Monitoring resistance to Bacillus thuringiensis subsp. israelensis in the field by performing bioassays with each Cry toxin separately

Bacillus thuringiensis subsp. israelensis (Bti) is increasingly used worldwide for mosquito control and is the only larvicide used in the French Rhône-Alpes region since decades. The artificial selection of mosquitoes with field-persistent Bti collected in breeding sites from this region led to a moderate level of resistance to Bti, but to relatively high levels of resistance to individual Bti Cry toxins. Based on this observation, we developed a bioassay procedure using each Bti Cry toxin separately to detect cryptic Bti-resistance evolving in field mosquito populations. Although no resistance to Bti was detected in none of the three mosquito species tested (Aedes rusticus, Aedes sticticus and Aedes vexans), an increased tolerance to Cry4Aa (3.5-fold) and Cry11Aa toxins (8-fold) was found in one Ae. sticticus population compared to other populations of the same species, suggesting that resistance to Bti may be arising in this population. This study confirms previous works showing a lack of Bti resistance in field mosquito populations treated for decades with this bioinsecticide. It also provides a first panorama of their susceptibility status to individual Bti Cry toxins. In combination with bioassays with Bti, bioassays with separate Cry toxins allow a more sensitive monitoring of Bti-resistance in the field.

Optimization of medium composition for the production of mosquitocidal toxins from Bacillus thuringiensis subsp. israelensis.

Optimization of chicken feather (CF) based culture medium for the production of Bacillus thuringiensis subsp. israelensis (Bti) biomass in combination with the agro industrial by-product (coconut cake, CC) and manganese chloride (MnCl2) has been evaluated. The biomass yield of Bti spore/crystal toxin was highest (12.06 g/L) from the test medium (CF+CC+MnCl2) compared to the reference medium (Luria Bertani, LB). Toxicity assay with Bti produced from the test medium against mosquito vectors (Culex quinquefasciatus, Anopheles stephensi and Aedes aegypti) was also satisfactory and results were comparable with bacteria produced from LB. The results suggest that Bti can be produced to the maximum extent possible as a potential mosquitocidal activity as suggested by the test medium (CF+CC+MnCl2).

Cloning and Expression of the CryIVD Gene of Bacillus thuringiensis subsp. israelensis and its Mosquito Larvicidal Activity / 中国寄生虫学与寄生虫病杂志

Objective To clone and express Bacillus thuringiensis subsp. isrealensis(B.t.i.) crystal protein CryIVD gene and determine its mosquito larvicidal activity. Methods The gene encoding CryIVD (2.0 kb or so) was amplified by PCR, the amplified fragment was inserted into E.coli plasmid pUC18 to construct the recombinant cloning and expression vector pUC18 CryIVD, which was named pUC18 1. The ligation was transformed into competent E.coli DH 5? and the recombinant vector pUC18 1 was confirmed by restriction enzyme digestion and DNA sequencing. After being inducted by IPTG, the expression of CryIVD gene in positive clone was detected by SDS PAGE and the mosquito larvicidal activity of CryIVD was also determined by standard bioassay. Results The results showed that the CryIVD gene was successfully cloned and expressed in E.coli DH 5? . Mosquito larvicidal activity of engineered E.coli (LC 50 ) to Cx.pipiens pallens and Ae.albopictus Ⅱ-Ⅲ instar larvae was 2.38?10 6 cells/ml and 1.6?10 7cells/ml respectively. Conclusion The CryIVD gene was successfully cloned and expressed, and a high mosquito larvicidal activity was observed.