Recombinant bacteria for mosquito control.

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.

Baseline susceptibility to bacterial insecticides in populations of Culex pipiens complex (Diptera: Culicidae) from California and from the Mediterranean Island of Cyprus.

Bacterial insecticides play an increasingly important role in mosquito control. To establish guidelines for detecting resistance at an early stage, information on natural variation in susceptibility of insect populations to these insecticides is needed. Between 1990 and 1993, the susceptibility of Culex pipiens L. complex to Bacillus thuringiensis subsp. israelensis de Barjac and/or Bacillus sphaericus Neide was determined in 31 collections from California. These collections were undertaken before the widespread use of B. thuringiensis subsp. israelensis and before the registration of B. sphaericus in California. Seven collections from the Mediterranean island of Cyprus, where no microbial insecticides have been used, also were tested. The 1990-1991 California collections exhibited limited variation in susceptibility to B. thuringiensis subsp. israelensis. LC50 and LC95 values spanned about a three-fold and four-fold range, respectively. The 1993 Cyprus collections exhibited both higher mean LC values, and greater variability in those values, than the California collections. The LC50s for the Cyprus collections varied over a 10-fold range, whereas the LC50s varied over a 12.5-fold range. Variation in susceptibility to B. sphaericus among the 1991 California collections was about five-fold at the LC50 and LC95. No significant geographic variation in susceptibility to B. thuringiensis subsp. israelensis was observed among regions within California. Although variation in susceptibility was limited among California collections, the greater variability observed among the Cyprus collections and between the Cyprus and California collections illustrates the importance of establishing regional baselines to monitor accurately for changes in susceptibility.

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).

The interaction of two cytolytic toxins, Cyt1Ab from Bacillus thuringiensis subsp. medellin and 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. sphaericus resistance in C. quinquefasciatus was suppressed from >17,000- to 2-fold with a 3:1 mixture of B. sphaericus and Cyt1Ab. This trait may prove useful for combating insecticide resistance and for improving the activity of microbial insecticides.

Lack of cross-resistance to Cry19A from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) resistant to cry toxins from Bacillus thuringiensis subsp. israelensis.

Culex quinquefasciatus mosquitoes with high levels of resistance to single or multiple toxins from Bacillus thuringiensis subsp. israelensis were tested for cross-resistance to the Bacillus thuringiensis subsp. jegathesan polypeptide Cry19A. No cross-resistance was detected in mosquitoes that had been selected with the Cry11A, Cry4A and Cry4B, or Cry4A, Cry4B, Cry11A, and CytA toxins. A low but statistically significant level of cross-resistance, three to fourfold, was detected in the colony selected with Cry4A, Cry4B, and Cry11A. This cross-resistance was similar to that previously detected with B. thuringiensis subsp. jegathesan in the same colony. These data help explain the toxicity of B. thuringiensis subsp. jegathesan against the resistant colonies and indicate that the Cry19A polypeptide might be useful in managing resistance and/or as a component of synthetic combinations of mosquitocidal toxins.

Cyt1A from Bacillus thuringiensis lacks toxicity to susceptible and resistant larvae of diamondback moth (Plutella xylostella) and pink bollworm (Pectinophora gossypiella).

We tested Cyt1Aa, a cytolytic endotoxin of Bacillus thuringiensis, against susceptible and Cry1A-resistant larvae of two lepidopteran pests, diamondback moth (Plutella xylostella) and pink bollworm (Pectinophora gossypiella). Unlike previous results obtained with mosquito and beetle larvae, Cyt1Aa alone or in combination with Cry toxins was not highly toxic to the lepidopteran larvae that we examined.

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

A previously untreated field population of Culex quinquefasciatus Say, collected near Bakersfield, CA, was subjected to intensive laboratory selection with the bacterial insecticide Bacillus sphaericus Neide (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 of B. sphaericus that 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 toward B. sphaericus strains 1593 and 2297, but little or no cross-resistance was observed toward B. sphaericus strains IAB59 or ISPC5 (= WHO 2173). Cross-resistance also was not detected toward the bacterial insecticide Bacillus thuringiensis subsp. israelensis, toward a recombinant strain expressing both B. thuringiensis subsp. israelensis and B. sphaericus (strain 1593) toxins, toward individual or multiple toxins from B. thuringiensis subsp. israelensis, or toward conventional synthetic insecticides. Genetic analysis revealed that B. sphaericus resistance 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 the Cx. pipiens (L.) complex.

Cyt1A from Bacillus thuringiensis synergizes activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae).

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 Culex and Anopheles mosquitoes, it is virtually nontoxic to Aedes aegypti, an important vector species. In the present study, we evaluated the capacity of the cytolytic protein Cyt1A from Bacillus 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.

Cyt1A from Bacillus thuringiensis restores toxicity of Bacillus sphaericus against resistant Culex quinquefasciatus (Diptera: Culicidae).

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.

Selection and characterization of temephos resistance in a population of Aedes aegypti from Tortola, British Virgin Islands.

A collection of Aedes aegypti from Tortola, British Virgin Islands, with a high level of temephos resistance (46.8-fold at the 95% lethal concentration [LC95]) was selected to higher resistance with temephos in the laboratory. After 13 generations of pressure, the temephos resistance ratio increased to 180.6 (LC95), whereas in the absence of selection pressure the resistance ratio declined to 8.5. Relatively low levels of resistance or cross-resistance to other organophosphate and carbamate insecticides, and a high level of resistance to the pyrethroid permethrin were also observed. Synergism tests implicated detoxifying esterases in temephos resistance and the presence of elevated esterase activity was confirmed by biochemical tests; however, no evidence was found of insensitive acetylcholinesterase. Mendelian crosses indicated that temephos resistance was inherited as a monofactorial trait. The presence of high levels of temephos and permethrin resistance in Ae. aegypti has important implications for Aedes control programs.

Variable cross-resistance to Cry11B from Bacillus thuringiensis subsp. jegathesan in Culex quinquefasciatus (Diptera: Culicidae) resistant to single or multiple toxins of Bacillus thuringiensis subsp. israelensis.

A novel mosquitocidal bacterium, Bacillus thuringiensis subsp. jegathesan, and one of its toxins, Cry11B, in a recombinant B. thuringiensis strain were evaluated for cross-resistance with strains of the mosquito Culex quinquefasciatus that are resistant to single and multiple toxins of Bacillus thuringiensis subsp. israelensis. The levels of cross-resistance (resistance ratios [RR]) at concentrations which caused 95% mortality (LC95) between B. thuringiensis subsp. jegathesan and the different B. thuringiensis subsp. israelensis-resistant mosquito strains were low, ranging from 2.3 to 5.1. However, the levels of cross-resistance to Cry11B were much higher and were directly related to the complexity of the B. thuringiensis subsp. israelensis Cry toxin mixtures used to select the resistant mosquito strains. The LC95 RR obtained with the mosquito strains were as follows: 53.1 against Cq4D, which was resistant to Cry11A; 80.7 against Cq4AB, which was resistant to Cry4A plus Cry4B; and 347 against Cq4ABD, which was resistant to Cry4A plus Cry4B plus Cry11A. Combining Cyt1A with Cry11B at a 1:3 ratio had little effect on suppressing Cry11A resistance in Cq4D but resulted in synergism factors of 4.8 and 11.2 against strains Cq4AB and Cq4ABD, respectively; this procedure eliminated cross-resistance in the former mosquito strain and reduced it markedly in the latter strain. The high levels of activity of B. thuringiensis subsp. jegathesan and B. thuringiensis subsp. israelensis, both of which contain a complex mixture of Cry and Cyt proteins, against Cry4- and Cry11-resistant mosquitoes suggest that novel bacterial strains with multiple Cry and Cyt proteins may be useful in managing resistance to bacterial insecticides in mosquito populations.

Isolation and characterization of two novel organophosphate resistance mechanisms in Culex pipiens from Cyprus.

Two novel mechanisms of organophosphate resistance were isolated and characterized from a population of Culex pipiens L. from Cyprus. Two strains, one expressing the novel, highly active esterases A5 and B5 (strain A5B5-R), and one expressing insensitive acetylcholinesterase (strain Ace-R), were developed by single pair crosses and selection with temephos and propoxur, respectively. The A5B5-R strain demonstrated resistance toward organophosphate insecticides that could be suppressed by the esterase inhibitor S,S,S-tributyl phosphorotrithioate (DEF). No cross-resistance to carbamates occurred. The Ace-R strain demonstrated resistance to organophosphate as well as to carbamate insecticides. Propoxur and temephos resistance was not affected by the monooxygenase inhibitor piperonyl butoxide or by DEF. The Ace-R strain possessed a novel toxicologic profile as well as a unique acetylcholinesterase inhibition pattern. Inheritance of temephos or propoxur resistance was codominant in F1 offspring. Backcrosses to a susceptible strain in both cases failed to fit a single gene model, suggesting that multiple loci may be involved. Combining the A5B5-R and the Ace-R strains resulted in high levels of temephos resistance, similar to that of the parents.

CytA enables CryIV endotoxins of Bacillus thuringiensis to overcome high levels of CryIV resistance in the mosquito, Culex quinquefasciatus.

Cry proteins produced by Bacillus thuringiensis are selective biodegradable insecticides used increasingly in bacterial insecticides and transgenic plants as alternatives to synthetic chemical insecticides. However, the potential for development of resistance and cross-resistance in target insect populations to Cry proteins used alone or in combination threatens the more widespread use of this novel pest control technology. Here we show that high levels of resistance to CryIV proteins in larvae of the mosquito, Culex quinquefasciatus, can be suppressed or reduced markedly by combining these proteins with sublethal quantities of CytA, a cytolytic endotoxin of B. thuringiensis. Resistance at the LC95 level of 127-fold for a combination of three CryIV toxins (CryIVA, B, and D), resulting from 60 generations of continuous selection, was completely suppressed by combining sporulated powders of CytA in a 1:3 ratio with sporulated powders of a CryIVA, CryIVB, and CryIVD strain. Combining the CytA strain with a CryIVA and CryIVB strain also completely suppressed mosquito resistance of 217-fold to the latter toxins at the LC95 level, whereas combination of CytA with CryIVD reduced resistance in a CryIVD-selected mosquito strain from greater than 1,000-fold to less than 8-fold. The CytA/CryIV model provides a potential molecular genetic strategy for engineering resistance management for Cry proteins directly into bacterial insecticides and transgenic plants.

Influence of Exposure to Single versus Multiple Toxins of Bacillus thuringiensis subsp. israelensis on Development of Resistance in the Mosquito Culex quinquefasciatus (Diptera: Culicidae).

The impending widespread use of transgenic crop plants encoding a single insecticidal toxin protein of Bacillus thuringiensis has focused attention on the perceived risk of rapid selection of resistance in target insects. We have used Bacillus thuringiensis subsp. israelensis toxins as a model system and determined the speed and magnitude of evolution of resistance in colonies of the mosquito Culex quinquefasciatus during selection for 28 consecutive generations with single or multiple toxins. The parental strain was synthesized by combining approximately 500 larvae from each of 19 field collections obtained from the states of California, Oregon, Louisiana, and Tennessee. At least 10,000 larvae were selected in each generation of each line at an average mortality level of 84%. The susceptibilities of the parental and selected lines were compared in parallel tests in every third generation by using fresh suspensions of toxin powders. The normal toxin complement of B. thuringiensis subsp. israelensis consists of four toxins, CryIVA, CryIVB, CryIVD, and CytA. Resistance became evident first in the line that was selected with a single toxin (CryIVD), attaining the highest level (resistance ratio [RR], >913 at 95% lethal concentration) by generation F(inf28) when the study was completed. Resistance evolved more slowly and to a lower level (RR, >122 by F(inf25)) in the line selected with two toxins (CryIVA+CryIVB) and lower still (RR, 91 by F(inf28)) in the line selected with three toxins (CryIVA+CryIVB+ CryIVD). Resistance was remarkably low (RR, 3.2) in the line selected with all four toxins. The results reveal the importance of the full complement of toxins found in natural populations of B. thuringiensis subsp. israelensis as an effective approach to resistance management.

Organophosphate resistance in Culex pipiens from Cyprus.

Populations of Culex pipiens were sampled from 8 locations in Cyprus between 1987 and 1993. All population samples generally revealed organophosphate resistance to malathion, temephos, chlorpyrifos, fenthion, dichlorvos, and pirimiphos methyl, in decreasing order of magnitude. Of 7 populations assessed with the carbamate propoxur, all proved to be resistant to different degrees. Of the 6 populations tested with permethrin, 2 were resistant to permethrin. Resistance was associated with the presence of 5 different overproduced esterases (esterases A1, A2, A5, B2, and B5) as well as an insensitive form of acetylcholinesterase. These results are discussed in relation to the ongoing mosquito abatement program in Cyprus and to similar programs in other parts of the world.

Esterases A2 and B2 in Culex quinquefasciatus (Diptera: Culicidae): role in organophosphate resistance and linkage.

Two highly active esterases, A2 and B2, were isolated in SeLax, a strain of Culex quinquefasciatus Say from California which demonstrated resistance levels of 19, 14, and 4.3 times to the organophosphate insecticides chlorpyrifos, temephos, and malathion, respectively. Selection of SeLax by temephos during 31 generations increased resistance to this insecticide to 65 times. This resistance was shown to be DEF-suppressible, monofactorial, and strongly associated with the presence of esterases A2 and B2. Although these two esterases are encoded by distinct structural genes, no disjunction was observed when SeLax was crossed to S-Lab, a susceptible strain, or to S54, a strain resistant to organophosphates by means of esterase A1. However, when SeLax was crossed to strain Tem-R, which is resistant to organophosphates because of a highly active esterase B1, all possible recombinants occurred. These results were discussed in relation to the recent discovery that increased activity of B esterases in the genus Culex is caused by gene amplification.