Molecular techniques for gene characterization: iPCR for identification of the 5'UTR of the ODC gene in the red macroalga Grateloupia imbricata

Máster Oficial en Gestión Costera

Identification of DNA motifs within the 5’UTR of the ornithine decarboxylase gene (GiODC) in the red seaweed Grateloupia imbricata

[ES]El desarrollo de las estructuras reproductoras (cistocarpos) en macroalgas rojas se ve afectado por factores abióticos (fotoperiodo, salinidad…) y endógenos (reguladores del desarrollo como el etileno o las poliaminas (PAs)); siendo estas últimas las principales responsables de la inducción del cistocarpo. Las PAs son moléculas ubícuas sintetizadas a través de una reacción enzimática mediada por la enzima ODC; por lo que conocer los mecanismos reguladores a nivel transcripcional del gen ODC es clave para el conocimiento del proceso reproductivo. Así, en el presente estudio se han identificado in silico diversos motivos conservados en la región 5 'UTR del gen ODC en la macroalga roja Grateloupia imbricata (GiODC) y se discute brevemente su presencia y posible implicación en diversos procesos metabólicos.

Genetic characterization and molecular identification of the bloodmeal sources of the potential bluetongue vector Culicoides obsoletus in the Canary Islands, Spain

[EN] Background: Culicoides (Diptera: Ceratopogonidae) biting midges are vectors for a diversity of pathogens including bluetongue virus (BTV) that generate important economic losses. BTV has expanded its range in recent decades, probably due to the expansion of its main vector and the presence of other autochthonous competent vectors. Although the Canary Islands are still free of bluetongue disease (BTD), Spain and Europe have had to face up to a spread of bluetongue with disastrous consequences. Therefore, it is essential to identify the distribution of biting midges and understand their feeding patterns in areas susceptible to BTD. To that end, we captured biting midges on two farms in the Canary Islands (i) to identify the midge species in question and characterize their COI barcoding region and (ii) to ascertain the source of their bloodmeals using molecular tools.Methods: Biting midges were captured using CDC traps baited with a 4-W blacklight (UV) bulb on Gran Canaria and on Tenerife. Biting midges were quantified and identified according to their wing patterns. A 688 bp segment of the mitochondrial COI gene of 20 biting midges (11 from Gran Canaria and 9 from Tenerife) were PCR amplified using the primers LCO1490 and HCO2198. Moreover, after selected all available females showing any rest of blood in their abdomen, a nested-PCR approach was used to amplify a fragment of the COI gene from vertebrate DNA contained in bloodmeals. The origin of bloodmeals was identified by comparison with the nucleotide-nucleotide basic alignment search tool (BLAST). Results: The morphological identification of 491 female biting midges revealed the presence of a single morphospecies belonging to the Obsoletus group. When sequencing the barcoding region of the 20 females used to check genetic variability, we identified two haplotypes differing in a single base. Comparison analysis using the nucleotide-nucleotide basic alignment search tool (BLAST) showed that both haplotypes belong to Culicoides obsoletus, a potential BTV vector. As well, using molecular tools we identified the feeding sources of 136 biting midges and were able to confirm that C. obsoletus females feed on goats and sheep on both islands.Conclusions: These results confirm that the feeding pattern of C. obsoletus is a potentially important factor in BTV transmission to susceptible hosts in case of introduction into the archipelago. Consequently, in the Canary Islands it is essential to maintain vigilance of Culicoides-transmitted viruses such as BTV and the novel Schmallenberg virus.