In order to initiate a genetic dissection of the Drosophila melanogaster larval response to light, two new single larva assays were designed: the Checker and ON/OFF assays. Each assay allows quantification of different aspects of the larval visual response by permitting the study of discrete behaviours in a single larva. Results of this study indicate that larvae respond to light by modulating their locomotion. In the Checker assay this can be seen as an increase in residence time spent in dark checks. In the ON/OFF assay this can be measured as a decrease in distance travelled during the light pulse, due at least in part to an increase in head swinging. Concomitantly, the larva exhibits a sharp change in direction from its original path when the lights are turned on. When the lights are turned off, the change in direction in the larval path, although smaller than at lights on, is still greater than in the absence of light transitions. Many of the components previously described to function in adult phototransduction and visual system specification, also have roles in the larval photoresponse as mutations in the genes that encode these components, are able to abolish light perception as measured in both the Checker and ON/OFF assays. However, these mutations disrupt only subsets of the behaviours associated with the larval perception of light, thus suggesting the existence of light detecting mechanism independent of the main visual pathway described for the adult visual system. / Thesis / Master of Science (MS)

No description available.

Muscarinic acetylcholine receptors (mAChRs) are G-protein-coupled receptors that are underutilized for controlling insect pests despite their involvement in various physiological functions. To-date, there are no commercialized insecticides targeting insect mAChRs. In this dissertation, effective target-site synergism was demonstrated in susceptible Drosophila melanogaster where mAChR agonism by pilocarpine enhanced the toxicity of insecticides targeting gamma-aminobutyric acid (GABA)-gated chloride channels, indicating the potential of insect mAChRs as a target for developing novel insecticides/synergists to control resistant pests. A point mutation (A301S) in the GABA-gated chloride channel confers resistance to dieldrin (Rdl), lindane, and fipronil, which I have confirmed using different routes of exposure. However, the same synergistic effect was not achieved in the resistant strain with the presence of this target-site mutation. This difference between two strains is perplexing because there is a change in the efficacy of several compound classes that do not directly act upon GABA-gated chloride channels. Specifically, a point mutation appears to influence how the insect central nervous system (CNS) responds to muscarinic compounds, type I pyrethroids, and acetylcholinesterase (AChE) inhibitors. In the case of acetylcholinesterase, the resistant insect increases the expression of Ace gene encoding this enzyme. Fully understanding how the CNS responds to receptor modifications is not well understood and could have a significant impact to pest management strategies. / Doctor of Philosophy / Insects significantly influence the food production, health, and the economy of the human world. Control of insect pest outbreaks relies on the proper use of insecticides. However, extensive application of insecticides has resulted in pests being able to adapt to these compounds, through insecticide resistance. Ultimately, this will affect currently used pest management strategies. To help alleviate this urgent problem, my dissertation provided an alternative strategy to control pests, which is to use a mixture of two molecules that influence different targets in the insect nervous system that could reduce the use of toxic or deleterious compounds that are the active ingredients. It is important to not solely rely on current insecticides on the market and find new insecticides that work differently. I used the fruit fly to help me understand how insecticide mixtures would work, but also understand how the complex nature of insect adaptations at the level of the nervous system continues to threaten pest management. Based on studies that were performed here, we now have a better understanding on how to investigate the failure of insecticides in the field, which will ultimately help us make new molecules.

Using a behavioural and genetic approach, we employed single-gene mutations and photoreceptor cell ablations to study the molecules and cell-types underlying larval response to light Drosophila larvae modulate their locomotion in response to light In the Checker Assay the response to light is defined as an increased residence time in dark quadrants versus lit. In the ON/OFF Assay, it is in part defined as a decreased path length in the light. Previously, mutations in genes, which function in the adult phototransduction cascade were found to abolish fight perception as defined by a reduction in path length. This response to light was reported to be mediated by rhodopsins, other than Rh1, via a pathway similar to the one present in the adult visual system. After undertaking a similar genetic approach in the Checker Assay, the response to light measured in this assay appears also to be mediated through a similar pathway. Mutations in sine oculis (so), a homeobox gene necessary for proper visual system development, and targeted expression of the cell death gene head involution defective (hid), to larval photoreceptor neurons, abolished light response as measured in the Checker Assay. Thus, mutations affecting development oflarval visual system suggest that this response to light is also housed in the larva’s main photoreceptor organ, the BO. The modular GAL4 system was used to target expression of cell death genes, rpr and hid, to Rh5 and Rh6 expressing larval photoreceptor cells. In strains tested in the ON/OFF Assay, in which Rh5 cells are missing, the response to light is abolished, as measured by both decreased path length and increased head swinging behaviour in the light. In a strain in which Rh6 photoreceptor cells are ablated, this response to light is not abolished. This suggests that Rh5 mediates responses in the ON/OFF Assay, which were previously abolished by mutations in genes operating in the adult phototransduction cascade. Thus Rh5, not Rh6, appears to be necessary in mediating the response to light carried out via a pathway similar to the operating in adult phototransduction. In both ablated strains, Rh5-gal4xUAS-rpr and Rh5-gal4xUAS-hid, the integrity of remaining photoreceptor cells is not compromised, and in the latter strain, the extent of ablation appears to be complete. Previously in the ON/OFF assay, mutations and ablations of cell-types were found to disrupt only a subset of behaviours associated with the larval perception of light. Based upon this evidence it was surmised that Rh1 mediates a basic independent visual system, which operates in the larva. However, uncovering the possible roles in this system was hindered, as parental control strains did not respond. / Thesis / Master of Science (MS)

Le vieillissement est un processus complexe accompagné par une capacité diminuée des cellules à tolérer et répondre aux formes différentes de stress causant des dommages comme l'agrégation de protéine dans les différentes composantes de la cellule. Les chaperons sont des joueurs probablement importants dans le processus de vieillissement en prévenant la dénaturation et l'agrégation des protéines. Chez Drosophila melanogaster, une petite protéine de choc thermique, Hsp22, localisée dans la matrice mitochondriale montre une expression elevée pendant le vieillissement. Sa surexpression chez la mouche augmente la durée moyenne de vie ainsi que la résistance au stress. Bien que Hsp22 montre une activité de chaperon dans des essais in vitro, les mécanismes par lesquels Hsp22 permet d’accroitre la durée de vie in vivo sont toujours inconnus. Une analyse transcriptionelle de tout le génome par microarrays et une analyse comparative du protéome mitochondrial par MALDI-TOF a été entreprise pour dévoiler les différences d’expression entre les mouches surexprimant Hsp22 et les contrôles appropriés. La surexpression générale de Hsp22 en utilisant le système GAL4/UAS dans Drosophila résulte en une augmentation de ~ 30% dans la durée de vie moyenne. L'analyse du transcriptome suggère que Hsp22 joue un rôle dans la détermination de durée de vie en changeant le processus général de vieillissement normal. Effectivement, les mouches surexprimant Hsp22 affichent une surexpression de gènes dont l’expression baisse normalement durant le vieillissement. Les gènes sont impliqués dans la production d'énergie, la biosynthèse des protéines, le taux de renouvellement des protéines et le métabolisme lipidique. L'analyse du protéome mitochondrial soutient aussi un rôle de Hsp22 sur la détermination de la durée de vie en maintenant la fonction mitochondriale et en favorisant la protéolyse. Les présentes données suggèrent l'importance de la maintenance de l’homéostasie protéique durant le vieillissement et discutent des mécanismes potentiels d’extension de la longévitié chez les mouches surexprimant Hsp22. / Aging is a complex process accompanied by a decreased capacity of cells to tolerate and respond to various forms of stresses leading to damages such as protein aggregation in various components of the cell. Chaperones are thus likely important players in the aging process by preventing protein denaturation and aggregation. In Drosophila melanogaster, a small heat shock protein Hsp22 localized in the mitochondrial matrix is preferentially up-regulated during aging. Its over-expression results in an extension of lifespan and an increased resistance to stress. Although Hsp22 has been shown to have a chaperone-like activity in vitro, the mechanisms by which it extends lifespan in vivo are still unknown. Genome-wide transcriptional analysis by microarray and comparative mitochondrial proteomic analysis by MALDI-TOF mass analysis have been performed to unveil differences in long-lived Hsp22 over-expressing flies and normal-lived control flies. Ubiquitous over-expression of Hsp22 using the GAL4/UAS system in Drosophila resulted in a ~ 30% increase in mean lifespan. The genomic analysis suggests that Hsp22 plays a role in lifespan determination by altering the regulation of the overall process of normal aging. Indeed, flies over-expressing Hsp22 display an up-regulation of genes normally down-regulated with age and involved in energy production, protein biosynthesis, protein turnover, and lipid metabolism. Mitochondrial proteomic analysis also supports a putative role of Hsp22 on lifespan determination by maintaining mitochondrial function and favoring proteolysis. The present data suggest the importance of the maintenance of protein homeostasis in aging and potential mechanisms of longevity in the Hsp22 over-expressing flies.

For animals to walk properly, motor neurons (MNs) need to reach their target muscles in the leg and they need to arborize properly. Cell adhesion molecules (CAMs) have pivotal roles in a variety of neural development processes including arborization. Two Immunoglobulin Super Family (IgSF) CAMs, DIP-α and its interacting partner Dpr10, have been shown to play key roles in leg MN arborization in flies. DIP-α is expressed by three leg MNs and dpr10 is expressed by leg muscles. When either DIP-α or dpr10 are removed in flies MNs reach their target but fail to arborize their branches. Interestingly, DIP-α and Dpr10 have some of the highest binding affinities relative to other DIPs and Dpr interacting pairs. Therefore, in this thesis I ask how important are these relatively high affinities and do the MNs have different affinity requirements? I show that decreasing and increasing the affinity between these two proteins in vivo has adverse effects on MN arborization and that different MNs rely on different thresholds of DIP-α::dpr10 binding affinities to arborize properly. Based on these experiments, I then ask how DIPs and Dprs interact when they are co-expressed by the same neuron. Here I provide evidence that altering the ratio of DIP-a::dpr10::dpr6 can inhibit DIP-α’s ability to bind in trans with Dpr10. Furthermore, together with Dr. Nick Morano in the Shapiro lab, we show that DIP-α has a longer linker domain compared to DIPs that are not able to bind in cis and that removing this linker domain in vivo phenocopies some of the effects seen by removing co-expressed dpr6 or dpr10 in DIP-α MNs. Therefore, although DIP-α and its partners Dpr6 and Dpr10 are expressed in MNs, relative concentrations and cis interactions in MNs dictate how DIP-α interacts in trans with Dpr10. Together, this thesis provides a better understanding as to how interactions between CAMs can be modified via affinities, concentration, and co-expression in vivo to ultimately shape axon morphology of Drosophila leg MNs.

Un tissu épithélial est composé de cellules polarisées, étroitement liées les unes aux autres par des jonctions adhérentes. La perte de ces jonctions adhérentes est la première étape dans le développement des cancers au niveau des tissus épithéliaux. Il est donc important de comprendre les mécanismes d’attachement inter-cellulaire. Pour étudier ces interactions, nous utilisons comme modèle l’embryon de drosophile, où une fine régulation des jonctions adhérentes est requise pour l’une des étapes précoces de développement. Durant cette étape du développement, les cellules épithéliales changent de voisines le long de l’axe antéro-postérieur sans perdre leur adhérence cellulaire. Ce processus d’intercalation cellulaire est dû au recrutement polarisé du moteur moléculaire Myosine II au niveau des jonctions qui se désassemblent. Il a été mis en évidence qu’au cours de ce processus la perte de fonction de la voie JAK/STAT perturbe la localisation de la Myosine II. Au cours de ma thèse, j’ai réalisé un crible génétique dans un contexte mutant pour le ligand de la voie JAK/STAT pour me permettre d’identifier des interacteurs potentiellement impliqués dans le contrôle spatial de Myosine II. J’ai pu mettre en évidence plusieurs gènes pouvant être impliqués dans cette intercalation. Parmi ces candidats, je me suis focalisée sur celui montrant le plus fort phénotype : le gène CG13992. La caractérisation de ce gène a fut la seconde étape de mon travail de thèse (car seules les séquences nucléotidiques et protéiques étaient connues). Les résultats obtenus ont permis de mettre en évidence l’implication de ce gène dans la localisation de la Myosine II mais ils restent à confirmer. / Epithelial tissue is composed of polarized cells, which are closely attached to each other by adherens junctions. The loss of adherens junctions is often a key step in the development of cancer in epithelial tissues. It is therefore important to understand the mechanisms of attachment between the cells. To study such epithelial plasticity, we use the Drosophila embryo as a model system, where a fine regulation of adherens junctions is required for one of the early processes of development: germ band elongation. During this process, epithelial cells change their neighbors along the anterior-posterior axis (cell cell intercalation) without loss of cell adhesion. Polarized recruitment of the molecular motor Myosin II at the junctions, that disassemble and reassemble, underlies the intercalation process. In part, intercalation relies on the normal activity of the the JAK / STAT pathway that is crucial for the spatial control of Myosin II. During my PhD, I conducted a genetic screen, in a mutant for the ligand of the JAK / STAT pathway, designed to identify second site interactors for Myosin II control. I identified several genes that appear to be involved in the intercalation process. Among these candidates, I focused on one with the strongest phenotype: the gene CG13992. The functional characterization of this gene was the second stage of my thesis (because only the nucleotide and the protein sequences were known). Preliminary results highlight the involvement of this gene in the localization of Myosin II that remain to be confirmed.

Spatial and temporal variation in the environment are ubiquitous. Traits are called clinal when they vary along an environmental gradient, and this is often interpreted as the result of spatially varying selection. Drosophila melanogaster is known to have many phenotypic and genotypic clines, replicated in many regions of the world. Recent studies have suggested that most clinal variation could be attributed to neutral, demographic processes. Because the environment varies in similar ways with latitude and across seasons, and seasonal variation is orthogonal to demography, one promising approach is to integrate clinal and seasonal variation to infer selection. Here, we test whether there is a genome-wide relationship between clinal and seasonal variation, and whether the pattern is consistent with selection. Also, we investigate the proportion of the variants that should be under latitudinal and seasonal selection to explain the pattern we uncovered. We estimate allele frequency from pooled samples of flies from eight different locations along the east coast of the US, and 13 samples collected in the spring and in the fall in Pennsylvania. We show that there is a genome-wide pattern of clinal variation mirroring seasonal variation. This pattern is stronger for exonic when compared to intergenic regions, consistent with natural selection. We find that the genome-wide relationship between clinal and seasonal variation could be explained by about 6.6% of our SNPs being under latitudinal and seasonal selection. Our results are consistent with the adaptive hypothesis of clinal variation and, together with other observations, leave little room for the role of demography in maintaining clines in D. melanogaster / Variação espacial e temporal são ubíquas. Caracteres são chamados clinais quando variam ao longo de um gradiente ambiental, e isso é interpretado como resultado de seleção estruturada no espaço. Drosophila melanogaster apresenta clinas em diversos caracteres fenotípicos e genotípicos, as quais são replicadas em diferentes regiões do mundo. Estudos recentes sugeriram que grande parte da variação clinal pode ser atribuída a processos neutros. Como o ambiente varia de maneira similar com a latitude e ao longo do ano, e essa variação sazonal é ortogonal à demografia, uma abordagem possível para inferir seleção natural é integrar variação clinal e sazonal. Neste trabalho, nós testamos se há uma relação entre variação clinal e sazonal ao longo do genoma de D. melanogaster. Também, investigamos a proporção de variantes que deveriam estar sob seleção espacial e sazonal para explicar o padrão encontrado. Estimamos a frequência alélica a partir de amostras de pools de moscas coletadas em diferentes oito localidades ao longo da costa leste dos Estados Unidos e em diferentes estações do ano na Pensilvânia, EUA. Nós encontramos um padrão genômico de variação clinal refletindo variação sazonal. Esse padrão é mais forte para variantes em regiões exônicas do que intergênicas, consistente com a ação de seleção. A relação entre variação clinal e sazonal encontrada pode ser explicada se 6,6% dos polimorfismos estiverem sob seleção espacial e sazonal. Nossos resultados são consistentes com a hipótese adaptativa de variação clinal e, junto com outras observações, revelam que o papel da demografia na manutenção de clinas em D. melanogaster é limitado

Une partie conséquente de mon travail a été d'effectuer un crible génétique en utilisant une bibliothèque de mutants générés par insertion aléatoire de Tn5-Sm, un minitransposon bactérien. Le crible a été réalisé dans un contexte défini: celui de mouches-hôtes auxquelles manquait le gène Eater, lequel code un récepteur de phagocytose (Kocks et al., 2005). Dans ces mouches, l'infection n'est plus contrôlée dans l'hémocoele par les hémocytes et les drosophiles mutantes succombent rapidement à une bactériémie. Plusieurs phénotypes bactériens étaient attendus à l'issue de ce crible. Une première catégorie de phénotype prévisible était une virulence accrue, par exemple si les bactéries mutantes devenaientcapables de traverser plus rapidement ou efficacement la barrière intestinale conséquemment à la perte d'un régulateur négatif. Un deuxième type de phénotype attendu était une virulence atténuée pouvant s'expliquer de plusieurs manières: 1- perte de résistance à l'environnement existant dans le lumen intestinal (enzymes digestives et lysozyme, radicaux libres et peptides antimicrobiens induits au niveau de l'épithélium intestinal dans le cadre d'une réponse immunitaire locale de l'hôte); 2- incapacité à traverser la matrice péritrophique; 3-incapacité à envahir les cellules épithéliales (adhésion, pénétration); 4- incapacité à résister aux défenses intracellulaires potentielles; 5- incapacité à sortir du côté basal des entérocytes 6- incapacité à proliférer dans l'hémolymphe ou perte de la résistance à l'action de la réponse immunitairesystémique qui est, quant à elle, fortement induite en l'absence de phagocytose, laquelle empêche chez les mouches sauvages la prolifération des bactéries ayant traversé la paroi intestinale. [...] Dans le cadre d'une infection intestinale, les mouches sauvages (et imd) succombaient en six jours alors que, de manière surprenante, les mouches mutantes de la voie Toll périssaient plus lentement, une situation opposée à celle du modèle de la piqûre septique. Quelques bactéries sont capables de traverser la paroi intestinale mais sont incapables de proliférer à moins que la réponse cellulaire ait été préalablement bloquée. L'épithélium intestinal apparaissait normal à la dissection et la presque totalité des bactéries ingérées étaient tuées dans l'intestin. Après avoir exclu l'hypothèse d'une toxine sécrétée dans le surnageant des bactéries adsorbées sur le filtre sur lequel viennent se nourrir les mouches, nous avons testé l'hypothèse qu'une suractivation de la réponse immunitaire était à l'origine du décès des mouches. La génétique mettant hors de cause les peptides antimicrobiens, la voie Toll n'étant apparemment pas activée dans l'épithélium intestinal, nous avons alors étudié laréponse oxydative induite par l'ingestion de bactéries (Ha et al., 2009), laquelle est capable de tuer les mouches lorsqu'elle n'est pas régulée correctement. Là-aussi, le résultat s'est avéré négatif. En fin de compte, j'ai pu établir que la mort des mouches était due à un état de famine, confirmé par des mesures des réserves métaboliques. Mes travaux ont permis d'établir un nouveau rôle de la voie Toll dans la résistance à la famine, en présence ou absence d'infection, qui sera peut-être à mettre en relation avec un rôle métabolique de la voie Toll consistant à bloquer la voie de réponse à l'insuline lors d'une infection. En conclusion, mes travaux permettent de mieux comprendre les relations hôte-pathogènequi s'établissent lors d'une infection intestinale. / For the systematic study of bacteri al virulence factors, we initially planned to screen the 12,000 mutant strains of the miniTn5-Sm tranposon-induced mutant bank in a wild-type S. marcescens strain Db10. Phagocytosis-deficient eater mutant flies (Kocks et al., 2005) were used in this screen to isolate the bacterial strains mutated for virulence factors and the genes responsible for crossing the gut barrier. In the eater mutant background, flies succumb to septicemia caused by the rapid proliferation of the bacteria in the hemolymph. Out of 1348 mutant strains screened, 58 candidate mutants have been isolated. Only 20% percent of the potential mutant strains displayed an increased virulence indicating that there are very few factor(s) that negatively control the virulence program of the bacterium. The fly survival phenotypes induced by the candidate mutants isolated in the first round of screen were retested. Only those bacterial strains that were consistent with the phenotype were chosen for the molecular identification of the transposon insertion sites using one primer PCR (Karlyshev et al., 2000). Once the genes impaired in each case had been identified, they were knocked off in the S. marcescens Db10 by site specific plasmid insertion mutagenesis. A mutant strain with the transposon inserted into the fliR gene, a component of the type III flagellar protein export system, exhibited attenuation of virulence. The plasmid insertionmutant strain generated to interrupt the gene fliR reproduced the fly survival phenotyp, indicating that the fliR gene is important for the virulence of S. marcescens. The fliR mutants are able to cross the peritrophic matrix, functionally similar to the human mucus. The bacteria were found in the vicinity of the epithelial cells but were not able to efficiently invade the intestinal epithelium as compared to the wild-type strain. Consequently lower titer of FliR mutants was found in the hemolymph. The inefficiency of the FliR mutants to invade cells was also confirmed in ex-vivo assay using insect cells.I thus demonstrated that the fliR gene which is important in the motility apparatus is also required by S. marcescens for the crossing of the epithelial barrier of D. melanogaster.[...]A strong oxidative response is triggered by D. melanogaster in the midgut against commensals and pathogens (Ha et al., 2009). In order to check whether the strong oxidative immune response is eventually killing the flies themselves, hydrogen peroxide was chemically neutralized in the midgut during the S. xylosus A. oral infection. No difference inthe fly survivals was observed with or without neutralization of the oxidative response indicating that over-production of reactive oxygen species (ROS) does not seem to be responsible for the fly death caused by a very low number of bacteria. Flies could efficiently survive to killed bacteria and filtered supernatant solution from overnight bacterial culture indicating that they do not die to the toxins released by the bacteria. Most surprisingly MyD88-, the Toll pathway-, mutant flies were surviving better to S. xylosus A. oral infection. A series of experiments lead us to the finding that the flies actually succumbed to starvation when orally infected with S. xylosus and that the MyD88 is required for the starvation susceptibility in microbiota-mediated manner. In conclusion my work has lead us to the better understanding of the host-bacterial interactions in the intestine.

A velocidade específica de respiração (QO2) é um parâmetro fundamental para entender-se o metabolismo e o estado fisiológico celular, fornecendo informações úteis para o processo e controle em biorreatores. Neste trabalho, cultivou-se diferentes células de insetos em ambiente controlado medindo-se o QO2 e concentração crítica de oxigênio (Ccrít). Foram utilizadas nos ensaios células de insetos Spodoptera frugiperda (Sf9) não infectadas e células de Drosophila melanogaster (S2) selvagem e recombinantes, utilizadas na expressão de diferentes proteínas. Todas as experiências foram realizadas em biorreator Inceltech com volume de trabalho de 1L, mantido a temperatura de 28ºC, agitação de 100 rpm e oxigênio dissolvido (OD) a 40% da saturação de ar, com difusão por membrana de silicone com mistura gasosa (O2 e N2) e vazão gasosa constante. Foi utilizado meio de cultura Sf900II sem soro fetal bovino. O QO2 foi medido pelo método dinâmico e pelo balanço de oxigênio na fase líquida. Neste trabalho foi implementado um novo processo durante o método dinâmico para interromper completamente a transferência gasosa durante a execução deste método. Implementou-se também uma metodologia para medição de Ccrít. Chegou-se a concentrações máximas celulares (Xm), velocidades máximas específicas de respiração (QO2) na fase exponencial e Ccrít, conforme segue: 1) Sf9 (ATCC 1711): Xm - 10,7.106 cel/mL; QO2 - 74,7.10-18 molO2/(cel.s); 2) S2 (Invitrogen): Xm - 51,2.106 cel/mL; QO2 - 3,4.10-18 molO2/(cel.s); Ccrít - 10%; 3) S2AcGPV2 (transfectadas para expressão de GPV): Xm - 26,6.106 cel/mL; QO2 -16,0.10-18 molO2/(cel.s); Ccrít - 10%; 4) S2MtEGFP (transfectadas para expressão de EGFP): Xm - 17,8.106 cel/mL; QO2 - 25,8.10-18 molO2/(cel.s); Ccrít - 5%; 5) S2AcHBsAgHy (transfectadas para expressão de HBsAg): Xm - 16,6.106 cel/mL; QO2 -33,6.10-18 molO2/(cel.s); Ccrít - 12%. Conclui-se que as linhagens selvagens e transfectadas de S2 possuem entre si uma atividade respiratória diferente e também que as novas metodologias implantadas verificaram-se satisfatoriamente. / Specific respiration rate (QO2) is a key parameter to understand cell metabolism and physiological state, providing useful information for process supervision and control. In this work, we cultivated different insect cells in a very controlled environment, being able to measure QO2 and critical oxygen concentration (Ccrit). Wild Spodoptera frugiperda (Sf9) and wild and transfected Drosophila melanogaster S2 cells (able to produce different proteins) were used. All experiments were performed in 1-liter working volume Inceltech bioreactor, maintaining temperature controlled at 28ºC, agitation rate at 100 rpm, and dissolved oxygen (DO) at 40% of air saturation, through membrane diffusion of mixed gases (O2 and N2) at constant total flow rate. SF900II serum free medium was used. QO2 was measured through dynamic method and oxygen mass balance in the liquid phase. In this work a new process was implemented during the dynamic method to interrupt completely the oxygen transfer during the execution of this method. It was also implemented a methodology for measurement of Ccrít (determined when DO reduces its decay rate, without oxygen transfer). Maximum cell concentration (Xm), maximum specific respiration rate (QO2) in the exponential phase and Ccrít were reached, as follows: 1) Sf9 (ATCC 1711): Xm - 10,7.106 cel/mL; QO2 - 74,7.10-18 molO2/(cel.s); 2) S2 (Invitrogen): Xm - 51,2.106 cel/mL; QO2 - 3,4.10-18 molO2/(cel.s); Ccrít - 10%; 3) S2AcGPV2 (transfected for GPV expression): Xm - 26,6.106 cel/mL; QO2 -16,0.10-18 molO2/(cel.s); Ccrít - 10%; 4) S2MtEGFP (transfected for EGFP expression): Xm - 17,8.106 cel/mL; QO2 - 25,8.10-18 molO2/(cel.s); Ccrít - 5%; 5) S2AcHBsAgHy (transfected for HbsAg expression): Xm - 16,6.106 cel/mL; QO2 -33,6.10-18 molO2/(cel.s); Ccrít - 12%. From these results, it can be concluded that the studied cell lines have different respiration activity and the new developed methodologies behave satisfactorily.