Carlos Saavedra

mRNA-Seq and microarray development for the Grooved carpet shell clam, Ruditapes decussatus: a functional approach to unravel host -parasite interaction

BackgroundThe Grooved Carpet shell clam Ruditapes decussatus is the autochthonous European clam and the most appreciated from a gastronomic and economic point of view. The production is in decline due to several factors such as Perkinsiosis and habitat invasion and competition by the introduced exotic species, the manila clam Ruditapes philippinarum. After we sequenced R. decussatus transcriptome we have designed an oligo microarray capable of contributing to provide some clues on molecular response of the clam to Perkinsiosis.ResultsA database consisting of 41,119 unique transcripts was constructed, of which 12,479 (30.3%) were annotated by similarity. An oligo-DNA microarray platform was then designed and applied to profile gene expression in R. decussatus heavily infected by Perkinsus olseni. Functional annotation of differentially expressed genes between those two conditionswas performed by gene set enrichment analysis. As expected, microarrays unveil genes related with stress/infectious agents such as hydrolases, proteases and others. The extensive role of innate immune system was also analyzed and effect of parasitosis upon expression of important molecules such as lectins reviewed.ConclusionsThis study represents a first attempt to characterize Ruditapes decussatus transcriptome, an important marine resource for the European aquaculture. The trancriptome sequencing and consequent annotation will increase the available tools and resources for this specie, introducing the possibility of high throughput experiments such as microarrays analysis. In this specific case microarray approach was used to unveil some important aspects of host-parasite interaction between the Carpet shell clam and Perkinsus, two non-model species, highlighting some genes associated with this interaction. Ample information was obtained to identify biological processes significantly enriched among differentially expressed genes in Perkinsus infected versus non-infected gills. An overview on the genes related with the immune system on R. decussatus transcriptome is also reported.

Phylogenetic relationships of commercial European and Australasian king scallops (Pecten spp.) based on partial 16S ribosomal RNA gene sequences

Abstract King scallops ( Pecten sp.) are commercially important in regions as distant as Europe, Australia, and Japan, and some species are cultured. The taxonomy of king scallops has been traditionally based in characteristics of the shell. Recently, the analysis of protein and DNA polymorphism has shown that the two forms of king scallops known from Europe, Pecten maximus and Pecten jacobaeus , are the same species in spite of showing clear morphological differences. This casts doubts on the distinction of other king scallop species based on morphology alone. We have studied the phylogenetic relationships of the two European species with two other Pecten taxa which show a striking morphological similarity with them in spite of living in the opposite side of the world: Pecten fumatus and Pecten novaezelandiae , from the southern Indo-Pacific around the coasts of Australia and New Zealand. We scored the nucleotide variability of a 501-base-pair-long fragment of the mitochondrial 16S ribosomal RNA gene in a total of 27 individuals and performed a phylogenetic analysis. Atlantic and Australasian taxa constituted two clearly separated lineages, which rules out an explanation for morphological similarity based on conspecificity due to human transfer, as has been proposed for other bivalve species-pairs in the studied regions. However, we found that the amount of differentiation observed between the two oceanic lineages was modest when compared with that expected under the generally accepted paleobiogeographical model of origin of Atlantic and Indo-Pacific king scallops, based on the closure of the Tethys Sea at the beginning of the Miocene, ca. 14 million years ago (average Kimura 2-parameter distance=0.0154±0.0053). We present an alternative model based on Beu and Darraghs [Proc. R. Soc. Victoria 113 (2001) 1] hypothesis of South Africa to Australia Pleistocene dispersal and Vermeijs [Mar. Biol. 112 (1992) 343] hypothesis of pre-Pleistocene transequatorial dispersal from Europe to South Africa. Finally, our phylogenetic analysis did not separate P. fumatus and P. novaezelandiae as different species, contrary to previous studies based on allozymes.

Inferring the demographic history of the Adriatic Flexopecten complex.

Phylogenetic analysis of DNA sequences can be a useful tool in taxonomical studies, which has important implications in terms of species traceability. The aim of our study is to solve the controversy regarding the taxonomical status of the Flexopecten group, which includes two putative sister species (F. glaber and F. proteus) that co-occur in the Adriatic Sea and are clearly distinguishable on the basis of morphometric characters. Our molecular analysis using three mitochondrial genes (COI, 12S rRNA and 16S rRNA) and one nuclear gene (Histone H3) suggest that F. glaber and F. proteus are the same species, as evidenced by both putative species appearing mixed in all genetic trees with no clustering according to species. Using a Bayesian approach, we inferred the demographic history of the Flexopecten group, which suggests that first F. glaber occurred in the Mediterranean, then F. glaber colonized the Adriatic sometime in the last 18,000 years, and finally the F. proteus morph appeared only recently in the Adriatic Sea. We propose F. proteus to be synonymized with F. glaber, which should have priority and be used in the future.

Phylogeographic analysis of introns and mitochondrial DNA in the clam Ruditapes decussatus uncovers the effects of Pleistocene glaciations and endogenous barriers to gene flow

Studies on the phylogeography of species inhabiting the Mediterranean and the nearby coasts of the NE Atlantic Ocean (MEDAT) have found subdivision and/or phylogeographic structure in one or more of the Atlantic, western Mediterranean and eastern Mediterranean basins. This structure has been explained as the result of past population fragmentation caused by Pleistocene sea level changes and current patterns of marine circulation. However, the increasing use of nuclear markers has revealed that these two factors alone are not enough to explain the phylogeographic patterns, and an additional role has been suggested for endogenous barriers to gene flow or natural selection. In this article we examined the role of these factors in Ruditapes decussatus, a commercial clam species native to MEDAT. A genetic analysis of 11 populations was carried out by examining 6 introns with a PCR-RFLP technique. We found subdivision in three regions: Atlantic (ATL), western Mediterranean plus Tunisia (WMED), and Aegean and Adriatic seas (AEGAD). Two introns (Ech and Tbp) showed alleles that were restricted to AEGAD. Sequencing a subsample of individuals for these introns indicated that AEGAD-specific alleles were separate clades, thus revealing a phylogeographic brake at the WMED-AEGAD boundary. Sequencing of the mitochondrial COI locus confirmed this phylogeographic break. Dating of the AEGAD mitochondrial haplotypes and nuclear alleles with a Bayesian MCMC method revealed that they shared common ancestors in the Pleistocene. These results can be explained in the framework of Pleistocene sea level drops and patterns of gene flow in MEDAT. An additional observation was a lack of differentiation at COI between the ATL and WMED, in sharp contrast with 4 introns that showed clear genetic subdivision. Neutrality tests did not support the hypothesis of a selective sweep acting on mtDNA to explain the contrasting levels of differentiation between mitochondrial and nuclear markers across the ATL-WMED transition, and we argue that the difference between markers is best explained by the existence of an endogenous genetic barrier, rather than by a physical barrier to larval migration alone.

Genetic and phenoptypic differentiation of zebra mussel populations colonizing Spanish river basins

Zebra mussel populations in Ebro and Mijares Rivers (northern Spain) were analyzed to study the mechanisms by which this aquatic species deals with pollution. Variability analyses of mitochondrial cytochrome oxidase I gene and of one nuclear microsatellite were performed for ten populations from the Ebro River and one from the Mijares River. Comparison of these results with those from five additional European populations indicated that the Spanish populations constitute a homogeneous gene pool. Transcriptome analyses of gill samples from a subset of the Spanish populations showed changes on expression levels that correlated with variations in general fitness and loads of heavy metals. The less polluted upstream Ebro populations showed overexpression of mitochondrial and cell proliferation-related genes compared to the more polluted, downstream Ebro populations. Our data indicate that heavy metals were the main factors explaining these transcriptomic patterns, and that zebra mussel is resilient to pollutants (like mercury and organochlorine compounds) proved to be extremely toxic to vertebrates. We propose that zebra mussel populations sharing a common gene pool may acclimate to different levels and forms of pollution through modulations in their transcriptomic profile, although direct selection on genes showing differential expression patterns cannot be ruled out.

Polymorphisms at Three Introns in the Manila Clam (Ruditapes philippinarum) and the Grooved Carpet-Shell Clam (R. Decussatus)

Abstract We have designed a set of exon-primed intron-crossing (EPIC) PCR primers to amplify introns at the genes TBP and SRP54 in the Manila clam (Ruditapes philippinarum) and the grooved carpet-shell clam (R. decussatus), and also one intron at a histone 3 homologous gene in the Manila clam. The primers were developed by using “universal” EPIC primers available in the literature and by searching for intron locations in cDNA sequences taken from public databases. The identity of the amplified products was checked by sequencing. The three introns of the Manila clam, and one in the carpet shell clam (TBP), exhibited length polymorphisms. The number of alleles was two at the TBP locus of the grooved carpet-shell clam, and ranged from three to five in the three loci of the Manila clam. The locus without length polymorphism in the carpet-shell clam exhibited polymorphism when digested with the restriction enzyme EcoR I (4 haplotypes). The variability of the markers was examined in two population samples in each species.

Genetic characterization of Argentinean Artemia species with different fatty acid profiles

The Anostracan genus Artemia is composed by several sibling species reproductively isolated, but identical or very similar in outward appearance. The genus shows also an underlying striking variability from the biochemical point of view, regarding especially the fatty acid profile of the cysts and nauplii. In Argentina, Artemia is represented by two bisexual species: A. franciscana and A. persimilis. Former studies have shown that A. franciscana is present in northern of 36º and that A. persimilis is constrained southwards of 37° S. In general, there is good agreement between morphological and cytogenetic comparisons of Argentinean populations with respect to species discrimination. However, new Argentinean Artemia populations are being analyzed morphologically and it becomes necessary to further investigate if the genetic adscription of these populations is congruent with the results obtained from the current morphological analyses. Restriction fragment length polymorphism (RFLP) analysis of a fragment of the 16S rRNA mitochondrial gene was used to investigate the genetic diversity and population structure of 10 new Artemia populations from Argentina. The mitochondrial DNA (mtDNA) results showed a similar pattern to that of previous cytogenetic and morphological analyses with the two Argentinean species appearing as highly divergent. The presence of A. persimilis in southern Argentina and the southernmost Chilean population was confirmed unveiling a novel picture of species distribution in the country. A. franciscana showed a unique haplotype. Populations of A. persimilis appeared highly structured, although their clustering did not follow a clear geographic pattern. The different Argentinean Artemia populations analyzed were characterized by high variability in their fatty acids, showing both marine- and freshwater-type profiles. For the first time, the investigation of the relatedness between the fatty acid composition in Artemia and genetic markers was attempted. The study aimed at the putative association of molecular markers with marine versus freshwater-type populations. A lack of correlation between RFLP patterns at mtDNA and the fatty acid (FA) profiles was found in the A. persimilis populations which was discussed from the point of view of two main genetic hypotheses and/or phenotypic plasticity.

Site‐selective modification of peptides: From “customizable units” to novel α‐aryl and α‐alkyl glycine derivatives, and components of branched peptides

The creation of peptide libraries by site‐selective modification of a few peptide substrates would increase the efficiency of discovery processes, but still is a real synthetic challenge. The site‐selective modification of small peptides at serine or threonine residues, by using a short scission–addition procedure, allows the preparation of peptides with unnatural α‐aryl glycines. In a similar way, the scission of hydroxyproline residues is the key step in the production of optically pure α‐alkyl glycines which are precursors or components of branched peptides. With these versatile processes, a single peptide can be transformed into a variety of peptide derivatives. The process takes place under mild conditions, and good global yields are obtained.

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

Abstract 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.

Unexpected mosaic distribution of two hybridizing sibling lineages in the teleplanically dispersing snail Stramonita haemastoma suggests unusual postglacial redistribution or cryptic invasion

Abstract Molecular approaches have proven efficient to identify cryptic lineages within single taxonomic entities. Sometimes these cryptic lineages maybe previously unreported or unknown invasive taxa. The genetic structure of the marine gastropod Stramonita haemastoma has been examined in the Western Mediterranean and North‐Eastern Atlantic populations with mtDNA COI sequences and three newly developed microsatellite markers. We identified two cryptic lineages, differentially fixed for alternative mtDNA COI haplogroups and significantly differentiated at microsatellite loci. The mosaic distribution of the two lineages is unusual for a warm‐temperate marine invertebrate with a teleplanic larval stage. The Atlantic lineage was unexpectedly observed as a patch enclosed in the north of the Western Mediterranean Sea between eastern Spain and the French Riviera, and the Mediterranean lineage was found in Macronesian Islands. Although cyto‐nuclear disequilibrium is globally maintained, asymmetric introgression occurs in the Spanish region where the two lineages co‐occur in a hybrid zone. A first interpretation of our results is mito‐nuclear discordance in a stable postglacial hybrid zone. Under this hypothesis, though, the location of genetic discontinuities would be unusual among planktonic dispersers. An alternative interpretation is that the Atlantic lineage, also found in Senegal and Venezuela, has been introduced by human activities in the Mediterranean area and is introgressing Mediterranean genes during its propagation, as theoretically expected. This second hypothesis would add an additional example to the growing list of cryptic marine invasions revealed by molecular studies.