This paper presents a detailed genetic study of Castanea sativa in El Bierzo, a major nut production region with interesting features. It is located within a glacial refuge at one extreme of the distribution area (northwest Spain); it has a centenary tradition of chestnut management; and more importantly, it shows an unusual degree of genetic isolation. Seven nuclear microsatellite markers were selected to analyze the genetic variability and structure of 169 local trees grafted for nut production. We analyzed in the same manner 62 local nuts. The selected loci were highly discriminant for the genotypes studied, giving a combined probability of identity of 6.1 x 10~6. An unprecedented density of trees was sampled for this project over the entire region, and nuts were collected representing 18 cultivars marketed by local producers. Several instances of misclassification by local growers were detected. Fixation index estimates and analysis of molecular variance (AMOVA) data are supportive of an unexpectedly high level of genetic differentiation in El Bierzo, larger than that estimated in a previous study with broader geographical scope but based on limited local sampling (Pereira-Lorenzo et al., Tree Genet Genomes 6: 701-715, 2010a). Likewise, we have determined that clonality due to grafting had been previously overestimated. In line with these observations, no significant spatial structure was found using both a modelbased Bayesian procedure and Mantel's tests. Taken together, our results evidence the need for more fine-scale genetic studies if conservation strategies are to be efficiently improved.

The present study examined whether animals attend to the individual components of the cue produced by a drug that stimulates different 5-hydroxytryptamine (5-HT) receptor populations, using a drug discrimination task based on the conditioned taste aversion (CTA) procedure. The training drug was indorenate (5-methoxytryptamine beta-methylcarboxylate) (INDO) that has been described as a 5-HT(1A/2C/1B) agonist able to exert discriminative control in both operant and CTA procedures. The principal objective was to examine generalization with the combined administration of agonists for the different receptor sites that may mimic the mechanism of action of the training drug. Male Wistar rats, deprived of water, were trained to discriminate INDO from saline; during the drug trials, the administration of INDO preceded saccharin-LiCl pairings, while, during the saline trials, the administration of saline preceded the saccharin-saline pairings. In generalization tests, INDO, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT, a 5-HT(1A) agonist), 1-(3-trifluoromethylphenyl)piperazine (TFMPP, a 5-HT(1B) agonist), alpha-methyl-5-HT (a 5-HT(2C) agonist) or 2-methyl-5-HT (a 5-HT(3) agonist), were administered alone or in combination. The results showed that 8-OH-DPAT, TFMPP and alpha-methyl-5-HT produced dose-dependent generalization, up to 88% in the case of 8-OH-DPAT. The combined administration of the following pairs of drugs, 8-OH-DPAT+TFMPP or 8-OH-DPAT+ alpha-methyl-5-HT, at doses that produced only 15-55% generalization when administered alone, produced greater than 80% generalization to INDO. However, the single administration of 2-methyl-5-HT produced only saline-like responding and its combined administration with 8-OH-DPAT did not modify the generalization produced by the single administration of 8-OH-DPAT. These results suggest that animals attend to the individual components of the drug cue; in the case of INDO, which has three elements, each mediated by a different receptor subpopulation (5-HT(1A), 5-HT(1B) and 5-HT(2C) ), the separate stimulation of at least two receptor subpopulations was 'interpreted' by the subject as the presence of the training drug.

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The pathogenic bacterium Pseudomonas aeruginosa possesses high metabolic versatility, with its effectiveness to cause infections likely due to its well-regulated genetic content. P. aeruginosa PAO1 has at least six fadD paralogous genes, which have been implicated in fatty acid (FA) degradation and pathogenicity. In this study, we used mutagenesis and a functional approach in P. aeruginosa PAO1 to determine the roles of the fadD4 gene in acyclic terpene (AT) and FA assimilation and on pathogenicity. The results indicate that fadD4 encodes a terpenoyl-CoA synthetase utilized for AT and FA assimilation. Additionally, mutations in fadD paralogs led to the modification of the quorum-sensing las/rhl systems, as well as the content of virulence factors pyocyanin, biofilm, rhamnolipids, lipopolysaccharides (LPS), and polyhydroxyalkanoates. In a Caenorhabditis elegans in vivo pathogenicity model, culture supernatants from the 24-h-grown fadD4 single mutant increased lethality compared to the PAO1 wild-type (WT) strain; however, the double mutants fadD1/fadD2, fadD1/fadD4, and fadD2/fadD4 and single mutant fadD2 increased worm survival. A correlation analysis indicated an interaction between worm death by the PAO1 strain, the fadD4 mutation, and the virulence factor LPS. Fatty acid methyl ester (FAME) analysis of LPS revealed that a proportion of the LPS and FA on lipid A were modified by the fadD4 mutation, suggesting that FadD4 is also involved in the synthesis/degradation and modification of the lipid A component of LPS. LPS isolated from the fadD4 mutant and double mutants fadD1/fadD4 and fadD2/fadD4 showed a differential behavior to induce an increase in body temperature in rats injected with LPS compared to the WT strain or from the fadD1 and fadD2 mutants. In agreement, LPS isolated from the fadD4 mutant and double mutants fadD1/fadD2 and fadD2/fadD4 increased the induction of IL-8 in rat sera, but IL1-β cytokine levels decreased in the double mutants fadD1/fadD2 and fadD1/fadD4. The results indicate that the fadD genes are implicated in the degree of pathogenicity of P. aeruginosa PAO1 induced by LPS-lipid A, suggesting that FadD4 contributes to the removal of acyl-linked FA from LPS, rendering modification in its immunogenic response associated to Toll-like receptor TLR4. The genetic redundancy of fadD is important for bacterial adaptability and pathogenicity over the host.