Journal of Chemical Ecology, Vol. 19, No. 11, 1993

XANTHINE TOXICITY TO CATERPILLARS SYNERGIZED BY ALLOPURINOL, A XANTHINE DEHYDROGENASE/OXIDASE INHIBITOR

FRANK

SLANSKY,

JR.

Department of Entomology and Nematology Institute of Food and Agricultural Sciences University of Florida, Gainesville, Florida 32611-0620 (Received May21, 1993; accepted July 8, 1993)

Abstraet--Xanthine (2,6-dioxypurine), which occurs in certain legumes and other plants, was fed in artificial diet to larvae of two noctuid moth species, a legume specialist, Anticarsia gemmatalis, and a generalist, Spodoptera frugiperda. In addition, diets either lacked or contained allopurinol (4-hydroxypyrazolo(3,4-d)-pyrimidine), an inhibitor of xanthine dehydrogenase and oxidase, enzymes that convert xanthine to uric acid. Xanthine alone (up to 2% fresh mass, fm) had little deleterious effect on either species, whereas allopurinol alone (up to 1% fm) had moderate but significant effects, increasing mortality, slowing development, and reducing insect biomass. At 0.5% fm allopurinol, the decrease in biomass-relative growth rate (RGR) was associated with reductions in the efficiency of conversion to biomass of digested food (ECD; both species) and in the biomass-relative consumption rate (RCR; A. gemmatalis). In addition, pupae of each species from allopurinol-fed larvae had increased water retention (i.e., lower percentage dry mass) compared with insects consuming control diet. When fed diet containing both compounds (1% fm xanthine + 0.5% fm allopurinol), no A. gemmatalis and only 40% of S. frugiperda larvae reached the prepupal stage; additionally for the latter species, there was a substantial slowing of growth and reductions in final biomass, RGR, RCR, and ECD. These results indicate a synergistic interaction, in which the effects of xanthine and allopurinol combined in the diet were significantly greater than the additive effects of each compound tested separately. Presumably, the inhibition of xanthine dehydrogenase by allopurinol prevented the absorbed xanthine from being converted to uric acid and excreted. In addition, this study expands the phenomenon of phytochemical detoxification by insects to include xanthine dehydrogenase, an enzyme generally not considered within this context. Key Words--Allelochemical, allopurinol, Anticarsia gemmatalis, detoxifi2635 0098-0331/93/1100-2635507.00/0 9 1993PlenumPublishingCorporation

2636

SLANSKV cation, dose-response,consumptionrate, foodutilization,Lepidoptera,Noctuidae, purine, Spodopterafrugiperda, synergism,uric acid, xanthine. INTRODUCTION

Polysubstrate monooxygenases, hydrolases, and group transferases, which typically increase the polarity and thus presumably the excretability of xenobiotics, are generally considered the primary enzyme systems responsible for the detoxification of allelochemicals by plant-feeding insects (Lindroth, 1991; Brattsten, 1992). However, other enzymes, including those involved in the formation of nitrogenous excretory compounds, may also contribute to allelochemical detoxification. For example, bruchid beetles specialized to feed on legume seeds containing the nonprotein amino acid canavanine metabolize it by arginase to urea, which in tum is metabolized by urease to ammonia (Rosenthal et al., 1978). In this case, rather than being excreted, the ammonia is used in the synthesis of protein amino acids (Rosenthal et al., 1982). Other nitrogen-containing allelochemicals, especially alkaloids, also may be detoxified by conversion to nitrogenous excretory compounds. For example, the naturally occurring purines, including hypoxanthine (6-oxypurine) in Lupinus and Solanum, xanthine (2,6-dioxypurine) in Beta, Medicago, Vicia, and Coffea, caffeine (1,3,7-trimethyl-2,6-dioxypurine) in Coffea, and zeatin (6-[4hydroxy-3-methyl-2-butenylamino]purine) in Zea (Gibbs, 1974; Duke, 1981; Robinson, 1983), have the same basic structure as uric acid (2,6,8-trioxypurine; Figure 1), the main nitrogenous excretory compound of most terrestrial insects (Cochran, 1985), and thus are likely candidates for detoxification and excretion as urates. However, there appear to be few published studies examining the effect on insect performance of dietary purines other than caffeine and related methylxanthines (Chovnick et al., 1980; Nathanson, 1984; Saul, 1984; Slansky and Wheeler, 1992 and references therein). Furthermore, although the metabolism of endogenous purines (e.g., adenine, guanine, hypoxanthine, and xanthine) is reasonably well understood (Cochran, 1985), little is known about the detoxification by insects of purines occurring in their food. I am aware of only one relevant study, that by Yu (1987) on larvae of Spodopterafrugiperda (J.E. Smith), showing a low rate of caffeine metabolism by microsomal oxidation in vitro. A key enzyme involved in the metabolism of nitrogenous compounds is xanthine oxidase (EC 1.1.3.22), although xanthine dehydrogenase (EC 1.1.1.204) is the common in vivo form of this enzyme in vertebrates as well as insects (the latter name will be used here) (Cochran, 1975; Hochstein et al., 1984; Parks and Granger, 1986). This enzyme oxidizes certain purines, including hypoxanthine to xanthine and xanthine to uric acid (Bergmann and Dikstein,

XANTHINE TOXICITY SYNERGIZED BY ALLOPURINOL

O

2637

0

II

N

II

N

C.HH3

O J[

CH3 N1&

I CH 3

HYPOXANTHINE

CAFFEINE

XANTHINE

CH

HNCH2CH:CCH2OH

~

o

ZEATIN

OH

N

I

:o

URIC ACID

/ ALLOPURINOL

FIG. 1. Chemical structures of some naturally occurring purines and of aUopurinol, an inhibitor of the enzymes (xanthine dehydrogenase and oxidase) that convert hypoxanthine to • and xanthine to uric acid.

1956; Cochran, 1985; Elion, 1989). An effective inhibitor of xanthine dehydrogenase, the hypoxanthine analog allopurinol [4-hydroxypyrazolo(3,4-d) pyrimidine; Figure 1] (Elion, 1966; Williams and Bray, 1981), has been used in studies of purine metabolism in vertebrates (e.g., Lohmann and Miech, 1976; Aldridge et al., 1977; Elion, 1989), as well as clinically to treat gout by preventing the buildup of urate crystals (Bartels, 1966). However, there are few studies examining allopurinol's effects on insects, although it has been used to investigate the role of stored urate in cockroaches (Engebretson and Mullins, 1986 and references therein to other allopurinol/insect studies; Suiter et al., 1992). This inhibitor should also make it possible to assess, in vivo, the involvement of xanthine dehydrogenase in allelochemical detoxification and excretion. Thus, in this study I examined whether the toxic effects of dietary xanthine could be synergized by aUopurinol in larvae of two species of noctuid moths. I studied the velvetbean caterpillar, Anticarsia gemmatalis Hfibner, which feeds on several legumes (Herzog and Todd, 1980), and the more generalized fall armyworm, S. frugiperda, which feeds on a variety of grasses as well as plants in over 20 other families (Tietz, 1972).

2638

SLANSKY METHODS AND MATERIALS

Experimental Protocol. Both species were obtained as eggs from laboratory colonies maintained at the Insect Attractants, Behavior, and Basic Biology Research Laboratory, ARS/USDA, Gainesville, Florida. Caterpillars were reared in groups on an artificial diet following Greene et al. (1976), except for the omission of formalin, methylparaben, and tetracycline. At the start of each experiment, individual late third-early fourth instars (15-26 mg fresh mass, fm) were weighed and placed in inverted 30-ml clear plastic cups capped with tightly fitting flexible plastic tops; a weighed portion of diet was then added to each cup. Fifteen caterpillars were set up in each treatment. Experimental diets were prepared by vigorously stirring each test compound along with the other dietary ingredients into the hot (ca. 65~ water-agar solution. Diets were allowed to cool and gel prior to slicing into portions fed to the larvae. Insects were maintained at 27 _+ I~ and 50 + 15% relative humidity, with a photoperiod of 14 : 10 hr light-dark. Chemical Tests. Initial experiments tested the separate effects on caterpillar growth and survival of dietary xanthine and allopurinol (both obtained from Sigma Chemical Co., St. Louis, Missouri) over a range of concentrations. Based on the results of these experiments, the diets for the synergism experiments were formulated with the concentrations of xanthine and allopurinol that had limited effects on caterpillar performance when tested separately (see Results). In the synergism experiments, survival, developmental time, biomass gain, and the consumption and utilization of food were assessed (see below). Quantifying Food Consumption and Utilization. Each caterpillar was fed a weighed quantity of diet sufficient to last the entire experiment, beginning with late third-early fourth instars and ending with pupation or death. A gravimetric technique was used to determine food consumption, frass egestion, and biomass gain, all based on oven-dry (60-65~ mass (dm), which allowed calculation of the following growth and consumption rates and food utilization efficiencies (Waldbauer, 1968; Slansky and Scriber, 1985): body mass-relative growth rate [RGR, (milligrams biomass gained) (milligram mean body mass • day)-~]; body mass-relative consumption rate [RCR, (milligrams food consumed) (milligram mean body mass • day)-~]; approximate digestibility [AD, 100 (food consumed - frass) (food consumed)-1]; and efficiency of conversion to biomass of digested (absorbed) food [ECD, 100 (biomass gained) (food consumed frass)-l]. In addition to the RCR based on dry mass of food, the RCR of xanthine was calculated [RCR .... mg xanthine ingested (gram mean body mass x day)-1]. Following Gordon (1968), biomass-relative consumption and growth rates were calculated based on a caterpillar's exponential mean biomass during the experiment, except that dry rather than live biomass was used. When larvae were fed, an additional 10 diet samples and 10 larvae were

X A N T H I N E T O X I C I T Y S Y N E R G I Z E D BY A L L O P U R I N O L

2639

weighed fresh, oven-dried (larvae were frozen prior to drying), and reweighed, providing fresh to dry mass conversion factors that allowed calculation of the dry mass of food provided to each larva and the initial dry mass of the experimental larvae, respectively. Frass was removed from the rearing cups after the first two days of an experiment and daily thereafter, oven-dried, and weighed. Developmental time was measured as the number of days from the start of an experiment until a larva reached the prepupal stage, when feeding stopped. Final dry mass was based on 1-day-old pupae, or, for larvae that reached the prepupal stage but failed to pupate, on prepupal mass. Most larvae reached the prepupal stage in approximately six days; any larvae remaining after 12 days were frozen and oven-dried (duration of development for these larvae was considered to be 12 days and their dry mass at this time was used as their final dry mass). Percentage dry mass of the insects was calculated only for 1-day-old pupae by dividing their dry mass by their fresh mass. All weighing occurred on electronic balances with 0.1 mg precision. Statistical Analysis. Performance data for the dosage experiments were analyzed using PC/SAS (SAS Institute, 1987) with one-way ANOVA (general linear models). If the F statistic was significant (P _< 0.05), Dunnett's test was used to compare each treatment mean with the control mean. In the synergism experiments, performance values for larvae fed each test diet were compared to values for control larvae using a two-tailed t test (PC/SAS; SAS Institute, 1987). To test for synergistic effects, performance values for individual larvae in the three treatments (i.e., xanthine alone, allopurinol alone, and the xanthine + allopurinol combination diet) were subtracted from the appropriate mean values for control larvae, and means of these differences were calculated for each treatment. Then, for each performance measure, the mean difference for larvae fed the combination diet was compared (using a one-tailed t test) with the sum of the mean differences for larvae fed each compound separately. If the mean difference for larvae fed the combination diet (compared with control larvae) was significantly different from the sum of the mean differences due to each compound separately, then a synergistic effect was indicated (i.e., the effect of the two compounds combined in the same diet was greater than that of the sum of each compound tested separately). If either or both of the compounds separately altered performance compared with Control larvae, but no significant difference occurred between the mean differences for larvae fed the combination diet (compared with control larvae) and the sum of the mean differences for larvae fed each compound separately, then an additive effect was indicated (i.e., the effect of the two compounds combined in the same diet was equivalent to the sum of the effect of each compound tested separately). In all experiments, mortality to the prepupal stage was analyzed with a G test of independence (Zar, 1984).

2640

SLANSKY RESULTS

Dose-Response to Xanthine and Allopurinol. Xanthine at dietary concentrations up to 2 % fresh mass (fm) had little deleterious effect on S. frugiperda (Table 1). Survival on the xanthine diets was comparable to that on the control diet (no added xanthine) and neither developmental time, RGR, nor percentage dry mass of the pupae was affected significantly by dietary xanthine. Although the A N O V A for final dry mass was significant, the Dunnett's test indicated that none of the values differed significantly from the control value (P > 0.05). Performance of A. gemmatalis also was generally unaffected by diets containing up to 1% fm xanthine (Table 1). Survival was equivalent on all three diets, and there was no significant effect of xanthine on either developmental TABLE 1. MEAN ( + S E M ) DURATION OF DEVELOPMENT, FINAL DRY MASS, BIOMASS-RELATIVE GROWTH RATE, AND % DRY MASS FOR LARVAE OF

S. frugiperda AND a . gemmatalis FED ARTIFICIAL DIET CONTAINING DIFFERENT CONCENTRATIONS OF XANTHINE a

Xanthine conc. (% fm) *(N)

Duration (days)

Final dry mass (mg)

RGR (mg/mg/day)

Dry mass (%)

S. frugiperda 0 (12) 0,5 (15) 1.o (lO) 2.0 (15) G = 0.17 h P > 0.50

6.8 6.7 6.9 7.1 F3.48 P

+ 0.3 _ 0.2 _+ 0.2 +_ 0.2 = 0.80 = 0.50

40.5 45.6 36.4 38.4 = =

+ 3.0 + 1.3 + 2.4 + 1.9 3.52 0.02

0.44 0.45 0.40 0.40 = =

___ 0.03 + 0.02 _ 0.01 + 0.02 2.10 0.11

26.2 + 0.4 27.2 _+ 0.9 26.3 + 1.0 26.5 + 0.5 = 0.34 = 0.79"

6.8 6.3 6.4 Fro38 P

• + + = =

66.2 57.5 66.0 = =

_+ 2.8 ___ 1.9" + 2.3 4.55 0.02

0.48 0.50 0.52 = =

_+ 0.01 + 0.01 + 0.01 1.69 0.20

23.5 ___ 0.6 23.9 + 0.3 24.9 + 0.6 = 2.06 = 0.15"

A. gemmatalis 0 (13) J 0.5 (14) 1.0 (14)

0.2 0.2 0.2 2.11 0.14

'~N = number of larvae surviving to the prepupal stage out of 15 initial larvae per treatment level. Asterisk indicates that a treatment mean was significantly different from the respective control mean based on Dunnett's test. b G test of independence with Yates correction (Zar, 1984) comparing mortality on the control and 1.0% xanthine diets. ~This P value is based on F3,30 (for S. frugiperda) and F2.21 (for A. gemmatalis) because % dry mass was calculated for 1-day-old pupae and not all larvae reaching the prepupal stage pupated (N = 6, 7, 8, 13 for S. frugiperda, and 6, 10, 8 for A. gemmatalis, for the control and increasing xanthine concentration diets, respectively). '~G test was not performed because mortality was the same in each treatment (i.e., one larva died). For the control diet, data for one developmentally abnormal larva were omitted from the analysis.

X A N T H I N E T O X I C I T Y S Y N E R G I Z E D BY ALLOPUILINOL

2641

time, RGR, or percentage dry mass. The only significant reduction occurred for final dry mass on the 0.5% xanthine diet. In a separate study examining the interaction of dietary water and xanthine, larvae of A. gemmatalis fed the same diet formulation used in the present study but containing 2 % fm xanthine had their developmental time prolonged about one day and final dry mass reduced about 10% compared with control larvae, resulting in a significant reduction in RGR (t26 ----- 3.39, P = 0.002; Slansky, unpublished data); percentage dry mass was not affected significantly, and survival was identical to that of larvae fed control diet. Allopurinol was tested at 0.5 and 1% fro. At these concentrations, survival of S. frugiperda declined significantly, developmental time was prolonged (statistically significant only on the 0.5% diet), final dry mass was reduced (significant only on the 1% diet), RGR declined significantly on both diets, and percentage dry mass was reduced (significant only on the 0.5 % diet) compared with larvae fed the control diet (Table 2). Allopurinol had similar effects on A. gemmatalis, causing significant reductions in survival, final dry mass, RGR, and percentage dry mass, and significantly prolonging duration of development at each concentration tested compared with control larvae (Table 2). Synergism between Allopurinol and Xanthine. In these experiments, S. frugiperda and A. gemmatalis were fed diets containing either 1% xanthine (no significant deleterious effects on performance in the dose-response experiments; Table 1), 0.5 % allopurinol (the lowest concentration tested in the dose-response experiments, but with deleterious effects on performance; Table 2), a combination of the two compounds at these concentrations, or a control diet with neither compound added. All S. frugiperda survived to the prepupal stage on the control, xanthine, and allopurinol diets (Table 3). Xanthine alone did not significantly affect duration of development or final dry mass, whereas allopurinol prolonged development and reduced final dry mass significantly. Addition of either compound to the diet significantly reduced percentage dry mass (Table 3). When S. frugiperda was fed the combination diet containing both xanthine and allopurinol, synergistic effects occurred. Only 40% of the larvae fed this diet formed prepupae (G = 11.74, P < 0.001, compared with control larvae); 20% died and 40% remained as larvae when the experiment was stopped after 12 days (Table 3). Developmental time of the S. frugiperda larvae fed the combination diet was prolonged and their final dry mass was reduced compared with larvae fed the control diet, and in addition, these changes were significantly greater than the additive effects for larvae fed diets containing the compounds separately (t4o = 4.44, P < 0.0005 and t4o = 4.78, P < 0.0005, respectively; Table 3). The percentage dry mass for larvae fed the combination diet also was reduced significantly compared with control larvae, but the effect was additive rather than synergistic (i.e., the reduction on the combination diet was not

2642

SLANSKY TABLE 2. MEAN ( _ SEM) DURATION OF DEVELOPMENT, FINAL DRY MASS, BIOMASS-RELATIVE GROWTH RATE, AND % DRY MASS FOR LARVAE OF

S. frugiperda AND A. gemmatalis FED ARTIFICIAL DIET CONTAINING DIFFERENT CONCENTRATIONSOF ALLOPURINOLa

Allopurinol conc. (% fro) (N)

Duration (days)

Final dry mass (mg)

RGR (mg/mg/day)

Dry mass (%)

7.3 _+ 0.3 8.8 _ 0.4* 8.3 + 0.3 F2.3! = 6.23 P = 0.005

39.7 _ 2.5 33.7 + 2.1 31.9 + 1.8' = 3.28 = 0.051

0.38 0.31 0.31 = =

___0.02 + 0.02* + 0.01" 6.45 0.005

27.3 _+ 0.5 24.8 + 0.7* 25.8 _ 0.5 = 5.07 = 0.02 d

5.5 _ 0.2 6.6 + 0.5* 7.2 + 0.5* Fz.z8 = 5.65 P = 0.009

57.3 + 2.5 32.7 _+ 2.5* 30.3 _ 3.5* = 30.8 = 0.0001

0.57 + 0.02 0.41 + 0.03* 0.33 + 0.03* = 24.4 = 0.0001

23.4 + 0.4 21.3 +_. 0.6* 20.9 + 1.1" = 7.07 = 0.004 J

S, frugiperda 0 (15) 0.5 (10) b 1.0 (9) b G = 4.61 c P < 0.05

A. gemmatalis 0 (15) 0.5 (11) 1.0 (5) G = 10.14 c P < 0.005

"N = number of larvae surviving to the prepupal stage out of 15 initial larvae per treatment level. Asterisk indicates that a treatment mean was significantly different from the respective control mean based on Dunnett's test. bData for two abnormally developing larvae fed the 0.5% allopurinol diet and one fed the 1% diet were omitted from the analysis. CG test of independence with Yates correction (Zar, 1984) comparing mortality on the control and 0.5% + 1.0% allopurinol diets. dThis P value is based on Fro21 for S. frugiperda and F2,24for A. gemmatalis because % dry mass was calculated for l-day-old pupae and not all larvae reaching the prepupal stage pupated (N = 9, 9, 6 for S. frugiperda and 14~ 9, 4 for A. gemmatalis, for the control, 0.5 % and 1.0% allopurinol diets, respectively).

s i g n i f i c a n t l y d i f f e r e n t f r o m t h e s u m o f t h e r e d u c t i o n s d u e to t h e t w o c o m p o u n d s w h e n t e s t e d s e p a r a t e l y ; t3o = 1 . 2 3 , P > 0 . 1 0 ; T a b l e 3). F o r S. frugiperda, x a n t h i n e a l o n e d i d n o t s i g n i f i c a n t l y a f f e c t R G R o r a n y o f its c o m p o n e n t s

(i.e., RCR,

AD,

and ECD;

F i g u r e 2). H o w e v e r ,

dietary

a l l o p u r i n o l c a u s e d a s i g n i f i c a n t r e d u c t i o n in R G R , a s s o c i a t e d w i t h a s i g n i f i c a n t l y l o w e r E C D ; n e i t h e r R C R n o r A D w e r e r e d u c e d s i g n i f i c a n t l y ( F i g u r e 2). R G R , R C R , a n d E C D f o r l a r v a e f e d t h e c o m b i n a t i o n d i e t w e r e all s i g n i f i c a n t l y less t h a n t h e v a l u e s f o r l a r v a e f e d c o n t r o l d i e t , a n d in a d d i t i o n , s y n e r g i s t i c e f f e c t s w e r e e v i d e n t . F o r l a r v a e f e d t h e c o m b i n a t i o n d i e t , t h e r e d u c t i o n s in R G R , R C R , a n d E C D ( c o m p a r e d w i t h c o n t r o l l a r v a e ) w e r e all s i g n i f i c a n t l y g r e a t e r t h a n t h e additive reductions from the xanthine and allopurinol diets tested separately ( F i g u r e 2). T h e A D o f S. frugiperda l a r v a e f e d t h e c o m b i n a t i o n d i e t w a s n o t a l t e r e d s i g n i f i c a n t l y c o m p a r e d w i t h that o f c o n t r o l l a r v a e .

2643

X A N T H I N E T O X I C I T Y S Y N E R G I Z E D BY A L L O P U R I N O L

TABLE 3. MEAN ( • SEM) DURATION OF DEVELOPMENT, FINAL DRY MASS, AND % DRY MASS FOR LARVAEOF S. frugiperda AND A. gemmatalis FED CONTROL ARTIFICIAL DIET OR ONE CONTAINING EITHER XANTHINE (1% FM), ALLOPURINOL (0.5% FM), OR BOTH AT THESE CONCENTRATIONSa

Diet (N)

Duration (days)

Final dry mass (rag)

Dry mass (%)

S. frugiperda Control (15) Xanthine (15) Allopurinol (15) Xanthine + allopurinol (12)"

7.1 6.8 7.7 10.7

• + • +

0.1 0.2 0.2* 0.5*t

44.9 42.1 38.0 26.0

• 2.4 • 1.7 +_ 2.0* _ 2.1*t

28.5 26.3 26.0 24.7

+ 0.4 b _+ 0.5* • 0.9* • 0.6*

A. gemmatalis Control (13) Xanthine (14) Allopurinol (9)a Xanthine + allopurinol (1)f

6.5 • 0.1 6.9 • 0.3 7.8 • 0.4*

52.5 • 2.4 47.1 • 2.9 36.2 • 1.4""

22.2 • 1.0e 22.8 + 0.4 20.5 + 0.8

"N = number of larvae surviving to the prepupal stage out of 15 initial larvae in each treatment. Asterisk indicates that a treatment mean was significantly different from the respective control mean, based on a two-tailed t-test, and t indicates that the effect of the two compounds combined in the same diet, compared with larvae fed the control diet, was significantly greater than that of the sum of the effects of each compound tested separately, based on a one-tailed t test (P < 0.05). See text for outcome of G tests. bBecause % dry mass was calculated for 1-day-old pupae and not all larvae reaching the prepupal stage pupated, these means are based on the following sample sizes: N = 15, 14, 13, 5 for S. frugiperda and 12, 10, 6, 0 for A. gemmatalis, respectively. "This number includes six larvae that had not reached the prepupal stage by day 12 when the experiment was stopped. JData for two developmentally abnormal larvae fed this diet were omitted from the analysis. "This mean is based on N = 8 because one pupa was lost during processing. fOnly one A. gemmatalis larva fed the combination diet did not die prior to the prepupal stage, precluding collection of meaningful data for this treatment. The one surviving insect remained in the larval stage on day 12 when the experiment was stopped.

F o r A. gemmatalis, survival o n the x a n t h i n e diet w a s not r e d u c e d c o m p a r e d w i t h that o n the c o n t r o l diet a n d , a l t h o u g h survival d e c l i n e d on the allopurinol diet, the r e d u c t i o n w a s not statistically significant (G = 0 . 4 4 , P > 0 . 2 5 ; T a b l e 3). X a n t h i n e alone h a d n o effect o n any o f the m e a s u r e s o f larval p e r f o r m a n c e , w h e r e a s allopurinol a l o n e p r o l o n g e d d e v e l o p m e n t and c a u s e d significant c h a n g e s in all o t h e r p e r f o r m a n c e m e a s u r e s ( r e d u c t i o n s in final dry m a s s , R G R , R C R , a n d E C D , and an i n c r e a s e in A D ) e x c e p t p e r c e n t a g e dry m a s s (Table 3 and F i g u r e 3). W h e n allopurinol and x a n t h i n e w e r e c o m b i n e d in the s a m e diet, 14 o f 15 A. gemmatalis larvae d i e d p r i o r to r e a c h i n g the p r e p u p a l stage, p r e v e n t i n g c o l l e c t i o n o f p e r f o r m a n c e data for insects in this t r e a t m e n t . T h e o n e r e m a i n i n g larva h a d not f o r m e d a p r e p u p a by day 12 w h e n the e x p e r i m e n t w a s s t o p p e d .

2644

SLANSKY Fall a r m y w o r m

[m RGR [ ]

RCR [ ] AD [ ] ECD]

2.5 "

60 iilili

~

50

2,0,,

4 0 >U

i i"i'l 30

E

,< 1.0,,

20 w

n,-

0.5 "

ol

CONTROL

10

XANTHINE

ALLOPURINOL XAN + ALLO

DIET

FIc. 2. Mean (+SEM) relative growth rate (RGR) and its three components [relative consumption rate (RCR), approximate digestibility (AD), and efficiency of conversion to biomass of digested food (ECD); see METHODS AND MATERIALS for formulae and Table 3 for N values] for larvae of the fall armyworm (S. frugiperda) fed a control artificial diet or diets containing xanthine (1.0% fm), allopurinol (0.5% fm), or the combination of these two compounds at the concentrations indicated. An asterisk indicates a significant difference from the control value based on a two-tailed t test and an " s " indicates a synergistic interaction in which the effect of the two compounds combined in the same diet was significantly greater than that of the sum of the effects of each compound tested separately, based on a one-tailed t test (P < 0.05).

DISCUSSION The results of this study clearly demonstrate a synergistic interaction between xanthine and allopufinol for both S. frugiperda and A. gemmatalis, although the latter species appeared to be more sensitive in terms of greater mortality when fed the combination diet. The deleterious effects of these two compounds when combined in the diet generally were much greater than the additive influence when each compound was tested separately. This synergism is particularly dramatic in that, for both species, xanthine alone (up to 2 % fm or 10% din) had little or no effect on performance and allopufinol alone (at 0.5% fm or 2.5% dm) had only moderate (albeit statistically significant) deleterious effects. Several studies of plant-feeding insects have demonstrated synergism of deleterious allelochemical activity using inhibitors of the enzymes traditionally considered to detoxify xenobiotics, such as polysubstrate monooxygenases and hydrolases (esterases) (reviewed in Lindroth, 1991; Brattsten, 1992; and Wheeler et al., 1993). The present study expands the phenomenon of phytochemical detoxification by insects, as demonstrated by synergism through

2645

X A N T H I N E T O X I C I T Y S Y N E R G I Z E D BY A L L O P U R I N O L

2..5 .

VeLvetbean

caterpillar

I 1 RGR [] RCR [] no [] EOD I

*

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.

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CONTROL

XANTHINE DIET

ALLOPURINOL

FI~. 3. Mean (+ SEM) quantitative performance measures for larvae of the velvetbean caterpillar (A. gemmatalis) fed a control artificial diet or diets containing xanthine (1.0% fm) or allopurinol (0.5% fm); all but one larva died when fed diet containing the combination of these two compounds at the concentrations indicated. An asterisk indicates a significant difference from the control value based on a two-tailed t test (see Figure 2 for additional information).

use of an enzyme inhibitor, to include xanthine dehydrogenase, an enzyme generally not considered within the context of xenobiotic detoxification. Xanthine alone does not appear to be a very potent defensive allelochemical against these caterpillars. In the experiments in which food consumption was measured, larvae of these two species ingested xanthine (dietary concentration = 1% fm) at a biomass-relative consumption rate (RCRxan) as high as 82 mg (539/~mol) (g x d) - l , and likely at twice this rate on the 2% xanthine diet in the experiments in which ingestion was not measured, with little reduction in performance. In terms of comparative toxicity to S. frugiperda, xanthine is similar to other allelochemicals such as phytic acid, oxalic acid, atropine, and chlorogenic acid (unpublished data). Other phytochemicals, including flavone (Wheeler et al., 1993), rotenone (G.S. Wheeler, unpublished data), benzaldehyde, caffeine, indole 3-acetonitrile, indole 3-carbinol, and certain simple coumatins (unpublished data) are more toxic, causing significant reductions in growth and/or survival at dietary concentrations lower than 1% fm. Less information is available on the sensitivity of A. gemmatalis larvae to allelochemicals, but caffeine (Slansky and Wheeler, 1992), fiavone, indole 3-acetonitrile, indole 3-carbinol, and certain simple coumarins (unpublished data) all reduce performance at dietary concentrations less than 1% fro. One reason why increased concentrations of dietary xanthine have little effect on larval performance may be that a greater rate of absorption of xanthine

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is accommodated by an increased rate of conversion to and excretion of uric acid. Enhanced uric acid excretion has been demonstrated in various insects when they were fed diets containing high levels of, or poor quality, protein, and diets lacking certain amino acids or the vitamin pyridoxine (Horie and Inokuchi, 1978; Horie and Watanabe, 1983a,b; Cochran, 1985). For example, when dietary protein (soybean meal) was increased from 20 to 60% dm, silkworms (Bombyx mori L.) absorbed about fourfold more nitrogen and excreted almost 18-fold more uric acid (Horie and Watanabe, 1983a; see also Kamioka et al., 1971), associated with induction of xanthine dehydrogenase activity (Ito and Mukaiyama, 1964). Similarly, larvae of Manduca sexta (L.) produced 2.5fold more uric acid when fed a low water (65% fm) versus high water (82% fm) diet despite similar biomass-relative consumption rates of nitrogen (Van't Hof and Martin, 1989). In that study, caterpillar growth was apparently limited by reduced water intake on the low-water diet, resulting in an excess of absorbed nitrogen that required excretion. If greater xanthine dehydrogenase activity and uric acid synthesis occurred in the present experiments, as is likely when larvae were fed increased concentrations of xanthine, then the lack of decline in ECD (efficiency of conversion to biomass of digested food) for either species fed diet supplemented with this purine suggests that there was no significant metabolic cost to these activities. This apparent lack of cost is supported by the results of studies on increased allelochemical processing by caterpillars, e.g., through greater detoxification enzyme activity (Neal, 1987; Appel and Martin, 1992). However, increased metabolic costs may not be reflected in a declining ECD when calculated based on dry mass (as used here); more appropriate measures include ECD based on energy values of insect, food, and frass and on direct measurement of metabolic rate (Stansky, 1985; Appel and Martin, 1992). Addition of up to 1% fm (5 % dm) of allopurinol to the standard diet consistently reduced the RGR (biomass-relative growth rate) and ECD of both species, suggesting that the presumed inhibition of xanthine dehydrogenase and consequent disruption of normal synthesis and excretion of uric acid exerted deleterious effects, although certain other enzymes also may have been inhibited (Glassman and Mitchell, 1959; Kelley and Beardmore, 1970; Jones et al., 1978; Chovnick et al., 1980). The reduction in percentage dry mass (i.e., increase in percentage of body water) for each species when fed diet containing allopurinol is consistent with this interpretation that normal excretion was disrupted, in that excretory activity and osmoregulation are interrelated (Cochran, 1985). Significant synergistic effects on the consumption and utilization of food (assessed only for S. frugiperda because only one A. gemmatalis larva fed the combination diet survived to the end of the experiment) included reductions in RCR, ECD, and RGR, associated with prolonged development and a decrease in final pupal mass; AD was not affected. It is evident that the deleterious effects of allopurinol

X A N T H I N E T O X I C I T Y S Y N E R G I Z E D BY A L L O P U R I N O L

2647

absorption were magnified when larvae consumed the combination diet, most likely because the dietary xanthine absorbed could not be converted to uric acid and excreted. Why A. gemmatalis appeared to be more sensitive than S. frugiperda to the dietary combination of xanthine and allopurinol, despite apparently similar ingested doses of these compounds, is unknown. Xanthine (1.0% fro) and allopurinol (0.5% fm) were used in both the dosage and synergism experiments. Most of the results for these compounds were consistent between experiments, showing either similar statistically significant differences or at least similar trends. Some variation in the outcome of a treatment when tested with larvae from different generations, even from the same laboratory colony, is to be expected and is likely associated with intergenerational changes in the "quality" of the insects (unpublished data; see also Clancy, 1991). Xanthine has been identified in certain plant species (see Introduction). Some of these are reported as food plants of the noctuid species studied here (Tietz, 1972), although there appear to be few published studies that have evaluated growth and survival of larvae of either species on such plants (e.g., Slansky, 1989). The results reported here suggest that this purine, by itself, in these plants would be basically innocuous, but that there could be substantial deleterious effects if it cooccurred with an inhibitor of xanthine dehydrogenase. To my knowledge, such an interaction involving a naturally occurring purine and an inhibitor of xanthine dehydrogenase has not been identified, although it is likely to occur, given the variety of modes of action already demonstrated for allelochemicals, e.g., digestive and detoxification enzyme inhibitors, nutrient absorption blockers, antivitamins, nutrient analogs, antihormones, and hormone analogs (Slansky, 1992). Studies on vertebrates indicate that xanthine dehydrogenase has a relatively broad range of substrates (Bergmann and Dikstein, 1956; Lohmann and Miech, 1976; Parks and Granger, 1986). Thus, this enzyme may be involved in the detoxification and excretion by insects of other allelochemicals in addition to xanthine, although further research is required to investigate this possibility. Acknowledgments--I thank B.L. Dawickeand K.A. Nesheimfor their technical assistance in carrying out these experiments and for data processing. I am grateful to G,S. Wheeler and D.R. Suiter for their reviewsof an early draft of this manuscript. This work was supportedby the National Science Foundation Award No. BSR-8918254and by state project ENY-03012. FloridaAgricultural Experiment Station Journal Series No. R-03174. REFERENCES ALDRIDGE,A,, PARSONS,W.D., and NEIMS,A.H. 1977. Stimulationof caffeinemetabolismin the

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specificity of Manduca sexta. Ecology 73:216-228.

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BARTELS,E.C. 1966. Allopurinol (xanthine oxidase inhibitor) in the treatment of resistant gout. JAMA 198:132-136. BERGMANN,F., and DIKSTEIN,S. 1956. Studies on uric acid and related compounds III. Observations on the specificity of mammalian xanthine oxidases. J. Biol. Chem. 223:765-780. BRATTSTEN, L.B. 1992. Metabolic defenses against plant allelochemicals, pp. 175-242, in G.A. Rosenthal, and M.R. Berenbaum (eds.). Herbivores: Their Interactions with Secondary Plant Metabolites, Vol. II: Evolutionary and Ecological Processes, 2nd ed. Academic Press, San Diego. CHOVNICK, A., MCCARRON, M., CLARK, S.H., HmLmER, A.J., and RUSHLOW,C.A. 1980. Structural and functional organization ofa gene in Drosophila melanogaster, pp. 3-23, in O. Siddiqi, P. Babu, L.M. Hall, and J.C. Hall (eds.). Development and Neurobiology of Drosophila. Plenum, New York. CLANCY, K.M. 1991. Multiple-generation bioassay for investigating western spruce budworm (Lepidoptera: Tortricidae) nutritional ecology. Environ. Entomol. 20:1363-1374. COCHRAN, D.G. 1975. Excretion in insects, pp. 177-281, in D.J. Candy and B.A. Kilby (eds.). Insect Biochemistry and Function. Chapman and Hall, London. COCHRAN,D.G. 1985. Nitrogenous excretion, pp. 467-506, in G.A. Kerkut, and L.I. Gilbert (eds.). Comprehensive Insect Physiology, Biochemistry and Pharmacology, Vol. 4. Regulation: Digestion, Nutrition, Excretion. Pergamon Press, Oxford. DUKE, J.A. 1981~ Handbook of Legumes of World Economic Importance, 1st ed. Plenum Press, New York. EuoN, G.B. 1966. Enzymatic and metabolic studies with allopurinol. Ann. Rheum. Dis. 25:608614. ELION, G.B. 1989. The purine path to chemotherapy. Science 244:41-47. ENGEBRETSOr~, J.A., and MULLINS, D.A. 1986. Effects of a purine inhibitor, allopurinol, on urate metabolism in the German cockroach, Blattella germanica L. (Dictyoptera: Blattellidae). Cutup. Biochem. Physiol. 83B:93-97. GIBBS, R.D. 1974. Chemotaxonomy of Flowering Plants. McGill-Queen's University Press, Montreal. GLASSMAN,E., and MITCHELL, H.K. 1959. Mutants of Drosophila melanogaster deficient in xanthine dehydrogenase. Genetics 44:153-162. GORDON, H.T. 1968. Quantitative aspects of insect nutrition. Am. Zool. 8:131-138. GREENE, G.L., LEPeLA, N.C., and DICKERSON, W.A. 1976. Velvetbean caterpillar: A rearing procedure and artificial medium. J. Econ. Entomol. 69:487-488. HERZOC, D.C., and TODD, J.W. 1980. Sampling velvetbean caterpillar on soybean, pp. 107-140, in M. Kogan, and D.C. Herzog (eds.). Sampling Methods in Soybean Entomology. SpringerVerlag, New York. HOCHSTEIN, P., HATCH, L., and SEVANIAN, A. 1984. Uric acid: Functions and determination. Methods Enzymol. 105:162-166. HORIE, Y., and tnoKucnt, T. 1978. Protein synthesis and uric acid excretion in the absence of essential amino acids in the silkworm, Bombyx mori. Insect Biochem. 8:251-254. HORm, Y., and WATANABE,K. 1983a. Effect of various kinds of dietary protein and supplementation with limiting amino acids on growth, haemolymph components and uric acid excretion in the silkworm, Bombyx mori. J. Insect Physiol. 29:187-199. HORIE, Y., and WATANABE,K. 1983b. Effects of dietary pyridoxine on larval growth, free amino acid pattern in haemolymph and uric acid excretion in the silkworm, Bombyx mori. Insect Biochem. 13:205-212. ITO, T., and MUKAIYAMA,F. 1964. Relationship between protein content of diets and xanthine oxidase activity in the silkworm, Bombyx mori. J. Insect Physiol. 10:789-796.

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