Endogenous and induced monooxygenase activity in gypsy moth larvae feeding on natural and artificial diets.код для вставкиСкачать
Archives of Insect Biochemistry and Physiology 10:47-56 (1 989) Endogenous and Induced Monooxygenase Activity in Gypsy Moth larvae Feeding on Natural and Artificial Diets Carol A. Sheppard and Stanley Friedman Department of Entomology, University of lllinois at Urbana-Champaign NADPH oxidase activity was measured in third to sixth instar gypsy moth larvae fed oak or pine foliage. Activity levels ranged from 400 t o 1,900 prnol NADPH oxidized/min/mg microsomal protein, but enzyme activity was not correlated with host plant ingested. Similarly, activity levels in larvae fed diets containing inducers, such as the terpenoid a-pinene or pentamethylbenzene, ranged from 700 to 1,500 pmol NADPH oxidized/min/rng protein, levels that were comparable to those measured for larvae fed control diets. 0-demethylase activity in older instar gypsy moth larvae fed pine averaged 109 pmol p-nitrophenol/min/mg protein, and activity levels in those fed diet containing apinene ranged from 22 to 55 pmol/min/mg protein. Although statistically significant, these induced 0-demethylase levels are well below those observed for Hehothis zea larvae. Our findings indicate that monooxygenases play a minor, if any, role i n the ability of later instar gypsy moth larvae to develop successfully on pine foliage. Key words: Lymantria dispar, Heliothis zea, Estigmene acrea, Diptera, a-pinene,pine, oak, MFO INTRODUCTION Larvae of the gypsy moth, Lyrnuntriu dispar, are major defoliators of northeastern United States shade and forest trees. Although generally considered to be polyphagous herbivores, the larvae show strong host plant preferences. These have been categorized by Mosher [l]as follows: class I, in which oaks predominate, species preferred by all larval instars; class 11, which includes the gymnosperms, favored ”after the early larval stages”; class I11 and IV species, least preferred. Recent research has shown Mosher’s class I and I1 categories to be correctly separated. First instar larvae suffer 100%mortality on coniferous foliage, whereas larvae switched at the fourth instar from black oak to pine foliage achieve development rates and female fecundities that are comparable to or better than those Received August 4,1988; accepted November 7,1988. Carol A. Sheppard’s current address is USDA, ARS, BARC-East, Insect Reproduction Laboratory, Building 306, Room 323, Beltsville, MD 20705. Address reprint requests there. 01989 Alan R. Liss, Inc. 48 Sheppard and Friedman of nonswitched larvae . It is well to note that this ontogenetic broadening of larval host plant range contains serious economic implications, in that deciduous trees can withstand two or three defoliations, but conifers die after a single such incident [ 3 ] . The striking contrast between oak and pine foliar chemistry raises the question of what larval functional changes underlie its ability in later life to utilize pine as an alternative host plant. Pine is characterized by high concentrations of potentially toxic terpenoids such as a- and P-pinene [4, and references therein], which may be detoxified enzymatically by cytochrome P-450-dependent monooxygenases(polysubstratemonooxygenases,mixed-function oxidases) [5,6]. In contrast, oak foliage is rich in tannins, compounds whose ability to form complexes with proteins accounts for their putative role as digestibility reducers [7,8]. However, it has been argued that the strongly alkaline pH conditions of gypsy moth larval midgut would dissociate such complexes . The insect literature is rife with reports that MFO* activity is induced following ingestion of natural or artificial diets containing terpenes [5,6,10-181. Therefore, to investigate the role played by these enzymes in gypsy moth utilization of pine foliage, we determined NADPH oxidase and O-demethylase activity levels in larvae fed oak and pine foliage and artificial diets containing known inducers of monooxygenase activity. The results of our studies, which include comparative assays performed with other lepidopterous larvae and adult Diptera, are the subject of this report. MATERIALS AND METHODS Insect Rearing and Feeding Gypsy moth larvae used in this study were maintained at 24"C, 40-50% relative humidity, on a 16:s L/D cycle. Eggs from which larvae were reared were obtained from facilities in Otis Air Force Base, Otis, MA. Other lepidopterous larvae used in these experiments were reared from matings among feral individuals-Hyalophora cecropia-or from laboratory colonies-Heliothis zea and Estigmenearea. For experiments in which larvae were fed fresh foliage, bur oak (Quercus macrocarpa), white pine (Pinus strobus), and wild cherry (Prunus serotina) were collected from the same location in a forest preserve, in which trees are free from direct chemical treatment and "drift" from agricultural fields is considered to be minimal. The cut ends of branches or stem ends of leaves were inserted into a water bottle to maintain turgidity, and foliage was replaced every 2 to 3 days. Since gypsy moths perish in the first instar if fed a diet of pine needles, in spite of feeding vigorously and producing copious amounts of feces for a limited period of time, larvae were reared through the first two instars on HWG diet, and transferred to either oak or pine as newly molted third instar larvae. When reared on artificial diet, the HWG diet formulated by Bell et al. , was used. Either 0.2% (+)-a-pineneor PMB was incorporated into *Abbreviations: HWG = high w h e a t germ; MFO = mixed-function oxidase; PMB methylbenzene; PMSF = phenlymethanesulfonyl fluoride. = penta- Monooxygenase Activity in Gypsy Moth larvae 49 the diet as an MFO inducer. The same batch of diet without addition served as a control in these experiments. Adult Diptera (Phorrnia regina and Musca dornestica) of both sexes were taken for experiments from laboratory stocks maintained on a diet of sucrose and water. Chemicals ( + )-a-Pinene, p-nitroanisole, p-nitrophenol, and PMB were purchased from Aldrich Chemical Co., Milwaukee. P-NADPH was obtained from the United States Biochemical Corp. , Cleveland. All other biochemicals were purchased from Sigma Chemical Co., St. Louis. Enzyme Preparation and Activity Assays Midgut tissue was obtained from ”full gut” larvae that had been feeding on a given diet or foliar type for 3 days prior to enzyme assay. The tissue was prepared by removing it whole and transferring it to ice-cold sucrose medium (0.25 M sucrose containing 1mM EDTA, 1%polyvinylpyrrolidone and 2 mM PMSF ). After cutting the gut lengthwise, the tissue was gently shaken to free it of its contents and was rinsed in fresh sucrose medium prior to homogenization in sucrose medium containing 0.5 mM PMSF. The method of Crankshaw et al.  yielded the most active larval enzyme preparations and was used to isolate microsomal fractions.For adult dipteran abdominal microsomes, the procedure described by Folsom and Hodgson  was employed. In assays of microsomal MFO activity as measured by NADPH oxidation, the following procedure was employed [21,22]. Duplicate amounts of microsomal protein ranging from 0.05 to 1.0 mg were incubated in 10-mlErlenmeyer flasks with 0.13 pmol NADPH contained in a final volume of 1.0 ml of 50 mM Tris buffer, pH 7.4. Flasks containing corresponding amounts of microsomal protein but no NADPH served as blanks. Incubations were carried out in a shaking water bath at 30°C. Absorbance at 340 nm was recorded on a dual beam spectrophotometer at zero time and at 30 min after initiation of the reaction, and NADPH oxidation was calculated from the decrease in absorption through that time span. 0-Demethylation of p-nitroanisole to p-nitrophenol was measured by slight modification of a procedure previously reported . Microsomal fractions were isolated as described above, and the reaction was carried out in 10-ml Erlenmeyer flasks for 30 min at 32°C in a shaking water bath. Microsomal protein ranging from 1.5 to 5 mg (in the case of gypsy moths) or 0.8 to 3 mg (H. zea) was added to initiate the reaction in “experimental” flasks, which contained the following in a final volume of 1.6ml: 0.5 mM NADP, 2.5 mM glucose6-phosphate, 2 units glucose-6-phosphatedehydrogenase, 7.5 mM MgCI2, 0.5 mM p-nitroanisole, and 100 mM Tris-HC1, pH 7.8. The reaction was terminated by the addition of 400 pl of 1 N HCl. “Control” flasks contained the same reaction mixture and final volume as did experimental flasks, except that HCl was added immediately after the addition of the microsomal protein at zero time. Upon termination of the reaction, the acidified reaction mixture was “vortexed” with 2 ml chloroform, and after centrifugation, 1 ml of the chloroform layer was “vortexed” with 1ml of 0.5 N NaOH. Following centrif- Sheppard and Friedman 50 ugation, the absorbance of the NaOH layer at 400 nm was determined for experimental vs. control samples, and results were calculated as mean p-nitrophenol produced/min/mg microsomal protein. Protein was determined by the method of Bradford , with human yglobulin Fraction I1 serving as a standard and protein dye reagent purchased from BioRad Corp., Richmond, CA. Experiments with adult Diptera were repeated at least twice, and all other experiments were replicated three or more times. A two-tailed Student's t-test was used for statistical analysis of the difference between the means of two groups, probability < .05 being considered significant. RESULTS Table 1summarizes the NADPH oxidase activities of three cohorts of larvae fed fresh oak or pine foliage through the 1985 season. In four comparisons, pine-fed larvae have significantlyhigher levels of endogenousNADPH-oxidation activity than do their oak-fed counterparts, and in three comparisons the reverse is true. Gypsy moth microsomal MFO activity also appears to be unaffected by seasonal variations in foliage [25, and references therein], since there is no evident correlation between the phenology of the ingested foliage and NADPH oxidase levels. NADPH oxidase activities in two species of Diptera and two additional species of Lepidoptera are shown in Table 2. Enzyme activity levels in the lepidopterous species, H. cecropia and E. acrea, fed cherry foliage are comparable, averaging about 3,600 pmol NADPH oxidized/min/mgprotein. The basal activities of microsomal enzymes isolated from housefly (M. domesticu) and blowfly (P. regina) abdomens range from nearly 5,000 to 13,000 pmoYmin/mg protein. For comparative purposes, NADPH oxidase levels in gypsy moth and saltmarsh caterpillar ( E . acrea) larvae fed a diet containing either of two known monooxygenase inducers, a-pinene or PMB, are presented in Table 3. No differences in enzyme activity are observed between gypsy moth larvae reared on control and inducer diets. In contrast, activity levels of salt marsh larvae TABLE 1. NADPH Oxidase Activity in Gypsy Moth Larvae Fed Fresh Foliage Cohort 1 2 3 Instar Date" 5th 6th 3rd 4th 5th 3rd 4th 5th 6th June 7 July 10 August 24 Enzyme Activityb Oak leaves Pine needles 1,933 567 1,400 600 767 867 600 833 1,400 t 203 * t k t * t t t 93 153 20 43 167 133 117 80 'Date when newly molted 3rd instar larvae were transferred from diet to foliage. bData expressed as pmol NADPH oxidized/min/mg protein S.E. 'Indicates P < .05%. Comparisons are within rows of a given cohort. x * 933 1,200 1,200 1,033 1,567 433 967 1,000 1,167 2 57' t 133* 2 t 2 t t * t 140 76* 107' 67* 100" 70 60" 51 MonooxygenaseActivity in Gypsy Moth larvae TABLE 2. NADPH Oxidase Activity in Representative Species of Diptera and Lepidoptera Insect Age M. dornestica (adult abdomen) P. regina (adult abdomen) H . cecropia (midgut) E . acrea (midgut) “Data expressed as 9 days 11days 2 days 4 days 4th instar 5th instar 5th instar 5th instar Diet Enzyme activity” Sucrose Sucrose Cherry foliage Cherry foliage Oak foliage 10,600 13,467 7,000 4,933 3,327 2,627 3,927 3,420 t 800 f 1,267 t f t f t f 333 300 233 99 567 467 x pmol NADPH oxidizediminlmgprotein t S.E. fed either of the inducer diets average 1.6 times more than those of their noninducer-fed counterparts. In addition, basal (noninduced)activity levels in salt marsh larvae are higher than the corresponding values for gypsy moth larvae fed the same batch of HWG diet. Measurements of 0-demethylase activity in older instar gypsy moth larvae fed fresh foliage or HWG diet with and without a-pinene are given in Table 4. The enzyme activity of fifth instar larvae fed pine foliage is significantly greater than that of larvae fed oak foliage. Fifth and sixth instar larvae fed HWG diet containing 0.2% a-pinene show a comparatively small, although statistically significant (see below and “Discussion”), increase in 0-demethylase activity over those fed HWG without inducer. 0-Demethylase activity in Heliofhiszeu larvae fed HWG diet containing 0.2% a-pinene is presented in Table 5 for comparative purposes. After 2 days of feeding, activity levels are 1.7 times greater in larvae fed the inducer-containing diet than those fed control diet. Enzyme activity levels in a-pinene-fed larvae rise from 290 to 800 pmol p-nitrophenollminlmgprotein prior to gut purging at day 3. Larvae in the empty gut stage fed a-pinene for 3 days have activity TABLE 3. NADPH Oxidase Activity in Larvae Fed Inducer Compounds Insect Instar L. dispar E . acrea 3rd 4th 5th 6th 4th Insect Instar L. dispar 4th 6th 4th 5th E. acrea x HWG diet 767 f 734 t 900 f 837 t 1,531 f Enzyme Activity HWG w/0.2% a-pinene 694 759 630 731 2,232 68 193 34 83 29 HWG diet t 200 f 230 t 103 f 50 t 91* Enzyme Activitf HWG w/0.2% PMB 1,030 t 137 1,531 f 328 4,234 t 430 313 3,587 * “Data expressed as pmol NADPH oxidizediminlmg protein f S.E. *IndicatesP < .05%. Comparisons are within rows. 1,397 1,301 7,268 5,020 f t f t 237 135 800* 386* 52 Sheppard and Friedman TABLE 4. 0-Demethylase Activity in Gypsy Moth Larvae Instar 5th 5th 6th Diet Enzyme activity Oak foliage Pine foliage HWG diet HWG w/0.2%a-pinene HWG diet HWG w/0.2%a-pinene 57 2 1 109 2 4* 13 2 4 22 2 4* 18 2 1 55 2 2* "Data expressed as X pmol p-nitrophenollminlmg protein i S.D. 'Indicates P < .05%. Comparisons are within larval instars, within a given dietary regime. levels comparable to those measured for larvae with full guts fed the pinene diet for 2 days. Table 6 shows development time and pupal weights for L. dispar larvae reared from egg eclosion to pupation on HWG diet or diet containing 0.2% a-pinene. In the first experiment, male pupae from HWG diet weighed more than those from the HWG w/pinene diet. Other than this, no appreciable differences were observed between insects reared on control and inducer-containingdiet. DISCUSSION The importance of microsomal cytochromeP-450-dependentmonooxygenases in the biotransformation of lipophilic xenobiotics, particularly drugs, became apparent with mammalian studies conducted in the mid-1950s. These enzymes are now known to be ubiquitous in aerobic organisms; in insects, MFOs play a primary role in hormone metabolism, pesticide resistance, and response to plant allelochemicals [see 11, 12,26-28 for review]. Although a large array of reactions is catalyzed by the cytochrome P-450 monooxygenases, e.g., epoxidation, hydroxylation, N-demethylation, 0demethylation, etc., all reactions require reducing equivalents in the form of NADPH. NADH is usually an ineffective substitute for NADPH, although it may have a synergistic effect when used in conjunction with the latter . Hence, demonstration of increased NADPH oxidation activity should serve TABLE 5. 0-Demethylase Activity in Fifth Instar Heliothis zea Larvae Day Gut lumen Diet Enzyme activityb 2 2 2.5 2.5 3 3 Full Full Full Full Empty Empty HWG HWG ~ 1 0 . 2 % a-pinene HWG HWG ~ 1 0 . 2 % a-pinene HWG HWG w/0.2% a-pinene 172 2 10 290 8" 268 2 6 818 2 7* 642 4 255 2 50* * "Day indicates number of days on diet, from day of eclosion to 5th instar to day of experiment. bData expressed as % pmol p-nitrophenoliminlmg protein 2 S.D. *IndicatesP < .05%. Comparisons are within a given day. Monooxygenase Activity in Gypsy Moth larvae 53 TABLE 6. Gypsy Moth Development on Normal and Inducer Diets Diet Experiment 1 HWG HWG w/0.2% a-pinene Experiment 2 HWG HWG w/0.2% a-pinene Days" 20.3 22.9 2 23.0 23.1 4 4 2 Malesb 1.8 1.7 451 & 83 358 2 67 1.3 1.4 376 343 2 F PuDal weights (N) Females (9)" (6) 52 (12) 31 (13) 1,047 993 ? k (N) 243 (16) 259 (20) 1,090 t 262 (12) 941 ? 267 (9) days t S1D. from day of egg eclosion to day of molt to 5th instar. bValues are X weight in mg & S.D.; (N) indicates number of individuals. *Indicates P < .05% in a two-tailed t-test. Comparisons are within columns, within a given experiment. as a reasonable indicator of monooxygenase activity. Given this, our results suggest that monooxygenases play only a minor role, if any, in the ability of later instar gypsy moth larvae to feed successfully on pine. A summary of experiments conducted through the 1985 season reveals no consistent pattern among NADPH oxidase activities of gypsy moths fed oak or pine foliage (Table 1). There is neither a correlation between NADPH oxidase activity and foliar type or phenology nor an ontogenetic increase in activity levels. The highest NADPH oxidase activity observed in fifth instar gypsy moth larvae fed oak leaves averages about 1,900pmol NADPH oxidizedmidmg microsoma1 protein (Table l), which is less than 50% that of fifth instar salt marsh larvae fed the same foliage (Table 2). That suitable assay conditions existed for NADPH oxidation may be seen by the values obtained for the cyclorrhaphous Diptera using the same assay system. These amounts, ranging from 5,000 to over 13,000 pmol NADPH oxidized/min/mgprotein (Table 2), are comparable to those reported in the literature . Artificial diet containing 0.2% a-pinene, the concentration commonly employed to induce monooxygenase activity in insects, fails to elicit enhanced activity levels in gypsy moth larvae from the third through the sixth instar (Table 3). In contrast, a 1.6-fold increase in endogenous NADPH oxidase activity is observed in fourth instar salt marsh larvae fed the pinene diet. Similarly, sixth instar southern armyworms fed this same dietary level of a-pinene exhibit significant increases in endogenous NADPH oxidation . In keeping with the results obtained with a-pinene in the diet, 0.2% PMB has no effect on gypsy moth NADPH oxidase activity (Table 3). However, fourth and fifth instar salt marsh larvae show 1.5- to 1.7-fold increases in activity following ingestion of the HWG w/PMB diet. And, as was expected from experiments involving Lepidoptera fed foliage (Tables 1and 2), salt marsh larvae fed HWG diet have higher basal activities than do gypsy moth larvae fed the same diet. Using larvae that originated from field-collected egg masses and that were fed pin oak foliage, Ahmad and Forgash  reported NADPH oxidase levels in fifth instar gypsy moth larvae that were significantly lower than those of larvae fed a basal, i.e., wheat germ diet. This would indicate, as have our stud- 54 Sheppard and Friedman ies, that feeding on pine foliage, which might be expected to induce higher levels of monooxygenase than would a HWG diet, does not do so in this insect. However, the specific activity levels reported in their paper were higher than those we found in our preparations. There is no reason to doubt their work, and the difference may lie in the fact that their strain, originating from fieldcollected egg masses, might be genetically different from ours, a laboratory strain obtained from the facility at Otis AFB. It should also be noted that in the same report they state that their wheat germ diets might be contaminated with DDT, a compound that would be expected to stress the larvae generally, leading to a number of metabolic changes. Since we were unable to obtain significant andlor consistent induction of NADPH oxidase levels with gypsy moths fed pine foliage or artificial diet containing monooxygenase inducers, we employed another assay commonly used to measure monooxygenase activity in insects. Results obtained with the 0-demethylation assay show that activity levels in fifth instar gypsy moth larvae fed pine foliage are more than 1.5 times greater than those of larvae fed oak foliage (Table 4).Fifth and sixth instar larvae feeding on artificial diet also show a significant increase in 0-demethylase activity following ingestion of 0.2% dietary a-pinene. The induced activity levels of fifth and sixth instar gypsy moth larvae using the 0-demethylase assay represent the highest we have observed for this insect under any conditions. However, using the same dietary regime and assay conditions, basal and induced activity levels in fifth instar "full gut" H. zeu larvae range from 172 to over 800 pmol p-nitrophenollminlmg microsomal protein, levels that are five- to 15-fold higher than the corresponding values for late instar gypsy moths (cf. Tables 4 and 5). In view of these comparative findings, it is questionable whether the 0-demethylase induction levels we observe in gypsy moth larvae are physiologically meaningful. Perusal of the literature reveals that the 0-demethylase activity levels we obtain with H . zeu larvae are within the range reported for other Lepidoptera with the exception of the gypsy moth. For example, after 2 days of feeding on corn foliage, 0-demethylase activity in sixth instar fall armyworms rises to 290 pmol p-nitrophenollminlmg protein, representing a 6.3-fold increase in activity over that of larvae fed soybean foliage . Similarly, basal activity levels in Munduca sextu larvae during fifth instar development to the prepupal stage range upward from 86 to 270 pmol p-nitrophenollminlmgprotein . Monooxygenase activity in gypsy moth larvae as measured by N-demethylation of p-chloro-N-methylaniline is also relatively low compared with that reported for other Lepidoptera. In gypsy moth larvae fed pin oak or a wheat germ-based artificial diet, peak activity levels occur in the final instar and average 0.13-0.15 nmol N-demethylated product (p-chloroani1ine)lminlmgmicrosoma1 protein . In contrast, basal activity levels in sixth instar southern armyworm larvae average 1.7 nmol p-chloroanilinelminlmg protein . Using the same substrate, N-demethylase activity in sixth instar fall armyworm larvae fed various host plants ranges from 0.31 to 0.96 nmol p-chloroanilinelminlmg protein , and activity levels in fifth instar H. cecropia larvae fed cherry foliage average 0.8 nmol p-chloroanilinelminlmg protein (C. Sheppard and S. Friedman, personal observations using assay conditions described in ). Monooxygenase Activity in Gypsy Moth Larvae 55 Normal larval development has been observed with gypsy moths reared on artificial diets containing terpenes and on pine foliage, which is rich in terpenoids. When reared from eclosion to pupation on a HWG diet containing 0.2% a-pinene, gypsy moth larvae have development rates and, except in one comparison, pupal weights that are not different from those for larvae fed HWG diet without addition (Table 6). Hence, little if any deleterious developmental effects are associated with this level of dietary terpenoid. Barbosa et al.  have reported that gypsy moth larvae switched at the fourth instar from black oak foliage to coniferous foliage may have faster larval development times and greater fecundities than larvae maintained on black oak foliage. Our own findings indicate, as well, that third instar larvae can, at times, feed successfully on coniferous foliage . Thus, it appears that if terpenoids are responsible for the restricted host plant range of very young gypsy moth larvae, some detoxication mechanism(s) other than MFOs must be utilized by gypsy moths. There are a number of ways in which insects may deal with toxins, ranging from sequestrationand immobilization, through various energyrequiring activities to nonabsorption. As Dowd et al. 1341 have stated, current research trends are biased toward the microsomal monooxygenases because of their induction by plant compounds. Our findings emphasize the need for investigations of alternative mechanisms whereby insect herbivores counteract plant defensive compounds. LITERATURE CITED 1. Mosher FH: Food plants of the gipsy [sic] moth in America. USDA Bull No. 250, pp 1-39 (1915). 2. Barbosa P, Martinat P, Waldvogel M: Development, fecundity and survival of the herbivore Lyrnuntriu dispur and the number of plant species in its diet. Ecol Entomol 22, 1 (1986). 3. Leonard DE: Bioecology of the gypsy moth. In: The Gypsy Moth: Research Toward Integrated Pest Management. Doane CC, McManus ML, eds. USDA, Washington, DC, pp 9-29 (1981). 4. Seigler DS: Secondary metabolites and plant systematics. In: The Biochemistry of Plants. Conn EE, ed. Academic Press, New York, Vol. 7, pp 139-176 (1981). 5. White RA Jr., Franklin RT, Agosin M: Conversion of a-pinene to a-pinene oxide by rat liver and the bark beetle, Dendroctonus terebruns, microsomal fractions. Pest Biochem Physiol 10, 233 (1979). 6. Brattsten LB: Cytochrome P-450 involvement in the interactions between plant terpenes and insect herbivores. ACS Symposium Series No. 208, pp 173-195 (1983). 7. Feeny PP: Effect of oak leaf tannins on larval growth of the winter moth Operophteru brumutu. J Insect Physiol24,805 (1968). 8. Feeny PF: Plant apparency and chemical defense. In: Biochemical InteractionsBetween Plants and Insects. Recent Advances in Phytochemistry, Vol. 10, pp 1-40 (1976). 9. Schultz JC, Leshowicz MJ: Hostplant, larval age, and feeding behavior influence midgut pH in the gypsy moth (Lymantriu dispar). Oecologia 72, 133 (1986). 10. Brattsten LB, Wilkinson CF, Eisner T: Herbivore-plant interactions: Mixed-function oxidases and secondary plant substances. Science 296,1349 (1977). 11. Brattsten LB: Ecological significance of mixed-function oxidations. Drug Metab Rev 10, 35 (1979). 12. Brattsten LB: Biochemical defense mechanisms in herbivores against plant allelochemicals. In: Herbivores: Their Interaction With Secondary Plant Metabolites. Rosenthal GA,Janzen DH, eds. Academic Press, New York, pp 199-270 (1979). 13. Yu SJ, Berry RE, Terriere LC: Host plant stimulation of detoxifying enzymes in a phytophagous insect. Pest Biochem Physiol22, 280 (1979). 56 Sheppard and Friedman 14. Berry RE, Yu SJ, Terriere LC: Influence of host plants on insecticide metabolism and management of variegated cutworm. J Econ Entomol73,771(1980). 15. Farnsworth DE, Berry RE, Yu SJ: Aldrin epoxidase activity and cytochrome P-450 content of microsomes prepared from alfalfa and cabbage looper larvae fed various plant diets. Pest Biochem Physiol25, 158 (1981). 16. Yu SJ: Induction of microsomal oxidases by host plants in the fall armyworm, Spodopteru frugiperdu (J.E.Smith). Pest Biochem Physiol27,59 (1982). 17. Brattsten LB, Evans CK, Bonetti S, Zalkow LH: Induction by carrot allelochemicals of insecticide-metabolising enzymes in the southern armyworm (Spodopteru eriduniu). Comp Biochem Physiol77(C),29 (1984). 18. Yu SJ, Ing RT: Microsomal biphenyl hydroxylase of fall armyworm larvae and its induction by allelochemicalsand host plants. Comp Biochem Physiol78(C),145 (1984). 19. Bell RA, Owens CD, Shapiro M, Tardif JR: Mass rearing and virus production. In: The Gypsy Moth: Research Toward Integrated Pest Management, Doane CC, McManus ML, eds. USDA, Washington, DC., pp 599-655 (1981). 20. Crankshaw DL, Hetnarski HK, Wilkinson CF: Microsomal NADPH-cytochrome c reductase from the midgut of the southern army worm (Spodopteru eriduniu).-Insect Biochem 9, 43 (1979). 21. Folsom MD, Hodgson E: Biochemical characteristics of insect microsomes: NADPH oxidation by intact microsomes from the housefly, Muscu domesticu. Comp Biochem Physiol 37, 301 (1970). 22. Ahmad S, Forgash AJ: NADPH oxidation by microsomal preparations of gypsy moth larval tissues. Insect Biochem 3,263 (1973). 23. Hansen LG, Hodgson E: Biochemical characteristicsof insect microsomes. Biochem Pharmacol 20,1569 (1971). 24. Bradford MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72,248 (1976). 25. Anelli CM: Mono-oxygenase activity and nutritional ecology of larvae of the gypsy moth, Lymuntriu dispur. Ph.D. dissertation, University of Illinois at Urbana-Champaign (1988). 26. Hodgson E: The significance of cytochrome P-450 in insects. Insect Biochem 13,237 (1983). 27. Hodgson E: Microsomal mono-oxygenases. In: Comprehensive Insect Physiology, Biochemistry and Pharmacology. Kerkut GA, Gilbert LI, eds. Pergamon Press. Vol. 11, pp 225-321. (1985). 28. Terriere, LC: Induction of detoxication enzymes in insects. Annu Rev Entomol29,71(1984). 29. Ahmad S, Forgash AJ: Gypsy moth mixed-function oxidases: gut enzyme levels increased by rearing on a wheat germ diet. Ann Entomol SOCAm 72,449 (1978). 30. Tate LG, Nakat SS, Hodgson E: Comparison of detoxication activity in midgut and fat body during fifth instar development of the tobacco hornworm, Munducu sextu. Comp Biochem Physiol72C, 75 (1982). 31. Ahmad S: Enzymatic adaptations of herbivorousinsects and mites to phytochemicals. J Chem Ecol22,533 (1986). 32. Yu SJ: Induction of detoxifying enzymes by allelochemicals and host plants in the fall armyworm. Pest Biochem Physiol29,330 (1983). 33. Brattsten LB, Wilkinson CF: Induction of microsomal enzymes in the southern armyworm (Prodeniu eriduniu). Pest Biochem Physiol3,393 (1973). 34. Dowd PF, Smith CM, Sparks TC: Detoxification of plant toxins by insects. Insect Biochem 23,453 (1983).