Detection of β-exotoxin synthesis in Bacillus thuringiensis using an easy bioassay with the nematode Caenorhabditis elegans.

Abstract

The insecticidal activity of Bacillus thuringiensis is owing to the action of Cry and Cyt proteins. In addition to the synthesis of insecticidal proteins, some strains are able to synthesize β-exotoxin, which is highly toxic to humans. In this regard, it is very important to have a simple method to detect β-exotoxin to avoid the commercial production of this type of strains. In this work, we developed a simple and fast method, using the nematode Caenorhabditis elegans to detect indirectly the synthesis of β-exotoxin by B. thuringiensis strain. Using this assay, we detected that ~60% of Mexican native strains (i.e. LBIT-471, 491, 492, 497, 507, 511, 515, 536 and 537) were toxic to the nematode (44-97% mortalities) and their β-exotoxin (βEx(+) ) production, including a positive control (NRD-12), was confirmed by HPLC. In addition, the negative controls (βEx(-) ) LBIT-436 (HD-1) and LBIT-438 and also the native strains LBIT-499, 500, 521, 522, 533 and 542, did not show a detrimental effect against nematodes larvae, neither the synthesis of β-exotoxin as determined by HPLC. Finally, we did not find a correlation between B. thuringiensis strains with similar plasmid patterns and the β-exotoxin production. In this work, we implemented a qualitative and fast bioassay using the nematode Caenorhabditis elegans to detect the production of β-exotoxin in different strains of Bacillus thuringiensis. We show that this assay is useful to detect β-exotoxin in B. thuringiensis with high reliability, helping to discriminate strains that could not be used as bioinsecticides because of their putative risk to humans. Data show that qualitative bioassay with nematodes is a potential alternative to fly larvae bioassays, and correlated with the determination of β-exotoxin by HPLC.