Effect of Diet on Detoxification Enzyme Activity of Platynota Idaeusalis (Walker) (Lepidoptera: Tortricidae) Larvae Strains

Rev. Fac. Agron. (LUZ). 2000, 17: 119-138

Effect of diet on detoxification enzyme activity of Platynota idaeusalis (Walker) (Lepidoptera: Tortricidae) larvae strains

Efecto de la dieta en la actividad enzimática de detoxificación de larvas de Platynota idaeusalis (Walker) (Lepidoptera: Tortricidae)

O. E. Domínguez-Gil y B. A. McPheron

Abstract

The effect of diet on detoxification enzyme capabilities in two genetically similar strains (one susceptible and other resistant to azinphosmethyl) of the tufted apple bud moth, Platynota idaeusalis (Walker) (Lepidoptera: Tortricidae), was studied by biochemical methods. The larvae were fed with a synthetic diet and four different host plant species: apple, Malus domestica (Bork.) cv Red Yorking; black raspberry, Rubus occidentalis L.; broad-leaved plantain, Plan- tago major L.; and dandelion, Taraxacum officinale Wiggers. Host plant affected larval detoxification enzyme activity in both the resistant and susceptible strain. Glutathione transferase and esterase activities, both implicated in P. idaeusalis resistance to azinphosmethyl, varied significantly between strains and among hosts. Diets of apple and plantain appeared to inhibit both enzyme systems compared to artificial diet in both insect strains. However, patterns of enzyme activity and azinphosmethyl susceptibility are not clearly linked, reinforcing the complex relationship of the insect with the chemistry of its host. Key words: diet-insect interaction, Platynota idaeusalis, resistance, detoxification enzyme activity.

Resumen

Se examinó el efecto de la dieta en la capacidad de la actividad enzimática de detoxificación a insecticida de dos razas similares genéticamente (una susceptible y una resistente a azinphosmethyl) de la “polilla de la manzana” Platynota idaeusalis (Walker) (Lepidoptera: Tortricidae), fue examinada. Las larvas objeto del estudio fueron alimentadas separadamente con una dieta artificial y cuatro diferentes especies de plantas hospederas: manzana, Malus domestica (Bork.) cv Red Yorking; mora, Rubus occidentalis L.; llantén, Plantago major L.; y diente

Recibido el 18-12-1996 l Aceptado el 21-11-1997 1 Departamento Fitosanitario. Facultad de Agronomía de LUZ. Apartado 15378. Maracaibo, Venezuela. (To whom correspondence should be addressed). 2 Department of Entomology, Pennsylvania State University, University Park, USA.

119 Domínguez-Gil y McPheron de león, Taraxacum officinale Wiggers. Las plantas hospederas afectaron la actividad enzimática de detoxificación de las larvas tanto resistentes como susceptibles. La actividad de las enzimas glutationa transferasa y esterasas, ambas implicadas en la resistencia de P. idaeusalis a azinphosmethyl difirieron significativamente entre razas y hospederas. Las dietas de manzana y llantén parecen inhibir ambos sistemas de enzimas cuando se compararon con la dieta artificial en ambas razas del insecto. Sin embargo, la actividad enzimática y la susceptibilidad a azinphosmethyl no están claramente ligadas, indicando la compleja relación que existe entre el insecto y la química de su hospedero. Palabras clave: interacción dieta-insecto, Platynota idaeusalis, resistencia, actividad enzimática de detoxificación.

Introduction

Currently, insecticide resistance ing of non-genetic influences (diet, age is a serious problem in Platynota and deve-lopment, temperature) on the idaeusalis (Walker) control. This tor- expre-ssion of insecticide resistance. tricid has developed resistance to many Previous studies in some insect spe- of the organophosphate (OP) insecti- cies have shown that host plants also cides most commonly used in apple affect le-vels of resistance (46, 6, 36, orchards during the past 30 years (32, 41, 37, 43, 17). Even though some in- 23, 24). These increased resistance le- sects have genes for insecticide resis- vels have resulted in field failures of tance, something in the plant appears OP insecticides (25). Fruit growers to modify the expression of resistance. have increased applications of carba- One important factor is the effect of mates (22) and pyrethroid insecticides larval host plant chemistry on detoxi- (23) in response to the development of fication capabilities. It has long been OP resistance. The use of these alter- known that feeding on certain host native insecticides can disrupt care- plants can induce and/or suppress en- fully balanced integrated pest manage- zymes involved in the detoxification of ment (IPM) programs developed in pesticides. Detoxification of plant apple orchards over the last 20 years allelochemicals by enzyme systems has (15, 7). Moreover, OPs, so-called IPM- been shown to be one of the most im- compatible materials, have been used portant ways that insects deal with to selectively control up to 15 other plant diets (27). At least 27 species of pests that attack apples (Hull 1991, arthropods have been found to have unpubl. data). Therefore, preserving detoxification systems inducible by P. idaeusalis susceptibility to cur- plant chemicals (47). Because P. rently available OP compounds is va- idaeusalis is a highly polyphagous spe- luable until we have other IPM-com- cies, there is a high probability for this patible control measures. It is impor- insect to feed on a wide range of plants tant to consider which factors influ- with unique se-condary compounds. ence the loss of P. idaeusalis suscepti- Platynota idaeusalis experiences a bility and to obtain a basic understand- wide array of different compounds in

120 Rev. Fac. Agron. (LUZ). 2000, 17: 119-138 its use of 17 plant families (30). Nu- host plants on levels of some enzyme merous studies with the highly systems that are related with patterns polyphagous gypsy moth, which uti- of resistance to OPs. The interaction lizes over 300 species of trees and of P. idaeusalis with its host plants shrubs from at least 14 plant families has not been extensively studied and (16, 26), have shown that food selec- affects many components of pest man- tion and utilization of both hosts and agement. Natural enemies may use nonhosts are related to plant non-crop plant species to feed in the allelochemicals (4, 38, 29). The same adult stage. Alternative hosts may enzymes that are involved in metabo- harbor susceptible or resistant P. lism and detoxification of pesticides are idaeusalis populations which can dis- also involved in the metabolism of plant perse into the orchard environment. allelochemicals. Thus, it is reasonable Results of this study can be combined to assume that OP insecticides and with other research results to help plant secondary chemicals may inter- design the next generation of pest act to produce positive or negative syn- management strategies. They may ergistic effects upon OP toxicity in P. also be used to preserve the effective- idaeusalis. Hunter et al. (21) deter- ness of currently recommended OP mined that an apple allelochemical, insecticides, which are IPM compat- phloridzin, influenced detoxification ible materials, until these new strate- activities of larval P. idaeusalis. Phlo- gies can be implemented. The present ridzin decreased GST activity in both study measured detoxification enzyme susceptible and resistant P. idaeusalis. activity of glutathione transferase and Also, phloridzin inhibited esterase and carboxylesterases (previously impli- aniline hydroxylation of the susceptible cated in pesticide resistance (12, 13, larvae, but induced higher esterase 11) on susceptible and resistant P. activity in resistant larvae. Therefore idaeusalis larvae reared on four host it is critical to investigate the effect of plants and artificial diet.

Materials and methods

Chemicals. Glutathione (98- Host Plants. Host plant species 100% reduced), a-naphthyl acetate (a- (table 1) were raised in the greenhouse NA), a-naphthyl butyrate (a-NB), and at the Pennsylvania State University, 1,2- dichloro-4-nitrobenzene (DCNB) University Park. Apple is the economi- (95% purity), were purchased from cally important host of P. idaeusalis; Sigma Chemical Co. (St. Louis, MO). the other three species are present in 1-chloro-2,4-dinitrobenzene (CDNB) the apple orchard and surrounding (99% purity) was obtained from Aldrich vegetation. The apple trees were from Chemical Co. (Milwaukee, WI). the cultivar Red Yorking on Emla 7 Coomassie Blue G-250 was purchased root stock. Black raspberries were from Serva Company. All other chemi- started from cuttings. The remaining cals were of analytical quality and host species were grown from seed in purchased from commercial suppliers. potting soil. All plants were trans-

121 Domínguez-Gil y McPheron Family Rosaceae Plantaginaceae Asteraceae Variety Red Yorking Allen Rosaceae . Source Aspers, PA. Canandaigua, NY. Port Matilda, PA. Fredonia, NY. Platynota idaeusalis Wiggers Stokes Seeds, Inc., L.Nurseries, Miller (Bork.). Adams County Nursery, L. B. McPheron house, Malus domestica Rubus occidentalis Plantago major Taraxacum officinale Table 1. Plant species used in fitness study for Common nameApple Scientific name Black raspberry Broad-leaved plantain Dandelion

122 Rev. Fac. Agron. (LUZ). 2000, 17: 119-138 planted to pots filled with Terra Lite for one generation to increase numbers Metro-Mix 250® Growing medium (E. (F2) and then selected (8.5 ppm C. Geiger, Harleysville, Pennsylvania). azinphosmethyl (50WP) directed at Plants were fertilized every two neonates in a diet incorporation bioas- months with water-soluble 15-30-15 say (7). Surviving larvae were reared (N-P-K) fertilizer (4.0 g/l). Plants were to pupation, sexed, and two reciprocal watered as needed and grown under a crosses with the susceptible line were 16-h photophase and ambient relative conducted. Progeny (F3) had, on ave- humidity. Whitefly, thrips, mite and rage, 75% genetic background from the aphid infestations in the greenhouse susceptible strain. This generation was necessitated the use of 2.5% safer in- interbreed for one generation to in- secticidal soap (50.5% potassium salts crease numbers (F4). Pupae were of fatty acids, AgroChem, Jamul, Cali- sexed, and two reciprocal crosses were fornia). As a precaution, all host spe- conducted. Progeny (F5) had, on ave- cies were treated at the same time. rage, 87% genetic background from the Test plants were selected arbitrarily susceptible strain. This generation was from a group of individual plants that interbreed for one generation to in- were similar in height and phenology. crease numbers (F6) and then selected For comparison with previous studies, (8.5 ppm as in F2). A bioassay (neo- P. idaeusalis larvae were also reared nate using diet-incorporated on a lima bean based artificial diet (31) azinphosmethyl) at this generation under identical environmental condi- showed an LC50 of 46 ppm. Surviving tions. larvae were reared to pupation, sexed, Insect cultures. The genotype and two reciprocal crosses with the resistant to azinphosmethyl used in susceptible line were conducted. Pro- this experiment is a line originally geny (F7) had, on average, 94% genetic derived from resistant larvae collected background from the susceptible in an apple orchard in Adams County, strain. This generation was interbreed Pennsylvania and made nearly for one generation to increase numbers isogenic relative to a laboratory sus- (F8). Pupae were sexed, and two re- ceptible line. The latter was accom- ciprocal crosses were conducted. Pro- plished by the protocol of backcross- geny (F9) had, on average, 97% genetic ing and selection detailed as followed: backgrounds from the susceptible larvae were collected from apple or- strain. This generation was interbreed chard with a history of resistance in to increase numbers (F10). The F10 Adams County, Pennsylvania. Field- neonates were selected (25 ppm collected larvae were reared to pupa- azinphosmethyl (50 WP) directed at tion, sexed, and mixed in two recipro- neonates in a diet incorporation bioa- cal crosses (male field × female labo- ssay). The survivors of this selection ratory susceptible and male laboratory constituted the resistant strain colony. susceptible × female field). Progeny No further crosses to the susceptible (F1) had, on average, 50% genetic line were made. The protocol for diet background from the susceptible incorporated azinphosmethyl selection strain. This generation was interbreed bioassay of neonates followed (7).

123 Domínguez-Gil y McPheron

Briefly, formulated azinphosmethyl After seven days on the host, lar- was applied (0.5 ml) in diluted aque- vae were removed, weighed individu- ous solution to the surface of 7-10 ml ally, and frozen at -80ºC. These larvae of lima bean based synthetic diet in and the larvae used in the previous plastic cups and allowed to air-dry for bioassays were reared at the same time 2-3 h. Test larvae were introduced into on the same plants. diet cups in groups of four. The larval Biochemical analyses. Detoxi- exposure period to the treated diet was fication enzyme activity was measured until pupation or death. Repeated back- in fifth instar larvae (19-days old) of crossing essentially creates an both azinphosmethyl-resistant and “isogenic” strain by gradually diluting susceptible strains reared on the four the fraction of the genome coming from plant species and artificial diet. Assays the resistant parent (39). This method were conducted for two different en- is used to move a major resistance zyme systems. Glutathione transferase gene into a susceptible genetic back- (GST) and carboxylesterase activities ground and thereby isolate it from were assayed according to the proto- other genes that affect the resistant cols of Carlini (13). GST activity was phenotype. determined because it is likely to be Treatment of insects. Twelve the most important enzyme in P. day old fourth instar larvae were used idaeusalis resistance to a for experiments. Larvae from the re- azinphosmethyl (13, 30) in Pennsylva- sistant strain were generation 16, ex- nia. Carboxylesterase activity was cept for the experiment on apple, where measured because this enzyme system generation 18 was used. Larvae were is a generally important detoxification maintained from hatching on a lima mechanism in insects, including P. bean based artificial diet (31) at 26.7ºC, idaeusalis (12, 13, 11). Ten to thirty 60% relative humidity with a photo- six individuals per treatment per en- period of 16:8 (L:D) h in the Depart- zyme substrate were used in each of ment of Entomology, Pennsylvania the enzyme assays. In most cases, GST State University. Twelve day old lar- and esterase activities were assayed vae were removed from artificial diet from single individuals. and transferred to one of four host Carboxylesterase and glu- plants apple, black raspberry, plantain, tathione transferase enzyme or dandelion or artificial diet for seven preparations. Whole larvae were ho- days. mogenized in 100 ml of 50 mM sodium Sleeve cages (5-10 larvae per phosphate buffer, pH 7.4, using a teflon cage) made of fine pore nylon meshed conical homogenizer. The homogenate and sealed with parafilm were used to was centrifuged at 12,000 x g at 4ºC prevent larval escape. Plants raised in for 10 min in a Brinkman fixed angle the greenhouse were moved to a walk Eppendorf 5415 centrifuge. The super- in type growth chamber at the Depart- natant was used to determine ment of Entomology, Pennsylvania carboxylesterase and glutathione State University set at 26.7ºC, day transferase activity. Supernatant not length: 16:8 (L:D) h and 50-80% R.H. used immediately was stored at -80ºC

124 Rev. Fac. Agron. (LUZ). 2000, 17: 119-138 until needed for analysis. tathione solution, and 387.5 ml of 50 In vitro enzyme assays. mM sodium phosphate buffer. The re- Carboxylesterase activity (3) was mea- action was started by adding 12.5-25 sured spectrophotometrically with a- ml of DCNB at a concentration of 40 naphthyl acetate and a-naphthyl bu- mM in ethanol to the solution (artifi- tyrate as substrates according to a cial diet and plantain: 12.5 ml; apple, method described by Gomori (18). The black raspberry and dandelion: 25 ml). incubation mixture (VF=1 ml) con- Absorbance change at 344 nm was tained 1 ml of supernatant, 1 ml of monitored for 5 min. substrate (0.25 mM final concentra- The procedure to determine GST tion), and 998 ml of 50 mM sodium activity using CDNB was based on a phosphate buffer, pH 7.4. The reaction method modified by Carlini (2, 13, 20). mixture was incubated for 10 min at In the case of larvae reared on artifi- 37ºC and ended by the addition of 2 ml cial diet and plantain, the reaction mix- of aqueous solution containing 0.9 mg ture consisted of 1 ml protein, 100 ml fast blue B salt and 7.5 mg sodium of 50 mM glutathione solution, and 874 dodecyl sulfate. The absorbance of the ml of 50 mM sodium phosphate buffer, reaction was read at 600 nm against a pH 7.4, which was incubated for 2 min reference lacking enzyme with a at 37ºC. For larvae reared on black Perkin-Elmer 3B Lambda spectropho- raspberry and dandelion, I used 1ml tometer equipped with a computer in- homogenate, 50 ml of 50 mM glu- terface to a Swan 286/12 and converted tathione solution, 436.5 ml of 50 mM to concentration of a-naphthol using a sodium phosphate buffer. The incuba- standard curve. tion mixture for apple was 10 ml ho- Glutathione transferase activity mogenate, 100 ml of 50 mM glu- was assayed spectrophotometrically tathione solution, and 865 ml of 50 mM with DCNB and CDNB as substrates. sodium phosphate buffer. The CDNB The procedure to determine GST ac- reaction was started by adding 12.5 to tivity using DCNB was based on a 25 ml CDNB at a concentration of 40 method reported by Siegfried and mM in ethanol to the solution (artifi- Mullin (41), modification of a method cial diet and plantain: 25ml; black described originally from Booth et al raspberry, apple, and dandelion: 12.5 (8). When DCNB was used as the sub- ml). The reaction was monitored for 3 strate, the reaction mixture for larvae min at 340 nm by recording change in reared on plantain and artificial diet absorbance. was 100 ml of homogenate added to a Enzyme activity (amount of con- solution containing 100 ml glutathione jugate formed) was converted to spe- at a concentration of 50mM in distilled cific activity using a millimolar extinc- water and 775 ml of 50mM sodium tion coefficient of 8.5 cm-1 for DCNB and phosphate buffer, and incubated for 2 9.6 cm-1 for CDNB (20). min at 37ºC. The incubation mixture Protein estimations. Protein for larvae reared on apple, black rasp- content of samples was determined by berry, and dandelion consisted of 50 ml the method of Bradford (9). Five ml of of homogenate, 50 ml of 50 mM glu- sample were added to 95 ml of 50mM

125 Domínguez-Gil y McPheron sodium phosphate buffer, pH 7.4, and by two way analysis of variance 5 ml of coomassie blue solution G-250. (ANOVA) using the Statview statisti- The absorbance was read 2 min after cal program (1), followed by Fisher’s addition of the dye at 595 nm against protected least significant difference a blank lacking protein and compared (PLSD) mean separation tests (33). to a bovine serum albumin (BSA) stan- Factors for the two-way analyses were dard curve. diet and strain. Specific comparisons Statistical Analyses. Activity between the resistant and the suscep- levels of detoxification enzymes among tible strain within each host were de- larvae fed different diets were analyzed termined by 2 tailed "t" tests.

Results

Bioassays in the laboratory con- tween strains in larvae reared on dan- firmed that the resistant line was re- delion, plantain, or apple (table 3, fi- sistant to azinphosmethyl. Results of gures 1 and 2). Susceptible larvae from topical bioassays on the F16 generation artificial diet and dandelion had sig- indicated a 3.4-fold level of resistance nificantly higher DCNB-specific GST to azinphosmethyl when compared activities than susceptible larvae on with the LD50 of the susceptible labo- apple, plantain, or black raspberry (fi- ratory strain. Host plant affected lar- gure. 1). GST activity toward DCNB val detoxification enzyme activity in was highest in resistant larvae fed both the resistant and susceptible artificial diet, followed by black rasp- strain. Glutathione transferase and berry, dandelion, plantain, and apple carboxylesterase activities, both impli- (figure 1). cated in P. idaeusalis resistance to Resistant larvae reared on black azinphosmethyl, varied significantly raspberry, artificial diet, and plantain between strains and among hosts. had significantly higher specific GST Glutathione transferase ac- activities toward CDNB than did their tivity. Activity levels of GST using the susceptible counterparts in pairwise model substrates DCNB and CDNB comparisons (table 3, figures 3 and 4). were significantly affected by diet, Resistant larvae fed apple had signifi- strain, and the interactions of these cantly reduced activity compared to two factors (table 2). Mean enzyme susceptible larvae when CDNB was activities of both the susceptible and used as a substrate. Although not sig- resistant strain on each diet are pre- nificantly different, resistant larvae on sented in table 3. dandelion also had lower activity than Resistant larvae fed artificial diet susceptible larvae for CDNB conjuga- and black raspberry exhibited signifi- tion (table 3, figures 3 and 4). Resis- cantly higher GST activity levels than tant larvae responded to a diet of black their susceptible counterparts (table 3, raspberry with the highest CDNB-spe- figures 1 and 2), when DCNB was cific GST enzyme activity and apple, used as the substrate. Little or no dif- the lowest, with the values for resis- ference in activities was detected be- tant larvae fed artificial diet, dande-

126 Rev. Fac. Agron. (LUZ). 2000, 17: 119-138

Table 2. Two-way analysis of variance indicating source of variation, degrees of freedom, F, and P values for data from enzyme analysis.

Detoxification Interaction enzyme Strain Diet (Strain × Diet) df F df F df F

Glutathione transferase- 1,250 52.8** 4,250 60.0** 4,250 36.7** DCNB/Protein GST- CDNB/Protein 1,296 75.6** 4,296 77.5** 4,296 27.4** EST- NA/Protein 1,278 27.2** 4,278 65.9** 4,278 10.9** EST- NB/Protein 1,277 6.5* 4,277 29.9** 4,277 7.9**

*F is significant at P< 0.01,** P< 0.001 lion, and plantain intermediate (table susceptible and resistant strain larvae 3, figure 3). reared on apple had the lowest acti- When susceptible larvae fed on vity levels among all treatments (table dandelion, their GST activity toward 3, figures 3 and 4). CDNB was the highest, with apple and Carboxylesterase activity. plantain the lowest and artificial diet General carboxylesterase activities to- and black raspberry intermediate ward a-naphthyl acetate and a-naph- (table 3, figures 3 and 4). thyl butyrate were significantly af- For CDNB conjugation, both the fected by diet, strain, and the interac-

Figure 1. DCNB-specific glutathione transferase activity of both the susceptible (S) and resistant strain (R) larvae of P. idaeusalis fed different diets. Vertical lines indicate 1 SE. Means within each strain indicated by the same letter are not significantly different from each other (P<0.05, Fisher´s protected LSD).

127 Domínguez-Gil y McPheron NB a- P. idaeusalis 87.6(9.3;18) 0.1554 NA a- 935(159;16) 770(112;16) 118(143;18) 271(34;36)0.011 256(31.2;36) 6,113(526;27) 523(128;15)2,998(241;36)7,298(454;36) 556(108;15) 157(15;35) 340(31;35) 134(16.0;35) 298(31.9;34) . DCNB CDNB 10.2(1.1;23)8.9(1.5;10) 717(62;33) 363(33;31) 347(43;31)26.9(2.1;27)20.0(2.4;30) 6,157(395;30) 261(54;31) 5,124(368;35) 1,221(84;30) 707(86;36) 567(53;30) 341(44;36) Glutathione transferase Carboxylesterase Strain strains fed different diets Diet Table 3. Mean enzyme activities [mM/min/mg] (± SEM; n) of susceptible (S) and resistant (R) Artificial dietArtificial dietP-level (t-test)Apple SApple RP-level (t-test) 29.7(2.3;16)Black raspberry 69.0(4.4;28) Black raspberry 0.0001P-level (t-test) S S RPlantain 3,174(343;16) R Plantain 0.5273P-level (t-test) 14.5(1.3;35) 50.9(4.0;32) Dandelion 0.0003Dandelion 0.0001P-level (t-test) S R 0.0001 S 13.9(1.6;24) 0.1461 R 11.2(1.0;35) 0.0001 0.055 0.034 1,237(77;34) 4,952(482;28) 0.0001 0.0002 172(18.1;36) 0.1818 0.0001 0.0603 0.0194 198(26.4;36) 0.0001 0.0001 0.0015

128 Rev. Fac. Agron. (LUZ). 2000, 17: 119-138

Figure 2. Boxplots of DCNB-specific glutathiones transferase activities of both the susceptible (S) and resistant strain (R) larvae of P. idaeusalis fed different diets. Box in the middle 50% of observations. The boxplot displays the 10th, 25th, 50th and 90th percentiles of a variable. All values for the variable above the 90th percentile and below the 10th percentile are plotted separately.

Figure 3. CDNB-specific glutathione transferase activity of both the susceptible (S) and resistant (R) larvae of P. idaeusalis fed different diets. Vertical lines indicate 1 SE. Means within each strain indicated by the same letter are not significantly different from each other (P < 0.05, Fisher´s protected LSD).

129 Domínguez-Gil y McPheron

Figure 4. Boxplots of CDNB-specific glutathione transferase activities of both the susceptible (S) and resistant strain (R) larvae of P. idaeusalis fed different diets. Box is middle 50% of observations. The boxplot displays the 10th, 25th, 50th, 75th and 90th percentiles of a variable. All values for the variable above the 90th percentile and below the 10th percentile are plotted separately.

Figure 5. a-naphthyl acetate-specific esterase activity of both the susceptible (S) and the resistant strain (R) larvae of P. idaeusalis fed different diets. Vertical lines indicate 1 SE. Means within each strain indicated by the same letter are not significantly different from each other (P < 0.05, Fisher´s protected LSD).

130 Rev. Fac. Agron. (LUZ). 2000, 17: 119-138 tions of these two factors (table 2). artificial diet, and apple following in Mean enzyme activities of both the decreasing level of activity. susceptible and resistant strain on Resistant larvae fed dandelion each diet are presented in table 3. and apple had significantly reduced a- Resistant larvae reared on dan- NB specific esterase activities com- delion, apple, artificial diet, and plan- pared to their susceptible counterparts tain had significantly lower a-NA spe- in pairwise comparisons (table 3, fi- cific esterase activities than their sus- gures 7 and 8), whereas resistant lar- ceptible counterparts in pairwise com- vae on black raspberry showed an op- parisons (table 3, figures 5 and 6), posite trend in activity with higher whereas resistant larvae on black activity than their susceptible coun- raspberry exhibited an opposite trend terpart in a pairwise comparison (table in activity with higher values than 3, Figures 7 and 8). Although not sig- susceptible larvae fed black raspberry nificantly different, resistant larvae fed (table 3, figures 5 and 6). Both resis- artificial diet and plantain had lower tant and susceptible larvae fed dande- levels than for the susceptible larvae, lion exhibited the highest esterase ac- for a-NB hydrolysis. Both resistant tivity toward a-NA among all treat- and susceptible larvae reared on arti- ments (table 3, figures 5 and 6). ficial diet exhibited the highest esterase Resistant larvae fed dandelion activities toward a-NB among all and artificial diet had significantly treatments. Among natural hosts, dan- higher levels of a-NA hydrolysis than delion had the esterase activities to- resistant larvae reared on plantain and ward a-NB. The lowest levels of a- apple, while resistant larvae fed black NB hydrolysis were observed in resis- raspberry had an intermediate value tant larvae fed apple, while resistant (figure 5). The lowest levels of a-NA larvae fed plantain and black raspberry hydrolysis in resistant larvae were had intermediate values (table 3, fig- observed in larvae fed on apple and ures 7 and 8). When susceptible lar- plantain (table 3, figures 5 and 6). vae ate artificial diet, their levels of When susceptible larvae fed on dande- a-NB hydrolysis were the highest, fol- lion, their levels of a-NA hydrolysis lowed by dandelion, apple, plantain, were the highest, with black raspberry, and black raspberry (figure 7).

Discussion

Plant allelochemicals modify le- with additional toxicant exposures (19, vels of detoxifying enzymes in herbi- 5). Even a reduction in lipid reserves vores and, therefore, their susceptibil- may increase the susceptibility of an ity to insecticides (6, 42, 10, 28, 40, insect to an insecticide such as DDT 29, 34). Although plant allelochemicals due to loss of sequestration sites (35). may directly induce or inhibit detoxi- Previous studies of the resistance fication enzymes, they may also im- of P. idaeusalis to azinphosmethyl pair the overall fitness of the herbivore have demonstrated that increased ac- thereby reducing its ability to cope tivities of both glutathione transferases

131 Domínguez-Gil y McPheron

Figure 6. Boxplots of a-naphtyl acetate-specific esterase activity of both the susceptible (S) and the resistant strain (R) larvae of P. idaeusalis fed different diets. Box is middle 50% of observations. The boxplot displays the 10th, 25th, 50th, 75th, and 90th percentiles of a variable. All values for the variable above the 90th percentile and below the 10th percentile are plotted separately.

Figure 7. a-naphtyl butyrate-specific activity of both the susceptible (S) and resistant (R) larvae of P. idaeusalis fed different diets. Vertical lines indicate 1 SE. Means within each strain indicated by the same letter are not significantly different from each other (P < 0.05, Fisher´s protected LSD).

132 Rev. Fac. Agron. (LUZ). 2000, 17: 119-138

Figure 8. Boxplots of a-naphtyl butyrate-specific esterase activity of both the susceptible (S) and the resistant strain (R) larvae of P. idaeusalis fed different diets. Box is middle 50% of observations. The boxplot displays the 10th, 25th, 50th, 75th, and 90th percentiles of a variable. All values for the variable above the 90th percentile and below the 10th percentile are plotted separately.

(13, 14, 30) and general esterases (11, larvae. The data reported here showed 12) are linked to azinphosmethyl re- that diet, genotype, and the interac- sistance in adults of P. idaeusalis. tions of these two factors affect the Hunter et al (21) reported that activity levels of GST and GST activity of both the susceptible carboxylesterases. These results mean and resistant strain P. idaeusalis lar- that activity in a given strain depended vae was decreased by the presence of on the diet upon which larvae were an apple allelochemical, phloridzin. reared. Also, phloridzin inhibited esterase ac- The use of two isogenic strains tivity and aniline hydroxylation in in this study, one susceptible to susceptible larvae. These previous azinphosmethyl and the other resistant studies were conducted with larvae to azinphosmethyl, enable us to at- reared on a lima-bean based synthetic tribute any differences in response to diet with or without phloridzin (21) or azinphosmethyl to resistance mecha- with male adults collected on phero- nisms (or possibly, closely-linked mone traps (13, 14, 30). genes) rather than unrelated strain Although P. idaeusalis is a differences. highly polyphagous insect, little is Susceptible larvae fed dandelion known about the effects of host plants had consistently high enzyme activi- on detoxification enzyme activity in ties (GST-CDNB, GST-DCNB, EST-

133 Domínguez-Gil y McPheron

NA) which correlates with the bioas- also because the mortality (LD50) is not say results. Susceptible larvae fed ar- significantly different from susceptible tificial diet tends to have high enzyme larvae fed artificial diet. These results activities but a low LD50. Susceptible agree with the report of Robertson et larvae fed apple, plantain, and black al (37), who found that resistant light raspberry all have low enzyme activi- brown apple moth larvae reared on ties. blackberry were significantly less re- Resistant larvae fed black rasp- sistant than resistant larvae reared on berry are more susceptible to any other diet. Enzyme analyses azinphosmethyl than on artificial diet, showed that GST activities were rela- associated with similar GST activities tively high resistant larvae fed black but lower esterase activities. There was raspberry. That increased activity was no consistent association between toxi- not clearly related to changes in city and enzyme activity on any of the azinphosmethyl sensitivity. The other hosts. likelyhood ratio results suggested that Resistant larvae reared on apple resistant larvae fed different diets had the lowest values of GST and might be responding to carboxylesterase activities (table 2). azinphosmethyl by different levels of The data support the studies of Hunter the same mechanism. These enzyme et al (21) using phloridzin. Hunter et assays suggested, however, that al (21) suggested that resistant larvae detoxification enzyme systems in ad- did not have higher mortality on diet dition to GST might be responsible for with phloridzin because the inhibition resistance, as previously suggested by of GST activity in resistant larvae may Bush et al (11). for P. idaeusalis. have been counterbalanced by the in- The data indicate that one can- duction of carboxylesterases. The in- not designate a population as resistant hibition observed was not clearly re- or susceptible without specifying the lated to changes in azinphosmethyl environment. The magnitude of resis- sensitivity because of wide confidence tance depends on the surrounding en- limits. However, the LD50 of resistant vironment. Furthermore, the data larvae fed apple was 2.5 times lower show that there are not single mecha- compared to resistant larvae fed arti- nisms responsible for the effect of host ficial diet. Robertson et al (37) also ob- plant on the toxicity of tained wide confidence limits in the azinphosmethyl. Some other mecha- calculation of LD50 for both resistant nisms should be examined to further and susceptible light brown apple moth explain the relationship between lar- larvae reared on apple. vae P. idaeusalis resistance to Resistant larvae fed on apple or azinphosmethyl and host plants includ- black raspberry might be erroneously ing aniline hydroxylation activity (21), classified as being genetically suscep- PSMO (44), existence of multiple forms tible because they are more susceptible of GST (45), and a decrease in the per- to azinphosmethyl compared with re- centage of penetration (44). sistant larvae fed any other diets, and No clear relationship between

134 Rev. Fac. Agron. (LUZ). 2000, 17: 119-138 toxicity and enzyme activity on diffe- stages. The fact that multiple forms rent hosts was found. One possibility of each enzyme respond to the model could be the complex chemistry of the substrates could mean that the effects plant could obscured the expression of of an enzyme dealing with the enzyme responsible for detoxifying azinphosmethyl might be obscured by the pesticide. It needs to look at the other enzymes measured by the as- effect of other isolated major plant com- says. If we had specific identification pounds like phloridzin on detoxification of one or more detoxification enzymes, and susceptibility to azinphosmethyl we could follow this in response to in some of the other plant species. Our hosts. It is also necessary to study the results suggest to study the combined relationship of enzyme detoxification effects of multiple plant compounds abilities with adult susceptibility or that could have antagonistic effect on neonate susceptibility. Finally, the re- the detoxification and susceptibility to sults of the present study clearly sug- azinphosmethyl. Additional work is gest that host plants might affect the needed to determine if there is any expression of resistance in P. carry-over of induction or inhibition of idaeusalis populations. detoxification abilities to other life

Acknowledgements

This research was partly funded ment of Entomology, Pennsylvania by the State Horticultural Association State University, USA for having a of Pennsylvania; special thanks to this participation when awarding the de- agency. Support was also obtained for partmental assistantship that allowed USDA-NE-IPM grant. We wish to ex- me to pursue this course of study. Spe- tend a grateful acknowledgement to cial gratitude to Ed Carlini for bioas- Dr. James L. Frazier and the Depart- says and biochemical analysis.

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