Sensitivity of LarvalOxythyrea funestaPoda (Coleoptera: Scarabaeidae, Cetoniinae) to Different Strains ofBacillus thuringiensis

JOURNAL OF INVERTEBRATE PATHOLOGY ARTICLE NO.

67, 187–189 (1996)

0029

NOTE Sensitivity of Larval Oxythyrea funesta Poda (Coleoptera: Scarabaeidae, Cetoniinae) to Different Strains of Bacillus thuringiensis Oxythyrea funesta Poda (Coleoptera: Scarabaeidae, Cetoniinae), a floral pest in certain mediterranean countries, has been shown to be sensitive to strains of Bacillus thuringiensis (Robert et al., 1994) known to be pathogenic to cerain Chrysomelidae larvae like Phaedon cochleariae F., Leptinotarsa decemlineata Say (Chaufaux et al., 1990). Nineteen new strains of B. thuringiensis, isolated from insects and soil (Chaufaux et al., 1994) which have proteins characteristic of strains with coleopteran larvicidal activity (Lereclus and Sanchis, 1989), enabled us to determine toxicity and persistence of larvicide activity to O. funesta. B. thuringiensis strains were cultivated in a casein and tryptone hydrolysate (HCT) medium, shaken for 48 hr until cell lyses at 30°C (Lecadet et al., 1980), and analyzed by SDS–PAGE (Lereclus et al., 1989). Protein quantity was estimated according to the Bradford method (1976). O. funesta was maintained in the laboratory at 25°C, using the technique developed by Hurpin (1959) for mass rearing of Cetonia aurata (Coleoptera). The larvae were reared in compost and the adults were fed bee pollen. Twenty-four hours after hatching, the larvae were placed in 20 cm3 of sifted nonsterile compost (treated or untreated) in nonaerated plastic containers measuring 50 mm in diameter and 40 mm high. The larvae were treated by incorporating 2 ml of a spore–crystal mixture (approximately 2 mg of protein) into the dry compost. Each treatment was carried out on batches of 10 larvae and repeated three to five times. Larval mortality was observed over 5 days and results are presented using Abbott’s correction (1925). Comparison of the effect of spores versus spore– crystal suspensions on larval mortality and bacterial multiplication was conducted. Spore pathogenicity was tested by 60-sec instar larvae treatment with suspensions of B. thuringiensis spore–crystals, heated at 80°C for 30 min; spore survival was then verified on HCT medium. Microscopic examination of the hemolymph of 74 dead larvae of all instars that were treated with spore–crystal suspensions was made to determine if the bacteria multiplied in the cadavers. The intoxicated larvae stopped feeding in the first 24 hr and mortality began the 2nd day. Previously reported for B. thuringiensis var. japonensis (130 kDa),

larval mortality of Anomala cuprea Hope and Popillia japonica Newman (Coleoptera: Scarabaeidae, Rutelinae) could occur up to the 21st day posttreatment (Suzuki et al., 1991). In our bioassays, the larvae (n 4 30) started to die after only 24 hr of feeding on compost treated with 100 mg of protein per gram. No bacilli were found upon microscopic examination of the treated cadavers, and no effect was exhibited for the heated spore–crystal suspensions on second instar larvae (6 death/60), indicating no septicemia. Table 1 presents the 19 strains of B. thuringiensis used in the experiment, the nature of the samples from which they were isolated, their electrophoretic characterization, and depending on the strains and their virulence to the three larval instars of O. funesta. First instar larvae were the most sensitive and 14 strains caused more than 50% mortality. However, only 8 strains containing proteins toxic to Coleopteran larvae TABLE 1 Mortality of 24-h-old O. funesta Larvae to Different B. thuringiensis strains Strains

Nature of sample

Protein bands (kDa)

% Mortality L1

63 79 1599 2518 2912 2915 2918 3354 3392 3556 3665 3670 3675 3736 3761 4274 4573 4635 5919

Soil Insect Soil Insect Soil Soil Soil Dung Insect Soil Insect Insect Soil Soil Insect Insect Insect Insect Insect

70,72,73 70,72,73 70,73,74,130 66,130,132 70,72,73,130,132 70,72,73,130,132 71,132 70,130,140 70,73 70,73,128,132 66,130 66,130 70,73 30,35,70,73 30,35,70,73 70 70,73 70,72,73,130,132 70,73,74

a

93.0 96.4 96.4 7.2 3.7 10.6 3.7 51.8 51.8 62.1 72.4 79.4 100.0 100.0 100.0 58.7 96.1 38.1 96.4

L2a

L3a

100.0 89.6 82.7 NT NT NT NT 0 0 7.2 14.0 0.0 100.0 100.0 100.0 7.2 96.4 3.7 79.4

93.0 82.7 82.7 NT NT NT NT NT NT NT NT NT 100.0 100.0 100.0 NT 100.0 NT 82.7

Note. Mortality using Abbott’s correction. NT, not tested. L1, first instar larvae; L2, second instar larvae; L3, third instar larvae (10 larvae × 3 per instar, 100 mg/cm3). a

187 0022-2011/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

188

NOTE

TABLE 2 Mortality of O. funesta by Larval Instar

TABLE 4 Persistence of Larvicidal Activity Utilizing 100 mg/cm3 B. thuringiensis Spore–Crystal Preparations

LC50 (mg/cm3)/instar (confidence limits, 95%)

tenebrionis 63 79 1599 3675 3736 3761 4573 5919

Instar

L1a

L2a

L3a

141.7 (110–184.4) 54.0 (39.1–69.4) 79.1 (62.7–99.7) 54.2 (42–70.1) 37.0 (28.3–48.2) 56.1 (44.5–70.7) 34.5 (27–44.2) 33.1 (27.5–42.8) 100.0 (79.3–129.4)

153.4 (126.2–186.7) 74.2 (59.9–88.1) 96.4 (76.6–121.1) 128.1 (104.8–156.5) 43.4 (33.6–56.2) 81.3 (67.2–98.2) 34.2 (26.1–45) 35.6 (26.6–47.5) 125.6 (103.2–160.7)

142.9 (122.7–167.8) 139.1 (114.1–173.1) 80.9 (63.2–103.6) 154.6 (123.9–204.2) 33.7 (25.2–45) 61.6 (48.3–78.5) 47.6 (38–59.7) 60.4 (48–75.8) 123.6 (99.1–163.3)

Strains

a L1, first instar larvae; L2, second instar larvae; L3, third instar larvae (three replications of 10 larvae each).

(70–73 kDa) were more than 80% pathogenic for third instar larvae. The activity of these strains is represented in Table 2 as lethal concentrations to cause 50% mortality (LC50). High sensitivity to B. thuringiensis strains was exhibited at all larval stages of O. funesta as opposed to other larval Coleoptera. For the same strain, the difference in sensitivity between L1 and L2 and between L1 and L3 was never greater than threefold, whereas for Leptinotarsa decemlineata Zehnder and Gelernter (1989) found a large difference in sensitivity between the second and third instars. Our results also indicate that the virulence of these new B.

TABLE 3 Mortality by Larval Age and Instar Utilizing 100 mg/cm3 B. thuringiensis Spore–Crystal Preparations (Four Replications of 10 Larvae Each) Larval age in days

5 6 7 8 9 10 11 12 13 14 15 a

Instar L1a

L2a

100.0 100.0 76.8 76.8 69.0 59.6 71.6 Ecdysis

100.0 100.0 69.0 51.0 53.6 61.3 48.4 74.2 Ecdysis

Differents strains were utilized for testing all instars.

L3a

Treated compost age (days)

a

L1

L2a

L3a

1 2 3 4 5

93.7 37.5 22.9 22.9 0

95.8 54.1 60.4 56.2 0

37.5 25 16.6 0 0

a Different strains were utilized for testing all instars (five replications of 10 larvae each).

thuringiensis strains is higher than that of the tenebrionis strain. In compost containing 100 mg of protein/cm3, larval mortality occurred for the duration of the first instar, that is, 12.4 days (±2.3, n 4 70); throughout the second instar 13.6 days (±2.3, n 4 30); and during at least the first 15 days of the 42 days of the third instar (±6.1, n 4 70) (Table 3). Larval activity rapidly declined over time. If the compost was treated on Day (D) 0, and healthy larvae of the different instars were placed on the substrate on any day from D1 to D5, it was observed that larvae of the first two instars only died through D4, that is, 4 days after the substrate was treated, and third instar larvae up till D3 (Table 4). Spores survival was maintained over the test period, as demonstrated by reisolation of B. thuringiensis 3 months later on HCT medium. In conclusion, these results indicated that O. funesta was particularly susceptible to several strains of B. thuringiensis which are furthermore pathogenic to Chrysomelidae. O. funesta is easy to rear year round and proved to be a test insect which enables good discrimination of B. thuringiensis strains. Studies concerning the potential exploitation of these strains are currently being conducted. K EY WORDS: Bacillus thuringiensis; Coleoptera; Scarabaeidae; Cetoniinae. ACKNOWLEDGMENT The authors thank J. Müller-Cohn and C. Young for help in translating the manuscript. REFERENCES

81.9 71.6 69.0 76.8 84.5 48.4

Abbott: 1925. J. Econ. Entomol. 18, 265–267. Bradford, M. M., 1976. Anal. Biochem. 72, 248–254. Chaufaux, J., Marchal, M., and Gilois, N., 9e Réunion des Microbiologistes INRA Biarritz, October 4–5. Chaufaux, J., Marchal, M., Robert, P., Gilois, N., Jehanno, I., and Buisson, C. 1994. 2nd Int. Conf. on Bacillus thuringiensis, Montpellier, August 28–September 2. Hurpin. 1959. Rev. Zool. Agric. Appl. N° 10–12. Lecadet, M., Blondel, O., and Ribier, J. 1980. J. Gen. Microbiol. 121, 203–212.

189

NOTE Lereclus, D., Arantes, O., Chaufaux, J., and Lecadet, M. M. 1989. FEMS Microbiol. Lett. 60, 211–218. Lereclus, D., and Sanchis, V. 1989. C.R. Acad. Agric. Fr. 75(6), 117– 126. Robert, P., Chaufaux, J., and Marchal, M. 1994. J. Invert. Pathol. 63, 99–100. Suzuki, N., Hori, H., Ogiwara, K., Asano, S., Sato, R., Ohba, M., and Iwahana, H. 1991. Biol. Control, 2, 138–142. Zehnder, G. W., and Gelernter, W. D. 1989. J. Econ. Entomol. 82/3, 756–761.

P. ROBERT J. CHAUFAUX M. MARCHAL INRA Station de Recherches de Lutte Biologique La Minière 78285 Guyancourt Cedex France Received March 14, 1995; accepted September 8, 1995