Insect Biochem. Vol. 20, No. 8, pp. 839-847, 1990 0020-1790/90 $3.00+ 0.00 Printed in Great Britain.All rights reserved Copyright © 1990PergamonPress plc DIGESTIVE ENZYMES ASSOCIATED WITH THE GLYCOCALYX, MICROVILLAR MEMBRANES AND SECRETORY VESICLES FROM MIDGUT CELLS OF TENEBRIO MOLITOR LARVAE CLf~LIAFERREIRA l, GISELAL. BELLINELLO 1, ALBERTOF. RIBE1RO 2 and WALTERR. TERRA l 'Departamento de Bioquimica, Instituto de Quimica, Universidade de Sgo Paulo, C.P. 20780, 01498--S~o Paulo and 2Departamento de Biologia, Instituto de Bioci6ncias, Universidade de Silo Paulo, C.P. 11461, 05499--Sfio Paulo, Brasil (Received 19 March 1990; revised and accepted 1 August 1990) Abstract--Acetylglucosaminidase, amylase, cellobiase and maltase are more active in anterior midgut cells, whereas aminopeptidase, carboxypeptidase and trypsin are more active in posterior midgut cells of Tenebrio molitor larvae. Differential centrifugation of midgut homogenates prepared in saline (or mannitol) isotonic buffered solutions revealed that aminopeptidase is associated with membranes, which occur in subcellular fractions displaying many microvilli. Carboxypeptidase, trypsin and the carbo- hydrases are mostly found in the soluble fraction, although significant amounts sediment together with cell vesicles. Data on differential calcium precipitation of midgnt homogenates and on partial ultrasound disruption of midgut tissue suggest that aminopeptidase is a microvillar enzyme and that the digestive enzymes recovered in the soluble fraction of cells are loosely bound to the cell glycocalyx. About 5% of the non-absorbable dye amaranth fed to T. molitor larvae remains in the midgnt tissue after rinsing. Most dye was recovered in the soluble fraction of midgut cells. This provided further support for the hypothesis that the digestive enzymes found in the soluble fraction are actually extracellular and that the true intracellular enzymes are those associated with cell vesicles. The results suggest that the carbohydrases are secreted by exocytosis from the anterior midgut and carboxypeptidase and trypsin from the posterior midgut. Key Word Index: microvillar enzymes, exocytosis, carbohydrase secretion, protease secretion, ultrasound disruption, differential centrifugation, differential precipitation, mealworm INTRODUCTION Studies of the compartmentalization of amino- peptidase, amylase, cellobiase, trehalase and trypsin along the midgut of Tenebrio molitor larvae led Terra et al. (1985) to propose that most carbohydrate digestion occurs in the lumen of the anterior midgut of this insect, whereas protein digestion takes place partly in the lumen and partly at the cell surface of the posterior midgut. This proposal assumes that the aminopeptidase found associated with membranes is a microviilar enzyme occurring mainly in the posterior midgut cells and does not advance any hypothesis on the site and mechanism of secretion of the soluble digestive enzymes. T. molitor larval midgut displays many mature cells rich in morphological signs of exocytosis (rough endoplasmic reticulum, Golgi complex and secretory vesicles) and some cells which are entirely or partly discharging into the lumen (Koefoed and Zerahn, 1972; Berdan et al., 1985; Tano et al., 1987; Ferreira, Ribeiro and Terra, unpublished observations). The first cells suggest that the main route of secretion is exocytosis, whereas the latter cells support a holocrine (or apocrine) route, in spite of many authors interpreting them as an artifact or as part of the cell renewal process (for review see Chapman, 1985). Subcellular fractionation of midgut cells, followed by enzyme assays, may be used to distinguish enzymes present in the cytosol from those occurring in secretory vesicles. Thus, this technique provides useful information to support proposals on the mechanisms by which midgut cells secrete digestive enzymes. Using these techniques, it was possible to support a model for the secretion of digestive enzymes by Erinnyis ello (Lepidoptera) (Santos et al., 1986) and Musca domestica (Diptera) (Espinoza- Fuentes et al., 1987) larval midgut cells, which in- cluded exocytosis and a microapocrine mechanism of secretion by extrusion of vesicles from the sides and tips of the cell microvilli. Based on the same techniques, digestive enzymes are only secreted in Pheropsophus aequinoctialis (Coleoptera) by exo- cytosis (Ferreira and Terra, 1989). In this paper we showed, by several criteria, that aminopeptidase is a microviUar enzyme of posterior midgut cells and that carbohydrases are secreted by exocytosis from the anterior midgut cells, whereas carboxypeptidase and trypsin are secreted by exocytosis from the posterior midgut cells of T. molitor larvae. Furthermore, we propose from our data that the dye amaranth may be used as a marker for molecules associated with the cell glycocalyx in subeeilular fractionation experiments. 839