The cuticle (the insect equivalent of skin) is one of the structural innovations responsible for the ascendancy and predominance of insects in nature. This research project is a molecular genetic and biochemical approach designed to identify specific components of the cuticle that are critical for its assembly. The organization of the cuticle in the maturing elytron, a modified wing structure of the red flour beetle, will serve as a model tissue system for cuticle morphogenesis, a critical step in arthropod development. This research will lead to a greater understanding of the molecular basis and the dynamics of how various proteins and chitin interact to assemble the multi-layered cuticle with its distinctive chemical and mechanical properties. In addition, this project will allow training of three graduate students, several undergraduate students and a post-doctoral fellow, and also encourage them to pursue careers in pure or applied science. As a result of this research team's efforts in training and outreach to high school biology teachers, many high schools across the country now use the red flour beetle for genetics experiments and promotion of its use in education will continue.
Results obtained from the pursuit of this research will be broadly disseminated in national and international meetings in the form of poster/oral presentations and in the form of refereed publications in mainstream journals. Resources (mutants and genetically engineered beetles) will be supplied to other researchers and high school teachers when requested. Details of available resources will also be uploaded in the popular Tribolium Genetics Website, which is maintained by this research group, and in the Tribolium genome database, "Beetlebase".
Chitin is a polysaccharide that provides structure and organization to the exoskeleton of insects. All insect species express enzymes that are devoted to the synthesis and degredation of chitin. Synthesis of chitin occurs during development when insects molt as they progress from egg to larvae to pupae, and on to adult. At these various molting stages a new exoskeleton is required to accommodate a growing body and elaborate adult structures, such as legs and wings. Likewise, degredation of chitin occurs during molting as the old exoskeletion is discarded. The beetle, Tribolium was used as a model insect to study the effects of reduced expression of proteins involved in chitin synthesis and the organization of chitin fibrils in the exoskeleton. A collaboration between researchers at the University of Kansas, the USDA and the University of Massachusets Medical School undertook the analysis of over 25 proteins for their importance to chitin deposition into the exoskeleton. Various methods were developed to biochemically analyze the amounts of chitin deposited and to microscopically define abnormal from normal deposition of chitin. Most of the proteins studied are conserved among insect species and only a few have been studied in other species. Central to these studies was a technique (RNAi) that specifically knocks-down expression of one protein and can be used at various times during devepment. Chitin found in the exoskeleton is synthesized by the protein, Chs-A. During a molt, the newly synthesized chitin is protected from degradation that is taking place in the old exoskeleton by the protein, Knk. And, to ensure that Knk is properly localized during this process requires the protein Rtv. Loss of expression of any one of these three proteins is lethal at the various stages of molting. The chitin level in the whole insect drops to less than 20% of normal and the layered organization of chitin fibrils in the exosketon is altered. These three proteins are shared and probably function similarly across insect species. What turned out to be a more species-specific group of proteins was also studied. A faimily of similar proteins called the CPAP proteins of which there are 17 expressed by Tribolium, are each predicted to bind to chitin fibrils. The effect of loss of expression was studied for each one. And the effects were not universal, ranging from being lethal, but only for particular molts, to affecting only certain structures of the insect exoskeleton, to having no observable effect on development. Chitin of the whole insect was shown to be diminished to about 20% for only one of the CPAP proteins and for two others diminished chitin could only be detected in the hindwing microscopically using a fluorescent probe that binds chitin. One of the CPAP proteins of Tribolium is homologous to one studied in the fruit fly (Drosophila). The effect that loss of function has, is however not the same. This suggests that the CPAP proteins play a more species specific role in the organization of the exoskeleton. These studies have begun to help us understand how the insect exoskeleton is synthesized with the focus being organization around chitin fibrils, how chitin in the newly synthesized exoskeleton is preserved, and how a group of proteins are important for organizing chitin fibrils in different parts of the insect anatomy. These proteins are also targets for developing means to refine control of insect pests and those that are carriers of disease without harm to those that are beneficial.
