Isolation and molecular characterization of Bacillus thuringiensis strains native to Saudi Arabia with naturally improved larvicidal toxicity against the mosquito vector Anopheles gambiae s.l.
M., El-Kersh, T. A; Ahmed, A. M; Al-Sheikh, Tripet, T.; Ibrahim, M. S and Metwally, A, . 2017
Background
Worldwide, mosquito vectors are transmitting several etiological agents of important human diseases, including malaria, causing millions of death every year. In Saudi Arabia as elsewhere, vector-control is based mostly on chemical insecticides which may be toxic and cause environmental deprivation. Here, to support the development of bio-pesticide alternatives, a study was conducted to identify native Bacillus thuringiensis (Bt) isolates with improved toxicity against the malaria vector, Anopheles gambiae.
Methods
Sixty-eight Bt isolates were obtained from 300 soil and other samples collected from 16 sites across Saudi Arabia. Bt identification was based on morphological characteristics of colonies, shape of parasporal crystals and biochemical profiles. After characterization of their mosquitocidal activity, larvicidal strains were described through 16S ribosomal DNA gene sequencing, cry, cyt and chi genes PCR-amplification profiles, and SDS-PAGE protein analyses.
Results
Spherical Bt crystals were predominant amongst the 68 isolates (34%), while irregular, bi-pyramidal and spore-attached crystals were found in 32, 13, and 21% of strains respectively. LC50 and LC90 bioassays showed that 23/68 isolates were larvicidal, with distinct biochemical activity profiles compared to non-larvicidal Bt strains. Eight larvicidal strains showed larvicidal activity up to 3.4-fold higher (LC50 range: 3.90-7.40µg/ml) than the reference Bti-H14 strain (LC50= 13.33µg/ml). Of these, 6 strains had cry and cyt gene profiles similar to Bti-H14 (cry4Aa, cry4Ba, cry10, cry11, cyt1Aa, cyt1Ab, cyt2Aa). The 7th strain (Bt63) displaying the highest larvicidal activity (LC50= 3.90 µg/ml) missed the cry4Aa and cyt1Ab genes and had SDS-PAGE protein profiles and spore/crystal sizes distinct from Bti-H14. The 8th strain (Bt55) with LC50 of 4.11µg/ml had cry and cyt gene profiles similar to Bti-H14 but gave a chi gene PCR product size of 2027bp. No strains harbouring cry2, cry17+27, cry24+40, cry25, cry29, cry30, or cyt2Ba were detected.
Conclusion
This study represents the first report of several Saudi indigenous Bt strain with significantly higher larvicidal efficacy against An. gambiae than the reference Bti-H14 strain. The very high toxicity of the Bt63 strain, combined with distinct cry and cyt genes and SDS-PAGE-protein profiles makes it a promising candidate for future applications in mosquito bio-control.
