Proteolytic gut activities in the rice water weevil, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae)

Archives of Insect Biochemistry and Physiology 53:19–29 (2003)

© 2003 Wiley-Liss, Inc.DOI: 10.1002/arch.10083Published online in Wiley InterScience (www.interscience.wiley.com)

Proteolytic Gut Activities in the Rice Water Weevil,Lissorhoptrus brevirostris Suffrian (Coleoptera:Curculionidae)

Carlos A. Hernández,1 Merardo Pujol,1 Julio Alfonso-Rubí,1 Raúl Armas,1 Yamilet Coll,1

Maylin Pérez,1 Annerys González,1 Marisa Ruiz,2 Pedro Castañera,2 and Félix Ortego2*

Digestive endoprotease activities of the rice water weevil, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae), werecharacterized based on the ability of gut extracts to hydrolyze specific synthetic substrates, optimal pH, and hydrolysis sensitiv-ity to protease inhibitors. Larvae of this species were found to use a complex proteolytic system that includes cathepsin D-,cathepsin B-, trypsin-, and chymotrypsin-like activities. Trypsin-like activity was evenly distributed among the anterior, middle,and posterior portions of the gut, whereas cathepsin B– and cathepsin D–like activities were mainly located in the anteriorand middle sections, and the chymotrypsin-like activity was highest in the middle and posterior sections. Gelatin-containingnative-PAGE gels indicated the presence of several aspartyl, cysteine, and serine protease forms and confirmed the spatialorganization of the proteolytic digestive process. Arch. Insect Biochem. Physiol. 53:19–29, 2003. © 2003 Wiley-Liss, Inc.

KEYWORDS: Lissorhoptrus brevirostris; rice water weevil; proteases; protease inhibitors

1Center for Genetic Engineering and Biotechnology, Sancti Spiritus, Cuba2CSIC, CIB, Dpto. de Biología de Plantas, Madrid, Spain

Contract grant sponsor: CSIC/CITMA; Contract grant number: 2001CU0016.

Abbreviations used: BApNa = Na-benzoyl-DL-arginine p-nitroanilide; DTT = dithiothreitol; E-64 = L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane;IAA = iodoacetamide; PMSF (phenylmethylsulfonyl fluoride); SA2PPpNa = N-succinyl-alanine-alanine-proline-phenylalanine p-nitroanilide; SA3pNa = N-succinyl-alanine-alanine-alanine p-nitroanilide; SBBI = Soybean Bowman-Birk inhibitor; TLCK = Na-p-tosyl-L-lysine chloromethyl ketone; TPCK = N-tosyl-L-phenylalanine chloromethyl ketone; ZAA2MNA = N-carbobenzoxy-alanine-arginine-arginine 4-metoxy-b-naphthyl amide.

*Correspondence to: Félix Ortego, CSIC, CIB, Dpto. de Biología de Plantas, Velázquez 144, 28006 Madrid, Spain. E-mail: [email protected]

Received 25 September 2002; Accepted 6 February 2003

INTRODUCTION

Water weevils are the most widely distributedand economically important root feeders on rice(Bowling, 1980). The rice water weevil, Lissorhoptrusbrevirostris (Coleoptera: Curculionidae), is the mostdestructive insect pest of rice in Cuba (Meneses etal., 1996). Both larvae and adults of this speciesfeed on the rice plant, but it is primarily the larvalstage that causes economic losses. Larvae feedwithin and upon rice roots, causing pruning dam-ages that reduce tillering and plant growth, and

delay maturity. Yield may be reduced by 30–60%when the infestation is severe (Meneses et al.,1996). Adults feeding on rice leaves may damageseedlings but generally do not cause economiclosses. A closely related species, L. oryzophilus, isthe main insect pest of rice in North America (Way,1990), and has been recently introduced to Japan,Korea, China, and Taiwan (Kobayashi et al., 1997).Other species of the genus Lissorhoptrus are impor-tant pests of rice in South and Central America(Pantoja et al., 1999), and pose a global threat torice production.

20 Hernández et al.

Archives of Insect Biochemistry and Physiology

Granular carbofuran has been the primarymeans for the control of rice water weevils sincethe early 1970s (Way, 1990). However, concernsover the environmental effects of carbofuran havelead to the adoption of alternative methods ofmanaging these pests. A biological control pro-gramme based on the use of entomopathogenicfungi (Beauveria bassiana and Metarhizium aniso-pliae) targeted at the adults of L. brevirostris has beendeveloped in Cuba, but carbofuran applications arestill necessary when the infestations are severe(Meneses et al., 1996). Recently, the United StatesEnvironmental Protection Agency revoked the reg-istration of carbofuran for rice protection. Althoughthe insecticides l-cyhalothrin (Karate) targeted atthe adults and diflubenzuron (Dimilin) targetedat the eggs are currently registered (Stout et al.,2000), a more diverse management program is de-sirable to avoid the development of resistance andthe potential risks of these insecticides to the en-vironment.

Plant resistance is an environmentally friendlyalternative to chemical control that can be used incombination with other control tactics in an inte-grated pest management approach. However, de-spite extensive screening, only a few rice genotypeswith moderate tolerance to rice water weevils havebeen identified (Heinrichs and Quisenberry, 1999).Genetic engineering aimed at production of insec-ticidal proteins within the rice plant is an alterna-tive for obtaining insect resistant plants. Cry genesencoding several variants of Bacillus thuringiensis(Bt) d-endotoxins have been expressed in the in-dica and japonica rice cultivars (Fujimoto et al.,1993; Nayak et al., 1997), and field testing hasshowed their effectiveness for protection againststem borers and leaffolders (Ye et al., 2001). How-ever, in order to obtain more durable plant resis-tance it will be necessary to combine Bt toxins withother insecticidal genes. Among those explored,hydrolase inhibitors derived from plants are of par-ticular interest because of their putative involve-ment in the natural defence system against insectpests (Reeck et al., 1997). The over-expression ofseveral protease inhibitors of different plant ori-gins in transgenic rice plants has resulted in an in-

crease of resistance against both insects feeding invegetative tissues (Duan et al., 1996; Xu et al.,1996; Lee et al., 1999) and storage pests (Irie etal., 1996; Alfonso-Rubí et al., 2002). However, be-cause of insect diversity with respect to digestiveproteases (Wolfson and Murdock, 1990) and dueto physiological adaptations of some insect spe-cies to the ingestion of hydrolytic enzyme inhibi-tors (Jongsma and Bolter, 1997), it is necessary togain a comprehensive knowledge of the major di-gestive enzymes present in a particular species andtheir interaction with appropriate inhibitors.

Complex proteolytic systems for protein diges-tion, based on the presence of endoproteases ofdifferent mechanistic classes and exopeptidases,appear to be widespread among curculionids(Ortego et al., 1998; Bonadé-Bottino et al., 1999).Apart from the cysteine protease activity identifiedin the larvae of L. oryzophilus (Mochizuki, 1998),the nature of the digestive proteases repertoire inLissorhoptrus species is largely unknown. The aimof this study is to characterize gut protease activi-ties in L. brevirostris larvae as a basis for selectingappropriate inhibitors for this species.

MATERIALS AND METHODS

Insects

Larvae of L. brevirostris were collected from therice roots in Sancti Spiritus (Cuba). The larvae wereplaced in ice and immediately transferred to thelaboratory and stored frozen (–20°C) until needed.

Chemicals and Equipment

All substrates and protease inhibitors were pur-chased from Sigma Chemical Co. (St. Louis, MO).Spectrophotometric measurements were made us-ing a Hitachi U-2000 spectrophotometer.

Gut Extracts

For the characterization of digestive proteases,third and fourth instar larvae were dissected in0.15 M NaCl, and the guts and contents removedand stored frozen (–20°C) until needed. The guts

Proteases From Lissorhoptrus brevirostris 21

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were subsequently homogenized in 0.15 M NaCl(5 guts/500 ml), centrifuged at 10,000g for 5 min,and the supernatants pooled (0.9 mg protein/ml)and kept on ice for enzymatic activity assays.

For the distribution of digestive proteases, theguts were dissected from 50 larvae of third andfourth instars and cut to three sections: anterior,middle, and posterior. The sections were pooledin groups of 16–17 to provide 3 samples of eachsection, sonicated, agitated in 1 ml of 0.15 M NaClfor 2 h at 4°C, and centrifuged at 15,000g for20 min. The supernatants were filtered through a0.2-mm membrane and stored frozen (–20°C) un-til needed. The pH was determined by squeezingfluid from five gut sections on pH indicator stripsNeutralit pH 5–10 from Merck (Darmstadt, Ger-many).

Enzyme Assays

All assays were carried out in triplicate andblanks were used to account for spontaneousbreakdown of substrates. Reaction buffers were:0.1 M citric acid-NaOH (pH 2.0–3.0), 0.1 M cit-rate (pH 3.0–6.0); 0.1 M phosphate (pH 6.0–7.0);0.1 M tris-HCl (pH 7.0–9.0); 0.1 M glycine-NaOH(pH 9.0–11.0); and 0.05 M Na2HPO4-NaOH (pH11.0–12.0). All buffers contained 0.15 M NaCl and5 mM MgCl2, except Na2HPO4-NaOH that onlycontained 0.15 M NaCl, since MgCl2 is not main-tained in highly alkaline solutions.

Unless otherwise stated, all protease activitieswere measured for 24 h at their optimum pH in 1ml reaction mixture that contained 20 ml of gut ex-tract. Non-specific protease activity was assayedwith 0.1% sulfanilamide-azocasein solution; tryp-sin-like activity with 1 mM BApNa (Na-benzoyl-DL-arginine p-nitroanilide); chymotrypsin-likeactivity with 0.25 mM SA2PPpNa (N-succinyl-ala-nine-alanine-proline-phenylalanine p-nitroanilide);and elastase-like activity with 0.5 mM SA3pNa (N-succinyl-alanine-alanine-alanine p-nitroanilide), asdescribed by Ortego et al. (1996). Cathepsin D-like activity was measured with 0.2% hemoglobinsolution during a 4-h incubation; and cathepsinB-like activity with 50 mM ZAA2MNA (N-carboben-

zoxy-alanine-arginine-arginine 4-methoxy-b-naph-thyl amide) during an incubation of 18 h, as de-scribed by Novillo et al. (1997a). Total protein inthe gut extracts was determined according to themethod of Bradford (1976) using bovine serumalbumin as the standard.

Effects of Protease Inhibitors and Activators In Vitro

The proteolytic activities of gut extracts were as-sayed in the presence of the following protease in-hibitors: the serine protease inhibitor, SBBI (Soy-bean Bowman-Birk inhibitor); the trypsin inhibitor,TLCK (Na-p-tosyl-L-lysine chloromethyl ketone);the chymotrypsin inhibitor, TPCK (N-tosyl-L-phenylalanine chloromethyl ketone); the cysteineprotease inhibitors, E-64 (L-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane) and IAA (iodo-acetamide); and the aspartic protease inhibitor,pepstatin-A. The cysteine protease activators L-cys-teine and DTT (dithiothreitol) were also tested.

Gut extracts were preincubated with the pro-tease inhibitors and activators for 15 min at 30°C,prior to addition of substrate. All compounds wereadded in 100 ml 0.15 M NaCl, except TPCK andpepstatin-A, which were added in 20 ml DMSO. Thedoses were selected in respect to the effective con-centrations recommended by Beynon and Salvesen(1989).

Zymograms

Electrophoretic detection of proteolytic formswas performed by 0.1% (w/v) gelatin-containing0.1% (w/v) 12% (w/v) polyacrylamide gel electro-phoresis under native conditions. Samples of thedifferent gut sections (containing approximately10 mg of protein) were preincubated for 15 min at30°C with 50 mM of the cysteine protease inhibi-tor E-64, 5 mM of the serine protease inhibitorPMSF (phenylmethylsulfonyl fluoride), or 50 mMof the aspartyl protease inhibitor pepstatin A. Con-trols were prepared by replacing the inhibitor so-lutions by their corresponding solvents. Afterelectrophoresis at 4°C, gels were placed in one ofthe following activation buffers for 24 h at 35°C:



0.1 M citric acid-NaOH, pH 3.0; 0.1 M citrate,5 mM L-cysteine, pH 5.0; and 0.1 M glycine-NaOH,pH 10.0. Proteolysis was stopped by transferringthe gels into a staining solution (0.3% (w/v)Coomassie Blue R-250 in 40% (v/v) methanol and10% (v/v) acetic acid). The gels were destained in25% (v/v) methanol and 10% (v/v) acetic acid.Bands of proteolytic activity were visualised againstthe blue background of the gel.

RESULTS

The pH dependence of proteolytic activities ofthe gut extracts from L. brevirostris larvae is pre-sented in Figure 1. The general proteinaceous sub-strate azocasein was hydrolyzed over a broad range

of pH, with three peaks of optimum activity at pH5.0, 7.5, and 9.5. Hydrolysis of hemoglobin wasmaximal at pH 2.5, and of ZAA2MNA at pHs 4.5and 6.0. Maximal hydrolysis of SA2PPpNa andBApNa occurred at pH 8.0 and 11.0, respectively.No hydrolysis of SA3pNa was detected after 24 hincubation with the gut extracts. Table 1 summa-rizes the activities of gut proteases against the testedsubstrates at the pH optima.

The proteolytic activity of larval gut extractsfrom L. brevirostris was further characterized by re-action with specific protease inhibitors (Table 2).The azocaseinolytic activity was inhibited by E-64,IAA and pepstatin-A at pH 5.0; by TLCK, TPCK, E-64, IAA, and DTT at pH 7.5; and by SBBI, TLCK,DTT, and L-cysteine at pH 9.5. The hydrolysis of

Fig. 1. The effect of pH on therate of hydrolysis of (A) theprotein substrates hemoglobinand azocasein and (B) the syn-thetic substrates ZAA2MNA,SA2PPpNa, and BApNa, by gutextracts from L. brevirostris lar-vae. Data are mean of triplicatemeasurements from a uniquepool of gut extracts, with stan-dard errors within 5% of themeans. Reaction buffers were0.1 M citric acid-NaOH (solidtriangles), 0.1 M citrate (opencircles), 0.1 M phosphate (solidsquares), 0.1 M tris-HCl (opentriangles), 0.1 M glycine-NaOH(solid circles), and 0.1 M Na2

HPO4-NaOH (open squares).


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hemoglobin was inhibited by pepstatin-A. ZAA2

MNA hydrolysis was inhibited by E-64 and IAA,and activated by DTT and L-cysteine at both the4.5 and the 6.0 pH optima. The hydrolysis ofBApNa was inhibited by SBBI, TLCK, and TPCK;whereas SA2PPpNa was inhibited by SBBI, TLCK,and TPCK, E-64, and IAA.

The pH of the lumen of the anterior, middle,and posterior portions of the gut were 5.0, 5.5,and 7.0, respectively. The amounts of total pro-tein were slightly higher in the middle and poste-rior gut sections, and the proteolytic activities werenot evenly distributed (Table 3). Thus, whereas thelevels of cathepsin-D- and catepsin-B-like activitiesin the anterior and middle sections of the gut were2–3 times and more than 10 times higher, respec-

tively, than in the posterior section, the levels ofchymotrypsin-like activity were almost double inthe middle and posterior sections than in the an-terior portion. The level of trypsin-like activity inthe three gut sections was alike.

Zymograms were used to elucidate the gela-tinolytic activity of the gut sections at different pH.No activity was detected when gelating-containingSDS-PAGE gels were performed. Thus, gelating-containing Native-PAGE gels were used instead,although some background degradation was ob-tained, particularly at alkaline pH, probably dueto the fact that some proteases displayed gela-tinolytic activity at Native-PAGE electrophoreticalconditions. At least four forms (a1–a4) were re-solved at pH 3.0 (Fig. 2A). The four protease formswere present in the anterior and middle sections,whereas in the posterior section, only a3 was

TABLE 1. Properties of Gut Proteases From Larvae of L. brevirostrisAgainst Protein and Synthetic Substrates

Substrate Optimum pH Specific activitya

Azocasein 5.0 1.4 ± 0.17.5 1.9 ± 0.29.5 2.5 ± 0.1

Hemoglobin 2.5 22.5 ± 0.9ZAA2MNA 4.5 3.4 ± 0.1

6.0 3.1 ± 0.2BApNa 11.0 6.8 ± 0.2SA2PPpNa 8.0 1.1 ± 0.1SA3pNa — No activity

aSpecific activities as nmol of substrate hydrolyzed/min/mg protein except for pro-teolytic activity against azocasein as mU D Abs 420 nm/min/mg protein and againsthemoglobin as mU D Abs 280 nm/min/mg protein. Figures are the mean ± stan-dard error of triplicate measurements from a unique pool of gut extracts.

TABLE 2. Effect of Protease Inhibitors and Activators on the Hydrolysis of Protein and Synthetic Substrates by Gut Extracts From L. brevirostris Larvaeat Their Optimum pH of Activity

% Relative activitya

Azocasein Hemoglobin ZAA2MNAbBApNa SA2PPpNa

pH 5.0 pH 7.5 pH 9.5 pH 2.5 pH 4.5 pH 6.0 pH 10.5 pH 8.0

Inhibitor (concentration)SBBI (10 mM) ne ne 31 ± 2 ne ne ne 54 ± 1 8 ± 1TLCK (1 mM) ne 48 ± 1 35 ± 3 ne c c 73 ± 2 61 ± 1TPCK (1 mM) ne 54 ± 3 ne ne c c 53 ± 2 59 ± 2E-64 (10 mM) 50 ± 5 74 ± 4 ne ne 57 ± 2 62 ± 2 ne 29 ± 1IAA (1 mM) 33 ± 2 69 ± 2 ne ne 49 ± 1 36 ± 2 ne 70 ± 2Pepstatin-A (10 mM) 43 ± 4 ne ne 57 ± 1 ne ne ne ne

ActivatorDTT (1 mM) ne 45 ± 4 25 ± 4 ne 270 ± 5 164 ± 6 ne neL-Cysteine (1 mM) ne ne 72 ± 2 ne 250 ± 7 182 ± 4 ne ne

aValues are mean ± SE of triplicate measurements from a unique pool of gut extracts treated with an inhibitor or activator vs. their corresponding controls without them.No effect (ne) was considered for activities between 80 and 120%.bThe reaction buffer contained 1 mM L-Cysteine, except when L-Cysteine and DTT were used as activators.cIt chemically interferes with the assay.

TABLE 3. Distribution of Total Protein and Proteolytic Activities Alongthe Gut Sections of L. brevirostris*

Anterior Middle Posterior

Total protein 15.3 ± 0.1a 18.4 ± 0.1b 20.6 ± 0.1c

Trypsin (BApNa) 17.9 ± 0.1a 18.0 ± 0.1a 17.7 ± 0.1a

Chymotrypsin (SA2PPpNa) 2.9 ± 0.1a 4.7 ± 0.4b 4.8 ± 0.2b

CTD (Hemoglobin) 0.14 ± 0.01a 0.16 ± 0.01a 0.06 ± 0.01b

CTB (ZAA2MNA) 29.1 ± 1.8a 30.4 ± 0.4a 2.5 ± 0.1b

*Total protein as mg protein/gut section, and proteolytic activities as pmol of sub-strate hydrolyzed/min/gut section, except for proteolytic activity against hemoglo-bin as mU D Abs 280 nm/min/gut section. Figures are the mean ± standard error(n = 3 independent gut extract pools). Row means followed by the same letter arenot significantly different from each other (Student-Newman-Keuls test, P £ 0.05).



Fig. 2. Gelatin-containing Native-PAGE gels of extractsfrom the anterior, middle and posterior portions ofLissorhoptrus brevirostris larval gut treated with buffer alone(C), PMSF, E-64, or pepstatin-A (Pst) and incubated for

24 h with (A) 0.1 M citric acid-NaOH, pH 3.0; (B) O.1Mcitrate, 5 mM L-cysteine, pH 5.0; and (C) 0.1 M glycine-NaOH, pH 10.0. Protease forms are named from lowestto highest mobility.


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present. None of these protease forms was inhib-ited by PMSF, E-64, or pepstatin-A. At pH 5.0, threeprotease forms (b1–b3) were resolved in the ante-rior and middle sections (Fig. 2B). Incubation ofthe samples with E-64 inhibited the three proteaseforms, whereas incubation with PMSF or pepstatin-A has no effect. Three bands (c1–c3) were presentin the anterior, middle, and posterior sections ofthe gut at pH 10.0, which were inhibited by PMSF(Fig. 2C).

DISCUSSION

We have found that gut extracts of L. brevirostrislarvae have azocaseinolytic activity within a broadrange of pH values, from acid to alkaline. Specificinhibitors of serine proteases were the most effec-tive inhibitors of azocasein hydrolysis at alkalinepH, whereas the azocaseinolytic activity was inhib-ited by cysteine and aspartyl protease inhibitors atacid pH, suggesting that this species has a diges-tive system based on proteases of different mecha-nistic classes. A wide pH range of proteolyticactivity against proteinaceous substrates has alsobeen reported for other curculionid species, suchas the boll weevil, Anthonomus grandis (Wolfsonand Murdock, 1990), the black vine weevil, Otio-rhynchus sulcatus (Michaud et al., 1995), the riceweevil, Sitophilus oryzae (Baker, 1982), the maizeweevil, S. zeamais (Baker, 1982), the cabbage seedweevil, Ceutorhynchus assimilis (Girard et al., 1998),a sugar beet weevil, Aubeonymus mariaefranciscae(Ortego et al., 1998), and the weevil Baris coerule-scens (Bonadé-Bottino et al., 1999).

Larvae and adults of most curculionid speciesexamined rely on complex proteolytic systems forprotein digestion. Serine-, cysteine-, and aspartyl-proteases have been identified in the guts of S.oryzae and S. zeamais (Baker, 1982; Matsumoto etal., 1997; Alfonso-Rubí et al., 2002). Both larvaeand adults of A. mariaefranciscae possess a pro-teolytic system based on at least trypsin-, chymot-rypsin-, elastase-, and cathepsin D-like proteases,and an azocaseinolytic activity at pH 5.0–7.0whose mechanistic class could not be established(Ortego et al., 1998). Protein digestion is provided

by both serine and cysteine proteases in the wee-vils B. coerulescens (Bonadé-Bottino et al., 1999)and C. assimilis (Girard et al., 1998). It has beenreported that the alfalfa weevil, Hypera postica(Elden, 1995; Wilhite et al., 2000), O. sulcatus(Michaud et al., 1995), and A. grandis (Murdocket al., 1987) have slightly acidic midguts and cys-teine proteases provide the major midgut endo-proteolytic activity. Nevertheless, aspartic and/orserine proteases have been identified in some ofthese species (Purcell et al., 1992; Wilhite et al.,2000). There are also some curculionids with serineprotease activity and alkaline pH optima, such asthe red palm weevil, Rhynchophorus ferrugineus(Alarcon et al., 2002), and the citrus weevil,Diaprepes abbreviatus (Yan et al., 1999).

The ability to hydrolyze specific synthetic sub-strates, the elucidation of the pH at which maxi-mal hydrolysis occurs, and their sensitivity toprotease inhibitors confirmed the presence ofcathepsin D-, cathepsin B-, chymotrypsin-, andtrypsin-like activities in gut extracts of L. brevirostrislarvae. Cysteine protease activity has been previ-ously identified in larvae of the related species L.oryzophilus (Mochizuki, 1998), but the methodol-ogy used for the preparation of digestive extracts(buffer containing the serine protease inhibitorPMSF) and the determination of proteolytic activ-ity (incubation at pH 8.0) were not favorable forthe detection of serine and aspartyl protease ac-tivities, respectively. We have found that the hy-drolysis of ZAA2MNA presented two peaks ofmaximum activity at pH 4.5 and 6.0, suggestingthe presence of at least two cysteine proteases. Gela-tin-containing Native-PAGE gels confirmed thepresence of at least three protease forms at pH 5.0that were inhibited by the cysteine protease inhibi-tor E-64. The zymograms also resolved three pro-teolytic forms at pH 10.0, which were inhibited bythe serine protease inhibitor PMSF, and fourgelatinolytic bands at pH 3.0 that, however, werenot inhibited by pepstatin-A. Multiple cysteine pro-teases have been found in the midguts of O. sulcatus(Michaud et al., 1995), and C. assimilis (Girard etal., 1998). In addition, four genes encoding cathe-psin L-like cysteine proteases have been isolated



from S. zeamays (Matsumoto et al., 1997). Unex-pectedly, we have found that the hydrolysis of thechymotrypsin substrate SA2PPpNa was inhibitedby the cysteine protease inhibitors E-64 and IAA.It is unlikely that the cysteine proteases character-ized in the gut extract of L. brevirostris larvae maybe involved in the hydrolysis of SA2PPpNa due tothe specificity of this substrate, and the fact thatSA2PPpNa hydrolysis was not enhanced by cysteineprotease activators (L-cysteine and DTT). In addi-tion, the hydrolysis of SA2PppNa occurred mostlyin the middle and posterior parts of the gut,whereas cathepsin B was located in the anterior andmiddle sections. Novillo et al. (1997b) reported thatE-64 inhibited the hydrolysis of commonly usedtrypsin substrates by digestive trypsin-like proteasesfrom several lepidopteran larvae, despite theinhibitor’s assumed complete specificity for cys-teine proteases. However, there are no previous re-ports of chymotrypsin inhibition by E-64.

Spatial organization of the proteolytic digestiveprocess has been described for several coleopteranspecies (Terra et al., 1996). We have found that inL. brevirostris larvae, cathepsin D- and cathepsin-B-like activities were mainly located in the anteriorand middle sections of the gut, whereas chymot-rypsin-like activity was higher in the middle andposterior sections. Taking into account that the pHin the anterior and middle sections of the gut wasslightly acid, in agreement with the optimum pHfor cathepsin D- and cathepsin-B-like activities, andthe posterior section has a pH of 7.0, close to op-timum for chymotrypsin-like activity, the regionaldifferentiation of these activities may be necessaryfor the proper digestion of dietary proteins. On theother hand, trypsin-like activity, with optima pHof activity more alkaline than the lumen, wasevenly distributed among the anterior, middle, andposterior portions of the gut. Gelatin-containinggels confirmed the spatial organization of the pro-teolytic digestive process. Our results suggest thatprotein digestion may be initiated in L. brevirostrislarvae by cathepsin D and cathepsin-B-like pro-teases, while serine protease activities would be in-volved in subsequent steps of the hydrolyticprocess. In the case of Tenebrio molitor larvae, whose

midgut also contains both serine and cysteine pro-teases, the differences in pH requirements formaximal activity of its proteases appear to be ac-commodated by the presence of cysteine proteasesin the acidic anterior midgut, while the serine pro-teases are mostly located in the alkaline posteriorregion (Thie and Houseman, 1990). This distribu-tion contrasts with that of Leptinotarsa decemlineatalarvae, in which all endo- and exoproteolytic ac-tivities studied are evenly distributed among themidgut sections, indicating that there is no clearregional differentiation in the digestion of proteinsalong the larval midgut (Novillo et al., 1997a).

Despite a digestive proteolytic system based onproteases of different mechanistic classes, cur-culionids are among the insects that have showedhigher susceptibility to protease inhibitors. It hasbeen reported that ingestion of cysteine proteaseinhibitors in natural or artificial diets increasedmortality and delayed developmental time in lar-vae of H. postica (Elden, 1995) and S. oryzae(Pittendrigh et al., 1997). Likewise, larvae of theweevil A. mariaefranciscae fed on diets containingthe serine protease inhibitors SBBI, soybean Kunitztrypsin inhibitor (STI), turkey egg white trypsin in-hibitor, or lima bean trypsin inhibitor endure lowersurvival rates and display significant delays in thedevelopmental time to pupation and to adult emer-gence (Ortego et al., 1998). Interestingly, the mostsignificant levels of mortality (about 90%) occurredwith larvae fed on diets containing a combinationof two or three inhibitors, suggesting a synergistictoxicity (Ortego et al., 1998).

Transgenic plants can be used as a valuable toolfor the evaluation of insecticide proteins againstL. brevirostris, due to the complexity of rearing thisspecies in the laboratory. Transgenic cultivars of ricehave already been proved useful against severalcurculionid pests. Thus, transgenic expression ofthe trypsin inhibitor BTI-CMe from barley in theindica and japonica rice cultivars confers resistanceto S. oryzae (Alfonso-Rubí et al., 2003). Likewise,Irie et al. (1996) reported that a corn cystatin en-coding gene expressed in rice seeds was able to in-hibit S. zeamais gut proteases. In addition, Grahamet al. (1997) observed that the expression of cow-


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pea trypsin inhibitor in strawberry is effective inprotecting roots from O. sulcatus larval feeding.Nevertheless, Bonadé-Bottino et al. (1999) showedthat larvae of B. coerulescens were insensitive to oil-seed rape transgenic plants expressing oryzocystatinI, because the inhibition of cysteine protease ac-tivity was compensated by insensitive serine pro-teases. Likewise, Girard et al. (1998) showed anincreased growth rate or no effect in two strains ofC. assimilis fed on transgenic oilseed rape seeds ex-pressing oryzacystatin I, despite inhibition of di-gestive proteases in vivo. Recently, it has beenreported that the expression of multiple insecticidalgenes confers broad resistance against a range ofdifferent rice pests (Maqbool et al., 2001). Thistechnology allows the possibility to obtain trans-genic rice plants that, expressing a suite of pro-tease inhibitors that match the digestive enzymesof L. brevirostris, would enhance their resistance tothis pest.

ACKNOWLEDGMENTS

We are grateful to Estación de Investigación deArroz del Sur del Jíbaro (Sancti Spiritus, Cuba),particularly Dr. Rafael Meneses, for technical assis-tance in collecting rice water weevils.

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Proteolytic gut activities in the rice water weevil, Lissorhoptrus brevirostris Suffrian (Coleoptera: Curculionidae)