Journal of Chemical Ecology, Vol. 17, No. 9, 1991

REASSESSMENT AND

OF T H E R O L E OF G U T A L K A L I N I T Y

DETERGENCY

IN I N S E C T H E R B I V O R Y 1

GARY W. FELTONz'* and SEAN S. DUFFEY Department of Entomology University of California Davis, California 95616 (Received February 28, 1991; accepted May 8, 1991)

Abstract--Previously it was reported that significant amounts of the tomato phenolic, chlorogenic acid, were oxidized in the digestive system of generalist feeders Spodoptera exigua and Helicoverpa zea. The covalent binding of the oxidized phenolic (i.e., quinone) to dietary protein exerts a strong antinutritive effect against larvae. In this study, we examined the lhte of ingested chlorogenic acid in larval Manduca sexta, a leaf-feeding specialist of solanaceous plants. Significant amounts of chlorogenic acid were bound to excreted protein by M. sexta when larvae fed on tomato foliage. However, in the case of M. sexta we suggest that the strong alkalinity and detergency of the midgut may minimize the antinutritive effects of oxidized phenolics. The solubility of tomato leaf protein is significantly greater at pH 9.7, representative of the midgut of M. sexta, than at pH 8.0, representative of the midguts of H. zea and S. exigua. We suggest that this increase in solubility would compensate for any loss in bioavailability of essential amino acids caused by the covalent binding of chlorogenic acid to amino acids. Furthermore, lysolecithin, a surfactant likely to contribute to the detergent properties of the midgut fluid, was shown to enhance protein solubility as well as inhibit polyphenol oxidase activity. The adaptive significance of gut alkalinity and detergency is discussed. Key Words--Manduca sexta, Lycoperskvn, phenolics, chlorogenic acid, polyphenol oxidase, midgut pH, surfactants, detergency, alkalinity, herbivore adaptations, plant defense, lysolecithin, insect nutrition. * To whom correspondence should be addressed. Approved by the Director of the Arkansas Agricultural Experiment Station. ZCurrent address: Department of Entomology, University of Arkansas, Fayettevitle, Arkansas 72701.

1821 0098-0331/91/0900-1821 $O6.5010 (r 1991 Plenum Publishing Corporution

1822

FELTON AND DUFFEY INTRODUCTION

Investigations continue to be conducted on the impact of plant phenolics and polyphenol oxidases on insect growth using the tomato plant, Lycopersicon esculentum, and the generalist noctuids Helicoverpa (=Heliothis) zea and Spodoptera exigua as a model system (Felton et al., 1989a,b; Felton and Duffey, 1990; Duffey and Felton, 1989). Upon damage to leaf tissue, liberated polyphenol oxidase oxidizes orthodihydroxyphenolics such as chlorogenic acid to the corresponding reactive, electrophilic quinone (Mayer, 1987). The quinones undergo rapid addition reactions with nucleophiles such as the thiol and amino groups of proteins (Hurrell et al., 1982; Pierpoint, 1983; Kalyanaraman et al., 1987). These reactions occur in the digestive systems of both H. zea and S. exigua, and, as a consequence, the utilization of dietary protein is impaired (Felton et al., 1989a,b; Duffey and Felton, 1989). In addition to H. zea and S. exigua, which consume a variety of plant tissues, the tomato herbivore complex includes other lepidopteran larvae such as the oligophagous Manduca sexta and the polyphagous Trichoplusia ni (Lange and Bronson, 1981). Of these species, only M. sexta and T. ni normally restrict their feeding to leaves (Lange and Bronson, 1981). The leaf-specialist M. sexta grows relatively more rapidly on tomato foliage than H. zea (Hare, 1983; Felton et al., t989a). Larval H. zea is better adapted to feeding on fruit where polyphenol oxidase activity is several orders of magnitude less than foliage (Felton et al., 1989a). One of the most striking differences in the digestive systems between the leaf specialists and the fruit-feeding lepidopteran species is the alkalinity of the midgut. The leaf-feeders, M. sexta and T. ni, have a gut pH (ca. 10.0 to 11.0) much greater than the generalist feeders Spodoptera and Helicoverpa (ca. 8.0) (Dow, 1984; Martin et al., 1987; Felton et al., 1989a). Our contention is that the highly alkaline gut conditions of the leaf-feeding herbivores represent one physiological strategy to enhance the acquisition of dietary protein, thus minimizing the impact of oxidative enzymes and their substrates on dietary protein. In this paper, we examine the effects of alkalinity and detergency upon the solubility of leaf protein, the activity of foliar oxidative enzymes, and the binding of phenolics to protein. The adaptive significance of gut alkalinity and detergency to leaf-feeding herbivores is discussed. METHODS AND MATERIALS

Insects. Eggs of M. sexta were obtained from Dr. Bruce Hammock, University of California, Davis. Neonate larvae were placed on artificial diet containing 4% fresh weight (fwt) casein (Chippendale, 1970). Isotope. Tritiated chlorogenic acid prepared by catalytic exchange was

G U T ALKALINITY AND DETERGENCY IN INSECT HERBIVORY

1823

obtained from Amersham Corp. and purified by thin-layer chromatography to remove labile tritium as described (Isman and Duffey, 1983) with a specific activity of 135 mCi/mmol. Extraction of Leaf Protein. To determine the effect of pH and oxidation on amino acid composition of extracted protein, four,leaves were excised at the petiole with a razor blade from each flowering-stage, greenhouse-grown, tomato plant (var. Ace). From each leaf, four leaflets were excised, weighed, and randomly placed in one of four extraction treatments. Each of the four treatments contained eight leaflets weighing approximately 3.0 g (fwt). Leaflets were frozen at - 2 0 ~ for 1 hr prior to homogenization and separately homogenized for 2 min in a Waring blender containing 100 ml ice-cold buffer from one of four treatments: 0.1 M sodium phosphate buffer at pH 8.0 and at pH 9.7 without diethyldithiocarbamate and at pH 8.0 and pH 9.7 with 2 mg/ml diethyldithiocarbamate. Diethyldithiocarbamate is an effective inhibitor of polyphenol oxidases (Mayer, 1987). Protein was extracted in the following manner. Following homogenization, the treatments were allowed to sit at 30~ for 2 hr to enhance solubilization of protein and to allow oxidation of phenolics. Each treatment was filtered through a single layer of cheesecloth and centrifuged at 10,000g for 30 min at 0-4~ The supernatant was used for all further enzyme and protein assays. A 50-ml aliquot of the supematant was removed from each treatment and placed on ice. Ten milliliters of 30 % trichloroacetic acid was added to each aliquot followed by centrifugation for 15 min at 10,000g. The pellets were washed with 90% methanol and recentrifuged, a process that was repeated several times. The methanol supernatant was discarded in each case. Finally, the pellets were frozen, lyophilized, and weighed to the nearest 0.1 rag. The extraction and quantification of protein was replicated three times after which the protein was pooled for each of the four treatments. Samples (ca. 50 rag) of the leaf protein were hydrolyzed in 6 N HC1 (1 mg protein/ml) for 24 hr at 100~ in vacuo and analyzed for amino acid content by automated amino acid analysis at the Protein Structure Laboratory, University of California, Davis. The total amount of amino acid per gram of foliage was calculated by multiplying the amino acid content of the protein by the average weight of precipitated protein per gram of foliage. Polyphenol oxidase and peroxidase activities from aliquots of the supernatant from the four treatment regimes were determined with chlorogenic acid and guaiacol-H202 as substrates, respectively (Ryan et al., 1982). One unit of polyphenol oxidase or peroxidase activity was defined as a change in 0D47o/ rain equal to 0,001/min. To examine the effect of pH on the solubility of leaf protein, leaves were excised from field-grown tomato plants (var. Castlemart) and immediately frozen at - 2 0 ~ for 1 hr. Leaves were lyophilized and ground to a fine powder.

1824

FELTON AND DUFFEY

Forty-milligram aliquots of leaf powder were added to centrifuge tubes containing 1 ml of 0.2 M sodium phosphate buffer at pH 7.0, 8.0, 9.0, or 10.0. The samples were placed on a shaking water bath for 60 rain at 30~ and centrifuged for 15 min at 16,000g. Ten-microliter aliquots from each sample were assayed for protein with 1.5 ml Coomassie blue protein reagent (BioRad Laboratories, Richmond, California) following the procedure of Jones et al. (1989). Ribulose-l,5-bisphosphate carboxylase/oxygenase (Sigma Chemical Co.) was used as a protein standard. Five replicates per pH concentration were tested. Effect of Alkalinity and Tomato Polyphenol Oxidase Activity on Binding of Chlorogenic Acid to Leaf Protein. To determine the effect of pH and polyphenol oxidase activity on binding of chlorogenic acid to leaf protein, partially purified tomato polyphenol oxidase was isolated from tomato foliage by precipitation with 35% ammonium sulfate following the method of Gentile et al. (1988). The enzyme preparation contained 9600 units/rag protein of chlorogenic acid oxidase activity. Less than 1% of the total peroxidase activity remained in the polyphenol oxidase enzyme preparation. Four treatments were performed: pH 8.0 with active polyphenol oxidase, pH 8.0 with heat-denatured polyphenol oxidase, pH 9.7 with polyphenol oxidase, and pH 9.7 with heat-denatured polyphenol oxidase. Each treatment contained 4 ml 0.1 M sodium phosphate buffer containing 14 /xmol unlabeled chlorogenic acid, 100,000 dpm [3H]chlorogenic acid, and 5 mg purified ribulose-l,5-bisphosphate carboxylase/oxygenase. At zero time, 0.3 mg of active or inactive polyphenol oxidase was added to the respective treatments. Treatments were placed on a water bath at 30~ and gently shaken for 2 hr. Reactions were stopped with the addition of ammonium sulfate to 95 % saturation. After vortexing, samples were centrifuged at 10,000g for 30 rain. Pelleted protein was washed repeatedly with 80% methanol and centrifuged until radioactivity remaining in the supernatant was equal to background levels. Finally, the protein pellet was solubilized in 0.1% SDS and added to ACS liquid scintillant for direct counting to calculate nanomoles of cblorogenic acid bound per milligram of protein. Determination of Binding of Chlorogenic Acid to Protein in Insect Gut. To determine if chlorogenic acid binds to protein in the digestive system when M. sexta larvae feed on tomato foliage, a 1.0/~Ci aliquot of [3H]chlorogenic acid in 50% methanol was applied to each excised tomato leaflet weighing 75-100 mg and the leaflet was allowed to air dry. A leaflet contained on average 2.2 /zmol chlorogenic acid/g fwt as determined spectrophotometrically (Broadway et al., 1986). Individual leaflets treated with the isotope were fed to individual day-old fifth instar M. sexta larvae that had been starved for 12 hr. After 3 hr of feeding each larva had ingested an entire leaflet. Feces were

G U T A L K A L I N I T Y A ND D E T E R G E N C Y IN I N S E C T H E R B I V O R Y

1825

collected as produced, pooled on an individual larval basis, and placed immediately in 5 ml ice-cold double distilled H20 containing 15 mg diethyldithiocarbamic acid to inhibit residual polyphenol oxidase activity. All larvae ceased defecating within 10 hr. After larvae (N = 10) stopped defecating, the pooled feces of each larva were homogenized for 1 min, centrifuged for 10 rain at 20,000g, and the supernatant was removed. Each pellet was reextracted in double distilled H20 containing diethyldithiocarbamic acid and 1% Triton X-100. The supernatants of a given pellet were pooled and placed on ice, and ammonium sulfate to 90% saturation was added to precipitate protein. After 30 rain, samples were centrifuged for 30 min at 20,000g, and the supernatant was removed for direct counting of [3H]chlorogenic acid by liquid scintillation. Each pellet was washed with 80% methanol and centrifuged to remove residual noncovalently bound chlorogenic acid. After several successive washings, the supernatants were pooled for each sample, and radioactivity was counted by liquid scintillation. Finally, the pelleted protein was solubilized in 0.1% sodium dodecylsulfate, and covalently bound [3H]chlorogenic acid was counted. The unbound chlorogenic acid represented combined radioactivity of the supernatants from the aqueous and methanolic extracts. A small aliquot also was applied to cellulose thin-layer plates and chromatographed as described (Felton et al., 1989a) to verify the formation of a chlorogenic acid-amino acid conjugate. A 50-/zl aliquot of hemolymph was removed from each larva 24 hr after it had ingested the leaflet. Radioactivity of the hemolymph was determined directly by liquid scintillation. Effect of Surfactant on Foliar Oxidases and Protein Solubility. To determine if lysolecithin, a surfactant that may contribute to midgut detergency, affects foliar polyphenol oxidase activity, tomato leaflets (16 g) were divided into four equal parts and homogenized separately in ice-cold 0.1 M potassium phosphate buffer, pH 6.8, containing one of the following treatments: 0.02%, 0.04%, 0.08% lysolecithin, and a control lacking added surfactant. Following homogenization, the treatments were centrifuged for 20 min at 10,000g and assayed in triplicate for polyphenol oxidase activity with chlorogenic acid by measuring increase in absorbance at 470 nm (Ryan et al., 1982). The experiment was replicated five times. To determine if lysolecithin affects protein solubility, 40 mg lyophilized leaf powder was added to 1 ml 0.1M sodium phosphate buffer, pH 7.0, with and without 0.04 % lysolecithin. The treatments were treated similarly to above experiments on effects of pH on protein extractability. The concentration of lysolecithin was selected because it stimulates the surfactant properties of M. sexta midgut fluid (Martin and Martin, 1984). Protein was quantified following Jones et al. (1989).

1826

FELTON AND DUFFEY RESULTS

Effect of pH and Oxidase Activity on Amount and Composition of Amino Acids in Extracted Protein. Both pH and polyphenol oxidase activity significantly affected the amount of extractable protein indexed as TCA-precipitable protein (Figure 1). Total amino acids are defined as the total molar content of amino acids in hydrolyzed protein. Essential amino acids are defined as essential amino acids required for larval growth of Helicoverpa zea (Rock and Hodgson, 1971). Significantly more total amino acids were extracted at pH 9.7 in both treatments (with or without polyphenol oxidase activity) than at the lower pH of 8.0 (Figure 1). Likewise, the inhibition of polyphenol oxidase resulted in greater amounts of extracted protein amino acids at both pH values. In treatments with polyphenol oxidase activity, nearly 42 % more total amino acids and 60% more essential amino acids were obtained at pH 9.7 than at 8.0 (Figure 1). Similar effects were observed in treatments lacking polyphenol oxidase activity, in which approximately 43 % more total and essential amino acids were extracted at the higher pH. The method of amino acid analysis does not take into account the essential amino acid, tryptophan, because it is destroyed during hydrolysis. The effects o f pH and polyphenol oxidase treatments on individual amino acids are shown in Table 1. Without exception, greater absolute amounts of all amino acids were obtained at pH 9.7 than at 8.0. In the case of the two most

30

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[ ] E s s e n t i a l Amino Acids

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o

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20 15

0

.~_

E

10

"6 E

5 0

pH 8,0 + PPO

pH 8.0

pH 9.7 + PPO

pH 9.7

Treatment

FIG. 1. The effect of pH and polyphenol oxidase activity on the extraction of essential and total amino acids from tomato foliage. PPO = polyphenol oxidase activity. Values are based upon single analyses of pooled replicates.

1827

GUT ALKALINITY AND DETERGENCY IN INSECT HERBIVORY

TABLE 1. EFFECT OF

pH

AND POLYPHENOL OXIDASE ACTIVITY ON EXTRACTABILITY OF

TR1CHLOROACETIC ACID-INSOLUBLE AMINO ACIDS"

Amino acid

pH 8.0 + PPO

pH 8.0

pH 9.7 + PPO

pH 9.7

ASP THR SER GLU PRO GLY ALA VAL MET ILE LEU TYR PHE LYS ARG HIS

1615 896 772 1757 694 1457 1394 1210 281 766 1474 600 684 1068 848 490

1837 1034 882 2156 885 1431 1544 1329 307 788 1712 687 803 1390 1007 617

2205 1245 1095 2435 1133 2486 1911 1684 327 1078 2013 853 978 1385 1174 672

2595 1479 1250 3109 1296 2061 2155 1925 513 1170 2510 1005 1179 1909 1332 860

"Values for amino acids are expressed as nanomoles/per gram of foliage (fresh weight).

limiting amino acids (i.e., essential amino acids at the lowest concentrations), 16% more methionine and 37% more histidine were obtained at the more alkaline pH. The presence o f polyphenol oxidase activity reduced the total quantity of amino acids at both pH levels. Apparently, the primary cause of this reduction is decreased solubility of phenolic-bound protein (Betschart and Kinsella, 1973). Oxidized chlorogenic acid is known to bind covalently to amino acids such as lysine, histidine, and methionine (Pierpoint, 1983; Barbeau and Kinsella, 1983). The presence of polyphenol oxidase activity reduced the relative amounts o f these amines. When the relative mole percent of amino acids for each protein treatment was taken into account, only methionine, lysine, and histidine were negatively affected by polyphenol oxidase activity (Figure 2). Methionine was reduced by 26%, lysine by nearly 16%, and histidine by 8% at pH 9.7 in the presence o f polyphenol oxidase activity. At pH 8.0, lysine was reduced by 11.7%, histidine by 8%, and methionine apparently was unaffected by oxidative activity. However, despite the greater relative loss in methionine, histidine, and lysine at pH 9.7, a greater absolute amount of these amino acids was soluble at the higher pH. Our amino acid analyses do not take into account potential loss of methionine to methionine sulfoxide formation via chlorogenoquinonemediated oxidation (Igarashi and Yasui, 1985).

1828

FELTON AND DUFFEY

30

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B lysine ~ histidine ~ methionine

Q

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pH 9.7 Treatment

FIG. 2. The effect of polyphenol oxidase activity on the loss of lysine, histidine, and methionine in tomato foliage. Reductions are based upon comparison of molar ratios of amino acids between treatments with and without polyphenol oxidase activity. Values are based upon single analyses of pooled replicates. The addition of the inhibitor, diethyldithiocarbamate, abolished polyphenol oxidase activity and reduced peroxidase activity by greater than 85 % at both pH levels (Table 2). The activity of both enzymes was markedly reduced by the higher pH, with nearly 35 % and 65 % reductions in polyphenol oxidase and peroxidase, respectively. When leaf powder was extracted at neutral and at alkaline pH levels, significantly more protein was soluble at alkaline pH (Figure 3; r = 0.880, P < 0.01). The relationship between pH of extraction buffer and percent protein was significantly correlated, and the slope of the regression line was significantly greater than zero (P < 0.01). Nearly 25% more soluble protein on a wet weight basis was observed at pH 10.0 compared to pH 7.0. Furthermore, in the bell pepper plant, Capsicum annuum, another solanaceous plant host of M. sexta, 45% more soluble foliar protein was obtained at the higher pH (unpublished data).

Effect of Alkalinity and Tomato Polyphenol Oxidase Activity on Binding of Chlorogenic Acid to Leaf Protein. Significantly more chlorogenic acid was bound to protein in the presence of polyphenol oxidase (Figure 4). In treatments containing polyphenol oxidase activity, pH had little effect on the amount of chlorogenic acid bound to protein (58.9 vs. 64.3 nmol/mg protein for pH 8.0 vs. 9.7, respectively). In the absence of polyphenol oxidase activity, pH significantly affected binding with nearly 60% more chlorogenic acid bound per milligram of protein at the higher pH of 9.7.

1829

GUT ALKALINITYAND DETERGENCY IN INSECT HERBIVORY

TABLE 2. EFFECTS OF pH AND DIETHYLDITHIOCARBAMICACID ON ACTIVITY OF FOLIAR OXIDASES

Treatment

PPO activity"

POD activity'

8.0 8.0 + inhibitor" 9.7 9.7 + inhibitor

100 0 65.5 0

100 15.0 33.8 4.1

"PPO activity = relativepolyphenoloxidase activity measured with chlorogenic acid as substrate. ~'PODactivity = relative peroxidase activity measured with guaiacol and hydrogen peroxide. 'Inhibitor = diethyldithiocarbamicacid at 2 mg/ml buffer.

Determination of Binding of Chlorogenic Acid to Protein in Insect Gut. Nearly 39% of the excreted [3H]chlorogenic acid was bound to protein when larvae were fed [3H]chlorogenic acid-treated leaflets (Figure 5). Thin-layer chromatographic analyses of aqueous aliquots of the protein precipitate also indicated that chlorogenic acid was bound to protein because greater than 90% of the radioactivity was associated with a single band (Rf = 0.85-0.95) that was both phenolic and amine positive (Felton et al., 1989a). A relatively large amount (ca. 14%) of the ingested radioactivity was retained in the hemolymph 24 hr following ingestion of the treated leaflet (Figure 5). Two to three times as much radioactivity was found in the hemolymph of M. sexta compared to S. exigua and H. zea larvae (Felton et al., 1989a). Radioactivity recovered in feces and hemolymph accounted for ca. 64% of the ingested radioactivity. Effect of Surfactant on Foliar Oxidases and Protein Solubility. The addition of the gut surfactant lysolecithin significantly decreased polyphenol oxidase activity (Figure 6).At a concentration of 0.04% lysolecithin, the activity of polyphenol oxidase was reduced by 24%. This concentration of lysolecithin corresponds to a critical micelle concentration equal to that reported in the midgut fluids of M. sexta larvae (Martin and Martin, 1984). Peroxidase activity was not affected at the surfactant concentrations tested (data not shown). The addition of 0.04% lysolecithin to the extraction buffer enhanced the solubility and/or extractability of leaf protein (Table 3). Approximately 9% more soluble protein occurred in the treatment with 0.04% lysolecithin. DISCUSSION

Alkalinity and detergency have been proposed as adaptations to avoid the antidigestive effects of tannins (Feeny, 1970; Berenbaum, 1980; Martin et al., 1987). Alkalinity reduces hydrogen bonding of tannins with proteins, thus pre-

1830

FELTON AND DUFFEY 3.2

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pH Fic. 3. Effect of pH on the extraction and solubility of tomato leaf protein. Each point represents the mean of three determinations. Error bars represent 95 % confidence limits. Points not followed by the same letter are significantly different at P < 0.05. venting protein precipitation (Martin and Martin, 1983; Pierpoint, 1983; Zucker, 1983; Hastam, 1988). Surfactants such as lysolecithin are highly effective at inhibiting the formation of insoluble tannin-protein complexes and appear to be widely present in insect midguts (Martin and Martin, 1984; Martin et al., 1987; Blytt et al., 1988). However, many lepidopteran larvae possess strongly alkaline, detergent gut fluids despite the apparent absence of tannins from their host plants (e.g., M. sexta; Martin et al., 1987). In these instances, it cannot be argued that alkalinity or detergency are adaptations for circumventing the antidigestive effects of tannins. We do not question the importance of alkalinity and detergency as adaptive factors mitigating tannin toxicity. These properties may play a significant role in enhancing the solubilization of dietary protein in the digestive tract. This is especially important considering that leaves are considered a protein-limited food source for most insect herbivores (White, 1978; Mattson, 1980; Brodbeck and Strong, 1987). The plant biochemistry literature is replete with examples that support our findings that alkalinity enhances solubility of leaf nitrogen (e.g., Singer et al., 1952; Betschart and Kinsella, 1973). Leaf protein is generally more soluble and chloroplasts are more effectively disrupted at higher pH than at acidic or neutral pH (Betschart and Kinsella, 1973; Jones et al., 1989). Moreover, alkali extraction is essential for solubilizing cytoplasmic proteins in tobacco leaves, resulting in 25-50% more protein than at neutral or acidic pH (Singer et al., 1952). We have observed that increased levels of dietary protein in H. zea or S. exigua may substantially or completely alleviate the growth inhibition caused by the

GUT ALKALINITY AND DETERGENCY IN INSECT HERBIVORY

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pH 9.7

pH 9.7 + PPO

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FIG. 4. Effect of pH and polyphenol oxidase activity on the binding of chlorogemc acid to ribulose-1,5-bisphosphate carboxylase/oxygenase. Each bar represents the mean of three determinations. Bars not followed by the same letter are significantly different at P < 0.05. PPO = polyphenol oxidase activity. binding of chlorogenoquinone to protein (Duffey and Felton, 1989; Felton et al., 1989b, unpublished data). Moreover, the nutritional quality of protein extracted at alkaline pH (8.0-12.0) is often superior to protein extracted at neutral pH (Woodham, 1983). Polyphenol oxidase activity is markedly reduced at alkaline pH from many solanaceous host plants of M. sexta, e.g., tomato, bell pepper, eggplant, and potato (see Table 1) (Felton et al., 1989a; Fujita and Tono, 1988; Sharma and Ali, 1980; Kang et al., 1983). Although the enzyme is active across a broad pH spectrum in many plant species, the pH optimum for most species is in the acidic to neutral range (i.e., 5.0-7.0; Mayer and Harel, 1979). It is noteworthy that none of the major lepidopteran herbivores of the tomato plant have a gut pH near the optimum of ca. 7.0 for enzyme activity (Felton et al., 1989a). Thus, alkalinity may improve herbivore performance on marginal food sources by reducing the activities of polyphenol oxidase and enhancing the acquisition of greater amounts of dietary protein. Although alkaline pH reduces polyphenol oxidase activity, the formation of chlorogenoquinone can occur nonenzymatically in basic conditions (Huh'ell et al., 1982; Barbeau and Kinsella, 1983, 1985; Cilliers and Singleton, 1989). However, Hurrell et al. (1982) found that significant nutritional damage to protein by nonenzymatically produced chlorogenoquinone was only observable under conditions of continuous oxygenation and stirring. Recently, Appel and Martin (1990) reported that the midgut of M. sexta larvae possessed strong

1832

FELTON AND DUFFEY 80 61.25 60

38.75 m o q)

4o

n

13.84

20

Percent Retained in Hemolymph

Percent Protein Bound in Feces

Percent Unbound in Feces

F~c. 5. The uptake and excretion of [3H]chlorogenic acid by fifth-instar Manduca sexta larvae.

reducing properties (redox potential = - 136 mV) and a slightly anaerobic environment, which may further minimize the antinutritive effects induced by the oxidation of phenolics such as chlorogenic acid. However, whether a specific quinone species will be reduced depends upon the difference in redox potentials of the midgut and the quinone. Also, it must be reiterated that once the quinones are formed and bound to nucleophilic portions of amino acids, changes in redox potentials will not disrupt the irreversible covalent bonds that form. The foreand hindguts of M. sexta were reported to be oxidizing, and it is possible that much of the binding that we observed took place in these regions of the gut. Binding occurring in the foregut will still impair the utilization of amino acids. Furthermore, the-activity of tomato polyphenol oxidase is so great that it is likely much of the phenolic binding to protein occurs in the initial stages of ingestion. The midgut reducing conditions may still serve to minimize nonenzymatic oxidations of other phenols. The amount of chlorogenic acid bound to excreted protein by M. sexta (i.e., 38.8%; Figure 4) was similar to that observed in S. exigua and H. zea (38 % and 49 %, respectively, Felton et al., 1989a). In the case of M. sexta, the antinutritive effect of quinone binding may be mitigated by the increased levels of soluble amino acids occurring at the higher midgut pH (Figure 1, Table 1). Our estimates on protein solubility in the digestive system of M. sexta may be quite conservative because the midgut pH has been reported as high as 11.3 (Dow, 1984). The upper limit in gut pH is restricted by the energetic costs required for maintaining a high pH and the negative consequences of high pH on protein quality and enzyme function. At pH levels of 12 or more, protein nutritional

1833

G U T A L K A L I N I T Y A ND D E T E R G E N C Y IN I N S E C T HERBI VORY

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Percent Lysolecithin FIG. 6. Effect of the surfactant, lysolecithin, on tomato foliar polyphenol oxidase activity. Each point represents the mean of one replicate performed in triplicate. PPO = polyphenol oxidase activity.

quality is degraded as a consequence of amino acid racemization, thiol oxidation, and formation of toxic cross-linked amines such as lysinoalanine (Friedman, 1982; Pierpoint, 1983; Whitaker and Feeney, 1983; Finot, 1983; Hurrell and Finot, 1985). At such a high pH, digestive enzymes would likely denature and be unable to function. These restrictions, therefore, would partially determine the upper limit for the evolution of gut pH. The surfactant lysolecithin also has the ability to enhance protein solubility (Table 3) and diminish polyphenol oxidase activity (Figure 6). The reduction in polyphenol oxidase activity may be due to protein denaturation or conformational changes. Certain detergents are known to inactivate polyphenol oxidase activity (Meyer and Biehl, 1981). The ability of surfactants to enhance extraction of leaf protein is due to the disruption of chloroplasts and other protein bodies (Betschart and KinseUa, 1973). Lysolecithin also has been shown to solubilize membrane-bound protein and phytosterols in plant cells (Sandstrom and Cleland, 1989). In addition, surfactants may play an important role in protein digestion by denaturing substrate protein and exposing more binding sites for proteolytic enzymes (Mole and Waterman, 1985). In conclusion, we concur with the theory that the alkalinity and detergency of midgut fluids of lepidopteran larvae represent adaptations to leaf-feeding. Whereas previous investigations have advocated these properties as adaptations to avoid toxicity from tannin-protein complexes (Feeny, 1970; Martin and Mar-

1834

FELTON AND DUFFEY TABLE 3. EFFECT OF LYSOLECITHIN ON PROTEIN EXTRACTION

Treatment

Protein ( % )"

95 % confidence limit

Control 0.04% lysolecithin

2.35 2.59

2.29-2.42 2.51-2.66

"Percent protein expressed as dry weight percent.

tin, 1984; Martin et al., 1987), we contend that they may have other significant functions for folivores. Both detergency and alkalinity were shown to reduce foliar polyphenol oxidase activity and enhance the extraction and/or solubility of leaf proteins. Moreover, surfactants may aid in the digestion of protein and in the acquisition of other key nutrients such as sterols (Sandstrom and Cleland, 1989). Gut alkalinity also may be important for extracting hemicelluloses of cell walls (Terra, 1988). Thus, gut alkalinity and detergency may represent important nutritional adaptations for facilitating the uptake of essential or limiting nutrients from host plants. Acknowledgments--Research was supported by USDA competitive grants awarded to S.S.D. (87-CRCR-I-2371) and S.S.D. and G.W.F. (89-37250-4639). The authors wish to thank Dr. D.T. Johnson and Mr. J. Workman for critical review of the manuscript. We also thank Dr. M.M. Marlin for kindly providing an unpublished manuscript by Appel and Marlin. This manuscript was published with the approval of the Director of the Arkansas Experiment Station.

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