Bacterial Control of Mosquitoes & Black Flies, 1990 Biochemistry, Genetics & Applications of Bacillus thuringiensis israelensis and Bacillus sphaericus

Mosquitoes and black flies are a constant threat to health and comfort, yet the modern chemical pesticides used to control them have cre­ ated serious ecological problems. Populations of resistant mosquitoes and black flies have evolved, beneficial insects and natural predators have been destroyed, and environmental pollution has increased worldwide. Therefore, scientists have energetically sought new, environmentally safe technologies to combat mosquitoes and black flies and the diseases they carry. Among the most effective alternative means of controlling these pests are the highly spe­ cific microbial agents derived from Bacillus tburingiensis or Bacillus spbaericus. The microbial control of mosquitoes and black flies is a very important, rapidly developing area of science. Entomologists and microbiologists have already achieved spectacular successes using B. tburingiensis and B. spbaericus against these pests. Recent discoveries of new bacterial isolates specific to new hosts and recent genetic improvements in these isolates have created the potential for wide-scale use of these biological control agents. Efficient microbial control of mosquitoes and black flies can now be achieved, but a proper knowledge of factors relating to the safe and effective use of these biological control agents is necessary. The efficacy of B. tburingiensis and B. spbaericus is influenced by the inherent differential tol­ erance of the target mosquitoes or black flies, by the formulation technology and application of these agents, and by environmental factors, especially sun­ light and temperature.

1 Bacillus thuringiensis subsp. israelensis (B.t.i.).- 1 Discovery of Bacillus thuringiensis israelensis.- 1.1 Introduction.- 1.2 Geography, Climate, and Environmental Conditions.- 1.3 Detection and Isolation.- 2 Characterization and Prospective View of Bacillus thuringiensis israelensis.- 3 Parasporal Body of Bacillus thuringiensis israelensis: Structure, Protein Composition, and Toxicity.- 3.1 Introduction.- 3.2 Synthesis.- 3.3 Structure.- 3.4 Purification and Solubilization.- 3.5 Protein Composition.- 3.6 Toxicity.- 3.6.1 Intact or Solubilized Parasporal Body.- 3.6.2 The 27-kDa Protein.- 3.6.3 The 65-kDa Protein.- 3.6.4 The 128- and 135-kDa Proteins.- 3.6.5 Synergistic Interaction of Toxic Proteins.- 3.7 Mosquiticidal Parasporal Bodies of Other Subspecies of B. thuringiensis.- 3.8 Discussion.- 4 Mechanism of Action of Bacillus thuringiensis israelensis Parasporal Body.- 4.1 Introduction.- 4.2 Mechanism of Action.- 4.2.1 Receptors.- 4.2.2 Toxin Structure and Membrane Insertion.- 4.2.3 Colloid-Osmotic Lysis Theory.- 4.2.4 Toxin Oligomerization.- 4.3 Discussion.- 5 Genetics of Bacillus thuringiensis israelensis.- 5.1 Introduction.- 5.2 Genetic Exchange Systems.- 5.2.1 Transformation.- 5.2.2 Transduction.- 5.2.3 Plasmid Transfer.- 5.3 Plasmids and Crystal Toxin Production.- 5.3.1 Plasmids and Plasmid Curing Analysis.- 5.3.2 Location of the ?-endotoxin Gene.- 5.4 Cloning of the Crystal Toxin Gene(s).- 5.5 Genetics and Biochemistry of the Crystal Toxin.- 5.6 Conclusions.- 6 Cloning of Bacillus thuringiensis israelensis Mosquito Toxin Genes.- 6.1 Introduction.- 6.2 Early Confusion in Cloning B.t.i. Toxin Protein Genes.- 6.3 Current Picture of B.t.i. Toxin Protein Genes.- 6.4 Discussion.- 7 Transfer of the Bacillus thuringiensis israelensis Mosquiticidal Toxin Gene into Mosquito Larval Food Sources.- 7.1 Introduction.- 7.2 Assignment of Toxic Activity.- 7.3 Cloning of the Mosquito Toxin Gene.- 7.4 Mosquito and Black Fly Larval Food Sources.- 7.5 Introduction of the Mosquito Toxin Gene into Larval Food Sources.- 7.6 Safety Aspects.- 8 Potential for Improved Formulations of Bacillus thuringiensis israelensis through Standardization and Fermentation Development.- 8.1 Introduction.- 8.2 History.- 8.2.1 Background.- 8.2.2 Discovery and Early History.- 8.2.3 Standardizing and Measuring B.t. Products.- 8.2.3.1 Early Concepts.- 8.2.3.2 The Spore Count.- 8.3 Bioassays and the International Unit.- 8.3.1 Philosophical Differences between Bioassays of Chemical and Microbial Insecticides.- 8.3.2 Bioassays and the LC50.- 8.3.3 Choice of Insect Species for Bioassay.- 8.3.4 The International Unit.- 8.3.5 The de Barjac Protocol for B.t.i.: Its Design and Principles.- 8.3.5.1 Preparation of Stock Suspension of the Standard.- 8.3.5.2 Preparation of Suspension of the Test Samples.- 8.3.5.3 Specifications for Larvae Used in Assay.- 8.3.5.4 Reading the Assay.- 8.3.5.5 Evaluations of Assays and Their Reproducibility.- 8.4 Activity Ratios.- 8.4.1 Definition of Activity Ratios.- 8.4.2 Use and Significance of Activity Ratios.- 8.4.2.1 Tn/Hv Ratios of subspecies kurstaki.- 8.4.2.2 Cq/Aa Ratios of subspecies israelensis.- 8.4.2.3 Reproducibility of Activity Ratios in Fermentation Studies.- 8.5 Potential for Improvements in Production of the B.t.i. Toxin.- 8.5.1 Fermentation.- 8.5.1.1 Strain Selection.- 8.5.1.2 Aeration.- 8.5.1.3 Selection of Nutrients.- 8.5.2 Recovery.- 8.5.2.1 Spore-crystal Formulations.- 8.5.2.2 Viable Spore-free Formulations.- 8.6 Summary.- 8.6.1 Bioassays and the Production of B.t..- 8.6.2 Bioassays and the Future of B.t..- 9 Activity, Field Efficacy, and Use of Bacillus thuringiensis israelensis against Mosquitoes.- 9.1 Introduction.- 9.2 Laboratory Evaluation.- 9.2.1 Screening Procedures.- 9.2.2 Preliminary Screening.- 9.2.3 Species Specificity.- 9.2.4 Instar Susceptibility.- 9.2.5 Biotic and Abiotic Factors Influencing Activity.- 9.2.6 Delayed Effects.- 9.3 Field Evaluation and Efficacy Trials.- 9.3.1 Spectrum of Field Activity.- 9 3.1.1 Floodwater Mosquitoes.- 9.3.1.2 Anopheles Mosquitoes.- 9.3.1.3 Culex Mosquitoes.- 9.3.2 Persistence and Recycling.- 9.4 Impact on Nontarget Organisms.- 9.5 Microbial Larvicides in Integrated Control of Mosquitoes.- 10 Progress in the Biological Control of Black Flies with Bacillus thuringiensis israelensis, with Emphasis on Temperate Climates.- 10.1 Nature of the Black Fly Problem in Temperate Climates.- 10.2 Advent of B.t.i..- 10.3 Trends in Recent Research Efforts.- 10.4 Factors Affecting the Efficacy of B.t i. against Black Flies.- 10.4.1 Environmental Parameters.- 10.4.1.1 Discharge.- 10.4.1.2 Stream Profile.- 10.4.1.3 Turbidity.- 10.4.1.4 Pollutants.- 10.4.1.5 Water Temperature.- 10.4.1.6 pH.- 10.4.1.7 Degree of Vertical Mixing in the Water Column.- 10.4.1.8 Reduced Water Velocity Due to Negative Relief.- 10.4.1.9 Attachment of B.t.i. to Benthic Substrates/Sediments.- 10.4.1.10 Other Factors.- 10.4.2 Black Fly Parameters.- 10.4.2.1 Larval Age.- 10.4.2.2 Species.- 10.4.2.3 Feeding Behavior.- 10.4.3 Formulation Parameters.- 10.4.3.1 Particle Size.- 10.4.3.2 Powdered versus Liquid Formulations.- 10.4.3.3 Formulation Additives.- 10.4.4 Treatment Parameters.- 10.4.4.1 Concentration.- 10.4.4.2 Duration of Application.- 10.4.4.3 Preparation of Powdered Formulations.- 10.5 Lack of Correlation in Formulation Potency against Black Flies and Mosquitoes.- 10.6 Effect of B.t.i. on Black Fly Populations.- 10.7 Effect of B.t.i. on Nontarget Populations.- 10.7.1 Toxicity.- 10.7.2 Inducement of Drift.- 10.8 Treatment and Assessment Methodologies.- 10.8.1 Discharge Calculation.- 10.8.2 Treatment.- 10.8.3 Assessments of Black Fly Mortality.- 10.9 Product/Formulation Improvement.- 10.10 Area-control Programs.- 10.11 Possibility of Resistance.- 10.12 Research Priorities in Temperate Climates.- 10.13 Conclusions.- 11 Use of Bacillus thuringiensis israelensis for Onchocerciasis Control in West Africa.- 11.1 Introduction.- 11.2 The Search for Suitable B.t.i. Formulations.- 11.3 Operational Use of B.t.i. in the OCP.- 11.4 Discussion and Prospects.- 12 Mammalian Safety of Bacillus thuringiensis israelensis.- 12.1 Introduction.- 12.2 Source and Preparation of Cultures.- 12.3 Results and Discussion.- 12.3.1 Oral and Intraperitoneal Administration.- 12.3.2 Subcutaneous Injection.- 12.3.3 Aerosol Exposure.- 12.3.4 Intracerebral Injection.- 12.3.5 Clearance.- 12.3.6 Ocular Irritancy.- 12.4 Summary.- 12.5 Summary of Other Studies.- 2 Bacillus sphaericus.- 3 Introduction to the Study of Bacillus sphaericus as a Mosquito Control Agent.- 13.1 Introduction.- 13.2 History of Extant Strains.- 13.3 Future Strains.- 14 Classification of Bacillus sphaericus Strains and Comparative Toxicity to Mosquito Larvae.- 14.1 Introduction.- 14.2 Different Approaches to the Classification and Characterization of Toxic Strains.- 14.3 Comparative Toxicity of Various Serotypes.- 14.4 Distribution of the Larvicidal Strains and Activity Ratios.- 15 The Mosquito Larval Toxin of Bacillus sphaericus.- 15.1 Location of the Toxin in the Bacterial Cell.- 15.2 Biochemical Nature of the Toxin.- 15.3 Pathology in the Host.- 15.4 Host Range of the Toxin.- 15.5 Pathology of the Toxin in Cultured Cells.- 15.6 Comparison of the B. sphaericus Toxin with the B.t.i. Toxin.- 16 Genetics of Bacillus sphaericus.- 16.1 Introduction.- 16.2 Evidence of Genetic Heterogeneity within the Species B. sphaericus.- 16.3 Naturally Occurring Antibiotic Resistances in B. sphaericus.- 16.4 Auxotrophic Mutants of B. sphaericus.- 16.5 Transformation of B. sphaericus with Plasmid DNA.- 16.6 B sphaericus Mutants with Limited Restriction Endonuclease Activity.- 16.7 Evidence for the Existence of a Bsp 1593 Modification System.- 16.8 Conjugal Transfer of pAMßl in B. sphaericus 1593.- 16.9 Construction of Plasmid Vectors for Cloning in B sphaericus 1593.- 16.10 Cloning of B. sphaericus Larvicidal Toxin Genes.- 17 Local Production of Bacillus sphaericus.- 17.1 Introduction.- 17.2 The Need for Local Production ofB. sphaericus.- 17.3 Growth and Cultivating Conditions for B. sphaericus.- 17.4 Formulation of B. sphaericus.- 17.5 Economics of Local Production of B. sphaericus.- 17.6 Conclusions.- 18 Persistence and Formulation of Bacillus sphaericus.- 18.1 Introduction.- 18.2 Factors Affecting Residual Activity.- 18.2.1 Definitions.- 18.2.2 Water Quality, Depth, and Feeding Behavior.- 18.2.3 Solar Radiation.- 18.2.4 Species Susceptibility.- 18.2.5 Recycling.- 18.3 Formulation: Field Efficacy.- 18.3.1 Granules.- 18.3.2 Flowable Concentrates.- 18.3.3 Sustained-release Formulations.- 18.3.4 Storage Considerations.- 18.4 Recommendations for Future Formulation Research.- 19 Formulations and Persistence of Bacillus sphaericus in Culex quinquefasciatus Larval Sites in Tropical Africa.- 19.1 Introduction.- 19.2 Field Evaluation.- 19.2.1 Wettable Powder 1593.- 19.2.2 Flowable Concentrate 2362.- 19.3 Bacteriological Tests.- 19.4 Discussion.- 19.4.1 Persistence of B. sphaericus in the Field.- 19.4.2 Recycling of B sphaericus in the Field.- 19.5 Conclusions.- 19.6 Summary.- 20 Field Trials of Bacillus sphaericus for Mosquito Control.- 20.1 Introduction.- 20.2 General Information on Field Trials.- 20.3 Field Trials against Culex.- 20.4 Field Trials against Aedes, Anopheles, Mansonia, andPsorophora.- 20.5 Discussion.- 21 Mammalian Safety of Bacillus sphaericus.- 21.1 Introduction.- 21.2 Source and Preparation of Cultures.- 21.3 Results and Discussion.- 21.3.1 Subcutaneous and Intraperitoneal Injection.- 21.3.2 Intradermal Injection.- 21.3.3 Ocular Irritancy.- 21.3.4 Intraocular Injection.- 21.3.5 Ocular Persistence.- 21.3.6 Intracerebral Injection.- 21.4 Summary of Other Studies.- 21.5 Conclusion.- 3 The Future.- 22 The Future of Bacterial Control of Mosquito and Black Fly Larvae.- 22.1 Introduction.- 22.2 Perspective for the Next Decade.- 22.2.1 Supportive Factors.- 22.2.2 Counterfactors.- 22.3 Prospects for the Future.