the digestive system of the “stick bug” cladomorphus phyllinus

7/22/2019 The digestive system of the stick bug Cladomorphus phyllinus

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The digestive system of the stick bug Cladomorphus phyllinus(Phasmida, Phasmatidae): A morphological, physiological andbiochemical analysis

Emiliano C. Monteiro a, Fbio K. Tamaki b, Walter R. Terra b, Alberto F. Ribeiro a,*

a Departamento de Gentica e Biologia Evolutiva, Instituto de Biocincias, Universidade de So Paulo, C.P. 11461, 05422-970 So Paulo, Brazilb 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 2013Accepted 20 November 2013

Keywords:

Midgut ultrastructure

Digestive enzymes

Midgutuxes

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 represen-tative, Cladomorphus phyllinus. Cells from anterior midgut exhibit a merocrine secretion, whereas pos-terior 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 dyeinjection 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 putativecounter-currentux ofuid 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 foregutand anterior midgut. Maltase is found along the midgut associated with the microvillar glycocalix, whileaminopeptidase 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 immu-

nocytochemical data.2013 Elsevier Ltd. All rights reserved.

1. Introduction

The Order Phasmida is composed of stick and leaf insects. Astheir common name imply, these animals are able to mimic withastonishing resemblance the stems or leaves of plants, upon whichthey 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 iscomposed solely of phytophagous insects and usually presentnocturnal habits (Bedford, 1978). The order Phasmida is closely

related to the Orthoptera, and both orders comprise a mono-

phyletic 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; Biagioet al., 2009), very little is known about this process occuring inPhasmida species. Anatomical descriptions of the digestive systemof Phasmida report that it is formed by a simple tubular structure

made up of a large foregut followed by a midgut lacking digestive

caeca (Bartheau, 1963; Gangrade, 1965; Beadle, 1972). Malpighiantubules are positioned between the midgut and hindgut. An

interesting aspect of the midgut is the presence of a complex sys-tem of appendices in its posterior region formed by small pro-tuberances connected to very thin tubules, morphologically verysimilar 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 unknownfunction (Ramsay, 1955;Savage, 1962). Scanty morphological datacan also be foundin 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 pre-

sented including the immunolocalization of digestive enzymes andpossible role of the midgut tubules in the process of digestion. Theresults show the occurrence of an endo-ectoperitrophic circulationof digestive enzymes, being the anterior midgut and the Malpi-

ghian tubules the main sites of water-absorption and water-secretion, respectively, in both fed and starved animals. Initialcarbohydrates digestion should occur in the foregut and anteriormidgut and protein digestion should take place initially in the

middle and posterior midgut, with nal digestion occurring in the* Corresponding author. Tel.: 55 11 3091 7579.

E-mail address: [email protected](A.F. Ribeiro).

Contents lists available atScienceDirect

Arthropod Structure & Development

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m/ l o c a t e / a s d

1467-8039/$ e see front matter 2013 Elsevier Ltd. All rights reserved.

http://dx.doi.org/10.1016/j.asd.2013.11.005

Arthropod Structure & Development 43 (2014) 123e134
mailto:[email protected]://www.sciencedirect.com/science/journal/14678039http://www.elsevier.com/locate/asdhttp://dx.doi.org/10.1016/j.asd.2013.11.005http://dx.doi.org/10.1016/j.asd.2013.11.005http://dx.doi.org/10.1016/j.asd.2013.11.005http://dx.doi.org/10.1016/j.asd.2013.11.005http://dx.doi.org/10.1016/j.asd.2013.11.005http://dx.doi.org/10.1016/j.asd.2013.11.005http://www.elsevier.com/locate/asdhttp://www.sciencedirect.com/science/journal/14678039http://crossmark.crossref.org/dialog/?doi=10.1016/j.asd.2013.11.005&domain=pdfmailto:[email protected]


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epithelial surface of the midgut. The midgut tubules are probablyinvolved in the alkalization of theposterior midgut, possibly related

to the digestion of hemicelluloses.

2. Materials and methods

2.1. Animals and preparation of midgut samples

Cultures of the stick insectC. phyllinus(Phasmida, Phasmatidae)were maintained in the laboratory at room temperature (25 C),

under natural photoregime conditions. The insects were kept inpasteboard 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. Theintestine was carefully isolated and separated into foregut, midgutand hindgut. The midgut was further divided into the followingsub-regions: anterior midgut (AMG), middle midgut (MMG),

proximal posterior midgut (PMG1) and distal posterior midgut(PMG2). Each midgut regionwas separated into tissue and contentsof the gut. The salivary glands, Malpighian tubules and midgut

tubules were also isolated.

The samples (tissues and isolated contents) were homogenized incold double distilled water with the help of a PottereElvehjem ho-mogenizer. Homogenates from midgut regions were centrifuged for

30 min at 13,000 gat 4 C. The supernatants of contents homoge-nates were recovered and named contents. The tissue supernatantswere recovered, and labeled soluble tissue fractions and the pelletswere resuspended in double-distilled water and named tissue

membrane fraction. The material was stored at20 C until use. Noenzyme inactivation was detected during storage.

2.2. Light and electron microscopy

For histological studies with light microscope, ten insects weredissected in the xative solution (Bouins) under the stereomicro-

scope and the digestive tract carefully isolated. The tissues werekept in the xative overnight at 4 C, dehydrated in graded ethanoland embedded in historesin (Leica, Heidelberg). Serial sections (4e5 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 membranein the midgut, samples fromthree specimenswere xed in Zambonis xative (Stefanini et al., 1967) overnight at

4 C, dehydrated in gradedethanol at room temperature,embeddedin parafn wax, and cut at 8 mm thickness. Sections were thencollected on glass slides and the parafn was removed with xylene.After hydration, thesections werewashed in PBS(20 mM phosphate

bufferpH 7.4,containing 0.15M NaCl),followed byimmersionin PBS

containing 0.2% Triton X- 100, for 1 h at room temperature. Thepreparations were incubated with WGA-FITC (wheat germ agglu-tinin coupled to uorescein isothiocyanate e SigmaeAldrich),

diluted 1:500 in PBS in the presence of excess N-acetylglucosaminefor 18 h at 4 C sheltered from light. Binding of WGA to chitin isspecic 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 moun-ted in Vectashield (Vector Labs, Inc. USA) mounting medium andexamined in a Zeiss LSM 410 confocal microscope.

For transmission electron microscopy, the midgut pieces fromten specimens were xed in 3% glutaraldehyde in 0.1 M cacodylatebuffer (pH 7.4) for 2 h at 4 C. After rinsing with 0.2 M sucrose in0.1 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. Afterdehydration in graded ethanol at room temperature, the material

was embedded in Spurrs resin (Spurr, 1969). The ultrathin sectionswere obtained using a Leica Ultracut UCT ultramicrotome, stainedwith lead citrate and examined in a Zeiss EM 900 electron micro-scope operated at 80 kV. For scanning electron microscopy, the

midgut pieces were xed and dehydrated as above, critical pointdried and gold coated according to standard procedures. Thepreparations were examined in a Zeiss DSM-940 electronmicroscope.

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 dilutionof a universal pH indicator (E. Merk, Darmstadt, pH 4e10). Theresulting 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 starvedanimals.

For dye experiments, 40 ml of 100 mM amaranth dye solution

was injected into the mouth or directly into the hemocoel ofC. phyllinusadult females either starved for 5 days or fedad 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 digestiveenzymes present in C. phyllinus with antibodies available in thelaboratory, electrophoresis experiments followed by Westernblotting and immunoassays were carried out.

Animals were dissected in cold 100 mM NaCl and their midgutisolated and sectioned as previously described. The epithelia ofeach sample were carefully separated from the contents of the

digestive system and homogenized in cold double-distilled waterby using a PottereElvehjem homogenizer.

Samples were mixed with sample buffer (2:1) containing60 mM Tris-HCl, pH 6.8, 2.5% (w/v) SDS, 0.36 mM 2-mercaptoethanol, 10% glycerol and 0.05% (w/v) bromophenol blue

and heated for 2 min at 95 C in a water bath. The samples werethen loaded in a 12% (w/v) polyacrylamide gel containing 0.1% (w/v) SDS. Electrophoresis was carried out on a discontinuous pHsystem (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 transferredelectrophoretically onto a nitrocellulose membrane lter (pore size0.45mm; 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 roomtemperature with non-fat milk in TBS (Tris-buffered saline: 50 mMTris-HCl buffer pH 7.4, containing 0.15 M NaCl), containing 0.05%

Tween 20 (TBS-T). After this step, lters were reacted with Muscadomesticaanti-trypsin antiserum (Jordo et al., 1996) or with Ten-ebrio 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-rabbitIgG coupled with peroxidase (Sigma, USA) diluted 1:1000 in TBS-Tfor 2 h at room temperature. After washing extensively with the

same buffer, the lters were incubated with 0.08% 4-chloro-1-naphthol in TBS containing 0.1% hydrogen peroxide until graybands appeared where antigens were recognized. The reagent wasprepared before use by the addition of one volume of 0.5% chloro-

naphthol in methanol to

ve volumes of TBS with hydrogen

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peroxide. Bands obtained in experiments with C. phyllinus ho-mogenates were compared with those obtained with M. domesticaandT. molitorgut 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 realizedwithout the use of 2-mercaptoethanol and performed at 4 C. Aftertheelectrophoresis, the gelwas removed fromthe equipment and thesubstrate Z-FR-MCA (carbobenzoxy-Phe-Arg-4-methylcoumarin-7-

amide) 1 mM was carefully spreaded throughout its surface. The

uorescence emitted by the liberation of MCA could be observed bythe utilization of a UV light thus identifying the band that containedtrypsinand comparing itsmolecular weightwith theone estimated in

the western blot experiments. Pre-stained markers were used toverify the mass of the activity band obtained.

2.5. Immunolocalization of trypsin and amylase

After being dissected and isolated the midgut regions were xedin 4% paraformaldehyde with 0.3% glutaraldehyde in 0.1 M phos-

phate buffer at pH 7.4 for 2 h at 4 C. The material was then rinsed

with phosphate buffer and dehydrated in graded ethanol solutionsat room temperature, and embedded in hard grade L. R. Whiteacrylic resin (Electron Microscopy Sciences, Ft. Washington, USA).

Ultrathin sections were cut on the ultramicrotome and collected on200 mesh collodion-coated nickel grids. The grids were then oa-ted on drops of TBS at pH 7.2 containing 1% BSA (Bovine SerumAlbumin, Sigma, USA) for 5 min, and placed on NGS (Normal Goat

Serum, Amersham, UK), diluted 1:30, for 30 min. The sections werethen incubated overnight in the primary antisera diluted 1:100 inTBS at pH 7.2 containing 1% BSA at 4 C. Both aforementionedantisera were utilized, namely M. domestica anti-trypsin (Jordo

et al., 1996) and T. molitoranti-amylase (Cristofoletti et al., 2001).As controls, sections were incubated with pre-immune serum usingthe 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 atpH8.2 with1% BSA and 0.05% NaN3 for 30 min at room temperatureand incubated with goat anti-rabbit IgG coupled to 15 nm goldparticles (Amersham, UK) diluted 1:20 in TBS at pH 8.2 plus 1% BSAand 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% NaN 3and 0.1%tween 20, followed by the same solutionwithout BSA. After xationin 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4 for10 min at room temperature, the samples were washed in double

distilled water, stained with uranyl acetate 2% and lead citrate, andexamined in a Zeiss EM 900 electron microscope.

2.6. Enzyme assays and protein determination

Protein was determined according toBradford (1976)with theuse of ovalbumin as a standard.

Amylase, maltase, trypsin, chymotrypsin and aminopeptidase

activities were determined as follows: amylase activity wasmeasured 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. Maltasewas assayed according to Terra et al. (1979) withthe use of4 mM p-nitrophenylb-D-glucoside (NPbGlu) in 10 mM phosphate buffer pH7.0. Trypsin activity was determined by the emission of uores-

cence of methyl-coumarin released from 10mM B-R-MCA (benzoyl-L-arginin-7-amido-4 methylcoumarin) in buffer 0.1 M TRIS-HCl, pH8.5. 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 timeperiods and initial rates of hydrolysis were calculated. All assayswere performed under conditions in which the activity was pro-portional 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 ofsubstrate (or bond) per min.

Carbonic anhydrase was assayed with a modication of the

method ofWilbur and Anderson (1948), as previously detailed byTerra et al. (1988). Tissue samples were dissected and rinsed in cold0.1 M NaCl as follows: AMG and MMG were separated from PMG1and PMG2. PMG1 was freed from midgut tubules. Contents of

midgut sections were removed before homogenizing. Midgut tu-bules were removed with scissors from PMG1 and Malpighian tu-bules from the region between midgut and hindgut. All tissueswere homogenized in double distilled water. An aliquot of 0.5 ml of

tissue homogenate was added to 1 ml of solution of 16 mM HEPESbuffer, pH 8.3 at 4 C, and immediately completed with 1 ml ofcarbon 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 andAnderson (1948) as: U (t0 tcat)/tcat, where t0 corresponds tothe time for the pH change (from 8.0 to 6.5) in the uncatalyzed

reaction, andtcat is the time necessary for the same pH change inthe catalyzed reaction.

3. Results

3.1. Anatomy and histology of the midgut of C. phyllinus

The gut ofC. phyllinusis formed by a simple tube without con-

volutions (Fig. 1A). It is constituted by a foregut, a midgut and ahindgut beginning at the pylorus, the place of insertion of theMalpighian 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 re-gions: the anterior midgut (AMG), exhibiting several foldings in itssurface at regular intervals, the middle midgut (MMG) presenting asmooth surface and a smaller diameter in relation to AMG and the

posterior midgut (PMG), which can be further subdivided intoproximal (PMG1) and distal (PMG2) sub-regions. In PMG1, thepresence of a complex system of midgut tubules can be observed.These structures are connected to the midgut through small pro-

tuberances, the midgut protuberances. The midgut tubules are verydelicate structures (with a diameter between 5 and 10 mm)morphologically similar to the Malpighian tubules. Midgut tubulesare 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 isformed by enterocytes, which are the main cell type, and regen-erative cells collected at nidi close to the basal lamina (Fig.1CeE). In

the AMG the epithelia display numerous epithelial folds and pre-sent slightly smaller and more stained cells in relation to the othermidgut regions (Fig.1C, D). In the MMGthe foldings of the epitheliacease 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 thatis continuous with the epithelium of the digestive tract. Cells fromthe 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 ofC. 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); 100mm(B and E); 300 mm (C); 50 mm (D, F and G).



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midgut tubules is similar to the one found in Malpighian tubules,differing only in cell size. Midgut tubule cells areatter and smallerthan Malpighian ones and both cell types are binucleate and exhibit

a brush border (Fig. 1F, G). Accumulations of an amorphous ma-terial, highlystained with eosin, are frequently seen in the openingsof the midgut protuberances to the gut lumen (Fig.1E). The hindgutis lined by cuboidal cells covered by a thin cuticle. In the rectum,

typical rectal papillae can be observed. Along the entire midgutlumen, a membranous structure can be detected surrounding thefood bolus. This structure is the peritrophic membrane asconrmed 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 occur-

rence of sites of water absorption and secretion along themidgut. Both starved and fed C. phyllinus adult females wereorally fed with amaranth solution. Twelve hours after dyeingestion, 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 theepithelium 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

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).



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amaranth solution into the hemocoel. Dye clearance by theMalpighian 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 he-molymph there was no observable staining on the haemocelside of the midgut epithelia. Thus, it was not possible to detect a

water-secreting region in the midgut through dye injection. Instarved animals, it was noted that the dye taken up by theMalpighian tubules not only passed backwards towards thehindgut, 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 midgutepithelium at ultrastructural level: The enterocytes, the regenera-

tive cells, organized in small basal clusters or nidi, and the endo-crine cells, at the base of the epithelia showing typical smallelectron dense vesicles. The enterocytes, the main cell type, are tallpolarized cells (Figs. 2, 3AeC) with glycocalyx-covered microvilli

extending to the lumen. Junctional desmosomes are present near tothe 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 membranepresents numerous infoldings that form a labyrinth of narrowchannels with associated mitochondria. These channels communi-

cate with the underlying extracellular space through openings closeto the basal lamina.

Several important morphological differences were observed inthe enterocytes of the different midgut regions ofC. phyllinus. In

AMG enterocytes, the secretory vesicles exhibit contents formingtwo distinct electron dense regions (Fig. 2A, B inset). The basallabyrinth in this region appears poorly developed in relation toother 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 exhibitinghomogeneous high electron density contents (Fig. 2C). The basalplasma membrane infoldings in this region are more developed

than in AMG, with a greater number of canals, abundant associ-ated mitochondria and a slightly larger number of openings to theextracellular medium (Fig. 2D). In contrast, PMG1 cells show adifferent morphology in having a low electron density cytoplasm

poor in organelles but rich in cytoskeleton elements. The micro-villi are smaller than the ones present in other midgut regions(around 5mm, 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 thismidgut region suggest the occurrence of microapocrine secretion.The invaginations of the basal plasma membrane are well devel-

oped in PMG1 cells, extending apically in the cytoplasm with alarge number of associated mitochondria (Fig. 3B). The PMG2 cellshave very long microvilli (around 20 mm) and also show tip ex-pansions with small vesicles inside (Fig. 3C). The basal labyrinth is

well developed as in PMG1 cells, forming long and narrow chan-nels with many associated mitochondria and openings to the basallamina. Secretory vesicles are not detected in the cytoplasm ofPGM1 and PGM2 cells.

3.4. Fine structure of the midgut tubules and Malpighian tubules

The epithelium of the midgut and the midgut protuberances iscontinuous, with a distinct transitional region between the twostructures. The cells of the protuberances exhibit apical microvillicontaining mitochondrial projections, a large number of cyto-

plasmic mitochondria, a well-developed basal labyrinth formed byinfoldings of the basal plasma membrane with associated mito-chondria, 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 Malpighiantubules (Fig. 4B, C). Both cell types are polarized and show apicalmicrovilli containing mitochondrial projections, and pleatedseptate junctions in the lateral membrane connecting adjacent

cells. They are rich in rough endoplasmic reticulum, Golgi areas andmitochondria, and the basal plasma membrane invaginations forman elaborate labyrinth with many openings to the basal lamina. Incontrast to the Malpighian tubules (Fig. 4C), the midgut tubules

cells show a shorter basal labyrinth with very few associatedmitochondria (Fig. 4B inset).

Scanning electron microscopy analysis (Fig. 4DeF) showed that

both structures are almost identical in their external morphology,

possessing helicoidal strands of musclebers organized around thetubules (Fig. 4E, F). Nevertheless, the midgut tubules present asmaller diameter than the Malpighian tubules (around 15 mm and

30mm, 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. domesticaanti-trypsin (Jordo

et al., 1996) serum recognize homologous digestive enzymes in

C. phyllinusgut. Amylase antiserum recognized a single band withsimilar migration to theT. molitoramylase (65 kDa). Trypsin anti-serum recognizes a single protein band (60 kDa), larger than the

M. domesticatrypsin (28.5 kDa), suggesting the dimerization of thisenzyme in C. phyllinus and conrmed using in-gel assays with

uorescent substrate (Z-FR-MCA) which shown a single activityband of 60 kDa.

Ultrastructural immunolabeling using anti-amylase serum

showed that AMG and MMG regions are the major places ofamylase secretion, since secretory vesicles and the Golgi areas arewell labeled (Fig. 3D, E, F). No signicant labeling is detected inPMG cells. Immunolabeling of amylase can also be detected in AMG

and MMG lumen, mainly in the peritrophic membrane, and in as-sociation with microvilli. Similar immunolabeling is observed withtrypsin antiserum (Fig. 3G).

3.6. Distribution of digestive enzymes and carbonic anhydrase

activities

Initial and nal sites of proteins (trypsin, chymotrypsin andaminopeptidase) and starch (amylase and maltase) digestion wereveried along the C. phyllinusgut.

Most amylase and trypsin activities were found in the foregutand in lesser amounts in the contents of AMG, presenting adecreasing 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 (De

G).



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concentrated in the AMG contents; besides a signicant activity isfound in the foregut. Aminopeptidase was detected in MMG and

PMG membrane fractions. On the other hand, maltase was presentin the AMG and PMG epithelia tissue soluble fraction, indicating itis weakly adhered to the epithelium (probably trapped in the gly-cocalyx) (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 midgutto the hindgut. It was observed that the excretion rate for all

assayed enzymes was equal or inferior to 35% for each midgutemptying (Table 1). No endogenous enzymatic inhibitors werefound in PMG.

Carbonic anhydrase, which forms carbonic acid that dissociates

into bicarbonate and a proton, may contribute to the mechanism ofPMG1 luminal alkalization. It is highly active in the Malpighiantubules and midgut tubules, presenting the highest specic activityin the latter (Table 2).

3.7. Studies of luminal pH and its effect over trypsin and amylase

Luminal pH measurements using both universal pH indicatorand pH meter indicated consistently the following pH values:foregut 5.3 0.5; AMG 5.6 0.1; proximal MMG 6.3 0.2; distalMMG 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 anddistal MMG, in order to obtain a higheraccuracy of the pH gradientsalong the digestive tube. The estimated optimum pH for amylaseand trypsin were 5.0 and 9.0, respectively.

4. Discussion

4.1. Secretory events inC. phyllinus midgut

The midgut enterocytes of C. phyllinus exhibit an intense

secretory activity of digestive enzymes and other proteins likeperitrophins (cf.Bolognesi et al., 2008). These cells are rich in or-

ganelles related to the secretory pathway, namely the roughendoplasmic reticulum, Golgi areas and secretory vesicles(Rothman and Orci, 1992). There are strong signs of occurrence of avery fast exocytic process in the AMG and MMG: close proximity of

the secretory vesicles to the apical plasma membrane and immu-nolocalization of both amylase and trypsin inside the secretoryvesicles and in the midgut lumen. On the other hand, the obser-vation of small expansions along and at the tip of the microvilli in

PMG cells showing small vesicles inside are indicative of a micro-apocrine mechanism of secretion in this midgut region, which in-volves 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 distinctsecretory mechanisms along the insect midgut is not unusual. Infact, this phenomenon has been detected in several insect species,

such as in the beetles T. molitor(Cristofoletti et al. 2001) andDer-mestes maculatus (Caldeira et al. 2007), in the lepidopteransErin-nyis ello(Santos et al. 1983) andSpodoptera frugiperda(Jordo et al.1999), and in the cricket Gryllodes supplicans (Biagio et al., 2009).

These results suggest that different midgut regions shouldcontribute in diverse ways to the digestive process.

In order to study the secretory pathways of digestive enzymes inthe midgut cells, we tested heterologous anti-amylase and anti-trypsin sera in western blot experiments: the rst one recognizeda single band of 65 kDa in C. phyllinus tissues, in accordance with

the expected molecular weight for insect amylases (Cristofolettiet al., 2001). On the other hand, the anti-trypsin serum recog-nized a single band of 60 kDa, which contrasted with the expectedmolecular weight of approximately 30 kDa for insect trypsins

(Jordo et al., 1996). To test the hypothesis thatC. phyllinustrypsinsare organized in dimers, we performed in-gel assays of midguthomogenates using uorescent substrate, which showed a singleactivity band of 60 kDa, conrming the dimerization of trypsins.

Immunocytolocalizations using the heterologous antibodieslabeled amylase and trypsin in both AMG and MMGGolgi areas andsecretory vesicles, as well as in the midgut lumen in associationwith microvilli. These data agree with the location of the corre-

sponding enzyme activities and indicate that both enzymes aresecreted through an exocytic process using the same secretorypathway. This is in accordance with the data obtained in Periplaneta

americana (Lima et al. 2001), but contrasts with the results ob-

tained in T. molitorandS. frugiperda, where amylase and trypsin aresecreted in different midgut regions. Thus, in this late situation, theinitial 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 andprotein digestion caused by the combination of different luminal

pH along the midgut and optimal pH for amylase and trypsin, asdiscussed below.

4.2. Compartmentalization of digestion and enzyme recycling inC. phyllinus

Digestive enzyme distribution showed that initial starch diges-

tion is carried out byamylase in the foregut and AMG, whereas nalcarbohydrate digestion takes place along the whole midgut on the

epithelial surface, where maltase activity was found. The initialprotein digestion is carried out by trypsin and chymotrypsin, whichconcentrate in the foregut and AMG. Nevertheless, protein diges-tion by trypsin should occur in higher levels in the MMG and PMG,

since the luminal pH in these regions is similar to the trypsin op-timum pH. This divergence between trypsin optimum pH (8.5) andthe luminal pH where the enzyme is secreted (5.6 in the AMG) hasalso been observed in other insects such as D. maculatus(Caldeira

et al., 2007) and P. americana (Lima et al. 2001). Final proteindigestion is carried out by membrane-bound aminopeptidase inthe surface of MMG and PMG cells.

In spite of the high activity of amylase, trypsin and chymo-

trypsin in the foregut, crop cells are covered by cuticle and do notpresent cytological features of typical secretory cells (cf.Rothmanand Orci, 1992). Enzyme assays of salivary gland homogenates did

not show any activity for the tested enzymes, suggesting that thepresence of digestive enzymes in the foregut can only be accountedby regurgitation from the midgut. Such mechanism is also observedin 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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et al., 1990; Marana et al., 1997; Woodring and Lorenz, 2007; Biagioet al., 2009). It is interesting to compare the results obtained in

C. phyllinus with data from other insect species, especially amongthe Orthoptera, which are closely related to the Phasmida (Grimaldi

and Engel, 2005). Studies of digestion in grasshoppers (Ferreira

et al., 1990; Marana et al., 1997) and crickets (Woodring et al.,2007; Biagio et al., 2009) showed that in these animals the carbo-hydrate digestion occurs mainly in the foregut and in the lumen ofthe anteriorly located gastric caeca, while protein digestion takes

place mainly in the caeca and in the midgut epithelia. In C. phyllinusthe caeca are absent and the main sites of carbohydrate digestion

are the foregut and the AMG, while protein digestion occurs mainlyin 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 Activity (U/animal) Specic activity (U/mg)

AMG/MMG 1500 200 1.30 0.1

PMG 1 4800 600 1.5 0.2

PMG 2 1500 200 1.0 0.2

Malpighian tubules 13,900 3000 7.0 1.0

Midgut tubules 25,000 3000 11.9 0.9

AMG: anterior midgut; MMG: middle midgut; PMG1: proximal posterior midgut;

PMG2: distal posterior midgut.

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

Hindgut contentb

(U/animal)

% Excretionc

Trypsin 1300 20 11.6 0.3 33.0

Amylase 11 1 0 0

Chymotrypsin 1100 100 15 2 5.0

Maltase 0.34 0.07 0.01 0.02 10.9

a Activity of soluble enzymes.b Activitywas measuredonly in thecolon,because therectumin manyinsects has

a role in water reabsorption that can inactivate digestive enzymes.c Excretion (%) was calculated withthe equation: (hindgutactivity 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.

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. De-

terminations were carried out in three different preparations obtained from one insect each. SEM were found to be 10 e25% 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; PMG2e 31.3 mg; hindgut e 102 mg.



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and spatial compartmentalization of different digestive enzymes ininsects.

All the assayed midgut soluble enzymes (amylase, maltase,trypsin and chymotrypsin) are excreted at low rates, as calculatedfrom 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, asobserved 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 watersecretion in the posterior midgut cells and

water absorption in anterior midgut cells. These results are alsoconsistent with the simple countercurrent model, as posited byBerridge (1970), and by Dow (1981). Thus, enzymes and products ofdigestion inside the endoperitophic space diffused into the ecto-

peritrophic space across the peritrophic membrane resulting inenzyme recycling and prevention of digestive enzyme excretionwith feces. The existence of such putative uid uxes inC. phyllinusmidgut is supported by dye experiments, which showed staining in

the luminal side of the AMG, 12 h after oral administration ofamaranth, suggesting that this region corresponds to the main siteof water absorption. The lackof 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 bythe Malpighian tubules, staining the hindgut and feces but not anyregion of the midgut hemal side. In starved animals, the dye enters

the midgut through the Malpighian tubules and diffuses bothbackwards (directed to the hindgut) and forwards (directed to themidgut lumen), suggesting the occurence of uid ux only instarved 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 absorptioninstead 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 digestiveenzymes in C. phyllinus may occur in both situations, thoughsecretion sites are not detected by dyeexperiments in the midgut of

fed animals.

4.3. Luminal pH

A very alkaline pH was observed in the PMG ofC. phyllinus,

especially in PMG1, where it reaches values around 9.0. Analkaline pH in insect midgut is described in several species,particularly in beetle larvae of the Scarabaeidae family, inphytophagous Lepidoptera, and in Diptera of the Nematocera

families (Terra and Ferreira, 2012). The high pH values present inLepidoptera larvae is believed to allow these animals to feed ontannin rich vegetables. The tannin binds to proteins and reducesthe 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 theextraction 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 beresponsible for the inactivation of potentially damaging enzymespresent in ingested plants (Felton et al., 1992), or it could help in

the extraction of vegetal proteins soluble at alkaline pH (Feltonand Duffey, 1991). C. phyllinus feeds preferentially on Myrtaceaeplant species (Sottoriva et al., 2008), which normally show lowconcentration of tannins in their leaves. Thus, it is unlikely that

the high pH present in PMG is preventing tannins from bindingto digestive enzymes in this case, where the high pH is found inthe posterior region of the midgut, while the rst contactsbetween 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 presentin the midgut of insects (Martin et al., 1985). Nevertheless, it is

possible that the high pH in C. phyllinus PMG may help in theextraction of hemicelluloses from plant cell walls, facilitatingdigestion by putative b-glucanases. Hemicelluloses are usuallyextracted in alkaline solutions for analytical purposes (Blake

et al., 1971), and some insect species such as E. ello are able todigest hemicelluloses efciently without affecting the cellulosefrom 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 aresome brief descriptions of these structures in literature, theirfunctional 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 tu-bules (Savage, 1962). Comparative ultrastructural analysis shows

that cells composing both structures have specializations related to

water and ion transport: abundant apical microvilli bearing mito-chondria and a very infolded basal plasma membrane with asso-ciated mitochondria (Martoja and Ballan-Dufranais, 1984). On the

other hand, some morphological differences were found betweenthese two structures. Cells of the midgut tubules are smaller andless rounded-shaped than Malpighian cells and show shorter basalplasma membrane infoldings with few associated mitochondria.

Dye injection experiments revealed that, unlike Malpighian tu-bules, the midgut tubules did not transport amaranth from thehaemolymph to the gut lumen, reinforcing the hypothesis that bothstructures have different physiological roles. Histological detection

of an acidophil compound at the midgut protuberance openingsand the occurrence of a highly luminal alkaline pH in the samemidgut region (PMG1) where the protuberances are present, sug-

gest that the midgut tubules may be involved in the alkalization ofthis region. A high activity of carbonic anhydrase in the midguttubules suggests that luminal alkalization maybe due to the midguttubular cell secretion of bicarbonate ions in the PMG. A similarphenomenon is observed in the mammalian duodenum in which

bicarbonate is transported through a putative ATPase activated bybicarbonate to neutralize the acid food bolus coming from thestomach (Sachs et al., 1982; Garner et al., 1983). Thus, according tothis hypothesis the carbonic anhydrase, present in the midgut tu-

bule cells, would produce bicarbonate ions (HCO3), via carbonic

acid dissociation (H2CO3), similar to the initially proposed mecha-nism of luminal pH buffering inM. 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 toconrm the mechanism involved in the midgut alkalization processinC. phyllinus. Another interesting example of high pH gradient in

the midgut lumen of an insect species occurs in the xylophagousbeetleOryctes nasicornis. The midgut of this insect presents threerows of caeca, with a ventral groove between the second and the

third row (Bayon, 1981). The pH in the midgut lumen is also veryalkaline especially in the region between the second and third rowsof caeca, where the pH reaches more than 11 (Bayon, 1981; Biggsand McGregor, 1996). Ultrastructural analysis showed morpholog-

ical evidences of ion and water transports in the groove cells and inthe second to third rows of caeca cells, similar to the ones observedin the cells of the midgut tubules ofC. phyllinus. Thus, the occur-rence 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 agencyFAPESP. We thank Dr. Evoneo Berti Filho for providing the phas-mids. We also thank W. Caldeira and M. V. Cruz for technicalassistance. E. C. Monteiro and F. K. Tamaki are graduated fellows of

CAPES and FAPESP, respectively. W. R. Terra and A. F. Ribeiro arestaff members of their departments and W. R. Terra is also researchfellow of CNPq.

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the digestive system of the “stick bug” cladomorphus phyllinus

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