Η μεσογειακή μύγα αποτελεί το κύριο παράσιτο πολλών καλλιεργούμενων φρούτων προκαλώντας ετησίως μεγάλες καταστροφές σε γεωργικές καλλιέργειες. Η μελέτη του εντόμου τόσο σε γενετικό όσο και σε πληθυσμιακό επίπεδο μπορεί να συμβάλλει σημαντικά στην ανάπτυξη ή και τη βελτίωση αποτελεσματικών και φιλικών προς το περιβάλλον μεθόδων ελέγχου. Οι μικροδορυφόροι είναι απλές επαναλήψεις ενός νουκλεοτιδικού μοτίβου που αποτελείται 1-6 ζεύγη βάσεων. Αποτελούν πολύ χρήσιμους γενετικούς δείκτες διότι είναι άφθονοι και διάσπαρτοι στο γονιδίωμα των ευκαρυωτκών οργανισμών. Επιπλέον είναι υψηλά πολυμορφικοί, κληρονομούνται ως συνυπερέχοντες Μεντελικοί δείκτες και αναλύονται εύκολα μέσω PCR, χαρακτηριστικά που τους καθιστούν πολύτιμα εργαλεία για πληθυσμιακές και εξελικτικές μελέτες. Από τους μικροδορυφορικούς δείκτες που αναπτύχθηκαν στο εργαστήριό μας, επιλέχθηκαν 10 με βάση το βαθμό πολυμορφισμού που έδειξαν σε εργαστηριακά στελέχη. Οι δείκτες αυτοί χρησιμοποιήθηκαν στην ανάλυση 481 ενηλίκων ατόμων που προέρχονταν από 19 διαφορετικά δείγματα φρούτων που συλλέχθηκαν από εννέα διαφορετικές περιοχές της Δυτικής Ελλάδας και της Βόρειας Πελλοπονήσου. Η γενοτυπική ανάλυση πραγματοποιήθηκε με ηλεκτροφόρηση των προϊόντων της PCR για κάθε γενετικό δείκτη σε πήκτωμα πολυακρυλαμιδίου και επακόλουθη αυτοραδιογραφία. Η στατιστική επεξεργασία των αποτελεσμάτων, με τη βοήθεια υπολογιστικών προγραμμάτων αποκάλυψε σημαντική γενετική διαφοροποίηση μεταξύ των δειγμάτων, που αποδίδεται εκτός των άλλων παραγόντων (κλιματολογικές συνθήκες, γεωγραφική προέλευση) στο είδος του ξενιστή. / C. capitata, is the main pest of many cultivable fruits and responsible for a significant loss in annual products, resulting in great economic damage. Studies on the genetic and population analysis will make a contribution towards the development or the improvement of environmental friendly control methods. Microsatellites are tandem simple sequence repeats of short (1-6) nucleotide motifs. They are very important genetic markers because they are dispersed and abundant in most eukaryotic genomes. They are highly polymorphic, inherited as co-dominant Mendelian markers and easily scored by PCR. Consequently, they have become one of the most popular molecular markers with application in many genetic studies, including genetic analysis of natural populations and evolutionary studies. From the available microsatellites in our laboratory, were selected ten (10), based on their polymorphism in laboratory strains. They were used for the screening of 481 adult flies in the medfly, collected from nineteen (19) different samples of fruits from nine (9) different areas in west Greece and north Peloponnesus. Analysis of genotype composition in the samples was achieved by polyacrylamide electrophoresis of the PCR products, for every genetic marker and then autoradiography. The statistic analysis of our results using software programs, revealed an important genetic differentiation in samples. Except for many factors (climatic conditions, geographic origin), the host origin will be responsible for this genetic differentiation.

Behavioural and chemical studies were conducted with Ceratitis capitata Wied. and Anastrepha obliqua Macq. and their host fruit, Averrhoa carambola L. (starfruit). The bioassays were performed to find out the age and time of day when the reproductive behaviour (calling, mating and oviposition) were more frequent as well as which maturation stage of the starfruit, females of A. obliqua and C. capitata would choose for oviposition. The results demonstrated that reproductive behaviour were more frequent among individuals which were eleven day-old for C. capitata and fifteen day-old for A. obliqua, except for oviposition that were more frequent among individuals which were seventeen day-old in this ultimate specie. For C. capitata, the onset of calling, courtship and oviposition were 6:00-9:00, 7:00-12:00 and 14:00-16:00 h, respectively, whereas for A. obliqua, these activities were highest at 5:00-9:00, 7:00-10:00 and 9:00-11:00 h, respectively. The maturation stages of starfruit in which females of C. capitata and A. obliqua would prefer to oviposit are ripe and unripe, respectively. The chemical analyses of extracts (secretions from the salivary glands and headspace) from calling males of of both species by gas chromatography coupled to mass spectrometry (GC-MS) revealed alcohols, esters, ketones, nitrogenated compounds and terpenoids. Comparative analyses of volatiles present in the salivary gland secretion of A. obliqua and C. capitata males with those which were found to be present in headspace extracts of calling males of these species, demonstrated that 50% of the compounds released by calling males are present in the secretion of salivary glands, suggesting that these glands maybe the storage site of these compounds. It worthy to note that among this mixture of compounds, there should be the sex pheromone components of both species, since the salivary gland extracts of A. obliqua males attracted co-specific females. The results also showed that unsaturated and saturated esters were the major componentes in extracts of ripe starfruits and mono- and sesquiterpenes characterized the mixture of unripe starfruits. It was also found that there is a similarity between the chemical composition of extracts from salivary glands of C. capitata and A. obliqua and the volatiles released by ripe and unripe starfruits. two compounds were common between salivary glands secretions of C. capitata calling males and ripe starfruit extracts whereas eight common compounds were found in salivary glands secretions of A. obliqua males and unripe starfruit extracts. These findings suggest that these coincident compounds may act as chemical cues which help females of both species to find starfruits in the appropriate maturation stage for oviposition. / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Estudos comportamentais e químicos foram conduzidos com Ceratitis capitata Wied. e Anastrepha obliqua Macq. e seu fruto hospedeiro, Averrhoa carambola L. (carambola). Os bioensaios visaram avaliar a idade e os horários de pico dos comportamentos reprodutivos (chamamento, cópula e oviposição) e os estágios de maturação da carambola selecionados pelas fêmeas de cada espécie para oviposição. Os resultados demonstraram que a idade de maior ocorrência das atividades reprodutivas foi aos 11 dias de vida adulta para C. capitata e aos 15 dias para A. obliqua, exceto para a oviposição cuja idade de maior ocorrência foi aos 17 dias para esta última espécie. Para C. capitata os horários de pico para as atividades de chamamento, cópula e oviposição foram 6:00 – 9:00, 10:00 – 12:00 e 14:00 – 16:00 h, respectivamente. Para A. obliqua esses horários foram: 6:00 – 9:00, 7:00 – 10:00 e 9:00 – 11:00 h. Os estágios de maturação da carambola preferidos por fêmeas de C. capitata e A. obliqua para oviposição foram, na mesma seqüência, maduro e verde. As análises químicas dos extratos (secreções de glândulas salivares e aeração) de machos em chamamento de ambas as espécies por cromatografia gasosa acoplada à espectrometria de massas (CG-EM) permitiram a identificação de álcoois, ésteres, cetonas, compostos nitrogenados e terpenóides. Análises comparativas entre os voláteis presentes nos extratos de secreções de glândulas salivares de machos de C. capitata e A. obliqua e aqueles presentes nos extratos de aeração de machos em chamamento das mesmas espécies, demonstraram que 50% dos compostos liberados pelos machos em chamamento estão presentes nas secreções de glândulas salivares, sugerindo que estas glândulas poderiam ser o sítio de estocagem desses compostos. Entre esses compostos similares provavelmente são encontrados componentes do feromônio sexual dessas espécie, uma vez que, os extratos de glândulas salivares de machos de A. obliqua foram atrativos a fêmeas coespecíficas. Os resultados também demonstraram que nos extratos de aeração de frutos de carambolas maduras predominaram ésteres saturados e insaturados, enquanto nos extratos de carambolas verdes os mono- e sesquiterpenos caracterizaram a mistura. Foram também observadas similaridades entre a composição química de extratos de glândulas salivares de C. capitata e A. obliqua e os voláteis liberados por carambolas maduras e verdes. Dois compostos foram comuns entre as secreções de glândulas salivares de machos de C. capitata e os extratos de carambolas maduras, enquanto oito compostos foram comuns entre as secreções de glândulas salivares de machos de A. obliqua e os extratos de carambolas verdes. Esses resultados sugerem que esses compostos coincidentes poderiam atuar como pistas que auxiliariam as fêmeas de ambas as espécies a encontrar carambolas no estágio de maturação adequado para oviposição.

The Ceratitis capitata is a very important agricultural pest, whose reproduction behaviour is controled by chemical signals. Males initiate mating by creating leks, where they release sexual pheromones to attract females. The main goal of this diploma thesis was to determine the influence of host plants on the composition of male sex-pheromones C. capitata and to compare emanations of wild males with those originating from laboratory population. We studied the chemical composition of volatiles, released by calling males C. capitata from laboratory and two wild populations, using two-dimensional gas chromatography with time-of-flight mass spectrometric detection (GC×GC-TOFMS), gas chromatography with electroantennographic and flame ionization detection (GC-EAD-FID). All data were statistically analyzed by multivariate data analyses. Significant differences were observed in the quantitative and qualitative composition of the chemical emanations among males from the three populations. The GC-EAD-FID analyses revealed fourteen antenally active compounds with a possible behavioral function. Isomenthone, geraniol, bornyl acetate, geranyl acetone and ethyl octanoate were newly identified antenally active compounds of C. capitata male sex pheromone. Statistical analyses indicated that males and females of...

[ES] Los viroides, genomas mínimos de RNA circular no codificante, monocatenarios y muy estructurados, parasitan factores celulares de las plantas para replicarse autónomamente, establecer infecciones sistémicas y usualmente causar enfermedades. Los de la familia Avsunviroidae se replican y acumulan en cloroplastos por un mecanismo de círculo rodante simétrico. Una RNA polimerasa cloroplástica produce concatémeros lineales de polaridad complementaria que son reducidos a monómeros por las ribozimas de cabeza de martillo (HHR) del concatémero. Producen extremos 5'-hidroxilo y 2',3'-fosfodiéster cíclico, que la isoforma cloroplástica de la tRNA ligasa convierte en enlaces 5',3'-fosfodiéster intramoleculares, generando viroides circulares de polaridad complementaria que pueden entrar en otra ronda de transcripción, simétrica a ésta. En esta Tesis se han analizado las secuencias y estructuras viroidales esenciales para su circularización, usando como modelo el viroide latente de berenjena (ELVd), que induce infecciones asintomáticas en berenjena. Expresamos en Escherichia coli precursores del ELVd(+) lineales flanqueados por dos copias de su HHR. Su procesamiento genera monómeros con los extremos adecuados para la ligación por la tRNA ligasa de la berenjena, que es coexpresada. Mutaciones puntuales y deleciones en el sitio nativo de ligación sugieren que solo el dominio HHR es esencial para la circularización. La conservación de la secuencia y estructura de la HHR con las del sustrato natural del enzima (los tRNAs) nos hacen proponer que la HHR del ELVd secuestra la ligasa mimetizando las características generales del bucle anticodón de los tRNAs. Este sistema de expresión permite también producir RNAs recombinantes, insertándolos en una posición particular del RNA del ELVd. Las quimeras son procesadas por las HHRs flanqueantes y sus extremos ligados por la tRNA ligasa. El andamiaje viroidal circular, compacto y posiblemente asociado a la ligasa, permite aumentar la vida media del RNA de interés y su acumulación en la bacteria. En esta Tesis adaptamos el sistema para producir RNAs de doble cadena (dsRNAs) que desencadenen interferencia por RNA (RNAi), un mecanismo de defensa y regulación génica eucariota basado en la complementariedad de bases entre RNAs. dsRNAs complementarios a genes endógenos reducen los niveles de sus transcritos y generan fenotipos de pérdida de función. Los insectos pueden tomar dsRNAs del ambiente, internalizarlos en sus células y distribuirlos sistémicamente, haciendo al RNAi una estrategia prometedora para el control de plagas. Para producir dsRNAs, separamos las repeticiones invertidas del gen diana que genera la horquilla con el cDNA de un intrón autocatalítico del grupo I de Tetrahymena thermophila, aumentando la estabilidad de los plásmidos de expresión. El intrón es eliminado tras la transcripción, resultando en una molécula viroidal de la que protruye el dsRNA de interés. Flanquear las repeticiones invertidas con una copia adicional permutada del intrón permite separar el ELVd del producto final, un dsRNA circular cerrado en ambos lados por pequeños bucles. Ambas moléculas poseen actividad reguladora: las quimeras viroide-dsRNA con homología al gen de la unión septada suave 1 del gusano de la raíz del maíz (Diabrotica virgifera virgifera) exhiben actividad insecticida oral contra las larvas similar a la de horquillas sintetizadas in vitro, y los dsRNAs circulares sin andamiaje viroidal homólogos al gen de la ATPasa vacuolar (subunidad A) y la proteína ribosomal S13 silencian eficientemente estos genes en adultos de la mosca del Mediterráneo (Ceratitis capitata); este caso es de especial relevancia al ser la primera demostración del RNAi para el control de esta plaga. En conclusión, a pesar de su limitada relevancia agrícola, el ELVd es útil para investigar la biología molecular de la familia Avsunviroidae y una poderosa herramienta biotecnológica en combinación con el sistema de expresión en E. coli. / [CA] Els viroides, genomes mínims d'RNA circular no codificant, monocatenaris i molt estructurats, parasiten factors cel·lulars de les plantes per a replicar-se autònomament, establir infeccions sistèmiques i usualment causar malalties. Els de la família Avsunviroidae es repliquen i acumulen en cloroplasts per un mecanisme de cercle rodant simètric. Una RNA polimerasa cloroplàstica produeix concatèmers lineals de polaritat complementària que són reduïts a monòmers per els ribozims de cap de martell (HHR) del concatèmer. Produeixen extrems 5'-hidroxil i 2',3'-fosfodièster cíclic, que la isoforma cloroplàstica de la tRNA lligasa converteix en enllaços 5',3'-fosfodièster intramoleculars, generant viroides circulars de polaritat complementària que poden entrar en una nova ronda de transcripció, simètrica a la primera. En aquesta Tesi s'han analitzat les seqüències i estructures viroidals essencials per a la seua circularització, emprant com a model el viroide latent d'albergínia (ELVd), que indueix infeccions asimptomàtiques en albergínia. Expressem en Escherichia coli precursors de l'ELVd(+) lineals flanquejats per dos còpies del seu HHR. El seu processament produeix monòmers amb els extrems apropiats per a la lligació mediada per la tRNA ligasa de l'albergínia, que és coexpressada. Mutacions puntuals i delecions en el lloc nadiu de lligació suggereixen que només el domini HHR és essencial per a la circularització. La conservació de la seqüència i estructura del HHR amb les del substrat natural de l'enzim (els tRNAs) ens fan proposar que el HHR de l'ELVd segresta la lligasa mimetitzant les característiques generals del bucle anticodó dels tRNAs. Aquest sistema d'expressió també permet produir RNAs recombinants, inserint-los en una posició particular de l'RNA de l'ELVd. Les quimeres són processades pels HHR flanquejants i els seus extrems lligats per la tRNA lligasa. L'RNA viroïdal circular, compacte i possiblement associat a la lligasa, permet augmentar la vida mitjana de l'RNA d'interés i la seua acumulació en els bacteris. En aquesta Tesi adaptem el sistema per a produir RNAs de doble cadena (dsRNAs) que desencadenen interferència per RNA (RNAi), un mecanisme de defensa i regulació gènica eucariota basat en la complementarietat de bases entre RNAs. dsRNAs complementaris a gens endògens redueixen els nivells dels seus transcrits i generen fenotips de pèrdua de funció. Els insectes poden prendre dsRNAs de l'ambient, internalitzar-los en les seues cèl·lules i distribuir-los sistèmicament, fent a l'RNAi una estratègia prometedora en el control de plagues. Per a produir dsRNAs, separem les repeticions invertides del gen diana que genera la forqueta amb el cDNA d'un intró autocatalític del grup I de Tetrahymena thermophila, augmentant l'estabilitat dels plasmidis d'expressió. L'intró és eliminat després de la transcripció, resultant en una molècula viroïdal de la qual protrueix el dsRNA d'interés. Flanquejar les repeticions invertides amb una còpia addicional permutada de l'intró permet separar l'ELVd del producte final, un dsRNA circular tancat als dos costats per xicotets bucles. Els dos tipus de molècules posseeixen activitat reguladora: les quimeres viroide-dsRNA amb homologia al gen de la unió septada suau 1 del cuc de l'arrel de la dacsa (Diabrotica virgifera virgifera) exhibeixen activitat insecticida oral contra les larves similar a la de forquetes sintetitzades in vitro, i els dsRNAs circulars sense l'RNA viroïdal homòlegs al gen de la ATPasa vacuolar (subunitat A) i la proteïna ribosomal S13 silencien eficientment aquests gens en adults de la mosca del Mediterrani (Ceratitis capitata); aquest cas és d'especial rellevància perquè és la primera demostració de l'RNAi per al control d'aquesta plaga. En conclusió, malgrat la seua limitada rellevància agrícola l'ELVd és útil per a investigar la biologia molecular de la família Avsunviroidae i una poderosa ferramenta biotecnològica en combinació amb el sistema d'expressió en E. coli. / [EN] Viroids, minimal genomes of non-coding circular RNA, single-stranded and highly structured, parasitize plant cellular factors to replicate autonomously, establish systemic infections, and typically cause disease. Those of the family Avsunviroidae replicate and accumulate in chloroplasts by a symmetrical rolling circle mechanism. A chloroplast RNA polymerase produces linear concatemers of complementary polarity that are reduced to monomers by the hammerhead ribozymes (HHR) of the concatemer. They produce 5'-hydroxyl and 2',3'-cyclic phosphodiester ends, which the chloroplastic isoform of tRNA ligase converts to intramolecular 5',3'-phosphodiester bonds, generating circular viroids of complementary polarity that can enter another round of transcription, symmetric to the first one. In this Thesis, the viroid sequences and structures essential for its circularization have been analyzed, using as a model the eggplant latent viroid (ELVd), which induces asymptomatic infections in eggplant. We expressed in Escherichia coli linear ELVd(+) precursors flanked by two copies of its HHR. Its processing generates monomers with suitable ends for ligation by the eggplant tRNA ligase, which is co-expressed. Point mutations and deletions at the wild-type ligation site suggest that only the HHR domain is essential for circularization. The conservation of the sequence and structure of the HHR with those of the natural substrate of the enzyme (the tRNAs) lead us to propose that the HHR of the ELVd hijacks the ligase, mimicking the general characteristics of the anticodon loop of the tRNAs. This expression system also allows the production of recombinant RNAs, inserting them into a particular position of the ELVd RNA. Chimeras are processed by flanking HHRs and their ends ligated by the tRNA ligase. The compact, circular viroidal scaffold, possibly associated with the ligase, allows increasing the half-life of the RNA of interest and its accumulation in the bacteria. In this Thesis we adapt the system to produce double-stranded RNAs (dsRNAs) that trigger RNA interference (RNAi), a eukaryotic gene regulation and defense mechanism based on base complementarity between RNAs. dsRNAs complementary to endogenous genes reduce the levels of their transcripts and generate loss-of-function phenotypes. Insects can take dsRNAs from the environment, internalize them into cells, and distribute them systemically, making RNAi a promising pest control strategy. To produce dsRNAs, we separated the inverted repeats of the target gene that generates the hairpin with the cDNA of a group-I autocatalytic intron from Tetrahymena thermophila, increasing the stability of the expression plasmids. The intron is removed after transcription, resulting in a viroidal molecule from which the dsRNA of interest protrudes. Flanking the inverted repeats with an additional copy of the intron in a permuted form allows the ELVd molecule to be separated from the final product, a circular dsRNA molecule capped on both sides by small loops. Both molecules have regulatory activity: the viroid-dsRNA chimeras with homology to the smooth septate junction 1 gene of the corn rootworm (Diabrotica virgifera virgifera) exhibit oral insecticidal activity against larvae similar to that of in vitro synthesized hairpins, and the circular dsRNAs without the viroid scaffold homologous to the vacuolar ATPase (subunit A) and ribosomal protein S13 genes efficiently silence those genes in adult Medfly (Ceratitis capitata); this case is of special relevance as it is the first demonstration of RNAi for the control of this pest. In conclusion, despite its limited agricultural relevance, the ELVd is useful for investigating the molecular biology of the Avsunviroidae family and a powerful biotechnological tool in combination with the E. coli expression system. / This work was supported by the Ministerio de Ciencia e Innovación (Spain; co-financed by the European Regional Development Fund) [BIO2017-83184-R] and [BIO2017‐ 91865‐EXP]; Universitat Politècnica de València [PAID-01-17]. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). / Ortolá Navarro, B. (2023). Heterologous expression of circular RNAs in Escherichia coli for analyzing the ligation process of chloroplastic viroids and producing double-stranded RNAs with insecticidal activity [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/192635