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Response of the Carrot Weevil,Listronotus oregonensis(Coleoptera: Curculionidae), to Strains ofBacillus thuringiensis
BIOLOGICAL CONTROL ARTICLE NO.
7, 293–298 (1996)
0097
Response of the Carrot Weevil, Listronotus oregonensis (Coleoptera: Curculionidae), to Strains of Bacillus thuringiensis F. E. SAADE´ ,* G. B. DUNPHY,*,1
AND
R. L. BERNIER†
*Department of Natural Resource Sciences, Entomology Unit, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste Anne de Bellevue, Quebec H9X 3V9, Canada; and †Imperial Chemical Industries Biological Products, North American Technical Centre, 2101 Hawden Road, Mississauga, Ontario L5K 2L3, Canada Received December 27, 1995; accepted May 13, 1996
Mortality and frass production bioassays were used to investigate the toxicity of seven strains of Bacillus thuringiensis against the adult carrot weevil, Listronotus oregonensis (Le Conte). A semi-artificial diet of carrot foliage with 4% agar was selected to maximize feeding by the insects. Bacillus thuringiensis subsp. tenebrionis (Krieg, Huger, Langenbruch, and Schnetter) (BTT) and two unidentified B. thuringiensis strains, A30 and A429, gave the lowest LC50 values. The frass bioassay supported the conclusions of the mortality assay. Mortality of adults continued after their removal from the insecticidal medium, with the highest mortality being caused by strains A429 and BTT. Survivors from the frass bioassay, initially exposed to strains A30, A429, and BTT, did not resume normal levels of feeding after their removal from the insecticidal medium. r 1996 Academic Press, Inc. KEY WORDS: Listronotus oregonensis; Bacillus thuringiensis; frass production.
INTRODUCTION
The carrot weevil, Listronotus oregonensis (Le Conte), is a major pest of umbelliferous plants such as celery, carrot, and wild carrots in the northeastern sectors of the United States of America (Whitcomb, 1965; Grafius and Otto, 1979) and Canada (Perron, 1971; Martel et al., 1975; Stevenson, 1976). Adult females lay eggs in petioles and the crown of carrot, the preferred host. The larvae, upon emergence, tunnel into the root, where they feed and develop until they exit into the soil, pupate, and overwinter as adults (Perron, 1971). Carrot root injury caused by the carrot weevil ranges from 2 to 22% of the total crop in Quebec and may reach up to 40% when left uncontrolled (Boivin, 1985a). 1 To whom correspondence should be addressed. Fax: (514) 3987990.
Packing and canning industries tolerate no more than 5% root injury (Perron, 1971), which makes efficient control measures necessary. The only control method available for the carrot weevil is the use of foliar insecticides against overwintered adults before ovipositioning (Boivin, 1985a). However, the development of resistance to chemical insecticides by many pest insects and the general concern for environmental damage have resulted in the development of an integrated pest management program with emphasis on alternative control methods. To date, an egg parasitoid, Anaphes sordidatus (Girault) (Boivin, 1985b), and the entomogenous nematodes Steinernema carpocapsaae [Steinernema feltiae (Poinar, 1989)], S. feltiae [Steinernema bibionis (Poinar, 1989)], and Heterorhabditis bacteriophora [Heterohabditis heliothidis (Poinar, 1990)] (Belair and Boivin, 1985; Boivin and Belair, 1989) have been examined for control of the carrot weevil. The Gram-positive, endospore-forming entomopathogenic bacterium Bacillus thuringiensis subsp. tenebrionis (Krieg, Huger, Langebrunch, and Schnetter) occupies an important position in the biological control of numerous pest insect species including Coleoptera (Krieg et al., 1983, 1987; Herrnstadt et al., 1986). The bacterium produces a crystalline protein during sporulation (Ho¨fte and Whiteley, 1989) which is proteolytically activated in the midgut to a smaller molecular weight toxin that inhibits insect feeding and eventually causes death (Li et al., 1991; Pietrantonio et al., 1993). B. thuringiensis subsp. tenebrionis (Krieg, Huger, Langebrunch, and Schnetter), a strain pathogenic to Coleoptera, together with six unidentified strains, isolated from Canadian soils, were shown to be active against various coleopteran insect species (Bernier, 1990). The present study was done to evaluate the toxicity of the aforementioned strains on the carrot weevil and to determine the survival of the insects after intoxication.
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1049-9644/96 $18.00 Copyright r 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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TABLE 1
MATERIALS AND METHODS
Bacterial Cultures, Spore-Crystal Suspensions, and Insect Rearing B. thuringiensis subsp. tenebrionis and the six unidentified strains of B. thuringiensis, labeled A30, A299, A311, A409, A410, and A429, (Imperial Chemical Industries Biological Products, North American Technical Centre, Ontario, Canada) were grown on nutrient agar buffered (pH 7.0) with an equimolar concentration (50 mM) of KH2PO4. After 5 days incubation at 30°C, the bacterial culture consisted of vegetative cells, sporangia, spores, and crystals. These stages, harvested by scraping, were lyophilized and stored at 220°C. The spore:crystal ratio of the strains was 1:1 (Saade´, 1993). Spore–crystal suspensions of each strain were used in the toxicity assays. However, because a public standard is not available (Ferro and Celernter, 1989) and to ensure that toxicity measurements would not reflect strains containing different amounts of proteins, the concentrations of extractable proteins for the crystal– spore mixture of each bacterial strain were determined and the spore–crystal mixtures adjusted to supply a given concentration of protein. Spore–crystal mixtures were suspended in 0.01% Triton X-100 (v/v) solution and washed three times by centrifugation (11,750 g, 122°C, 3 min). The final pellet was resuspended in a putatively selective crystal solubilizing buffer [Na2CO3, 40.5 mM; phenylmethylsulfonylfluoride, 0.5 mM; dithiothreitol, 0.1 m M, pH 10.0] and solubilized at 42°C by incubating for 2 h and vortexing every 30 min, which effectively extracted the proteins (Gringorten et al., 1990). The suspension was centrifuged (11,750 g, 22°C, 3 min) and the supernatant was assayed for total protein five times per sample according to Bradford (1976) using a Bio-Rad protein assay kit (Bio-Rad, Ontario, Canada) with bovine serum albumin as the standard. The spore pellet was examined for residual crystals using phase-contrast microscopy; no crystals were found. Recognizing that the solubilizing buffer might extract spore proteins, the final protein solution was referred to as extractable protein. The relationship between extractable proteins and crude dry weight of the spore and crystal mixture varied with the Bt strain (Table 1). The laboratory cultures of the carrot weevil were periodically supplemented with weevils from the Agriculture Canada Research Station (St. Jean-sur-Richelieu) to maintain hybrid vigor. The colony was reared in an incubator at 24°C during the 16 h light and at 16°C during the 8 h of darkness according to Martel et al. (1975). Diet Selection Maximizing Insect Feeding To select a constantly available diet that would act as a gustatory stimulant ensuring uptake of the toxins
The Relationship between Total Extractable Proteins and Crude Dry Weight of the Spore and Crystal Mixtures from B. thuringiensis subsp. tenebrionis Dry weight of mixtures (mg) for the following protein concentrations Strain
Extractable protein (µg/mg mixture)
75 µg
150 µg
225 µg
300 µg
A30 A299 A311 A409 A410 A429 BTT
19.0 7.7 55.6 7.7 100.6 4.9 2.1
3.95 9.69 1.35 9.69 0.74 15.24 35.54
7.00 19.38 2.70 19.38 1.48 30.48 71.08
11.85 29.07 4.05 29.07 2.22 45.72 106.62
15.80 38.76 5.40 38.76 2.96 60.96 142.16
placed on it, six diets were fed to adult weevils: (1) carrot root, (2) softened (steamed) carrot root with 2% (w/v) agar, (3) fresh carrot foliage, (4) high-fiber foliage suspension containing 4% (w/v) agar, (5) low-fiber foliage suspension in 4% (w/v) agar, and (6) 4% (w/v) agar. Diet 4 was prepared by suspending 22 g of chopped carrot foliage in 70 ml of water. Diet 5, formulated like diet 4, was rendered low in fiber by gravity sedimentation. An equal volume of autoclaved agar suspension containing different amounts of agar was added to given volumes of diet to produce the 2% (w/v) and 4% (w/v) concentrations of agar. Abundant food was placed in plastic petri dishes (100 mm diameter) with moist filter paper to which 10 adults were added. Three replicate plates per treatment were placed in the incubators, and the number of frass pellets on filter paper, changed daily, was counted daily for 3 days. Tukey’s test was used to compare group means. Insecticidal Bioassays Two different bioassays were used to measure the toxicity of the extractable proteins of the bacterial strains against the carrot weevil: adult mortality and a modification of the frass production assay of van Frankenhuyzen and Gringorten (1991). The latter is regarded as a rapid method to determine the specificity of numerous B. thuringiensis toxins against Lepidoptera. Diet 5, the optimum medium for insect feeding, was offered to the insects as food cylinders (6 mm in length 3 6 mm in diameter; 10 cylinders/plate) cut from foliage homogenate supplemented with agar. The cylinders were coated with test spore–crystal suspensions by dipping them into distilled water containing different concentrations of protein from the rehydrated spore–crystal mixtures, agitating the preparations for 5 s, and allowing them to air-dry. The concentration of extractable proteins included 75, 150, 225, and 300 µg protein/ml (higher concentrations produced thick spore–
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crystal slurries that immediately deterred insect feeding) with distilled water as a control. Adults, collected 3–8 days after eclosion (during which time they were starved), were added to the diet in petri dishes (15 3 100 mm diameter) containing moistened filter paper. Insects were allowed to feed for 8 days in the mortality bioassay and for 6 days in the frass bioassay at 25°C in darkness. Four replicates containing 15 adult insects (8 females and 7 males) per plate per dose were used. The sex ratio was maintained to preclude the possibility of differences in sex influencing the results. There was no evidence of a confluent bacterial lawn on the agar surface, indicating limited if any growth by the bacterial strains. Viable plate counts of the agar using Bt selective media (Saade´) also did not show bacterial growth, and thus no additional sporulation that might influence assay results was evident. Mortality was monitored daily over the assay period and the median lethal concentration (LC50) for each test strain was calculated by probit analysis. Where LC50 values of selected strains were not statistically different but the regression slopes were different, these bacterial strains were ranked by the magnitude of the slopes according to Burges and Thomson (1971). The total number of frass pellets per insect exposed to the same extractable protein concentrations over a period of 6 days was calculated. Because there was a linear relationship between the percentage decrease in frass production relative to the control groups and the log of protein concentration (Fig. 1), the concentration of protein required to inhibit frass production by 50% (FP50) was determined using linear regression analysis (SAS, 1988).
TABLE 2 Effect of Selected Diets on the Rate of Defectation by the Adult Carrot Weevil, Listronotus oregonensis, at 25°C Number of fecal pellets/10 insects/day a
Diet Carrot root Steamed carrot root 1 2% (w/v) agar Fresh carrot foliage High-fiber carrot foliage: suspension 1 4% (w/v) agar Low-fiber carrot foliage: suspension 1 4% (w/v) agar
24.4 6 1.2 47.0 6 3.3 61.5 6 4.9 72.8 6 7.2 130.0 6 15.6
a Mean 6 standard error of the mean; N 5 4 replicates of 15 insects per replicate.
Insect Survival and Frass Production After Removal from the Diet Containing Insecticidal Proteins Although lepidopteran larvae are known to survive sublethal intoxication by B. thuringiensis (Milne et al., 1990) that would influence insecticide efficacy, the consequence of limited intoxication for beetles is not known. To determine the long-term effect of B. thuringiensis strains on adults previously exposed to the insecticidal extractable proteins at the LC50 values, survivors of a given treatment (at 8 days for mortality and 6 days for frass production) were consolidated on untreated food cylinders in petri plates with moistened filter paper. Control insects not exposed to extractable proteins were similarly treated. The insects were incubated as previously described. Mortality of both treatment and control groups was recorded over a period of 7 days and the frass for insects was counted for 5 days from their respective assay groups with proportional adjustment of frass output of the surviving insects. Tukey’s test was used to compare the means. Treatment mortality was adjusted to control mortality using Abbott’s formula. RESULTS
Diet Selection Maximizing Insect Feeding
FIG. 1. Effect of increasing extractable protein concentration from spore–crystal mixtures of strains of B. thuringiensis on the extent of frass production by carrot weevil adults.
To select an optimum diet conducive to maximizing food consumption by the carrot weevil, the effect of food formulations on adult defecation was determined. Carrot roots did not elicit frass production to the same extent as fresh carrot foliage (Table 2). Homogenizing the foliage and adding agar strongly enhanced frass production, with the low-fiber foliage suspensions with 4% agar diet being optimum for frass production. Agar alone was the least favorable for frass production. Although not quantified, it was observed that insects fed on diet 5 more avidly than the other formulations. The foliage suspension could be stored at 220°C for 8 months without losing its effect.
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TABLE 4
To standardize the amount of insecticidal proteins in the diet from different Bt strains for use in the bioassays, the protein content of the spore–crystal mixture was determined. The concentration of protein/mg of the spore–crystal mixture varied with isolates (A30, 19.0 6 1.1 µg; A299, 7.7 6 0.6 µg; A311, 55.6 6 4.4 µg; A409, 7.7 6 0.5 µg; A410, 100.6 6 11.1 µg; A429, 4.9 6 0.1 µg; and BTT 2.1 6 0.1 µg). The LC50 for strains of B. thuringiensis on adult carrot weevil mortality was lowest for A429 and highest for A311 (Table 3). The rank order of toxicity for the different strains was: A429 5 BTT 5 A30 . A299 5 A410 . A409 5 A311. Among the most toxic strains, BTT displayed the steepest slope. Toxicity of the most and least virulent B. thuringiensis strains was assessed further in terms of their effect on feeding inhibition as indicated by a decrease in frass production. Strains A429 (FP50 5 10.67 6 1.70 µg protein/ml) and BTT (FP50 5 41.94 6 4.20 µg protein/ml) caused the greatest decrease in frass pellet production followed by strain A30 (FP50 5 94.58 6 9.80 µg protein/ ml). Weevils feeding on the least virulent isolate, strain A311 (FP50 5 1234.37 6 185.15 µg protein/ml), did not produce significantly less frass than control weevils. There was a significant correlation between insect mortality (LC50, this study) and reduction in frass production (r 5 0.99; P , 0.05). At all dosages tested in feeding in this study, strains A30, BTT, and A429 resulted in a significant decrease in frass pellet production per insect over a period of 6 days compared with the less toxic strain A311, which paralleled the decline in frass by the control weevil (Table 4; representative data for the dose of 225 µg/ml). The number of frass pellets per insect exposed to strains A30, BTT, and A429 exhibited a three- to fourfold decrease after 6 days of intoxication.
Effect of 225 µg/ml Protein from Bacillus thuringiensis Strains on Defecation of the Adult Carrot Weevil, Listronotus oregonensis Number of frass pellets/insect/day a Strain
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
A30 A429 BTT A311 Control
1.1 6 0.9 0.6 6 0.1 0.8 6 0.1 1.2 6 0.1 1.4 6 0.1
NC b 0.4 6 0.1 0.5 6 0.1 1.0 6 0.1 1.2 6 0.1
0.4 6 0.0 0.4 6 0.1 0.3 6 0.0 0.9 6 0.1 1.2 6 0.1
0.4 6 0.1 0.4 6 0.0 0.2 6 0.0 0.8 6 0.1 0.1 6 0.1
0.3 6 0.0 0.3 6 0.1 0.2 6 0.2 0.6 6 0.0 0.8 6 0.1
2.2 6 0.4 2.9 6 0.4 1.3 6 0.2 5.8 6 0.4 6.4 6 0.4
a Mean 6 standard error of the mean; N 5 4 replicates containing 15 insects per replicate. b NC - not calculated.
Insect Survival and Frass Production after Removal from Diet Containing the Extracts When adult weevils surviving an LC50 dosage for a given bacterial strain were placed onto untreated diet 5 for 7 days, mortality continued. For weevils feeding on strains A299, A311, A409, A30, and A410, mortality was not significantly different from the mortality experienced by control weevils and ranged from 10 to 20%. However, mortality of surviving weevils previously exposed to strains A429 and BTT was significantly greater than the survival of weevils feeding on other strains and ranged between 45 and 55%. Surviving adults previously subjected to LC50 of extracts of strains A30, BTT, and A429 displayed a similar and a continuously lower defecation rate (3–6 frass pellets/insect/day) than control insects (10–14 frass pellets/insect/day) during the 5 days of exposure to the untreated diet (Table 5). Frass production by weevils feeding on strain A311 was comparable to frass production by control insects (9–12 frass pellets/insect/ day).
TABLE 3 Median Lethal Concentration (LC50 µg Protein/ml) of Bt Strains of Adults of the Carrot Weevil, Listronotus oregonensis, Exposed for 8 Days Strain
LC50 (µg protein/ml)
95% fiducial limits (µg protein/ml)
Slope a
A429 BTT A30 A299 A410 A409 A311
102 118 143 230 255 429 461
52–200 89–155 101–204 143–370 196–331 201–914 266–800
0.71 6 0.07 1.80 6 0.25 1.38 6 0.11 1.01 6 0.05 1.90 6 0.08 0.64 6 0.02 0.90 6 0.03
a Mean 6 standard error of the mean. N 5 4 replicates containing 15 insects per replicate.
TABLE 5 Rate of Frass Production by Listronotus oregonensis Previously Exposed to Strains of Bacillus thuringiensis and Subsequently Incubated on Toxin-Free Medium Number of frass pellets/10 insects/day a Strain
Day 1
Day 2
Day 3
Day 4
A30 5.3 6 0.9 6.1 6 0.8 3.5 6 0.4 4.0 6 0.4 A429 3.8 6 0.6 5.1 6 0.3 4.5 6 0.5 5.1 6 0.3 BTT 4.6 6 0.6 5.8 6 0.6 5.0 6 0.5 3.5 6 0.5 A311 11.2 6 1.4 11.0 6 0.2 10.3 6 0.9 10.1 6 0.5 Control 12.5 6 0.9 13.4 6 0.8 12.2 6 0.8 12.1 6 1.1
Day 5 3.0 6 1.5 3.0 6 1.3 2.1 6 0.3 8.2 6 0.7 9.8 6 0.4
a Mean 6 standard error of the mean; N 5 4 replicates containing 15 insects per replicate.
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DISCUSSION
The discovery of B. thuringiensis isolates with toxic activity against coleopterous insects (Krieg et al., 1983; Herrnstadt et al., 1986) has initiated an interest in evaluating the toxicity of insecticidal extractable proteins and identifying and explaining susceptibility in pest species of Coleoptera. At least five species of coleopterans display varying degrees of sensitivity to B. thuringiensis (Herrnstadt et al., 1986). Our results show that three of the seven strains tested are pathogenic to adults of the carrot weevil. The lack of constant availability of the natural diet (fresh foliage) of weevil adults had led to the formulation of an alternative diet. The preferred food source in terms of fecal production (as a measure of consumption rate) was found to be a diet composed of chopped carrot foliage with 4% agar. The foliage can be chopped and stored with water at 220°C until needed. Carrot foliage contains essential oils and resins (Berenbaum, 1990) that may have been released by chopping, enhancing the attraction of the insect to the diet and thus creating a high consumption rate. Agar does play a nutritional role but it may have helped preserve the texture of the foliage and minimized water loss from the diet which, collectively, would be conducive to feeding and/or frass production. The strains BTT, A429, and A30, based on low LC50 values, were most toxic to carrot weevil adults. The ranking order of toxicity for these three strains was a function of the percentage of insects killed. The steepness of the slopes for these strains dictated the rank of the strains; e.g., the rank order of toxicity for a 50% kill was BTT . A30 . A429. The mortality of surviving weevils previously exposed to the extracted proteins at LC50 value concentrations of the strains other than BTT and A429 was not significantly different from the mortality of control weevils. This further suggests that the BTT and A429 strains are more toxic than the other strains and that the effect of strains A429 and BTT at the LC50 concentration is irreversible. The long-term mortality effect of strains BTT and A429 at the LC50 concentration might be of economic importance under field conditions. An alternative procedure for assessing the toxicity of the extractable protein against beetles in this study was the frass production assay as an indication of feeding inhibition. In general, frass production curves traced a concentration-dependent response with respect to the log concentration of extractable Bt proteins and reduced feeding. These concentration–response curves correlated well with those of the mortality bioassay. Mortality of insects caused by the toxic proteins is generally slow and mediated by cessation of feeding, development of septicemia from germinating B. thuringiensis spores and/or other opportunistic mi-
croorganisms, and an irreparably damaged gut, which eventually cause insect mortality (Hall and Dunn, 1958; Heimpel and Angus, 1959; Somerville et al., 1970; van Frankenhuyzen and Gringorten, 1991). The frass production assay offers an expedient means of reliability determining bacterial isolated toxicity against beetles. At all test concentrations of the highly toxic strains of Bt (BTT, A30, and A429), weevil feeding was significantly reduced by the third day of treatment. Beyond that period, feeding remained very low, which may indicate that the gut was severely damaged by Day 3. Following intoxication and subsequent exposure to untreated diet, frass production remained very low for insects exposed to the more effective strains, suggesting that the gut had undergone irreversible paralysis. Our results were unlike those with the spruce budworm, Choristoneura fumiferana (Clem.), in which sublethal levels of B. thuringiensis did not inhibit midgut tissue regeneration (Milne et al., 1990). This study leads to the questions of why the extractable proteins of different strains differ in toxicity for carrot weevil adults and the long-term effect of survival on fecundity and F2 survivorship. Both are currently under investigation. REFERENCES Be´lair, G., and Boivin, G. 1985. Susceptibility of the carrot weevil (Coleoptera; Curculionidae) to Steinernema feltiae, S. bibionis and Heterorhabditis heliothidis. J. Nematol. 17, 363–366. Berenbaum, M. R. 1990. Evolution of specialization in insectumbellifer associations. Annu. Rev. Entomol. 35, 319–343. Bernier, R. L. 1990. Personal communication. Boivin, G. 1985a. Evaluation of monitoring techniques for the carrot weevil, Listronotus oregonensis (Coleoptera; Curculionidae). Can. Entomol. 177, 927–933. Boivin, G. 1985b. Anaphes sordidatus (Girault) (Hymenoptera: Mymaridae), an egg parasite of the carrot weevil, Listronotus oregonensis (Le Conte). Can. Entomol. 118, 393–394. Boivin, G., and Be´lair, G. 1989. Infectivity of two strains of Steinernema feltiae (Rhabditida; Steinernematidae) in relation to temperature, age and sex of carrot weevil (Coleoptera; Curculionidae) adults. J. Econ. Entomol. 82, 762–765. Bradford, M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Burges, H. D., and Thomson, E. M. 1971. Standardization and assay of microbial insecticides. In ‘‘Microbial Control of Insects and Mites’’ (H. D. Burges and N. W. Hussey, Ed.), pp. 591–622. Academic Press, New York. Ferro, D. N., and Gelernter, W. D. 1989. Toxicity of a new strain of Bacillus thuringiensis to Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 82, 750–755. Goldberg, L. J., and Margalit, J. 1977. A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaemia unguiculata, Culex univeritattus, Aedes aegyptii and Culex pipiensis. Mosq. News. 37, 355–358. Grafius, E., and Otto, M. 1979. Detection and control of the carrot weevil. Michigan State University. Ext. Bull. E-890.
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Gringorten, J. L., Witt, D. P., Milne, R. E., Past, P. G., Sohi, S. S., and van Frankenhyyzen, K. 1990. An in vitro system for testing Bacillus thuringiensis toxins: The lawn assay. J. Invertebr. Pathol. 56, 237–242. Hall, I. M., and Dunn, P. H. 1958. Susceptibility of some insect pests to infection by Bacillus thuringiensis Berkliner in laboratory tests. J. Econ. Entomol. 51, 296–298. Heimpel, A. M., and Angus, T. A. 1959. The site of action of crystalliferous bacteria in Lepidoptera larvae. J. Insect Pathol. 1, 152–170. Herrnstadt, C., Soares, G. G., Wilcox, E. R., and Edwards, D. L. 1986. A new strain of Bacillus thuringiensis with activity against coleoptera insects. BioTechnology 4, 305–308. Ho¨fte, H., and Whiteley, H. R. 1989. Insecticidal crystal proteins of Bacillus thuringiensis. Microbiol. Rev. 53, 242–255. Krieg, A., Huger, A. M., and Schnetter, W. 1987. Bacillus thuringiensis var. tenebrionis strain M-7 is identical to the formerly in Germany isolated strain BI 256-82 B.T. ssp. tenebrionis which is pathogenic to coleopteran insects. J. Appl. Entomol. 104, 417–424. Krieg, V. A., Huger, A. M., Langenbrunch, G. A., and Schnetter, W. 1983. Bacillus thuringiensis var. tenebrionis, a new pathotype effective against larvae of Coleoptera. Z. Angew. Entomol. 96, 500–508. Li, J., Carroll, J., and Ellar, D. J. 1991. Crystal structure of insecticidal endotoxin from Bacillus thuringiensis at 2.5A resolution. Nature (London) 353, 815–821. Martel, P., Svec, H. J., and Harris, C. R. 1975. Mass rearing of the carrot weevil, Listronotus oregonensis (Coleoptera; Curculionidae) under controlled environmental conditions. Can. Entomol. 107, 95–98. Milne, R., Ge, A. Z., Rivert, D., and Dean, D. H. 1990. Specificity of insecticidal crystal proteins. In ‘‘Analytical Chemistry of Bacillus thuringiensis.’’ (L. A. Hickel and W. L. Fitch, Eds.) pp. 22–35. American Chemical Society, Washington, D.C. Perron, J. P. 1971. Insect pests of carrots in organic soils of southwest-
ern Que´bec with special reference to the carrot weevil. Listronotus oregonensis (Coleoptera; Curculionidae). Can. Entomol. 103, 1441– 1446. Pietrantonio, P. V., Federici, B. A., and Gill, S. S. 1993. Interaction of Bacillus thuringiensis endotoxins with the insect midgut epithelium, In ‘‘Parasites and Pathogens of Insects’’ (N. E. Beckage, S. N. Thompson, and B. A. Federici Eds.), Vol. 2. pp. 55–80. Academic Press, New York. Poinar, G. O., Jr. 1989. Examination of the neoaplectanid species feltiae Filipjev, carpocapsae Weiser and bibionis bovien (Nematoda: Rhabditidae). Rev. Nematol. 12, 375. Poinar, G. O., Jr. 1990. Taxonomy and biology of Steinernematidae and Heterorhabditidae. In ‘‘Entomopthogenic Nematodes in Biological Controls’’ (R. Gaugler and H. K. Kaya, Eds.), pp. 23–61. CRC Press, Boston. Saade´, F. E. 1993. ‘‘Evaluation of Strains of Bacillus thuringiensis as Biological Control Agents of the Adult Stages of the Carrot Weevil, Listronotus oregonensis (Coleoptera; Curculionidae).’’ M.Sc. Thesis. National Library, Ottawa. SAS Institute. 1988. SAS/STAT Users Guide, release 6.03 edition. SAS Institute Inc., Cary, NC. Somerville, H. J., Tamada, Y., and Omi, E. M. 1970. Lethal effect of purified spore and crystalline endotoxin preparations of Bacillus thuringiensis on several lepidopteran insects. J. Invertebr. Pathol. 16, 241–248. Stevenson, A. B. 1976. Seasonal history of the carrot weevil, Listronotus oregonensis (Coleoptera: Curculionidae) in the Holland Marsh, Ontario. Proc. Entomol. Soc. Ontario 107, 71–78. van Frankenhuyzen, K., and Gringorten, J. L. 1991. Frass failure and pupation failure as quantal measurements of Bacillus thuringiensis toxicity to Lepidoptera. J. Invertebr. Pathol. 58, 465–467. Whitcomb, W. D. 1965. The carrot weevil in Massachusetts. Biology and control. University Massachusetts Agric. Exp. Str. Bull. 550, 1–30.
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Response of the Carrot Weevil, Listronotus oregonensis (Coleoptera: Curculionidae), to Strains of Bacillus thuringiensis F. E. SAADE´ ,* G. B. DUNPHY,*,1
AND
R. L. BERNIER†
*Department of Natural Resource Sciences, Entomology Unit, McGill University, Macdonald Campus, 21,111 Lakeshore Road, Ste Anne de Bellevue, Quebec H9X 3V9, Canada; and †Imperial Chemical Industries Biological Products, North American Technical Centre, 2101 Hawden Road, Mississauga, Ontario L5K 2L3, Canada Received December 27, 1995; accepted May 13, 1996
Mortality and frass production bioassays were used to investigate the toxicity of seven strains of Bacillus thuringiensis against the adult carrot weevil, Listronotus oregonensis (Le Conte). A semi-artificial diet of carrot foliage with 4% agar was selected to maximize feeding by the insects. Bacillus thuringiensis subsp. tenebrionis (Krieg, Huger, Langenbruch, and Schnetter) (BTT) and two unidentified B. thuringiensis strains, A30 and A429, gave the lowest LC50 values. The frass bioassay supported the conclusions of the mortality assay. Mortality of adults continued after their removal from the insecticidal medium, with the highest mortality being caused by strains A429 and BTT. Survivors from the frass bioassay, initially exposed to strains A30, A429, and BTT, did not resume normal levels of feeding after their removal from the insecticidal medium. r 1996 Academic Press, Inc. KEY WORDS: Listronotus oregonensis; Bacillus thuringiensis; frass production.
INTRODUCTION
The carrot weevil, Listronotus oregonensis (Le Conte), is a major pest of umbelliferous plants such as celery, carrot, and wild carrots in the northeastern sectors of the United States of America (Whitcomb, 1965; Grafius and Otto, 1979) and Canada (Perron, 1971; Martel et al., 1975; Stevenson, 1976). Adult females lay eggs in petioles and the crown of carrot, the preferred host. The larvae, upon emergence, tunnel into the root, where they feed and develop until they exit into the soil, pupate, and overwinter as adults (Perron, 1971). Carrot root injury caused by the carrot weevil ranges from 2 to 22% of the total crop in Quebec and may reach up to 40% when left uncontrolled (Boivin, 1985a). 1 To whom correspondence should be addressed. Fax: (514) 3987990.
Packing and canning industries tolerate no more than 5% root injury (Perron, 1971), which makes efficient control measures necessary. The only control method available for the carrot weevil is the use of foliar insecticides against overwintered adults before ovipositioning (Boivin, 1985a). However, the development of resistance to chemical insecticides by many pest insects and the general concern for environmental damage have resulted in the development of an integrated pest management program with emphasis on alternative control methods. To date, an egg parasitoid, Anaphes sordidatus (Girault) (Boivin, 1985b), and the entomogenous nematodes Steinernema carpocapsaae [Steinernema feltiae (Poinar, 1989)], S. feltiae [Steinernema bibionis (Poinar, 1989)], and Heterorhabditis bacteriophora [Heterohabditis heliothidis (Poinar, 1990)] (Belair and Boivin, 1985; Boivin and Belair, 1989) have been examined for control of the carrot weevil. The Gram-positive, endospore-forming entomopathogenic bacterium Bacillus thuringiensis subsp. tenebrionis (Krieg, Huger, Langebrunch, and Schnetter) occupies an important position in the biological control of numerous pest insect species including Coleoptera (Krieg et al., 1983, 1987; Herrnstadt et al., 1986). The bacterium produces a crystalline protein during sporulation (Ho¨fte and Whiteley, 1989) which is proteolytically activated in the midgut to a smaller molecular weight toxin that inhibits insect feeding and eventually causes death (Li et al., 1991; Pietrantonio et al., 1993). B. thuringiensis subsp. tenebrionis (Krieg, Huger, Langebrunch, and Schnetter), a strain pathogenic to Coleoptera, together with six unidentified strains, isolated from Canadian soils, were shown to be active against various coleopteran insect species (Bernier, 1990). The present study was done to evaluate the toxicity of the aforementioned strains on the carrot weevil and to determine the survival of the insects after intoxication.
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TABLE 1
MATERIALS AND METHODS
Bacterial Cultures, Spore-Crystal Suspensions, and Insect Rearing B. thuringiensis subsp. tenebrionis and the six unidentified strains of B. thuringiensis, labeled A30, A299, A311, A409, A410, and A429, (Imperial Chemical Industries Biological Products, North American Technical Centre, Ontario, Canada) were grown on nutrient agar buffered (pH 7.0) with an equimolar concentration (50 mM) of KH2PO4. After 5 days incubation at 30°C, the bacterial culture consisted of vegetative cells, sporangia, spores, and crystals. These stages, harvested by scraping, were lyophilized and stored at 220°C. The spore:crystal ratio of the strains was 1:1 (Saade´, 1993). Spore–crystal suspensions of each strain were used in the toxicity assays. However, because a public standard is not available (Ferro and Celernter, 1989) and to ensure that toxicity measurements would not reflect strains containing different amounts of proteins, the concentrations of extractable proteins for the crystal– spore mixture of each bacterial strain were determined and the spore–crystal mixtures adjusted to supply a given concentration of protein. Spore–crystal mixtures were suspended in 0.01% Triton X-100 (v/v) solution and washed three times by centrifugation (11,750 g, 122°C, 3 min). The final pellet was resuspended in a putatively selective crystal solubilizing buffer [Na2CO3, 40.5 mM; phenylmethylsulfonylfluoride, 0.5 mM; dithiothreitol, 0.1 m M, pH 10.0] and solubilized at 42°C by incubating for 2 h and vortexing every 30 min, which effectively extracted the proteins (Gringorten et al., 1990). The suspension was centrifuged (11,750 g, 22°C, 3 min) and the supernatant was assayed for total protein five times per sample according to Bradford (1976) using a Bio-Rad protein assay kit (Bio-Rad, Ontario, Canada) with bovine serum albumin as the standard. The spore pellet was examined for residual crystals using phase-contrast microscopy; no crystals were found. Recognizing that the solubilizing buffer might extract spore proteins, the final protein solution was referred to as extractable protein. The relationship between extractable proteins and crude dry weight of the spore and crystal mixture varied with the Bt strain (Table 1). The laboratory cultures of the carrot weevil were periodically supplemented with weevils from the Agriculture Canada Research Station (St. Jean-sur-Richelieu) to maintain hybrid vigor. The colony was reared in an incubator at 24°C during the 16 h light and at 16°C during the 8 h of darkness according to Martel et al. (1975). Diet Selection Maximizing Insect Feeding To select a constantly available diet that would act as a gustatory stimulant ensuring uptake of the toxins
The Relationship between Total Extractable Proteins and Crude Dry Weight of the Spore and Crystal Mixtures from B. thuringiensis subsp. tenebrionis Dry weight of mixtures (mg) for the following protein concentrations Strain
Extractable protein (µg/mg mixture)
75 µg
150 µg
225 µg
300 µg
A30 A299 A311 A409 A410 A429 BTT
19.0 7.7 55.6 7.7 100.6 4.9 2.1
3.95 9.69 1.35 9.69 0.74 15.24 35.54
7.00 19.38 2.70 19.38 1.48 30.48 71.08
11.85 29.07 4.05 29.07 2.22 45.72 106.62
15.80 38.76 5.40 38.76 2.96 60.96 142.16
placed on it, six diets were fed to adult weevils: (1) carrot root, (2) softened (steamed) carrot root with 2% (w/v) agar, (3) fresh carrot foliage, (4) high-fiber foliage suspension containing 4% (w/v) agar, (5) low-fiber foliage suspension in 4% (w/v) agar, and (6) 4% (w/v) agar. Diet 4 was prepared by suspending 22 g of chopped carrot foliage in 70 ml of water. Diet 5, formulated like diet 4, was rendered low in fiber by gravity sedimentation. An equal volume of autoclaved agar suspension containing different amounts of agar was added to given volumes of diet to produce the 2% (w/v) and 4% (w/v) concentrations of agar. Abundant food was placed in plastic petri dishes (100 mm diameter) with moist filter paper to which 10 adults were added. Three replicate plates per treatment were placed in the incubators, and the number of frass pellets on filter paper, changed daily, was counted daily for 3 days. Tukey’s test was used to compare group means. Insecticidal Bioassays Two different bioassays were used to measure the toxicity of the extractable proteins of the bacterial strains against the carrot weevil: adult mortality and a modification of the frass production assay of van Frankenhuyzen and Gringorten (1991). The latter is regarded as a rapid method to determine the specificity of numerous B. thuringiensis toxins against Lepidoptera. Diet 5, the optimum medium for insect feeding, was offered to the insects as food cylinders (6 mm in length 3 6 mm in diameter; 10 cylinders/plate) cut from foliage homogenate supplemented with agar. The cylinders were coated with test spore–crystal suspensions by dipping them into distilled water containing different concentrations of protein from the rehydrated spore–crystal mixtures, agitating the preparations for 5 s, and allowing them to air-dry. The concentration of extractable proteins included 75, 150, 225, and 300 µg protein/ml (higher concentrations produced thick spore–
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crystal slurries that immediately deterred insect feeding) with distilled water as a control. Adults, collected 3–8 days after eclosion (during which time they were starved), were added to the diet in petri dishes (15 3 100 mm diameter) containing moistened filter paper. Insects were allowed to feed for 8 days in the mortality bioassay and for 6 days in the frass bioassay at 25°C in darkness. Four replicates containing 15 adult insects (8 females and 7 males) per plate per dose were used. The sex ratio was maintained to preclude the possibility of differences in sex influencing the results. There was no evidence of a confluent bacterial lawn on the agar surface, indicating limited if any growth by the bacterial strains. Viable plate counts of the agar using Bt selective media (Saade´) also did not show bacterial growth, and thus no additional sporulation that might influence assay results was evident. Mortality was monitored daily over the assay period and the median lethal concentration (LC50) for each test strain was calculated by probit analysis. Where LC50 values of selected strains were not statistically different but the regression slopes were different, these bacterial strains were ranked by the magnitude of the slopes according to Burges and Thomson (1971). The total number of frass pellets per insect exposed to the same extractable protein concentrations over a period of 6 days was calculated. Because there was a linear relationship between the percentage decrease in frass production relative to the control groups and the log of protein concentration (Fig. 1), the concentration of protein required to inhibit frass production by 50% (FP50) was determined using linear regression analysis (SAS, 1988).
TABLE 2 Effect of Selected Diets on the Rate of Defectation by the Adult Carrot Weevil, Listronotus oregonensis, at 25°C Number of fecal pellets/10 insects/day a
Diet Carrot root Steamed carrot root 1 2% (w/v) agar Fresh carrot foliage High-fiber carrot foliage: suspension 1 4% (w/v) agar Low-fiber carrot foliage: suspension 1 4% (w/v) agar
24.4 6 1.2 47.0 6 3.3 61.5 6 4.9 72.8 6 7.2 130.0 6 15.6
a Mean 6 standard error of the mean; N 5 4 replicates of 15 insects per replicate.
Insect Survival and Frass Production After Removal from the Diet Containing Insecticidal Proteins Although lepidopteran larvae are known to survive sublethal intoxication by B. thuringiensis (Milne et al., 1990) that would influence insecticide efficacy, the consequence of limited intoxication for beetles is not known. To determine the long-term effect of B. thuringiensis strains on adults previously exposed to the insecticidal extractable proteins at the LC50 values, survivors of a given treatment (at 8 days for mortality and 6 days for frass production) were consolidated on untreated food cylinders in petri plates with moistened filter paper. Control insects not exposed to extractable proteins were similarly treated. The insects were incubated as previously described. Mortality of both treatment and control groups was recorded over a period of 7 days and the frass for insects was counted for 5 days from their respective assay groups with proportional adjustment of frass output of the surviving insects. Tukey’s test was used to compare the means. Treatment mortality was adjusted to control mortality using Abbott’s formula. RESULTS
Diet Selection Maximizing Insect Feeding
FIG. 1. Effect of increasing extractable protein concentration from spore–crystal mixtures of strains of B. thuringiensis on the extent of frass production by carrot weevil adults.
To select an optimum diet conducive to maximizing food consumption by the carrot weevil, the effect of food formulations on adult defecation was determined. Carrot roots did not elicit frass production to the same extent as fresh carrot foliage (Table 2). Homogenizing the foliage and adding agar strongly enhanced frass production, with the low-fiber foliage suspensions with 4% agar diet being optimum for frass production. Agar alone was the least favorable for frass production. Although not quantified, it was observed that insects fed on diet 5 more avidly than the other formulations. The foliage suspension could be stored at 220°C for 8 months without losing its effect.
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TABLE 4
To standardize the amount of insecticidal proteins in the diet from different Bt strains for use in the bioassays, the protein content of the spore–crystal mixture was determined. The concentration of protein/mg of the spore–crystal mixture varied with isolates (A30, 19.0 6 1.1 µg; A299, 7.7 6 0.6 µg; A311, 55.6 6 4.4 µg; A409, 7.7 6 0.5 µg; A410, 100.6 6 11.1 µg; A429, 4.9 6 0.1 µg; and BTT 2.1 6 0.1 µg). The LC50 for strains of B. thuringiensis on adult carrot weevil mortality was lowest for A429 and highest for A311 (Table 3). The rank order of toxicity for the different strains was: A429 5 BTT 5 A30 . A299 5 A410 . A409 5 A311. Among the most toxic strains, BTT displayed the steepest slope. Toxicity of the most and least virulent B. thuringiensis strains was assessed further in terms of their effect on feeding inhibition as indicated by a decrease in frass production. Strains A429 (FP50 5 10.67 6 1.70 µg protein/ml) and BTT (FP50 5 41.94 6 4.20 µg protein/ml) caused the greatest decrease in frass pellet production followed by strain A30 (FP50 5 94.58 6 9.80 µg protein/ ml). Weevils feeding on the least virulent isolate, strain A311 (FP50 5 1234.37 6 185.15 µg protein/ml), did not produce significantly less frass than control weevils. There was a significant correlation between insect mortality (LC50, this study) and reduction in frass production (r 5 0.99; P , 0.05). At all dosages tested in feeding in this study, strains A30, BTT, and A429 resulted in a significant decrease in frass pellet production per insect over a period of 6 days compared with the less toxic strain A311, which paralleled the decline in frass by the control weevil (Table 4; representative data for the dose of 225 µg/ml). The number of frass pellets per insect exposed to strains A30, BTT, and A429 exhibited a three- to fourfold decrease after 6 days of intoxication.
Effect of 225 µg/ml Protein from Bacillus thuringiensis Strains on Defecation of the Adult Carrot Weevil, Listronotus oregonensis Number of frass pellets/insect/day a Strain
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
A30 A429 BTT A311 Control
1.1 6 0.9 0.6 6 0.1 0.8 6 0.1 1.2 6 0.1 1.4 6 0.1
NC b 0.4 6 0.1 0.5 6 0.1 1.0 6 0.1 1.2 6 0.1
0.4 6 0.0 0.4 6 0.1 0.3 6 0.0 0.9 6 0.1 1.2 6 0.1
0.4 6 0.1 0.4 6 0.0 0.2 6 0.0 0.8 6 0.1 0.1 6 0.1
0.3 6 0.0 0.3 6 0.1 0.2 6 0.2 0.6 6 0.0 0.8 6 0.1
2.2 6 0.4 2.9 6 0.4 1.3 6 0.2 5.8 6 0.4 6.4 6 0.4
a Mean 6 standard error of the mean; N 5 4 replicates containing 15 insects per replicate. b NC - not calculated.
Insect Survival and Frass Production after Removal from Diet Containing the Extracts When adult weevils surviving an LC50 dosage for a given bacterial strain were placed onto untreated diet 5 for 7 days, mortality continued. For weevils feeding on strains A299, A311, A409, A30, and A410, mortality was not significantly different from the mortality experienced by control weevils and ranged from 10 to 20%. However, mortality of surviving weevils previously exposed to strains A429 and BTT was significantly greater than the survival of weevils feeding on other strains and ranged between 45 and 55%. Surviving adults previously subjected to LC50 of extracts of strains A30, BTT, and A429 displayed a similar and a continuously lower defecation rate (3–6 frass pellets/insect/day) than control insects (10–14 frass pellets/insect/day) during the 5 days of exposure to the untreated diet (Table 5). Frass production by weevils feeding on strain A311 was comparable to frass production by control insects (9–12 frass pellets/insect/ day).
TABLE 3 Median Lethal Concentration (LC50 µg Protein/ml) of Bt Strains of Adults of the Carrot Weevil, Listronotus oregonensis, Exposed for 8 Days Strain
LC50 (µg protein/ml)
95% fiducial limits (µg protein/ml)
Slope a
A429 BTT A30 A299 A410 A409 A311
102 118 143 230 255 429 461
52–200 89–155 101–204 143–370 196–331 201–914 266–800
0.71 6 0.07 1.80 6 0.25 1.38 6 0.11 1.01 6 0.05 1.90 6 0.08 0.64 6 0.02 0.90 6 0.03
a Mean 6 standard error of the mean. N 5 4 replicates containing 15 insects per replicate.
TABLE 5 Rate of Frass Production by Listronotus oregonensis Previously Exposed to Strains of Bacillus thuringiensis and Subsequently Incubated on Toxin-Free Medium Number of frass pellets/10 insects/day a Strain
Day 1
Day 2
Day 3
Day 4
A30 5.3 6 0.9 6.1 6 0.8 3.5 6 0.4 4.0 6 0.4 A429 3.8 6 0.6 5.1 6 0.3 4.5 6 0.5 5.1 6 0.3 BTT 4.6 6 0.6 5.8 6 0.6 5.0 6 0.5 3.5 6 0.5 A311 11.2 6 1.4 11.0 6 0.2 10.3 6 0.9 10.1 6 0.5 Control 12.5 6 0.9 13.4 6 0.8 12.2 6 0.8 12.1 6 1.1
Day 5 3.0 6 1.5 3.0 6 1.3 2.1 6 0.3 8.2 6 0.7 9.8 6 0.4
a Mean 6 standard error of the mean; N 5 4 replicates containing 15 insects per replicate.
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DISCUSSION
The discovery of B. thuringiensis isolates with toxic activity against coleopterous insects (Krieg et al., 1983; Herrnstadt et al., 1986) has initiated an interest in evaluating the toxicity of insecticidal extractable proteins and identifying and explaining susceptibility in pest species of Coleoptera. At least five species of coleopterans display varying degrees of sensitivity to B. thuringiensis (Herrnstadt et al., 1986). Our results show that three of the seven strains tested are pathogenic to adults of the carrot weevil. The lack of constant availability of the natural diet (fresh foliage) of weevil adults had led to the formulation of an alternative diet. The preferred food source in terms of fecal production (as a measure of consumption rate) was found to be a diet composed of chopped carrot foliage with 4% agar. The foliage can be chopped and stored with water at 220°C until needed. Carrot foliage contains essential oils and resins (Berenbaum, 1990) that may have been released by chopping, enhancing the attraction of the insect to the diet and thus creating a high consumption rate. Agar does play a nutritional role but it may have helped preserve the texture of the foliage and minimized water loss from the diet which, collectively, would be conducive to feeding and/or frass production. The strains BTT, A429, and A30, based on low LC50 values, were most toxic to carrot weevil adults. The ranking order of toxicity for these three strains was a function of the percentage of insects killed. The steepness of the slopes for these strains dictated the rank of the strains; e.g., the rank order of toxicity for a 50% kill was BTT . A30 . A429. The mortality of surviving weevils previously exposed to the extracted proteins at LC50 value concentrations of the strains other than BTT and A429 was not significantly different from the mortality of control weevils. This further suggests that the BTT and A429 strains are more toxic than the other strains and that the effect of strains A429 and BTT at the LC50 concentration is irreversible. The long-term mortality effect of strains BTT and A429 at the LC50 concentration might be of economic importance under field conditions. An alternative procedure for assessing the toxicity of the extractable protein against beetles in this study was the frass production assay as an indication of feeding inhibition. In general, frass production curves traced a concentration-dependent response with respect to the log concentration of extractable Bt proteins and reduced feeding. These concentration–response curves correlated well with those of the mortality bioassay. Mortality of insects caused by the toxic proteins is generally slow and mediated by cessation of feeding, development of septicemia from germinating B. thuringiensis spores and/or other opportunistic mi-
croorganisms, and an irreparably damaged gut, which eventually cause insect mortality (Hall and Dunn, 1958; Heimpel and Angus, 1959; Somerville et al., 1970; van Frankenhuyzen and Gringorten, 1991). The frass production assay offers an expedient means of reliability determining bacterial isolated toxicity against beetles. At all test concentrations of the highly toxic strains of Bt (BTT, A30, and A429), weevil feeding was significantly reduced by the third day of treatment. Beyond that period, feeding remained very low, which may indicate that the gut was severely damaged by Day 3. Following intoxication and subsequent exposure to untreated diet, frass production remained very low for insects exposed to the more effective strains, suggesting that the gut had undergone irreversible paralysis. Our results were unlike those with the spruce budworm, Choristoneura fumiferana (Clem.), in which sublethal levels of B. thuringiensis did not inhibit midgut tissue regeneration (Milne et al., 1990). This study leads to the questions of why the extractable proteins of different strains differ in toxicity for carrot weevil adults and the long-term effect of survival on fecundity and F2 survivorship. Both are currently under investigation. REFERENCES Be´lair, G., and Boivin, G. 1985. Susceptibility of the carrot weevil (Coleoptera; Curculionidae) to Steinernema feltiae, S. bibionis and Heterorhabditis heliothidis. J. Nematol. 17, 363–366. Berenbaum, M. R. 1990. Evolution of specialization in insectumbellifer associations. Annu. Rev. Entomol. 35, 319–343. Bernier, R. L. 1990. Personal communication. Boivin, G. 1985a. Evaluation of monitoring techniques for the carrot weevil, Listronotus oregonensis (Coleoptera; Curculionidae). Can. Entomol. 177, 927–933. Boivin, G. 1985b. Anaphes sordidatus (Girault) (Hymenoptera: Mymaridae), an egg parasite of the carrot weevil, Listronotus oregonensis (Le Conte). Can. Entomol. 118, 393–394. Boivin, G., and Be´lair, G. 1989. Infectivity of two strains of Steinernema feltiae (Rhabditida; Steinernematidae) in relation to temperature, age and sex of carrot weevil (Coleoptera; Curculionidae) adults. J. Econ. Entomol. 82, 762–765. Bradford, M. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254. Burges, H. D., and Thomson, E. M. 1971. Standardization and assay of microbial insecticides. In ‘‘Microbial Control of Insects and Mites’’ (H. D. Burges and N. W. Hussey, Ed.), pp. 591–622. Academic Press, New York. Ferro, D. N., and Gelernter, W. D. 1989. Toxicity of a new strain of Bacillus thuringiensis to Colorado potato beetle (Coleoptera: Chrysomelidae). J. Econ. Entomol. 82, 750–755. Goldberg, L. J., and Margalit, J. 1977. A bacterial spore demonstrating rapid larvicidal activity against Anopheles sergentii, Uranotaemia unguiculata, Culex univeritattus, Aedes aegyptii and Culex pipiensis. Mosq. News. 37, 355–358. Grafius, E., and Otto, M. 1979. Detection and control of the carrot weevil. Michigan State University. Ext. Bull. E-890.
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