Midgut adaptation and digestive enzyme distribution in a phloem feeding insect, the pea aphid Acyrthosiphon pisum

Journal of Insect Physiology 49 (2003) 11–24 www.elsevier.com/locate/jinsphys

Midgut adaptation and digestive enzyme distribution in a phloem feeding insect, the pea aphid Acyrthosiphon pisum Plinio T. Cristofoletti a, Alberto F. Ribeiro b, Celine Deraison c, Yvan Rahbe´, Walter R. Terra a,∗ a

Departamento de Bioquı´mica, Instituto de Quı´mica, Universidade de Sa˜o Paulo, C.P. 26077, 05513-970 Sa˜o Paulo, Brazil Departamento de Biologia, Instituto de Biocieˆncias, Universidade de Sa˜o Paulo, C.P. 11461, 05422-970 Sa˜o Paulo, Brazil INRA-INSA de Lyon, UMR Biologie Fonctionnelle Insectes et Interactions, Bat. Louis-Pasteur, 20 ave. A. Einstein, 69621 Villeurbanne, France b

c

Received 5 June 2002; accepted 23 September 2002

Abstract Transmission electron micrographs of the pea aphid midgut revealed that its anterior region has cells with an apical complex network of lamellae (apical lamellae) instead of the usual regularly-arranged microvilli. These apical lamellae are linked to one another by trabeculae. Modified perimicrovillar membranes (MPM) are associated with the lamellae and project into the lumen. Trabeculae and MPM become less conspicuous along the midgut. The most active A. pisum digestive enzymes are membranebound. An aminopeptidase (APN) is described elsewhere. An α-glucosidase (α-Glu) has a molecular mass of 72 kDa, pH optimum 6.0 and catalyzes in vitro transglycosylations in the presence of an excess of the substrate sucrose. There is a major cysteine proteinase activity (CP) on protein substrates that has a molecular mass of 40 kDa, pH optimum 5.5, is inhibited by E-64 and chymostatin and is activated by EDTA+cysteine. The enzyme is more active against carbobenzoxy-Phe-Arg-4-methylcoumarin-7amide (ZFRMCA) than against ZRRMCA. These features identify the purified CP as a cathepsin-L-like cysteine proteinase. Most CP is found in the anterior midgut, whereas α-Glu and APN predominate in the posterior midgut. With the aid of antibodies, αGlu and CP were immunolocalized in cell vesicles and MPM, whereas APN was localized in vesicles, apical lamellae and MPM. The data suggest that the anterior midgut is structurally reinforced to resist osmotic pressures and that the transglycosylating αGlu, together with CP and APN are bound to MPM, thus being both distributed over a large surface and prevented from excretion with honeydew. α-Glu frees glucose from sucrose without increasing the osmolarity, and CP and APN may process toxins or other proteins occasionally present in phloem.  2003 Elsevier Science Ltd. All rights reserved. Keywords: Cysteine proteinase; α-Glucosidase; Perimicrovillar membranes; Transglycosylation; Aminopeptidase; Midgut ultrastructure; Midgut function

1. Introduction Corresponding author. Fax: +55-11-3091-2186. Abbreviations: DTT, dithiotreitol; EDTA, ethylenediaminetetracetic acid; E-64, transepoxysuccinyl-l-leucyl-amido (4-guanidino butane); HEPES, 4-(2hydroxyethyl)-1-piperazineethanesulfonic acid; LpNA, l-leucine-pnitroanilide; NPGlu, p-nitrophenyl-d-glucoside; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; SucAAFMCA, succinyl-alanyl-alanyl-phenylalanine-7-amido-4-methyl coumarin; ZFRMCA, carbobenzoxy-phenylalanine-arginine-7-amido-4-methyl coumarin, ZRRMCA, carbobenzoxy-arginine-arginine-7-amido-4methyl coumarin E-mail address: [email protected] (W.R. Terra). ∗

Aphids are successful insects that feed on plant phloem sap, which is composed of large amounts of sucrose (0.15–0.73 M), some amino acids (15–65 mM), minerals and usually negligible quantities of peptides and proteins. Although some phloem sap may contain large amounts of proteins (Ziegler, 1975), aphids are traditionally viewed as insects with no luminal digestion of polymers, having as an important digestive event only the hydrolysis of sucrose, and putatively absorbing phloem amino acids with the aid of membranes associated with the apical midgut cell membranes (Terra, 1990).

0022-1910/03/$ - see front matter  2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0022-1910(02)00222-6

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The traditional picture of aphid midgut function is becoming challenged. It is now clear that other phloemfeeding hemipterans (whiteflies) are able to use dietary proteins, although no proteinase activity was found in their guts (Salvucci et al., 1998). Furthermore, the aphid Acyrtosiphon pisum has a strong midgut aminopeptidase, but like whiteflies it seems to lack proteinase activity (Rahbe´ et al., 1995). In addition to protein and polypeptide digestion, other aspects of aphid midgut physiology deserve attention. Thus, it is not clear how the aphid midgut withstands the osmotic pressures caused by the phloem sap ingested, in spite of the fact that these pressures decrease along the midgut, due to fructose absorption and transglycosylation reactions catalysed by a not-yet characterized αglucosidase (Ashford et al., 2000). In this paper, a morphological, immunocytochemical and enzymological study of the A. pisum midgut is described. The results show for the first time that the cells in the anterior midgut have trabeculae between their apical lamellae reinforcing the tissue. Furthermore, the membranes associated with the apical lamellar membranes (MPM, Modified Perimicrovillar Membranes, see Section 3.3) are shown to originate from multimembrane vesicles. Finally, a transglycosylating α-glucosidase and a previously unknown cathepsin-L-like cysteine proteinase have been purified and found to be associated with MPM, thus avoiding being excreted with honeydew.

2. Materials and methods 2.1. Animals and preparation of midgut samples Aphids (Acyrthosiphon pisum, Hemiptera: Aphididae) were maintained in the laboratory on broad bean seedlings (Vicia faba) in ventilated plexiglass cages (21 °C, 70% r.h., L16:D8). For the experiments, a limited number of mass-reared adults were allowed to lay for 24 h on young Vicia plants, and the resulting apterous insects were used as adults 9 days old. The insects were immobilized on a flat surface, using adhesive tape, and their guts were removed under a stereomicroscope in Yeager’s physiological solution (Dreux, 1963). The midguts were separated and homogenized in double distilled water with the aid of a Potter-Elvehjem homogenizer. After being filtered through a nylon net of 100 µm pore size, the homogenates were designated crude homogenate and stored. Crude homogenates were used to assay enzymes or were centrifuged at 100,000g for 1 h at 4 °C, resulting in a supernatant (designated midgut soluble fraction) and a pellet (midgut cell membranes). Washed midgut cell membranes were prepared by dispersing the midgut cell membranes in water, followed by three freezing and thawing cycles and recentrifugation at 100,000g for 1 h at 4 °C.

2.2. pH of midgut contents Rinsed midguts were transferred to a glass slide and divided into V1, V2, V3, and V4+R (see Fig. 5). The contents of each section were dispersed in 5 µl of dissecting medium and then added to 5 µl of a five-fold dilution of universal pH indicator (E. Merck, Darmstadt, pH 4– 10). The resulting coloured solutions were compared with suitable standards. Other procedure consisted in dipping the dissected digestive tract in a drop of pH indicator and observing the midgut luminal colours after penetration of the chromogene. 2.3. Protein determination and enzyme assays Protein was determined according to Bradford (1976) using ovalbumin as a standard. When samples contained detergent, protein was determined according to Smith et al. (1985) as modified by Morton and Evans (1992), using bovine serum albumin as a standard. General proteolytic activity was determined using a [125I]globulin substrate in 100 mM citrate–sodium phosphate buffer (pH 4–7) or 100 mM sodium phosphate buffer (pH 6.5-9), as adapted from Sarath et al. (1989). Labeled globulin was obtained by coupling 125Iodide to commercial purified γ-globulin using the standard iodobead protocol (Pierce, USA). For assays, one gut equivalent was mixed with 5 µg (5×105 dpm) of labeled globulin, plus additions in buffer for a final volume of 300 µl. Inhibitors/activators were used in the following final concentrations (E64, 10 µM; benzamidine, 2 mM; EDTA, 5 mM; pepstatin A, 1 µM; chymostatin, 50 µM; elastatinal, 2 µg/ml; DTT/EDTA, 1.5/3 mM) and were pre-incubated with gut extract at room temperature for 15 min before adding substrate. The reaction mixture was incubated at 37 °C for 24 h. 500 µl of 20% trichloroacetic acid and 250 µl 2% casein were added to stop the reaction; centrifuging samples at 10,000g removed precipitated proteins, and radioactivity recovered in the supernatant was measured. Aminopeptidase was assayed in 100 mM Tris–HCl buffer (pH 7.0) using 1 mM LpNA according to Erlanger et al. (1961). Cysteine proteinase was routinely assayed with 10 µM ZFRMCA in 100 mM citrate–sodium phosphate buffer pH 6.0 containing 3 mM EDTA and 3 mM cysteine. Other substrates used were 1 µM ⑀-amino-caproyl-Leucyl-(S-benzyl) Cysteinyl-MCA (Alves et al., 1996) and 10 µM ZRRMCA. With all these substrates, cysteine proteinase activity was measured by methylcoumarin fluorescence (excitation 380 nm and emission 460 nm). α-Glucosidase activity was measured determining the appearance of p-nitrophenolate (Terra et al., 1979) from 5 mM NPαGlu in 100 mM sodium phosphate pH 6.5. Trehalase was assayed according to Dahlqvist (1968) using 10 mM trehalose in 100 mM sodium phosphate buffer pH 6.5.

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Incubations were carried out at 30 °C for at least four different time periods, and initial rates of hydrolysis were calculated. All assays were performed under conditions so that product was proportional to enzyme concentration and to incubation time. Controls without enzyme and others without substrate were included. One unit of enzyme (U) is defined as the amount that hydrolyses 1 µmol of substrate (or bond) per minute. Enzyme activities were expressed in milli units (mU). Kinetic parameters of the α-glucosidase, which shows inhibition by excess substrate, were determined by fitting the data to the equation below (Segel, 1975), using SigmaPlot (Jandel Scientific, USA.). Michaelis equation for reactions where substrate S is also a competitive inhibitor with a dissociation constant KiS is: v⫽

Vmax[S] Km ⫹ [S] ⫹

[S]2 KiS

2.4. Gradient ultracentrifugation of midgut cell membranes Midgut cell membranes (Section 2.1) were isolated and their densities were estimated by sucrose gradient ultracentrifugation. For this, samples of 100 µl of a suspension of midgut cell membranes (corresponding to 100 animals/ml) were layered on the top of sucrose gradients (40–70%, w/v) prepared in 50 mM sodium phosphate buffer pH 6.5. Other gradients used were 10–40%, 10– 60%, 10–70%, and 30–60%. The gradients were centrifuged (4 °C) at 96,000g for 25.5 h and fractions (0.25 ml) were collected from the bottom of the tube with the aid of a peristaltic pump. Marker enzyme activity and the refractive index were determined in each fraction. A plot of refractive index versus sucrose densities was used to estimate densities of isolated membrane fractions. 2.5. Purification of the membrane-bound aglucosidase Washed midgut cell membranes (Section 2.1) were maintained in the presence of 10 mM Triton X-100 in 20 mM HEPES buffer pH 7.4 for 2 h at 4 °C, before being centrifuged at 100,000g for 1 h at 4 °C. The pellet was stored for cysteine proteinase studies (see Section 2.7), whereas a supernatant sample (0.2 mg protein from 100 animals) was applied onto a Mono Q column (FPLC System, Pharmacia-LKB Biotechnology, Sweden) equilibrated in 20 mM HEPES buffer pH 7.5 and eluted with a gradient of 0–600 mM NaCl in the same buffer. The flow was 1 ml/min and fractions of 0.4 ml were collected. Fractions 43–47 were pooled and designated partially purified α-glucosidase. Complete purification was achieved by loading the latter fractions onto a Superose 12 column (FPLC System) equilibrated and eluted with

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20 mM HEPES buffer pH 7.5 containing 0.1% Triton X-100 and 200 mM NaCl. 2.6. Electrophoretic resolution of membrane-bound cysteine proteinases The pellet remaining after Triton X-100 treatment of washed midgut cell membranes (Section 2.5) was used as a source of membrane-bound cysteine proteinases. The pellet was suspended in 2.5% SDS in 400 mM sodium borate buffer pH 6.5, or alternatively with 100 mM sodium phosphate buffer pH 6.5, and after 1 h at 30 °C was centrifuged at 100,000g for 1 h at room temperature. The supernatant was the source of solubilized cysteine proteinase. Aliquots (150 µl, corresponding to 15 midguts) of this SDS-containing sample were applied to 12% polyacrylamide gels (Hedrick and Smith, 1968), using the system of Davis (Davis, 1964) in glass tubes of 5 mm i.d. and 10 mm length. Separation was achieved with a current of 2.5 mA/column at 4 °C. The gels were fractionated in a gel fractionator (Autogeldivider Savant Instruments, USA) with 100 mM citrate–sodium phosphate buffer pH 6.0 containing 3 mM EDTA and 3 mM cysteine. Fractions were obtained by pushing the gel rod against a steel net, followed by washing out the fragments with buffer. Fractions of 0.4 ml (corresponding to about 1.5 mm of gel) were collected with the aid of a fraction collector. After standing overnight at 4 °C, the fractions were assayed with different substrates. 2.7. Chromatographic resolution of membrane-bound cysteine proteinases A sample of SDS-solubilized cysteine proteinases (Section 2.6) was loaded onto a Mono Q column equilibrated in 20 mM Tris–HCl buffer pH 7.0, containing 0.1% SDS. Elution was accomplished with a gradient of 0600 mM NaCl in the same buffer. The flow was 1 ml/min and fractions of 0.4 ml were collected. 2.8. SDS-PAGE and Western blotting SDS-PAGE of A. pisum samples was carried out in 12% (w/v) polyacrylamide gels containing 0.1% (w/v) SDS, on a discontinuous pH system (Laemmli, 1970), using Bio-Rad (USA) Mini-protein II equipment. Samples were mixed with sample buffer (2:1) containing 60 mM Tris–HCl, pH 6.8, 2.5% (w/v) SDS, 0.336 mM β-mercaptoethanol, 10% glycerol and 0.05% (w/v) bromophenol blue and heated for 2 min at 95 °C in a water bath before being loaded onto the gels. Electrophoresis was carried out at 200 V and the gel was silver-stained for proteins (Blum et al., 1987). Molecular-weight markers: lysozyme (14 kDa), soybean trypsin inhibitor (21 kDa), carbonic anhydrase (31 kDa), ovalbumin (45 kDa),

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bovine serum albumin (66 kDa), and phosphorylase B (97 kDa). Western blotting was performed essentially as previously described (Cristofoletti et al., 2001). After SDSPAGE, the proteins were electrophoretically transferred onto a nitrocellulose filter, which after a blocking step reacted with the appropriate antiserum and then with anti-rabbit IgG coupled with peroxidase. After washings, the strips were treated with an ECL Western blotting kit (Amersham, UK), according to the manufacturer’s instructions, and then exposed to a high-performance luminescence detection film (Hyperfil–ECL, Amersham, UK). The transfer efficiency was evaluated using prestained molecular weight markers (Bio-Rad or Sigma, USA). Pre-immune serum was used in control experiments to show that antisera were specific. 2.9. HPLC of mono- and oligosaccharides Samples of the reaction media containing purified (or partially purified) A. pisum α-glucosidase and sucrose as substrate, after being added to acetonitrile and filtered, were injected into an AsahiPak NH2P-50 (4.6×250 mm) column (Shimadzu) and were analysed in an Shimadzu HPLC system provided with a refractive index detector (RID 3A). Elution was accomplished with 70% acetonitrile in water with a flow of 0.8 ml/min. 2.10. Electron microscopy For transmission-electron microscopy, animals were dissected in their own hemolymph and midgut sections were fixed in 3% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 2 h at 4 °C. The midgut pieces were post-fixed in 1% osmium tetroxide in the cacodylate buffer. In-block staining was performed in 1% aqueous uranyl acetate for 16–18 h. After dehydration, the material was embedded in Spurr resin, cut into ultrathin sections, stained with lead citrate, and examined in a Zeiss EM 900 electron microscope operated at 80 kV. For immunolabeling of aminopeptidase, cysteine proteinase, and α-glucosidase at the ultrastructural level, midgut pieces were fixed (paraformaldehydeglutaraldahyde), embedded in L.R. White acrylic resin, incubated with the primary and secondary (goat anti-rabbit IgG coupled to 15 nm Gold) antibodies and examined in a Zeiss EM 900 electron microscope as detailed elsewhere (Silva et al., 1995). As controls, sections were incubated with nonimmune serum under the same conditions. 3. Results 3.1. Properties of the most active enzymes in A. pisum Assays of midgut homogenates with 125I-radiolabeled globulin as substrate showed the presence of a major

proteinase maximally active at pH 4.5, which was strongly inhibited by E-64 and chymostatin, not affected by benzamidine and pepstatin and that was activated by EDTA plus cysteine (not shown). The homogenate was not active on the chymotrypsin substrate SucAAFMCA, but was active on ZFRMCA, substrate described for trypsin and cysteine proteinase (Fig. 1), and also on ⑀amino-caproyl-leucyl-(S-benzyl)-cysteinyl-MCA, substrate for cysteine proteinase (Alves et al., 1996) (not shown). The activity on ZFRMCA was activated by EDTA + cysteine, was inhibited by E-64 (Fig. 1) and chymostatin and was not affected by benzamidine (not shown). Elastatinal (5 µM) inhibited 35% of this activity (not shown). EDTA+cysteine activation and E-64 inhibition indicated the enzyme was a cysteine proteinase, whereas chymostatin inhibition suggested it was mainly cathepsin-L-like (see proteinase substrates and inhibitors in Barrett et al., 1998). Thus, the data showed that the major proteinase present in the aphid midgut is a cathepsin-L-like cysteine proteinase with pH optimum 5.5 (Fig. 1). The proteinase pH optimum measured with protein substrate is (Section 4.5, see above) probably an artifact. Below substrate saturation, enzyme activity depends on pH and substrate concentration (Segel, 1975). Since the protein substrate unfolds at extreme pH values increasing the concentration of proteinase susceptible bonds, pH optima determined with synthetic substrate are more reliable, as their concentrations are not affected by pH. In addition to the cysteine proteinase, other major activities found in A. pisum midgut were: aminopeptidase, α-glucosidase, and trehalase. Midgut homogenates were centrifuged at 100,000g for 1 h at 4 °C and assays were carried out in supernatants and pellets. The activities remaining in the pellets (% of total, mean and SEM,

Fig. 1. Effect of pH on the hydrolase active on ZFRMCA from A. pisum midgut. Midgut homogenates were assayed with no addition (䊏), in the presence of 3 mM EDTA plus 3 mM cysteine (䊊) or with EDTA plus cysteine with 1 mM E-64 (쎲). Buffers: 100 mM citratesodium phosphate pH 3-7 and 100 mM sodium phosphate buffer pH 6.5–8.5.

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N=3) were: aminopeptidase 100±20; cysteine proteinase, 80±20; α-glucosidase, 100±20; trehalase, 50±10. This means that trehalase is partly soluble and partly membrane-bound, whereas the other activities are mostly membrane-bound. Table 1 shows that membrane-bound aminopeptidase, α-glucosidase and trehalase were solubilized in Triton X-100, whereas cysteine proteinase needed an SDS-borate buffer to be put into solution. The properties of solubilized aminopeptidase are described elsewhere (Cristofoletti et al., in preparation) and those of cysteine proteinase and α-glucosidase are presented below. Resolution in native SDS-PAGE of solubilized membrane-bound cysteine proteinase resulted in a single peak more active on ZFRMCA than on ZRRMCA [Fig. 2(A)]. The activity was completely inhibited by 1 µM E-64 (not shown). This means that the membrane-bound cysteine proteinase in A. pisum midgut is cathepsin-L-like (prefered ZFRMCA, over ZRRMCA, see Barrett et al., 1998). The occurrence of a single membrane-bound cysteine proteinase was confirmed by chromatography on a Mono Q column (FPLC system) (not shown). The active fractions of the FPLC Superose chromatography of solubilized midgut cell membranes [Fig. 2(B)] were separately submitted to SDS-PAGE and the gel were silver stained [Fig. 2(C)]. The activity of fractions in Fig. 2(B) was proportional to the intensity of a single 40 kDa band in Fig. 2(C). The data suggested that A. pisum has a membrane-bound cathepsin-L-like enzyme sized 40 kDa. Mono Q chromatography of solubilized midgut cell membranes showed a single peak of α-glucosidase activity against maltose, sucrose or NPαGlu substrates (not shown). The active fractions of this chromatography Table 1 Solubilization of membrane-bound aminopeptidase, cysteine proteinase, α-glucosidase, and trehalase from A. pisum midgut cell membranes Enzyme

Solubilizing agent

% Solubilization % Recovery

Aminopeptidase Cysteine proteinase α-Glucosidase Trehalase

Triton X-100 99±6 SDS 85±5

114±9 100±8

Triton X-100 70±4 Triton X-100 84±2

73±6 90±20

Cell membranes were maintained in the presence of 10 mM Triton X100 in 20 mM HEPES buffer pH 7.4 for 2h at 4 °C, before being centrifuged at 100,000g for 1 h at 4 °C. The supernatant was colleted and the pellet was suspended in 2.5% SDS in 400 mM sodium borate buffer pH 6.6 and, after 1 h at 30 °C, the suspension was centrifuged at 100,000g for 1 h at room temperature. % Solubilization is the ratio of activity in the supernatant and the activity in pellet plus activity in supernatant. % Recovery is the ratio of activity in supernatant and in the original membrane sample. Figures are means and SEM calculated from determinations in each of four experiments.

Fig. 2. Physical properties of SDS-solubilized cysteine proteinase from A. pisum midgut cell membranes. (A) Resolution in native SDSPAGE of solubilized cysteine proteinase. The samples did not contain β-mercaptoethanol and were not boiled. The gel fractions were assayed with ZFRMCA or ZRRMCA. The recoveries of the applied activities were about 30%. In the presence of 1 µM E-64 the activity with both substrates was abolished. (B) Chromatography on Superose 12 of SDSsolubilized cysteine proteinase. The Superose column was equilibrated and eluted with 20 mM HEPES buffer pH 7.5 containing 0.1% SDS and 200 mM sodium chloride. (C) SDS-PAGE of fractions 13, 14, 15, and 16 from the chromatography on Superose. Proteins were revealed by silver staining.

were pooled, loaded onto a Superose column and a single active peak on NPαGlu was eluted [Fig. 3(A)]. The fractions of the peak were separately submitted to SDSPAGE and the gel was silver-stained [Fig. 3(B)]. The activity of the chromatographic fractions in Fig. 3(A) was proportional to the intensity of a single 72 kDa band in Fig. 3(B). This means that the α-glucosidase was pure

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Table 2 Kinetic parameters of the partially purified A. pisum α-glucosidase Substrate

Km (mM)

kcat (s⫺1)

kcat/Km (mM⫺1 KiS (mM) s⫺1)

NPαGlu Maltose Sucrose

0.43±0.02 40±5 33±10

10.7±0.3 4.4±0.3 7±9

25±2 0.11±0.02 0.2±0.3

10.4±0.8 270±40 32±10

The enzyme source was the pool of fractions of the Mono Q chromatography. Kinetic parameters were calculated taking into account that excess substrate inhibits the enzyme. KiS is the dissociation constant of the complex enzyme with a substrate acting as competive inhibitor. kcat values are underestimated because partially purified enzyme may have up to 20% (w/w) of contaminants. Due to high inhibition by excess sucrose [see Fig. 3(C)], the corresponding kcat value are uncertain. Further details in Section 2.

Substrate inhibition in glycosidase reactions may result from transglycosylation reactions, where part of the product is not released into solution, but is linked to the substrate forming an oligosaccharide. This occurs in A. pisum α-glucosidase reactions since on incubation with excess sucrose, tri-, and tetrasaccharides are formed (Fig. 4). 3.2. Distribution of pH values and enzyme activities along A. pisum midgut

Fig. 3. Physical and kinetic properties of a solubilized α-glucosidase from A. pisum midgut cell membranes. (A) Chromatography on Superose 12 of the active fractions (fractions 43 to 47) previously obtained by Mono Q chromatography of Triton X-100 solubilized midgut homogenates. The Superose column was equilibrated and eluted with 20 mM HEPES buffer pH 7.5 containing 0.1% Triton X-100 and 200 mM sodium chloride. (B) Silver-stained SDS-PAGE of fractions 8, 9, and 13 from the chromatography on Superose. (C) Effect of sucrose concentration on α-glucosidase activity. The enzyme source was the pool of active fractions of the Superose chromatography.

and has a 72 kDa molecular weight. The purified α-glucosidase has a pH optimum of 6.0 (not shown), is inhibited by excess substrate [Fig. 3(C)] and is twice as active on sucrose as on maltose (Table 2). It is remarkable that the enzyme binds sucrose as an inhibitor and as a substrate with equal strength (Km=KiS, Table 2).

The pH range in A. pisum midgut contents is shown in Fig. 5. Identical results were obtained when aphid guts were dipped in a universal pH indicator or when isolating midgut regions and mixing their individual contents with the indicator. Most cysteine proteinase activity occurred in V1, aminopeptidase activity was found preferentially in V1–V3 and α-glucosidase in V2–V4 (Fig. 6). As mentioned previosly, these enzymes are membrane bound. Centrifugation of midgut homogenates (previously filtered through a piece of Nylon mesh of 45 µm pore size) at 600g for 10 min results in a pellet containing most of the activities of aminopeptidase, cysteine proteinase, and α-glucosidase (not shown). This suggests that these enzymes are present in large membranes, such as the plasma membrane. In order to see if those enzymes occur in the same membranes, washed midgut membranes were resolved by gradient ultracentrifugation. The following sucrose gradients were used (%, w/v): 10–40, 10–60, 10–70, 30– 60, and 40–70. The best resolution was obtained with the gradient 40–70%. The results showed that aminopeptidase was present mainly in a heavy membrane fraction (d=1.153), cysteine proteinase and α-glucosidase were distributed between a heavy and a light (d=1.135–1.142) membrane fraction and trehalase was abundant in a very light (d=1.117) membrane fraction (Fig. 7 and Table 3).

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Fig. 4. High-pressure liquid-chromatography of the products of A. pisum α-glucosidase. Samples of Triton X-100 solubilized A. pisum midgut cell membranes were incubated with 1 M sucrose in 50 mM phosphate buffer pH 6.5. After different periods of time, samples were injected into an AsahiPak NH2P-50 (4.6×250mm) column (HPLC system) and eluted with 70% acetonitrile in a HPLC system with Refractive Index detection (RI units). Incubation times (min): 0, 90, 930, and 1440. G3, and G4, are peaks with retention times similar to maltotriose and maltotetraose, respectively. Inset: experimental conditions as described above but using purified A. pisum α-glucosidase (active fractions obtained by Mono Q chromatography) with incubation times of 0, 930, and 1440 min.

Fig. 5. pH of gut contents at different sites in A. pisum. Ranges correspond to determinations performed on guts coloured by a universal pH indicator. Parentheses refer to averages of at least four determinations (reproducible within 0.2 pH units) carried out in isolated gut contents. FG, foregut; V1–V4 are sections of the ventriculus; R, rectum. Aphids have no Malpighian tubules.

3.3. Morphology of A. pisum midgut cells

Fig. 6. Distribution of the major hydrolases along the gut of A. pisum. APN, aminopeptidase; CP, cysteine proteinase; Glu, α-glucosidase. Determinations were carried out in three different preparations obtained from 20 insects each. SEM were found to be 10–20% of the means. The specific activities (mU/mg protein) in whole midgut homogenates were: APN, 192±11; CP, 0.39±0.04; Glu, 1180±80. Amount of proteins in gut sections (µg/animal) were: V1, 4.8±0.9; V2, 5±1; V3, 5.4±0.9; V4, 3.8±0.5.

The morphology of the aphid midgut and major ultrastructural features of the midgut cells have been described before (O’Loughlin and Chambers, 1972; Ponsen, 1987, 1991 and references therein), and therefore only a brief description will be given, emphasizing details not taken into account before. The midgut of A. pisum consists in a dilated anterior region, frequently named the stomach (V1 in Fig. 5), and a slender and longer region called the intestine (V2–V4 in Fig. 5). Regenerative cells are very rare and will not be described here. V1 cells have in their apex a complex network of lamellae (apical lamellae), instead of the regularly-arranged microvilli observed in most insect

midgut cells [Fig. 8(A)]. The lamellae do not have evident core microfilaments and are associated with one another through 15 nm-length trabeculae, resembling septate junctions [Fig. 8(A,B)]. Trabeculae are found between lamellae of the same cell and also between lamellae of adjacent cells (not shown). Associated with the lamellae, especially those similar to microvilli, occur amorphous membranous masses [Fig. 8(A,B)]. As these masses associate with microvilli-like membranes, they resemble the perimicrovillar membranes found in most hemipteran cells (Terra and Ferreira, 1994). Accordingly these structures were named modified perimicrovillar

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Table 3 Densities (g/cm3) of A. pisum midgut cell membranes associated with marker enzymes Enzyme

Peak 1

Peak 2

Peak 3

Aminopeptidase Cysteine proteinase α-Glucosidase Trehalase

1.153±0.006 1.153±0.007 1.153±0.008 1.154±0.005

1.135±0.008 1.137±0.007 1.142±0.009 –

– – – 1.117±0.003

Densities were calculated from figures similar to Fig. 7. Data are means and SEM corresponding to four independent experiments.

Fig. 7. Typical resolution of membranes from A. pisum midguts by ultracentrifugation in sucrose gradients. Fractions were collected from the bottom of the tubes. Densities were determined with the aid of a refractometer. (䊊), activity; (쎲), density (g/cm3). (A), aminopeptidase; (B), cysteine proteinase; (C), α-glucosidase; (D), trehalase. The arrows indicate the peaks which densities are shown in Table 3.

Fig. 8. Fine structure of A. pisum midgut cells. (A) Apical surface of V1 cells showing the apical lamellar system with associated modified perimicrovillar membranes (MPM) projecting into the lumen. Note trabecullae (small arrows) between lamellae and MPM masses moving among lamellae (large arrows). (B) Detail of modified perimicrovillar membranes associated with lamellae in V1 cells. Note trabecullae (arrows) between lamellae. (C) Apical cytoplasm of a V1 cell showing a multi-membranous vesicle. (D) Golgi area of a V1 cell. Note Golgi vesicles being surrounded by rough endoplasmic elements (arrows), forming a multi-membranous vesicle. (E) Golgi complex buding vesicles in a V1 cell. (F) Apical surface of V2 cells showing an apical lamellar system less developed than in V1 cells. Note in the detail the tubular structures on the lamellar surface (arrows) that are thought to correspond to trabecullae not linking adjacent lamellae. Abbreviations: ALS, apical lamellar system; G, Golgi area; L, lumen; Mi, mitochondria; MMV, multi-membranous vesicle; MPM, modified perimicrovillar membranes; V, Golgi vesicle. Bars: 1.0 µm (A), 0.1 µm (B, C, E, and insert in F), 0.5 µm (D, F).

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membranes (MPM). These membranes seem to move through the apical lamellar system [Fig. 8(A)] inside vesicles visible in the cytoplasm [Fig. 8(C)] and apparently formed with membranes from Golgi and rough endoplasmic reticulum [Fig. 8(D,E)]. At the base of the apical lamellar system there are many mitochondria [Fig. 8(A)]. The basal plasma membrane invaginates forming narrow and ramified channels with openings to the underlying hemolymph spaces. These infoldings show many associated mitochondria (not shown). V2 cells are similar to V1 cells, except that the apical lamellar system is less developed, showing lamellae that are not linked by trabeculae and less conspicuously modified perimicrovillar membranes [Fig. 8(F), Ponsen, 1987, 1991]. The trabeculae not linking two adjacent lamellae form tubular structures [Fig. 8(F)] previously described (O’Loughlin and Chambers, 1972) as extracellular microtubules. Although O’Loughlin and Chambers (1972) described the extracellular microtubules as running parallel to the microvilli, close inspection in their micrographs (e.g. button of Fig. 1 and Fig. 3) shows that their orientation varies, with microtubules also placed transversally. Although V3 and V4 cells were not studied in any detail, their general morphology seem to be similar to V2 cells (cf. Ponsen, 1987, 1991).

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ies, Aphis gossypii, from which a cathepsin-L was recently cloned, but not characterized biochemically (Deraison et al., unpublished). Furthermore, sucrose gradient data (see Section 3.2) show that α-glucosidase and cysteine proteinase have the same distribution in A. pisum midgut cells in agreement with immunolocalization results (see below). Using the antibodies described above, immunocytochemical experiments were performed with V1 and V2 cells. α-Glucosidase was detected in V1 cells in the modified perimicrovillar membranes and in cell vesicles, including those in the Golgi area, and was absent from the apical lamellar membranes [Fig. 9(C,D)]. Labeling for this enzyme in V2 cells was similar to that in V1 cells (not shown). Cysteine proteinase labeling paralleled that of the α-glucosidade in V1 cells [Fig. 9(B)], although no labeling was observed in V2 cells. Aminopeptidase occurred in the modified perimicrovillar membranes and in internal vesicles, but also in the apical lamellar membranes [Fig. 9(A)]. As observed for α-glucosidase and

3.4. Immunocytochemical localization of aminopeptidase, a-glucosidase, and cysteine proteinase in A. pisum V1 cells Antibodies raised in a rabbit against purified A. pisum aminopeptidase (purification described in Cristofoletti et al., in preparation) recognized, in Western blots, a single protein in midgut homogenates that co-migrated with purified aminopeptidase (not shown). Pre-immune serum was unable to react with A. pisum midgut proteins. These data prove that the aminopeptidase antiserum recognizes only aminopeptidase molecules in A. pisum midguts. A Western blot of A. pisum midgut homogenate after SDS-PAGE and incubated with antiserum against Dysdercus peruvianus α-glucosidase (Silva et al., 1995) showed only one band with 72 kDa (not shown). This agrees with A. pisum α-glucosidase molecular weight. These data together with the finding that the pre-immune serum is not reactive indicate the antiserum is specific. Antibodies raised against a recombinant cathepsin-Llike cysteine proteinase from Sitophilus zeamais (a gift from Dr. I. Matsumoto, University of Tokyo) (Matsumoto et al., 1997) gave, in Western blots, a strong (band around 48 kDa) and a faint (band around 30 kDa) reaction. It is possible that the strong reaction corresponds to the cysteine proteinase, molecular weight 40 kDa, whose properties are described above, and the faint reaction to a processed fragment of that cysteine proteinase. This fits the results obtained for another aphid spec-

Fig. 9. Immunocytochemical localization of aminopeptidase, cysteine proteinase and α-glucosidase in A. pisum anterior (V1) midgut cells. Although heterologous anti-cysteine proteinase serum was not shown with certainty to react only with cysteine proteinase, evidence discussed in text favors this view. (A) Aminopeptidase labeling in multimembranous vesicles, apical lamellar system and modified perimicrovillar membranes. (B) Cysteine proteinase labeling in multi-membranous vesicles and modified perimicrovillar membranes. (C) α-Glucosidase labeling in multi-membranous vesicles and modified perimicrovillar membranes. (D) α-Glucosidase labeling in Golgi area. (E) Aminopeptidase labeling in Golgi vesicles being surrounded by RER elements (arrows). Abbreviations as in Fig. 8. Bar=0.5 µm.

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cysteine proteinase (not shown) aminopeptidase labeling was also observed in Golgi vesicles [Fig. 9(E)]. Similar labeling was observed in V2 cells (not shown). 4. Discussion 4.1. Organization and origin of A. pisum apical midgut cell membranes. The apical surface of the midgut cells of most hemipterans is formed by microvilli ensheathed by an outer (perimicrovillar) lipid membrane which is almost free from intramembranous particles. The perimicrovillar membrane maintains a constant distance from the microvillar membrane, extends toward the luminal compartment with a dead end, and limits a closed compartment (the perimicrovillar space) (Ferreira et al., 1988; Silva et al., 1995). The surface of aphid midgut cells departs from this model, however, apparently as a result of adaptation to sucking highly-concentrated sucrosecontaining phloem sap (see Section 4.4). The A. pisum midgut cell surface consists of an apical lamellar system in which the lamellae are linked to one another through 15 nm-length trabeculae. This should increase the tissue’s stretch resistance, because the trabeculae also occur between adjacent cells. The face of the lamellae exposed to the luminal contents is frequently associated with a membrane, here named modified perimicrovillar membrane, that extends toward the luminal compartment and has an amorphous structure. These amorphous structures are seen filling the lumen of the V1 (stomach) region, and are supposed to be linked to the apical lamellar system of V1 cells. Along the midgut, the number of those amorphous structures decreases and the apical lamellar system becomes less compact. In more posterior regions, trabeculae are frequently seen associated with a single lamella. In this case, trabeculae resemble cell microtubules, although they are narrower. Typically, the microtubule diameter is 24 nm (Dustin, 1984), whereas A. pisum microtubulelike structures have a diameter of 15 nm. Hemipteran perimicrovillar membranes are formed in the Golgi areas of gut cells, migrate as the internal membrane of double membrane vesicles, which finally fuse at the cell apex—the outer vesicle membrane with the microvillar membrane and the inner vesicle membrane with the perimicrovillar membrane (Silva et al., 1995). Data presented in this paper support a model for the origin of the A. pisum modified perimicrovillar membrane that is different from that of previously described perimicrovillar membranes. According to this model (Fig. 10), vesicles budded from Golgi cisternae are surrounded by ribosome-depleted rough endoplasmic reticulum forming a multi-membranous vesicle. These steps resemble the formation of autophagic vacuoles from ribosome-free

Fig. 10. Model for the origin of the membrane masses associated with the apical lamellar system, the modified perimicrovillar membranes. Golgi cisternae bud vesicles (a) that become surrounded by RER membranes (b). These, after losing the ribosomes, form a vesicle enveloping membranes (c) that eventually fuse with the base of the apical lamellae emptying its membrane content into the interlamellar space (d). The membrane mass moves along the interlamellar space, reordering the trabecullae so that the lamellae do not set apart (e) and finally become associated with the luminal surface of the lamellae, projecting into the midgut lumen (f). Abbreviations as in Fig. 8.

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regions of rough endoplasmic reticulum (Dunn, 1990a, b). The multimembranous vesicle fuses at the base of the apical lamellae and as it progresses between them, the trabeculae are set apart in the front of the vesicle and reassembled back at its rear end. It is noteworthy that this system maintains the stretching resistance of the tissue during movement of vesicles between lamellae [Fig. 8(A)]. As soon as the vesicle attains the apical surface, their membranes become bound at the luminal surface of the lamellae. According to this model, the lamellar membranes are derived from rough endoplasmic reticulum membranes, whereas the modified perimicrovillar membranes originate from Golgi cisternae. Thus, α-glucosidase and the cysteine proteinase are expected to be found in the vesicles budding from Golgi and aminopeptidase is expected in these vesicles but also in rough endoplasmic reticulum membranes surrounding the Golgi cisternae (Fig. 10). In agreement with this, α-glucosidase, cysteine proteinase, and aminopeptidase were found in Golgi vesicles. Nevertheless, no aminopeptidase was detected in rough endoplasmic reticulum membranes. Although this apparently challenges the model, it is more likely that this results from the low titres (or incomplete maturation and folding) of aminopeptidase in those membranes. 4.2. Distribution of aminopeptidase, a-glucosidase, and cysteine proteinase among A. pisum midgut cell membranes Most aminopeptidase labeling is observed in the apical lamellar membranes [Fig. 9(A)]. Thus, the membrane with density=1.153 [Fig. 7(A), Table 3) should correspond to the lamellar membranes, whereas that with density=1.135 probably represents the modified perimicrovillar membranes. This is confirmed by the finding that α-glucosidase, which labels mostly the modified perimicrovillar membranes [Fig. 9(C)], sediments mostly associated with the lighter membrane [Fig. 7(C)]. The unexpectedly high amount of α-glucosidase bound to the heavier membrane may result from the association (during midgut homogenization) of fragments of the modified perimicrovillar membranes with apical lamellar membranes. Work done with other hemipteram midguts lends support to this hypothesis. α-Glucosidase was found to be associated with the midgut perimicrovillar membranes in Rhodnius prolixus (Ferreira et al., 1988) and Dysdercus peruvianus (Silva et al., 1996). In Dysdercus peruvianus, electron micrographs showed that the lighter membrane preparations contained mainly single membrane vesicles, whereas the heavier membrane preparations had numerous double membrane structures (Silva et al., 1996). In other words, perimicrovillar membrane structures (perhaps also modified perimicrovillar membranes) are not much contaminated by high-density membranes (microvillar or apical lamellar membranes),

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the contrary being true for the microvillar (or apical lamellar) membrane preparations. Cysteine proteinase labeling parallels that of α-glucosidase, implying that it occurs associated with the modified perimicrovillar membranes. Since the profile of cysteine proteinase activity is identical to that of αglucosidase in the gradients of midgut membranes, it is likely that the S. zeamais antiserum is recognizing the membrane-bound cysteine proteinase from A. pisum midguts. Further support for this conclusion is the observation that the S. zeamais antiserum fails to label V2 tissue, in agreement with the much lower cysteine proteinases activity in this region. The very high sequence similarity between aphid and weevil cysteine proteinases (Deraison et al., unpublished) is consistent with the recognition by the beetle antibody of the aphid proteinase. The densities of the midgut apical lamellar membranes and modified perimicrovillar membranes of A. pisum are higher than those of Rhodnius prolixus (Ferreira et al., 1988) and Dysdercus peruvianus (Silva et al., 1996) midgut microvillar and perimicrovillar membranes, respectively. This means that the protein content in A. pisum midgut membranes is higher than in those of the other hemipterans. This is so probably because of the presence of trabeculae in A. pisum lamellar membranes and the presence of a higher enzyme content in the A. pisum modified perimicrovillar membranes. 4.3. The role of a-glucosidase in transglycosylation reactions The reaction path of glycosidases (including αglucosidases) consists of both glycosylation and deglycosylation steps. During the glycosylation step, the glycone part of the substrate becomes covalently linked to the enzyme (forming a glycosyl-enzyme), the glycosylenzyme is attacked by water, releasing the glycone, or by the substrate (if present in sufficiently high concentration), forming a trisaccharide if the substrate is a disaccharide (Hays et al., 1998). According to this mechanism, α-glucosidase is expected to react with sucrose, releasing fructose and forming a glycosylenzyme that in sequence is attacked by water (freeing glucose) or by sucrose, if present in high concentration, releasing a trisaccharide. When the concentration of the trisaccharide increases it may in turn attack the glycosylenzyme resulting in the release of a tetrasaccharide and so on. This was observed with A. pisum purified α-glucosidase incubated with a concentrated sucrose solution (this paper). The failure of Ashford et al. (2000) to detect fructose residues in the oligosaccharides formed by A. pisum midgut homogenates incubated with sucrose is unexpected. Chains of glucose should be formed only in the presence of excess glucose, implying extensive sucrose hydrolysis (no transglycosylation), which does not agree with current the data. It is possible that the

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amount of fructose was under the detection limit of Ashford et al.’s assay or, less likely, that A. pisum midguts contain a β-fructosidase able to remove fructose residues from oligosaccharides. If this β-fructosidase exists, our assays accomplished in A. pisum solubilized midgut cell membranes, after chromatography on Mono Q columns, should have detected an activity on sucrose that differed from that on maltose and NPαGlu. As this result was not seen, we conclude that a β-fructosidase is lacking in A. pisum. The A. pisum α-glucosidase KiS value of sucrose is substantially lower than for maltose. This means that A. pisum α-glucosidase architecture favors the reaction of sucrose with the glucosyl-enzyme, thus promoting transglycosylation reactions more efficiently with sucrose than with maltose. Differences in the architecture of the enzymes may also explains the appearance of trehalulose in the honeydew of some whiteflies sucking sap lacking this disaccharide (Hendrix et al., 1992). The α-glucosidase in these cases probably orients fructose in such a way that the C-1 hydroxyl attacks the glucosyl-enzyme, originating trehalulose. This kind of reaction is not energy-demanding. Nevertheless, there is evidence with the silverleaf whitefly that trehalulose is synthesized by a specific enzyme (Salvucci, 2000). 4.4. Midgut physiology of A. pisum The midgut physiology of A. pisum is better understood in the light of work done with other Hemiptera. Due to that, an overview of current knowledge on hemipteran midgut morphology and function will be presented before discussing A. pisum. The ancestor of the entire order Hemiptera is supposed to have been a sap sucker similar to present day cicadas and fulgorids. Sap-sucking Hemiptera may suck phloem or xylem sap. These food sources have very low contents of protein (with the exception of few phloem saps, see below) and carbohydrate polymers and are relatively poor in free essential amino acids. In contrast to xylem sap, phloem sap is very rich in sucrose (Terra, 1990). Thus, except for dimer (sucrose) hydrolysis, no food digestion is usually necessary in sap-suckers. Upon adapting to this food, hemipteran ancestors would lose the enzymes involved in initial and intermediate digestion and associated with the lack of luminal digestion also lose the peritrophic membrane. The major problem facing a sap-sucking insect is to absorb nutrients, such as essential amino acids, that are present in very low concentrations in sap. Amino acids may be absorbed according to a hypothesized mechanism that depends on perimicrovillar membranes (Terra and Ferreira, 1994). Aphids may suck more-or-less continuously phloem sap of sucrose concentration up to 1.0 M and osmolarity up to three times that of the insect hemolymph (Ashford et al., 2000). As the ingested phloem sap passes along

the aphid midgut, its osmolarity decreases, resulting in a honeydew isoosmotic with hemolymph (Fisher et al., 1984). As a consequence, the anterior parts of the midgut must withstand higher hydrostatic pressions (caused by the tendency of water to move from the hemolymph into midgut lumen) than the posterior ones. Midgut stretching resistance is apparently helped by the existence of links between apical lamellae that become less conspicuous along the midgut (Ponsen, 1991; this paper, Fig. 8). We suggest that following the appearance during evolution of apical lamellar links, the perimicrovillar membranes were replaced by the membranes seen associated with the tips of the lamellae, the modified perimicrovillar membranes. The physiological role of the modified perimicrovillar membranes may include: (a) making amino acid absorption easier by increasing the concentration of amino acids by binding them in a reversible way, especially in V1, where those membranes are more conspicuous; (b) immobilizing α-glucosidase in a large area, so that this enzyme is not lost in the honeydew and is able to efficiently release fructose from sucrose without increasing the osmolarity of midgut contents (the sucrose glucosyl moiety is transferred to another sucrose molecule); and (c) immobilizing a cathepsin-L-like cysteine proteinase in a large area in V1 (which almost lacks αglucosidase), thus avoiding its excretion. These putative roles deserve further comment. A role in midgut amino acid absorption depends on the presence of amino acid binding proteins on the surface of the modified perimicrovillar membranes and of amino acid carriers in the apical lamellar membranes. Although amino acid carriers have been found in the microvillar membranes of several insects (Wolfersberger, 2000), no attempts have been made to study the other postulated proteins. It is interesting to note that amino acid absorption in A. pisum midguts is influenced by the presence of the bacteria Buchnera in the mycetocytes of the mycetomes occurring in the aphid hemocoel (Prosser et al., 1992). The molecular mechanisms underlying this phenomenon are not known, in spite of the fact that there is strong evidence showing that Buchnera uses the non-essential amino acids absorbed by the host in the synthesis of essential amino acids (Prosser and Douglas, 1992; Shigenobu et al., 2000). It is likely that amino acid absorption through apical lamellar carriers depends on the amino acid concentration gradient between midgut lumen and hemolymph, whereas hemolymph titres vary widely according to Buchnera metabolic activity (Liadouze et al., 1995). The presence of an active cysteine proteinase in the midgut of a phloem-sucking insect is at first sight puzzling, since proteins are not considered to be major components of phloem, in spite of some exceptions to this rule. One possible function of this enzyme maybe to cope with toxic proteins, which may be present in

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phloem as part of the plant’s defenses against predators (e.g. lectins, Gatehouse et al., 1994). It should be noted, however, that lectins are frequently resistant to midgut action both in A. pisum (Rahbe´ et al., 1995) and in lepidopterans (Gatehouse et al., 1994). Another potential role for the A. pisum cysteine proteinase may be as an agent of true protein digestion together with aminopeptidase, which occurs in the apical lamellar membranes and in the modified perimicrovillar membranes. As commented before, phloem sap may contain relatively high concentrations of protein. Having calculated that sap with concentrations in excess of about 5 mg/ml could provide an adequate source of amino acids for phloemfeeding insects, Salvucci et al. (1998) fed whiteflies with labeled proteins and found that these were digested and incorporated by the insects. These workers failed to demonstrate in vitro proteolysis using whitefly homogenates, probably because they worked with the supernatants of Triton X-100-treated samples, under which conditions the cysteine proteinase (if similar to that of A. pisum) would remain in the pellet. The same explanation probably also holds for all the previous failures to demonstrate proteinase activity in aphid midguts (Mochizuki, 1998). A midgut membrane-bound cysteine proteinase similar to that of A. pisum was found in Brevicoryne brassicae (Silva et al., 2000), and a candidate cathepsinL was cloned from Aphis gossypii (Deraison et al., unpublished), suggesting that its occurrence may be general among aphids and perhaps also among other phloem feeders.

Acknowledgements This work was supported by the Brazilian research agencies FAPESP and CNPq (PRONEX program) and by travel grants from a Brazil/France Program (CAPESCOFECUB). We thank Dr. C. Ferreira for helpful discussions and L.Y. Nakabayashi, W. Caldeira, M.V. Cruz and G. Duport for technical assistance. We also thank Dr. I. Matsumoto from the University of Tokyo for a cysteine proteinase antiserum and Dr. L. Juliano from UNIFESP-EPM (Brazil) for the substrate ⑀-amino-caproyl-leucyl-(s-benzyl)-Cysteinyl-MCA. P.T. Cristofoletti is a post-doctoral fellow of FAPESP and A.F. Ribeiro and W.R. Terra are staff members of their respective departments and research fellows of CNPq.

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