Genetic differences within and between species of deep-sea crabs (chaceon) from the North Atlantic Ocean

The deep-sea red crab Chaceon quinquedens is a commercially important crustacean on the Atlantic continental shelf and slope of North America. To assess genetic subdivision in C. quinquedens, we examined the nucleotide sequence of the mitochondrial 16S rDNA gene and the internal transcribed spacers (ITS) of the nuclear ribosomal repeat in samples from southern New England and the Gulf of Mexico. We compared those data to sequences from two congeners, a sympatric species from the Florida coast, C. fenneri, and an allopatric eastern Atlantic species, C. affinis. The 16S rDNA data consisted of 379 aligned nucleotides obtained from 37 individuals. The greatest genetic difference among geographical groups or nominal species was between C. quinquedens from southern New England and C. quinquedens from the Gulf of Mexico. Haplotypes from these two groups had a minimum of 10 differences. All 11 C. fenneri samples matched the most common haplotype found in C. quinquedens from the Gulf of Mexico, and this haplotype was not detected in C. quinquedens from southern New England. The three haplotypes of C. affinis were unique to that recognized species, but those haplotypes differed only slightly from those of C. fenneri and C. quinquedens from the Gulf of Mexico. Based on 16S rDNA and ITS data, genetic differences between C. quinquedens from southern New England and the Gulf of Mexico are large enough to conclude that these are different fishery stocks. Our results also indicate that the designation of morphological species within the commercially important genus Chaceon is not congruent with evolutionary history. The genetic similarity of C. affinis from the eastern Atlantic and C. quinquedens from the Gulf of Mexico suggests these trans-Atlantic taxa share a more recent common history than the two populations of "C. quinquedens" that we examined.     

Exploitation of a turbot (Scophthalmus maximus L.) immune-related expressed sequence tag (EST) database for microsatellite screening and validation

In this study, we identified and characterized 160 microsatellite loci from an expressed sequence tag (EST) database generated from immune-related organs of turbot (Scophthalmus maximus). A final set of 83 new polymorphic microsatellites were validated after the analysis of 40 individuals of Atlantic origin including both wild and farmed individuals. The allele number and the expected heterozygosity ranged from 2 to 18 and from 0.021 to 0.951, respectively. Evidences of null alleles at moderate-high frequencies were detected at six loci using population data. None of the analysed loci showed deviations from Mendelian segregation after the analysis of five full-sib families including approximately 92 individuals/family. The markers are used to consolidate the turbot genetic map, and because they are mostly EST-derived, they will be very useful for comparative genomic studies within flatfishes and with model fish species. Using an in silico approach, we detected significant homologies of microsatellite sequences with the EST databases of the flatfish species with highest genomic resources (Senegalese sole, Atlantic halibut, bastard halibut) in 31% of these turbot markers. The conservation of these microsatellites within Pleuronectiformes will pave the way for anchoring genetic maps of different species and identifying genomic regions related to productive traits.     

Phylogeography and cryptic introduction of the ragworm Hediste diversicolor (Annelida,Nereididae) in the Northwest Atlantic

Many benthic marine invertebrates show striking range disjunctions across broad spatial scales. Without direct evidence for endemism or introduction, these species remain cryptogenic. The common ragworm Hediste diversicolor plays a pivotal role in sedimentary littoral ecosystems of the North Atlantic as an abundant prey item and ecosystem engineer, but exhibits a restricted dispersal capacity that may limit connectivity at both evolutionary and ecological time scales. In Europe, H. diversicolor is subdivided into cryptic taxa and genetic lineages whose distributions have been modified by recent invasions. Its origin in the northwest Atlantic has not been adequately addressed. To trace the age and origin of North American ragworm populations, we analyzed mtDNA sequence data (COI) from the Gulf of Maine and Bay of Fundy (n=73 individuals) and compared our findings with published data from the northeast Atlantic. Our results together with previous data indicate that two species of the H. diversicolor complex have independently colonized the northwest Atlantic at least three different times, resulting in two distinct conspecific assemblages in the Bay of Fundy and Gulf of Maine (respectively) that are different from the species found in the Gulf of St. Lawrence. North American populations had significantly lower genetic diversity compared with populations in the northeast Atlantic, and based on patterns of shared identity, populations in the Bay of Fundy originated from the Baltic Sea and North Sea. Populations from the Gulf of Maine were phylogenetically distinct and most likely originated from unsampled European populations. Analyses of the North American populations revealed patterns of post‐colonization gene flow among populations within the Gulf of Maine and Bay of Fundy. However, we failed to detect shared haplotypes between the two regions, and this pattern of complete isolation corroborates a strong phylogeographic break observed in other species.     

Determination of plasma melatonin levels by enzyme-linked immunosorbent assay (EIA) in turbot (Scophtalmus maximus L.) and tench (Tinca tinca L.)

An enzymoimmunoassay (EIA) kit for plasma melatonin (MLT) measurements was employed in tench (Tinca tinca) and in turbot (Scophtalmus maximus). Tench and turbot plasma samples were purified with a C18 reversed phase extraction columns because this kit is designed for human serum measurements. The lowest detection limit of the technique was 11.48 pg/well with a sensitivity at 50% binding of 100 pg/well. Intra-assay and inter-assay CV (%) were always less than 5% (n=8), and 9% (n=6) in tench plasma samples, and less than 5% (n=8) and 13% (n=5) in turbot plasma samples, respectively. Correlation coefficients between EIA and RIA measurements in tench and turbot plasma samples were 0.93 and 0.89 (p<0.001) respectively. Diurnal and nocturnal plasma melatonin mean levels were 14.7+/-2.1 pg/ml and 87.4+/-11 pg/ml in tench (n=15), and 3.5+/-0.4 pg/ml and 28.1+/-2.1 pg/ml in turbot (n=15). These species showed a melatonin circadian rhythm as in other animals studied. The results suggest that the commercial kit used in this experiment could be a suitable and alternative method to RIA for plasma MLT determinations in tench and turbot although it is necessary to increase volumes (1ml) and concentrate daytime samples.     

Panmictic population structure in the hooded seal (Cystophora cristata)

Two putative populations of hooded seals (Cystophora cristata) occur in the North Atlantic. The Greenland Sea population pup and breed on the pack ice near Jan Mayen ('West Ice') while the Northwest Atlantic population is thought to pup in the Davis Strait, in the Gulf of St. Lawrence (the 'Gulf'), and off southern Labrador or northeast Newfoundland (the 'Front'). We used microsatellite profiling of 300 individuals at 13 loci and mitochondrial DNA sequencing of the control region of 123 individuals to test for genetic differentiation between these four breeding herds. We found no significant genetic differences between breeding areas, nor evidence for cryptic nor higher level genetic structure in this species. The Greenland Sea breeding herd was genetically most distant from the Northwest Atlantic breeding areas; however, the differences were statistically nonsignificant. Our data therefore suggest that the world's hooded seals comprise a single panmictic genetic population.     

Cathepsin D from Atlantic cod (Gadus morhua L.) liver. Isolation and comparative studies

The isolated cathepsin D-like enzyme from Atlantic cod (Gadus morhua L.) liver was shown to be a monomer with a molecular mass of approximately 40 kDa. It was inhibited by Pepstatin A and had an optimum for degradation of haemoglobin at pH 3.0. The purified enzyme had lower temperature stability than bovine cathepsin D. Antibodies raised against the purified enzyme and against two C-terminal peptides of cod cathepsin D recognized a 40 kDa protein in immunoblotting of the samples from the purification process. Both antisera showed cross reactivity with a similar sized protein in liver from cod, saithe (Pollachius virens L.), Atlantic herring (Clupea harengus L.) and Atlantic salmon (Salmo salar L.). A protein of same size was detected in wolffish (Anarhichas lupus L.) liver with the antibody directed against the purified enzyme. This antibody also recognized the native enzyme and detected the presence of cathepsin D in muscle of cod, saithe, herring and salmon. These antibodies may be useful in understanding the mechanisms of post mortem muscle degradation in fish by comparing immunohistochemical localization and enzyme activity, in particular in cod with different rate of muscle degradation. They may also be used for comparing muscle degradation in different fish species.     

POPULATION STRUCTURE OF THE BOTTLENOSE DOLPHIN (TURSIOPS TRUNCATUS) AS DETERMINED BY RESTRICTION ENDONUCLEASE ANALYSIS OF MITOCHONDRIAL DNA

Abstract: Restriction fragment length polymorphisms of mitochondrial DNA (mtDNA) were used to test for population subdivision in the bottlenose dolphin (Tursiops truncatus). Atlantic and Pacific dolphin mtDNA samples exhibited distinctly different haplotypes (approximately 2.4% sequence divergence), indicating a lack of gene exchange. Within the Atlantic Ocean, mtDNA samples from the Gulf of Mexico and the Atlantic Coast were also found to be distinct, with a sequence divergence of approximately 0.6%. The Atlantic Coast–Gulf of Mexico dichotomy is consistent with patterns of genetic variation from other marine and coastal organisms from this region, and supports the hypothesized role of bio-geographic events in promoting the divergence of these and other forms. Regional differentiation was identified along the Atlantic Coast, whereas low sequence divergences among haplotypes and consistent haplotype frequencies across populations suggested considerable gene exchange among Gulf of Mexico populations. A highly divergent haplotype found in two individuals from two localities in the Gulf of Mexico is best explained by dispersal from either a distinct offshore Gulf stock or an unsampled Atlantic Coast stock. Additional samples are required to test for the existence of a distinct offshore race and, if it exists, to identify its distribution and contribution to population structure.     

Isolation and characterization of eight microsatellite loci for white hake (Urophycis tenuis)

Eight tetranucleotide microsatellite loci were characterized for white hake, Urophycis tenuis, a commercially exploited demersal gadoid fish distributed in the Northwest Atlantic. The eight loci are polymorphic with number of alleles and observed heterozygosities ranging from seven to 40 and from 0.544 to 0.978, respectively, in samples of between 39 and 47 individuals collected from throughout the species range. These loci were developed for population genetic studies.     

槐尺蠖多酚氧化酶的纯化及酶学特征

经40%饱和度硫酸铵分级沉淀,Sephadex G-100凝胶过滤等步骤,将槐尺蠖Semiothisa cinerearia Bremer et Grey 多酚氧化酶纯化,纯化倍数为6.96倍。该酶对焦性没食子酸,邻苯二酚和L多巴的Km值分别为0.23 mmol/L, 0.48 mmol/L和0.49 mmol/L。多酚氧化酶在pH 7.0,37℃时活性最高,并在40℃以上条件下,随着保温时间的延长酶活力下降。用槲皮苷和硫脲作抑制剂对该酶活性的抑制结果表明,这两种抑制剂分别属于竞争性和非竞争性抑制剂。     

Female philopatry in coastal basins and male dispersion across the North Atlantic in a highly mobile marine species, the sperm whale (Physeter macrocephalus)

The mechanisms that determine population structure in highly mobile marine species are poorly understood, but useful towards understanding the evolution of diversity, and essential for effective conservation and management. In this study, we compare putative sperm whale populations located in the Gulf of Mexico, western North Atlantic, Mediterranean Sea and North Sea using mtDNA control region sequence data and 16 polymorphic microsatellite loci. The Gulf of Mexico, western North Atlantic and North Sea populations each possessed similar low levels of haplotype and nucleotide diversity at the mtDNA locus, while the Mediterranean Sea population showed no detectable mtDNA diversity. Mitochondrial DNA results showed significant differentiation between all populations, while microsatellites showed significant differentiation only for comparisons with the Mediterranean Sea, and at a much lower level than seen for mtDNA. Samples from either side of the North Atlantic in coastal waters showed no differentiation for mtDNA, while North Atlantic samples from just outside the Gulf of Mexico (the western North Atlantic sample) were highly differentiated from samples within the Gulf at this locus. Our analyses indicate a previously unknown fidelity of females to coastal basins either side of the North Atlantic, and suggest the movement of males among these populations for breeding.     

Purification and characterization of a novel peroxidase from bitter gourd (Momordica charantia)

Peroxidase from bitter gourd was purified by three step purification scheme; ammonium sulphate fractionation, gel filtration and affinity chromatography. The enzyme was purified 42 fold with the retention of 67% of the initial activity. The enzyme exhibited its maximum activity at pH 5.6 and 40 degrees C. The enzyme retained half of its activity even after 1 h incubation at 60 degrees C. Molecular weight of the purified glycosylated bitter gourd peroxidase determined by Sephacryl S-100 and SDS-PAGE was 43 kDa. The stokes radius, diffusion coefficient and sedimentation coefficient of the purified peroxidase were 27.3 A, 8.17 x 10(-7) cm(2)/sec and 3.74 S, respectively. K(m) for o-dianisidine and ABTS were 1.3 and 4.9 mM, respectively. The activity of the enzyme was inhibited by sulfide, azide and L-cysteine. The carbohydrate content and sulfydryl groups of the enzyme were 25% (w/w) mass of the protein and 16 mmoles/mole of the protein, respectively.     

Purification and characterization of an acetylcholinesterase from the infective juveniles of Heterorhabditis bacteriophora

Acetylcholinesterases (AChEs) have been estimated in the infective juveniles (IJs) of eight different strains of heterorhabditid nematodes. The enzyme content ranged from 45.6 to 421.3 units/10(5) IJs with specific activity 34.0 to 82.6 units/mg protein. The isoenzyme patterns revealed the existence of two-slow-moving isoforms. Heterorhabditis bacteriophora AChE1A has been purified from the IJs of the heterorhabditid nematode strain of the highest enzymatic activity to homogeneity by ammonium sulfate precipitation, gel filtration on Sephacryl S-200 and DEAE-Sepharose. The specific activity of the purified enzyme was 1378.1 units/mg protein with purification fold 17.5 over crude extract. The enzyme has a pH optimum at 7.5. The optimum temperature for enzyme activity and stability was 35 degrees C. The activation energy was calculated to be 9.0 kcal/mol. The enzyme hydrolyzes acetylthiocholine (AcSCh), propionylthiocholine (PrSCh), S-butyrylthiocholine (BuSCh) and benzoylthiocholine (BzSCh) iodides with relative rate 100, 74.6, 41.7 and 22.2%, respectively. It displayed an apparent Michaelis-Menten behavior in the concentration range from 0.1 to 2 mM for the three former substrates with Km values 0.27, 0.42 and 0.59 mM, respectively. H. bacteriophora ChE1A is an AChE since it hydrolyzed AcSChI at higher rate than the other substrates and displayed excess substrate inhibition with AcSChI at concentrations over 2 mM. It was inhibited by eserine and BW284C51, but not by iso-OMPA. Its biochemical properties were compared with those reported for different species of insects as target hosts for heterorhabditid nematodes and animal parasitic nematodes.     

Comparative study of the purification and characterization of the cytosolic glutathione S-transferases from two salmonid species: Atlantic salmon (Salmo salar) and brown trout (Salmo trutta)

In the present report, an efficient method for isolating multiple cytosolic forms of glutathione S-transferases from liver and kidney cytosolic samples of two salmonid species (brown trout and Atlantic salmon) is described, and some of the multiple properties of these enzymes are presented. Glutathione S-transferases were partially purified by low-pressure affinity chromatography on a column with glutathione coupled to agarose, which retained an average of 89.47% of the total activity. The GST activity was appropriated towards CDNB and ETHA as substrates. The application of an HPLC system associated to elestrospray ionization mass spectrometry allowed the identification of five GST cytosolic isoforms, corresponding to subunits with M(r) between 23,700 and 26,900 Da being the main form, with retention time of 17 min, a pi-class-related GST isoenzyme.     

Population Structure and Variation in Red Snapper (<Emphasis Type="Italic">Lutjanus campechanus</Emphasis>) from the Gulf of Mexico and Atlantic Coast of Florida as Determined from Mitochondrial DNA Control Region Sequence

The mitochondrial DNA control regions of red snapper (Lutjanus campechanus) from the Gulf of Mexico (n = 140) and Atlantic coast of Florida (n = 35) were sequenced to generate a prestocking genetic baseline for planned stock enhancement. Intrasample haplotype and nucleotide diversities ranged from 0.94 to 1.00 and 1.8% to 2.5%, respectively. All population analyses were consistent with the hypothesis that red snapper constitute a single, panmictic population over the sampled range. A ubiquitous, predominant haplotype, shared by 23% of the specimens, appeared to be evolutionarily recent, in contrast to previous findings based on restriction fragment length polymorphism data. Tajimas D values were suggestive of a recent bottleneck. Mismatch distributions from Gulf samples were smooth and unimodal, characteristic of recent population expansion. However, the Atlantic sample exhibited a comparatively broader, possibly multimodal distribution, suggestive of a more stable population history. Additional control-region data may clarify potentially disparate demographic histories of Gulf and Atlantic snapper.     

Allozyme differentiation within and between red drum (Sciaenops ocellatus) from the Gulf of Mexico and Atlantic Ocean

Nine polymorphic nuclear-gene (allozyme) loci were surveyed among 491 red drum ( Sciaenops ocellatus ) sampled in 1988 and 1989 from nearshore localities in the northern Gulf of Mexico (Gulf) and the Atlantic coast of the southeastern United States (Atlantic). Data were combined with those from a previous study to generate a data set of 762 individuals representing 11 sample localities in the Gulf and 175 individuals representing five sample localities in the Atlantic. The combined data set included individuals from the 1986 and 1987 year classes and permitted rigorous testing of both temporal and spatial genetic heterogeneity. Average heterozygosity-per-locus values (estimated using 33 assumed monomorphic loci) were 0·048 (Gulf red drum) and 0·046 (Atlantic red drum). Tests of heterogeneity in allele frequencies between year classes at individual localities and across regions (Gulf and Atlantic) were non-significant. Tests of spatial (geographic) heterogeneity indicated that red drum are weakly subdivided: genetically-differentiated subpopulations occur in the northern Gulf and along the south-eastern Atlantic coast. Genetic data were consistent with the hypothesis that red drum within the Gulf and along the Atlantic coast comprise singie subpopulalions. Genetic differences between Gulf and Atlantic red drum seem likeiy to stem from historical or recent interactions between dispersal and impediments to gene flow.     

Biliverdin reductase from the liver of Atlantic salmon (Salmo salar)

Biliverdin reductase was characterized and purified from the liver of Atlantic salmon (Salmo salar) using a novel enzymatic staining method. The properties of the enzyme are quite different from those of mammals. The purified enzyme is a monomeric protein with a molecular weight of approximately 68 kD and an isoelectric point of around 3.8. The enzyme can utilize both NADH and NADPH as coenzyme, but the kinetic properties of the NADH-dependent and the NADPH-dependent enzyme activities are different: K _m value for biliverdin IX is 0.6 M in the NADPH system, while it is 6.8 M in the NADH system. Both enzyme activities are inhibited by excess biliverdin IX, but the NADPH-dependent enzyme activity is far more susceptible. The optimum pH for activity is 5.5 with NADPH and 6.0 with NADH. The optimum reaction temperature is 35°C.     

Hierarchical analyses of genetic variation of samples from breeding and feeding grounds confirm the genetic partitioning of northwest Atlantic and South Atlantic populations of swordfish (Xiphias gladius L.)

In species with high migratory potential, the genetic signal revealing population differentiation is often obscured by population admixture. To our knowledge, the explicit comparison of genetic samples from known spawning and feeding areas has not been conducted for any highly migratory pelagic fish species. This study examines the geographic heterogeneity of swordfish mitochondrial DNA (mtDNA) lineages within the Atlantic Ocean using 330 base pairs of sequence of the control region from 480 individuals. Hierarchical analyses of sequence variation were conducted to test whether samples from areas identified as the corresponding spawning and feeding grounds for the northwest (NW) Atlantic (Caribbean and Georges Banks-US northeast) and the South Atlantic (Brazil-Uruguay and Gulf of Guinea), were more closely related to each other than to samples from any other region, including the Mediterranean Sea, the Indian Ocean, and the Pacific Ocean. Phylogeographic analyses reveal that swordfish mtDNA phylogeny is characterized by incomplete lineage sorting and secondary contact of two highly divergent clades. However, despite this complex phylogenetic signature, results from an analysis of nucleotide diversity and from an analysis of molecular variance (AMOVA) were for the most part concordant and indicate that NW Atlantic and South Atlantic swordfish belong to separate populations. The mtDNA distinctiveness of NW Atlantic and South Atlantic swordfish populations is indicative of philopatric behavior in swordfish towards breeding and feeding areas.     

Worldwide phylogeography of the blacktip shark (Carcharhinus limbatus) inferred from mitochondrial DNA reveals isolation of western Atlantic populations coupled with recent Pacific dispersal

Although many coastal shark species have widespread distributions, the genetic relatedness of worldwide populations has been examined for few species. The blacktip shark, (Carcharhinus limbatus), inhabits tropical and subtropical coastal waters throughout the world. In this study, we examined the genetic relationships of blacktip shark populations (n = 364 sharks) throughout the majority of the species' range using the entire mitochondrial control region (1067-1070 nucleotides). Two geographically distinct maternal lineages (western Atlantic, Gulf of Mexico, and Caribbean Sea clades, and eastern Atlantic, Indian, and Pacific Ocean clades) were identified and shallow population structure was detected throughout their geographic ranges. These findings indicate that a major population subdivision exists across the Atlantic Ocean, but not the Pacific Ocean. The historical dispersal of this widespread, coastal species may have been interrupted by the rise of the Isthmus of Panama. This scenario implies historical dispersal across the Pacific Ocean (supported by the recovery of the same common haplotype from the Philippines, Hawaii, and the Gulf of California reflecting recent/contemporary dispersal abilities) and an oceanic barrier to recent migration across the Atlantic. Genetic structure within the eastern Atlantic/Indo-Pacific (Phi(ST) = 0.612, P < 0.001) supports maternal philopatry throughout this area, expanding previous western Atlantic findings. Eastern Atlantic/Indo-Pacific C. limbatus control region haplotypes were paraphyletic to Carcharhinus tilstoni haplotypes in our maximum-parsimony analysis. The greater divergence of western Atlantic C. limbatus than C. tilstoni from eastern Atlantic/Indo-Pacific C. limbatus reflects the taxonomic uncertainty of western Atlantic C. limbatus.     

不同生境两种生态型芦苇叶片质膜H~ -ATPase的比较(英文)

利用两相法纯化质膜微囊,研究了分布于西北沙漠地区的两种生态型芦苇(Phragmites communis Trin.)(水生芦苇和重度盐化草甸芦苇,分别简称为水芦和盐芦)叶片质膜H -ATPase的部分性质。结果显示,与水芦相比,盐芦质膜H -ATPase的ATP水解活性升高,Km值由1.27 mmol/L降至0.30 mmol/L,但Vmax没有显著差异。并且该酶活性对温度的敏感性和pH谱型也发生了变化。以对硝基苯磷酸盐为底物,低浓度时盐芦的质膜H -ATPase水解活性高于水芦,高浓度时则没有差异。Km在水芦和盐芦中分别为3.61 mmol/L和1.92 mmol/L,但Vmax在两种生态型中没有差异。钒酸盐抑制实验表明,两种生态型的质膜H -ATPase磷酸-酶区的催化性质不同。胰酶对质膜H -ATPase活性的活化谱型也存在差异,说明该酶C末端的结构或性质发生了变化。此外,与水芦相比,盐芦质膜H -ATPase的质子泵活性及与水解活性的耦联程度也升高了。以上结果说明,当芦苇从水生环境向盐渍环境过渡时,质膜H -ATPase的催化性质发生了变化,这些变化可能是由酶结构的修饰和不同的同工酶谱引起的。H -ATPase催化性质的变化可能是对盐渍生境的适应性反应。