Indian Journal of BiotechnologyVol 2, April 2003, pp 268-270

Short Communications

Characterization of Mosquito Larvicidal Ba-cillus thuringiensis Isolated from Soils

of India

Banani Sur!", Nitu Nigam', A K Joshil and Vinod Biharil

IFermentation Technology Division,Central Drug Research Institute, Lucknow 226 001, India2Medical Genetics, Sanjay Gandhi Post Graduate Institute,

Lucknow 226 014, India

Received 4 February 2002; accepted 14 August 2002

It has been established now that insects are acquiring resis-tance to commercial products of Bacillus thuringiensis. Sothere is a continuous search for bacteria producing new tox-ins. With this in view, an insecticidal B. thuringiensis was iso-lated from soil sample obtained from the vicinity of a penicil-lin factory in Vadodara, India. The isolate belongs to serotypeH-14. Phenotypic characters of the isolate were identical tostandard B. thuringiensis var. israelensis IPS-82. It was foundto possess good larvicidal activity against Anopheles stephensiand Culex pipiens and exhibited high resistance for penicillin(500 ug/ml), The electrophoretic protein profiles of purifiedcrystals with standard B. thuringiensis (IPS-82) were studied.The isolate apparently showed the same protein profile as that

•of B. thuringiensis var. israelensis. Soil with natural selectivepressure of antibiotic penicillin thus appears to be a goodecological niche for the isolation of B. thuringiensis.Keywords: Bacillus thuringiensis, bioinsecticide, ecological

niche, parasporal inclusion, serotype

The quest for non-hazardous environmentallycompatible pest control measures has spurred the in-terest in Bacillus thuringiensis, which is now recog-nized as one of the most promising bioinsecticides. B.thuringiensis is a spore forming bacteria, which pro-duces insecticidal crystal proteins toxic to insect-pestsbelonging to Lepidoptera, Diptera and Coleoptera. Apotent strain belonging to variety israelensis and ac-tive against mosquitoes and black fly larvae was dis-covered accidentally by Goldberg and Margalit in1977 from the soil of mosquito breeding site in Israel.Thereafter, many workers have reported the occur-rence of highly mosquitocidal B. thuringiensis strainsbelonging to serovars israelensis (Balaraman et ai,

*Author for correspondence:Tel: 0522-2212411-18 ext 4356; Fax: 0522-2223405E-mail: info@cdriindia.org.

1981; Abdel-Hameed et ai, 1990); kyushuensis (Ohba& Aizawa, 1979) thompsoni and canadensis, respec-tively (Ragni et al, 1996). Soil is the natural reservoirfor insecticidal B. thuringiensis with moderate to lowtoxicities (Ishii & Ohba, 1993; Ohba & Aizawa,1979).

Because of possible development of resistance ininsects to the commercialized products based onB. thuringiensis, there is a continuous search for bac-teria producing new toxins. The present work aimedto screen novel insecticidal isolate from soil reportsthe isolation of an insecticidal isolate of B. thur-ingiensis.

Soil samples (4) were collected from grasslands,orchards and urban lands in Lucknow and one soilsample originated from a damp shadowed place nearthe penicillin factory in Vadodara, Iridia. The sampleswere collected by the removal of surface soil with asterile spatula and then collections were made from adepth 2-5 ern below the surface and processed withinone week. Sample areas were devoid of spraying withany commercial formulation of B. thuringiensis.

One gram of soil sample was placed in a tube (6xW') containing 9 ml of sterilized distilled water andwas vortexed for 5 min and heated at 60°C for 10 minto allow only sporulating bacteria to survive. Finally100 ul of the same was plated in starch nitrate me-dium (soluble starch, 2; yeast extract, 0.4; KN03. 0.2;and agar, 2%; pH 7.0) and observed for 72 hrs for theappearance of bacterial colonies. Colonies similar inmorphology of B. thuringiensis were selected andstreaked in Luria agar (HIMEDIA) plate fortified withpenicillin (0.005%) and were examined under a phasecontrast microscope for the presence of parasporalinclusion bodies and spores.

All the isolates were grown in 10 ml of Luria broth(HIMEDIA) in (6XY2") tube for 48 hrs with shakinguntil sporulation. Qualitative bioassays were carriedon 3'd instar larvae of Anopheles stephensi using 25numbers of larvae suspended in 50 ml of distilledwater in a beaker containing 100 ul of bacterialgrowth.

Quantitative toxicity tests were performed onlywith insecticidal isolate confirmed earlier by qualita-tive bioassays. Quantitative bioassay of 48 hrs-oldactive culture was done by serial dilution. Six-fold

SUR et a/: ISOLATION OF B.THURINGIENSIS

serial dilution of the active culture was made in dis-tilled water. Twenty 3rd instar larvae of either A.stephensi or 4th instar larvae of Culex pipiens wereintroduced at 10 ml of each dilution in beakers andkept at 28°C. The 24-hour mortalities were scored andthe LCso values were determined and compared withLCso values of standard B. thuringiensis IPS-82. Tox-icity was expressed in International Units (IU) usingthe formula proposed by Lacey (1984).

LCso IPS 82, .ul ml' )--'--'-------xI5000=ICmrLCso of test material

Insecticidal isolate (B-5) was sent to Pasteur Insti-tute of Paris for serological confirmation and preser-vation.

A loopful of sporulated culture grown on Luriaagar (HIMEDIA) was subcultured in 200 ml of nutri-ent broth (HIMEDIA) and shaken at 250 rpm for 5days at 30°C to ensure sporulation and completeautolysis. The spore crystal mixture was thoroughlyshaken in 1M NaCI containing 0.01 % Triton X-IOOand sedimented by centrifugation at 12,000 g for 10min. Sedimented crystals were then solubilised at

Table I-Standard identification card of strain 8-5

Origin: India, 1997 No. 8-5 Soil

Spores: elliptical, in

non-swollen sporangia

Parasporal inclusion bodies: +

Nutrient agar: Smooth

Gram: +

Size: (5-7.5) x (1.25-2) urn

Motility: +Acid from:

Adonitol: -

Inositol: -

Xylose: -

Mannitol: -

Arabinose: -

Dextrose: +Lactose: -

Alkaline phosphatase: + Salicin: +Indoline (in urea-indole): - Trehalose: +AMC (Voges-Proskauer): - Sucrose: -

MIC to penicillin: 500 ug/rnl Agglutination: H-14

Endotoxin: + (Anopheles stephensi, Titre: 10,000 (IU)

Culex pipiens) IC pipiens8000 (IU)IA. stephensi

Methyl red: +Nitrate reduction: +Starch hydrolysis: +Egg-yolk lecithinase: +

Esterase: +

Exotoxin: -ve

AMC, Acethyl-methyl-carbinol. MIC, Minimum inhibitory con-centration

269

60°C for 3 hrs in 50 mM NaOH and 10 mM EDTA.Sodium dodecyl sulphate-polyacrylamide gel electro-phoresis (SDS-PAGE) was performed by the methodof Laemmli (1970) by using 10% running and 4%stacking gels. Standard strain of B. thuringiensis varisraelensis (IPS-82) was used for comparison of pro-tein profile.

A total of 50 isolates recovered from five soil sam-ples were analyzed for mosquito larvicidal assayagainst Anopheles stephensi. Only one isolate (B-5)originated from the vicinity of penicillin factoryproved to possess mosquitocidal activity. Organismhad the typical morphology of B. thuringiensis strain,centrally occupied elliptical spore in non swollen spo-rangia and measured 1.25 x 5-7.5 us». InternationalUnit (IU) was determined by quantitative bioassayusing IPS-82 as standard. The isolate was found topossess 8,000 IV against Anopheles stephensi and10,000 IU against Culex pipiens. It was highly resis-tant to penicillin and sugar arabinose, xylose andmannitol were not fermented to produce acid (Ta-ble 1), which is one of the confirmatory test for B.thuringiensis (Buchanan & Gibbons, 1975). Acidproduction from salicin represents strain variation (deBarjac & Bonnefoi, 1968).

During the present survey, recovery of B. thur-ingiensis strain with mosquitocidal activity is 2% (lin 50) probably due to use of selective isolation me-dium. B. thuringiensis is known for hydrolyzingstarch and reducing nitrate salts to nitrite. So the useof starch nitrate medium preferentially selects B.thuringiensis culture to grow. In the present surveyinsecticidal assay did not include coleoptera and lepi-doptera. Probably incidence of insecticidal B. thur-ingiensis could be more from Indian soil providedcoleoptera and lepidoptera larvae were also includedin this study. It would be apparent that non-toxic iso-lates were more frequent in soil. Earlier researchersalso reported non-toxic isolates from soil of Japanbelonging to serotype 14 (serovar israelensisi (Kim etal, 1988; Ohba et al, 1988). In B. thuringiensis varisraelensis (H-14) insecticidal toxicity is attributed to72 mDa transmissible plasmid (Gonzalez & Carlton,1984). Possibly in soil due to adverse environmentalcondition loss of plasmid is more frequent resulting indominance of non-toxic isolates in the soil. On theother hand, it was observed that the soil with naturalselection pressure of penicillin is more fertile for B.thuringiensis probably due to advantageous environ-ment.

270 INDIAN J BIOTECHNOL, APRIL 2003

a b c

65-KDa

29-KDa19-KDa •.••••_~_ ••

Fig. I-Protein profile of Bacillus thuringiensis alkali-solubilizedtoxins on SDS-PAGE: lanes a SIGMA MW-SDS-70L standardprotein b, B-5, C, IPS-82.

Purified parasporal inclusion of the type strain ofserovar israelensis ·(IPS-82) and the isolated B-5strain were examined for their protein component bySDS-PAGE (Fig. 1). Parasporal inclusion of thestrains consisted of highly heterogenous multipleprotein components with molecular masses rangingfrom 14 to 65 kDa in B-5 and IPS-82. In both thestrains, 29 kDa and 65 kDa protein were the majorprotein component as evidenced by SDS-PAGE(Fig. 1). A number of other minor protein bands lesserthan 29 kDa were more prominent in isolate B-5.

At present, little information is available regardingthe actual mosquitocidal toxin in the isolate B-5strain. However, it is likely that 65 kDa protein isprimarily responsible for mosquitocidal activity be-cause the protein of this class is mostly insecticidal instrains of B. thuringiensis var israelensis (H-14) (Ar-onson et al, 1986).

It is well accepted that 25 to 29 kDa proteins hav-ing mosquito specific activity in B. thuringiensis var.israelensis are cytolytic for vertebrate and insect cells(Thomas & Ellar, 1983; Knowles et ai, 1992). In thepresent study, protein of the size, 29 kDa is also de-tected in B-5 isolate. However, 29 kDa protein of theisolate, whether haemolytic or not will be the subjectof future work. Further peR analysis will foretell thecrystal type gene in the isolate.

The study concludes that the soil with natural se-lective pressure of antibiotic like soil near the antibi-otic producing factory or medical colleges may provea good ecological niche for the organism, as B. thur-ingiensis is naturally resistant for penicillin. It has

also provided evidence that B. thuringiensis strainswith low to moderate mosquitocidal activities arecommon in natural environments as reported earlierby other researchers (Ohba & Aizawa, 1979; Ishii &Ohba, 1993).

ReferencesAbdel-Hameed A et ai, 1990. Studies on Bacillus thuringiensis H-

14 strains isolated in Egypt. I. Screening for active strains.World J Microbiol Biotechnol, 6,299-304.

Aronson A I et ai, 1986. Bacillus thuringiensis and related insectpathogens. Microbial Rev, 50, 1-24.

Balaraman K et al, 1981. An indigenous virulent strain of Bacillusthuringiensis, highly pathogenic and specific to mosquitoes.Curr Sci, 50, 199-200.

Buchanan R E & Gibbons N E, 1975. Bergey's Manual of Deter-minative Bacteriology, edited by R E Buchanan & N E Gib-bons, s" edn. Williams & Wilkins Co, Baltimore, USA. Pp535-536.

de Barjac H & Bonnefoi A, 1968. A classification of strains ofBacillus thuringiensis Berliner with a key to their differen-tiation. J Invertebr Pathol, 11,335-347.

Goldberg L J & Margalit J, 1977. A bacterial spore demonstratingrapid larvicidal activity against Anopheles sergentii, Urano-taenia unguiculata, Culex univitattus, Aedes aegypti andCulex pipiens. Mosq News, 37,355-358.

Gonzalez J M J & Carlton B C, 1984. A large transmissible plas-mid is required for crystal toxin production in Bacillus thur-ingiensis variety israelensis. Plasmid, 11,28-38.

Ishii T & Ohba M, 1993. Diversity of Bacillus thuringiensis envi-ronmental isolates showing larvicidal activity specific formosquitoes. J Gen Microbiol, 139, 2849-2854.

Kim S H et al, 1998. Distribution, serological identification andPCR analysis of Bacillus thuringiensis isolated from soils ofKorea. Curr Microbial, 37, 195-200.

Knowles B H et al, 1992. A broad-spectrum cytolytic toxin fromBacillus thuringiensis var. kyushuensis. Proc R Sac Lond BBioi Sci, 248, 1-7.

Lacey L A, 1984. Production and formulation of Bacillus sphaeri-cus. Mosq News, 44, 153-159.

Laemmli U K, 1970. Cleavage of structural proteins during theassembly of the head of bacteriophage T4. Nature (Lond),227,680-685.

Ohba M & Aizawa K, 1979. A new subspecies of Bacillus thur-ingiensis possessing lla: llc flagellar antigenic structure :Bacillus thuringiensis subsp. kyushuensis. J Invertebr Pathol,33,387-388.

Ohba M et ai, 1988. Occurrence of non-insecticidal Bacillusthuringiensis flagellar serotype 14 in the soil in Japan. SysAppl Microbiol, 11,85-89.

Ragni A et al, 1996. Characterization of six highly mosquitocidalBacillus thuringiensis strains that do not belong to H-14 se-rotype. Curr Microbiol, 32,48-54.

Thomas W E & Ellar D J, 1983. Bacillus thuringiensis var. is-raelensis crystal d-endotoxin: Effects on insect and mam-malian cells in vitro and in vivo. J Cell Sci, 60, 181-197.