The surface topography of <Emphasis Type=Italic>Eudiplozoon nipponicum</Emphasis> (Monogenea) developmental stages parasitizing carp (<Emphasis Type=Italic>Cyprinus carpio</Emphasis> L.)

Using scanning electron microscopy (SEM), the external morphology of all developmental stages (egg, oncomiracidium, diporpa, just fused juvenile and adult) of the parasite, Eudiplozoon nipponicum (Monogenea, Diplozoidae), from the gills of carp was studied. During the ontogeny, the tegument, tegumentary and sensory structures are subsequently developed. The tegument of free swimming oncomiracidium occurs in two types — the ciliated and non-ciliated with numerous uniciliated sensory structures. An attachment apparatus starts to form during the oncomiracidium stage. Further developmental stages are adapted to the environment of the gills. Tegumentary folds become more apparent later in development and assist to the parasite’s attachment. In connection with its reproductive strategy, the two morphological structures of diporpa (ventral sucker and dorsal papilla) appear to play important role. On the gills, two individuals need to meet and these structures mediate the fusion between two diporpae. The hindbody of adult parasite is highly modified for attachment. The haptor, folds and lobular extensions are most developed. The forebody is flexible and able to interact with host gill tissue via the mouth and associated mouth structures. The process of food intake of the parasite was discussed.     

Mutation rate and pattern of microsatellites in common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

Microsatellites are popular molecular markers in genetic and evolutionary studies. Their mutational dynamics have been extensively studied in humans and fruit flies, but few data were available in fish. By genotyping 55 individuals of a F1 pedigree, we investigated the mutation rates and patterns of 49 microsatellites in one of the most important fresh water fish species, the common carp (Cyprinus carpio L.). The overall mutation rate of the 49 loci was 5.56×10⁻⁴/locus/generation (95% confidence interval 1.52×10⁻⁴ and 1.63×10⁻³). The change of allele size was between +2 to −5 repeat units, assuming that the mutation allele arose from the parental allele most similar in size to the mutant.     

Selection of growth-related genes and dominant genotypes in transgenic Yellow River carp <Emphasis Type=Italic>Cyprinus carpio</Emphasis> L.

Transgenic Yellow River carp is characterized by rapid growth rate and high feed-conversion efficiency and exhibits a great application prospect. However, there is still a significant separation of growth traits in the transgenic Yellow River carp family; as such, growth-related genotypes must be screened for molecular marker-assisted selection. In this study, 23 growth-related candidate genes containing 48 SNP markers were screened through bulked segregant analysis (BSA) among transgenic Yellow River carp family members showing significant separation of growth traits. Then, two growth-related genes (Nos. 17 and 14 genes) were identified through combined genome-wide association study (GWAS) of candidate genes and validation of the full-sibling family approach. Nos. 17 and 14 genes encode BR serine/threonine-protein kinase 2 (BRSK2) and eukaryotic translation-initiation factor 2-alpha kinase 3 (Eif2ak3), respectively. The average body weight of three subgroups carrying the genotypes 17GG, 17GG?+?14CC, and 17GG?+?14TT of these two genes increased by 27.96, 38.28, and 33.72%, respectively, compared with the controls. The proportion of individuals with body weight >?500 g in these subgroups increased by 19.22, 26.82, and 30.92%, respectively. The results showed that appropriate genotype carriers can be selected from the progeny population through BSA sequencing combined with simplified GWAS analysis. Hence, basic population for breeding can be constructed and transgenic Yellow River carp strains with stable production performance and uniform phenotypic properties can be bred.     

The effect of olfactory deprivation on the thresholds of sensitivity of skin chemoreceptors of carp <Emphasis Type=Italic>Cyprinus carpio</Emphasis>

Using method of registration of electrical potentials caused by chemical stimulation of the skin surface of the head of the carp Cyprinus carpio, the effect of prolonged olfactory deprivation (3, 9, and 12 months after operation) and partial blinding of anosmiated fish (9 months) on the sensitivity thresholds of external chemoreceptors was studied. It was found that, 3 months following anosmiation, the thresholds decreased by orders of 0.1–0.5, and, after 9 and 12 months, they decreased by orders of 0.1–0.8 depending on the kind of the chemical stimulus and the zone of registration. The highest increase in the sensitivity of skin chemoreceptors was recorded in anosmiated fish deprived also of object vision, by orders of 0.3–1.5. The results obtained indicate the existence of a close functional relationship between the olfactory and visual systems, on the one hand, and the skin chemosensory system, on the other hand. The compensatory processes taking place in these systems in the case of the loss of one of them have an adaptive pattern and are directed at provision of reliability of behavioral contacts and survival of fish in the environment.     

Food web overlap among native axolotl (<Emphasis Type=Italic>Ambystoma mexicanum</Emphasis>) and two exotic fishes: carp (<Emphasis Type=Italic>Cyprinus carpio</Emphasis>) and tilapia (<Emphasis Type=Italic>Oreochromis niloticus</Emphasis>) in Xochimilco,Mexico City

Two exotic fishes, common carp (Cyprinus carpio) and tilapia (Oreochromis niloticus), were introduced more than 20 years ago into Xochimilco for aquaculture, and now dominate the system in terms of biomass and numbers. Over this same period, wild populations of the microendemic axolotl salamander (Ambystoma mexicanum) have been dramatically reduced, and it currently persists in isolated areas of this aquatic system, which is situated at the edge of Mexico City. In this study, we examine potential trophic interactions and niche overlap among two exotic fishes: carp and tilapia, and the native axolotl. Axolotl had more diverse diets and a higher trophic position compared to the exotics. Stable isotope analysis revealed substantial trophic niche overlap among axolotl and the exotics. The two exotics occupied a larger niche area than the axolotl, suggesting higher levels of omnivory and diet specialization. Current exotic fish removal efforts will further our understanding of interactions between the axolotl and exotic species, and are expected to benefit axolotl recovery efforts.     

Fertility of triploid hybrids of Prussian carp (<Emphasis Type="Italic">Carassius gibelio</Emphasis>) with common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

Fertility of backcross triploid hybrids containing one genome of Prussian carp and two genomes of common carp is investigated. The females of hybrids of Prussian carp and common carp (Prussian × common carp) are prolific and produce diploid gametes. Since males of such hybrids are sterile, their reproduction is realized by means of induced gynogenesis. Triploid progeny is obtained by backcrossing female Prussian × common carp with carp males. Among triploids obtained from hybrids F₁ and among hybrids of the first gynogenetic generation, there were no prolific specimens. However, in reproduction of diploid hybrids by means of gynogenesis during six generations, the female fertility in the backcross progeny is restored. From backcross triploid females (daughters of Prussian × common carp of the sixth gynogenetic generation), a viable triploid gynogenetic progeny and a tetraploid backcross (by carp) progeny are obtained. The obtained data may be considered as the experimental proof of the hypothesis of reticular speciation.     

The effect of starvation on taste preferences and testing behavior of food items in the carp <Emphasis Type=Italic>Cyprinus carpio</Emphasis>

The present study is aimed at recording changes in taste preferences and behavior related to testing of artificial agar-agar (2%) pellets with free amino acids (21 amino acids, L-isomers, 0.1–0.001 M) in the carp Cyprinus carpio at increasing duration of starvation (2, 12, 18, 24, 72, and 144 h). Two hours after the end of feeding until satiation, the fish manifest a low foraging activity and, in most cases, they do not grasp the offered pellet. Later on, the feeding motivation in fish increases rapidly and up to 24 h of starvation each of the offered pellets is grasped. In the first hours of starvation the fish refuse from consumption of the grasped pellets even with highly attractive taste stimuli (extract of chironomids, cysteine). Up to 24 h of starvation, the fish restore the differentiated attitude to taste properties of amino acids. Of the latter, four amino acids increase, six decrease, and the other 11 do not render significant influence on consumption of pellets. Up to 72 h of starvation, at the background of continuing increase in the absolute level of consumption of pellets of all types, the taste preferences change but weakly. However, up to 144 h of starvation the number of deterrent amino acids was reduced to one (phenylalanine). At increasing duration of starvation, the number of repeated grasping of pellets changes weakly while duration of testing by fish of pellets of most types increases, especially abruptly in the first 24 h. It is assumed that the decrease or complete loss in fish of the negative reaction to substances, initially having aversive taste properties may be considered as the main mechanism providing expansion of the feeding spectrum and/or transfer of fish onto consumption of new items, initially consumed just occasionally or fully avoided.     

Polymorphism of the cytochrome oxidase b gene (<Emphasis Type="Italic">cyt b</Emphasis>) in Russian populations of common carp and wild common carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis> L.)

Polymorphism of the mitochondrial cyt b gene was examined in 35 individuals of common carp and wild common carp (Cyprinus carpio L.). The fish examined represented two natural populations from Khabarovsk krai (Ac and Am), Volga wild common carp, Don wild common carp, and two common carp breeds, Ropsha (strains BB and MM) common carp and Hungarian common carp. The highest level of nucleotide (π) and haplotype (h) diversity was detected in two strains of Ropsha common carp (MM, π = 0.67%, h = 0.7; and BB, π = 0.21%, h = 0.9) and in one population (Am) of Amur wild common carp (π = 0.26%; h = 0.6). The second population of Amur wild common carp (Ac) and Hungarian common carp were characterized by lower variation estimates (π = 0.035%, h = 0.4; and π = 0.09%, h = 0.7, respectively). Genetic homogeneity was demonstrated for the populations of Volga and Don wild common carp (π = 0, h = 0). In the sample of the cyt b sequences examined, three lineages were identified. Lineages I and II united all haplotypes of the Am Amur wild common carp along with two haplotypes of Ropsha common carp, strain MM. The third lineage (III) was formed by the haplotypes of three individuals of Ropsha common carp strain MM, all representatives of Ropsha common carp strain BB, Hungarian common carp, Ac Amur wild common carp, and Don and Volga wild common carps. Statistically significant amino acid differences were observed only for the sequences, corresponding to haplotypes of lineage III, and the sum of sequences of lineages I and II. Effectiveness of different types of markers to differentiate the two subspecies of European and Amur wild common carp (C. c. carpio and C. c. haematopterus) is discussed, as well as the issues of the origin and dispersal of Russian common carp and wild common carp breeds.     

<Emphasis Type=Italic>Agrobacterium</Emphasis>-mediated transformation of cotton (<Emphasis Type=Italic>Gossypium hirsutum</Emphasis> L.) via vacuum infiltration

AnAgrobacterium-mediated gene transfer system with recovery of putative transformants was developed for cotton (Gossypium hirsutum L.) cv. Cocker-312. Two-month-old hypocotyl-derived embryogenic calli were infected through agroinfiltration for 10 min at 27 psi in a suspension ofAgrobacterium tumefaciens strain GV3101 carrying tDNA with theGUS gene, encoding β-glucuronidase (GUS), and the neomycin phosphotransferase II (nptII) gene as a kanamycin-resistant plant-selectable marker. Six days after the histochemicalGUS assay was done, 46.6% and 20%GUS activity was noted with the vacuum-infiltration and commonAgrobacterium-mediated transformation methods, respectively. The transformed embryogenic calli were cultured on selection medium (100 mg/L and 50 mg/L kanamycin for 2 wk and 10 wk, respectively) for 3 mo. The putative transgenic plants were developed via somatic embryogenesis (25 mg/L kanamycin). In 4 independent experiments, up to 28.23% transformation efficiency was achieved. PCR amplification and Southern blot analysis fo the transformants were used to confirm the integration of the transgenes. Thus far, this is the only procedure available for cotton that can successfully be used to generate cotton transformants.     

Genetic monitoring and effects of stocking practices on small <Emphasis Type=Italic>Cyprinus carpio</Emphasis> populations

The ability to detect genetic differences both in space and time is crucial for conserving genetic variation. It can reveal genetic diversity and genetic composition changes of declining native populations that are supported through stocking with captive bred individuals. The present study was designed to analyse the temporal stability of a declining common carp (Cyprinus carpio) population from Lake Volvi (North Greece). Polymorphism was evaluated using seven microsatellite loci at two sampling time points (separated by 12 years). The genetic variability of four additional populations (from two rivers and two lakes) in Northern Greece was also investigated for comparison. Heterozygosity values (0.692–0.868) and allelic richness (8.530–11.148) were high for all studied populations and comparable to other European populations. However, the analysis of temporal common carp samples from Lake Volvi revealed a significant change in their genetic composition and admixture analysis demonstrated significant introgression of stocked individuals into the native population. Both temporal and point estimate methods revealed low effective size (Ne = 61–171.3) for this population, possibly a result of an ancient genetic bottleneck that led to population decline and/or recent anthropogenic interventions. This low Ne has rendered the native population vulnerable to alteration of its genetic composition. Our study demonstrates that enhancement programs should be applied cautiously, especially for small populations. Moreover, it underlines the need for temporal analyses, which may contribute to the evaluation of previous management policies and to future decision making.     

Damaging effect of the fish pathogen <Emphasis Type=Italic>Aeromonas salmonicida</Emphasis> ssp. <Emphasis Type=Italic>salmonicida</Emphasis> on intestinal enterocytes of Atlantic salmon (<Emphasis Type=Italic>Salmo salar</Emphasis> L.)

In fish, bacterial pathogens can enter the host by one or more of three different routes: (a) skin, (b) gills and (c) gastrointestinal tract. Bacteria can cross the gastrointestinal lining in three different ways. In undamaged tissue, bacteria can translocate by transcellular or paracellular routes. Alternatively, bacteria can damage the intestinal lining with extracellular enzymes or toxins before entering. Using an in vitro (Ussing chamber) model, this paper describes intestinal cell damage in Atlantic salmon (Salmo salar L.) caused by the fish pathogen Aeromonas salmonicida ssp. salmonicida, the causative agent of furunculosis. The in vitro method clearly demonstrated substantial detachment of enterocytes from anterior region of the intestine (foregut) upon exposure to the pathogen. In the hindgut (posterior part of the intestine), little detachment was observed but cellular damage involved microvilli, desmosomes and tight junctions. Based on these findings, we suggest that A. salmonicida may obtain entry to the fish by seriously damaging the intestinal lining. Translocation of bacteria through the foregut (rather than the hindgut) is a more likely infection route for A. salmonicida infections in Atlantic salmon.Financial support from the Commission of the European Communities, quality of Life and Management of Living Resources programme, project Q5RT-2000-31656 Gastrointestinal Functions and Food Intake Regulation in Salmonids: Impact of Dietary vegetable Lipids (GUTINTEGRITY) and from Magnus Bergvalls Stiftelse for KS, is acknowledged.This work does not represent the opinion of the European Community, which is thus not responsible for any use of the data presented.     

Characterization of AT-rich microsatellites in common bean (<Emphasis Type=Italic>Phaseolus vulgaris</Emphasis> L.)

Polymorphism of microsatellite markers is often associated with the simple sequence repeat motif targeted. AT-rich microsatellites tend to be highly variable and this appears to be notable, especially in legume genomes. To analyze the value of AT-rich microsatellites for common bean (Phaseolus vulgaris L.), we developed a total of 85 new microsatellite markers, 74 of which targeted ATA or other AT-rich motif loci and 11 of which were made for GA, CA or CAC motif loci. We evaluated the loci for the level of allelic diversity in comparison to previously characterized microsatellites using a panel of 18 standard genotypes and genetically mapped any loci polymorphic in the DOR364 × G19833 population. The majority of the microsatellites produced single bands and detected single loci, however, 15 of the AT-rich microsatellites produced multiple or double banding patterns; while only one of the GA or CA-rich microsatellites did. The polymorphism information content (PIC) values averaged 0.892 and 0.600 for the AT and ATA motif microsatellites, respectively, but only 0.140 for the CA-rich microsatellites. GA microsatellites, which had a large average number of repeats, had high to intermediate PIC, averaging 0.706. A total of 45 loci could be genetically mapped and distribution of the loci across the genome was skewed towards non-distal locations with a greater prevalence of loci on linkage groups b02, b09 and b11. AT-rich microsatellites were found to be a useful source of polymorphic markers for mapping and diversity assessment in common bean that appears to uncover higher diversity than other types of simple sequence repeat markers.     

Efficient separation and purification of allophycocyanin from <Emphasis Type=Italic>Spirulina</Emphasis> (<Emphasis Type=Italic>Arthrospira</Emphasis>) <Emphasis Type=Italic>platensis</Emphasis>

Allophycocyanin (APC) is a minor component of phycobiliproteins in cyanobacteria and red algae. This paper describes a simple and inexpensive extracting method for isolating APC from Spirulina (Arthrospira) platensis with high efficiency. The crude phycobiliprotein extract was pretreated by ammonium sulfate fractionation. Then, by adding hydroxylapatite into crude phycobiliprotein extract dissolved in 20 mM phosphate buffer (pH 7.0), APC was selectively adsorbed by hydroxylapatite but C-phycocyanin (C-PC) was not. The hydroxylapatite was collected and APC was extracted from the crude phycobiliprotein extract. Then, the enriched APC was washed off from the hydroxylapatite using 100 mM phosphate buffer (pH 7.0). In this simple extracting method it was easy to remove C-PC and isolate APC in large amounts. The absorbance ratio A ₆₅₀/A ₂₈₀ of extracted APC reached 2.0. The recovery yield was 70%, representing 4.61 mg · g⁻¹ wet weight. The extracted APC could be further purified by a simple anion-exchange chromatography with a pH gradient from 5.6 to 4.0. The absorbance ratio A ₆₅₀/A ₂₈₀ of the purified APC reached 5.0, and the overall recovery yield was 43%, representing 2.83 mg · g⁻¹ wet weight. Its purity was confirmed by native polyacrylamide gel electrophoresis (PAGE) and sodium dodecyl sulfate-PAGE.     

Micro-morphology of common bean (<Emphasis Type=Italic>Phaseolus vulgaris</Emphasis> L.) nodules undergoing senescence

We studied morphological changes over time by nodules formed on the root system of the common bean (Phaseolus vulgaris L.). Two cultivars, Bayomex and Cacahuate 72 with growth habit Type I and the Rhizobium etli strain CE-3 were used. The results showed the collapse of the infected zone, degradation of the cell walls and membranes, changes in the number and distribution of the starch granules, appearance of protein granules, and disintegration of the central tissue of the nodule with ageing. Additionally, we describe the influence of time on the progress of the nodular senescence.     

Cryopreservation of <Emphasis Type=Italic>Robinia</Emphasis><Emphasis Type=Italic>pseudoacacia</Emphasis>

Cryopreservation of Robinia pseudoacacia explants by vitrification achieved 78% survival following the stepwise preculture of shoot tips in (0.3 + 0.5 + 0.7 M) sucrose with a 80 min incubation in PVS2; compared to 87% survival after desiccation of explants to 30% water content, following 3 days alginate bead (with glycerol and sucrose treatments) preculture in 0.7 M sucrose.     

High-density AFLP map of nonbrittle rachis 1 (<Emphasis Type=Italic>btr1</Emphasis>) and 2 (<Emphasis Type=Italic>btr2</Emphasis>) genes in barley (<Emphasis Type=Italic>Hordeum vulgare</Emphasis> L.)

Wild relatives of barley disperse their seeds at maturity by means of their brittle rachis. In cultivated barley, brittleness of the rachis was lost during domestication. Nonbrittle rachis of occidental barley lines is controlled by a single gene (btr1) on chromosome 3H. However, nonbrittle rachis of oriental barley lines is controlled by a major gene (btr2) on chromosome 3H and two quantitative trait loci on chromosomes 5HL and 7H. This result suggests multiple mutations of the genes involved in the formation of brittle rachis in oriental lines. The btr1 and btr2 loci did not recombine in the mapping population analyzed. This result agrees with the theory of tight linkage between the two loci. A high-density amplified fragment-length polymorphism (AFLP) map of the btr1/btr2 region was constructed, providing an average density of 0.08 cM/locus. A phylogenetic tree based on the AFLPs showed clear separation of occidental and oriental barley lines. Thus, barley consists of at least two lineages as far as revealed by molecular markers linked to nonbrittle rachis genes.Electronic Supplementary Material Supplementary material is available for this article at An erratum to this article can be found at     

Expression and functional analysis of <Emphasis Type=Italic>ZmDWF4</Emphasis>, an ortholog of <Emphasis Type=Italic>Arabidopsis DWF4</Emphasis> from maize (<Emphasis Type=Italic>Zea mays</Emphasis> L.)

DWF4 encodes a rate-limiting mono-oxygenase that mediates 22α-hydroxylation reactions in the BR biosynthetic pathway and it is the target gene in the BR feedback loop. Knockout of DWF4 results in a dwarfed phenotype and other severe defects in Arabidopsis. Here we report on the isolation of the ZmDWF4 gene in maize. Sequence analysis revealed that the open reading frame of ZmDWF4 was 1,518 bp, which encodes a protein composed of 505 amino acid residues with a calculated molecular mass of 57.6 kD and a predicated isoelectric point (pI) of 9.54. Phylogenetic analysis indicated that ZmDWF4 was very close to the Arabidopsis DWF4. In young maize seedlings, the expression of ZmDWF4 in shoots was much higher than that in roots. The highest expression of ZmDWF4 was observed in husk leaves and the lowest in silks during flowering stage. The expression of ZmDWF4 in maize was significantly down regulated by exogenous brassinolide. A heterogeneous complementary experiment demonstrated that the defects of three Arabidopsis DWF4 mutants could be rescued by constitutive expression of ZmDWF4, with leaf expandability, inflorescence stem heights and fertile capabilities all restored to normal levels. Increases in seed and branch number as well as the height of florescence stem were observed in the over-expressed transformants. These findings suggest that ZmDWF4 may be an ortholog gene of Arabidopsis DWF4 and responsible for BR biosynthesis in maize. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.