Species identification of imported and Japanese commercial green algal products based on phylogenetic analyses using the nrITS2 and 5S rDNA spacer regions

The identification of imported green algal products represents a critical challenge to the Japanese Customs Service in terms of ensuring effective control of imported quota items (species of genera Monostroma and Enteromorpha and their products). To assist officials in this difficult task, we tested the applicability of phylogenetic analyses using the nuclear-encoded ribosomal DNA internal transcribed spacer 2 (nrITS2) and 5S ribosomal DNA (rDNA) spacer regions to identify green algal products at the species level. Our nrITS2-based phylogenetic analysis was able to roughly separate the Ulva-related samples into three clades: the Ulva linza–procera–prolifera (LPP) complex and two other clades, and identified the Monostroma-related samples as M. nitidum. Subsequent detailed analysis within the LPP complex using the 5S rDNA spacer region divided the samples into two groupings (the U. linza clade and U. prolifera clade), while it detected possible hybrid samples of U. linza and U. prolifera from a number of commercial green algal products.     

PCR扩增特异性16SrDNA和溶血素基因检测致病性嗜水气单胞菌

根据已发表的气单胞菌16S rDNA基因序列及气单胞菌气溶素(aerolysin)基因序列，设计了2对引物，建立了检测致病性嗜水气单胞菌的PCR方法。通过对12株气单胞菌的检测，发现16S rDNA引物具有高度的特异性，仅对嗜水气单胞菌扩增阳性。而Aero基因引物检测结果与采用生物学方法(鲜血平板法)检测的结果符合率为97．2％，且具有高度的敏感性，可检测最低1fg的模板。将16S rDNA与Aero基因结合PCR方法检测致病性嗜水气单胞菌与用致病性嗜水气单胞菌检测试剂盒的符合率为94．4％。该方法的建立为致病性嗜水气单胞菌的检测提供了一种简便、快速的途径，是一种比较实用的致病性嗜水气单胞菌的检测方法。     

Multiplex species-specific PCR identification of native and non-native oysters (Crassostrea) in Brazil: a useful tool for application in oyster culture and stock management

In an effort to develop molecular tools for oyster identification, this study reveals the usefulness of a multiplex polymerase chain reaction (PCR) for the reliable, rapid and low-cost identification of the four oyster species found along the Brazilian coast: Crassostrea gasar, Crassostrea rhizophorae, Crassostrea gigas and Crassostrea sp. Canela originally found at Pará, Brazil. In order to perform a simultaneous identification of these species, we used a set of five primers developed and adapted from the cytochrome oxidase subunit I (COI) and internal transcribed spacer 1 (ITS1) fragments, respectively. Amplification was successful in all four species and PCR products were visualized in agarose gel. A single reaction was capable of distinguishing between the species: C. gigas, with two fragments (236 bp for COI and 718 bp for ITS1); C. gasar, with one fragment (718 bp for ITS1); Crassostrea sp., with one fragment (621 bp for ITS1) and C. rhizophorae with two fragments (377 bp for COI and 718 bp for ITS1). This molecular approach provides a simple and rapid identification of the oyster species from the Brazilian coast, thus increasing the efficient and quality of oyster culture programs by reducing the risk of wrong species identification.     

PCR-based segregation of one hybrid variety of Labeo rohita and Catla catla from their wild-types

High consumer preference together with its polyculture potential has undoubtedly driven Rohu (Labeo rohita) and Catla (Catla catla) to top the list of the most preferred fishes among the Indian major carps. Commonly found in these fishes are hybrids that can be natural or man-made. Increasing emphasis on biodiversity issues has necessitated proper stock management of these through molecular genetics techniques. Also with few morphological differences that can be used to differentiate wild types and hybrids properly, the problem demands a straightforward molecular approach. Here, we report a simple PCR-based technique that can differentiate the hybrid variety from wild types easily using three different microsatellite markers. Three sets of primers were used to amplify three different microsatellite markers from the genomic DNA isolated from pectoral fins. When the PCR products using all three primer sets were analyzed, ‘hybrid–Rohu’ could be distinguished from wild types. Whereas the hybrid–Rohu DNA yielded specific PCR products with all three primer pairs, only two PCR products were obtained either from wild-type Catla DNA (by primer sets 1 and 2) or from wild-type Rohu DNA (by primer sets 1 and 3). This study clearly demonstrates that a simple PCR-based technique will help the fish breeders and hatcheries to identify and differentiate suspected hybrid–Rohu carp from the wild types within a few hours.     

Molecular identification of macroalgal fragments in gut contents of the sea urchin <Emphasis Type="Italic">Hemicentrotus pulcherrimus</Emphasis>

In order to improve the efficiency of stock enhancement programs for the sea urchin Hemicentrotus pulcherrimus, information on food algal species, which affect growth and gonad production greatly, is necessary. Since it is difficult to identify species from the macroalgal fragments within the gut contents of the sea urchin by microscopic observation, we tried to apply a DNA barcoding method for gut contents analysis. We used a partial rbcL gene sequence for taxonomic section and newly designed primer sets, respectively, for brown algae and for red algae. Direct sequencing of the PCR products was carried out. Species identification was based on the phylogenetic relationship. We could objectively identify four species and two taxonomic groups (genus or family) in brown algae, and two species and four taxonomic groups in red algae from the gut contents. Sargassum hemiphyllum was the most abundant brown alga in the gut contents but was not dominant in the study site. The result showed the importance of identification to the species level. In addition, red algal epiphytes were detected with brown algal fragments. The DNA barcoding method will enable the researchers to verify the important role of epiphytes as a potential food source.     

Detection of Vibrio cholerae and Vibrio vulnificus by duplex PCR specific to the groEL gene

Vibrio cholerae and V. vulnificus are of major concern due to their effect on public health throughout the world. It is therefore imperative to identify a gene and method that are suitable for the accurate species-specific detection of these two species. A duplex polymerase chain reaction (PCR) assay was developed using two sets of primers targeting the groEL gene for the accurate simultaneous detection of V. cholerae and V. vulnificus. The nucleotide sequence of the groEL gene was compared with the sequences of other Vibrio and non-Vibrio species. The specificity of two primer sets for duplex PCR was checked using 24 Vibrio and 8 non-Vibrio species. The primer sets were found to be specific for these two species and could detect both of the target bacterial species without any ambiguity, even when comparing closely related species. For both species, the detection limit was 100 pg of purified genomic DNA. The duplex PCR showed high specificity and sensitivity for each species and was sufficient for the detection of V. cholerae and V. vulnificus from artificially infected shellfish tissue, flounder, and even inoculated seawater. This method is simple and cost-effective, and can be utilized for the simultaneous detection of both species, thus representing an effective tool for both epidemiologist and ecologist.     

Development of real-time PCR assays for detection of megalocytiviruses in imported ornamental fish

Megalocytiviruses have been associated globally with severe systemic disease and economic loss in farmed food fish and ornamental fish. The viruses have been spread internationally by translocation of live fish. In New Zealand, megalocytiviruses are regarded as exotic. A potential pathway for introduction has been identified, namely imported ornamental fish. In the present study, real‐time PCR assays were developed for detection of megalocytiviruses using a conserved major capsid protein gene. A SYBR green assay was developed to target all known megalocytiviruses. A second real‐time PCR assay using a molecular beacon was developed to specifically target gourami, Trichogaster trichopterus, iridovirus, a species of iridovirus previously linked to ornamental fish imports in Australia. The analytical sensitivity for the SYBR green and molecular beacon assays were 10 and 100 fg, respectively. The analytical specificity of the real‐time PCR assays determined using genomic DNA templates from three target viruses, 12 non‐target viruses and 25 aquatic bacterial species were 100%. The intra‐run and inter‐run coefficients of variation of both assays were <5%. The real‐time PCR assays developed in this study provide rapid, sensitive, and specific detection of megalocytiviruses and gourami iridovirus.     

Automatic segmentation method for live fish eggs microscopic image analysis

Automatic visual inspection (AVI) is a popular tool for object detection and analysis in many fields. In fisheries, AVI systems designed for fish egg development analysis are used for fish egg assessments in commercial and experimental fish production systems and in ecological monitoring. The first step in AVI systems is to subdivide an image into meaningful regions. The accuracy of this segmentation directly influences the success of subsequent image analysis operations such as feature selection and classification. At present, a lot of image segmentation techniques are available, but there is no universal segmentation technique suitable for all kinds of images.This paper presents a cascading automatic segmentation method for microscopic digitised images of live common carp (Cyprinuscarpio) eggs in Petri dishes filled with water. The proposed method involves five main steps:1) image pre-processing, 2) image initial segmentation by Otsu’s method, 3) segmentation post-processing based on morphological operations, 4) image edge detection and morphological operations to remove incomplete eggs, and finally 5) a Watershed method was used to resolve the problem of separating adjoining fish eggs. Testing the method in Matlab software verified that fish eggs were recognized, segmented and counted with 100% accuracy compared to manual observation in all 96 test cases.     

16种商品海参16S rRNA的PCR-RFLP鉴定方法

基于570 by左右的线粒体16S rRNA基因片段,利用3种核酸内切酶(Dde I,Hae III和Sty I)对16种海参PCR产物进行酶切,分别产生10种、5种和5种单倍型,通过单倍型的组合,即能有效区分16种海参的种类.运用此方法对19种商品海参(包括冻品和干品)进行鉴定,结果表明,9种产品属于错误贴标.本研究建立的PCR-RFLP方法方便、有效,可靠,可为海参产品评估、进出口种类鉴定提供实用高效的方法.本研究旨在为市场海参产品贴标情况的评估提供技术支撑.     

Identification of Vibrio harveyi,Vibrio ichthyoenteri,and Photobacterium damselae isolated from olive flounder Paralichthys olivaceus in Korea by multiplex PCR developed using the rpoB gene

Major fish bacterial diseases in Korea are edwardsiellosis, streptococcosis, and vibriosis. Among vibrionaceae, Listonella anguillarum, Vibrio harveyi, V. ichthyoenteri, and Photobacterium damselae were identified as causative organisms of vibriosis in flounder. In this study, we developed a multiplex PCR method using the RNA polymerase β subunit (rpoB) gene, known as a housekeeping gene for identification of Vibrio spp. causing vibriosis in flounder. Three pairs of PCR primers were designed based on the rpoB sequence of three species, V. harveyi, V. ichthyoenteri, and P. damselae. The PCR assay, using a mixture of six primers, yielded amplicons of 601, 434, and 533 bp in V. harveyi, V. ichthyoenteri, and P. damselae. None of the untargeted species yielded an amplicon. The detection limits for pure culture in kidney were 2.5 × 10⁴ cfu/g kidney for V. harveyi, 2.5 × 10⁵ cfu/g kidney for V. ichthyoenteri, and 2.5 × 10⁶ cfu/g kidney for P. damselae. From the colonies on TCBS agar plates of different samples, 632 Vibrio spp. isolated from aquacultured flounder between 2004 and 2010 were identified by the multiplex PCR method. As a result, 265 strains (41.9 %) were V. ichthyoenteri; 115 strains (18.2 %) were V. harveyi and 72 strains (11.4 %) were P. damselae.     

Ontogenetic habitat and dietary shifts in Japanese turban snail <Emphasis Type="Italic">Turbo cornutus</Emphasis> at Nagai,Sagami Bay,Japan

A field survey and a laboratory experiment were conducted to examine ontogenetic shifts in habitat and diet of the turban snail Turbo cornutus. The main habitat of turban snail juveniles smaller than 10 mm shell height (SH) was turfs of articulated coralline algae, and that of adults larger than 50 mm SH was kelp beds of Ecklonia bicyclis and Ecklonia cava. However, the ontogenetic habitat shift during the juvenile stage of 20–50 mm SH was unclear. From the results of a long-term feeding experiment and stable isotope analysis, the gelidiacean alga Gelidium elegans was assumed to be more important as a food source for juvenile turban snail than E. cava in the field. However, the frequency of individuals inhabiting gelidiacean algal turfs was low in both juvenile and adult stages. Thus, the ontogenetic shifts in habitat and diet did not coincide and other factors, i.e., daytime refuge, are important. As the growth rate was higher in the juveniles fed on the two algal species than those fed on single algal species, co-occurrence of various algal habitats in rocky shore ecosystems as a coastal ecosystem complex may enhance growth of turban snail juveniles feeding on a combination of different algal species.     

环介导恒温扩增技术检测血卵涡鞭虫

血卵涡鞭虫病是海水甲壳类的重要寄生虫病,其流行范围广、死亡率高、危害非常严重。本研究根据GenBank中已登录的血卵涡鞭虫(Hematodinium sp.)ITS1序列设计了1套引物,该引物可识别目标基因中6个不同区段。以此套引物建立了一种基于环介导恒温扩增技术(Loop-mediated isothermal amplification,LAMP)的血卵涡鞕虫病诊断方法。特异性试验结果表明,该套引物对血卵涡鞕虫检测具有较高的特异性,能有效检出血卵涡鞕虫。敏感度试验结果表明,该LAMP技术的灵敏度比常规PCR技术高4个数量级。分别运用LAMP和常规PCR技术对25份临床疑似病例进行检测,LAMP方法共检出感染病例25份,常规PCR检出感染病例23份。该技术能在65℃恒温条件下45～60min完成目的DNA的扩增,可直接通过肉眼观察反应产物中是否产生白色沉淀或经SYBRGreenI染色后通过颜色变化、扩增产物的琼脂糖凝胶电泳来定性判断结果,这将为血卵涡鞕虫病的临床诊断提供一种更加简便、快速、实用的方法。     

Neuropeptides isotocin and arginine vasotocin in urophysis of three fish species

In this study, for the first time, both neuropeptides isotocin (IT) and arginine vasotocin (AVT) have been identified and measured in urophysis, the neurohaemal organ of the caudal neurosecretory system of teleost fish. So far, AVT, but not IT, was quantified by radioimmunoassay (RIA) in urophysis of several fish species. We have used high-performance liquid chromatographic assay with fluorescence detection (HPLC-FL) preceded by solid-phase extraction (SPE) and liquid chromatography-electrospray ionization triple-quadrupole tandem mass spectrometry (LC-ESI MS/MS) technique to determine both neuropeptides in urophysis of three fish species. The efficiency of peptide’s SPE extraction was 79–85 %. In HPLC-FL method, the limits of detection (LOD) and quantification (LOQ) were estimated as 1.0 and 3.4 pmol/mL for IT and 0.25 and 2.20 pmol/mL for AVT. In LC–MS/MS method, LOD and LOQ were estimated as 0.4 and 1.2 pmol/mL for IT and 0.06 and 0.2 pmol/mL for AVT. The chromatographic methods are good alternative for RIA, because enable to measure both nonapeptides simultaneously in one sample. In round goby (Neogobius melanostomus), three-spined stickleback (Gasterosteus aculeatus) and sea bream (Sparus aurata), urophysial IT concentrations ranged between 0.056 and 0.678 pmol/mg tissue and AVT concentrations ranged between 0.0008 (or even below detection threshold) and 0.084 pmol/mg tissue.     

采用种间特异性PCR对海参产品的物种鉴定研究

运用设计出的11个种特异性PCR反向引物,通过与通用正向引物16Sar进行PCR扩增,分别在11种海参中扩增得到大小为269～406 bp的种特异性16S rRNA基因产物,而在非特异种类中扩增不出任何产物,即可有效鉴定这11种海参.此法成功检测出2种冷冻海参替代产品,即用等刺参代替仿刺参,用玉足海参代替棘辐肛参,并且...     

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.     

Application of real-time PCR for early diagnosis of diseases caused by Aeromonas salmonicida,Vibrio anguillarum,and Tenacibaculum maritimum in turbot: A field study

In the present study a multiplex real-time PCR method was developed for early detection of diseased fish infected by Aeromonas salmonicida, Vibrio anguillarum, and/or Tenacibaculum maritimum. The method consisted of the detection of three species-specific genes after DNA extraction with a commercial kit. Three types of samples were tested, and the results were compared with those of traditional diagnosis. The method obtained a limit of detection of 10⁴ cfu/mL (2 x 10² cfu/tube). Additionally, 27 samples from fish showing signs of disease were correctly diagnosed by the developed methodology, demonstrating its suitability for implementation in aquaculture.     

A technique for the isolation of microscopic algae suitable for feeding to specific invertebrate larvae

A technique is described for extracting algal cells from the gut of wild Nassarius pauperatus larvae. These cells were subsequently cultured and proved a suitable food for Nassarius pauperatus larvae cultured in the laboratory. It is suggested that the application of this technique may supply suitable food species for many species of herbivorous zooplankton, and therefore be important aquaculturally.     

Analysis of the α-amylase gene sequence and the enzyme activity of Indian rock oyster Saccostrea forskali

Indian rock oyster Saccostrea forskali is an important commercial species in Thailand. In this study, its full-length α-amylase (SfAmy) cDNA nucleotide sequence was investigated. The SfAmy cDNA was 1,689 bp long and contained a 1,563-bp open reading frame encoding 520 amino acid residues, including a 17-amino acid signal peptide. The molecular mass and the estimated isoelectric point (pI) of the deduced mature S. forskali α-amylase (SfAMY) were 55.948 kDa and 6.45, respectively. The deduced protein sequence showed 45–88 % identity to other mollusk AMYs. The molecular weight was confirmed by the weight of the purified native enzyme. The specific activities of crude and purified native enzymes toward 1 % starch were 29.53 and 187.42 U/mg. In addition, the obtained recombinant SfAMY also showed activity in digesting 1 % starch. The specific activities of the crude and purified recombinant proteins were 11.8 and 46 U/mg. Both enzymes showed optimal activity temperature at 40 °C but their optimum pH values were different, 6.0 for the native and 5.0 for the recombinant. The expression of SfAmy examined by RT-PCR showed the highest levels in the digestive gland but none was observed in the adductor muscle.     

罗氏沼虾(Macrobrachium rosenbergii)幼体病原肠杆菌PCR检测技术的建立与应用

根据罗氏沼虾(Macrobrachium rosenbergii)幼体培育期主要细菌性病原阴沟肠杆菌omp A基因序列、产气肠杆菌gry B基因序列设计特异性引物,通过对PCR扩增产物进行测序鉴定与特异性和敏感性试验,建立了两种病原菌的PCR快速检测方法,并对发病样品进行了检测。结果显示,设计的阴沟肠杆菌与产气肠杆菌检测引物能分别扩增出与预计大小一致的385 bp和201 bp的特异性片段,与其余供试菌株无交叉反应。两种检测方法的灵敏度分别为103 CFU/ml和102 CFU/ml。罗氏沼虾幼体样品的检测结果与实际发病情况一致,建立的检测方法也可直接对样品进行PCR检测,而无需细菌分离培养。本研究建立的阴沟肠杆菌与产气肠杆菌PCR检测方法具有较高的特异性与灵敏度,可缩短检测时间,该方法的建立对罗氏沼虾幼体病原的快速诊断、分子流行病学的调查及无特定病原(SPF)群体的建立具有重要意义。     

Fish and seafood traceability based on AFLP markers: Elaboration of a species database

Several sociological, health and conservation arguments request a correct labelling of seafood products. Nowadays, molecular genetics is a useful tool for food chain traceability, particularly in regards to species identification. Among the variety of PCR-based molecular markers, AFLPs (Amplified Fragment Length Polymorphisms) have recently been used to investigate genomes of different complexities. This paper assesses the potential use of the AFLP technology to determine fish and seafood species in processed commercial products and domestic stocks. In particular a species database of fish, molluscs and crustaceans has been created with the aim to identify species of origin of seafood products by previously defined AFLP patterns. Different EcoRI and TaqI primer combinations were selected from 20 screened combinations in relation to the total number of detected fragments and polymorphic ones. Most informative combinations were E32/T32, E32/T33, E33/T33, E33/T37, E33/T38, E40/T33, E40/T37, E42/T32, E42/T37. The comparison of informative markers between unknown frozen or fresh products and reference samples has enabled the accurate identification of 32 different species. The taxonomic characterization has been performed either at the species or at the population level depending on the number of available individuals. AFLP variation at the population level is particularly helpful for the stock traceability of domestic strains. Size homoplasy was also investigated in one species to assess the rate of non-homologous comigrating fragments and to detect additional polymorphic markers to be used in stock identification. Results of Band Sharing Index (BSI) and percentage of polymorphic fragments are presented and are discussed in relation to the wide applicability of AFLPs both for fish and seafood safety and authenticity testing in such fields as food traceability and restocking management. The database, available upon request at nonnis@biol.unipr.it, will be continuously updated.