Genetic variability in Ruditapes decussatus clam combined with Perkinsus infection level to support founder population selection for a breeding program.

Clam farmers worldwide face several challenges, including irregular seed supply and high mortalities due to pathogenic organisms such as Perkinsus olseni. In Europe, there is a high unmet consumer demand for native clam species such as Ruditapes decussatus. The high market value of R. decussatus makes the culture of this species potentially more attractive than that culture of the alien species Ruditapes philippinarum. Thus, there is a market opportunity in breeding and producing R. decussatus at an industrial scale. A selective breeding program to improve R. decussatus performance will be carried out in Portugal; and the first critical step to develop such a breeding program is the establishment of a founder population. In this study, intra- and interpopulation genetic diversity was assessed using 13 microsatellite markers in eight natural beds located in Portugal, Spain and Italy. Also, allele and genotypic frequencies of each microsatellite locus were assessed discriminating between clams infected and non-infected by P. olseni. All locations showed similar values for several genetic diversity parameters. Analyses of population differentiation (F ST, Bayesian clustering and AMOVAs) revealed five genetically differentiated regions: Rías Altas and Rías Baixas (NW Spain), North/Central Coast of Portugal, Gulf of Cadiz and Adriatic Sea. Significant differences in the allelic and genotypic frequency distribution between infected clams and non-infected ones at four microsatellite loci are reported suggesting that resistance to the disease could have a genetic basis. Moreover, a positive or negative relationship between the frequency of certain alleles and the parasite infection was inferred. Further studies should confirm the potential use of those alleles as genetic markers for P. olseni infection. Integrating results of genetic diversity within and between populations and Perkinsus infection levels, a founder population for a R. decussatus breeding program is proposed, composed by individuals from Barallobre (Rías Altas), Pontevedra or Cangas (Rías Baixas), Óbidos (North/Central Coast of Portugal), Algarve (Gulf of Cadiz) and Venice (Adriatic Sea).

Assessing the geographic scale of genetic population management with microsatellites and introns in the clam Ruditapes decussatus.

The clam Ruditapes decussatus is commercially important in southwestern Europe, suffering from population decline and hybridization with exotic Manila clam (R. philippinarum). Previous studies with intronic markers showed a genetic subdivision of the species in three races (Atlantic, West Mediterranean, and Adriatic-Aegean). However, detailed population genetic studies to help management of the main production areas in the southwest of Europe are missing. We have analyzed eight Atlantic and two Mediterranean populations from the Spanish coasts using 14 microsatellites and six intronic markers. Microsatellites confirmed the Atlantic and West Mediterranean races detected with introns and showed that genetic variability was higher in Mediterranean than in Atlantic populations. Both marker types showed that genetic differentiation of Atlantic populations was low and indicated that populations could be managed at the regional level in the case of Cantabrian and Gulf of Cadiz areas, but not in the case of Rias Baixas and the Mediterranean. This study shows the interest of including different types of markers in studies of genetic population structure of marine organisms.

Development and multiplex PCR amplification of microsatellite markers in the commercial clam Venerupis rhomboides (Mollusca: Bivalvia).

Venerupis rhomboides is a commercial clam whose production could be enhanced through effective management of natural and hatchery stocks. This study provides the first panel of microsatellite markers for the exploitation of this species according to genetic criteria. A total of 22 polymorphic microsatellite loci were isolated and characterized from two genomic libraries enriched for different motifs. The number of alleles per locus ranged from 2 to 14 in a sample of 20 clams from Spain, and the observed and expected heterozygosity from 0 to 0.95 and 0.05-0.901, respectively. Sixteen loci were in agreement with Hardy-Weinberg equilibrium after sequential Bonferroni correction and linkage disequilibrium between loci pairs was not detected. To reduce the cost of the genotyping process, tri- and pentaplex PCRs, amplifying a total of 13 microsatellites loci were optimized. The microsatellites developed here represent the first nuclear markers described in V. rhomboides and will be useful tools for genetic studies involving assessment of genetic variation and population structure of natural and cultivated populations, assignment testing, construction of genetic linkage maps and dissection of production traits.

Identification, inheritance, and variation of microsatellite markers in the black scallop Mimachlamys varia.

Five polymorphic microsatellite loci were identified in the black scallop Mimachlamys varia after construction of a genomic library enriched for (GT)n. To examine the transmission pattern of microsatellite alleles, several families were created and genotypes scored for three loci. The expected Mendelian ratios were found in 12 of 14 segregations examined. Unexpected segregations may be explained by a genotyping error (allelic dropout), given that when a specific allele was treated as dominant, the phenotypic ratios conformed to Mendelian expectations. The five loci were also examined in two samples from the Spanish coast. The two localities displayed similar mean values for the number of alleles per locus (7.2-8.4), allelic richness (7.2-7.9), and observed (0.389-0.484) and expected heterozygosity (0.545-0.618). Significant Hardy-Weinberg deviations were observed at three loci, with heterozygote deficiency occurring in all cases. Global multilocus θ value and allele frequencies at one locus revealed significant differentiation between the two localities.

Evolutionary dynamics of the 5S rDNA gene family in the mussel Mytilus: mixed effects of birth-and-death and concerted evolution.

In higher eukaryotes, the gene family encoding the 5S ribosomal RNA (5S rRNA) has been used (together with histones) to showcase the archetypal example of a gene family subject to concerted evolution. However, recent studies have revealed conspicuous features challenging the predictions of this model, including heterogeneity of repeat units, the presence of functional 5S gene variants as well as the existence of 5S rDNA divergent pseudogenes lacking traces of homogenization. In the present work, we have broadened the scope in the evolutionary study of ribosomal gene families by studying the 5S rRNA family in mussels, a model organism which stands out among other animals due to the heterogeneity it displays regarding sequence and organization. To this end, 48 previously unknown 5S rDNA units (coding and spacer regions) were sequenced in five mussel species, leading to the characterization of two new types of units (referred to here as small-beta 5S rDNA and gamma-5S rDNA) coexisting in the genome with alpha and beta rDNA units. The intense genetic dynamics of this family is further supported by the first description of an association between gamma-5S rDNA units and tRNA genes. Molecular evolutionary and phylogenetic analyses revealed an extensive lack of homology among spacer sequences belonging to different rDNA types, suggesting the presence of independent evolutionary pathways leading to their differentiation. Overall, our results suggest that the long-term evolution of the 5S rRNA gene family in mussels is most likely mediated by a mixed mechanism involving the generation of genetic diversity through birth-and-death, followed by a process of local homogenization resulting from concerted evolution in order to maintain the genetic identities of the different 5S units, probably after their transposition to independent chromosomal locations.

Intron characterization and their potential as molecular markers for population studies in the scallops Aequipecten opercularis and Mimachlamys varia.

Exon-primed intron-crossing PCR was used on the European commercial scallops Aequipecten opercularis and Mimachlamys varia to characterize introns of four nuclear genes and to identify DNA markers useful for population studies. The primers used yielded the expected product, except those for the lysozyme gene that failed to work in M. varia and amplified a fragment of a proteasome subunit gene (APSM) in A. opercularis. According to the sequences characterized, A. opercularis has at least four calmodulin genes, one of arginine kinase and two of beta-tubulin, and M. varia five, one and one, respectively. Length polymorphism or/and restriction fragment length polymorphism was detected at two loci of A. opercularis (arginine kinase and APSM) and four of M. varia (calmodulin and beta-tubulin), distinguishing in each case two or three alleles. The polymorphic loci were not closely linked. The population survey included four localities from Spain and one from Northern Ireland for A. opercularis and two Spanish localities for M. varia. Observed heterozygosity (H(o)) per locus was 0.276 and 0.296 in A. opercularis. The Northern Ireland sample had the lowest H(o) value (0.200) and the Mediterranean Spanish sample the highest (0.350). In M. varia, H(o) per locus ranged from 0.172 to 0.391 and the two localities showed similar H(o) values (0.255 and 0.293). All population-locus combinations were in agreement with Hardy-Weinberg equilibrium, except two loci of M. varia that showed a strong heterozygote deficit in the two localities examined. Evidence for genetic differentiation among samples was not found.

Sequence characterization and phylogenetic analysis of the 5S ribosomal DNA in some scallops (Bivalvia: Pectinidae).

This study was designed to characterize the 5S ribosomal DNA repeat unit and to evaluate its phylogenetic informativeness in Mimachlamys varia, Hinnites distortus, Aequipecten opercularis and Pecten maximus, scallops of the bivalve family Pectinidae. The repeat unit was PCR amplified and several products cloned and sequenced. The deduced coding region covered 120 bp, showed 52-53% GC content and an identifiable internal promoter. The spacer region was 313-345 bp in length with 30-40% GC and the ends contained elements showing similarity with upstream and downstream sequences involved in the transcription. The sequences of P. maximus were identical and those of M. varia and H. distortus differed only at 2-3 positions. However, the sequences of A. opercularis showed a percentage of differences of 0.69-9.15%, distinguishing two types of units differing in sequence and length of the spacer region. All scallops displayed an identical gene sequence, except M. varia that differed by one nucleotide substitution, but a highly variable spacer. In the phylogenetic trees the sequences grouped by species with two well supported clades, one containing the sequences of M. varia and H. distortus and the other those of P. maximus and A. opercularis, the latter grouped according to the unit type. The topology obtained was in accordance with scallop evolutionary relationships inferred from previous phylogenetic studies.

Karyotype and chromosomal location of 18S-28S and 5S ribosomal DNA in the scallops Pecten maximus and Mimachlamys varia (Bivalvia: Pectinidae).

This work describes the karyotype and chromosomal location of the ribosomal DNA (rDNA) of Pecten maximus and Mimachlamys varia, two commercial scallop species from Europe. According to the chromosome centromeric index values found, the karyotype of P. maximus is composed of 1 metacentric, 2 metacentric-submetacentric, 1 telocentric-subtelocentric and 15 telocentric pairs, and that of M. varia of 4 metacentric, 2 subtelocentric-submetacentric, 9 subtelocentric, 3 subtelocentric-telocentric and 1 telocentric-subtelocentric pairs. In P. maximus, 18S-28S rDNA was located by FISH on a metacentric-submetacentric pair, and in M. varia on a subtelocentric-submetacentric pair using both silver staining and FISH. PCR amplification of the 5S rDNA unit yielded a single product of about 460 bp (P. maximus) and 450 bp (M. varia), that used as probe revealed a 5S rDNA site on a telocentric pair in P. maximus and a subtelocentric pair in M. varia. Two-color FISH or sequential silver staining of 5S rDNA-FISH-metaphases corroborated that the two gene families are located on different chromosomes in both species. A comparative analysis of the data allowed the inference of karyotypic relationships within scallops.

Cerastoderma glaucum 5S ribosomal DNA: characterization of the repeat unit, divergence with respect to Cerastoderma edule, and PCR-RFLPs for the identification of both cockles.

The 5S rDNA repeat unit of the cockle Cerastoderma glaucum from the Mediterranean and Baltic coasts was PCR amplified and sequenced. The length of the units was 539-568 bp, of which 120 bp were assigned to the 5S rRNA gene and 419-448 bp to the spacer region, and the G/C content was 46%-49%, 54%, and 44%-47%, respectively. Two types of units (A and B), differing in the spacer, were distinguished based on the percentage of differences and clustering in phylogenetic trees. A PCR assay with specific primers for each unit type indicated that the occurrence of both units is not restricted to the sequenced individuals. The 5S rDNA units of C. glaucum were compared with new and previously reported sequences of Cerastoderma edule. The degree of variation observed in C. edule was lower than that in C. glaucum and evidence for the existence of units A and B in C. edule was not found. The two cockles have the same coding region but displayed numerous fixed differences in the spacer region and group separately in the phylogenetic trees. Digestion of the 5S rDNA PCR product with the restriction enzymes HaeIII and EcoRV revealed two RFLPs useful for cockle identification.

Sequence analysis of the ribosomal DNA internal transcribed spacer region in some scallop species (Mollusca: Bivalvia: Pectinidae).

The internal transcribed spacer (ITS) region of the ribosomal DNA from the European scallops Aequipecten opercularis, Mimachlamys varia, Hinnites distortus, and Pecten maximus was PCR amplified and sequenced. For each species, three or five clones were examined. The size ranged between 636 and 713 bp (ITS1, 209-276 bp; 5.8S rRNA gene, 157 bp; ITS2, 270-294 bp) and GC content ranged between 47 and 50% (ITS1, 43-49%; 5.8S rRNA gene, 56-57%; ITS2, 44-49%). Variation within repeats was minimal; only clones from M. varia and P. maximus displayed a few variable sites in ITS2. Among scallops, including Chlamys farreri whose ITS sequence appears in databases, significant variation was observed in both ITS1 and ITS2. Phylogenetic analysis using ITS1, ITS2, or both spacer sequences always yielded trees with similar topology. Aequipecten opercularis and P. maximus grouped in one clade and the other three scallops (C. farreri, M. varia, and H. distortus) in another, where M. varia and H. distortus are the more closely related species. These results provide new insights into the evolutionary relationships of scallop species and corroborate the close evolutionary relationship between the tribes Aequipectinini and Pectinini previously deduced from 18S rDNA sequences.

Fenofibrate of gemfibrozil for treatment of types IIa and IIb primary hyperlipoproteinemia: a randomized, double-blind, crossover study.

OBJECTIVE: To compare the hypolipidemic effects of gemfibrozil and micronized fenofibrate in patients with primary hyperlipoproteinemia, phenotypes IIa and IIb, with emphasis on their cholesterol-lowering effectiveness. METHODS: A randomized, double-blind, double-dummy, crossover study was performed to assess the effects of gemfibrozil (900 mg) and micronized fenofibrate (200 mg), administered once daily, to 21 patients (45 to 70 years old)-16 with type IIa and 5 with type IIb primary hyperlipidemia. The two treatment periods lasted 6 weeks each; the run-in and washout periods were 4 weeks. RESULTS: Both drugs significantly reduced total cholesterol, calculated low-density lipoprotein (LDL) cholesterol, triglycerides, apolipoprotein B, and fibrinogen (P<0.01 for all calculations, except P<0.05 for fibrinogen with gemfibrozil therapy) and increased high-density lipoprotein (HDL) cholesterol (P<0.01). Neither drug affected Lp(a) lipoprotein, whereas uric acid was reduced only by fenofibrate (P<0.01). The percentage decrease in total cholesterol and LDL cholesterol was greater with fenofibrate than with gemfibrozil (-22% versus -15%, P<0.02; and -27% versus -16%, P<0.02, respectively). In contrast, reductions in levels of triglycerides (-54% versus -46.5%), apolipoprotein B, and fibrinogen, as well as the increase in HDL (+9% for both drugs), showed no significant difference between treatments. Separate analysis of patients with type IIb hyperlipoproteinemia showed essentially the same plasma lipid changes as for the overall group, but with greater modifications in triglyceride and HDL concentrations. CONCLUSION: Fenofibrate and gemfibrozil induced similar variations from baseline values in triglycerides, HDL cholesterol, apolipoprotein B, and fibrinogen, but the decreases in total and LDL cholesterol levels were greater with fenofibrate, in this group of patients with primary hyperlipidemia.

Quejas sobre la memoria y deficits mnemicos en ancianos sanos y deprimidos / Memory complaints and memory deficits in healthy and depressed elderly individuals

El objetivo de este estudio es: a) determinar si las quejas sobre la memoria están relacionadas con la depresión y b) comparar el rendimiento de ancianos depresivos y normales en pruebas objetivas para evaluar memoria. El Test California de Aprendizaje Verbal (CVLT) se utilizó para evaluar a la memoria. Con el fin de cuantificar la apreciación subjetiva del déficit mnémico por parte de los sujetos estudiados, se empleó un cuestionario sobre quejas de la memoria (CQSM) y la intensidad del trastorno depresivo se midió utilizando el Inventario de Beck sobre Depresión (BDI). Los resultados mostraron que los pacientes deprimidos no tenían deficiencias en la memoria, comparándolos con un grupo control sano. Se observó un asociación entre depresión y quejas sobre la memoria. Los pacientes tenían un puntaje significativamente más alto en quejas y en síntomas depresivos, con respecto al grupo control. Los dos grupos no se diferenciaban en las medidas objetivas de memoria y aprendizaje.

Quejas sobre la memoria y deficits mnemicos en ancianos sanos y deprimidos / Memory complaints and memory deficits in healthy and depressed elderly individuals

El objetivo de este estudio es: a) determinar si las quejas sobre la memoria están relacionadas con la depresión y b) comparar el rendimiento de ancianos depresivos y normales en pruebas objetivas para evaluar memoria. El Test California de Aprendizaje Verbal (CVLT) se utilizó para evaluar a la memoria. Con el fin de cuantificar la apreciación subjetiva del déficit mnémico por parte de los sujetos estudiados, se empleó un cuestionario sobre quejas de la memoria (CQSM) y la intensidad del trastorno depresivo se midió utilizando el Inventario de Beck sobre Depresión (BDI). Los resultados mostraron que los pacientes deprimidos no tenían deficiencias en la memoria, comparándolos con un grupo control sano. Se observó un asociación entre depresión y quejas sobre la memoria. Los pacientes tenían un puntaje significativamente más alto en quejas y en síntomas depresivos, con respecto al grupo control. Los dos grupos no se diferenciaban en las medidas objetivas de memoria y aprendizaje. (AU)

Evaluación de los signos de organicidad, segun Piotrowsky, en el Rorschach de pacientes con un comienzo de demencia / Evaluation of the Piotrowski's organic signs in the Rorschach test of patients with an early stage of dementia

El propósito de esta investigación es observar la frecuencia con la cual se presentan los signos orgánicos de Piotrowsky en los Rorschach de un grupo de personas que padecen de una probable demencia. El grupo orgánico estaba formado por quince pacientes y fue comparado con un grupo normal. Ambos grupos eran similares en edad, cociente intelectual y educación. Observamos que, solamente, Perplejimiento y Frases Automáticas - se acercaron a un nivel convencional de significación. Nuestros resultados fueron cocmparados con los de otros investigadores. Concluimos que Perplejidad, Movimiento y Frases Automáticas pueden ser elementos útiles para ayudar a detectar los comienzos de un proceso demencial

Evaluación de los signos de organicidad, segun Piotrowsky, en el Rorschach de pacientes con un comienzo de demencia / Evaluation of the Piotrowskis organic signs in the Rorschach test of patients with an early stage of dementia

El propósito de esta investigación es observar la frecuencia con la cual se presentan los signos orgánicos de Piotrowsky en los Rorschach de un grupo de personas que padecen de una probable demencia. El grupo orgánico estaba formado por quince pacientes y fue comparado con un grupo normal. Ambos grupos eran similares en edad, cociente intelectual y educación. Observamos que, solamente, Perplejimiento y Frases Automáticas - se acercaron a un nivel convencional de significación. Nuestros resultados fueron cocmparados con los de otros investigadores. Concluimos que Perplejidad, Movimiento y Frases Automáticas pueden ser elementos útiles para ayudar a detectar los comienzos de un proceso demencial (AU)