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Evaluation of Bacillus thuringiensis and Bacillus sphaericus Strainsfrom Chinese Soils Toxic to Mosquito Larvae

MING SUN, XIXIA LUO, JINGYUAN DAI, KEHUI QU, ZIDO LIU, LING YU, YAHUA CHEN, AND ZINIU YU1

Department of Microbial Sciences and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, People’s Republic of China

Received February 3, 1995; accepted February 21, 1996

During our research to isolate and screen microbialagents, eight Bacillus thuringiensis isolates and fiveBacillus sphaericus isolates were shown to have hightoxicity to mosquito larvae. Comparing the LC50 val-ues, four B. sphaericus isolates with LC50 values rang-ing from 0.50 to 1.47 ng/ml were about two to six timesmore toxic than strain 1593 (LC50 3.00 ng/ml) againstlarvae of Culex quinquefasciatus. Four B. thuringien-sis isolates (LC50 values ranging from 3.80 to 7.54 ng/ml) and four B. sphaericus isolates with LC50 of 17.0 to43.7 ng/ml were more toxic to Aedes aegypti than strain1897 (LC50 8.46 ng/ml) and strain 1593 (LC50 67.3 ng/ml). As to Anopheles hyrcanus, the LC50 values of threeB. sphaericus isolates ranging from 3.63 to 5.73 ng/mlwere three to five times smaller than that of strain1593 (LC50 16.1 ng/ml). Two B. sphaericus isolatesshowed high toxicity against mosquito larvae in thethree genera Culex, Aedes, and Anopheles. © 1996 Aca-

demic Press, Inc.

KEY WORDS: Bacillus thuringiensis; Bacillus sphaeri-cus; Culex quinquefasciatus; Aedes aegpyti; Anopheleshyrcanus.

INTRODUCTION

Mosquitoes are known as vectors of several humandiseases (Mattingly, 1969). Development of resistanceto chemical pesticides and environmental concernslimit the use of some chemicals for mosquito control(Brattsten et al., 1986). Since the discovery and iden-tification of Bacillus thuringiensis subsp. israelensis(serotype H14) in 1978 (de Barjac, 1978), this agent hasbeen developed as an alternative tool for mosquito con-trol (Goldberg and Margalit, 1977). As early as in 1979,China started the production of Jie-Jue-Ling, a B.t.israelensis preparation controlling mosquito larvae (Yuand Dai, 1980), and efforts were made to assess thetoxicity and field efficiency of B.t. israelensis in mos-quito control (Yu, 1983; Yu and Chen, 1983; Yu andWang, 1987; Yu and Shen, 1990).

The laboratory resistance in mosquitoes has beendemonstrated to some isolates of B. thuringiensis(Georghious et al., 1983; Gill et al., 1989; Goldman etal., 1986; Vazquez-Garcia, 1983). Increasing attentionhas been devoted to search for alternative mosquito-active Bacillus strains, B. thuringiensis or B. sphaeri-cus. B. sphaericus has the advantage over B.t. israelen-sis in that it appears to persist in the environmentlonger than B.t. israelensis and thus can provide long-lasting control (WHO, 1985).In this paper we report the screening of B. thuring-

iensis and B. sphaericus isolates against mosquito lar-vae, providing data for further research on the devel-opment of bacterial agents having potential for mos-quito control.

MATERIALS AND METHODS

Bacillus spp. A number of B. thuringiensis and B.sphaericus isolates were selected for this research froma large collections isolated in this laboratory. Strain1897 and strain 1593 were used as B. thuringiensis andB. sphaericus reference materials, respectively.

Bacillus preparations. B. thuringiensis or B.sphaericus isolates were cultured on nutrient agarslant (180 × 18 mm test tube containing 8 ml nutrientmedium) 5–7 days at 30°C. The mixtures of spores andcrystals were floated into 10 ml of sterile distilled wa-ter and shaken vigorously. The spore–crystal suspen-sion was considered as stock suspension, which wasdiluted later for preliminary screening of active iso-lates.For products by shaking culture, the B. thuringiensis

isolates were inoculated into a 500-ml flask containing50 ml PM liquid medium (10 g peptone, 5 g glucose, 2g yeast extract, 0.3 g MgSO4·7 H2O, 0.02 g FeSO4·7H2O, 0.02 g ZnSO4·7 H2O, 0.02 g MnSO4·7 H2O, 1 gKH2PO4, in 1 liter water, pH7.2). B. sphaericus isolateswere grown in the nutrient medium (peptone 3.0 g,beef extract 5 g, yeast extract 0.5 g, MnCl20.006 g,CaCl2 0.08 g, MgCl2 0.07 g, H2O 1000 ml) and cultured1 To whom correspondence should be addressed.

JOURNAL OF INVERTEBRATE PATHOLOGY 68, 74–77 (1996)ARTICLE NO. 0060

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

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with shaking at 30°C for 45–55 hr. The products fromeach 50-ml culture were centrifuged, washed free ofmedium, and suspended in 5 ml distilled water for dryweight determination and toxicity assay.

Insects. Three species of mosquitoes were used astarget insects, Culex quinquefasciatus Say, Aedes ae-gypti L., and Anopheles hyrcanus (Pallas). The mosqui-toes were maintained in an insectary with the methodsdescribed by Weiser (1991).

Bioassay. In priminary screening, all of the B. thur-ingiensis and B. sphaericus isolates selected weretested against the second instar larvae of Cx. quinque-fasciatus. Twenty-five larvae were transferred intoeach enamel cup (7 × 9cm) with 50 ml sterile distilledwater. The stock suspension of cultures from agarslants was diluted to 10−1, 10−2, and 10− 3, and 0.5 ml ofeach dilution was added to each of the three cups. Thecups were kept at 30°C. Larval mortality was scored 48hr after treatment. Those isolates that caused mortal-ity the same as or higher than the reference were keptfor further evaluation.After the promising isolates were found, further

screening was carried out for the Bacillus spp. againstthe larvae of Cx. quinquefasciatus, Ae. aegypti, and An.hyrcanus. All the larvae were treated as describedabove at 10−4, 10− 5, and 10−6 dilution of shaking flaskcultures.Based on the results of further screening, the fer-

mentation products were diluted twofold to get a seriesof dilutions of shaking flask cultures. The larvae of Cx.quinquefasciatus, Ae. aegypti, and An. hyrcanus were

treated, and the LC50 values of spores and crystal com-plex were determined by using methods of regressionanalysis described previously (Luo et al., 1993) for eachisolate.

Identification of B. thuringiens is isolates. Identifi-cation of flagella antigen of B. thuringiensis isolateswas conducted by using H antisera against serotypesH1 to H27 of the standard B. thuringiensis. H antiseraand H antigens were prepared by following the conven-

TABLE 1Mortality of Cx. quinquefasceiatus Caused by Diluted Bacterial Cultures

Slant Cultures Shaken flask cultures

Strains 10−1 10−2 10−3 10−4 10−5 10−6

B. thuringiensis 1897 100 80 26 100 13X3 100 92 18 100 12903 100 100 45917 100 100 30 100 25767 100 100 50 100 28909 100 100 30 100 16967 100 80 152J107 100 96 258I 100 100 42 98 1025B 100 100 16 100 3253-2 100 100 45 100 209L (1) 100 98 36 97 17Q23 100 95 8 100 29

B. sphaericus 1593 100 80 0 95 63 2050A 98 95 42 100 68 838B 98 93 38 98 93 35726 100 95 29 100 98 3850A9 95 95 61 98 73 1041A 96 91 63 95 88 2080-I 98 95 56 95 65 3Bs8 100 96 30 96 95 43

TABLE 2Mortality of Ae. aegypti Caused by Diluted Shaking Flask

Cultures of Bacteria

Strains 10−4 10−5 10−6

B. thuringiensis 1897 100 48 858-2 100 23 6909 100 83 2917 100 98 139L(1) 100 38 5767 100 32 025B 93 87 08I 100 28 0Q23 100 98 0X3 100 73 0967 100 14 0

B. sphaericus 1598 62 8 080-I 28 0 0Bs8 96 70 0726 70 24 050A9 54 0 038B 80 26 050A 52 0 041A 69 16 0

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tional process (Norris, 1964). The tube agglutinationtest was carried out to serotype each isolate (Dai et al.,1994).

RESULTS

Of hundreds of Bacillus spp. tested, 12 B. thuring-iensis isolates and 7 B. sphaericus isolates showedequal or higher toxicity against Cx. quinquefasciatusthan strain 1897 or strain 1593 in the preliminaryscreening (Table 1). These strains were kept for furtherscreening.In the further screen, 9 B. thuringiensis isolates and

7 B. sphaericus isolates caused mortality of Cx. quin-quefasciatus the same as or higher than strain 1897and strain 1593 (Table 1); the mortalities of Ae. aegypticaused by 5 B. thuringiensis isolates (909, 917, 25B,Q23, X3) were about the same as that of strain 1897,

while 4 B. sphaericus isolates (Bs8, 726, 38B, 41A) pro-duced apparently higher mortality than strain 1593(Table 2); the activity of 3 B. thuringiensis isolatesagainst An. hyrcanus was comparable with that ofstrain 1897, and the toxicity of 7 B. sphaericus isolateswas slightly higher than that of strain 1593 (Table 3).In Table 4, the LC50 values of 6 B. thuringiensis iso-

lates against Cx. quinquefasciatus, ranging from 2.94to 4.87 ng/ml, were near that of strain 1897 (LC50 value4.11 ng/ml). Two B. sphaericus isolates (38B and Bs8)with LC50 values of 0.50 ng/ml were six times moretoxic and 2 B. sphaericus strains (726 and 41A) withLC50 values of 1.14 and 1.41 ng/ml were about threetimes more toxic than strain 1593 (LC50 3.00 ng/ml)against Cx. quinquefasciatus. It was noted that LC50values of B. sphaericus against Cx. quinquefasciatuswere much lower than that of B. thuringiensis.Four B. thuringiensis isolates with LC50 values of

3.80 to 7.54 ng/ml and 4 B. sphaericus isolates withLC50 of 17.0 to 43.7 ng/ml were more toxic to Ae. ae-gypti than strains 1897 (LC50 8.46 ng/ml) and 1593(LC50 67.3 ng/ml), while the LC50 values of B. sphaeri-cus were larger than that of B. thuringiensis.Three B. sphaericus isolates with LC50 values rang-

ing from 3.63 to 5.73 ng/ml were three to five timesmore toxic to An. hyrcanus than strain 1593 (LC50 16.1ng/ml).It was noted that two isolates (726 and Bs8) showed

remarkably high toxicity against mosquito larvae inthe three genea Culex, Aedes, and Anopheles.Based on the immunologic identification, the 8 most

active B. thuringiensis strains (9L(1), 53-2, Q23, 25B,767, 917, 909, and X3) belong to serotype H14, and the5 most active B. sphaericus strains (Bs8, 50A, 726, 41A,and 38B) belong to serotype H5. SDS–PAGE showed

TABLE 3Mortality of An. hyrcanus Cause by Diluted Slant Cultures

of Bacteria

Strains 10−1 10−2 10−3

B. thuringiensis 1897 100 100 09L (1) 100 100 025B 100 100 0767 100 100 053-2 100 100 0

B. sphaericus 1593 75 0 0726 100 0 041A 90 0 050A 100 0 038B 85 0 050A9 100 0 080-I 100 0 0Bs8 85 55 0

TABLE 4LC50 of Spore–Crystal Complex to Mosquito Larvae

Cx. quinquefasciatus Ae. aegypti An. hyrcanus

StrainNo

LC50

(ng/ml)

95% FLaLC50

(ng/ml)

95% FLLC50

(ng/ml)

95% FL

Lower Upper Lower Upper Lower Upper

B. thuringiensis 1897 4.11 3.55 4.74 8.46 7.62 9.41909 3.80 3.57 4.05X3 6.07 3.52 6.81767 2.94 2.60 3.34917 3.10 2.69 3.58Q23 3.46 2.95 4.07 6.71 6.30 7.1825B 3.94 3.41 4.58 7.54 6.79 9.419L (1) 3.98 3.35 4.9153-2 4.87 4.20 5.69

B. sphaericus 1593 3.00 2.46 3.68 67.3 49.0 84.2 16.1 13.4 18.8Bs8 0.50 0.39 0.62 17.0 14.6 19.8 3.63 3.13 4.1338B 0.50 0.39 0.62 23.8 19.8 28.250A 5.25 3.99 6.36726 1.14 0.94 1.37 32.4 27.5 37.8 5.73 4.78 6.5741A 1.41 1.05 1.80 43.7 34.4 52.9

a FL, fiducial limits.

SUN ET AL.76

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that the protein profile of the 8 B. thuringiensis strains’inclusions was similar to that of B.t. israelens HD-567,except that strains 53.2 and 9L(1) demonstrated anadditional protein, z115 and z140 kDa, respectively(data not shown).

CONCLUSION AND DISCUSSION

The toxicity of 8 B. thuringiensis isolates and 5 B.sphaericus isolates against mosquito larvae were de-termined. Five B. thuringiensis and 4 B. sphaericusisolates tested showing high toxicity against Cx. quin-quefasciatus were selected on the basis of the LC50 val-ues. As to Ae. aegypti, 4 B. thuringiensis strains and 4B. sphaericus strains demonstrated greater toxicitythan reference materials, three B. sphaericus strainsshowing 3 to 7 times more toxicity than strain 1593against An. hyrcanus.It is interesting to note that 2 B. sphaericus strains,

726 and Bs8, showed high toxicity against these threetarget mosquitoes.The different species of mosquitoes showed various

susceptibility to B. sphaericus. The susceptibility rangewas: Cx. quenquefasciatus > Ae. aegypti > An. hyrca-nus. Comparing the LC50 values, B. thuringiensisstrains showed higher insect specificity, while B.sphaericus strains indicated wider insect spectrum.This might indicate that B. sphaericus has high poten-tial as a control agent of mosquito larvae.

ACKNOWLEDGMENTS

This research received financial support from UNDP/WORLDBANK/WHO Special Program for Research and Training in TropicalDisease. We sincerely thank Dr. Mir S. Mulla, University of Califor-nia, Riverside, for revising this paper, and Dr. M.-M. Lecadet, Pas-teur Institute, for confirming the serotype of all the B. thuringiensisand B. sphaericus isolates list in Table 4.

REFERENCES

Brattsten, L. B., Holyoke, C. W., Jr., Leeper, J. R., and Raffa, K. F.1986. Insecticide resistance: Challenge to pest management andbasic research. Science 231, 1255–1260.

Dai, J., Wan, B., Luo, X., Yu, L., and Yu, Z. 1994. Studies on 410Bacillus thuringiensis isolated from soils. J. Huazhong Agric.Univ. 13, 144–152.

de Barjac, H. 1978. Une nouvelle variete de Bacillus thuringiensistre toxique pour les moustiques: B. thuringiensis var. israelensisserotype H14. C. R. Seances Acad. Sci. D. 286, 797–800.

Georghious, G. P., Baker, J., Ai-khatib, Z., Mellon, R., Murray, C.,Tran, H., Vazquez, M., Pelsue, F., and Hazelrigy, J. 1983. Insec-ticide resistance in mosquitoes: research on new chemicals andtechniques for management. In “Mosquito Control Research An-nual Report,” pp. 86–91. Univ. of California, Riverside.

Gill, S. S., Dai, S. M., Chang, C., Georghious, G. P., and Chow, E.1989. Mosquito Resistance to the 72 kDa Toxin of Bacillus thur-ingiensis israelensis, In “Mosquito Control Research Annual Re-port,” pp. 24–25. Univ. of California, Riverside.

Goldberg, L., and Margalit, J. 1977. A bacterial spore demonstratingrapid larvicidal against Anopheles sergentii, Uranotaenia inguicu-lata, Culex univitattus, Aedes aegypti and Culex pipiens. MosquitoNews 37, 355–358.

Goldman, I. F., Arnold, J., and Carlton, B. C. 1986. Selection forresistance to Bacillus thuringiensis subspecies israelensis in fieldand laboratory populations of the mosquito Aedes aegpyti. J. In-vertebr. Pathol. 47, 317–324.

Luo, X., Gu, B., Jiang, H., Shen, J., and Yu, Z. 1993. Assay of toxicityand standardization of Bacillus thuringiensis products. In “Pro-duction and Application of Bacillus thuringiensis Preparations” (Z.Yu, Ed.), pp. 167–202. Agricultural Pub. House, Beijing.

Mattingly, P. F. 1969. The biology of mosquito borne disease. In “TheScience of Biology Series I” (J. D. Carthy, and J. D. Sutcliff, Eds.),pp. 13–183, Allen & Unwin, London.

Norris, J. R. 1964. The Classification of Bacillus thuringiensis. J.Appl. Bacteriol. 27, 439–447.

Vazquez-Garcia, M. 1983. Investigation of potentiality of resistanceto Bacillus thuringiensis ser H14 in Culex quinguefasciatusthrough accelerated selection pressure in the laboratory. Ph.D.dissertation, Univ. of California Riverside.

Weiser, J. 1991. Evaluation of insecticidal and Molluscicidal activity.In “Biological Control of Vector,” p. 37. Wiley, New York.

Yu, Z., and Dai, D. 1980. The effects of Jie-Jue-Ling preparation(Bacillus thuringiensis H14) against mosquito larvae. Microbiol-ogy (Chinese) 7, 198–199.

Yu, Z. 1983. Advanced studies on Bacillus thuringiensis against mos-quito larvae. Biol. Control 5, 66–75.

Yu, Z., and Chen, Y. 1983. Bioassay of Bacillus thuringiensis refer-ence standard IPS. 78. J. Huazhong Agri. Univ. 2, 79–80.

Yu, Z., and Wang, Y. 1987. Bioassay of bacterial preparation againstmosquito larvae. I. A base to determine biological unit. InsecticideMicrobiol. 1, 102–109. Beijing Agricultural Univ. Press, Beijing.

Yu, Z., and Shen, J. 1990. Bioassay of bacterial preparations againstmosquito larvae—The relationship between biological potency andlethal dosage per larvae. Bull. Soc. Vector Ecol. 15, 48–52.

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