Arthropod Structure & Development 43 (2014) 123e134

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Arthropod Structure & Development

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The digestive system of the stick bug Cladomorphus phyllinus (Phasmida, Phasmatidae): A morphological, physiological and biochemical analysis
Emiliano C. Monteiro a, Fbio K. Tamaki b, Walter R. Terra b, Alberto F. Ribeiro a, *
a b

Departamento de Gentica e Biologia Evolutiva, Instituto de Biocincias, Universidade de So Paulo, C.P. 11461, 05422-970 So Paulo, Brazil Departamento de Bioqumica, Instituto de Qumica, Universidade de So Paulo, C.P. 26077, 05513-970 So Paulo, Brazil

a r t i c l e i n f o
Article history: Received 25 September 2013 Accepted 20 November 2013 Keywords: Midgut ultrastructure Digestive enzymes Midgut uxes Midgut luminal alkalization Immunolabeling

a b s t r a c t
This work presents a detailed morphofunctional study of the digestive system of a phasmid representative, Cladomorphus phyllinus. Cells from anterior midgut exhibit a merocrine secretion, whereas posterior midgut cells show a microapocrine secretion. A complex system of midgut tubules is observed in the posterior midgut which is probably related to the luminal alkalization of this region. Amaranth dye injection into the haemolymph and orally feeding insects with dye indicated that the anterior midgut is water-absorbing, whereas the Malpighian tubules are the main site of water secretion. Thus, a putative counter-current ux of uid from posterior to anterior midgut may propel enzyme digestive recycling, conrmed by the low rate of enzyme excretion. The foregut and anterior midgut present an acidic pH (5.3 and 5.6, respectively), whereas the posterior midgut is highly alkaline (9.1) which may be related to the digestion of hemicelluloses. Most amylase, trypsin and chymotrypsin activities occur in the foregut and anterior midgut. Maltase is found along the midgut associated with the microvillar glycocalix, while aminopeptidase occurs in the middle and posterior midgut in membrane bound forms. Both amylase and trypsin are secreted mainly by the anterior midgut through an exocytic process as revealed by immunocytochemical data. 2013 Elsevier Ltd. All rights reserved.

1. Introduction The Order Phasmida is composed of stick and leaf insects. As their common name imply, these animals are able to mimic with astonishing resemblance the stems or leaves of plants, upon which they live and feed, in a remarkable defense mechanism against predators. There are more than 3000 phasmid species described, distributed in all tropical and temperate ecosystems. The order is composed solely of phytophagous insects and usually present nocturnal habits (Bedford, 1978). The order Phasmida is closely related to the Orthoptera, and both orders comprise a monophyletic group termed Orthopterida (Kristensen, 1981). Although some data can be found concerning the digestive process in the related order Orthoptera (Dow, 1981; Ferreira et al., 1990; Marana et al., 1997; Woodring and Lorenz, 2007; Biagio et al., 2009), very little is known about this process occuring in Phasmida species. Anatomical descriptions of the digestive system of Phasmida report that it is formed by a simple tubular structure made up of a large foregut followed by a midgut lacking digestive

* Corresponding author. Tel.: 55 11 3091 7579. E-mail address: aribeiro@ib.usp.br (A.F. Ribeiro). 1467-8039/$ e see front matter 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.asd.2013.11.005

caeca (Bartheau, 1963; Gangrade, 1965; Beadle, 1972). Malpighian tubules are positioned between the midgut and hindgut. An interesting aspect of the midgut is the presence of a complex system of appendices in its posterior region formed by small protuberances connected to very thin tubules, morphologically very similar to the Malpighian tubules (Ramsay, 1955; Savage, 1962). These midgut tubules, apparently found only in Phasmida species, are simply regarded as modied Malpighian tubules with unknown function (Ramsay, 1955; Savage, 1962). Scanty morphological data can also be found in the literature with histological (Bartheau, 1963; Gangrade, 1965) and ultrastructural (Beadle, 1972) descriptions of the digestive system of stick bugs. In this work, a detailed morpho-physiological study of the digestive system of the stick bug Cladomorphus phyllinus is presented including the immunolocalization of digestive enzymes and possible role of the midgut tubules in the process of digestion. The results show the occurrence of an endo-ectoperitrophic circulation of digestive enzymes, being the anterior midgut and the Malpighian tubules the main sites of water-absorption and watersecretion, respectively, in both fed and starved animals. Initial carbohydrates digestion should occur in the foregut and anterior midgut and protein digestion should take place initially in the middle and posterior midgut, with nal digestion occurring in the

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epithelial surface of the midgut. The midgut tubules are probably involved in the alkalization of the posterior midgut, possibly related to the digestion of hemicelluloses. 2. Materials and methods 2.1. Animals and preparation of midgut samples Cultures of the stick insect C. phyllinus (Phasmida, Phasmatidae) were maintained in the laboratory at room temperature (25  C), under natural photoregime conditions. The insects were kept in pasteboard boxes containing guava tree (Psidium guava) branches, sprayed daily with water. Three adult females were immobilized by placing them on crushed ice and then dissected in cold 100 mM NaCl solution. The intestine was carefully isolated and separated into foregut, midgut and hindgut. The midgut was further divided into the following sub-regions: anterior midgut (AMG), middle midgut (MMG), proximal posterior midgut (PMG1) and distal posterior midgut (PMG2). Each midgut region was separated into tissue and contents of the gut. The salivary glands, Malpighian tubules and midgut tubules were also isolated. The samples (tissues and isolated contents) were homogenized in cold double distilled water with the help of a PottereElvehjem homogenizer. Homogenates from midgut regions were centrifuged for 30 min at 13,000 g at 4  C. The supernatants of contents homogenates were recovered and named contents. The tissue supernatants were recovered, and labeled soluble tissue fractions and the pellets were resuspended in double-distilled water and named tissue membrane fraction. The material was stored at 20  C until use. No enzyme inactivation was detected during storage. 2.2. Light and electron microscopy For histological studies with light microscope, ten insects were dissected in the xative solution (Bouins) under the stereomicroscope and the digestive tract carefully isolated. The tissues were kept in the xative overnight at 4  C, dehydrated in graded ethanol and embedded in historesin (Leica, Heidelberg). Serial sections (4e 5 mm thickness) were obtained using a Leica RM2145 microtome, stained with hematoxylin and eosin, and mounted in glass slides with entellan (Merck, Darmstadt). For uorescent visualization of chitin, in order to detect the peritrophic membrane in the midgut, samples from three specimens were xed in Zambonis xative (Stefanini et al., 1967) overnight at 4  C, dehydrated in graded ethanol at room temperature, embedded in parafn wax, and cut at 8 mm thickness. Sections were then collected on glass slides and the parafn was removed with xylene. After hydration, the sections were washed in PBS (20 mM phosphate buffer pH 7.4, containing 0.15 M NaCl), followed by immersion in PBS containing 0.2% Triton X- 100, for 1 h at room temperature. The preparations were incubated with WGA-FITC (wheat germ agglutinin coupled to uorescein isothiocyanate e SigmaeAldrich), diluted 1:500 in PBS in the presence of excess N-acetylglucosamine for 18 h at 4  C sheltered from light. Binding of WGA to chitin is specic in the presence of excess N-acetylglucosamine (Peters and Latka, 1986). As controls, sections were incubated with PBS buffer. After rinsing in PBS at room temperature, the sections were mounted in Vectashield (Vector Labs, Inc. USA) mounting medium and examined in a Zeiss LSM 410 confocal microscope. For transmission electron microscopy, the midgut pieces from ten specimens were xed in 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 h at 4  C. After rinsing with 0.2 M sucrose in 0.1 M cacodylate buffer, the tissues were post-xed in 1% osmium tetroxide in the same buffer for 1 h at 4  C. En-bloc staining was

performed in 1% aqueous uranyl acetate for 16e18 h at 4  C. After dehydration in graded ethanol at room temperature, the material was embedded in Spurrs resin (Spurr, 1969). The ultrathin sections were obtained using a Leica Ultracut UCT ultramicrotome, stained with lead citrate and examined in a Zeiss EM 900 electron microscope operated at 80 kV. For scanning electron microscopy, the midgut pieces were xed and dehydrated as above, critical point dried and gold coated according to standard procedures. The preparations were examined in a Zeiss DSM-940 electron microscope. 2.3. pH of midgut contents and dye experiments The content of gut sections of dissected animals was dispersed in 5 ml of dissecting saline and then added to 5 ml of a 5-fold dilution of a universal pH indicator (E. Merk, Darmstadt, pH 4e10). The resulting colored solution was compared with suitable standards. Measurements were also performed on dilutions (100) of gut contents in double distilled water with a pH meter (Digimed, DHPH-1). Measurements were performed both on fed and starved animals. For dye experiments, 40 ml of 100 mM amaranth dye solution was injected into the mouth or directly into the hemocoel of C. phyllinus adult females either starved for 5 days or fed ad libitum. The insects were then dissected at different time intervals and the gut inspected for dye adsorption. 2.4. SDS-polyacrylamide gel electrophoresis and western blotting In order to detect a possible homology between the digestive enzymes present in C. phyllinus with antibodies available in the laboratory, electrophoresis experiments followed by Western blotting and immunoassays were carried out. Animals were dissected in cold 100 mM NaCl and their midgut isolated and sectioned as previously described. The epithelia of each sample were carefully separated from the contents of the digestive system and homogenized in cold double-distilled water by using a PottereElvehjem homogenizer. Samples were mixed with sample buffer (2:1) containing 60 mM Tris-HCl, pH 6.8, 2.5% (w/v) SDS, 0.36 mM 2mercaptoethanol, 10% glycerol and 0.05% (w/v) bromophenol blue and heated for 2 min at 95  C in a water bath. The samples were then loaded in a 12% (w/v) polyacrylamide gel containing 0.1% (w/ v) SDS. Electrophoresis was carried out on a discontinuous pH system (Laemmli, 1970) with the use of Bio-Rad (USA) Mini Protein II equipment, at 200 V until the front marker (bromophenol blue) reached the end of the gel. Proteins in the gel were then transferred electrophoretically onto a nitrocellulose membrane lter (pore size 0.45 mm; Bio-Rad, USA) (Towbin et al., 1979). The transfer efciency was evaluated by observing the pre-stained molecular weight markers (Sigma, USA). The lters were blocked for 1 h at room temperature with non-fat milk in TBS (Tris-buffered saline: 50 mM Tris-HCl buffer pH 7.4, containing 0.15 M NaCl), containing 0.05% Tween 20 (TBS-T). After this step, lters were reacted with Musca domestica anti-trypsin antiserum (Jordo et al., 1996) or with Tenebrio molitor anti-amylase antiserum (Cristofoletti et al., 2001), both of them diluted 100 fold in TBS-T for 2 h at room temperature. After washing with TBS-T, the lters were reacted with anti-rabbit IgG coupled with peroxidase (Sigma, USA) diluted 1:1000 in TBS-T for 2 h at room temperature. After washing extensively with the same buffer, the lters were incubated with 0.08% 4-chloro-1naphthol in TBS containing 0.1% hydrogen peroxide until gray bands appeared where antigens were recognized. The reagent was prepared before use by the addition of one volume of 0.5% chloronaphthol in methanol to ve volumes of TBS with hydrogen

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peroxide. Bands obtained in experiments with C. phyllinus homogenates were compared with those obtained with M. domestica and T. molitor gut homogenates, that served as positive control. In order to conrm the specicity of the anti-trypsin antiserum, samples that had not been previously heated were loaded in a 12% polyacrylamide gel in conditions identical to those previously described, except for the fact that the experiment was realized without the use of 2-mercaptoethanol and performed at 4  C. After the electrophoresis, the gel was removed from the equipment and the substrate Z-FR-MCA (carbobenzoxy-Phe-Arg-4-methylcoumarin-7amide) 1 mM was carefully spreaded throughout its surface. The uorescence emitted by the liberation of MCA could be observed by the utilization of a UV light thus identifying the band that contained trypsin and comparing its molecular weight with the one estimated in the western blot experiments. Pre-stained markers were used to verify the mass of the activity band obtained. 2.5. Immunolocalization of trypsin and amylase After being dissected and isolated the midgut regions were xed in 4% paraformaldehyde with 0.3% glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 for 2 h at 4  C. The material was then rinsed with phosphate buffer and dehydrated in graded ethanol solutions at room temperature, and embedded in hard grade L. R. White acrylic resin (Electron Microscopy Sciences, Ft. Washington, USA). Ultrathin sections were cut on the ultramicrotome and collected on 200 mesh collodion-coated nickel grids. The grids were then oated on drops of TBS at pH 7.2 containing 1% BSA (Bovine Serum Albumin, Sigma, USA) for 5 min, and placed on NGS (Normal Goat Serum, Amersham, UK), diluted 1:30, for 30 min. The sections were then incubated overnight in the primary antisera diluted 1:100 in TBS at pH 7.2 containing 1% BSA at 4  C. Both aforementioned antisera were utilized, namely M. domestica anti-trypsin (Jordo et al., 1996) and T. molitor anti-amylase (Cristofoletti et al., 2001). As controls, sections were incubated with pre-immune serum using the same conditions. After rinsing in TBS at pH 7.2 with 0.2% BSA, 0.05% NaN3 and 0.1% tween 20, the samples were placed in TBS at pH 8.2 with 1% BSA and 0.05% NaN3 for 30 min at room temperature and incubated with goat anti-rabbit IgG coupled to 15 nm gold particles (Amersham, UK) diluted 1:20 in TBS at pH 8.2 plus 1% BSA and 0.05% NaN3 for 1 h at room temperature. The grids were then washed in TBS at pH 7.2, containing 0.2 BSA, 0.05% NaN3 and 0.1% tween 20, followed by the same solution without BSA. After xation in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 for 10 min at room temperature, the samples were washed in double distilled water, stained with uranyl acetate 2% and lead citrate, and examined in a Zeiss EM 900 electron microscope. 2.6. Enzyme assays and protein determination Protein was determined according to Bradford (1976) with the use of ovalbumin as a standard. Amylase, maltase, trypsin, chymotrypsin and aminopeptidase activities were determined as follows: amylase activity was measured determining the appearance of reducing groups (Noelting and Berneld, 1948) in 50 Mm citrate-phosphate buffer at pH 6.0 with 0.5% (w/v) starch as substrate and 10 mM NaCl. Maltase was assayed according to Terra et al. (1979) with the use of 4 mM pnitrophenyl b-D-glucoside (NPbGlu) in 10 mM phosphate buffer pH 7.0. Trypsin activity was determined by the emission of uorescence of methyl-coumarin released from 10 mM B-R-MCA (benzoylL-arginin-7-amido-4 methylcoumarin) in buffer 0.1 M TRIS-HCl, pH 8.5. Chymotrypsin was assayed by the utilization of S-AAPF-MCA (Chymotrypsin substrate II, Calbiochem) 10 mM in Tris-HCl buffer, pH 8.5. Aminopeptidase was assayed according to Erlanger et al.

(1961), with the use of 1 mM LpNA (L-Leucyl-p-nitroanilide, SigmaeAldrich), in 100 mM Tris-HCl buffer, pH 7.8. Incubations were carried out at 30  C for four different time periods and initial rates of hydrolysis were calculated. All assays were performed under conditions in which the activity was proportional to protein concentration and time. Controls without enzyme and without substrate were included. An enzyme unit (U) is dened as the amount of enzyme that hydrolyzes 1 mmol of substrate (or bond) per min. Carbonic anhydrase was assayed with a modication of the method of Wilbur and Anderson (1948), as previously detailed by Terra et al. (1988). Tissue samples were dissected and rinsed in cold 0.1 M NaCl as follows: AMG and MMG were separated from PMG1 and PMG2. PMG1 was freed from midgut tubules. Contents of midgut sections were removed before homogenizing. Midgut tubules were removed with scissors from PMG1 and Malpighian tubules from the region between midgut and hindgut. All tissues were homogenized in double distilled water. An aliquot of 0.5 ml of tissue homogenate was added to 1 ml of solution of 16 mM HEPES buffer, pH 8.3 at 4  C, and immediately completed with 1 ml of carbon dioxide-saturated water (4  C). The time necessary for the pH to change from pH 8.0 to pH 6.5 was measured. The units of enzymatic activity were estimated according to Wilbur and Anderson (1948) as: U (t0 tcat)/tcat, where t0 corresponds to the time for the pH change (from 8.0 to 6.5) in the uncatalyzed reaction, and tcat is the time necessary for the same pH change in the catalyzed reaction. 3. Results 3.1. Anatomy and histology of the midgut of C. phyllinus The gut of C. phyllinus is formed by a simple tube without convolutions (Fig. 1A). It is constituted by a foregut, a midgut and a hindgut beginning at the pylorus, the place of insertion of the Malpighian tubules. The foregut is formed by the buccal cavity, a pharynx and an esophagus that ends in a muscular proventriculus. The midgut can be divided into three morphologically distinct regions: the anterior midgut (AMG), exhibiting several foldings in its surface at regular intervals, the middle midgut (MMG) presenting a smooth surface and a smaller diameter in relation to AMG and the posterior midgut (PMG), which can be further subdivided into proximal (PMG1) and distal (PMG2) sub-regions. In PMG1, the presence of a complex system of midgut tubules can be observed. These structures are connected to the midgut through small protuberances, the midgut protuberances. The midgut tubules are very delicate structures (with a diameter between 5 and 10 mm) morphologically similar to the Malpighian tubules. Midgut tubules are not observed in PMG2, which show a smooth surface. The hindgut is divided into an ileum and a rectum that ends in the anus (Fig. 1A). The foregut is lined by a simple epithelium, which is composed of attened cells and covered by a cuticle layer that forms small spines. The midgut is also lined by a simple epithelium which is formed by enterocytes, which are the main cell type, and regenerative cells collected at nidi close to the basal lamina (Fig. 1CeE). In the AMG the epithelia display numerous epithelial folds and present slightly smaller and more stained cells in relation to the other midgut regions (Fig. 1C, D). In the MMG the foldings of the epithelia cease becoming smooth. In the PMG1 sub-region is characterized by the presence of midgut protuberances and associated tubules (Fig. 1E, F). These structures are formed by a simple epithelium that is continuous with the epithelium of the digestive tract. Cells from the midgut protuberances are larger, showing a round shape and a conspicuous brush border. The cubic epithelium that forms the

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Fig. 1. (A) Anatomical diagram of the digestive system of C. phyllinus. (B) Fluorescence microscopy of the midgut showing a conspicuous labeled multilayer peritrophic membrane in the lumen. (C and D) Histological aspects of the midgut epithelium. (C) General view of the AMG region; note the presence of a brush border (arrows). (D) Detailed view of the enterocytes and regenerative nidi in the midgut. (E) Image of the PMG region showing a midgut protuberance; the arrow points to an accumulation of an acidophil substance at the protuberance opening. (F) Detailed image of midgut tubules. (G) Detailed aspect of a Malpighian tubule. Abbreviations: AMG anterior midgut; Col Colon; Ep midgut epithelium; Es Esophagus; L midgut lumen; MgP midgut protuberances; MgT midgut tubules; MMG middle midgut; MT Malpighian tubules; N nucleus; Ni nidi; Pha Pharynx; PM peritrophic membrane; PMG posterior midgut; PMG1 proximal posterior midgut; PMG2 distal posterior midgut; Rec Rectum. Bar 1 cm (A); 100 mm (B and E); 300 mm (C); 50 mm (D, F and G).

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Fig. 2. Fine structure of the AMG (A and B), and MMG (C and D). (A) Apical cytoplasm exhibiting microvilli and secretory vesicles. (B) Basal cytoplasm of an enterocyte showing basal membrane infoldings with few openings (arrow) to the underlying basal lamina; the inset shows a Golgi area with associated secretory vesicle. (C) Apical cytoplasm with microvilli and electron dense secretory vesicles. (D) Basal cytoplasm showing well developed membrane infoldings and many openings (arrows) to the basal lamina. Abbreviations: BL basal lamina; D desmosome; G Golgi area; Mit mitochondria; Mv microvilli; SJ septate junction; SV secretory vesicle. Bar 0.5 mm (A, B inset and C); 1 mm (B and D).

midgut tubules is similar to the one found in Malpighian tubules, differing only in cell size. Midgut tubule cells are atter and smaller than Malpighian ones and both cell types are binucleate and exhibit a brush border (Fig. 1 F, G). Accumulations of an amorphous material, highly stained with eosin, are frequently seen in the openings of the midgut protuberances to the gut lumen (Fig. 1E). The hindgut is lined by cuboidal cells covered by a thin cuticle. In the rectum, typical rectal papillae can be observed. Along the entire midgut lumen, a membranous structure can be detected surrounding the food bolus. This structure is the peritrophic membrane as conrmed by the uorescent visualization of chitin with WGA-FITC conjugates (Fig. 1B).

3.2. Dye experiments Dye experiments were realized in order to verify the occurrence of sites of water absorption and secretion along the midgut. Both starved and fed C. phyllinus adult females were orally fed with amaranth solution. Twelve hours after dye ingestion, the animals show dye in the hindgut and feces, which indicates its passage through the whole midgut. It was observed, both in starved and fed animals, that the luminal side of the epithelium in the AMG region of the midgut is stained with dye, suggesting that this region is water-absorbing. Insects were also dissected in different intervals after injection of the same

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amaranth solution into the hemocoel. Dye clearance by the Malpighian tubules was inferred by the presence of dye in the tubular lumen followed by dye accumulation on the hindgut. Even in animals dissected 16 h after dye injection into the hemolymph there was no observable staining on the haemocel side of the midgut epithelia. Thus, it was not possible to detect a water-secreting region in the midgut through dye injection. In starved animals, it was noted that the dye taken up by the Malpighian tubules not only passed backwards towards the hindgut, but also moved forward being present in the midgut lumen. 3.3. Fine structure of midgut regions cells Three distinct cell types can be clearly identied in midgut epithelium at ultrastructural level: The enterocytes, the regenerative cells, organized in small basal clusters or nidi, and the endocrine cells, at the base of the epithelia showing typical small electron dense vesicles. The enterocytes, the main cell type, are tall polarized cells (Figs. 2, 3AeC) with glycocalyx-covered microvilli extending to the lumen. Junctional desmosomes are present near to the lateral apex, followed by smooth septate junctions (Fig. 2A). They have abundant rough endoplasmic reticulum, several Golgi areas (dictyosomes) and are rich in polymorphic mitochondria, mainly in the apical and basal regions. The basal plasma membrane presents numerous infoldings that form a labyrinth of narrow channels with associated mitochondria. These channels communicate with the underlying extracellular space through openings close to the basal lamina. Several important morphological differences were observed in the enterocytes of the different midgut regions of C. phyllinus. In AMG enterocytes, the secretory vesicles exhibit contents forming two distinct electron dense regions (Fig. 2A, B inset). The basal labyrinth in this region appears poorly developed in relation to other regions, showing fewer associated mitochondria as well as less openings to the basal lamina (Fig. 2B). Enterocytes in the MMG, on the other hand, present secretory vesicles exhibiting homogeneous high electron density contents (Fig. 2C). The basal plasma membrane infoldings in this region are more developed than in AMG, with a greater number of canals, abundant associated mitochondria and a slightly larger number of openings to the extracellular medium (Fig. 2D). In contrast, PMG1 cells show a different morphology in having a low electron density cytoplasm poor in organelles but rich in cytoskeleton elements. The microvilli are smaller than the ones present in other midgut regions (around 5 mm, in contrast to around 9 mm in the AMG and MMG). The presence of dilated microvillar tips, containing small vesicles inside (Fig. 3A), and free similar vesicles in the lumen of this midgut region suggest the occurrence of microapocrine secretion. The invaginations of the basal plasma membrane are well developed in PMG1 cells, extending apically in the cytoplasm with a large number of associated mitochondria (Fig. 3B). The PMG2 cells have very long microvilli (around 20 mm) and also show tip expansions with small vesicles inside (Fig. 3C). The basal labyrinth is well developed as in PMG1 cells, forming long and narrow channels with many associated mitochondria and openings to the basal lamina. Secretory vesicles are not detected in the cytoplasm of PGM1 and PGM2 cells.

3.4. Fine structure of the midgut tubules and Malpighian tubules The epithelium of the midgut and the midgut protuberances is continuous, with a distinct transitional region between the two structures. The cells of the protuberances exhibit apical microvilli containing mitochondrial projections, a large number of cytoplasmic mitochondria, a well-developed basal labyrinth formed by infoldings of the basal plasma membrane with associated mitochondria, with many openings to the extracellular matrix (Fig. 4A). The midgut tubules are thin blind ended tubules exhibiting morphological features very similar to that found in the Malpighian tubules (Fig. 4B, C). Both cell types are polarized and show apical microvilli containing mitochondrial projections, and pleated septate junctions in the lateral membrane connecting adjacent cells. They are rich in rough endoplasmic reticulum, Golgi areas and mitochondria, and the basal plasma membrane invaginations form an elaborate labyrinth with many openings to the basal lamina. In contrast to the Malpighian tubules (Fig. 4C), the midgut tubules cells show a shorter basal labyrinth with very few associated mitochondria (Fig. 4B inset). Scanning electron microscopy analysis (Fig. 4DeF) showed that both structures are almost identical in their external morphology, possessing helicoidal strands of muscle bers organized around the tubules (Fig. 4E, F). Nevertheless, the midgut tubules present a smaller diameter than the Malpighian tubules (around 15 mm and 30 mm, respectively). 3.5. Trypsin and amylase western blots and immunocytolocalization Western blot conrmed that both T. molitor anti-amylase (Cristofoletti et al., 2001) and M. domestica anti-trypsin (Jordo et al., 1996) serum recognize homologous digestive enzymes in C. phyllinus gut. Amylase antiserum recognized a single band with similar migration to the T. molitor amylase (65 kDa). Trypsin antiserum recognizes a single protein band (60 kDa), larger than the M. domestica trypsin (28.5 kDa), suggesting the dimerization of this enzyme in C. phyllinus and conrmed using in-gel assays with uorescent substrate (Z-FR-MCA) which shown a single activity band of 60 kDa. Ultrastructural immunolabeling using anti-amylase serum showed that AMG and MMG regions are the major places of amylase secretion, since secretory vesicles and the Golgi areas are well labeled (Fig. 3D, E, F). No signicant labeling is detected in PMG cells. Immunolabeling of amylase can also be detected in AMG and MMG lumen, mainly in the peritrophic membrane, and in association with microvilli. Similar immunolabeling is observed with trypsin antiserum (Fig. 3G). 3.6. Distribution of digestive enzymes and carbonic anhydrase activities Initial and nal sites of proteins (trypsin, chymotrypsin and aminopeptidase) and starch (amylase and maltase) digestion were veried along the C. phyllinus gut. Most amylase and trypsin activities were found in the foregut and in lesser amounts in the contents of AMG, presenting a decreasing gradient along the gut. Most chymotrypsin activity was

Fig. 3. Ultrastructural aspects of the PMG (AeC) and immunolocalization of digestive enzymes (DeG). (A) Apical cytoplasm of PMG1 cells showing small microvilli presenting dilated tips with small vesicles inside (arrow). (B) Basal cytoplasm of an enterocytes of the PMG1 region; note the extremely developed infoldings of the basal plasma membrane with many openings to the basal lamina (arrows). (C) Apical cytoplasm of PMG2 cells with long microvilli presenting expansions along their length (arrows). (DeF) Images of midgut regions treated with T. molitor anti-amylase. (D) Detail of AMG enterocyte, showing labeling in Golgi areas and associated secretory vesicles. (E) Apical region of AMG cell; note labeled microvilli and secretory vesicles. (F) Apical region of MMG showing labeling in microvilli and secretory vesicles. (G) Image of the apex of an AMG enterocyte treated with M. domestica anti-trypsin; note labeling in secretory vesicles and in association with microvilli. Abbreviations: BL basal lamina; G Golgi area; Mit mitochondria; Mv microvilli; SV secretory vesicle. Bar 1 mm (A); 2 mm (B and C); 0.5 mm (DeG).

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concentrated in the AMG contents; besides a signicant activity is found in the foregut. Aminopeptidase was detected in MMG and PMG membrane fractions. On the other hand, maltase was present in the AMG and PMG epithelia tissue soluble fraction, indicating it is weakly adhered to the epithelium (probably trapped in the glycocalyx) (Fig. 5). Hindgut activities, as well as gut volume measurements of C. phyllinus were used to calculate enzymatic excretory rates, providing that enzymes do not inactivate en route from the midgut to the hindgut. It was observed that the excretion rate for all assayed enzymes was equal or inferior to 35% for each midgut emptying (Table 1). No endogenous enzymatic inhibitors were found in PMG. Carbonic anhydrase, which forms carbonic acid that dissociates into bicarbonate and a proton, may contribute to the mechanism of PMG1 luminal alkalization. It is highly active in the Malpighian tubules and midgut tubules, presenting the highest specic activity in the latter (Table 2). 3.7. Studies of luminal pH and its effect over trypsin and amylase Luminal pH measurements using both universal pH indicator and pH meter indicated consistently the following pH values: foregut 5.3 0.5; AMG 5.6 0.1; proximal MMG 6.3 0.2; distal MMG 8.0 0.5; PMG1 9.1 0.1; PMG2 8.5 0.1; hindgut 7.3 0.3. The MMG region was further subdivided in proximal MMG and distal MMG, in order to obtain a higher accuracy of the pH gradients along the digestive tube. The estimated optimum pH for amylase and trypsin were 5.0 and 9.0, respectively. 4. Discussion 4.1. Secretory events in C. phyllinus midgut The midgut enterocytes of C. phyllinus exhibit an intense secretory activity of digestive enzymes and other proteins like peritrophins (cf. Bolognesi et al., 2008). These cells are rich in organelles related to the secretory pathway, namely the rough endoplasmic reticulum, Golgi areas and secretory vesicles (Rothman and Orci, 1992). There are strong signs of occurrence of a very fast exocytic process in the AMG and MMG: close proximity of the secretory vesicles to the apical plasma membrane and immunolocalization of both amylase and trypsin inside the secretory vesicles and in the midgut lumen. On the other hand, the observation of small expansions along and at the tip of the microvilli in PMG cells showing small vesicles inside are indicative of a microapocrine mechanism of secretion in this midgut region, which involves the pinching off of the dilated microvillar tips containing these vesicles (cf. Santos et al., 1986; Ribeiro et al., 1990; Jordo et al., 1999; Silva et al. 2013). The occurrence of two distinct secretory mechanisms along the insect midgut is not unusual. In fact, this phenomenon has been detected in several insect species, such as in the beetles T. molitor (Cristofoletti et al. 2001) and Dermestes maculatus (Caldeira et al. 2007), in the lepidopterans Erinnyis ello (Santos et al. 1983) and Spodoptera frugiperda (Jordo et al. 1999), and in the cricket Gryllodes supplicans (Biagio et al., 2009).

These results suggest that different midgut regions should contribute in diverse ways to the digestive process. In order to study the secretory pathways of digestive enzymes in the midgut cells, we tested heterologous anti-amylase and antitrypsin sera in western blot experiments: the rst one recognized a single band of 65 kDa in C. phyllinus tissues, in accordance with the expected molecular weight for insect amylases (Cristofoletti et al., 2001). On the other hand, the anti-trypsin serum recognized a single band of 60 kDa, which contrasted with the expected molecular weight of approximately 30 kDa for insect trypsins (Jordo et al., 1996). To test the hypothesis that C. phyllinus trypsins are organized in dimers, we performed in-gel assays of midgut homogenates using uorescent substrate, which showed a single activity band of 60 kDa, conrming the dimerization of trypsins. Immunocytolocalizations using the heterologous antibodies labeled amylase and trypsin in both AMG and MMG Golgi areas and secretory vesicles, as well as in the midgut lumen in association with microvilli. These data agree with the location of the corresponding enzyme activities and indicate that both enzymes are secreted through an exocytic process using the same secretory pathway. This is in accordance with the data obtained in Periplaneta americana (Lima et al. 2001), but contrasts with the results obtained in T. molitor and S. frugiperda, where amylase and trypsin are secreted in different midgut regions. Thus, in this late situation, the initial digestion of starch (amylase) and proteins (trypsin) are spatially separated (Jordo et al., 1999; Cristofoletti et al., 2001). Although both enzymes are secreted in the same place in C. phyllinus, it seems to occur a functional separation of starch and protein digestion caused by the combination of different luminal pH along the midgut and optimal pH for amylase and trypsin, as discussed below. 4.2. Compartmentalization of digestion and enzyme recycling in C. phyllinus Digestive enzyme distribution showed that initial starch digestion is carried out by amylase in the foregut and AMG, whereas nal carbohydrate digestion takes place along the whole midgut on the epithelial surface, where maltase activity was found. The initial protein digestion is carried out by trypsin and chymotrypsin, which concentrate in the foregut and AMG. Nevertheless, protein digestion by trypsin should occur in higher levels in the MMG and PMG, since the luminal pH in these regions is similar to the trypsin optimum pH. This divergence between trypsin optimum pH (8.5) and the luminal pH where the enzyme is secreted (5.6 in the AMG) has also been observed in other insects such as D. maculatus (Caldeira et al., 2007) and P. americana (Lima et al. 2001). Final protein digestion is carried out by membrane-bound aminopeptidase in the surface of MMG and PMG cells. In spite of the high activity of amylase, trypsin and chymotrypsin in the foregut, crop cells are covered by cuticle and do not present cytological features of typical secretory cells (cf.Rothman and Orci, 1992). Enzyme assays of salivary gland homogenates did not show any activity for the tested enzymes, suggesting that the presence of digestive enzymes in the foregut can only be accounted by regurgitation from the midgut. Such mechanism is also observed in other insects, particularly in some Orthoptera species (Ferreira

Fig. 4. Fine structure of the midgut tubules and Malpighian tubules. (A) Fine structure of a midgut protuberance cell. Note the abundance of mitochondria and the invaginations of the basal plasma membrane with many openings to the basal lamina (arrows). (B) Oval shaped cell of the midgut tubule, with apical microvilli with mitochondrial projections; inset: detail of the invaginations of the basal plasma membrane in midgut tubule cells with many openings to the basal lamina (arrows) and few associated mitochondria. (C) Cell of the Malpighian tubule. (DeF) Scanning electron micrographs. (D) Image of a midgut protuberance connected to a midgut tubule. (E) Detail of a midgut tubule. (F) Detail of a Malpighian tubule; note the associated helicoidal muscle bers present in (E) and (F) (arrows). Abbreviations: BL basal lamina; MgT midgut tubule; Mit mitochondria; MgP midgut protuberance; MT Malpighian tubule; Mv microvilli; N nucleus. Bar: 2 mm (A, B); 0.5 mm (inset); 5 mm (C); 150 mm (D); 20 mm (E and F).

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Fig. 5. Distribution of the major hydrolases along the gut of C. phyllinus. C: contents of the gut; M: membrane fraction of the tissues; S: soluble fractions of the tissues. Determinations were carried out in three different preparations obtained from one insect each. SEM were found to be 10e25% of the means. The activities (U/animal) in whole gut homogenates were: trypsin 25; chymotrypsin 1500; aminopeptidase 12; maltase 0.414. Amount of proteins in gut sections (per animal) were: foregut e 675 mg; AMG e 101 mg; MMG e 76 mg; PMG1 e 51.4 mg; PMG2 e 31.3 mg; hindgut e 102 mg.

et al., 1990; Marana et al., 1997; Woodring and Lorenz, 2007; Biagio et al., 2009). It is interesting to compare the results obtained in C. phyllinus with data from other insect species, especially among the Orthoptera, which are closely related to the Phasmida (Grimaldi and Engel, 2005). Studies of digestion in grasshoppers (Ferreira

Table 1 Excretion (%) of midgut enzymes at each midgut emptying (data taken from Fig. 5). ENZYME Soluble cell fraction plus ventricular contents (U/animal)a 1300 11 1100 0.34 20 1 100 0.07 Hindgut contentb (U/animal) 11.6 0.3 0 15 2 0.01 0.02 % Excretionc

et al., 1990; Marana et al., 1997) and crickets (Woodring et al., 2007; Biagio et al., 2009) showed that in these animals the carbohydrate digestion occurs mainly in the foregut and in the lumen of the anteriorly located gastric caeca, while protein digestion takes place mainly in the caeca and in the midgut epithelia. In C. phyllinus the caeca are absent and the main sites of carbohydrate digestion are the foregut and the AMG, while protein digestion occurs mainly in the MMG and PMG, reinforcing the importance of a functional
Table 2 Results of activity (U/animal), and specic activity (U/mg of protein) of carbonic anhydrase in the epithelium of the midgut regions, as well as in the midgut tubules and Malpighian tubules. Regions AMG/MMG PMG 1 PMG 2 Malpighian tubules Midgut tubules Activity (U/animal) 1500 4800 1500 13,900 25,000 200 600 200 3000 3000 Specic activity (U/mg) 1.30 1.5 1.0 7.0 11.9 0.1 0.2 0.2 1.0 0.9

Trypsin Amylase Chymotrypsin Maltase

a

33.0 0 5.0 10.9

Activity of soluble enzymes. b Activity was measured only in the colon, because the rectum in many insects has a role in water reabsorption that can inactivate digestive enzymes. c Excretion (%) was calculated with the equation: (hindgut activity 3.7)/(soluble plus midgut content activity) 100. Hindgut activity was multiplied by 3.7 because on emptying, the midgut lls 3.7 times the colon.

AMG: anterior midgut; MMG: middle midgut; PMG1: proximal posterior midgut; PMG2: distal posterior midgut.

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and spatial compartmentalization of different digestive enzymes in insects. All the assayed midgut soluble enzymes (amylase, maltase, trypsin and chymotrypsin) are excreted at low rates, as calculated from enzyme activities recovered in the midgut and hindgut (Table 1). These results are in accordance with the existence of a posterioreanterior water ux in the ectoperitrophic space, as observed in most studied insects (see reviews in Terra and Ferreira, 1994; Terra, 2001; Bolognesi et al. 2008; Terra and Ferreira, 2012), which is caused by water secretion in the posterior midgut cells and water absorption in anterior midgut cells. These results are also consistent with the simple countercurrent model, as posited by Berridge (1970), and by Dow (1981). Thus, enzymes and products of digestion inside the endoperitophic space diffused into the ectoperitrophic space across the peritrophic membrane resulting in enzyme recycling and prevention of digestive enzyme excretion with feces. The existence of such putative uid uxes in C. phyllinus midgut is supported by dye experiments, which showed staining in the luminal side of the AMG, 12 h after oral administration of amaranth, suggesting that this region corresponds to the main site of water absorption. The lack of signicant staining in the MMG and PMG (as well as in the midgut tubules) shows that these structures are not absorptive. Haemolymph-injected amaranth is cleared by the Malpighian tubules, staining the hindgut and feces but not any region of the midgut hemal side. In starved animals, the dye enters the midgut through the Malpighian tubules and diffuses both backwards (directed to the hindgut) and forwards (directed to the midgut lumen), suggesting the occurence of uid ux only in starved animals, in which the Malpighian tubules are the main site of water secretion. Similar results were observed in grasshoppers, with the difference that the caeca are the site of water absorption instead of the anterior midgut (Dow, 1981; Marana et al., 1997). Nevertheless, once the enzyme excretion rates are low in both starved and fed animals, it seems that the recycling of digestive enzymes in C. phyllinus may occur in both situations, though secretion sites are not detected by dye experiments in the midgut of fed animals. 4.3. Luminal pH A very alkaline pH was observed in the PMG of C. phyllinus, especially in PMG1, where it reaches values around 9.0. An alkaline pH in insect midgut is described in several species, particularly in beetle larvae of the Scarabaeidae family, in phytophagous Lepidoptera, and in Diptera of the Nematocera families (Terra and Ferreira, 2012). The high pH values present in Lepidoptera larvae is believed to allow these animals to feed on tannin rich vegetables. The tannin binds to proteins and reduces the efciency of digestion at lower pH (Berembaum, 1980). This might also be the case for Nematoceran larvae. In Scarabaeidae beetles, on the other hand, the high pH value may facilitate the extraction of hemicellulose of the cell wall of vegetables (Terra, 1988). There are other possible explanations for the alkaline pH found in insect species. A highly alkaline pH may also be responsible for the inactivation of potentially damaging enzymes present in ingested plants (Felton et al., 1992), or it could help in the extraction of vegetal proteins soluble at alkaline pH (Felton and Duffey, 1991). C. phyllinus feeds preferentially on Myrtaceae plant species (Sottoriva et al., 2008), which normally show low concentration of tannins in their leaves. Thus, it is unlikely that the high pH present in PMG is preventing tannins from binding to digestive enzymes in this case, where the high pH is found in the posterior region of the midgut, while the rst contacts between enzymes and the food bolus occurs in the foregut and anterior midgut. Also, tannin can be solubilized at lower pH,

especially in the presence of surfactant substances often present in the midgut of insects (Martin et al., 1985). Nevertheless, it is possible that the high pH in C. phyllinus PMG may help in the extraction of hemicelluloses from plant cell walls, facilitating digestion by putative b-glucanases. Hemicelluloses are usually extracted in alkaline solutions for analytical purposes (Blake et al., 1971), and some insect species such as E. ello are able to digest hemicelluloses efciently without affecting the cellulose from the leaves they ingest to any degree (Terra and Ferreira, 2012). 4.4. The midgut tubules The presence of midgut tubules seems to be an anatomical characteristic found only in Phasmida species. Though there are some brief descriptions of these structures in literature, their functional role is unknown (Ramsay, 1955; Savage, 1962; Bartheau, 1963; Gangrade, 1965; Beadle, 1972). Due to the morphological similarity between the midgut tubules and the Malpighian tubules, the former are sometimes described as modied Malpighian tubules (Savage, 1962). Comparative ultrastructural analysis shows that cells composing both structures have specializations related to water and ion transport: abundant apical microvilli bearing mitochondria and a very infolded basal plasma membrane with associated mitochondria (Martoja and Ballan-Dufranais, 1984). On the other hand, some morphological differences were found between these two structures. Cells of the midgut tubules are smaller and less rounded-shaped than Malpighian cells and show shorter basal plasma membrane infoldings with few associated mitochondria. Dye injection experiments revealed that, unlike Malpighian tubules, the midgut tubules did not transport amaranth from the haemolymph to the gut lumen, reinforcing the hypothesis that both structures have different physiological roles. Histological detection of an acidophil compound at the midgut protuberance openings and the occurrence of a highly luminal alkaline pH in the same midgut region (PMG1) where the protuberances are present, suggest that the midgut tubules may be involved in the alkalization of this region. A high activity of carbonic anhydrase in the midgut tubules suggests that luminal alkalization may be due to the midgut tubular cell secretion of bicarbonate ions in the PMG. A similar phenomenon is observed in the mammalian duodenum in which bicarbonate is transported through a putative ATPase activated by bicarbonate to neutralize the acid food bolus coming from the stomach (Sachs et al., 1982; Garner et al., 1983). Thus, according to this hypothesis the carbonic anhydrase, present in the midgut tubule cells, would produce bicarbonate ions (HCO 3 ), via carbonic acid dissociation (H2CO3), similar to the initially proposed mechanism of luminal pH buffering in M. domestica midgut (Terra et al., 1988). Nevertheless, later studies showed that, in M. domestica, the pH buffering is likely to occur due to the secretion of ammonia ions (Terra and Regel, 1995). Thus, further studies are required to conrm the mechanism involved in the midgut alkalization process in C. phyllinus. Another interesting example of high pH gradient in the midgut lumen of an insect species occurs in the xylophagous beetle Oryctes nasicornis. The midgut of this insect presents three rows of caeca, with a ventral groove between the second and the third row (Bayon, 1981). The pH in the midgut lumen is also very alkaline especially in the region between the second and third rows of caeca, where the pH reaches more than 11 (Bayon, 1981; Biggs and McGregor, 1996). Ultrastructural analysis showed morphological evidences of ion and water transports in the groove cells and in the second to third rows of caeca cells, similar to the ones observed in the cells of the midgut tubules of C. phyllinus. Thus, the occurrence of such midgut structures in insects seems to be related to the alkalinization of the midgut lumen.

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Acknowledgments This work was supported by the Brazilian research agency FAPESP. We thank Dr. Evoneo Berti Filho for providing the phasmids. We also thank W. Caldeira and M. V. Cruz for technical assistance. E. C. Monteiro and F. K. Tamaki are graduated fellows of CAPES and FAPESP, respectively. W. R. Terra and A. F. Ribeiro are staff members of their departments and W. R. Terra is also research fellow of CNPq. References
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