Les femelles moustiques peuvent être vectrices de nombreux agents infectieux (virus, protozoaires, helminthes) pour l'Homme, qui peuvent être la cause de maladies graves comme le paludisme et la dengue. Ces maladies menacent respectivement 50 et 40% de la population mondiale ; le paludisme étant responsable de près d'un million de décès par an. Les méthodes de lutte anti-vectorielle destinées à limiter les populations vectrices et stopper la transmission de maladies, se heurtent au développement incessant de résistances de la part des moustiques et des agents infectieux vis-à-vis des traitements employés. Bien que certaines régions du monde aient réussi à stopper efficacement la transmission de certaines de ces maladies, une grande partie des régions tropicales reste menacée. De plus l'expansion rapide de certaines espèces vectrices, telles qu'Aedes albopictus, accroît les risques sanitaires dans de nouvelles régions du globe. La technique de l'insecte stérile (TIS), qui a permis l'éradication ou la suppression des populations de nombreux insectes nuisibles aux cultures et à l'Homme, représente un moyen de lutte prometteur contre les moustiques. Cette technique s'appuie sur le lâcher en masse de mâles stérilisés par rayonnements ionisants qui, en transférant un sperme stérile aux femelles sauvages, vont permettre une diminution progressive de la population cible. Suite à l'épidémie de chikungunya à l'île de la Réunion en 2005 et face aux menaces permanentes de recrudescence de la dengue et du paludisme, les services de lutte anti-vectorielle réunionnais mettent en place d'importants moyens de lutte contre les populations de moustiques concernées. Toutefois, ces mesures ne permettant pas une diminution durable des densités de vecteurs, une étude de faisabilité est en cours quant à l'utilisation de la TIS pour diminuer et contrôler les populations d’Aedes albopictus, vecteur de la dengue et du chikungunya, et d’Anopheles arabiensis, vecteur du paludisme.Ce travail de thèse s'inscrit dans le cadre du projet TIS Réunion, dans le but d'étudier la biologie et le comportement des souches destinées aux lâchers de mâles stériles. Dans un premier temps, cette étude s'intéresse à la comparaison entre les souches d'élevage d’Anopheles arabiensis et les souches sauvages, ainsi qu'aux modalités de stérilisation des mâles de la souche à sexage génétique. Une seconde partie est consacrée à l'étude de l'effet de l'irradiation sur les mâles d’Aedes albopictus, en étudiant plus particulièrement leur stratégie de reproduction, leur capacité d'insémination en laboratoire, ainsi que leur compétitivité sexuelle et longévité face aux mâles sauvages en conditions semi-contrôlées. / Mosquito females are potential vectors of numerous pathogens (viruses, protozoa, helminths), which can cause serious diseases such as malaria and dengue in humans. These two infectious diseases are threatening 50 and 40% of the world population respectively. Malaria is responsible for nearly one million deaths per year, and is considered by many experts as the most important insect-transmitted disease. Antivectorial control methods, intended to limit the vector populations and to stop the disease transmission have to face many challenges such as the development of mosquitoes' and pathogens' resistance to the treatments employed to control them. Although various regions of the world have succeeded in efficiently stopping the transmission of some diseases, most of the tropical regions remain under threat. In addition, the rapid expansion of some vector species, such as Aedes albopictus, increases the risks in previously safe areas of the world. The sterile insect technique (SIT) has allowed the eradication or suppression of various insect pest populations threatening crops, animal, and human health, and could offer a promising control tool against mosquitoes. The classical SIT relies on the mass releases of males sterilized by ionizing radiation;they transfer sterile sperm to wild females, which results in a progressive reduction of the target population.Following the chikungunya outbreak in Reunion Island in 2005 and considering the constant threat of arecrudescence of dengue and malaria, the anti-vectorial services in Reunion Island are deploying important means to control the relevant mosquito populations. However, these measures do not confer a permanent, or long-lasting reduction of vector densities. A feasibility study is ongoing, evaluating the use of the SIT to diminish and control the populations of Ae. albopictus, a vector of dengue and chikungunya, and Anopheles arabiensis, a vector of malaria. This PhD work was developed in the context of the SIT Reunion project, with the aim of studying the biology and the behaviour of some strains intended for the sterile male releases. Firstly, this study endeavours to compare colonized and wild strains of An. arabiensis, and to determine the sterilisation procedures of the genetic sexing strain males. The second part of this work studies the effect of irradiation on male Ae. albopictus, and most notably their reproductive strategy, the insemination capacity in laboratory, and finally their sexual competitiveness and longevity against wild males under semi-field conditions.

As moscas-das-frutas se constituem no maior obstáculo para a produção, processamento e comércio de frutas frescas em todo o mundo. O Brasil não é exceção e, embora as condições brasileiras sejam bastante adequadas à produção de frutas tropicais e temperadas, a fruticultura enfrenta, continuamente, o problema das moscas-das-frutas. Dentre as espécies de maior importância econômica e quarentenária, destaca-se a mosca-do-mediterrâneo Ceratitis capitata (Wied., 1824) que tem, como centro de origem, a África Equatorial, e é uma das pragas mais destrutivas, causando elevados prejuízos à fruticultura mundial. No Brasil, onde sua presença foi registrada no início do século passado, esta praga se encontra amplamente distribuída em todo o território nacional. Os processos integrados de controle de pragas e doenças, com emprego de controle biológico, monitoramento populacional da praga, sistema de informação geográfica e aplicação de técnicas de controle das populações têm se desenvolvido significativamente nos últimos anos, permitindo a obtenção de frutas de alta qualidade. Com o sucesso da aplicação da Técnica do Inseto Estéril (TIE) na erradicação da Cochliomyia hominivorax (Coquerel, 1858) da Ilha de Curaçao e da região Sudeste dos Estados Unidos, houve um estímulo para o uso de programa contra várias espécies de moscas-das-frutas. A necessidade de se buscar respostas para o uso de fontes alternativas na esterilização de insetos, notadamente o uso de raios X, em escala industrial e a busca de novos modelos que possam inovar desde o ponto de vista tecnológico a TIE, com métodos que dispensem, por completo, o uso de radiação ionizante é o desafio do presente estudo, que teve como objetivo avaliar, através de testes em laboratório e em gaiola de campo, a linhagem transgênica de Ceratitis capitata (Wied.), OX3864A, comparada à linhagem tsl Vienna 8 da mesma espécie, esterilizada por raios X, visando a sua possível aplicação em programas de controle de moscas-dasfrutas, com foco na TIE. Os resultados obtidos mostram que a criação massal da linhagem transgênica esta dominada e que a dieta adotada pela BIOFÁBRICA MOSCAMED BRASIL (BMB), para a linhagem tsl Vienna 8, pode ser usada para a criação da linhagem transgênica OX3864A sem afetar o rendimento e os padrões de qualidade requeridos para o uso da linhagem na TIE. No que diz respeito aos bioensaios em gaiola de campo, os machos de ambas linhagens apresentaram uma boa performance sexual sendo capazes de competir com machos coespécificos selvagens pela cópula com fêmeas selvagens virgens. O desempenho dos machos transgênicos OX3864A não deferiu do desempenho dos machos irradiados de tsl Vienna 8. A fim de aprofundar os estudos, necessário se faz a realização de novas pesquisas para avaliar o desempenho de linhagens transgênicas em campo aberto / Fruit flies are within the main barriers to fresh fruit production, processing and trade throughout the world. In that sense, Brazil is not an exception, and although Brazilian conditions are favorable to the production of tropical and temperate fruits, this industry continually faces problems related to the fruit fly. From an economical and quarantine point of view, Ceratitis capitata (Wied., 1824) can be considered the most important fruit fly specie, which has the Equatorial Africa as center of origin, is also one of the most destructive pests, causing high losses to the world fruit industry. In Brazil, where its presence has been first noticed in the beginning of the past century, nowadays this pest is widespread throughout the whole national territory. With the latest developments on biological control, pest monitoring, geographical information systems and application of population control techniques it is seen that the integrated pest management systems are allowing the production of high quality fruits. Since the successful application of the sterile insect technique on the eradication of Cochliomyia hominivorax (Coquerel, 1858) in Curaçao and in the US Southeast region, there has been an incentive to the use of this technique in programs against various fruit fly species. On the search for alternative sources for insect sterilization especially the use of X-ray in high scale and the search for new models able to innovate the sterile insect technique from a technological point of view with methods that exempt the use of ionizing irradiation is the challenge of the present study, whose aim was to evaluate through lab and field cage tests the transgenic OX3864A RIDL of Ceratitis capitata (Wied.) strain, compared to X-ray sterilized tsl Vienna 8 strain, looking for a possible application on fruit fly control programs focused on the Sterile Insect Technique (SIT). Results obtained show that the mass rearing of the transgenic strain is dominated and the diet adopted by MOSCAMED BRAZIL rearing facility, for tsl Vienna 8 can also be used for the OX3864A transgenic strain without affecting its yield and quality standards required for its use in SIT. Regarding the field cage bioassays, males from both strains presented a good sexual performance, being able to compete with wild co-specific to mate with virgin wild females. Transgenic males did not differ from irradiated tsl Vienna 8 males. Further studies on sexual performance evaluation on open field of transgenic strain is recommended in order to provide more knowledge

As moscas-das-frutas se constituem no maior obstáculo para a produção, processamento e comércio de frutas frescas em todo o mundo. O Brasil não é exceção e, embora as condições brasileiras sejam bastante adequadas à produção de frutas tropicais e temperadas, a fruticultura enfrenta, continuamente, o problema das moscas-das-frutas. Dentre as espécies de maior importância econômica e quarentenária, destaca-se a mosca-do-mediterrâneo Ceratitis capitata (Wied., 1824) que tem, como centro de origem, a África Equatorial, e é uma das pragas mais destrutivas, causando elevados prejuízos à fruticultura mundial. No Brasil, onde sua presença foi registrada no início do século passado, esta praga se encontra amplamente distribuída em todo o território nacional. Os processos integrados de controle de pragas e doenças, com emprego de controle biológico, monitoramento populacional da praga, sistema de informação geográfica e aplicação de técnicas de controle das populações têm se desenvolvido significativamente nos últimos anos, permitindo a obtenção de frutas de alta qualidade. Com o sucesso da aplicação da Técnica do Inseto Estéril (TIE) na erradicação da Cochliomyia hominivorax (Coquerel, 1858) da Ilha de Curaçao e da região Sudeste dos Estados Unidos, houve um estímulo para o uso de programa contra várias espécies de moscas-das-frutas. A necessidade de se buscar respostas para o uso de fontes alternativas na esterilização de insetos, notadamente o uso de raios X, em escala industrial e a busca de novos modelos que possam inovar desde o ponto de vista tecnológico a TIE, com métodos que dispensem, por completo, o uso de radiação ionizante é o desafio do presente estudo, que teve como objetivo avaliar, através de testes em laboratório e em gaiola de campo, a linhagem transgênica de Ceratitis capitata (Wied.), OX3864A, comparada à linhagem tsl Vienna 8 da mesma espécie, esterilizada por raios X, visando a sua possível aplicação em programas de controle de moscas-dasfrutas, com foco na TIE. Os resultados obtidos mostram que a criação massal da linhagem transgênica esta dominada e que a dieta adotada pela BIOFÁBRICA MOSCAMED BRASIL (BMB), para a linhagem tsl Vienna 8, pode ser usada para a criação da linhagem transgênica OX3864A sem afetar o rendimento e os padrões de qualidade requeridos para o uso da linhagem na TIE. No que diz respeito aos bioensaios em gaiola de campo, os machos de ambas linhagens apresentaram uma boa performance sexual sendo capazes de competir com machos coespécificos selvagens pela cópula com fêmeas selvagens virgens. O desempenho dos machos transgênicos OX3864A não deferiu do desempenho dos machos irradiados de tsl Vienna 8. A fim de aprofundar os estudos, necessário se faz a realização de novas pesquisas para avaliar o desempenho de linhagens transgênicas em campo aberto / Fruit flies are within the main barriers to fresh fruit production, processing and trade throughout the world. In that sense, Brazil is not an exception, and although Brazilian conditions are favorable to the production of tropical and temperate fruits, this industry continually faces problems related to the fruit fly. From an economical and quarantine point of view, Ceratitis capitata (Wied., 1824) can be considered the most important fruit fly specie, which has the Equatorial Africa as center of origin, is also one of the most destructive pests, causing high losses to the world fruit industry. In Brazil, where its presence has been first noticed in the beginning of the past century, nowadays this pest is widespread throughout the whole national territory. With the latest developments on biological control, pest monitoring, geographical information systems and application of population control techniques it is seen that the integrated pest management systems are allowing the production of high quality fruits. Since the successful application of the sterile insect technique on the eradication of Cochliomyia hominivorax (Coquerel, 1858) in Curaçao and in the US Southeast region, there has been an incentive to the use of this technique in programs against various fruit fly species. On the search for alternative sources for insect sterilization especially the use of X-ray in high scale and the search for new models able to innovate the sterile insect technique from a technological point of view with methods that exempt the use of ionizing irradiation is the challenge of the present study, whose aim was to evaluate through lab and field cage tests the transgenic OX3864A RIDL of Ceratitis capitata (Wied.) strain, compared to X-ray sterilized tsl Vienna 8 strain, looking for a possible application on fruit fly control programs focused on the Sterile Insect Technique (SIT). Results obtained show that the mass rearing of the transgenic strain is dominated and the diet adopted by MOSCAMED BRAZIL rearing facility, for tsl Vienna 8 can also be used for the OX3864A transgenic strain without affecting its yield and quality standards required for its use in SIT. Regarding the field cage bioassays, males from both strains presented a good sexual performance, being able to compete with wild co-specific to mate with virgin wild females. Transgenic males did not differ from irradiated tsl Vienna 8 males. Further studies on sexual performance evaluation on open field of transgenic strain is recommended in order to provide more knowledge

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.

Tanto as técnicas de controle biológico quanto a Técnica do inseto estéril (TIE), são utilizadas em vários países para o controle, supressão e até mesmo erradicação de moscas-das-frutas e outras pragas da agricultura, pecuária e saúde publica. O uso de tais técnicas minimiza o emprego contínuo de inseticidas, protege o ambiente e se adequa aos padrões de segurança alimentar. No entanto, é necessário para a implementação de tais programas, tecnologia para produzir milhões de parasitóides e a própria praga em laboratório com qualidade biológica similar aos insetos encontrados na natureza e com custo competitivo com o controle químico. Os objetivos deste trabalho foi estabelecer protocolos para criação artificial de A. sp. 1 aff. fraterculus em dieta larval líquida que permita atingir níveis de criação massal, para uma possível diminuição no custo da criação e determinar a dose de radiação esterilizante para adultos de A. sp. 1 aff. fraterculus atendendo os parâmetros de qualidade exigidos pela Técnica do Inseto Estéril com insetos provenientes da criação do Laboratório de Radioentomologia do CENA/USP. Foram testadas sete dietas em relação à dieta convencional utilizada no laboratório de Radioentomologia do CENA/USP, que foi usada como controle. Todas as sete dietas tiveram em comum a exclusão do ágar na sua formulação. Somente duas das dietas testadas foram adequadas para o desenvolvimento larval da mosca, estas quando comparadas com a dieta padrão, apresentaram resultados inferiores com relação ao volume de larvas recuperadas, emergência e o peso das pupas, porém, não apresentaram diferenças significativas quanto aos períodos de desenvolvimento, recuperação pupal, razão sexual e longevidade sob estresse. Concluí-se que é possível a substituição da dieta com ágar por dietas líquidas para criação artificial de A. sp.1 aff. fraterculus, de custo reduzido e com maior praticidade de manejo,porém devidos aos seus parâmetros de qualidade inferiores aos da dieta padrão, são necessários maiores testes principalmente quanto à adaptabilidade do inseto ao novo meio. Para determinar a dose esterilizante o presente trabalho analisou a fertilidade, fecundidade, emergência, habilidade de vôo e longevidade sob estresse de A. fraterculus irradiadas com doses de 40, 50, 60, 70 e 80 Gy. A fonte de radiação gama foi um irradiador de Co-60 (Gammabeam-650) localizado no mesmo laboratório. Os testes foram realizados sob as seguintes condições ambientais: 26 ± 1 ° C, 70 ± 5% RH, e fotofase de 12:12. A dose de r adiação gama esterilizante para machos de A. sp. 1 aff. fraterculus foi determinada sendo 70 Gy. Fêmeas irradiadas, mesmo com a dose mais baixa de 40 Gy, não ovipositaram, possivelmente pela atrofia dos ovários causada pela radiação. A radiação não afetou os demais parâmetros de qualidade do inseto como emergência, longevidade e habilidade de vôo / Both the biological control techniques as the Sterile Insect Technique (SIT), are used in many countries to control, suppress and even eradicate fruit flies and other pests in agriculture and public health. The use of such techniques minimizes the continuous employment of insecticides, protects the environment and conforms to standards for food safety. However, it is necessary to implement such programs, technology to produce millions of parasitoids and the pest in its own laboratory with biological quality similar to the insects found in nature and cost competitive with chemical control. The objectives of this study was to establish protocols for artificial rearing of A. sp. 1 aff. fraterculus in liquid larval diet that will achieve levels of mass production for a possible reduction in the cost of establishing and determining the dose of radiation sterilization of adult A. sp. 1 aff. fraterculus meeting the quality parameters required by the Sterile Insect Technique with insects from the creation of Radioentomologia Laboratory of CENA / USP. Seven experimental diets compared to the conventional diet used in laboratory Radioentomologia of CENA / USP, which was used as control. All seven diets have in common the exclusion of agar in its formulation. Only two of the diets tested were suitable for larval development of the fly, they compared with the standard diet, showed inferior results with respect to the volume of recovered larvae, pupae and weight of emergency, however, no significant differences regarding the periods of development , pupal recovery, sex ratio and longevity under stress. It is possible to replace the diet with agar for liquid diets for artificial creation of A. sp.1 aff. fraterculus, reduced cost and greater convenience of handling, but due to their quality standards lower than the standard diet, more tests are needed especially regarding the adaptability of the insect to the new environment. To determine the sterilizing dose this study examined the fertility, fecundity, emergence, flight ability and longevity under stress in A. fraterculus irradiated with 40, 50, 60, 70 and 80 Gy. The radiation source was a gamma irradiator with a Co-60 (Gammabeam-650) located in the same laboratory. The tests were performed under the following environmental conditions: 26 ± 1 ° C, 70 ± 5% RH and photoperiod of 12:12. The dose of gamma radiation sterilization for male A. sp. 1 aff. fraterculus was determined with 70 Gy. Irradiates females, even with the lower dose of 40 Gy, layed no eggs, possibly due to atrophy of the ovaries caused by radiation. The radiation did not affect the other quality parameters such as insect emergence, longevity and ability to fly

A dengue é atualmente uma das doenças tropicais com maior rapidez de propagação e de grande alcance mundial (OMS, 2013). No Brasil, o governo federal, bem como os estados e os municípios gastam montantes significativos em sua prevenção e controle. O presente trabalho analisa aspectos econômicos e sociais da implementação de uma nova técnica de combate ao mosquito Aedes aegypti, a TIE, vis-à-vis os métodos convencionais. Essa técnica é avaliada com base no estudo de caso da Biofábrica Moscamed, responsável pelo desenvolvimento da mesma no Brasil. A TIE consiste na esterilização dos insetos e posterior soltura no ambiente, com a finalidade de diminuir a população de insetos selvagens, estes vetores do vírus da dengue. Dois cenários são analisados: cenário atual de medidas de prevenção e controle adotadas pelas prefeituras e o cenário de adoção da TIE, em substituição aos métodos atuais. São tomados como estudos de caso a aplicação da TIE em Jacobina e Juazeiro, na Bahia, cujos dados foram extrapolados para uma simulação também para o município de Piracicaba, em São Paulo. Calcula-se o VPL para o cenário da TIE, considerando um horizonte temporal de quatro anos e a discussão é conduzida no escopo simplificado de uma Análise de Impacto Regulatório (AIR). Foram analisados ainda os gastos das prefeituras para o controle da dengue e de seu vetor, com base no ano de 2013. Realizou-se uma análise dos aspectos sociais e econômicos concernentes à dengue, que apontou como impactos relevantes as mortes, o afastamento dos doentes de suas atividades e o alto número de notificações mesmo após aplicação dos métodos de controle vigentes. Por se tratar de uma questão de saúde pública a análise tem foco majoritariamente social, uma vez que para se implementar projetos com benefícios sociais a intervenção e os investimentos públicos são de fundamental importância, suplantando a visão econômica de viabilidade do projeto. A mensuração dos benefícios da TIE foi limitada pelo curto período em que os experimentos de campo estão sendo aplicados, não havendo ainda resultados publicados sobre sua eficácia. Do mesmo modo, não se tem dados que permitam calcular os benefícios dos atuais métodos de controle do vetor e da doença. Assim, não foi possível conduzir uma comparação de eficácia e uma estimativa da possibilidade de associação entre os métodos tradicionais e a TIE. Em suma, mediante análise fundamentada em um roteiro de referência proposto para análise de impacto regulatório conclui-se que projetos com avaliação positiva do ponto de vista social, mesmo com indicadores econômicos negativos, são geralmente financiados pela esfera pública e, no caso aqui analisado da dengue, devem buscar melhorar os resultados dos atuais programas que não têm sido capazes de evitar os prejuízos sociais e econômicos dessa doença no Brasil. / Dengue has been one of the most important tropical diseases that has showed a fast and worldwide propagation according to the WHO (2013). In Brazil, the federal government as well as states and municipalities are spending significant amounts of resources of all kinds on its prevention and control. Yet it seems worthless. This paper examines the economic feasibility of implementing a new technique to fight the mosquito Aedes aegypti, the TIE (Técnica do Inseto Estéril-Sterile Insect Technique), vis-à-vis the conventional methods employed. The TIE is evaluated through the case study of the Moscamed, a biofirm which is responsible for its development and propagation in Brazil. The TIE consists on the sterilization of insects and their release into the environment, aiming to reducing the population of susceptible wild insects, which can transmit the virus of dengue among population. Two scenarios are analyzed: 1) the current scenario of prevention and control adopted by municipalities, and 2) the scenario that describes the adoption of the TIE, replacing the current methods. The cities of Jacobina and Juazeiro in Bahia are used as case\'s study, and whose data were extrapolated for a simulation also for the city of Piracicaba, located in the state of São Paulo. The Net Present Value (NPV) is calculated for the scenario of TIE, considering a four years\' timeline, and additionally a discussion is conducted in the scope of a simplified Regulatory Impact Analysis (RIA). It was also analyzed the spending of municipalities for the control of dengue and its vector, based on the year of 2013. It was conducted an analysis of the social and economic aspects related to dengue, which identified as relevant impacts: deaths, absence of patients of their labor activities and the high number of notifications even after implementation of the existing control methods. Because it is a public health problem, this analysis had mostly a social focus. It is well known that to implement projects with social benefits, the government intervention and public investment are of fundamental importance, despite the economic view of the project feasibility. The measurement of the benefits from the TIE was limited by the short period in where the field experiments were taken place. In fact, there are no enough published results about its effectiveness. Similarly, there is no data allowing calculation of the benefits of the current vector control and disease. Thus, it was not possible to conduct a comparison of efficiency and an estimation of a possible association between traditional methods and TIE. In summary, this analysis, based on a proposed reference roadmap for Regulatory Impact Analysis, concludes that projects with positive assessment from a social point of view, even if they have negative economic indicators, are usually funded by the public sphere. In the specific dengue case, the government should seek to improve the results of current programs that have not been able to avoid the social and economic losses of this disease in Brazil.

Genetic control strategies are a novel method for reducing populations of pest insects such as the yellow fever mosquito Aedes aegypti, a major vector of several important arboviral diseases. This thesis describes efforts to develop new tools to engineer the Ae. aegypti genome and to better understand existing tools, and furthermore to use these to engineer a gene drive system in Ae. aegypti. The piggyBac transposon was found to be extremely stable in the germline of Ae. aegypti, and transposons engineered into the germline could not be remobilized with either an endogenous or exogenous source of piggyBac transposase. Conversely, somatic remobilization of piggyBac transposons was found to be readily detectable in the presence of a source of active transposase, the first report of such remobilization in Ae. aegypti. Toward new tools for genome engineering, the site-specific integrase from the phage φC31 was successfully used to promote exchange between a transgene cassette inserted into the genome of Ae. aegypti and a cassette in a plasmid vector, in the first demonstration of recombinase mediated cassette exchange technology in a pest insect species. The integrases from phages φRV1 and Bxb1 were not found to be active in the germline of the mosquito. Finally, development of a gene drive system in Ae. aegypti using an RNAi-mediated killer-rescue mechanism was attempted. Tissue-specific expression of tTAV-regulated-toxic effectors genes, using the promoter regions of the blood meal induced genes Carboxypeptidase A-1, 30Kb and Vitellogenin A, was possible, but sex-specificity was not achieved. A blood meal inducible lethal phenotype was not possible using the chosen promoters, with expression of the effectors either leading to death in early development or to a sublethal phenotype. RNAi against tTAV fused to the Mnp fragment of the dengue virus’ genome was tissue specific, but was found to be highly effective in the fat body suggesting that the Vitellogenin A was the best candidate for the engineering of killer-rescue systems in the mosquito.

En Afrique sub-saharienne, près de 10 millions de km2 de terres, les plus fertiles en productions fourragères et agricoles, sont infestées de mouches tsé-tsé limitant ainsi les initiatives de développement d’une agriculture durable. Les tsé-tsé transmettent des trypanosomes qui sont responsables des trypanosomoses animales et humaines africaines. En 2000, les Chefs d’Etats et de Gouvernements africains ont décidé de redoubler d’efforts pour lutter contre les mouches tsé-tsé et les trypanosomoses en créant la Pan African Tsetse and Trypanosomosis Eradication Campaign (PATTEC). Dans ce contexte, le gouvernement sénégalais a initié un programme d’éradication des glossines dans la zone des Niayes en utilisant une souche de Glossina palpalis gambiensis originaire du Burkina Faso. La présente thèse visait à optimiser l’utilisation de la technique de l’insecte stérile (TIS) en Afrique de l’Ouest pour lutter contre les glossines. Un dispositif de transport sur de longues distances de pupes matures a été développé et validé à partir de pupes mâles de G. p. gambiensis produites et irradiées à Bobo-Dioulasso, Burkina Faso et à Bratislava, Slovaquie (irradiation faite à Seibersdorf, Autriche) et transportées par voie aérienne jusqu’à Dakar, Sénégal. Le dispositif constitué d’une boîte isotherme et des packs S8 a permis de maintenir les pupes à une température de 10 ± 3°C et de les transporter pendant 2-3 jours jusqu’au centre d’émergence de l’ISRA, pour produire des mâles stériles utilisables pour la technique de l’insecte stérile. Un contrôle qualité a été réalisé sur un échantillon de 50 pupes prélevé dans chaque lot de pupes (minimum 2 lots par envoi) pour déterminer l’aptitude d’envol des mâles stériles et leur survie sous stress (à jeun). Le reste des pupes utilisé pour les lâchers sur le terrain a été considéré comme témoin. Le protocole qualité décrit permettra un suivi précis de la qualité des mâles stériles utilisés dans les programmes opérationnels d’éradication dans le cadre de la PATTEC. Un outil moléculaire de discrimination de mâles stériles lâchés et sauvages a également été développé à partir du gène mitochondrial COI (cytochrome oxydase) et a montré que les séquences COI des mouches lâchées (produites en insectarium) sont 100% identiques entre elles et différentes de celles des mouches sauvages. Par ailleurs, afin de déterminer les conditions optimales d’élevage de souches de G. p. gambiensis et de déterminer la souche qui sera la plus adaptée à tel ou tel environnement ou pays dans le cadre d’une lutte avec une composante lâcher de mâles stériles, les traits de vie (survie et fécondité) de trois souches de G. p. gambiensis (souches originaires du Burkina Faso (BKF), Sénégal (SEN) et souche introgressée (SENbkf) ont été évalués dans différentes conditions de températures et d’humidités relatives. La température optimale d’élevage en masse a été de 25 ± 1°C, 24,6 ± 1°C et 23,9 ± 1°C pour BKF, SENbkf et SEN respectivement. La variation de l’humidité relative (entre 40 et 75%) a eu très peu d’influence sur la survie et la fécondité. La souche BKF a mieux résisté à de fortes températures que les souches SEN et SENbkf, mais la température limite de survie a été de 32°C pour les trois souches. / In sub-Saharan Africa, nearly 10 million km² of land, the most fertile for animal and agricultural productions, are infested with tsetse flies limiting all development initiatives for sustainable agriculture. Tsetse flies transmit trypanosomes that cause human and animal african trypanosomosis, a debilitating disease of humans (sleeping sickness) and livestock (nagana). In 2000, the African Heads of State and Government decided to increase efforts to address the tsetse and trypanosomosis problem on the African continent and created the Pan-African Tsetse and Trypanosomosis Eradication Campaign (PATTEC). In this context, the Government of Senegal initiated a tsetse eradication program in the Niayes area using a Glossina palpalis gambiensis strain originating from Burkina Faso. The objective of this thesis was to optimize the use of the sterile insect technique (SIT) in West Africa in order to control the tsetse flies. A system to transport mature pupae over long distances has been developed and validated for male G. p. gambiensis pupae produced and irradiated either in Bobo-Dioulasso, Burkina Faso or in Bratislava, Slovakia (irradiation done in Seibersdorf, Austria) and then transported by air to Dakar, Senegal. The system, constituted of an insulated box and S8 packs, allowed the maintenance of pupae at a temperature of 10 ± 3°C and their transport during 2-3 days to the emergence center of ISRA, where they molted into sterile males which were used for the sterile insect technique.A quality control was carried out on a sample of 50 pupae from each batch (at least 2 batches per shipment) to determine the flight ability of sterile males and their survival under stress conditions (without feeding). The remaining emerging pupae were released in the target area of the eradication programme and were considered as control group. The described protocol for quality control will allow accurate monitoring of the quality of sterile males used in operational eradication programs organized in the context of PATTEC.A molecular tool to distinguish between sterile and wild males was also developed using the mitochondrial gene COI (cytochrome oxidase). We showed that COI sequences of released flies (reared in insectary) are 100% identical and different from those of wild flies.Furthermore, in order to determine the optimal rearing conditions for G. p. gambiensis strains and to identify the strain that would be the best adapted to a particular environment or country in the context of a control with an SIT component, life history (survival and fecundity) of three G. p. gambiensis strains (strains originating from Burkina Faso (BKF), Senegal (SEN), and an introgressed strain (SENbkf)) were investigated at different temperatures and relative humidity conditions. The optimal temperature for the mass-rearing was 25 ± 1°C, 24.6 ± 1°C and 23.9 ± 1°C for BKF, SENbkf and SEN respectively. The relative humidity ranging from 40 to 75% had very little influence on the survival and fecundity. The BKF strain resisted better at higher temperatures than the SENbkf and SEN strains but the temperature limit for survival was about 32°C for all three strains.

Várias espécies de moscas-das-frutas têm sido alvo de investigações visando sua criação massal, incluindo Ceratitis capitata (Wiedemann) (mosca-do-Mediterrâneo ou medfly) e, mais recentemente, Anastrepha fraterculus (Wiedemann) (mosca-das-frutas sul americana), sendo as espécies de maior importância econômica e quarentenária em várias áreas produtoras de frutas do Brasil, Uruguai, Argentina e Peru. Um dos fatores determinantes para que a Técnica do Inseto Estéril (TIE) seja aplicada contra uma determinada praga é a existência de métodos de criação massal da espécie. Para se alcançar a criação em larga escala dessas espécies de moscas-das-frutas, é necessário, inicialmente, o preparo de uma dieta larval adequada. Como a dieta de criação pode oferecer uma quantidade enorme de substâncias nutritivas, as larvas criadas artificialmente amadurecem mais cedo do que as selvagens, o que acelera a sucessão de gerações. Essas mudanças detectadas durante o estágio larval podem formar adultos com maiores reservas nutritivas, atingindo a maturidade sexual mais cedo. Existem várias biofábricas de mosca-das-frutas no mundo capazes de produzir machos estéreis que são utilizados para a erradicação ou supressão populacional em área-ampla. Visando-se diminuir os custos de produção dos insetos, pesquisas voltadas para a otimização e desenvolvimento de novas dietas não podem parar. Portanto, os objetivos deste trabalho foram determinar a adequação e densidade larval ideal das moscas C. capitata e A. fraterculus em novas dietas à base de farinha de milho (FM), bagaço de cana-de-açúcar (BC), pó de cenoura liofilizada (PC) e carragenina (MC), escolhendo a dieta mais eficiente e viável economicamente para produção de cada uma dessas espécies com base na quantidade e qualidade de insetos produzidos e no custo de produção. As densidades larvais ideais para a criação de C. capitata nas dietas larvais FM, BC e PC foram, respectivamente, 0,5; 2,0; e 1,0 mL de ovos/kg de dieta. A dieta larval com maior produtividade foi a dieta BC, produzindo uma média de 6.514 pupas/kg de dieta, seguida das dietas FM e PC, que produziram, respectivamente, uma média de 5.018 e 4.135 pupas por kg de dieta. A ordem decrescente de viabilidade econômica entre as três dietas larvais para C. capitata foi a mesma (BC>FM>PC), sendo necessário um investimento médio de R$ 471,29; R$ 482,26; e R$ 1.085,85, respectivamente, para a produção de 1 milhão de pupas. Os resultados com A. fraterculus mostraram que as densidades larvais ideais nas dietas FM e MC foram de 2,0/kg de dieta e 1,5 mL de ovos/L de dieta, respectivamente. A dieta larval PC foi considerada inviável devido à baixa produtividade e peso de pupas. Em termos de produtividade, a dieta larval MC (produção média de 22.451 pupas/L de dieta), foi mais eficiente do que a FM (6.688 pupas/kg de dieta). Quanto à viabilidade econômica, a dieta larval MC foi mais vantajosa, demandando um investimento médio de R$ 312,67 para a produção de 1 milhão de pupas, enquanto que a dieta FM custou R$ 1.266,36 para produzir a mesma quantidade de pupas / Several species of fruit flies have been the subject of research for their mass rearing, including Ceratitis capitata (Wiedemann) (Mediterranean fruit fly or medfly) and, more recently, Anastrepha fraterculus (Wiedemann) (South American fruit fly), being these species the ones with most economic and quarantine importance in several fruit producing areas of Brazil, Uruguay, Argentina and Peru. One of the requirements for the Sterile Insect Technique (SIT) is the existence of mass rearing methods of the target species. In order to achieve the large-scale rearing of these species of fruit flies, it is necessary initially to develop a suitable larval diet. As the rearing diet can offer a huge amount of nutrients, artificially reared larvae mature earlier than wild ones, what accelerates the succession of generations. These changes during the larval stage may form adults with higher nutritional reserves and they can reach sexual maturity earlier. There are several mass-rearing facilities for fruit flies in the world capable of producing sterile males that are used for population eradication and suppression in area-wide. In order to reduce insect production costs, the optimization or development of new diets cannot stop. Therefore, the objectives of this study were to determine the adequacy and optimal larval density of C. capitata and A. fraterculus flies in new diets based on corn flour (FM), sugarcane bagasse (BC), lyophilized carrot powder (PC) and carrageenan (MC), choosing the most efficient and less costly diet for the production of each of the species, based on the quantity and quality of insects reared and production costs. The optimal larval densities for C. capitata in larval diets FM, BC and PC were, respectively, 0.5; 2.0 and 1.0 mL of eggs/kg of diet. The larval diet that reached the highest productivity was the BC diet, giving an average of 6,514 pupae/ kg of diet, followed by FM and PC diets, which produced an average of 5,018 and 4,115 pupae per kg of diet, respectively. The decreasing order of economic viability among the three larval diets used was the same (BC>FM>PC), requiring a mean investment of R$ 471.29; R$ 482.26; and R$ 1,085.85, respectively, for the production of 1 million pupae. The results with A. fraterculus showed that the ideal larval densities in FM and MC diets were 2.0 eggs/kg of diet and 1.5 mL of eggs/L of diet, respectively. The PC larval diet was considered infeasible based on the low production and weight of pupae. In terms of productivity, the MC larval diet, which resulted in an average of 22,451 pupae produced per L of diet, was more efficient than FM, which produced a mean of 6,688 pupae per kg of diet. Considering the economic costs, the MC larval diet was also superior, requiring R$ 312,67.00 for the production of 1 million pupae, while the FM diet required R$ 1,266.36 to produce the same amount of pupae

No description available.