Related Experiment Videos
Isolation and Identification of novel toxins from a new mosquitocidal isolate from Malaysia, Bacillus thuringiensis
M D Kawalek1, S Benjamin, H L Lee
1Department of Entomology, University of California, Riverside 92521, USA.
Abstract:
A new mosquitocidal Bacillus thuringiensis subsp., jegathesan, has recently been isolated from Malaysia. Parasporal crystal inclusions were purified from this strain and bioassayed against fourth-instar larvae of Culex quinquefasciatus, Aedes aegypti, Aedes togoi, Aedes albopictus, Anopheles maculatus, and Mansonia uniformis. The 50% lethal concentration of crystal inclusions for each species was 0.34, 8.08, 0.34, 17.59, 3.91, and 120 ng/ml, respectively. These values show that parasporal inclusions from this new subspecies have mosquitocidal toxicity comparable to that of inclusions isolated from B. thuringiensis subsp. israelensis. Solubilized and chymotrypsin-activated parasporal inclusions possessed low-level hemolytic activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the crystals were composed of polypeptides of 77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa. Analysis by Western blotting (immunoblotting) with polyclonal antisera raised against toxins purified from B. thuringiensis subsp. israelensis reveals that proteins in parasporal inclusions of subsp. jegathesan are distinct, because little cross-reactivity was shown. Analysis of the plasmid content of B. thuringiensis subsp. jegathesan indicates that the genes for toxin production may be located on 105- to 120-kb plasmids. Cry- clones that have been cured of these plasmids are nontoxic. Southern blot analysis of plasmid and chromosomal DNA from subsp. jegathesan showed little or low homology to the genes coding for CryIVA, CryIVB, and CryIVD from B. thuringiensis subsp. israelensis.
Insights
A new Bacillus thuringiensis subspecies, jegathesan, isolated from Malaysia, demonstrates potent mosquitocidal activity against various mosquito species. Its parasporal crystal inclusions show toxicity comparable to B. thuringiensis subsp. israelensis, with distinct protein profiles.
Area of Science:
- Microbiology
- Molecular Biology
- Entomology
Background:
- Bacillus thuringiensis (Bt) is a soil bacterium known for producing insecticidal crystal proteins.
- Bt subspecies israelensis (Bti) is widely used for mosquito control.
- The search for novel Bt strains with improved or distinct mosquitocidal properties is ongoing.
Purpose of the Study:
- To characterize a newly isolated mosquitocidal Bacillus thuringiensis subsp. jegathesan from Malaysia.
- To evaluate the toxicity of its parasporal crystal inclusions against key mosquito vectors.
- To determine the protein composition and genetic basis of its insecticidal activity.
Main Methods:
- Purification and bioassay of parasporal crystal inclusions against larvae of Culex quinquefasciatus, Aedes aegypti, Aedes togoi, Aedes albopictus, Anopheles maculatus, and Mansonia uniformis.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting to analyze crystal proteins.
- Plasmid analysis, including curing experiments and Southern blot hybridization, to investigate toxin gene location.
Main Results:
- Parasporal inclusions of B. thuringiensis subsp. jegathesan exhibited significant mosquitocidal toxicity, with 50% lethal concentrations (LC50) ranging from 0.34 ng/ml to 120 ng/ml across tested species.
- The toxicity was comparable to B. thuringiensis subsp. israelensis.
- SDS-PAGE revealed crystal polypeptides of 77, 74, 72, 68, 55, 38, 35, 27, and 23 kDa. Western blot analysis indicated distinct proteins compared to Bti toxins.
- Toxin genes are likely located on 105- to 120-kb plasmids, as cry- clones cured of these plasmids were nontoxic.
Conclusions:
- Bacillus thuringiensis subsp. jegathesan represents a novel source of mosquitocidal toxins with potential applications in vector control.
- The distinct protein composition and genetic basis suggest a unique mechanism of action compared to existing Bti strains.
- Further research into these novel toxins could lead to new strategies for managing mosquito populations.