Toxicity of Bacillus thuringiensis var. israelensis crystals to Aedes aegypti larvae: carbonate reversal

The toxicity of purified Bacillus thuringiensis var. israelensis crystals to larvae of Aedes aegypti could be reversed 100-fold by levels of K(2)CO(3) as low as 0.15%.     

Toxicity of Bacillus thuringiensis subsp. israelensis to adult Aedes aegypti mosquitoes

Adult female Aedes aegypti mosquitoes were killed by the parasporal crystals of Bacillus thuringiensis subsp. israelensis (ONR-60A) when the crystals were introduced into the insect midgut as an enema. The 50% lethal dose for intact parasporal crystals was 0.21 microgram/mg of mosquito (wet weight), and for solubilized crystals the 50% lethal dose was 0.04 microgram/mg. These values were compared with 50% lethal concentrations in a free-feeding larval mosquito bioassay of 0.018 and 1.28 microgram/ml for intact and solubilized crystals, respectively. Preparations from B. thuringiensis subsp. kurstaki were ineffective against both adult and larval mosquitoes. An adult mosquito bioassay is suggested as a direct means of screening potential mosquito control agents.     

Digestion of Bacillus thuringiensis var. israelensis spores by larvae of Aedes aegypti.

The larvicidal activity of Bacillus thuringiensis var. israelensis against mosquitoes and the blackfly is included in parasporal crystalline bodies which are produced during sporulation. Following ingestion, the crystals are solubilized in the larval midgut and induce death within a short time; the spores germinate in the dead larvae and complete a growth cycle. The fate of the spores in surviving live larvae was elucidated by using a nonlarvicidal B. thuringiensis var. israelensis mutant. When introduced as the only food source, spores of this mutant support development to the adult stage of newly hatched Aedes aegypti larvae at a rate directly related to spore concentration. The conclusion that spores of B. thuringiensis var. israelensis are digested in the larval gut was substantiated by following the incorporation of [35S]methionine-labeled spores into larval tissues.     

The fate of Bacillus thuringiensis var. israelensis in B. thuringiensis var. israelensis-killed pupae of Aedes aegypti

Carcasses of mosquito larvae killed by Bacillus thuringiensis var. israelensis allow its complete growth cycle (germination, vegetative growth, and sporulation), thus becoming toxic themselves to scavenging larvae. In this study, we demonstrate that the bacterium is capable of inducing death of Aedes aegypti pupae and of recycling in the resulting carcasses. B. thuringiensis var. israelensis-killed pupae were obtained by treating 40-hr-old synchronized fourth instar larvae with a low dose of spores (8000/ml). The fraction of dead pupae was reduced by higher or lower spore concentrations as well as by treating younger or older larval populations (both fourth instar): Increased proportions of dead larvae were obtained at higher concentration or by earlier treatment, whereas lower concentrations or later treatment resulted in more living pupae. Multiplication of B. thuringiensis var. israelensis is shown to occur in the carcasses of dead pupae. The number of spores in each pupal carcass followed a similar kinetic as in larval carcasses, but the final yield was about 10-fold higher, apparently reflecting the difference in dry weight between the two mosquito developmental stages (426 micrograms vs 83 micrograms, respectively). The specific larvicidal activity in a homogenized dead pupa was similar to that of B. thuringiensis var. israelensis powder, LC50 of about 600 spores/ml.     

Toxicity of Bacillus thuringiensis toward Aedes aegypti larvae.

Among six strains of Bacillus thuringiensis and five other species of Bacillus, only two strains of B. thuringiensis, strains HD-1 and BA-068, were toxic to Aedes aegypti larvae within 24 hr. The LC₅₀s were 5.6 × 10⁴ and 2.4 × 10⁵ spores/ml for strains HD-1 and BA-068, respectively. The toxic factor(s) was heat sensitive and γ ray resistant and preliminary evidences indicated that it was associated with the crystalline body of B. thuringiensis.     

Spray-dried Bacillus thuringiensis Serovar israelensis formulations for control of Aedes aegypti larvae

Suspensions containing 0.25 and 1.25 g/liter of Bacillus thuringiensis subsp. israelensis (Bti) spore-toxin complex were spray-dried by using maltodextrin DE-6, corn starch, and nixtamalized corn flour (25 g/liter) as materials to entrap active delta-endotoxin. The inlet air temperature of the drier was kept constant at 141 degrees C and the outlet temperature was maintained at 60 or 70 degrees C. The Probit analysis of the concentration-mortality response of third instars of Aedes aegypti (L.) larvae of the spray-dried products at 60 degrees C showed that LC50 values for maltodextrin DE-6 with 1 and 5% spore-toxin complex were 4 and 10% higher in toxicity, respectively, than that for the unformulated spore-toxin complex without drying. The LC50 value for corn starch with 1 and 5% of spore-toxin complex were also higher in toxicity (7 and 8% respectively). However, LC50 values for nixtamalized corn flour with one and 5% spore-toxin complex were 81 and 55% higher in toxicity, respectively. Dried products contain an a(w) < or = 0.7, suggesting that they are able to keep the products without microorganism growth for longer periods. The scanning electron microscope of Bti spray-dried formulations with nixtamalized corn flour showed smooth spherical particles entrapping the active ingredient. These results suggested that Bti spore-toxin complex formulated with maltodextrin DE-6, corn flour, and nixtamalized corn flour, and then spray-dried may increase larval feeding and thus increase activity against Ae. aegypti larvae.     

Effect of proteolytic and detoxification enzyme inhibitors on Bacillus thuringiensis var. israelensis tolerance in the mosquito Aedes aegypti

Bacillus thuringiensis var. israelensis (Bti) is highly pathogenic to mosquito larvae and is widely used for mosquito control. Its mosquitocidal activity however is relatively low compared to many chemical insecticides. The detoxification mechanisms in the mosquito, among other things, might neutralize the Bti activity, resulting in resistance or tolerance. We tested whether or not the detoxification mechanisms against chemical insecticides might also operate against Bti, rendering it less effective. We targeted four enzymes in Aedes aegypti larvae involved in detoxification with inhibitors that have been used in resistance studies in chemical insecticides and assayed their effects on Bti toxicity. Results revealed that phenylmethanesulphonyl fluoride (PMSF), diethyl maleate, phenobarbital (PB), and piperonyl butoxide (PBO) altered Bti toxicity to various degrees. PMSF is a serine protease inhibitor that prevents Bti digestion and improves Bti activity. PB that induces several detoxifying enzymes had two different effects depending on the method of treatment. Mortality was higher when treatment with PB was discontinuous (149%) whereas with continuous treatment it was lower (101%). PBO, a typical cytochrome P450 inhibitor, increased Bti effect (159%). The combination of discontinuous pretreatment of larvae with PB followed by PBO had a synergistic effect and showed increased activity (146%). It appears that the mechanism for Bti resistance in mosquitoes is similar to that of chemical insecticides. Our studies indicate that we may be able to increase Bti activity by inhibiting some of the detoxification systems as active as broad spectrum chemical insecticides.     

Cadherin Fragments from Anopheles gambiae Synergize Bacillus thuringiensis Cry4Ba's Toxicity against Aedes aegypti Larvae

A peptide from cadherin AgCad1 of Anopheles gambiae larvae was reported as a synergist of Bacillus thuringiensis subsp. israelensis Cry4Ba''s toxicity to the Anopheles mosquito (G. Hua, R. Zhang, M. A. Abdullah, and M. J. Adang, Biochemistry 47:5101-5110, 2008). We report that CR11 to the membrane proximal extracellular domain (MPED) (CR11-MPED) and a longer peptide, CR9 to CR11 (CR9-11), from AgCad1 act as synergists of Cry4Ba''s toxicity to Aedes aegypti larvae, but a Diabrotica virgifera virgifera cadherin-based synergist of Cry3 (Y. Park, M. A. F. Abdullah, M. D. Taylor, K. Rahman, and M. J. Adang, Appl. Environ. Microbiol. 75:3086-3092, 2009) did not affect Cry4Ba''s toxicity. Peptides CR9-11 and CR11-MPED bound Cry4Ba with high affinity (13 nM and 23 nM, respectively) and inhibited Cry4Ba binding to the larval A. aegypti brush border membrane. The longer CR9-11 fragment was more potent than CR11-MPED in enhancing Cry4Ba against A. aegypti.Mosquitoes are vectors of human and animal infectious diseases. Aedes (Stegomyia) aegypti can transmit viruses that cause dengue fever and yellow fever. Mosquitoes have shown a rapid increase in resistance to various chemical insecticides (16). Nonchemical larvicides based on the bacterium Bacillus thuringiensis subsp. israelensis de Barjac are used to control mosquitoes. The specific toxicity of B. thuringiensis subsp. israelensis to Anopheles, Culex, and Aedes spp. is due to the protein components of the parasporal crystal (reviewed in reference 9). The Cry4Ba insecticidal protein is one of at least four types of parasporal crystals expressed in B. thuringiensis subsp. israelensis. The Cry4Ba insecticidal protein is highly toxic to Anopheles and Aedes larvae but not to Culex larvae (2, 6).Synergists of B. thuringiensis subsp. israelensis, another strategy to improve the efficacy of Cry4Ba and B. thuringiensis subsp. israelensis, would lead to the reduced quantity needed to obtain control, lengthen residual activity, and possibly delay the onset of resistance in target insects (7, 8, 10, 21). In the case of mosquitocidal Cry11Aa, synergistic cytolytic toxin functions as an adventitious receptor, inducing prepore formation and subsequent membrane insertion (20). Recently, a new type of synergist based on peptide fragments of host insect cadherins was shown to enhance Cry1A, Cry3, and Cry4Ba toxicities to lepidopteran, coleopteran, and dipteran larvae, respectively (5, 11, 18, 19). A fragment of the Anopheles gambiae larva midgut cadherin AgCad1 was shown to enhance Cry4Ba against A. gambiae (11). Here we show that the C-terminal cadherin repeat (CR) CR11 to the membrane proximal extracellular domain (MPED) (CR11-MPED) of AgCad1 and another fragment (CR9 to CR11 [CR9-11]) also enhance Cry4Ba against another important mosquito species, A. aegypti.The CR9-11 and CR11-MPED regions of AgCad1 were overexpressed in Escherichia coli according to Chen et al. (5) and tested for the ability to enhance Cry4Ba toxicity to A. aegypti larvae. The CR11-MPED plasmid has been described previously (11), and CR9-11 in pET30a was constructed using the same method, with primers 5′-CGA GCA TAT GGG GTC CCC G TT GCC GAA ATT and 5′-CGC TCT CGA GAA ACA C GA ACG TCA CGC GGT TC. To determine the extent that CR9-11 and CR11-MPED could enhance a low dose of Cry4Ba inclusion body form (IBF), we added increasing amounts of CR9-11 and CR11-MPED IBFs to a Cry4Ba IBF concentration predicted to cause about 35% larval mortality. Bioassays were conducted with fourth-instar A. aegypti larvae as previously described (11). Each treatment was replicated four times, each replicate contained 10 larvae, and larval mortality was recorded after 16 h. The enhancement effect reached a plateau at a 1:25 (Cry4Ba/peptide) mass ratio for both AgCad1 fragments (data not shown). To determine the specificity of the cadherin effect, we included the partial cadherin-like protein WCR8 to WCR10 (WCR8-10) from western corn rootworm Diabrotica virgifera virgifera (18), using a Cry4Ba/WCR8-10 mass ratio of 1:100. The control bioassay using the WCR8-10 cadherin fragment from D. virgifera virgifera showed no synergistic effect with Cry4Ba (data not shown).To assess the relative increase in toxicity when cadherin fragments were present, larvae were fed the Cry4Ba IBF alone or with a fixed 1:25 mass ratio of AgCad1 peptide. The calculated 50% lethal concentration (LC₅₀) of the Cry4Ba IBF was 20.34 ng/ml (16.37 to 25.93 ng/ml) (Table ​(Table1).1). The addition of CR9-11 and CR11-MPED IBFs to Cry4Ba IBF reduced the Cry4Ba LC₅₀s to 3.43 ng/ml (1.66 to 5.80 ng/ml) and 7.35 (5.94 to 9.07 ng/ml), respectively (Table ​(Table1);1); furthermore, soluble forms (SF) of CR9-11 and CR11-MPED also reduced the Cry4Ba IBF LC₅₀s, to 5.79 ng/ml (4.42 to 6.73 ng/ml) and 9.23 ng/ml (7.53 to 11.33 ng/ml), respectively (Table ​(Table1).1). The increased synergistic levels of longer cadherin fragments that are involved with toxin binding were also observed with cadherin fragments from Manduca sexta (3). The use of the SF led to a lower level of enhancement than those of the IBFs of the cadherin peptides. This might be explained by the fact that mosquito larvae are filter feeders; thus, more peptides are ingested if they can be filtered by the mosquito (22).

TABLE 1.

Toxicity of Cry4Ba protoxin IBF alone and in combination with A. gambiae cadherin fragments to fourth-instar larvae of A. aegypti

Toxicity of protease-resistant domains from the delta-endotoxin of Bacillus thuringiensis subsp. israelensis in Culex quinquefasciatus and Aedes aegypti bioassays.

The mosquitocidal glycoprotein endotoxin of Bacillus thuringiensis subsp. israelensis was digested with chymotrypsin to yield protease-resistant domains which were then separated from smaller protease digestion products by high-performance liquid chromatography. Once purified, the domains no longer bound wheat germ agglutinin, a lectin which binds N-acetylglucosamine (GlcNAc) and GlcNAc oligomers. Purified protease-resistant domains were as toxic for Culex quinquefasciatus larvae as intact solubilized toxin. In separate experiments, the toxicity of chymotrypsin-digested endotoxin for Aedes aegypti larvae was reduced fivefold or more. A model is presented in which GlcNAc-containing oligosaccharides are required for toxicity for A. aegypti larvae but not C. quinquefasciatus larvae.     

Cytotoxicity of a cloned Bacillus thuringiensis subsp. israelensis CryIVB toxin to an Aedes aegypti cell line

A cloned CryIVB toxin was purified from a cured strain of Bacillus thuringiensis (BT) containing the cryIVB gene on the recombinant plasmid Cam135. Solubilized protoxin was treated with Aedes gut extract or trypsin for varying times and tested for toxicity in vitro on three dipteran and one lepidopteran cell line. Treatment with the Aedes extract but not trypsin, produced an active toxin which lysed only Aedes aegypti cells out of those tested. This activation was time-dependent reaching a maximum after 6 h. Both the Aedes extract-treated and trypsin-treated toxin killed A. aegypti larvae, but this toxicity declined rapidly with increasing time of exposure to the proteolytic preparations.     

Influence of Bacillus thuringiensis var. israelensis on oviposition of Aedes albopictus (Skuse).

The effect of Bacillus thuringiensis var israelensis (B.t.i.) on the oviposition behavior of Aedes albopictus was evaluated in the field and laboratory in Clemson, SC, U.S.A. In the field, water taken from containers in which mosquito larvae were reared (conditioned water) was placed in 16 containers. Eight containers received 50 jld of B.t.i., and eight with water only were kept as controls. In the laboratory, field-collected females of Ae. albopictus were placed in rearing chambers and provided two containers for oviposition, one with 50 microl of B.t.i and one a control with water only. Eight cage experiments were conducted, five using filtered tap water and three with conditioned water. In the field over the 13 trials, more eggs were laid in the containers with B.t.i. although no significant difference was found in the number of eggs between the treatment and controls over 72 h. In the laboratory, more eggs were laid in the containers with B.t.i. versus the controls. The containers had filtered tap water and B.t.i. had significantly more eggs laid in them compared to the controls.     

Cytopathological effect of Bacillus thuringiensis israelensis endotoxins on the intestines of Aedes aegypti mosquito larvae

The dynamics of pathological changes in the intestine of Aedes aegypti larvae under the influence of toxins Cry11A and Cry4B produced by Bacillus thuringiensis israelensis was studied by means of electron microscope. Most significant ultrastructure changes in the intestine of the second instar larvae were observed in the midgut. The cytoplasm of cells disintegrated, and elongated lacunae appeared. The number of microvilli decreased, or they disappeared in the result of destruction. The peritrophic membrane displaced to the lumen of midgut. Any changes in epithelial cells and cuticle in time of foregut and hindgut were not observed in a comparison to control. The toxin Cry4B caused the most effective destruction of the midgut epithelium.     

Combination of Mesocyclops thermocyclopoides and Bacillus thuringiensis var. israelensis: a better approach for the control of Aedes aegypti larvae in water containers.

The efficacy of a local Thai-strain of the copepod, Mesocyclops thermocyclopoides and the larvicide, Bacillus thuringiensis var. israelensis (Bti), used jointly and singly, was studied against Aedes aegypti in water containers. In a laboratory test, copepods alone produced mortality of 98-100% in 1st instar larvae of Ae. aegypti at copepod:larvae ratios ranging from 1:1 to 1:4. In an outdoor field simulated experiment that ran for 16 wk, after a single inoculation, the treatment of copepods and Bti combined yielded the better, more sustainable results than the agents used individually. Numbers of mosquito larvae per sample in the combined treatment were zero during the first 8 wk; larval numbers then increased but were maintained at a very low level for the next 4 wk after which the larval numbers increased moderately but still remained below numbers in the control. Bti alone kept the larvae at the zero level for the first 4 wk after which their numbers increased slightly and were at low levels up to 12 wk. Copepods alone maintained larval numbers at a low level as compared with those of the control. During the course of the experiment the larval numbers in the control were greater than 20 per sample. Statistically significant differences were noted among treatment means (F = 23.083, df = 3/60, P<0.01) over the total period of the study. The number of copepods in the joint treatment was significantly higher than in the copepod alone treatment for the first 8 wk (t = -4.97, df = 14, P<0.01). The density of copepods, however, for the whole 16-wk period was not significantly different in these two treatments (t = -1.51, df = 30, P>0.1).     

Toxicity of parasporal crystals of Bacillus thuringiensis subsp. israelensis to mosquitoes.

Toxicity of Bacillus thuringiensis subsp. israelensis (ONR-60A/WHO 1897) parasporal crystals to three medically important mosquito larvae is described. The numbers of larvae killed are in relation to crystal dry weight. The crystals are lethally toxic to Aedes aegypti Linnaeus (mean 50% lethal concentration [LC50] = 1.9 x 10(-4) micrograms/ml), Culex pipiens var. quinquefasciatus Say (LC50 = 3.7 x 10(-4) micrograms/ml), and Anopheles albimanus Wiedemann (LC50 = 8.0 x 10(-3) micrograms/ml). Purfied crystals of B. thuringiensis subsp. kurstaki, which are toxic to lepidopteran insects, are ineffective against the mosquito larvae. Likewise, B. thuringiensis subsp. israelensis parasporal crystals are not efficacious for larvae of the lepidopteran, Manduca sexta.     

Isolation and characterization of mutants of Bacillus thuringiensis var. israelensis

Several mutants of Bacillus thuringiensis var. israelensis HD-500 (Bti) were obtained after treatment with N -methyl-n'-nitro- N -nitrosoguanidine. On the basis of the production (+) or absence (-) of spore (Spo) and crystal (Cry) the strains were grouped into three categories: Spo + Cry +, Spo + Cry - and Spo - Cry + . NGI-22, a Spo + Cry - mutant lacked all crystal proteins. Both NGI-23 and NGI-23-1 were Spo - Cry + . NGI-23-1, however, produced multiple crystals per cell. These mutants could prove useful not only for analysing sporulation and crystal formation as well as the linkage between the two genetic processes but also for improving the potential of Bti as a microbial insecticide.     

Evaluation of a new tablet formulation based on Bacillus thuringiensis sorovar. israelensis for larvicidal control of Aedes aegypti

The effect of sunlight on the efficacy and persistence of an experimental tablet formulation based on Bacillus thuringiensis sorovar. israelensis (C4P1) was evaluated against Aedes aegypti larvae under simulated field conditions. The initial mortality ranged from 93 to 100%, and the residual activity (> or = 70% mortality) recorded in containers exposed to sunlight or shade were, respectively, 13-35 days and 40-54 days. The results suggest that C4P1 can provide long-term larvicidal effect and operational advantages.     

Development of selective media for Bacillus sphaericus and Bacillus thuringiensis var. israelensis

Summary The resistance of 8 strains of Bacillus sphaericus and of 2 strains of Bacillus thuringiensis var. israelensis (B.t.i.) to various antibiotics and antibiotic combinations were tested. All B. sphaericus strains were resistant to streptomycin, lincomycin and bacitracin, and six strains were resistant to combinations of these antibiotics. This antibiotic resistance could be utilised to establish selective media to identify and follow the fate of B. sphaericus and of B.t.i. in the field.     

Bacillus thuringiensis var. israelensis production involving re-use of the supernatant

The supernatant arising after biomass separation of Bacillus thuringiensis var. israelensis by flocculation/sedimentation was re-used after being supplemented with 25, 50 and 75% (w/v) of the original culture medium, based on corn steep liquor, glucose and mineral salts. Supplementation at 75% gave a spore concentration (1 x 10(10) c.f.u. ml(-1)) five times greater than that obtained with the other supplements.