RESUMO
Mercury (Hg) is extremely toxic for all living organisms. Hg-tolerant symbiotic rhizobia have the potential to increase legume tolerance, and to our knowledge, the mechanisms underlying Hg tolerance in rhizobia have not been investigated to date. Rhizobial strains of Ensifer medicae, Rhizobium leguminosarum bv. trifolii and Bradyrhizobium canariense previously isolated from severely Hg-contaminated soils showed different levels of Hg tolerance. The ability of the strains to reduce mercury Hg2+ to Hg0, a volatile and less toxic form of mercury, was assessed using a Hg volatilization assay. In general, tolerant strains displayed high mercuric reductase activity, which appeared to be inducible in some strains when grown at a sub-lethal HgCl2 concentration. A strong correlation between Hg tolerance and mercuric reductase activity was observed for E. medicae strains, whereas this was not the case for the B. canariense strains, suggesting that additional Hg tolerance mechanisms could be playing a role in B. canariense. Transcript abundance from merA, the gene that encodes mercuric reductase, was quantified in tolerant and sensitive E. medicae and R. leguminosarum strains. Tolerant strains presented higher merA expression than sensitive ones, and an increase in transcript abundance was observed for some strains when bacteria were grown in the presence of a sub-lethal HgCl2 concentration. These results suggest a regulation of mercuric reductase in rhizobia. Expression of merA genes and mercuric reductase activity were confirmed in Medicago truncatula nodules formed by a sensitive or a tolerant E. medicae strain. Transcript accumulation in nodules formed by the tolerant strain increased when Hg stress was applied, while a significant decrease in expression occurred upon stress application in nodules formed by the Hg-sensitive strain. The effect of Hg stress on nitrogen fixation was evaluated, and in our experimental conditions, nitrogenase activity was not affected in nodules formed by the tolerant strain, while a significant decrease in activity was observed in nodules elicited by the Hg-sensitive bacteria. Our results suggest that the combination of tolerant legumes with tolerant rhizobia constitutes a potentially powerful tool in the bioremediation of Hg-contaminated soils.
RESUMO
La identificación de especies de Anopheles es compleja debido a la presencia de varios complejos de especies o especies cripticas cuyos ejemplares son de difícil distinción morfológica. Se corroboró o se corrigió la identificación de especies de Anopheles spp. capturadas en Ecuador, utilizando 393 secuencias de ADN mitocondrial, Citocromo Oxidasa I (COI) de 36 especies de Anofelinos neotropicales depositadas en GenBank y mediante la construcción de árboles filogenéticos usando parsimonia máxima. Se analizaron las identificaciones moleculares de cinco especies de Anopheles mediante los criterios de monofília y topología del árbol, secuencias provenientes de la localidad tipo y la divergencia genética intra/inter especies. Las topologías de los árboles resultantes corroboran la identificación de secuencias/especies de Anopheles (Anopheles) pseudopunctipennis Theobald, An. (Ano.) eiseni Coquillett y An. (Nyssorhynchus) albimanus Wiedemann; se corrige la identificación molecular de An. (Nys.) rangeli Gabaldón, Cova-García y López (no oswaldoi) y permanece en duda la identificación correcta de las secuencias relacionadas el grupo Punctimacula, hasta tener disponibles un mayor número de secuencias COI de especies relacionadas. La matriz de ADN-COI de 241 secuencias/haplotiposx36 especies construida para estos análisis resultará de utilidad para la identificación molecular de especies de Anofelinos de Ecuador y del Neotrópico con base en la porción de 520 pb entre 2.272 a 2.792pb de COI.
The Anopheles species identification is complicated by the presence of several complexes of species and species whose specimens are difficult morphological distinction. We use 393 mitochondrial DNA sequences, Cytochrome Oxidase I (COI) of 36 Neotropical species of Anophelinae deposited in GenBank and by constructing phylogenetic trees using maximum parsimony the objective was to corroborate or correct the molecular identification of five sequences/species of Anopheles collected in Ecuador and also availables in Genbank. We identified the taxonomic units, based on monophyly, phylogenetic species definition, tree topology, ocurrence of sequences from the type locality and genetic intra/inter divergence of clades. The topologies of the resulting trees corroborate the identification of sequences of Anopheles (Anopheles) pseudopunctipennis Theobald, An. (Ano.) eiseni Coquillett y An. (Nyssorhynchus) albimanus Wiedemann, while the molecular identification of An. (Nys.) rangeli Gabaldón, Cova-García y López (not oswaldoi) is corrected and remains in doubt the correct identification of related sequences for sequences belonging to Punctimacula group, until have available a greater number of COI sequences from related species. However according to the criteria of type locality, these are not punctimacula in sensu stricto. Finally, the matrix of alignment DNA-COI haplotypes sequences of 241x36 species built for these analyzes will be useful for molecular identification of Anophelinae species from Ecuador and the Neotropics based on the portion of 520 bp COI (between 2,272- 2,792 bp).
