1995). BT was first detected in 1902 in dying larvae of the Silkworm, Bombyx mori, and first used as an insecticide against lepidop- tera in 1938 (Worthing et al.
How-To-Do-It
Toxicity of Bacillus thuringiensis var. Kurstaki to the Painted Lady Butterfly, Vanessa cardui Richard Stalter Gerard Nadal Dwight Kincaid Bacillus thuringiensis var. Kurstaki (BT) is a Gram-positive, sporulating, rod-shaped bacterium that is widely used as a microbial insecticide against a variety of lepidoptera targets, especially the Gypsy Moth, Lymantra dispar (Johnson et al. 1995). BT was first detected in 1902 in dying larvae of the Silkworm, Bombyx mori, and first used as an insecticide against lepidoptera in 1938 (Worthing et al. 1987). Microbial insecticides have long been thought to be a more effective and specific control of forest defoliators such as the Gypsy Moth and Western Spruce Budworm, Choristoneura occidentalis Freeman, than chemical pesticides such as malathion that kill a wide spectrum of arthropods. In agricultural systems, many of the nontarget arthropods are readily killed by chemical pesticides, including ‘‘useful’’ arthropods that prey upon harmful or target insect pests. Most students are aware of the injurious effects on nontarget organisms of the use of pesticides such as DDT to control insect pests. Man-made pesticides such as DDT were nearly responsible for the demise of many of our top food chain avian predators, including the eagle and osprey. It was Rachel Carson’s Silent Spring that first brought this to the attention of the American public. Since that time scientists have developed new insecticides that are more specific for controlling insect pests. The use of bacteria such
Richard Stalter, Ph.D., is Professor of Biology and Gerard Nadal is a Teaching Assistant in the Biology Department at St. John’s University, Jamaica, NY 11439. Dwight Kincaid is in the Biology Department at Lehman College of The City University of New York, Bronx, NY 10468.
as BT is the most recent attempt to develop host-specific control. Unfortunately BT is toxic to a much wider group of insects than originally believed. In the present laboratory excercise we present methods for testing the null hypothesis that BT is not harmful to nontarget moths and butterflies. In this laboratory exercise there are a number of teaching applications pertaining to ecology, microbiology and introductory biology—including integrated pest management, aseptic technique, serial dilution, LD 50 , bioassay, experimental design, statistical analysis of 2x2 contingency tables, and insect development. The supposed benefits of microbial insecticides such as BT, are that they are not harmful to humans, unlike chemical pesticides, and that they exhibit some degree of target specificity. Both assumptions have recently begun to be investigated. In an epidemiological study in Oregon, Green et al. (1990) showed that some patients with preexisting medical conditions have developed infections from which BT has been cultured. Johnson et al. (1995) demonstrated that the lethal effects of BT are not restricted to target lepidoptera in field studies. The specific concern for the unintended effects of BT against nontarget butterflies has been discussed in detail by Wagner & Miller (1995). They report a recent United States Forest Service study on the susceptibility to BT of six native butterflies: Diana Fritillary, Eastern Tiger Swallowtail, Hedgerow Hairstreak, Redspotted Purple, Spring Azure, and Tawny Emperor. All were shown to be highly susceptible to BT. Moths, too, are susceptible to BT, though laboratory tests indicate a wide variation in susceptibility. The Giant Silk Moth is highly susceptible to BT treatment; however,
the Pinion (Lithophane) and Underwing (Catocala) moths show a wide range of tolerance. Susceptibility may vary within larval development with younger caterpillars generally more susceptible than mature ones (Wagner & Miller 1995). The leading biorational insecticide, Bacillus thuringiensis, is a ubiquitous Gram-positive, spore-forming bacterium that forms a parasporal crystal during the stationary phase of its growth cycle. . . B. thuringiensis is already a useful alternative or supplement to synthetic chemical pesticide application in commercial agriculture, forest management, and mosquito control. It is also a key source of genes for transgenic expression to provide pest resistance in plants (Schnepf et al. 1998). The Painted Lady butterfly is one our most common butterflies. It can be found in almost all of the 48 states. The adult butterfly’s upper surface is beautifully mottled in orange, brown and black. Blue, red and white spots occur on the wings. The underside of the adult is gray with red and white markings. Each larva builds a webbed nest on leaves of the Malvaceae family, especially the hollyhock or mallow, its favorite host plants. Mature larvae can be recognized by the presence of a prominent yellow stripe on each side of their dark bodies. Just prior to pupation the larvae will hang from the underside of the leaf to develop a golden brown crysalid. We report here the effects of BT against Painted Lady larvae (Tables 1 and 2) and propose a series of experiments that are appropriate for a class of advanced high school students or for college level students in microbiology, ecology or population biology. The following experiments are powerful in demonstrating the highly lethal
TOXICITY OF BACILLUS THURINGIENSIS VAR. KURSTAKI 207
effects of BT against a nontarget organism, the Painted Lady butterfly, Vanessa cardui.
Materials & Methods Larval Growth & Maintenance Painted Lady butterfly larvae are used in these exercises as the host organism in the LD50 experiment and bioassay (infectivity assay) experiment. The larvae and food are commercially available from Carolina Biological Supply Co., 278 York Rd., Burlington, NC 27215. The food comes in solid, paste form. Culture the larvae on the specially prepared food. Place ten larvae in a 250-ml beaker with an ample supply of food. The beaker should be covered with either cheesecloth or aluminum foil to prevent the larvae from escaping. Small holes should be poked in the foil to allow for air exchange. Transfer of the larvae should be done with a paintbrush, as the larvae are very susceptible to injury. Larvae should never be picked up by hand. The larvae should be transferred to new containers every 48 hours, as their waste products accumulate rapidly. The soiled vessels should be cleaned with soapy water.
Preparation of BT On the day of the experiment, prepare a standard solution of BT according to the instructions on the container. Preparations of BT spores are sold by several companies and are widely available in most nurseries and garden supply centers. The BT used in these experiments is sold by Safer Inc., a subsidiary of the Ringer Corp., 9959 Valley View Rd., Eden Prairie, MN 55534, and marketed under the name, ‘‘Caterpillar Killer.’’ County and State Agriculture Stations can provide information on stores that carry BT. Shake the BT concentrate well before adding it to tap water for dilution. What we refer to as 100% BT is a dilution of 1 ml concentrate/250 ml water (⬃3 teaspoons/gallon). Mix the 100% BT solution thoroughly to ensure an even distribution of spores. It is important to keep the experimental and control organisms in separate rooms to prevent the possibility of contamination. Always wash your hands thoroughly with warm soapy water if latex gloves are not used. Clean and wash beakers and other containers contaminated with BT with soapy water and autoclave the vessels before they are used again in the
experiment. If an autoclave is not available, use disposable plastic cups to house the larvae. Inoculate blocks, 1 cm3, of Painted Lady culture medium with 0.2 ml of the BT dilution using a standard 1.0 ml pipet. In the dilution endpoint experiment, inoculate the media blocks with the most dilute solution first. This will enable the student to use the same pipet for all treatments, which saves time and cost. Slowly add the solution by drops. Then place the cube in the appropriate beaker (three cubes per beaker). To avoid mistakes, arrange the experiment vessels in a row (0.5% to 100% BT), beginning with the control, to which plain water is added to make it the weakest treatment.
Data Analysis The concentration of BT required to kill 50% of the Painted Lady butterfly larvae is the lethal dose, abbreviated as the LD50. The lethal dose varies with the species of host, the stage of the organism’s life history, the age of development of the larvae (in the case of the Painted Lady), and the variety or strain of the microorganism. Typically, many test organisms are used in each experiment, and the endpoint is the smallest or most dilute number of organisms that will kill one-half of the organisms tested. For any particular bioassay experiment, the frequencies of outcomes are placed into a 2x2 contingency table with the frequencies labeled a,b,c,d as below. BT
Control
兺
Living
a
b
ab
Dead
c
d
cd
ac
bd
N
兺
Under the null hypothesis of independence (no relationship between outcome and treatment), the expected frequency for any cell is calculated by multiplying its row total by its column total and then dividing by the grand total (N). For instance, the expected frequency for d (Control, insect died) is (c d) (b d) / N. It is useful to have students calculate these expected frequencies for comparisons to observed frequencies. Although the chi-square test of independence may be used, the G-test is preferred (Sokal & Rohlf 1995). Compute a G-test statistic using Box 17.6 on page 731 of Sokal & Rohlf (1995). The steps are: 1. a(ln a) b(ln b) c(ln c) d(ln d)
208 THE AMERICAN BIOLOGY TEACHER, VOLUME 62, NO. 3, MARCH 2000
2. (ab) [ln (ab)] (cd) [ln (cd)] (ac) [ln (ac)] (bd) [ln bd)] 3. N ln N 4. G 2 (quantity 1 quantity 2 quantity 3) 5. Compare G to chi-square at 1 degree-of-freedom (df) to complete test. Critical values from the chi-square table at 1 df are 2.706, 3.841, 5.024, 6.636, 7.879, and 10.828 for P-values of 0.1, 0.05, 0.025, 0.01, 0.005, and 0.001, respectively.
Exercise I. BT Toxicity Objective To inoculate media with BT and determine the toxic effects of BT on Painted Lady larvae. Students will learn about aseptic techniques, media preparation, the importance of controls and replications in an experiment, and care in the maintenance of stock cultures.
Materials • Painted Lady larvae (10/vessel for a total of 4 vessels, e.g. beakers) • Painted Lady butterfly larvae media, 12 1-cm cubes, 3 per vessel • 4 vessels with appropriate covers • 1-ml pipets • Tape to mark vessels • Marking pencil • Graduated cylinder • Small paint brush
Procedure 1. Wash hands thoroughly before and after each treatment. Pipet 0.2 ml of 100% BT solution onto each of nine cubes. 2. On a separate bench, pipet 0.2 ml water onto 3 cubes of food and place into a vessel labeled ‘‘Control.’’ Add 10 larvae to the control vessel and cover the top with either cheesecloth or aluminum foil with small air holes. The holes must be small enough to prevent the larvae from escaping. 3. Place cubes of treated Painted Lady butterfly medium into 3 labeled (experimental) vessels. Add 10 larvae to each vessel and cover with appropriate covers (with air holes).
Comments 1. The number of organisms per container can be varied below 10 larvae. No more than 10 larvae
2. 3.
4.
5.
6. 7.
should be used per control container, to keep them from outgrowing their containers and to ensure an adequate amount of food. Controls will need to be fed increasing amounts of food as they grow. Experimental organisms should die within 24 hours, especially larvae in early instar stages. Older larvae may take up to 48 hours to die. A comparative experiment of BT’s time-to-kill as a function of developmental stage could be performed to demonstrate this principle. The experiment should begin on a Monday or Tuesday to allow adequate time for observation during the school week. An adequate amount of food should be placed in the control vessel on Friday to carry the controls through the weekend. The larvae should be kept a safe distance from heat sources and direct sunlight. The data in Table 1 indicate that the Painted Lady butterfly larvae raised on media dosed with a 100% BT solution, the dose recommended for garden lepidopteran pests, die within 24 hours, while those treated with a 10% solution die within 48 hours.
The instructor should discuss the possible effect of BT treatment on nontarget organisms, especially a BT treatment intended for garden (Cabbage Looper) and yard (Gypsy Moth) pests.
mined by weight loss and death) in the nontarget organism. In addition to the learning objectives of Exercise I, students will learn about the preparation of serial dilutions in experiments, the use of analytical balances, preparation of a figure to show survival of organisms as a function of time, the concept of LD50, and how to perform a G-test of significance for cross-tabulated data with a hand calculator.
and weigh to obtain the average weight of each organism. 5. Add 0.2 ml of appropriate dilution to each of three 1-cm cubes of Painted Lady butterfly larvae media, and place in an appropriately labeled container with 10 larvae and cover. Repeat this for each set of larvae. It is essential to work with the control organisms first to prevent contamination. It is equally important to begin with the least concentrated solution and work upward to prevent the effects of carry-over contamination. 6. Before the organisms receive fresh food, they must consume all of the initial food. This may require adding a drop of water to each cube daily in order to keep it hydrated and palatable to the larvae. 7. The surviving larvae should be weighed daily. Because the larvae are extremely delicate, transfer all of the surviving larvae into a clean, dry, preweighed vessel using a paint brush. Weigh the vessel and the larvae. Subtract the weight of the empty vessel from the weight of the vessel plus larvae to determine the weight of the surviving organisms. Divide the total weight of the surviving organisms by the number of surviving organisms to determine the average weight of each organism. Plots of weight as a function of time for each group (Figure 1b) should be prepared as well as plots showing surviving organisms as a function of time (Figure 1a).
Materials • Same as in Experiment 1 above, plus 12 test tubes. • Balance, accurate to 0.01 g.
Procedure 1. Label the test tubes 100% BT, 10% BT, 8% BT, 6%BT, 4%BT, 3%BT, 2%BT, 1%BT, 0.5%BT, and Control (100% water). 2. Prepare the 100% BT by adding 1 ml BT concentrate to 250 ml water. 3. Prepare the dilutions according to Table 2. BT Dilution 10% 8% 6% 4% 3% 2% 1% 0.5% 0%
Water (ml) 9 9.2 9.4 9.6 9.7 9.8 9.9 9.95 10
100%BT (ml) 1 0.8 0.6 0.4 0.3 0.2 0.1 0.05 0
4. Add 10 larvae of approximately equal size to a preweighed beaker
Exercise II. Effect of Diluted Concentrations of BT on Painted Lady Butterfly Larvae
Table 2. Toxicity of BT solutions to Painted Lady butterfly larvae at 48 and 72 hours. Only 60% of the larvae survived at 0.5% treatment at 72 hours.
Objectives
Hour 0
To demonstrate that BT spores can be diluted to an ‘‘infectivity endpoint’’ and to determine the range at which BT solutions lose toxicity (as deter-
BT solution Number Alive Average Wet Wgt., mg.
Table 1. 82 Painted Lady larvae treated with 100% BT in a comparison with controls treated with 100% water. G 97, P ⬍ 0.0001. BT
Control
Living
0
43
Dead
37
2
37
45
10% 10
6% 15
5% 15
4% 15
3% 15
2% 15
1% 15
0.5% Control 15 15
12.0
10.1
14.4
11.2
12.3
15.6
11.5
12.7
10% 0
6% 2
5% 4
4% 3
3% 4
2% 6
1% 4
16.5
10.5
12.0
14.5
20.8
26.5
6% 0
5% 0
4% 2
3% 4
2% 5
1% 4
0
0
19.0
12.8
19.6
45.3
6.7
Hour 48 BT solution Number Alive Average Wet Wgt., mg.
0
0.5% Control 9 14 62.6
46.6
Hour 72 BT solution Number Alive Average Wet Wgt., mg.
10% 0 0
0.5% Control 9 14 100.7
103.4
TOXICITY OF BACILLUS THURINGIENSIS VAR. KURSTAKI 209
cantly higher than those treated with a higher BT concentration, 2 to 6%. A BT solution between 0.5 and 1% is lethal to half of the treated Painted Lady butterfly larvae, 7.5 larvae at 48 hours. Thus BT is a powerful insecticide to Painted Lady butterfly larvae. Might the lethal effects of BT be similar when tested on other nontarget butterflies and moths? BT has a wider range of insecticidal activity than might have been previously thought. The need for pesticides that are nontoxic to humans must be weighed against the spectrum of activity possessed by BT and the downstream ecological effects of this broad range of activity.
References Green, M., Heumann, M., Sokolow, R., Foster, L.R., Bryant, R. & Skeels, M. (1990). Public health implications of the microbial pesticide Bacillus thuringiensis: An epidemiological study, Oregon, 1985–86. American Journal of Public Health, 80(7), 848–852. Johnson, K.S., Schriber, J.M., Nitao, J.K. & Smitley, D.R. (1995). Toxicity of Bacillus thuringiensis var. Kurstaki to three nontarget Lepidoptera in field studies. Environmental Entomology, 24, 288–297. Schnepf, E., Crickmore, N., van Rie, J., Lereclus, D., Baum, J., Feitelson, J., Zeigler, D.R. & Dean, D.H. (1998). Bacillus thuringiensis and its pesticidal crystal proteins. Microbiology and Molecular Biology Reviews, 62, 775–806. Sokal, R.R. & Rohlf, F.J. (1995). Biometry, 3rd ed. New York: W.H. Freeman. Wagner, D. & Miller, J.C. (1995). Must butterflies die for the gypsy moth’s sins? American Butterflies, 3, 19–23. Worthing, C.R. & Walker, S.B. (Eds.). (1987). The Pesticide Manual, A World Compendium, 8th ed. British Crop Protection Council. Figure 1. a. Survival of Painted Lady butterfly larvae treated with 0.5%, 1% and 10% BT solutions over a period of 72 hours. b. Average weight of Painted Lady butterfly larvae reared on media with 0.5%, 1% and 10% BT solutions. Controls received 100% water.
Conclusions The data in this experiment indicate that BT kills some Painted Lady butterfly larvae at the lowest (0.5%) dilution tested after 48 hours (LD50 7.5) (Figure 1a). However, the average weight of the controls as well as the larvae fed
0.5% BT solution is not significantly different after 72 hours. The data in Table 2 show that there are significant differences in average weight of the larvae raised on media treated with a 1 to 6% BT solution by 48 hours. However, the average weight of the larvae treated with a 1% solution is signifi-
210 THE AMERICAN BIOLOGY TEACHER, VOLUME 62, NO. 3, MARCH 2000
Selected References for Further Study Houston, D.R. (1981). Mortality and factors affecting disease development. The Gypsy Moth: Research toward integrated pest management. Forest Service Technology Bulletin, 1584, pp. 281–293. Jacobs, S.E. (1951). Bacteriological control of the Flour Moth, Ephestia kuehniella A. Proceedings of the Society for Applied Bacteriology, 13, 83–89.
