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’