Species identification method for marine products of Seriola and related species

The complete nucleotide sequences of mitochondrial DNA (mtDNA) from four Seriola spp. (S. quinqueradiata, S. lalandi, S. dumerili, and S. rivoliana) were determined with the aim of developing a species identification analysis method for discriminating between commercially important Seriola spp. and other related species. In addition, the nucleotide sequences of the mitochondrial cytochrome b gene (Cytb) from five related but less expensive species in terms of market value (Seriolella brama, S. caerulea, S. punctata, Hyperoglyphe japonica, and Rachycentron canadum), which are often used as substitutes for Seriola spp., were determined. Restriction enzyme sites were examined by comparing the nucleotide sequences, and species-specific primers were designed for PCR-based restriction fragment length polymorphism (RFLP) analysis. Based on the results of the PCR amplification studies, the four Seriola spp. and the five related species tested could be categorized into three groups according to their PCR product pattern: a 373-bp product from the four Seriola spp., a 513-bp product from three Seriolella spp. and H. japonica, and a 204-bp product from R. canadum. In addition, RFLP analysis of the PCR products was able to differentiate these fish species.     

A simple method to distinguish two commercially valuable eel species in Japan <Emphasis Type="Italic">Anguilla japonica</Emphasis> and <Emphasis Type="Italic">A. anguilla</Emphasis> using polymerase chain reaction strategy with a species-specific primer

In order to distinguish the two eel species Anguilla japonica and A. anguilla irrespective of unprocessed and processed samples, a quick, convenient method using polymerase chain reaction (PCR) with a species-specific primer was established. The comparison of partial sequences of the mitochondrial 16S ribosomal RNA gene between A. japonica and A. anguilla facilitated designing of a primer pair common to both A. japonica and A. anguilla and a primer specific to A. japonica. PCR was carried out with the three primers for total 110 specimens of A. japonica and A. anguilla. PCR products with the species-specific primer showed two bands for A. japonica and one band for A. anguilla in an agarose gel electrophoresis. This analytical procedure required only a short period and is more convenient than PCR-restriction fragment length polymorphism, one of the standard methods employed for fish species identification.     

How “The Blob” affected groundfish distributions in the Gulf of Alaska

We investigated the distributional shifts of groundfish in response to anomalous ocean conditions, particularly the recent anomalously warm period (2014–2016; “The Blob”), based on data from ten Gulf of Alaska bottom trawl surveys conducted by the Alaska Fisheries Science Center during 1996–2015. Six groundfish species were considered: Pacific cod (Gadus macrocephalus), arrowtooth flounder (Atheresthes stomias), walleye pollock (Gadus chalcogrammus), Pacific ocean perch (Sebastes alutus), northern rock sole (Lepidopsetta polyxystra), and southern rock sole (Lepidopsetta bilineata). Ontogenetic differences were examined by dividing data for each fish species into size classes. Our study demonstrated that after accounting for size‐specific depth preferences, the spatial responses of groundfish to anomalous ocean conditions differed by species and foraging guild in the central Gulf of Alaska. Pacific cod and arrowtooth flounder showed similar responses to ocean warming, but different responses to cooling. In general, Pacific cod moved to deeper depths in warmer years and moved to shallower depths in colder years. Arrowtooth flounder also moved deeper in warmer years. However, in colder years, large arrowtooth flounder (>40 cm) shifted toward shallower depths while smaller‐sized fish shifted toward deeper depths. In warmer years, large pollock (>30 cm) moved to deeper waters while smaller pollock (10–20 cm) moved to shallower waters. Pacific ocean perch exhibited an opposite response to thermal changes in habitat compared with Pacific cod and arrowtooth flounder. They moved deeper in colder years, but there was no clear change in depth as a function of size in response to warmer habitat.     

Use of juvenile salmon growth and temperature change indices to predict groundfish post age‐0 yr class strengths in the Gulf of Alaska and eastern Bering Sea

Juvenile marine growth (SW1) of salmon and a new temperature change (TC) index were evaluated as ecosystem indicators and predictors for the post age‐0 year class strength (YCS) of groundfish in the Gulf of Alaska (GOA) and eastern Bering Sea (EBS). Our hypothesis was that SW1, as measured on the scales of adult Pacific salmon (Oncorhynchus spp.), is a proxy for ocean productivity on the continental shelf, a rearing area for young salmon and groundfish. Less negative TC index values are the result of a cool late summer followed by a warm spring, conditions favorable for groundfish YCS. In the GOA, SW1 was a positive predictor of age‐1 pollock (Theragra chalcogramma), but not age‐2 sablefish (Anoplopoma fimbria) YCS, indicating that the growth of the Karluk River sockeye salmon that enter Shelikof Strait is a proxy for ocean conditions experienced by age‐0 pollock. Contrary to our hypotheses, the TC index was a negative predictor of GOA pollock YCS; and the SW1 a negative predictor of EBS pollock and cod YCS since the 1980s. Recent fisheries oceanography survey results provide insight into possible mechanisms to support the inverse SW1 and YCS relationship. For the EBS, the TC index was a significant positive predictor for pollock and cod YCS, supporting the hypothesis that a cool late summer followed by a warm spring maximizes the over‐wintering survival of pollock and cod (Gadus macrocephalus), especially since the 1980s. The TC and SW1 index showed value for the assessment of pollock and cod, but not sablefish.     

Comparison of the Nutritional Quality of Fish Meals Made from By-products of the Alaska Fishing Industry in Diets for Pacific Threadfin (Polydactylus sexfilis)

The nutritional quality of 13 commercial fish meals (10 from pollock, two from salmon, and one from cod) made from by-products of the Alaska fish processing industry was determined in diets for the marine fish Pacific threadfin Polydactylus sexfilis . Pelleted feeds were manufactured containing these meals as complete replacement of a low-temperature processed Norwegian fish meal (control). These feeds were fed to triplicate tanks of fish at the facilities of the Oceanic Institute, Hawaii for a period of 9 wk. At the end of the trial, the final weight, feed efficiency, protein efficiency ratio and survival data were subjected to ANOVA procedures. The performance parameters of the fish fed the white fish meals (pollock and cod) were not significantly different from the control. Of the two salmon meals, one promoted good growth and efficiency and one did not. It is concluded that the nutritional quality of Alaska white (pollock and cod) fish meals is equivalent to that of the low-temperature Norwegian fish meal and are of suitable quality for inclusion in commercial feeds for Pacific threadfin.     

Methods for identification and differentiation of different Shewanella spp. isolates for diagnostic use

Shewanella spp. are Gram‐negative, rod‐shaped, motile bacteria that are widely distributed in marine and freshwater environments. The bacteria are present in the physiological microflora of fish from temperate waters and are known as fish spoilage species. From clinically healthy fish and from fish with skin ulcerations, Shewanella spp. is regularly isolated, indicating a possible role as fish pathogen. In this study, 74 isolates of Shewanella spp. were analysed. For species identification, biochemical techniques, 16S rRNA sequencing, MALDI‐TOF MS and the Sherlock Microbial Identification System (MIS) based on the composition of fatty acid ethyl esters were compared. The phylogenetic relationship, cytotoxicity in vitro and resistance against antibiotics were tested. The most reliable method for species identification was 16S rRNA sequencing. From diseased fish, clinically healthy fish and the aquatic environment, different Shewanella species were isolated. This indicates that Shewanella spp. is widespread in the aquatic milieu and acts as a secondary pathogen. The virulence of Shewanella spp. is probably not depending on the species but on the isolate itself. Many isolates of Shewanella spp. were showing multiresistances against antibiotic substances, especially in samples derived from retailers and in routine diagnostics, all Shewanella spp. should therefore be tested for resistances against antibiotic agents.     

Integrating seabird dietary and groundfish stock assessment data: Can puffins predict pollock spawning stock biomass in the North Pacific?

Information on the annual variability in abundance and growth of juvenile groundfish can be useful for predicting fisheries stocks, but is often poorly known owing to difficulties in sampling fish in their first year of life. In the Western Gulf of Alaska (WGoA) and Eastern Bering Sea (EBS) ecosystems, three species of puffin (tufted and horned puffin, Fratercula cirrhata, Fratercula corniculata, and rhinoceros auklet, Cerorhinca monocerata, Alcidae), regularly prey upon (i.e., “sample”) age-0 groundfish, including walleye pollock (Gadus chalcogramma, Gadidae) and Pacific cod (Gadus microcephalus, Gadidae). Here, we test the hypothesis that integrating puffin dietary data with walleye pollock stock assessment data provides information useful for fisheries management, including indices of interannual variation in age-0 abundance and growth. To test this hypothesis, we conducted cross-correlation and regression analyses of puffin-based indices and spawning stock biomass (SSB) for the WGoA and EBS walleye pollock stocks. For the WGoA, SSB leads the abundance of age-0 fish in the puffin diet, indicating that puffins sample the downstream production of the WGoA spawning stock. By contrast, the abundance and growth of age-0 fish sampled by puffins lead SSB for the EBS stock by 1–3 years, indicating that the puffin diet proxies incoming year class strength for this stock. Our study indicates connectivity between the WGoA and EBS walleye pollock stocks. Integration of non-traditional data sources, such as seabird diet data, with stock assessment data appears useful to inform information gaps important for managing US fisheries in the North Pacific.     

Ichthyophonus‐infected walleye pollock Theragra chalcogramma (Pallas) in the eastern Bering Sea: a potential reservoir of infections in the North Pacific

In 2003, the Alaska walleye pollock industry reported product quality issues attributed to an unspecified parasite in fish muscle. Using molecular and histological methods, we identified the parasite in Bering Sea pollock as Ichthyophonus. Infected pollock were identified throughout the study area, and prevalence was greater in adults than in juveniles. This study not only provides the first documented report of Ichthyophonus in any fish species captured in the Bering Sea, but also reveals that the parasite has been present in this region for nearly 20 years and is not a recent introduction. Sequence analysis of 18S rDNA from Ichthyophonus in pollock revealed that consensus sequences were identical to published parasite sequences from Pacific herring and Yukon River Chinook salmon. Results from this study suggest potential for Ichthyophonus exposures from infected pollock via two trophic pathways; feeding on whole fish as prey and scavenging on industry‐discharged offal. Considering the notable Ichthyophonus levels in pollock, the low host specificity of the parasite and the role of this host as a central prey item in the Bering Sea, pollock likely serve as a key Ichthyophonus reservoir for other susceptible hosts in the North Pacific.     

Causes for Soft Flesh in Giant Grenadier (Albatrossia pectoralis) Fillets

Abstract

The causes of soft flesh in giant grenadier (Albatrossia pectoralis) were investigated. The softness of giant grenadier flesh was related to its high moisture and low protein content, averaging 91.4% and 6.8%, respectively, and its muscle structure. The ratio of water to protein (13.4) was almost three times higher than for Dover sole (5.9) and Alaska pollock (4.7). Compared to Alaska pollock, giant grenadier muscle had large myotomes in loose bundles, characteristic of soft fleshed fish. Protease activity was low and as in Alaska pollock not considered a factor in soft flesh.     

An Evaluation of the Suitability of Selected Waste Products in Feeds for Three Fish Species

ABSTRACT

Five types of aquatic food industry waste products (carp offal, carp roe, fish frames, trout offal and surimi processing waste) together with fish meal were evaluated for their suitability as potential fish meal replacements, partially or wholly, in diets for three species (rainbow trout, Murray cod and shortfin eel) cultured in Australia, using a number of criteria.

The proximate composition of the ingredients on a dry matter basis including protein content, lipid and ash, varied considerably. The essential amino acid (EAA) contents of the waste products and fish meal decreased in the order: carp roe > fish meal > carp offal > ‘surimi’ processing waste > fish frames > trout offal. The results of cluster analysis of A/E ratios of waste products and fish whole body fell within three clusters. The EAAI of whole body tissue of Murray cod, rainbow trout and Australian shortfin eel however, were closest to fish meal, followed by fish frame waste and/or ‘surimi’ waste. The results on A/E ratios and EAAI did not conform to the raw data on TAA and EAA. Therefore, the study emphasizes the need to have a multi-prong approach to determine the suitability of ingredients for incorporation into fish feeds.     

Isolation of the groESL cluster from Vibrio anguillarum and PCR detection targeting groEL gene

Vibrio anguillarum is a major pathogenic bacterium that causes vibriosis and septicemia in fish and shellfish. In this study, we identified the groESL genes, which encode bacterial chaperonins, from V. anguillarum. The groE gene cluster consisted of a 291-bp groES gene, a 69-bp intergenic spacer region, and a 1,635-bp groEL gene order. Sequence analysis with the groESL gene of Vibrio species exhibited that the groEL gene was more species-specific and suitable than the groES gene for V. anguillarum detection. Owing to the difficulty in distinguishing V. anguillarum from the closely related V. ordalii, we compared the sequences of groEL from V. anguillarum and the groEL homolog hsp60 from V. ordalii, in order to design a primer set based on a region dissimilar between the two. PCR with the groEL primer set produced a clear 195-bp amplicon in six serotypes of V. anguillarum, whereas 23 Vibrio species of 39 samples, including V. ordalii, and 10 species of enteric bacteria gave no bands. PCR using the groEL primers also amplified a unique product from V. anguillarum-infected flounder and oyster tissues. These results demonstrate that the groEL-target PCR assay is a sensitive and species-specific tool for the detection of V. anguillarum infection.     

Characterization of vitamin B12 compounds from commercially available fish roe products

Although fish roes (or the egg-laden ovary) are seafood products with high nutritional value and are considered abundant in vitamins including vitamin B₁₂, nevertheless, the detailed properties of vitamin B₁₂ have not been fully elucidated in fish roe products. Vitamin B₁₂ content was determined using C18-reversed-phase high-performance liquid chromatography after purification of samples from immunoaffinity columns. Salmon egg-laden ovary products (sujiko), salmon roe products (ikura), dried mullet roe products (bottarga), and white sturgeon roe products (caviar) were found to contain substantial levels of vitamin B₁₂ (more than approximately 15 µg/100 g wet weight). Interestingly, high levels of vitamin B₁₂ per 100 g wet weight have been measured in pollack roe products (tarako) and flying fish roe products (tobiko). Liquid chromatography–electrospray-ionization/tandem mass spectrometry analysis revealed that vitamin B₁₂ was the predominant corrinoid in the evaluated fish roe products examined, while no inactive corrinoid compounds were identified. These results suggest that commercially available fish roe products could be an important source of vitamin B₁₂ for use as a dietary product for humans.

     

Rapid identification of eels <Emphasis Type="Italic">Anguilla japonica</Emphasis> and <Emphasis Type="Italic">Anguilla anguilla</Emphasis> by polymerase chain reaction with single nucleotide polymorphism-based specific probes

ABSTRACT:   Standard molecular techniques, such as sequencing and restriction fragment length polymorphism analysis after polymerase chain reaction (PCR) amplification are relatively complicated, and species identification can take a long time when using such techniques. We established a quick method, using PCR with species-specific TaqMan Minor Groove Binder (MGB) probes based on single nucleotide polymorphism (SNP) to distinguish the two eel species Anguilla japonica and Anguilla anguilla . This method can be used in processed products. Partial sequences of the mitochondrial 16S rRNA gene were compared between A. japonica and A. anguilla to design a primer pair common to both A. japonica and A. anguilla and probes specific to A. japonica and A. anguilla . Different fluorescence intensities were produced in two PCR microtubes each containing A. japonica - and A. anguilla -specific probes for one target sample. We observed the fluorescence intensity of PCR products in microtubes under ultraviolet transillumination, with similar results to those obtained by real-time PCR. Therefore, SNP-based PCR is a powerful tool for identifying materials of processed foods from either A. japonica or A. anguilla .     

Genotypic diversity among Shewanella spp. collected from freshwater fish

Different Shewanella species are isolated both from healthy and from diseased fish. To date, contemporary methods do not provide sufficient insight to determine species and detail differentiation between tested strains. Bacteria isolated from cultured (n = 33), wild (n = 12) and ornamental (n = 6) fish, as well as several reference strains, were tested by 16S rRNA gene sequencing, ERIC‐PCR and pulsed‐field gel electrophoresis (PFGE) assays. Our study indicates that isolates collected from freshwater fish were genetically diverse. Based on 16S rRNA gene sequences, bacteria were clustered into groups S. putrefaciens, S. xiamenensis and S. oneidensis. Some isolates were classified only to genus Shewanella; thus, 16S rRNA gene analyses were not enough to determine the species. ERIC‐PCR revealed 49 different genotype profiles indicating that the method might be useful for differentiation of Shewanella isolates irrespectively to species identification, contrary to PFGE which is not suitable for Shewanella typing.     

A direct multiplex loop-mediated isothermal amplification method to detect three CITES-listed shark species

1. Species identification of sharks under catch or trade regulations is important for law enforcement and species conservation. Rapid detection of Convention on the International Trade in Endangered Species (CITES)-listed species is needed for on-site screening.
2. Species-specific primers were designed to target three mitochondrial genes (ND2, COI, and CytB) in both the simplex and multiplex loop-mediated isothermal amplification (LAMP) assay for the pelagic thresher shark (Alopias pelagicus), the bigeye thresher shark (Alopias superciliosus), and the scalloped hammerhead shark (Sphyrna lewini), respectively. Another primer set designed to target S. lewini was used for detection-limit testing of the LAMP assay.
3. The refined direct multiplex LAMP was used to detect the three CITES-listed shark species and omitted the lengthy DNA extraction process. A homogenizer was used to release the DNA from the shark tissues, and a simplex or multiplex LAMP reaction was conducted for 30 min in an incubator at 65°C using species-specific primer sets.
4. Positive LAMP reactions showed a colour change from pink to yellow, whereas negative reactions showed no colour change. Multiplex LAMP assays were performed using 84 samples, which successfully identified the target and non-target species and provided a fast (<1 h), simple, and reliable method to distinguish three CITES-listed shark species from the other non-target species, for either fresh or dry fin products.
5. Results of this study and the method developed will play a critical role in assisting fishery agencies and customs officials in identifying the illegal catch and trade of CITES-listed shark species.

     

Universal PCR primers for ribosomal protein gene introns of fish

Human ribosomal protein (RP) gene sequences with respect to intron/exon structures and corresponding cDNA or genomic data of fish species were obtained from the GenBank database. Based on conserved exon sequences, 128 primer pairs for 41 genes were designed for exon-primed intron-crossing (EPIC) polymerase chain reaction (PCR). In reference to the draft genome sequences of the Pacific bluefin tuna (Thunnus orientalis), 12 primer pairs expected to amplify introns of the bluefin tuna with lengths of 500–1000 bp were selected and applied to six distantly related fish species belonging to the Orders Clupeiformes, Tetraodontiformes, Pleuronectiformes, Perciformes, Scorpaeniformes, and Anguilliformes. PCR amplification was observed for at least four species in each primer pair, and all fragments were larger than those expected for intronless amplification. Single fragment amplification was observed for at least seven primer pairs per species. Fragment sizes of the bluefin tuna for nine primer pairs corresponded to those expected from the genomic data. Thus, our primer pairs are potentially applicable to a wide variety of fish species and serve as an initial step for isolating single-copy nuclear DNA sequences.     

Seasonal changes in pelagic fish biomass around the Chiswell Island Steller sea lion rookery in 2003

Fisheries acoustics surveys were conducted around the Chiswell Island rookery in the northern Gulf of Alaska at night in April and August 2003 to assess seasonal changes in prey available to Steller sea lions (Eumetopias jubatus) foraging around the rookery. Adult walleye pollock (Theragra chalcogramma) ≥28 cm fork length was the dominant biomass in the upper 50 m of the water column in both months, increasing from 122.8 kg/nmi² in April to 457.9 kg/nmi² in August. A similar pattern was observed for Pacific herring (Clupea pallasii), which averaged 2.8 and 65.6 kg/nmi² in April and August, respectively. Incidental trawl catch suggested the appearance of age-0 pollock and juvenile salmonids (Oncorhynchus spp.) around the rookery in August as well. The increased biomass of these key prey species is linked to increased foraging trip durations by lactating sea lions from Chiswell Island, and supports the general view that sea lions in the northern Gulf of Alaska are not food limited during summer months.     

Chum salmon (Oncorhynchus keta) growth and temperature indices as indicators of the year–class strength of age‐1 walleye pollock (Gadus chalcogrammus) in the eastern Bering Sea

Ecosystem‐based fisheries management requires the development of physical and biological time series that index ocean productivity for stock assessment and recruitment forecasts for commercially important species. As recruitment in marine fish is related to ocean condition, we developed proxies for ocean conditions based on sea surface temperature (SST) and biometric measurements of chum salmon (Oncorhynchus keta) captured in the walleye pollock (Gadus chalcogrammus) fishery in the eastern Bering Sea in three periods (July 16–30, September 1–15 and September 16–30). The main purpose of this paper was to evaluate Pacific salmon (Oncorhynchus spp.) growth as a possible indicator of ocean conditions that, in turn, may affect age‐1 walleye pollock recruitment. Marine growth rates of Pacific salmon are the result of a complex interplay of physical, biological and population‐based factors that fish experience as they range through oceanic habitats. These growth rates can, therefore, be viewed as indicators of recent ocean productivity. Thus, our hypothesis was that estimated intra‐annual growth in body weight of immature and maturing age‐4 male and female chum salmon may be used as a biological indicator of variations in rearing conditions also experienced by age‐0 walleye pollock; consequently, they may be used to predict the recruitment to age‐1 in walleye pollock. Summer SSTs and chum salmon growth at the end of July and September explained the largest amount of variability in walleye pollock recruitment indicating that physical and biological indices of ocean productivity can index fish recruitment.     

Development of a novel PCR assay based on the 16S-23S rRNA internal transcribed spacer region for the detection of Lactococcus garvieae

Lactococcus garvieae is recognized as an emerging pathogen in fish. Several PCR‐based methods have been developed for the detection and identification of L. garvieae; however, the sensitivity of these methods is still in question regarding the discrimination of this organism from other closely related species. Two primers, ITSLg30F and ITSLg319R, were designed from the sequence in the 16S–23S internal transcribed spacer (ITS) region and used for the specific detection of L. garvieae. L. garvieae strains including fish isolates were positive by this method. In contrast, previously developed PCR methods showed false‐positive results with non‐L. garvieae species. Our results indicate that a PCR method using the newly designed ITS primer set provides a sensitive and efficient tool for the detection of L. garvieae in fish and aquaculture environments.     

青岛地区常见鱼类过敏原的鉴定及其交叉反应研究

选择青岛地区喜食的阿拉斯加狭鳕、蓝点马鲛、大黄鱼、真鲷、鲑、大菱鲆、鲤 等7种鱼为研究对象,利用鱼类过敏患者血清,通过SDS-PAGE及免疫印迹方法对每种鱼的过敏原蛋白进行鉴定。利用兔抗鲅鱼小清蛋白多克隆抗体,通过间接ELISA和抑制性ELISA等对7种鱼的小清蛋白之间的交叉反应进行探讨。结果表明,青岛地区居民鱼类过敏原主要为48~57、33~41、28 及17 ku的蛋白,和传统公认的小清蛋白为主要过敏原的共识不同。而针对小清蛋白的交叉反应研究也表明,不同鱼类蛋白之间存在强烈的交叉反应。因此,虽然鱼类过敏原可能不同,但如果对以上7种鱼中的一种鱼过敏,最好不要食用其他任何一种鱼。