Due to concerns with synthetic neurotoxic insecticides used in insect pest management, alternative control methods are sought. Methoprene is a juvenile hormone analogue, which is well-known for its lethal effects on immature insects, and is registered as a stored-grain insecticide in the U.S.A. and Australia. However, less is known about its sub-lethal effects. Experiments were conducted to investigate the effects of methoprene (Diacon II) on heat tolerance, cold tolerance and progeny production of Tribolium castaneum (Herbst) when late-instar larvae or adults were exposed to wheat treated with methoprene. Methoprene at 3.33 ppm or higher, reduced heat tolerance of adults at 46ºC. In contrast, methoprene did not affect the heat tolerance of larvae. Cold tolerance and cold acclimation of both adults and larvae was not affected by methoprene. Exposure of larvae to 0.001 or 0.0165 ppm of methoprene on wheat had 37 and 72% reduction in adult emergence, respectively. The surviving adults had normal movement, but their progeny production was reduced by 71%. Males were affected more than females. Exposure of adults to methoprene (66.6 ppm) did not reduce progeny production. Methoprene is used as a surface treatment to control insects in empty grain bins, processing facilities and warehouses. Experiments were conducted to assess the effect of surface material, temperature and cleaning practices on methoprene residual efficacy. A bioassay with late-instar larvae at 30ºC until adult emergence was used to measure methoprene residual efficacy. Methoprene applied at the label rate (0.0003 mg/cm2) on varnished wood, held at 20, 30 or 35ºC for 24 weeks, prevented all development of larvae into adults. Conversely, concrete surfaces allowed 22% adult emergence after 8 weeks and 69% after 24 weeks. Temperatures, 20, 30 or 35ºC, that the concrete surfaces were held before the bioassay, did not affect this decline in efficacy. Presence of flour or repeated removal of flour (cleaning) reduced the efficacy of methoprene on concrete surfaces, but not on varnished wood. Maintaining concrete surfaces at 65ºC for 48 hours did not reduce efficacy. The implications of these results are discussed in the context of controlling stored-product insects, as well as insect physiology.

No abstract.

Group B Sox genes play a critical developmental role in both vertebrates and insects. Within the model species Drosophila melanogaster, two SoxB genes, Dichaete and SoxNeuro, have been shown to act as ‘master regulators’ in the early development of the central nervous system. SoxB genes have only been characterised in a handful of arthropod species thus far, with most work to date focusing on drosophilids. The purpose of this investigation was twofold. First, I set out to resolve the phylogenetic origins of arthropod SoxB genes, as mutually exclusive models explaining their emergence are still contested. I have identified and annotated the SoxB of several invertebrate taxa. In total, my investigation includes 24 different metazoan taxa, and represents the largest investigation of arthropod SoxB phylogeny to date. In light of this research, I have proposed a new model of SoxB evolution which resolves the conflicting elements of the two primary competing models. Second, to study the evolution of SoxB in terms of functional conservation/divergence, I selected the emerging model organism Tribolium castaneum to draw a comparative analysis with Drosophila melanogaster. I first began by characterising the spatiotemporal expression patterns of SoxNeuro mRNA in early Tribolium embryos using whole mount in situ hybridisation, and examined published Dichaete expression patterns in the context of central nervous system development in T. castaneum. Using these data, I draw a comparison to the expression profiles of Dichaete and SoxNeuro orthologues in Drosophila melanogaster and other species. I have found that both Dichaete and SoxNeuro expression patterns in the developing central nervous system are remarkably well-conserved across species. I also attempted to characterise genome-wide binding for both Dichaete and SoxNeuro proteins in Tribolium in what would have represented the first genomic investigation of its kind in this emerging species. Using a tethered DNA adenine methyltransferase (Dam) enzyme for both SoxNeuro and Dichaete, I hoped to characterise the genomic loci with which each protein interacts within the beetle genome (a technique known as DamID). Unfortunately, these last set of experiments have proved unsuccessful, despite several attempts which have made use of different promoters, different DNA enrichment methodologies, and tackling unforeseen DNA contamination issues. Nevertheless, the troubleshooting experiments that I have carried out will pave the way for further genomic experiments in Tribolium, easing the establishment of genomic research in this emerging organism.

No description available.

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Submitted by Mario BC (mario@bc.ufrpe.br) on 2017-05-17T14:57:52Z No. of bitstreams: 1 Amaury Soares de Brito.pdf: 1041809 bytes, checksum: 74b076dde16ba738e59a5ac81be89a70 (MD5) / Made available in DSpace on 2017-05-17T14:57:52Z (GMT). No. of bitstreams: 1 Amaury Soares de Brito.pdf: 1041809 bytes, checksum: 74b076dde16ba738e59a5ac81be89a70 (MD5) Previous issue date: 2015-03-23 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Tribolium castaneum is a secondary pest that causes quantitative and qualitative losses in grain stocks of various cereals and derivatives. Your control is performed with several applications of insecticides and the consequence of this practice is the environmental contamination of people and the selection of resistant insects populations. New methods of control, such as the use of essential oils are gaining strength to mitigate negative impacts. Following this line of reasoning the objective of this study was to evaluate the fumigant action, contact and repellent of essential oils in control of T. castaneum. Were used essential oils of plants Eucalyptus citriodora Hook (Myrtaceae), Foeniculum vulgare Mill (Lamiaceae), Cymbopogon winterianus Jowitt (Poaceae), Citrus sinensis (L) Osbeck (Rutaceae), Citrus aurantium, Croton helitropiifolius Kunth, Croton blanchetianus Baill and Croton pulegiodorus Baill (Euphorbiaceae). In fumigation different concentrations of test oils were applied on filter paper strips attached on the bottom of the fumigation chamber cover (0.5 L), which contained 20 grams of food substrate and 20 adults of T. castaneum unsexed. The insect mortality was recorded after 120 hours of exposure in B.O.D kiln incubator, at 30 °C and RH 70±5%. For the contact test, different concentrations of the oils were diluted in acetone and applied to filter paper disks (9 cm ∅) and the control was applied only acetone. After evaporation of the solvent, the disks were placed in petri dishes and 20 adult insects released unsexed, and the mortality was assessed after 24 hours of exposure in BOD. The repellency tests were performed in arenas composed of two pots connected to a central box. In a pot of one end moistened feed substrate was mixed deposited with concentrations of the essential oil diluted in acetone, in the other pot (control) was deposited only food; Twenty insects do not sexed adult were released in the central box, being exposed for 48 hours to evaluate the preference. Was used a completely randomized design with four replications; data fumigation tests were submitted to regression analysis, and the contact tests submitted to probit analysis. The repellent rate was calculated by the formula IR=2*(G+P) and the percentage of repellency (PR) by the formula [PR=(NC-NT)/(NC+NT)*100]. In fumigation tests C. aurantium oil showed 100% mortality at a concentration of 490 μL/L air, and F. vulgare 93% mortality at the concentration of 900 μL/L air. In contact tests for E. citriodora were obtained LC50 =0.5035 μL/cm2 and CL99 = 0.8478 μL/cm2. For C. winterianus were estimated LC50 = 0.9760 and 5.1983 CL99 = μL/cm2. F.vulgare got the lowest concentrations with LC50 = 0.0157 and CL99 = 0,0216 μL/cm2. The oils of E. citriodora and C. winterianus showed repellent effect and C. winterianus and F. vulgare reduced adult emergence. / Tribolium castaneum é uma praga secundária que causa perdas quantitativas e qualitativas nos estoques de grãos de diversos cereais e seus derivados. O seu controle é realizado com diversas aplicações de inseticidas e a consequência dessa prática é a contaminação ambiental, das pessoas e a seleção de populações de insetos resistentes. Novos métodos de controle, como o uso de óleos essenciais estão adquirindo força por mitigar impactos negativos. Seguindo esta linha de raciocínio o objetivo deste trabalho foi avaliar o efeito fumigante, de contato e repelente de óleos essenciais no controle do T. castaneum. Foram usados óleos essenciais das plantas Eucalyptus citriodora Hook (Myrtaceae), Foeniculum vulgare Mill (Lamiaceae), Cymbopogon winterianus Jowitt (Poaceae), Citrus sinensis (L) Osbeck (Rutaceae), Citrus aurantium L., Croton helitropiifolius Kunth, Croton blanchetianus Baill e Croton pulegiodorus Baill (Euphorbiaceae). Nos testes de fumigação diferentes concentrações dos óleos foram aplicadas em tiras de papel filtro presas na parte inferior da tampa da câmara de fumigação (0,5L), na qual continha 20 gramas de substrato alimentar e 20 insetos adultos de T. castaneum não sexados. A mortalidade dos insetos foi avaliada após 120 horas de exposição em estufa incubadora tipo B.O.D, a 30ºC e 70 % UR. Para os testes de contato, diferentes concentrações dos óleos foram diluídas em acetona e aplicados em discos de papel filtro (9 cm Ø), e na testemunha foi aplicado apenas acetona P.A. Após a evaporação do solvente os discos foram colocados em placas de Petri e liberados 20 insetos adultos não sexados, e a mortalidade avaliada após 24 horas de exposição, em estufa incubadora tipo B.O.D. Os testes de repelência foram efetuados em arenas compostas por dois potes ligados a uma caixa central. Em um pote de uma extremidade foi depositado substrato alimentar umedecido com concentrações do óleo essencial diluído em acetona, e no pote da outra extremidade (testemunha) foi depositado apenas alimento; vinte insetos adultos não sexados foram liberados na caixa central, ficando expostos por 48 horas para avaliação da preferência. Utilizou-se o delineamento inteiramente casualizado com quatro repetições; os dados dos testes de fumigação foram submetidos à análise de regressão, e os dos testes de contato submetidos à análise de probit. O índice de repelência foi calculado pela formula IR=2*G(G+P) e a porcentagem de repelência (PR) pela formula [PR=(NCNT)/(NC+NT)*100]. Nos testes de fumigação o óleo de C. aurantium apresentou 100% de mortalidade na concentração de 490 μL/L ar e F. vulgare 93% de mortalidade com a concentração de 900 μL/L. ar. Nos testes de contato para E. citriodora foram obtidas CL50=0,5035 μL/cm2 e CL99=0,8478 μL/cm2. Para C. winterianus foram estimadas CL50=0,9760 e CL99= 5,1983 μL/cm2. F. vulgare obteve as menores concentrações com CL50=0,0157 e CL99= 0,0216μL/cm2. Os óleos de E. citriodora C. winterianus apresentaram efeito repelente e C. winterianus e F. vulgare reduziram a emergência de adultos.

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Doctor of Philosophy / Department of Entomology / James F. Campbell / James R. Nechols / Theoretical and empirical ecological research has emphasized the need for understanding how animals perceive and respond to landscape structure and the importance of integrating both behavioral and landscape approaches when studying movement behavior. Knowledge of insect movement behavior is essential for understanding and modeling dispersal and population structure and developing biologically-based integrated pest management programs. My dissertation research addresses questions concerning how insects respond to landscape structure by examining movement behavior of an important stored-product pest, red flour beetle (Tribolium castaneum), in experimental landscapes. Results show that beetles modify movement behavior depending on landscape structure. Edge effects and interpatch distances may influence landscape viscosity, or the degree to which landscape structure facilitates or impedes movement, resulting in significant differences in velocity and tortuosity (amount of turning) of movement pathways, as well as retention time in landscapes with different levels of habitat abundance and aggregation. Perceptual range, or the distance from which habitat is detected, appears to be limited while beetles are moving in a landscape as they did not respond to a flour resource before physical encounter. Beetles showed differential responses to patches with various characteristics, entering covered patches more quickly than uncovered patches with more resource or the same amount of resource. Permeability of patches changed with subsequent encounters suggesting that full evaluation of patch quality may only occur after entering a patch. Beetles responded to landscape structure differently depending on the activity in which they were engaged. Distribution of movement pathways was similar to that of the habitat, but distribution of oviposition sites were significantly more aggregated than pathways and habitat. Oviposition site choice may be influenced by a complex set of factors which include previous visitation, amount of resource, travel costs, and edge effects. Insights were gained concerning how red flour beetle perceives resources, modifies search strategies, responds to boundaries, and chooses reproductive sites in patchy landscapes. This research provides new information regarding how red flour beetle interacts with landscape structure that has implications in the areas of behavioral and landscape ecology and applications in stored-product insect ecology.

Submitted by Nathália Faria da Silva (nathaliafsilva.ufv@gmail.com) on 2017-07-13T13:51:37Z No. of bitstreams: 1 texto completo.PDF: 406966 bytes, checksum: abd8716c362cf52ad877eda05b8eaefc (MD5) / Made available in DSpace on 2017-07-13T13:51:37Z (GMT). No. of bitstreams: 1 texto completo.PDF: 406966 bytes, checksum: abd8716c362cf52ad877eda05b8eaefc (MD5) Previous issue date: 2001-03-30 / Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Atualmente, têm-se encontrado grandes entraves no controle de pragas em unidades armazenadoras e processadoras de grãos. A necessidade de formas de controle rápidas, de baixo custo e com menor impacto ambiental tem induzido a geração de novas tecnologias e o melhor manejo das já existentes. A futura saída do brometo de metila do mercado gera a necessidade de se estudarem tecnologias que permitam um controle efetivo de pragas de grãos armazenados em período inferior a 24 h, especialmente em unidades processadoras de grãos. O trabalho desenvolvido teve por objetivo estudar a interação da utilização de fosfina (PH3) e dióxido de carbono (CO2) no controle de Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) em diferentes temperaturas. Foram utilizados 40 adultos de T. castaneum em três repetições. Os tratamentos testados constituíram -se de uma atmosfera contendo 1 g m^-3 de fosfina (PH3) associada a 5% de CO2, nas temperaturas de 20, 25, 30, 35 e 40ºC. Em cada temperatura, foram avaliados cinco diferentes períodos de exposição, de acordo com os dados obtidos em testes preliminares. Os testes foram realizados em três câmaras metálicas, acondicionadas no interior de uma câmara climática, com controle de temperatura e umidade relativa. Após a avaliação de mortalidade em cada tratamento, realizou-se uma análise de próbite dos dados para determinar os tempos letais (TL) para controlar 50 e 95% da população de T. castaneum em cada temperatura estudada. Através de análise de regressão, obtiveram-se equações para determinar os TL50 e TL95 em função da temperatura. Ao utilizar-se a temperatura de 40ºC, verificou- se que é possível controlar 95% da população do inseto testado em aproximadamente 23 h. De acordo com os resultados obtidos, efetuou-se uma análise da viabilidade econômica da tecnologia proposta: considerando um aquecimento da estrutura a ser tratada, a tecnologia mostrou-se viável a 40ºC, em volumes superiores a 1.447 m³. / The control of stored grains and processing unities pests is going through great difficulties. The need of rapid, low cost and environmentally safe ways of controlling such pests induces the generation of new technologies and a better handling of the existing ones. The phasing out of methyl bromide is sparking the search for possible alternatives for effective pest control within a 24 hours period, especially for grain processing units. The accomplished work aimed to study the combination of phosphine (PH3) with carbon dioxide (CO2) in different temperatures on the control of Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae). Forty adults of T. castaneum were used in three replicates, and the treatments were modified atmospheres with 1 g m^-3 of phosphine combined with 5% of CO2 in the temperatures of 20, 25, 30, 35 and 40ºC. Five exposure periods for each temperature were used, according to results obtained in preliminary tests. The experiment was carried out in metallic chambers placed inside a climatic chamber with air temperature and relative humidity control. After the mortality evaluation of each treatment, probit analyses were carried out in order to determine the lethal times (LT) values to control 50 and 95% of T. castaneum population for each studied temperature. Afterwards, a regression analysis was used to obtain the equations of the LT50 and LT95 as a function of the temperature. For the temperature of 40ºC, it was possible to control 95% of the insect population with the proposed treatment in 23.2 hours. An analysis of the economical viability was then made using the results obtained on the research, comparing the proposed technology with two other one available, an increasing of the environment temperature was also studied. In the economical analysis, the treatment of 40ºC was better than the available one when the facilities to be treated are greater than 1447 m³.

Embryonic development entails a series of morphogenetic events which require a precise coordination of molecular mechanisms coupled with cellular dynamics. Phyla such as arthropods show morphological and gene expression similarities during middle embryogenesis (at the phylotypic germband stage), yet early embryogenesis adopts diverse developmental strategies. In an effort towards understanding patterns of conservation and divergence during development, investigations are required beyond the traditional model systems. Therefore, in the past three decades, several insect species representing various insect orders have been established as experimental model systems for comparative developmental studies. Among these, the red flour beetle Tribolium castaneum has emerged as the best studied holometabolous insect model after the fruit fly Drosophila melanogaster. Unlike Drosophila, Tribolium is a short-germ insect that retains many ancestral characters common to most insects. The early embryogenesis of Tribolium shows dynamic epithelial rearrangements with an epibolic expansion of the extraembryonic tissue serosa over the embryo, the folding of the embryo in between the serosa and the second extra embryonic tissue amnion and the folding of the amnion underneath the embryo. These extensive tissues are evolutionarily conserved epithelia that undergo different tissue movements and are present in varying proportions in different insects, providing exceptional material to compare and contrast morphogenesis during early embryogenesis. However, most of the previous work on insects including Tribolium have largely focused on the conservation and divergence of gene expression patterns and on gene regulatory interactions. Consequently, very little studies on dynamic cell behaviour have been done and we lack detailed information about the cellular and tissue dynamics during these early morphogenetic events. During my PhD, I first established a live imaging and data analysis pipeline for studying Tribolium embryogenesis in 4-D. I combined live confocal and lightsheet imaging of transgenic or transiently labelled embryos with mechanical or genetic perturbations using laser ablations and gene knockdowns. Using this pipeline quantifications of cell dynamics and tissue behaviours can be done to compare different regions of the embryo as the development proceeds. In the second and third part of my thesis, I describe the actomyosin dynamics and associated cell behaviours during the stages of serosa epibolic expansion, amniotic fold formation and serosa window closure. I cloned and characterised the cellular dynamics of the Tribolium spaghetti squash gene (Tc-squash) - the non-muscle Myosin II regulatory light chain, which is the main molecular force generator in epithelial cells. Interestingly, the analysis of Tc-squash dynamics indicates a conserved role of Myosin II in controlling similar cell behaviours across short germ and long germ embryos. In the last part of the thesis, I report the dynamics of an actomyosin cable that emerges at the interface of the serosa and amnion. This cable increases in tension during development, concomitant with serosa tissue expansion and increased tensions in the serosa. It behaves as a modified purse string as it’s circumference shrinks due to a decrease in the number of cable forming cells over time. This shrinkage is an individual contractile property of the cells forming the cable. This indicates that a supracellular and contractile actomyosin cable might be functional during serosa window closure in insects with distinct serosa and amnion tissues. Further, the tension in the cable might depend on the relative proportion of the serosa, amnion and embryonic regions. Using these integrated approaches, I have correlated global cellular dynamics during early embryogenesis with actomyosin behaviours, and then performed a high-resolution analysis and perturbations of selected events. The established imaging, image processing and perturbation tools can serve as an important basis for future investigations into the tissue mechanics underlying Tribolium embryogenesis and can also be adapted for comparisons of morphogenesis in other insect embryos. More broadly, correlating the existing genetic, mechanical and biochemical understanding of developmental processes from Drosophila with species such as Tribolium, could help identify deeply conserved design principles that lead to different morphologies through differences in underlying regulation.:Page List of Tables v List of Figures vii 1 Introduction 1 1.1 Evo-Devo of insects 3 1.2 Tribolium castaneum 5 1.3 Fluorescence live imaging and lightsheet microscopy 10 1.4 Morphogenesis 15 1.5 Thesis objective 29 2 4D lightsheet imaging and analysis pipeline of Tribolium embryos 33 2.1 Standardisation of an injection protocol for sample mounting and imaging with the Zeiss LZ1 SPIM 35 2.2 Double labelling of Tribolium embryos 37 2.3 Image processing with Fiji 37 2.4 Long term timelapse imaging of Tribolium embryogenesis with SPIM 44 2.5 2D cartographic projections of 3D data as a method to visualise and analyse SPIM data 47 2.6 Summary 59 3 Cellular dynamics of the non muscle Myosin II regulatory light chain - Tc-Squash 61 3.1 Tc-Squash dynamics during Tribolium embryogenesis 64 3.2 Myosin drives basal cell closure during blastoderm cellularisation 66 3.3 Myosin shows planar polarity in the embryonic tissue 69 3.4 Myosin accumulation and apical constriction of putative germ cells at the posterior pole 71 3.5 Myosin pulses during apical constriction of mesoderm cells 74 3.6 Myosin accumulates at the extraembryonic-embryonic boundary to form a contractile supracellular cable 77 3.7 Summary 77 4 A supracellular actomyosin cable operates during serosa epiboly 79 4.1 Actin and Myosin accumulate at the extraembryonic-embryonic boundary 81 4.2 The actomyosin assembly migrates ventrally till it forms the rim of the serosa window 82 4.3 The actomyosin cable shows dynamic shape changes during serosa window closure 87 4.4 Serosa cells increase in area till circular serosa window stage 89 4.5 Tension in the serosa tissue increases during epibolic expansion 89 4.6 Serosa cells decrease their apical areas after laser ablation 92 4.7 Tension in the actomyosin cable increases during serosa epiboly 93 4.8 Myosin dynamics at the cable changes between early and serosa window stage 96 4.9 Individual cell membrane shrinkage and cell rearrangements decrease the cable circumference 98 4.10 Myosin dynamics at the cable during serosa window closure 101 4.11 Tension in the cable is not relieved after multiple laser cuts 103 4.12 Analysis of the actomyosin cable in Tc-zen 1 knockdown 105 4.13 Summary 109 5 Discussion 111 5.1 Reconstruction of insect embryogenesis using lightsheet microscopy and tissue cartography 111 5.2 Conserved Myosin II behaviours and its implications on morphogenesis across insects 114 5.3 A contractile supracellular actomyosin cable functions serosa window closure in Tribolium 119 6 Materials and Methods 123 6.1 Tribolium stock maintenance 123 6.2 RNA extraction and cDNA synthesis 124 6.3 Cloning of templates for mRNA synthesis and transgenesis 124 6.4 dsRNA synthesis for RNAi experiments 126 6.5 Capped, single stranded RNA synthesis 126 6.6 Fluorescence image acquisition 27 A Appendix 131 Bibliography 143