Spinosad is an insecticide widely used for the control of insect pest species, including Mediterranean fruit fly, Ceratitis capitata . Its target site is the α6 subunit of the nicotinic acetylcholine receptors, and different mutations in this subunit confer resistance to spinosad in diverse insect species. The insect α6 gene contains 12 exons, with mutually exclusive versions of exons 3 (3a, 3b) and 8 (8a, 8b, 8c). We report here the selection of a medfly strain highly resistant to spinosad, JW-100 s, and we identify three recessive Ccα6 mutant alleles in the JW-100 s population: (i) Ccα6 3aQ68* containing a point mutation that generates a premature stop codon on exon 3a (3aQ68*); (ii) Ccα6 3aAG > AT containing a point mutation in the 5′ splicing site of exon 3a (3aAG > AT); and (iii) Ccα6 3aQ68*-K352* that contains the mutation 3aQ68* and another point mutation on exon 10 (K352*). Though our analysis of the susceptibility to spinosad in field populations indicates that resistance has not yet evolved, a better understanding of the mechanism of action of spinosad is essential to implement sustainable management practices to avoid the development of resistance in field populations.

Infections caused by multidrug-resistant (MDR) bacteria, including Vancomycin-Resistant Enterococcus (VRE), have become one of the greatest clinical challenges of the 21 st century. Particularly, VRE strains of E. faecium, a natural member of the gastrointestinal consortia, have recently emerged as one of the most problematic cases of MDR nosocomial pathogens. It is widely known that the establishment of high-level intestinal colonization of this bacterium, triggered by antibiotic treatment of the host, potentially leads to bacteremia, endocarditis and surgical wound, urinary tract, and devicerelated infections in hospitalized patients. However, the unique determinants possessed by these strains, which enable them to benefit from the antibiotic-induced perturbations of the gut microbiota and host intestinal immune defenses, remain to be identified.Essentially, what needs to be revealed is the differential gene expression of E. faecium in its niche at different time points; before and during antibiotic treatment of the host, and very interestingly, in the presence of probiotic bacteria. Differentially expressed genes will highlight how and why E. faecium is able to dominate the gut, as well as how it interacts with its surroundings, possibly pinpointing its potential weaknesses, as well as provide hints on what specific properties should be prerequisites for bacteria to be used as probiotics.Since the global expression intended to be analyzed is that of E. faecium alone, a method to separate this bacteria from the rest of the gut microbiota is required. Failure to do so would result in the study of the gut microbiota's transcriptome as a whole: metatranscriptomics. In practice, the separation VRE. faecium from the rest of the commensal microbiota could be carried out by means of flow cytometry if the generation of a fluorescent E. faecium was achieved. Additionally, it would serve as a great tool to study a series of facets of VRE faecium colonization which remain virtually unknown. It would enable the monitorization of VRE infection through whole-body imaging of infected mice, fluorescence microscopy analysis of histological cuts to determine its most predominant locations, the use of FbFP as a translational or transcriptional reporter, etc.Consequently, the main objective of this Final Degree Project has been to attempt the generation of a Vancomycin-Resistant Enterococcus faecium clinical isolate (C68) expressing a fluorescent protein whose chromophore can form under the anaerobic conditions found in the guts of animals: (FMN)-based fluorescent protein (FbFP).In order to fulfill the main objective of this work, first, the FbFP gene was synthesized with the appropriate features for its expression in E. faecium and for its cloning. Next, the plasmid construction aimed to confer E. faecium fluorescent properties (pBT2-FbFP), was generated. This was done by inserting the FbFP gene into the pBT2 plasmid. As a previous step to the transformation of E. faecium, pBT2-FbFP was transformed into E.coli DH5a in or...

The Mediterranean fruit fly, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), is considered one of the most economically damaging pests of citrus orchards in Spain. The characterization of C. capitata population structure, at a large geographical scale, by using a combination of RAPD and ISSR markers can allow analyzing the genetic variability of this species, and provide some insight in decision making for resistance management, recently recorded in Spain. We compared six Spanish populations along the Mediterranean area (Gerona, Amposta, Tortosa, Castellón, Valencia and Málaga) with populations from other geographical areas where this pest is widely distributed (Africa, Middle East, South America and Atlantic Islands) and two laboratory strains. The results obtained with both types of molecular markers were similar. A dendrogram based on Nei genetic distances showed that all Mediterranean Spanish populations, except the population collected in Gerona, were clearly separated from the rest. However, no clear differentiation among Spanish populations was found, probably as a result of the high levels of gene flow (Nm value of 2.8 for RAPD and 3.9 for ISSR). Implications of these findings on resistance management of C. capitata are discussed.Additional key words: gene flow; medfly; molecular markers. ResumenEstudio de la estructura genética de poblaciones españolas de Ceratitis capitata mediante marcadores RAPD e ISSR: implicaciones en el manejo de la resistencia La mosca mediterránea de la fruta, Ceratitis capitata (Wiedemann) (Diptera: Tephritidae), está considerada como una de las plagas económicamente más perjudiciales de los cultivos de cítricos en España. La caracterización de la estructura poblacional de C. capitata, a gran escala, mediante el uso conjunto de marcadores RAPD e ISSR puede permitir analizar la variabilidad genética de esta especie, y proporcionar alguna información en la toma de decisiones del manejo de la resistencia, registrada recientemente en España. Se compararon seis poblaciones españolas del área mediterránea (Gerona, Amposta, Tortosa, Castellón, Valencia y Málaga) con poblaciones de otras zonas geográficas donde esta plaga está ampliamente distribuida (África, Oriente Medio, América del Sur e Islas del Atlántico) y dos líneas de laboratorio. Los resultados obtenidos con ambos tipos de marcadores moleculares fueron similares. Un dendrograma basado en las distancias genéticas de Nei mostró que todas las poblaciones mediterráneas españolas, a excepción de la población recogida en Gerona, estaban claramente separadas del resto. Sin embargo, no se encontró una diferenciación clara entre las poblaciones españolas, probablemente como resultado de los altos niveles de flujo géni-co (un valor de Nm de 2,8 para RAPD y 3,9 para ISSR). Se discuten las implicaciones de estos resultados en el manejo de la resistencia de C. capitata.Palabras clave adicionales: flujo génico; marcadores moleculares; mosca mediterránea de la fruta.*Corresponding author: bberoiz@cib.csic.es Received: 30-12-...

The Mediterranean corn borer, Sesamia nonagrioides (Lefebvre) (Lepidoptera: Noctuidae), is a major pest of maize in the Mediterranean area. Transgenic Bt maize expressing the Cry1Ab toxin from the bacterium Bacillus thuringiensis can effectively control this pest. The characterization of S. nonagrioides population structure, at a large geographical scale, would provide some insight in decision making for resistance management. The genetic relationships among nine populations from Spain, one from France, one from Italy, three from Greece, and one from Turkey were assessed using Random Amplyfied Polymorphic DNA (RAPD) markers. Populations from France and Spain formed a cluster independent from a cluster of populations collected in Italy, Turkey, and Greece in a unweighted pair-group method with arithmetic average dendrogram constructed from Nei's genetic distances. Average genetic differentiation among samples was significant for all geographical groupings analyzed (F (ST) = 0.160 +/- 0.014 for Spanish populations; 0.133 +/- 0.022 for Spanish and French populations; and 0.095 +/- 0.010 for Greek, Italian, and Turkish populations). Genetic differentiation was also significant for all paired comparisons of populations, including two Spanish populations separated by only 15 km with no apparent geographical barriers. No pattern of isolation by distance was observed among Mediterranean corn borer populations collected in Spain and France. These results suggest a limited genetic exchange between relatively distant S. nonagrioides populations in Europe, which might contribute to decreased rate of spread of resistance alleles once resistance has developed at a certain site.

Key-words:Austropotamobius italicus was once widely distributed throughout most of the country's limestone basins in Spain. But its populations have shown a very strong decline over the last thirty years, due to different factors. Thus, the species now enjoys protection under regional, national and international legislation. Therefore, knowledge of the levels and patterns of distribution of genetic diversity in crayfish populations is critical when making conservation management decisions. In the present work, the current genetic structure of Spanish populations of white-clawed crayfish, A. italicus, was analyzed. Eleven Spanish populations and an Italian sample were studied through an 1184 bp-lentgh sequence of cytochrome oxidase subunit I mitochondrial gene. Data analysis revealed the existence of eight haplotypes in the Iberian Peninsula, the highest diversity reported to date in Spanish crayfish. Also a substantial genetic differentiation among populations was found, with a clear geographic pattern. The genetic variability found in these populations is similar to, and even higher, than that reported in previous studies on other Spanish and European populations of A. italicus. Thus, given the current risk status of the species across its range, this variability in certain populations offers some hope for the species from a management point of view. RÉSUMÉ La variabilité de l'ADN mitochondrial des populations espagnoles d' A. italicus déduite de l'analyse d'une région COI Mots-clés :Austropotamobius italicus, mtDNA, Austropotamobius italicus était autrefois largement distribué dans la plupart des régions des bassins calcaires en Espagne. Mais ces populations ont montré une baisse très forte au cours des trente dernières années, en raison de différents facteurs. Ainsi, l'espèce bénéficie aujourd'hui d'une protection législative régionale, nationale et internationale. Par conséquent, la connaissance des niveaux et des schémas de distribution de la diversité des ressources génétiques dans les populations d'écrevisses est essentielle pour prendre des décisions de bioconservation. Dans le présent travail, la structure génétique actuelle des populations espagnoles de l'écrevisse à pattes blanches, A. italicus, a été analysée. Onze populations

ABSTRACT1. Spanish populations of the white-clawed crayfish have declined sharply over the last three decades. Although Austropotamobius pallipes was once widely distributed and very abundant in most of the limestone basins of the country, outbreaks of crayfish plague since 1978 have reduced its populations, and now only some 500-600 small populations are left.2. Consequently, the species now enjoys protection under national legislation. Management decisions regarding the conservation of a threatened species require an understanding of the genetic structure of its populations.3. Using random amplified polymorphic DNA (RAPD) fingerprinting the genetic variability of 11 populations of A. pallipes was assessed over the species' range in Spain, and their phylogenetic relationships determined.4. Substantial genetic differentiation was detected among the populations tested; no clear relationship was found between patterns of genetic variability and hydrological basin. The RAPD markers showed the degree of genetic variability of these populations to be similar to, and in some cases slightly higher than, that reported in previous studies on other Spanish and European populations of A. pallipes.5. The results offer hope for the recovery of this species in Spain, and provide information that might be useful in the management of crayfish reintroduction programmes.

The identification of allergy-causing mites is conventionally based on morphological characters. However, molecular taxonomy using ribosomal DNA (rDNA) may be particularly useful in the analysis of mite cultures and purified mite fractions in the production of allergenic extracts. Full-length internal transcribed spacers (ITS1 and ITS2) were obtained from Dermatophagoides farinae, Dermatophagoides pteronyssinus, Dermatophagoides microceras and Euroglyphus maynei (Astigmata: Pyroglyphidae), Glycyphagus domesticus and Lepidoglyphus destructor (Astigmata: Glycyphagidae), Tyrophagus fanetzhangorum, Tyrophagus putrescentiae, Tyrophagus longior, Tyrophagus neiswanderi, Acarus farris and Acarus siro (Astigmata: Acaridae), and Blomia tropicalis (Astigmata: Echymopodidae), using mite-specific primers. Polymerase chain reaction (PCR) products were digested with HpaII and RsaI restriction enzymes in order to produce species-specific PCR restricted fragment length polymorphism (RFLP) profiles. A semi-nested re-amplification step was introduced before the RFLP in order to apply the method to environmental samples. Results demonstrate that rDNA sequences can be used for the unambiguous identification of mite species. The PCR-RFLP system allows the identification of species in purified mite fractions when the availability of intact adult mite bodies for morphological identification is limited. This reliable and straightforward PCR-RFLP system and the rDNA sequences obtained can be of use in the identification of allergy-causing mite species.

The banana weevil (BW), Cosmopolites sordidus (Coleoptera: Curculionidae), is one of the most important insect pests of bananas and plantains. The mobility and the origin of BW infestations at the Canary Islands (Tenerife, La Gomera and La Palma) have been analysed using Random Amplified Polymorphic DNA (RAPD) as molecular markers. Populations from Costa Rica, Colombia, Uganda and Madeira were also included for comparison. One hundred and fifteen reproducible bands from eight primers were obtained. The level of polymorphism in the populations from the Canary Islands (40-62%) was in the range of those found in other populations. Nei's genetic distances, pair-wise fixation index (FST) values indicate that the closest populations are Tenerife populations among themselves (Nei's genetic distance=0.054-0.100; FST=0.091-0.157) and Costa Rica and Colombia populations (Nei's genetic distance=0.049; FST=0.113). Our results indicate the existence of BW local biotypes with limited gene flow and affected by genetic drift. These results are compatible with a unique event of colonization at Tenerife; whereas, the outbreaks in La Gomera and La Palma may come from independent introductions. The Madeira population is phylogenetically and geographically closer to the Canary Islands populations, suggesting that it is the most likely source of the insects introduced in the Canary Islands.