Submitted by Vasti Diniz (vastijpa@hotmail.com) on 2017-09-06T13:22:53Z No. of bitstreams: 1 arquivototal.pdf: 1661491 bytes, checksum: 603ad8044463f6291792eef191227188 (MD5) / Made available in DSpace on 2017-09-06T13:22:54Z (GMT). No. of bitstreams: 1 arquivototal.pdf: 1661491 bytes, checksum: 603ad8044463f6291792eef191227188 (MD5) Previous issue date: 2015-04-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The lectins are proteins capable of recognize and bond reversibly to carbohydrates without modifying them by a non-catalytic domain. In vegetables, they are synthetized as resistance factors against phytopathogens, and since they recognize structures present in the parasites, for their structural, catalytic and carbohydrate-specific characteristics give information on the action mechanisms against different parasitic species. Our study feature its catalytic activity of the lectin extracted from Abelmoschus esculentus (AEL) and evaluated their effects on phytophagous and phytopathogens for its application as a potential as crop protection and. We performed peptide analysis from AEL using mass spectrometry to determine conservative domains inherent to its molecular properties and protease activity inhibition toxicity, essays toxicity on the fruit fly Ceratitis capitata and root knot nematodes Meloidogyne javanica and Meloidogyne incognita on their different development stages through topical application, fungistatic activity on phytopathogenic fungi. AEL presents in its composition regions that are homologous to kunitz-type protease inhibitors, with the identification of conserved peptides for this class of proteins and protease inhibition activity (92%), which reveal its catalytic function and direct their action mechanisms on the different phytopathogens. It´s displayed insecticide effect on 3rd instar larvae and pupae, nematicide effect for the root knot nematodes acting mainly in larve hatched but did not presented fungistatic activity. / Lectina é um grupo de proteínas capaz de reconhecer e se ligar a carboidratos de forma reversível sem modifica-los por domínio não catalítico. Em vegetais, são sintetizadas como fator de resistência contra fitopatógenos por reconhecer estruturas presentes nestes parasitas e e suas características estruturais, catalíticas e carboidrato-específicos fornecem informações sobre seus mecanismos de ações em diferentes espécies de parasitas. O objetivo deste estudo foi caracterizar a atividade catalítica da lectina extraída de sementes de Abelmoschus esculentus (AEL) e avaliar seus efeitos sobre organismos fitófagos e fitopatógenos para sua utilização como um possível defensivo agrícola. Foram realizadas análises dos peptídeos da AEL por espectrometria de massas para detecção de domínios conservados inerentes a sua propriedade molecular e realização de atividade de inibição de proteases e ensaios de toxicidade em mosca de fruta Ceratitis capitata e nematoides de galha Meloydogine javanica e Meloydogine incógnita, nos seus diferentes estágios de desenvolvimento através de aplicação tópica e avaliação de sua atividade fungiostática sobre fungos fitopatogênicos, AEL apresentou em sua composição regiões homólogas à inibidores de protease do tipo kunitz, com identificação de 03 peptídeos conservados nesta classe de proteínas e atividade de inibição de protease (92%), os quais revelam sua função catalítica e direcionam seus mecanismos de ações nos diferentes parasitas. Apresentou efeito inseticida sobre larvas 3º instar e pupa, apresentou efeito nematicida sobre os nematoides de galhas, atuando principalmente sobre a eclosão das larvas, mas não apresentou atividade fungiostática.

Made available in DSpace on 2014-12-17T14:03:33Z (GMT). No. of bitstreams: 1 CleysyvanSM_DISSERT.pdf: 1173359 bytes, checksum: 82a094c436e4d9b81948c390ed5d9ed1 (MD5) Previous issue date: 2010-02-26 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / Chitin-binding vicilins from legume seeds (Erythrina velutina. Canavalia ensiformes and Phaseolus vulgares) were isolated by ammonium sulfate followed by affinity chromatography on a chitin column. Effect of these vicilins on female adults of Ceratitis capitata was examined by bioassay and in a semi-field assay model. Mechanism of action of the vicilins was determined by in vivo digestibility and chitin affinity. Among the tested vicilins, E. velutina when added to diet caused strong effect on mortality at 10% dose. This insecticidal property was tested in a semi-field assay which showed the same effect observed in laboratory conditions, where doses of 10% and 15% were lethal to female adults of C. capitata. These deleterious effects were not only associated to the binding to chitin structures present in peritrophic membrane, but principally to its low digestibility in the C. capitata digestive tract. This fact was confirmed because chiting binding proteins as WGA and the other tested vicilins were not toxic to female adults of C. capitata due susceptibility of these proteins to digestive enzymes of the insects. By other side EvV was more resistant to digestive enzymes, causing deleterious effects on female adults of C. capitata. These results showed that EvV may be part of the pest management programs or an alternative in plant improvement program in the population control of this fruticulture pest / Vicilinas ligantes a quitina de sementes de Erythrina velutina, Canavalia ensiformis e Phaseoulus vulgaris foram isoladas atrav?s de procedimento que consistiu de precipita??o com sulfato de am?nio seguida de cromatografia de afinidade em coluna de quitina. O efeito dessas vicilinas sobre f?meas adultas de Ceratitis capitata foi examinada por bioensaios em condi??es de laborat?rio e por bioensaios em condi??es de semi-campo. O mecanismo de a??o dessas vicilinas foi determinado por um ensaio de digestibilidade in vivo e pela propriedade de liga??o a uma matriz de quitina. Entre as vicilinas testadas, a vicilina de E. velutina foi a ?nica letal, causando mortalidade de 100% quando adicionada ? dieta das moscas das frutas adultas na concentra??o de 10% (p/p). Este efeito inseticida da vicilina de E. velutina foi tamb?m testado em condi??es de semi-campo, onde concentra??es de 10 % e 15% (p/p) de vicilina adicionada ? dieta foram letais para f?meas adultas de C. capitata. Esse efeito delet?rio das prote?nas ligantes ? quitina n?o foi associado somente ? propriedade de liga??o ? quitina, visto que as outras vicilinas, al?m da lectina de g?rmen de trigo (WGA) foram t?xicas quando adicionadas ? dieta das moscas das frutas. Entretanto, a resist?ncia/susceptibilidade dessas prote?nas aos processos digest?rios desses insetos poderia explicar os diferentes efeitos observados. Das prote?nas ligantes ? quitina, apenas aquelas refrat?rias ? a??o das enzimas digestivas nos processos digest?rios teve a??o inseticida. Esses resultados mostraram que vicilina de E. velutina ? um bioinseticida com potencial de uso nos programas de controle de pragas diminuindo a popula??o de moscas das frutas no campo

Made available in DSpace on 2014-07-29T16:24:18Z (GMT). No. of bitstreams: 1 Dissertacao Lilian Rosana S Rabelo.pdf: 1111316 bytes, checksum: 3f59633e68c556a639d849cab559a09d (MD5) Previous issue date: 2010-08-18 / Fruit flies (Diptera, Tephritidae) are important insect pests in world fruit orchards; responsible for significant yield losses, and an obstacle to the fruit market due to quarantine restrictions imposed by importing countries. The knowledge of their population dynamics in a specific region is an important factor in insect pest management programs. Fruit flies were monitored by surveys conducted in five counties of the state of Goiás, aiming to increase the knowledge of the species, the faunistic index, the population fluctuation, as well as to elaborate a pictorial identification key for the species catalogued. Collecting expeditions were conducted weekly, using traps model McPhail, with corn hydrolyzed protein at 5% as food attractant. The most common species population fluctuation was assessed by the FTD (fly/trap/day) index. Surveys took place from November 2004 to October 2009 in cucurbit crops and family orchards; and from September 2008 to August 2009 in fragments of the native vegetation of the Cerrado (Brazilian Savanna), at the Vale do São Patrício - GO region, in four counties belonging to the area of Mitigating Risk System (MRS) of A. grandis. This study was also carried out in a guava commercial orchard in the Cristalina county GO, from November 2008 to October 2009. In the Vale do São Patrício 173 Anastrepha females were captured, and 14 species identified as belonging to five infrageneric groups. The largest number of flies captured took place in the Uruana county (37%), followed by Itapuranga (30%), Carmo do Rio Verde (26%), and Jaraguá (7%) counties. A. manihati species was the most common (34%), followed by A. obliqua (19.65%), and A. pickeli (13.87) A. dissimilis, A. quiinae and A. pickeli were recorded for the first time in the state of Goiás. A. grandis was captured for the first time in the counties of Uruana and Jaraguá with FDI within the contents accepted for cucurbits fruit export. In the Vale do São Patricio region the genus Anastrepha occurred in all seasons, with population peaks coinciding with the fructification period of most of the host species of the Cerrado fauna. At the guava commercial orchard of Cristalina 170,812 male and female individuals of C. capitata, and 1, 654 female individuals of Anastrepha were captured. C. capitata was the most abundant, with 98.24% of the total, while genus Anastrepha was the least, with 1.76%, and seven species identified, in which A. fraterculus (1.46%) and A. sororcula (0.175%) were the most common. C. capitata and A. fraterculus were associated with the class super of dominance, abundance, frequency and constancy. The seven species of Anastrepha sampled in this work matched the 1.09 index of diversity. C. capitata and A. fraterculus population peaks coincided with the guava production season, with a population outburst occurring in January 2009. C. capitata population levels remained relatively high during the collecting period, except from March to July 2009, with peaks in December 2008. There was a significant correlation of C. capitata and A. fraterculus population fluctuation with weather. A pictorial identification key was elaborated for the 22 species of fruit flies recorded in Goiás: A. amita, A. bahiensis, A. bistrigata, A. dissimilis, A. distincta, A. fraterculus, A. grandis, A. leptozona, A. manihoti, A. montei, A. obliqua, A. pseudoparallela, A. pickeli, A. quiinae, A. serpentina, A. sororcula, A. striata, A. turpiniae, A. zenildae, A. zerny, Ceratitis Capitata and Anastrepha sp. (new species - R. A. Zucchi personal information). / As moscas-das-frutas (Diptera, Tephritidae) são consideradas importantes pragas da fruticultura mundial, pois causam perdas significativas à produção e limitam o livre transporte de frutas devido às restrições quarentenárias impostas por países importadores. O conhecimento da dinâmica populacional das moscas-das-frutas de uma determinada região é um fator importante para o manejo desta praga. As moscas-das-frutas foram estudadas a partir de levantamentos realizados em cinco municípios do Estado de Goiás, com os objetivos gerais de aumentar o conhecimento sobre as espécies ocorrentes, índices faunísticos, flutuações populacionais e elaborar uma chave de identificação pictórica para as espécies registradas neste Estado. As coletas foram realizadas semanalmente, em armadilhas modelo McPhail, com atrativo alimentar (proteína hidrolisada de milho a 5%). A flutuação populacional das espécies mais frequentes foi avaliada por meio do índice MAD (mosca/armadilha/dia). De novembro de 2004 a outubro de 2009, os levantamentos foram realizados em cultivos de cucurbitáceas e em pomares domésticos e, de setembro de 2008 a agosto de 2009, em fragmentos de vegetação nativa do Cerrado, na Região do Vale do São Patrício (GO), em quatro municípios pertencentes à área com Sistema de Mitigação de Risco (SMR) de A. grandis. O estudo também foi conduzido em um pomar comercial de goiaba no município de Cristalina (GO), de novembro de 2008 a outubro de 2009. Na Região do Vale do São Patrício, foram capturadas 173 fêmeas de Anastrepha e identificadas 14 espécies pertencentes a cinco grupos infragenéricos. Em Uruana, foi capturado o maior número de moscas-das-frutas (37%), seguido de Itapuranga (30%), Carmo do Rio Verde (26%) e Jaraguá (7%), sendo que A. manihoti foi a espécie mais frequente (34%), seguida da A. obliqua (19,65%) e da A. pickeli (13,87%). Quanto às espécies A. dissimilis, A. quiinae e A. pickeli, elas foram referidas pela primeira vez no Estado de Goiás. Já A. grandis foi capturada pela primeira vez nos municípios de Uruana e Jaraguá com MAD, dentro dos índices aceitos para exportação de frutos de cucurbitáceas. Na Região do Vale do São Patrício, as moscas-dasfrutas do gênero Anastrepha ocorrem em todas as estações do ano, e os picos populacionais coincidem com o período de frutificação da maioria das espécies de plantas hospedeiras dos Cerrados. No pomar comercial de goiaba em Cristalina, foram capturados 170.812 exemplares (machos e fêmeas) de C. capitata, dos quais 1.654 são fêmeas de Anastrepha. C. capitata (a espécie mais abundante), representando 98,24% do total. Já o gênero Anastrepha foi representado por 1,76%, sendo identificadas sete espécies, das quais A. fraterculus (1,461%) e A. sororcula (0,175%) foram as mais frequentes. C. capitata e A. fraterculus foram associadas à classe super de dominância, abundância, frequência e constância. As sete espécies de Anastrepha amostradas neste estudo correspondem ao índice de diversidade de 1,09. O pico populacional de C. capitata e A. fraterculus coincide com a época de produção de frutos da goiaba, com uma explosão populacional em janeiro de 2009. Os níveis populacionais de C. capitata mantiveram-se relativamente altos em todo o período de coleta, exceto de março a julho de 2009, com pico em dezembro de 2008. Houve correlação significativa da flutuação populacional de C. capitata e de A. fraterculus com os fatores meteorológicos estudados. Foi elaborada uma chave de identificação pictórica para as 22 espécies de moscas-das-frutas registradas em Goiás: A. amita, A. bahiensis, A. bistrigata, A. dissimilis, A. distincta, A. fraterculus, A. grandis, A. leptozona, A. manihoti, A. montei, A. obliqua, A. pseudoparallela, A. pickeli, A. quiinae, A. serpentina, A. sororcula, A. striata, A. turpiniae, A. zenildae, A. zerny, Ceratitis Capitata e Anastrepha sp. (espécie nova - R. A. Zucchi - inf. pessoal).

This work covers stages one through seven (of nine stages) of a classical biological control program for Mediterranean fruit fly (=medfly), Ceratitis capitata (Wiedemann). Major research objectives concentrate on stage five (exploration and collection of natural enemies), and stage seven (testing and selecting natural enemies for additional work). Coffee was collected monthly from three locations in Kenya from November 1997 through July 1999. Four species of tephritid flies and ten parasitoid species were recovered. Four guilds of parasitoids were recorded, and two egg-prepupal endoparasitoids, Fopius caudatus (Szépligeti) and F. ceratitivorus (Wharton), were discovered. The oviposition behavior of these two species is contrasted. Domination of this tropical parasitoid assemblage by koinobionts is discussed relative to the dominance of temperate fruit-infesting tephritid systems by idiobionts. Fruit handling procedures were examined for impact on overall percent emergence and specifically percent emergence of flies versus parasitoids. It was determined that stirring samples had a significant positive effect on overall emergence, however daily misting of fruit did not. The only treatment without a significant bias in fly emergence over parasitoids was the stirred/dry treatment. Effects of these results on rearing procedures are discussed. Host specificity and host suitability of parasitoids reared from coffee were examined via: (1) association of parasitoids with host flies based on characteristics of the fly puparia from which parasitoids emerged, (2) rearing of cucurbit infesting tephritids and their parasitoids in Kenya, (3) rearing of flowerhead infesting tephritids and their parasitoids in Kenya and Hawaii, and (4) host range testing of Psyttalia species in Kenya and Hawaii. These results are discussed in terms of their utility for predicting nontarget effects. Psyttalia concolor (Szépligeti) was shipped to Hawaii and tested against the nontarget gall forming tephritid Procecidochares utilis Stone introduced to control the weed Ageretina adenophora (Maui pamakani). Psyttalia concolor failed to attack the gall-forming P. utilis both in choice and no-choice tests, but readily attacked tephritid larvae offered in fruit in choice tests. Recommendations for further testing and release of the parasitoids from Kenya are discussed for Hawaii and Latin America.

Differential gene expression plays a key role in developmental pathways within an organism. Examples of such pathways include primary sex determination signaling and the formation of secondary sexual characteristics. This dissertation is focused on the use of suppression subtractive hybridization (SSH) to identify genes that are differentially expressed and involved in some aspect of sexual development in the Mediterranean fruit fly (medfly), Ceratitis capitata. In the course ofthis project, a method for sexing individual specimens from pre-adult stages was developed. This method was used to collect sex-specific RNAs at different developmental stages for use in SSH. A total of25 subtraction products were obtained across all the stages examined. Analysis of these products revealed that approximately half were similar to cytoplasmic ribosomal proteins and mitochondrial ribosomal RNA The remaining products represent putative medfly homologs of other previously identified genes or potentially novel genes One ofthe subtraction products, representing a potentially novel gene, was characterized in detail. This gene, named mapotge', represents a novel medfly gene that appears to encode a polypeptide of 299 amino acids. The N-terminus of this polypeptide contains a BTB-POZ domain. This domain functions as a protein-protein interaction motif found in a wide range of organisms from humans to Drosophila that mediates protein dimerization and oligomerization. The temporal expression pattern of mapotge' was determined using RT-PCR and Northern blot analysis. These revealed that the transcript is expressed throughout embryogenesis in both females and males, and in adult females that are > 0.5 days post-eclosion. Minimal expression is observed in female and male third instar larvae, early pupae, and in adult males. Studies were also initiated to characterize the representation of additioual sequences containing a BTB-POZ domain in the medfly genome. This was performed using Southern blot analysis and degenerate primers for the polymerase chain reaction (PCR). These results indicate the presence of at least three sequences in the medfly, in addition to 'mapotge', that contain a BTB-POZ domain. Potential evolutionary relationships ofthe BTB-POZ domain sequences from the medfly and other insect species were also analyzed.

Made available in DSpace on 2014-10-09T12:53:57Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:09:27Z (GMT). No. of bitstreams: 0 / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

Made available in DSpace on 2014-10-09T12:53:57Z (GMT). No. of bitstreams: 0 / Made available in DSpace on 2014-10-09T14:09:27Z (GMT). No. of bitstreams: 0 / Este trabalho teve por objetivo investigar a hipótese de que as ondas acústicas provocam algum efeito mensurável no desenvolvimento de uma população de moscas das frutas, tratamento denominado sonicação. A radiação ionizante, que é o agente físico causal do tratamento denominado irradiação, foi utilizada como referência pois seus efeitos sobre os seres vivos e em especial sobre os insetos são amplamente conhecidos. Uma etapa da pesquisa verifica possíveis efeitos de interação entre ondas acústicas e raios gama. Os experimentos de sonicação foram realizados no laboratório de Entomologia do Instituto Biológico de São Paulo. Os experimentos de irradiação foram realizados no Centro de Tecnologia das Radiações do IPEN, situado no campus da USP em São Paulo. Foi utilizada a fonte Gammacell modelo 220, com atividade média de 3757,069 ± 293,484 curies (Ci) e taxa de dose média de 3,106 ± 0,245 quilograys por hora (kGy/h). Os níveis do tratamento sonicação aplicados, em hertz e quilohertz, foram 0 Hz (controle) ; 5,0 Hz ; 10,0 Hz ; 20,0 Hz ; 40,0 Hz ; 60,0 Hz ; 80,0 Hz ; 1,0 kHz ; 2,0 kHz ; 10,0 kHz ; 15,0 kHz e 20,0 kHz ; Os níveis do tratamento irradiação, aplicados em grays foram: 5,0 Gy ; 7,5 Gy ; 10,0 Gy ; 12,5 Gy ; 15 Gy ; 50 Gy ; 100 Gy ; 150 Gy e 200 Gy. Foi utilizado um tubo acústico de vidro com dimensões de 40,6 cm de comprimento por 9,1 cm de diâmetro e ondas senoidais com respostas em decibéis, de intensidade sonora de 93,60 ± 1,51 dB até 123,96 ± 0,23 dB. Os resultados mostraram que há evidencias para rejeitarmos a hipótese de nulidade H0, de que as médias dos tratamentos devido as ondas acústicas não diferem significativamente. / Tese (Doutoramento) / IPEN/T / Instituto de Pesquisas Energéticas e Nucleares - IPEN-CNEN/SP

Los semioquímicos son sustancias implicadas en la comunicación entre seres vivos. En el caso de los insectos, pueden ser herramientas de gran utilidad en la lucha contra plagas, como alternativas a la aplicación de insecticidas convencionales. Son sustancias de gran selectividad y muy activas a baja dosis. Los semioquímicos (feromonas y aleloquímicos) se emplean en el control de plagas tanto para la detección y seguimiento de poblaciones como, de forma directa, en las técnicas de confusión sexual, trampeo masivo y atracción y muerte, entre otras. En la presente tesis se describe la búsqueda y aplicación de semioquímicos para el control de diversas plagas de importancia agronómica, estudiando tres aspectos fundamentales. En primer lugar, el desarrollo y aplicación de la técnica de confusión sexual contra Aonidiella aurantii y Tuta absoluta. En el caso de A. aurantii, se trata de la primera aplicación eficaz de esta técnica contra una plaga de diaspinos y en el caso de T. absoluta, se establecen las condiciones para la aplicación de tratamientos eficaces de confusión sexual. En segundo lugar, se ha estudiado la existencia de valores óptimos de emisión para sistemas basados en la atracción de insectos, aplicado a las plagas Chilo suppressalis, Lobesia botrana, Bactrocera oleae y Ceratitis capitata. En el caso de los lepidópteros C. suppressalis y L. botrana y del tefrítido B. oleae se demostró que la atracción de machos se ve afectada por el nivel de emisión de feromona, de forma que velocidades de emisión por encima y por debajo del nivel óptimo obtenido tienen menor poder atrayente. Sin embargo, respecto a C. capitata, y su paraferomona trimedlure, su respuesta es de tipo asintótico, no se encontró un nivel óptimo y por tanto, mayores dosis de paraferomona atraían al mismo número de machos. La obtención de emisores con velocidades de emisión óptimas es un aspecto poco estudiado en la literatura científica que, sin embargo, es esencial para el desarrollo de los métodos de control basados en la atracción. Como tercera parte de esta tesis, se estudiaron detalladamente los compuestos emitidos por machos y hembras de C. capitata en diversos estadios, para intentar establecer compuestos y mezclas con posible poder atrayente. Para este trabajo se ha utilizado la técnica de microextracción en fase sólida, no empleada hasta el momento para este problema, y la metodología estadística del análisis de componentes principales. La disponibilidad de atrayentes eficaces para hembras de C. capitata supondría una gran ventaja para el control de esta plaga. / Vacas González, S. (2011). Uso de semioquímicos en el control de plagas. Estudios básicos y de aplicación [Tesis doctoral no publicada]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/12499 / Palancia

Με στόχο την απομόνωση του γονιδίου hsp83 της μεσογειακής μύγας, πραγματοποιήθηκε διαλογή μιας cDNA βιβλιοθήκης από προνύμφες 3ου σταδίου, με ανιχνευτή το δεύτερο εξώνιο του hsp83 ομόλογου γονιδίου της Drosophila auraria. Από τη διαλογή αυτή προέκυψαν αρκετοί αλληλεπικαλυπτόμενοι κλώνοι, ο μεγαλύτερος των οποίων (CM-1) είχε μέγεθος 2.593 kb και περιελάμβανε ένα ανοιχτό αναγνωστικό πλαίσιο 715 αμινοξέων, από τη μετάφραση του οποίου προκύπτει ένα πολυπεπτίδιο προβλεπόμενου μοριακού βάρους 81,74 kDa. Η προβλεπόμενη αμινοξική αλληλουχία έδειξε πολύ μεγάλη ταυτότητα με όλα τα μέλη της οικογένειας HSP90 και ειδικότερα με τις HSP83 ομόλογες πρωτεΐνες της Drosophila. Επιπλέον, η προβλεπόμενη αμινοξική αλληλουχία περιείχε όλες τις συντηρημένες περιοχές των μελών της οικογένειας των HSP90 και στο καρβοξυτελικό της άκρο, έφερε το πενταπεπτίδιο MEEVD, το οποίο είναι χαρακτηριστικό όλων των κυτταροπλασματικών ισομορφών αυτής της οικογένειας. Με βάση αυτά τα αποτελέσματα ο κλώνος CM-1, ονομάστηκε Cchsp83. Ο κλώνος αυτός, εκτός από ολόκληρη την κωδική περιοχή του γονιδίου, περιείχε μέρος της 5’ μη μεταφραζόμενης περιοχής και ολόκληρη την 3’ μη μεταφραζόμενη περιοχή του hsp83 γονιδίου της μεσογειακής μύγας. Ανάλυση κατά Southern σε γονιδιωματικό DNA με διάφορα ένζυμα περιορισμού και κατάλληλους cDNA ανιχνευτές, έδειξε ότι το Cchsp83 γονίδιο υπάρχει σε ένα μόνο αντίγραφο στο γονιδίωμα της μεσογειακής μύγας. Ανάλυση “Northern” έδειξε την ύπαρξη ενός μεταγράφου με μέγεθος 2,7 kb περίπου. Το Cchsp83 γονίδιο, χαρτογραφήθηκε με in situ υβριδοποίηση σε μία θερμοεπαγόμενη χρωμοσωματική διόγκωση των πολυταινικών χρωμοσωμάτων των σιελογόνων αδένων του εντόμου (6R:94C). Η μελέτη του προτύπου έκφρασης του γονιδίου Cchsp83 έγινε σε επίπεδο RNA με RT-PCR σε ολικό RNA και σε επίπεδο πρωτεΐνης με ανάλυση Western σε ολικά πρωτεϊνικά εκχυλίσματα. Αντισώματα για την CcHSP83 αναπτύχθηκαν μετά από έκφραση ενός σημασμένου με 6 His τμήματος του Cchsp83 cDNA (490-690aa) σε βακτήρια E. coli. Η αντίδραση του Cchsp83 γονιδίου στην αύξηση της θερμοκρασίας είναι πολύ γρήγορη και ευαίσθητη. Μετάγραφα του γονιδίου ανιχνεύονται μετά από 5 λεπτά θερμικού στρες και σε θερμοκρασίες αρκετά χαμηλές (300C). Η επαγωγή του γονιδίου γίνεται μέγιστη μετά από 30-60 λεπτά θερμικού στρες στους 37-390C. Η επαναφορά της έκφρασης του γονιδίου στα φυσιολογικά επίπεδα μετά από θερμικό στρες είναι αργή, αφού για να γίνει αυτό απαιτούνται 4 ώρες ανάκαμψης στους 250C, μετά από μόλις 30 λεπτά θερμικού στρες στους 380C. Αναπτυξιακή μελέτη του προτύπου έκφρασης του Cchsp83 έδειξε ότι το γονίδιο εκφράζεται σε όλη τη διάρκεια της ανάπτυξης της μεσογειακής μύγας. Στους 250C τόσο τα επίπεδα του RNA, όσο και τα επίπεδα της πρωτεΐνης, είναι υψηλά στα εμβρυικά στάδια, χαμηλά στα προνυμφικά και μέτρια στα νυμφικά και ενήλικα στάδια. Μετά από θερμικό στρες στους 380C, τα επίπεδα των μεταγράφων αυξάνονται αρκετά, ιδιαίτερα στα στάδια που σε φυσιολογικές συνθήκες είναι χαμηλά. Όσον αφορά στα επίπεδα της πρωτεΐνης, τα αποτελέσματα ήταν παρόμοια μετά από θερμικό στρες στους 350C, αλλά όχι στους 37-390C, όπου δεν παρατηρήθηκε καθόλου επαγωγή. Το γεγονός αυτό υποδηλώνει ότι σε υψηλές θερμοκρασίες καταστέλλεται η ωρίμανση ή και η μετάφραση του Cchsp83 RNA. Με στόχο την απομόνωση των ρυθμιστικών περιοχών του γονιδίου Cchsp83 της μεσογειακής μύγας, πραγματοποιήθηκε διαλογή μιας χρωμοσωματικής λEMBL-4A βιβλιοθήκης, με ανιχνευτή το δεύτερο εξώνιο του hsp83 ομόλογου γονιδίου της Drosophila auraria. Από τη διαλογή αυτή απομονώθηκαν δύο κλώνοι, ένας από τους οποίους περιελάμβανε μέρος της κωδικής περιοχής, την 5’ μη μεταφραζόμενη περιοχή και 3,5 kb της 5’ ανοδικής περιοχής του γονιδίου Cchsp83. Σύγκριση της γονιδιωματικής αλληλουχίας με τη cDNA αλληλουχία, αποκάλυψε την ύπαρξη ενός μικρού εσωνίου 275 bp ανάμεσα στην 5’ μη μεταφραζόμενη περιοχή και στην αρχή της κωδικής περιοχής του γονιδίου. Βιοπληροφορική ανάλυση και πειράματα 5’ RACE, υποδηλώνουν ότι το σημείο έναρξης της μεταγραφής του γονιδίου βρίσκεται 144 bp ανοδικά του 5’ άκρου του εσωνίου και 23 bp καθοδικά ενός τυπικού στοιχείου TATA (ΤΑΤΑΑΑΤΑ). Δύο πιθανά στοιχεία απόκρισης στη θερμοκρασία (HSEs) εντοπίστηκαν στην εγγύς 5’ περιοχή του γονιδίου, 35 και 330 bp ανοδικά του στοιχείου TATA. Επιπλέον, βρέθηκαν 4 ακόμα πιο απομακρυσμένα HSEs, 1.595, 2.861, 2.880 και 2.890 bp, ανοδικά του σημείου έναρξης της μεταγραφής, καθώς και ένα HSE μέσα στο εσώνιο. Λειτουργική ανάλυση της εγγύς 5’ ανοδικής περιοχής του γονιδίου Cchsp83 πραγματοποιήθηκε με τη μέθοδο του γενετικού μετασχηματισμού. Τρία αλληλεπικαλυπτόμενα τμήματα του υποκινητή του γονιδίου Cchsp83, μήκους 519 bp (-380/+139, PL), 230 bp (-86/+144, PM) και 193 bp (-55/+139, PS) τοποθετήθηκαν μπροστά από το γονίδιο αναφοράς lacZ και εισήχθησαν στο γονιδίωμα της μεσογειακής μύγας με το σύστημα μετασχηματισμού Minos. Η έκφραση του γονιδίου αναφοράς, ελέγχθηκε σε όλες τις διαγονιδιακές σειρές που προέκυψαν με την ποσοτική ενζυματική μέθοδο της β-γαλακτοζιδάσης. Οι PM-lacZ και PS-lacZ σειρές δεν έδειξαν ανιχνεύσιμα επίπεδα έκφρασης του lacZ γονιδίου τόσο σε φυσιολογικές συνθήκες, όσο και σε συνθήκες θερμικού στρες. Αντιθέτως, οι περισσότερες από τις PL-lacZ σειρές έδειξαν σημαντικά επίπεδα συστατικής έκφρασης. Το αναπτυξιακό πρότυπο έκφρασης του γονιδίου αναφοράς μελετήθηκε σε μία PL-lacZ σειρά και βρέθηκε παρόμοιο με εκείνο του ενδογενούς γονιδίου, υποδηλώνοντας ότι η -380/+139 περιοχή του υποκινητή περιλαμβάνει όλα τα ρυθμιστικά στοιχεία που απαιτούνται για την ορθή χρονική έκφραση του Cchsp83 γονιδίου σε φυσιολογικές συνθήκες. Αν και η περιοχή αυτή του υποκινητή περιλαμβάνει δύο δυνητικά στοιχεία απόκρισης στη θερμοκρασία καθώς και την 5’ UTR, εντούτοις δεν είχε την ικανότητα να οδηγήσει σε θερμοεπαγόμενη έκφραση το γονίδιο αναφοράς. Τα αποτελέσματά μας υποδεικνύουν ότι η περιοχή -380/+139 του Cchsp83 γονιδίου μπορεί να χρησιμοποιηθεί για την ανάπτυξη διαγονιδιακών συστημάτων σήμανσης της μεσογειακής μύγας, τα οποία αναμένεται να συμβάλλουν στη διατήρηση και ανίχνευση κατάλληλων στελεχών που χρησιμοποιούνται σε προγράμματα βιολογικού ελέγχου του επιβλαβούς αυτού εντόμου. / By using the second exon of the D. auraria hsp83 gene as a probe, a number of overlapping cDNA clones were isolated from a Ceratitis capitata (medfly) cDNA library. The longest cDNA had a size of 2,593 bp and contained an open reading frame coding for a putative polypeptide of 715 amino acids with a predicted molecular weight of 81.74 kDa. In addition, it contained a part of the 5’-untranslated region and the complete 3’-untranslated region of a medfly hsp83 homolog, named Cchsp83. The predicted amino acid sequence showed a high degree of identity to all known sequences of the HSP90 family and was more closely related to the Drosophila HSP83 homologs. The putative medfly HSP83 contained all the conserved sequences of the members of the HSP90 family and was ended, at the C-terminal, with the pentapeptide MEEVD that characterizes all the cytosolic members of this family. Genomic Southern blot analysis, with several restriction enzymes and appropriate cDNA probes, indicated that the Cchsp83 gene exists as a single copy in the medfly genome. Northern blot hybridization revealed a single transcript of approximately 2.7 kb. In situ hybridization analysis showed that the Cchsp83 gene maps at the 94C region of the 6th chromosome (6R:94C), which corresponds to one of the major heat shock puffs of the medfly salivary gland polytene chromosomes. Evaluation of the Cchsp83 gene transcript and protein levels was performed by RT-PCR and western analysis, using total RNA and protein from synchronized animals. Anti-HSP83 antibodies were prepared against a histidine tag purified chimeric polypeptide from E. coli cells, transformed with a part of the coding region (aa 490-690) of the Cchsp83 cDNA. The response of the Cchsp83 gene to heat was fast and sensitive. Heat-induced transcript levels could be detected within 5 min at temperatures as low as 300C. Maximum transcript levels were obtained after 30-90 min treatments at 35-390C. Following recovery at 250C, after a 30 min heat shock, the accumulated transcripts remained at high levels for approximately 3h and declined to the non-induced levels 1h later. Developmental studies showed that the Cchsp83 gene is expressed constitutively throughout medfly development. At 250C, both transcript and protein levels were high in embryonic stages, low in larval stages and moderate in pupal and adult stages. Following heat shock at 380C, the transcript levels increased approximately 3- to 5-fold, depended on the developmental stage. Similar results were obtained for the protein levels after a heat shock at 350C, but not at 380C, suggesting that the Cchsp83 mRNAs are not translated efficiently at high temperatures. Screening of a genomic λEMBL-4A library from 24-h-old medfly embryos with the second exon of the D. auraria hsp83 gene, resulted in the isolation of a genomic clone containing part of the coding region, the untranslated leader region (5’ UTR) and 3.5 kb from the 5’ flanking region of the gene. Comparison of the genomic and cDNA nucleotide sequences revealed the presence of a small intron of 275 bp between the 5’ untranslated and coding regions of the gene. Thus the Cchsp83 gene is organized into two exons separated by a small intron. Computational analysis and 5’ RACE experiments, suggested that the putative transcription initiation site of the gene is located 144 bp upstream of the 5’ splicing site of the intron and 23 bp downstream of a typical TATA box (TATAΑΑΤΑ). Two putative heat shock elements (HSEs) were identified in the proximal 5’ flanking region of the gene, 35 and 330 bp, upstream of the TATA box. In addition to them, four distal HSEs, 1,595, 2,861, 2,880 and 2,890 bp, upstream of the putative transcription initiation site and one HSE inside the intron were identified. Functional analysis of the proximal 5’ flanking region of the Cchsp83 gene was performed by germline transformation. Three overlapping promoter fragments PL (-380/+139), PM (-86/+144) and PS (-55/+139), were fused to the lacZ reporter gene and the resulting constructs were introduced into the medfly genome via Minos-element mediated germline transformation. The expression of the reporter gene, in at least 8 homozygous transformed lines for each construct, was evaluated by quantitative β-galactosidase assays. The PM-lacZ and PS-lacZ lines did not show detectable levels of lacZ expression at neither normal or heat shock conditions. On the other hand, most of the PL-lacZ lines showed significant levels of constitutive lacZ expression. Developmental expression studies in one of these lines showed that the reporter gene exhibited similar developmental expression pattern to the endogenous one, suggesting that the PL promoter region includes all the necessary regulatory elements for driving correct temporal expression of the Cchsp83 at normal conditions. Although this promoter region contained the two proximal HSEs and the 5’ UTR, it was unable to drive heat-induced expression of the reporter gene suggesting that additional upstream and/or downstream sequences are necessary for the heat-induced expression of the Cchsp83 gene. Our data indicate that the PL promoter region of the Cchsp83 gene can be used as a driver for the development of robust transgenic marker systems in medfly. Such systems are important for detecting, maintaining and recognizing medfly strains that are used today in population control programs of this agricultural pest.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2017-02-15T10:50:25Z No. of bitstreams: 1 texto completo.pdf: 1856190 bytes, checksum: 405040c338d9d0453f53e6b127a24ccd (MD5) / Made available in DSpace on 2017-02-15T10:50:25Z (GMT). No. of bitstreams: 1 texto completo.pdf: 1856190 bytes, checksum: 405040c338d9d0453f53e6b127a24ccd (MD5) Previous issue date: 2016-03-29 / O Brasil está entre os maiores produtores de frutas do mundo, ocupando a terceira posição no ranking, com produção anual de 40 milhões de toneladas. O Vale de São Francisco manteve em 2014 o patamar de 85% das exportações brasileiras de manga. Entre as frutas, a manga apresenta produção nacional ascendente, com quase 1,2 milhão de toneladas em 2014. Entre os obstáculos, a maior participação do setor no mercado internacional está nos aspectos fitossanitários, principalmente as moscas- das-frutas (Diptera: Tephritidae). O objetivo deste trabalho foi analisar a flutuação populacional de moscas-das-frutas em pomares comerciais de mangueira, bem como estudar a influência dos fatores abióticos na flutuação populacional. A partir dos resultados, discute-se a viabilidade de ampliação do número de propriedades aptas a exportar mangas in natura para países que exigem que os frutos sejam oriundos de Áreas de Baixa Prevalência de Pragas (ABPP). O estudo foi desenvolvido a partir de dados coletados de 2009 a 2014 em 44 pomares comerciais de manga localizados nos Projetos de Irrigação Senador Nilo Coelho, Maria Tereza e Bebedouro, em Petrolina, PE. A espécie exótica Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) foi detectada em todos os pomares estudados, e os dados sugerem que ela tenha deslocado as espécies nativas do gênero Anastrepha (Diptera: Tephritidae). A população apresentou níveis elevados em todas as áreas monitoradas, principalmente de outubro a janeiro, em todos os períodos estudados. A temperatura e a umidade relativa influenciaram fortemente a captura de insetos adultos, em que a temperatura apresentou correlação positiva e a umidade, correlação negativa. A população de C. capitata esteve em níveis acima do admitido para implantação de ABPP, de forma que o aumento de exportações de manga para mercados mais exigentes dependerá grandemente da execução de um programa para supressão da população. / Brazil is a major fruit grower worldwide, occupying the third position and harvesting 40 x 106 ton. In 2014, Sao Francisco Valley responded for 85% of mango exports of the country. Mango production is going through a period of increase in production and approximately 1.2 million tons were harvested in 2014. Phytosanitary measures are major constraints to export, mainly those established to avoid the entry of fruit flies (Diptera: Tephritidae). This work aimed to analyse the population fluctuation of fruit flies in commercial mango orchards, as well as study the influence of abiotic factors in population density. The feasibility of increasing the number of properties certified according to international standards of areas of low pest prevalence (ALPP) is discussed, as this is a measure established by several international markets. Data gathered from 2009 to 2014 for 44 commercial orchards in the districts Senador Nilo Coelho, Maria Tereza, and Bebedouro, in Petrolina, PE, were used. The exotic species Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) was detected in all orchards and data suggest that it may have displaced the native species of the genus Anastrepha (Diptera: Tephritidae). Population density was high in all considered areas, mainly from October to January. Temperature and relative humidity had significant impact on population density, the former showing a positive correlation and the later, a negative one. C. capitata population was higher than the threshold implementation in standards for LPPA so that the increase of mango exports for more restrictive countries will largely depend on the conduction of a suppression program. / Não foi localizado o currículo lattes do autor.