MATERIALS AND METHODS

Bacterial Strains and Culture Conditions
Two B. thuringiensis strains were used in this study. B. thuringiensis israelensis (977) was kindly supplied by Prof. Priest, FG, Heriot University, England). B. thuringiensis israelensis (NRRL HD-522) was kindly supplied by Prof. Nakamura, LK, United State Department of Agriculture. All experiments of this study have been carried during the year 2006. These strains were cultured in NB (nutrient broth) medium for 5 days at 30°C under continuous shaking at 200 rpm.

Preparation of Soluble Toxin Protein
Spore-crystal complex was suspended in 0.1 M Na₂CO₃ buffer (pH 10) (De Leon and Ibarra, 1995). The suspension was incubated for 3 h at 37°C under continuous shaking at 200 rpm, followed by centrifugation at 12000 rpm for 30 min. Supernatant was dialyzed against deionized water overnight at 4°C. Protein concentration was determined by measurement the absorbance at 280 nm and the protein analyzed using 10% SDS-polyacrylamide gel according to previous report (Laemmli, 1970).

Oncomelania Hupensis Snail Bioassay
The intermediate host of Schistosoma japonicum was collected from Xichang City of Sichuan Province in China. To detect possible infections by Schistosoma japonicum, snails were kept in 200 mL jars in tap water and under light for 3 h. The water was examined under light microscope for the existence of cercariae. The snails were allowed to adapt to laboratory conditions for 1-3 weeks.

The soluble toxin proteins were added to 20 mL dechlorinated tap water to a final concentration of about 20 ng mL^-1 and 0.9 μg mL^-1 for both tested strains, immersion of groups of 10 snails of uniform size in these concentrations for 24 h. And then, the snails were washed with and reimmersed in dechlorinated tap water to recover them for 24 h. Two replicates were tested for each strain. The mortality percentage calculated as the number of dead snails to the starting number. The sample was replaced by dechlorinated tap water in the control group.

RESULTS AND DISCUSSION

It was observed that the concentration of the soluble protein was an important factor in determining their toxicity against snails. Where the low toxin concentration of 20 ng mL^-1 for both strains (977) and (NRRL HD-522), resulted in 0% mortality after 48 h, but the high concentration of 0.9 μg mL^-1 resulted in 70% mortality after 24 h. SDS-PAGE polypeptide pattern of the soluble proteins investigated against snails, with a predominant protein bands at about 66, 53, 35 and 28 kDa for B. thuringiensis israelensis (977) and (NRRL HD-522), (Fig. 1).

In our previous studies, it has been shown that B. thuringiensis strain that has been selected for being toxic against Biomphalaria alexandrina snails (Salem, 2004).The spore and crystal suspensions of the same strain was highly toxic (80% mortality) for both non-infected field collected and laboratory-reared Biomphalaria alexandrina snails in Egypt. The same strain and other B. thuringiensis standard strains were preliminary bioassayed for toxicity against infected, non-infected Chinese Oncomelania snails and the emitted cercariae (Salem et al., 2006). The new isolate reacted positively to the universal primers specific for the entire coding regions of 7 cry genes including cry1, cry3 and cry4. Moreover, the results of SDS-PAGE displayed common protein bands at about 66, 43 and 35 kDa in the protein patterns of strains from different serovars as well as the new isolate in addition to the distinct protein bands for each strain.

In the present study, bioassays indicated that non-infected Chinese Oncomelania snails are susceptible for the soluble protein of the test strains at high concentration only.

The dose which cause 70% mortality in this study is greater than those seen with mosquitoes and this variation is presumably may be due to individual species sensitivity (Tyrell et al., 1979).

Fig. 1:	SDS-PAGE of alkali soluble protein of two B. thuringiensis israelensis strains: Lane M: Protein molecular weight marker; Lanes 1 and 2: Strains 977 and HD-522, respectively

The molluscicidal activity of alkali-soluble protein from B. thuringiensis israelensis strains may attributed to a 66 kDa protein alone or in combination with other obtained polypeptides. It has been reported that purified crystal proteins of Bacillus thuringiensis var. israelensis by FPLC on a Mono Q column yielded 130, 65, 28, 53, 30-35 and 25 kDa proteins and all the purified proteins killed Aedes aegypti larvae after citrate precipitation, but the 65 kDa protein was the most toxic. The precipitated mixture of 27 and 130 kDa proteins was almost as toxic as solubilized crystals. Moreover, the activated form (25 kDa) of the 27 kDa protein was generally cytotoxic with the lowest LCs0 values in vitro assays against a range of insect cell lines while they found the activated forms of the 130 and 65 kDa protoxins (53 and 30-35 kDa proteins, respectively) were much more specific than the 25 kDa protein in their action on dipteran cells reported that a 65 kDa protein responsible for mosquitocidal activity (Chilcott and Ellar, 1988). Other researchers separated three major protein components, 130, 65 and 26 kDa from solubilized Bacillus thuringiensis subsp. israelensis crystal by gel filtration and the results showed that bioassays of mixtures of the individual toxins towards mosquito larvae revealed a number of synergistic interactions which explained in part why the native crystal is considerably more toxic than any of the individual toxins (Park et al., 2001). The dipteran-specific insecticidal protein Cry4A is produced as a protoxin of 130 kDa in Bacillus thuringiensis subsp. israelensis. Yamagiwa et al. (1999) processed it into the two protease-resistant fragments of 20 and 45 kDa through the intramolecular cleavage of a 60 kDa intermediate in vitro and in vivo. The functional properties of the two fragments, GST (glutathione S-transferase) fusion proteins of the 60 kDa intermediate showed that neither the GST-20 kDa fusion protein (GST-20) nor the GST-45 kDa fusion protein (GST-45) was actively toxic against mosquito larvae of Culex pipiens, whereas the GST-60 kDa intermediate fusion protein (GST-60) exhibited significant toxicity.

Based on the data presented here, it could be concluded that the soluble proteins of the test strains have molluscicidal activity towards non-infected Chinese Oncomelania snails at high concentrations only and the molluscicidal activity of alkali-soluble protein from B. thuringiensis israelensis strains may attributed to a 66 kDa protein alone or in combination with other obtained polypeptides.

ACKNOWLEDGMENTS

This research was supported by grant from the National post-doctor Foundation of China (20080440780), the company of Combest of Guangzhou, the Natural Science Foundation of Shenzhen University (2005027, 4wxmA), Natural science youth foundation of Guangdong Province (04300440).