Dissertations / Theses on the topic 'Sterile insect technique'

The false codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae), is an important pest of citrus in South Africa and challenging to manage due to its inconspicuous nature. An effective method currently being employed for the area-wide suppression of the FCM is the Sterile Insect Technique (SIT) and the effective dispersal of sterile moths is very important for success with SIT. This study was conducted in the Addo area of the Sundays River Valley (Eastern Cape) where the programme is commercially used. In this study, sterile male moths were released in different orchards on a citrus farm, and in nearby veld at different times of the year, and their dispersal was monitored through the use of pheromone traps. Various climatic factors were monitored. This provided insight into the local dispersal of sterile male FCM adults in response to abiotic cues (particularly climatic factors). The movement of the FCM in four citrus cultivars, namely lemons, navel and Valencia oranges and mandarins and in the nearby veld (open field), was determined at six different stages of the year. Results clearly indicated that sterile FCM movement is concentrated within citrus orchards, as very few moths were trapped beyond 30 m from the release point, particularly in navel and Valencia orchards. Of the climatic factors measured, minimum and maximum temperatures had the most significant influence on FCM dispersal, and based on the results, various recommendations are made for the releases of sterile FCM in an area-wide SIT management programmes on citrus. A better understanding of the dispersal capabilities of the FCM in an agricultural system, under different conditions and at different times of the year, is invaluable not only in improving release strategies in an SIT programme but in planning future control strategies against the FCM.

The tomato leafminer Liriomyza bryoniae (Kaltenbach) (Diptera: Agromyzidae) is an economically important pest of greenhouse tomato crops in the UK, which at high infestations can reduce the value of the crop by up to 20% . Sterile insect technique, SIT, is the release of sterile insects to overflood and mate with the pest population. Wild females that mate with sterile males lay eggs that contain dominant lethal mutations and are unable to develop into larvae. SIT has been proposed as a novel, alternative method of L.bryoniae pest suppression that could be used in conjunction with the current biological controls, such as Diglyphus isaea (Walker) (Hymenoptera: Eulophidae). This thesis presents information on the development of SIT against L.bryoniae and examines the feasibility and compatibility with existing pest management methods. Work was carried out that determined high quality, sterile adult L.bryoniae were produced when late stage pupae were irradiated with a dose of 160 Gy gamma radiation. Both male and female L.bryoniae were sterile after irradiation with 160 Gy, which is important given the current inability to separate pupae by sex. A multi-treatment cage experiment was used to compare bi-sex and male-only releases and showed that the release of sterile females did not have a detrimental effect and did not add to the pest problem. The multi-treatment cage experiment also compared the use of D.isaea with sterile male releases both separately and concurrently. Whilst the study conditions favoured the optimal environment for D.isaea oviposition and development, the concurrent release of sterile male L.bryoniae and D.isaea were compatible. SIT is a suitable method for L.bryoniae suppression; but further work to develop a more time and cost-efficient mass-rearing technique and greater knowledge of the market are required in order for it to become a financially viable pest management option. Overall, the irradiation of L.bryoniae pupae with 160 Gy produced sterile adults that have comparable fitness to wild-type adults, do not produce viable offspring and have the potential to suppress a L.bryoniae infestation.

Thesis (PhD)--Stellenbosch University, 2013.
ENGLISH ABSTRACT: Two mathematical models are formulated in this dissertation for the population growth of an Eldana saccharina Walker infestation of sugarcane under the influence of partially sterile released insects. The first model describes the population growth of and interaction between normal and sterile E. saccharina moths in a temporally variable, but spatially homogeneous environment. The model consists of a deterministic system of difference equations subject to strictly positive initial data. The primary objective of this model is to determine suitable parameters in terms of which the above population growth and interaction may be quantified and according to which E. saccharina infestation levels and the associated sugarcane damage may be measured. The second model describes this growth and interaction under the influence of partially sterile insects which are released in a temporally variable and spatially heterogeneous environment. The model consists of a discretized reaction-diffusion system with variable diffusion coefficients, subject to strictly positive initial data and zero-flux Neumann boundary conditions on a bounded spatial domain. The primary objectives in this case are to establish a model which may be used within an area-wide integrated pest management programme for E. saccharina in order to investigate the efficiency of different sterile moth release strategies in various scenarios without having to conduct formal field experiments, and to present guidelines by which release ratios, frequencies and distributions may be estimated that are expected to lead to suppression of the pest. In addition to the mathematical models formulated, two practical applications of the models are described. The first application is the development of a user-friendly simulation tool for simulating E. saccharina infestation under the influence of sterile insect releases over differently shaped spatial domains. This tool provides the reader with a deeper understanding as to what is involved in applying mathematical models, such as the two described in this dissertation, to real-life scenarios. In the second application, an optimal diversification of sugarcane habitats is considered as an option for minimising average E. saccharina infestation levels, and as a further consequence, improving the cost-efficiency of sterile insect releases. Although many special cases of the above model classes have been used to model the sterile insect technique in the past, few of these models describe the technique for Lepidopteran species with more than one life stage and where F1-sterility is relevant. In addition, none of these models consider the technique when fully sterile females and partially sterile males are being released. The models formulated in this dissertation are also the first to describe the technique applied specifically to E. saccharina, and to consider the economic viability of applying the technique to this species. Furthermore, very few examples exist of such models which go beyond a theoretical description and analysis towards practical, real-life applications as illustrated in this dissertation.
AFRIKAANSE OPSOMMING: Twee wiskundige modelle word in hierdie proefskrif vir die populasiegroei van ’n Eldana saccha- rina Walker infestasie van suikerriet onder die invloed van gedeeltelik steriele, vrygelate insekte daargestel. Die eerste model beskryf die populasiegroei van en -interaksie tussen normale en steriele E. saccharina motte in ’n dinamiese, maar ruimtelik-homogene omgewing. Die model is ’n stelsel deterministiese verskilvergelykings onderhewig aan streng positiewe aanvangswaardes. Die primˆere doelstelling met hierdie model is om geskikte parameters te bepaal in terme waarvan die bogenoemde groei en interaksie gekwantifiseer kan word, en waarvolgens E. saccharina infestasievlakke en die gepaardgaande suikerrietskade gemeet kan word. Die tweede model beskryf hierdie groei en interaksie onder die invloed van gedeeltelik steriele insekte wat in ’n dinamiese en ruimtelik-heterogene omgewing vrygelaat word. Die model is ’n gediskretiseerde stelsel reaksie-diffusievergelykings met veranderlike diffusieko¨effisi¨ente onderhewig aan streng positiewe aanvangswaardes en zero-vloei Neumann-randwaardes op ’n begrensde ruimtelike gebied. Die primˆere doelstellings in hierdie geval is om ’n model tot stand te bring wat in ’n area-wye, ge¨ıntegreerde pesbestrydingsprogram vir E. saccharina gebruik kan word om die doeltreffendheid van verskillende steriele motvrylatingstrategie¨e te bepaal sonder om daadwerklik veldeksperimente uit te voer, en om riglyne daar te stel waarvolgens vrylatingsverhoudings, -frekwensies en -verspreidings bepaal kan word wat na verwagting na ’n onderdrukking van die pes sal lei. Bykomend tot die wiskundige modelle in hierdie proefskrif, word twee praktiese toepassings van die modelle ook beskryf. In die eerste toepassing word ’n gebruikersvriendelike simulasie hulpmiddel ontwikkel om E. saccharina infestasie onder die invloed van steriele insekvrylatings in verskillende ruimtelike gebiede te simuleer. Hierdie toepassing fasiliteer ’n dieper begrip van wat ter sprake is in die toepassing van wiskundige modelle, soos die twee modelle in hierdie tesis, tot werklike scenario’s. In die tweede toepassing word ’n optimale diversifisering van suikerriet habitats as ’n opsie vir die vermindering van die gemiddelde E. saccharina infestasie vlakke beskou, en gevolglik word die verbetering van die koste-doeltreffendheid van steriele insekvrylatings afgeskat. Alhoewel verskeie spesiale gevalle van die bogenoemde twee klasse van modelle reeds in die verlede gebruik is om die doeltreffendheid van die steriele-insektegniek te modelleer, beskryf weinig van hierdie modelle die tegniek vir Lepidopteriese spesies met meer as een lewensfase en waar F1-steriliteit ter sprake is. Verder beskryf geen van hierdie modelle die tegniek waar algeheel steriele wyfies en gedeeltelik steriele mannetjies vrygelaat word nie. Die modelle in hierdie tesis is ook die eerste waar die tegniek spesifiek op E.saccharina toegepas word, en waar die ekonomiese lewensvatbaarheid van die tegniek vir hierdie spesie oorweeg word. Verder bestaan daar min voorbeelde van soortgelyke modelle wat verder gaan as ’n teoretiese beskrywing en wiskundige ontleding na praktiese, werklike toepassings, soos in hierdie proefskrif ge¨ıllustreer.

In the light of increasing pesticides resistance in agricultural pests and in insect vectors of human diseases, leading to the rise in occurrence of mosquito-borne diseases, new, efficient and environmentally friendly methods of pest control are needed. Sterile Insect Technique (SIT), relying on mass releases of radiation sterilised males to reduce reproductive potential of target pest populations, although not new, offers an alternative to the use of pesticides and is an environmentally non-polluting method of insect control. Many insect species, however, are not very amenable to classical SIT, due to detrimental side-effects of radiation treatment. We propose a new method, a genetically engineered modification of classical SIT, replacing radiation with genetically induced sterility. Based on conditional expression of male-germline targeted nucleases which introduce double strand breaks into the male germline DNA to render males sterile, this method emulates SIT mechanism, at the same time eliminating radiation and associated detrimental side-effects. Different variants of such a system were investigated in this project, eventually leading to the creation of functional conditional male-sterility systems in two model organisms – the Yellow fever mosquito, Aedes aegypti and the Mediterranean fruit fly, Ceratitis capitata. Both systems utilise chimeric nuclease composed of protamine and FokI cleavage domain fusion. The sperm-specificity and the conditionality of the sterile phenotype have been achieved through the use of tetracycline repressible expression system driven by the β2-tubulin promoter in Ceratitis capitata and by the Topi promoter in Aedes aegypti.

Desde a década de 50, os cientistas tem utilizado radiação ionizante para esterilizar insetos, que são liberados na natureza para acasalar, mas sem nenhuma descendência. Conhecida como a técnica do inseto estéril (TIE), este método de controle de inseto vem tradicionalmente utilizando radiações ionizantes para esterilizar os insetos, sendo uma técnica que não gera resíduo, e pode agir em sinergia com as demais técnicas dentro do manejo integrado de pragas. O Brasil por vários anos vem lutando contra o aumento de pragas, introduzindo novas táticas e técnicas dentro dos programas de MIP, para driblar a resistência dos produtos químicos, como: diminuir os resíduos dos agrotóxicos; para algumas culturas importantes do o nosso país, temos um largo espectro de pragas ocorrendo do inicio ao fim da colheita, uma delas é a cultura do algodoeiro e dentre as pragas chave dessa cultura, temos algumas lagartas extremamente importantes, entre elas Heliothis virescens e Helicoverpa armigera. Essas espécies são parecidas morfologicamente, sendo a segunda identificada a poucos anos aqui no Brasil. Ainda não há trabalhos no Brasil utilizando TIE como ferramenta adicional para lepidópteros, portanto objetivou-se com esse estudo avaliar o efeito de doses de radiação gama nas diferentes fases do ciclo evolutivo de Heliothis virescens e Helicoverpa armigera, bem como avaliar a esterilidade na geração P e a capacidade de competição dos insetos irradiados com os não irradiados. A fase pupa foi a que apresentou um melhor resultado, pois com 75 Gy atingiu a esterilidade em Heliothis virecens e 100 Gy esterilizou Helicoverpa armigera, portanto contemplou a fase e a dose escolhida para avaliar a competição entre os insetos irradiados e os insetos normais de ambas as espécies. E tanto Heliothis virecens como Helicoverpa armigera apresentaram um resultado satisfatório, pois os insetos irradiados conseguiram reduzir significativamente a viabilidade dos ovos na proporção de 9: 1: 1
Since the 1950s, scientists have used ionizing radiation to sterilize insects, which are released in nature to mate, but without any progeny. Known as the sterile insect technique (TIE), this method of insect control has traditionally used ionizing radiation to sterilize insects, a technique that does not generate residues, and can act in synergy with the other techniques within integrated pest management. For several years, Brazil has been fighting against the increase of pests, introducing new tactics and techniques within the IPM programs, to overcome the resistance of chemical products, such as: reducing the residues of agrochemicals; For some important crops of our country, we have a wide spectrum of pests occurring from the beginning to the end of the harvest, one of them is the cotton crop and among the key pests of this crop, we have some extremely important caterpillars, among them Heliothis virescens and Helicoverpa armigera These species are morphologically similar, the second being identified a few years ago in Brazil. There are still no studies in Brazil using TIE as an additional tool for Lepidoptera, therefore the purpose of this study was to evaluate the effect of doses of gamma radiation in the different phases of the evolutionary cycle of Heliothis virescens and Helicoverpa armigera, as well as to evaluate the sterility in generation P And the ability of insects to irradiate with non-irradiated insects. The pupal phase presented the best result because 75 Gy achieved sterility in Heliothis virecens and 100 Gy sterilized Helicoverpa armigera, therefore it contemplated the phase and dose chosen to evaluate the competition between the irradiated insects and the normal insects of both species. Both Heliothis virecens and Helicoverpa armigera presented a satisfactory result, as the irradiated insects managed to significantly reduce the viability of the eggs in a ratio of 9: 1: 1

Ceratitis capitata est une espèce nuisible dont l'histoire est jalonnée d'invasions. La principale technique utilisée, pour contrôler cette espèce est celle du SIT (Sterile Insect Technique). Cette technique qui a un faible impact sur l'environnement, consiste à relâcher périodiquement de grandes quantités d'individus, généralement des mâles qui vont entrer en compétition avec les mâles de la nature pour s'accoupler aux femelles. L'amélioration de cette technique nécessite, d'une part une meilleure connaissance de la dynamique des populations et de la bioclimatologie pour programmer les lâchers et d'autre part, des données sur le comportement sexuel des mâles stériles. Enfin, une analyse du génome de cette espèce pour un élément transposable a été entreprise afin de déterminer si cet élément pourrait être utilisé pour vecteur de transgenèse. Plus précisément, mon travail a porté sur les questions suivantes: 1) L'origine des populations Californienne ; 2) Est-ce que les femelles peuvent s'accoupler plusieurs fois? Si oui, est-ce que la fréquence des ré-accouplements varie avec les conditions écologiques et la taille des populations? 3) Cette espèce est la première à être modifiée par éléments transposables. Actuellement, les meilleurs résultats sont obtenus avec l'élément PiggyBac. Dans la mesure où la présence d'élément de ce type dans le génome hôte peut influencer la stabilité du transgène, j'ai recherché la présence d'éléments de type PiggyBac chez cette espèce ainsi que chez 13 autres espèces de Tephritidae
The medfly, Ceratitis capitata, is a pest species with a long history of invasion success. The major environmentally benign method of pest control is the Sterile Insect Technique. SIT relies on the periodic release into the field of mass-reared sterile flies, preferentially males that should compete with wild males in mating with females. SIT improvement requires knowledge of population dynamics and bioclimatology to program fly releases and knowledge of sexual behavior to have competitive sterile male. My PhD research activity was mainly focused on medfly and covered three fields: population dynamics, sexual behavior and transposable elements. Specifically: 1. Medfly population dynamics: what is the status of medfly in California? 2. Sexual behavior: do medfly females remate in the wild? Does the frequency of remating vary with the varying of ecological condi1ions and population sizes? 3. On genome. Medfly was the first non-drosophilid insect to be genetically modified by transposable elements. Nowadays, the most promising transformation system is based on the element piggyBac. As the presence, in the host species, of endogenous piggyBac-like elements could influence transgene stability, I analysed the presence of piggyBac-like elements in medfly genome. The analysis was extended to other 13 tephritid species. The answers to some of these questions have already been submitted to the attention of the international scientific community, consequently the corresponding papers are included in this thesis after a general introduction of the discussed subject

False codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is considered the most important indigenous pest of citrus in southern Africa. Major concerns such as progressive insecticidal resistance, the negative impact of insecticides on the environment, as well as the influence of consumers opposed to chemical residues on fruit, created opportunities for biological control methods such as Sterile Insect Technology (SIT). This technology is now established in the Western and Eastern Cape provinces of South Africa as an effective, sustainable alternative to conventional FCM control methods. Due to the prevalence of the pest in all citrus producing areas of South Africa, potential for SIT to expand is enormous. Success of an SIT programme is highly dependent on efficient application of the technology to achieve its objectives in a timeous manner. The aim of this study was to advance the application of SIT for control of FCM on citrus in South Africa, by investigating the effect of certain critical stages in the process. The effect of long-distance transportation on fitness of irradiated FCM was determined, showing reduced performance with cold-immobilized transport. A significant decrease in flight ability and longevity of irradiated FCM was found, although critically, realized fecundity was not affected. The effect of two different insecticides in the pyrethroid and organophosphate chemical groups were investigated for their residual effect on mortality of released irradiated FCM, to determine if these pest control programmes could be integrated. Both chlorpyrifos and tau-fluvalinate were effective in killing irradiated FCM for a number of days after application, after which degradation of the active ingredient rendered it harmless. This effect was found to be similar for irradiated and non-irradiated males, consequently ratios of sterile : wild male FCM should be retained regardless of whether sprays are applied or not. The modes for release of sterile FCM in an SIT programme were investigated. Efficacy of ground and aerial release platforms were tested by evaluating the recovery of released irradiated male FCM in these orchards. More irradiated FCM were recovered in orchards released from the ground compared to air. However, an economic analysis of both methods shows application of irradiated insects over a large geographical area is more cost-effective by air. Depending on the terrain and size of the target area, a combination of both methods is ideal for application of SIT for control of FCM in citrus. Development of application technology for advance of the programme is discussed and recommendations for future research and development are offered.

Thesis (PhD(Agric))--Stellenbosch University, 2012.
The sugarcane borer, Eldana saccharina Walker (Lepidoptera: Pyralidae) is the most limiting factor in the South African sugar industry with losses to this insect pest estimated to be at least ZAR60 million per annum. Because of its cryptic nature as well as the fact that E. saccharina is both indigenous to Africa and occurs on several host plants, attempts to control or eradicate it using several available methods have not been very successful. However, the sterile insect technique (SIT) is one of the newer control methods that can be incorporated into an area-wide integrated pest management (AW-IPM) programme to achieve better control or eradication. The implementation of the SIT program needs to go through a series of well-researched phases in order to be successful. In the first of this multi-phase project, it was determined that E. saccharina is susceptible to ionizing radiation, and is thus a suitable candidate for the SIT development against it and that a sub-sterilizing dose of 200 Gy is sufficient to induce F1(inherited) sterility in male and complete sterility in female moths respectively. The results presented here are discussed in the context of further development of the SIT as an addition to the arsenal of tactics in an AW-IPM programme against E. saccharina. Based on these initial findings, the study examined the lek and mating behavior of male moths subjected to three radiation doses (150, 200, 250 Gy) against normal non-irradiated/fertile moths. Both mass-rearing and irradiation of E. saccharina led to a quantitative departure of male mating behavior away from that exhibited by their wild counterparts. However, treated males are still able to form leks and mate with wild females. Male E. saccharina irradiated at all three doses tested were found to be as active and competitive as wild males, but in some of the traits measured, performance diminished significantly with an increase in the radiation dosage. In general, the performance of moths treated at 200 Gy did not differ significantly from that of moths treated at 150 Gy and therefore the former dose is ideal for SIT development since it results in a lower residual F1 fertility than the latter. The level of mating competitiveness and compatibility was assessed under both laboratory and semi-field conditions in pairwise comparisons consisting of laboratory reared vs. wild (L-W), 200 Gy irradiated vs. wild (S-W) and laboratory reared vs. irradiated moths (L-S). Based on the results from the more robust field cage assays, the mating indices generated indicated that the mass-reared E. saccharina strain produced in South Africa has not yet evolved sexual behaviours suggestive of incipient pre-mating isolation barriers with local wild strains. Wild moths did not discriminate against either the partially sterile or laboratory reared moths and most importantly, the irradiated males mated significantly more than their wild counterparts regardless of the type of female. The irradiated insects could therefore achieve the purpose for which they are intended upon release into the field. Third, the critical thermal limits (CTLs) to activity at high and low temperatures (i.e. critical thermal maxima “CTmax” and minima “CTmin”) of different E. saccharina strains/treatments were investigated under standard experimental conditions. The effect of laboratory rearing and increasing radiation dosage on thermal tolerance of the adult stage of E. saccharina was explored. There were highly significant differences between the laboratory-reared and wild strain and also between non-irradiated and irradiated strains in both CTmax and CTmin. Laboratory reared E. saccharina moths were more heat tolerant compared to wild moths for both genders while in the case of CTmin, the reverse was true. Irradiation had a negative effect on both CTmax and CTmin. Moths treated at the lowest radiation dose were more cold and heat tolerant than those treated at higher dosages thereby reinforcing the importance of lower dosages rather than those that induce full sterility against E. saccharina. In general, gender effects on the CTLs were non-significant. Pilot sterile male releases in shade house trials to measure the impact of sustained releases of partially sterile adult males at an over-flooding moth ratio of 10T: 1U (treated to untreated),were conducted to measure their efficacy to stop E. saccharina incursions and suppress populations prior to testing in pilot studies under true season-long and area wide conditions. Results from the current study demonstrated that releasing partially irradiated (200 Gy) adult male moths at the afore mentioned release rate significantly reduced sugarcane stalk damage as well as lowered the number of fertile progeny from F1to succeeding generations in a stable E. saccharina population initiated in a cage house. There were more damaged internodes per stalk in the control than in the sugarcane receiving regular releases of partially sterile male moths. Overall, there were significantly more undamaged stalks in the treated sugarcane than the untreated control. Furthermore, there were significantly more larvae per stalk retrieved from the control compared to the treated sugarcane suggesting that the sustained release of steriles was efficacious in reducing emergence of fertile larvae in the succeeding generations. The results of this study indicate that there is considerable scope for the SIT against E. saccharina.

As moscas-das-frutas são consideradas importantes pragas da fruticultura mundial, causando perdas significativas à produção e ao livre trânsito de frutas. Com a preferência por alimentos com baixos níveis de agrotóxicos, pesquisas estão buscando alternativas ecológica, social e econômica viáveis para o controle destas pragas. A Técnica do Inseto estéril (TIE) é uma técnica onde os insetos são criados, esterilizados e liberados em áreas infestadas, reduzindo a fertilidade da população selvagem, além de ser um método não poluente e não agressivo ao ambiente. Com o desenvolvimento de novos estudos da metodologia da TIE, foi introduzida uma nova linhagem mutante a Sensibilidade a Letal Temperatura (tsl) Viena-8, cujas fêmeas são sensíveis à alta temperatura, podendo ser eliminadas já na fase embrionária, no início da criação massal, agregando mais benefícios a este método de controle e/ou supressão de praga. Com diferentes testes de incubação de ovos da linhagem tsl, com temperaturas e tempos combinados e variados, comprovou-se a extrema sensibilidade às mudanças de temperatura. A temperatura ideal para a incubação dos ovos dessa linhagem está entre 24ºC a 26ºC pelo período de 24 a 48 horas. Deve-se tomar o máximo de cuidado com a elevação da temperatura acima de 26ºC durante a manutenção e multiplicação dessa linhagem em condições de laboratório ou massalmente. Acima de 26ºC, há sérios riscos de comprometer a qualidade dos insetos produzidos. O curto período de 6 horas a uma temperatura de 34ºC, já é suficiente para alterar negativamente a proporção de fêmeas de ovos tratados termicamente
The fruit flies are considered important pests of the world horticulture, causing significant loses to the production and the free traffic of fruits. With the preference for foods with low levels of pesticides, researches are looking for alternatives ecological, social and economical viable for the control of these pests. The Sterile Insect Technique (SIT) is a technique where the insects are reared, sterilized and release in infested areas, reducing the fertility of the wild population, and is a method no pollutant and no aggressive to the environment. With the development of new studies of the methodology of SIT, a new mutation, Temperature Sensitive Lethal (tsl) Vienna 8, whose females are sensitive to the high temperature, could already be eliminated in the embryonic phase, in the beginning of the mass-rearing, resulting in more benefits to the control/suppression this pests. Different tests of incubation with eggs of tsl strain, with variation of time and temperature was done and proved a very sensitive to temperature changes. The ideal temperature for the incubation of the eggs is among 24ºC to 26ºC by the period from 24 to 48 hours. The maximum of care should be taken with the elevation of the temperature above 26ºC during the maintenance and multiplication in laboratory conditions or mass-rearing. Above this temperature it can commit the quality of the produced insects. The short period of 6 hours with temperature to 34ºC, is enough to change negatively the proportion of females of eggs in the thermal treatment

The mosquito Aedes polynesiensis, a member of the Aedes scutellaris complex, is the main vector in the South Pacific region of the Wuchereria bancrofti parasite, the causative agent of lymphatic filariasis (LF), and is also a major nuisance biter. Decades of Mass Drug treatment (MDA) have not been successful in elimination LF. Two non-vector species in the Ae. scutellaris complex were introgressed with Ae. polynesiensis to attempt to obtain lines that would produce cytoplasmic incompatibility (CI) with wild populations and/or LF-refractoriness. Despite selection of progeny from Brugia-challenged, non-infective females at each backcross, no refractory line was acquired. However, three lines from crosses between aposymbiotic Ae. polynesiensis and Ae. riversi displayed CI and male mating competiveness suitable for the purpose of population suppression using the incompatible insect technique (IIT). A population study was conducted of potential release sites and the evaluation of monitoring tools for Ae. polynesiensis on Moorea and Tetiaroa, French Polynesia. There was no evidence of active migration between selected islets on the atoll of Tetiaroa, suggesting it is a suitable site for field releases of CI males. The BioGents Sentinel trap was shown to be an efficient and convenient trap suitable for Ae. polynesiensis monitoring. The effects of temperature and larval density on life-table parameters relevant to IIT were examined, including: larval survivorship, developmental time to pupation, male to female ratio, male pupae yield, male size and adult male survival. These findings were used to design and conduct a 14-week field experiment testing CI male strain against an isolated population, using optimized rearing conditions. Approximately 8000 males were released weekly on motu Onetahi, Tetiaroa atoll. Significant sterility was induced by Wolbachia in the targeted female population, supporting the development and scale-up of this approach toward Ae. polynesiensis nuisance and LF transmission reduction.

False codling moth (FCM), Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is considered the most important indigenous pest of citrus in southern Africa. It is recognized by several markets as a phytosanitary organism and the efficient control of this pest is now more important than ever. The pheromone communication between the male and female moths has been exploited in order to control FCM through the sterile insect technique (SIT). The sterilized males used for all SIT programmes across South Africa come from a colony that originates from wild material collected from the Citrusdal area of the Western Cape Province. The aim of this study was to determine if any differences in attractiveness of females to males exist between different geographical populations of FCM and if so what impact this would have on the male’s ability to locate females from other populations via the volatile sex pheromone released by the female. Laboratory trials with Y-tube olfactometers and flight tunnels tested the attraction of male moths to virgin females, but did not yield any consistent results. Field experiments were conducted with sterile male Citrusdal moths released and recaptured in yellow delta traps in two separate trials. For one trial, the traps were baited with live virgin females from five different geographical populations including Addo, Nelspruit, Marble Hall, Citrusdal and the Old colony, which is a mixture of several populations. For the other trial traps were baited with various synthetic pheromone blends including three regional blends which included South Africa, Ivory Coast and Malawi and three commercial blends including Pherolure, Isomate and Checkmate. For the virgin female trial the Citrusdal males showed a significant preference for females from their own population. There was also a significant difference in the recaptures from the different synthetic pheromones. The South African blend was the most attractive of all the regional and commercial blends. A cross-mating trial was also conducted under laboratory conditions in petri dishes with five different FCM populations including Citrusdal, Addo, Marble Hall, Nelspruit and Old (mixed origin). Females produced more eggs when mated with males from the same population for the Addo, Marble Hall, Nelspruit and Old (mixed origin) populations. The only case in which this was statistically significant was for the Marble Hall population. All the crosses produced viable eggs and the origin of the male or female did not influence egg hatch. The results from this study may lead to improvements in both the control and monitoring of FCM populations. The control methods include mating disruption, attract-and-kill and SIT. Tailoring these methods for a specific growing area with a pheromone blend originating from the area or releasing sterile moths from a colony that originates from the area may optimize the available monitoring and control options.

The mosquito Aedes aegypti is responsible for 50 million dengue fever infections in humans each year. A novel control strategy, named RIDL (Release of Insects carrying a Dominant Lethal) relies on releasing large numbers of genetically sterile male insects in order to control pest populations. This thesis describes the development of new tools to improve the efficiency of RIDL against Ae. aegypti and assessment of candidate strains for field release. Chapter 3 describes a new selection system for Ae. aegypti based on ethanol susceptibility conferred by the alcohol dehydrogenase gene (Adh) from Drosophila melanogaster. I observed that the susceptibility of Ae. aegypti larvae to ethanol can be triggered by expression of Adh in larvae. Chapters 4 and 5 focus on RIDL strains with a genetic sexing mechanism, for easy and stringent selection for males before mass-releases, eliminating disease- transmitting females. In Chapter 4, I describe the creation of a late-acting sexing strain of Ae. aegypti based on the Ae. aegypti doublesex (Aedsx) alternative splicing system. In Chapter 5, I describe an attempt to create an early-acting sexing system. Killing the females of the release generation early would free space and resources for the production of males. This was done by combining the Adh gene and the Aedsx alternative splicing system described in Chapters 3 and 4. Chapter 6 reports the results of a comparison, in terms of quality and productivity, between an existing Ae. aegypti RIDL strain and a wild-type control. Results showed equivalent female quality and productivity between the two strains, while RIDL males were less fertile in comparison with wild-type males. RIDL eggs also seemed more susceptible to long- term storage. The results of this work show promise for development of novel RIDL strains that may be used in the field to control disease-transmitting mosquitoes.

No mundo todo, o manejo integrado de moscas-das-frutas é feito com associação do controle biológico aplicado e técnica do inseto estéril. Além da boa eficiência no campo, a associação dessas técnicas é também favorecida pelo fato de ambos os organismos, insetos estéreis e parasitoides, poderem ser produzidos massalmente na mesma fábrica com menor custo. Visando à produção massal do parasitoide de moscas-das-frutas Diachasmimorpha longicaudata e de insetos estéreis para atender a programas de manejo integrado de Ceratitis capitata e Anastrepha fraterculus, o presente trabalho foi desenvolvido no Centro de Energia Nuclear na Agricultura (CENA), da Universidade de São Paulo, entre os anos de 2006 a 2012. Durante esse período, metodologias de criação em laboratório foram implementadas e permitiram o desenvolvimento da tecnologia necessária para a produção desses insetos em grande escala no Brasil. Dados de 25 gerações do parasitoide produzido em grande escala em C. capitata tsl-Viena 8 e 51 gerações em A. fraterculus, bem como os efeitos e diferenças desses hospedeiros na qualidade do parasitoide foram analisados. É possível criar o parasitoide D. longicaudata em ambos os hospedeiros, C. capitata linhagem tsl-Viena 8 e A. fraterculus, entretanto essa linhagem de C. capitata não é indicada para produção massal do parasitoide, pois a emergência é muito baixa devido à debilidade do hospedeiro, possivelmente acarretada pelas manipulações genéticas que permitem a sexagem da mosca na fase embrionária. Caso essa seja a linhagem com melhor custo/ benefício disponível, recomenda-se o uso de larvas hospedeiras oriundas de ovos tratados termicamente para a produção do parasitoide. A criação em grande escala de A. fraterculus foi estabelecida com sucesso após o período de domesticação da espécie, obtendo-se larvas de boa qualidade com baixo custo, devido à dieta larval com baixa concentração de ágar e total uso de ingredientes nacionais nas dietas de adultos e larvas, por mais de 50 gerações. O uso de larvas irradiadas de A. fraterculus para a produção de D. longicaudata promove maior emergência do parasitoide, com maior quantidade e melhor qualidade de fêmeas que larvas de C. capitata.
The integrated management of fruit flies is worldwide done with the association of augmentative biological control with parasitoids and the sterile insect technique. Beyond the efficiency in the field, the joint of both techniques is also favored by the easy production of organisms, both parasitoids and sterile insects, in the same facility with low costs. Aiming for the mass production of the fruit fly parasitoid Diachasmimorpha longicaudata and sterile insects to support integrated management programs for Ceratitis capitata and Anastrepha fraterculus, this work was developed at the Center of Nuclear Energy in Agriculture (CENA) of the University of São Paulo, between 2006 and 2012. Along this period, laboratory rearing methods were improved which allowed the development of the required technology for a large scale production of those insects in Brazil. Data of 25 generations of the parasitoid large scale production using C. capitata tsl-Vienna 8 strain and 51 generations using A. fraterculus as hosts are presented. Moreover, the effect and differences between hosts in parasitoid\'s quality were analyzed. The parasitoid D. longicaudata can be large scale produced using both C. capitata tsl- Vienna 8 or A. fraterculus as hosts, but this strain of C. capitata is not recommended for D. longicaudata mass production once the parasitoid emergency is very low due to the host weakness. If this strain is the best cost/benefit available, the use of host larvae originating from heat treated eggs might be considered to parasitoids production. The A. fraterculus large scale rearing was established successfully after the domestication period of the specie, producing a large amount of insects with excellent quality using local ingredients and little agar in the diet composition over 50 generations. A. fraterculus irradiated larvae as host for large scale producing D. longicaudata promotes greater parasitoid emergence with greater female biased sex ratio and better female quality than C.capitata larvae.

Le specie aliene invasive sono specie animali o vegetali che molto spesso vengono introdotte involontariamente in ecosistemi non autoctoni, con impatto negativo per l'ambiente e le attività umane. La Drosophila suzukii Matsumura (Diptera: Drosophilidae), nota anche come Spotted Wing Drosophila, è una specie aliena invasiva originaria dell'Asia orientale, che si è ampiamente diffusa nelle Americhe e in Europa, dove è divenuta un grave parassita delle colture frutticole, causando notevoli perdite economiche. In Trentino la prima segnalazione di questo insetto su ospiti coltivati è avvenuta nel 2009. La sua rapida diffusione in tutto il mondo è attribuita a delle caratteristiche peculiari di D. suzukii, come l'ovopositore seghettato che gli consente di deporre le uova in frutti sani in maturazione, l’ampia gamma di piante ospiti, l’elevata fecondità e la tolleranza nei confronti di un ampio spettro di condizioni climatiche, fattori che ne ha aumentano significativamente l'invasività. Gli attuali approcci per il controllo di D. suzukii si basano principalmente su strategie di gestione integrata dei parassiti, tra cui il controllo chimico, le reti anti-insetto, la cattura massale e pratiche culturali, come la potatura delle chiome, l'irrigazione a goccia e il sollecito allontanamento della frutta infestata dai campi nel corso della raccolta. Analogamente ad altri parassiti invasivi, D. suzukii non viene efficacemente controllata da parte dei nemici naturali nelle aree invase, consentendogli di riprodursi rapidamente e diffondersi illimitatamente, aumentando la pressione dei parassiti sulle coltivazioni. Il controllo biologico inondativo e quello classico, sono in corso di valutazione e potrebbero diventare dei promettenti candidati per limitare i focolai stagionali di D. suzukii e i relativi danni alle colture. Tuttavia, nonostante i notevoli passi in avanti sulle conoscenze della biologia e l'ecologia di D. suzukii, questo insetto rappresenta ancora una seria minaccia per le colture e vi è la necessità di ulteriori studi al fine di aggiornare gli strumenti di gestione a disposizione per far fronte a questo importante parassita delle colture. Nel corso dei monitoraggi faunistici in provincia di Trento, abbiamo riscontrato e segnalato per la prima volta in Europa la presenza del parassitoide larvale asiatico Leptopilina japonica Novkovic & Kimura (Hymenoptera: Figitidae), uno dei più efficaci nemici naturali di D. suzukii nell’areale nativo. L’esteso monitoraggio in campo successivo al primo ritrovamento ha permesso di stabilire l'elevata capacità di questo parassitoide di parassitare il suo ospite su un ampio spettro di specie vegetali, appartenenti a diverse famiglie tassonomiche come Moraceae, Rosaceae, Glossulariaceae, Rhamnaceae, Cornaceae, Ericaceae, Phytolaccaceae, Dioscoreaceae, Adoxaceae e Solanaceae. Questi risultati offrono nuove prospettive per il controllo biologico di D. suzukii nelle aree di recente introduzione, anche per quanto concerne i futuri programmi di controllo biologico classico con Ganaspis brasiliensis Ihering (Hymenoptera: Figitidae). Il controllo biologico per mezzo di parassitoidi o predatori può essere facilmente integrato con la tecnica dell'insetto sterile (SIT), potenziando il controllo sulle popolazioni di parassiti. Tuttavia, il successo della SIT è in gran parte influenzato dal sistema riproduttivo del parassita bersaglio, per cui l’approfondimento dei tratti riproduttivi è un prerequisito fondamentale per verificare l’effettiva applicabilità di questa tecnica di controllo. L'analisi della paternità mediante la genotipizzazione di loci microsatellite della progenie di femmine catturate in natura, ha rilevato elevati livelli di paternità multipla nelle covate genotipizzate, stabilendo il comportamento poliandro di D. suzukii, sebbene non sia stata rilevata alcuna distorsione della paternità nell'allocazione dello sperma, ovvero in termini di maschio prevalenza. Oltre al controllo biologico e alla SIT, le strategie basate sulla manipolazione dell'habitat potrebbero utilmente integrare le comuni pratiche culturali messe in atto dagli agricoltori per il controllo di D. suzukii. Le nostre prove in campo hanno dimostrato che la consociazione con Mentha x piperita (Menta piperita, Lamiaceae) Origanum vulgare L. (Origano, Lamiaceae), Thymus vulgaris L. (Timo, Lamiaceae) o Ocimum gratissimum L. (Basilico africano, Lamiaceae), non risulta avere un impatto positivo nel limitare i danni causati da D. suzukii su colture di mirtillo, mentre la realizzazione di una bordura di Prunus padus (ciliegio europeo, Rosacee) si è dimostrata in grado di ridurre l'infestazione su di una coltura di lampone, agendo come pianta trappola. Inoltre, il controllo biologico conservativo mediante la tecnica dell’augmentorium può offrire un’ulteriore integrazione agli strumenti di controllo. Abbiamo infatti potuto osservare come il nostro prototipo si è dimostrato adatto per una sua applicazione su D. suzukii e sui relativi parassitoidi indigeni, favorendo la sanificazione delle colture dai frutti infestati e sostenendo i più comuni parassitoidi pupali. Con questa ricerca abbiamo inteso verificare l’efficacia di nuovi strumenti di controllo di D. suzukii, quali il controllo biologico, sia conservativo che classico, la SIT e la manipolazione dell'habitat, la cui opportuna integrazione offrirebbe la possibilità di implementare un approccio agroecologico di gestione di questo parassita invasivo.
Invasive alien species are animal or plant species that are very often unintentionally introduced into non-native ecosystems, with negative impact for the environment and human activities. Drosophila suzukii Matsumura (Diptera: Drosophilidae), also known as Spotted Wing Drosophila, is an invasive alien species native to East Asia, which has widely established in Americas and Europe, where it has become a serious pest of fruit crops, causing considerable economic losses. In Trentino Province the first oviposition on crop hosts was reported in 2009. The rapid worldwide spread is attributed to peculiar features of D. suzukii, such as the serrated ovipositor that allows it to oviposit into healthy ripening fruits, broad host plants range, high fecundity potential and tolerance of wide climatic conditions. Current approaches for controlling D. suzukii rely primarily on integrated pest management strategies, including chemical control, insect-proof netting, mass trapping and cultural practices, such as canopy pruning, drip irrigation, and field sanitation. Similarly to other invasive pest, D. suzukii lacks of an effective suppression by natural enemies in the recently invaded areas, enabling it to reproduce quickly and spread unlimitedly, increasing pest pressure on cropping systems. Augmentative and classical biological control are still under investigation as promising candidates for limiting D. suzukii seasonal outbreaks and related damage on crops. However, despite the remarkable steps forward in the knowledge on biology and ecology of D. suzukii, it is still a serious threat to crops and further investigations are urgently required in order to update the availability of management tools to cope with this crop pest. During our faunistic surveys in Trentino province, we found and reported for the first time in Europe, the presence of the Asian larval parasitoid Leptopilina japonica Novkovic & Kimura (Hymenoptera: Figitidae), one of the most effective natural enemies of D. suzukii in the native range. The following extensive field monitoring based on fruit sampling revealed the high capacity of this parasitoid to parasitize its host on a complex of several plant species, belonging to Moraceae, Rosaceae, Glossulariaceae, Rhamnaceae, Cornaceae, Ericaceae, Phytolaccaceae, Dioscoreaceae, Adoxaceae and Solanaceae. These findings offer new perspective for biological control of D. suzukii in the introduced areas, also in relation to the classical biological control programmes with Ganaspis brasiliensis Ihering (Hymenoptera: Figitidae). Biological control by means of parasitoids or predators can be easily integrated with sterile insect technique (SIT), synergizing the control effects on pest population. Nevertheless, the success of SIT is largely influences by mating system of target pest, thus deepen reproductive traits is a fundamental step forward. Paternity analysis by means of microsatellite loci genotyping of the progeny of wild-caught females, detected high levels of multiple paternity in genotyped broods, stating the polyandrous behaviour of D. suzukii, but no found evidence of a strong paternity skew in sperm allocation, in terms of prevailing male. Beyond biological control and SIT, habitat manipulation strategies may further help local farmers to deal with D. suzukii in addition to common applied cultural practices. Our field trials have shown that intercropping with Mentha x piperita (Peppermint, Lamiaceae) Origanum vulgare L. (Oregano, Lamiaceae), Thymus vulgaris L. (Thyme, Lamiaceae), or Ocimum gratissimum L. (African basil, Lamiaceae), has no effect in limiting the damage of D. suzukii on blueberries, whilst hedges of Prunus padus (European bird cherry, Rosaceae) was able to reduce the infestation on raspberry, having a potential role as dead-end trap plant for crop protection against D. suzukii. Furthermore, conservative biological control by means of the augmentorium technique may be further explored, as we observed that our prototype has shown to be well adapted to D. suzukii and its indigenous parasitoids, favouring crop sanitation from infested fruits and sustaining the most common Drosophila pupal parasitoids. This research aims to give new insights for a more sustainable management of D. suzukii, offering new solutions that, by combining biological control, both conservative and classical, SIT and habitat manipulation, may move towards an agroecological approach for controlling this invasive pest.

Thesis (MSc (Agric) (Conservation Ecology and Entomology))--University of Stellenbosch, 2010.
ENGLISH ABSTRACT: Ambient temperature plays a key role in insect-physiology, -population dynamics and ultimately -geographic distribution. Here, I investigate the survival of codling moth, Cydia pomonella (Linnaues) (Lepidoptera: Tortricidae), which is a pest of economic importance in pome fruit production, to a wide range of temperature treatments. In this thesis, I first explore how temperature affects the survival and limits to activity of codling moth and secondly investigate if thermal acclimation can improve field performance of moths used in sterile insect technique control programmes under ecologically relevant conditions. First, I found that absolute temperature as well as the duration of temperature exposure significantly affects adult C. pomonella survival. Lethal temperatures, explored between -20 °C to -5 °C and 32 °C to 47 °C over a range of durations, showed that 50% of the adult C. pomonella population killed at -12 °C and at 44 °C after 2 hrs for each treatment. At high temperatures a pretreatment at 37 °C for 1 hr dramatically improved survival at 43 °C for 2 hrs from 20% to 90% (p<0.0001). Furthermore, high temperature pre-treatments (37 °C for 1 hr) significantly improved low temperature survival at -9 °C for 2 hrs. In sum, my results suggest pronounced plasticity of acute high temperature tolerance in adult C. pomonella, but limited acute low temperature responses. Secondly, low-temperature acclimated laboratory-reared moths were recaptured in significantly higher numbers (d.f. = 2, χ2 = 53.13 p<0.001), by sex pheromone traps, under cooler conditions in the wild relative to warm-acclimated or non-acclimated moths. However, these improvements in low temperature performance in cold-acclimated moths came at a cost to performance under warmer conditions in the wild. This novel study demonstrates the importance of thermal history on C. pomonella survival and clear costs and benefits of thermal acclimation on field and laboratory performance, and thus, the potential utility of thermal pre-treatments for improved efficacy in the sterile insect technique programme for C. pomonella control under cooler, springtime conditions. Finally, on a global scale, this study highlights that low and high temperatures could play a role in CM adult survival through direct mortality and thus, may influence, or have influenced in the pest, population dynamics.
AFRIKAANSE OPSOMMING: Temperatuur speel ‘n belangrike rol in die fisiologie, populasiedinamika en geografiese verspreiding van insekte. In hierdie tesis ondersoek ek die rol van ‘n wye reeks temperature op die oorlewing van kodlingmot Cydia pomonella (Linnaues) (Lepidoptera: Tortricidae), ‘n sagtevrug pes-spesie van ekonomiese belang. Ek ondersoek hoofsaaklik die effek van temperatuur op die fisiologie en fiksheid van kodlingmot, asook die mate waartoe termiese akklimasie (‘n mate van aanpassing) die veldgedrag van die steriele insek beheer-metode (SIT), d.m.v. kodlingot, in relevante omgewingstemperature kan verbeter. Ek het (i) gevind dat die temperatuur en duur van die temperatuur toediening ‘n betekenisvolle toename in volwasse C. pomonella oorlewing tot gevolg het. In die deel van die studie is temperature tussen -20 °C en -5 °C and tussen 32 °C en 47 °C ondersoek oor ‘n reeks van 0.5, 1, 2, 3 en 4 ure van duur. In kort lei -12 °C en 44 °C vir 2 uur onderskeidelik tot die uitsterf van 50% van die volwasse C. pomonella populasie. Indien die motte vooraf gehou is by 37 °C vir ongeveer 1 uur, is oorlewing by 43 °C vir 2 ure betekenisvol verbeter van 20% tot 90% (p<0.0001). Hoër temperatuur vooraf-blootstellings (akklimasie), by 37 °C vir 1 uur, het daartoe gelei dat lae temperatuur lae-temperatuur-oorlewings by -9 °C vir 2 ure betekenisvol verbeter het. Oor die algemeen het die resultate gedui dat hoër akute temperatuurstoleransie in C. pomonella bestaan, maar beperkte akute lae-temperatuur reaksies bestaan. Verder het lae-temperatuur akklimasie (laboratorium geteelde) motte ‘n betekenisvolle hoër getal hervangste deur geslagsferomone in koeler omgewings opgelewer (v.i. = 2, χ2 = 53.13, p<0.001) in vergelyking met warmer-temperatuur geakklimatiseerder motte. Hierdie verbeteringe in laetemperatuur reaksies vanaf lea-temperatuur akklimasie groepe is teen ‘n koste teen warmer reaksie-toestande in die natuur geïs. Hierdie eersdaagse studie demonstreer die belang van historiese temperatuur op die oorlewing van C. pomonella. Die kostes- en voordele van termiese akklimasie op veld- en laboratoriumpopulasie reaksies en die potensiële gebruik daarvan in die verbetering van steriele insek tegniek programme, onder koeler omstandighede, is uitgelig. Laastens, beklemtoon hierdie studie die belangrikheid van temperatuur as bepalende faktor van kodlingmot-oorlewing en die invloed daarvan op die vrugte-pes populasiedinamika.

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

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.

The Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), a major pest of horticulture in eastern Australia, is a relatively poor coloniser of new habitat. This thesis examines behavioural properties that might limit the ability of B. tryoni to establish new populations. As the potential for B. tryoni to establish an outbreak population may be most directly limited by mechanisms associated with dispersal and mating behaviour, these two factors were the focus of this research project. The relevance of dispersal and mating behaviour for control of outbreak populations was assessed. Dispersal (i) Dispersal patterns of males and females are not different. Dispersal of post-teneral male B. tryoni from a point within an orchard near Richmond, New South Wales, was monitored following temporally replicated releases. Application of sterile insect technique (SIT) requires knowledge of dispersal from a release point so that effective release rates can be determined. In addition, dispersal following introduction to new habitat can lead to low or negative population growth and an Allee effect. In Spring and Autumn, 2001 – 2003, three different strains of B. tryoni were released: (1) wild flies reared from infested fruit collected in the Sydney Basin; (2) a laboratory-reared strain with a colour mutation (white marks); and (3) sterile flies obtained by gamma-irradiation of a mass-reared strain. Dispersal was monitored using a grid of traps baited with the male attractant, cuelure. During the majority of releases, flies were massmarked using a self-marking technique and fluorescent pigment powder to enable identification of recaptured flies. A preliminary study found that fluorescent pigment marks had no effect on adult survival and marks did not fade significantly in the laboratory over a period of five weeks after eclosion. As cuelure repels inseminated sexually mature female B. tryoni, unbaited, coloured flat sticky traps, and black and yellow sticky sphere traps baited with a food lure (protein autolysate solution) were used to supplement traps baited with cuelure. The effectiveness of these two sticky trap types was assessed, and recaptures used to compare patterns of dispersal from a release point by male and female B. tryoni. Fluorescent yellow (chartreuse), green, and clear unbaited flat sticky traps were relatively ineffective for monitoring dispersal of sterile male and female B. tryoni, recapturing only 0.1% of released sterile flies. Monitoring dispersal with sticky ball traps baited with protein autolysate solution was more successful, with yellow spheres and black spheres recapturing 1.7% and 1.5%, respectively. Trap colour had no effect on recaptures on flat sticky traps or sticky spheres. Equal recapture rates on yellow and black sticky sphere traps suggests that the odour of yeast autolysate solution was more important than colour for attraction of post-teneral flies to traps. Using the results of recaptures on odoriferous black and yellow sticky sphere traps within one week of release, regression equations of male and female recaptures per trap were found to be similar (Figure 4-3). This is the first study to clearly indicate that post-teneral dispersal patterns of male and female B. tryoni released from a point do not differ, enabling the use of existing models to predict density of both sexes of B. tryoni following post-teneral dispersal. (ii) Males disperse further in Spring than in Autumn, but this is not temperature-related. Analysis of replicated recaptures in traps baited with cuelure revealed that dispersal of male B. tryoni in an orchard near Richmond, New South Wales, was higher in Spring than in Autumn (Figure 5-6). As the maximum daily temperature was significantly higher in Spring than in Autumn this result was unexpected, since earlier studies have found that B. tryoni disperse at the onset of cool weather in search of sheltered over-wintering sites. Dispersal of post-teneral B. tryoni may have been affected by habitat suitability; it was found that seasonal trends in dispersal could have been influenced by local habitat variables. Low mean dispersal distances in Autumn may be explained by the presence of fruiting hosts in the orchard, or the availability of resources required by over-wintering flies. There was no significant correlation between temperature and mean dispersal distance, suggesting that higher rates of dispersal cannot be explained by temperature-related increases in activity. Recapture rate per trap was significantly negatively correlated with increasing daily maximum and average temperature. This may have consequences for detection of B. tryoni outbreaks in quarantine areas due to reduced cuelure trap efficiency. (iii) Maturity and source variation affect dispersal and response to cuelure. This research indicated that most male and female B. tryoni do not disperse far from a release point, suggesting that an invading propagule would not spread far in the first generation. However, there is considerable variation in flight capability among individuals. Comparison of wild, laboratory-reared white marks, and gamma-irradiated sterile male B. tryoni indicated that mean dispersal distance and redistribution patterns were not significantly affected by fly origin. Despite no difference in dispersal distance from the release point, recaptures of wild and sterile males per Lynfield trap baited with cuelure were highest within one week after release, whereas recaptures of white marks males per trap increased in the second week. This result may offer evidence to support the hypothesis that sterile male B. tryoni respond to cuelure at an earlier age. Rearing conditions used to produce large quantities of males for sterilisation by gamma-irradiation may select for earlier sexual maturity. Mating Behaviour (i) Density and sex ratio do not affect mating, except at low densities. Demographic stochasticity in the form of sex ratio fluctuations at low population density can lead to an observed Allee effect. The effect of local group density and sex ratio on mating behaviour and male mating success of a laboratory-adapted strain of B. tryoni was examined in laboratory cages. In the laboratory-adapted strain of B. tryoni used in this study, a group of one female and one male was sufficient for a good chance of mating success. The proportion of females mated and male mating success was not significantly affected by density or sex ratio, although variability in male mating success was higher at low density. This could indicate that mating success of B. tryoni can be reduced when local group density is low owing to decreased frequency in encounters between males and females. (ii) Mass-reared males exhibit aberrant mating behaviour, but this does not reduce mating success. Strong artificial selection in mass-rearing facilities may lead to decreased competitiveness of sterile males released in SIT programs as a result of alteration or loss of ecological and behavioural traits required in the field. The effects of domestication and irradiation on the mating behaviour of males of B. tryoni were investigated by caging wild, mass-reared and sterile (mass-reared and gammairradiated) males with wild females. Mating behaviour of mass-reared males was different from that of wild males, but behaviour of wild and sterile males was similar. Mass-reared males were found to engage in mounting of other males much more frequently than wild and sterile males, and began calling significantly earlier before darkness. Male calling did not appear to be associated with female choice of mating partners, although this does not exclude the possibility that calling is a cue used by females to discriminate between mating partners. Conditions used to domesticate and rear large quantities of B. tryoni for SIT may select for an alternative male mating strategy, with mass-reared males calling earlier and exercising less discrimination between potential mating partners. Despite differences in behaviour of wild, mass-reared and sterile males, frequency of successful copulations and mating success were similar. (iii) Pheromone-calling by males was increased in larger aggregations but this did not result in significantly more female visits. Finally, large laboratory cages with artificial leks were used to investigate the importance in B. tryoni of male group size for female visitation at lek sites and initiation of male pheromone-calling. Calling propensity of male B. tryoni was increased by the presence of conspecific males. Females visited the largest lek more frequently than single males, but there was no correlation between lek size and female visitation. Female B. tryoni had a limited capacity to perceive a difference between the number of calling males; female visitation at leks was only weakly associated with male calling, suggesting that lek size and the number of pheromone-calling males may not be the only factor important in locating mates in B. tryoni. The weak, but positive correlation between male calling and female visitation may indicate that passive attraction maintains lek-mating in B. tryoni. Further studies are essential on mating behaviour of B. tryoni, including identification of male mating aggregations in the field, measurement of habitat variables associated with male aggregations, the influence of density on wild B. tryoni mating success, and the role of pheromone-calling, in order to optimise use of SIT for control of this pest.

The Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), a major pest of horticulture in eastern Australia, is a relatively poor coloniser of new habitat. This thesis examines behavioural properties that might limit the ability of B. tryoni to establish new populations. As the potential for B. tryoni to establish an outbreak population may be most directly limited by mechanisms associated with dispersal and mating behaviour, these two factors were the focus of this research project. The relevance of dispersal and mating behaviour for control of outbreak populations was assessed. Dispersal (i) Dispersal patterns of males and females are not different. Dispersal of post-teneral male B. tryoni from a point within an orchard near Richmond, New South Wales, was monitored following temporally replicated releases. Application of sterile insect technique (SIT) requires knowledge of dispersal from a release point so that effective release rates can be determined. In addition, dispersal following introduction to new habitat can lead to low or negative population growth and an Allee effect. In Spring and Autumn, 2001 � 2003, three different strains of B. tryoni were released: (1) wild flies reared from infested fruit collected in the Sydney Basin; (2) a laboratory-reared strain with a colour mutation (white marks); and (3) sterile flies obtained by gamma-irradiation of a mass-reared strain. Dispersal was monitored using a grid of traps baited with the male attractant, cuelure. During the majority of releases, flies were massmarked using a self-marking technique and fluorescent pigment powder to enable identification of recaptured flies. A preliminary study found that fluorescent pigment marks had no effect on adult survival and marks did not fade significantly in the laboratory over a period of five weeks after eclosion. As cuelure repels inseminated sexually mature female B. tryoni, unbaited, coloured flat sticky traps, and black and yellow sticky sphere traps baited with a food lure (protein autolysate solution) were used to supplement traps baited with cuelure. The effectiveness of these two sticky trap types was assessed, and recaptures used to compare patterns of dispersal from a release point by male and female B. tryoni. Fluorescent yellow (chartreuse), green, and clear unbaited flat sticky traps were relatively ineffective for monitoring dispersal of sterile male and female B. tryoni, recapturing only 0.1% of released sterile flies. Monitoring dispersal with sticky ball traps baited with protein autolysate solution was more successful, with yellow spheres and black spheres recapturing 1.7% and 1.5%, respectively. Trap colour had no effect on recaptures on flat sticky traps or sticky spheres. Equal recapture rates on yellow and black sticky sphere traps suggests that the odour of yeast autolysate solution was more important than colour for attraction of post-teneral flies to traps. Using the results of recaptures on odoriferous black and yellow sticky sphere traps within one week of release, regression equations of male and female recaptures per trap were found to be similar (Figure 4-3). This is the first study to clearly indicate that post-teneral dispersal patterns of male and female B. tryoni released from a point do not differ, enabling the use of existing models to predict density of both sexes of B. tryoni following post-teneral dispersal. (ii) Males disperse further in Spring than in Autumn, but this is not temperature-related. Analysis of replicated recaptures in traps baited with cuelure revealed that dispersal of male B. tryoni in an orchard near Richmond, New South Wales, was higher in Spring than in Autumn (Figure 5-6). As the maximum daily temperature was significantly higher in Spring than in Autumn this result was unexpected, since earlier studies have found that B. tryoni disperse at the onset of cool weather in search of sheltered over-wintering sites. Dispersal of post-teneral B. tryoni may have been affected by habitat suitability; it was found that seasonal trends in dispersal could have been influenced by local habitat variables. Low mean dispersal distances in Autumn may be explained by the presence of fruiting hosts in the orchard, or the availability of resources required by over-wintering flies. There was no significant correlation between temperature and mean dispersal distance, suggesting that higher rates of dispersal cannot be explained by temperature-related increases in activity. Recapture rate per trap was significantly negatively correlated with increasing daily maximum and average temperature. This may have consequences for detection of B. tryoni outbreaks in quarantine areas due to reduced cuelure trap efficiency. (iii) Maturity and source variation affect dispersal and response to cuelure. This research indicated that most male and female B. tryoni do not disperse far from a release point, suggesting that an invading propagule would not spread far in the first generation. However, there is considerable variation in flight capability among individuals. Comparison of wild, laboratory-reared white marks, and gamma-irradiated sterile male B. tryoni indicated that mean dispersal distance and redistribution patterns were not significantly affected by fly origin. Despite no difference in dispersal distance from the release point, recaptures of wild and sterile males per Lynfield trap baited with cuelure were highest within one week after release, whereas recaptures of white marks males per trap increased in the second week. This result may offer evidence to support the hypothesis that sterile male B. tryoni respond to cuelure at an earlier age. Rearing conditions used to produce large quantities of males for sterilisation by gamma-irradiation may select for earlier sexual maturity. Mating Behaviour (i) Density and sex ratio do not affect mating, except at low densities. Demographic stochasticity in the form of sex ratio fluctuations at low population density can lead to an observed Allee effect. The effect of local group density and sex ratio on mating behaviour and male mating success of a laboratory-adapted strain of B. tryoni was examined in laboratory cages. In the laboratory-adapted strain of B. tryoni used in this study, a group of one female and one male was sufficient for a good chance of mating success. The proportion of females mated and male mating success was not significantly affected by density or sex ratio, although variability in male mating success was higher at low density. This could indicate that mating success of B. tryoni can be reduced when local group density is low owing to decreased frequency in encounters between males and females. (ii) Mass-reared males exhibit aberrant mating behaviour, but this does not reduce mating success. Strong artificial selection in mass-rearing facilities may lead to decreased competitiveness of sterile males released in SIT programs as a result of alteration or loss of ecological and behavioural traits required in the field. The effects of domestication and irradiation on the mating behaviour of males of B. tryoni were investigated by caging wild, mass-reared and sterile (mass-reared and gammairradiated) males with wild females. Mating behaviour of mass-reared males was different from that of wild males, but behaviour of wild and sterile males was similar. Mass-reared males were found to engage in mounting of other males much more frequently than wild and sterile males, and began calling significantly earlier before darkness. Male calling did not appear to be associated with female choice of mating partners, although this does not exclude the possibility that calling is a cue used by females to discriminate between mating partners. Conditions used to domesticate and rear large quantities of B. tryoni for SIT may select for an alternative male mating strategy, with mass-reared males calling earlier and exercising less discrimination between potential mating partners. Despite differences in behaviour of wild, mass-reared and sterile males, frequency of successful copulations and mating success were similar. (iii) Pheromone-calling by males was increased in larger aggregations but this did not result in significantly more female visits. Finally, large laboratory cages with artificial leks were used to investigate the importance in B. tryoni of male group size for female visitation at lek sites and initiation of male pheromone-calling. Calling propensity of male B. tryoni was increased by the presence of conspecific males. Females visited the largest lek more frequently than single males, but there was no correlation between lek size and female visitation. Female B. tryoni had a limited capacity to perceive a difference between the number of calling males; female visitation at leks was only weakly associated with male calling, suggesting that lek size and the number of pheromone-calling males may not be the only factor important in locating mates in B. tryoni. The weak, but positive correlation between male calling and female visitation may indicate that passive attraction maintains lek-mating in B. tryoni. Further studies are essential on mating behaviour of B. tryoni, including identification of male mating aggregations in the field, measurement of habitat variables associated with male aggregations, the influence of density on wild B. tryoni mating success, and the role of pheromone-calling, in order to optimise use of SIT for control of this pest.

Thesis (MSc)--Stellenbosch University, 2011.
ENGLISH ABSTRACT: The Mediterranean fruit fly (medfly), Ceratitis capitata, is a highly invasive species throughout the world and considered as one of the most successful agricultural and economical pests. The increase of global trade in fruit and human travel combined with the biology of the medfly has allowed the species to spread from its proposed Afrotropical origin, to a number of locations throughout the world. In the Western Cape various control strategies have been implemented to control medfly populations, including insecticides and more environmentally-friendly techniques such as the Sterile Insect Technique (SIT). In order to be effective, however, an SIT program requires some knowledge of the population structure and the movement of individuals between pest-occupied sites. The identification of sites from which re-invasion is most likely to occur and knowledge regarding the primary routes through which pests are likely to re-establish are critical to ensure successful SIT programmes. To provide this important information to SIT and area-wide pest control programs in South Africa, sampling at two different spatial scales (regional- and fine-scale) in South Africa was undertaken. Regional scale sampling was done at 13 locations in the Western Cape and fine scale sampling was done at 13 locations within the Ceres-valley. All individuals were genotyped at 11 polymorphic microsatellite markers and selected individuals from the regional scale were sequenced for the mitochondrial gene COI. Our results show that populations at regional- and fine-scale in the Western Cape are characterized by high levels of genetic diversity (HEregional = 0.805; HEfine = 0.803). Little or weak population differentiation was detected at the regional- and fine-scales, suggesting overall high levels of gene flow among sampling locations. These findings were supported by coalescent based methods indicating sufficient levels of gene flow to prevent population differentiation between neighbouring (200m) and distant (350km) populations. However, natural dispersal in C. capitata has been shown to rarely exceed 10 km. As such, high levels of gene flow between distant populations are more likely the result of humanmediated dispersal, linked to the movement of fresh produce within South Africa. This high level of gene flow has important implications for pest management practices, as my results suggests that area-wide pest management should be undertaken at a regional scale, rather than on a farm or valley scale. My results are placed within a management framework, and I argue for more stringent control when fruit are transported within South Africa. Of particular interest for future studies is the investigation of gene flow at broader spatial scales (i.e. the whole of South Africa) and a comparison of the genetic diversity, population differentiation and gene flow patterns of C. capitata with that of Ceratitis rosa will be important to establish a successful pest management strategy in South Africa.
AFRIKAANSE OPSOMMING: Die Mediterreense vrugtevlieg (medvlieg), Ceratitis capitata, is ‘n indringerspesie wêreldwyd en word beskou as een van die mees suksesvolle ekonomiese en landbou peste. Die medvlieg het ‘n Afrotropiese oorsprong, maar die toename in wêreldwye handel en reis, gekombineer met die biologie van die medvlieg het gelei tot die verspreiding van die spesie na ‘n groot aantal bestemmings regoor die wêreld. Die Wes-Kaap provinsie van Suid-Afrika implementeer tans verskeie strategieë om medvlieg bevolkings te beheer. Hierdie strategieë sluit in die gebruik van plaagdoders sowel as meer omgewingsvriendelike tegnieke soos die Steriele Insektegniek (SIT). Om ‘n effektiewe SIT program te implementeer vereis basiese kennis ten opsigte van die genetiese struktuur van die bevolking sowel as van die beweging van individue tussen verskillende pesvoorkomsgebiede. Die identifisering van areas van waar herkolonisering mees waarskynlik kan plaasvind en kennis in verband met die primêre roetes waardeur pes spesies hervestig, is van kritiese belang om ‘n suksesvolle SIT program te verseker. Medvlieg individue is op twee verskillende ruimtelike skale (streeks- en plaaslike-skaal) versamel om die nodige inligting aan SIT en area-wye pes beheer programme in Suid-Afrika te verskaf. Streeks-skaal individue is by 13 lokaliteite regoor die Wes-Kaap versamel en plaaslike-skaal individue by 13 lokaliteite in die Ceres-vallei. Alle versamelde individue is vir 11 polimorfiese mikrosatelliet merkers gegenotipeer en DNS volgordebepaling van geselekteerde individue vanuit die streek-skaal is gedoen vir die mitochondriale geen COI. My resultate toon dat bevolkings op beide skale gekarakteriseer word deur hoë vlakke van genetiese diversiteit (HEstreeks = 0.805; HEplaaslik = 0.803) en geen of swak bevolkings differensiasie. Hierdie resultate dui daarop dat daar hoë vlakke van geenvloei tussen bevolkings is. Hierdie bevindinge word verder ondersteun deur metodes gebaseer op die statistiese eienskappe van die genealogiese verhouding tussen allele onder sekere mutasie en demografiese modelle, wat voldoende vlakke van geenvloei aandui tussen nabye (200m) sowel as verafgeleë (350km) bevolkings om bevolkings differensiasie te verhoed. Natuurlike beweging in C. capitata is egter selde meer as 10 km, sodanig kan die hoë vlakke van geenvloei toegeskryf word aan die verspreiding van individue met menslike hulp, spesifiek in die vervoer van varsprodukte in Suid-Afrika. Hierdie hoë vlak van geenvloei het verreikende implikasies vir pes beheer praktyke, omdat my resultate voorstel dat area-wye pes beheer onderneem moet word op ‘n streeks-skaal eerder as op ‘n plaas-tot-plaas of vallei wye area. Ek plaas my resultate in ‘n bestuursraamwerk, waarin ek streng beheer van vrugtevervoer in Suid-Afrika beklemtoon. Verdere navorsing moet fokus op die ondersoek van geenvloei op ‘n landswye skaal (hele Suid-Afrika) in C. capitata, sowel as die vergelyking van die genetiese diversiteit, bevolkings differensiasie en geenvloei patrone van C. capitata met die van Ceratitis rosa om ‘n suksesvolle pes beheer strategie vir Suid-Afrika te formuleer.

The olive fruit fly, Bactrocera oleae, (Rossi) (Diptera:Tephritidae), is a key pest of olive crops. The sterile insect technique (SIT) is an environmentally benign and species-specific method of pest control, aiming to reduce the reproductive potential of a wild population through the mass-release of sterile insects. Previous olive fly SIT trials, involving the release of gamma-ray sterilised mixed-sex populations, achieved limited success. Key problems included altered diurnal mating rhythms of the laboratory-reared insects, leading to assortative mating between released and wild populations, and low competitiveness of the radiation sterilised mass-reared flies. Consequently, the production of competitive, male-only release cohorts is seen as essential. The RIDL (Release of Insects carrying a Dominant Lethal) system is a transgene-based derivative of SIT, one version of which involves the mass release of insects carrying a female specific lethal transgene (fsRIDL). This thesis describes: 1) the development of fsRIDL olive fly strains and the molecular analysis of transgene insertion and function; 2) the analysis of strain life-history parameters; 3) studies into sexual selection and mating compatibility; 4) a caged proof-of-principle population suppression trial; and, 5) selection dynamics on the fsRIDL trait in caged populations. Olive fly fsRIDL strains were developed with full female-lethal penetrance and repressibility. The lead strain displayed similar life-history and sexual competitiveness traits to those of the wild-type strain from which they were derived. In addition, transgenic males showed photoperiod compatibility and strong sexual competitiveness with field-collected wild olive flies. The feasibility of the fsRIDL approach was demonstrated when repeated male releases caused eradication of caged olive fly populations. Although needing field confirmation, these results suggest that fsRIDL olive fly strains may help to mitigate key problems experienced in previous olive fly SIT trials, and could help form the basis of a renewed effort towards olive fly SIT control.

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

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

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.

In order to employ the Sterile Insect Technique (SIT), biologically fit mosquitoes able to compete with their wild counterparts, suitable field sites for mass release of sterilized male mosquitoes, and appropriate methods of rearing genetic sex-separation (GSS) mosquito strains are required. The life history traits and biological fitness of four laboratory-reared, southern African Anopheles arabiensis strains were investigated. Despite being colonized at different times, the four strains demonstrated comparable levels of biological fitness. Three sites in the Kruger National Park were assessed as possible SIT field sites. Mosquito collections were conducted at each site during three summer months. Anopheles arabiensis was predominant at Malahlapanga during each collection period, establishing Malahlapanga as the most appropriate site for SIT field trials. A standard larval diet was shown to be appropriate for rearing An. arabiensis GSS. This work formed the laboratory basis for the evaluation of a SIT strategy for South Africa.

The digestive tracts of pest fruit flies (Tephritidae: Diptera) contain a diverse range of bacteria. Since the 1980’s there has been increasing interest in the role of microbial symbionts in tephritid fruit fly performance, rising sharply in the past decade. The sterile insect technique (SIT) is an environmentally safe insect pest management method that has been implemented against several tephritids and other pest insects of economic significance. The efficacy of SIT relies upon sterile males outcompeting field males to copulate with field females, which then fail to reproduce, resulting in suppression of pest populations. Mass production and sterilisation by irradiation can adversely affect several male fly traits. SIT has also been developed and deployed for the control of Queensland fruit fly, Bactrocera tryoni (Froggatt), Australia’s most significant horticultural pest species. Research has shown that bacteria play an important role in tephritid biology, and some bacterial isolates can improve performance traits, including mating competitiveness. However, little is known about the application of symbiotic bacteria in enhancing tephritid performance in SIT operational programs, and this is particularly the case for B. tryoni. Symbiotic bacteria supplied to mass-reared fruit flies may help overcome some of these issues. However, the effects of tephritid ontogeny, sex, diet and irradiation on their microbiota are not well known. The aim of this PhD was to establish the diversity and abundance of bacterial symbionts in the gut of B. tryoni collected from different laboratory and field environments. Overall my research contributes to the wider research effort on the microbiota of tephritid pest fruit flies. Recent advances in sequencing technology have enabled more insights into the diversity and dynamics gut bacterial communities of insects and the roles they play in insect development. Gut bacteria have been demonstrated to improve the performance of tephritid fruit flies and thus are a promising target in improving the sterile insect technique used in tephritid fruit fly management.

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.