Insights on the seasonal variations of reproductive features in the Eastern Atlantic Bluefin Tuna.

The Atlantic Bluefin Tuna (ABFT, Thunnus thynnus) is one of the most intensely exploited fisheries resources in the world. In spite of the years of studies on ABFT, basic aspects of its reproductive biology remain uncertain. To gain insight regarding the seasonal changes of the reproductive characteristics of the eastern stock of ABFT, blood and tissue samples were collected from mature specimens caught in the Mediterranean basin during the reproductive (May-June) and non-reproductive season (Oct-Nov). Histological analysis of the gonads of May-June samples indicated that there were females which were actively spawning (contained post-ovulatory follicles) and females that were not actively spawning that had previtellogenic and fully vitellogenic oocytes. In males, testis were at early or late stage of spermatogenesis during the reproductive season. In Oct-Nov, ovaries contained mostly previtellogenic oocytes as well as ß and α atretic follicles while the testis predominantly contained spermatogonia and few cysts with spermatocytes and spermatozoa. Gonadosomatic index (GSI) in females was highest among the actively spawning individuals while in males GSI was higher in early and late spermatogenic individuals compared to those that were spent. Plasma sex steroids levels varied with the reproductive season. In females, estradiol (E2), was higher in May-June while testosterone (T) and progesterone (P) did not vary. In males, E2 and T were higher in May-June while P levels were similar at the two sampling points. Circulating follicle stimulating hormone (FSH) was higher in Oct-Nov than in May-June both in males and females. Vitellogenin (VTG) was detected in plasma from both males and females during the reproductive season with levels in females significantly higher than in males. VTG was undetected in Oct-Nov samples. Since choriogenesis is an important event during follicle growth, the expression of three genes involved in vitelline envelope formation and hardening was measured and results showed significantly higher levels in ovaries in fish caught in May-June with respect to those sampled in Oct-Nov. In addition, a set of genes encoding for ion channels that are responsible for oocyte hydration and buoyancy, as well as sperm viability, were characterized at the two time points, and these were found to be more highly expressed in females during the reproductive season. Finally, the expression level of three mRNAs encoding for different lipid-binding proteins was analyzed with significantly higher levels detected in males, suggesting sex-specific expression. Our findings provide additional information on the reproductive biology of ABFT, particularly on biomarkers for the assessment of the state of maturation of the gonad, highlighting gender-specific signals and seasonal differences.

Moving cages further offshore: effects on southern bluefin tuna, T. maccoyii, parasites, health and performance.

The effects of offshore aquaculture on SBT health (particularly parasitic infections and haematology) and performance were the main aim of this study. Two cohorts of ranched Southern Bluefin tuna (SBT) (Thunnus maccoyii) were monitored throughout the commercial season, one maintained in the traditional near shore tuna farming zone and one maintained further offshore. SBT maintained offshore had reduced mortality, increased condition index at week 6 post transfer, reduced blood fluke and sealice loads, and haematological variables such as haemoglobin or lysozyme equal to or exceeding near shore maintained fish. The offshore cohort had no Cardicola forsteri and a 5% prevalence of Caligus spp., compared to a prevalence of 85% for Cardicola forsteri and 55% prevalence for Caligus spp. near shore at 6 weeks post transfer. This study is the first of its kind to examine the effects of commercial offshore sites on farmed fish parasites, health and performance.

Effects of immunostimulants on ranched southern bluefin tuna Thunnus maccoyii: immune response, health and performance.

Ranched southern bluefin tuna Thunnus maccoyii were fed baitfishes supplemented with vitamins (predominantly E and C) or vitamins and immunostimulants, nucleotides and ß-glucans, over 12 weeks after transfer and monitored for enhancement in immune response, health and performance through their 19 week grow-out period. Fish from two different tows were sampled separately at three different sampling points: at transfer to grow-out pontoons, at 8 weeks post-transfer and at harvest, 19 weeks post-transfer. Lysozyme activity was enhanced during vitamin supplementation compared to control fish. Performance (i.e. survival, condition index and crude fat), health (i.e. blood plasma variables including pH, osmolality, cortisol, lactate and glucose) and alternative complement activity were not commonly improved through diet supplementation. There were some tow-specific improvements in performance through vitamin supplementation including survival, selected parasite prevalence and intensity, and alternative complement activity. Immunostimulant supplementation also showed a tow-specific improvement in plasma cortisol level. Tow-specific responses may suggest that life history, previous health condition and husbandry can affect the success of vitamin and immunostimulant enhancement of immune response, health and performance of ranched T. maccoyii.

Identification of a novel selenium-containing compound, selenoneine, as the predominant chemical form of organic selenium in the blood of bluefin tuna.

A novel selenium-containing compound having a selenium atom in the imidazole ring, 2-selenyl-N(alpha),N(alpha),N(alpha)-trimethyl-L-histidine, 3-(2-hydroseleno-1H-imidazol-5-yl)-2-(trimethylammonio)propanoate, was identified from the blood and other tissues of the bluefin tuna, Thunnus orientalis. The selenium-containing compound was purified from the tuna blood in several chromatographic steps. High resolution mass spectrometry and nuclear magnetic resonance spectroscopy showed that the exact mass of the [M+H](+) ion of the compound was 533.0562 and the molecular formula was C(18)H(29)N(6)O(4)Se(2). Its gross structure was assigned as the oxidized dimeric form of an ergothioneine selenium analog in which the sulfur of ergothioneine is replaced by selenium. Therefore, we named this novel selenium-containing compound "selenoneine." By speciation analysis of organic selenium compounds using liquid chromatography inductively coupled plasma mass spectrometry, selenoneine was found widely distributed in various tissues of the tuna, with the highest concentration in blood; mackerel blood contained similar levels. Selenoneine was measurable at 2-4 orders of magnitude lower concentration in a limited set of tissues from squid, tilapia, pig, and chicken. Quantitatively, selenoneine is the predominant form of organic selenium in tuna tissues.

Proliferation and apoptosis of male germ cells in captive Atlantic bluefin tuna (Thunnus thynnus L.) treated with gonadotropin-releasing hormone agonist (GnRHa).

The effects of administration of gonadotropin-releasing hormone agonist (GnRHa) on proliferation and apoptosis of male germ cells were evaluated on Atlantic bluefin tuna (Thunnus thynnus L.) reared in captivity. Fish (n=19) were treated with a sustained-release delivery system loaded with GnRHa during the natural spawning season of 2004 and 2005 (June-July). Untreated Control fish (n=17) and adult wild spawners were used for comparison. Fish were sacrificed 2-8 d after GnRHa implantation and body weight and gonad weight were recorded, and gonads and blood were taken. Germ cell proliferation and apoptosis were evaluated through the immunohistochemical detection of proliferating cell nuclear antigen (PCNA) and the terminal deoxynucleotidyl transferase-mediated d'UTP nick end labelling (TUNEL) method, respectively. Plasma 11 ketotestosterone (11-KT) levels were measured using an ELISA method. Mean gonado-somatic index and seminiferous lobule diameter did not differ between GnRHa-treated and Control fish, and were significantly lower in captive-reared individuals than in wild spawners. Significant increases in 11-KT plasma levels and spermatogonial mitosis, along with a reduction of germ cell apoptosis were demonstrated in GnRHa-treated fish compared to Controls. The results suggest that GnRHa administration was effective in enhancing germ cell proliferation and reducing apoptosis in captive males through the stimulation of luteinizing hormone (LH) release and testicular 11-KT production.

Thermal effects on the blood respiratory properties of southern bluefin tuna, Thunnus maccoyii.

Thermal effects on the blood respiratory properties of southern bluefin tuna (Thunnus maccoyii) at 10, 23 and 36 degrees C, and at 0.5 and 1.5% CO(2) were investigated. A reversed temperature effect occurred as the oxygen partial pressure required for 50% haemoglobin saturation (P(50)) at 0.5% CO(2) decreased from 2.9 kPa at 10 degrees C to 1.7 kPa at 23 degrees C (apparent heat of oxygenation, DeltaH degrees , =+27 kJ mol(-1)). However, oxygen binding was essentially independent of temperature at warmer temperatures (P(50)=1.7-2.0 kPa from 23-36 degrees C at 0.5% CO(2); DeltaH degrees =-6.5 kJ mol(-1)). Hill's coefficient (n(H)) ranged from 1.3 to 1.6, and there was a large effect of temperature on the Bohr factor (DeltalogP(50)/DeltapH=-1.6 at 10 degrees C and -0.9 at 36 degrees C). This is the first study of whole blood to demonstrate the thermal dependence of DeltaH degrees itself, whereby the oxygen equilibrium curve is more sensitive to temperature in the lowest thermal range examined. We suggest that the functional basis for these observations lies in the necessity to ensure a sufficient oxygen supply to all tissues, including the heart and liver, without suffering from premature or excessive oxygen unloading around the heat exchanger prior to delivery of oxygen to organs and tissues that lie efferent to the exchanger.

Characterization of transthyretin in the Pacific bluefin tuna, Thunnus orientalis.

A cDNA encoding transthyretin was cloned from the Pacific bluefin tuna (Thunnus orientalis). This cDNA contains a complete open reading frame encoding 151 amino acid residues. The deduced amino acid sequence is 81% and 55% identical to the gilthead seabream and common carp forms, respectively, and 33-39% to mammalian, reptilian, and amphibian forms. A 1.0-kb transcript was found in the the liver and ovary; the liver is the main source of this protein. Analysis of triiodo-L-thyronine (T(3)) and L-thyroxine (T(4)) binding demonstrated that both T(3) and T(4) bind to bluefin transthyretin. The binding activity of T(3) for bluefin transthyretin is higher than that of T(4). These results indicate that bluefin transthyretin acts as a transporter of thyroid hormones (THs) in the plasma, and plays an important role in the function of THs in target cells.

Development and validation of a radioimmunoassay for fish insulin-like growth factor I (IGF-I) and the effect of aquaculture related stressors on circulating IGF-I levels.

This paper describes the development and validation of a commercially available radioimmunoassay (RIA) for the detection of fish insulin-like growth factor-I (IGF-I). The assay was developed using recombinant barramundi IGF-I as antigen and recombinant tuna IGF-I as radiolabelled tracer and standard. Assay sensitivity was 0.15 ng/ml, inter-assay variation was 16% (n = 9) and intra-assay variation was 3% (n = 10). Cross reactivity of less than 0.01% was found with salmon insulin, salmon IGF-II and barramundi IGF-II, less than 0.5% with human IGF-I and less than 1% with human IGF-II. Parallel dose-response inhibition curves were shown for barramundi (Lates calcarifer), coho salmon (Oncorhynchus kisutch), Southern Bluefin tuna (Thunnus maccoyii), tilapia (Oreochromis mossambicus), and seabream (Pagrus auratus) IGF-I. The assay was then used to measure stress related changes in different aquacultured fish species. Salt water acclimated Atlantic salmon smolts (Salmo salar) bathed for 2 h in fresh water showed significantly lower IGF-I concentrations than control smolts two days after the bath (53.1 compared to 32.1 ng/ml), with levels of IGF-I also lower in smolts exhibiting stunted growth (stunts). Capture and confinement of wild tuna in sea-cages resulted in a significant decrease in IGF-I levels (28 ng/ml) when compared to tuna captured and sampled immediately (48 ng/ml), but had recovered to starting levels after 3 weeks (43 ng/ml). Handling and isolation in silver perch (Bidyanus bidyanus) led to a gradual decline in IGF-I over a 12 h period (36-19 ng/ml) but showed signs of recovery by 24 h (24 ng/ml) and had recovered fully 72 h after treatment (40 ng/ml). A similar trial in black bream (Acanthopagrus butcherii) showed comparable results with IGF-I levels gradually decreasing (40-26 ng/ml) over 24 h, results that were mirrored by cortisol concentrations which increased during this time (1-26 ng/ml). In the studies presented here changes in IGF-I levels were not observed for at least 3 h after exposure to the stressor. We suggest this is due to the endocrine nature of IGF-I regulation and the clearance rate of IGF-I in vivo.

Comparative analysis of autoxidation of haemoglobin.

Autoxidation of oxyhaemoglobin (oxyHb) to methaemoglobin was measured at different temperatures in haemoglobin solutions from Atlantic hagfish, river lamprey, common carp, yellowfin tuna and pig. The aims were to evaluate the impact of the absent distal histidine in hagfish haemoglobin, the importance of oxyHb being either monomeric (hagfish and lamprey) or tetrameric (carp, tuna and pig) and to gain information on the temperature-sensitivity of autoxidation. The rate of autoxidation was lower in hagfish than in carp, yellowfin tuna and lamprey haemoglobins at any given temperature. Substitution of the distal histidine residue (His E7) with glutamine in hagfish haemoglobin was therefore not associated with an accelerated autoxidation, as might be expected on the basis of the normal protective role of His E7. Glutamine may have similar qualities to histidine and be involved in the low susceptibility to autoxidation. The low oxidation rate of hagfish haemoglobin, together with an oxidation rate of lamprey haemoglobin that did not differ from that of carp and yellowfin tuna haemoglobins, also revealed that autoxidation was not accelerated in the monomeric oxyhaemoglobins. Pig haemoglobin was oxidised more slowly than fish haemoglobins, demonstrating that fish haemoglobins are more sensitive to autoxidation than mammalian haemoglobins. The rate of autoxidation of hagfish haemoglobin was, however, only significantly greater than that of pig haemoglobin at high temperatures. Autoxidation was accelerated by rising temperature in all haemoglobins. Arrhenius plots of carp and yellowfin tuna haemoglobin revealed a break at 25 degrees C, reflecting a lower temperature-sensitivity between 5 and 25 degrees C than between 25 and 40 degrees C.

Hydrogen ion binding properties of tuna haemoglobins.

Tunas are very active fish with a high aerobic capacity, but they also regularly perform burst swimming with massive production of lactic acid. The present study examines whether H(+) buffering by tuna haemoglobin (Hb) is elevated to cope with metabolic acidoses (by analogy with the high buffer capacity of tuna white musculature) or whether the Hb-H(+) binding properties resemble other teleosts that have low buffer values and high Haldane effects. H(+) titration of oxygenated and deoxygenated composite Hb from yellowfin tuna, skipjack tuna and bigeye tuna in 0.1 M KCl revealed low Hb-specific buffer values in all three tunas. Values at physiological pH were comparable to those reported in less active species such as carp and eel. The fixed acid Haldane effect was large (maximal uptakes of close to 4 mol H(+) per mol Hb tetramer upon deoxygenation). Thus, the Hb-H(+) binding properties of very active tuna species correspond to other teleosts. Low Hb buffer values may be a pre-requisite for the regulation of red blood cell pH via Na(+)/H(+) exchange. Approximately nine "neutral" groups were titratable in tuna Hbs, suggesting that two alpha-amino groups and seven histidine residues are titrated within each tetramer.

Responses of the red blood cells from two high-energy-demand teleosts, yellowfin tuna (Thunnus albacares) and skipjack tuna (Katsuwonus pelamis), to catecholamines.

In fishes, catecholamines increase red blood cell intracellular pH through stimulation of a sodium/proton (Na+/H+) antiporter. This response can counteract potential reductions in blood O2 carrying capacity (due to Bohr and Root effects) when plasma pH and intracellular pH decrease during hypoxia, hypercapnia, or following exhaustive exercise. Tuna physiology and behavior dictate exceptionally high rates of O2 delivery to the tissues often under adverse conditions, but especially during recovery from exhaustive exercise when plasma pH may be reduced by as much as 0.4 pH units. We hypothesize that blood O2 transport during periods of metabolic acidosis could be especially critical in tunas and the response of rbc to catecholamines elevated to an extreme. We therefore investigated the in vitro response of red blood cells from yellowfin tuna (Thunnus albacares) and skipjack tuna (Katsuwonus pelamis) to catecholamines. Tuna red blood cells had a typical response to catecholamines, indicated by a rapid decrease in plasma pH. Amiloride reduced the response, whereas 4,4'diisothiocyanatostilbene-2,2'-disulphonic acid enhanced both the decrease in plasma pH and the increase in intracellular pH. Changes in plasma [Na+], [Cl-], and [K+] were consistent with the hypothesis that tuna red blood cells have a Na+/H+ antiporter similar to that described for other teleost red blood cells. Red blood cells from both tuna species were more responsive to noradrenaline than adrenaline. At identical catecholamine concentrations, the decrease in plasma pH was greater in skipjack tuna blood, the more active of the two tuna species. Based on changes in plasma pH, the response of red blood cells to catecholamines from both tuna species was less than that of rainbow trout (Oncorhynchus mykiss) red blood cells, but greater than that of cod (Gadus morhua) red blood cells. Noradrenaline had no measurable influence on the O2 affinity of skipjack tuna blood and only slightly increased the O2 affinity of yellowfin tuna blood. Our results, therefore, do not support our original hypothesis. The catecholamine response of red blood cells from high-energy-demand teleosts (i.e., tunas) is not enhanced compared to other teleosts. There are data on changes in cardio-respiratory function in tunas caused by acute hypoxia and modest increases in activity, but there are no data on the changes in cardio-respiratory function in tunas accompanying the large increases in metabolic rate seen during recovery from exhaustive exercise. However, we conclude that during those instances where high rates of O2 delivery to the tissues are needed, tunas' ability to increase cardiac output, ventilation volume, blood O2 carrying capacity, and effective respiratory (i.e., gill) surface area are probably more important than are the responses of red blood cells to catecholamines. We also use our data to investigate the extent of the Haldane effect and its relationship to blood O2 and CO2 transport in yellowfin tuna. Yellowfin tuna blood shows a large Haldane effect; intracellular pH increases 0.20 units during oxygenation. The largest change in intracellular pH occurs between 40-100% O2 saturation, indicating that yellowfin tuna, like other teleosts, fully exploit the Haldane effect over the normal physiological range of blood O2 saturation.

Homeothermic fish and hemoglobin: primary structure of the hemoglobin from bluefin tuna (Thunnus thynnus, Scromboidei).

Some fish are warm-bodied, e.g. the bluefin tuna (Thunnus thynnus), which has a muscle temperature 12-17 degrees C higher than its environment. This endothermy is achieved by aerobic metabolism and conserved by means of a heat-exchanger system. The hemoglobins of bluefin tuna are adapted to these conditions by their endothermic oxygenation, thus contributing to the preservation of the body energy. This is a new and so far unique property of tuna hemoglobin. The primary structure of the alpha and beta chains of bluefin tuna hemoglobins is presented. The sequence was determined after enzymatic and chemical cleavages of the chains and sequencing of the peptides in gas- and liquid-phase sequencers. The alpha chains consists of 143 residues and are N-terminally acetylated. The beta chains have 146 amino acids and show two ambiguities at positions 140 and 142. The alpha chains differ from the human alpha chains in 65 amino-acid residues, the beta chains in 76. The hemoglobins of bluefin tuna, carp and man are compared and their different physiological properties are discussed in relation to the sequence data. From the primary structure of tuna hemoglobins, it is possible to propose a molecular basis for their peculiar endothermic transition from the T to the R structure.

Effects of temperature change on acid-base regulation in skipjack tuna (Katsuwonus pelamis) blood.

The effects of temperature change (in vitro) on acid-base balance of skipjack tuna blood were investigated. By examining the relationship between blood pH and temperature (in vitro) under conditions of constant CO2 tension (open system), it was observed that dpH/dT = -0.013 U/degrees C. This value falls well within the range of in vivo values reported for other ectothermic vertebrates, and is only slightly different than results obtained in vitro under conditions of constant CO2 content (closed system; dpH/dT = -0.0165 U/degrees C). It is concluded that changes in pH following temperature changes can be accounted for solely by the passive, in vitro behaviour of the chemical buffer system found in the blood, so that active regulatory mechanisms of pH adjustment need not be postulated for skipjack tuna.

The effect of evolution on the structure of tuna myoglobin.

The circular dichroic activity of tuna myoglobin in the far ultraviolet has been found to be lower than that of mammalian myoglobin, thus indicating a lower content of alpha-helix. Fluorescence and absorption studies have indicated that the structure of the N-terminal region of the molecule is essentially the same in all the examined apomyoglobins, whereas differences have been observed in the heme microenvironment. The prediction of secondary structure has revealed that the alpha-helical segments of tuna myoglobin, especially those involved in the formation of the heme pocket, are shorter than those of sperm whale myoglobin.

Oxygen equilibrium studies on hemoglobin from the bluefin tuna (Thunnus thynnus).

Oxygen equilibrium curves of the purified hemoglobin component I from the Atlantic bluefin tuna (Thunnus thynnus) have been determined between pH 6.5 and 8.75 at 25 degrees C, and for five temperatures between 10 and 30 degrees C at pH 7.0 and 7.5. From the equilibrium data oxygen equilibrium constants for four oxygenation steps, Ki (i = 1 to 4) were estimated. The number of the Bohr protons released on the ith oxygenation (delta Hi+), and the enthalpy and entropy changes at each oxygenation step (delta Hi and delta Si) were calculated. The Hill plot for oxygenation below neutral pH is biphasic; the top asymptote lies to the right of the bottom one and the linking limb between them exhibits a slope less than unity, exhibiting apparent negative co-operativity. The values of K1 and K2 exhibit little pH dependence, while those of K3 and K4 increase by two orders of magnitudes as the pH is changed from 6.5 to 8.75. In consequence, oxygen equilibrium above neutral pH exhibits a normal positive co-operativity. The oxygen equilibrium at lower temperature is biphasic as is that below neutral pH. The shape of the Hill plot is temperature-dependent. The affinity at low saturation decreases, and that at high saturation increases upon raising the temperature from 10 to 30 degrees C, resulting in crossing of the middle portion of the equilibrium curves at different temperatures. The delta H1 and delta H2 values are negative as are those of most other hemoglobins, but the delta H3 and delta H4 values are positive. Consideration of these results in a framework of the allosteric model extended to take account of differences between subunits has indicated that the deoxy quaternary structure is stabilized at low pH or low temperature, and that subunit heterogeneity gives rise to the biphasic oxygen equilibrium curve. An analysis of delta Hi+ suggests that the large number of the Bohr groups is responsible for the biased allosteric equilibrium towards the deoxy quaternary structure. The positive delta H3 and delta H4 values are also considered to arise from the large endothermic contribution of the Bohr protons released at the third and fourth steps of oxygenation.

Circular dichroism and conformation of fish hemoglobins.

The circular dichroism spectrum of fully liganded CO hemoglobin from the Atlantic bluefin tuna (Tunnus thynnus) shows a pH- and temperature-dependent feature at 416 nm. It is half-developed at pH 5.9 and 20 degrees C and its change with temperature corresponds to a heat of 34 kcal/mol (tetramer) for the transition. Correlation with studies on function (Morris, R. J., and Gibson, Q. H. (1982) J. Biol. Chem. 257, 4869-4874) shows that the dichroism feature changes at about 1 pH unit below the R-T transition. There is a close correlation between the 416 nm band and changes in circular dichroism at 287 nm. The new 416 nm band is seen in several fish hemoglobins, but not with human hemoglobin. With hemoglobin from Brevoortia tyrannus, which has been sufficiently studied to permit the comparison, there is a smaller gap between the change in dichroism spectrum and the functional R-T transition. So far, no change in function has been associated with the appearance of the 416 nm circular dichroism band.