Detection of Kudoa hexapunctata and Kudoa neothunni from retail raw tuna in Japan using a novel duplex polymerase chain reaction

Kudoa hexapunctata was taxonomically separated from Kudoa neothunni, but their main host is tuna. K. hexapunctata has been identified as causative agent of foodborne diseases associated with the ingestion of raw Pacific bluefin tuna (PBT) in Japan, but K. neothunni has not. Therefore, it is clinically and epidemiologically important to detect and distinguish these two species. In the present study, we developed a novel duplex polymerase chain reaction (dPCR) targeting the 28S rRNA gene sequences of K. hexapunctata and K. neothunni. The dPCR amplified the desired genetic regions of each species, and the detection limit was 10 copies/reaction. A total of 36 retail tuna samples from different fishing ports were purchased and tested by dPCR. Thirty-one tested positive for K. hexapunctata and four tested positive for K. neothunni. Several retail PBT samples were examined in some of the fishing ports, and among these samples, the detection rates of K. hexapunctata was higher than 85%, and the rates were similar between wild and farmed PBT. The detection rates of K. hexapunctata in wild and farmed retail PBT were 75% and 71%, respectively, in May. However, the rates in June and July were 100% for both. K. hexapunctata and K. neothunni myxospores were not observed in the dPCR-positive samples, except in juvenile PBT, suggesting that the number of parasites was insufficient to cause foodborne disease. Thus, dPCR is a useful method for detecting and distinguishing K. hexapunctata and K. neothunni, and can be used in epidemiological studies of these parasites.     

Detection and characterization of Kudoa thunni from uncooked yellowfin tuna (Thunnus albacares) in Southeast Asia

Myxosporean parasites Kudoa spp. have been reported in several marine fish species worldwide. However, little is known about the contamination of these parasites in raw fish in Southeast Asia, where the consumption demand of uncooked fish is increasing. In 2019, the occurrence of several cases of raw yellowfin tuna (Thunnus albacares) obtained from retail shops with the presence of unknown white, nodular cysts within the musculature have raised public health concerns for the consumption of raw marine fish in Vietnam. Microscopic examination revealed numerous myxospores with the quadratic shape of the Kudoidae. Morphologically, stained spores detected in this study are suspected to Kudoa thunni. To confirm the suspected Kudoa species, further examination of the 18S small-subunit (SSU) was conducted and the results of nucleotide sequence analysis obtained from nodular cysts revealed 99.18–100% identity to that of Kudoa thunni sequences available in GenBank. Detection of K. thunni infection in tuna in Southeast Asia highlights the need for appropriate surveillance and control measures to ensure high quality standards and safety on raw fish production and consumption.     

Kudoa prunusi n. sp. (Myxozoa: Multivalvulida) from the brain of Pacific bluefin tuna Thunnus orientalis (Temminck & Schlegel, 1844) cultured in Japan

Kudoa prunusi n. sp. (Myxozoa; Multivalvulida) is described from the brain of Pacific bluefin tuna Thunnus orientalis cultured in Japan. Numerous white cysts, up to 0.5mm in size, were found on and in the brain. Spores having typically five spore valves and five polar capsules resembled a five-petal cherry blossom in apical view and were conical shape with a round bottom in side view. Average spore size was 9.63 (8.5-10.3) μm in width and 7.50 (6.7-8.6) μm in length. The spore dimensions of K. prunusi overlapped with those of Kudoa yasunagai ex Sillago ciliata having five to six spore valves, but they were clearly distinct in spore shape, 18S rDNA and 28S rDNA sequences (0.3% and 1.7% differences, respectively). Phylogenetic analysis of 18S rDNA revealed that K. prunusi grouped with the brain-infecting multivalvulid species, K. yasunagai, K. chaetodoni, K. lethrini and K. neurophila, rather than five-valved Kudoa spp. Combined with morphological, molecular and biological differences, K. prunusi was proven to be a new species.     

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.     

A possible explanation for the population size discrepancy in tuna (genus Thunnus) estimated from mitochondrial DNA and microsatellite data

A recent study using both mitochondrial DNA (mtDNA) and microsatellite data reported on a population size discrepancy in the eastern tiger salamander where the effective population size (N_e) estimate of the former exceeded that of the latter. That study suggested, among other hypotheses, that homoplasy of microsatellite alleles is responsible for the discrepancy. In this investigation, we report 10 new cases of a similar discrepancy in five species of tuna. These cases derive from our Bayesian inferences using data from Pacific Bluefin Tuna (Thunnus orientalis) and Yellowfin Tuna (Thunnus albacares), as well as from published estimates of genetic diversity for additional populations of Yellowfin Tuna and three other tuna species. Phylogenetic character analyses of inferred genealogies of Pacific Bluefin and Yellowfin Tuna reveal similar reduced levels of mtDNA and microsatellite homoplasy. Thus, the discrepancy between inferred population sizes from mtDNA and microsatellite data in tuna is most likely not an artifact of the chosen mutation models used in the microsatellite analyses, but may reflect behavioral differences between the sexes such as female-biased philopatry and male-biased dispersal. This explanation now warrants critical testing with more local populations of tuna and with other animal and plant groups that have different life histories.     

Intra-specific variation of Kudoa spp. (Myxosporea: Multivalvulida) from apogonid fishes (Perciformes), including the description of two new species, K. cheilodipteri n. sp. and K. cookii n. sp., from Australian waters

Kudoa spp. from the musculature and intestinal mucosa of species of the teleost family Apogonidae were examined for their taxonomic identity. Two novel species are characterised: Kudoa cheilodipteri n. sp. from the musculature of Cheilodipterus quinquelineatus Cuvier, Ostorhinchus cyanosoma (Bleeker) and O. aureus (Lacépède); and Kudoa cookii n. sp. from the submucosa of the intestines of O. cookii (Macleay) only. Both species are characterised using morphology, small subunit ribosomal DNA (SSU rDNA), large subunit ribosomal DNA (LSU rDNA), and biological characters. Three new host records, O. cyanosoma, O. aureus and Apogon doederleini, and associated geographical, morphological and genetic data are also provided for Kudoa whippsi Burger & Adlard, 2010. Morphological and molecular intra-specific variation of all isolates assigned to K. whippsi is also examined. Phylogenetic analyses further support the idea that tissue tropism is a distinguishing character between morphologically similar species; species reported here display close relatedness to morphologically similar species infecting the same tissue within their hosts.     

SSU rDNA analysis of Kudoa rosenbuschi (Myxosporea) from the Argentinean hake Merluccius hubbsi

The cloning and sequencing of the small subunit (SSU) ribosomal DNA gene from Kudoa rosenbuschi (myxosporean species associated with post-mortem myoliquefaction process in the Argentinean hake Merluccius hubbsi) is reported. The SSU rDNA was found to contain 1740 bp with a single polymorphic site with either a C or T at position 221. The sequence data obtained in this study and those known sequences of Kudoa species deposited in the GenBank were all analyzed to construct a phylogenetic tree. Nucleotide sequences showed the highest degree of identity with K. funduli, followed by K. miniauriculata, K. clupeidae and K. dianae. Phylogenetic analysis placed K. rosenbuschi in the same branch of K. clupeidae and K. funduli, and showed it to be closely related to K. dianae, K. paniformis and K. miniauriculata.     

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).     

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.     

Cookie‐cutter shark Isistius spp. predation upon different tuna species from the south‐western Atlantic Ocean

The predation of cookie‐cutter sharks Isistius spp. upon the early life stages of yellowfin tuna Thunnus albacares, skipjack tuna Katsuwonus pelamis and little tunny Euthynnus alletteratus are described. New evidence suggesting a connection between commercial fishing and predation by Isistius sp. is presented, with these sharks biting tunas hooked in surface waters during daylight. The healing patterns of the wounds made by the sharks are described in detail and, although such damage is known to negatively influence market price elsewhere, it is not the case on the south‐east Brazilian coast.     

210Po Activity and Concentrations of Selected Trace Elements (As, Cd, Cu, Hg, Pb, Zn) in the Muscle Tissue of Tunas Thunnus albacares and Katsuwonus pelamis from the Eastern Pacific Ocean

Daily mineral intake (DMI) of Cu and Zn, percentage weekly intake (PWI) of As, Cd, Hg, Pb, and doses of ²¹⁰Po were estimated by using their elemental concentration in muscle of two tuna species and the average tuna consumption in Mexico. Skipjack tuna Katsuwonus pelamis had significantly (p?<?0.05) higher levels of As (1.38???g?g^?1 dw) and Cu (1.85???g?g^?1 dw) than yellowfin tuna Thunnus albacares, whereas Pb concentrations (0.18???g?g^?1 dw) were significantly (p?<?0.05) higher in T. albacares. The sequence of elemental concentrations in both species was Zn?>?Cu?>?As?>?Hg?>?Pb?>?Cd. In T. albacares, concentrations of Cd and Pb in muscle tissue were positively correlated (p?<?0.05) with weight of specimens, while Cu was negatively correlated. DMI values were below 10?%. PWI figures (<2?%) are not potentially harmful to human health. ²¹⁰Po concentration in T. albacares and K. pelamis accounts for 13.5 to 89.7?% of the median individual annual dose (7.1???Sv) from consumption of marine fish and shellfish for the world population.     

Estimation of overall fishing intensity of tuna longline fishery-yellowfin tuna (Thunnus albacares) fishery in the indian ocean as a case study

According to the FAO catch statistics, the total catch of yellowfin tuna (Thunnus albacares) from the Indian Ocean is characterised by decline in the longline fishery and rapid increase in the surface fishery. In the present communication, an attempt has been made to estimate the overall effective fishing intensity of longline fishery for yellowfin tuna by the Japanese longliners during the years 1973–1975. The results on areas and seasons of effective effort expended are presented, along with estimates of tuna availability, effective fishing intensity and the relative gear efficiency.     

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.     

Food consumption of yellowfin tuna,Thunnus albacares, in Sri Lankan waters

Synopsis A total of 4181 stomachs of yellowfin tuna,Thunnus albacares (22–164 cm FL), mainly caught by gillnets in the period from July 1984 to June 1986 were analyzed. Food consumption of yellowfin tuna in nature was determined using the values of the average stomach content, incorporating laboratory measurements of gastric evacuation rates in a feeding model. The diet of yellowfin tuna around Sri Lanka comprised a variety of macro zooplanktonic and nektonic organisms. Juveniles < 39 cm FL, are planktivores. Tuna > 40 cm FL gradually increase their consumption of fishes with increasing size. Among fish speciesAuxis sp. are the most important. The daily food consumption of juvenile yellowfin tuna (22–59 cm FL) range from 1.8 g to 136.2 g, increasing to about 284.7 g to 551.9 g for the medium size tuna (60–99 cm FL). The adult tuna (100–>130 em FL) consume around 513 g to 538.8 g of prey per day. Daily ration estimates increase from 2.1% to 5.5% of body weight with increasing size up to 70 cm FL, beyond which it decreases. The predatory pressure of yellowfin tuna on commercially important fishes and other species is discussed.     

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.     

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

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

Mitochondrial DNA analysis reveals three stocks of yellowfin tuna Thunnus albacares (Bonnaterre, 1788) in Indian waters

Yellowfin tuna (Thunnus albacares) is an epipelagic, oceanic species of family Scombridae found in tropical and subtropical region of Pacific, Indian and Atlantic Ocean. It is commercially important fish and accounts for 19 % of total tuna catches in Indian waters. In present study, population structure of yellowfin tuna was examined using sequence analysis of mitochondrial DNA from seven geographically distinct locations along the Indian coast. A 500 bp segment of D-loop region was sequenced and analysed for 321 yellowfin samples. Hierarchical analysis of molecular variance showed significant genetic differentiation among three groups (VE); (AG); (KO, TU, PO, VI, PB) analyzed (Φ _ST  = 0.03844, P ≤ 0.001). In addition, spatial analysis of molecular variance identified three genetically heterogeneous groups of yellowfin tuna in Indian waters. Results were further corroborated by significant value of nearest neighbour statistic (S _nn = 0.261, P ≤ 0.001). Thus finding of this study rejects the null hypothesis of single panmictic population of yellowfin tuna in Indian waters.     

基于栖息地指数的东太平洋黄鳍金枪鱼渔场预报

黄鳍金枪鱼是东太平洋海域重要的金枪鱼种类之一,也是我国金枪鱼延绳钓的主要捕捞对象之一。根据2011年东太平洋海域(20°N—35°S、85°W—155°W)延绳钓生产统计数据,结合表温(SST)和海面高度(SSH)的遥感数据,采用频次分析法获得黄鳍金枪鱼分布的SST和SSH适宜范围;运用一元非线性回归方法,以渔获量为适应性指数,按季度分别建立了基于SST和SSH的长鳍金枪鱼栖息地适应性指数,采用算术平均法获得基于SST和SSH环境因子的栖息地指数综合模型,并用2012年各月实际作业渔场进行验证。结果显示,黄鳍金枪鱼渔场多分布在SST为24—29℃、SSH为0.3—0.7 m的海域。采用一元非线性回归建立的各因子适应性指数模型在统计上均为显著(P0.05)。经与2012年实际生产情况比较,作业渔场预报准确性达66%以上。研究获得栖息地指数模型可为金枪鱼延绳钓渔船寻找中心渔场提供参考。