Digestion in insects: a molecular, cellular, physiological, and evolutionary approach

• Grant number: 98/10458-2
• Support Opportunities: Research Projects - Thematic Grants
• Start date: November 01, 1998
• End date: October 31, 2003
• Field of knowledge: Biological Sciences - Biochemistry - Enzymology
• Principal Investigator: Walter Ribeiro Terra
• Grantee: Walter Ribeiro Terra
• Host Institution: Instituto de Química (IQ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Pesquisadores principais: Alberto Augusto Gonçalves de Freitas Castro Ribeiro ; Clelia Ferreira Terra
• Associated research grant(s): 01/13938-0 - Roderick James Dillon | University of Bath - Inglaterra, AV.EXT

Abstract

The use of chemical pesticides to control insects causes environmental damages. To avoid these damages, plants have been transformed in order to express proteins that act through the insect gut, thus becoming resistant to insects (Alstad & Andow, Science268: 1894-1896, 1995). The development of new transgenic plants needs a more sophisticated understanding of insect midgut physiology than is available. Otherwise, taking into account the variety of these animals, it is necessary to find out general principles from which hypotheses can be advanced in relation to the digestive process in an insect not previously studied. Studies carried out in different insect orders, mainly at our laboratory, showed that the spatial organization of the digestive process depends more on phylogeny than on feeding habits. These studies supported a hypothesis on how insect digestive systems evolved (review: Terra W. R., Evolution of digestive systems of insects. Annu. Rev.Entomol. 35.- 181-200, 1990). The improvement of this hypothesis needs that more information is gathered on insects not considered up to now. The characterization of digestive enzymes, microviliar membranes from midgut cells, and of peritrophic membranes (chitin-protein membranes enveloping the food inside the insect midgut) are necessary steps toward a description in molecular detail of digestive events. Besides the importance of these studies to basic science, they may give support to applied science, as digestive enzymes and proteins from microviliar membranes and peritrophic membranes have been used as targets in insect control strategies (Felton & Gatehouse in Biology of the insect Midgut, pp, 373-416, 1996,Chapman, London). This project deals with the problems advanced above along 3 main research lines: a) evolution of digestive systems; b) digestive enzymes and their secretory mechanisms-1c) microvillar and peritrophic membrane proteins. The insects for the different experiments were chosen because of their phylogenetic position and/or because they are important Brazilian pests.For improving and testing the hypothesis on the evolution of digestive systems, studies will be performed with insects not considered before, such as Dyctyoptera(cockroaches), Hemiptera (spittie bugs), Phthiraptera (lice), Hymenoptera Symphyta (sawflies) and some families (Cerambycidae, Curculionidae and Dermestidae) of Coleoptera (beetles), which is the largest insect order. The studies will include morphological descriptions of insect midguts, as well as enzyme determinations indifferent midgut compartments. In the case of large insects, additional experiments will be performed, such as the identification of absorptive sites for water (with dyes) and leucine (or glucose) by using mixtures of dyes and leucine (or glucose), followed by the determination of the mass ratio of these compounds aiong the midgut. The methods to be employed are those described by Terra (op. cit.). The results, together with literature data, will be used to make a cladogram. This cladogram, compared with the phylogenyand distribution of feeding habits of the insects, will permit to decide to which of these factors the digestive characteristics depend more.The aims in relation to the digestive enzymes are (procedures reviewed by Terra & Ferreira, Comp. Biochem. Physiol. lO9B-. 1-62, 1994): a) aminopeptidases, dipeptidases, chymotrypsins, and trypsins from several insects, as well as Musca domestica aspartic proteinase and Tenebrio molitor cysteine proteinase, will be characterized with emphasis on substrate specificity, which seems to differ from mammalian enzymes, thus explaining the different responses of insects toward bacterial a -endotoxin and protease inhibitor ingestion. b) The trypsins from Peripianeta americana and T molitor, as well as, P. americana amylase can be purified in large amounts and for this their 3-D structures will be defined by crystallography. c) M. domestica midgut and salivary lysozymes will be isolated, microsequenced and compared with those from ruminants, which also digest bacteria. d) a -Glucosidases, laminarinases, lichenases, trehalases, and b -glycosidases from several insects will be isolated and characterized to define their substrate specificity and/or physiological function, whereas the role of amino acyl residues from Spodoptera frugiperda b -glucosidase will be determined by site-directed mutagenesis. e) Attempts to clone and sequence C-DNA stretches coding isolated and favorable enzymes will be performed.Insect digestive enzyme secretory mechanisms seem to include aspects not found in other animals (Terra & Ferreira, op. cit.). Studies of these unique processes may provide the basis for a more detailed understanding of the physiology of digestion in insects and may result in valuable contribution to the field of cell biology. With this aim, serum reacting with favorable digestive enzymes will be used to follow by immunocytochemical techniques their secretory routes. Double immunolabeling will be employed to compare the routes of usual enzymes with those similar to cathepsins. Furthermore, a study of the midgut microvillar cytoskeleton of S. frugiperda will be carried out in relation to secretory mechanisms.Finally, midgut microviliar proteins and peritrophic membrane proteins will be characterized (Capelia et al. Insect Biochem. Molec. Biol.27: 793-801, 1997; Teliam in Biology of the Insect Midgut, pp. 86-114, 1996, Chapman, London), microsequenced and compared with sequence data in an attempt to find structural and functional correlations. During the development of this project, several people are expected to finish their training: 2 post-docs, 4 Ph.D. students, 4 M.Sc. students and at least 9 graduate students, from which about 4 should start their studies to get a M.Sc. degree. (AU)