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Ultrastructure and secretory activity of Abracris flavolineata (Orthoptera: Acrididae) midguts
Pergamon
PII: SOO22-1910(96)00117-5
J. Insect Physrol. Vol. 43, No. 5, pp. 465413, 1997 0 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0022-1910197 $17.00 + 0.00
Ultrastructure and Secretory Activity of Abracris Jtavolineata (Orthoptera: Acrididae) Midguts SANDRO
R. MARANA,*
ALBERT0
F. RIBEIRO,?
WALTER
R. TERRA,*
CLkLIA
FERREIRA*$
Received 15 July 1996; revised 7 October 1996
The midgut of Abracris Jlavofineatu adults comprises a ventriculus and six anteriorly placed caeca each displaying an anterior and a posterior lobe. Columnar cells in the caeca and anterior ventriculus present secretory vesicles originating from abundant Golgi areas, which seem to result (through exocytosis) in dark granules among the microvilli. A. Jlavolineatu males were starved for 24 h, fed for 20 min at noon and dissected at 0, 1, 3 and 5 h after the meal. Enzyme assays were accomplished on crop and caecal contents and in subcellular fractions obtained from the isolated anterior caeca. Subcellular fractions putatively containing secretory vesicles were recognized. Digestive enzyme activity is usually low (amylase is high) in the secretory vesicles in starving insects, decreases 1 h after the meal, increases at 3 h, and thereafter decreases again (amylase remains constant). In caecal contents, digestive enzymes decrease at 1 h and increase at 3 h after the meal, the contrary being true for crop contents. Thus, in A. flavolineutu caecal cells, digestive enzymes (/3-glucosidase is an exception) are synthesized and secreted by exocytosis in response to feeding. Also in response to feeding, digestive enzymes are transferred from caecal contents to the crop and, after about 3 h following the meal, crop-caecal dispersed material with accompanying enzymes are translocated to the caeca, where digestion ends and absorption occurs. 0 1997 Elsevier Science Ltd Columnar Trypsin
cells
Midgut
caeca
Secretory
vesicles
Exocytosis
INTRODUCTION
Maltase
/3-Glucosidase
carbohydrates and proteins are digested mainly in the crop and caecal contents, respectively, although at least part of the intermediate and final digestions of protein occur at the surface of the caecal and ventricular cells. Compartmentalization data also support the hypothesis that digestive enzymes are synthesized and secreted by all midgut cells (mainly in the caeca) and then passed forward into the crop (Ferreira et al., 1990a). The role of the grasshopper midgut caeca in nutrient and water absorption has been known for many years (Treheme, 1967). More recently these studies were detailed by Dow (1981a, b), who showed that in starving grasshoppers a counter-current flow is formed caused by the secretion of fluid by the Malpighian tubules and by its absorption in the caeca. Similar fluid fluxes were found in most insects and are responsible for digestive enzyme recycling, thus minimizing enzyme excretion (Terra, 1990). Feeding grasshoppers present remarkable salivation, which saturates the water absorption site in the caeca, and then abolishes counter-current flows (Dow, 1981a, b). This was confirmed by the finding that
Although grasshoppers have been studied for a long time (Uvarov, 1966; Wigglesworth, 1972; Chapman and Joem, 1990), only recently have they became the subjects of quantitative work regarding digestive physiology. Except for amylase and trehalase, the salivary glands display negligible amounts of digestive enzymes. Carbohydrases (except trehalase) predominate in the crop, and their pH optima agree with the pH prevailing in the crop contents. Aminopeptidase (soluble and membrane bound) and trypsin predominate in the cells and contents of the caeca, respectively (Droste and Zebe, 1974; Anstee and Charnley, 1977; Ferreira et al., 1990a). Thus, enzyme compartmentalization data suggest that
*Departamento Slo Paula, ?Departamento Slo Paula, :To whom all
Amylase
de Bioquimica, lnstituto de Quimica, Universidade de C.P. 26077, 05599-970, SZo Paula, Brazil de Biologia, Institute de BiociEncias, Universidade de C.P. 11461, 05422-970, S%o Paula, Brazil correspondence should be addressed. 465
SANDRO R. MARANA
466
feeding grasshoppers excrete digestive enzymes at a rate similar to that of undigested food (Ferreira et ul., 1990a). In spite of some light- and electron-microscope studies of grasshopper midguts (Hodge, 1936, 1939; Heinrich and Zebe, 1973; Bemays, 1981), sufficient data have not been produced to relate midgut cell features to actual midgut fluid fluxes or secretory events. In this paper, ultrastructural data on the midgut are presented supporting the current model of midgut fluid fluxes and secretion. In addition, biochemical data are given confirming the secretory activity of caecal cells. MATERIAL
AND METHODS
Animals Abrucris Jlavolineuta (Orthoptera, Acrididae, subfamily Ommatolampinae, tribe Abracrini) adults were collected in a small forest close to the University campus in Rio Claro, State of Sao Paulo, Brazil. They were maintained in our laboratory in cages (3 1x3 1x40 cm) under natural photoperiod conditions at relative humidity of 50-60% at 24f2”C. The grasshoppers had free access to water and were fed ad libitum with collard (Brassica oleracea acephalu) leaves which were maintained with their stems in water and were changed twice a week. Preparation
of samples
of gut sections
Grasshoppers at different feeding conditions were immobilized by placing them on ice, after which they were dissected in cold 250 mM NaCI. Caecal and ventricular tissues, after being thoroughly rinsed with 250 mu NaCl, were homogenized in double distilled water using a Potter-Elvehjem homogenizer. The preparations were then passed through a nylon mesh of 100 pm pore size. Peritrophic membranes and contents were homogenized similar to gut preparations, but without previous rinsing with saline, and centrifuged at 20 OOOxg for 30 min at 4°C to remove undigested material. After collection with the aid of a capillary, the caecal contents were dispersed in a known volume of double distilled water. All samples were stored at -20°C until use. Light and electron
microscopy
For studies with the light microscope, animals were dissected in their own hemolymph and midgut regions were fixed in 4% paraformaldehyde in 20 mM phosphate buffer (pH 7.4) containing 0.15 M NaCl. The materials were embedded in Historesin, cut into 34 pm sections and stained with hematoxylin and eosin. For transmission-electron microscopy, animals were dissected in their own hemolymph and midgut sections were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 h at 4°C. The midgut pieces were post-fixed in 1% osmium tetroxide in the cacodylate buffer. In-block staining was performed in 1% aqueous uranyl acetate for 16-18 h. After dehydration, the
et 01.
material was embedded in Spurr resin, cut into ultrathin sections, stained with lead citrate, and examined in a Zeiss EM 900 electron microscope operated at 80 kV. D@zrential
CentriJingation of’cuecal
homogenates
Caecal anterior lobes were homogenized in 2 mM sodium phosphate buffer (pH 7.0) containing 471 mM mannitol, with the aid of a Potter-Elvehjem (10 strokes). The resulting homogenates were passed through a nylon mesh of 45 pm pore size and its volume adjusted to contain material corresponding to one animal/ml. Differential centrifugation of the caecal homogenates were performed according to two different procedures. The first is a simplified method which was used to study tissue digestive enzymes at different times after a meal. For this, the homogenates were centrifuged at 100 OOOxg for 60 min. The supematant (S) was recovered and the resulting pellet was resuspended in the homogenizing buffer and, after three cycles of freezing and thawing, was centrifuged again at 100 OOOxg for 60 min. The resulting supematant (SM) and pellet (M) were collected and, along with FS, were assayed for several enzymes. The second procedure is somewhat more complex, as it involves a larger number of steps, the aim being to assign enzymes to specific cell compartments. For this, the homogenates were centrifuged and the following fractions collected: P,, pellet 6OOxg, 10 min; P2, pellet 33OOxg, 10 min; P,, pellet 25 OOOxg, 10 min; Pz, pellet 100 OOOxg, 60 min, FS, final supematant. After this differential centrifugation the collected pellets were homogenized with the aid of a Potter-Elvehjem homogenizer in the homogenization medium and, following three freezing and thawing cycles, were centrifuged at 100 OOOxg for 60 min. Thus, fraction P, resulted in a supematant SP, and a pellet PP,, fraction PI in SP2 and PP,, fraction P.3 in SP, and PP, and fraction P, in SP, and PP4. The supematants and pellets corresponding to each fraction were assayed for several enzymes. The following enzyme markers for subcellular fractions were assayed: succinate dehydrogenase (mitochondria) and lactate dehydrogenase (cytosol). Results are presented according to De Duve et al. (1995) where the height of the histogram is proportional to the specific activity, and the area of the histogram is proportional to the total activity of the enzyme. Protein
determination
and enzyme
assays
Protein was determined according to Bradford (1976) using ovalbumin as a standard. Aminopeptidase was assayed in 50 mu sodium phosphate buffer (pH S.O), using 1 mu leucinep-nitroanilide as a substrate (Erlanger et ul., 1961). Amylase was assayed in 50 mM sodium citrate-phosphate (pH 6.5) containing 10 KIM NaCl with 0.5% soluble starch as a substrate (Noelting and Bemfeld, 1948). Aryl P-glucosidase activity was determined in 50 mu sodium citrate-phosphate buffer (pH 6.0), using 1 ITIM p-nitrophenyl P-D-glucoside as a substrate (Terra et al., 1979). Cellobiase and maltase were assayed
AHHACKIS
FLA VOLINEATA
in 50 mM sodium citrate-phosphate buffer (pH 6.0) with 7 mM cellobiose and maltose as substrates, respectively, (Dahlqvist, 1968). Dipeptidase activity was assayed in 50 mM Tris+HCl buffer pH 8.0 with Gly-Leu as a substrate (Nicholson and Kim, 1975). y-Glutamyl transferase was assayed in 50 mM Tris-HCl pH 8.8 with 1 mM yglutamyl p-nitroanilide and SO mM Gly-Gly as substrates (Erlanger et ul., 1961). Lactate dehydrogenase and succinate dchydrogenase were assayed according to Bergmeyer and Bemt (1974) and Ackrell et ul. (1978), respcctively, as detailed elsewhere (Santos and Terra, 1984). Trypsin was assayed in 50 mM sodium phosphate pH 7.5 with 0.8 mM a-N-benzoyl-Dr_-arginine-/2-nitroanilide as a substrate (Erlanger ef ul., 1961). Incubations were carried out for at least four different time periods, and initial rates of hydrolysis were calculated. All assays were performed under conditions such that activity was proportional to protein concentration and to time. Controls without enzyme or without substrate were performed. One U of enzyme is defined as the amount of the enzyme that hydrolyzes 1 pmol of substrate (or bond) per min.
467
MlDGlJTS
the same region. Details are provided below. Regenerative cells are collected in nidi at the base of the epithelium and occur along the caeca and ventriculus. Endocrine cells are rare, lack a basal labyrinth and are situated at the base of the epithelium. These cells, which occur in insects of several different orders, typically show a cytoplasm with plenty of small vesicles with either clear or dark contents (Nishiitsuji-Uwo and Endo, 1981; Zitman et al., 1993; Raes and Verbeke, 1994). Fine structure
of columnar
midgut curcal
cells
Columnar cells are similar in the two lobes of the midgut caeca. The apical membrane is modified into cylindrical microvilli in a compact, regular arrangement. The microvilli are about 8.1 (anterior caeca) or 12.2 pm long (posterior caeca) and are covered with a conspicuous glycocalyx. Among the microvilli there are dense granules, which may change in shape when pressed by the neighboring microvilli [Fig. l(A)]. The extracellular dense granules seem to derive from secretory vesicles close to the base of the microvilli. The contents of the vesicles are, however, less dense than the granules. The
RESULTS General
morphologv
qf the gut
The general morphology of A. ,flavolineata adult gut is similar to that of other grasshoppers (Hodge, 1936, 1939) and therefore only a brief description will be given. The gut of adults is composed of a large crop (66.4% of gut length), a relatively short ventriculus (13.5% of gut length) with six anteriorly placed caeca, and a hindgut (20.1% of gut length) differentiated into ileum, colon and rectum. The caeca have anterior and posterior lobes, which are referred to here as anterior and posterior caeca. The posterior caeca corresponds to 8% of gut length. whereas the anterior caeca equated 12.6%. The cpithelium of the anterior caeca forms folds which permits distensibility and increases the surface area. The posterior caeca present at their proximal end discrete pockets similar to those described in detail in Schistoccwu grcguriu (Bemays, 1981). The height of the cells in the anterior caeca are about 64 pm, whereas those in the posterior caeca are about 100 pm. Although different regions along the ventriculus cannot be distinguished under the stereoscopic microscope, the pH of the contents does vary along its length (anterior, pH 6.5; posterior, pH 7.4) (Ferreira et ul., 1990a). The heights of the cells along the ventriculus change from 78 Frn in the anterior region to 103 pm in the posterior region. In order to describe better the differences found in the morphology of the cells along the A. ,fluvolineata midgut, the lobes of the caeca were separated, whereas the ventriculus was divided into three sections of approximately the same length (Y,, VI, V,). Morphologically columnar cells can be recognized in all midgut regions, in spite of the fact that their morphology can change along the midgut and even within
FIGURE 1. Ultrastructural features of adult A. .flu~~jlinrcltu midgut cells. (A) Detail of the apex of an anterior caecal cell. Note secretory vesicles and dark granules outside the cells. (B) Anterior caecal cell. Observe Golgi areas with associated secretory vesicles. (C) Detail of the base of an anterior caecal cell showing basal membrane infoldings. (D) Detail of the apex of a Vz cell. Note the lack of granules outside the cell. (E) Detail of the base of V, cell showmg a basal labyrinth poorly dcvelopcd. Abbreviations: BL, basal lamina: BMI, basal membrane mfoldings: G, Golgi area; Gr, granules; Mi. mitochondria; Mv, microvilli; N. nucleus; RER, rough endophasmx reticulum; SV. secretory vesicle. Bar = I pm.
468
SANDRO R. MARANA
secretory vesicles may be traced back to the abundant Golgi areas observed mainly close to the nucleus [Fig. l(B)]. In the vicinity of Golgi areas, rough endoplasmic reticulum is plentiful, and occurs as groups of elongated cystemae. At the base of the microvilli there are also abundant mitochondria and well developed smooth endoplasmic reticulum cistemae [Fig. l(A)]. Multivesicular bodies and lipid spheres are scattered throughout the cell cytoplasm. The basal plasma membrane, covered with a prominent basal lamina, invaginates and forms narrow and branched channels which open to the underlying hemolymph spaces [Fig. l(C)]. Fine structure
of columnar
ventricular
cells
V, columnar cells are very similar to caecal cells, with microvilli about 7.6 pm long. V2 columnar cells have secretory vesicles with dark contents, but no dense granules are observed outside the cells [Fig. l(D)]. The microvilli are about 12.5 pm long. The basal labyrinth is remarkably developed. V, columnar cells are taller than V, and V, cells, have microvilli about 12.7 pm long and resemble V, cells, except for a basal labyrinth not so well developed [Fig. l(E)]. Subcellular distribution of digestive enzymes in the cells of the anterior lobe of caeca at different times after a meal A.$avoZineata males were starved for 24 h to ensure that they will eat during the 20 mm/meal. The meals were offered at noon, because at this time, in the field, A.Jlavolineata grasshoppers are most active (Dr Alejo Mesa, personal communication). At 0, 1, 3 and 5 h after the meal, groups of animals were dissected and their anterior caeca isolated and homogenized in isotonic conditions. ALfEavolineata anterior caeca were chosen because these tissues are the richest in digestive enzymes (Ferreira et al., 1990a). The simplified subcellular fractionation procedure was employed. Three subcellular fractions were obtained: a final soluble fraction (fraction S) corresponding to the supematant of the centrifugation; a soluble fraction (fraction SM) freed by freezing-thawing the pellet of the centrifugation, and the remaining pellet (fraction M) after freezing-thawing. Some of the digestive enzymes which are found in major amounts in the caecal cells (Ferreira et al., 1990a) were assayed in S, SM and M, and the results are displayed in Fig. 2. The data showed that maltase and cellobiase specific activities increase in SM 3 h after the meal and then decrease. Amylase specific activity in SM is high in starving insects, decreasing on feeding (1 h after the meal), and then increasing to an approximately constant value (3 and 5 h after the meal). The aryl @glucosidase specific activity remained constant during the whole experiment. Fraction SM probably contains the enzymes from
et
al.
inside the secretory vesicles which are disrupted on freezing and thawing. Thus, the experiment suggested that digestive enzymes in the secretory vesicles reached maximum activities at 3 h after the meal. Due to this, detailed subcellular experiments were performed using males starved for 24 h, fed for 20 min at noon and dissected 3 h after the meal. Only the anterior caeca were homogenized in isotonic conditions. The results are displayed in Fig. 3. y-Glutamyl transferase is found mainly in particulate fractions both in membrane-bound (shaded areas in Fig. 3) and soluble forms, whereas aminopeptidase and dipeptidase are recovered mainly in the final supematant, although significant amounts also occur in the particulate fractions, mainly in soluble form (Fig. 3). About 70% of the alkaline phosphatase of anterior caeca homogenates is soluble (not shown). Lactate dehydrogenase and trypsin occur almost exclusively in the final supematant. Succinate dehydrogenase is mostly recovered in membranes, although significant amounts are also found in soluble form derived from the particulate fractions (P,, Pz, P, and P4) and in the final supematant (Fig. 3). To test the possibility that the viscosity of medium was leading to incomplete pelleting of succinate dehydrogenase-carrying membranes, subcellular fractionations were performed as described in Fig. 3, except that the 100 OOOxgcentrifugations lasted for 180 min rather than 60 min. The results (not shown) were essentially similar to those presented in Fig. 3. Amylase and maltase are found in major amounts in the final supematant. Nevertheless, the specific activities of these enzymes are higher in the soluble part derived from the particulate fractions. Digestive enzymes after a meal.
in A. flavolineata gut compartments
Amylase activity in the caecal cells depends on the feeding stage, whereas for aryl P-glucosidase activity this is not true (see above). Due to this, these enzymes were chosen to study the distribution of digestive enzymes between caecal and crop contents at different times after a meal. This experiment was performed in an attempt to detect movements of caecal and crop contents. The results are shown in Fig. 4. Amylase activity decreases in caecal contents at 1 h after a meal, increases at 3 h and returns to the initial activity after 5 h. In crop contents, amylase activity increases at 1 h and then decreases from 3 h on to a constant value similar to the initial activity (Fig. 4). The behavior of aryl P-glucosidase activity is similar to that of amylase activity, except that after 5 h its activity in caeca contents does not return to the initial value (Fig. 4). DISCUSSION
Synthesis and secretion of digestive enzymes by A. flavolineata in response to a meal Caecal and V, cells present secretory vesicles coming from plentiful Golgi areas, and show dense granules
ABRACRIS
Maltase
FLAVOLINEATA
MIDGUTS
Amylase
469
Cellobiase
Aryl p-glucosidase
lh
:f 50
100
.
0
Y!
ToiZ
, .
d-+
0
1
50
100
&ySh ,
0
I
50
100
Protein FIGURE 2. Subcellular distribution of digestive enzymes from A. Jinvolineata anterior caeca after a meal (simplified procedure). Tissues originating from starved males (0 h) and from males after 1, 3 and 5 h after a meal were homogenized in isotonic buffer. Fractions from left to right are: M, cell membranes; SM, secretory vesicle contents; S, soluble fraction. The data are means and SEM based on four assays performed in each of three different preparations obtained from two insects. The recovery of each hydrolase activity in subcellular fractions was between 60 and 100% of the homogenate activity. The length of horizontal bars (with or without histogram bars) is proportional to the percentage of total protein. The height of the histogram is proportional to the relative specific activity (ratio of the specific activity of the sample to that of the homogenate, which is established as one), whereas the area of the histogram is proportional to the percentage amount (% total activity) of the enzyme. The absence of a histogram means that no activity was found in the corresponding sample.
among the microvilli. These dense granules, which may change in shape when pressed by neighboring microvilli, seem to be derived from vesicles situated close to the base of the microvilli. The release of long-standing granules may be an adaptation to enhance the dispersion of the secretory vesicle contents into the caecal lumen. The movement of fluid from the ventriculus and crop into the caeca (see below) would prevent a uniform diffusion of normal vesicle contents after exocytosis. V2 and V, cells display the same secretory process as caecal and V, cells, except for the lack of extracellular dense granules. The absence of these granules may be a consequence of their quick dispersion in the posterior ventricular contents, where the pH is higher than that in the posterior region (Ferreira et al., 1990a). Taking into account the amounts of digestive enzymes recovered in midgut cells (Ferreira et al., 1990a), the anterior caecal cells would be the most active secretory region. Nevertheless, before accepting this hypothesis it is necessary to show that digestive enzymes are actually enclosed in secretory vesicles in A. JEavolineatu anterior caecal cells. Subcellular fractionation is one of the techniques which may be used for this purpose. Since it is known that the feeding stage may influence digestive enzyme synthesis and secretion, the simplified subcellular fractionation procedure was employed with
A. jhzvohzeata anterior caecal cells from insects at 0, 1, 3 and 5 h after the meal. The data suggested (see section 3) that maltase and cellobiase activities increase in secretory vesicles 3 h after the meal and then decrease. Amylase activity is high in secretory vesicles in starving insects, decreases 1 h after the meal and then increases to a constant value. The aryl /3-glucosidase activity remained constant during the whole experiment. Although cellobiase and aryl /3-glucosidase are both pglucosidases, they correspond to different enzymes in A. flavolineatu midguts (Marana et al., 1995). Although these results were consistent, a confirmation of the subcellular distribution of enzymes is still necessary, based on the fact that the cell fractions which supposedly contain secretory vesicles may also contain glycocalyx associated enzymes. For this, A. Jlavolineatu males were starved for 24 h, fed for 20 min at noon and dissected 3 h after the meal. The anterior caeca were then homogenized in isotonic conditions followed by differential centrimgation. The enzyme markers used were: lactate dehydrogenase (cytosol) and succinate dehydrogenase (mitochondria). Attempts to use y-glutamyl transferase, alkaline phosphatase and aminopeptidase as microvillar membrane markers were unsuccessful. These enzymes are mostly soluble in A. jhwolineata anterior caeca, in contrast to what is observed in several other
SANDRO
R. MARANA
et al.
Amylase
Aryl p-glucosidase
3,
IL SDH
Caeca
LDH
3
2 .
2 1
1 .
‘_n
h s ‘j;
4
$
3
.=
z
-
JJ 1
3
:__F,
Caeca
5
3_
1 al
of
I
Crop 2.
2_
1 .
1 .
Crop L
4
I
Dip
3
3
AmY
. 3
.
I 5
FIGURE4. Amylasc and aryl /3-glucosidase activities in caecal and crop contents after a meal. Activities are expressed as relative specific activities to discount differences in grasshoppers sizes, and incomplete recoveries of luminal contents, which is frequent in the case of the caecal contents. Each data point corresponds to four assays in two different samples obtained from two insects.
4 I
.
Hours after a meal
GIlkIm
4
. 1
Mal
3 2 1 JIII!CI
50
100
PrOotein FIGURE 3. Distribution of enzymes among the subcellular fractions of A. ,fluvolineata anterior caeca. Fractions from left to right are: SP,, PP,, SP,, PP,, SP,, PP,, SP,, PP, and final supematant. The data are means and SEM based on four assays performed in each of three different preparations obtained from two insects, The recovery of each hydrolase activity in subcellular fractions was between 60 and 100% of the homogenate activity. Other details as in legend of Fig. 2. Mat, maltase; Amy, amylase; AP, aminopeptidase; Dip, dipeptidase; y-Glut, y-glutamyl transferase; LDH, lactate dehydrogenase; SDH, succinate dehydrogenase; Try; trypsin.
insects (Terra and Ferreira, 1994). The occurrence of succinate dehydrogenase in soluble fractions (Fig. 3) may result from its partial solubilization in the conditions employed. The high specific activities observed in the soluble fractions argue against accidental contamination whereas the identical results by pelleted material, observed after extending the 100 OOOxg centrifugations to 180 min rule out a viscosity effect. Amylase and maltase are found in major amounts in the final supematant, although their specific activities are higher in the soluble subfraction part derived from the particulate fractions. Activities recovered in membranes (PP) are probably artifacts as a result of the adsorption of soluble enzymes (SP). Digestive enzyme activities recovered in SP, and SP, correspond to enzyme mol-
ecules enclosed inside secretory vesicles, whereas those in SP, and FS may correspond to enzymes entrapped in the cell glycocalyx in either weak or very weak form, respectively (Ferreira et al., 1990b; Terra and Ferreira, 1994). Thus, the data suggest that at least amylase and maltase (Fig. 3), and probably also cellobiase (Fig. 2), occur partly in secretory vesicles and partly associated with the caeca cell glycocalyx. Aminopeptidase is recovered in PP, in sufficient amounts to rule out an artifact. Since similar PP, preparations obtained from different insects contain microvillar membranes (Terra and Ferreira, 1994), it is probable that part of the A. jkvolineatu caecal aminopeptidase activity corresponds to a microvillar enzyme. Nevertheless, most of the aminopeptidase activity is soluble and is distributed among subcellular fractions in a way resembling amylase and maltase. Thus, soluble aminopeptidase should occur in secretory vesicles and associated with the cell glycocalyx. Membrane-bound dipeptidase activity seems to be an artifact. Although the specific activity of soluble dipeptidase is higher in FS, its subcellular distribution resembles that of amylase and maltase. Trypsin is found in FS, with negligible activity being recovered in SP, and SP,. In the light of the previous discussion, it is possible that trypsin activity in the secretory vesicles is low, in contrast to the major amounts recovered in FS, presumably associated with the glycocalyx. In summary, the results of the detailed cell fractionation confirmed the conclusions drawn from the obser-
A BRA CRIS FLA VOLINEA TA MIDGUTS
vations of the simplified procedure. Thus, the data on A. caecal cells show that, in this insect, digestive enzymes are synthesized and secreted by exocytosis in response to feeding. An exception seems to be aryl pglyucosidase, whose intracellular activities do not change, regardless of the feeding stage. Some enzymes are stored in secretory vesicles even during fasting (e.g. amylase), whereas others are not (e.g. maltase and cellobiase). These results apparently diverge from others. Secretory vesicles were not observed in L. migrutoria midguts, which show, on the other hand, cell extrusions as well as small and large vesicles budding from the cell microvilli (Heinrich and Zebe, 1973). The occurrence of secretory vesicles in L. migratoria midgut cells may depend on the insect feeding stage. As shown in this paper, the amount of digestive enzymes in secretory vesicles in A. Jtavolineata midguts is highly dependent on the feeding stage. Although frequently interpreted as apocrine secretion, cell extrusions are more probably manifestations of cell renewal (see review: Terra and Ferreira, 1994). Small vesicles budding from sides of midgut cell microvilli may correspond to microapocrine secretion. This consists of secretory-like vesicles budding laterally from the microvilli as double membrane vesicles. Secretion of digestive enzymes by this route has been established in Erinnyis ello (Santos et al., 1986). Earlier description of microapocrine secretion of digestive enzymes in Musca domestica (Espinoza-Fuentes et al., 1987) was not confirmed (Jordao et al., 1996). M. domestica presents only single membrane budding-vesicles in their midgut cell microvilli. This kind of microvillar vesiculation has also been described in other Diptera (De Priester, 1971; Nopanitaya and Misch, 1974; Lehane, 1976) as well as in Collembola (Humbert, 1979) and Orthoptera (Heinrich and Zebe, 1973). It may correspond to a kind of microvillar membrane renewal. Large vesicles budding from the top of midgut cell microvilli are probably associated with the formation of the peritrophic membrane (Heinrich and Zebe, 1973; Santos et al., 1986).
jlavolineata
Digestive physiological tion: a model
events
during
Acrididae
diges-
Immediately after a meal, A. jlavolineata caecal cells secrete digestive enzymes into the caecal contents, which pass on to the crop. Based on the work of Baines (1979) with dyes, it is likely that in A.Jiavolineata the discharge of the caecal contents into the anterior ventriculus is caused by gross contractions of the caeca, and that digestive enzymes are passed forward from the anterior midgut into the crop moved by antiperistalsis. Ingested saliva, which contains small amounts of amylase (Ferreira et al., 1990a), may disperse the food in the crop. Solid food entering the midgut is prevented from penetrating into the caeca (Baines, 1978; Ferreira et al., 1990a), probably due to the small apertures maintained
471
by the sphincter-like muscle placed near the point of caecal attachment to the midgut (Hodge, 1939; this paper). In contrast, crop fluid with dissolved food molecules and accompanying digestive enzymes passes into the caeca. A. Javolineatu caeca, V, and V, cells present morphological characteristics of fluid-absorbing cells (see discussion in Ribeiro et al., 1990). Nevertheless, caeca are the most important water-absorbing sites in grasshoppers due to their larger luminal surface. According to Chapman and Brandenburger (cited in Chapman, 1988a), the surface area of anterior caeca is more than seven times greater than the posterior caeca and twice as great as that of the ventriculus. Apparently, most A. Javolineata crop digestion has finished by about 3 h after a meal, since at this time digestive enzymes decrease in crop and increase and in the caecal contents, suggesting a transference of material from crop to caeca. This is in accordance with several studies of crop emptying performed in other grasshoppers (Baines et al., 1973; Simpson, 1983). In the caeca, most initial protein digestion and most final protein and carbohydrate digestion should take place, followed by the absorption of the resulting amino acids and sugars. Since the crop fluid entering the midgut overcomes the water absorption capacity of the caeca (Dow, 1981b), excess fluid moves down the ventriculus together with solid food. Further digestion of solid food occurs along the ventriculus and, as soon as the food molecules become sufficiently small to pass through the peritrophic membrane, digestion is finished by microvillar enzymes, such as aminopeptidase, and absorption presumably occurs (Ferreira et al., 1990a). Between 3 and 5 h after a meal, digestive enzymes in A. Javolineata caeca contents decrease, suggesting they are discharged into the ventriculus. In S. gregaria, caecal contents are emptied into the ventriculus 3 h after taking a meal. Thereafter, caecal luminal volume increases again due to the forward movement of Malpighian tubule secretion in the absence of salivation (Dow, 1981b). It is probable that the same occurs in A. jlavolineata. The data discussed above showed that the anterior caeca are the main sites of digestive enzyme secretion, of the intermediary and final stages of digestion and, finally, of nutrient absorption, The lack of obvious differences in the sizes of the anterior caeca among grasshoppers adapted to different diets may indicate that different food sources do not impose different constraints on the system (Chapman, 1988b). A. flavolineata posterior caeca have the same digestive functions as the anterior caeca, although on a smaller scale due to their smaller sizes (see above). The fact that the posterior caeca change in relative size among different grasshoppers suggest they may have functions other than digestive ones. Posterior caeca are more developed in forbivorous than in graminivorous species (Chapman, 1988b). Since forbs are richer in allelochemicals than grasses, Chapman (1988b) suggested that posterior caeca have a major role in detoxification. The epithelial pockets
472
SANDRO
R. MARANA
present in the posterior caeca of many grasshoppers (including A. flavolineata) may have a role in the removal of phenolics from the gut (Bernays, 1981; Chapman, 1988b). REFERENCES Ackrell B. A. C., Keamey E. B. and Singer T. P. (1978) Mammalian succinate dehydrogenase. Meth. Enzym. 53, 466483. Anstee J. H. and Charnley A. K. (1977) Effects of frontal ganglion removal and starvation on activity and distribution of six gut enzymes in Locusta. J. Insect Physiol. 23, 9655974. Baines D. M. (1978) Observations on the peritrophic membrane of Locusta migratoriu migratorioides (RandF) nymphs. Acrida 7, 1I-22. Baines D. M. (1979) Studies of weight changes and movements of dyes in the caeca and midgut of fifth-instar Locusta migratoria migratorioides (RandF) in relation to feeding and food deprivation. Acrida 8, 95-108. Baines D. M., Bemays E. A. and Leather E. M. (1973) Movement of food through the gut of fifth-instar males of Locusta migratoria migratorioides. Acrida 2, 3 19-332. Bemays E. A. (1981) A specialized region of the gastric caeca in the locust Schistocerca gregaria. Physiol. Entomol. 6, l-6. Bergmeyer, H. U. and Bemt, E. (1974) Calorimetric assay with Llactate, NAD, phenazine methosulfate and INT. In Method.7 of Enzymatic Analysis (Ed. Bergmeyer H. U.), 2nd edn, pp. 579-582. Chemie, Weinheim. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Chapman R. F. (1988a) Variations in the size of the midgut caeca during the fifth instar of the grasshopper Schistocerca americana (Drury). J. Insect. Physiol. 34, 329-335. Chapman R. F. (1988b) The relationship between diet and the size of the midgut caeca in grasshoppers (Insecta: Orthoptera: Acridoidea). Zool. J. Linn. Sot. 94, 319-338. Chapman, R. F. and Joem, A., Biology of Grasshoppers. Wiley, New York ( 1990). Dahlqvist A. (1968) Assay of intestinal disaccharidases. Analyt. Biochem. 22, 99- 107. De Duve C., Pressman B. C., Gianetto R., Wattiaux R. and Appelmans F. (1955) Tissue fractionation studies-6. Intracellular distribution patterns of enzymes in rat liver. Biochem. J. 60, 604-617. De Priester W. (1971) Ultrastructure of the midgut epithelial cells in the fly Calliphora erythrocephala. J. Ultrastruct. Res. 36, 783-805. Dow J. A. T. (1981a) Localization and characterization of water uptake from the midgut of the locust, Schistocerca gregaria. J. Expl Biol. 93, 269-28 1. Dow J. A. T. (198 I b) Countercurrent flow, water movements and nutrient absorption in the locust midgut. J. Insect Physiol. 27, 579-585. Droste H. J. and Zebe E. (1974) Carbohydrasen und Kohlenhydratverdauung im Darmtrakt von Locusta migratoria. J. Insect Physiol. 20, 1639-1657. Erlanger B. F., Kokowsky N. and Cohen W. (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch. Biochem. Biophys. 95, 271-278. Espinoza-Fuentes F. P., Ribeiro A. F. and Terra W. R. (1987) Microvillar and secreted digestive enzymes from Musca domestica larvae. Subcellular fractionation of midgut cells with electron microscopy monitoring. Insect B&hem. 17, 8 199827. Ferreira C., Oliveira M. C. and Terra W. R. (1990a) Compartmentalization of the digestive process in Ahrucris,Ravolineata (Orthoptera: Acrididae) adults. Insect Biochem. 20, 267-274. Ferreira C., Bellinello Cl. L., Ribeiro A. F. and Terra W. R. (1990b) Digestive enzymes associated with the glycocalyx, microvillar membranes and secretory vesicles from midgut cells of Tenehrio molitor larvae. Insect Biochem. 20, 8399847.
et al.
Heinrich D. and Zebe E. (I 973) Zur Feinstrukttu der Mitteldarmzellen von Locusta migratoria in verschiedenen Phasen der Verdauung. Cytobiologie 7, 3 15-326. Hodge C. (1936) The anatomy and histology of the alimentary tract of the grasshopper, Melanoplus differentialis Thomas. J. Morphol. 59, 423439. Hodge C. (I 939) The anatomy and histology of the alimentary tract of Locusta migratoria L. (Orthoptera: Acrididae). J. Morphol. 64, 3755399. Humbert W. (1979) The midgut of Tomocerus minor Lubbock (Insecta, Collembola): ultrastructure, cytochemistry, ageing and renewal during a molting cycle. Cell Tiss. Res. 196, 39-57. Jordlo B. P., Terra W. R., Ribeiro A. F., Lehane M. J. and Ferreira C. (1996) Trypsin secretion in Musca domestica larval midguts: a biochemical and immunocytochemical study. Insect Biochem. Molec. Biol. 26, 337-346. Lehane M. J. (1976) Digestive enzyme secretion in Stomoxys calcitrans (Diptera: Muscidae). Cell Tiss. Res. 170, 2755287. Marana S. R., Terra W. R. and Ferreira C. (1995) Midgut p-D-ghCOsidases from Ahracris~avolineata (Orthoptera: Acrididae). Physical properties, substrate specificities and function. Insect Biochem. Molec. Biol. 25, 8355843. Nicholson J. A. and Kim Y. S. (1975) A one-step L-amino acid oxidase assay for intestinal peptide hydrolase activity. Analyt. Biochem. 63, 110-117. Nishiitsuji-Uwo J. and Endo Y. (1981) Gut endocrine cells in insects: the ultrastructure of the endocrine cells in the cockroach midgut. Biomed. Res, 2, 3044. Noelting G. and Bemfeld P. (1948) Sur les enzymes amylolytiques. III. La /3-amylase: dosage activite et controle de I’absence d’oamylase. Helv. Chim. Acta 31, 2866290. Nopanitaya W. and Misch D. W. (1974) Developmental cytology of the midgut in the flesh-fly Sarcophaga bullata (Parker). Tiss. Cell. 6, 4877502. Raes H. and Verbeke M. (1994) Light and electron microscopical study of two types of endocrine cell in the midgut of the adult worker honeybee (Apis mellifrra). Tiss. Cell 26, 223-230. Ribeiro, A. F., Ferreira, C. and Terra, W. R. (1990) Morphological basis of insect digestion, In Animal Nutrition and Transport Processes (Edited by J. Mellinger), Vol. 1, pp. 966105. Karger, Basel. Santos C. D. and Terra W. R. (1984) Plasma membrane-associated amylase and trypsin: intracellular distribution of digestive enzymes in the midgut of the cassava homworm Erinn_)k ello. Insect Biochem. 14, 5877595. Santos C. D., Ribeiro A. F. and Terra W. R. (1986) Differential centrifugation, calcium precipitation and ultrasonic disruption of midgut cells of Erinnyis ello caterpillars. Purification of cell microvilh and inferences concerning secretory mechanisms. Can. J. Zool. 64, 490-500. Simpson S. J. (1983) Changes during the 5th instar of Locusta migratoria in the rate of crop emptying and their relationship to feeding and food utilization. Entomol. Expl Appl. 33, 2355243. Terra W. R. (1990) Evolution of digestive systems of insects. Ann. Rev. Entomol. 35, 181-200. Terra W. R. and Ferreira C. (1994) Insect digestive enzymes: properties, compartmentalization and function. Camp. B&hem. Ph.vsiol. 109B, l-62. Terra W. R., Ferreira C. and De Bianchi A. G. (1979) Distribution of digestive enzymes among the endo and ectoperitrophic spaces and midgut cells of Rhynchosciara and its physiological sigmficance. J. Insect Ph,vsiol. 25, 487494. Treheme J. (1967) Gut absorption. Ann. Rev. Entomol. 12, 45558. Uvarov, B. (I 966) Grasshoppers and Locusts, Vol. 1. Cambridge, London. Wigglesworth, V. B. (1972) The Principles of’ Insect Physiology. 7th edn. Methuen, London. Zitnan D., Saman 1. and Sehnal F. (1993) Peptidergic innervation and endocrine cells of insect midgut. Arch. Insect Biochem. Physiol. 22, 113-132.
ABRACRIS FLAVOLINEATA Alejo
MIDGUTS Mesa (UNESP,
413 campus
of Rio Clara) for collecting Ahracris to the technicians L.Y. Nakabayashi, M.V. Cruz and W. Caldeira. S.R. Marana is a graduate fellow of FAPESP. A.F. Ribeiro, W.R. Terra and C. Ferreirra arc staff members of their respective departments and research fellows of CNPq.
~flavolineutu. We are also indebted
Acknowledgements-This work was supported by the Brazilian Research Agencies FAPESP and CNPq. We arc much indebted to Dr
PII: SOO22-1910(96)00117-5
J. Insect Physrol. Vol. 43, No. 5, pp. 465413, 1997 0 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0022-1910197 $17.00 + 0.00
Ultrastructure and Secretory Activity of Abracris Jtavolineata (Orthoptera: Acrididae) Midguts SANDRO
R. MARANA,*
ALBERT0
F. RIBEIRO,?
WALTER
R. TERRA,*
CLkLIA
FERREIRA*$
Received 15 July 1996; revised 7 October 1996
The midgut of Abracris Jlavofineatu adults comprises a ventriculus and six anteriorly placed caeca each displaying an anterior and a posterior lobe. Columnar cells in the caeca and anterior ventriculus present secretory vesicles originating from abundant Golgi areas, which seem to result (through exocytosis) in dark granules among the microvilli. A. Jlavolineatu males were starved for 24 h, fed for 20 min at noon and dissected at 0, 1, 3 and 5 h after the meal. Enzyme assays were accomplished on crop and caecal contents and in subcellular fractions obtained from the isolated anterior caeca. Subcellular fractions putatively containing secretory vesicles were recognized. Digestive enzyme activity is usually low (amylase is high) in the secretory vesicles in starving insects, decreases 1 h after the meal, increases at 3 h, and thereafter decreases again (amylase remains constant). In caecal contents, digestive enzymes decrease at 1 h and increase at 3 h after the meal, the contrary being true for crop contents. Thus, in A. flavolineutu caecal cells, digestive enzymes (/3-glucosidase is an exception) are synthesized and secreted by exocytosis in response to feeding. Also in response to feeding, digestive enzymes are transferred from caecal contents to the crop and, after about 3 h following the meal, crop-caecal dispersed material with accompanying enzymes are translocated to the caeca, where digestion ends and absorption occurs. 0 1997 Elsevier Science Ltd Columnar Trypsin
cells
Midgut
caeca
Secretory
vesicles
Exocytosis
INTRODUCTION
Maltase
/3-Glucosidase
carbohydrates and proteins are digested mainly in the crop and caecal contents, respectively, although at least part of the intermediate and final digestions of protein occur at the surface of the caecal and ventricular cells. Compartmentalization data also support the hypothesis that digestive enzymes are synthesized and secreted by all midgut cells (mainly in the caeca) and then passed forward into the crop (Ferreira et al., 1990a). The role of the grasshopper midgut caeca in nutrient and water absorption has been known for many years (Treheme, 1967). More recently these studies were detailed by Dow (1981a, b), who showed that in starving grasshoppers a counter-current flow is formed caused by the secretion of fluid by the Malpighian tubules and by its absorption in the caeca. Similar fluid fluxes were found in most insects and are responsible for digestive enzyme recycling, thus minimizing enzyme excretion (Terra, 1990). Feeding grasshoppers present remarkable salivation, which saturates the water absorption site in the caeca, and then abolishes counter-current flows (Dow, 1981a, b). This was confirmed by the finding that
Although grasshoppers have been studied for a long time (Uvarov, 1966; Wigglesworth, 1972; Chapman and Joem, 1990), only recently have they became the subjects of quantitative work regarding digestive physiology. Except for amylase and trehalase, the salivary glands display negligible amounts of digestive enzymes. Carbohydrases (except trehalase) predominate in the crop, and their pH optima agree with the pH prevailing in the crop contents. Aminopeptidase (soluble and membrane bound) and trypsin predominate in the cells and contents of the caeca, respectively (Droste and Zebe, 1974; Anstee and Charnley, 1977; Ferreira et al., 1990a). Thus, enzyme compartmentalization data suggest that
*Departamento Slo Paula, ?Departamento Slo Paula, :To whom all
Amylase
de Bioquimica, lnstituto de Quimica, Universidade de C.P. 26077, 05599-970, SZo Paula, Brazil de Biologia, Institute de BiociEncias, Universidade de C.P. 11461, 05422-970, S%o Paula, Brazil correspondence should be addressed. 465
SANDRO R. MARANA
466
feeding grasshoppers excrete digestive enzymes at a rate similar to that of undigested food (Ferreira et ul., 1990a). In spite of some light- and electron-microscope studies of grasshopper midguts (Hodge, 1936, 1939; Heinrich and Zebe, 1973; Bemays, 1981), sufficient data have not been produced to relate midgut cell features to actual midgut fluid fluxes or secretory events. In this paper, ultrastructural data on the midgut are presented supporting the current model of midgut fluid fluxes and secretion. In addition, biochemical data are given confirming the secretory activity of caecal cells. MATERIAL
AND METHODS
Animals Abrucris Jlavolineuta (Orthoptera, Acrididae, subfamily Ommatolampinae, tribe Abracrini) adults were collected in a small forest close to the University campus in Rio Claro, State of Sao Paulo, Brazil. They were maintained in our laboratory in cages (3 1x3 1x40 cm) under natural photoperiod conditions at relative humidity of 50-60% at 24f2”C. The grasshoppers had free access to water and were fed ad libitum with collard (Brassica oleracea acephalu) leaves which were maintained with their stems in water and were changed twice a week. Preparation
of samples
of gut sections
Grasshoppers at different feeding conditions were immobilized by placing them on ice, after which they were dissected in cold 250 mM NaCI. Caecal and ventricular tissues, after being thoroughly rinsed with 250 mu NaCl, were homogenized in double distilled water using a Potter-Elvehjem homogenizer. The preparations were then passed through a nylon mesh of 100 pm pore size. Peritrophic membranes and contents were homogenized similar to gut preparations, but without previous rinsing with saline, and centrifuged at 20 OOOxg for 30 min at 4°C to remove undigested material. After collection with the aid of a capillary, the caecal contents were dispersed in a known volume of double distilled water. All samples were stored at -20°C until use. Light and electron
microscopy
For studies with the light microscope, animals were dissected in their own hemolymph and midgut regions were fixed in 4% paraformaldehyde in 20 mM phosphate buffer (pH 7.4) containing 0.15 M NaCl. The materials were embedded in Historesin, cut into 34 pm sections and stained with hematoxylin and eosin. For transmission-electron microscopy, animals were dissected in their own hemolymph and midgut sections were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 h at 4°C. The midgut pieces were post-fixed in 1% osmium tetroxide in the cacodylate buffer. In-block staining was performed in 1% aqueous uranyl acetate for 16-18 h. After dehydration, the
et 01.
material was embedded in Spurr resin, cut into ultrathin sections, stained with lead citrate, and examined in a Zeiss EM 900 electron microscope operated at 80 kV. D@zrential
CentriJingation of’cuecal
homogenates
Caecal anterior lobes were homogenized in 2 mM sodium phosphate buffer (pH 7.0) containing 471 mM mannitol, with the aid of a Potter-Elvehjem (10 strokes). The resulting homogenates were passed through a nylon mesh of 45 pm pore size and its volume adjusted to contain material corresponding to one animal/ml. Differential centrifugation of the caecal homogenates were performed according to two different procedures. The first is a simplified method which was used to study tissue digestive enzymes at different times after a meal. For this, the homogenates were centrifuged at 100 OOOxg for 60 min. The supematant (S) was recovered and the resulting pellet was resuspended in the homogenizing buffer and, after three cycles of freezing and thawing, was centrifuged again at 100 OOOxg for 60 min. The resulting supematant (SM) and pellet (M) were collected and, along with FS, were assayed for several enzymes. The second procedure is somewhat more complex, as it involves a larger number of steps, the aim being to assign enzymes to specific cell compartments. For this, the homogenates were centrifuged and the following fractions collected: P,, pellet 6OOxg, 10 min; P2, pellet 33OOxg, 10 min; P,, pellet 25 OOOxg, 10 min; Pz, pellet 100 OOOxg, 60 min, FS, final supematant. After this differential centrifugation the collected pellets were homogenized with the aid of a Potter-Elvehjem homogenizer in the homogenization medium and, following three freezing and thawing cycles, were centrifuged at 100 OOOxg for 60 min. Thus, fraction P, resulted in a supematant SP, and a pellet PP,, fraction PI in SP2 and PP,, fraction P.3 in SP, and PP, and fraction P, in SP, and PP4. The supematants and pellets corresponding to each fraction were assayed for several enzymes. The following enzyme markers for subcellular fractions were assayed: succinate dehydrogenase (mitochondria) and lactate dehydrogenase (cytosol). Results are presented according to De Duve et al. (1995) where the height of the histogram is proportional to the specific activity, and the area of the histogram is proportional to the total activity of the enzyme. Protein
determination
and enzyme
assays
Protein was determined according to Bradford (1976) using ovalbumin as a standard. Aminopeptidase was assayed in 50 mu sodium phosphate buffer (pH S.O), using 1 mu leucinep-nitroanilide as a substrate (Erlanger et ul., 1961). Amylase was assayed in 50 mM sodium citrate-phosphate (pH 6.5) containing 10 KIM NaCl with 0.5% soluble starch as a substrate (Noelting and Bemfeld, 1948). Aryl P-glucosidase activity was determined in 50 mu sodium citrate-phosphate buffer (pH 6.0), using 1 ITIM p-nitrophenyl P-D-glucoside as a substrate (Terra et al., 1979). Cellobiase and maltase were assayed
AHHACKIS
FLA VOLINEATA
in 50 mM sodium citrate-phosphate buffer (pH 6.0) with 7 mM cellobiose and maltose as substrates, respectively, (Dahlqvist, 1968). Dipeptidase activity was assayed in 50 mM Tris+HCl buffer pH 8.0 with Gly-Leu as a substrate (Nicholson and Kim, 1975). y-Glutamyl transferase was assayed in 50 mM Tris-HCl pH 8.8 with 1 mM yglutamyl p-nitroanilide and SO mM Gly-Gly as substrates (Erlanger et ul., 1961). Lactate dehydrogenase and succinate dchydrogenase were assayed according to Bergmeyer and Bemt (1974) and Ackrell et ul. (1978), respcctively, as detailed elsewhere (Santos and Terra, 1984). Trypsin was assayed in 50 mM sodium phosphate pH 7.5 with 0.8 mM a-N-benzoyl-Dr_-arginine-/2-nitroanilide as a substrate (Erlanger ef ul., 1961). Incubations were carried out for at least four different time periods, and initial rates of hydrolysis were calculated. All assays were performed under conditions such that activity was proportional to protein concentration and to time. Controls without enzyme or without substrate were performed. One U of enzyme is defined as the amount of the enzyme that hydrolyzes 1 pmol of substrate (or bond) per min.
467
MlDGlJTS
the same region. Details are provided below. Regenerative cells are collected in nidi at the base of the epithelium and occur along the caeca and ventriculus. Endocrine cells are rare, lack a basal labyrinth and are situated at the base of the epithelium. These cells, which occur in insects of several different orders, typically show a cytoplasm with plenty of small vesicles with either clear or dark contents (Nishiitsuji-Uwo and Endo, 1981; Zitman et al., 1993; Raes and Verbeke, 1994). Fine structure
of columnar
midgut curcal
cells
Columnar cells are similar in the two lobes of the midgut caeca. The apical membrane is modified into cylindrical microvilli in a compact, regular arrangement. The microvilli are about 8.1 (anterior caeca) or 12.2 pm long (posterior caeca) and are covered with a conspicuous glycocalyx. Among the microvilli there are dense granules, which may change in shape when pressed by the neighboring microvilli [Fig. l(A)]. The extracellular dense granules seem to derive from secretory vesicles close to the base of the microvilli. The contents of the vesicles are, however, less dense than the granules. The
RESULTS General
morphologv
qf the gut
The general morphology of A. ,flavolineata adult gut is similar to that of other grasshoppers (Hodge, 1936, 1939) and therefore only a brief description will be given. The gut of adults is composed of a large crop (66.4% of gut length), a relatively short ventriculus (13.5% of gut length) with six anteriorly placed caeca, and a hindgut (20.1% of gut length) differentiated into ileum, colon and rectum. The caeca have anterior and posterior lobes, which are referred to here as anterior and posterior caeca. The posterior caeca corresponds to 8% of gut length. whereas the anterior caeca equated 12.6%. The cpithelium of the anterior caeca forms folds which permits distensibility and increases the surface area. The posterior caeca present at their proximal end discrete pockets similar to those described in detail in Schistoccwu grcguriu (Bemays, 1981). The height of the cells in the anterior caeca are about 64 pm, whereas those in the posterior caeca are about 100 pm. Although different regions along the ventriculus cannot be distinguished under the stereoscopic microscope, the pH of the contents does vary along its length (anterior, pH 6.5; posterior, pH 7.4) (Ferreira et ul., 1990a). The heights of the cells along the ventriculus change from 78 Frn in the anterior region to 103 pm in the posterior region. In order to describe better the differences found in the morphology of the cells along the A. ,fluvolineata midgut, the lobes of the caeca were separated, whereas the ventriculus was divided into three sections of approximately the same length (Y,, VI, V,). Morphologically columnar cells can be recognized in all midgut regions, in spite of the fact that their morphology can change along the midgut and even within
FIGURE 1. Ultrastructural features of adult A. .flu~~jlinrcltu midgut cells. (A) Detail of the apex of an anterior caecal cell. Note secretory vesicles and dark granules outside the cells. (B) Anterior caecal cell. Observe Golgi areas with associated secretory vesicles. (C) Detail of the base of an anterior caecal cell showing basal membrane infoldings. (D) Detail of the apex of a Vz cell. Note the lack of granules outside the cell. (E) Detail of the base of V, cell showmg a basal labyrinth poorly dcvelopcd. Abbreviations: BL, basal lamina: BMI, basal membrane mfoldings: G, Golgi area; Gr, granules; Mi. mitochondria; Mv, microvilli; N. nucleus; RER, rough endophasmx reticulum; SV. secretory vesicle. Bar = I pm.
468
SANDRO R. MARANA
secretory vesicles may be traced back to the abundant Golgi areas observed mainly close to the nucleus [Fig. l(B)]. In the vicinity of Golgi areas, rough endoplasmic reticulum is plentiful, and occurs as groups of elongated cystemae. At the base of the microvilli there are also abundant mitochondria and well developed smooth endoplasmic reticulum cistemae [Fig. l(A)]. Multivesicular bodies and lipid spheres are scattered throughout the cell cytoplasm. The basal plasma membrane, covered with a prominent basal lamina, invaginates and forms narrow and branched channels which open to the underlying hemolymph spaces [Fig. l(C)]. Fine structure
of columnar
ventricular
cells
V, columnar cells are very similar to caecal cells, with microvilli about 7.6 pm long. V2 columnar cells have secretory vesicles with dark contents, but no dense granules are observed outside the cells [Fig. l(D)]. The microvilli are about 12.5 pm long. The basal labyrinth is remarkably developed. V, columnar cells are taller than V, and V, cells, have microvilli about 12.7 pm long and resemble V, cells, except for a basal labyrinth not so well developed [Fig. l(E)]. Subcellular distribution of digestive enzymes in the cells of the anterior lobe of caeca at different times after a meal A.$avoZineata males were starved for 24 h to ensure that they will eat during the 20 mm/meal. The meals were offered at noon, because at this time, in the field, A.Jlavolineata grasshoppers are most active (Dr Alejo Mesa, personal communication). At 0, 1, 3 and 5 h after the meal, groups of animals were dissected and their anterior caeca isolated and homogenized in isotonic conditions. ALfEavolineata anterior caeca were chosen because these tissues are the richest in digestive enzymes (Ferreira et al., 1990a). The simplified subcellular fractionation procedure was employed. Three subcellular fractions were obtained: a final soluble fraction (fraction S) corresponding to the supematant of the centrifugation; a soluble fraction (fraction SM) freed by freezing-thawing the pellet of the centrifugation, and the remaining pellet (fraction M) after freezing-thawing. Some of the digestive enzymes which are found in major amounts in the caecal cells (Ferreira et al., 1990a) were assayed in S, SM and M, and the results are displayed in Fig. 2. The data showed that maltase and cellobiase specific activities increase in SM 3 h after the meal and then decrease. Amylase specific activity in SM is high in starving insects, decreasing on feeding (1 h after the meal), and then increasing to an approximately constant value (3 and 5 h after the meal). The aryl @glucosidase specific activity remained constant during the whole experiment. Fraction SM probably contains the enzymes from
et
al.
inside the secretory vesicles which are disrupted on freezing and thawing. Thus, the experiment suggested that digestive enzymes in the secretory vesicles reached maximum activities at 3 h after the meal. Due to this, detailed subcellular experiments were performed using males starved for 24 h, fed for 20 min at noon and dissected 3 h after the meal. Only the anterior caeca were homogenized in isotonic conditions. The results are displayed in Fig. 3. y-Glutamyl transferase is found mainly in particulate fractions both in membrane-bound (shaded areas in Fig. 3) and soluble forms, whereas aminopeptidase and dipeptidase are recovered mainly in the final supematant, although significant amounts also occur in the particulate fractions, mainly in soluble form (Fig. 3). About 70% of the alkaline phosphatase of anterior caeca homogenates is soluble (not shown). Lactate dehydrogenase and trypsin occur almost exclusively in the final supematant. Succinate dehydrogenase is mostly recovered in membranes, although significant amounts are also found in soluble form derived from the particulate fractions (P,, Pz, P, and P4) and in the final supematant (Fig. 3). To test the possibility that the viscosity of medium was leading to incomplete pelleting of succinate dehydrogenase-carrying membranes, subcellular fractionations were performed as described in Fig. 3, except that the 100 OOOxgcentrifugations lasted for 180 min rather than 60 min. The results (not shown) were essentially similar to those presented in Fig. 3. Amylase and maltase are found in major amounts in the final supematant. Nevertheless, the specific activities of these enzymes are higher in the soluble part derived from the particulate fractions. Digestive enzymes after a meal.
in A. flavolineata gut compartments
Amylase activity in the caecal cells depends on the feeding stage, whereas for aryl P-glucosidase activity this is not true (see above). Due to this, these enzymes were chosen to study the distribution of digestive enzymes between caecal and crop contents at different times after a meal. This experiment was performed in an attempt to detect movements of caecal and crop contents. The results are shown in Fig. 4. Amylase activity decreases in caecal contents at 1 h after a meal, increases at 3 h and returns to the initial activity after 5 h. In crop contents, amylase activity increases at 1 h and then decreases from 3 h on to a constant value similar to the initial activity (Fig. 4). The behavior of aryl P-glucosidase activity is similar to that of amylase activity, except that after 5 h its activity in caeca contents does not return to the initial value (Fig. 4). DISCUSSION
Synthesis and secretion of digestive enzymes by A. flavolineata in response to a meal Caecal and V, cells present secretory vesicles coming from plentiful Golgi areas, and show dense granules
ABRACRIS
Maltase
FLAVOLINEATA
MIDGUTS
Amylase
469
Cellobiase
Aryl p-glucosidase
lh
:f 50
100
.
0
Y!
ToiZ
, .
d-+
0
1
50
100
&ySh ,
0
I
50
100
Protein FIGURE 2. Subcellular distribution of digestive enzymes from A. Jinvolineata anterior caeca after a meal (simplified procedure). Tissues originating from starved males (0 h) and from males after 1, 3 and 5 h after a meal were homogenized in isotonic buffer. Fractions from left to right are: M, cell membranes; SM, secretory vesicle contents; S, soluble fraction. The data are means and SEM based on four assays performed in each of three different preparations obtained from two insects. The recovery of each hydrolase activity in subcellular fractions was between 60 and 100% of the homogenate activity. The length of horizontal bars (with or without histogram bars) is proportional to the percentage of total protein. The height of the histogram is proportional to the relative specific activity (ratio of the specific activity of the sample to that of the homogenate, which is established as one), whereas the area of the histogram is proportional to the percentage amount (% total activity) of the enzyme. The absence of a histogram means that no activity was found in the corresponding sample.
among the microvilli. These dense granules, which may change in shape when pressed by neighboring microvilli, seem to be derived from vesicles situated close to the base of the microvilli. The release of long-standing granules may be an adaptation to enhance the dispersion of the secretory vesicle contents into the caecal lumen. The movement of fluid from the ventriculus and crop into the caeca (see below) would prevent a uniform diffusion of normal vesicle contents after exocytosis. V2 and V, cells display the same secretory process as caecal and V, cells, except for the lack of extracellular dense granules. The absence of these granules may be a consequence of their quick dispersion in the posterior ventricular contents, where the pH is higher than that in the posterior region (Ferreira et al., 1990a). Taking into account the amounts of digestive enzymes recovered in midgut cells (Ferreira et al., 1990a), the anterior caecal cells would be the most active secretory region. Nevertheless, before accepting this hypothesis it is necessary to show that digestive enzymes are actually enclosed in secretory vesicles in A. JEavolineatu anterior caecal cells. Subcellular fractionation is one of the techniques which may be used for this purpose. Since it is known that the feeding stage may influence digestive enzyme synthesis and secretion, the simplified subcellular fractionation procedure was employed with
A. jhzvohzeata anterior caecal cells from insects at 0, 1, 3 and 5 h after the meal. The data suggested (see section 3) that maltase and cellobiase activities increase in secretory vesicles 3 h after the meal and then decrease. Amylase activity is high in secretory vesicles in starving insects, decreases 1 h after the meal and then increases to a constant value. The aryl /3-glucosidase activity remained constant during the whole experiment. Although cellobiase and aryl /3-glucosidase are both pglucosidases, they correspond to different enzymes in A. flavolineatu midguts (Marana et al., 1995). Although these results were consistent, a confirmation of the subcellular distribution of enzymes is still necessary, based on the fact that the cell fractions which supposedly contain secretory vesicles may also contain glycocalyx associated enzymes. For this, A. Jlavolineatu males were starved for 24 h, fed for 20 min at noon and dissected 3 h after the meal. The anterior caeca were then homogenized in isotonic conditions followed by differential centrimgation. The enzyme markers used were: lactate dehydrogenase (cytosol) and succinate dehydrogenase (mitochondria). Attempts to use y-glutamyl transferase, alkaline phosphatase and aminopeptidase as microvillar membrane markers were unsuccessful. These enzymes are mostly soluble in A. jhwolineata anterior caeca, in contrast to what is observed in several other
SANDRO
R. MARANA
et al.
Amylase
Aryl p-glucosidase
3,
IL SDH
Caeca
LDH
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1 .
Crop L
4
I
Dip
3
3
AmY
. 3
.
I 5
FIGURE4. Amylasc and aryl /3-glucosidase activities in caecal and crop contents after a meal. Activities are expressed as relative specific activities to discount differences in grasshoppers sizes, and incomplete recoveries of luminal contents, which is frequent in the case of the caecal contents. Each data point corresponds to four assays in two different samples obtained from two insects.
4 I
.
Hours after a meal
GIlkIm
4
. 1
Mal
3 2 1 JIII!CI
50
100
PrOotein FIGURE 3. Distribution of enzymes among the subcellular fractions of A. ,fluvolineata anterior caeca. Fractions from left to right are: SP,, PP,, SP,, PP,, SP,, PP,, SP,, PP, and final supematant. The data are means and SEM based on four assays performed in each of three different preparations obtained from two insects, The recovery of each hydrolase activity in subcellular fractions was between 60 and 100% of the homogenate activity. Other details as in legend of Fig. 2. Mat, maltase; Amy, amylase; AP, aminopeptidase; Dip, dipeptidase; y-Glut, y-glutamyl transferase; LDH, lactate dehydrogenase; SDH, succinate dehydrogenase; Try; trypsin.
insects (Terra and Ferreira, 1994). The occurrence of succinate dehydrogenase in soluble fractions (Fig. 3) may result from its partial solubilization in the conditions employed. The high specific activities observed in the soluble fractions argue against accidental contamination whereas the identical results by pelleted material, observed after extending the 100 OOOxg centrifugations to 180 min rule out a viscosity effect. Amylase and maltase are found in major amounts in the final supematant, although their specific activities are higher in the soluble subfraction part derived from the particulate fractions. Activities recovered in membranes (PP) are probably artifacts as a result of the adsorption of soluble enzymes (SP). Digestive enzyme activities recovered in SP, and SP, correspond to enzyme mol-
ecules enclosed inside secretory vesicles, whereas those in SP, and FS may correspond to enzymes entrapped in the cell glycocalyx in either weak or very weak form, respectively (Ferreira et al., 1990b; Terra and Ferreira, 1994). Thus, the data suggest that at least amylase and maltase (Fig. 3), and probably also cellobiase (Fig. 2), occur partly in secretory vesicles and partly associated with the caeca cell glycocalyx. Aminopeptidase is recovered in PP, in sufficient amounts to rule out an artifact. Since similar PP, preparations obtained from different insects contain microvillar membranes (Terra and Ferreira, 1994), it is probable that part of the A. jkvolineatu caecal aminopeptidase activity corresponds to a microvillar enzyme. Nevertheless, most of the aminopeptidase activity is soluble and is distributed among subcellular fractions in a way resembling amylase and maltase. Thus, soluble aminopeptidase should occur in secretory vesicles and associated with the cell glycocalyx. Membrane-bound dipeptidase activity seems to be an artifact. Although the specific activity of soluble dipeptidase is higher in FS, its subcellular distribution resembles that of amylase and maltase. Trypsin is found in FS, with negligible activity being recovered in SP, and SP,. In the light of the previous discussion, it is possible that trypsin activity in the secretory vesicles is low, in contrast to the major amounts recovered in FS, presumably associated with the glycocalyx. In summary, the results of the detailed cell fractionation confirmed the conclusions drawn from the obser-
A BRA CRIS FLA VOLINEA TA MIDGUTS
vations of the simplified procedure. Thus, the data on A. caecal cells show that, in this insect, digestive enzymes are synthesized and secreted by exocytosis in response to feeding. An exception seems to be aryl pglyucosidase, whose intracellular activities do not change, regardless of the feeding stage. Some enzymes are stored in secretory vesicles even during fasting (e.g. amylase), whereas others are not (e.g. maltase and cellobiase). These results apparently diverge from others. Secretory vesicles were not observed in L. migrutoria midguts, which show, on the other hand, cell extrusions as well as small and large vesicles budding from the cell microvilli (Heinrich and Zebe, 1973). The occurrence of secretory vesicles in L. migratoria midgut cells may depend on the insect feeding stage. As shown in this paper, the amount of digestive enzymes in secretory vesicles in A. Jtavolineata midguts is highly dependent on the feeding stage. Although frequently interpreted as apocrine secretion, cell extrusions are more probably manifestations of cell renewal (see review: Terra and Ferreira, 1994). Small vesicles budding from sides of midgut cell microvilli may correspond to microapocrine secretion. This consists of secretory-like vesicles budding laterally from the microvilli as double membrane vesicles. Secretion of digestive enzymes by this route has been established in Erinnyis ello (Santos et al., 1986). Earlier description of microapocrine secretion of digestive enzymes in Musca domestica (Espinoza-Fuentes et al., 1987) was not confirmed (Jordao et al., 1996). M. domestica presents only single membrane budding-vesicles in their midgut cell microvilli. This kind of microvillar vesiculation has also been described in other Diptera (De Priester, 1971; Nopanitaya and Misch, 1974; Lehane, 1976) as well as in Collembola (Humbert, 1979) and Orthoptera (Heinrich and Zebe, 1973). It may correspond to a kind of microvillar membrane renewal. Large vesicles budding from the top of midgut cell microvilli are probably associated with the formation of the peritrophic membrane (Heinrich and Zebe, 1973; Santos et al., 1986).
jlavolineata
Digestive physiological tion: a model
events
during
Acrididae
diges-
Immediately after a meal, A. jlavolineata caecal cells secrete digestive enzymes into the caecal contents, which pass on to the crop. Based on the work of Baines (1979) with dyes, it is likely that in A.Jiavolineata the discharge of the caecal contents into the anterior ventriculus is caused by gross contractions of the caeca, and that digestive enzymes are passed forward from the anterior midgut into the crop moved by antiperistalsis. Ingested saliva, which contains small amounts of amylase (Ferreira et al., 1990a), may disperse the food in the crop. Solid food entering the midgut is prevented from penetrating into the caeca (Baines, 1978; Ferreira et al., 1990a), probably due to the small apertures maintained
471
by the sphincter-like muscle placed near the point of caecal attachment to the midgut (Hodge, 1939; this paper). In contrast, crop fluid with dissolved food molecules and accompanying digestive enzymes passes into the caeca. A. Javolineatu caeca, V, and V, cells present morphological characteristics of fluid-absorbing cells (see discussion in Ribeiro et al., 1990). Nevertheless, caeca are the most important water-absorbing sites in grasshoppers due to their larger luminal surface. According to Chapman and Brandenburger (cited in Chapman, 1988a), the surface area of anterior caeca is more than seven times greater than the posterior caeca and twice as great as that of the ventriculus. Apparently, most A. Javolineata crop digestion has finished by about 3 h after a meal, since at this time digestive enzymes decrease in crop and increase and in the caecal contents, suggesting a transference of material from crop to caeca. This is in accordance with several studies of crop emptying performed in other grasshoppers (Baines et al., 1973; Simpson, 1983). In the caeca, most initial protein digestion and most final protein and carbohydrate digestion should take place, followed by the absorption of the resulting amino acids and sugars. Since the crop fluid entering the midgut overcomes the water absorption capacity of the caeca (Dow, 1981b), excess fluid moves down the ventriculus together with solid food. Further digestion of solid food occurs along the ventriculus and, as soon as the food molecules become sufficiently small to pass through the peritrophic membrane, digestion is finished by microvillar enzymes, such as aminopeptidase, and absorption presumably occurs (Ferreira et al., 1990a). Between 3 and 5 h after a meal, digestive enzymes in A. Javolineata caeca contents decrease, suggesting they are discharged into the ventriculus. In S. gregaria, caecal contents are emptied into the ventriculus 3 h after taking a meal. Thereafter, caecal luminal volume increases again due to the forward movement of Malpighian tubule secretion in the absence of salivation (Dow, 1981b). It is probable that the same occurs in A. jlavolineata. The data discussed above showed that the anterior caeca are the main sites of digestive enzyme secretion, of the intermediary and final stages of digestion and, finally, of nutrient absorption, The lack of obvious differences in the sizes of the anterior caeca among grasshoppers adapted to different diets may indicate that different food sources do not impose different constraints on the system (Chapman, 1988b). A. flavolineata posterior caeca have the same digestive functions as the anterior caeca, although on a smaller scale due to their smaller sizes (see above). The fact that the posterior caeca change in relative size among different grasshoppers suggest they may have functions other than digestive ones. Posterior caeca are more developed in forbivorous than in graminivorous species (Chapman, 1988b). Since forbs are richer in allelochemicals than grasses, Chapman (1988b) suggested that posterior caeca have a major role in detoxification. The epithelial pockets
472
SANDRO
R. MARANA
present in the posterior caeca of many grasshoppers (including A. flavolineata) may have a role in the removal of phenolics from the gut (Bernays, 1981; Chapman, 1988b). REFERENCES Ackrell B. A. C., Keamey E. B. and Singer T. P. (1978) Mammalian succinate dehydrogenase. Meth. Enzym. 53, 466483. Anstee J. H. and Charnley A. K. (1977) Effects of frontal ganglion removal and starvation on activity and distribution of six gut enzymes in Locusta. J. Insect Physiol. 23, 9655974. Baines D. M. (1978) Observations on the peritrophic membrane of Locusta migratoriu migratorioides (RandF) nymphs. Acrida 7, 1I-22. Baines D. M. (1979) Studies of weight changes and movements of dyes in the caeca and midgut of fifth-instar Locusta migratoria migratorioides (RandF) in relation to feeding and food deprivation. Acrida 8, 95-108. Baines D. M., Bemays E. A. and Leather E. M. (1973) Movement of food through the gut of fifth-instar males of Locusta migratoria migratorioides. Acrida 2, 3 19-332. Bemays E. A. (1981) A specialized region of the gastric caeca in the locust Schistocerca gregaria. Physiol. Entomol. 6, l-6. Bergmeyer, H. U. and Bemt, E. (1974) Calorimetric assay with Llactate, NAD, phenazine methosulfate and INT. In Method.7 of Enzymatic Analysis (Ed. Bergmeyer H. U.), 2nd edn, pp. 579-582. Chemie, Weinheim. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Chapman R. F. (1988a) Variations in the size of the midgut caeca during the fifth instar of the grasshopper Schistocerca americana (Drury). J. Insect. Physiol. 34, 329-335. Chapman R. F. (1988b) The relationship between diet and the size of the midgut caeca in grasshoppers (Insecta: Orthoptera: Acridoidea). Zool. J. Linn. Sot. 94, 319-338. Chapman, R. F. and Joem, A., Biology of Grasshoppers. Wiley, New York ( 1990). Dahlqvist A. (1968) Assay of intestinal disaccharidases. Analyt. Biochem. 22, 99- 107. De Duve C., Pressman B. C., Gianetto R., Wattiaux R. and Appelmans F. (1955) Tissue fractionation studies-6. Intracellular distribution patterns of enzymes in rat liver. Biochem. J. 60, 604-617. De Priester W. (1971) Ultrastructure of the midgut epithelial cells in the fly Calliphora erythrocephala. J. Ultrastruct. Res. 36, 783-805. Dow J. A. T. (1981a) Localization and characterization of water uptake from the midgut of the locust, Schistocerca gregaria. J. Expl Biol. 93, 269-28 1. Dow J. A. T. (198 I b) Countercurrent flow, water movements and nutrient absorption in the locust midgut. J. Insect Physiol. 27, 579-585. Droste H. J. and Zebe E. (1974) Carbohydrasen und Kohlenhydratverdauung im Darmtrakt von Locusta migratoria. J. Insect Physiol. 20, 1639-1657. Erlanger B. F., Kokowsky N. and Cohen W. (1961) The preparation and properties of two new chromogenic substrates of trypsin. Arch. Biochem. Biophys. 95, 271-278. Espinoza-Fuentes F. P., Ribeiro A. F. and Terra W. R. (1987) Microvillar and secreted digestive enzymes from Musca domestica larvae. Subcellular fractionation of midgut cells with electron microscopy monitoring. Insect B&hem. 17, 8 199827. Ferreira C., Oliveira M. C. and Terra W. R. (1990a) Compartmentalization of the digestive process in Ahrucris,Ravolineata (Orthoptera: Acrididae) adults. Insect Biochem. 20, 267-274. Ferreira C., Bellinello Cl. L., Ribeiro A. F. and Terra W. R. (1990b) Digestive enzymes associated with the glycocalyx, microvillar membranes and secretory vesicles from midgut cells of Tenehrio molitor larvae. Insect Biochem. 20, 8399847.
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ABRACRIS FLAVOLINEATA Alejo
MIDGUTS Mesa (UNESP,
413 campus
of Rio Clara) for collecting Ahracris to the technicians L.Y. Nakabayashi, M.V. Cruz and W. Caldeira. S.R. Marana is a graduate fellow of FAPESP. A.F. Ribeiro, W.R. Terra and C. Ferreirra arc staff members of their respective departments and research fellows of CNPq.
~flavolineutu. We are also indebted
Acknowledgements-This work was supported by the Brazilian Research Agencies FAPESP and CNPq. We arc much indebted to Dr