Chinook salmon NADP+-dependent cytosolic isocitrate dehydrogenase: Electrophoretic and genetic dissection of a complex isozyme system and geographic patterns of variation

Species in the genus Oncorhynchus express complicated isocitrate dehydrogenase (IDHP) isozyme patterns in many tissues. Subcellular localization experiments show that the electrophoretically distinct isozymes of low anodal mobility expressed predominantly in skeletal and heart muscle are mitochondrial forms (mIDHP), while the more anodal, complex isolocus isozyme system predominant in liver and eye is cytosolic (sIDHP). The two loci encoding sIDHP isozymes are considered isoloci because the most common allele at one of these loci cannot be separated electrophoretically from the most common allele of the other. Over 12 electrophoretically detectable alleles are segregating at the two sIDHP* loci in chinook salmon. Careful electrophoretic comparisons of the sIDHP isozyme patterns of muscle, eye, and liver extracts of heterozygotes reveal marked differences between the tissues with regard to both relative isozyme staining and the expression of several common alleles. Presumed single-dose heterozygotes at the sIDHP isolocus isozyme system exhibit approximate 9:6:1 ratios of staining intensity in liver and eye, while they exhibit approximate 1:2:1 ratios in skeletal muscle. The former proportions are consistent with the equal expression of two loci (isolocus expression), while the latter are consistent with the expression of a single locus. Screening of over 10,000 fish from spawning populations and mixed-stock fishery samples revealed that certain variant alleles (*127, *50) are detectable only in liver and eye, while other alleles (*129, *94, and *74) are strongly expressed in muscle, eye, and liver. The simplest explanation for these observations is that the "isolocus" sIDHP system of chinook salmon (and that of steelhead and rainbow trout) results from the expression of two distinct loci (sIDHP-1* and sIDHP-2*) that have the same common allele (as defined by electrophoretic mobility). IDHP expression in skeletal muscle is due to the nearly exclusive expression of the sIDHP-1* locus, while IDHP expression in eye and liver tissues is due to high levels of expression of both sIDHP-1* and sIDHP-2*--giving rise to the isolocus situation in these latter tissues. Direct inheritance studies confirm this model of two genetically independent (disomic) loci encoding sIDHP in chinook salmon. Extensive geographic surveys of chinook salmon populations from California to British Columbia reveal marked differences in allele frequencies at both sIDHP-1* and sIDHP-2* and considerably more interpopulation differentiation than was recognized previously when sIDHP was treated as an isolocus system with only five recognized alleles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Partial characterization of transferrins of some species of the family Cyprinidae

1. Transferrins of five cyprinid fishes (Tinca tinca, Ctenopharyngodon idella, Chondrostoma nasus. Hypophthalmichthys molitrix and Aristichthys nobilis) were isolated in a pure state using Sephadex G-100, DEAE-Sephadex and SP-Sephadex. 2. All the transferrins are heterogeneous. In H. molitrix and A. nobilis the heterogeneity is caused partly, but not exclusively, by the presence of sialic acid; in the other species it is independent of sialic acid. 3. Molecular weights ranging from 69,900 (T. tinca) to 78,800 (H. molitrix) were obtained by sedimentation equilibrium ultracentrifugation. SDS-polyacrylamide gel electrophoresis with beta-mercaptoethanol indicated that the transferrins are composed of a single polypeptide chain. 4. Amino acid compositions of all species were similar, the greatest similarity being in the transferrins of H. molitrix and A. nobilis. 5. Carbohydrate was not found in transferrins of T. tinca and C. idella, but was present in A. nobilis (2.63%) and H. molitrix (5.01%). 6. Alanine was N-terminal in C. idella. No N-terminal amino acid was found in T. tinca, H. molitrix and A. nobilis.     

Allozyme Polymorphisms and Biochemical-Genetic Taxon Markers in Lampreys (Lampetra, Eudontomyzon, Petromyzon)

Variation at 23 putative enzyme-coding loci was scored in 424 lampreys, including 321 European brook lampreys (Lampetra planeri), 83 European river lampreys (L. fluviatilis), 11 Ukrainian brook lampreys (Eudontomyzon mariae), and nine sea lampreys (Petromyzon marinus). Twelve polymorphisms are described for Lampetra species (LDH*, SOD-2*, PNP*, AAT-1*, AK-1*, ES-2*, LGL*, MPI*, GPI-1*, GPI-2*, PGM*, IDHP-2*), and two each for E. mariae (GPI-1*, ME-2*) and P. marinus (MDH-1*, ME-2*). Diagnostic allozymes are presented for the discrimination of lamprey taxa, some of which are difficult to recognize from the morphology of ammocoetes larvae, the life stage usually encountered when collecting cyclostomes. The allelic markers described permit the clear allocation to a genus, except for the species L. fluviatilis and L. planeri, which are not differentiated by qualitative allozyme analysis.     

Population structure of seabass in Portugal: evidence from allozymes

Starch-gel electrophoresis was used to assess the level and distribution of genetic variation in juvenile European seabass sampled from five different nursery grounds along the coast of Portugal between November 1992 and February 1994. Thirty-eight loci were examined, of which six ( AAT-3*, ADA*, GPI-1*, GPI-2*, G3PDH-2*, SOD* ) were polymorphic at the 99% level and were used in population surveys. Low but statistically significant multilocus F _ST values suggest that population structuring exists along the Portuguese coast. There is some restriction in gene flow between the population at Faro and all other sites to the north.     

洱海鲢、鳙的食物组成及与太湖新银鱼的食性重叠研究

为了研究鲢（Hypophthalmichthys molitrix）、鳙（Aristichthys nobilis）两种鱼食物组成的空间变化,分析了2010年7月至2011年10月在洱海古生和北部两采样点逐月采集或收集的共93尾鲢和71尾鳙的肠道内含物,对其食物组成的季节变化、空间变化及与太湖新银鱼（Neosalanx taihuensis）的食物重叠进行了研究。结果表明,鲢、鳙全年摄食的饵料生物数分别为75 属和 64属。根据相对重要性指数（IRI%）,微囊藻（Microcystis）和象鼻溞（Bosmina）为两种鱼的主要饵料类群。鲢、鳙的食物组成呈明显的季节变化,这与饵料生物的季节变化密切相关。在空间上,两种鱼在古生摄食的浮游植物比例大于北部,这是由不同区域饵料生物组成差异导致的结果。Schoener重叠指数显示鲢、鳙与太湖新银鱼之间的食性重叠不显著（E0.6）。       

Biochemical genetic differentiation between Pomatoschistus marmoratus and P. tortonesei

Several diagnostic genetic markers were identified in Pomatoschistus marmoratus and P. tortonesei using polyacrylamide gel electrophoresis (PAGE) of allozymes. Twenty-one loci were resolved, including the electrophoretic pattern of muscle proteins. The MDH*, PGM-,2*, EST-1,2*, FUM* and PGI-2* loci exhibited different alleles which were fixed for the two species being analysed. Genetic distance, as calculated by Nei's index, showed a value of 0·413. Environmental hypersalinity, could have influenced the geographical distribution of P. tortonesei .     

Taxonomic study of the Iberian Cobitis (Osteichthyes, Cobitidae), with description of a new species

Morphometric and electrophoretic variation are analysed among Iberian and African populations of Cobitis (Cobitidae). Three discrete morphotypes are distinguished, showing a remarkable genetic differentiation among them. Each of the three morphotypes is judged to represent a species, one being described as a new species. Both approaches agree on the separate status of the Iberian populations from the Alagón drainage, Tagus basin of west central Spain. This population is described as a new species, Cobitis vettonica sp. nov., a sexually dimorphic spined loach was assigned to the subgenus Iberocobitis Bacescu, 1961. Morphometric and electrophoretic data placed this new species phenetically close to Cobitis maroccana Pellegrin, 1929 and Cobitis pahdica De Buen, 1930, but differs from these species in overall shape, male coloration pattern, shorter barbels and fins, posterior lamina of the cleithrum, seven branched rays on the dorsal fin and in having a unique alleles at sAAT-2*, EST-1* and MPI* loci. The new species range described in this paper is more restricted than that of C. pahdica. Both inhabit the Tagus basin but have not been reported to be sympatric.     

The inner ear morphology and hearing abilities of the Paddlefish (Polyodon spathula) and the Lake Sturgeon (Acipenser fulvescens)

Concern regarding the spread of silver carp (Hypopthalmichthys molitrix) and bighead carp (Aristichthysc nobilis) through the Illinois River has prompted the development of an Acoustic Fish Deterrent (AFD) system. The application of this technology has resulted in a need to understand the auditory physiology of fish other than the target species, in order to minimise the effect of the AFD barrier on the ecology of indigenous fish populations. To this end, both the structures involved in sound reception and the hearing abilities of the paddlefish (Polyodon spathula) and the lake sturgeon (Acipenser fulvescens) are studied here using a combination of morphological and physiological approaches, revealing that both fish are responsive to sounds ranging in frequency from 100 to 500 Hz. The lowest hearing thresholds from both species were acquired from frequencies in a bandwidth of between 200 and 300 Hz, with higher thresholds at 100 and 500 Hz. The rationale for studying hearing in P. spathula and A. fulvescens in particular, is the value placed on them by both the commercial caviar producing industry and by the recreational fisheries sector. The hearing abilities of twelve P. spathula and twelve A. fulvescens were tested in sound fields dominated by either sound pressure or particle motion, with the results showing that acipenseriform fish are responsive to the motion of water particles in a sound field, rather than the sound pressure component. In this study, we measure the intensity of the sound field required to evoke threshold responses using a pressure sensitive hydrophone, as pressure dominated sound fields are the most audible acoustic condition for specialists like H. molitrix and A. nobilis (the target species). The results of the auditory examination clearly show that P. spathula and A. fulvescens are not sensitive to sound pressure, and will therefore have a significantly higher deterrent threshold than H. molitrix and A. nobilis in a pressure dominated sound field.     

Genetic variation at isozyme loci in blue whiting from the north-east Atlantic

Genetic variation in blue whiting Micromesistius poutassou was investigated by starch gel electrophoresis of enzymes in tissue extracts. A total of 130 specimens from the spawning areas west of the British Isles were collected from trawl catches in 1990 ( n = 30) and 1992 ( n = 100). In 25 tissue enzyme loci screened for genetic variants in the 1990 sample, polymorphisms (0.95 criterion) were found at IDDH-2 *, IDHP-2 * and PGM-1 *, giving a frequency of polymorphic loci of P _0.95= 0.12. In pooled samples the average heterozygosity per locus was estimated at H _e =0.043 and the effective number of alleles per locus at 2.20, 1.45 and 1.53, respectively, for IDDH-2 *, IDHP-2 * and PGM-1 *.     

A comparison of complete mitochondrial genomes of silver carp Hypophthalmichthys molitrix and bighead carp Hypophthalmichthys nobilis: implications for their taxonomic relationship and phylogeny

Based upon morphological characters, Silver carp Hypophthalmichthys molitrix and bighead carp Hypophthalmichthys nobilis (or Aristichthys nobilis ) have been classified into either the same genus or two distinct genera. Consequently, the taxonomic relationship of the two species at the generic level remains equivocal. This issue is addressed by sequencing complete mitochondrial genomes of H. molitrix and H. nobilis , comparing their mitogenome organization, structure and sequence similarity, and conducting a comprehensive phylogenetic analysis of cyprinid species. As with other cyprinid fishes, the mitogenomes of the two species were structurally conserved, containing 37 genes including 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA (tRNAs) genes and a putative control region (D-loop). Sequence similarity between the two mitogenomes varied in different genes or regions, being highest in the tRNA genes (98·8%), lowest in the control region (89·4%) and intermediate in the protein-coding genes (94·2%). Analyses of the sequence comparison and phylogeny using concatenated protein sequences support the view that the two species belong to the genus Hypophthalmichthys . Further studies using nuclear markers and involving more closely related species, and the systematic combination of traditional biology and molecular biology are needed in order to confirm this conclusion.     

The hearing abilities of the silver carp (Hypopthalmichthys molitrix) and bighead carp (Aristichthys nobilis)

Concern regarding the spread of silver carp (Hypopthalmichthys molitrix) and bighead carp (Aristichthys nobilis) through the Illinois River has prompted the development of a Bio-acoustic Fish Fence (BAFF) to act as an acoustic fish deterrent. The application of this technology has resulted in a need to understand the auditory physiology of the target species, in order to maximise the effect of the barrier in preventing the migration of the non-indigenous carp species into Lake Michigan, whilst minimising the effect on indigenous fish populations. Therefore, the hearing thresholds of 12 H. molitrix and 12 A. nobilis were defined using the Auditory Brainstem Response (ABR) technique, in a pressure-dominated sound field generated by submerged transducers of the type used in the construction of the BAFF system. The results clearly show that these fish are most sensitive to sounds in a frequency bandwidth of between 750 Hz and 1500 Hz, with higher thresholds below 300 Hz and above 2000 Hz.     

Protein variation in wild Atlantic salmon, with particular reference to southern Ireland

The extent of genetic variation in wild Atlantic salmon parr, Sulmo salur L., from river systems in Ireland, Iceland and eastern Canada, was investigated using starch gel electrophoresis. Within Ireland, seven polymorphic enzyme loci ( sAAT-4 *, GPI-1 *, IDDH-1 *, IDDH-2 *, IDHP-3 *, MDH-3 * and mMEP-2 *) were screened in nine different rivers and nine tributaries from the River Blackwater. Significant heterogeneity in gene frequencies occurred between riverine samples and between samples from tributaries of the River Blackwater. Variation between tributaries was as great as between rivers elsewhere in the country. Levels of population differentiation were comparable to those found in other regions throughout the range of the species, and temporal stability in gene frequencies was apparent when the results were compared with previously published data. Screening of riverine samples from Iceland and eastern Canada (Newfoundland and New Brunswick) allowed the Irish results to be considered in a broader context. Irish salmon cluster in the western European group, to which may be added Icelandic populations. Salmon from eastern Canada show a high level of genetic distinctiveness from the European group.     

花鲈群体的遗传变异

采用水平淀粉凝胶电泳技术对花鲈群体的遗传结构进行了研究 ,共检测了中国沿海花鲈 14 6尾和 40尾日本东京湾花鲈。其群体的多态位点比例为 0 2 667— 0 53 3 3 ,观测杂合度和预期杂合度分别为 0 0 2 11— 0 0 515和0 0 3 98— 0 0 797。中日花鲈在LDH ,GPI 1 ,GPI 2 基因位点上的等位基因接近完全置换。中国花鲈各群体之间的根井遗传距离为 0 0 0 0 4— 0 0 0 11,平均值约为 0 0 0 80 ;而中日花鲈间的根井遗传距离为 0 1870— 0 1954,平均值为 0 192 6。以上结果表明中国花鲈群体间遗传变异很小 ,中日花鲈间遗传变异远大于中国花鲈群体间的遗传距离。     

Genetic comparison of salmon from the White Sea and north-western Atlantic Ocean

Samples of salmon Salmo salar from the River Kachkovka and the River Nilma in northern Russia were analysed by starch gel electrophoresis and compared to three Norwegian stocks, the Neiden river in northern Norway and Øyreselv and Hopselv rivers on the west coast. The comparison included the following polymorphic loci: AAT-4 *, IDDH-2 *, IDHP-3 *, MDH- 3,4 *, MEP-2 *, ESTD * as well as the newly discovered polymorphic loci FBALD-3 * and TPI-3 *. Samples were run side by side on gels, and the alleles found in the Russian stocks were the same as those found in the Norwegian stocks, although the electrophoretic methods used lead to differences in designations of alleles. A polymorphism in ESTD * which involves a slow allele was commonly observed in the three northern populations of the Nilma, Kachkovka and Neiden rivers. This allele was absent in the other Norwegian stocks and in a major brood stock of farmed salmon in Norway. The IDHP-3 * 116 allele was found in unusually high frequencies in the northern populations. Thus, the variability observed at these two loci indicates a barrier to gene flow between the northern salmon stocks and the more southern stocks in the East Atlantic area.     

Polymorphism and inheritance of seed storage protein in sunflower

The data on polymorphism and inheritance of the seed storage protein helianthinin are presented. The results of hybrid analysis indicate that in the annual sunflower Helianthus annuus, helianthinin synthesis is controlled by at least three loci: HelA, HelB, HelB, and HelC. Codominant alleles controlling different electrophoretic variants of polypeptides were identified at each of the loci. The HelA locus was inherited independently of HelB and HelC in a series of dihybrid crosses. The frequencies of recombination between loci HelB and HelC estimated in F2 and BC of two crossing combinations were respectively 21.8 and 19.0%. Segregation of the Hel-C-controlled variants in the progenies from the crosses of cultured sunflower with annual wild species and forms corresponded to that theoretically expected for Mendelian inheritance. The maternal type of helianthinin inheritance was observed in the progenies from the crosses of inbred H. annuus lines with perennial diploid and polyploid Helianthus species. Altered expression of the HelC locus was detected in some hybrid combinations. These alterations appeared in early (F1, F2) hybrid generations and were similar in different hybrid combinations. They did not depend on the perennial paternal species being more influenced by the maternal genotype and by the mode of obtaining hybrids (in an embryo culture or in the field). These results are explained by "genomic shock" generated by hybridization of genetically incompatible species.     

Genetic similarity between species of Akodon (Rodentia, Cricetidae)

Genetic similarity between species of rodents of the Akodon genus (A. dolores, A. molinae, and A. azarae) has been estimated by analysis of electrophoretic zymograms corresponding to 23 loci. Nei's coefficient between A. dolores and A. molinae was within the range usually found in conspecific populations. This evidence plus the successful production of "hybrids" (Merani et al., J. Exp. Zool., 206:343-346, '78) suggests that A. dolores and A. molinae may represent geographic races of the same species.     

Allozyme variability and genetic divergence of Pacific trout (species Parasalmo) from western Kamchatka

Genetic variation in several populations of Parasalmo (Onchorhyncus) mykiss from Kamchatka was examined on the basis of data obtained by the author and results from literature. Three (sSOD-1*, LDH-C*, and EST-1*) out of 44 protein-coding loci were highly polymorphic. Low-frequency alternative alleles occurred at sAAT-1,2*, LDH-A2*, EST-5*, IDDH-1,2*, and sMDH-1,2*. The results of the present work and comparison with evidence on North American species indicated that, in the Kamchatka part of the range, P. mykiss is represented by several populations carrying unique alleles and forming a genetically independent group. Gene exchange between North American and Western Kamchatka populations is mainly determined by straying in the feeding stock of the American coastal form entering the Sea of Okhotsk. The genetic divergence and mean heterozygosities in the Kamchatka populations were low (D magnitude of 0.0002-0.0275; HS magnitude of 0.011-0.0371). The difference between the Western Kamchatka populations from the North American coastal form was so small (D magnitude of 0.0109-0.0241) that these forms clustered together. Genetic divergence of the Kamchatka populations and the inland North American P. mykiss is an order of magnitude higher (D magnitude of 0.1973-0.2367).     

NAD(P)H oxidase mediates the endothelial barrier dysfunction induced by TNF-alpha

We tested the hypothesis that the NAD(P)H oxidase-dependent generation of superoxide anion (O2-*) mediates tumor necrosis factor-alpha (TNF)-induced alterations in the permeability of pulmonary microvessel endothelial monolayers (PMEM). The permeability of PMEM was assessed by the clearance rate of Evans blue-labeled albumin. The NAD(P)H oxidase subcomponents p47phox and p22phox were assessed by immunofluorescent microscopy and Western blot. The reactive oxygen species O2-* was measured by the fluorescence of 6-carboxy-2',7'-dichlorodihydrofluorescein diacetatedi(acetoxymethyl ester), 5 (and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate-acetyl ester, and dihydroethidium. TNF treatment (50 ng/ml for 4.0 h) induced 1) p47phox translocation, 2) an increase in p22phox protein, 3) increased localization of p47phox with p22phox, 4) O2-* generation, and 5) increased permeability to albumin. p22phox antisense oligonucleotide prevented the TNF-induced effect on p22phox, p47phox, O2-*, and permeability. The scrambled nonsense oligonucleotide had no effect. The TNF-induced increase in O2-* and permeability to albumin was also prevented by the O2-* scavenger Cu-Zn superoxide dismutase (100 U/ml). The results indicate that the activation of NAD(P)H oxidase, via the generation of O2-*, mediates TNF-induced barrier dysfunction in PMEM.     

Identification of endangered Hawaiian ducks (Anas wyvilliana), introduced North American mallards (A. platyrhynchos) and their hybrids using multilocus genotypes

Hawaiian ducks (Anas wyvilliana), or koloa, are endemic to the Hawaiian Islands and are listed as a federal and state endangered species. Hybridization between koloa and introduced mallards (A. platyrhynchos) is believed to be a primary threat to the recovery of koloa. We evaluated the utility of two sets of nuclear markers (microsatellite loci and amplified fragment length polymorphisms) and a variable portion of the mitochondrial DNA control region to distinguish among koloa, mallards, and hybrids. We show that microsatellite and AFLP markers can be used to distinguish between koloa and mallard-koloa hybrids with a high degree of confidence. For all but one of the putative koloa in our sample, the posterior probability of belonging to the koloa category was >0.90. Similarly all but one of the mallard-koloa hybrids were assigned to the hybrid category with posterior probabilities >0.98. Subsets of markers led to poorer resolution among koloa, mallard and hybrid categories. Among a sample of 61 koloa, hybrids and mallards, we found 25 different mtDNA haplotypes, belonging to two groups of haplotypes (A and B) identified previously in mallards and their relatives. All putative koloa samples exhibited group B haplotypes, of which 65% comprised one haplotype, while the rest were divided among four haplotypes. All Hawai’i mallard samples exhibited haplotypes that belonged to group A. Hybrids and California mallards exhibited haplotypes belonging to both groups, but a majority were of group A, suggesting that hybridization may more commonly involve mating between Hawai’i mallard females and koloa males.     

Molecular and structural analysis of electrophoretic variants of soybean seed storage proteins

Soybean (Glycine max L.) storage proteins are composed mainly of two major components, beta-conglycinin and glycinin. Electrophoretic variants of the beta subunit of beta-conglycinin and the A3 polypeptide of glycinin were detected on SDS-PAGE, and designated them as beta* and A3*, respectively. beta* and A3* exhibited higher and lower mobilities, respectively, than the common beta subunit and A3 polypeptide. The N-terminal nine and 10 amino acid sequences of beta* and A3* were completely identical to the previously reported sequences of the beta subunit and the A3 polypeptide, respectively. Analysis using concanavalin A-horseradish peroxidase and treatment with N-glycosidase indicated that glycans were not responsible for the difference in electrophoretic mobility of beta* or A3*. Furthermore, five clones of beta* or beta and three clones of A3*, respectively, were sequenced but we could not detect deletions and insertions except for a single or a few amino acid substitutions as compared with the common beta subunit and A3 polypeptide. These results indicate that a single or a few amino acid substitution affects the electrophoretic mobilities of beta* and A3*.