Showing papers in "Marine Biotechnology in 2001"

Generating transparent zebrafish: a refined method to improve detection of gene expression during embryonic development.

Abstract: In zebrafish (Danio rerio) pigmentation is initiated during embryogenesis and begins in the retinal epithelium and in the melanophores. The pigment cells develop rapidly, and within hours they constitute a prominent feature of the embryo. In order to improve signal detection by whole mount in situ hybridization, confocal microscopy, or expression of GFP, embryos may be treated with 1-phenyl 2-thiourea (PTU) during embryogenesis. PTU inhibits melanogenesis by blocking all tyrosinase-dependent steps in the melanin pathway but can be toxic at high concentrations. The embryos remain transparent as long as the PTU treatment is continued. However, PTU treatment must be initiated before the initial pigmentation because it does not remove already formed pigment. Here we provide a protocol for generating transparent zebrafish while avoiding the toxic and teratogenic effects of PTU treatment.

Identification of antihypertensive peptides from peptic digest of two microalgae, Chlorella vulgaris and Spirulina platensis.

Abstract: The peptidic fractions that inhibited angiotensin I-converting enzyme (ACE) were separated from the peptic digests of 2 microalgae, Chlorella vulgaris and Spirulina platensis, by ion exchange chromatography and gel filtration. Oral administration of peptidic fractions into spontaneously hypertensive rats at 200 mg/kg of body weight resulted in marked antihypertensive effects. Further separation of the peptidic fractions by ODS high-performance liquid chromatography furnished the following active peptides: Ile-Val-Val-Glu (inhibitory against ACE with an IC(50) of 315.3 microM), Ala-Phe-Leu (63.8 microM), Phe-Ala-Leu (26.3 microM), Ala-Glu-Leu (57.1 microM), and Val-Val-Pro-Pro-Ala (79.5 microM) from C. vulgaris; Ile-Ala-Glu (34.7 microM), Phe-Ala-Leu, Ala-Glu-Leu, Ile-Ala-Pro-Gly (11.4 microM), and Val-Ala-Phe (35.8 microM) from S. platensis.

Assessing the health of grass shrimp (Palaeomonetes pugio) exposed to natural and anthropogenic stressors: a molecular biomarker system.

Abstract: We developed a molecular biomarker system (MBS) to assess the physiological status of Palaomenetes pugio (grass shrimp) challenged with exposure to heat stress, cadmium, atrazine, and the water-accommodating fraction of either diesel fuel or bunker fuel No. 2. The MBS assayed 9 specific cellular parameters of shrimp that are indicative of a nonstressed or stressed condition: heat-shock protein 60, heat-shock protein 70, alpha B-crystallin homologue, lipid peroxide, total glutathione level, ubiquitin, mitochondrial manganese superoxide dismutase, metallothionein, and cytochrome P-450 2E homologue. Using these 9 parameters, the MBS can distinguish between the responses to each stressor, and to the nonstressed control conditions. The MBS was able to determine the structural integrity of the cell as defined by protein turnover, protein chaperoning, and lipid composition via lipid peroxide levels, and the status of key metabolic processes such as cytoskeletal integrity and glutathione redox potential. This technology aids in the accurate diagnosis of the health of shrimp because the physiological significance of changes of each parameter is well known. This technology is particularly relevant for environmental monitoring because grass shrimp are used as key indicator species in many estuarine ecosystems. Finally, this system is easy to implement, precise, and can be quickly adapted to an automated high-throughput system for mass sample analysis.

Characterization of the Myostatin Gene in the Gilthead Seabream ( Sparus aurata ): Sequence, Genomic Structure, and Expression Pattern

Abstract: We report on the sequence and expression analysis of the myostatin gene (MSTN) in the gilthead seabream Sparus aurata. A 2189-bp transcript was isolated, encoding an open reading frame (385 amino acids) that showed 74% to 60% protein similarity with other vertebrate myostatins. Phylogenetic analysis of MSTN and other related genes confirmed the evolutionary relationships of the isolated sequence. The complete sequences of two introns were also determined. Intron-exon boundaries were conserved when compared with those of mammalian MSTN genes, whereas intron size was smaller. Reverse transcriptase polymerase chain reaction on total RNA extracted from different tissues and developmental stages revealed MSTN expression in the skeletal muscle, but also in other tissues. The observed expression profile differed from that in mammals, suggesting possible additional functions of myostatin in the teleost fish.

Genetic diversity of Ostreopsis ovata (Dinophyceae) from Malaysia.

Abstract: The genus Ostreopsis is an important component of benthic and epiphytic dinoflagellate assemblages in coral reefs and seaweed beds of Malaysia. Members of the species may produce toxins that contribute to ciguatera fish poisoning. In this study, two species have been isolated and cultured, Ostreopsis ovata and Ostreopsis lenticularis. Analyses of the 5.8S subunit and internal transcribed spacer regions ITS1 and ITS2 of the ribosomal RNA gene sequences of these two species showed that they are separate species, consistent with morphological designations. The nucleotide sequences of the 5.8S subunit and ITS1 and ITS2 regions of the rRNA gene were also used to evaluate the interpopulation and intrapopulation genetic diversity of O. ovata found in Malaysian waters. Results showed a low level of sequence divergence within populations. At the interpopulation level, the rRNA gene sequence distinguished two groups of genetically distinct strains, representative of a Malacca Straits group (isolates from Port Dickson) and a South China Sea group (isolates from Pulau Redang and Kota Kinabalu). Part of the sequences in the ITS regions may be useful in the design of oligonucleotide probes specific for each group. Results from this study show that the ITS regions can be used as genetic markers for taxonomic, biogeographic, and fine-scale population studies of this species.

Invasion without a bottleneck: Microsatellite variation in natural and invasive populations of the brown mussel Perna perna (L).

Abstract: Population-level genetic diversity of the brown mussel Perna perna was investigated using nuclear microsatellite markers in 6 natural and 6 invasive populations. A total of 448 individuals from 12 populations spanning the natural and introduced ranges of the brown mussel were scored for 2 polymorphic microsatellite loci. Wright's hierarchical F statistics (FST), Hardy-Weinberg equilibrium, Nei's genetic distance, and other descriptive statistics were used to quantify geographic population subdivision, and to estimate the number of migrants per generation. FST values (0.007–0.042) revealed that genetic partitioning among populations was low. Microsatellite data revealed a slight difference in observed heterozygosity and no statistically significant differences in expected heterozygosity or allelic diversity between natural and introduced populations. Effective numbers of migrants (Nem) per generation ranged from 6 to 35 individuals. The potential significance of an invasive species with high genetic variation in terms of the risk of establishment and conservation implications is discussed.

The first internal transcribed spacer (ITS-1) of ribosomal DNA as a molecular marker for phylogenetic and population analyses in crustacea.

Abstract: The objective of the present study is to explore the feasibility of using the first internal transcribed spacer (ITS-1) of ribosomal DNA as a molecular marker for studying the interspecific and intraspecific genetic variations among crustaceans. We designed primers that could amplify ITS-1 from a majority of taxonomic groups of crustaceans. The gene was found to exhibit a high degree of length polymorphism among different groups, ranging from 182 bp in the barnacle Balanus amphitrite to approximately 820 bp in the spiny lobster Panulirus japonicus. With respect to differences between congeneric species, it was found that the ITS-1 sequences of 3 mitten crabs, Eriocheir sinensis, Eriocheir leptognathus, and Eriocheir formosa, exhibit 5.4% to 16.3% nucleotide divergence, suggesting that ITS-1 is informative for phylogenetic analysis at the species level. Yet there are extensive (0.9%–2.3%) variations within individual E. formosa, so that phylogenetic analyses could be obscured. ITS-1 was found to vary between 2 geographical populations of the shrimp Penaeus japonicus. The variations involved substitutions as well as insertions/deletions between shrimp from Australia and South China Sea. These results show that ITS-1 is highly divergent among different crustaceans and could be an appropriate marker for molecular systematic studies at the species and population levels, although the presence of intragenomic variation needs to be taken into consideration.

The complete primary structure of molluscan shell protein 1 (MSP-1), an acidic glycoprotein in the shell matrix of the scallop Patinopecten yessoensis.

Abstract: The complete primary structure of MSP-1, a major water-soluble glycoprotein in the foliated calcite shell layer of the scallop Patinopecten yessoensis, is reported. The full-length complementary DNA for MSP-1 isolated by polymerase chain reaction contained a sequence for a signal peptide of 20 amino acids followed by a polypeptide of 820 amino acids with calculated molecular mass of 74.5 kDa. The deduced amino acid sequence of MSP-1 includes a high proportion of Ser (32%), Gly (25%), and Asp (20%), and the predicted isoelectric point is 3.2; in these respects, MSP-1 is a typical acidic glycoprotein of mineralized tissues. A repeated modular structure characterizes MSP-1, with a sequence unit between 158 and 177 amino acids in length being repeated 4 times in tandem in the middle part of the protein. The repeated unit comprises 3 modules (SG, D, and K domains), each having a distinct amino acid composition and sequence. The SG domain is almost exclusively composed of Ser and Gly residues. The D domain is rich in Asp residues, potential N-glycosylation and phosphorylation sites. The K domain is rich in Gly residues and has a core of basic residues. The Asp residues are arranged more or less regularly in the D domains, exhibiting some repeated motifs such as Asp-Gly-Ser-Asp and Asp-Ser-Asp. Further, the 4 D domains indicate remarkable overall sequence similarities to each other. These observations suggest that the regular arrangements of COO− groups in the D domain side chains may be important for specific control of crystal growth.

Rapid isolation of DNA from fresh and preserved fish scales for polymerase chain reaction.

Abstract: We developed a simple and inexpensive method to extract DNA from fresh and preserved fish scales. The procedure is based on boiling the scales in 5% Chelex 100, followed by digestion with proteinase K and subsequent absorption of genomic DNA using silica. A single fresh scale from larger species (e.g., tilapia) or a few scales from smaller species (e.g., 4 scales from zebrafish) provide over 200 ng of DNA, enough for at least 40 polymerase chain reaction amplifications. The procedure is applicable for DNA isolation not only from fresh and ethanol-preserved scales, but also from dried and formaldehyde-treated samples, and thus might be useful for investigating specimens stored in museums and other collections. Since the removal of a few scales is a gentle means of sample collection, this technique will allow analysis of genetic diversity, mating systems, and parentage in populations of endangered or ornamental fish with minimal experimental influence.

Rapid and Simultaneous Identification of Body Parts from the Morphologically Similar Sharks Carcharhinus obscurus and Carcharhinus plumbeus (Carcharhinidae) Using Multiplex PCR

Abstract: Many commercially exploited carcharhinid sharks are difficult to identify to species owing to extensive morphological similarities. This problem is severely exacerbated when it comes to identifying detached shark fins, and the finless and headless shark carasses typically sold in markets. To assist in the acquisition of urgently needed conservation and management data on shark catch and trade, we have developed a highly streamlined approach based on multiplex polymerase chain reaction (PCR) that uses species-specific primers derived from nuclear ribosomal ITS2 sequences to achieve rapid species identification of shark body parts. Here we demonstrate the utility of this approach for identifying fins and flesh from two globally distributed, morphologically very similar carcharhinid sharks (Carcharhinus obscurus and Carcharhinus plumbeus) intensively targeted in fisheries worldwide, and often confused for each other even as whole animals. The assay is conducted in a 4-primer multiplex format that is structured to simultaneously achieve the following efficiency and cost-reduction objectives: it requires only a single-tube amplification reaction for species diagnosis, it incorporates an internal positive control to allow detection of false-negative results, and it is novel in that it allows species identification even when DNAs from two species are combined in the same tube during the PCR reaction. The latter innovation reduces the required effort for screening a set of unknown samples by 50%. The streamlined approach illustrated here should be amenable for use in a shark conservation and management context where large numbers of samples typically need to be screened; the approach shown may also provide a model for a rapid diagnostic method applicable to species identification in general.

Dioxin toxicology and the aryl hydrocarbon receptor: insights from fish and other non-traditional models.

Abstract: Fish are established models in biology. Recent findings suggest that fish and other nontraditional species also may serve as valuable model systems for understanding receptor-dependent signaling pathways and their interactions with environmental chemicals. Because they are highly sensitive to chlorinated dioxins and related halogenated aromatic hydrocarbons, fish are being used to elucidate the role of chemical signaling pathways in the developmental and molecular toxicology of dioxin-like compounds. Much of this work is focused on the aryl hydrocarbon receptor (AHR), a ligand-activated, bHLH-PAS transcription factor through which dioxins cause altered gene expression and toxicity. In contrast to mammals, which appear to express a single dioxin-binding AHR, many fish species possess at least two AHR genes. Studies of these two fish AHRs may help to reveal the multiple functions of the single mammalian ``AHR,'' its physiological ligand, and the molecular mechanisms involved in differential sensitivity to dioxin-like compounds. In addition, fish have great potential as models for understanding the in vivo functions and interactions of bHLH-PAS proteins, the evolutionary history of their diversification in vertebrates, and their role in human physiology and disease.

Detection and phylogenetic analysis of novel crenarchaeote and euryarchaeote 16S ribosomal RNA gene sequences from a Great Barrier Reef sponge.

Abstract: The presence of Archaea in the Great Barrier Reef marine sponge Rhopaloeides odorabile was investigated by 16S ribosomal RNA community analysis of total DNA extracted from the sponge tissue. The 16S rRNA gene sequences corresponding to group I crenarchaeotes and group II euryarchaeotes were recovered from R. odorabile tissue. The location of archaeal cells within the sponge tissue was investigated using fluorescently labeled oligonucleotide probes. The presence of Archaea was confirmed within all regions of the sponge tissue from R. odorabile, with a significantly higher number of archaeal cells located in the pinacoderm than the mesohyl region. This is the first report of euryarchaeaotes associated with marine sponges.

Polymerase chain reaction in detection of Gymnodinium mikimotoi and Alexandrium minutum in field samples from southwest India.

Abstract: Polymerase chain reaction (PCR) primers were constructed for the detection of two toxic dinoflagellate species, Gymnodinium mikimotoi and Alexandrium minutum. The primers amplified a product of expected size from cultured cells of G. mikimotoi and A. minutum. The species-specific primers targeting G. mikimotoi did not yield any product with a wide range of other cultured algae used as negative controls. Primers designed for A. minutum were species-group-specific since it PCR yielded a product from the closely related species A. ostenfeldii and A. andersonii, but not from other species of this genus tested. The confirmation of PCR products was performed by digestion of the products with restriction enzymes. Sensitivity analyses of the primers on DNA template from cultured cells was positive by PCR at a DNA template concentration of 1.5 x 10(-4) ng/microl (0.3 cells/L) for A. minutum, and at a DNA concentration of 2.5 x 10(-2) ng/microl (697 cells/L) for G. mikimotoi. The PCR method for detection of G. mikimotoi and A. minutum was applied on field samples collected with a plankton net. Gymnodinium mikimotoi could be detected in 11 field samples by microscopy, and all these field samples were positive by PCR. The cell counts of G. mikimotoi in simultaneously collected water samples ranged from 306 to 2077/L. Alexandrium minutum could be detected by microscopy in 3 different field samples. The cell counts in water samples collected at the same time as the net samples ranged from 115 to 1115 cells/L. Alexandrium minutum was detected by PCR in these field samples, with the exception of the sample displaying the lowest cell count (115 cells/L). Plankton samples that were negative by microscopy for any of the two target species were also negative by PCR. All the PCR products from field samples were confirmed by restriction enzyme digestion. The application of PCR-based detection of harmful algal bloom species for aquaculture and monitoring purposes in natural field samples is discussed.

Genetic heterogeneity of the giant tiger shrimp (Penaeus monodon) in Thailand revealed by RAPD and mitochondrial DNA RFLP analyses

Abstract: Genetic diversity of the giant tiger shrimp (Penaeus monodon) collected from 5 areas, Chumphon and Trat (Gulf of Thailand), and Phangnga, Satun, and Trang (Andaman Sea), was examined by randomly amplified polymorphic DNA (RAPD) and mitochondrial DNA (16S ribosomal DNA and an intergenic COI-COII) polymorphism. A total of 53 polymorphic fragments from UBC299, UBC273, and UBC268 was consistently scored across all samples. From the respective primers 26, 32, and 30 genotypes were generated. A 260-bp RAPD fragment generated by the primer UBC268 was specifically observed in 95.8% of Trat P. monodon, suggesting that this RAPD could be used as a marker for comparing phenotypic performance of P. monodon from Trat and other geographic samples. In addition, 37 mtDNA composite haplotypes were observed from restriction analysis of the same P. monodon samples. High haplotype diversity (0.855) and nucleotide diversity (3.328%) of Thai P. monodon were observed. Population differentiation of P. monodon between the Andaman Sea and Gulf of Thailand was clearly illustrated by both techniques (P <.0001). Nevertheless, contradictory results on patterns of differentiation were observed between P. monodon within the Gulf of Thailand. Analysis of nuclear DNA polymorphism (RAPD) indicated a genetically significant difference between Chumphon and Trat ( P <.0001), whereas mtDNA polymorphism did not show differentiation between these samples (P =.0497). Under the presumption of selective neutrality of these markers, biased female gene flow between Trat and Chumphon P. monodon may exist and be responsible for an anomalous differentiation pattern between these geographic samples.

Microsatellite genotyping of individual abalone larvae: parentage assignment in aquaculture.

Abstract: In aquaculture, microsatellite DNA markers are used to genotype parental broodstock, to assess fertilization success, and to maintain pedigree information for selective breeding. In this study we genotyped individual Haliotis asinina larvae by analyzing a suit of polymorphic microsatellite loci. At least 10 loci can be analyzed from a single abalone veliger larva. We assayed 5 polymorphic loci to identify the parents of individual larvae produced in 3 separate crosses. In all cases, the parents of an individual veliger could be determined from as few as 3 loci. The microsatellite analysis revealed that, in each of our crosses, a single male fathered most of the veligers, despite efforts to normalize the amount of sperm contributed by competing males. These observations suggest that highly controlled breeding practices may be required to ensure that the genetic diversity of an abalone population produced for aquaculture is maintained at the level of diversity of the original broodstock.

Sterol and squalene content of a docosahexaenoic-acid-producing thraustochytrid: influence of culture age, temperature, and dissolved oxygen.

Abstract: Thraustochytrid strain ACEM 6063, rich in omega-3 polyunsaturated fatty acids, was cultured at 15°C and 20°C in high (>40%) and low (<5%) dissolved oxygen (DO), and at 25°C in low-DO media. Samples were taken 4, 2, and 0 days before each culture reached peak biomass (T−4, T−2, and Tp, respectively). Twenty sterols, 13 of which were identified, were detected. Predominant were cholest-5-en-3β-ol, 24-ethylcholesta-5,22E-dien-3β-ol, 24-methylcholesta-5,22E-dien-3β-ol, and 2 coeluting sterols, one of which was 24-ethylcholesta-5,7,22-trien-3β-ol. These 4 sterols comprised 50% to 90% of total sterols. Cultures grown at high DO had simpler sterol profiles than those grown at low DO. Only the 4 sterols mentioned above were present at more than 3% of total sterols in high-DO cultures. In low-DO cultures, up to 6 additional sterols were present at more than 3% of total sterols. Culture age, temperature, and DO influenced squalene and sterol content. Total sterols (as a proportion of total lipids) decreased with increasing culture age. If organisms such as ACEM 6063 are to be used for commercial production of lipid products for human consumption, both their sterol content and factors influencing sterol production need to be characterized thoroughly.

Presence of a nonhydrolyzable biopolymer in the cell wall of vegetative cells and astaxanthin-rich cysts of Haematococcus pluvialis (Chlorophyceae).

Abstract: The green alga Haematococcus pluvialis accumulates massive amounts of the red pigment astaxanthin in response to stimuli inducing it to form cysts. During the encystment process the cell wall undergoes a clear hardening and thickening. In this work, a cell wall fraction withstanding successive acid and basic hydrolysis was isolated and proves to be algaenan by Fourier transform infrared spectroscopy. This compound is equally abundant in nonmotile vegetative cells and astaxanthin-rich cysts. This finding indicates that the synthesis of algaenan does not require the activation of the machinery for the massive production of secondary carotenoids. We conclude that algaenan cannot cause the changes occurring in the cell wall during the encystment process without the involvement of other cell wall components.

Application of microsatellite markers to population genetics studies of Japanese flounder Paralichthys olivaceus.

Abstract: We examined population genetic structure by means of microsatellite analysis among 7 Japanese flounder (Paralichthys olivaceus) populations collected from coastal sea areas around Japan. As was expected, all of the 11 microsatellite loci examined were variable in all populations (number of alleles per locus, 15.2–18.2; average of expected heterozygosity, 0.74–0.78). Eleven population pairs in 21 possible pairwise comparisons showed significant genetic heterogeneity associated with allele frequency distributions or fixation index (F ST ). Modified Cavalli-Sforza chord distance (D A ) and Nei's standard genetic distance (D ST ) ranged from 0.051 to 0.090, and from 0.000 to 0.025, respectively. There was evidence that the populations assessed in this study were not drawn from a single panmictic population; however, it appears that Japanese flounder populations around Japan are not well-structured, as an estimate of the fixation index value among the 7 localities was very low (F ST = 0.0025).

Amphitrite ornata, a Marine Worm, Contains Two Dehaloperoxidase Genes

Abstract: Amphitrite ornata, a terebellid polychaete, inhabits marine environments that are contaminated by biogenically produced halometabolites. These halogenated organic compounds are toxic and quite diverse. To survive in this environment, A. ornata produces a novel dehaloperoxidase (DHP I) that detoxifies haloaromatic compounds. In this study we identified and characterized two dehaloperoxidase genes, designated dhpA and dhpB, from an A. ornata complementary DNA library. The deduced amino acid sequences (DHP A and DHP B) of the two dhp genes both contain 137 amino acid residues, but they differ at 5 amino acid positions. Allelic variation was observed for both genes as well. Polymerase chain reaction-restriction fragment length polymorphism assays of genomic DNA from 19 in individuals showed that each individual contains both the dhpA and the dhpB genes. Therefore, the two types of DHP are encoded by separate genes and are not alleles of a single gene. Furthermore, DHP A and DHP B may have different substrate specificities since they have amino acid differences in the active site.

The 60-kDa heat shock protein (HSP60) of the sea anemone Anemonia viridis: a potential early warning system for environmental changes.

Abstract: Expression of heat shock proteins (HSPs) is often correlated with adaptation to environmental stress. We examined the role of HSP60 (60 kDa) in acclimatization to thermal stress in the sea anemone Anemonia viridis. Using monoclonal antibodies, we identified HSP60 in sea anemones for the first time, and showed that its expression varied with changes in seawater temperature (SWT). Anemonia viridis displayed high levels of HSP60 when extreme temperatures prevailed in stressful habitats such as tidal pools. Specimens sampled from different temperature layers in the same tidal pool differed in their levels of HSP60. Specimens from subtidal zones exhibited a seasonal pattern of expression of HSP60, according to the seasonal SWT. The level of HSP60 was significantly higher in the summer (SWT, 31°C) than in other seasons throughout the year. This study suggests the use of HSP60 expression as a tool for stress detection in marine invertebrates.

Effects of insert size on transposition efficiency of the sleeping beauty transposon in mouse cells.

Abstract: Transposon vectors are widely used in prokaryotic and lower eukaryotic systems. However, they were not available for use in vertebrate animals until the recent reconstitution of a synthetic fish transposon, Sleeping Beauty (SB). The reacquisition of transposability of the SB transposase fostered great enthusiasm for using transposon vectors as tools in vertebrate animals, particularly for gene transfer to facilitate accelerated integration of transgenes into chromosomes. Here, we report the effects of insert sizes on transposition efficiency of SB. A significant effect of insert size on efficiency of transposition by SB was found. The SB transposase enhanced the integration efficiency effectively for SB transposon up to approximately 5.6 kb, but lost its ability to enhance the integration efficiency when the transposon size was increased to 9.1 kb. This result indicates that the SB transposon system is highly applicable for transferring small genes, but may not be applicable for transferring very large genes.

Characterization of Microsatellites in the IGF-2 and GH Genes of Asian Seabass (Lates calcarifer)

Abstract: Four microsatellites were identified by screening the DNA sequences of Asian seabass (Lates calcarifer) deposited to GenBank. Two markers each are located in the growth hormone gene (GH) and in the insulin-like growth factor II gene (IGF-2), respectively. The markers were characterized by genotyping 34 Asian seabass individuals. All 4 microsatellites showed polymorphism: the number of alleles per locus ranged from 2 to 11 (average, 5.0), while the expected heterozygosity ranged from 0.51 to 0.85 (average, 0.63) at the 4 loci. Cross-priming with all 4 primer pairs was tested in species belonging to 5 different genera, but no bands were amplified. These microsatellites are the first genomic DNA markers characterized in L. calcarifer; thus they may be valuable for research and aquaculture production of this species.

Microsatellite Variation Among Red Snapper (Lutjanus campechanus) from the Gulf of Mexico

Abstract: Allelic variation at a total of 20 nuclear-encoded microsatellites was examined among adult red snapper (Lutjanus campechanus) sampled from 4 offshore localities in the Gulf of Mexico. The number of alleles at the 20 microsatellites ranged from 5 to 20; average (± SE) direct count heterozygosity values ranged from 0.148 ± 0.025 to 0.902 ± 0.008. No significant departures from expectations of Hardy-Weinberg equilibrium were found for any locus within samples, and genotypes at pairs of microsatellites appeared to be randomly associated, i.e., in genotypic equilibrium. Tests of homogeneity in allele distributions among the 4 localities were nonsignificant for 19 of the microsatellites. Allele distribution at microsatellite Lca 43 was heterogeneous among localities before (but not after) Bonferroni corrections for multiple tests executed simultaneously. Tests of homogeneity in the distribution of individual alleles at Lca 43 gave similar results: one low frequency allele was distributed heterogeneously among samples before, but not after, Bonferroni correction. Molecular analysis of variance indicated that more than 99% of variation at each microsatellite was distributed within sample localities. These results generally are consistent with the hypothesis of a single population (stock) of red snapper in the northern Gulf of Mexico.

Utility of natural populations for microarray analyses: isolation of genes necessary for functional genomic studies.

Abstract: How much variation is there in gene expression? How is this variation partitioned within and among populations? How much variation is biologically important? That is, how much of this variation affects longevity, reproductive fitness, or probability of survival? Microarray analyses can be used to accurately quantify the expression of most, if not all, genes expressed in a tissue and thus address the first question The latter questions can be investigated by examining the patterns of variation within and among natural populations of Fundulus These populations are large and affected by historical, demographic, and selective constraints, providing a framework for the partition of variation in gene expression within and among populations Additionally, the well established, phylogenetic relationship among Fundulus species can be used to discern adaptive change A phylogenetic perspective reveals changes that are produced by natural selection and therefore indicates whether this variation affects longevity, reproductive fitness, or probability of survival, ie, whether the variation is biologically important However, a Fundulus microarray requires DNAs encoding specific Fundulus genes This paper provides information on the production, isolation, and characterization of 4440 Fundulus cDNAs used in microarrays Our approach was to pick random colonies from a normalized cDNA library and then PCR amplify and sequence these genes in a 96-well format Periodically, the isolated and sequenced cDNAs were subtracted from the normalized library Normalization reduced the number of redundant genes from 33% to 11%, increasing the effectiveness of this screening process From 4440 sequenced cDNAs, 49% (2173) had a match in GenBank using BlastX searches Of these, 53% were nonredundant, yielding 1149 identified genes These data suggest that cDNAs necessary for microarray analyses can be produced effectively from most organisms

Characterization and quantification of chitosan extracted from nacre of the abalone Haliotis tuberculata and the oyster Pinctada maxima.

Abstract: This study was performed to characterize and quantify chitosan by simple physicochemical methods (infrared spectroscopy and potentiometric measurements). These procedures were validated with well-characterized chitosan before being used to investigate chitosan in nacre of the abalone Haliotis tuberculata and of the giant oyster Pinctada maxima. Potentiometric study revealed a chitosan extract from the nacre of H. tuberculata with a degree of deacetylation of around 88% and an intrinsic pK of 6.5. According to infrared and potentiometric data, a low yield (η) of extraction was calculated (η= 0.064%). For experiments performed on the nacre of P. maxima, and in spite of more stringent deacetylation conditions, results suggested that a chitin-protein complex (η= 0.053%) was isolated rather than chitosan.

Characterization of psychrotrophic bacteria in the surface and deep-sea waters from the northwestern Pacific Ocean based on 16S ribosomal DNA analysis.

Abstract: Seventy-eight 4°C-culturable bacteria were isolated using ZoBell 2216E medium from surface (0–200 m) and deep-sea (1000–9671 m) waters in the northwestern Pacific Ocean. Growth studies indicated that all 4°C-culturable bacteria were psychrotrophs. Six phylotypes were observed in the surface water samples and 8 phylotypes in the deep-sea waters. Phylogenetic characterization based on 16S ribosomal DNA sequence analysis of the representative phylotypes revealed that some bacterial genera, Pseudoalteromonas, Photobacterium, and Vibrio, were common to surface and deep-sea waters, and others, Pseudomonas and Halomonas, specifically occurred in surface water. Overall, the members of Vibrionaceae appear to be dominant in both habitats.

Growth Rate of the microalga Tetraselmis suecica changes the biochemical composition of Artemia species.

Abstract: The impact of different microalgal semicontinuous cultures on growth and biochemical composition in the next link of the food chain was tested using the filter feeder Artemia species as a model. The marine microalga Tetraselmis suecica was cultured semicontinuously with renewal rates between 10% and 50% and used to feed Artemia. Microalgal cultures maintained with a low renewal rate that had biochemical composition similar to that of the stationary-phase cultures commonly used in aquaculture produced poor growth and survival and low food-conversion efficiency compared to cultures maintained with a high renewal rate. Changes in the renewal rate in microalgal cultures also resulted in important changes in the gross biochemical composition of the filter feeder. The gross biochemical composition of the Artemia resembled that of the microalgae used as food except for total lipid content. The percentage of protein in the organic fraction of Artemia increased from 45% to 65% of the organic weight with increasing renewal rates in the microalgal cultures, while the carbohydrate percentage decreased under the same conditions. Higher renewal rates resulted in higher lipid percentages in the microalga, but in Artemia the percentage of lipids decreased from 19% of the organic weight with a renewal rate of 10%, to 13% with a renewal rate of 50%. The percentage of all polyunsaturated fatty acids in Artemia, including 20:5n-3, increased slightly with increasing renewal rates in the microalgal cultures. Results emphasize the importance of controlling microalgal nutritional value for the success of aquaculture food chains in which filter feeders are involved.

Multiple tissue transformation in adult zebrafish by gene gun bombardment and muscular injection of naked DNA.

Abstract: The efficiency of two direct gene transfer methods, gene gun (or particle bombardment) and intramuscular injection, in transforming adult zebrafish tissues in vivo was examined by a noninvasive approach using green fluorescent protein (GFP) reporter gene driven by the ubiquitously expressed human cytomegalovirus promoter. Particle bombardment of adult zebrafish caused internalization and expression of the plasmid only in the superficial layer such as epithelial cells, pigment cells, endothelial cells, and neurons, whereas direct injection primarily transformed muscle fibers of several bundles near or around the injection site. Expression was also evident in several nonmuscle tissues, such as skin epithelia, pigment cells, blood vessel cells, and neuron-like cells. GFP expression persisted for more than 50 days with both methods. These observations indicate the potential of these methods for functional analysis of tissue-specific promoters, delivery of DNA vaccine, and muscular expression of other useful genes.

Microsatellite Analysis of 6-Hour-Old Embryos Reveals No Preferential Intraspecific Fertilization Between Cupped Oysters Crassostrea gigas and Crassostrea angulata

Abstract: Experimental examination of reproductive isolation is the first step in understanding hybridization processes. Here, we studied preferential fertilization between 2 cupped oyster taxa, Crassostrea angulata and Crassostrea gigas, as a potential prezygotic reproductive isolation. Early examination of sperm competition is now possible by molecular analysis of oyster embryos. This avoids the confounding effect of differential mortality during the larval stage. Six hundred embryos were sampled from 2 crosses. Three microsatellite loci were enough to determine without ambiguity the taxa of contributing sires of embryos. No evidence of preferential fertilization between gametes from the same taxa was shown. A significantly higher contribution of the C. gigas males was revealed with the C. angulata females, but not with the C. gigas females, which might suggest early heterosis or interaction differences between gametes. In the light of these results, natural hybridization between both taxa can be expected in cases of their geographical coexistence, as in the Southern European populations in which both taxa are in contact as a result of aquaculture development.

Cryopreservation in Aquarium Fishes

Abstract: Few studies have addressed sperm cryopreservation in aquarium fishes (body sizes of 10 cm or less). There are several challenges inherent in developing cryopreservation procedures for these fishes. First, their small body size and sperm volume limit experimental replication and the numbers of treatments possible without pooling of samples. This hinders research, especially if many experimental variables are evaluated. The small sample volume necessitates identification of optimal sperm-to-egg ratios to maximize fertilization potential and places greater emphasis on increasing and maintaining sperm viability after thawing. Other technical problems include the use of 0.25-ml French straws, which increase difficulties in sample handling (automated straw fillers are more common for the 0.5-ml straw) and labeling. Sperm cryopreservation of live-bearing fishes (with internal fertilization) is essentially unexplored. The sperm of these fishes is sufficiently different in structure (e.g., head shape) and physiology (e.g., energy metabolism) from the sperm of other fishes that the need to develop specialized techniques is almost assured. The requirement for artificial insemination also introduces a new variable complicating the collection of data (e.g., assessing fertilization is not straightforward with internally held eggs). Cryopreservation in aquarium fishes will assist the development (e.g., through selective breeding), protection (e.g., through germplasm repositories), and distribution (e.g., through shipment of frozen sperm) of research lines and offers benefits for restoration of endangered species.