Applied Microbiologyand Biotechnology

Eur J Appl Microbiol Biotechnol (1983) 18:168-173

9 Springer-Verlag 1983

Growth, Sporulation and Larvicidal Activity of Bacillus sphaericus A. Kalfon, I. Larget-Thi6ry, J.-F. Charles, and H. de Barjac Unit6 de Lutte Biologique contre les Insectes II, Institut Pasteur, 75724 Paris Cedex 15, France Summary. A new medium (MBS) for optimal sporulation of Bacillus sphaericus was defined. With the two main mosquito pathogenic strains grown in this medium, 1593-4 and 2297, highest cell and spore yields were obtained, concomitantly with an highest larvicidal activity against Culexpipiens. Study of both strains asporulated mutants showed a decrease in larvicidal power. After plasmid curing treatments, toxicity of strain 1593-4 did not decrease, neither toxic parasporal inclusion bodies of strain 2297 disappear.

Myers and Yousten 1978; Singer 1981). This led us to study sporulation under different culture conditions, in order to achieve both high growth levels and high spore yields and to thereby increase larvicidal activity. After selecting an optimal, we used it to compare final whole cultures of B. sphaericus wild type strains with those of asporulated mutants and also with final whole cultures of B. thuringiensis var.

israelensis. A n o t h e r point considered here involves the function of B. sphaericus plasmids (B. thuringiensis plasmids are known to code for the crystal toxin). We approached this question by trying to cure our strains in order to obtain non-toxic or inclusion free clones.

Introduction

Bacillus sphaericus is a spore-forming bacterium which is very commonly found in soil. This species contains a few strains which are toxic for mosquito larvae (Kellen et al. 1965, Singer 1974); larvicidal activity of B. sphaericus varies according to the different strains. For example, the larvicidal power of strain SSII-1, appears not to be related to sporulation (Myers and Yousten 1978), while in another strain, 1593-4, toxicity increases with sporulation (Myers et al. 1979, Bourgouin and de Barjac 1980). A m o n g the strains toxic for Culex sp. larvae, we selected no. 1593-4 and no. 2297 for their particularly high larvicidal activity. F u r t h e r m o r e strain 2297 can be characterized by its parasporal inclusions which are readily noticeable with either phase contrast or electron microscopy (Wickremesinghe and Mendis 1980, Davidson and Myers 1981, Yousten and Davidson 1982) and which have been shown to be toxic (de Barjac and Charles 1983). B. sphaericus is known for its limited sporulation in most liquid culture media (Singer et al. 1966; Offprint requests to: A. Kalfon

Materials

and Methods

Bacterial Strains. B. sphaericus and B. thuringiensis israelensis strains have been sent by the intermediary of the WHO and are now part of our Laboratory Collection of Entomopathogenic Bacillus, which serves as an International Reference Collection. All have been isolated from mosquito larvae in natural breeding sites: Culexfatigans in Indonesia by Singer 1974 for B. sphaericus no. 1593-4, Culex quinquefasciatus in Sri Lanka 1979 by Wickremesinghe for B. sphaericus no. 2297 (= MR4) and Culex sp. in Central Negev Desert, Israel, 1977, by Goldberg and Margalit, for B. thuringiensis israelensis no. 1884. Media. A mineral solution is used as a basis, (gram per liter): KHzPO4 6.8, MgSO4 7 H20 0.3, MnSO4 0.02, Fe~.(SO4)3 0.02, ZnSO4 7 H~O 0.02, CaC12 0.2, pH adjusted to 7.2. Different compounds were added to this solution (gram per liter): A = Tryptose soy broth (Difco) 10 - B = Tryptose (Difco) 10 + Yeast extract (Difco) 2 - C = Casein hydrolysate 7 + Glucose 3 - D = Proteose peptone 3 (Difco) 10 + Yeast extract 2 and E = Nutrient broth (Difco) 8. All these media have been compared to the following ones (gram per liter): - Usual = K H 2 P O 4 6.8, MgSO4 7 H20 0.123, MnSO4 4 H20 0.002, ZnSO4 7 H/O 0.014, Fez(SO4)3 0.02, CaCI2 4H20 0.183 - pH 7.2. - Poly = Peptone 5, yeast extract 10, meat extract 5, NaC1 3 - pH 7 to 7.2 (with or without glucose 10).

A. Kalfon et al.: Growth, Sporulation and Larvicidal Activity of Bacillus sphaericus

Bioassays were m a d e in plastic cups containing 150 ml of various dilutions from 48 h whole cultures. O n e h u n d r e d young L4 Culexpipiens larvae were distributed in four cups per dilution. Five dilutions per culture were tested, in order to determine the LDs0 and LDg0 on log-probit paper. Mortality was recorded at 24 and 48 h.

10.

Results and Discussion

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Sporulation Media

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1. Growth curves of B. sphaericus 2297 in MBS or Poly m e d i u m (Zeiss apparatus) Fig,

P e p t o n e 5, meat extract 1, yeast extract 5, NaC1 5, p H 7.4 or 9. In addition, three other formula have been tested (see Table 1). Cultures were incubated at 30 ~ C with shaking for 24 or 48 h. Sporulation was examined with phase contrast microscopy. Optical density was m e a s u r e d in a C o l e m a n or a Zeiss apparatus at 650 nm. Total viable cell counts were m a d e by plating dilutions on nutrient agar in Petri dishes. Spore counts were m a d e after heating the cultures 15 min at 80 ~ C. For strain 2297, growth curves have been established in two media, MBS and Poly. -

169

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Many strains of B. sphaericus have been tested against mosquito larvae in our laboratory. Up until now, cultures were grown in Poly medium, which gives a high growth level but a poor sporulation. Results obtained with the other media are recorded in Table 1. From these results, we selected the medium "B" or "MBS" (medium B. sphaericus) which is made with the basic mineral solution supplemented by 1% tryptose and 0.2% yeast extract. This medium gives the best combination of growth and spore yield for the two B. sphaericus strains tested. Comparing cultures of strain 2297 grown either in MBS or in Poly medium shows that for about the same growth rate, a higher spore yield was obtained with MBS (Fig. 1). In this medium, sporulation began at about the 16th or 18th h of incubation and reached about 60% at 24 h.

Table

1. Definition of an optimal sporulation m e d i u m for Bacillus

sphaericus Medium

Strain 2297

Strain 1593-4

Sporulation ~

O,D. b

Sporulation a

O.D.

s

0.75 0.67 0.56

e

0.78 0.8 0.23

e ++ + + ++ s

0.92 0.85 0.4 0.44 0.72 0.35 0.27 0.68

e ++ + + ++ ~

0.9 0.95 0.21 0.75 0.34 0.28 0.30 -

+ +

0.45 -

++ ++

0.58 0.74

Asporogenous Mutants. Mutagenesis was achieved on spore suspensions by ethyl-methyl sulfonate 0.2 volume, 1 h at 30 ~ C, according to the procedure described by Lecadet et al. (1974). Strains thus obtained which did not sporulate after sub-culturing, were considered asporogenous mutants. These strains were stored at - 7 0 ~ in 18% a q u e o u s glycerol.

Plasmid Curing. In order to look for a relationship between plasmids and larvicidal activity found in strains 1593 and 2297 (Davidson et al. i982), we tried to obtain p l a s m i d 4 r e e strains by curing treatment. W e used different m e t h o d s (Okada et al. 1981): culture at 4 1 ~ culture with 1% SDS or 10gg/ml ethidium bromide. Dilutions of these cultures were then spread on nutrient agar in Petri dishes. Two h u n d r e d isolated colonies of strain 2297 were checked by phase contrast microscopy for the presence of parasporal inclusions. O n e h u n d r e d isolated colonies of strain 1593-4 were grown for 48 h in MBS m e d i u m , examined for sporulation and assayed against Culex larvae, to find clones where the toxicity had been lost. Bioassays. Beside cell and spore counts and optical density m e a s u r e m e n t s , the larvicidal activity of the strains was determined.

Poly Poly + glucose 1% Usual (without glucose) A B = MBS C C + malate 1% D t F p H 7.2 F pH 9 Yeast extract 0,5% + malate 0.5% Yeast extract 1% Casein hydrolysate 1% + yeast extract 0.2%

a e = poor sporulation around 2% + = 1 0 - 6 0 % sporulation + + = > 60% b M e a s u r e d in C o l e m a n apparatus

A. Kalfon et al.: Growth, Sporulation and Larvicidal Activity of Bacillus sphaericus

170

Rather large characteristic inclusions appeared simultaneously. These inclusions were even larger when cells were grown on solid MBS medium (Fig. 2). When strain 2297 was grown in Poly medium, the growth rate was slightly higher than in MBS but spore yield was lower. Cell counts and larvicidal activities on Culex pipiens of B. sphaericus strains 1593-4 and 2297 and of B. thuringiensis israelensis strain 1884, when grown on various media for 48 h are recorded in Table 2 and Fig. 3 and 4. LCs0 and LC90 were determined both for dilutions of final whole cultures and in terms of CFU or spores/ml. Growth of both B. sphaericus strains was similar

in both media, but the sporulation rate is much higher in MBS than in Poly medium (Table 2). This can be related to the observation that both strains developed the highest toxicity in MBS medium, as shown by the LCs0 which is 10 or 100 times lower than that obtained with Poly cultures. Likewise, both strains are most toxic when grown in MBS medium. In MBS medium, strain 2297 was more toxic than strain 1593-4, since at 24 h it was lethal at dilutions in the range of 10 .6 , 10 .7 (Table 2). This could be the result of either a higher sporulation rate, or a difference in the mode of action of the toxins of the different strains, as suggested by the slopes of regression lines. Indeed, in either medium, strain

Fig. 2. Electronic micrography of strain 2297 in the sporulation stage

Table 2. Cell counts and larvicidal activity of B. sphaericus and B. Thuringiensis israelensis grown in different media Medium

B. sphaericus

B. sphaericus

B. thuringiensis

Strain 2297

Strain 1593-4

Strain 1884

MBS

CFU/mP Spores/ml

LCso (24 h)b: dil. CFU/ml

Spores/ml

1.36 x 109 8.8 x 108 (65%)

dil.

8.6 x 108 9 x 106 (1%)

MBS

Poly

8 x 10 ~ 4.5 x 108 (56%)

Spores/ml

8.6 x 10 -~ 1.16 x 102 75.6

LC90 (24 h) dil FWC (48 h)

4.75 x 10 .6 7,5 • 10 -7

x 10 -6 • 103

1.24 • 109 8.7 x 105 (0.07%)

Usual + glucose

1.1 x 109 7.65 x 108 (70~

1.4 x 109 7.5 x 108 (54 ~ )

3.3 x 108 1.74 x 108 (52%)

2.9 3.1 2.2

x 10 -6 x 103 x 103

1.15 x 10 -6 1.6 • 103 8.6 x 102

1,41 x 10 .6 4.65 x 102 2.45 • 102

2.1 2.3

x 10 -6 • 103

6.2 x 10 -7 2 x 10z 1 xl0 z 3.43 • 10 -6 1.6 x 10 -6

4.3 3.4

-

1.93x103

-

1.6 x 1 0 3

6.4 • 10 -7 8.9 x 10z 4.8 x l 0 a

2.46 x 10 -5

_ 4.5

1.17 x 10 -2

7.2 x 10 -6 6.42 • 10 -6

3.3 x 10 6 2 • 10 -6

a CFU: Colony forming unit b LCs0 and LC90: after 24 h or 48 h exposure of Culex pipiens larvae

x 10 -5 x 104

MBS +

6.1 x 10 -6 5.2 X 1 0 3

• 10 -5

3.2 3.9

glucose

MBS

9 x 10 -7 1.2 • 103 1.92 x 102

LCso (48 h): CFU/ml

Poly

A. Kalfon et al.: Growth, Sporulation and Larvicidal Activity of Bacillus sphaericus

t~

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Fig. 3. Larvicidal activity against Culex pipiens of B. sphaericus strains grown in MBS or Poly medium

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Fig. 4. Larvicidal activity on Culex pipiens of B. thuringiensis israelensis grown in different media 107

2297 gives slightly more vertical slopes (Fig. 3). The presence in strain 2297 of characteristic inclusions supports this hypothesis. For strain 1593-4, the slope obtained from Poly culture was more horizontal than the slope from MBS culture: this could imply the interference of different toxic factors issuing from vegetative cells in Poly medium and from spores in MBS medium. However, interpretation is difficult because, after 48h of incubation in Poly medium, cells appeared to be at look like a presporulation stage, with swollen sporangia but no individualized forespores. Bacillus thuringiensis israelensis is usually grown in medium containing glucose, where strain 1884 gives a good cell yield and a high level of sporulation. In MBS medium, with or without glucose, its growth rate was a bit higher for about the same rate of sporulation (Table 2). This suggests that MBS medium may also be valuable to increase sporulation for B. thuringiensis as well as for other Bacillus species. The toxicity of B. thuringiensis israelensis is directly related to crystals and not to spores (Larget and de Barjac 1981). For the usual medium, we

m bs + g l u c o s e

10-6

105

log(dilution)

obtained higher toxicity per spore; this could be due either to more crystals or bigger ones.

Plasmid-Curing Treatments Since in B. thuringiensis plasmids are involved in both toxicity and inclusion formation (Gonzalez et al. 1981, Klier and Rapoport 1982), we first attempted to detect such an involvement in the two strains of

B. sphaericus. In screening by bioassays on Culex pipiens larvae more than 100 sub-cultures of B. sphaericus strain 1593-4, that had been subjected to curing treatment, we never found a significant decrease in toxicity level. Microscopic examination of 200 colonies of strain 2297 after various curing procedures invariably revealed the presence of parasporal inclusions. In some colonies, inclusions were initially absent, but they re-appeared when the same colony was sub-cultured. The plasmid pattern of the sub-cultures after the curing treatment was not determined, due to our failure to get non toxic mutant after curing treatment,

A. Kalfon et al.: Growth, Sporulation and Larvicidal Activity of Bacillus sphaericus

172

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Fig. 5. Larvicidal activity at 48 h, on Culex

pipiens, of asporogenous B. sphaericus strains I 0 _'6

10 - 5'

10 - 4'

log(dilution)

All this fails to demonstrate in B. sphaericus a relationship between plasmids and toxins or inclusions.

Asporogenous Mutants

Fig. 6. Electronic micrography of the asporogenous mutant 2297-7 (24-h-old MBS culture)

as happens in B. thuringiensis. The temporary loss of the parasporal inclusions after curing treatment in strain 2297, might be explained as physiological stress of the bacteria after the drastical curing treatment, or due to plasmid dilution. Further study of isolate 2297-7, which was reported by Davidson et al. (1982) to have lost a large plasmid and to produce fewer inclusion bodies, showed that this strain was as toxic as the wild type strain and contained inclusion bodies. By using the technique of Klier et al. (1982), we found no plasmids in strain 1593-4 and only two small ones in strain 2297.

Four asporogenous mutant strains from B. sphaericus 1593-4 and two from strain 2297 were bioassayed on Culex pipiens larvae, after 24 h of incubation in MBS medium. Results are given in Fig. 5. Asporogenous mutants had different levels of toxicity. The two mutants of strain 2297 (aspo 7 and aspo 16) developed real larvicidal activity, but this was 10 times lower than that in the wild type. The LCs0 of these strains were 0.27 x 10.4 and 0.85 • 10 -4, respectively whereas the wild type strain 2297 had an LCs0 or 0.24 x 10-s. This could suggest either that another toxin is present in vegetative cells or that these mutants develop late, such that the derepression of sporulation affords concomitantly some expression of toxicity. The observation by electron microscopy of the asporogenous mutant 7 favors the second hypothesis, since in the sporangium the beginning of spore individualization and the presence of a small characteristic inclusion (Fig. 6) were observed. Three of four asporogenous mutants of strain 1593-4 were not toxic for Culex pipiens larvae, producing in 48 h a maximum mortality of about 10% at dilutions 10-3 and 10 -4. One of these mutants, aspo 7, was found by electron microscopy to have completely asporogenous cells (Fig. 7). On the other hand, traces of an abortive sporulation process were detected in another 1593-4 mutant, aspo 12, which

A. Kalfon et al.: Growth, Sporulation and Larvicidal Activity of Bacillus sphaericus

173

ability is g o o d advantage and could p r o m o t e its use, especially in such cases as polluted waters.

Acknowledgements. This investigation received support from the Vector Biology and Control Component of the UNDP/World Bandk/World Health Organization Special Programme for Research and Training in Tropical Disease.

References

Fig. 7. Electronic micrography of the asporogenous mutant 1593-4aspo7 (24-h-old MBS culture)

was still s o m e w h a t toxic against m o s q u i t o larvae. This m u t a n t had an LCs0 of 0.53 x 10 -5, which is close to that of the wild type strain (0.45 x 10-5), but an LC90 10 times lower. In fact the slope of its regression line is nearly horizontal (Fig. 5). In this case, then, as for strain 2297, a relationship appears to exist b e t w e e n toxicity and the sporulation process.

Conclusion T h e potential of Bacillus sphaericus as a biocontrol agent for m o s q u i t o larvae is shown to be greatly increased by culture in a p p r o p r i a t e conditions. T h e M B S m e d i u m used in this study induces a higher spores yield for a b o u t the same growth and a higher larvicidal activity on Culexpipiens than Poly m e d i u m . Toxicity appears both quicker and higher with strain 2297 which thus looks n o w like o n e of the m o s t promising B. sphaericus strains. In this strain, toxic crystal-like inclusions are f o r m e d during sporulation. S o m e relation b e t w e e n sporulation and toxicity seems also to exist in the o t h e r strain studied, no. 1593. O n the o t h e r hand, preliminary results of plasmid-curing t r e a t m e n t s on both B. sphaericus strains did not point to a relationship b e t w e e n plasmids and toxicity. T h e larvicidal activity of B. sphaericus strain 2297 in M B S m e d i u m , expressed as dilutions of whole culture, was a b o u t the same level as that of B. thuringiensis israelensis in usual m e d i u m . This opens the possibility of large scale application of B. sphaericus 2297 for v e c t o r control, and emphasizes the i m p o r t a n c e of developing a p p r o p r i a t e sporulation m e d i a commercially. A limiting factor in the use o f B. sphaericus as a biocontrol agent is its n a r r o w s p e c t r u m of activity. It is effective against Culex and Anopheles larvae, but not against Aedes larvae. O n the side, its persistence

de Barjac H, Charles J-F (1983) Une nouvelle toxine active sur les moustiques, prdsente dans des inclusions cristallines produites par Bacillus sphaericus. CR Acad Sci Paris (s6rie III) 296:905-910 Bourgouin C, Barjac H de (1980) Evaluation du potentiel de Bacillus sphaericus comme larvicide anti-moustiques. WHO mimeogr, doc. WHO/VBC/80.792 Davidson EW, Myers P (1981) Parasporal inclusions in B. sphaericus. FEMS Microbiology Letts 10:261-265 Davidson EW, Spizizen J, Yousten AA (1982) Recent advances in the genetics of Bacillus sphaericus. In: Proceeding IlIrd international colloquium on invertebrate pathology. Brighton, Sussex, GB pp 14-17 Gonzalez JM, Dulmage HI, Carlton BC (1981) Correlation between specific plasmids and 6-endotoxin production in Bacillus thuringiensis. Plasmid 5:351-365 Kellen WR, Clark TB, Lindegren JE, Ho BC, RogoffMH, Singer S (1965) Bacillus sphaericus Neide as a pathogen of mosquitoes. J Invertebr Pathol 7:442-448 Klier A, Fargette F, Ribier J, Rapoport G (1982) Cloning and expression of the crystal protein genes from Bacillus thuringiensis strain berliner 1715. The EMBO Journal 1 : 791-799 Larget I (1980) Etude du pouvoir pathog~ne du Bacillus thuringiensis var. israelensis sur les larves de Culicidae (Dipt~res N6matoc~res). Mise au point d'un titrage biologique. Th~se de 3~me Cycle (Octobre), Universit6 Paris-Sud/Orsay, 148 pages Larget I, Barjac H de (1981) Sp6cificit6 et principe actif de Bacillus thuringiensis var. israelensis. Bull Soc Pathol Exot 74:216-227 Lecadet MM, Klier A, Ribier J (1974) Isolation and characterization of two asporogenous Rifampycin resistant mutant of B. thuringiensis. Biochimie 56:1471 Myers P, Yousten AA (1978) Toxin activity of Bacillus sphaericus SSII-1 for mosquito larvae. Infection and immunity. Am Soc Microbiol 19 : 1047-1053 Myers P, Yousten AA, Davidson EW (1978) Comparative studies of mosquito larval toxin of Bacillus sphaericus SSII-1 and 1593. Can J Microbiol 11:1227-1231 Okada H, Kinoshita S, Negoro S (1981) Nylon oligomer degradating enzymes and their control by a plasmid in Flavobacterium sp. K 172. In: Microbial utilization of renewable resources, vol 2, pp 191-199 Singer S (1974) Entomogenous bacilli against mosquito larvae. Dev Ind Microbiol 15:187-194 Tinelli R, Bourgouin C (1982) Larvicidal toxin from Bacillus sphaericus spores. FEBS Letts 142:155-158 Wickremesinghe RSB, Mendis CL (1980) Bacillussphaericus spore from Sri Lanka demonstrating rapid larvicidal activity on Culex quinquefasciatus. Mosquito News 40:387-389 Yousten AA, Davidson EW (1982) Ultrastructural analysis of spores and parasporal crystals formed by Bacillus sphaericus 2297. Appl Environ Microbiol 44:1449-1455 Received March 30, 1983