Plant cell wall polysaccharide-degrading enzymes of Melanoplus bivittatus

J. Insect Physiol., 1969, Vol. 15,pp. 2273 to 2283. Pergamon Press. Printed in Great Britain

PLANT CELL WALL POLYSACCHARIDE-DEGRADING ENZYMES OF MELANOPLUS BIVITTATUS* KENNETH

W. TALMADGEt

and PETER

ALBERSHEIM

University of Colorado, Boulder, Colorado 80302 (Received 30 May 1969)

Abstract-Enzymes in freeze-dried extracts and acetone powder preparations of whole grasshoppers, Melanoplus bivittutus, are capable of degrading hypocotyl cell walls isolated from S- and 8-day-old red kidney bean plants. Over 40 per cent of the neutral sugars in the cell walls are solubilized by 4 hr incubation with the enzyme preparations. This extraction of cell wall constituents includes 64 per cent of the galactose, 48 per cent of the glucose, and 46 per cent of the arabinose recoverable from untreated walls. The following p-nitrophenyl glycosidase activities are detectable in grasshopper extracts: OL-and /%galactosidase, (Y- and j?-glucosidase, /I-xylosidase, or-fucosidase, and ar-mannosidase. Enzymes are also present which degrade commercial xylan and carboxymethylcellulose. A correlation is made between the ability of the grasshopper enzymes to extract wall polymer and to degrade model substrates.

INTRODUCTION

THE OCCURRENCE of one or more polysaccharide-degrading enzymes has been reported in a variety of phytophageous insects, including aphids (MCALLAN and CAMERON,1956; NUORTEVA and LAUREMA,1961; ADAMSand DREW, 1963), locusts (EVANSand PAYNE, 1964; ROBINSON,1964), silkworms (ITO and TANAKA, 1959), cotton stainers (KHAN and FORD, 1967), and grasshoppers (DAVIS, 1963). The enzymes reported include (Y-and @glucosidase, cellulase, OL-and p-galactosidase, @glucuronidase, or-arabinosidase, polygalacturonase, and polymethylgalacturonase. In the majority of these investigations, the enzymes have been localized in the salivary glands and the alimentary tract. This location of the polysaccharidedegrading enzymes suggests that they are involved in the digestion of plant polymers. The plant cell wall constitutes about 50 per cent of the dry weight of a cell, and about 90 per cent of the wall is polysaccharide. This wall polymer could constitute an important nutritional source for plant-eating insects. If these polysaccharidedegrading enzymes enable insects to digest and nutritionally utilize cell walls, the enzymes should be capable of degrading the polysaccharides of isolated cell walls. However, the previous work on the substrate specificity of insect-produced * Supported in part by a grant from the United States Atomic Energy Commission No. AT(ll-l)-1426. t Recipient of a National Science Foundation predoctoral fellowship. 2273

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KENNETHW. TALMADGE ANDPETERALBER~HEIM

polysaccharide-degrading enzymes has been limited to measuring their ability to hydrolyse model substrates, oligosaccharides, and sometimes isolated naturally occurring polymers. A sensitive gas-chromatographic method has recently been developed for determining the sugar composition of cell walls (ALBERSHEIMet al., 1967). This method has been used successfully to study the ability of polysaccharide-degrading enzymes produced by fungi to degrade plant cell wall polymers (MCNAB et al., 1967; BATEMANet al., 1969; ENGLISH and ALBERSHEIM,1969). The present paper describes experiments which use this method and demonstrate that the enzymes in freeze-dried extracts and acetone powder preparations from whole grasshoppers, Melanoplus biwittatus, are capable of degrading isolated plant cell walls. A correlation is made between the ability of the grasshopper enzymes to extract wall polymers and to degrade model substrates. MATERIALS AND METHbDS Enzyme preparations The enzymes studied in this investigation were obtained from grasshoppers, MeZanopZusbivittatus. The grasshoppers, collected during the summer of 1968, were predominantly adults with a small number of late instars occasionally included. The following two types of preparations were utilized during the course of this study, both of which were prepared within several hours of collecting the grasshoppers. (a) An acetone powder was made by blending in a Waring Blendor for 1 min 30 to 50 whole grasshoppers with 50 ml of cold ( - 20°C) acetone. The suspension was filtered, the precipitate resuspended in 50 ml of cold acetone, and the mixture reblended. The procedure was repeated once more and the acetone powder air dried. Enzyme solutions were obtained by extracting 1 g of acetone powder with 50 ml of 0.05 M sodium phosphate buffer, pH 6.0, at 0°C. The suspension was centrifuged for 30 min at 2O,OOOg,and the supernatant fluid dialysed for 18 hr at 2°C against 0.01 M sodium phosphate buffer, pH 6.0. (b) Freeze-dried preparations were obtained by homogenizing in a Waring Blendor for 1 min 40 to 50 grasshoppers with 50 ml of 0.05 M sodium phosphate buffer, pH 6.0, at 0°C. The suspension was centrifuged for 40 min at 34,000g. The supernatant solution was dialysed for about 18 hr at 2°C against 0.01 M sodium phosphate buffer, pH 6.0, and the dialysed solution lyophilized. Enzyme solutions were obtained by suspending 0.2 g of the freeze-dried powder in 25 ml of 0.05 M sodium phosphate buffer, pH 6.0, at 0°C. The insoluble material was removed by centrifuging the suspension for 30 min at 12,000g. Enzyme assays a-and /3-Galactosidase, LY-and /3-glucosidase, /3-xylosidase, OL-and p-fucosidase, and or-mannosidase activities were measured by their ability to hydrolyse the corresponding p-nitrophenyl glycoside (purchased from Sigma Chemical Co.,

POLYSACCHARIDE-DEGRADING

RNZYMRS OF MELANOPLUS

BIVITTATUS

2275

Calbiochem, or Koch Light Lab.). The rates of hydrolysis were determined by measuring the increase in optical density at 400 rnp resulting from the release of pnitrophenol. Assay mixtures typically consisted of 3-O ml of O-1 M citrate-sodium phosphate buffer, pH 5.0, containing 0.5 mg/ml of the proper substrate, and 0.05-0.3 ml of enzyme solution from freeze-dried or acetone powder preparations. a-fucosidase was routinely assayed in O-1 M citrate-sodium The p-nitrophenyl phosphate buffer at pH 6.4. Reaction mixtures were incubated at 30°C for 1 to 2 hr. The reactions were stopped and maximum colour developed by addition of 1.0 ml of 1 M NH,OH-NH&l buffer, pH 9% Controls were included in all experiments and duplicate assays were run for all samples. The controls were obtained either by adding enzyme to substrates after the reactions were made alkaline or by adding heat-denatured enzyme. Xylanase, cellulase, polygalacturonase, and polymethylgalacturonase activities were assayed by determining the ability of the enzyme preparations to increase the reducing groups in solutions containing polymeric substrates. Reducing groups were assayed with the aid of dinitrosalicylic acid reagent (MILLER, 1959). Reaction mixtures contained 2-O ml of O-1 M sodium acetate buffer, O-1 ml of the enzyme solution, and 1-Oml of one of the following polymer solutions adjusted to pH 6.8: 0.5% carboxymethylcellulose (Hercules Inc.), 0.2% xylan (City Chemical Corp.), 0.4% polymethylgalacturonic acid (Sunkist Growers Inc.), or 0.2% polygalacturonic acid (Sunkist Growers Inc.). Reaction mixtures were incubated for 1 to 2 hr at 3 l”C, and the reactions stopped by the addition of 3.0 ml of dinitrosalicylic acid reagent (1% dinitrosalicylic acid, 0.2% phenol, 0.05% sodium sulphite, and 1% sodium hydroxide). The solutions were immediately placed in a boiling water-bath for exactly 10 min to permit colour development. The solutions were then removed from the water bath, and 1.0 ml of 40% Rochelle salt solution was added to each. After cooling to room temperature, the optical density of each solution was measured at 575 rnp. The increase in reducing groups was determined from the appropriate standard curve. Endo-polygalacturonase, endo-polymethylgalacturonase, and endo-cellulase were qualitatively assayed using a falling ball viscometer. Reaction mixtures contained 10 ml of 1% solution of the respective polymers dissolved in water and adjusted with NaOH to pH 6.8, and 1.0 ml of enzyme extracted from acetone powder. Other methods The hypocotyl cell walls utilized in this study were obtained from 5- or &day-old red kidney bean plants. Growth conditions and isolation of hypocotyl cell walls have previously been described (NEVINSet al, 1967, 1968). The cell wall degradation studies were carried out by incubating 10 mg of cell wall with 1-Oml of freezedried extract for periods from 30 min to 4 hr at 31°C. The reactions were stopped by adding 3.0 ml of absolute ethanol. The precipitate was isolated by centrifugation at 1500 g and subsequently washed twice with 3 ml portions of water and twice with 3 ml portions of 70% ethanol. The method used for the quantitative

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KENNETHW. TALMADGEANDPETFZ ALBEFBHEIM

analysis of the component sugars of the cell wall residue by gas-liquid chromatography has been previously described (ALBERSHEIM et al., 1967; NEVINS et al., 1967). Protein was determined by the method of LOWRY et al. (1951) with crystalline bovine albumin as the standard. RESULTS Ceil

wall degradation

A typical gas-chromatographic separation of the alditol acetates of the seven neutral sugars most frequently encountered in higher plants is depicted by the solid line in Fig. 1. The sample contains the sugars liberated from approximately 10 mg of hypocotyl cell walls isolated from 8-day-old red kidney bean plants. Each sample also contains as an internal standard 1 mg of myo-inositol which is used to calculate the absolute amounts of the various sugars. The dotted line illustrates the GO.

)S.

$1

M

o-

1

30

I

40 Retention

I I

50

time,

I

i

1

60

70

min

FIG. 1. Gas-chromatographic separation of the alditol acetates prepared from the sugars obtained by 2 N trifluoroacetic acid hydrolysis of hypocotyl cell walls isolated from %day-old red kidney bean plants is represented by the solid line. The gas-chromatographic separation of the alditol acetates obtained by acid hydrolysis of similar walls, which were pretreated with 1.0 ml of freeze-dried grasshopper extract for 4 hr at 3O”C, pH 6.0, is represented by the dotted line. The samples contain in 1 ml of acetic anhydride the sugars liberated from approximately 10 mg of cell wall and 1 mg of myo-inositol which is an internal standard. Approximately 1~1 of the sample is injected into the gas-chromatograph. The monosaccharides represented are: rhamnose (R), fucose (F), arabinose (A), xylose (X), mannose (M), galactose (Ga), glucose (Gl), and myo-inositol (Inos).

POLYSACCHARIDE-DEGRADING

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sugars remaining after the cell walls are subjected to the action of 1 ml of freezedried grasshopper extract. The data obtained in kinetic studies of the ability of the grasshopper enzymes to degrade cell walls are presented in Fig. 2. After incubation with the enzyme mixture for 4 hr at 30°C there is a 40zper cent decrease in the acid-hydrolysable

I

2

3

I

4

hr

FIG. 2. Changes in sugar composition, as determined gas-chromatographically, of cell walls when treated for increasing periods of time with freeze-dried extract obtained from the grasshopper Melanoplus bivittatus. Approximately 10 mg portions of hypocotyl cell wall isolated from %day-old red kidney bean plants were incubated for the times indicated with 1.0 ml of freeze-dried extract at 3O”C, pH 6.0.

neutral sugar recovered from the cell walls. Most of this decrease is attributed to the removal of galactose, arabinose, and glucose. These are removed in the following amounts, in mg/lOO mg of cell wall: galactose, 5.1; arabinose, l-3; and glucose, 1.1. These amounts represent the following percentages of the sugar recoverable from untreated cell walls: galactose, 64; arabinose, 46; and glucose, 48. Degradation studies were carried out at pH 4.0, 5.0, and 7.0 for a 2-hr period at 31°C on hypocotyl cell walls isolated from 5-day-old red kidney bean plants. The total decrease in the sugars recovered by acid hydrolysis of cell walls subjected to enzyme degradation at pH 7-O and 5.0 is respectively 13 and 18 per cent. At pH 4.0, the total decrease by enzyme treatment is only 2 per cent, with several

2278

KENNETHW. TALMADCEANDPETER ALBERSHEIM

sugars showing a small increase; decrease.

glucose,

however,

exhibited

a 17 per cent

Model substrates The degree of hydrolysis of the p-nitrophenyl glycosides is proportional to incubation time for periods up to 2 hr. Table 1 lists the specific activities obtained TABLE ~-ACTIVITIES OF P-NITROPHENYLGLYCOSIDASES

Enzyme a-Glucosidase /?-Glucosidase /%Galactosidase or-Mannosidase or-Galactosidase /?-Xylosidase c+Fucosidase /%Fucosidase

Specific activity &moles p-nitrophenol liberated/hr per mg protein) 11.3 7.7 5.5 1.8 1.0 0.4 0.2 0.0

Specific activity &moles p-nitrophenol liberated/hr per mg protein) of p-nitrophenyl glycosidases. Reaction mixtures contained 3.0 ml of O-1 M citrate-sodium phosphate buffer, pH 5.0, containing 0.5 mg/ml of the proper substrate and from 0.05 to 0.2 ml of freeze-dried extract. Para-nitrophenyl-ol-fucosidase was determined in 0.1 M citrate-sodium phosphate buffer, pH 6.4. Reaction mixtures were incubated for 1 hr at 30°C.

from freeze-dried extracts for the eight glycosidases. These were determined at pH 5.0 for all the enzymes except the p-nitrophenyl-a-fucosidase which was measured at pH 6.4. Of those assayed, the glucosidases are the most active. There is approximately 20 mg of protein in the freeze-dried extract of a single adult grasshopper. Thus, the values in this table represent approximately 5 per cent of an insect’s total activity @moles of p-nitrophenol liberated/hr per insect). The curves of Fig. 3 illustrate the pH dependency for the various p-nitrophenyl glycosidase activities when assayed in O-1 M citrate-sodium phosphate buffer over a pH range of 2.8 to 7.5. Each of the enzymes exhibited a rather broad pH maximum in the range of 4.5-5.5 ; the only exception was p-nitrophenyl-a-fucosidase which has a maximum at 6.4. 01- and /&glucosidases of these grasshoppers exhibit optimal activity between pH 5.1 to 5.4 which is similar to the value reported by /3-glucosidase obtained from adult ROBINSON (1956) f or a 4-methylumbelliferone locust, Locusta migratoria, and by EVANS and PAYNE (1964) for an ol-glucosidase obtained from the adult desert locust, Schistocerca gregaria Forsk. Table 2 illustrates the inhibitory effect of 10 mM concentrations of several sugars on the p-nitrophenyl glycosidase activities. The substrate concentrations were 1.1 mM. With the possible exception of p-nitrophenyl+-xylosidase, each of the enzymes is inhibited to some degree by its product. It is interesting that galactose strongly inhibits both the 01- and /3-galactosidases, while glucose inhibits

POLYSACCHARIDE-DEGRADING

OF MELANOPLUS

ENZYMES

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2279

PH Zl-

(b)

3-

I

3

I

4

I

5

I

6

/

7

I

6

PH

FIG. 3. Effect of pH on the activity of seven p-nitrophenyl glycosidases and carboxymethylcellulase. Reaction mixtures for the@-nitrophenyl glycosidase assays contained 1.0 ml of the substrate in 0.05 M sodium acetate buffer, pH 4.5, 1 ml of 0.2 M citratiodium phosphate, and O-1 ml of enzyme from freeze-dried extracts. Reaction mixtures for carboxymethylcellulase assays contained 1.0 ml of 0.5% carboxymethylcellulose, 2.0 ml of 0.1 M sodium acetate buffer, and 0.1 ml of enzyme from freeze-dried extracts. The various enzymes are designated as follows: D-p-nitrophenyl-/?-glucosidase; l(a), haitrophenol-a-glucosidase; (b), B--$-nitroA-carboxymethylcellulase; p-nitrophenyl-/?-xylosidase; phenyl-p-galactosidase; A-@-nitrophenyl-a-galactosidase; O-p-nitrophenyl-amannosidase; O-p-nitrophenyl-a-fucosidase.

2280

KENNETH

p-nitrophenyl-cll-glucosidase only 13 per cent. ROBINSON

W. TALMADGBANDPETBRALBERSHEIM

46 per cent, but inhibits p-nitrophenyl-/3-glucosidase (1964)h

as reported

similar effects of glucose on a /3-

glucosidaseandofgalactoseona/3-galactosidaseobtained

TABLE

2--PERCENTAGE

INHIBITION

OF P-NITROPHENYL

from

GLYCOSIDASES

Locusta migratoriu. BY VARIOUS

SUGARS

Inhibitors (concentrations 10 mM) Enzyme &GIucosidase or-Glucosidase /?-Galactosidase m-Galactosidase or-Mannosidase j%Xylosidase

Glucose

Galactose

Mannose

13 46 6 0

0 0 64 67 0 0

0 3 6 0 19 0

Xylose 0 6.7 0 12 0 4.5

Percentage inhibition ofp-nitrophenyl glycosidases by several sugars (10 mM). Reaction mixtures contained 1.0 mg (1.1 mM) of the proper substrate in 2.0 ml of 0.1 M citratesodium phosphate buffer, 5 mg of inhibitor in 1-O ml of 0.1 M citrate-sodium phosphate buffer, and 0.05 to 0.2 ml of freeze-dried extracts. Reaction mixtures were incubated for 1 hr at 30°C.

Acetone powder extracts do not reduce the viscosity of either polygalacturonic acid or polymethylgalacturonic acid solutions. However, such extracts, when incubated with a 1% carboxymethylcellulose solution for 2-S min at 25”C, result in a 75 per cent decrease in the viscosity, as measured by the reduction in the time required for a ball to descend a given distance in a viscometer. The degradation of carboxymethylcellulose by acetone powder extracts could also be followed with the reducing group assay. The enzyme activity is found to be optimal between pH 3.4 to 3.8. This is distinctly different from the pH maximum of 5-O to 5.6 for p-nitrophenyl+glucosidase. The carboxymethylcellulose hydrolysing enzyme releases l-2 pmole glucose equivalentsfmg protein in 2 hr at 31°C. This corresponds to cleaving approximately 15 per cent of the glycosidic linkages in the polymer. Acetone powder extracts are able to hydrolyse a commercial soluble xylan, but do not hydrolyse appreciably polygalacturonic acid or polymethylgalacturonic acid. The xylanase activity, as measured by the reducing group assay, possesses maximal activity between pH 5-5 to 6-5. This enzyme releases 1.7 pmole xylose equivalents/ mg protein in 2 hr at 31°C. This corresponds to the hydrolysis of about 50 per cent of the glycosidic bonds in the polymer. No attempt was made in this investigation to determine if any of the degradative enzymes found in the grasshopper extracts were of microbial origin. The exact source of the enzymes should have little effect on the physiological functions of these enzymes in the insect.

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2281

DISCUSSION The grasshopper, Melanoplus bivittatus, has been shown to contain a variety of polysaccharide-degrading enzymes. In addition to an ability to degrade model substrates, these enzymes can degrade hypocotyl cell walls isolated from .5- and S-day-old red kidney bean plants. Glucose and galactose, as well as arabinose, are the sugars most effectively removed from isolated cell walls by the enzymes. This correlates well with the observation that the glycosidases most active on model substrates are the w and fl-glucosidases and p-galactosidase. When cell degradation is carried out at pH 4.0, glucose constitutes 90 per cent of the sugar removed. This observation is supported by the fact that carboxymethylcellulase is the only enzyme identified while using model substrates which is optimally active in the region of pH 4. The enzymes examined in the freeze-dried extracts and acetone powder preparations appear to be stable in storage at 0°C for extended periods of time. These preparations offer a convenient source for the purification and characterization of enzymes which could be utilized in structural analysis of plant cell wall polysaccharides. This approach has been important in the elucidation of the structure of bacterial cell wall polysaccharides (STROMINGER and GHUYSEN, 1967). Polysaccharide-degrading enzymes, which are present in a wide variety of phytophagous insects and are commonly found in the digestive tract, undoubtedly aid in the digestion of plant polymers. This conclusion is supported by the observation that the class of degradative enzymes (protease, lipase, carbohydrase) which is prominent in a particular insect species generally reflects the type of diet (herbivorous, carnivorous) on which the species feeds (HOUSE, 1965). Another important function of these polysaccharide-degrading enzymes might be in the selection of host plants and plant parts. MCALLAN and ADAMS(1961) have examined the occurrence of pectinase in the saliva of a number of species of aphids, and compared the presence of these enzymes with the types of plant penetration used by the aphids. They suggest that pectinase aids the stylet in intercellular penetration of the host plant. Pectinase is not needed for intracellular penetration. Thus, the ability to secrete pectinase could be part of host selection by this insect. MCALLAN and ADAMS(1961) h ave found also that the secretion of pectinase by a particular species depends upon the host plant from which the aphids are obtained. This same dependence of enzyme production on the plant being used as a food source has been noted in a study of cellulase production by aphids (ADAMS and DREW, 1965). ADAMS(1967) has measured the carboxymethylcellulose-hydrolysing activity in the salivary secretion of four species of aphids. He compared the cellulase activity secreted by aphids raised for 18 hr on intact plants with the activity secreted by similar aphids grown on excised leaves for a comparable period of time. He has found that 60 to 80 per cent of the aphids of four different species grown on intact plants secrete cell&se; of the same species raised on excised leaves, none of the aphids of two species produce measurable cellulase, while only about 40 per cent of the aphids in the other two species secrete cellulase. Thus, it would appear that the nature of the plant upon which the insect is feeding has

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KENNETH

W. TALMADGEANDPETERALBERSHEIM

some control over the production of polysaccharide-degrading enzymes in the insect. Recent work in this laboratory with fungal pathogens of plants has demonstrated that isolated cell walls are able to control the production of polysaccharidedegrading enzymes by pathogens grown in culture using these walls as their sole carbon source (ENGLISH and ALBERSHEIM,1969). Thus, a pathogen grown on cell walls isolated from a susceptible variety secretes greater amounts of a particular degradative enzyme than when the pathogen is grown on cell walls isolated from a resistant variety. Although there appear to be many factors affecting the host plant and plant part selection by insects (MULKERN, 1967), the control by the plant of degradative enzyme production by the insect could be an important factor in some plant-insect interactions. Acknowledgement-The authors express their thanks to Dr. GORDON making the grasshopper identifications and for his helpful suggestions.

ALEXANDER

for

REFERENCES ADAMSJ. B. (1967) A physiologic difference in aphids (Homoptera) raised on excised leaves and on intact plants. Can.J. Zool. 45,588-589. ADAMSJ. B. and DREW M. (1963) A cellulose-hydrolyzing factor in aphids. Cun.J. Zool. 41, 1205-1211. ADAMS J, B. and DREW M. (1965) A cellulose-hydrolyzing factor in aphid saliva. Can. J. Zoot. 43,489-496. ALBERWEIM P., NEVINSD., ENGLISHP., and KARR A. (1967) A method for the analysis of sugars in plant cell-wall polysaccharides by gas-liquid chromatography. Carbohydrate Res. 5,34O-345. BATEMAND. F., VANETTEN H. D., ENGLISHP. D., NEVINSD. J., and ALBER~HEIMP. (1969) Susceptibility to enzymatic degration of cell walls from bean plants resistant and susceptible to Rhizoctonia solani Kuhn. Plant Physiol. 44, 641-648. DAVISG. F. (1963) Carbohydrase activity of alimentary canal homogenates of five grasshopper species (Orthoptera: Acrididae). Archs int. Physiot. Biochim. 71,166-174. ENGLISH P. D. and ALBER~HEIMP. (1969) Host-pathogen interactions-I. A correlation between or-galactosidase production and virulence. Plant Physiol. 44, 217-224. EVANS W. L. and PAYNED. (1964) Carbohydrases of the alimentary tract of the desert locust Schistocerca gregaria Forsk. J. Insect Physiol. 10, 657-674. HOUSEH. L. (1965) Digestion. In The Physiology Insects (Ed. by ROCKSTEINM.) 2, 81% 858. Academic Press, New York. ITO T. and TANAKAM. (1959) Beta-glucosidase of the midgut of the silkworm Bonbyx mori. Biol. Bull., Woods Hole 116, 95-105. KHAN M. and FORD J. (1967) The distribution and localization of digestive enzymes in alimentary canal and salivary glands of the cotton stainer, Dysdercus fasciatus. J. Insect Physiol. 13, 1619-1628. LOWRY 0. H., ROSEBROUGH N., FARR A., and RANDALLR. J. (1951) Protein measurements with Folin phenol reagent. J. biol. Chem. 193,265-275. MCALLAN J. W. and ADAMSJ. B. (1961) The significance of pectinase in plant penetration by aphids. Can.J. Zool. 39,305-309. MCALLANJ. W. and CAMERONM. (1956) Determination of pectin polygalacturonase in four species of aphids. Can.J. Zool. 34,559-564.

of

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MCNAB J. M., NJSXINSD., and ALBERSHEIMP. (1967) Differential resistance of cell walls of Acer pseudoplatanus, Triticum vulgare, Hordeum v&are and Zea mays to polysaccharidedegrading enzymes. Phytopathology 57,625-631. MILLER G. L. (1959) Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analyt. Chem. 31,426-428. MULKERNG. B. (1967) Food selection by grasshoppers. A. Rev. Ent. 12,59-78. NEVINS D., ENGLISH P., and ALBERSHEIMP. (1967) The specific nature of plant cell wall polysaccharides. Plant Physiol. 42,900-906. NEVINS D., ENGLISH P., and ALBERSHEIMP. (1968) Changes in cell wall polysaccharides associated with growth. Plant Physiol. 43,914-922. NUORTEVA P. and LAUREMAS. (1961) On the occurrence of pectin polygalacturonase in the salivary glands of Heteroptera and Homoptera Auchenorrhyncha. Ann. Ent. fenn. 27, 89-93. ROBINSOND. (1956) Fluorimetric determination of p-glucosidase: its occurrence in the tissues of animals, including insects. Bi0chem.J. 63, 39-M. ROBINSOND. (1964) Fluorimetric determination of glycosidases in the locust Locusta migratoria and other insects. Comp. Biochem. Physiol. 12,95-105. STROMINGERJ. L. and GHUYSEN J. M. (1967) Mechanisms of enzymatic bacteriolysis. Science, N. Y. 156, 213-221.