Identification of subpopulations in pelagic marine fish species using amino acid composition

The spatial stock complexity of marine fish species requires that population structure is taken into account in fisheries management. The aim of this study was to determine whether the amino acid composition (AAC) of the adult fish allows the identification of subpopulations within the stock. During a cruise in November 2003 along the entire Mediterranean coast of Spain, individuals were collected of the following pelagic species: Sardina pilchardus, Sardinella aurita, Engraulis encrasicolus, Trachurus trachurus, Trachurus mediterraneus, Scomber scombrus and Scomber colias. Individuals of S. pilchardus and E. encrasicolus were also collected from the waters of the Strait of Sicily in 2002 and 2003. The AAC of the fish eyes was seen to be species specific, and therefore, the differences in AAC among species may be based on inherited characters. Moreover, a clear differentiation was seen between the Spanish and Sicilian populations of S. pilchardus and E. encrasicolus. Furthermore, in the Spanish waters of the Mediterranean Sea, discriminant analysis revealed a substantial separation between the northern and southern subpopulations of S. pilchardus, S. aurita and E. encrasicolus. Temporal variations in AAC within species in each area were lower than the spatial variations observed among areas for each species, probably reflecting the influence on the AAC of the contrasting environmental characteristics of each area. Our results indicate that the ACC of the eyes in adult fish is a good tool for discriminating among subpopulations in pelagic marine fish species.     

Production of reactive oxygen species (ROS) by various marine fish species during the larval stage

Previous studies have indicated that the devil stinger produces reactive oxygen species (ROS) during early development from fertilized egg to larva. To determine whether ROS generation is a common feature in marine fish species, we conducted chemiluminescence analysis using ROS specific probe (L012) on larvae of six marine fish species. Marbled rockfish, black rockfish, and devil stinger showed higher levels of chemiluminescence response (CR), whereas the levels of CR of sevenband grouper, tiger puffer, and red seabream were fairly lower. These CRs were inhibited by the addition of superoxide dismutase. Hypersensitive photon-counting microscopic observation of black rockfish suggested that ROS production was concentrated in the head area. Our results suggest that the larvae of these six marine fishes produce ROS to considerably different extents depending on species, and that rockfish species, belonging to ovoviviparous fish, tend to produce much higher levels of ROS especially at the later larval stage.     

核糖体转录间隔子2应用于鱼类种属的鉴别

为了防止珍稀鱼类的非法捕捞和销售, 鱼类种属的鉴别就成为非常关键的问题, 特别是形态学方法无法区分的样品(如鱼苗、鱼鳞、鱼卵、鱼肉及其加工产品等)。为了帮助珍稀鱼类资源的管理和保护, 文章报道了一种利用核糖体基因的转录间隔子2鉴别鱼类种属的分子遗传学方法: (1) 利用同一目鱼类5.8S rRNA和28S rRNA基因的保守性, 设计出扩增鲤形目鱼类这两个基因间转录间隔子2 DNA片段, 测序获得它们的碱基排列顺序; (2) 再根据不同鱼类转录间隔子2序列的差异, 设计出每种鱼的种属特异引物、种属鉴别标准物, 构建鱼类分子分类图谱, 利用PCR复合扩增技术鉴别鱼类种属。通过对国内不同地方采集的5种鲤形目鱼类的210个单一品种样本和40个混合样本的鉴别检验, 该方法能够准确、灵敏和快速鉴别这5种鱼, 可用于鱼类资源保护和评估、管理和开发, 特别是在渔业管理人员渔业执法、海关打击珍稀鱼类走私、防止商业欺诈和外来有害生物入侵等方面非常有用     

Identification of different species ofPenicillium causing deterioration of the Moroccan table grapes during storage

Penicillium glabrum, P. purpurescens, P. decumbens, P. expansum, P. chrysogenum, P. crustosum andP. aurantiogriseum are responsible for some of the alterations noticed on Moroccan table grapes cold stored. Contamination of these table grapes withPenicillium species occurs in the vineyard and inside the cooling station where other fruits which are often fungus-contaminated are also kept. All of thesePenicillium species were able to grow at 0°C apart fromP. glabrum andP. purpurescens. Consequently, refrigeration of grapes during long-term storage is not sufficient in itself in preserving their initial qualities.     

Nutritional value and bioaccumulation of heavy metals in muscle tissues of five commercially important marine fish species from the Red Sea

The study evaluated the nutritional quality and investigated the heavy metals concentration in muscle tissues of five commercially important marine fish species, including brownspotted grouper (Epinephelus chlorostigma), squaretail coralgrouper (Plectropomus areolatus), black pomfret (Parastromateus niger), goldbanded jobfish (Pristipomoides multidens), and blueskin seabream (Polysteganus coeruleopunctatus) from the Red Sea, Jeddah Coast, Saudi Arabia. Significant differences (p < 0.05) were observed in the proximate chemical composition of fish muscles in these species. The highest protein content (17.66 ± 0.58%) was achieved in blueskin seabream while the lowest (15.28 ± 0.46%) was observed in brownspotted grouper. The highest lipid content (2.97 ± 0.45%) was recorded in squaretail coralgrouper while the lowest (1.52 ± 0.26%) was observed in blueskin seabream. Heavy metal concentrations varied significantly within and between fish species under study (p < 0.05). Significant differences in the concentration of heavy metals among fish species were recorded. Results revealed that the bioaccumulation of Cr, Fe, Ni, and Cd in muscles of fish species under study was higher than the standard concentration, but that of Mn, Cu, and Pb were less than the standard concentration recommended in the EU, FAO, and WHO guidelines. In conclusion, these fish species represent a high-quality food source but is unsafe due to the level of certain minerals in their tissues. Results also indicated that the Red Sea environment is contaminated with heavy metals, which was reflected in the tissues of fishes used in this study.     

Piezotolerance of the respiratory terminal oxidase activity of the piezophilic Shewanella violacea DSS12 as compared with non-piezophilic Shewanella species

The facultative piezophile Shewanella violacea DSS12 is known to alter its respiratory components under the influence of hydrostatic pressure during growth, suggesting that it has a respiratory system that functions in adaptation to high pressure. We investigated the pressure- and temperature-dependencies of the respiratory terminal oxidase activity of the membrane of S. violacea relative to non-piezophilic Shewanella species. We observed that the activity in the membrane of S. violacea was more resistant to high pressure than those of non-piezophilic Shewanella even though DSS12 was cultured under atmospheric pressure. On the other hand, the temperature dependency of this activity was almost the same for all of the tested strain regardless of optimal growth temperature. Both high pressure and low temperature are expected to lower protein flexibility, causing a decrease in enzyme activity, but the results of this study suggest that the mechanism maintaining enzyme activity under high hydrostatic pressure is different from that at low temperature. Additionally, the responses of the activity to the pressure- and temperature-changes were independent of membrane lipid composition. Therefore, the piezotolerance of the respiratory terminal oxidases of S. violacea is perhaps dependent on the properties of the protein itself and not on the lipid composition of the membrane. Our observations suggest that S. violacea constitutively express piezotolerant respiratory terminal oxidases that serve adaptation to the deep-sea environment.     

Identification and characterization of N-acylhomoserine lactone-acylase from the fish intestinal Shewanella sp. strain MIB015

N-Acylhomoserine lactones (AHLs) are used as quorum-sensing signal molecules by many gram-negative bacteria. We have reported that Shewanella sp. strain MIB015 degrades AHLs. In the present study, we cloned the aac gene from MIB015 by PCR with specific primers based on the aac gene in Shewanella oneidensis strain MR-1, which showed high homology with the known AHL-acylases. Escherichia coli expressing Aac showed high degrading activity of AHLs with long acyl chains. HPLC analysis revealed that Aac worked as AHL-acylase, which hydrolyzed the amide bond of AHL. In addition, expression of Aac in fish pathogen Vibrio anguillarum markedly reduced AHL production and biofilm formation. In conclusion, this study indicates that Aac might be effective in quenching quorum sensing of fish pathogens.     

Exploitation-related reef fish species richness depletion in the epicenter of marine biodiversity

The central Visayan region of the Philippines historically has the highest concentration of coral reef fishes than any other large marine area in the world. This well-supported biogeographic phenomenon is contradicted by recent transect observations on coral reefs that indicates that the Visayan region and the southern Philippine Sea region have the lowest species richness in the Philippines. The Visayan region has unusually low counts of species typically exploited in fisheries and the aquarium trade. This evidence, coupled with numerous reports of intense fishing and habitat degradation and subsequent species declines at local scales suggests that this exploitation is having a cumulative effect on the overall species richness of the Visayan region. Successes in Marine Protected Areas in this region in increasing species richness at local scales suggests that improved management of these protected areas coupled with much more intensive fisheries management will be key to reviving a healthy biodiversity in the Visayas.     

Identification of RNase HII from psychrotrophic bacterium, Shewanella sp. SIB1 as a high-activity type RNase H

The gene encoding RNase HII from the psychrotrophic bacterium, Shewanella sp. SIB1 was cloned, overexpressed in Escherichia coli, and the recombinant protein was purified and biochemically characterized. SIB1 RNase HII is a monomeric protein with 212 amino acid residues and shows an amino acid sequence identity of 64% to E. coli RNase HII. The enzymatic properties of SIB1 RNase HII, such as metal ion preference, pH optimum, and cleavage mode of substrate, were similar to those of E. coli RNase HII. SIB1 RNase HII was less stable than E. coli RNase HII, but the difference was marginal. The half-lives of SIB1 and E. coli RNases HII at 30 degrees C were approximately 30 and 45 min, respectively. The midpoint of the urea denaturation curve and optimum temperature of SIB1 RNase HII were lower than those of E. coli RNase HII by approximately 0.2 M and approximately 5 degrees C, respectively. However, SIB1 RNase HII was much more active than E. coli RNase HII at all temperatures studied. The specific activity of SIB1 RNase HII at 30 degrees C was 20 times that of E. coli RNase HII. Because SIB1 RNase HII was also much more active than SIB1 RNase HI, RNases HI and HII represent low- and high-activity type RNases H, respectively, in SIB1. In contrast, RNases HI and HII represent high- and low-activity type RNases H, respectively, in E. coli. We propose that bacterial cells usually contain low- and high-activity type RNases H, but these types are not correlated with RNase H families.     

Comparative analysis of genetic structure of 2 species of marine fish Dicentrarchus labrax and Dicentrarchus punctatus

We present here the genetic structure existing among five samples of the spotted sea bass Dicentrarchus punctatus, and we compare it to what prevails in the common sea bass D. labrax, a congeneric species sampled on almost the same geographical range. A genetic distance tree inferred from the polymorphism at six microsatellite loci shows a distinct pattern for the two species. D. labrax samples appears to be genetically more homogeneous with a global Fst of 3% as compared to the 10% observed at D. punctatus, indicating a lesser level of gene flow in the latter species. While appearing more differentiated, D. punctatus presents no clear geographical organisation of its genetic variability in opposition to D. labrax samples. This allows us to propose this pair of closely relative species as a good candidate for the study by comparative analysis of the biological and/or historical factors affecting genetic differentiation in marine environment.     

Identification and typing of fish pathogenic species of the genus Tenacibaculum

Tenacibaculosis is a major bacterial disease that causes severe fish outbreaks and losses and limits the culture of a variety of commercially valuable anadromous and marine fish species in Europe, America, Asia and Oceania. Fish affected by tenacibaculosis have external lesions and necrosis that affect different areas of the body surface, reducing their commercial value. Several species of Tenacibaculum have been identified as the causal agent of tenacibaculosis in fish, including Tenacibaculum maritimum, Tenacibaculum soleae, Tenacibaculum discolor, Tenacibaculum gallaicum, Tenacibaculum dicentrarchi and “Tenacibaculum finnmarkense” (quotations marks denote species that have not been validly published). Diagnosis of tenacibaculosis is usually based on culture-dependent detection and identification techniques which are time-consuming and do not allow to differentiate closely related species. The development of reliable techniques for studying the relationships between members of the genus Tenacibaculum and for distinguishing fish-pathogenic species of Tenacibaculum genus is, therefore, a key step in understanding the diversity and incidence of tenacibaculosis in global aquaculture, designing effective prevention strategies and early implementation of infection control measures. In this review, recent advances in molecular, serological, proteomic and chemotaxonomic techniques developed for the identification and differentiation of Tenacibaculum species, as well as for the analysis of their genetic and epidemiological relationships are discussed. Key features of current diagnostic methods likely to facilitate control and prevention of tenacibaculosis and to avoid the spread of its aetiological agents are also outlined.

     

Identification and isolation of a Castellaniella species important during biostimulation of an acidic nitrate- and uranium-contaminated aquifer

Immobilization of uranium in groundwater can be achieved through microbial reduction of U(VI) to U(IV) upon electron donor addition. Microbial community structure was analyzed in ethanol-biostimulated and control sediments from a high-nitrate (>130 mM), low-pH, uranium-contaminated site in Oak Ridge, TN. Analysis of small subunit (SSU) rRNA gene clone libraries and polar lipid fatty acids from sediments revealed that biostimulation resulted in a general decrease in bacterial diversity. Specifically, biostimulation resulted in an increase in the proportion of Betaproteobacteria (10% of total clones in the control sediment versus 50 and 79% in biostimulated sediments) and a decrease in the proportion of Gammaproteobacteria and Acidobacteria. Clone libraries derived from dissimilatory nitrite reductase genes (nirK and nirS) were also dominated by clones related to Betaproteobacteria (98% and 85% of total nirK and nirS clones, respectively). Within the nirK libraries, one clone sequence made up 59 and 76% of sequences from biostimulated sediments but only made up 10% of the control nirK library. Phylogenetic analysis of SSU rRNA and nirK gene sequences from denitrifying pure cultures isolated from the site indicate that all belong to a Castellaniella species; nearly identical sequences also constituted the majority of biostimulated SSU rRNA and nirK clone libraries. Thus, by combining culture-independent with culture-dependent techniques, we were able to link SSU rRNA clone library information with nirK sequence data and conclude that a potentially novel Castellaniella species is important for in situ nitrate removal at this site.     

Universal primers for amplification of the complete mitochondrial control region in marine fish species

Through multiple alignment analysis of mitochondrial tRNA-Thr and tRNA-Phe sequences from 161 fishes, new universal primers specially targeting the entire mitochondrial control region were designed. This new primer set successfully amplified the expected PCR products from various kinds of marine fish species, belonging to various families, and the amplified segments were confirmed to be the control region by sequencing. These primers provide a useful tool to study the control region diversity in economically important fish species, the possible mechanism of control region evolution, and the functions of the conserved motifs in the control region.     

Molecular pathways during marine fish egg hydration: the role of aquaporins

The pre‐ovulatory hydration of the oocyte of marine teleosts, a unique process among vertebrates that occurs concomitantly with meiosis resumption (oocyte maturation), is a critical process for the correct development and survival of the embryo. Increasing information is available on the molecular mechanisms that control oocyte maturation in fish, but the identification of the cellular processes involved in oocyte hydration has remained long ignored. During the past few years, a number of studies have identified the major inorganic and organic osmolytes that create a transient intra‐oocytic osmotic potential for hydrating the oocytes, whereas water influx was believed to occur passively. Recent work, however, has uncovered the role of a novel molecular water channel (aquaporin), designated aquaporin‐1b (Aqp1b), which facilitates water permeation and resultant swelling of the oocyte. The Aqp1b belongs to a teleost‐specific subfamily of water‐selective aquaporins, similar to mammalian aquaporin‐1 (AQP1) that has possibly evolved by duplication of a common ancestor and further neofunctionalization in oocytes of marine teleosts for water uptake. Strikingly, Aqp1b shows specific regulatory domains at the cytoplasmic tail, which are key to the vesicular trafficking and temporal insertion of Aqp1b in the oocyte plasma membrane during the phase of maximal hydration. These findings are revealing that the mechanism of oocyte hydration in marine teleosts is a highly regulated process based on the interplay between the generation of inorganic and organic osmolytes and the controlled insertion of Aqp1b in the oocyte surface. The discovery of Aqp1b in teleosts provides an important insight into the molecular basis of the production of viable eggs in marine fish.     

Identification of functionally important cysteine residues of the human renin-binding protein as the enzyme N-acetyl-D-glucosamine 2-epimerase

Renin-binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney. It was recently identified as the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353] and its active site residue was determined to be cysteine 380 by site-directed mutagenesis [Takahashi, S. et al. (1999) J. Biochem. 126, 639-642]. To further investigate the relationship between structure and function of recombinant human (rh) RnBP as a GlcNAc 2-epimerase, we have constructed several C-terminal deletion and multi-cysteine/serine mutants of rhGlcNAc 2-epimerase and expressed them in Escherichia coli cells. The expression was detected by Western blotting using anti-rhRnBP antiserum. The C-terminal deletion mutant, Delta400-417, had approximately 50% activity relative to the wild-type enzyme, but other C-terminal deletion mutants, Delta380-417, Delta386-417, and Delta390-417, had no enzymatic activity. Mutational analysis of multi-cysteine/serine mutants revealed that cysteines 41 and 390 were critical for the activity or stabilization of the enzyme, while cysteine residues in the middle of the enzyme, cysteines 125, 210, 239, and 302, had no essential function in relation to the activity.