Master of Science / Biochemistry and Molecular Biophysics Interdepartmental Program / Subbaratnam Muthukrishnan / The exoskeleton of insects, the cuticle, functions as a support structure and a physical barrier that protects insects from mechanical damage and dehydration. The exoskeleton is mainly made of chitin and proteins, some cross-linked to one another into certain patterns to form the rigid and resistant cuticle. In previous studies from our laboratory, cuticular proteins analogous to peritrophins (CPAPs) and Knickkopf (Knk) were identified and characterized mainly at the pharate adult stage during insect development. However, the dynamic assembly of both CPAP and Knk into the cuticle and the functions of the CPAPs are still not fully understood. Our study is to investigate how these cuticular proteins are assembled into the cuticle during different developmental stages and carry out their functional characterizations in the red flour beetle, Tribolium castaneum. RNA interference (RNAi) experiments that resulted in down-regulation of transcripts for CPAP 1-C, CPAP1-H, CPAP 1-J, CPAP 3-C and Knk genes resulted in molting defects. Confocal and transmission electron microscopic analysis examined protein expression at twelve stages of development, as well as the span from young larva through adult day 3 stages. The results suggested that the CPAP 3-C protein is present in the lower part of endocuticle in the so-called assembly zone and it was not distributed thoughout the procuticle with chitin. Down-regulation of CPAP 3-C transcripts revealed a disorganized assembly zone; however, no loss of chitin content or the laminar architecture of the procuticle was found. Knk protein was present throughout the procuticle and some of the protein was found inside of the epithelial cells.

No description available.

Un objectif important de la biologie du développement est de comprendre la base cellulaire de la morphogenèse, notamment le destin des différentes cellules souches dans l'embryon en développement. En décrivant la morphogenèse des espèces représentatives des différents groupes ou embranchements, on fournit une base solide pour comparer les processus similaires dans les différents organismes, et pour tirer des conclusions sur l'évolution des plans d'organisation des animaux. À cette fin, les scientifiques développent des techniques chez des espèces sélectionnées - celles-ci comprennent la manipulation de la fonction des gènes, le marquage et le suivi des populations distinctes de cellules, et l'imagerie in vivo.Dans cette thèse, je présente mes efforts pour améliorer la boîte à outils génomique du coléoptère Tribolium castaneum. J'utilise une technique d'édition du génome récemment découverte, CRISPR/Cas, pour introduire des modifications précises dans le génome de Tribolium, y compris l'introduction de grands fragments par recombinaison homologue. Je montre que l'expression de tous les composants de CRISPR/Cas est induite de manière efficace par des promoteurs endogènes de Tribolium. En me basant sur ces résultats, je développe VALCYRIE, une approche transgénique pourmarquer des clones de cellules uniques dans l'embryon de Tribolium.Ce travail me permet d'enquêter sur le devenir des cellules dans la région terminale postérieure du blastoderme de Tribolium. En utilisant une approche de marquage clonal, je montre que ces cellules donnent naissance à des cellules germinales primordiales et aumésoderme postérieur. Avec la même méthode, je montre que l'intestin postérieur de Tribolium se développe à partir d'une population distincte de cellules au début de la bandelette germinale. En utilisant une technique de microscopie timelapse haute résolution, je décris le sort de cellules individuelles dans le blastoderme de Triboliumet je fais la lumière sur le plan de développement des segments gnathal et thoracique de l'embryon à ce stade. En outre, je montre que l'amnios de Tribolium augmente considérablement au cours du développement précoce. En me basant sur des données d'imagerie, je passe en revue la cartographie du devenir de la bandelette germinale en ce qui concerne l'amnios et l'ectoderme embryonnaire / An important objective of developmental biology is to understand the cellular basis of morphogenesis, including fates of distinct progenitor cells in the developing embryo. Describing morphogenesis in representative species of different groups or phyla provides a solid basis for comparing similar processes in different organisms, and for drawing conclusions about the evolution of animal body plans. To this end, scientists develop techniques in selected species - these include manipulation of gene function, marking and tracking of distinct populations of cells, and in vivo imaging.In this thesis, I present my efforts to enhance the genomic toolkit of the beetle Tribolium castaneum. I use a recently discovered genome editing technique, CRISPR/Cas, to introduce precise alterations in the Tribolium genome, including the introduction of large fragments by homologous recombination. I show that all CRISPR/Cas components are driven efficiently by endogenous Tribolium promoters. Based on these results, I develop VALCYRIE, a transgenic approach to mark single cell clones in developing Tribolium embryos.This work allows me to investigate the fates of the cells in the posterior terminal region of the Tribolium blastoderm. Using a clonal labeling approach, I demonstrate that these cells give rise to primordial germ cells and posterior mesoderm. With the same technique, I demonstrate that the hindgut of Tribolium develops from a distinct cell population in the early germband. Using high-resolution time lapse microscopy, I describe the fates of single cells in the Triboliumblastodermand shed new light on the fatemap of gnathal and thoracic segments of the embryo at this stage. Furthermore, Ishow that the amnion of Tribolium expands greatly during early development. Based on imaging data, I review the fate map of the early germ band with regard to the amnion and the embryonic ectoderm

Master of Science / Department of Biochemistry / Michael R. Kanost / Resilin is a rubber-like cuticular protein found in many insect species. Resilin is important for jumping and flying of those insects due to the properties of high elasticity and efficient energy storage. Some recombinant proteins or peptides derived from resilin sequences have been synthesized to produce biomaterials that mimic the remarkable properties of resilin. This research focused on resilin in the red flour beetle, Tribolium castaneum. A cDNA for T. castaneum resilin was inserted into plasmid vectors for expression of resilin in Escherichia coli or Bacillus subtilus. Resilin produced in E. coli was used as antigen to produce a rabbit antiserum. Resilin synthesized by B. subtilis as a secreted protein was purified and used for biochemical studies. Resilin is highly expressed in the late pupal stage, and in hind wings, but not found in elytra of pharate adults, indicated by RT-PCR and immunoblot analysis. Recombinant resilin could be cross-linked in the presence of horseradish peroxidase and hydrogen peroxide, detected by appearance of a high molecular weight band on SDS-PAGE, which had blue fluorescence under ultraviolet light, presumably due to dityrosine linkages. RNA interference was used to knock down resilin expression in T. castaneum. Immunoblot and RT-PCR analyses indicated that resilin expression was successfully decreased by RNAi. However, the knockdown adults exhibited no apparent differences in morphology, behavior or life span from control beetles. Blue fluorescence under ultraviolet illumination has frequently been used as an indication of the presence of resilin containing dityrosine cross-links in insect tissues such as wings, wing tendons and leg joints. A similar blue fluorescence was observed in hind wings of T. castaneum. However, this fluorescence was not decreased in hind wings of beetles in which resilin expression was knocked down by RNA interference. There was a blue fluorescence in the hind wings of knockdown beetles, which was similar in distribution to that in wings of control insects. This result suggests that the observed blue fluorescence in T. castaneum hind wings is derived not only from cross-linked resilin but also from components other than resilin, perhaps other cuticular proteins that contain dityrosine cross-links.

Master of Science / Department of Biochemistry / Gerald Reeck / Aphids mount a remarkable salivary secretion to overcome plant host defenses. Our group has previously reported a gene unique to aphids enriched in the salivary glands of the pea aphid A. pisum, C002, which is required for successful feeding on its host plant Vicia fava. Here I present an analysis of genetic variation within the available EST data for C002 in pea aphids. From 596 total ESTs, 332 are full-length, and segregate into 8 validated haplotypes based on the criteria I set in place to access the quality of EST data. Additionally, Armet, is a putative multi-functional gene implicated as a neurotrophic factor during development, and as a part of the unfolded protein response during stress. I employ RNA interference in the model organism T. castaneum to determine the effect of transcript knockdown during development from early in-star larval stages, through pupation, and its effect on adult emergence. I report that knockdown of Armet transcript significantly hinders the ability for beetles to emerge from the pupae.

No description available.

Doctor of Philosophy / Division of Biology / Susan J. Brown / A segmented body plan is conserved in vertebrates and arthropods. Comparative studies implicate a conserved mode of A-P axis patterning and segmentation among vertebrates: Wnt signaling is involved in fate map patterning along the A-P axis and in segmentation in the posterior region of the embryo. On the other hand, comparative studies in arthropods have revealed distinct modes of development between long and short germ insects, which differ both morphologically and genetically. In the short germ insect Tribolium, a Wnt activity gradient contributes to A-P axis patterning and generates a posterior Tc-cad expression gradient that regulates segmentation through a pair-rule gene clock, forming segments sequentially as in vertebrates. In contrast, instead of Wnt activity, a hierarchy of regulatory genes regionalizes the blastoderm and defines segments simultaneously in the long germ insect Drosophila. In Tribolium, Tc-zen-1, Tc-otd-1 and Tc-cad play key roles in patterning serosa, head and trunk regions, respectively, of the blastoderm fate map, which are impacted by changes in Wnt activity levels. However, interactions between these genetic factors have not been described. My work revealed that cross talk between the Wnt and Dpp signaling pathways regulates the expression of Tc-zen-1 to determine the serosa. Furthermore, mutually repression between Tcotd-1 and Tc-cad defines the head and trunk regions while mutual repression between Tc-zen-1 and cad determines the posterior extent of the dorsal serosa. Analysis of target genes of the posterior Tc-cad gradient indicates that the Tc-cad gradient regulates the serial expression of gap genes, which are predominately regulators of Hox genes. Thus the posterior Tc-cad gradient determines segment formation through regulation of pair-rule genes in the insect segmentation clock, and defines segmental identity through regulation of gap genes. Given its effect on Tc-zen-1 and Tc-cad, the Wnt activity gradient is a key organizer of the Tribolium blastoderm fate map. Since homologs of these genes as well as the Wnt signaling pathway have also been identified in several other short germ band insects, and affect cell fates along the A-P body axis, this work provides a new paradigm for the short germ mode of development to guide studies in other arthropods.

Made available in DSpace on 2015-03-26T13:23:50Z (GMT). No. of bitstreams: 1 texto completo.pdf: 269289 bytes, checksum: fda931f23e88417c20515b21a0ea64cb (MD5) Previous issue date: 2005-02-18 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The current work aimed to assess the susceptibility of adults of Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae) and Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), subjected to ozone treatment at different depths in the grain mass, thus estimating the lethal time for 50% and 95% of each species population, and also assess the physiological quality of the maize grains subjected to the ozone treatment. Maize grains with moisture level around 13% (wb) were distributed in cylindrical PVC containers with 20 cm diameter, 100 cm high and connections for gas injection and exhaustion. At 10 cm from the base of the container, a metallic net was placed to sustain the grain and to form a plenum for better gas distribution. Maize grains infested with adults of Sitophilus zeamais and Tribolium castaneum, obtained from laboratory colonies from climatic chamber of the B.O.D. type, were placed in cages of 3.0 cm high and 15.0 cm diameter with top and bottom of voil to evaluate the efficiency of ozone as fumigant. These cages were placed at different grain mass depths (over the plenum, middle and top of grain column) and subjected to a modified atmosphere of 50 ppm ozone under different exposure periods. The ozone was injected in a continuous flow of 8.0 L min-1 in connection located at the base (plenum) of the container. Tests of electrical conductivity, germination potential and humidity level were carried out in the maize grains to assess the ozone effect on them. The grain mass temperature was maintained around 25 oC throughout the experiment. To do that, a temperature controlled chamber was built where the containers were placed. The temperature was monitored through a data acquisition and store system called f-wire. Six cylindrical containers were used in all of the assays and in three of these ozone was injected, while the remaining ones were injected with atmospheric air (control). It was concluded that in general the increase in exposure period increased the efficiency of the treatments with 50 ppm ozone for adults S. zeamais and T. castaneum. The species S. zeamais was more susceptible. The highest exposure period to control 95% of the insects was of 240.75 h for S. zeamais and 390.18 h for T. castaneum. The greatest exposure period to control 50% of the adult insects was 124.20 h for S. zeamais and 234.75 h for T. castaneum. In general, the treatments with modified atmosphere containing 50 ppm ozone and atmospheric air did not affect the physiological quality of the maize grains. / O presente trabalho teve por meta avaliar a suscetibilidade dos adultos de Sitophilus zeamais (Motschulsky) (Coleoptera: Curculionidae) e Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), submetidos ao tratamento com ozônio em diferentes camadas da massa de grãos, estimando-se, assim, os tempos letais (TL) para 50% e 95% da população de cada espécie e, ainda, avaliar a qualidade fisiológica dos grãos de milho submetidos aos tratamentos com ozônio. Grãos de milho com teor de umidade em torno de 13% (b.u.) foram distribuídos em recipientes cilíndricos, construídos em PVC, com 20 cm de diâmetro, 100 cm de altura e conexões para injeção e exaustão de gás. A 10 cm da base do recipiente, colocou-se uma tela metálica para sustentação dos grãos e formação de um plenum para melhor distribuição do gás. Para avaliar a eficácia do ozônio como fumigante, grãos de milho infestados com adultos de Sitophilus zeamais e Tribolium castaneum, obtidos de criação contínua em câmara climática do tipo B.O.D., foram distribuídos em gaiolas de 3,0 cm de altura e 15,0 cm de diâmetro, também em PVC, sendo o fundo e a tampa confeccionados em tecido do tipo organza. Estas gaiolas foram dispostas em diferentes camadas da massa de grãos (sobre o plenum, mediana e superior) e submetidas a uma atmosfera modificada com 50 ppm de ozônio em diferentes períodos de exposição. O ozônio foi injetado em fluxo contínuo de 8,0 L min-1, em conexão localizada na base (plenum) do recipiente. Para avaliar o efeito da fumigação com o ozônio na qualidade do milho, foram realizados testes de condutividade elétrica, potencial de germinação e teor de umidade. Em todo o experimento, a temperatura da massa de grãos foi mantida próxima de 25 oC. Para tanto, construiu-se uma câmara com controle de temperatura onde foram acondicionados os recipientes cilíndricos, sendo esta monitorada por meio de um sistema de aquisição e armazenamento de dados denominado 1-wire. Em todos os ensaios foram utilizados seis recipientes cilíndricos, sendo que em três destes injetou-se ozônio e nos outros foi injetado ar atmosférico (testemunha). Concluiu-se que, em geral, o aumento do período de exposição resultou no aumento da eficácia dos tratamentos com 50 ppm de ozônio para os adultos de S. zeamais e T. castaneum. A espécie que se mostrou mais susceptível foi S. zeamais. O maior período de exposição para o controle de 95% dos insetos foi de 240,75 h para o S. zeamais e de 390,18 h para o T. castaneum. O maior período de exposição para o controle de 50% dos insetos adultos foi de 124,20 h para o S. zeamais e de 234,75 h para o T. castaneum. Em geral, os tratamentos com atmosfera modificada com 50 ppm de ozônio e com ar atmosférico, não afetaram a qualidade fisiológica dos grãos de milho.

No description available.

No description available.