Serological evidence of an antibody response in farmed southern bluefin tuna naturally infected with the blood fluke Cardicola forsteri

In this study, adaptive immune response was investigated in farmed southern bluefin tuna, Thunnus maccoyii, infected with a sanguinicolid Cardicola forsteri. A cohort (Cohort₂₀₀₅) of southern bluefin tuna was sampled between March 2005 and August 2006. Samples were taken at the transfer of wild caught tuna to sea cages and then at regular intervals. Parasite intensity, abundance and prevalence data were recorded. An ELISA was developed to detect and quantify an antibody response against the blood fluke in southern bluefin tuna serum. Intensity and prevalence of the blood fluke were shown to peak in May 2005 at 10.9 flukes per infected fish (SE = 1.72) and 97.5% prevalence and then decreased to low prevalence (10%) and intensity (1.0). There were no significant changes in prevalence or intensity in 2006. Antibody titres and seroprevalence increased from 1.37 U μl⁻¹ and 10% at transfer in March 2005 to reach a peak in December 2005 of 25.86 U μl⁻¹ (SE = 6.26 U μl⁻¹) and 66.66%. No significant changes were observed in antibody titres for the same cohort of fish during 2006. Parasitological and serological values from Cohort₂₀₀₅ were compared to a 2006 cohort (Cohort₂₀₀₆) in March 2006 and August 2006 to determine if prior infection in Cohort₂₀₀₅ elicited any protection against infection in 2006. Although significant differences were not observed in intensities between cohorts it was shown that Cohort₂₀₀₅ had significantly lower abundances and prevalences of blood fluke infection than Cohort₂₀₀₆. Although there was no significant difference in mean antibody titres between cohorts in March 2006, the mean antibody titre of Cohort₂₀₀₆ was significantly greater than that of Cohort₂₀₀₅ in August 2006. No significant differences were observed in seroprevalence. This is one of the few studies to demonstrate the development of acquired resistance in fish against a parasite in an aquaculture environment under natural infection conditions.     

The Real maccoyii: Identifying Tuna Sushi with DNA Barcodes – Contrasting Characteristic Attributes and Genetic Distances

Background

The use of DNA barcodes for the identification of described species is one of the least controversial and most promising applications of barcoding. There is no consensus, however, as to what constitutes an appropriate identification standard and most barcoding efforts simply attempt to pair a query sequence with reference sequences and deem identification successful if it falls within the bounds of some pre-established cutoffs using genetic distance. Since the Renaissance, however, most biological classification schemes have relied on the use of diagnostic characters to identify and place species.

Methodology/Principal Findings

Here we developed a cytochrome c oxidase subunit I character-based key for the identification of all tuna species of the genus Thunnus, and compared its performance with distance-based measures for identification of 68 samples of tuna sushi purchased from 31 restaurants in Manhattan (New York City) and Denver, Colorado. Both the character-based key and GenBank BLAST successfully identified 100% of the tuna samples, while the Barcode of Life Database (BOLD) as well as genetic distance thresholds, and neighbor-joining phylogenetic tree building performed poorly in terms of species identification. A piece of tuna sushi has the potential to be an endangered species, a fraud, or a health hazard. All three of these cases were uncovered in this study. Nineteen restaurant establishments were unable to clarify or misrepresented what species they sold. Five out of nine samples sold as a variant of “white tuna” were not albacore (T. alalunga), but escolar (Lepidocybium flavorunneum), a gempylid species banned for sale in Italy and Japan due to health concerns. Nineteen samples were northern bluefin tuna (T. thynnus) or the critically endangered southern bluefin tuna (T. maccoyii), though nine restaurants that sold these species did not state these species on their menus.

Conclusions/Significance

The Convention on International Trade Endangered Species (CITES) requires that listed species must be identifiable in trade. This research fulfills this requirement for tuna, and supports the nomination of northern bluefin tuna for CITES listing in 2010.

–The question is not what you look at, but what you see Thoreau

     

Discovery of intermediate hosts for two species of blood flukes Cardicola orientalis and Cardicola forsteri (Trematoda: Aporocotylidae) infecting Pacific bluefin tuna in Japan

Fish blood flukes (Aporocotylidae) are important pathogens of farmed finfish around the world. Among them, Cardicola spp. infecting farmed tuna are considered to be serious threats to tuna farming and have received tremendous attention. We conducted periodical samplings at a tuna farming site in Japan between January and May, 2015 to determine the life cycle of Cardicola spp. We collected over 4700 terebellid polychaetes from ropes, floats and frames of tuna culture cages and found nearly 400 infected worms. Sporocysts and cercariae found in Nicolea gracilibranchis were genetically identified as Cardicola orientalis by 28S and ITS2 ribosomal DNA sequences. This was the first discovery of the intermediate host for this parasite species. Infection prevalence and the abundance of N. gracilibranchis significantly varied between sampling points and the highest number of infected terebellids were collected from ropes. We also demonstrated morphologically and molecularly that asexual stages found in a single Amphitrite sp. (Terebellidae) and adult worms isolated from farmed juvenile tuna were Cardicola forsteri. This is the first report of C. forsteri in Pacific bluefin tuna (PBT) Thunnus orientalis in Japan. Our results demonstrated that all three species of Cardicola orientalis, C. forsteri and Cardicola opisthorchis exist in Japanese farmed PBTs and that they all use terebellid polychaetes as the intermediate hosts.     

Effects of hypoxia on oxygen consumption,swimming velocity and gut evacuation in southern bluefin tuna (<Emphasis Type="Italic">Thunnus maccoyii</Emphasis>)

Of the few measurements of the behavioural and physiological responses of tuna to hypoxia, most are restricted to shallow diving tropical species. Furthermore, when wild tuna experience low dissolved oxygen, they are likely to have an increased oxygen demand associated with the metabolic cost of food digestion and assimilation (specific dynamic action). However the response of postprandial tuna to hypoxia has never been examined. This study focuses on the metabolic and behavioural responses of both fasted and postprandial southern bluefin tuna (Thunnus maccoyii) to low dissolved oxygen. Fasted T. maccoyii were exposed to dissolved oxygen levels of 4.44, 3.23, 2.49 and 1.57 mg·l⁻¹ for 20–21 h. In moderate hypoxia (4.44 and 3.23 mg·l⁻¹), swimming speed was enhanced (1.5 and 1.3 times normoxic speed, respectively) presumably to increase ventilation volume. Routine metabolic rate (R _r ) was similarly elevated (1.3 and 1.2 times normoxic R _r , respectively), most likely due to increased metabolic demand of faster swimming. At 2.49 mg·l⁻¹, swimming speed increased to over double the normoxic speed, possibly as an escape response. At 1.57 mg·l⁻¹, both swimming speed and R _r were reduced (0.8 and 0.9 times normoxic level, respectively), and tuna failed to survive the entire 20 h exposure period. This reveals that the critical oxygen level of T. maccoyii is between 1.57 and 2.49 mg·l⁻¹, demonstrating that they are remarkably well adapted to low dissolved oxygen. Feeding did not greatly influence their hypoxia tolerance with tuna surviving exposure to dissolved oxygen levels of 2.96 and 1.81 mg·l⁻¹ for 21 h, after ingesting a ration of 6.7% body weight of sardines (Sardinops sagax). In a subsequent experiment to determine the effects of hypoxia on digestion rate, T. maccoyii were fed to satiation and exposed to a dissolved oxygen level of 2.84 mg·l⁻¹ for 6.5–8 h. There was no significant difference in swimming speed, R _r and gastric evacuation rates of tuna in hypoxia compared to those in normoxia. This demonstrates that in moderate to severe hypoxia, T. maccoyii are still capable of aerobically supporting maintenance metabolism, routine swimming and specific dynamic action. It is hypothesized that adaptations which support the large metabolic scope of tuna are also likely to be beneficial for oxygen extraction and delivery in conditions of hypoxia.     

Phylogenetic relationships between tuna species of the genus Thunnus (Scombridae: Teleostei): Inconsistent implications from morphology,nuclear and mitochondrial genomes

In order to infer phylogenetic relationships between tuna species of the genus Thunnus, partial sequences of the mitochondrial cytochrome b and ATPase genes were determined in all eight species. Supplemental restriction analysis on the nuclear rRNA gene was also carried out. Pacific northern bluefin tuna (Thunnus thynnus orientalis) was found to have mtDNA distinct from that of the Atlantic subspecies (T. t. thynnus) but very similar to that from the species albacore (T. alaluga). In contrast, no differentiation in nuclear genome was observed between the Atlantic and Pacific northern bluefin tunas. The Atlantic northern bluefin and southern bluefin tunas possessed mtDNA sequences very similar to species of yellowfin tuna group and not so similar to albacore and bigeye tunas which were morphologically assigned to the bluefin tuna group. The molecular data indicate that (1) mtDNA from albacore has been incorporated into the Pacific population of northern bluefin tuna and has extensively displaced the original mtDNA, and (2) albacore is the earliest offshoot, followed by bigeye tuna in this genus, which is inconsistent with the phylogenetic relationships between these tuna species inferred from morphology. Correspondence to: S. Chow     

Postprandial metabolic increment of southern bluefin tuna Thunnus maccoyii ingesting high or low‐lipid sardines Sardinops sagax

This study examined the postprandial metabolism and swimming speed of southern bluefin tuna Thunnus maccoyii when fed sardines Sardinops sagax of either high‐lipid and high‐energy content or low‐lipid and low‐energy content. Five groups of two or three T. maccoyii (mean ±s.e. mass = 19·8 ± 0·5 kg, n = 14) were fed either low [2·2% lipid, 5·5 MJ kg^?1 gross energy (GE)] or high‐lipid (12·9%, 9·2 MJ kg^?1 GE) S. sagax. Before feeding, T. maccoyii swam at 0·74 ± 0·03 body lengths s^?1 (n = 5) and their routine metabolic rate was 305 ± 15 mg kg^?1 h^?1. Swimming speed and metabolic rate of T. maccoyii increased following feeding. Thunnus maccoyii swam 1·3 and 1·8 times faster during digestion of low and high‐lipid S. sagax, respectively. Postprandial peak metabolic rate, duration of elevated metabolism and total postprandial metabolic increment were all greater for T. maccoyii that ingested high‐lipid S. sagax. When total postprandial increment is represented as a proportion of ingested energy, there was no difference between high and low‐lipid meals, equating to between 30 and 35% of ingested energy. It was estimated that increased postprandial swimming costs account for 25 and 46% of the total postprandial metabolic response for low and high‐lipid S. sagax meals, respectively. Specific dynamic action (SDA) accounts for c. 20% of ingested energy regardless of S. sagax lipid level. These results confirm that the postprandial metabolic increment of T. maccoyii is greater than most other fish species. Much of the high cost of postprandial metabolic increment can be attributed to increased postprandial swimming costs. For T. maccoyii, it appears that activity and SDA are not independent, which complicates bioenergetic evaluation. High postprandial metabolic costs accentuate the great energetic requirements of T. maccoyii.     

DNA barcodes reveal species-specific mercury levels in tuna sushi that pose a health risk to consumers

Excessive ingestion of mercury—a health hazard associated with consuming predatory fishes—damages neurological, sensory-motor and cardiovascular functioning. The mercury levels found in Bigeye Tuna (Thunnus obesus) and bluefin tuna species (Thunnus maccoyii, Thunnus orientalis, and Thunnus thynnus), exceed or approach levels permissible by Canada, the European Union, Japan, the US, and the World Health Organization. We used DNA barcodes to identify tuna sushi samples analysed for mercury and demonstrate that the ability to identify cryptic samples in the market place allows regulatory agencies to more accurately measure the risk faced by fish consumers and enact policies that better safeguard their health.     

A new blood fluke of the genus Cardicola (Trematoda: Sanguinicolidae) from Pacific bluefin tuna Thunnus orientalis (Temminck & Schlegel, 1844) cultured in Japan

A new sanguinicolid blood fluke, Cardicola orientalis n. sp., is described from the afferent branchial artery and heart of Pacific bluefin tuna Thunnus orientalis (Temminck & Schlegel) cultured in Mie and Nagasaki Prefectures, Japan. The new species is most similar to C. ambrosioi Braicovich, Etchegoin, Timi et Sardella, 2006 from the Brazilian flathead, Percophis brasiliensis Quoy & Gaimard, but can be differentiated by the position of the female genital pore (in midline or slightly sinistral in C. orientalis vs. sinistral in C. ambrosioi) and much longer distance between male and female genital pore (101 μm vs. 27 μm). In wet mount preparations of infected fish, eggs were accumulated in great numbers in the gill lamellae and afferent filament arteries. Importance of this blood fluke infection of cultured Pacific bluefin tuna in Japan is discussed.     

Fast versus slow growing tuna species: age,growth, and implications for population dynamics and fisheries management

Growth models describe the change in length or weight as a function of age. Growth curves in tunas can take different forms from relatively simple von Bertalanffy growth curves (Atlantic bluefin, albacore tunas) to more complex two- or three-stanza growth curves (yellowfin, bigeye, skipjack, southern bluefin tunas). We reviewed the growth of the principal market tunas (albacore, bigeye, skipjack, yellowfin and the three bluefin tuna species) in all oceans to ascertain the different growth rates among tuna species and their implications for population productivity and resilience. Tunas are among the fastest-growing of all fishes. Compared to other species, tunas exhibit rapid growth (i.e., relatively high K) and achieve large body sizes (i.e., high L _∞ ). A comparison of their growth functions reveals that tunas have evolved different growth strategies. Tunas attain asymptotic sizes (L _∞ ), ranging from 75 cm FL (skipjack tuna) to 400 cm FL (Atlantic bluefin tuna), and reach L _∞ at different rates (K), varying from 0.95 year^?1 (skipjack tuna) to 0.05 year^?1 (Atlantic bluefin tuna). Skipjack tuna (followed by yellowfin tuna) is considered the “fastest growing” species of all tunas. Growth characteristics have important implications for population dynamics and fisheries management outcomes since tunas, and other fish species, with faster growth rates generally support higher estimates of Maximum Sustainable Yield (MSY) than species with slower growth rates.     

Genetic identification of Thunnus orientalis, T. thynnus, and T. maccoyii by a cytochrome b gene analysis

The three species of bluefin tunas, Thunnus orientalis, T. maccoyii, and T. thynnus, are morphologically similar, which can pose problems for fisheries management and marketing. We examined intraspecific genetic diversity and interspecific genetic boundaries among these three species by analyzing the cytochrome (Cyt) b gene. The full lengths of the nucleotide sequences were 1,141 bp in T. orientalis and T. thynnus and ranged 1,138?~?1,141 bp in T. maccoyii. Mean nucleotide diversities were 0.0019?±?0.0002 in T. thynnus (n?=?8), 0.0063?±?0.0005 in T. orientalis (n?=?22), and 0.0059?±?0.0007 in T. maccoyii (n?=?24). Average numbers of nucleotide differences and nucleotide substitutions per site among the three species were 18.748?±?2.879 and 0.017?±?0.003, respectively. The Neighbor-joining and minimum-evolution trees showed distinct clades with high bootstrapping value support, and the high Fst value indicated significant differentiation among the three species. T. thynnus, T. orientalis, and T. maccoyii could be individually distinguished from each other Thunnus tunas by the 132nd, 375th, and 1,023rd sites of the Cyt b sequences. In the mismatch analysis, Fu??s and Tajima??s tests of sequences from T. orientalis and T. maccoyii provided evidence of their population expansion dating to the middle Pleistocene.     

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 β-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.     

A glutathione peroxidase 4 (GPx4) homologue from southern bluefin tuna is a secreted protein: first report of a secreted GPx4 isoform in vertebrates

The antioxidant enzyme glutathione peroxidase 4 (GPx4) is capable of reducing complex lipid hydroperoxides in addition to hydrogen peroxide and organic hydroperoxides. Mammals express three GPx4 isoforms that are targeted to nucleoli, mitochondria or cytosol via variable amino termini. To better understand the role of this important antioxidant enzyme in marine finfish, we determined the subcellular localisation of a GPx4 homologue from southern bluefin tuna (Thunnus maccoyii; SBT). We created constructs for the expression of the selenocysteine-to-cysteine mutant of SBT GPx4 (GPx4C) tagged with enhanced green fluorescent protein (EGFP), including or lacking a putative amino-terminal signal peptide, and expressed the fusion proteins in a fish cell line. Fluorescence microscopy revealed that the full-length GPx4C-EGFP fusion protein localised to the trans-Golgi, suggesting that tuna GPx4 may be directed to the secretory pathway. Anti-GFP immunoblotting of cell lysates and proteins from culture media showed that the secretion of SBT GPx4 into the culture medium required an amino-terminal signal peptide. According to available sequence data, the SBT GPx4 isoform studied here is representative of other piscine GPx4 isoforms, suggesting that the secretion of at least one GPx4 isoform may be common amongst teleost fish.     

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).     

Profiling the glycome of Cardicola forsteri,a blood fluke parasitic to bluefin tuna

Infections by blood flukes (Cardicola spp.) are considered the most significant health issue for ranched bluefin tuna, a major aquaculture industry in Japan and Australia. The host–parasite interfaces of trematodes, namely their teguments, are particularly rich in carbohydrates, which function both in evasion and modulation of the host immune system, while some are primary antigenic targets. In this study, histochemistry and mass spectrometry techniques were used to profile the glycans of Cardicola forsteri. Fluorescent lectin staining of adult flukes indicates the presence of oligomannose (Concanavalin A-reactive) and fucosylated (Pisum sativum agglutinin-reactive) N-glycans. Additionally, reactivity of succinylated wheat germ agglutinin (s-WGA) was localised to several internal organs of the digestive and monoecious reproductive systems. Glycan structures were further investigated with tandem mass spectrometry, which revealed structures indicated by lectin reactivity. While O-glycans from these adult specimens were not detectable by mass spectrometry, several oligomannose, paucimannosidic, and complex-type N-glycans were identified, including some carrying hexuronic acid and many carrying core xylose. This is, to our knowledge, the first glycomic characterisation of a marine platyhelminth, with broader implications for research into other trematodes.     

Demographic Structure,Sex Ratio and Growth Rates of Southern Bluefin Tuna (Thunnus maccoyii) on the Spawning Ground

The demographics of the southern bluefin tuna (SBT) Thunnus maccoyii spawning stock were examined through a large-scale monitoring program of the Indonesian longline catch on the spawning ground between 1995 and 2012. The size and age structure of the spawning population has undergone significant changes since monitoring began. There has been a reduction in the relative abundance of larger/older SBT in the catch since the early 2000s, and a corresponding decrease in mean length and age, but there was no evidence of a significant truncation of the age distribution. Pulses of young SBT appear in the catches in the early- and mid-2000s and may be the first evidence of increased recruitment into the spawning stock since 1995. Fish in these two recruitment pulses were spawned around 1991 and 1997. Size-related variations in sex ratio were also observed with female bias for fish less than 170 cm FL and male bias for fish greater than 170 cm FL. This trend of increasing proportion of males with size above 170 cm FL is likely to be related to sexual dimorphism in growth rates as male length-at-age is greater than that for females after age 10 years. Mean length-at-age of fish aged 8–10 years was greater for both males and females on the spawning ground than off the spawning ground, suggesting that size may be the dominant factor determining timing of maturation in SBT. In addition to these direct results, the data and samples from this program have been central to the assessment and management of this internationally harvested stock.     

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.     

Test of an antifouling treatment on tuna fish-cages in Boston Bay,Port Lincoln,South Australia

Abstract

A test of the antifouling properties of Wattyl NetClear® was conducted on fish farms containing southern bluefin tuna (Thunnus maccoyii) during a 6-month period (February-July 2002). Wattyl NetClear® is a water-based synthetic latex-based coating where the active antifouling agent is a mixture of two isothiazolinones likely to affect both biochemical conditioning and bacterial colonisation. The development of fouling was monitored using underwater stereo-photogrammetry on 3 treated and 3 untreated net cages with the main factors treatment, depth and cage nested within treatment. A significant treatment and depth effect was found but variable through time and in some instances with significant cage effects. By the end of the study, the difference in fouling load between treated and untreated nets was 14.7%. In total 72% of all free-space data points were on treated nets. The dominating fouling organisms were Enteromorpha sp. and sponges with low settlement of blue mussel and paper oysters. Passive deposition of tuna faeces contributed significantly to the cover. The largest cover of fouling was observed in April-May with a dominance of sponges in June-July. Enteromorpha sp. dominated shallow depths while sponges dominated at deeper levels. Tuna faeces were distributed independent of depth but varied with time. The results showed that fouling of fish cages consisted of both active settlement and passive deposition, the latter independent on antifouling treatment.