Performance of the gypsy moth, Lymantria dispar, on quaking aspen, Populus tremuloides, is strongly affected by foliar concentrations of phenolic glycosides. Because the microbial insecticide Bacillus thuringiensis is widely used against gypsy moths and has a mode of action similar to that of phenolic glycosIdes, we investigated the combined effects of the two toxins on gypsy moth larvae. The experimental design was a 2 x 2 factorial: two levels (0, +) of phenolicglycosides for each of two levels (0, +) of B. thuringiensis. The toxins were incorporated into artificial diets and bioassayed against first and fourth instars. Bacillus thuringiensis and phenolic glycosides ne~atively and addi· tively affected larval survival, growth and development tImes. Both agents slightly reduced consumption rates. In addition, B. thuringiensis reduced diet digestibility whereas phenolic glycosides decreased the efficiency with which food was converted to biomass. These results suggest that the efficacy of B. thuringiensis applications in aspen forests is likely to be affected by the allelo· chemical composition of foliage

Arteel, Gavin E

Lindroth, Richard L

English

Valparaiso University

The Great Lakes Entomologist Volume 25 Number 4 - Winter 1992 Number 4 - Winter 1992 Article 1 October 1992 Effects of Aspen Phenolic Glycosides on Gypsy Moth (Lepidoptera: Lymantriidae) Susceptibility to Bacillus Thuringiensis Gavin E. Arteel University of Wisconsin Richard L. Lindroth University of Wisconsin Follow this and additional works at: https://scholar.valpo.edu/tgle  Part of the Entomology Commons Recommended Citation Arteel, Gavin E. and Lindroth, Richard L. 1992. "Effects of Aspen Phenolic Glycosides on Gypsy Moth (Lepidoptera: Lymantriidae) Susceptibility to Bacillus Thuringiensis," The Great Lakes Entomologist, vol 25 (4) Available at: https://scholar.valpo.edu/tgle/vol25/iss4/1 This Peer-Review Article is brought to you for free and open access by the Department of Biology at ValpoScholar. It has been accepted for inclusion in The Great Lakes Entomologist by an authorized administrator of ValpoScholar. For more information, please contact a ValpoScholar staff member at scholar@valpo.edu. 1992 THE GREAT LAKES ENTOMOLOGIST 239 EFFECTS OF ASPEN PHENOLIC GLYCOSIDES ON GYPSY MOTH (LEPIDOPTERA: LYMANTRIIDAE) SUSCEPTIBILITY TO BACILLUS THURINGIENSIS Gavin E. Arleel and Richard L. Lindroth1 ABSTRACT Performance of the gypsy moth, Lymantria dispar, on quaking aspen, Populus tremuloides, is strongly affected by foliar concentrations of phenolic glycosides. Because the microbial insecticide Bacillus thuringiensis is widely used a~ainst gypsy moths and has a mode of action similar to that of phenolic glycosIdes, we investigated the combined effects of the two toxins on gypsy moth larvae. The experimental design was a 2 x 2 factorial: two levels (0, +) of phenolicglycosides for each of two levels (0, +) of B. thuringiensis. The toxins were incorporated into artificial diets and bioassayed against first and fourth instars. Bacillus thuringiensis and phenolic glycosides ne~atively and addi· tively affected larval survival, growth and development tImes. Both agents slightly reduced consumption rates. In addition, B. thuringiensis reduced diet digestibility whereas phenolic glycosides decreased the efficiency with which food was converted t  biomass. These results suggest that the efficacy of B. thuringiensis applications in aspen forests is likely to be affected by the allelo· chemical composition of foliage. The western leading edge of the range of the gypsy moth, Lymantria dispar L., is now passing through the Great Lakes states. In Michigan, defo­liation increased nearly 10-fold from 1988 to 1991, with over 600,000 acres affected in 1991 (F. Sapio, Michigan Dept. Natural Resources, pers. comm.). Widespread defoliation has ot yet occurred in Wisconsin, but gypsy moth populations are now established in the eastern part of th  state and control programs are underway. Establishment of the gypsy moth in the Great Lakes region is of concern for many reasons, notable among them being its impact on the aspen resource. Quaking aspen (Populus tremuloides) and bigtooth aspen (P. grandidentata) are preferred food plants of the gypsy moth (Lechowicz and Maufette 1986). Unlike northeastern forests, aspens are a dominant component of forests in the Great Lakes area; Wisconsin alone has over 3 million acres of the aspen forest type (Spencer et al. 1990). Moreover, aspen is harvested extensively for production of a variety of pulpwo d and plywood commodities (Maass et al. 1990). Extensive defoliation of aspen forests is also of concern because of potential impact on outdoor recreational activities in this area. Although considered a preferred host, quaking aspen is not uniformly susceptible to defoliation by the gypsy moth. Clonal differences in foliar defensive chemistry are great, and have been implicated as one cause of this lDepartment of Entomology, University of Wisconsin, Madison, WI 53706. 1Arteel and Lindroth: Effects of Aspen Phenolic Glycosides on Gypsy Moth (Lepidoptera:Published by ValpoScholar, 1992240 THE GREAT LAKES ENTOMOLOGIST Vol. 25, No.4 variation (Lindroth and Hemming 1990, Hemming and Lindroth, unpubL data). The defensive chemistry of aspen consists almost entirely of phenolic compounds, particularly phenolic glycosides and condensed tannins (Palo 1984, Bryant et al. 1987, Lindroth et al. 1987a). Phenolic glycosides exhibit biological activity toward a variety of Lepidoptera (Bryant et al. 1987, Lin­droth et al. 1988, Lindroth and Peterson 1988) and are toxic t  ~psy moth larvae at moderate to high doses (> 2% dietary concentration; Lmdr th and Hemming 1990, Lindroth and Weisbrod 1991, Hemming and Lindroth, unpubl. data). The putative mode of action of phenolic glycosides involves formation of degenerative lesions in the midgut (Lindroth et al. 1988, Lin­droth and Peterson 1988). Bacillus thuringiensis subspecies kurstaki (strain HD-l) is widely used in commercial formulations for control of the gypsy moth. The mode of action of B. thuringiensis also involves formation of gut lesions. Endotoxins produced by B. thuringiensis are activated in insect guts by proteases, whereupon they bind to brush-border membrane vesicles. Resulting lesions cause lysis of the gut epithelium and death due to septicemia and starvation (Li et al. 1991). The purpose of this study was to evaluate the combined effects of aspen phenolic glycosides and B. thuringiensis on gypsy moth performance. Because the two toxins have similar modes of action. we predicted they would exert additive or synergistic effects when administered together. If such results are found. then differential effects of B. thuringiensis are likely to occur for gypsy moths feeding on aspen clones of widely varying defensive chemistry. MATERIALS AND METHODS Our feeding experiments were designed as 2 x 2 factorials, with two levels (0, +) of phenolic glycosides for each of two levels (0, +) of B. thuringiensis. Gypsy moth larvae used in th  experiments were obtained from egg masses provided by the Otis Methods Development Center. Otis Air National Guard Base. MA. Artificial diets. The four artificial diets used in this study were adapted from the standard high wheat germ formulation of ODell et al. (1985). Methyl paraben, a diet preservative, was not used so diets were autoclaved to inhibit growth of mold. Vitamins, B. thuringiensis and phenolic glycosides were incorporated after diets had cooled o 45-50 0 C. The B. thuringiensis product used was Foray 48B (Novo Nordisk Bioindustrials, Danbury, CT). Phenolic glycosides were semi-purified from quaking aspen foliage by the extraction and fractionation procedure of Lindroth et al. (1986). Analysis by HPLC indi­cated that 90% of the crude preparation consisted of the phenolic glycosides salicortin and tremulacin. Because we were interested in potential interactive effects between B. thuringiensis and phenolic glycosides. each toxin was incorporated at a level less than that causing substantial mortality. Based on preliminary experi­ments, we selected 100 and 500 IU/ml diet as appropriate concentrations for first and fourth instar gypsy moths, respectively. Phenolic glycosides were incorporated at 2% diet wet weight. Bioassays. We conducted two types of bioassays, first in ar survival trials nd fourth instar feeding trials. to assess effects of B. thuringiensis and phenolic glycosides on gypsy moth performance. For each bioassay. larvae from individual egg masses were distributed across treatments to equalize potential maternal or genetic effects (Rossiter et al. 1990). Larvae were main­tained at 25° C, with a 15:9 light-dark cycle. For survival trials. each of ten replicates per treatment consisted of 15 2The Great Lakes Entomologist, Vol. 25, No. 4 [1992], Art. 1https://scholar.valpo.edu/tgle/vol25/iss4/11992 THE GREAT LAKES ENTOMOLOGIST 241 newly hatched larvae placed into 28 ml plastic rearing cups containing a cube of experimental diet. Insects were provided with diets containing B. thurin­giensis for only 48 hours, in order to mimic the short-lived viability of the pathogen under field conditions. Afterwards these larvae were switched to corresI;onding diets without B. t uringiensis (i.e., the control and phenolic glycosIde diets). All food was replaced at 2-3 day intervals to maintain fresh­ness. We checked rearing containers three times a day for dead larvae or new second instars. We calculated survival rates as the proportion of larvae that survived the first larval stadium, and development times as the time elapsed from onset of a trial to the time at which half of the living larvae molted into the second stadium. We conducted fourth instar feeding trials in order to measure standard nutritional indices of larval performance. Larvae were reared on control diet through the third stadium. As newly molted fourth ins tars, larvae were assigned to one of the four experimental diets (10 replicates per treatment). Each replicate consisted of a single larva in a 28 ml plastic cup containing an experimental diet. As for the survival trials, diets containing B. thuringiensis were provided for only 48 hours. All diets were replaced at 1-2 day intervals as required. At the end of each trial, we froze the larva, then dried (50 0 C) and weighed the larva, frass and uneaten food. Dry weights of larvae t the onset of the trials were stimated based on proportional dry weights of a sample of 10 newly molted fourth instars. We calculated nutritional indices on the basis of dry weights, using standard formulas' (Waldbauer 1968, Scriber 1977) for relative growth rate (RGR), relative consumption rate (RCR), approximate digestibility (AD), efficiency of conversion of digested food (ECD), and effi­ciency of conv rsion of ingested food (ECI). We analyzed results from the bioassays by two-way analysis of variance (ANOVA), using SAS statistical software. Nutritional indices varied little between male and female larvae, so data from both sexes were pooled prior to analysis. RESULTS AND DISCUSSION First instar performance was reduced by both B. thuringiensis and pheno­lic glycosides (Fig. 1). Survival rates decreased 11% in the presence of B.thuringi nsis, 24% in the presence of phenolic glycosides, and 34% in the presence of both, indicating a simple additive effect. First instar development times were prolonged 40% by B. thuringiensis and 27% by phenolic glyco­sides. The combined effect of both toxins on development time was somewhat less than additive, as indicated by the nearly significant B. thuringiensis x phenolic glycoside interaction term. Fourth instar development times and growth rates were also significantly affected by he dieta y toxins (Table 1). In both cases the ffects of B. thurin­giensis and phenolic glycosides were additive. For example, relative g ow h rates of larvae were reduced 33,24 and 52% on the B. thuringiensis, phenolic glycoside and B. thuringiensis plus phenolic glycoside diets, respectively. Effects of the experimental diets on larval growth can be ttributed to changes in consumption rates and digestion and conversion efficiencies (Table 1). That both toxins independently reduced food consumption by larvae is indicated by the fact that the main effects of B. thuringiensis and phenolic glycosides were significant, whereas their interaction was not. Bacillus thurin­giensis decreased the ability of larvae to digest diets (ADs) but phenolic glyco­sides did not; moreover, the negative effect of B. thuringiensis on AD values was ameliorated in the presence of phenolic glycosides. In contrast, phenolic 3Arteel and Lindroth: Effects of Aspen Phenolic Glycosides on Gypsy Moth (Lepidoptera:Published by ValpoScholar, 1992242 THE GREAT LAKES ENTOMOLOGIST Vol. 25, No.4 a.100 -80 P values: Bt .061~ PG <.00160ca BtxPG .905> .~ 40:::s CJ) 20 0 o 100 8,-----------------------------~b. 6 4 2 0+--"" Pvalues: Bt <.001 PG <.001 BtxPG .069 o 100 Bt concentration (IU/ml) Figure 1. Effects of B. thuringiensis and phenolic glycosides on survival (a) and devel­opment time (bl of first instar gypsy moth larvae. Error bars indicate ± 1 SE. Hatched and shaded bars indicate diets lacking and containing phenolic glycosides, respectively. glycosides reduced the efficiency with which larvae converted digested food into biomass (ECD values) but B. thuringiensis did not. Finally, because ECl is the mathematical product of AD and ECD, it follows that this parameter was significantly reduced by both B. thuringiensis and phenolic glycosides, and that the reduction due to B. thuringiensis was less pronounced in the presence of phenolic glycosides. Results from this study illustrate that both aspen phenolic glycosides and dietary B. thuringiensis negat vely affect the performance of gypsy moth larvae. The effects of these toxins are, however, primarily additive rather than synergistic in nature. This additive effect is consistent with the mode of action of both toxins, which appears to involve the formation of degenerative lesions in larval gut epithelia. T  modes of action of the compounds are not 4The Great Lakes Entomologist, Vol. 25, No. 4 [1992], Art. 1https://scholar.valpo.edu/tgle/vol25/iss4/11992 THE GREAT LAKES ENTOMOLOGIST 243 Table L - Effects of lJ. thuringiensis and phenolic glycosides on nutritional indices of f urth instar gypsy moths (X ± 1 SE). Development RGR RCR AD ECD ECI Diet time (days) (mg'mg'day-1) (mg-mg-day-1) (%) (%) (o/d 5.4±O.l O.2l±.01 O.56±O.2 52.7±O.l 71.9±3.3 37.6 ± 1.2 B. thuringiensis 7.2±O.4 O.14±.Ol O.52±.O2 39.9±2.2 70.5±4.9 27.8±2.1 Phenolic glycoside 7.1 ±O.6 O.16±.01 0.54±.02 47.4±0.8 59.7 ±2.2 28.3±1.0 B. thuringiensis lO.0±O.5 O.lO±.Ol 0.42±.03 44.4±3.0 55.9±5.5 23.8 ± 1.2 ± phenolic glycoside P values: B. thuringiensis <.001 <.001 .002 <.001 .520 <.001 Phenolic glycoside <.001 <.001 .022 .868 .003 <.001 Interaction .200 .747 .120 .029 .763 .051 entirely similar, however, because B. thuringiensis suppressed ADs whereas phenolic glycosides reduced ECDs. Few studies have investigated the effects of specific plant allelochemicals on the efficacy of B. thuringiensis as an insecticidal agent, and results have differed for various compounds. Trumble et al. (1991) found that psoralens and B. thuringiensis had an additive effect in prolonging development time in Spodoptera exigua. Krischik et al. (1988) showed that the alkaloid nicotine inhibited the toxicity of B. thuringiensis against Manduca sexta, whereas theflavonoid rutin had no effect. Felton and Dahlman (1984) found that effects of B. thuringiensis on M. sexta were enhanced by the nonprotein amino acid L· canavanine. The authors suggest that these results may be due to the com­bined effects of both B. thuringiensis and L-canavanine on gut permeability and function, biological activity similar to that which we have proposed for the action of B. thuringiensis and phenolic glycosides on the gypsy moth. Gypsy moth performance on aspen is strongly and inversely correlated with phenolic glycoside concentrations (Hemming and Lindroth, unpubl. data) and these concentrations vary by over an order of magnitude among aspen clones (Lindroth et al. 1987b, Hemming and Lindroth, unpubL data). Given that phenolic glycosides and B. thuringiensis have additive effects on gypsy moths, it follows that the efficacy of B. thuringiensis applications in aspen forest types may be influenced by the chemical composition of foliage. Future research will address that possibility. ACKNOWLEDGMENTS We thank Novo Nordisk Bioindustrials for the gift of F ray 48B. This research was supported by USDA Competitive Grant 91-37302-6294 and by the College of Agricultural and Life Sciences (Hatch Project 3211), University of Wisconsin, Madison. LITERATURE CITED Bryant, J. P., T. P. Clausen, P. B. Reichardt, M. C. McCarthy, and R. A. Werner. 1987. Effects of nitrogen fertilization upon the secondary chemistry and nutritional value of quaking aspen (Populus tremuloides Michx.lleaves for the large aspen tortrix (Choris­toneura conflictana [Walker]). Oecologia 73:513-517. Felton, G. W., and D. L. Dahlman. 1984. Allelochemical induced stress: effects of L· 5Arteel and Lindroth: Effects of Aspen Phenolic Glycosides on Gypsy Moth (Lepidoptera:Published by ValpoScholar, 1992244 THE GREAT LAKES ENTOMOLOGIST Vol. 25, No.4 canavanine on the pathogenicity of Bacillus thuringiensis in Manduca sexta. J. Invert. Patho!. 44:187-191. Krischik, V. A., P. Barbosa, and C. F. Reichelderfer. 1988. Three trophic level interac­tions: allelochemicals, Manduca sexta (L.) and Bacillus thuringiensis var. Kurstaki Berliner. Environ. Entomol. 17:476-482. Lechowicz, M. J., and Y. Mauffette. 1986. Host preferences of the gypsy moth in eastern North American versus European forests. Rev. D'entomol. Quebec 31:43-51. Li, J., J. Carroll and D. J. Ellar. 1991. Crystal structure of insecticidal o-endotoxin from Bacillus thuringiensis at 2.5 A resolution. Nature 353:815-821. Lindroth, R. L., and J. D. C. Hemming. 1990. Responses of the gypsy moth (Lepidop­tera: Lymantriidae) to tremulacin, an aspen phenolic glycoside. Environ. Entomol. 19:842-847. Lindroth, R. L., and S. S. Peterson. 1988. Effects of plant phenols on performance of southern armyworm larvae. Oecologia 75:185-189. Lindroth, R.L., and A.V. Weisbrod. 1991. Genetic variation in response of the gypsy moth to aspen phenolic glycosides. Biochem. Syst. Ecol. 19:97-103. Lindroth, R. L., M. T. S. Hsia, nd J. M. Scriber. 1987a Characterization of phenolic glyoosides from quaking aspen (Populus tremuloidesi. Biochem. Syst. EcoL 15:677-680. __. 1987b. Seasonal patterns in the phytochemistry of three Populus species. Bio­chern. Syst. Eco!. 15:681-686. Lindroth, R. L., J. M. Scriber, and M. T. S. Hsia. 1986. Differential responses of tiger swallowtail subspecies to secondary metabolites from tUlip tree and quaking aspen. Oecologia 70:13-19. __. 1988. Chemical ecology of the tiger swallowtail: mediation f host use by phenolic glycosides. Ecology 69:814-822. Maass, D. I., L. C. Irland, and S. D. Salisbury. 1990. Aspen utilization in the northern United States, pp. 79-89 In: R. D. Adams [ed.] Aspen Symposium 1989, Proceedings. USDA Forest Service, North Central Forest Experiment Station. ODell, T. M., C. A. Butt, and A. W. Bridgeforth. 1985. Lymantria dispar, pp. 355-367 In: P. Singh and R. F. Moore [eds.] Handbook of Insect Rearing. Elsevier, New York. Palo, R. T. 1984. Distribution of birch (Betula spp.), willow (Salix spp.l, and poplar (Populus spp.) secondary metabolites and their potential role as chemical defense against herbivores. J. Chern. Ecol. 10:499-520. Rossiter, M. C., W. G. Yendol, a  N. R. Dubois. 1990. Resistance to Bacillus thuringien­sis in gypsy moth (Lepidoptera; Lymantriidae): genetic and environmental causes. J. Eoon. Entomol. 83:2211-2218. Scriber, J. M. 1977. Limiting effects of low leaf· water content on the nitrogen utilization, energy budget, and larval growth of Hyalophora cecropia (Lepidoptera: Saturniidae). Oecologia 28:269-287. Spencer, J. S., Jr., E  C. Leatherberry, and N. P. Kingsley. 1990. The Lake States' aspen resource revisited: mid1960s-1987, pp. 243-252 In: R. D. Adams led.] Aspen Sympo­sium 1989, Proceedings. USDA Forest Service, North Central Forest Experiment Station. Trumble, J. T., W. J. Moar, M. J. Brewer, and W. G. Carson. 1991. Impact of UV radiation on activity of linear furanocoumarins and Bacillus thuringiensis var. kul'­staki against Spodoptera exigua: implications for tritrophic interactions. J. Chern. Eco!. 17:973-987. Waldbauer, G. P. 1968. The consumption and utilization of food by insects. Adv. Insect Physiol. 5:229-288. 6The Great Lakes Entomologist, Vol. 25, No. 4 [1992], Art. 1https://scholar.valpo.edu/tgle/vol25/iss4/1

Effects of Aspen Phenolic Glycosides on Gypsy Moth (Lepidoptera: Lymantriidae) Susceptibility to \u3ci\u3eBacillus Thuringiensis\u3c/i\u3e

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Effects of Aspen Phenolic Glycosides on Gypsy Moth (Lepidoptera: Lymantriidae) Susceptibility to \u3ci\u3eBacillus Thuringiensis\u3c/i\u3e

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