Cardicola opisthorchis n. sp. (Trematoda: Aporocotylidae) from the Pacific bluefin tuna, Thunnus orientalis (Temminck & Schlegel, 1844), cultured in Japan

A new aporocotylid blood fluke is described, based on specimens from the ventricle of the Pacific bluefin tuna, Thunnus orientalis (Temminck et Schlegel), cultured in Wakayama and Nagasaki Prefectures, Japan. The new species is morphologically similar to the members of the genus Cardicola Short, 1953, but shows distinct differences in the body form, location of the testis and the orientation of the ootype. The body of the new species is long and slender, whereas other Cardicola species are small and generally lanceolate. The testis is mostly located posterior to the caeca and anterior to the ovary, occupying 31–45% of body length, in contrast to the known Cardicola species, whose testis is typically intercaecal. The ootype is oriented anteriorly, while in most congeners, it is directed posteriorly or horizontally. Phylogenetic analyses of this aporocotylid, together with Cardicola orientalis Ogawa, Tanaka, Sugihara et Takami, 2010 from the same host, were conducted based on DNA sequences of the ITS2 rDNA and the 28S region of ribosomal RNA. The analyses revealed that the new blood fluke belongs to the genus Cardicola despite the marked morphological differences. Thus, this aporocotylid is named Cardicola opisthorchis n. sp. and the generic diagnosis is emended in this paper. In addition, 100% identity among the ITS2 sequences from the present species, Cardicola sp. from T. orientalis in Mexico and Cardicola sp. from the northern bluefin tuna, Thunnus thynnus (Linnaeus) in Spain suggests that C. opisthorchis n. sp. has a broad geographical distribution and that it infects both the Pacific and northern bluefin tuna.     

Assessment of the nutritional status of field-caught larval Pacific bluefin tuna by RNA/DNA ratio based on a starvation experiment of hatchery-reared fish

RNA/DNA ratio is a useful and reliable indicator of the nutritional status of fish larvae and juveniles. In order to assess the nutritional status of field-caught larval Pacific bluefin tuna Thunnus orientalis (Temminck et Schlegel), starvation experiments of hatchery-reared larvae were conducted and changes in the RNA/DNA ratio of fed and starved larvae were analyzed. Starvation experiments were conducted every 3 days after first feeding. The survival rate of Pacific bluefin tuna larvae ranged 10-50% after 1 day of starved conditions and growth retardation was observed immediately. These results suggest that Pacific bluefin tuna larvae have a very low tolerance to starvation. The RNA/DNA ratios of fed larvae were approximately 2.0-4.0. On the other hand, the value of starved larvae significantly decreased to 1.0-3.0. The nutritional status of 3 cohorts of field-caught tuna larvae collected in the northwestern Pacific Ocean was examined based on the value of the RNA/DNA ratio of the 1 day starved larvae. 4.35-25.77% of the cohorts were regarded as the “starving condition”, which was negatively correlated to the ambient prey densities. These findings suggest that the nutritional condition of larval Pacific bluefin tuna was influenced by the ambient prey density, and starvation itself and starvation-induced predation could greatly contribute to mortality in the larval period of Pacific bluefin tuna.     

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.     

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     

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.     

First estimates of metabolic rate in Atlantic bluefin tuna larvae

Atlantic bluefin tuna is an iconic scombrid species with a high commercial and ecological value. Despite their importance, many physiological aspects, especially during the larval stages, are still unknown. Metabolic rates are one of the understudied aspects in scombrid larvae, likely due to challenges associated to larval handling before and during respirometry trials. Gaining reliable estimates of metabolic rates is essential to understand how larvae balance their high growth needs and activity and other physiological functions, which can be very useful for fisheries ecology and aquaculture. This is the first study to (a) estimate the relationship between routine metabolic rate (RMR) and larval dry weight (DW) (mass scaling exponent) at a constant temperature of 26°C, (b) measure the RMR under light and darkness and (c) test whether the interindividual differences in the RMR are related to larval nutritional status (RNA/DNA and DNA/DW). The RMR scaled nearly isometrically with body size (b = 0.99, 0.60–31.56 mg DW) in contrast to the allometric relationship observed in most fish larvae (average b = 0.87). The results show no significant differences in larval RMR under light and darkness, suggesting similar larval activity levels in both conditions. The size explained most of the variability in RMR (97%), and nutritional condition was unrelated to the interindividual differences in routine metabolism. This is the first study to report the metabolic rates of Atlantic bluefin tuna larvae and discuss the challenges of performing bioenergetic studies with early life stages of scombrids.     

Further evidence of spawning of bluefin tuna (Thunnus thynnus L., 1758) and the tuna species (Auxis rochei Ris., 1810, Euthynnus alletteratus Raf., 1810) in the eastern Mediterranean Sea: preliminary results of TUNALEV larval survey in 2004

A tuna larval survey (TUNALEV) in the Northern Levantine Basin (Cilician Basin) was conducted onboard a trawler from 5 to 18 June 2004. To determine the spatial distribution and abundance of tuna larvae, Bongo 60 and Bongo 90 nets were used. Ichtyoplankton samples from 104 stations were taken. In total, 121 bluefin tuna (Thunnus thynnus), 94 bullet tuna (Auxis rochei) and 22 Atlantic black skipjack (Euthynnus alletteratus) larvae were collected. In comparison with the other tuna larvae, the concentration of bluefin tuna larvae was highest in the Bay of Mersin. The collected larvae in this area were composed mainly of 5–9 mm size specimens.     

Correlation of Humoral Immune Response in Southern Bluefin Tuna,T. maccoyii,with Infection Stage of the Blood Fluke,Cardicola forsteri

The blood fluke, Cardicola forsteri, is a prevalent infection in ranched southern bluefin tuna. This project aimed to define the timing and intensity of the various developmental stages of C. forsteri within southern bluefin tuna as well as to relate infection to host pathology and immune response. Archival samples from several cohorts of T. maccoyii sampled from 2008 to 2010 were used in this study. The prevalence and intensity of C. forsteri infection was described using heart flushes and histological examination. Humoral immune response, i.e. C. forsteri specific antibody, lysozyme activity, and alternative complement activity, was also described. Based on the validated and detailed C. forsteri infection timeline, relationships between infection events, physiological response, and diagnosis were proposed. Immune response developed concurrently with C. forsteri infection, with the majority of physiological response coinciding with commencing egg production. Further research is needed to confirm the origin of C. forsteri antigen which is responsible for immune response development and how T. maccoyii immune response works against infection. To aide this research, further diagnostic methods for confirmation of infection need to be developed.     

Projections of climate‐driven changes in tuna vertical habitat based on species‐specific differences in blood oxygen affinity

Oxygen concentrations are hypothesized to decrease in many areas of the ocean as a result of anthropogenically driven climate change, resulting in habitat compression for pelagic animals. The oxygen partial pressure, pO₂, at which blood is 50% saturated (P₅₀) is a measure of blood oxygen affinity and a gauge of the tolerance of animals for low ambient oxygen. Tuna species display a wide range of blood oxygen affinities (i.e., P₅₀ values) and therefore may be differentially impacted by habitat compression as they make extensive vertical movements to forage on subdaily time scales. To project the effects of end‐of‐the‐century climate change on tuna habitat, we calculate tuna P₅₀ depths (i.e., the vertical position in the water column at which ambient pO₂ is equal to species‐specific blood P₅₀ values) from 21st century Earth System Model (ESM) projections included in the fifth phase of the Climate Model Intercomparison Project (CMIP5). Overall, we project P₅₀ depths to shoal, indicating likely habitat compression for tuna species due to climate change. Tunas that will be most impacted by shoaling are Pacific and southern bluefin tunas—habitat compression is projected for the entire geographic range of Pacific bluefin tuna and for the spawning region of southern bluefin tuna. Vertical shifts in P₅₀ depths will potentially influence resource partitioning among Pacific bluefin, bigeye, yellowfin, and skipjack tunas in the northern subtropical and eastern tropical Pacific Ocean, the Arabian Sea, and the Bay of Bengal. By establishing linkages between tuna physiology and environmental conditions, we provide a mechanistic basis to project the effects of anthropogenic climate change on tuna habitats.     

Tissue Turnover Rates and Isotopic Trophic Discrimination Factors in the Endothermic Teleost,Pacific Bluefin Tuna (Thunnus orientalis)

Stable isotope analysis (SIA) of highly migratory marine pelagic animals can improve understanding of their migratory patterns and trophic ecology. However, accurate interpretation of isotopic analyses relies on knowledge of isotope turnover rates and tissue-diet isotope discrimination factors. Laboratory-derived turnover rates and discrimination factors have been difficult to obtain due to the challenges of maintaining these species in captivity. We conducted a study to determine tissue- (white muscle and liver) and isotope- (nitrogen and carbon) specific turnover rates and trophic discrimination factors (TDFs) using archived tissues from captive Pacific bluefin tuna (PBFT), Thunnus orientalis, 1–2914 days after a diet shift in captivity. Half-life values for ¹⁵N turnover in white muscle and liver were 167 and 86 days, and for ¹³C were 255 and 162 days, respectively. TDFs for white muscle and liver were 1.9 and 1.1‰ for δ ¹⁵N and 1.8 and 1.2‰ for δ ¹³C, respectively. Our results demonstrate that turnover of ¹⁵N and ¹³C in bluefin tuna tissues is well described by a single compartment first-order kinetics model. We report variability in turnover rates between tissue types and their isotope dynamics, and hypothesize that metabolic processes play a large role in turnover of nitrogen and carbon in PBFT white muscle and liver tissues. ¹⁵N in white muscle tissue showed the most predictable change with diet over time, suggesting that white muscle δ ¹⁵N data may provide the most reliable inferences for diet and migration studies using stable isotopes in wild fish. These results allow more accurate interpretation of field data and dramatically improve our ability to use stable isotope data from wild tunas to better understand their migration patterns and trophic ecology.     

Euryphorus brachypterus (Copepoda: Caligidae) on wild pacific bluefin tuna from the Tsugaru Strait,northern Japan

Parasitic copepods infecting large scombrid fishes have been known for a long time because their hosts are economically important. Most studies, however, have focused on their morphology or their infection status in aquaculture from pathological viewpoints, and very few quantitative surveys have been conducted under conditions in the wild. This study therefore investigated the prevalence of Euryphorus brachypterus (Caligidae) in wild Pacific bluefin tuna (PBF). Results of sampling from August to September 2014 at the western area of the Tsugaru Strait, Japan showed that 13.2% of the PBF individuals (n = 1978) were infected with this copepod. The prevalence of infections was highest in larger fish but varied among landing dates, which were classified into three clusters and in all smaller fish, the prevalence of infections was zero. This suggests that E. brachypterus mainly uses the larger PBF, which becomes sources of further infections in other seas, and that at least two host populations with different infection statuses at the strait.     

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.     

A Validated Methodology for Genetic Identification of Tuna Species (Genus Thunnus)

Background

Tuna species of the genus Thunnus, such as the bluefin tunas, are some of the most important and yet most endangered trade fish in the world. Identification of these species in traded forms, however, may be difficult depending on the presentation of the products, which may hamper conservation efforts on trade control. In this paper, we validated a genetic methodology that can fully distinguish between the eight Thunnus species from any kind of processed tissue.

Methodology

After testing several genetic markers, a complete discrimination of the eight tuna species was achieved using Forensically Informative Nucleotide Sequencing based primarily on the sequence variability of the hypervariable genetic marker mitochondrial DNA control region (mtDNA CR), followed, in some specific cases, by a second validation by a nuclear marker rDNA first internal transcribed spacer (ITS1). This methodology was able to distinguish all tuna species, including those belonging to the subgenus Neothunnus that are very closely related, and in consequence can not be differentiated with other genetic markers of lower variability. This methodology also took into consideration the presence of introgression that has been reported in past studies between T. thynnus, T. orientalis and T. alalunga. Finally, we applied the methodology to cross-check the species identity of 26 processed tuna samples.

Conclusions

Using the combination of two genetic markers, one mitochondrial and another nuclear, allows a full discrimination between all eight tuna species. Unexpectedly, the genetic marker traditionally used for DNA barcoding, cytochrome oxidase 1, could not differentiate all species, thus its use as a genetic marker for tuna species identification is questioned.     

Differential replication of ribosomal DNA during larval development in Calliphora (Diptera)

The hybridization of ribosomal RNA with DNA extracted from whole insects has been compared at six stages during the development of the blowfly Calliphora stygia. Total DNA from hatching larvae contains approximately 20% more rDNA than does total DNA from either mid-embryonic or adult insects. DNA from late third-instar larvae close to pupariation contains about 30% less rDNA than that from hatching larvae. The proportion of rDNA to total DNA in larvae of intermediate ages falls between these two extremes. Ribosomal RNA cistrons are thus replicated more rapidly than the remainder of the genome in the late embryonic or earliest larval stages, but more slowly during the later cycles of DNA synthesis in those larval tissues which develop high levels of polyteny.     

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.     

Description and genetic characterisation of Hysterothylacium (Nematoda: Raphidascarididae) larvae parasitic in Australian marine fishes

Nematodes belonging to the genus Hysterothylacium (family Raphidascarididae) infect various species of marine fish in both the larval and adult stages. Humans can be accidentally infected upon eating infected seafood. In spite of their importance, relatively little is known of their occurrence and systematics in Australia. An examination of various species of marine teleosts in Australian waters revealed a high prevalence of Hysterothylacium larval types. In the present study, seven previously undescribed Hysterothylacium larval morphotypes (V to VII and IX to XII) were discovered. In total we found 10 different morphotypes and we genetically characterised nine morphotypes identified. A morphological dichotomous identification key has been established to differentiate these morphotypes. Since some larvae of Hysterothylacium from marine fishes cannot be differentiated morphologically from other nematode larvae, such as Paraheterotyphlum, Heterotyphlum, Iheringascaris and Lapetascaris, the first and second internal transcribed spacers (ITS-1 and ITS-2) of nuclear ribosomal DNA (rDNA) of these larvae were characterised to confirm their taxonomic status. This genetic characterisation implied that some distinct morphotypes belong to different developmental stages of the same species. In addition, it revealed that some morphotypes can comprise distinct genotypes. No match was found between ITS-1 and ITS-2 sequences obtained from larvae in the present study and those from adults available in the GenBank, highlighting the lack of knowledge on occurrence of adult nematodes infecting Australian fish.