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.

Como qualquer inseto holometábolo, a abelha Apis mellifera sofre metamorfose completa, apresentando grandes mudanças na forma e fisiologia quando passa do estágio larval para o estágio de pupa (muda metamórfica). Após esta muda, com o prosseguimento do desenvolvimento, o tegumento pupal (cutícula e a epiderme subjacente), extensivamente remodelado, é substituído pelo tegumento adulto, definitivo, que passa por intensa melanização e esclerotização. Eletroforese bidimensional e espectrometria de massas foram utilizadas neste trabalho para caracterizar as mudanças do padrão proteico no tegumento em desenvolvimento de operárias e zangões. No total foram identificadas 51 proteínas diferentes no tegumento torácico extraído de larvas, pupas e adultos (adultos-faratos). Quatorze proteínas foram identificadas como genuinamente cuticulares: Apidermina-3,1-like, Apidermina-2, Cuticular proteins analogous to peritrophins-3C e 3D, AmelCPR3, 12, 16 e 27, Glicoproteína SgAbd-2-like, e cinco outras proteínas homólogas à proteínas cuticulares de outras espécies de insetos contendo um domínio de ligação à quitina. As proteínas diferiram principalmente quantitativamente entre as fases de desenvolvimento e sexo, e poucas diferenças qualitativas foram observadas. Por exemplo, Apidermina-2 é típica de tegumentos fortemente esclerotizados e pigmentados. As diferenças quantitativas foram destacadas pela comparação da abundância de algumas proteínas e seus respectivos RNA mensageiros (utilizando RT-PCR em tempo real) entre as fases de desenvolvimento e entre os sexos. Várias proteínas cuticulares mostraram mais de uma forma molecular, aparentemente derivadas de modificações pós-traducionais. Além de conferir suporte experimental para a validação de genes de A. mellifera preditos, ou não-anotados, nossos dados forneceram novas informações sobre as proteínas que atuam no tegumento em desenvolvimento. / As a holometabolous insect, the honey bee undergoes complete metamorphosis, displaying a marked change in shape and physiology when passing from the larval to the pupal stage (metamorphic molt). As development progresses, the extensively remodeled pupal integument (cuticle and subjacent epidermis) is replaced by the adult integument, which undergoes intense sclerotization and melanization. Two-dimensional electrophoresis and mass spectrometry were here used to characterize the changing protein patterns in the developing integument of workers and drones. Overall, we identified 51 different proteins in the thoracic integument extracted from larvae, pupae and adults (pharate adults). Fourteen proteins were identified as genuine cuticular proteins: Apidermin-3,1-like protein, Apidermin-2, Cuticular Proteins Analogous to Peritrophins-3C and 3D, AmelCPR3, 12, 16 and 27, Glycoprotein SgAbd-2-like, and 5 other proteins homologous to cuticular proteins from other insect species, and containing the chitin-binding domain. Integument proteins mainly differed quantitatively among the developmental stages and sexes, although few qualitative differences have also been detected. For example, Apidermin-2 is typical of the heavily pigmented and sclerotized integument. The quantitative differences were highlighted by comparing the levels of some of these proteins and their respective mRNAs (using RT-qPCR) among the developmental phases and between sexes. It is noteworthy that several cuticle proteins showed more than one molecular form, apparently derived from post-translational modifications. In addition to give experimental support for validation of predicted, or unannotated, honey bee genes, our data provided new information on proteins acting in the metamorphosing integument.

Como qualquer inseto holometábolo, a abelha Apis mellifera sofre metamorfose completa, apresentando grandes mudanças na forma e fisiologia quando passa do estágio larval para o estágio de pupa (muda metamórfica). Após esta muda, com o prosseguimento do desenvolvimento, o tegumento pupal (cutícula e a epiderme subjacente), extensivamente remodelado, é substituído pelo tegumento adulto, definitivo, que passa por intensa melanização e esclerotização. Eletroforese bidimensional e espectrometria de massas foram utilizadas neste trabalho para caracterizar as mudanças do padrão proteico no tegumento em desenvolvimento de operárias e zangões. No total foram identificadas 51 proteínas diferentes no tegumento torácico extraído de larvas, pupas e adultos (adultos-faratos). Quatorze proteínas foram identificadas como genuinamente cuticulares: Apidermina-3,1-like, Apidermina-2, Cuticular proteins analogous to peritrophins-3C e 3D, AmelCPR3, 12, 16 e 27, Glicoproteína SgAbd-2-like, e cinco outras proteínas homólogas à proteínas cuticulares de outras espécies de insetos contendo um domínio de ligação à quitina. As proteínas diferiram principalmente quantitativamente entre as fases de desenvolvimento e sexo, e poucas diferenças qualitativas foram observadas. Por exemplo, Apidermina-2 é típica de tegumentos fortemente esclerotizados e pigmentados. As diferenças quantitativas foram destacadas pela comparação da abundância de algumas proteínas e seus respectivos RNA mensageiros (utilizando RT-PCR em tempo real) entre as fases de desenvolvimento e entre os sexos. Várias proteínas cuticulares mostraram mais de uma forma molecular, aparentemente derivadas de modificações pós-traducionais. Além de conferir suporte experimental para a validação de genes de A. mellifera preditos, ou não-anotados, nossos dados forneceram novas informações sobre as proteínas que atuam no tegumento em desenvolvimento. / As a holometabolous insect, the honey bee undergoes complete metamorphosis, displaying a marked change in shape and physiology when passing from the larval to the pupal stage (metamorphic molt). As development progresses, the extensively remodeled pupal integument (cuticle and subjacent epidermis) is replaced by the adult integument, which undergoes intense sclerotization and melanization. Two-dimensional electrophoresis and mass spectrometry were here used to characterize the changing protein patterns in the developing integument of workers and drones. Overall, we identified 51 different proteins in the thoracic integument extracted from larvae, pupae and adults (pharate adults). Fourteen proteins were identified as genuine cuticular proteins: Apidermin-3,1-like protein, Apidermin-2, Cuticular Proteins Analogous to Peritrophins-3C and 3D, AmelCPR3, 12, 16 and 27, Glycoprotein SgAbd-2-like, and 5 other proteins homologous to cuticular proteins from other insect species, and containing the chitin-binding domain. Integument proteins mainly differed quantitatively among the developmental stages and sexes, although few qualitative differences have also been detected. For example, Apidermin-2 is typical of the heavily pigmented and sclerotized integument. The quantitative differences were highlighted by comparing the levels of some of these proteins and their respective mRNAs (using RT-qPCR) among the developmental phases and between sexes. It is noteworthy that several cuticle proteins showed more than one molecular form, apparently derived from post-translational modifications. In addition to give experimental support for validation of predicted, or unannotated, honey bee genes, our data provided new information on proteins acting in the metamorphosing integument.

The Common bed bug, Cimex lectularius L., suddenly reappeared in developed countries in the past 15 years. The factor contributing to the sudden resurgence of the bed bugs is insecticide resistance. In this study, we investigated the mechanisms of reduced cuticular penetration type insecticide resistance in bed bugs. First, we determined and compared the lethal dosage (LD50) of a pyrethroid insecticide using topical and injection application. The resistant strain not only had significantly greater resistance ratios, but also demonstrated significantly greater penetration resistance ratios. This provided the evidence of the reduced cuticular penetration in bed bugs. Second, we determined the levels of gene transcription (CPR-type cuticle protein genes) using real-time quantitative polymerase chain reaction (qRT-PCR). We identified 62 putative bed bug cuticle protein-encoding contigs based on the presence of the Chitin-binding 4 (CB4) domain. Based on the qRT-PCR analysis of the mRNAs, we found many of the genes were up-regulated in the resistant strain suggesting thickening of the cuticle or increasing the cuticular proteins might be involved in the reduced cuticular penetration. Third, we identified and described the cuticular proteins using the matrix-assisted laser desorption/ionization (MALDI) time-of-flight/time-of-flight (TOF/TOF) high-resolution tandem mass spectrometry (MALDI-TOF/TOF). The total of 265 peptides were identified, among which 206 belonged to one of 50 confidently identified proteins. We identified the CPRL, CPF, CPFL, TWDL, and CPAP1 family proteins. The profile of the cuticular proteins between the resistant and the susceptible strains bed bugs were almost identical. Fourth, we determined and compared the cuticular thickness using Scanning Electron Microscopy (SEM). We found statistical differences of the cuticular thickness among different strains (populations), however, correlation between the levels of insecticide resistance and cuticular thickness were not found. Finally, we identified and described bed bug cuticular hydrocarbon profiles using Gas-Chromatography and Mass-Spectrometry (GC-MS). The total of 87 compounds in addition to n-alkanes were extracted and identified. There were no correlation found with the concentration and the levels of insecticide resistance. However, several additional compounds exhibited the correlation between the concentration of the compounds and the levels of insecticide resistance. Overall, we found three lines of evidence to support reduced cuticular penetration as a mechanism of insecticide resistance in some bed bug populations. This study provides additional evidence of the reduced cuticular penetration type resistance in bed bugs. / Ph. D.