Chapter 21
Impact of Bacillus thuringiensis on Nontarget Lepidopteran Species in Broad-Leaved Forests 1
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R. C. Reardon and D. L. Wagner 1
National Center of Forest Health Management, Forest Service, U.S. Department of Agriculture, Morgantown, W V 26505 Department of Ecology and Evolutionary Biology, University of Connecticut, Storrs, CT 06269
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Bacillus thuringiensis variety kurstaki (Btk) is the only commercially produced biological insecticide available for use in suppression and eradication programs against the gypsy moth, Lymantria dispar (L.). There have been few multi-year laboratory and field studies designed specifically to evaluate the impacts of Btk on non-target native lepidopteran species. The susceptibility of lepidopteran larvae to Btk must often be evaluated on a species-by-species basis. Since 1980, approximately 1.7 million hectares (ha) have been treated with Bacillus thuringiensis variety kurstaki (Btk) in the eastern United States as part of the Federal and State Gypsy Moth, Lymantria dispar (L.), Cooperative Suppression Program During this interval, one application of Btk was applied per year to suppress populations of the European strain of the gypsy moth that was introduced and established in the United States since the 1860's, however, two or three applications per year were used in eradication efforts in Oregon (1985-87) and Utah (1988-1993) against the European strain and, in Washington and Oregon (1992), on approximately 200,000 ha against the Asian strain. Also, two applications of Btk were used in 1994 to eradicate an infestation of European, Asian and hybrid strains of the gypsy moth (introduced via military cargo shipped from Germany) on approximately 50,000 ha in eastern North Carolina. In Ontario, Canada, between 1985 and 1994, approximately 250,000 ha were treated with Btk to control the European Strain. No doubt, Btk usage will continue to increase because gypsy moth is already established in approximately 30 million ha of forest land in North America and about 240 million ha are believed to be susceptible to gypsy moth infestation. Here we provide an overview of: (1) Btk characteristics that are likely to influence non-target species, (2) documented impacts of Btk on non-target lepidopteran species in association with gypsy moth populations; and (3) recommendations for improving Btk efficacy for target pest species while minimizing impact to non-target species. 0097-6156/95/0595-0284$12.00/0 © 1995 American Chemical Society
Hall and Barry; Biorational Pest Control Agents ACS Symposium Series; American Chemical Society: Washington, DC, 1995.
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Bacillus thuringiensis var. kurstaki characteristics Commercial formulations. In 1970, Dulrnage (1) isolated the HD-1 strain of Btk and it became commercially available shortly thereafter. It is used today for production of most Btk formulations used to control defoliating forest Lepidoptera in North America. The HD-1 strain is a serotype 3a3b, and the crystal has a fairly broad spectrum of activity against a large number of Lepidoptera. Four companies produce various types of formulations (e.g., aqueous flowable suspension, nonaqueous emulsifiable suspension, oil flowable) of the HD-1 strain of Btk for use against gypsy moth. Each formulation contains inert ingredients which are unique and various additives (e.g., stickers) can be included to produce the final tank mix (2). Mode of action. The mode of action of Btk is complex and poorly understood. Commercial formulations of Btk contain both the spore (or endospore) and crystal (or parasporal body). The crystal is a protein matrix of large molecules of inactive protoxins that are not toxic to insects until solubilized in the gut. In many lepidopteran pests, the toxin subunits, when ingested separately, are the major cause of mortality; the spore effect is believed to be minimal, hi susceptible insects, the alkaline midgut environment (pH>8.0) and proteolytic (proteinsplitting) enzymes, dissolve ingested crystals and release smaller delta-endotoxins. These proteins, also known as the insecticidal crystal proteins (ICPs), bind to and force through specific receptor sites on the midgut membrane forming an ionselective channel This results in a perforation of the gut and leakage of gut contents, including spores, into the hemolymph. At this point, gut paralysis occurs, the larva stops feeding, and death follows in a few hours to a few days. In less susceptible insects, the spore penetrates into the hemolymph where conditions permit spore germination and bacterial (vegetative cell) multiplication to take place, resulting in a septicemia, that contributes to or causes larval death (2). If a sublethal dose is ingested, the larva may stop feeding; * weight gain and development may also be slowed. In some cases, damaged cells in the midgut are replaced and the larva eventually recovers and resumes feeding (3). As part of its mode of action, Btk can germinate, multiply and resporulate in the infected insect's hemolymph; however, vegetative cells, spores and crystals are not abundantly produced under such conditions. Since the insect integument does not rupture, spores and crystals are not released to contaminate foliage that might be consumed by other susceptible species. Usually the diseased caterpillars fall to the ground, and the Btk toxins are degraded in the soil. Under favorable conditions, Btk spores can germinate and grow in moist soil, deriving essential nutrients from decaying plants. The spore can persist in soil (and other protected sites) longer than the crystal toxins (4) staying viable for several months and, under ideal conditions, for years. Natural Btk epizootics have never been observed in a forest insect population. Consequently, to control forest insect pests, Btk must be
Hall and Barry; Biorational Pest Control Agents ACS Symposium Series; American Chemical Society: Washington, DC, 1995.
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applied annually in the manner of a conventional stomach-poison type of insecticide. Btk cannot be expected to infect subsequent generations of the gypsy moth (2). The insecticidal crystal proteins (ICPs) of Btk were first classified according to the genes that encode them (5). The cryl (A to E) groups with their subgroups, e.g., crylA (a, b or c) are toxic to lepidopteran larvae. The commercial formulations produced with the HD-1 strain generally contain one or up to three of the crylA ICPs. Recent studies of these purified ICPs against the gypsy moth showed that cryIA(a) and cryIA(b) are significantly more toxic than cryIA(c). This is not necessarily the case with all lepidopteran larvae. Spores alone have no effect on gypsy moth larvae. The addition of a very small amount of spores to a low concentration of the ICPs, significantly increases mortality to 100 percent as a result of lethal septicemia. This interaction between Btk and the ICPs does not appear to be specific. Other bacteria that are part of the forest microflora also show significant synergism with the cryIA(a) and cryIA(c) ICPs. These observations suggest that once the midgut is perforated, these insects become susceptible to nonspecific infections by bacterial opportunists and that other forest microflora can act synergistically with Btk. Potency, The potency of Btk preparations is determined by parallel bioassays with the HD-l-S-1980 standard on artificial diet with 4-day-old cabbage loopers (Trichoplusia ni). Since insecticidal activity varies greatly among insect species this method often results in a misrepresentation of the actual efficacy against a given pest species. Nevertheless, all bioassays should include this international standard, primarily because differences in larval batches and variation in fermentation dramatically effect formulation potency. Comparisons of preparations for efficacy need to take into consideration not only the LC50 (that dose needed to kill at least 50 percent of the larvae) but the slope of the regression. The slope shows the dose-response relationship over a range of doses, i.e., the regression coefficient. This information is important because many preparations will have similar LC50 but differ significantly at the 95 percent level of effectiveness (LC95) - the level of insecticidal activity often required to significantly reduce a pest population to acceptable levels. Radcliffe and Yendol (6) documented that for third instar gypsy moth larvae the LC50 was 2.7 (range 1.9-3.4) international units (IU)/larva and the LC95 was 21.1 (13.648.5)IU/larva. Efficacy. The timing of Btk application for gypsy moth is generally dictated by (1) the degree of foliar expansion, (2) larval stage of development, and (3) the size (biomass) of the larvae within an instar (there is an inverse relationship between susceptibility to Bt and larval size). In general, the application timing requires a subjective judgment and varies at a particular site between years due to differences in weather and population density (7). In the laboratory, Yendol et al (8) showed that when given a choice, gypsy
Hall and Barry; Biorational Pest Control Agents ACS Symposium Series; American Chemical Society: Washington, DC, 1995.
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moth larvae consumed more untreated leaf disks or those sprayed with the lowest Btk concentrations than those receiving the highest concentrations. Bryant and Yendol (9) showed that a given dose of Btk per unit oak leaf surface area (cm ) was more effective against gypsy moth when applied at a higher density of small drops (50 to 150 jam) than at a lower density of larger drops (>150 um).
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The optimal drop size and drop density of Btk on foliage needed to control gypsy moth have not yet been determined. During application, however, a wide range of drop sizes from 50-500 um can usually be generated and deposited with different types of nozzles and atomizers. Typically, drop sizes between 75 and 250 um volume median diameter (VMD) are used in gypsy moth suppression. Also, data is insufficient to support the exclusive use of a particular atomizer or nozzle (e.g., flat fan, hollow cone, Micronair, Beecomist) over another. Presently, a wide range of drop sizes and types of nozzles are used for both rotary and fixed-wing aircraft (7). The current trend is towards increasing the dose and decreasing the total volume of Bt applied. Typically, doses of 50-90 BlU/ha are applied undiluted in volumes of 1.8 to 4.7 L/ha for one application. Since there exists only minimal replicated results supporting the effectiveness of one dose and volume combination over others, there is a broad range of doses and diluted and undiluted volumes presently applied in suppression programs. Yendol et al (10) showed that the distribution of Btk deposit vdthin a broadleaved forest canopy following aerial application was Hghly variable; however, deposit differences between upper and lower canopy levels or directionally within canopy level, were not significant. Deposition tends to be log normal, where many leaves contain less than the average dose, balanced by relatively few highly dosed leaves. For a typical application of undiluted Bt at a dose of 60 BlU/ha and rate of 4.7 L/ha, droplet densities can range from 1.3 to 6.0 droplets/cm of foliage. Droplet sizes during these applications commonly ranged from 80 to 226 um V M D . 2
Technology. The aerial application technology used to apply Btk to broadleaved forests for suppressing gypsy moth populations was developed during the 1960's for application of chemical insecticides. In general, that technology was not very efficient at maximizing deposit on target foliage; therefore, with present technology approximately 50% of the applied Btk does not reach the target. The results of spray tower and laboratory bioassays using Btk indicate that small droplets (
