The Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae) digestive tract: Histology, ultrastructure and its physiological significance

Journal of Insect Physiology xxx (2010) xxx–xxx

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IP-2398; No of Pages 8

Subcellular fractionation of midgut cells of the sunn pest Eurygaster integriceps(Hemiptera: Scutelleridae): Enzyme markers of microvillar and perimicrovillarmembranes

M. Allahyari a, A.R. Bandani b,*, M. Habibi-Rezaei c

a Department of Plant pests and Diseases, Fars Agriculture and Natural Resources Research Center, Shiraz, Iranb Plant Protection Department, Agricultural and Natural Resources Campus, University of Tehran, Karaj, Iranc School of Biology, College of Science, University of Tehran, Tehran, Iran

A R T I C L E I N F O

Article history:

Received 30 September 2009

Received in revised form 10 December 2009

Accepted 16 December 2009

Keywords:

Sunn pest

Enzyme marker

Microvillar membrane

Perimicrovillar membrane

A B S T R A C T

The subcellular distributions of six digestive and non-digestive enzymes (a-glucosidase, b-glucosidase,

alkaline phosphatase, acid phosphatase, aminopeptidase and lactate dehydrogenase) of Eurygaster

integriceps have been studied. The subcellular distributions of acid phosphatase and a-glucosidase are

similar and the gradient ultracentrifugation profiles of these two enzymes overlap. Two partially

membrane-bound enzymes, alkaline phosphatase and b-glucosidase have similar distributions in

differential centrifugation fractions, which are different from that of a-glucosidase. Sucrose gradient

ultracentrifugation of membranes from luminal contents showed that b-glucosidase carrying

membranes are heavier. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the profile

of proteins extracted from b-glucosidase carrying membranes is different from that of a-glucosidase

carrying membranes. We conclude that b-glucosidase and aminopeptidase are markers of microvillar

membrane (MM) and perimicrovillar space, respectively, while a-glucosidase and acid phosphatase are

perimicrovillar markers. In E. integriceps V1 luminal content is a rich source of PMM and MM and that is

used to resolve these membranes.

� 2009 Elsevier Ltd. All rights reserved.

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1. Introduction

The sunn pest Eurygaster integriceps Puton (Hemiptera:Scutelleridae) is a major pest of wheat and barley in wide areasof the near and middle east, west and central Asia, north Africa, andeastern and south Europe (Brown, 1965; Critchley, 1998; Praker etal., 2002). Sunn pest infestations in some areas can lead to 100%crop loss in the absence of control measures. Pesticide spraying isthe main method of sunn pest control in areas where infestation ishigh. In addition to the high cost of chemical control, insecticidespose a risk to the balance of nature, human health, water quality,wildlife, and the environment as a whole (Javahery, 1995). Thusnew control methods are needed to diminish reliance oninsecticides for control of this serious crop pest. Among possiblenew control strategies, insect-resistant transgenic crops arepromising, and commercial releases of first-generation maizeand cotton expressing a single modified Bacillus turingiensis (BT)toxin have been successful (Christou et al., 2006; Ferry et al., 2006).

Membrane-bound proteins of insect midgut play importantroles in digestion, absorption and other aspects of gut physiology,

* Corresponding author. Tel.: +98 261 2818705; fax: +98 261 2238529.

E-mail address: [email protected] (A.R. Bandani).

Please cite this article in press as: Allahyari, M., et al., Subcellular fra(Hemiptera: Scutelleridae): Enzyme markers of microvillar and perj.jinsphys.2009.12.010

0022-1910/$ – see front matter � 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.jinsphys.2009.12.010

and are potential targets for crop plant transgenes. Some of theseproteins are glycosylated and play important roles in interactingwith exogenous particles such as BT toxins, lectins, and otherinsecticidal proteins. Thus characterizing lumen side membrane-bound proteins of midgut epithelial cells provides prerequisiteknowledge for developing new control methods (Wilkins andBillingsley, 2001). Hemipteran midgut epithelial cells are coveredwith perimicrovillar membranes (PMM) that have a physiologicalrole similar to the peritrophic membrane seen in the midgut ofother insects. Like the peritrophic membrane, the PMM is involvedin food digestion and absorption and is in close contact withingested external materials (Terra and Ferreira, 2005). Lectins andinsecticidal proteins are known to interact with midgut microvillarmembrane (MM) by binding to glycoproteins (Powell et al., 1998;Sauvion et al., 2004; Trung et al., 2006; Mehlo et al., 2005).

PMM and MM can be isolated from gut tissue homogenates andidentified using suitable marker enzymes. Several methods havepreviously been used for MM isolation, among which the differentialdivalent cation precipitation has been the method of choice (Terra etal., 2006). However, this method cannot be used for Hemipteranmidgut MM and PMM (Terra, personal communication). Moreover,it is important to check the subcellular distributions of putativeenzyme markers as enzymes previously used in other species are notnecessarily always suitable (Terra et al., 2006).

ctionation of midgut cells of the sunn pest Eurygaster integriceps

imicrovillar membranes. J. Insect Physiol. (2010), doi:10.1016/

mailto:[email protected]

http://dx.doi.org/10.1016/j.jinsphys.2009.12.010


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M. Allahyari et al. / Journal of Insect Physiology xxx (2010) xxx–xxx2

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So far the subcellular distribution of digestive enzymes of onlytwo heteropteran insect species, Rhodnius prolixus and Dysdercus

pruvianus, have been studied. Ferreira et al. (1988) showed that a-glucosidase and a-mannosidase are, respectively perimicrovillarand microvillar membrane markers in R. prolixus, while in D.

pruvianus, a-glucosidase and b-glucosidase are, respectively peri-microvillar and microvillar membrane markers (Silva et al., 1996).

Sunn pest gut physiology and biochemistry have not beeninvestigated. The current study was conducted to identify markerenzymes for MM and PMM of the sunn pest using differential anddensity gradient centrifugation. The isolated membranes can beused to study proteins and glycoproteins that have important rolesin midgut cell physiology. Considering the importance of membranephysiology and food digestion as a target for sunn pest control, it isclear that understanding function of digestive enzymes, theirsecretory mechanisms, microvillar and perimicrovillar physiologyand biochemistry needs more attention. The knowledge thus gainedwill lead to new management strategies for pest control.

2. Materials and methods

2.1. Animals

Adult female E. integriceps were collected from wheat fields ofShiraz, Iran in the spring, over-wintering and aestivating sitesduring winter and summer, respectively. Collected diapausingfemales (summer and winter collected females) were stored in adark incubator at 14 8C until use. Diapausing females were treatedtopically with a juvenile hormone mimics, pyriproxyfen(10,000 ppm of pyriproxyfen 10EC, Admiral1 Sumitomo ChemicalCo., Ltd., Japan) and kept in plastic containers on wheat grains at14:10 photoperiod, 25 � 2 8C and 40% RH in order to terminatediapause before dissection. Wet cotton pieces were put in the rearingcontainer to provide a water source for the insects.

2.2. Sample preparation

Sample preparation was based on the method of Silva et al.(1996) with slight modifications. The insects were immobilized bykeeping them in the freezer (approximately �20 8C) for 5 min. Themidgut was dissected in a Petri dish containing ice-cold 260 mMNaCl.

The midgut is the major part of the sunn pest alimentary tract,having four distinct regions which are referred to 1st (V1), 2nd(V2), 3rd (V3), and 4th (V4) ventriculus.

The first ventriculus (V1) was opened lengthwise and itscontents were washed in 260 mM NaCl tree times. Groups of V1tissues equal to 10 V1/ml were homogenized in a motorizedpotter-elvehjem homogenizer (Teflon pestle, 0.1 mm clearance)for 3 min at 500 rpm. Two homogenizing buffers were used (toensure that membrane-bound enzymes are truly membranebound) isotonic (260 mM KCl, pH 7.0) and hypotonic (2 mMTris–HCl buffer, pH 7.0 containing 50 mM mannitol) providingmild and vigorous condition, respectively (see Ferreira and Terra,1980). The resulting homogenate was passed through a 47.0 mmpore size nylon filter mesh to remove connective tissues andunbroken cells. This filtrate was subjected to fractionation bydifferential centrifugation procedures.

To prepare a membrane fraction for sucrose gradient centrifu-gation, V1 tissues were homogenized in distilled water at 10 V1/mlas mentioned above and centrifuged at 25,000 � g for 30 min at4 8C. The pellet was suspended in distilled water and subjected tothree cycles of freezing and thawing to release proteins trappedbetween membranes during centrifugation and centrifuged againas described above. Subsequently, the pellet was resuspended indistilled water and stored in �20 8C until use.


To prepare a membrane fraction from V1 luminal contents, V1was dissected and opened lengthwise and its contents werecollected by washing it in a known volume of distilled water.Fifteen V1 contents were diluted in 15 ml distilled water andsonicated for 20 s (4 � 5 s) followed by placing on ice for 30 min toprecipitate food particles. The supernatant was centrifuged at25,000 � g for 30 min, the pellet was suspended in 5 ml of distilledwater, centrifuged as before, and resuspended in 1 ml of double-distilled water, then being stored at �20 8C.

Enzyme activity was measured in V1 luminal contents, PMMand V1 tissue. V1 were dissected in distilled water, then openedlengthwise and the contents dispersed in a known volume ofdistilled water. To separate PMM from V1 tissue, empty V1 waskept in deionized water at 4 8C for approximately 15 min and thenswollen PMM was separated from V1 tissue using fine forceps. V1tissue and PMM were homogenized using a potter-elvehjemhomogenizer (glass pestle). The V1 tissue homogenate was passedthrough a 47 mm pore size nylon filter mesh to remove connectivetissue and unbroken cells. All three fractions were adjusted to aconcentration of 10 guts per ml of distilled water and stored in�20 8C for further use as an enzyme source.

2.3. Differential centrifugation

Four fractions were collected from the V1 homogenate bydifferential centrifugation. These fractions were designated as P1(pellet of 600 � g for 10 min), P2 (pellet of 3300 � g for 10 min), P3(pellet of 25,000 � g for 10 min) and P4 (pellet of 100,000 � g for1 h). Pellets were homogenized in the buffer (pellets obtained fromhypotonic buffer homogenized in the hypotonic buffer and pelletsobtained from isotonic buffer homogenized in the isotonic buffer)using a potter-elvehjem homogenizer. After three freezing–thawing cycles, suspensions were centrifuged at 100,000 � g for1 h and supernatant and pellet of each fraction used for enzymeassay experiments.

2.4. Gradient ultracentrifugation of V1 contents

Sucrose gradients ultracentrifugation was performed accordingto Silva et al. (1996) with some modifications. 200 ml of membranefraction was loaded on to a 4.6 ml sucrose gradient (10–40%, w/v)in 5 mM imidazol buffer containing 40 mM KCl and 5 mM EDTAand then centrifuged at 100,000 � g for 15 h. Fractions (200 ml)were collected from the top of the pelleted material with the aid ofa peristaltic pump and assayed for enzyme activity. The density ofeach fraction was measured with the aid of a refractometer.

2.5. Electron microscopy

Dissected V1 (freed from luminal contents in 215 mM NaCl)and subcellular fractions were fixed in 4% glutaraldehyde in0.15 M sodium cacodylate buffer (pH 7.0) for 4 h at 4 8C. Afterrinsing with cacodylate buffer, they were postfixed in 1% OSO4 for1.5 h and washed in cacodylate buffer. After dehydration ingraded ethanol, samples were cleared 20–30 min in propyleneoxide and embedded in resin. Ultrathin sections (0.05 mm) werecut using a U3 ultra-microtome followed by staining with uranylacetate and lead citrate and examined in a Philips CM10 Electronmicroscope.

2.6. Enzyme assay

a- and b-glucosidase activity were measured using 5 mM a-and b-D, 4-nitrophenyl glucopyranoside in 50 mM citrate–phosphate buffer pH 5.0, respectively, based on the appearanceof p-nitrophenol in the solution according to Terra et al. (1979).





Fig. 1. (A) Diapausing adult female dissected without treatment. Note inactive V3,

V4 and ovaries. (B) Diapausing female after treatment with pyriproxyfen that

caused V3, V4 activation. (C) V4 and ovaries containing eggs. Abbreviations are (V1)

first ventriculus; (V2) second ventriculus; (V3) third ventriculus; (V4) fourth

ventriculus; (R) rectum; (O) ovary; (E) eggs. Scale bar in A (2 mm).

M. Allahyari et al. / Journal of Insect Physiology xxx (2010) xxx–xxx 3

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Aminopeptidase activity was measured using 2 mM L-leucinep-nitroanilide (LPNA) in 50 mM Tris–HCl buffer pH 7.0 as substrate(Spungin and Blumberg, 1989). The activity of acid phosphatasewas determined using 5 mM 4-nitrophenyl phosphate in 50 mMcitrate–phosphate buffer pH 4.5 as substrate. Alkaline phosphatasewas determined using 50 mM glycine–NaOH pH 10.4 containing1 mM MgSO4 (Terra et al., 1979). Lactate deydrogenase activitywas measured according to Krieg et al. (1967).

In all assays reaction volumes were incubated at 30 8C for atleast four different periods of time and initial rates of hydrolysiswere calculated. Controls without enzyme and substrate wereincluded. One unit of enzyme (U) is defined as the amount thathydrolyses 1 mmol of substrate per min.

2.7. Protein determination

Protein concentration was measured according to the methodof Bradford (1976), using bovine serum albumin (Bio-Rad,Munchen, Germany) as a standard.

2.8. SDS-PAGE electrophoresis

Proteins of membrane fractions were solubilized in 2% Triton X-100 solution for 15 h at 4 8C followed by centrifugation at100,000 � g for 1 h. The solubilized proteins (supernatant) werecombined with sample buffer to give final concentration of 10 mMTris–HCl buffer pH 6.8, 1% (w/v) SDS, 2% b-mercaptoethanol, 10%glycerol (v/v). Samples were heated in boiling water for 5 minbefore being loaded onto a 7.5% (w/v) polyacrylamide gelcontaining 0.1% SDS (Laemmli, 1970). Three micrograms of proteinwere loaded in each lane and the gel was run at 0.07 mA/cm2 andstained with silver nitrate (Yan et al., 2000).

3. Results

3.1. Terminating adult diapause

The midgut is the major part of the E. integriceps alimentarytract, having four distinct regions which are referred to 1st (V1),2nd (V2), 3rd (V3), and 4th ventriculus (V4). Diapausing femaleinsects have small and empty V2, V3, and V4. Application ofpyriproxyfen terminated diapause and accelerated food transitthrough the midgut as shown in Fig. 1.

3.2. Subcellular fractionation of digestive enzymes

The subcellular distribution of enzymes in the V1 cells of E.

integriceps is shown in Fig. 2 (isotonic condition) and Fig. 3(hypotonic condition) and electron micrographs of the fractionsare shown in Fig. 5. All the assayed enzymes except aminopepti-dase were generally present in particulate fractions (pelletfractions). In isotonic homogenizing conditions, a-glucosidasewas present in major amount in particulate of first fraction(600 � g) but in hypotonic homogenizing conditions the distribu-tion of this enzyme extended to lighter particulate fractions(Fig. 3). Acid phosphatase subcellular distribution was similar tothat of a-glucosidase. These enzymes were not found insubstantial amounts in the final supernatant.

Alkaline phosphatase and b-glucosidase are partly membrane-bound enzymes and they were found to be associated with lighterstructures, microvillar vesicles. The subcellular distribution oflactate dehydrogenase did not change according to whetherisotonic or hypotonic homogenizing condition were used, andmajor amounts of this enzyme were bound to cell fragments(Figs. 2 and 3).


Aminopeptidase occurred in the final supernatant with onlytrace activity in particulate fractions regardless of the homogeniz-ing conditions. Aminopeptidase recovery in the fractionatedhomogenate was much larger than for the other enzymes (about200%) in both homogenizing condition.

Fig. 4A and B shows the ultrastructure of V1 midgut cellsbearing microvillar and perimicrovillar structures. Fig. 4C is a





Fig. 2. Subcellular distribution of V1 E. integriceps homogenate in isotonic condition (260 mM KCl pH 7.0). Collected fractions were 600 � g, 3300 � g and 25,000 � g each for

10 min and 100,000 � g for 60 min. After differential centrifugation each fraction were homogenized in the buffer and centrifuged at 100,000 � g for 1 h. Supernatant and

pellet of each fraction were assayed for enzyme activity. White bars represent supernatants; dark bars represent pellets. Fractions P1, P2, P3, P4 and final supernatant are

showed from left to right. Bars show mean values together with the standard error of mean based on three independent preparations obtained from 70 females each. The

horizontal width of each bar is proportional to the protein content of the supernatant or pellet that it represents.


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closer view showing a double-membrane vesicle. Fig. 4D shows across-section of microvilli, which are seen as thick-walled circlescontaining cytoskeleton like internal structures.

Transmission electron micrographs were produced from twoparticulate fractions that were prepared during differentialcentrifugation (P1 and P3) (Fig. 5). Large membranous structures(Fig. 5B) were enriched in first fraction (600 � g), which may befragments of PMM. Also, vesicles bearing cytoskeleton likestructures were observed in this pellet (P1) (Fig. 5C). Fig. 5Dshows membranes present in P3.

3.3. Enzyme activities in different parts of V1

Table 1 shows the activities of aminopeptidase, a and b-glucosidases and acid phosphatase in luminal content, PMM andV1 tissue. PMM has the highest specific activity of a-glucosidase,whereas the highest specific activity of b-glucosidase wasobserved in V1 luminal contents. V1 tissue has the greatestaminopeptidase specific activity.

3.4. Resolving V1 luminal content membranes

Membranes in the V1 luminal contents can be resolved intomembrane fractions that are associated with a-glucosidase, acidphosphatase and b-glucosidase activities (Fig. 6). The a-glucosi-dase rich fraction has the lowest density (1.0564 � 0.009 g/cm2),while the b-glucosidase rich fraction has the highest density


(1.104 � 0.001 g/cm2). Acid phosphatase carrying membranes coin-cided with a-glucosidase carrying membranes especially at higherspeeds (130,000 � g) that completely overlap each others (data arenot shown). Unlike the b-glucosidase, most of the a-glucosidase andacid phosphatase activity occurred in the pellet after density gradientultracentrifugation (�20% in pellet and �5% in a-glucosidase/acidphosphatase rich membranes).

3.5. Gel electrophoresis

As shown in Fig. 7. membranes recovered from the pellet aftergradient ultracentrifugation in lane 1 (pellet) and lane 2 (b-glucosidase rich membrane) are to some extent are similar. Lane 4shows a-glucosidase carrying membranes that have fewermembrane-bound proteins. Lane 3 shows acid phosphatase richmembrane proteins that are more similar to lane 4.

4. Discussion

Acid phosphatase and a-glucosidase show similar subcellulardistribution. When extracted in isotonic conditions they occurmainly in P1, a fraction that is rich in large membranous structures.More severe homogenizing condition (hypotonic) caused morefragmentation and smaller vesicles, thus extending specificactivity to lighter fractions. The same trends were seen for themembrane-bound digestive enzymes of Rhyncosiara americana

(Ferreira and Terra, 1980). In E. integriceps there were only minor





Fig. 3. Subcellular distribution of V1 E. integriceps homogenate in hypotonic conditions (Tris–HCl 2 mM buffer pH 7.0 containing 50 mM mannitol). Other details are as in the

legend of Fig. 2.


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activities of acid phosphatase and a-glucosidase in the finalsupernatant indicating that these two enzymes are membrane-bound. The distributions of these two enzymes in gradientultracentrifugation profiles substantially overlapped. Acid phos-phatase carrying membranes are a little heavier than a-glucosi-dase carrying membranes perhaps because these enzymes are ontwo different domains on the same membrane. Analysis of theprotein content of membranes acquired through sucrose gradientultracentrifugation showed that a-glucosidase carrying mem-branes have less membrane-bound proteins than b-glucosidasecarrying membranes (Fig. 7). a-Glucosidase is a general marker ofPMM in Hemiptera and Thysanoptera (Ferreira et al., 1988; Silvaet al., 1995, 1996, 2004) thus in E. integriceps acid phosphatase isanother PMM enzyme marker. Terra hypothesized that evolutionof Heteroptera from sap-feeding ancestors had been associatedwith regaining lysosomal enzymes for polymer digestion (Terraand Ferreira, 2005). Acid phosphatase in R. prolixus and R.

americana is a soluble enzyme in major amount (Ferreira andTerra, 1980) and resembles those found in mammals as marker oflysosomes (Ferreira et al., 1988).

Alkaline phosphatase and b-glucosidase also have similardistributions in differential centrifugation fractions. In mildhomogenizing conditions they are seen in particulate fractionsbut in hypotonic condition some activity appears in solublefractions and in the final supernatant. Thus these two enzymes arepartially membrane bound and their locations are different froma-glucosidase. Sucrose gradient ultracentrifugation showed thatb-glucosidase carrying membranes are heavier and the profile oftheir extracted proteins is different from that of a-glucosidasecarrying membranes. Alkaline phosphatase is a putative MMmarker in many insects (Terra and Ferreira, 2005; Terra et al., 2006)


and b-glucosidase was reported as MM marker in D. peruvianus

(Silva et al., 1996). Thus b-glucosidase can be a marker of MM in E.

integriceps.Aminopeptidase seems to be a soluble enzyme because major

activity of this enzyme appeared in the final supernatantregardless of the homogenizing condition. Ferreira et al. (1988)proposed that an aminopeptidase with activity towards LPNA in R.

prolixus posterior midgut cells may be a perimicrovillar spacemarker because after several freezing and thawing cycles and inhypotonic homogenization conditions its activity was prominentin the soluble part of the particulate fractions. Silva et al. (1996)obtained similar results when fractionating D. peruvianus V1 cells,from which aminopeptidase was released more easily than lactatedehydrogenase (LDH) (a cytosol marker). In E. integriceps, however,those trends (release from membranes after freeze–thaw cycles)were not confirmed and specific activity of aminopeptidase wasprominent in final supernatant and V1 luminal contents (Figs. 2and 3). Recovery of aminopeptidase in these fractions was highrelative to the homogenate (about 135% in isotonic condition and250% for hypotonic condition), indicating the probable presence ofan endogenous inhibitor in the original homogenate. Thus, thedifferences between particulate fractions cannot be seen clearly.However, comparing aminopeptidase distribution with that of LDHit can be concluded that aminopeptidase is probably a perimicro-villar space marker.

The fact that specific activity of aminopeptidase in V1 luminalcontent is lower than in V1 tissue homogenate (Table 1) and finalsupernatant, appears to rule out the hypothesis that aminopepti-dase is secreted into the gut lumen. According to Terra and Ferreira(2005) polymers in ingested materials (food particles) can haveinhibitory effects on aminopeptidase activity. In our experiments,





Fig. 4. Transmission electron microscope image from E. integriceps V1 epithelial cells. (A and B) A midgut epithelial cell, perimicrovillar membrane (white arrow head),

microvillus (black arrow head). (C) Double-membrane vesicle (arrow), (D) microvilli cross-section. Scale bars: A (0.66 mm), B (0.63 mm), C (0.35 mm) and D (0.2 mm).


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although such materials (food particles) may have close contact tothe enzyme during homogenization process, they were not presentin final supernatant.

To verify that if E. integriceps has perimicrovillar structure andto visualize pelleted material during differential centrifugation,TEM images were prepared from V1 midgut cells and the P1 and P3pellets. Silva et al. (1996) were unsuccessful in separating thesetwo membranes from each other in V1 tissue. They used V1luminal contents for membrane resolution and their ultrastruc-tural works showed that PMM of D. pruvianus was shorter inlength than those of R. prolixus. Our preliminary works usingsonication and sucrose gradient density ultracentrifugation also

Table 1Enzyme activities in different parts of V1 (first part of Eurygaster integriceps midgut).

Aminopeptidase b-Glucos

Luminal contents 2.46�0.45 53.33�2

PMM 2.04�0.17 24.46� 0

V1 tissue 4.56�0.26 27.93� 0

Data are means� SE of specific activities in mU/mg protein based on three replication of


found that PMM and MM could not be resolved from V1 tissues.Accordingly, we used E. integriceps V1 luminal content as a richsource of PMM and MM that could be used to resolve thesemembranes.

It has been proposed that MM is rich and PMM is poor inintegral proteins (Lane and Harrison, 1979; Terra and Ferreira,2005). Thus, PMM should have lower buoyant density than MM(Evans, 1979). In agreement with this, we found that the E.

integriceps a-glucosidase (PMM marker) rich fraction hadthe lowest buoyant density (1.056 g/cm2) and b-glucosidase(MM marker) rich fraction had the greatest buoyant density(1.104 g/cm2). Two main functions have been proposed for

idase a-Glucosidase Acid phosphatase

.64 115.33�3.91 10.64�0.49

.50 453.25�16.56 36.66�1.24

.81 316.97�15.36 41.15�0.95

50 animals.





Fig. 5. Light microscope and electron microscope images from P1 and P3 particulate fractions resulting from differential centrifugation. (A) Light microscope image of the

pellet of 600 � g centrifugation of V1 homogenized in 260 mM KCl buffer pH 7.0 (magnified 16�with a digital camera), membrane vesicles aggregated around food particles

(arrow head). (B and C) TEM of the pellet of first fraction (600 � g), large membranous structures probably fragments of perimicrovillar membranes (white arrow head),

vesicles having cytoskeleton structures probably microvillar vesicles (black arrow head). (D) TEM of the pellet of third fraction (25,000 � g), membrane vesicles from

unknown intracellular source. Scale bars: B (2 mm), C (0.7 mm) and D (0.2 mm).


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PMM, including presumed roles in amino acid absorption andcompartmentalization of digestion process (Ferreira et al., 1988;Silva et al., 1995; Terra and Ferreira, 2005). The efficientabsorption of organic compound (mainly amino acids) presentin minor concentration in phloem sap is particularly importantin plant-sucking Hemiptera. It is supposed that non sap-suckingheteropteran insects (predators, seed suckers, hematophagousbugs) have been evolved from phloem sap-sucking insectswhich have lost their peritrophic membrane and acquired PMM(Goodchild, 1966). In these insects K+ ions are activelytransported by MM from the perimicrovillar space (PMS) intomidgut cells resulting in a K+ concentration gradient between

Fig. 6. Total membrane fraction resolution from E. integriceps V1 luminal contents

by ultracentrifugation in sucrose gradients. 0.2 ml fractions were collected from the

supernatant above the sedimented materials (pellet) and were assayed for a-

glucosidase, b-glucosidase and acid phosphatase. The density of each fraction was

determined using a refractometer. Other experiments showed a similar profile.

Fig. 7. Polyacrylamide gel electrophoresis of polypeptides from membranes

resolved from luminal membrane contents (SDS-PAGE 7.5%). (M) Protein markers:

160, 110, 90, 75, 55, 45, 35, 25 and 15 kDa. (1) Solubilized proteins from the pellet of

sucrose gradient ultracentrifugation. (2) Protein profile of b-glucosidase rich

membrane fraction. (3) Acid phosphatase rich fraction. (4) a-Glucosidase rich

fraction. The heavy band at 75 kDa is 1 mM BSA in buffer added to conserve enzyme

activity. Three micrograms proteins were loaded and the gel was run at 0.07 mA/

cm2 and stained with silver nitrate.

Please cite this article in press as: Allahyari, M., et al., Subcellular fractionation of midgut cells of the sunn pest Eurygaster integriceps

(Hemiptera: Scutelleridae): Enzyme markers of microvillar and perimicrovillar membranes. J. Insect Physiol. (2010), doi:10.1016/j.jinsphys.2009.12.010




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the gut luminal sap and the PMS. This concentration gradientprovides the driving force for absorption of organic compounds(amino acids) from the PMS by appropriate protein transporterspresent in the PMM. Uptake into the midgut cells is enhanced bythe simultaneous transfer of water from the midgut lumen intomidgut cells.

The other function of PMM is the compartmentalization ofdigestion process, which in other insects is accomplished by theperitrophic membrane. Thus, in E. integriceps oligopeptides formedin the luminal gut through the action of salivary and gutproteinases are transported into the PMS where further digestionand absorption are taking place. Also, the PMM prevents non-specific binding of undigested materials onto MM hydrolyticenzymes and transport proteins.

Acknowledgments

This research was funded by a grant (No. 86025.11) from theIran National Science Foundation (INSF). Authors would like tothank Mr. M.K. Mossallaee for his support and assistance.

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The Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae) digestive tract: Histology, ultrastructure and its physiological significance

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