Margaret C. Wirth

Recombinant bacteria for mosquito control

SUMMARY Bacterial insecticides have been used for the control of nuisance and vector mosquitoes for more than two decades. Nevertheless, due primarily to their high cost and often only moderate efficacy, these insecticides remain of limited use in tropical countries where mosquito-borne diseases are prevalent. Recently, however, recombinant DNA techniques have been used to improve bacterial insecticide efficacy by markedly increasing the synthesis of mosquitocidal proteins and by enabling new endotoxin combinations from different bacteria to be produced within single strains. These new strains combine mosquitocidal Cry and Cyt proteins of Bacillus thuringiensis with the binary toxin of Bacillus sphaericus, improving efficacy against Culex species by 10-fold and greatly reducing the potential for resistance through the presence of Cyt1A. Moreover, although intensive use of B. sphaericus against Culex populations in the field can result in high levels of resistance, most of this can be suppressed by combining this bacterial species with Cyt1A; the latter enables the binary toxin of this species to enter midgut epithelial cells via the microvillar membrane in the absence of a midgut receptor. The availability of these novel strains and newly discovered mosquitocidal proteins, such as the Mtx toxins of B. sphaericus, offers the potential for constructing a range of recombinant bacterial insecticides for more effective control of the mosquito vectors of filariasis, Dengue fever and malaria.

Cyt1A of Bacillus thuringiensis Delays Evolution of Resistance to Cry11A in the Mosquito Culex quinquefasciatus

ABSTRACT Insecticides based on Bacillus thuringiensis subsp. israelensis have been used for mosquito and blackfly control for more than 20 years, yet no resistance to this bacterium has been reported. Moreover, in contrast to B. thuringiensis subspecies toxic to coleopteran or lepidopteran larvae, only low levels of resistance to B. thuringiensis subsp. israelensis have been obtained in laboratory experiments where mosquito larvae were placed under heavy selection pressure for more than 30 generations. Selection of Culex quinquefasciatus with mutants of B. thuringiensis subsp. israelensis that contained different combinations of its Cry proteins and Cyt1Aa suggested that the latter protein delayed resistance. This hypothesis, however, has not been tested experimentally. Here we report experiments in which separate C. quinquefasciatus populations were selected for 20 generations to recombinant strains of B. thuringiensis that produced either Cyt1Aa, Cry11Aa, or a 1:3 mixture of these strains. At the end of selection, the resistance ratio was 1,237 in the Cry11Aa-selected population and 242 in the Cyt1Aa-selected population. The resistance ratio, however, was only 8 in the population selected with the 1:3 ratio of Cyt1Aa and Cry11Aa strains. When the resistant mosquito strain developed by selection to the Cyt1Aa-Cry11Aa combination was assayed against Cry11Aa after 48 generations, resistance to this protein was 9.3-fold. This indicates that in the presence of Cyt1Aa, resistance to Cry11Aa evolved, but at a much lower rate than when Cyt1Aa was absent. These results indicate that Cyt1Aa is the principal factor responsible for delaying the evolution and expression of resistance to mosquitocidal Cry proteins.

Cyt1A from Bacillus thuringiensis Synergizes Activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae)

ABSTRACT Bacillus sphaericus is a mosquitocidal bacterium recently developed as a commercial larvicide that is used worldwide to control pestiferous and vector mosquitoes. Whereas B. sphaericus is highly active against larvae of Culexand Anopheles mosquitoes, it is virtually nontoxic toAedes aegypti, an important vector species. In the present study, we evaluated the capacity of the cytolytic protein Cyt1A fromBacillus thuringiensis subsp. israelensis to enhance the toxicity of B. sphaericus toward A. aegypti. Various combinations of these two materials were evaluated, and all were highly toxic. A ratio of 10:1 of B. sphaericus to Cyt1A was 3,600-fold more toxic to A. aegypti than B. sphaericus alone. Statistical analysis showed this high activity was due to synergism between the Cyt1A toxin and B. sphaericus. These results suggest that Cyt1A could be useful in expanding the host range of B. sphaericus.

Laboratory selection for resistance to Bacillus sphaericus in Culex quinquefasciatus (Diptera: Culicidae) from California, USA.

Abstract A previously untreated field population ofCulex quinquefasciatusSay, collected near Bakersfield, CA, was subjected to intensive laboratory selection with the bacterial insecticideBacillus sphaericusNeide (strain 2362) at a level producing 95% mortality. Resistance rapidly appeared and resistance levels increased such that fourth instars of generation 12 were able to survive a concentration ofB. sphaericusthat was 7,000 times higher than the median lethal concentration (LC50) of the susceptible reference colony. Similar resistance levels were detected in first instars. Cross-resistance in the selected colony was detected towardB. sphaericusstrains 1593 and 2297, but little or no cross-resistance was observed towardB. sphaericusstrains IAB59 or ISPC5 (=WHO 2173). Cross-resistance also was not detected toward the bacterial insecticideBacillus thuringiensissubsp.israelensis,toward a recombinant strain expressing bothB. thuringiensissubsp.israelensisandB. sphaericus(strain 1593) toxins, toward individual or multiple toxins fromB. thuringiensissubsp.israelensis,or toward conventional synthetic insecticides. Genetic analysis revealed thatB. sphaericusresistance was inherited as a recessive trait and controlled by a single major locus. These data are discussed in relation to cases of field resistance toward this biopesticide in theCx. pipiens(L.) complex.

DEVELOPING RECOMBINANT BACTERIA FOR CONTROL OF MOSQUITO LARVAE

ABSTRACT Genetic engineering techniques have been used to significantly improve mosquito larvicides based on the bacteria Bacillus thuringiensis (Bt) subsp. israelensis (Bti) and Bacillus sphaericus (Bs). These new larvicides hold excellent promise for providing better and more cost-effective control of nuisance mosquitoes and vectors of important diseases, including the anopheline vectors of malaria and culicine vectors responsible for filariasis and viral encephalitides. The toxicity of Bti and Bs is due primarily to endotoxin proteins produced during sporulation. After ingestion by larvae, these are activated and destroy the larval stomach, quickly resulting in death. By cloning the genes encoding various endotoxins from Bt and Bs species, and engineering these for high levels of synthesis, we have been able to generate recombinant bacterial strains based on Bti that are more than 10 times as effective as the conventional strains of Bti or Bs that serve as the active ingredients of commercial bacterial larvicides currently used for mosquito control. The best of these recombinants contain all major Bti endotoxins, specifically, Cry4A, Cry4B, Cry11A, and Cyt1A, plus the binary (Bin) endotoxin of Bs, the principal mosquitocidal protein responsible for the activity of this species. The presence of Cyt1A in these recombinants, which synergizes Cry toxicity and delays resistance to these proteins and Bs Bin, should enable long term use of these recombinants with little if any development of resistance. In the field, these new recombinants should be particularly effective larvicides against most important vectors and nuisance species of the genus Culex, the malaria vectors Anopheles gambiae and An. arabiensis, and species of Aedes and Ochlerotatus sensitive to Bs.

Cyt1A from Bacillus thuringiensis Restores Toxicity of Bacillus sphaericus Against Resistant Culex quinquefasciatus (Diptera: Culicidae)

Abstract The 2362 strain of Bacillus sphaericus, which produces a binary toxin highly active against Culex mosquitoes, has been developed recently as a commercial larvicide. It is being used currently in operational mosquito control programs in several countries including Brazil, France, India, and the United States. Laboratory studies have shown that mosquitoes can develop resistance to B. sphaericus, and low levels of resistance have already been reported in field populations in Brazil, France, and India. To develop tools for resistance management, the Cyt1A protein of Bacillus thuringiensis subsp. israelensis De Barjac was evaluated for its ability to suppress resistance to B. sphaericus in a highly resistant population of Culex quinquefasciatus Say. A combination of B. sphaericus 2362 in a 10:1 ratio with a strain of B. thuringiensis subsp. israelensis that only produces Cyt1A reduced resistance by >30,000-fold. Resistance was suppressed completely when B. sphaericus was combined with purified Cyt1A crystals in a 10:1 ratio. Synergism was observed between the Cyt1A toxin and B. sphaericus against the resistant mosquito population and accounted for the marked reduction in resistance. However, no synergism was observed between the toxins against a nonresistant mosquito population. These results indicate that Cyt1A could be useful for managing resistance to B. sphaericus 2362 in Culex populations, and also provide additional evidence that Cyt1A may synergize toxicity by enhancing the binding to and insertion of toxins into the mosquito microvillar membrane.

Synergy between Toxins of Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus

Abstract Synergistic interactions among the multiple endotoxins of Bacillus thuringiensis subsp. israelensis de Barjac play an important role in its high toxicity to mosquito larvae and the absence of insecticide resistance in populations treated with this bacterium. A lack of toxin complexity and synergism are the apparent causes of resistance to Bacillus sphaericus Neide in particular Culex field populations. To identify endotoxin combinations of the two Bacillus species that might improve insecticidal activity and manage mosquito resistance to B. sphaericus, we tested their toxins alone and in combination. Most combinations of B. sphaericus and B. t. subsp. israelensis toxins were synergistic and enhanced toxicity relative to B. sphaericus, particularly against Culex quinquefasciatus Say larvae resistant to B. sphaericus and Aedes aegypti (L.), a species poorly susceptible to B. sphaericus. Toxicity also improved against susceptible Cx. quinquefasciatus. For example, when the Cyt1Aa toxin from B. t. subsp. israelensis was added to Bin and Cry toxins, or when native B. t. subsp. israelensis was combined with B. sphaericus, synergism values as high as 883-fold were observed and combinations were 4–59,000-fold more active than B. sphaericus. These data, and previous studies using cytolytic toxins, validate proposed strategies for improving bacterial larvicides by combining B. sphaericus with B. t. subsp. israelensis or by engineering recombinant bacteria that express endotoxins from both strains. These combinations increase both endotoxin complexity and synergistic interactions and thereby enhance activity and help avoid insecticide resistance.

Mtx Toxins Synergize Bacillus sphaericus and Cry11Aa against Susceptible and Insecticide-Resistant Culex quinquefasciatus Larvae

ABSTRACT Two mosquitocidal toxins (Mtx) of Bacillus sphaericus, which are produced during vegetative growth, were investigated for their potential to increase toxicity and reduce the expression of insecticide resistance through their interactions with other mosquitocidal proteins. Mtx-1 and Mtx-2 were fused with glutathione S-transferase and produced in Escherichia coli, after which lyophilized powders of these fusions were assayed against Culex quinquefasciatus larvae. Both Mtx proteins showed a high level of activity against susceptible C. quinquefasciatus mosquitoes, with 50% lethal concentrations (LC50) of Mtx-1 and Mtx-2 of 0.246 and 4.13 μg/ml, respectively. The LC50s were 0.406 to 0.430 μg/ml when Mtx-1 or Mtx-2 was mixed with B. sphaericus, and synergy improved activity and reduced resistance levels. When the proteins were combined with a recombinant Bacillus thuringiensis strain that produces Cry11Aa, the mixtures were highly active against Cry11A-resistant larvae and resistance was also reduced. The mixture of two Mtx toxins and B. sphaericus was 10 times more active against susceptible mosquitoes than B. sphaericus alone, demonstrating the influence of relatively low concentrations of these toxins. These results show that, similar to Cyt toxins from B. thuringiensis subsp. israelensis, Mtx toxins can increase the toxicity of other mosquitocidal proteins and may be useful for both increasing the activity of commercial bacterial larvicides and managing potential resistance to these substances among mosquito populations.

Cyt1Ab1 and Cyt2Ba1 from Bacillus thuringiensis subsp. medellin and B. thuringiensis subsp. israelensis Synergize Bacillus sphaericus against Aedes aegypti and Resistant Culex quinquefasciatus (Diptera: Culicidae)

ABSTRACT The interaction of two cytolytic toxins, Cyt1Ab fromBacillus thuringiensis subsp. medellinand Cyt2Ba from Bacillus thuringiensis subsp.israelensis, with Bacillus sphaericus was evaluated against susceptible and resistant Culex quinquefasciatus and the nonsensitive species Aedes aegypti. Mixtures of B. sphaericus with either cytolytic toxin were synergistic, and B. sphaericusresistance in C. quinquefasciatus was suppressed from >17,000- to 2-fold with a 3:1 mixture of B. sphaericusand Cyt1Ab. This trait may prove useful for combating insecticide resistance and for improving the activity of microbial insecticides.

Evolution of resistance to the Bacillus sphaericus Bin toxin is phenotypically masked by combination with the mosquitocidal proteins of Bacillus thuringiensis subspecies israelensis

Two insecticidal bacteria are used as larvicides to control larvae of nuisance and vector mosquitoes in many countries, Bacillus thuringiensis ssp. israelensis and B. sphaericus. Field studies show both are effective, but serious resistance, as high as 50 000-fold, has evolved where B. sphaericus is used against Culex mosquitoes. To improve efficacy and deal with even greater potential problems of resistance, we previously developed several recombinant larvicidal bacteria that combine the best mosquitocidal proteins of these bacteria. In the present study, we report laboratory selection studies using our best recombinant strain against larvae of Culex quinquefasciatus. This recombinant, Bti/BsBin, is a strain of B. thuringiensis ssp. israelensis engineered to produce a large amount of the B. sphaericus binary (Bin) toxin, which makes it more than 10-fold as mosquitocidal as the its parental strains. Here we show that larvae exposed to Bti/BsBin failed to develop significant resistance after 30 successive generations of heavy selection pressure. The highest level of resistance obtained at the LC(95) level was 5.2-fold, but declined to less than two-fold at the 35th generation. Testing the selected populations against B. sphaericus alone showed resistance to Bin evolved, but was masked by combination with B. thuringiensis ssp. israelensis. These results suggest that recombinant bacterial strains have improved mosquito and vector management properties compared with the wild-type strains used in current commercial formulations, and should prove useful in controlling important human diseases such as malaria and filariasis on a long-term basis, even when used intensively under field conditions.