ABSTRACT
The bacteria Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus produce insecticidal toxins used to control mosquito larvae throughout the world. Unfortunately, there are few alternative insecticides with similar activity and environmental safety, which may limit the long-term success of these insecticides. Bacillus thuringiensis subsp. jegathesan is another bacterium with toxins that are active against mosquitoes and has potential for development as a commercial product. B. t. subsp. jegathesan would be ineffective if cross-resistance was detected or if treated mosquito populations evolved resistance. B. t. subsp. jegathesan was evaluated for its potential for selecting insecticide resistance in Culex quinquefasciatus Say. Susceptibility changes in mosquitoes selected with the wild-type strain were compared with susceptibility changes in mosquitoes selected with Cry11B, a component toxin of B. t. subsp. jegathesan. Resistance was detected in generation 18 in the Cry11B-selected colony, reached a maximum of 38-fold, and was present through generation 40. The B. t. subsp. jegathesan-selected colony evolved 13-fold resistance in generation 22, but resistance declined to 2.3-fold in generation 26 and remained low throughout the study. Cry11B-selected mosquitoes showed no significant resistance to the wild-type bacterium, whereas B. t. subsp. jegathesan-selected mosquitoes expressed significant resistance to Cry11B. Both colonies displayed cross-resistance to component toxins of B. t. subsp. israelensis, but they lacked cross-resistance to that wild-type strain. The patterns of resistance and cross-resistance in this study are consistent with the patterns previously observed in mosquitoes selected with B. t. subsp. israelensis and suggest that B. t. subsp. jegathesan might also be at low risk for resistance.
Subject(s)
Bacillus thuringiensis/pathogenicity , Bacterial Proteins/toxicity , Bacterial Toxins/toxicity , Culex , Animals , Insecticide Resistance , Pest Control, Biological/methods , Structure-Activity RelationshipABSTRACT
Two novel crystal protein genes from a highly mosquitocidal Bacillus thuringiensis serovar medellin strain were cloned and sequenced. The corresponding proteins, Cry29A and Cry30A, were nontoxic when tested individually against the mosquito species bioassayed (Aedes aegypti, Culex pipiens and Anopheles stephensi). However, Cry29A synergized the toxicity of Cry11Bb against Aedes aegypti by a four-fold factor.
Subject(s)
Bacillus thuringiensis , Bacterial Proteins/genetics , Bacterial Proteins/toxicity , Bacterial Toxins/genetics , Bacterial Toxins/toxicity , Endotoxins/genetics , Endotoxins/toxicity , Amino Acid Sequence , Animals , Bacillus thuringiensis/genetics , Bacillus thuringiensis/physiology , Bacillus thuringiensis Toxins , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Bacterial Toxins/chemistry , Bacterial Toxins/isolation & purification , Bacterial Toxins/metabolism , Blotting, Southern , Culicidae/drug effects , DNA, Bacterial/chemistry , DNA, Bacterial/isolation & purification , Electrophoresis, Polyacrylamide Gel , Endotoxins/chemistry , Endotoxins/isolation & purification , Hemolysin Proteins , Molecular Sequence Data , Pest Control, Biological , Polymerase Chain Reaction , Sequence Analysis, DNASubject(s)
Bacterial Proteins/genetics , Bacterial Proteins/pharmacology , Bacterial Toxins , Culex/drug effects , Culex/physiology , Endotoxins/genetics , Endotoxins/pharmacology , Gene Deletion , Animals , Bacillus thuringiensis/genetics , Bacillus thuringiensis/physiology , Bacillus thuringiensis Toxins , Culex/microbiology , Drug Resistance , Hemolysin Proteins , Larva/drug effects , Larva/microbiology , Larva/physiology , Pest Control, BiologicalABSTRACT
We cloned and sequenced a new cytolysin gene from Bacillus thuringiensis subsp. medellin. Three IS240-like insertion sequence elements and the previously cloned cyt1Ab and p21 genes were found in the vicinity of the cytolysin gene. The cytolysin gene encodes a protein 29.7 kDa in size that is 91.5% identical to Cyt2Ba from Bacillus thuringiensis subsp. israelensis and has been designated Cyt2Bc. Inclusions containing Cyt2Bc were purified from the crystal-negative strain SPL407 of B. thuringiensis. Cyt2Bc reacted weakly with antibodies directed against Cyt2Ba and was not recognized by an antiserum directed against the reference cytolysin Cyt1Aa. Cyt2Bc was hemolytic only upon activation with trypsin and had only one-third to one-fifth of the activity of Cyt2Ba, depending on the activation time. Cyt2Bc was also mosquitocidal against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus, including strains resistant to the Bacillus sphaericus binary toxin. Its toxicity was half of that of Cyt2Ba on all mosquito species except resistant C. quinquefasciatus.
Subject(s)
Bacillus thuringiensis/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/toxicity , Bacterial Toxins , Culicidae/drug effects , Endotoxins/genetics , Endotoxins/toxicity , Animals , Bacillus thuringiensis/genetics , Bacillus thuringiensis Toxins , Bacterial Proteins/metabolism , Cloning, Molecular , Endotoxins/metabolism , Hemolysin Proteins , Hemolysis , Molecular Sequence Data , Restriction Mapping , Sequence Analysis, DNAABSTRACT
Bacillus thuringiensis (Bt) subsp. medellin (Btmed) produces parasporal crystalline inclusions which are toxic to mosquito larvae. It has been shown that the inclusions of this bacterium contain mainly proteins of 94, 68 and 28-30 kDa. EcoRI partially digested total DNA of Btmed was cloned by using the Lambda Zap II cloning kit. Recombinant plaques were screened with a mouse policlonal antibody raised against the 94 kDa crystal protein of Btmed. One of the positive plaques was selected, and by in vitro excision, a recombinant pBluescript SK(-) was obtained. The gene encoding the 94 kDa toxin of Btmed DNA was cloned in a 4.4 kb DNA fragment. Btmed DNA was then subcloned as a EcoRI/EcoRI fragment into the shuttle vector pBU4 producing the recombinant plasmid pBTM3 and used to transform by electroporation Bt subsp. israelensis (Bti) crystal negative strain 4Q2-81. Toxicity to mosquito larvae was estimated by using first instar laboratory reared Aedes aegypti, and Culex quinquefasciatus larvae challenged with whole crystals. Toxicity results indicate that the purified inclusions from the recombinant Bti strain were toxic to all mosquito species tested although the toxicity was not as high as one produced by the crystal of the Btmed wild type strain. Poliacrylamide gel electrophoresis indicate that the inclusions produced by the recombinant strain Bti (pBTM3) were mainly composed of the 94 kDa protein of Btmed, as it was determined by Western blot.
