Digestion in Arachnida and the control of proteolytic activity

Abstract

Arthropoda (main classes: Crustacea, Arachnida and Insecta) is the largest phylum representing 70% of all species already described. Besides that, this group is very important economically, including agriculture pests as well as harmful species to human, animal and plant health, being involved in the transmission of several diseases and with poisoning accidents. The digestive tract of arthropods is a good target to the development of control methods because it is an important interface between these animals and their environment. Some current control attempts rely on strategies that can affect the digestive systems and are able to reduce the digestion rates, insect growth and reproduction. Specific targets of these control strategies are structural components of the digestive system (gut epithelium, peritrophic membrane) and molecular components (digestive enzymes). Depolymerase inhibitors, mainly peptidase inhibitors (PIs) are widely used. Plants are the most common sources of these molecules. Surprisingly, other sources, such as inhibitors present in animal tissues, are not explored, otherwise good results were obtained with Manduca sexta (tobacco hornworm) serpin expressed on transgenic cotton in the development of Bemisia tabaci (sweet potato whitefly). A comparative study of digestion in Arthropoda, the characterization of the molecular aspects of digestion in these animals, the interaction of peptidases and PIs, as well as, the study of peptidases specificity are very important to the development of better control strategies. Studies with insect trypsins have allowed a better understanding of insect resistance to plant PIs (Lopes et al., 2004).There is a lot of data about Crustacea and Insect digestion but data about digestion in Arachnida are scarce. The present project aims: (a) to characterize digestion in Arachnida, studying the enzymes involved in this process and gut morphology. The data obtained will be compared with Crustacea and Insect literature on digestion in order to understand digestion evolution in Arthropoda; (b) to search for peptidase inhibitory activities in Insecta and Arachnida.In this project, Arachnida digestion will be focused using as models the regurgitate and the midgut from the spider Nephilengys cruentata (Araneae: Tetragnatidae), the midgut from the scorpion Tityus serrulatus (Scorpiones: Buthidae) and the midgut from the tick Amblyomma cajennense (Acari: Ixodidae). Histological studies and wheat-germ aglutinin immunolocalization experiments will be used in order to characterize the morphological aspects of Arachnida digestion and the presence of peritrophic membrane in their midgut. Western-blotting experiments will allow the analysis of cross-reaction of Arachnida digestive enzymes (samples: regurgitate and midgut from N. cruentata and midgut from scorpion T. serrulatus and the tick A. cajjennense) with antibodies raised against Tenebrio molitor amylase, trypsin, cysteine proteinase and b-glycosidase. The cross-reaction will permit the use of immunogold techniques to study the secretion of these enzymes in Arachnida. Besides that, the quantification of enzyme activities involved in protein and carbohydrate digestion will permit the characterization of some molecular aspects of Arachnida digestion.The study of the control of proteolytic activity in different Arthropoda species (Arachnida: N. cruentata, T. serrulatus e A. cajennense and Insecta: Periplaneta americana, Tenebrio molitor, Dysdercus peruvianus, Musca domestica, Diatraea saccharalis and Spodoptera frugiperda) will be started by the determination of the presence of inhibitory activity in the midgut and hemolymph from these animals on purified proteinases (bovine trypsin and chymotrypsin and insect trypsin, chymotrypsin and cysteine proteinase). (AU)