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

Στην παρούσα διατριβή μελετήθηκε μια φυλοσύνδετη θερμοευαίσθητη μετάλλαξη, η wiser, η οποία προέκυψε από μια δυσγενική διασταύρωση με το 23.5 hobo MRF στέλεχος και οφείλεται σε ένθεση Ρ στοιχείου στη θέση 7Ε του Χ χρωμοσώματος. Μελέτες βιωσιμότητας έδειξαν ότι τα κρίσιμα στάδια στα οποία η μετάλλαξη επιφέρει το θάνατο των ατόμων είναι της προνύμφης και της νύμφης. Στο στάδιο της νύμφης στους 29οC τα άτομα πεθαίνουν λίγο πριν ή κατά τη διάρκεια της εκκόλαψης των ακμαίων ατόμων. Η Ρ ένθεση εντοπίζεται στην 5’ ρυθμιστική περιοχή του γονιδίου CG32711. Το γονίδιο CG32711 δίνει δύο μετάγραφα που προκύπτουν από εναλλακτική συρραφή και όχι ένα όπως προβλέπεται στη Flybase. Η προβλεπόμενη μεταφραζόμενη περιοχή είναι κοινή και για τα δύο μετάγραφα. Η ανάλυση κατά Western αποκάλυψε ότι το γονίδιο μεταφράζεται και δίνει δύο πρωτεϊνικές ισομορφές με μοριακό βάρος περίπου 8 και 9 kD. Με βάση τη δομή της πρωτεΐνης, η ισομορφή των 9 kD πρέπει να οφείλεται σε μετα-μεταφραστική τροποποίηση. Στα κανονικά άτομα στα στάδια της προνύμφης εκφράζεται η ισομορφή των 8 kD, της νύμφης και οι δύο και στα ακμαία η ισομορφή των 9 kD. Ο μεταλλαγμένος φαινότυπος στο στέλεχος wiser πρέπει να οφείλεται στην μη σωστή έκφραση της ισομορφής των 9 kD κατά τη διάρκεια της ανάπτυξης. Από τα αποτελέσματα της RNA in situ υβριδοποίησης, της ανοσοϊστοχημείας και της εκτοπικής έκφρασης προκύπτει ότι το γονίδιο εμπλέκεται στη γλοιογένεση, την ανάπτυξη του τραχειακού συστήματος, των αιμοκυττάρων, τη μορφογένεση των φτερών, των ποδιών και των ματιών και είναι απαραίτητο για τη βιωσιμότητα των ατόμων. Η πρωτεΐνη CG32711 μπορεί να δρα ως ενδιάμεσο μόριο σε ένα ευρύτερο μονοπάτι ενεργοποίησης άλλων μορίων που επηρεάζουν τις παραπάνω λειτουργίες. Εκτοπική έκφραση της προβλεπόμενης μεταφραζόμενης περιοχής του γονιδίου οδήγησε σε λειτουργική πρωτεΐνη. Εκφραζόμενη υπό τον έλεγχο της ρυθμιστικής περιοχής του γονιδίου apmd544 οδήγησε στην εκκόλαψη ακμαίων ατόμων wiser στους 29οC. Υπερέκφραση της προβλεπόμενης μεταφραζόμενης περιοχής του γονιδίου CG32711 υπό τον έλεγχο των ρυθμιστικών περιοχών των αλληλομόρφων των γονιδίων apmd544 και elavC155 οδήγησε σε υπερέχουσες φαινοτυπικές ανωμαλίες στα φτερά, τα πόδια, τα μάτια και το θώρακα. Γεγονός που δείχνει ότι η έκφραση του γονιδίου είναι δοσοευαίσθητη. Τέλος, στην 5΄ρυθμιστική περιοχή του γονιδίου CG32711 υπάρχουν περιοχές με διαφορετικές ιδιότητες. Δύο άλλες Ρ ενθέσεις (οι PL26 και PL28) σε διαφορετικές θέσεις της οδηγούν σε θανατογόνες μεταλλάξεις με διαφορετικές ιδιότητες ως προς το πρότυπο έκφρασης. / In the present study we describe the genetic, molecular and developmental properties of an X-linked temperature sensitive mutation in Drosophila melanogaster. The mutant flies emerged at 19oC have incised wings, smaller and rough eyes. For this reason we have named the mutation wiser (wings scalloped-eyes rough). At 29oC the mutation is lethal. The lethality occurs at the stage of late pupae, just before or during the eclosion of imago. The mutation occurred from a dysgenic cross with the 23.5 hobo MRF strain and is due to a P element insertion at the locus 7E. Viability studies showed that the mutation causes the death of the mutants also at the stage of 3rd instar larvae at 29oC and acts as semi-lethal. The P insertion is located 490bp upstream the predicted coding region of the gene CG32711. The gene is transcribed in two mRNA, not in one as predicted in Flybase. The two transcripts have shown quantitative differences among the developmental stages that where examined in both the mutant strain wiser and the wild-type Canton-s both in 19oC and 29oC. The longer transcript derives from alternative splicing in 260nt of the first intron. The predicted translated region is identical to both transcripts. Western analysis revealed that the gene is translated in two protein isoforms of 8 kD and 9 kD probably due to post-translational modification. The pattern expression of the two isoforms in the wild-type strain is the following: at the stages of 3rd instar larvae and 1st instar pupae the isoform of 8 kD, at the stages of 2nd and 3rd instar pupae both isoforms and in adults just the protein isoform of 9 kD is expressed. The mutant phenotype and properties are due to the abnormal expression of the protein isoform of 9 kD during the development of the mutants. RNA in situ hybridization, immunohistochemistry and ectoping expression of the predicted translated region of the gene CG32711 revealed that the gene plays a functional role in gliogenesis, tracheal and hemocyte development, wing, eye and leg formation and is essential for the survival of the individuals. Ectoping expression of the predicted translated region of the gene CG32711 revealed that the translated protein is functional and when expressed under control of the regulatory region of the gene apterous rescues the mutant flies at 29oC. The function of the gene CG32711 is also dosage-sensitive due to the fact that many individuals carrying phenotype abnormalities in the wings, thorax, eyes and legs were developed by the ectoping expression. Two other lethal mutations PL26 and PL28 are located at the 5´ regulatory region of the gene CG32711. The differences concerning the pattern expression and the properties of these two mutations in relation to those of the wiser mutation, revealed that the three mutations affect the same gene but in a different way.

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