DIGESTION IN ADULT FEMALES OF THE LEAF-FOOTED BUG Leptoglossus zonatus (HEMIPTERA: COREIDAE) WITH EMPHASIS ON THE GLYCOSIDE HYDROLASES α-AMYLASE, α-GALACTOSIDASE, AND α-GLUCOSIDASE

A r t i c l e

DIGESTION IN ADULT FEMALESOF THE LEAF-FOOTED BUGLeptoglossus zonatus(HEMIPTERA: COREIDAE) WITHEMPHASIS ON THE GLYCOSIDEHYDROLASES �-AMYLASE,�-GALACTOSIDASE, AND�-GLUCOSIDASEAriane A. Rocha and Carlos J. C. PintoDepartamento de Microbiologia e Parasitologia, Centro de CienciasBiologicas, Universidade Federal de Santa Catarina, Florianopolis, SantaCatarina, Brazil

Richard I. SamuelsDepartment of Entomology and Plant Pathology, State University of NorthFluminense-UENF, Campos dos Goytacazes, Rio de Janeiro, Brazil

Daniel Alexandre and Carlos P. SilvaDepartamento de Bioquımica, Centro de Ciencias Biologicas, UniversidadeFederal de Santa Catarina, Florianopolis, Santa Catarina, Brazil

The leaffooted bug, Leptoglossus zonatus (Hemiptera: Coreidae) is anemerging pest of several crops around the World and up to now very little isknown of its digestive system. In this article, glycoside hydrolase(carbohydrase) activities in the adult midgut cells and in the luminalcontents of L. zonatus adult females were studied. The results showed thedistribution of digestive carbohydrases in adults of this heteropteran speciesin the different intestinal compartments. Determination of the spatialdistribution of α-glucosidase activity in L. zonatus midgut showed onlyone major molecular form, which was not equally distributed between

Correspondence to: Carlos P. Silva, Departamento de Bioquımica, Centro de Ciencias Biologicas, UniversidadeFederal de Santa Catarina, 88040-900, Florianopolis, SC, Brazil. E-mail: [email protected]

ARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY, Vol. 85, No. 3, 152–163 (2014)Published online in Wiley Online Library (wileyonlinelibrary.com).C© 2014 Wiley Periodicals, Inc. DOI: 10.1002/arch.21149

Digestion in Adult Females of the Leaf-Footed bug Leptoglossus zonatus � 153

soluble and membrane-bound isoforms, being more abundant as amembrane-bound enzyme. The majority of digestive carbohydrases werefound in the soluble fractions. Activities against starch, maltose and thesynthetic substrate NPαGlu were found to show the highest levels ofactivity, followed by enzymes active against galactosyl oligosaccharides.Based on ion-exchange chromatography elution profiles and bandingpatterns in mildly denaturing electrophoresis, both midgut α-amylases andα-galactosidases showed at least two isoforms. The data suggested that themajority of carbohydrases involved in initial digestion were present in themidgut lumen, whereas final digestion of starch and of galactosyloligosaccharides takes place partially within the lumen and partially at thecell surface. The complex of carbohydrases here described was qualitativelyappropriate for the digestion of free oligosaccharides and oligomaltodextrinsreleased by α-amylases acting on maize seed starch granules. C© 2014 WileyPeriodicals, Inc.

Keywords: Plant-insect interaction; �-glucosidase; insect digestion; �-amylase;�-galactosidase; digestive enzymes

INTRODUCTION

The leaf-footed bug Leptoglossus zonatus (Hemiptera: Heteroptera: Coreidae) is a highlypolyphagous true bug with a wide host range including economically important cropssuch as cashew nut, citrus, cotton, guava, maize, melon, oranges, passion fruit, pithaya,pomegranate, satsuma mandarin, sorghum, and tomato among others (Grimm andSomarriba, 1999; Schaefer and Panizzi, 2000; Xiao and Fadamiro, 2009, 2010; Tepole-Garcıa et al., 2012). This insect has a wide distribution in the Americas, ranging from theSouth-Western United States throughout Mexico and Central America to Southern Brazil.This species is becoming a serious problem in several crops in Brazil because both adultsand nymphs feed on the ripe and unripe fruits of several plants causing direct damage orby transmitting plant pathogens, for example, the trypanosomatid Herpetomonas macgheeiand the yeast Nematospora coryli (Clarke and Wilde, 1970; Jankevicius et al., 1993; Schaeferand Panizzi, 2000). Like many other Leptoglossus species, L. zonatus has recently risen instatus as a pest of a wide range of crops in the Southern United States. L. zonatus is nowconsidered the major emerging pest of satsuma mandarin in Southern Alabama (Xiaoand Fadamiro, 2010).

Most of the phytophagous heteropterans are intermittent feeders, spending most oftheir time away from the food source. During their feeding activities, they use a styletbundle composed of two inner maxillary and two outer mandibular stylets giving rise totwo canals, one through which saliva is pumped and the other through which the food issucked. Due to the short period of contact of the saliva with the plant food outside theintestinal tract, little digestion occurs before ingestion and normally low levels of digestiveenzymes (mainly carbohydrases, such as polygalacturonases and amylases) are found inthe saliva compared to the midgut (Agusti and Cohen, 2000; Walker and Allen, 2010;Mehrabadi et al., 2012). This situation is in contrast to what is observed in predaceousbugs, where pre-oral digestion is normal and high concentrations of digestive enzymes(specially peptidases) can be found in the saliva (Zeng and Cohen, 2000; Zibaee et al.,2012).

Archives of Insect Biochemistry and Physiology

154 � Archives of Insect Biochemistry and Physiology, March 2014

Figure 1. Photography of the female Leptoglossus zonatus midgut showing the first ventricular segment (V1),second ventricular segment (V2), third ventricular segment (V3), and fourth ventricular segment (V4). Scalebar: 1 cm.

Considering the probable importance of carbohydrate digestion for the success of thispest, the composition, spatial distribution and the role of digestive glycoside hydrolasesin this species deserve attention and clarification, as the digestive physiology of thisinsect could be a target for future control strategies. The main purpose of this studywas to provide information concerning the number and spatial distribution of variousglycoside hydrolases, with emphasis on the enzymes responsible for the initiation ofstarch digestion in L. zonatus. In this paper, the partial purification and localization of themajor carbohydrases from L. zonatus midgut cells and luminal contents were described.The importance of these enzymes in this heteropteran gut physiology was also discussed.

MATERIALS AND METHODS

Rearing of Insects

Adult L. zonatus were obtained from maize plantations in Florianopolis/SC Brazil and weremaintained on a diet of ripe corn grains (Zea mays) that were replaced on a daily regime.The insects were kept in wooden cages 29 cm × 29 cm × 39 cm with plastic gauze wallsunder natural photoperiod conditions at a relative humidity of 60–70% and at 26 ± 1◦C.The insects had free access to water.

Preparation of Samples from Insects

Adult females were cold immobilized by placing them on crushed ice and dissected toremove the whole midgut in cold 250 mM NaCl. Females were chosen because of theirlarger size. Only actively feeding animals with food filling the gut tracts were chosenfor dissection. After the removal of the whole gut, the adhering unwanted tissues wereremoved. The midgut was divided according to its gross morphology (Fig. 1) into threesections: first ventriculus (V1), second ventriculus (V2) and third ventriculus (V3). Dueto its small size, V4 was not used. Midgut walls were pulled apart and the luminal contentscorresponding to each of the midgut sections were collected with the aid of a capillaryand dispersed in a known volume of distilled water. Midgut epithelium sections, afterbeing freed from the luminal contents, were thoroughly rinsed in 215 mM NaCl prior tohomogenization in cold distilled water using a Potter-Elvehjem homogenizer immersedin ice. Midgut tissue homogenates were centrifuged at 20,000 × g for 30 min at 4◦C andthe supernatant (soluble fraction) was collected, and the sediment (membrane fraction)was homogenized in distilled water with the aid of the Potter–Elvehjem homogenizer.



Conditions for Hydrolase Assays and Protein Determination

Activities against cellobiose (20 mM) and maltose (10 mM) were determined by measuringthe release of glucose according to the method of Dahlqvist (1968). The activities againstraffinose (20 mM) and starch (0.5%, w/v) were measured by determining the increasein reducing groups in the media following the method of Noelting and Bernfeld (1948).Activities against: p-nitrophenyl derivative of glucose, p-nitrophenyl α-D-glucopyranoside(NPαGlu) and p-nitrophenyl β-D-glucopyranoside (NPβGlu), galactose p-nitrophenylα-D-galactopyranoside (NPαGal) (10 mM for all subtrates) were measured following theappearance of p-nitrophenolate according to the method of Terra et al. (1979).

All assays for carbohydrases were performed in 50 mM citrate-phosphate buffer, pH5.5, except for α-amylase, where 50 mM sodium-acetate buffer pH 5.5 containing 20 mMNaCl and 0.2 mM CaCl2 was used. All assays were performed at 30◦C. Buffers (50 mM)used in determination of pH optima were: sodium acetate, citrate-sodium phosphate, Tris-HCl, with pH values ranging from 3 to 9, with intervals of 0.2 pH units. Incubations werecarried out for at least four different time periods, unless otherwise stated and initial ratesof hydrolysis were calculated. One unit of enzyme was defined as the amount that catalysesthe cleavage of 1 μmol of substrate/min. Low levels of enzyme activity were expressed inmilliunits (mU). The enzyme activity calculations were expressed per midgut section orper mg of protein.

Protein was determined according to Smith et al. (1985), as modified by Morton andEvans (1992), using bovine serum albumin as a standard.

In Gel Assays

α-Galactosidases from whole midgut preparations were detected and partially charac-terised by SDS-polyacrylamide gel electrophoresis essentially as described by Silva et al.(1999). Samples containing appropriate protein concentrations were diluted two-fold inelectrophoresis sample buffer [2.1 mL distilled water, 0.5 mL 0.5 M Tris-HCl, pH 6.8,0.4 mL glycerol, 0.8 mL 10% (w/v) SDS, 0.2 mL 1% (w/v) bromphenol blue] (note theabsence of 2-mercaptoethanol) and subjected to electrophoresis without boiling the sam-ples (Laemmli, 1970) in a slab mini-gel (10 cm × 7 cm × 1.0 mm) using a BioRad MiniProtean 3 apparatus at 150 V. The electrophoresis runs were carried out at 4◦C using pre-cooled buffers. After electrophoresis, the gels were treated to an extensive renaturationstep in 2.5% (w/v) Triton X-100 solution. The gels were washed three times (15 min pereach step). After renaturation and reduction of tris concentration, gels were transferredto substrate/buffer solution (100 mM citrate-phosphate) containing 2 mM of the fluo-rogenic substrate 4-methylumbelliferyl α-D-galactopyranoside (pH 5.5) and incubated for15 min at 30◦C. After incubation, fluorescent bands were visualized using a UV lamp andgels were photographed.

Ion-exchange Chromatography

Samples containing 25 whole midgut equivalents were homogenized in 2 mL of a solutioncontaining 10 μM E-64 and 5 μg/mL pepstatin A by using a Potter-Elvehjem homogenizer.The homogenate was centrifuged at 20,000g for 30 min at 4◦C. The supernatant wasapplied to a DEAE-Sepharose Fast Flow column (5 × 0.5 cm id) equilibrated with 10 mMimidazole buffer, pH 6.0 using an Akta (GE Health Care) apparatus. The column waswashed with 5 mL of the same buffer and then eluted with 25 mL of a linear gradient



Table 1. Spatial distribution of carbohydrase activities along the different midgut segments of adultLeptoglossus zonatus

Substrates SV1 MV1 SV2 MV2 SV3 MV3

Cellobiose 38 ± 1.3c 9 ± 0.2b 77 ± 5.3d 3 ± 0.5a 9 ± 0.1b 12 ± 0.9bMaltose 105 ± 8.3a 254 ± 12b 135 ± 11a 930 ± 35c 160 ± 10a 240 ± 9bMelibiose 101 ± 9.5b 91 ± 8b 268 ± 27c 48 ± 5a 237 ± 30c 64 ± 7aNPαGal 258 ± 30b 776 ± 55d 840 ± 70d 110 ± 10a 402 ± 35c 120 ± 9.5aNPαGlu 850 ± 75a 2,700 ± 100b 1,620 ± 80a 3,800 ± 90c 1,650 ± 75a 1,220 ± 50aNPβGlu 26 ± 1.5a 27 ± 2a 87 ± 4c 57 ± 3b 20 ± 0.9a 25 ± 3aStarch 2,635 ± 95b 2,945 ± 100b 4,390 ± 350c 2,030 ± 80b 990 ± 60a 2,300 ± 300b

Results (expressed as milli Units/segment) are means calculated from five independent cohorts containing five midgutsegments each. Results followed by the same letter indicate no significant differences when using Student’s t-test(P < 0.05). SV1: soluble fraction from the first ventricular segment; MV1: particulate fraction obtained from the firstventricular segment, and so forth for the other segments: SV2, MV2, SV3, and MV3.

(0–1M NaCl) in imidazole buffer, followed by 5 mL of isocratic elution in this NaClcontaining buffer. The flow rate was 1.0 mL/min and fractions of 0.4 mL were collected.Recoveries of the activities from the column were 70–80%.

Kinetic Studies

The effect of substrate concentrations on the activity of isolated α-amylases was deter-mined in 50 mM acetate, 20 mM NaCl, 0.2 mM CaCl2, pH 5.5 at 30◦C, using 10 differentsubstrate concentrations. KM and VMax values were determined by weighted linear regres-sion (Wilkinson, 1961) using the program Enzfitter R©. Enzyme preparations were poolsfrom the fractions obtained after the DEAE-Sepharose ion-exchange chromatography.Plots of Lineweaver–Burk were verified and the determinations were performed withconcentrations of the substrate in the range of 0.2 to 2 KMs.

RESULTS

Morphology of L. zonatus Midgut

The midgut of L. zonatus is the major part of the alimentary canal. It is divided into threesections or ventricular segments (V1−V3), which are linked through V4 to the hindgut(Fig. 1). As common in heteropteran species, following a short and narrow oesophagus,the first and the most voluminous segment (V1) marks the beginning of the midgut. V1is an expandable compartment where the food bolus is first stored. V2 is a convolutedtubular compartment which is separated from V1 by a narrow region which acts to reducethe flow between V1 and V2. A second narrowing can be observed between V2 and V3.The later is an oval shaped compartment which varies in volume during the passage ofthe food bolus. V4 is a short tubular segment linking V3 to the hidgut.

Spatial Distribution of Digestive Carbohydrases in L. zonatus

The Table 1 shows the spatial distribution of carbohydrase activities in the ventricularsegments of L. zonatus from two different fractions: (1) soluble fractions and (2) partic-ulate fractions containing membrane proteins. The highest midgut glycoside hydrolase



Table 2. Substrate specificity of isolated midgut Leptoglossus zonatus soluble α-amylases

Fraction VMax (mU/mg protein) KM (%, m/v) VMax/KM (mU/mg protein/%) Ratio VMax/KM

A1 49.2 ± 2.18 0.057 ± 0.006 863 ± 70 1.0A2 17.0 ± 0.04 0.018 ± 0.002 945 ± 83 1.09

Isolated α-amylases obtained after the ion-exchange chromatography (see Fig. 5) were incubated with ten differentconcentrations of gelatinized starch in 50 mM sodium-acetate buffer pH 5.5 containing 20 mM NaCl and 0.2 mM CaCl2.Kinetic parameters (mean and SEM) were determined by a weighted linear regression using the software Enzfitter R©.

activities were found in V1 and V2. α-Galactosidase activity was higher in the solublefractions compared with the membrane fractions obtained from V2 and V3, but in thefirst midgut compartment V1, α-galactosidase activity, assayed against the synthetic sub-strate NPαGal, was significantly higher in the membrane fraction. The distribution ofα-galactosidase activity assayed against melibiose was similar to the activity toward thesynthetic substrate NPαGal in the other midgut compartments, except for SV1/MV1.Conversely, α-glucosidase activity assayed against NPαGlu from V1 and V2 membranefractions was higher than the soluble counterpart, but in V3 the difference between solu-ble and membrane-associated activity was not so great. The distribution of α-glucosidaseactivity assayed against maltose parallels the activity toward the synthetic substrate NPαGlu.The activities against synthetic (NPβGlu) and natural (cellobiose) β-glucosidase substrateswere much lower than α-galactosidases and α-glucosidases activities. β-Glucosidases werepredominant in V2, associated to both membranes and soluble fractions. α-Amylase ac-tivity was detected in all midgut compartments, but the highest activities were seen in thesoluble V2 fractions, although a significant percentage of the amylase activity was foundin the particulate fractions of all midgut compartments.

Partial Characterization of L. zonatus Midgut Carbohydrases

Two molecular species of midgut soluble α-amylases were revealed by the ion-exchangechromatography, where it was possible to separate two peaks of activity, peak A1 andpeak A2 (Fig. 2). L. zonatus soluble V2 α-amylase activities peaked around pH 5.6 againstsolubilised starch (data not shown). Ratios of VMax/KM using gelatinised starch as asubstrate demonstrated that both peaks (A1 and A2) hydrolyse starch with the sameefficiency, but peak A2 had a higher affinity for the substrate than peak A1 (Table 2).Determinations of α-amylase activity carried out on homogenates from salivary glandsshowed negligible enzyme activity. Comparisons of activities recovered from seed flourwith mass equivalent to the midgut mass showed that a very low percentage of the luminalα-amylase activity could be derived from the seeds, whereas the other enzymes are onlyproduced by the insects themselves.

There is only one molecular species of α-glucosidase, as demonstrated by the ion-exchange chromatography and assays with NPαGlu and maltose as substrates (Fig. 3).L. zonatus soluble V2 α-glucosidase activity peaks around pH 5.6 against NPαGlu andmaltose (data not shown).

The major midgut soluble α-galactosidases of adult L. zonatus were isolated throughion-exchange chromatography and by semi-denaturant polyacrilamide gel electrophore-sis followed by assays against a fluorogenic substrate (Figs. 4 and 5). Two molecularspecies of soluble α-galactosidases, were demonstrated by the ion-exchange chromatogra-phy (Fig. 4), and electrophoresis in semi-denaturing conditions (Fig. 5). L. zonatus midgut



Figure 2. Ion-exchange chromatography of Leptoglossus zonatus α-amylase activities on a DEAE-Sepharose FastFlow column equilibrated with 10 mM imidazole buffer, pH 6.0 using an Akta (GE Health Care) apparatus.The column was washed with 5 mL of the same buffer and then eluted with 25 mL of a linear gradient (0–1MNaCl) in imidazole buffer, followed by 5 mL of isocratic elution in this NaCl containing buffer. The flow rate was1.0 mL/min and fractions of 0.4 mL were collected. Fraction A1 corresponds to the pool of fractions 24–27 andfraction A2 contains the pool of fractions 32–36.

α-galactosidase activity peaked around pH 5.6 against NPαGal and melibiose (data notshown). These enzymes were also active on the disaccharide melibiose and on the trisac-charide raffinose, but with different specificities (Fig. 4). In Fig. 4, it is possible to seethat there is a largest peak with a shoulder when NPαGal was used as the substrate. Thehighest peak coincides with the activity against raffinose, while the shoulder coincideswith the peak against the substrate melibiose. Curiously, the α-galactosidase active againstmelibiose had a very low activity against raffinose and enzyme active towards raffinose hada very low activity against melibiose.

DISCUSSION

In this study, the spatial distribution and the physiological roles of digestive glycosidehydrolases of the leaf-footed bug L. zonatus were investigated. Similar to other heteropter-ans, the L. zonatus midgut has four distinct regions V1–V4), (Goodchild, 1963; Silva et al.,1994). As already described for other seed-feeding bugs, L. zonatus adults use the flow ofsaliva generated in their stylets to suspend and solubilize seed nutrients that are suckedup into the pharynx and oesophagous and them into the first ventriculus. As the saliva



Figure 3. Ion-exchange chromatography of Leptoglossus zonatus α-glucosidase activities on the DEAE-SepharoseFast Flow column. α-Glucosidase activity assayed with NPαGlu, ( ); α-glucosidase activity assayed withmaltose ( ). More details as in legend of Fig. 2.

Figure 4. Ion-exchange chromatography of Leptoglossus zonatus α-galactosidase activities on a DEAE-SepharoseFast Flow column equilibrated with 10 mM imidazole buffer, pH 6.0 using an Akta (GE Health Care) apparatus.Activity assayed against NPαGal ( ); activity assayed against melibiose ( ); activity assayed towardraffinose ( ). More details can be seen in the Fig. 2 legend.



Figure 5. Mildly denaturing SDS-PAGE demonstrating α-galactosidase activities after in gel assays of Leptoglossuszonatus adult midguts samples. Samples containing 0.75 midgut equivalents were loaded onto 7.5% polyacry-lamide gels. After the run and subsequent renaturation in a 2.5% aqueous solution of Triton X-100, the gelwas incubated in a substrate/buffer solution (100 mM citrate-phosphate containing 2 mM 4-methylumbelliferylα-D-galactopyranoside, pH 5.5) and incubated for 15 min at 30◦C. Arrows indicate the position of two activeα-galactosidases.

is in contact for a relatively short period with the liquefied food outside the insect, thefirst major digestive events take place in V1. As also observed for other seed-feedingheteropteran species, L. zonatus saliva lacks or has very low α-amylase activity (data notshown), similar to other seed-feeding Heteroptera (Silva and Terra, 1994; Woodring et al.,2007). However, in contrast with Dysdercus peruvianus (Silva and Terra, 1994) and Oncopel-tus fasciatus (Woodring et al., 2007), L. zonatus midgut compartments have relatively highα-amylase activity, interestingly with high levels of activity associated with the particulatefractions (Table 1). In insects, α-amylases (α-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1)hydrolyze starch and other polysaccharides to maltose, maltotriose and maltodextrinsinitializing the digestion of the starch granules (Silva et al., 2001; Terra and Ferreira,2005). All carbohydrase activities assayed in this study showed pH optima of 5.6, which isclose to the values of pH found in luminal contents of adult guts of heteropterans such asD. peruvianus (Silva and Terra, 1994), O. fasciatus (Woodring et al., 2007) and Eurygasterintegriceps (Mehrabadi et al., 2009).



Maize starch granule digestion may be initiated by the action of the two α-amylasespresent in the luminal contents of V1 and mainly in V2, where they are more abundant,liberating oligomaltodextrins. In L. zonatus, the two amylase isoforms have similar effi-ciency against gelatinized starch, but isoform A2 has a higher affinity due to its lowerKm (Table 2). The intermediate and final digestion of starch probably takes place at thesurface of the perimicrovillar membranes throughout V1 to V2 and less important in V3,carried out by the major membrane-bound α-glucosidase acting on the liberated oligoma-ltodextrins to produce glucose (Table 1 and Fig. 3). As seen in other heteropteran species,L. zonatus also has only one molecular form of the major α-glucosidase (Silva and Terra,1994, 1995; Silva et al., 2004). The similar distribution along the midgut compartmentsof the enzyme activities against maltose and the synthetic substrate NPαGlu (Table 1) andthe profiles from the ion-exchange chromatographies support this assumption (Fig. 3).

Adult L. zonatus females have at least two digestive α-galactosidases (Figs. 4 and 5)that are distributed throughout the three major midgut compartments (Table 1). Theprobable target for these enzymes may be trisaccaride raffinose, the major member ofthe so-called raffinose family of oligosaccharides, which are a group of soluble galactosyl-sucrose oligosaccharides thought to be involved in desiccation tolerance (in quiescentseeds) and as a reserve of rapidly metabolizable sugar, which produce carbons and energyduring germination (Zhou et al., 2012). To serve as an energy source, raffinose has tobe hydrolyzed into its constituent monosaccharides. Raffinose is a nonreducing trisac-caride, and as such it can be hydrolyzed at the two nonreducing ends by α-galactosidases(at the galactose moiety) or by β-fructosidases (at the fructose moiety). In animals,β-fructosidases have been detected only in Lepidoptera (Insecta) (Santos and Terra, 1986;Carneiro et al., 2004; Daimon et al., 2008). In L. zonatus, raffinose may be hydrolyzed byα-galactosidases liberating galactose and sucrose, which can be further hydrolyzed by themajor membrane-bound α-glucosidase and also by the minor soluble α-glucosidase. Theoccurrence of at least two aryl α-galactosidases in L. zonatus, which are active against thesynthetic substrate NPαGal, but have different specificities toward raffinose and melibiose,(Figs. 4 and 5), suggests that these enzymes are involved in the digestion of substratesother than galactosyl-oligosaccharides. As suggested for the heteropteran D. peruvianus,glycoproteins, and glycolipids may be the most likely natural substrates for these enzymes(Silva and Terra, 1997). As observed in other insect species, in L. zonatus the activity ofα-galactosidases is lower than the activity of α-glucosidases, but higher than the activityof β-glucosidases (Table 1), suggesting that starch and galactosyl oligosaccharides maybe more important as nutrients than cellulose or hemicellulose. The partial digestionof hemicelluloses and glycoproteins by the minor β-glucosidases may also occur in themidgut lumen and at the perimicrovillar membranes, as the β-glucosidases are equallydistributed between the particulate and soluble fractions.

ACKNOWLEDGMENTS

This work was supported by the Brazilian research agencies CNPq, FAPESC, and FAPERJ.R.I. Samuels and C.P. Silva are CNPq research fellows.

LITERATURE CITED

Agusti N, Cohen A. 2000. Lygus hesperus and L. lineolaris (Hemiptera: Miridae): the salivary andmidgut enzymes. J Entomol Sci 35:175–182.



Carneiro CN, Isejima EM, Samuels RI, Silva CP. 2004. Sucrose hydrolases from the midgut of thesugarcane stalkborer Diatraea saccharalis. J Insect Physiol 50:1093–1101.

Clarke RG, Wilde GE. 1970. Association of the green stink bug and the yeast spot disease organismof soybeans. II. Frequency of transmission to soybeans, transmission from insect to insect,isolation from field population. J Econ Entomol 63:355–357.

Dahlqvist A. 1968. Assay of intestinal disaccharidases. Anal Biochem 22:99–107.Daimon T, Taguchi T, Meng Y, Katsuma S, Mita K, Shimada T. 2008. β-Fructofuranosidase genes

of the silkworm, Bombyx mori: insights into enzymatic adaptation of B. mori to toxic alkaloids inmulberry latex. J Biol Chem 283:15271–15279.

Goodchild AJP. 1963. Studies on the functional anatomy of the Heteroptera. Proc Zool Soc (Lond.)141:851–910.

Grimm C, Somarriba A. 1999. Suitability of physic nut (Jatropha curcas L.) as single host plant forthe leaf-footed bug Leptoglossus zonatus Dallas (Het., Coreidae). J Appl Ent 123:347–350.

Jankevicius SI, de Almeida ML, Jankevicius JV, Cavazzana JrM, Attias M, de Souza W. 1993. Axeniccultivation of trypanosomatids found in corn (Zea mays) and in phytophagous hemipterans(Leptoglossus zonatus, Coreidae) and their experimental transmission. J Eukar Microbiol 4:576–581.

Laemmli UK. 1970. Cleavage of structural proteins during the assembly of the bacteriophage T4.Nature 227:680–685.

Mehrabadi M, Bandani AR, Mehrabadi R, Alizadeh H. 2012. Inhibitory activity of proteinaceousα-amylase inhibitors from Triticale seeds against Eurygaster integriceps salivary α-amylases: Inter-action of the inhibitors and the insect digestive enzymes. Pestic Biochem Physiol 102:220–228.

Mehrabadi M, Bandani AR, Saadati F, Ravan S. 2009. Sunn pest, Eurygaster integriceps Putton(Hemiptera: Scutelleridae), digestive α-amylase, α-glucosidase and β-glucosidase. J Asia PacEntomol 12:79–83.

Morton RE, Evans TA. 1992. Modifications of the bicinchoninic acid protein assay to eliminate lipidinterference in determining protein content. Anal Biochem 204:332–334.

Noelting G, Bernfeld P. 1948. Sur les enymes amylolytiques. III. La β-amylase: dosage d’activite etcontole de l’absence d’α-amylase. Helv Chim Acta 31:286–290.

Santos CD, Terra WR. 1986. Midgut α-glucosidase and β-fructosidase from Erinnyis ello larvae andimagoes. Physical and kinetic properties. Insect Biochem 16:819–824.

Schaefer CW, Panizzi AR. 2000. Economic importance of Heteroptera: A general view. In: SchaeferCW, Panizzi AR, editors. Heteroptera of economic importance. Boca Raton: CRC Press, 828 p.

Silva CP, Silva JR, Vasconcelos FF, Petretski DA, DaMatta RA, Ribeiro AF, Terra WR. 2004. Occur-rence of perimicrovillar membranes in paraneopteran insect orders with comments on theirfunction and evolutionary significance. Arthropod Struct Dev 33:139–148.

Silva CP, Terra WR, Xavier-Filho J, de Sa MFG, Lopes AR, Pontes EG. 1999. Digestion in larvaeof Callosobruchus maculatus and Zabrotes subfasciatus (Coleoptera: Bruchidae) with emphasis onα-amylases and oligosaccharidases. Insect Biochem Mol Biol 29:355–366.

Silva CP, Terra WR, Xavier-Filho J, de Sa MFG, Isejima EM, DaMatta RA, Miguens FC, BifanoTD. 2001. Digestion of legume starch granules by larvae of Zabrotes subfasciatus (Coleoptera:Bruchidae) and the induction of α-amylases in response to different diets. Insect Biochem MolBiol 31:41–50.

Silva CP, Terra WR. 1994. Digestive and absorptive sites along the midgut of the cotton seed suckerbug Dysdercus peruvianus (Hemiptera: Pyrrhocoridae). Insect Biochem Mol Biol 24:493–505.

Silva CP, Terra WR. 1995. An α-glucosidase from perimicrovillar membranes of Dysdercus peruvianus(Hemiptera: Pyrrhocoridae) midgut cells. Purification and properties. Insect Biochem MolBiol 25:487–494.

Silva CP, Terra WR. 1997. α-Galactosidase activity in ingested seeds and in the midgut of Dysdercusperuvianus (Hemiptera: Pyrrhocoridae). Arch Insect Biochem Physiol 34:443–460.



Smith PR, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provezano MD, Fujimoto EK, GoekeNM, Olson BJ, Klenk DC. 1985. Measurements of protein using bicinchoninic acid. AnalBiochem 150:76–85.

Tepole-Garcıa RE, Pineda-Guillermo S, Martınez-Herrera J, Castrejon-Gomez VR. 2012. Recordsof Two Pest Species, Leptoglossus zonatus (Heteroptera: Coreidae) and Pachycoris klugii (Het-eroptera: Scutelleridae), Feeding on the Physic Nut, Jatropha curcas, in Mexico. Fla Entomol95:208–210.

Terra WR, Ferreira C. 2005. Biochemistry of digestion. In: Gilbert LI, Iatrou K, and Gill SS, editors.Comprehensive molecular insect science. Elsevier, Oxford. p 171–224.

Terra WR, Ferreira C, de Bianchi AG. 1979. Distribution of digestive enzymes among the endo- andectoperitrophic spaces and midgut cells of Rhynchosciara and its physiological significance. JInsect Physiol 25:487–494.

Walker W, Allen ML. 2010. Expression and RNA interference of salivary polygalacturonase genesin the tarnished plant bug, Lygus lineolaris. J Insect Sci 10:173. doi: 10.1673/031.010.14133.

Wilkinson GN. 1961. Statistical estimations in enzyme kinetics. Biochem J 80:324–332.Woodring J, Hoffmann K, Lorenz MW. 2007. Feeding, nutrient flow, and digestive enzyme release

in the giant milkweed bug, Oncopeltus fasciatus. Physiol Entomol 32:328–335.Xiao YF, Fadamiro HY. 2009. Host preference and development of Leptoglossus zonatus on satsuma

mandarin. J Econ Entomol 102:1908–1914.Xiao YF, Fadamiro HY. 2010. Evaluation of damage to satsuma mandarin (Citrus unshiu) by the

leaffooted bug, Leptoglossus zonatus (Hemiptera: Coreidae). J Appl Entomol 134:694–703.Zeng F, Cohen AC. 2000. Comparison of α-amylase and protease activities of a zoophytophagous

and two phytozoophagous Heteroptera. Comp Biochem Physiol A 126:101–106.Zhou M-L, Zhang Q, Zhou M, Sun Z-M, Zhu X-M, Shao J-R, Tang Y-X, Wu Y-M. 2012. Genome-wide

identification of genes involved in raffinose metabolism in Maize. Glycobiology 22:1775–1785.Zibaee A, Hoda H, Fazeli-Dinan M. 2012. Role of proteases in extra-oral digestion of a predatory

bug, Andrallus spinidens. J Insect Sci 12:51. doi: 10.1673/031.012.5101.


Documents

DIGESTION IN ADULT FEMALES OF THE LEAF-FOOTED BUG Leptoglossus zonatus (HEMIPTERA: COREIDAE) WITH EMPHASIS ON THE GLYCOSIDE HYDROLASES α-AMYLASE, α-GALACTOSIDASE, AND α-GLUCOSIDASE