSI) increased progressively from May, during early vitellogenesis, until June during mid and late vitellogenesis, where the highest values were reached. Subsequently, throughout the maturation-spawning phases (July), GSI decreased progressively reaching the minimal values during recovering-resting period (October).     

Geographic variation of body morphology of the Atlantic bluefin tuna, (Thunnus thynnus,Linnaeus, 1758)

Geometric morphometric methods were used to explore body shape morphology in 260 Atlantic bluefin tuna, Thunnus thynnus, collected in Sardinia (Western Mediterranean) during the breeding phase and in the Bay of Biscay (North Eastern Atlantic) during the feeding phase. The shape of each specimen was captured by high resolution digital images and recording the 2‐D coordinates of seven morphological landmarks. A general procruste analysis (GPA) was applied in order to eliminate any morphological variations resulting from size, position or orientation of specimens. A thin plate‐spline (TPS) method was then used to provide a graphical representation of the shape conformation between two sets of data. Results of the regression model between the direct and indirect measurements accounted for a R² = 0.98. The Principal Components Analysis shows differences linked to the two sampling areas, accounting for 37% and 19.97% of the body shape variation in the first (PC1) and second (PC2) principal component, respectively. Specifically, the deformation grid projection highlights the major differences regarding the anterior‐ventral part of the body (landmark 5‐6‐7). These differences might not necessarily be linked to an actual population substructure. Instead, it was hypothesized that such body shape differences were due to the diverse life phases during which specimens were collected, since the reproductive specimens show a ‘pot‐bellied’ shape, which was larger than for the feeding specimens that showed a ‘slimmer’ shape. Analyses of likely sexual dimorphism conducted on Sardinian specimens did not reveal any significant differences; whereas body shape differences related to the pre‐ and post‐reproductive sizes were detected.     

Expression of vitellogenin receptor gene in the ovary of wild and captive Atlantic bluefin tuna (Thunnus thynnus)

The cDNA sequences of vitellogenin receptor proteins (VgR(+) and VgR(-)), containing or lacking the O-linked sugar domain, were determined in Atlantic bluefin tuna (Thunnus thynnus L.). VgR(-) gene expression in the ovary was compared in captive-reared and wild Atlantic bluefin tuna during the reproductive cycle. Gonad samples from adult fish were sampled from 2008 to 2010 from stocks reared in captivity at different commercial fattening operations in the Mediterranean Sea and from wild individuals caught either by traditional tuna traps during their migration towards the spawning grounds in the Mediterranean Sea or by the long-line artisanal fishery. In addition, juvenile male and female Atlantic bluefin tuna were sampled from a farming facility, to obtain baseline information and pre-adulthood amounts of VgR(-). The total length of VgR(+) cDNA was 4006 nucleotides (nt) and that of VgR(-) was 3946 nt. Relative amounts of VgR(-) were greater in juvenile females and in those adults having only previtellogenic oocytes (119 ± 55 and 146 ± 26 folds more than juvenile males, respectively). Amounts of VgR(-) were less in individuals with yolked oocytes (ripening stage, May-June) and increased after spawning in July (92 ± 20 and 113 ± 13 folds more than juvenile males in ripening and post-spawning fish, respectively). These data suggest that regulation of VgR(-) is not under oestrogen control. During the ripening period, greater VgR(-) gene expression was observed in wild fish than in fish reared in captivity, possibly because of (a) differences in water temperature exposure and/or energy storage, and/or (b) an inadequate diet in reared Atlantic bluefin tuna.     

Quantification of ovarian follicles in bluefin tuna Thunnus thynnus by two stereological methods

The numbers of different types of ovarian follicles (developing, degenerating and postovulatory follicles) were estimated in bluefin tuna Thunnus thynnus using two stereological procedures: the model‐based method of Weibel & Gomez, which has become a tool of broad application in the quantification of oocytes in fishes, and the assumption‐free ‘disector’ (sic) method of Sterio. The estimates of developing follicles (follicles containing lipid‐stage, vitellogenic and migratory‐nucleus oocytes) made by the model‐based method tended to be lower than those obtained with the disector, though significant differences were not observed except for vitellogenic follicles. Counts of atretic follicles by the model‐based method were higher than those made using the disector, the differences being remarkable between both techniques, particularly in the case of β‐atresia, where the statistical analysis indicated significantly unequal estimations with the two methods. In contrast, the amount of postovulatory follicles estimated by the disector, which would stand for the realized batch fecundity, was somewhat larger than that calculated with the model‐based method.     

Feeding habits of the Atlantic bluefin tuna, Thunnus thynnus (L. 1758), in the central Mediterranean Sea (Strait of Messina)

The study of feeding habits of the Atlantic bluefin tuna was carried out in 123 specimens, ranging from 115 to 222 cm fork length (FL) and collected during spring seasons of 2010 and 2011 in the central Mediterranean Sea (Strait of Messina). The analysis of stomach contents allowed us to identify 91 taxa of prey items, mainly belonging to Teleostea (54), Cephalopoda (20) and Crustacea (13). The percentage of index of relative abundance (IRI) shows the highest values for the myctophid Hygophum benoiti (%IRI = 22.854) and the stomiid Chauliodus sloani (%IRI = 15.124), followed by the oegopsid squid Illex coindetii (%IRI = 14.316). The broad spectrum of prey items could suggest a generalist behavior of this predator, with several species that occasionally occurs in its diet. However, if prey are grouped into food categories, the importance of mesopelagic and benthopelagic fishes can be appreciated (54.41 % of %IRI). The assessment of the hypothetical foraging rhythm of the Atlantic bluefin tuna highlighted that its feeding activity is concentrated on diel migrating fauna during night and on larger preys upon daylight. The predation on the high-energetic food as mesopelagic and bathypelagic fishes during the pre-spawning and the spawning period may bring an energetic advantage in tuna metabolism and gonadal maturation     

Diet composition of bluefin tuna (Thunnus thynnus L. 1758) in the Eastern Mediterranean Sea,Turkey

This study gives relevant information on the diet composition of the bluefin tuna (Thunnus thynnus) during the spawning period in the eastern Mediterranean Sea. The stomach contents of 218 bluefin tuna were sampled from 2003 to 2006 during the fishing season (May–June) aboard purse seiners operating in the northern Levantine Sea off the coast of Turkey. Stomachs were removed from the fish soon after landing and kept frozen at ?18°C until analysis. Prey items were classified into large taxonomic categories and preserved in 70% ethanol. A total of 745 different prey specimens belonging to 47 taxa were identified, including 34 species of fish, 11 of squid, and two of crustaceans. The most important fish and cephalopod prey belonged to the families Myctophidae, Carangidae, Chauliodontidae, Paralepididae, and Octopoda. This study marks the observation of myctophid fish in the stomach contents of bluefin tuna from the Mediterranean Sea. The paper offers some new information of regional importance and compares the feeding habits of the species to other regions, bringing confirmation on the opportunistic feeding ecology of the species in the enclosed Mediterranean Sea, where bluefin tuna seasonally occur as a strong cohort. New information on the diet composition of T. thynnus in the eastern Mediterranean Sea is revealed; the findings indicate that, depending on the abundance of the different prey species in the habitat, the dominant prey species can be distinctive.