title: Isolation and characterization of native Bacillus thuringiensis strains from Saudi Arabia with enhanced larvicidal toxicity against the mosquito vector Anopheles gambiae (s.l.).
abstract: Worldwide, mosquito vectors are transmitting several etiological agents of important human diseases, including malaria, causing millions of deaths 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 (s.l.).
  
  
  
  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.
  
  
  
  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. LC50and LC90bioassays 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 (LC50range: 3.90-7.40 μg/ml) than the reference Bti-H14 strain (LC50= 13.33 μg/ml). Of these, 6 strains had cry and cyt gene profiles similar to Bti-H14 (cry4Aa, cry4Ba, cry10, cry11, cyt1Aa, cyt1Ab, cyt2Aa). The seventh 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 eight strain (Bt55) with LC50of 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.
  
  
  
  This study represents the first report of several Saudi indigenous Bt strains 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.
authors.affiliation: Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia.
authors.forename: Talaat A
authors.initials: TA
authors.lastname: El-Kersh
authors.affiliation: Department of Zoology, College of Science, King Saud University, Riyadh, Saudi Arabia.
authors.forename: Ashraf M
authors.initials: AM
authors.lastname: Ahmed
authors.affiliation: Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia.
authors.forename: Yazeed A
authors.initials: YA
authors.lastname: Al-Sheikh
authors.affiliation: Centre for Applied Entomology and Parasitology, School of Life Sciences, Keele University, Staffordshire, ST5 5BG, UK. f.tripet@keele.ac.uk.
authors.forename: Frédéric
authors.initials: F
authors.lastname: Tripet
authors.affiliation: Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Saud University, Riyadh, Saudi Arabia.
authors.forename: Mohamed S
authors.initials: MS
authors.lastname: Ibrahim
authors.affiliation: Department of Food Science & Nutrition, College of Food and Agricultural Sciences, King Saud University, Riyadh, Saudi Arabia.
authors.forename: Ali A M
authors.initials: AA
authors.lastname: Metwalli
mesh_terms: D000818:Animals; D000852:Anopheles / Q000187:drug effects*; D001413:Bacillus thuringiensis / Q000145:classification / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism / Q000494:pharmacology*; D004731:Endotoxins / Q000378:metabolism / Q000494:pharmacology*; D006460:Hemolysin Proteins / Q000378:metabolism / Q000494:pharmacology*; D006801:Humans; D007306:Insecticides / Q000378:metabolism / Q000494:pharmacology*; D007814:Larva / Q000187:drug effects*; D009032:Mosquito Control; D000072138:Mosquito Vectors / Q000187:drug effects*; D010802:Phylogeny; D012529:Saudi Arabia; D012988:Soil Microbiology
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis
keywords: 16S rRNA; Anopheles gambiae; Bacillus thuringiensis; Biochemical type; Isolation; Parasporal crystals; SDS-PAGE; cry and cyt genes