Nutrient deficiencies and the gypsy moth, Lymantria dispar: Effects on larval performance and detoxication enzyme activities

J. Inserrfhysiol.Vol.37,hio. I, pp. 45-52, 1991 Printedin Great Britain. All rights reserved

0022-1910/91 $3.00+ 0.00 Copyright0 1991PergamonPressplc

NUTRIENT DEFICIENCIES AND THE GYPSY MOTH, LYA4ANTRL4 DISPAR: EFFECTS ON LARVAL PER.FORMANCE AND DETOXICATION ENZYME ACTIVITIES RICHARDL. LINDROTH,‘.~ MIEL A. BARMAN’and ANNEV. WEISBROD’ ‘Department of Entomology and 2Environmental Toxicology Center, University of Wisconsin, Madison, WI 53706, U.S.A. (Received I June 1990; revised 21 September 1990)

Abstract-We

investigated the consequences of specific nutrient deficiencies for growth performance, food processing efficiencies and detoxication enzyme activities in larvae of the gypsy moth, Lymantria dispar. Larvae were reared on one of four artificial diets, including a low wheat-gelm control diet, and protein-, mineral- and vitamin-deficient diets. Growth of fourth instars was reduced on each nutrient-deficient diet; reductions were attributable to decreased efficiencies of conversion of digested food. Larvae on the low protein diet exhibited compensatory feeding responses, but not great enough to offset the reduction in protein intake. The low protein and low mineral diets prolonged development time of females, and reduced pupal weights of males and females. All larvae fed the low vitamin diet succumbed to a nuclear polyhedrosis virus late in the fifth larval stadium. We observed few significant effects and no clear-cut patterns of response of detoxication enzymes to nutrient limitation. Polysubstrate monooxygenase and glutathione transferase activities were unaffected. Soluble esterase and carbonyl reductase activities tended to increase in response to protein deficiency, but decrease in response to’ vitamin deficiency. Phytophagous insects evolutionarily adapted to feeding on nutrient-poor but allelochemical-rich host plants may have evolved biochemical/physiological mechanisms that serve to maintain effective enzyme function in the context of nutrient deficiency. Key

Word Index: Carbonyl reductase; detoxication; esterase; feeding trials; glutathione transferase; gypsy moth; Lymantria dispar; nutrient deficiencies; nutritional indices; polysubstrate monooxygenase

ingly low growth rates (Mattson and Scriber, 1987; Van? Hof and Martin, 1989). In addition to the direct effects of malnutrition on primary biochemical and physiological systems, low nutrient diets may reduce insect performance by effecting changes in detoxication systems, thereby altering susceptibility to allelochemicals. Numerous studies with vertebrates have documented that nutrient deficiencies may decrease, or at times increase, the activity of particular detoxication enzymes. Such effects are likely to occur in insects as well, but literature on this topic is virtually nonexistent. The gypsy moth, Lymantriu dispar, is a major forest pest in the northeastern United States, and is continuing to expand its range westward and southward. The insect is highly polyphagous, utilizing over 300 species of trees and shrubs from at least 14 plant families (Doane and McManus, 1981; Lechowicz and Mauffette, 1986). Extraordinary polyphagy notwithstanding, numerous studies have shown that food selection and utilization of both hosts and nonhosts are related to plant allelochemicals (Miller and

lNTRODUCTION The nutrient compos.ition of plant tissues strongly influences performance parameters (e.g. growth, development, survival, reproduction) associated with fitness in phytophagous insects (Scriber and Slansky, 1981; Slansky and Scriber, 1985; Mattson and Scriber, 1987). Indeed, differences in nutrient composition among various plant taxa have likely been a driving force in insect evolution. For instance, Mattson and Scriber (1987) argue that insects adapted to feeding on nutrient-poor foliage are fundamentally different (physiologically, morphologically and behaviourally) from those adapted to feeding on nutrient-rich foliage. As the culminant expression of many such traits, body growth rates tend to be inherently lower for insects adapted to nutrient-poor foods than for insects adapted to nutrient-rich foods. .Folivores of woody plants, in particular, are confronted with food containing especially low levels of essential nutrients such as protein, water and minerals, and exhibit correspond45

RICHARD

46 Table I. Composition

of standard

(control)

Wet weight (%)

Ingredient Wheat-germ Casein (vitamin free) Cellulose (alphacel) Mineral mix (Wesson’s) Vitamin mix (HoffmannLaRoche No. 26862) Sorbic acid AgU Water (double distilled)

L. LINDROTH et al.

diet Dry weight (%)

2.00 2.00 9.40 0.74 0.93

12.01 12.01 56.46 4.44 5.59

0.19 1.39 83.36

I.14 8.35

Feeny, 1983; Barbosa and Krischik, 1987; Meyer and Montgomery, 1987; Rossiter et al.. 1988; Lindroth and Hemming, 1990; Lindroth et al., 1990). In contrast, surprisingly little is known about the effects of foliar nutrients on the physiology and ecology of gypsy moths. The purpose of the research described here was twofold: (1) to determine the consequences of nutrient deficiencies for larval food processing efficiencies and attendent performance parameters and (2) to assess the effects of nutrient deficiencies on detoxication enzyme activities. Because protein, minerals and vitamins are critical for normal function of detoxication enzymes, we hypothesized that nutrient deficiencies would reduce activities of a suite of enzymes, including polysubstrate monooxygenases, esterases. carbonyl reductases and glutathione transferases. MATERIALS

AND METHODS

Insects and art$cial diets

Gypsy moth egg masses were obtained from the Beneficial Insects Research Laboratory (USDA), Newark, Del. All egg masses were surface sterilized in dilute formaldehyde solution (ODell et al., 1985) prior to use. Newly hatched larvae from each egg mass were maintained for 10 days in 142ml plastic containers, in groups of approx. 35. We then transferred the larvae to 600 ml plastic containers, in which they were maintained in groups of 20-40 until completion of the third larval stadium. For stadia 1-3, all larvae were fed a standard artificial diet (Table 1). All experiments were conducted at 25°C under 16 h light-8 h dark. Artificial diets were adapted from the high wheatgerm formulation of ODell et al. (1985). The preservative methyl paraben was not used in order to avoid potential induction of detoxication enzymes by the compound. We autoclaved diet mixtures to inhibit growth of mould; vitamins were added to the mixtures only after cooling to below 70°C. To more closely approximate protein concentrations in gypsy moth food plants, our standard (control) diet contained 2.0 and 2.0% (wet weight) wheat-germ and casein, respectively, in contrast to the 11.1 and 2.3% values of the standard formulation (Table 1). We substituted cellulose for the difference. The standard control diet was modified to produce diets deficient in protein, minerals or vitamins. We

then conducted preliminary feeding trials, using fourth-instar growth rates as a performance index, to determine the extent to which each nutrient would need to be reduced to effect significant and similar growth reduction among insects fed the three diets. Thus for the experiments reported here, the low protein diet contained 0.5% casein (25% of control value), the low mineral diet contained 0.0925% mineral mix (12.5% of control value), and the low vitamin diet contained no vitamin mix. For each diet formulation, we compensated for loss of a particular nutrient by adding in an equivalent amount of cellulose. To assess the mineral composition of the diets, we had nutrient analyses performed by the Soil and Plant Analysis Laboratory of the University of Wisconsin. Control and low protein diets were assayed for total nitrogen by a total Kjeldahl nitrogen technique. Control and low mineral diets were assayed for total minerals by acid digestion followed by inductively coupled plasma spectroscopy. Bioassays

We conducted fourth-instar feeding trials and fourth-fifth-instar survival/development trials to determine the effects of nutrient deficiencies on larval performance. For the feeding trials, individual newly moulted fourth instars (6&75 mg fresh weight) were placed into 28-ml plastic cups containing a cube of one of the experimental diets. Larvae from each egg mass were represented only once among the replicates for each treatment. Cups were cleaned and food replenished at 2-3 day intervals until completion of the fourth stadium. At the conclusion of each trial, we froze the larva, then dried (50°C) and weighed the larva, frass and uneaten food. Dry weights of larvae at the onset of the trials were estimated from proportional dry weights of 12 newly moulted larvae from the same egg masses as experimental larvae. Similarly, dry weight of food administered was determined from a subset of diet cubes for which proportional dry weights were measured. We calculated nutritional indices on the basis of dry weights, using standard formulas (Waldbauer, 1968; Scriber, 1977) for relative growth rate, relative consumption rate, approximate digestibility, efficiency of conversion of digested food and efficiency of conversion of ingested food. For the survival/development trials, newly moulted fourth instars were assigned to the four test diets; each replicate consisted of 12 larvae in a 600 ml rearing container. We monitored survival rates (stadia 4-5) development times (egg hatch to pupation) and measured pupal weights 34 days after pupation. Enzyme assays

Larvae to be used for enzyme assays were assigned to one of the experimental diets as newly moulted fourth instars, and reared in groups of 12-15 to the

Nutrient

deficiencies

and gypsy moth performance

47

Table 2. Nitrogen and mineral composition of experimental diets (proportion of diet dry weight) Diet Component

Control

Low protein

r\ (%) P (%) K (%) Ca (%) Mg (%) s (%) ha (%) B (ppm) k[n (ppm) Fe (ppm) Cu (ppm) Zo (ppm)

2.44 0.70 0.8 1 0.72 0.11 0.28 0.32 12.50 29.20 293.40 12.80 31.70

I .26 -

Low mineral 0.30 0.24 0.14 0.05 0.20 0.23 13.80 25.50 114.70 8.90 27.80

Minimally optima1 dietary level” 0.25-0.65 0.80-0.90 0.03 0.10 0.001 148 20 20-60

Values represent means of duplicate analyses. Mineral concentrations of the low protein diet were not assessed; these should be equivalent to values for the control diet. Similarly, nitrogen concentration of the low mineral diet should be equivalent to that of the control diet. “For Lepidoptera (folivores), from Mattson and Scriber (1987).

fifth larval stadium. Actively feeding larvae (fifth instars for 2-5 days) were used to prepare enzyme solutions. Midguts (15-30 per replicate) were removed, washed in 0.2 M potassium phosphate buffer (pH 7.8, with 1 mM EDTA), and homogenized by 10 strokes in a Ten Broeck tissue grinder. We centrifuged the homogenate at 10,000 g (10 min) to remove cellular and mitochondrial debris, then centrifuged the supernatant at 100,OOOg (60min) to separate soluble (cytosolic) and microsomal (membrane-bound) (enzymes. We flash-froze the enzyme solutions in liquid nitrogen and stored them at - 65°C until used Yor enzyme assays. All preparative procedures were performed at O-4”C. Many different enzyme systems are involved in detoxication of xenobiotics in insects. On the basis of the reactions catalyzed, these are generally categorized into the functional classes of oxidases, hydrolases, reductases and transferases (Ahmad et al., 1986). We assayed activity of one major enzyme system from each ‘class, including polysubstrate monooxygenases (oxidases), general esterases (hydrolases), carbonyl reductases and glutathione transferases. All of these enzyme systems play critical roles in the nutritional ecology of at least some Lepidoptera (Lindroth, 1991), although their relTable 3. Vitamin compositi#>n of control and low vitamin diets (per 100 P drv diet)

evance to gypsy moth-host interactions is still poorly known. We measured microsomal polysubstrate monooxygenase activities by the cytochrome c reductase and NADPH oxidation assays. Soluble esterase enzyme activity was determined by the I-naphthyl acetate assay, and soluble glutathione transferase activity was quantified as the conjugation of l-chloro2,4_dinitrobenzene (CDNB) with glutathione. Soluble and microsomal carbonyl reductase activities were determined by the juglone (quinone)-dependent NADPH oxidation method. Details of all procedures are provided by Lindroth et al. (1990). Protein concentrations of the enzyme solutions were measured by the Folin-phenol procedure of Schacterle and Pollack (1973). Statistics Results from the bioassays and enzyme assays were analysed by one-way analysis of variance (ANOVA), using SAS statistical software. When the ANOVA F statistic was significant (P < 0.05) we compared treatment means by the Student-Newman-Keuls multiple range test (SAS Institute, 1982). Data on development time and pupal weight were pooled, by sex, within each replicate. Proportional data (survival rates, nutritional indices) were transformed (arcsin JL) prior to analysis.

Diet

RESULTS

Vitamin

Control

Low vitamin

Vitamin A (IU) Vitamin E (IU) Vitamin B,, (pg) Riboflavin (mg) Niacin (mg) Ascorbic acid (mg) d-Pantothenic acid (mg) Choline (mg) Folic acid (pg) Pyridoxine (mg) Thiamine (mg) d-Biotin cue) ~._, Inositol (mg)

123,252.8

15.61 4.62 0.03 0.10 0.66 0.00 0.17 NA 0.05 0.08 0.22 3.24 NA

NA = information not available.

49.3 11.2 2.9 6.3 2,800.O 5.3 243.1 1,397.5 1.3 1.4 115.0 118.8

Artificial diet analyses Nitrogen and mineral compositions of the experimental diets are summarized in Table 2. The level of total nitrogen in the low protein diet was approximately half that of the control diet. Mineral concentrations in the low mineral diet were substantially lower than in the control diet, although concentrations were not reduced uniformly. In general, macronutrient concentrations declined more than did micronutrient concentrations, iron being the single exception.

RICHARD L. LINDROTH et al.

48 Table 4. Dietary

effects on nutritional

indices of fourth-instar

gypsy moths (2 & 1 SE; N = 8 for each mean)

Diet

Duration (days)

RGR (ma/ma/day)

(mdmaiday)

Control Low protein Low mineral Low vitamin

5.0 6.3 5.7 5.8

0.22 0.15 0.16 0.16

1.83 i 0.07sb 2.33 + 0.11’ 1.72+ 0.05’ 2.04 f 0.04b

ANOVA

P value

+ f + +

0.3” 0.3b 0.2Ub 0.3ab

0.019

* i + +

RCR

O.Olb 0.01a 0.02’ 0.01’


ECD 18.3 19.8 22.9 19.6


+ 0.7’ + I.lsb + I .4b + 0.7’b

Nutrient-deficient diets significantly altered the performance parameters of fourth-instar gypsy moths (Table 4). The low protein diet prolonged fourth stadium duration 26% over that of larvae on the control diet; the low mineral and low vitamin diets also tended to increase stadium duration but not significantly so. Larval growth on all the deficient diets was suppressed to a level about 30% less than that of control animals. Larvae fed the low protein diet exhibited a compensatory feeding response, elevating intake rates by 27%. Food consumption rates were unaffected by mineral- or vitamin-deficient diets. Similarly, overall diet digestibility was not affected by deficiencies in protein, minerals or vitamins. Conversion efficiencies, however, were strongly affected. Values for efficiency of conversion of digested food were reduced similarly for all larvae fed the deficient diets, registering 40-47% lower than those for larvae fed the control diet. Values for efficiency of conversion of ingested food were correspondingly reduced. Nutrient-deficient diets also influenced performance characteristics of fourth-fifth instars (Table 5). Survival rates were similar for larvae reared on the control, low protein and low mineral diets, but not a

ANOVA

P

91.7 91.7 87.5 0.0 value

For columns with significant different (P < 0.05).

* 4.3b * 3.7b f I .9b + 0.0’


ANOVA

11.9 6.7 9.1 7.8


0.3’ 0.4” I.Ib 0.2”b


ECD = efficiency of conversion

of

Effects of nutrient deficiencies on detoxication enzyme activities (Table 6) did not parallel those observed for growth performance parameters. Differences in NADPH oxidation rates among treatment groups were only marginally significant, and none was significantly different from the control. Likewise, no differences occurred for cytochrome c reductase activities; mean values for larvae fed each of the nutrient-deficient diets were two-fold higher than those for control larvae, but within-treatment vari-

(days)

Weight

I SE;

(mg)

__ Males

Females

37.3 * 0.9 39.1 f 0.7 39.8 + 0.7

42.6 + 1.2” 46.3 f l.lb 48.6 k 0.7b

0.125

0.010

F statistics,

f f + f

Enzyme assays

Duration

Control Low protein Low mineral Low vitamin

Wo)

3.1b 2.9” 2.3’ 2.2’

rates, development times and pupal weights of gypsy moths (R f N = 6 for each mean)

Survival (%)

Diet

f + + +

single larva on the low vitamin diet survived to pupation. In the latter case the immediate cause of death was infection by a nuclear polyhedrosis virus. Late in the fifth stadium, these larvae ceased feeding, became lethargic, melanized, and flaccid. Death ensued within 24-48 h of the onset of symptoms. Microscopic examinations of body fluid revealed large numbers of polyhedral inclusion bodies. Any rearing containers with larvae exhibiting signs of viral infection were immediately transferred to a separate environmental control chamber to reduce the potential for contamination of other larvae. Only larvae on the low vitamin diet succumbed to viral infection; no such symptoms were observed for the few larvae that died in other treatments. Consumption of low protein or low mineral diets during stadia 4-5 did not alter larval development times for males, but prolonged development times for females. Pupal weights were 13% less for males on the low protein and low mineral diets than for those fed control diet. Effects on female pupal weights were more pronounced. The low protein diet reduced pupal weights by 22%, whereas the low mineral diet did so by 39%. Thus, in contrast to males, females were more deleteriously affected by the low mineral diet than by the low protein diet.

Bioassays

Table 5. Dietary effects on survival

65.4 34.4 39.4 39.8

0.022

Within a column, means bearing different superscripts are significantly different (P < 0.05). RGR = relative growth rate, RCR = relative consumption rate, AD = approximate digestibility, digested food, EC1 = efficiency of conversion of ingested food.

Vitamin composition of the control and low vitamin diets (Table 3) was calculated from lot analyses for the vitamin mix (Hoffmann-LaRoche, Inc., Nutley, N.J.) and nutritive values for Kretschmer wheat-germ (Quaker Oats Co., Chicago, Ill.). Several major differences are readily apparent. Concentrations of vitamin A and folic acid were 8,000- and 28,000-fold higher, respectively, in the control diet than in the low vitamin diet. Moreover, the control diet contained substantial ascorbic acid, whereas the low vitamin diet contained none.

EC1

C%)

means bearing

Males 509 f Bb 44x + 13” 443 * 17” 0.013

different

superscripts

Females 1623 f 87’ 1273 i 65b 996 k 51”
Nutrient deficiencies and gypsy moth performance Table 6. Dietary elk:s

on detoxication enzyme activities of fifth-instar gypsy moths (2 k

49 1SE; sample sizes in parentheses)

Carbonyl reductase Diet Control Low protein Low mineral Low vitamin ANOVA P value

NADI’H oxidatl on I .96 + 0.:!7 (6)

1.74& 0.30 (9) I .55 * 0.31 (7) 2.79 + O.Z!2(5)

Cytochrome c reductase 32.1 f 60.9 f 70.7 f 74.3 *

0.062

5.2 (6) 14.0 (8) 18.9 (6) 5.3 (5)

0.156

Esterase 1474 + 28 1906 f 94 1622 + 86 1218 k49

Soluble

(6)b (5) (5)b (5)


40.2 k 60.4 + 51.6 + 25.1 *

1.0 (6yb 9.7 (5) 4.8 (5)k 1.9 (5)

0.002

Microsomal 84.4 + 7.4 (6) 87.0 f 8.7 (4) 59.1 f 13.2 (5) 68.5 rt 2.8 (5) 0.126

Glutathione transferase 372.7 & 38.9 321.3 f 27.4 293.9 f 40.0 349.6 f 26.3

(6) (5) (5) (5)

0.412

All specific activities shown as nmol/min/mg protein. For columns with significant ANOVA F statistics, means bearing different superscripts are significantly different (P < 0.05).

ation was also high. Esterase activities varied, but not in a consistent manner across diets. Esterase activities of larvae fed the low protein and low vitamin diets were 29% higher and 17% lower, respectively, than those of larvae fed the control diet. Similarly, soluble carbonyl reductase activities were elevated in larvae reared on the low protein diet, but tended to decline in larvae on the low vitamin diet. Diet did not significantly alter microsomal carbonyl reductase or glutathione transferase activities. DISCUSSION

Plant constituents utilized as nutrients by phytophagous insects vary qualitatively and quantitatively, both among and within plants and through time. Of these, protein is generally recognized as the nutrient most limiting to insect growth under natural conditions (Mattson, 1980; White, 1984; Mattson and Scriber, 1987). In our artificial diet, protein was provided in the form of wheat-germ and casein. Total nitrogen (the standard index of protein) in the control diet was similar to levels reported for mature foliage of numerous deciduous trees of eastern North America [l&4.5% dry weight (Scriber and Feeny, 1979; Slansky and Scriber, 1985; Lindroth et al., 1987; Mattson and Scriber, 1987)]. In contrast, total nitrogen of the low protein diet was slightly less than the lowest values reported. Values for minimally optimal dietary levels of minerals for insects were tabulated by Mattson and Scriber (1987; Table 2). The specific mineral requirements of folivorous inslects remain largely unknown, yet several researchers have suggested that performance of insects may at times be limited by low mineral concentrations in host tissues (Mattson et al., 1982; Mattson and Scriber, 1987). Our mineral-deficient diet differed most substantially from the control with respect to the macronutrients phosphorus, potassium, calcium, and magnesium, and the micronutrient iron. Mattson and Scriber (1987) compiled average mineral concentrations for foliage of temperate deciduous trees from several locations. Levels of both phosphorus and iron in foliage were similar to or less than levels in our low mineral diet. Given that these are average values for many tree species, and that the gypsy moth performs well on a variety of deciduous trees, phosphorus and iron are unlikely candidates for the cause of growth suppression observed in our

study. Thus potassium, calcium and magnesium are the minerals most likely responsible for reduced larval performance. Vitamins comprise a quantitatively minor yet essential component of insect diets. The water-soluble B vitamins serve as cofactors for enzymes involved in energy metabolism, and most members of the B complex are required by insects (Reinecke, 1985). The requirement of insects for ascorbic acid, another water-soluble vitamin, is associated with phytophagy. It is essential for insects that naturally feed on fresh plant tissues, although some exceptions exist (Dadd, 1985). Nutritional requirements of insects for the fat-soluble vitamins (A, D, E, K) remain unclear, although none appears critical for larval growth per se (Dadd, 1985). Reduced performance of larvae fed the low vitamin diet may have resulted from overall low vitamin levels, or from the complete absence of one constituent, ascorbic acid. Given that ascorbic acid is an essential nutrient for most phytophagous insects, the latter possibility is likely. Not surprisingly, nutrient deficient diets significantly reduced larval growth rates and pupal weights, and prolonged female development times. The greater effect on females is consistent with the fact that the gypsy moth is sexually dimorphic with respect to size-female pupae are three-four-fold heavier than male pupae (Rossiter, 1987; Lindroth et al., 1990). Our data suggest that if nutrient deficiencies are encountered by gypsy moths in the field, females will be more deleteriously affected than males. By altering food consumption rates, gypsy moth larvae can maintain consistent growth rates on diets with substantially different protein concentrations. Growth of larvae fed our low wheat-germ control diet was the same as that of larvae reared on a high wheat-germ diet [ 11.1% wheat-germ, 2.3% casein (Lindroth and Hemming, 1990)], and of larvae on a high wheat-germ diet lacking casein (Lindroth et af., 1990). Consumption rates, however, were nearly three-fold higher for control larvae in this study than for larvae in the earlier studies. Moreover, our results reveal that this compensatory response is limited. Larvae were unable to completely offset the reduction in casein from 2.0 to 0.5% simply by increasing consumption rates. Under natural conditions additional constraints, such as simultaneous

50

RICHARDL. LINDROTHel al.

consumption of allelochemicals, may further reduce the efficacy of compensatory feeding responses. Compensatory increases in food consumption in response to nutrient deficiencies have commonly been observed in insects (e.g. Slansky and Feeny, 1977; Timmins et al., 1988; Slansky and Wheeler, 1989). Many of these studies have involved deficient diets created via dilution of a standard diet with water or a nonnutritive substrate such as cellulose. With the exception of protein, little is known about how deficiencies of particular nutrients may induce compensatory feeding responses in insects (Slansky and Wheeler, 1989). Interestingly, we found that gypsy moth larvae altered feeding rates in response to decreased protein, but not in response to decreased minerals or vitamins. Conceivably, gypsy moth larvae have evolved physiological mechanisms enabling “evaluation” of the protein quality of a diet. The quantitative nutritional index most closely coupled to reduced growth of larvae fed the nutrientdeficient diets was efficiency of conversion of digested food. Values for larvae on the deficient diets were approximately half of the value for larvae on the control diet. Efficiencies of conversion of digested food are influenced by both “acquisition costs” (costs associated with foraging/feeding) and “processing costs” (e.g. costs of biochemical and physiological processes involved in digestion. assimilation, transformation, detoxication, and elimination of food) (sensu Martin and Van? Hof, 1988). An increase in appetitive movements associated with feeding on suboptimal diets may reduce values for efficiency of conversion of digested food because of elevated metabolic (respiratory) demands. Lindroth and Hemming (1990) observed greater movement among first-instar gypsy moths fed diets containing a phenolic glycoside toxin, but no similar response was detected in this study with fourth instars fed nutrient-deficient diets. This does not preclude the possibility of elevated acquisition costs, but we believe that increased processing costs are more likely responsible for reduced efficiencies of conversion of digested food. Protein, minerals and vitamins are essential components for the ions or biomolecules required for food processing mechanisms involved in the maintenance of ion/water gradients and gut pH, assimilation of nutrients, introduction to and transformation of nutrients in the metabolic pool, and biosynthesis and elimination of waste products. As the efficiency of these mechanisms declines due to nutrient deficiencies, efficiencies of conversion of digested food, and consequently relative growth rates, will decrease. An unexpected and interesting result of our studies was the interaction between vitamin deficiency and viral susceptibility. The source of the virus is unknown. No wild-collected gypsy moths (of any stage), or foliage from gypsy moth infected regions, have entered our quarantine rearing facility. Our egg masses were obtained from a laboratory culture in which symptoms of nuclear polyhedrosis virus are

occasionally, but infrequently, detected. Thus the virus probably entered with the egg masses, was not entirely killed by our sterilization procedure, and produced an epizootic in vitamin-deficient larvae. Immune dysfunction was clearly a result of vitamin deficiency-all larvae reared through the fifth stadium on the low vitamin diet developed symptoms as late fifth instars, but siblings reared on other diets showed no signs of infection. Increased susceptibility to the virus may have resulted from ascorbic acid deficiency, a possibility that warrants further investigation. Given the high degree of polyphagy of the gypsy moth, enzymatic detoxication systems are likely to play important roles in adaptation to particular hosts. General activity levels of the major detoxication enzymes are, however, unimpressive. Polysubstrate monooxygenase activity, as determined by NADPH oxidation, was similar to that reported for the gypsy moth by Ahmad and Forgash (1978) and Sheppard and Friedman (1989) but much lower than values reported for Spodoptera eridania and S. frugiperdu (Gunderson et al., 1986). Cytochrome c reductase activity of larvae on our control diet was only about 30% of the level measured in a previous study in our laboratory (larvae reared on a high wheat-germ diet [Lindroth et al., 19901) and much less than values reported for Heliothis virescens (Brattsten, 1987) and S. eridania and S. frugiperdu (Gunderson et al., 1986). Esterase, carbonyl reductase and glutathione transferase activities were all low to moderate in comparison to activities conducted with a variety of other Lepidoptera (Brattsten, 1987; Yu, 1987; Lindroth, 1989a, 1989b). Numerous researchers have observed dietmediated changes in detoxication enzyme activity in insects. Changes occur in response to consumption of particular allelochemicals, or as a result of feeding on different food plants (Yu, 1982, 1983, 1986; Lindroth, 1989a, 1989b, 1989~). Most research has focused on induction of enzyme activity. Almost nothing is known about the consequences of specific nutrient deficiencies for detoxication enzyme activity in insects. In contrast, a host of studies with vertebrates has documented significant effects of nutrient deficiencies on the activity of detoxication enzymes, and subsequent changes in susceptibility to xenobiotics (Chhabra, 1981; Dauterman, 1980; Parke and Ioannides, 1981; Boyd and Campbell, 1983; Guengerich, 1984). For example, reductions in the quality or quantity of dietary protein generally depress polysubstrate monooxygenase activity. Conjugation reactions are also often affected, but the changes are less consistent; increases, decreases, and zero effects have been observed. Trace nutrient (vitamin and mineral) deficiencies generally reduce activity of polysubstrate monooxygenases. This is not unexpected, given that nicotinic acid, riboflavin, pantothenic acid, copper and iron are integral

Nutrient deficiencies and gypsy moth performance components of the enz:yme system. A number of trace elements are also required for nonoxidative detoxication reactions, but little is known about effects of nutrient deficiencies on these enzymes. We observed surprisingly few changes in the activity of gypsy moth detoxication enzymes, and no clear-cut patterns. None of the nutrient-deficient diets produced polysubstrate monooxygenase activities significantly different from those of control animals. Esterase and soluble carbonyl reductase activities were low in larvae fed the low vitamin diet, but high in larvae fed the low protein diet. Similar increases associated with reduction of dietary protein were observed for esterase
51

have responded to nutrient limitations over evolutionary time by development of enzyme systems with particularly high affinity for limiting substrates. Their argument centred on enzymes required for nutrient extraction and transport, but it is feasible that analogous evolutionary adaptations have occurred with enzymes involved in detoxication metabolism. Acknowledgements-We

thank Mark Bloomer for technical assistance, and the Beneficial Insect Research Laboratory (USDA-ARS), Newark, Del., for providing gypsy moth egg

masses. This research was supported by USDA Competitive Research Grant 87-CRCR-2581, by the College of Agricultural and Life Sciences (Hatch Project 3211), and by the Graduate School of the University of Wisconsin, Madison, Wis. REFERENCES

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Barbosa P. and Krischik V. A. (1987) Influence of alkaloids on feeding preference of eastern deciduous forest trees by the gypsy moth Lymantria dispar. Am. Nat. 130, 53-69. Bovd J. N. and Campbell T. C. (1983) Impact of nutrition on detoxication. In Biological Basis of Detoxication (Eds Caldwell J. and Jakoby W. B.), pp. 287-306. Academic Press, New York. Brattsen L. B. (1987) Metabolic insecticide defenses in the boll weevil compared to those in a resistance-prone species. Pest. Biochem. Physiol. 27, l-12. Chhabra R. S. (1981) Effect of dietary factors and environmental chemicals on intestinal drug metabolizing enzymes. Toxicol. Environ. Chem. 3, 173-199. Dadd R. H. (1985) Nutrition: organisms. In Comprehensive Insect Physiology, Biochemistry and Pharmacology (Eds Kerkut G. A. and Gilbert L. I.) Vol. IV, pp. 313-390. Pergamon Press, Oxford. Dauterman W. C. (1980) Physiological factors affecting metabolism of xenobiotics. In Introduction to Biochemical Toxicology (Eds Hodgson E. and Guthrie F. E.), pp. 133-142. Elsevier, New York. Doane C. C. and McManus M. L. (1981) The Gypsy I

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