Bacillus thuringiensis: Laboratory tests against four species of biting lice (mallophaga: Trichodectidae)

JOURNAL

OF INVERTEBRATE

fk~cillos

PATHOLOGY

23, 232-236

thoringiensis: of Biting R. E.

Laboratory

Tests Against

Lice (Mallophaga: Laboratory, of Agriculture,

Received

Four

Species

Trichodectidae)l

N. ALLAN,

GINGRICH,

Livestock Insects U.S. Department

U.S.

(1974)

AND

D. E. HOPKINS

Agricultural Kerrville,

September

Research Service, Texas ‘78028

5. 1973

Four species of mammalian biting lice, Bovicola bovis, B. crassipes, B. limbata, and susceptible in the laboratory to the spored-endotoxin complex of than to the j%exotoxin. The relative potency of these active principles was thus reversed from that for avian biting lice. The activity of the preparations tested was not homologous with that of E-61, the international standard for &endotoxin activity. B. ovi.s, were more Bacillus thuringiensis

INTRODUCTION

Hoffman and Gingrich (1968) first reported that biting lice of the order Mallophaga were susceptible to Bacilllti thurin&ens& when they controlled shaft lice, Menopon gallinae, wing lice, Lipeurus capork, and chicken body lice, Menacanthus stramineus, on chickens with a commercial product containing a mixture of active principles from B. thuringiensis. In subsequent tests with purified preparations, the p-exotoxin appeared to be responsible for this control; the S-exotoxin was ineffective (Gingrich, unpubl.) . However, critical experimentation was not possible at that time to determine which fractions of B. thuringiensis were active against chicken lice because the insects are difficult to rear and handle in vitro. Recently, methods became available for in vitro rearing of four species of Mallophaga of the genus Bovicola. We therefore used laboratory-reared lice of these species to bioassay several preparations of B. thur‘This paper reflects the results of research only. Mention of a proprietary product or a pesticide in this paper does not constitute an endorsement or a recommendation of this product by the U.S. Department of Agriculture.

ingiensis. The results of these tests and also our attempts to relate the activity of these preparations with those of the proposed international standard, E-61, are reported here. MATERIALS

0 1974byAoedemia

of reproduotion

in any

Press, Inc. form

reserved.

METHODS

Lice were reared by the procedures described by Hopkins and Chamberlain (1969) for B. crassipes and B. limbata, by Hopkins (1970) for B. ozlis, and by Hopkins and Chamberlain (1972) for B. bovis. The preparations of B. thuringiensis were selected on the basis of their presumed principal active ingredients. Included were HD-1 and HD-73 from Serotype 3a, 3b (var. lcurstaki) that contained the sporecrystal complex but no exotoxin. Both materials were developed by H. T. Dulmage, Cotton Insects Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, Brownsville, Texas, who also supplied the sample of HD-73. The sample of HD-1 was commercially formulated and supplied by Abbott Laboratories, North Chicago, Illinois. The remaining materials tested, all from Serotype I (var. thuringiensis), and their suppliers were: 1,0001.2 (International Minerals and

232 Copyright All rights

AND

Bacillus

th~urinyiensis

AGAINST

RESULTS

Comparative

Bacillus

thuringiensis LTeo

(hr)

B. ovis Microbial

LTbo

HD-1 HD-73 BTB-183 L-69 1,0001.2 E-61

27.9 44.9 69.6 62.7 104.3 128.1

3.59 4.69 4.71 3.02 12.3 7.3

LTso 13.2 59.4 28.6 58.9 65.6 136.6

of Materials

1

MATERIALS and slope B. bovis

b

Toxicity

Table 1 shows that while the susceptibilities of the four species of biting lice to the various microbial insecticides varied greatly, the two HD materials were usually the most toxic. The least active material was E-61, and the next least active was the purified exotoxin 1,0001.2. Generally, the materials containing mixtures of active principles were intermediate in activity compared with the purified materials. An additional test was made in which HD-1 and 1,0001.2, representing Sendotoxin and ,B-exotoxin principal ingredients, respectively, were autoclaved at 120°C for 15 min and then bioassayed against B. ovis in the usual manner. HD-1 was not active after autoclaving, but 1,0001.2 was not affected. Because of the obvious variation in the slopes of the regression lines obtained, few comparisons of potency among materials were attempted. However, HD-1 and HD73, because of their similarities in serotype and production, were analyzed comparatively to determine some of the possible causes of variation. In the overall comparison, the b values for the species of lice

TABLE OF SIX

233

LICE

mula were then treated by weighted probit/log time transformation analysis. Analysis of covariance (Snedecor and Cochran, 1967) was used to compare regression lines in slopes, elevations, and residual variances.

Chemical Corporation, Libertyville, Illinois) ; Biotrol BTB-183@ (Nutrilite Products, Lakeview, Califorina) ; Bakthane L-69@ (Rohm and Haas Co., Philadelphia, PasPennsylvania) ; and E-61 (Institute teur, Paris). The sample of 1,0001.2 had been purified to contain only the p-exotoxin; the other materials contained spores, cryetals, and exotoxin. Equal numbers of adult males and females of each species, except B. bovis, were used for the bioassays. B. bovis reproduces parthenogenetically, and only females were available. The lice ranged in age from 2 to 21 days when tested. For testing, 1 part of each microbial material was blended with 9 parts of goatskin scrapings, the diet for lice. Diluents similar to those with the test material were mixed into diet at the same proportion for the appropriate controls. Then 25 mg of treated diet was placed in a 1-dr shell vial with 10 insects and held at 76% RH and 35°C. Time of death was taken as the end point because of the wide variation in susceptibilities of the lice and the need to keep a constant ratio of insecticide to edible diet. Thus, mortality counts were taken for male and female lice scparately at 8, 16, and/or 24 hr intervals, beginning when lice were put on the treated diet. Two or more tests were made at different times with each material, the number depending on the number of replicates per test. However, a total of at least 150 lice were exposed to each. The data corrected for deaths in the control by Abbott’s for-

TOXICITY

BITING

TO FOUR

(b) of regression

SPECIES lines

OF Bovicola,

for

B. limb&a b

4.52 5.23 6.37 7.61 8.29 10.01

LTSO 39.6 37.5 46.6 80.1 66.9 165.0

B. b 5.36 3.44 4.87 6.85 7.77 11.48

LTso

-

42.2 76.2 77.4 108.8 102.0 123.9

crass&s

b 4.47 3.43 8.34 10.17 17.13 7.92

234

GINGRICH,

ALLAK

ANI) HOPKINS

TABLE COMPARATNE

TOXICITY

OF

Bacillus

2

thuringiensis

TO MALE

LT50 for B. thuringiensis HD-1

Sex

HD-73

E-61

AND

FEMALE

Bovicola

materialsn~b 1,0001.2

BTB-183

B. ovis

23.2 a 35.4 b

41.3a 49.2 a

39.5 a 45.6 b

72.5 a 80.6 a

34.8 a 45.0 a

38.7 a 39.9 a

115.0 a 145.9 a

108.6 a 104.7 a

69.1 a 70.1 a

104.8 a 101.1 a

ND

ND

ND

B. crassipes

110.6 a 133.9 b B. limbata

160.2 a 172.4 a

a Paired means within a column for each species of lice not followed different at the 5 % level. b ND, not done.

treated with HD-1 did not differ significantly. However, they did differ significantly with HD-73: the slopes for regression of B. ovi.s and B. bovis were not parallel to those of B. limbata and B. crassipes. Also, when HD-1 was compared with HD-73 against individual species of lice, we found that the regression lines were parallel only when B. bovis was the test louse. The slopes were significantly different when the tests involved the three species exposed in bisexual tests. Differential

Susceptibility

by same letter are significantly

existed among the other species. The comparison of regression lines for selected materials (Table 2) showed that such differential susceptibility was greatest for HD-1 and E-61 ; in each instance where it occurred, male lice were more susceptible than female lice, When we used international units to compare the potencies of HD-1 and E-61 (Table 3)) the reaction was homologous between males and females of the same species, but it was hetereologous between some species of the same sex.

of MaEe and FeDISCUSSION

male Lice

The four species of biting lice tested here Since only females of B. bovis were tested, we examined the possibility that varied in susceptibility to a given treatdifferences in susceptibility due to sex ment and also the different treatments

RELATIVE

POTENCIES”

TABLE 3 OF HD-1 AGAINST

IU/mg Sex

B. ovis

Male Female

4956 4121

a Potencies expressed in IU/mg assigned a value of 1000 IU/mg. b ND, not done.

SEXES

of HD-1 as determined B.

limbata 4603 3831

OF Bovicola

against

B. crassipes

2800 2936

relative to the proposed international

B. bovisb

ND 1035 standard E-61, which has been

Baci2Zus thuringiensis varied in their effects on a given species. These variations are not surprising in view of the specific host preferences of the species and the variety of B. thuringiensis materials tested. Burgerjon and Martouret (1971) have reviewed the diverse factors that determine the host spectrum of B. thuringiensis. Obviously, from our results, specific factors for both the bacterium and the insect were interacting. There were no consistent qualitative or quantitative differences in the responses of insects, but among the treatments, the purified spore-crystal materials were consistently more toxic than the materials containing ,R-exotoxin, either purified or mixed with sporesand crystals. The demonstration of the thermostable character of p-exotoxin was made to establish its presence and, therefore, to provide a basis for the activity of 1,0001.2. Similarly, the autoclaving demonstrated the thermolabile character of the active principle in HD-1. The relationship between the active ingredients and toxicity to mammalian lice reported here seemsto be the reverse of the relationship for chicken lice (Gingrich, unpubl.), which were tested only on living hosts. The fact that products containing exotoxin were more active against lice on chickens than products containing only the spore-crystal complex is probably attributable to the action of exotoxin against immature insects during or soon after ecdysis. On the living hosts, all stages of lice occurred; in the laboratory bioassays, only adults were treated. However, the inverse relationship of activity involving sporecrystal products against chicken and mammalian biting lice can be explained only on the basis of differential susceptibility of the insects to the active principles. This finding is particularly interesting because the spore-crystal complex is generally considered active per OSonly to lepidopteran insects. Our results may therefore show that mammalian lice are also sensitive to the spore-crystal complex, but they also open

AGAINST

BITING

LICE

235

up the possibility that a new insecticidal entity may have been discovered. Differences caused by strain and method of production can also be important in the activity spectra of B. thuringiensis. Dulmage (1971), for example, used Heliothis virescens to demonstrate variation in activity among 18 isolates of Serotype 3 grown on different media. In our test, the potency to three speciesof Bovicola of two of the materials he tested after special fermentation (32,000 IU/mg for HD-73 and 13,000 IU/mg for HD-1 when medium B-4 was used) was reversed from that which he found (HD-73 was more potent than HD1). Moreover, the differences for H. virestens were only quantitative ; the differences for Bovicola were qualitative. In Bovicola, both the slopes of regression and also the differential activity toward male and female differed for HD-1 and HD-73. The possibility that these variations may be caused by different active principles needs examination. The variation in ratios of potency for the different materials with E-61 confirm the heterologous nature of the active ingredients. These differences and the apparently diverse susceptibility of the lice for B. thuringiensis mean that a single microbial material can probably not be developed for standardizing commerical materials for use against Bovicola lice; thus, individual bioassays will be needed to evaluate new materials. REFERENCES A., AND MARTOURET, C. 1971. Determination and significance of the host spectrum of Bacillus thuringiensis. In “Microbial Control of Insects and Mites” (H. D. Burges and N. W. Hussey, eds.), pp. 305-325. Academic Press, New York. DULMAGE, H. T. 1971. Production of B-endotoxin by 18 isolates of Bacillus thuringiensis Serotype 3, in 3 fermentation media. J. Znvertebr. Pathol., 18, 353358. HOFFMAN, R. A., AND GINGRICH, R. E. 1968. Dusts containing Bacillus thuringiensis for control of chicken body, shaft, and wing lice. J. Econ. Entomol., 61, 85-88. BURGERJON,

236

GINGHICH,

ALLAN

D. E. 1970. In vitro colonization of the sheep biting louse, Bovicola 0vi.s. Ann. Entomol. Sot. AmeT., 63, 1196-1197. HOPKINS, D. E., AND CHAMBERLAIN, W. F. 1969. In vitro colonization of the goat biting lice, Bovicola crassipes and B. limbata. Ann. Entomol. Sot. Amer., 62, 526-528. HOPKINS,

AA-D

HOPKINS

D. E., AND CHAMBERLAIN, W. F. 1972. In vitro colonization of the cattle biting louse, Bovicola bovk. Ann. Entomol. Sot. Amer., 65, 771-772. SNEDECOR, G. W., AND COCHRAN, W. G. 1967. “Statistical Methods,” 593 pp. Iowa State Univ. Press, Ames, Iowa. HOPKINS,