ISSN 0003�6838, Applied Biochemistry and Microbiology, 2011, Vol. 47, No. 8, pp. 762–766. © Pleiades Publishing, Inc., 2011.Original Russian Text © M.V. Zubasheva, L.A. Ganushkina, T.A. Smirnova, R.R. Azizbekyan, 2010, published in Biotekhnologiya, 2010, No. 6, pp. 27–33.

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INTRODUCTION

As mosquitoes are known to be a carrier of a series ofinfectious diseases, the problem of their control is con�sidered to be presently challenging. Use of chemical lar�vicides is not sufficiently effective and, in some cases,leads to tolerance of mosquito larvae. Biological meth�ods of mosquito control using the bacteria Bacillus thu�ringiensis spp. israelensis (Bti) and Bacillus sphaericus,whose larvicidal effects are due to protein crystals, aresupposed to be more ecologically safe [1–4].

However, it is necessary to extend the spectrum oflarvicidal effect producing bacteria, because the mos�quito larvae are capable to develop tolerance to thebiopreparations prepared on the basis of B. sphaericus.It was previously shown that some strains of Breviba�cillus laterosporus possess larvicidal activity withrespect to a variety of insects [5–8]. We have describedseveral crystal�forming strains of Brevibacillus lat�erosporus so far. It was shown that the specific larvi�cidal activity of the strain LAT006 with respect toDiptera species was comparable with that of crystalsproduced by B. thuringiensis and B. sphaericus thatallowed considering Brevibacillus laterosporus to be apotential producer of biological means for mosquitocontrol [9–12].

One of the disadvantages of the biological means ofmosquito control is their relatively short�term residualeffect that may be due to sedimentation of a preparationon the water pool bottom, absorption by silt particles

and organic matter, and a series of other biological andphysical factors. We previously showed that the incap�sulation of B. thuringiensis (Bti) and B. sphaericus cellsby the infusorian Tetrahymena pyriformis providedenhanced larvicidal effect of the bacteria [13–16].

Protozoa occupy an important niche in naturalecological systems. They appear to work as the poten�tial reservoirs of microorganisms pathogenic forhumans and animals, inducing infectious diseases.Fast distribution of Protozoa by a variety of ways, shortgeneration period, and ability to form stable, calmforms favor their transformation into epidemiologi�cally dangerous factors.

Interaction of the free�living infusorian T. pyrifor�mis with a series of pathogenic bacteria subfamilies hasa complicated symbiotic nature. In nonsterile sub�strates, such as soil or water, in the absence of Proto�zoa, a bacterial population is intensively suppressed bymicrobial cenosis, while higher abundance and stabil�ity of bacterial population is observed in a long�termmanner in association with infusoria [17–21].

The study of T. pyriformis infusorian relationshipswith viruses revealed that one and the same strain maytotally inactivate the influenza virus but appear to bethe permanent host for other RNA� and DNA�viruses[21]. Apparently, for the majority of viruses, there is acorresponding Protozoa species, in whose organismthe virus can either replicate or persist, as well as aProtozoa species, which specifically inactivates thisvirus. For example, the poliovirus can replicate inT. pyriformis cells [22], but it is inactivated in an envi�ronment containing Entamoeba moshkovskii [23].

Enhancement of Larvicidal Activity of Brevibacillus laterosporusby Bioincapsulation in Protozoa Tetrahymena pyriformis

and Entamoeba moshkovskiiM. V. Zubashevaa, L. A. Ganushkinab, T. A. Smirnovaa, and R. R. Azizbekyana

a State Research Institute for Genetics and Selection of Industrial Microorganisms, Moscow, 117545 Russiab Martsinovski Institute for Parasitology and Tropical Medicine, Sechenov Moscow Medical Academy, Moscow, 119435 Russia

e�mail: raziz@genetika.ruReceived June 8, 2010

Abstract—Conditions for bioincapsulation of crystal�forming strain Brevibacillus laterosporus LAT 006spores and crystals by using Tetrahymena pyriformis and Entamoeba moshkovskii Protozoa have been devel�oped. Increase in the larvicidal activity of the incapsulated bacteria was demonstrated. Fractions of purespores and crystals and intact spore�crystal preparation of LAT 006 were shown not to have toxic effect on theprotozoa cells.

Keywords: bioincapsulation, Brevibacillus laterosporus, mosquitoes, Protozoa.

DOI: 10.1134/S0003683811080126

Abbreviations: SCS—spore�crystal suspension; PMSF—phenyl�methylsulfonylfluoride; EDTA—ethylenediamine tetraacetate.

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ENHANCEMENT OF LARVICIDAL ACTIVITY OF Brevibacillus laterosporus 763

Based on these speculations and taking intoaccount relatively low larvicidal activity of B. lat�erosporus, the present study was aimed at the investiga�tion of interactions of Protozoa T. pyriformis andE. moshkovskii with this bacterium and assessment ofthe possibility to increase its larvicidal activity by itsincapsulation by Protozoa cells.

EXPERIMENTAL

B. laterosporus Strain

In order to perform the research, we chose the crys�tal�forming strain B. laterosporus LAT006, which wasobtained from the collection of entomopathogenicbacteria of IEBC (Institute Pasteur, Paris, France).The strain was cultivated on BP solid medium at 37°Cfor 96 h in order to reach maximal spore and crystalformation. Spore�crystal mass was washed out of agarsurface and used for isolation of pure fractions ofLAT006 spores and crystals.

Obtainment of Cultural Supernatant and Cell Fractions

Bacterial cells were precipitated by 15 min centrif�ugation at 10000g, 4°C, of B. laterosporus culture atdifferent stages of cultivation. The pellet was thricewashed from the medium by 15 min centrifugation at10000g, 4°C, in a physiological solution and thenresuspended in the same solution and brought up tothe initial volume. The obtained supernatant was ster�ilized by pushing it through 0.45 μm bacterial mem�brane filters.

Isolation and Purification of B. laterosporus Crystals

LAT006 strain was cultivated in the liquid BPmedium at 37°C until mature sporangia were devel�oped (light microscopy control). The liquid phase waswithdrawn by 15 min centrifugation at 15000g. Thespore mass was resuspended in 0.005 M Tris�HClbuffer, pH 7.2. After that the suspension was supple�mented with 1 mM phenylmethylsulfonylfluoride(PMSF), 1 mM EDTA, and 2% Triton X�100 solutionto a final concentration of 0.02%, and then it wasincubated at 20°C for 1 h [20]. Spore�crystal suspen�sion (SCS) prepared in this way was disintegrated bysonication on ice (two 30 sec cycles with 30 sec pausesat 40 kHz) using a VCD16�850 sonicator (Ultrasonic,United Kingdom). The crystals and spores of B. lat�erosporus were purified by ultracentrifugation of thespore�crystal strain suspension in a nonlinear densitygradient of sodium bromide (20%, 30%, 40% and50%) at 150000g for 2 h at 4°C [26, 27]. The obtainedfractions were washed thrice from sodium bromide by15 min centrifugation at 15000g in distilled water andthen resuspended in the initial volume of distilledwater and analyzed by microscopy. The crystal andspore preparation purity was controlled by optical and

phase�contrast microscopy of smears, which werestained by the method of Smirnov [28].

Protozoa Strains

Axenic strain of Tetrahymena pyriformis wasobtained at the Department of Molecular Biology(Biological Faculty, Moscow State University). Theculture was sustained in a nutrient medium and sub�cultured every 10 days. To perform the subculturing,2 ml of the culture were transferred into 15 ml of themedium, containing 300 units/ml of penicillin and15 μg/ml streptomycin. Infusoria were cultivated intubes and kept in the dark at 24°C.

Nutrient medium for infusorian cultivation wasprepared as follows: 720 mg of K2HPO4 ⋅ H2O and930 mg KH2PO4 of the high purity grade (Russia) weredissolved in 150 ml of distilled water and the pH to wasbrought to 6.48. The 15 ml aliquots of the obtainedsolution were distributed into the tubes. After that, 1 gof fresh beef liver was added and the lube was closedwith a cotton�gauze plug and autoclaved at 0.5 atm for15 min in a vertical autoclave. The prepared mediumwas stored in a cold room.

Four�day infusorian culture was used for experi�ments. Infusorians cells were washed twice with sterilewater and precipitated by 10 min centrifugation at3000g. The obtained pellet was resuspended in water.The number of infusoria was counted microscopically.The initial mixture contained 1.5–2.0 × 104 infusoriancells per ml.

Entamoeba moshkovskii was cultivated in Pavlova’smedium (8.5 g of sodium chloride, 0.590 g of sodiumphosphate, and 0.450 g of potassium phosphate weredissolved in 1 liter of water supplemented with 1 : 15bovine serum (v/v)). The 15 ml aliquots of thismedium together with E. moshkovskii cells were putinto tubes containing 0.5 g of sterile rice starch. Theculture was stored in tubes in a dark room at 25°C andwas subcultured every 10 days. The seven�day amoebacultures were used for experiments. To perform theexperiments, cells were washed twice with physiologi�cal solution by centrifugation at 2000g for 10 min. Theobtained pellet was resuspended in physiological solu�tion and the number of cells was counted microscopi�cally. The initial mixture contained 5–8 × 103 amoebacells.

To perform the biological tests, 4.5 ml of washedProtozoa suspension was supplemented with 0.5 ml ofthe tested B. laterosporus sample. The obtained mix�ture was mixed with a shaker for 2 h and then intro�duced into the flasks containing the mosquito larvae.The reference control tubes contained 4.5 ml of Pro�tozoa suspension, 0.5 ml of distilled water (for T. pyri�formis) and 0.5 ml of physiological solution (forE. moshkovskii) instead of bacteria.

The concentration that killed 50% of the mosquitolarvae (LD50) was estimated by the Probit�analysis

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program. The period in which 50% death of the mos�quito larvae occurred (LT50) was estimated as well.

RESULTS AND DISCUSSION

We have shown that the larvicidal activity of somestrains of B. laterosporus is connected with their abilityto synthesize crystalline protein toxins [10]. It shouldbe noted that the ability of B. laterosporus to crystal�formation was first described in [10]. It was also ofinterest to study the possibility to increase B. lat�erosporus efficacy by bioincapsulation. On the firststage of research, we developed conditions for bioin�capsulation of spores and crystals of the effectiveB. laterosporus strains using T. pyriformis and E. mosh�kovskii Protozoa strains.

It was preliminarily shown that the fractions ofpurified spores and crystals, as well as spore�crystal

mixture of LAT006, did not produce a toxic effect onT. pyriformis and E. moshkovskii cells (Table 1).

When it became obvious that separate fractions ofB. laterosporus LAT006 were not lethal for Protozoacells, we used bioincapsulation approach to assess thelarvicidal effect of the bacteria.

The 1 ml of spore�crystal mass of B. laterosporus pre�pared for bioincapsulation contained 1–3 × 106 FLAT006spores (1–1.2 μg of total protein) and 1.5–2.0 × 104

T. pyriformis cells. The 1 ml mixture of purified spores ofB. laterosporus prepared for bioincapsulation contained1–3 × 107 spores and 1.3–1.5 × 104 T. pyriformis cells.The 1 ml mixture of purified crystals contained 0.9–1 μgof the total protein, corresponding to the crystal fraction,and 1.5–2.0 × 104 T. pyriformis cells. To perform the bio�incapsulation, mixtures of either spore�crystal mass orthe purified fractions of LAT006 spores and crystals were

Table 1. Effect of spore and LAT006 crystal fraction on Protozoa cells

Time, h

Survival of protozoa, %

Control* Water suspension of spores and crystals Purified crystals Purified spores

T** E** T E T E T E

0 100 100 100 100 100 100 100 100

2 110 104 103 107 97 103 93 106

24 150 112 163 111 195 107 117 113

48 160 121 130 118 200 125 113 120

* See “Experimental”** T. pyriformis*** E. moshkovskii

Table 2. Effect of different LAT006 fractions incapsulated into the T. pyriformis cells on the mosquito larvae

Sample

LC50 represented as

The studied LAT006* sample dilution

Protein concentra�tion in the LAT006

sample, µg/ml

The studied LAT006* sample dilution

Protein concentra�tion in the LAT006

sample, µg/ml

24h 48h

SCS 3.1 × 10–2

(1.8 × 10–2–6.0 × 10–2)3.090 4.3 × 10–4

(2.3 × 10–4–9.0 × 10–4)0.040

SCS + Infusorian 2.4 × 10–3

(1.8 × 10–3–4.9 × 10–30.237 1.0 × 10–4

(1.5 × 10–5–2.9 × 10–40.012

Purified crystals 1.2 × 10–2

(4.4 × 10–3–8.1 × 10–21.163 2.1 × 10–4

(1.3 × 10–4–5.6 × 10–40.022

Purified crystals + Infusoria 2.4 × 10–4

(1.3 × 10–4–1.6 × 10–40.024 1.9 × 10–5

(1.4 × 10–5–2.6 × 10–50.003

Purified spores – – – –

Purified spores + Infusorian – – – –

* 95% significance values for the LC50 calculation using Probit software are shown in parentheses; dashes mean the absence of the larvalethality

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ENHANCEMENT OF LARVICIDAL ACTIVITY OF Brevibacillus laterosporus 765

incubated in the presence of infusoria at 25°C for 2 h atconstant stirring. After that, the B. laterosporus–T. pyri�formis mixture was introduced into test flasks containingAnopheles stephensi mosquito larvae of the age III. Thereference control mosquito flasks were supplementedwith nonincapsulated B. laterosporus specimens. Thenumber of dead larvae was counted in 2 h, 5 h, 18 h, 28 h,and 48 h after B. laterosporus introduction (Table 2). BothLC50 and LT50 were estimated for each repeat.

As is shown in Table 2, bioincapsulation of thespore�crystal suspension into the T. pyriformis cellssignificantly increased the larvicidal activity comparedto nonincapsulated specimens (48 h after the contactwith the larvae, the LD50 values were 0.012 and 0.040 μgprotein/ml respectively; see Table 2, the columns are interms of protein). Similar increase in larvicidal activitywas observed for bioincabsulated purified crystals incomparison with nonincapsulated specimens (LD50 was0.003 and 0.022 μg protein/ml, respectively).

Neither separate nor incapsulated purified sporesof LAT006 strain produced larvicidal effect onA. stephensi larvae.

Similar experiments were carried out with E. mosh�kovskii cells. Bioincapsulation mixture (1 ml) of the

spore�crystal mass of B. laterosporus contained 1–3 ×106 LAT006 strain spores (1–1.2 μg of total protein)and 1.0–1.3 × 104 of E. moshkovskii cells. Bioincapsu�lation mixture (1 ml) of purified spores of B. lat�erosporus contained 1–3 × 107 spores and 1.0–1.3 ×104 cells. Bioincapsulation mixture (1 ml) of purifiedLAT006 crystals contained 0.9–1 μg of total proteinand 1.0–1.3 × 104 cells. The incubation of bioincapsu�lation mixtures of spore�crystal mass, as well as puri�fied spores and LAT006 crystals with amoeba culture,was performed as described above for T. pyriformis(Table 3).

Table 3 shows that bioincapsulation of spore�crys�tal suspension and purified crystals into the E. moshk�ovskii cells increased their larvicidal activity againstthe mosquito larvae.

Purified spores of the LAT006 strain did not showlarvicidal activity with respect to A. stephensi neitherprior nor after incapsulation into T. pyriformis orE. moshkovskii (Tables 2, 3).

The LT50 after incapsulation of B. laterosporus bythe amoeba E. moshkovskii and the infusorian T. pyri�formis was decreased 2.5–3.3�fold (Table. 4).

Table 3. Effect of different LAT006 fractions incapsulated into the E. moshkovskii cells on the mosquito larvae

Sample

LC50 represented as

The studied LAT006* sample dilution

Protein concentra�tion in the LAT006

sample, µg/ml

The studied LAT006* sample dilution

Protein concentra�tion in the LAT006

sample, µg/ml

24h 48h

SCS 2.6 × 10–2

(1.1 × 10–2–5.2 × 10–2)2.560 3.8 × 10–4

(2.5 × 10–4–5.0 × 10–40.045

SCS + Amoeba 6.3 × 10–3

(1.5 × 10–3–1.0 × 10–2)0.626 2.6 × 10–4

(1.6 × 10–4–3.9 × 10–40.026

Purified crystals 1.1 × 10–3

(8.5 × 10–5–1.8 × 10–40.130 2.9 × 10–4

(8.8 × 10–5–3.7 × 10–40.028

Purified crystals + Amoeba 1.8 × 10–4

(8.6 × 10–5–4.7 × 10–40.018 5.1 × 10–5

(1.6 × 10–5–2.4 × 10–50.005

Purified spores – – – –

Purified spores + Amoeba – – – –

* 95% significance values for the LC50 calculation using Probit software are shown in parentheses; dashes mean the absence of the larvalethality

Table 4. Period required for 50% lethality of A. stephensi larvae

Protozoa

LT50, h

spore�crystal suspension of LAT006 Purified crystals of LAT006

nonincapsulated incapsulated nonincapsulated incapsulated

T. pyriformis 18 5 18 5

E. moshkovkii 18 7 18 7

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The obtained data suggest that incapsulation ofB. laterosporus into infusorian and amoeba cells leadsnot only to rapid death of the larvae but also providespossibility to decrease the doses of entomopathogenicbacteria, which is likely due to accumulation of con�siderable amount of crystals and spores of ento�mopathogenic toxin within the infusorian cells (seethe figure).

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SC

DV

Spores and crystals (SC) of B. laterosporus in digestive vac�uole (DV) of T. pyriformis. Zoom × 1350