Elucidating mechanisms of tobacco budworm resistance to allelochemicals by dietary tests with insecticide synergists

PESTlCiDE

BIOCHEMISTRY

AND

PHYSIOLOGY

32, 55-61 (1988)

Elucidating Mechanisms of Tobacco Budworm Resistance to Allelochemicals by Dietary Tests with Insecticide Synergists’ PAUL A. HEDIN,~,* WILLIAM L. PARROTT,* JOHNIE N. JENKINS,* JOSEPHE. MULROONEY,* AND JULIUS J. MENN~ *Crop Science Research Laboratory, VS. Department of Agriculture, ARS, Mississippi State, Mississippi 39762-5367 and fPlant Sciences institute, U.S. Department of Agriculture, BARC-W, Beltsville, Maryland 20705 Received April 5, 1988; accepted June 2, 1988 Three major cotton (Gossypium hirsutum L.) plant allelochemicals-gossypol, a mixture of condensed tannins, and isoquercitrin-were fed to l-, 3-, and 5-day-old tobacco budworm (Heliothis virescens Fab.) larvae at the 0.06% level with and without 0.02, 0.06, and 0.60% piperonyl butoxide, an insecticide synergist that inhibits the activity of an insect’s existing detoxifying enzymes. All three allelochemicals were toxic to the l- and 3-day-old larvae, but they were not toxic or were only marginally toxic to the 5-day-old insects. Piperonyl butoxide was toxic to the l-, 3-, and 5-day-old insects, although less so to the 5-day-old insects. With both the allelochemicals and piperonyl butoxide in the diet, further decreases of growth were observed. Seven additional synergists representing several classes of compounds known to inhibit mixed function oxidases (MFOs; polysubstrate macrooxygenases, PSMOs) or esterases in the insect were also added to the diet at the 0.02 and 0.06% levels, with and without allelochemicals at the 0.06% level. However, only piperonyl butoxide decreased the growth rate of 5-day larvae fed the three allelochemicals. Thus, one effect of gossypol and other allelochemicals on insect feeding can be inferred to be toxicity, but gossypol may also act as an antifeedant. o 1988Academic press, hc.

Gossypol and related terpenoids, condensed tannins, anthocyanin, flavonoids, and other compounds in cotton (Gossypium hirsutum L.) have been shown to be the major sources of resistance to the tobacco budworm (Heliothis virescens Fab.) by us and others (14). These showed that the compounds are toxicants for Heliothi$ spp. in dietary studies, and other workers (1, 5) have found evidence that the compounds also affect feeding behavior. Larvae avoided feeding on the gossypol glands that were surrounded by an anthocyanin envelope. Further work in this laboratory (6) revealed that 5-day-old larvae fed gossypolcontaining diets continued to grow nor’ Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply approval to the exclusion of other vendors that may also be suitable. * To whom correspondence should be addressed.

mally but that l-day-old larvae fed those same diets grew very little. This indicated that as the larvae matured, their ability to adapt to gossypol increased, the ability most likely being due to the mechanism reported by Mullin (7). He showed that insects cope with allelochemicals by inducing the biosynthesis of detoxifying enzymes such as mixed function oxidases (MFOS;~ polysubstrate macrooxygenases, PSMOs), hydrolases , and esterases. Synergists have been employed to maintain or improve toxicant activity of insecticide action by inhibiting the activity of insects’ existing detoxifying enzymes (8). It would seem, therefore, that if gossypol manifests its activity against Heliothis larvae by toxic action, the addition of an ap-

3 Abbreviations used: MFO, mixed function oxidase; PSMO, polysubstrate macrooxygenase; TPB, O,O,O-tributyl phosphorothioate; TCP, tri-0-cresyl phosphate; PP, propyl paraoxon.

55 0048-3575/88 $3.00 Copyri&t 0 1988 by Academic Press, Inc. Au rights of reproduction in any form reserved.

56

HEDIN

ET

propriate synergist to a gossypol diet fed to the larvae should further reduce their growth by inhibiting the activity of the insect’s detoxifying enzymes. MFO inhibitors provide an experimental method of altering toxic action while probably having less of an effect on antifeedant action. On the other hand, reduction of growth by the synergist would not preclude a second (antifeedant) role of gossypol. SYNEROIST

PIPERONYL

AL.

In this study, three major cotton plant allelochemicals-gossypol, a mixture of condensed tannins, and isoquercitrinwere fed to l-, 3-, and 5-day-old larvae at the 0.06% level, previously established to arrest the growth of l-day-old, but not 5day-old, larvae (6). In a second series of tests, eight synergists representing several classes of compounds known to inhibit either MFOs or ksterases (8) were added at ACTIVITY

CLASS

METHYLENEDIOXYPHENYL

MIXED

FUNCTION

OXIDASE

METHYLENEDIOXYPHENYL

MIXED

FUNCTION

OXIDASE

METHYLENEDIOXYPHENYL

MIXED

FUNCTION

OXIDASE

MIXED

FUNCTION

OXIDASE

BUTOXIDE

SESAME 0IL:TRIGLYCERIDES. SESAMIN ~C2,$i,&l.SESAMOLlN ‘%#I SW

SESAMOL

N-ALKYL

N-OCTYLBICYCLOHEPTENE DICARBOXIWDE WOK-2641

ORGANOPHOSPHATE

ESTERASE

ORGANOPHOSPHATE

ESTERASE

ORGANOPHOSPHATE

ESTERASE

m&Q-TRIBUTYL PHoSPHOROTMOATE

TRI-o-CRESYL

PROPYL

PHOSPHATE

PARAOXON

AROMATIC

H.C.

ESTERASE

PENTAMETHYLZBENZENE

FIG.

1. Names, structures, functional classes, and modes of action of the candidate synergists.

EFFECT

OF SYNERGISTS

ON TBW RESISTANCE

the 0.02, 0.06, and 0.60% levels to diets containing the allelochemicals. MATERIALS

AND METHODS

Insects and Diets

Tobacco budworm larvae used in the various tests were neonate larvae from adults maintained as described by Jenkins et al. (9). Larvae (20 per test) were placed in 30ml clear plastic cups containing 10g per cup of a Nutri-Soy (Flavor-Rite Corp., Memphis, TN) wheatgerm diet. All tests were performed with insects from a colony obtained at the Mississippi State University

57

TO ALLELOCHEMICALS

rearing facility. In the various tests, l-, 3-, and S-day-old larvae, previously maintained on the stated control diet, were transferred to the test diets, incubated at 27.6”C until they were 10 days old, and weighed. Typically, 200- to 600-g batches of diet were required for each replicated test. Allelochemicals and synergists in quantities suffkient for the test were either added to the diet without dilution or solvation, if liquid, or dissolved in ethanol or ethyl ether and then added to the warmed diet during mechanical stirring. Following mixing, the diet was poured into the vials in which gelation occurred upon cooling.

TABLE 1 Mean Larval Weights of IO-Day-Old Tobacco Budworm Larvae (Mississippi State Colony) after Placement as 1, 3, and 5 Day Olds on Diets Containing 0 and 0.06% of Allelochemicals and 0, 0.02, 0.06, and 0.60% Piperonyl Butoxide

% Allelochemical 0 0 0 0 0.06 0.06 0.06 0.06

% Fiperonyl butoxide 0 0.02 0.06 0.60 0 0.02 0.06 0.60

LSD(O.05) 0 0 0 0 0.06 0.06 0.06 0.06

0 0.02 0.06 0.60 0 0.02 0.06 0.60

LSD(O.05) 0 0 0 0 0.06 0.06 0.06 0.06

0 0.02 0.06 0.60 0 0.02 0.06 0.60

LSD(O.05)

Larval weight (mg) Gossypol l-day-old larvae 290.4 bc* 134.3 de %.9 ef 1.2 j 71.6 fg 14.3 hij 5.2 j 0.4 j 34.1

cd de hi 0 k 142.9 gh 71.0 ij 22.8 jk 0 k

287.1 247.3 92.4

42.4

3-day-old larvae 371.2 a 360.9 a 170.3 d 12.2 hij 167.1 d 28.2 hij 19.5 hij 8.0 ij 30.2

5-day-old larvae 316.8 b 378.5 a 271.8 c 51.6 gh 306.3 bc 174.5 d 93.1 f 50.7 48.3

Tannins

ghi

294.8 297.4 229.3 0.7

0 0 0 0

-

bc bc ef i i i i i

53.3

317.3 bc 189.4 fg 78.2 ij 13.4 jk 205.9 ef 206.2 ef 98.6 hi 11.3 jk

71.4 292.2 382.2 357.2 43.5 388.7

Isoquercitrin

215.3 211.6 137.9 11.1 217.8 186.9 166.9

efg efg h i efg fgh gh 8.7 i

48.9 c

a ab ijk

a 316.0 bc 174.8 fg 47.7 ijk 37.5

351.7 307.4 333.5 50.6 294.3 278.6 243.6 36.2

a abc ab i bc cd

de i

48.0

* Means followed by the same letter are not significantly different (P > 0.05; Duncan’s multiple range test).

58

HEDIN

Allelochemicals

Gossypol was provided by the U.S. Department of Agriculture, Agricultural Research Service’s Southern Research Center (New Orleans, LA). The identity was confirmed by MS and NMR. The condensed tannins and isoquercitrin were extracted together from cotton leaves with CHClJ MeOH& (2/1/l). The more polar fraction (containing both the mixture of tannins and the isoquercitrin) was repeatedly chromatographed on Sephadex LH-20 with aqueous methanol and methanol until each of the two desired products was chromatographitally homogenous (1,2). The methanol was removed by evaporation, and the aqueous residue was diluted with water. Upon freeze dehydration, multigram quantities of the crystalline products were obtained. The condensed tannins were characterized by the procedures of Czochanska et al. (10) and were found to consist of related polymers, the molecular weights ranging from 1500 to 6000. The prodelphinidin:procyanidin ratio ranged from 1.8 to 3.7, and the monomer units were primarily (81-95%) of the cis configuration (1, 2). The isoquer-

ET AL.

citrin also was isolated from cotton leaves by extraction and chromatography, and its structure verified by procedures we have described (11). Synergists

The following compounds were procured as indicated and used without further purification: piperonyl butoxide (Pfaltz and Bauer, Waterbury, CT); sesame oil (Sigma Chemical Co., St. Louis, MO); sesamol, 98%, and pentamethylbenzene, 99% (Aldrich Chemical Co., Milwaukee, WI); trio-cresyl phosphate and O,O,O-tributyl phosphorothioate (ICN Biomedicals, Inc., Plainview, NY); MGK-264; N-octylbicycloheptenedicarboximide (CAS = N-[2ethylhexyllbicyclo-[2,2,1]-5-heptane-2,3dicarboximide) (Chem Service, West Chester, PA); propyl paraoxon, synthesized by reflux of 4nitrophenyl phosphorodichloridate (Aldrich Chemical Co.) in npropanol. The structure of propyl paraoxon was confirmed by mass spectrometry. The structures of the synergists and their classesand activities are given in Fig. 1.

TABLE 2 Weights of IO-Day-Old Tobacco Budworm Larvae after Placement as 5 Day Olds on Diets Containing Gossypol and Synergists Synergist and amount

Larval weight (% of control) m

None

0% Gossypol

0.06% Gossypol

100.0 e-h*

60.8 h-l

O,O,O-Tributyl phosphorothioate

0.02 0.06

91.2 e-i 59.1 i-l

46.8 j-l 26.3 1

Sesame oil

0.02 0.06

124.0 c-e 140.9 c-d

56.7 i-l 117.5 d-f

Sesamol

0.02 0.06

191.8 ab 206.4 a

114.6 d-f 47.3 i-l

Tri-o-cresyl phosphate

0.02 0.06

157.3 b-c 211.1 a

56.1 i-l 73.1 g-k

MGK-264

0.02 0.06

100.6 d-h 81.3 f-k

114.1 d-g 86.0 e-j

Propyl paraoxon

0.02 0.06

66.1 h-l 43.9 i-l

42.1 k-l 51.5 i-l

* Means followed by the same letter are not significantly different (P > 0.05; Duncan’s multiple range test).

EFFECT

Statistical

OF SYNERGISTS

ON TBW RESISTANCE

Procedures

Data obtained from the determination of larval weights were subjected to analysis of variance. Means were compared with Duncan’s multiple range test (12), and LSD values were calculated for the further analysis of one test. The statistical design was completely randomized with 20 replications (1 larva/rep). Data were analyzed using the general linear model procedure (13). The age by allelochemical interaction was significant in all tests. Therefore, each age was analyzed separately to simplify interpretation of results. RESULTS

AND DISCUSSION

The mean weights of lo-day-old tobacco budworm larvae after placement as l-, 3-, and 5-day-olds on test diets containing 0 and 0.06% gossypol, the tannins, and isoquercitrin individually and in combination with 0, 0.02,0.06, and 0.60% piperonyl butoxide are given in Table 1, along with statistical data. Tests with the l-day-old larvae TABLE Weights

of IO-Day-Old

Synergist and amount

Tobacco

Budworm

59

TO ALLELOCHEMICALS

showed that piperonyl butoxide was in itself toxic, and that all the allelochemicals were toxic. The growth of larvae placed as 3day-olds on the treated diets showed that piperonyl butoxide alone was toxic at 0.06 and 0.60% and toxic in one of three replicates at 0.02%. Among the three allelochemicals, only gossypol and the tannins were clearly toxic to the larvae. Of these two, only gossypol showed increased toxicity in the presence of piperonyl butoxide, and only when the piperonyl butoxide concentration was 0.02 and 0.06%. That is, gossypol plus 0.60% piperonyl butoxide was not significantly more toxic than 0.60% piperonyl butoxide alone. Clearly, neither the allelochemicals nor piperonyl butoxide was as toxic to the 3-day-old larvae as to the 1-day-old larvae. Larvae placed on treated diets as 5 day olds were only marginally susceptible to piperonyl butoxide at 0.02 and 0.06% and also to the allelochemicals in the absence of piperonyl butoxide. The severity of the 3

Larvae afrer Placement Tannins and Synergists

as 5 Day

Olds

on Diets

Containing

Larval weight (% of control) m

None

0% Tannins

0.06% Tannins

100.0 d-f*

28.1 i-k

O,O,O-Tributyl phosphorothioate

0.02 0.06

113.6 c-e 59.9 g-i

22.8 20.3

j-k j-k

Sesame oil

0.02 0.06

142.8 bc 147.9 b

34.1 28.8

h-rk i-k

0.02 0.06

182.6 a 126.4 b-d

63.8 gh 51.3 h-j

Tri-o-cresyl phosphate

0.02 0.06

131.2 bc 135.6 bc

21.5 jk 30.1 i-k

MGK-264

0.02 0.06

142.1 bc 95.6 ef

34.9

Propyl paraoxon

0.02 0.06

114.1 c-e 49.8 h-j

0.02 0.06

84.1 fg 92.4 ef

Pentamethylbenzene

h-k 17.7 k

33.1 i-k jk

24.3

20.0 jk 44.1 h-j

* Means followed by the same letter are not significantly different (P > 0.05; Duncan’s multiple range test).

60

HEDIN

growth inhibition increased as the percentage of piperonyl butoxide added with the allelochemicals was increased. However, at 0.60% piperonyl butoxide, the contribution of the three allelochemicals was essentially zero. The weights (expressed as percentages of control) and statistical data for lo-day-old tobacco budworm larvae after placement as 5 day olds on diets containing gossypol, the tannins, or isoquercitrin with one of several synergists (7) other than piperonyl butoxide are given in Tables 24. However, none of these candidates significantly synergized further reductions in growth although there was a consistent downward trend when TBP was added to the diet along with each of the allelochemicals. Three of the candidates, sesame oil, sesamol, and TCP, may have been feeding stimulatory because in the absence of the allelochemicals, they caused significant increases in growth when incorporated at either one or both levels. Two others, TBP

ET AL.

and PP, may have inhibited feeding, because growth was generally depressed upon their incorporation. In summary, piperonyl butoxide was the only candidate synergist, among eight representing four classes and two types of activities, that showed significant activity with all three employed allelochemicals. From the tests that were conducted, it can be inferred that all three allelochemicals acted as toxicants, because piperonyl butoxide, a known inhibitor of mixed function oxidase activity in insects, further suppressed the growth of larvae. These tests show that the allelochemicals are potentially very toxic if the insects are unable to metabolize them and also that the insects have been able to adapt to these toxic materials . With regard to piperonyl butoxide, the most convincing evidence of its synergistic action is the following, obtained with insects placed on the treated diets as 5 day olds: Growth was normal in insects fed any

TABLE 4 Weights of IO-Day-Old Tobacco Budworm Larvae after Placement as 5 Day Olds on Diets Containing Isoquercitrin and Synergists Synergist and amount

Larval weight (% of control) (%I

None

0% Isoquercitrin

0.06% Isoquercitrin

100.0 f-h*

35.5 j-k

O,O,O-Tributyl phosphorothioate

0.02 0.06

181.9 b-d 110.8 f-h

27.1 k 22.6 k

Sesame oil

0.02 0.06

179.7 t+d 207.4 a-c

25.2 k 30.0 jk

Sesamol

0.02 0.06

160.6 cd 219.4 ab

49.0 h-k 91.4 f-i

Tri-o-cresyl phosphate

0.02 0.06

233.1 a 82.4 g-j

31.3 jk 52.4 h-k

MGK-264

0.02 0.06

151.4 de 111.1 e-g

67.4 g-k 71.1 g-k

Propyl paraoxon

0.02 0.06

93.5 f-g 56.8 h-k

38.9 i-k 58.3 g-k

Pentamethylbenzene

0.02 0.06

187.8 a-d 138.2 d-f

61.8 g-k 62.3 g-k

* Means followed by the same letter are not significantly different (P > 0.05; Duncan’s multiple range test).

EFFECT

OF SYNERGISTS

ON TBW RESISTANCE

one of the allelochemicals at 0.06% and nearly normal in those fed 0.02 or 0.06% piperonyl butoxide, but growth was suppressed in insects fed both an allelochemical and 0.02 or 0.06% piperonyl butoxide. On the other hand, the failure of three other MFO inhibitors to depress growth in concert with the allelochemicals clouds the assumption that PB acted by inhibiting MFO activity. Furthermore, evidence for a toxic effect by whatever mechanism does not negate other evidence for the allelochemicals having an antifeedant effect. REFERENCES 1. P. A. Hedin, J. N. Jenkins, D. H. Collum, W. H. White, W. L. Parrott, and M. W. MacGown, Cyandin-3-Bglucoside, a newly recognized basis for resistance in cotton to the tobacco budworm, Experientia 39, 799 (1983). 2. P. A. Hedin, J. N. Jenkins, D. H. Collum, D. H. White, and W. L. Parrott, Multiple factors in cotton contributing to resistance to the tobacco budworm, in “Plant Resistance to Insects” (P. A. Hedin, Ed.), p. 347, Amer. Chem. Sot. Symposium Series No. 208, Amer. Chem. Sot., Washington, DC, 1983. 3. A. A. Beh and R. D. Stipanaovic, The chemical composition, biological activity and genetics of pigment glands in cotton, in “Proceedings, Behwide Prod. Res. Conf., Atlanta, GA,” p. 244, 1977.

TO ALLELOCHEMICALS

61

4. B. G. Chart, A. C. Waiss, and M. J. Lukefahr, Condensed tannin, an antibiotic chemical from Gossypium hirsutum, J. Insect Physiol. 24, 113 (1972). 5. W. L. Parrott, USDA, Mississippi State, unpublished data (1983). 6. W. L. Parrott, USDA, Mississippi State, unpublished data (1984). 7. C. A. Mullin, Detoxification enzyme relationships in arthropods of differing feeding strategies, in “Bioregulators for Pest Control” (P. A. Hedin, Ed.), p. 267, Amer. Chem. Sot. Symposium Series No. 276, Amer. Chem. Sot., Washington, DC, 1985. 8. J. E. Casida, Mixed function oxidase involvement in the biochemistry of insecticide synergists, J. Agric. Food Chem. 18, 783 (1970). 9. J. N. Jenkins, W. L. Parrott, J. C. McCarty, and W. H. White, Breeding cotton for resistance to the tobacco budworm: Techniques to achieve uniform field infestations, Crop Sci. 23, 970 (1982). 10. Z. Czochanska, L. P. Foo, R. H. Newman, L. J. Porter, and W. A. Thomas, Direct proof of a homogenous polytlavan-3-01 structure for polymeric proanthocyanidins, J. Chem. Sot. Chem. Commun. 375 (1979). 11. P. A. Hedin, W. W. Neel, M. L. Burks, and E. Grimley, Evaluation of plant constituents associated with pecan phylloxora gall formation, J. Chem. Ecol. 11, 473 (1985). 12. D. B. Duncan, Multiple range and multiple F tests, Biometrics 11, 1 (195.5). 13. SAS Institute, “SAS User’s Guide: Statistics,” SAS Institute, Gary, NC, 1982.