Identification of <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> and <Emphasis Type="Italic">Undaria undarioides</Emphasis> Laminariales,Phaeophyceae using mitochondrial 23S ribosomal DNA sequences

ABSTRACT:   Mitochondrial 23S ribosomal (r) DNA were sequenced from two Undaria species. The 23S rDNA from seven Undaria pinnatifida individuals were 2707 bp in length, whereas the 23S rDNA from four Undaria undarioides individuals were 2708–2709 bp in length. We found 15–20 nucleotide substitutions and 1–2 gaps between U. pinnatifida (the major haplotype) and U. undarioides . Based on the differences in sequences, we carried out PCR/RFLP analyses to distinguish between U. pinnatifida and U. undarioides , which showed different PCR/RFLP patterns upon Hin fI digestion. Sequence differences and PCR/RFLP analyses of mt 23S rDNA are useful for species identification of Undaria species .     

Identification of razor clams <Emphasis Type="Italic">Ensis arcuatus</Emphasis> and <Emphasis Type="Italic">Ensis siliqua</Emphasis> by PCR-RFLP analysis of ITS1 region

ABSTRACT:   Ensis arcuatus and Ensis siliqua are the most economically important species of razor clams in the European Union. Due to similarities between their shell morphology, and the differing retail value, these species are often misidentified. Therefore, it is necessary to develop an appropriate protocol to allow accurate differentiation between these species of razor clam in order to protect consumer rights, avoid commercial fraud (whether intentional or unintentional), and to enforce labeling and safety regulations. With the aim of developing a rapid and reliable method of differentiation, individuals of E. arcuatus and of E. siliqua were examined by polymerase chain reaction restriction fragment polymorphism (PCR–RFLP) using the internal transcribed spacer region 1 (ITS1). A species-specific restriction endonuclease pattern was found with the enzyme Ksp I for both species, allowing their exact identification. Thus, this work provides a simple, reliable and rapid protocol for accurate discrimination between E. arcuatus and E. siliqua , which proves useful for traceability and enabling the enforcement of labeling regulations.     

Molecular attempt to identify prey organisms of lobster phyllosoma larvae

ABSTRACT:   A molecular approach was adopted to investigate the natural diets of palinurid and scyllarid lobster phyllosoma larvae. The central domain of the 18S rDNA gene was amplified using nested polymerase chain reaction (PCR) and genomic DNA extracted from the larval hepatopancreas. The resulting 18S rDNA clones were screened using restriction fragment length polymorphism (RFLP) analysis, and then FASTA homology search and phylogenetic analysis were performed on the nucleotide sequences to identify the source of the eukaryotic organisms. The feasibility of this method was confirmed using the laboratory-reared phyllosoma larvae of the Japanese spiny lobster Panulirus japonicus that were fed either with common mussel Mytilus edulis gonads or with Artemia nauplii exclusively. Among the 804 clones isolated from five wild-caught mid- to late-stage phyllosoma larvae (three palinurids and two scyllarids), 0–132 clones per sample possessed restriction profiles distinct from those of the hosts. The Cnidaria and Urochordata DNA were identified from both the palinurid and the scyllarid larvae, which were thought to be prey animals for the mid- to late-stage phyllosoma larvae.     

Phylogenetic analysis of noxious red tide flagellates Chattonella antiqua, C. marina, C. ovata, and C. verruculosa (Raphidophyceae) based on the rRNA gene family

ABSTRACT:   Four species of Chattonella , which are well known to form red tides that are lethal to fish, were subjected to phylogenetic analysis on the basis of the ribosomal RNA genes (rDNA), 5.8S rDNA, 18S rDNA, 28S rDNA, and the flanking internal transcribed spacers 1 and 2 (ITS1 and ITS2). The 18S rDNA sequences of C. antiqua , C. marina , and C. ovata isolated from different regions in Japan were compared. They were found to be identical with each other in a sequence 1818 bp long. The sequences of the D1/D2 region in the 28S rDNA, 5.8S rDNA, and ITS region that are known to be more variable regions were also found to be identical. These homogeneities of the rRNA gene family revealed the extremely close relatedness of C. antiqua , C. marina , and C. ovata . The sequences of C. verruculosa were different from those of these three species , resulting in an 89.2% homology in the 18S rDNA sequences, 70.4% homology in the D1/D2 region in the 28S rDNA sequences, and an 81.5% homology in 5.8S rDNA sequences and the ITS regions. Chattonella verruculosa was grouped within a single cluster composed of Dictyochophyceae rather than the other species of Raphidophyceae.     

Seasonal dynamics in a coastal Vibrio community examinedby a rapid clustering method based on 16S rDNA

ABSTRACT:   A rapid clustering scheme for Vibrio specieswas developed based on the analysis of 16S rDNA, which compriseda group-specific polymerase chain reaction (PCR) and restriction fragmentlength polymorphisms (RFLP) of the PCR-amplified 16S rDNA. The group-specificPCR, using a specific primer Vib1F, clustered the Vibrio speciesinto two large groups: Vib1F⁺ group and Vib1F^– group.The PCR-RFLP, using Sca I and Bln I, further dividedthese groups into smaller groups. Finally, 46 Vibrio speciesand eight related species could be clustered into 14 groups usingthis rapid clustering method. Seasonal dynamics of the Vibrio communityin Yoshimi Bay, Hibiki-nada Sea, Japan, were examined based on therapid clustering method. In both seawater and sediments, the groupcomprised Vibrio parahaemolyticus , Vibrio alginolyticus , Vibriocampbellii , Vibrio carchariae , Vibrio harveyi and Vibrionatriegens and was predominant when the seawater temperaturewas above 20°C, whereas the group of Vibrio splendidus biotypeI and Vibrio lentus was abundant when the temperature wasaround or below 20°C.     

Species identification method for <Emphasis Type="Italic">Scombrops boops</Emphasis> and <Emphasis Type="Italic">Scombrops gilberti</Emphasis> based on polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial DNA

ABSTRACT:   Gnomefish Scombrops boops and Scombrops gilberti are commercially important fishes in Japan, but these species are often confused in the markets because of their morphological similarity. To identify these two species, we performed nucleotide sequencing and restriction fragment length polymorphism (RFLP) analysis on 16S ribosomal RNA (rRNA) gene and the control region in mitochondrial DNA. Five and 12 nucleotide substitutions were observed between species in the 777-bp 16S rRNA gene and 471-bp control region, respectively. Diagnostic restriction sites for discriminating between S. boops and S. gilberti were found in the 16S rRNA gene, but not in the control region. Polymerase chain reaction (PCR)–RFLP analysis using two enzymes, Eco NI and Mva I, clearly discriminated between S. boops and S. gilberti identified by meristic characters. The PCR–RFLP analysis identified most of the 168 Scombrops young caught in the coastal waters of the Izu and Miura peninsulas as S. boops , suggesting that S. gilberti juveniles are rare in this area.     

Application of the rpoS gene for the detection of Vibrio anguillarum in flounder and prawn by polymerase chain reaction

Vibrio anguillarum , an opportunistic fish pathogen, is the main species responsible for vibriosis, a disease that affects feral and farmed fish and shellfish, and causes considerable economic losses in marine aquaculture. In this study, we used polymerase chain reaction (PCR) to detect V. anguillarum . PCR specificity was evaluated by amplifying the rpoS gene, a general stress regulator, in six strains of V. anguillarum and 36 other bacterial species. PCR amplified a species-specific fragment (689 bp) from V. anguillarum . Furthermore, the PCR assay was sensitive enough to detect rpoS expression from 3 pg of genomic DNA , or from six colony-forming units (CFU) mL⁻¹ of cultured V. anguillarum . However, the assay was less sensitive when genomic DNA from the infected flounder and prawn was used (limit of detection, 50 ng and 10 ng g⁻¹ tissue, respectively). These data demonstrate that PCR amplification of the rpoS gene is a sensitive and species-specific method to detect V. anguillarum in practical situations.     

Spat availability of commercial bivalve species recruited on artificial collectors from the northern Gulf of California. Seasonal changes in species composition

This study reports a year‐round recruitment of spat of four commercial bivalve species; Pteria sterna, Euvola vogdesi, Pinctada mazatlanica and Pinna rugosa collected in the region of Puerto Peñasco, north‐eastern coast of the Gulf of California. Bimonthly recruitment of commercial bivalve spat on netlon^® collectors was evaluated for six sites from June 2007 to August 2008. To describe spat recruitment abundances with environmental parameters, sea surface temperature (°C) and surface chlorophyll a concentration (mg m^?3) were characterized by means of monthly Aqua/MODIS satellite data. For each species a repeated measures anova was used to evaluate differences in the number of spat between months, sites and depths. Maximum sea surface temperature was recorded in August–September (~31.5°C) and the minimum in January–February (~15°C), while the minimum surface chlorophyll a was observed in June–September (mean range = 1.5–2 mg m^?3) and the maximum in January–March (mean range = 2–5 mg m^?3). Spat recruitment showed distinct patterns; P. sterna can be characterized as having a Winter–Spring pattern, E. vogdesi a winter pattern, while P. mazatlanica and P. rugosa a summer spat recruitment pattern. This information constitutes part of the fundamental data needed for the development of aquaculture and conservation initiatives in the region based on wild spat supply.     

Identification of ommastrephid squid paralarvae collected in northern Hawaiian waters and phylogenetic implications for the family Ommastrephidae using mtDNA analysis

ABSTRACT:   A total of 110 adult individuals from four ommastrephid (family Ommastrephidae) squid species ( Ommastrephes bartramii, Sthenoteuthis oualaniensis, Eucleoteuthis luminosa, and Hyaloteuthis pelagica ) were used to obtain diagnostic DNA markers for species identification. Restriction fragment length polymorphism (RFLP) analysis of a partial segment (855 bp) of the mitochondrial DNA cytochrome oxidase I (COI) gene amplified by polymerase chain reaction (PCR) revealed that the restriction profiles of two endonucleases ( Alu  I and Tsp 509 I) were diagnostic for species identification. The restriction assay partially supplemented with nucleotide sequence analysis successfully assigned 69 damaged and morphologically equivocal ommastrephid paralarvae collected in northern Hawaiian waters, identifying 60 O. bartramii , eight S. oualaniensis , and one E. luminosa . The family Ommastrephidae appears to be monophyletic. Although the phylogenetic relationships among genera were not resolved well due to apparent homoplasy and large genetic divergence between species, COI sequence data without transitions provided support for subfamily level relationships.     

A herpes-like virus infecting Crassostrea gigas and Ruditapes philippinarum larvae in France

Concomitant sporadic high mortalities were reported in June 1997 among batches of larval Pacific oyster, Crassostrea gigas, and Manila clam, Ruditapes philippinarum , in a French commercial hatchery. Histological observation showed the presence of cellular abnormalities in affected animals. Electron transmission microscopy revealed the presence of herpes-like virus particles in infected larvae of both bivalve species. Viruses observed in C. gigas and R. philippinarum are closely related with respect to ultrastructure and morphogenesis. They were detected simultaneously in both bivalve species larvae indicating possible interspecific transmission. Moreover, PCR analysis using oyster herpes-like virus specific primers allowed amplification of fragments of expected sizes for both bivalve species and demonstrated the presence of viral DNA. The PCR products obtained for both bivalve species and digested by restriction enzymes displayed the same patterns. These data suggest that the same herpes-like virus may infect larval oysters and clams.     

Possible cytotoxic effects of the dinoflagellate, Gyrodinium aureolum, on juvenile bivalve molluscs

Juveniles of eight commercially important species of bivalve molluscs (Spisula solidissima, Argopecten irradians, Crassostrea virginica, Mytilus edulis, Mya arenaria, Ostrea edulis, Mercenaria mercenaria, Placopecten magellanicus) were exposed in the laboratory to the commonly occurring dinoflagellate, Gyrodinium aureolum. Histological analyses of gut tissues indicated that the impact of G. aureolum on the shellfish was species-specific. High rates of mortality were noted in the bay scallop, A. irradians, but not in other molluscan species. There were no pathological differences between control animals and animals fed G. aureolum in S. solidissima, M. arenaria, or M. mercenaria. The most severely affected molluscs were C. virginica and A. irradians. C. virginica did not exhibit differences in digestive gland parameters between control and experimental animals; however, several animals did show significant mantle and gill lesions. Bay scallops exhibited decreased height of absorptive cells and increased lumen diameter after exposure to Gyrodinium suggesting, at least, poor food quality of Gyrodinium. Evidence of toxic effects was not identified in the digestive gland. Several bay scallops also showed variable amounts of inflammation in the kidney associated with protozoal infestations and variable amounts of predominately rod-shaped bacteria within the urinary space. Aquaculturists, especially of scallop species, should monitor for the presence of G. aureolum. Given its large size (25-30 m), G. aureolum could be filtered from incoming water to hatcheries, thus avoiding mass mortalities of spat and juvenile scallops.     

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 .     

贝类对对虾养殖池塘沉积物中小型底栖动物的影响

通过分析凡纳滨对虾单养及其与泥蚶混养实验的沉积物的理化特性、小型底栖动物种类组成及其生物量的变化,研究贝类对对虾池中小型底栖动物的影响。实验中对虾放养密度均为17×10~4个/hm~2,泥蚶密度分别为0粒/m~2(S)、60粒/m~2(SC1)、120粒个/m~2(SC2)和180粒/m~2(SC3)。结果显示,随养殖时间增加:(1)沉积物中有机物含量呈上升趋势,pH与氧化还原电位逐渐下降;随贝类放养密度增加,底质环境恶化程度趋缓;(2)小型底栖动物的丰度和生物量呈下降趋势,随着贝类放养密度增加,小型底栖动物的群落结构变化逐渐减少;(3)回归分析表明,介形类与线虫比值与对虾产量呈较好的相关性,一定密度贝类混养有利于底泥中介形类与线虫比值的提高,初步结果为养殖过程中该比值平均要达到6,单次值不低于3.5。研究表明,对虾与泥蚶混养有利于底质的改善和小型底栖动物的生长,较高密度的泥蚶(80~140个/m~2)有明显净化底质的作用。     

Complete mitochondrial DNA sequence of Atlantic horse mackerel Trachurus trachurus and molecular identification of two commercially important species T. trachurus and T. japonicus using PCR-RFLP

ABSTRACT:   To characterize and identify mitochondrial DNA (mtDNA) nucleotide sequence variation in two commercially important Trachurus species, Trachurus trachurus and T. japonicus , the complete mtDNA sequence of T. trachurus was determined. The T. trachurus mtDNA consists of 16 559 bp, containing 22 transfer RNA (tRNA) genes, two rRNA genes, and 13 protein-coding genes. Comparing the mtDNA nucleotide sequences of the Trachurus species, a polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP) method was developed to differentiate these two commercially important species. The primer pair Lt1-ND5 and Ht1-ND5, corresponding to ND5 , was designed to amplify a 360-bp fragment. Following digestion with Eco  RI, the PCR product for T. japonicus resulted in 93- and 267-bp fragments, while T. trachurus lacked a restriction site for Eco  RI. In contrast, after digestion with Hin  fI, the T. trachurus PCR product yielded 44-, 84-, and 232-bp fragments, while the T. japonicus product was not digested. The PCR-RFLP analysis established in the present study was useful for identifying T. trachurus and T. japonicus .     

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.     

Two monoclonal antibodies for the recognition of Mytilus spp. larvae: studies on cultured larvae and tests on plankton samples

Monoclonal antibodies (mAbs) were generated against 2-day-old mussel larvae in an attempt to develop a rapid and rigorous method for the identification of mussel larvae in field plankton samples. Previously, we have shown that two of these mAbs recognised Galician Mytilus galloprovincialis obtained from monospecific cultures, but did not recognise the larvae of other bivalve species present in that area. To assess the possibility of using these mAbs in routine assays for measuring the abundance of mussel larvae in plankton, studies on cultured mussel larvae, at different stages of development, and tests on bivalve larvae from plankton samples were carried out. Initially, to see whether the two mAbs also recognise other mussel larval stages, they were tested against mussel larvae of different ages obtained from monospecific cultures. The results indicate that both antibodies stain all the stages tested, even 1-month-old postlarvae. In addition, we also demonstrate that these mAbs also recognise other forms of Mytilus. Both antibodies bind to M. galloprovincialis larvae from the Mediterranean Sea and M. edulis larvae. Finally, and more significantly, studies on field plankton samples were performed to confirm if both mAbs are really mussel-specific, and do not cross-react with larvae of any other bivalve species existing in the plankton. The results presented here clearly indicate that our two monoclonal antibodies specifically recognise the mussel larvae in field plankton samples from different geographical regions, but not the larvae of any other bivalve species. Thus, these monoclonal antibodies could be used for routine monitoring of mussel larvae in plankton samples from different sources.     

Identification of the Fish Species Processed to Fish Meal

Abstract

As a consequence of the BSE crisis in Europe, the components of meal produced from fish or warm-blooded animals are under stricter control. PCR-based techniques for species identification of fish meal have been developed and applied to meals produced from a single species each, including herring, capelin, anchovy, horse mackerel and blue whiting. DNA was extracted by means of the cationic detergent cetyltrimethylammonium bromide and a region of the mitochondrial cytochrome b gene was amplified using universal primers. The amplicon was further characterized by restriction fragment length polymorphism (RFLP) and single strand conformation polymorphism (SSCP) analysis. A number of fish meals could be differentiated due to their species-specific DNA patterns.     

珠母贝属6个种的ITS 2分子标记研究

对珠母贝属的大珠母贝、珠母贝、白珠母贝、黑珠母贝、长耳珠母贝、黑珠母贝和合浦珠母贝6个种的内部转录间隔区2(ITS2)序列及其两侧的5.8S和28S的部分序列进行了比较分析。其中黑珠母贝的序列来自GenBank。PCR扩增片段大小为600bp左右,测序结果表明,ITS2长211～254bp,两端的5.8S和28S分别长84bp和272bp(均含引物)。序列比对分析结果表明,5.8S和28S序列高度保守,不适合于种类鉴定,而ITS2序列高度变异,270个比对位点中有146个位点发生突变,其中72个位点发生插入/缺失突变。除白珠母贝和黑珠母贝之间的遗传距离较小外,其余种类之间的遗传距离远远大于种内遗传距离。基因型分析表明,每个种具有各自特有的基因型。基因型和序列变异分析表明ITS2序列可作为珍珠贝种类鉴定的分子标记。可用于种间、杂交育种、幼体和珍珠贝肉等材料的种类鉴定与遗传分析。     

Herpes-like virus infections in hatchery-reared bivalve larvae in Europe: specific viral DNA detection by PCR

Periodic losses in oyster hatcheries are regularly reported in Europe. Herpes-like virus infections seem to play a key role. Polymerase chain reaction (PCR) was used to investigate the presence of herpes-like virus DNA in larval samples belonging to different bivalve species from different geographical origins. Seventeen samples of the 81 analysed appeared positive for the herpes-like virus DNA by PCR. These results confirm previous data indicating that herpes-like virus infections occur in commercial French hatcheries. Polymerase chain reaction positive results were also obtained for bivalve larval samples originating from Spain and the UK. The number of virus DNA positive samples depended on the primer pair used. The primer pair C2/C6 appears well adapted for herpes-like virus DNA detection because of processing ease and great sensitivity. Positive samples were observed in four bivalve species: Crassostrea gigas , Ostrea edulis , Ruditapes decussatus and Ruditapes philippinarum . Herpes-like virus DNA detection is reported in larval R. decussatus for the first time. Many samples in which viral DNA was detected by PCR correspond to larval batches presenting mortalities. Herpes-like viruses may be one of the causative agents of mortalities observed in bivalve hatcheries.     

Development of primers and a procedure for specific identification of the diatom <Emphasis Type="Italic">Thalassiosira weissflogii</Emphasis>

A recent study showed Thalassiosira weissflogii to be a diatom containing suitable nutrition for larviculture of the black tiger shrimp, Penaeus monodon. Accurate and practical identification of this diatom species is therefore important for commercial hatcheries. The purpose of this study was to establish a DNA-based method of identification to supplement morphological examination, avoiding confusion with other Thalassiosira sp. Primers, 18SF/28SR1, specific for ribosomal DNA genes (3′-end of 18S rDNA through 5′-end of 28S rDNA, covering two internal transcribed spacers), were employed as a first-step polymerase chain reaction, followed by a second nested amplification using specifically designed primers, ITS1-F-D/ITS1-R-D. The nested-PCR result revealed specificity in the detection, distinguishing T. weissflogii from T. pseudonana, Cyclotella meneghiniana, and Chaetoceros sp., and the PCR fragment of the amplified region had a sequence that was 99% identical to the T. weissflogii sequence held by GenBank.