Document formatted into pages; contains 78 pages. / Title from PDF of title page. / Thesis (M.S.P.H.)--University of South Florida, 2003. / Includes bibliographical references. / Text (Electronic thesis) in PDF format. / ABSTRACT: Malathion is a commonly used organophosphate pesticide on field crops, fruits, nut trees, vegetables, livestock, agricultural premises, and land. The approved uses also include mosquito and medfly control. These uses can result in human skin contact. The purpose of this study is to evaluate the human skin absorption of malathion for the purpose of assessing the risks associated with aqueous solution exposures following applications. Aerial applications can result in solubilized malathion in swimming pools and other waters that may be contacted. Human volunteers were selected and exposed to aqueous solutions of malathion at various concentrations. Participants submerged their arms and hands in twenty liters of dilute malathion solution in either a stagnant or stirred environment. The "disappearance method" was applied by measuring malathion concentrations in the water before and after human subject exposure to the water for various periods of time. Malathion was measured using Gas Chromatography. No measurable skin absorption was detected in 42% of the participants. Measurable skin absorption among the remaining 58% of participants resulted in doses that were more than an order of magnitude less than the minimal dose necessary to cause a measurable change in red blood cell acetylcholinesterase (RBC-AChE). Extrapolation of these results to a mathematical model for recreational swimmers and bathers exposed to contaminated swimming pools and surface waters typically detected after bait application again are an order of magnitude below the doses needed to cause a detectable change in RBC-AChE. These data indicate that exposure to aqueous malathion following usual aerial bait applications is not appreciably absorbed, and therefore, it is not a public health hazard. / System requirements: World Wide Web browser and PDF reader. / Mode of access: World Wide Web.

Τα έντομα και άλλοι ποικιλόθερμοι οργανισμοί έχουν αναπτύξει διάφορους μοριακούς μηχανισμούς για την προστασία τους από ακραίες θερμοκρασίες και άλλες στρεσογόνες καταστάσεις που συμβαίνουν κατά τη διάρκεια της ζωής τους. Ένας από τους σημαντικότερους μηχανισμούς είναι η παραγωγή των θερμοεπαγόμενων πρωτεϊνών οι οποίες, ως μοριακοί συνοδοί, προστατεύουν τα κύτταρα από τη μετουσίωση και συσσωμάτωση των πρωτεϊνών τους. Πολλές από τις θερμοεπαγόμενες πρωτεΐνες συνθέτονται και σε φυσιολογικές συνθήκες και παίζουν σημαντικούς ρόλους στην ορθή αναδίπλωση, μεταφορά και αποικοδόμηση των πρωτεϊνών του κυττάρου. Το εργαστήριο μας ενδιαφέρεται για το ρόλο των θερμοεπαγόμενων πρωτεϊνών και τη ρύθμιση των γονιδίων τους στη Μεσογειακή μύγα. Τέσσερα θερμοεπαγόμενα γονίδια, hsp90, hsp70, hsp27 και hsp26, έχουν κλωνοποιηθεί και χαρακτηριστεί στο εργαστήριό μας. Στόχοι αυτής της μεταπτυχιακής εργασίας είναι α) η λειτουργική ανάλυση της 5’ ανοδικής περιοχής του γονιδίου hsp90 της Μεσογειακής μύγας και β) η έκφραση του γονιδίου σε συνθήκες ψυχρού στρες. Για να μελετήσουμε την ρύθμιση του γονιδίου hsp90, δημιουργήσαμε διαγονιδιακά στελέχη που έφεραν το γονίδιο της λουσιφεράσης (ως γονίδιο αναφοράς) υπό τον έλεγχο διαφορετικών 5’ περιοχών του γονιδίου hsp90. Η ανάλυση των στελεχών αυτών έδειξε ότι η ευρύτερη περιοχή του υποκινητή που εκτίνεται έως και το -3536 περιλαμβάνει όλα τα απαραίτητα στοιχεία για τη θερμοεπαγόμενη έκφραση του γονιδίου. Για να μελετήσουμε εάν η χαμηλή θερμοκρασία (0 oC) έχει κάποια επίδραση στη βιωσιμότητα του εντόμου και στην έκφραση του hsp90, ομάδες αρσενικών ενήλικων εντόμων επωάστηκαν για διαφόρους χρόνους στους 0 oC και ακολούθως μελετήθηκε η επιβίωσή τους και η έκφραση του θερμοεπαγόμενου γονιδίου με RT-PCR και qRT-PCR. Τα αποτελέσματά μας υποδηλώνουν ότι οι χαμηλές θερμοκρασίες αυξάνουν την έκφραση του hsp90 σχεδόν στα ίδια επίπεδα με εκείνα του θερμικού στρες. Επίσης η βιωσιμότητα των εντόμων βρέθηκε πολύ υψηλή στους 0 oC. / Insects and other poikilotherms have multiple molecular mechanisms for their protection from extreme environmental and other stressful conditions which happens during their life. One of these mechanisms is the production of heat shock proteins. These proteins, being molecular chaperones, protect the cells from protein denaturation and aggregation. Some of these proteins are produced under normal conditions, and they play important role in proper protein folding, transport and degradation. We are interested in the role of heat shock proteins and the regulation of their genes in Mediterranean fruit fly (medfly). Four heat shock genes have been cloned and characterized in our laboratory. These are hsp90, hsp70, hsp27 and hsp26. The aim of this master thesis is: a) to study the normal and heat-induced regulation of the medfly’s hsp90 gene and b) to study the expression of this gene under cold shock conditions. For the first aim we created transgenic flies which they had the lusiferase gene (as a reference gene) under the control of different 5’ upstream regions of hsp90 gene. The analysis of these transgenic strains showed that the region between the first transcription base pair and -3536 base pair is crucial for the expression of the gene under normal and heat shock conditions. In order to examine whether the low temperature (0 oC) has any effect in the medfly’s viability and the expression of hsp90 gene, male groups were exposed at different intervals at 0 oC and then it was examined the viability and the expression of the gene. Our results suggest that the low temperature increases the expression of hsp90 nearly to the same levels of the thermal stress and this temperature does not affect the viability of the insect.

Η απόκριση των κυττάρων στο στρες συνδέεται με την επαγωγή μιας ομάδας πρωτεϊνών που ονομάζονται θερμοεπαγόμενες πρωτεΐνες (Hsps). Οι πρωτεΐνες αυτές ανήκουν στην κατηγορία των κυτταρικών συνοδών μορίων (chaperons) και προστατεύουν τα κύτταρα από τη μετουσίωση και συσσωμάτωση των πρωτεϊνών τους. Οι περισσότερες Hsps συντίθενται και σε φυσιολογικές συνθήκες και παίζουν σημαντικούς ρόλους στην ορθή αναδίπλωση, μεταφορά και αποικοδόμηση των πρωτεϊνών του κυττάρου. Οι Hsps κατηγοριοποιούνται σε διακριτές οικογένειες σύμφωνα με τα μοριακά τους βάρη. Από τις οικογένειες αυτές η οικογένεια των μικρών Hsps (sHsps), με μοριακά βάρη12-43 kDa, χαρακτηρίζονται από την παρουσία μιας πολύ συντηρημένης επικράτειας της α-κρυσταλλίνης στο καρβoξυτελικό τους άκρο. Μία από τις πιο καλά μελετημένες sHsps είναι η Hsp27. Η Hsp27, εκτός από το κύριο ρόλο της ως μοριακού συνοδού, εμπλέκεται και σε άλλες κυτταρικές λειτουργίες όπως η διαφοροποίηση, η απόπτωση, ο σχηματισμός του κυτταροσκελετού και η ρύθμιση της οξειδωτικής ισορροπίας των κυττάρων. Από τα αποτελέσματα της παρούσας εργασίας φάνηκε ότι το γονίδιο Cchsp27 εκφράζεται κάτω από φυσιολογικές συνθήκες κατά την ανάπτυξη της μεσογειακής μύγας και ότι η έκφραση του ρυθμίζεται στα διάφορα αναπτυξιακά στάδια του εντόμου. Πειράματα στον εγκέφαλο, στις ωοθήκες και στους όρχεις ενηλίκων ατόμων έδειξαν ότι στον εγκέφαλο των αρσενικών ατόμων το Cchsp27 mRNA παρουσιάζει σημαντικά υψηλότερα επίπεδα έκφρασης συγκριτικά με τον εγκέφαλο των θηλυκών ατόμων. Ακόμη παρατηρήθηκαν υψηλότερα επίπεδα έκφρασης του Cchsp27 mRNA στους όρχεις από ότι στις ωοθήκες. Τα αποτελέσματα από την έκφραση του γονιδίου στους σιελογόνους αδένες προνυμφών έδειξαν υψηλά επίπεδα έκφρασης στους σιελογόνους αδένες 24 ώρες πριν την εκτίναξη των προνυμφών, ενώ σταδιακά η έκφραση μειώνεται μέχρι το στάδιο της εκτινασσόμενης προνύμφης. Η έκφραση του Cchsp27 mRNA αυξάνεται στο στάδιο του λευκού προβομβυκίου ενώ στη συνέχεια σταδιακά μειώνεται. Ύστερα από την καλλιέργεια σιελογόνων αδένων παρουσία εκδυσόνης φάνηκε ότι συμβαίνει καταστολή της έκφρασης γονιδίου σε υψηλές συγκέντρωσης της ορμόνης, ενώ παρατηρείται επαγωγή σε συγκέντρωση 10-6 M. Σε ότι αφορά στην καλλιέργεια ωοθηκών και όρχεων παρουσία εκδυσόνης, παρατηρήθηκε μέγιστη επαγωγή του Cchsp27 γονιδίου στις ωοθήκες στα νεοεκκολαπτόμενα θηλυκά, ενώ μέγιστη επαγωγή στους όρχεις παρατηρήθηκε στα αρσενικά άτομα δύο ημερών. Ακολούθως, δημιουργήθηκε ένα διαγονιδιακό στέλεχος που φέρει το υβριδικό γονίδιο hsp27-GFP υπό τον έλεγχο της ευρύτερης 5΄ περιοχής του hsp27 γονιδίου, με σκοπό την μελλοντική μελέτη της κυτταρικής και ενδοκυτταρικής κατανομής της πρωτεΐνης Hsp27 κατά την ανάπτυξη της μεσογειακής μύγας. Τέλος, μελετήθηκε η έκφραση των γονιδίων Cchsp27 και Cchsp23 σε συνθήκες ψυχρού στρες. Και τα δύο γονίδια έδειξαν σημαντική έκφραση αυξανόμενου χρόνου επώασης στους 0 οC, με το Cchsp27 να παρουσιάζει υψηλότερα επίπεδα έκφρασης σε σχέση με το Cchsp23. / The stress response in cells is connected with the induction of heat shock proteins (Hsps). The Hsps are part of the molecular chaperons system and their role is to protect the cells from the protein denaturation and aggregation. Most Hsps are produced under non-stress conditions and they play a significant role in the correct folding, transmission and degradation of the cell’s proteins. Hsps are being divided into families according to their molecular mass. One of these families is the small heat shock proteins (sHsps) with molecular mass 12-43 kDa. This family is being characterized with a highly conservative domain called α-crystallin, which is located in the C-terminal domain. One of the most well studied sHsp is Hsp27. Hsp27 has an important role as molecular chaperone but also it is implicated in other events such differentiation, apoptosis, cytoskeleton’s formation and the regulation of the oxidized balance of the cell. The results that had arisen from this project, have shown that the Cchsp27 gene is expressed under non-stress conditions during the medfly’s development and that its expression is being regulated during the insect’s developmental stages. Experiments that were performed in brain, ovaries and testes which were removed from individual adults, have shown that the Cchsp27 gene is expressed highly in the male brain and in testes, comparing with female brain and ovaries respectively. Experiments that were performed in larvae salivary glands have shown significant expression levels of the Cchsp27 gene, 24 hours before jumping stage. The expression levels of the Cchsp27 gene are being reduced until the jumping stage and then they increased in the white pupa stage. The expression levels of the Cchsp27 gene are being reduced for the next three stages. The salivary glands were incubated with the hormone ecdysone and the results from these cultures have shown that in high ecdysone concentrations there is a repression in the gene’s expression whereas in concentration of 10-6 M there has been an induction. Ovaries and testes were incubated also with ecdysone and the results indicate that the maximum Cchsp27 gene induction happens in ovaries that have been removed from newborn females, and in testes that have been removed from males of the age of two days. Also, part of this project was the construction of a transgenic strain that it carries the hybrid gene hsp27-GFP under the regulation of the 5΄upstream region of the hsp27 gene. This strain will be used in the future for the study of the cellular distribution of the Hsp27 protein during the medfly’s development. For the last part of this project we study the expression levels of Cchsp27 and Cchsp23 genes under cold shock conditions. Both genes are expressed and their expression levels are being raised as the time of incubation increases in 0 οC. The expression levels of Cchsp27 gene were higher in comparison with the expression levels of Cchsp23 gene.

Malathion is a commonly used organophosphate pesticide on field crops, fruits, nut trees, vegetables, livestock, agricultural premises, and land. The approved uses also include mosquito and medfly control. These uses can result in human skin contact. The purpose of this study is to evaluate the human skin absorption of malathion for the purpose of assessing the risks associated with aqueous solution exposures following applications. Aerial applications can result in solubilized malathion in swimming pools and other waters that may be contacted. Human volunteers were selected and exposed to aqueous solutions of malathion at various concentrations. Participants submerged their arms and hands in twenty liters of dilute malathion solution in either a stagnant or stirred environment. The "disappearance method" was applied by measuring malathion concentrations in the water before and after human subject exposure to the water for various periods of time. Malathion was measured using Gas Chromatography. No measurable skin absorption was detected in 42% of the participants. Measurable skin absorption among the remaining 58% of participants resulted in doses that were more than an order of magnitude less than the minimal dose necessary to cause a measurable change in red blood cell acetylcholinesterase (RBC-AChE). Extrapolation of these results to a mathematical model for recreational swimmers and bathers exposed to contaminated swimming pools and surface waters typically detected after bait application again are an order of magnitude below the doses needed to cause a detectable change in RBC-AChE. These data indicate that exposure to aqueous malathion following usual aerial bait applications is not appreciably absorbed, and therefore, it is not a public health hazard.

O Submédio do Vale do São Francisco (VSF), que compreende parte dos estados da Bahia e de Pernambuco, é considerado atualmente o principal polo de produção de uvas frescas para exportação do Brasil. Entre as principais pragas que afetam essa cultura na região destaca-se a mosca-das-frutas, Ceratitis capitata, conhecida como moscamed ou mosca-do-mediterrâneo. Para a viticultura do VSF, populações elevadas de C. capitata representam o principal problema quarentenário para as exportações de uvas in natura, e tratamento quarentenário a frio, na pós-colheita, é obrigatório para uvas que se destinam aos Estados Unidos, Japão e Mercosul, onerando o custo final de produção. Visando aperfeiçoar as estratégias de manejo de C. capitata nessa cultura, o presente trabalho teve como objetivos estudar a biologia e comportamento desse inseto em cultivares de uvas de mesa produzidas no VSF, assim como avaliar o uso da radiação X como tratamento pós-colheita de cachos de uvas. Dados de estudos de preferência de oviposição e biologia de C. capitata em quatro cultivares de uvas de mesa, duas vermelhas (?Benitaka\' com semente e ?Crimson\' sem semente) e duas brancas (?Itália\' com semente e ?Festival\' sem semente) mostraram que esse inseto manifesta preferência de oviposição por bagas de cultivares vermelhas (Benitaka e Crimson), em condições de livre escolha. A cultivar de uva utilizada pelas larvas de C. capitata como alimento influenciou no desenvolvimento e sobrevivência dos estágios imaturos biológico, bem como no comportamento reprodutivo, longevidade e parâmetros demográficos das coortes de C. capitata recuperadas. Os valores estimados de Ro, rm e ? para coortes estudadas reafirmam que C. capitata se caracteriza por ser uma espécie com um alto potencial de crescimento e que as cultivares avaliadas constituem hospedeiros favoráveis à multiplicação desse inseto. Além disso, os resultados obtidos mostraram que C. capitata manifesta preferência de oviposição em cachos de uva Itália com maturação fisiológica mais avançada, em condições de escolha. No entanto, as fêmeas podem iniciar o ataque nas bagas, em parreirais desta variedade, aos 60 dias após a poda das plantas, quando os frutos ainda apresentam características físico-químicas desfavoráveis para a oviposição e desenvolvimento das fases imaturas, tais como: elevada firmeza e acidez e baixo teor de SST. As doses letais, DL99,9968, para inibir a emergência de adultos, quando aplicadas sobre ovos de 48h e larvas jovens (L1) infestando bagas de uvas Itália, foram estimadas em 12,08 e 20,44 Gy, respectivamente. Parâmetros químicos e sensoriais não foram afetados pela exposição das uvas às diferentes doses de raios X testadas, após 72h de armazenamento. Os conhecimentos biológicos, comportamentais e demográficos obtidos na presente pesquisa contribuirão ao aperfeiçoamento das estratégias de manejo de C. capitata nesta cultura, nas condições do VSF / The Lower Basin of the San Francisco Valley (VSF), which includes part of the states of Bahia and Pernambuco, is currently considered the major exporter and producer of table grapes in Brazil. Ceratitis capitata, also known as Moscamed or Mediterranean fly is among the main pests affecting fruit crops in the region. For the viticulture of the VSF, high populations of C. capitata are considered the main quarantine problem for exports of fresh table grapes, and for that reason post-harvest quarantine cold treatment is compulsory for grapes aimed to markets like the United States, Japan and Mercosur, adding additional charges to the final production cost. In order to improve the management strategies against C. capitata in this crop, this research aimed to study the biology and behavior of this insect in some table grape varieties grown in the VSF, as well as evaluate the use of Xrays as post-harvest treatment in table grape bunches. Studies on C. capitata oviposition preference and biology in four varieties of table grapes, two red (\'Benitaka\' seeded and \'Crimson\' seedless) and two white (\'Itália\' seeded and \'Festival\' seedless) showed that this insect expresses an oviposition preference for berries on red cultivars (Benitaka and Crimson), under free choice conditions. The table grape cultivar used for medfly larvae as food also influenced the development and survival of biologically immature stages as well as on reproductive performance, longevity and demographic parameters of C. capitata cohorts originating from the studied table grape varieties studied. The estimated values of Ro, rm and ? for the studied cohorts reaffirm that C. capitata is characterized by being a specie with a high growth potential and that the cultivars used in this research are favorable hosts for the multiplication of this insect. In addition, the results showed that C. capitata under choice conditions has clear preference of oviposition in \'Itália\' bunches in advanced physiological maturity. However, females can start attacking berries of this variety in vineyards, only 60 days after pruning, when the fruits still have unfavorable physicochemical characteristics for oviposition and immature stages development, such as high firmness and acidity and low in SST. The lethal dose, DL99,9968 to inhibit the emergence of adults when applied on 48 hours eggs and young larvae (L1) infesting \'Itália\' grape berries were estimated at 12,08 and 20,44 Gy, respectively. Chemical and sensory parameters were not affected by the exposure of table grapes to the different X-ray doses tested after 72 hours of storage. Biological, behavioral and demographic knowledge obtained in this study will contribute to the improvement of C. capitata management strategies in this crop under the VSF conditions

O Submédio do Vale do São Francisco (VSF), que compreende parte dos estados da Bahia e de Pernambuco, é considerado atualmente o principal polo de produção de uvas frescas para exportação do Brasil. Entre as principais pragas que afetam essa cultura na região destaca-se a mosca-das-frutas, Ceratitis capitata, conhecida como moscamed ou mosca-do-mediterrâneo. Para a viticultura do VSF, populações elevadas de C. capitata representam o principal problema quarentenário para as exportações de uvas in natura, e tratamento quarentenário a frio, na pós-colheita, é obrigatório para uvas que se destinam aos Estados Unidos, Japão e Mercosul, onerando o custo final de produção. Visando aperfeiçoar as estratégias de manejo de C. capitata nessa cultura, o presente trabalho teve como objetivos estudar a biologia e comportamento desse inseto em cultivares de uvas de mesa produzidas no VSF, assim como avaliar o uso da radiação X como tratamento pós-colheita de cachos de uvas. Dados de estudos de preferência de oviposição e biologia de C. capitata em quatro cultivares de uvas de mesa, duas vermelhas (?Benitaka\' com semente e ?Crimson\' sem semente) e duas brancas (?Itália\' com semente e ?Festival\' sem semente) mostraram que esse inseto manifesta preferência de oviposição por bagas de cultivares vermelhas (Benitaka e Crimson), em condições de livre escolha. A cultivar de uva utilizada pelas larvas de C. capitata como alimento influenciou no desenvolvimento e sobrevivência dos estágios imaturos biológico, bem como no comportamento reprodutivo, longevidade e parâmetros demográficos das coortes de C. capitata recuperadas. Os valores estimados de Ro, rm e ? para coortes estudadas reafirmam que C. capitata se caracteriza por ser uma espécie com um alto potencial de crescimento e que as cultivares avaliadas constituem hospedeiros favoráveis à multiplicação desse inseto. Além disso, os resultados obtidos mostraram que C. capitata manifesta preferência de oviposição em cachos de uva Itália com maturação fisiológica mais avançada, em condições de escolha. No entanto, as fêmeas podem iniciar o ataque nas bagas, em parreirais desta variedade, aos 60 dias após a poda das plantas, quando os frutos ainda apresentam características físico-químicas desfavoráveis para a oviposição e desenvolvimento das fases imaturas, tais como: elevada firmeza e acidez e baixo teor de SST. As doses letais, DL99,9968, para inibir a emergência de adultos, quando aplicadas sobre ovos de 48h e larvas jovens (L1) infestando bagas de uvas Itália, foram estimadas em 12,08 e 20,44 Gy, respectivamente. Parâmetros químicos e sensoriais não foram afetados pela exposição das uvas às diferentes doses de raios X testadas, após 72h de armazenamento. Os conhecimentos biológicos, comportamentais e demográficos obtidos na presente pesquisa contribuirão ao aperfeiçoamento das estratégias de manejo de C. capitata nesta cultura, nas condições do VSF / The Lower Basin of the San Francisco Valley (VSF), which includes part of the states of Bahia and Pernambuco, is currently considered the major exporter and producer of table grapes in Brazil. Ceratitis capitata, also known as Moscamed or Mediterranean fly is among the main pests affecting fruit crops in the region. For the viticulture of the VSF, high populations of C. capitata are considered the main quarantine problem for exports of fresh table grapes, and for that reason post-harvest quarantine cold treatment is compulsory for grapes aimed to markets like the United States, Japan and Mercosur, adding additional charges to the final production cost. In order to improve the management strategies against C. capitata in this crop, this research aimed to study the biology and behavior of this insect in some table grape varieties grown in the VSF, as well as evaluate the use of Xrays as post-harvest treatment in table grape bunches. Studies on C. capitata oviposition preference and biology in four varieties of table grapes, two red (\'Benitaka\' seeded and \'Crimson\' seedless) and two white (\'Itália\' seeded and \'Festival\' seedless) showed that this insect expresses an oviposition preference for berries on red cultivars (Benitaka and Crimson), under free choice conditions. The table grape cultivar used for medfly larvae as food also influenced the development and survival of biologically immature stages as well as on reproductive performance, longevity and demographic parameters of C. capitata cohorts originating from the studied table grape varieties studied. The estimated values of Ro, rm and ? for the studied cohorts reaffirm that C. capitata is characterized by being a specie with a high growth potential and that the cultivars used in this research are favorable hosts for the multiplication of this insect. In addition, the results showed that C. capitata under choice conditions has clear preference of oviposition in \'Itália\' bunches in advanced physiological maturity. However, females can start attacking berries of this variety in vineyards, only 60 days after pruning, when the fruits still have unfavorable physicochemical characteristics for oviposition and immature stages development, such as high firmness and acidity and low in SST. The lethal dose, DL99,9968 to inhibit the emergence of adults when applied on 48 hours eggs and young larvae (L1) infesting \'Itália\' grape berries were estimated at 12,08 and 20,44 Gy, respectively. Chemical and sensory parameters were not affected by the exposure of table grapes to the different X-ray doses tested after 72 hours of storage. Biological, behavioral and demographic knowledge obtained in this study will contribute to the improvement of C. capitata management strategies in this crop under the VSF conditions

La mosca mediterránea de la fruta, Ceratitis capitata, es la principal plaga de la fruticultura en el mundo. El desarrollo de métodos ambientalmente seguros de control de plagas, como Bacillus thuringiensis (Bt), adquiere cada vez mayor importancia. Esta tesis doctoral evalúa la validez de Bt y sus delta-endotoxinas como agentes de control de C. capitata. El análisis de la biodiversidad de Bt ha reflejado la gran riqueza presente en el agroecosistema de los cítricos. Sin embargo, ninguna de las cepas ensayadas (905) muestra alta toxicidad sobre C. capitata cuando esporas/cristales o sobrenadantes de su cultivo son ensayados. En cambio, la solubilización de los cristales de una selección de 42 cepas de Bacillus sp. ha demostrado que, para las cepas de la subespecie israelensis (Bti), este tratamiento produce una ganancia de función biológica. Adicionalmente, la predigestión de dichas protoxinas con proteasas de otro díptero, Culex pipiens, aumenta su actividad larvicida (CL50 31.26 µg/cm2). Por otro lado, se ha demostrado que, entre las delta-endotoxinas producidas por Bti, la protoxina Cyt1Aa es el factor determinante, con una CL50 sobre larvas de 4.93 µg/cm2. Sobre adultos, Cyt1Aa produce efectos subletales. Complementariamente, esta tesis propone un nuevo método de control basado en el desarrollo de toxinas recombinantes de fusión Cry-anticuerpo. Se ha puesto en práctica un sistema modelo para evaluar esta estrategia: se han desarrollado 4 variantes proteicas por la fusión entre partes de la protoxina Cry1Ab y un anticuerpo específico contra GFP (VHH anti-GFP) y éstas se han ensayado sobre larvas transgénicas de Drosophila melanogaster que expresan GFP en su intestino. Deficiencias en la unión de las toxinas de fusión a GFP, han impedido demostrar, por el momento, la estrategia propuesta. Por último, se han detectado al menos 160 proteínas distintas en las membranas intestinales de C. capitata y se han identificado las siguientes: V-ATPasa subunidades A y B, y alfa-tubulina. / Vidal Quist, JC. (2010). Estrategias para la utilización de la bacteria entomopatógena Bacillus thuringiensis (Berliner) en el control de Ceratitis capitata (Wiedemann) [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/8336

Os experimentos de campo foram realizados em 2002 e 2003 em três pomares no município de Monte Alegre do Sul, SP, representados por uma coleção de linhagens de goiabeiras (janeiro a abril), uma coleção de cultivares de nespereiras (agosto a setembro) e uma coleção de cultivares de pessegueiros (setembro a outubro). Nos ensaios de infestação, foram utilizadas três linhagens de goiaba (Guanabara, L7P28 e 252), duas cultivares de nêspera (Precoce Campinas e a Precoce 264-54) e três cultivares de pêssego (Aurora 2, Dourado 1 e Régis). Para a determinação do período de infestação, aplicou-se o processo de ensacamento e desensacamento quinzenal e semanal da goiaba e nêspera, respectivamente, e apenas o ensacamento semanal no pêssego. Cada experimento iniciou-se com os frutos ainda no início de seu desenvolvimento (frutos verdes). Para o processo de desensacamento, no início dos experimentos foram ensacados 500 e 400 frutos de goiaba e nêspera, respectivamente. Em cada ensaio, desde o início (frutos verdes pequenos) até a completa maturação, quinzenalmente (goiaba) ou semanalmente (nêspera e pêssego) foi ensacada/desensacada uma amostra de 30 frutos, os comprimentos e diâmetro eram mensurados e retirava-se amostras para realização das análises físico-químicas em laboratório. Após o completo amadurecimento, os frutos foram colhidos e levados ao laboratório, onde foram pesados, medidos os diâmetro e comprimento e depois acondicionados individualmente em copos plásticos com areia+vermiculita até aproximadamente a metade e cobertos com "voil" preso com fita elástica. Para o conhecimento da flutuação populacional das moscas, foram instaladas três armadilhas modelo McPhail com torula (atraente alimentar) em cada pomar, de janeiro/2002 a janeiro/2004. Das moscas-das-frutas originadas dos frutos, foram identificadas cinco espécies de tefritídeos e oito espécies de lonqueídeos. Os parasitóides emergidos foram das famílias Braconidae e Figitidae, representados por cinco espécies de cada uma. Ceratitis capitata apresentou comportamento sazonal com picos populacionais durante o segundo semestre. Verificou-se que a população de moscas-das-frutas (Anastrepha spp. e lonqueídeos) se mantém na área dos pomares (goiaba, nêspera e pêssego) durante o ano todo. Os três hospedeiros sofreram o ataque de moscas-das-frutas nos seus primeiros estádios de desenvolvimento, a partir de 2 cm de diâmetro. Ao longo do desenvolvimento, em todos os hospedeiros (goiaba, nêspera e pêssego), a infestação foi ascendente, apresentando as maiores elevações no final do amadurecimento. Os parâmetros físicos (tamanho, firmeza, brix e cor) mostraram-se mais confiáveis para o prognóstico da suscetibilidade do fruto ao ataque das moscas-das-frutas do que os parâmetros químicos (pH, acidez e umidade) para todos os hospedeiros. Os aspectos ecológicos da infestação dos frutos pela moscas-das-frutas e a relação tritrófica (parasitóide/mosca/fruto) foram discutidos. / Field experiments were conducted in three orchards in Monte Alegre do Sul, SP, each containing either a collection of strains of peach (September to October), guava (January to April) or loquat (August to September), during 2002 and 2003. Three strains of guava (Guanabara, L7P28 and 252), two of loquat (Precoce Campinas and Precoce 264-54) and three of peach (Aurora 2, Dourado 1 and Régis) were used for infestation assessment. Guava and loquat fruits were bagged and unbagged biweekly and weekly, respectively, for the assessment of the infestation period and only weekly for the peach. In all cases, fruits were bagged at the beginning of their development, while still green. A sample of 30 bagged fruits were unbagged weekly (loquat and peach) or biweekly (guava) until fruits were completely ripe, from a total of 500 (guava) and 400 (loquat) fruits. Fruits were measured and a sample was taken for physicochemical analysis. Fruits were harvested once they were ripe, taken to the laboratory, weighted and measured. They were placed into plastic cups half-filled with sand and covered with a fine fabric to allow for field-collected fly development. Three McPhail traps containing Torula yeast (food attractant) were hung from January/2002 to January/2004 to assess the population fluctuation of fruit flies in each orchard. Five tephritids and eight lonchaeids species emerged from field-collected fruits maintained in the lab. Five species each of braconid and figitid fruit fly parasitoids were also observed emerging from these fruits. Ceratitis capitata showed a sazonal behavior with population density peaking at the second semester. It was also observed that Anastrepha spp. and lonchaeids remained in the orchards throughout the year. All three host fruits larger than 2 cm in diameter were infested by fruit flies at the early stages of development. Fruit infestation increased in all orchards as fruit development occurred with the highest infestation level observed at the end of the ripening. Physical parameters, such as size, firmness, brix and color were more reliable in estimating fruit susceptibility to fruit-flies than the chemical parameters evaluated (pH, acidity and humidity) for all three host fruits. The ecological aspects of the infestation of fruits by fruit flies and the tritrophic interactions (parasitoid/fruit fly/fruit) were discussed.

O zoneamento ecológico exploratório de Anastrepha fraterculus e de Ceratitis capitata foi baseado nos dados climáticos de 497 estações meteorológicas de todo o Brasil para o clima recente (1961 - 1990) e nos dados climáticos previstos pelo IPCC para o clima futuro (2080 no cenário A2). Com esses dados, foram confeccionados evapluviogramas para calcular o índice de desenvolvimento das duas espécies, com base em suas necessidades térmicas e de umidade de solo. Os índices de desenvolvimento foram espacializados pelo sistema de informações geográficas SPRING 4.1.1 e divididos em cinco classes. As localidades mais propícias ao desenvolvimento de A. fraterculus, no clima recente, localizam-se nas regiões Sudeste e Sul. Essas regiões apresentam temperaturas médias mensais dentro da faixa mesotérmica de desenvolvimento da espécie e período curto de estiagem. Por outro lado, as localidades com condições menos favoráveis estão no semi-árido da região Nordeste, onde as temperaturas ficam sempre acima da temperatura-limite de desenvolvimento da mosca-das-frutas sul-americana e as condições hídricas são inadequadas ao desenvolvimento da fase de pupa. Com o aumento da temperatura, devido ao aquecimento global previsto para 2080 (cenário A2), as regiões Norte, Nordeste e Centro-Oeste serão completamente desfavoráveis ao desenvolvimento de A. fraterculus. Na região Sul e parte da Sudeste, a temperatura ficará dentro da faixa mesotérmica de desenvolvimento de A. fraterculus, mesmo com o aumento da temperatura, mas em alguns meses, excederá a temperatura-limite superior de desenvolvimento. No entanto, as condições hídricas serão desfavoráveis em grande parte do ano. No cenário do clima recente, as regiões Norte e Nordeste, com as temperaturas elevadas, são desfavoráveis ao desenvolvimento de C. capitata durante o ano todo. No Nordeste, também as condições hídricas só são favoráveis em curto período do ano. Na região Centro-Oeste, de março a agosto, parte do Mato Grosso do Sul e de Goiás são desfavoráveis ao desenvolvimento de C. capitata, em razão da precipitação pluvial baixa. Na região Sudeste, o período de setembro a março é favorável ao desenvolvimento, em razão da temperatura e condição hídrica adequadas. Na região Sul, apenas em maio e junho, quando ocorrem as menores temperaturas, o período é desfavorável ao desenvolvimento de C. capitata. Na previsão para 2080, devido principalmente ao aumento da temperatura causado pelo aquecimento global, apenas nos estados de Santa Catarina e do Rio Grande do Sul, haverá condições climáticas apropriadas ao desenvolvimento da mosca-do-mediterrâneo. Portanto, as mudanças climáticas previstas serão menos favoráveis ao desenvolvimento de C. capitata (espécie exótica) do de A. fraterculus (espécie nativa). / Exploratory ecological zoning for the South American fruit fly, Anastrepha fraterculus, and the medfly, Ceratitis capitata, were established based on data of 497 meteorological stations throughout Brazil, considering recent (1961 - 1990) and future climate forecast by IPPC (2080, A2 scenario). Climograms were used to estimate the South American fruit fly and medfly development indexes (Id) based on their thermal and soil moisture requirements. The development index was divided into five classes and spacialized by using the SPRING 4.1.1 geographical information system. Based on development index the Southwest and South regions were the most suitable for Anastrepha fraterculus in the recent climate. These regions have average monthly temperatures within the mesothermic range of A. fraterculus development and a short period of drought, while the unfavorable localities were situated in the semi-arid of the Northwest region, where temperatures are always above the threshold temperature of development and soil moisture are unsuitable for the pupa stage. For the increase of temperature due to global warming foreseen for 2080 (scenario A2), the North, Northwest and Center-West regions will become entirely unsuitable for the South American fruit fly development. Temperatures within the mesothermic range of development will still be available in part of Southwest region and in the South region, even with the increase of the temperature. However, the hydric conditions will be unfavorable for most of the year, and the upper temperature threshold will be exceeded in warmer months. For Ceratitis capitata, the recent climate of the North and Northwest regions were unsuitable for its development due to the high temperatures year-round. In the Northwest region, the hydric conditions will be favourable for a short period of the year. The low rainfall from March to August will make part of the state of Mato Grosso do Sul and Goiás unfavorable for medfly development in the Center-West region. In the Southwest region, temperature and hydric conditions from September to March are inadequate for medfly development. In the South region, conditions will be unfavorable to medfly development only during the months of May and June, when low temperatures occur. In the prediction for 2080, due to the temperature increase, only Santa Catarina and Rio Grande do Sul states will have adequate climatic conditions to allow for C. capitata development. Therefore, the foreseen climate changes will be more unfavorable for the development of C. capitata (exotic species) than A. fraterculus (native species).

Μελέτες στη Μεσογειακή μύγα έχουν οδηγήσει στον χαρακτηρισμό πέντε άρρενο-ειδικών πρωτεϊνών (MSSPs) οι οποίες είναι ομο- και ετερο- διμερή δύο συγγενών, τύπου α και β, πολυπεπτιδίων (MSSP-α και –β). Τα πολυπεπτίδια αυτά κωδικοποιούνται από τουλάχιστον 7 γονίδια τα οποία με βάση την ομοιότητα των αλληλουχιών τους κατατάσσονται σε 3 ομάδες: msspα (α1 και α2), msspβ (β1, β2 και β3) και msspγ (γ1 και γ2). Λειτουργική ανάλυση των υποκινητών των γονιδίων msspα2 και msspβ2 έδειξε ότι το γονίδιο msspα2 εκφράζεται σε υψηλά επίπεδα στο λιπώδη ιστό των ενήλικων αρσενικών ατόμων ενώ το γονίδιο msspβ2 εκφράζεται σε χαμηλά επίπεδα στο έντερο και των δύο φύλων. Με στόχο την αρρενο- ειδική υπερέκφραση τής αλκοολικής αφυδρογονάσης τής D. melanogaster σε άτομα C. Capitata για την κατασκευή ενός Στελέχους Διαλογής Φύλου στη Μεσογεική μύγα στο οποίο παρουσία υψηλών επιπέδων αλκοόλης θα πεθαίνουν τα θηλυκά άτομα, ενώ τα αρσενικά θα επιβιώνουν, κατασκευάστηκαν διαγονιδιακά στελέχη τα οποία έφεραν το γονίδιο DmadhFAST υπό τον έλεγχο της περιοχής -522/+37 (α2PL) του γονιδίου msspα2. Η κατασκευή των διαγονιδιακών στελεχών έγινε με γενετικό μετασχηματισμό χρησιμοποιώντας το σύστημα μετασχηματισμού Minos. Από την ανάλυση 9 μετασχηματισμένων σειρών σε επίπεδο RNA (Northern ανάλυση και RT-PCR) και σε επίπεδο πρωτεΐνης (Western ανάλυση και Adh assay) προέκυψαν τα εξής συμπεράσματα: α) Ο υποκινητής α2PL είναι κατάλληλος για υψηλή φύλο-ειδική έκφραση διαγονιδίων στα ενήλικα αρσενικά άτομα της Μεσογειακής μύγας και β) Το γονίδιο της ADH της D. melanogaster πιθανόν δεν είναι το κατάλληλο γονίδιο επιλογής για τη δημιουργία στελεχών γενετικού διαχωρισμού του φύλου στη Μεσογειακή μύγα. Επιπλέον, στην παρούσα διατριβή επιλέχθηκε να μελετηθεί ο τρόπος έκφρασης του γονιδίου msspβ1. Όπως και με το msspα2 γονίδιο μελετήθηκε η λειτουργία του msspβ1 γονιδίου, in vivo, σε μετασχηματισμένες σειρές χρησιμοποιώντας το τμήμα -485/+35 (β1PL) του υποκινητή και το γονίδιο lacZ ως γονίδιο αναφοράς. Οι αναλύσεις έκφρασης του διαγονιδίου lacZ, τόσο στο επίπεδο του RNA (RT-PCR) όσο και στο επίπεδο της πρωτεΐνης (β-gal assay), σε 7 διαφορετικές σειρές έδειξαν ότι το τμήμα β1PL είναι αρκετό για την ορθή χρονο- ειδική έκφραση του διαγονιδίου σύμφωνα με το φυσιολογικό πρότυπο έκφρασης των mssp-α και -β γονιδίων, εντούτοις, δεν παρουσιάζει αυστηρά άρρενο-ειδικό πρότυπο έκφρασης, αν και η έκφραση στα αρσενικά άτομα ήταν εντονότερη από ότι στα θηλυκά. Από τη σύγκριση των επιπέδων μεταγραφής του διαγονιδίου lacZ υπό τον έλεγχο του υποκινητή β1PL με εκείνα του διαγονιδίου DmadhFAST υπό τον έλεγχο του υποκινητή α2PL, φαίνεται ότι η ισχύς του υποκινητή του γονιδίου msspβ1 είναι εκατοντάδες φορές μικρότερη από εκείνη του msspα2. Τα αποτελέσματα αυτά σε συνδυασμό με προηγούμενη μελέτη υποδηλώνουν ότι το δεύτερο γονίδιο mssp που εκφράζεται σε υψηλά επίπεδα και με αρρενο-ειδικό τρόπο θα πρέπει να είναι το msspβ3 γονίδιο ή κάποιο άλλο mssp γονίδιο του οποίου το προϊόν είναι ένα MSSP πολυπεπτίδιο τύπου β. / Studies in the medfly have led to the characterization of five male-specific serum proteins (MSSPs) that are homo- and hetero- dimmers of two major polypeptide types, MSSP-α and –β. These polypeptides are coded by at least 7 distinct genes which, based on their homology, are classified in three subgroups, MSSP-α (α1 and α2), MSSP-β (β1, β2 and β3) and MSSP-γ (γ1 and γ2). Functional analysis of the msspa2 and msspβ2 promoters showed that msspa2 is expressed in high levels in the fat body of adult male individuals, whereas msspβ2 is expressed in low levels in the midgut of both sexes. In order to overexpress in a male-specific manner the D. melanogaster alcohol dehydrogenase (ADH) in C. capitata for the construction of a Genetic Sexing Strain (GSS) in the medfly, which in the presence of high alcohol concentration will lead to the death of female and the survival of the male individuals, we constructed transgenic medfly strains using DmadhFAST under the control of the -522/+37 fragment (α2PL) of the msspa2 promoter, via Minos-mediated germline transformation. The RNA analysis (Northern and RT-PCR) and the protein analysis (Western and Adh assay) of 9 transgenic strains showed that: a) The α2PL fragment is sufficient for high levels of male-specific expression of transgenes in the medfly and b) The D. melanogaster ADH gene may not be the appropriate gene for the construction of GSS strains in the medfly. In addition in the present study we performed functional analysis of the msspβ1promoter in vivo in medfly transgenic adults generated by Minos-mediated germ line transformation. For this analysis we used the -485/+37 fragment of the msspβ1 promoter and the lacZ reporter gene. The RNA analysis (RT-PCR) and the protein analysis (b-gal assay) performed on 7 transgenic strains showed that the β1PL fragment is sufficient for the proper time-specific expression of the transgene in accordance to the expression pattern of mssp-a and –β genes, not in male-specific manner, although the level of expression in males was higher than in females. The comparison of the expression levels of the lacZ and the DmadhFAST transgenes showed that the α2PL promoter fragment is a hundred time stronger than the β1PL. These results indicate that msspβ3 or another β type mssp gene may be expressed in high levels in the male adults of the medfly.