Co-expression of Bacillus thuringiensis Cry4Ba and Cyt2Aa2 in Escherichia coli revealed high synergism against Aedes aegypti and Culex quinquefasciatus larvae

Cry4Ba is a delta-endotoxin produced by Bacillus thuringiensis subsp. israelensis and Cyt2Aa2 is a cytolytic delta-endotoxin produced by B. thuringiensis subsp. darmstadiensis. Cry4Ba produced in Escherichia coli was toxic to Aedes aegypti larvae (LC(50)=140 ng ml(-1)) but virtually inactive to Culex quinquefasciatus larvae. Cyt2Aa2 expressed in E. coli exhibited moderate activity against A. aegypti and C. quinquefasciatus larvae with LC(50) values of 350 and 250 ng ml(-1), respectively. Co-expression of both toxins in E. coli dramatically increased toxicity to both A. aegypti andC. quinquefasciatus larvae (LC(50)=7 and 20 ng ml(-1), respectively). This is the first report to demonstrate that Cry4Ba and Cyt2Aa2 have high synergistic activity against C. quinquefasciatus larvae.     

Chemical composition and anti-mosquito potential of rhizome extract and volatile oil derived from Curcuma aromatica against Aedes aegypti (Diptera: Culicidae).

Crude rhizome extracts and volatile oils of Curcuma aromatica were evaluated for chemical composition and anti-mosquito potential, including larvicidal, adulticidal, and repellent activities against the Aedes aegypti mosquito. Chemical identification achieved by GC/MS analysis revealed that xanthorrhizol, 1H-3a, 7-methanoazulene and curcumene at 35.08 and 13.65%, 21.81 and 30.02%, and 13.75 and 25.71%, were the main constituents in hexane extracts and volatile oils, respectively. Volatile oil of Cu. aromatica possessed a significantly higher larvicidal activity against the 4th instar larvae of Ae. aegypti than that of hexane extracts, with LC50 values of 36.30 and 57.15 ppm, respectively. In testing for adulticidal activity, on the other hand, hexane-extracted Cu. aromatica (LC50: 1.60 microg/mg female) was found to be slightly more effective against female Ae. aegypti than volatile oil (LC50: 2.86 microg/mg female). However, the repellency of these two products against Ae. aegypti adult females differed significantly. The hexane-extracted Cu. aromatica, with a median complete protection time of 1 h (range = 1-1.5 h) when applied at a concentration of 25%, appeared to have significantly higher repellency than that of distillate oil (0.5 h, range = 0-0.5 h). The different results obtained from both products of Cu. aromatica were probably due to variety in quantity and type of active ingredients as well as the biological and physiological characteristics that differed between both developmental stages of mosquitoes, larvae, and adults.     

Ovicidal and larvicidal activity against Aedes aegypti and Anopheles gambiae complex mosquitoes of essential oils extracted from three spontaneous plants of Burkina Faso

Essential oils extracted from dried leaves of three spontaneous plants naturally growing in Burkina Faso, i.e. Cymbopogon proximus, Lippia multiflora and Ocimum canum, exhibited larvicidal activity by the WHO standard protocol against 3rd and 4th instar F1-larvae of field-collected mosquitoes vectors of human disease, namely Aedes aegypti and members of the Anopheles gambiae complex, An. arabiensis and An. gambiae. The median lethal concentration (LC50) for Ae. aegypti and An. gambiae s.l. larvae ranged between 53.5-258.5 ppm and 61.9-301.6 ppm, respectively. The LC90 estimates ranged 74.8-334.8 ppm for Ae. aegypti, and 121.6-582.9 ppm for An. gambiae s.l. Ovicidal activity against eggs of An. gambiae s.l. was also demonstrated. The LC50 values for An. gambiae s.l. eggs ranged between 17.1-188.7 ppm, while LC90 values ranged between 33.5-488 ppm. Lippia multiflora showed the highest activity against An. gambiae s.l. eggs and Ae. aegypti larvae, whereas no difference was found among C. proximus and L. multiflora in their activity against An. gambiae s.l. larvae. Of the three plants, essential oils from O. canum had the lowest activity against both eggs and larvae. Eggs were more susceptible than larvae. Ae. aegypti larvae were more susceptible than larvae of An. gambiae s.l.     

Larvicidal activities against Aedes aegypti of some Brazilian medicinal plants

Larvicidal activities against Aedes aegypti have been determined in the ethanolic extracts obtained from 51 Brazilian medicinal plants. Eleven of the 84 extracts studied showed significant (LC50 < 100 microg mL(-1)) activities against larvae, with extracts from Annona crassiflora (root bark, LC50 = 0.71 microg mL(-1); root wood, LC50 = 8.94 microg mL(-1)) and Annona glabra (seed, LC50 = 0.06 microg mL(-1)) showing the highest activities. The results obtained should be of value in the search for new natural larvicidal compounds.     

Larvicidal activity of Tagetes patula essential oil against three mosquito species

Larvicidal activity of Tagetes patula essential oil was tested against the fourth instar larvae of Aedes aegypti, Anopheles stephensi, and Culex quinquefaciatus. Five different concentrations of essential oil were studied and the results were compared with that of synthetic insecticide, malathion. A. aegypti (LC(50) 13.57, LC(90) 37.91) was most susceptible followed by An. stephensi (LC(50) 12.08, LC(90) 57.62) and C. quinquefaciatus (LC(50) 22.33, LC(90) 71.89).     

Comparative delta-endotoxins of Bacillus thuringiensis against mosquito vectors (Aedes aegypti and Culex pipiens)

Pure crystals of seven Bacillus thuringiensis field isolates from the Lower Silesia region (Poland) were tested against larvae of Aedes aegypti L. and Culex pipiens L. (Culicidae, Diptera). The crystals of OpQ3 phylloplane isolate (belonging to the first biochemical type of B. thuringiensis subsp. japonensis, yoso, jinghongiensis) killed from 68 +/- 7% to 84 +/- 7% of the fourth instar larvae of A. aegypti. The crystals of two other strains (KpF3 and KpC1) of this group caused mortality between 3 +/- 2% and 70 +/- 7%. The LC50 ranged from 3.2 +/- 0.4 to 34.1 +/- 4.8 microg/ml. The effect of B. thulringiensis wratislaviensis H-47 crystals was the lowest with larval mortality from 0% to 17 +/- 3%. No significant (0%-37 +/- 6%) effect of B. thuringiensis crystals on the larvae of C. pipiens was observed. Our results show that the delta-endotoxins of B. thuringiensis act very specifically.     

Bioactivity of selected plant essential oils against the yellow fever mosquito Aedes aegypti larvae

The bioactivity of 14 essential oils from five plants has been studied using the brine shrimp lethality test and the Aedes aegypti larvicidal assay. All essential oils screened had LC50 values smaller than 200 microg/ml, showing significant lethality against brine shrimp. In addition, nine of the 14 essential oils tested showed toxicity against the fourth-instar A. aegypti larvae in 24 h (LC50<100 microg/ml). Of these, the leaf and bark essential oils of Cryptomeria japonica demonstrated high larvicidal activity, the most active being the leaf essential oil of C. japonica, with a LC50=37.6 microg/ml (LC90=71.9 microg/ml), followed by the bark essential oil of C. japonica also showing high activity against A. aegypti larvae, with a LC50=48.1 microg/ml (LC90=130.3 microg/ml). The results obtained from this study suggest that the leaf and bark essential oils of C. japonica are promising as larvicides against A. aegypti larvae and could be useful in the search for new natural larvicidal compounds.     

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.     

Two insecticidal tetranortriterpenoids from Azadirachta indica

Two new triterpenoids, 6 alpha-O-acetyl-7-deacetylnimocinol [24,25,26,27-tetra-norapotirucalla-(apoeupha)-6 alpha-acetoxy-7 alpha-hydroxy-1,14,20,22-tetraen-21,23-epoxy-3-one] (1) and meliacinol [24,25,26,27-tetranorapotirucalla-(apoeupha)-1 alpha-trimethylacryloxy-21,23-6 alpha,28-diepoxy-16-oxo-17-oxa-14,20,22-trien-3 alpha,7 alpha-diol] (2) were isolated from the methanolic extract of the fresh leaves of Azadirachta indica (neem). Their structures have been elucidated through spectral studies, including 2D-NMR (COSY-45, NOESY, HMQC and HMBC). The bioactivity of these as well as of nimocinol, reported earlier from the same source, is reported. The first compound and nimocinol showed toxicity on fourth instar larvae of mosquitoes (Aedes aegypti) with LC50 values of 21 and 83 ppm, respectively. The second compound had no effect upto 100 ppm.     

The naturally derived insecticide spinosad is highly toxic to Aedes and Anopheles mosquito larvae

Spinosad is a naturally derived biorational insecticide with an environmentally favourable toxicity profile, so we investigated its potency against mosquito larvae (Diptera: Culicidae). By laboratory bioassays of a suspension concentrate formulation of spinosad (Tracer), the 24 h lethal concentration (LC50) against Aedes aegypti (L.) third and fourth instars was estimated at 0.025 p.p.m. following logit regression. The concentration-mortality response of third- and fourth-instar Anopheles albimanus Weidemann did not conform to a logit model. The LC50 value of spinosad in Anopheles albimanus was 0.024 p.p.m. by quadratic linear regression. A field trial in southern Mexico demonstrated that spinosad 1 p.p.m. compared with the standard temephos (Abate) 1% granules 100 g/m3 water prevented Ae. aegypti breeding in plastic containers of water for 8 weeks; at 10 p.p.m. spinosad prevented breeding for > 22 weeks. In another field trial, spinosad at 5 p.p.m. and temephos both completely eliminated reproduction of Ae. aegypti for 13 weeks. In contrast, the bacterial insecticide Bacillus thuringiensis var. israelensis (Bti, Vectobac) AS) performed poorly with just 2 weeks of complete inhibition of Ae. aegypti breeding. Spinosad also effectively prevented breeding of Culex mosquitoes and chironomids in both trials to a degree similar to that of temephos. We conclude that spinosad merits evaluation as a replacement for organophosphate or Bti treatment of domestic water tanks in Mesoamerica. We also predict that spinosad is likely to be an effective larvicide for treatment of mosquito breeding sites.     

Larvicidal and repellent potential of Albizzia amara Boivin and Ocimum basilicum Linn against dengue vector, Aedes aegypti (Insecta:Diptera:Culicidae)

Investigations were made to test the larval toxicity and smoke repellent potential of Albizzia amara and Ocimum basilicum at different concentration (2%, 4%, 6%, 8% and 10%) against the different instar (I, II, III and IV) larvae and pupae of Aedes aegypti. The LC50 values of A. amara and O. basilicum for I instar larvae was 5.412 and 3.734, II instar 6.480 and 4.154, III instar 7.106 and 4.664, IV instar 7.515 and 5.124, respectively. The LC50 and LC90 values of pupae were 6.792%, 5.449% and 16.925%, 15.474%. The smoke toxicity of A. amara was more effective against A. aegypti than the O. basilicum.     

Larvicidal activity of major essential oils from stems of Allium monanthum Maxim. against Aedes aegypti L

The stems of Allium monanthum were extracted, and the major essential oil composition and larvicidal effects were studied. The analyses were conducted by gas chromatography and mass spectroscopy revealed that the essential oils of A. monanthum stems. The A. monanthum essential oil yield was 4.25%, and gas chromatography and mass spectroscopy analysis revealed that its major constituents were dimethyl trisulfide (23.21%), dimethyl tetrasulfide (11.24%) and methlyl propyl trisulfide (8.21%). The essential oil had a significant toxic effect against early fourth-stage larvae of Aedes aegypti L with an LC(50) value of 23.14 ppm and an LC(90) value of 36.31 ppm. Also, dimethyl trisulfide (≥95.0%), dimethyl tetrasulfide (≥95.0%) and methlyl propyl trisulfide (≥95.0%) were tested against the F(21) laboratory strain of A. aegypti. Methlyl propyl trisulfide (≥95.0%) has good activity with an LC(50) value of 19.38 ppm. Also, the above indicates that other major compounds may play a more important role in the toxicity of essential oil.     

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.     

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.     

Introduction of Culex toxicity into Bacillus thuringiensis Cry4Ba by protein engineering

Bacillus thuringiensis mosquitocidal toxin Cry4Ba has no significant natural activity against Culex quinquefasciatus or Culex pipiens (50% lethal concentrations [LC(50)], >80,000 and >20,000 ng/ml, respectively). We introduced amino acid substitutions in three putative loops of domain II of Cry4Ba. The mutant proteins were tested on four different species of mosquitoes, Aedes aegypti, Anopheles quadrimaculatus, C. quinquefasciatus, and C. pipiens. Putative loop 1 and 2 exchanges eliminated activity towards A. aegypti and A. quadrimaculatus. Mutations in a putative loop 3 resulted in a final increase in toxicity of >700-fold and >285-fold against C. quinquefasciatus (LC(50) congruent with 114 ng/ml) and C. pipiens (LC(50) 37 ng/ml), respectively. The enhanced protein (mutein) has very little negative effect on the activity against Anopheles or AEDES: These results suggest that the introduction of short variable sequences of the loop regions from one toxin into another might provide a general rational design approach to enhancing B. thuringiensis Cry toxins.     

Lethal and behavioural effects of three synthetic repellents (DEET, IR3535 and KBR 3023) on Aedes aegypti mosquitoes in laboratory assays

The knock-down, mortality and 'irritancy' effects of three synthetic repellents (DEET, IR3535 and KBR 3023) on Aedes aegypti (L) (Diptera: Culicidae) were evaluated in the laboratory in the absence of animal bait. Filter paper tests were carried out to assess the knock-down effect (KDt(50) and KDt(95)) and mortality (LC(50) and LC(95)) induced by each repellent. 'Irritancy' tests were carried out to compare the flight response (time to first take-off, or FT) to increasing concentrations of repellents (2-7%) and at five distances from the treated surface (0-40 mm). DEET had an insecticidal effect (KDt(50) = 9.7 min at 7%; CL(50)= 1165 mg/m(2)), whereas IR3535 and KBR 3023 did not. Relative to an untreated control, IR3535 was an irritant (relative irritancy or RI > 1) at doses of 5% and 7% (RI = 17.7 and 9.9, respectively), whereas DEET was an irritant at lower concentrations (RI = 12.3 at 2% DEET). KBR 3023 was the weakest irritant over the same range of concentrations (RI(max) = 3.6 at 6%). DEET was more of an irritant (RI(20) = 9.4) than IR3535 (RI(20) = 2.9) over a range of distances (0-20 mm), and KBR 3023 was not an irritant unless mosquitoes made contact with the treated surface. All three repellents had a significant effect on mosquitoes, but DEET exhibited a more complex mode of action than the others due to its insecticidal properties. The repellents do not behave as a single class of compounds with a common mode of action, but most probably affect different physiological systems in insects. The physiological and molecular mechanisms of repellents, especially DEET, should be investigated to ensure a better use of these molecules for skin applications and/or for treating materials against mosquitoes.     

Mosquito larval consumption of toxic arborescent leaf-litter,and its biocontrol potential

Previously we described the mosquito larvicidal properties of decomposed leaf-litter from deciduous trees, especially the alder Alnus glutinosa (L) Gaertn., due to toxic polyphenols and other secondary compounds. To further examine the biocontrol potential of toxic leaf-litter for mosquito control, feeding rates of third-instar mosquito larvae were assessed for examples of three genera: Anopheles stephensi Liston, Aedes aegypti (L) and Culex pipiens L. (Diptera: Culicidae). When immersed in a suspension of non-toxic leaf-litter particles (approximately 0.4 mm), pre-starved larvae of all three species ingested sufficient material in 30 min to fill the anterior gut lumen (thorax plus two to three abdominal segments). Gut filling peaked after 1-2 h ingestion time, filling the intestine up to six to seven abdominal segments for Ae. aegypti, but maxima of five abdominal segments for Cx. pipiens and An. stephensi. Using three methods to quantify consumption of three materials by third-instar larvae of Ae. aegypti, the average amount of leaf-litter (non-toxic 0.4 mm particles) ingested during 3 h was determined as approximately 20 microg/larva (by dry weight and by lignin spectrophotometric assay). Consumption of humine (approximately 100 microm particles extracted from leaf-litter) during 3 h was approximately 80 microg/larva for Ae. aegypti, but only approximately 30 microg/larva for Cx. pipiens and 15 microg/larva for An. stephensi, with good concordance of determinations by dry weight and by radiometric assay. Cellulose consumption by Ae. aegypti was intermediate: approximately 40 microg/larva determined by radiometric assay. Apparent differences between the amounts of these materials ingested by Ae. aegypti larvae (humine four-fold, cellulose two-fold more than leaf-litter) may be attributed to contrasts in palatability (perhaps related to particle size or form), rather than technical discrepancies, because there was good concordance between results of both methods used to determine the amounts of humine and leaf-litter ingested. Bioassays of toxic leaf-litter (decomposed 10 months) with 4-h exposure period (ingestion time) ranked the order of sensitivity: Ae. aegypti (LC50 < 0.03 g/L) > An. stephensi (LC50 = 0.35 g/L) > Cx. pipiens (LC20 > 0.4 g/L). When immersed in the high concentration of 0.5 g/L toxic leaf-litter (0.4 mm particles), as little as 15-30 min ingestion time (exposure period) was sufficient to kill the majority of larvae of all three species, as soon as the gut lumen was filled for only the first few abdominal segments. Possibilities for mosquito larval control with toxic leaf-litter products and the need for standardized ingestion bioassays of larvicidal particles are discussed.     

Cloning and characterization of a cytolytic and mosquito larvicidal delta-endotoxin from Bacillus thuringiensis subsp. darmstadiensis

The cytolytic delta-endotoxin gene from Bacillus thuringiensis subsp. darmstadiensis was amplified from genomic DNA by PCR by using primers designed from the sequence of cyt2Aa1 gene of B. thuringiensis subsp. kyushuensis. DNA sequence analysis revealed an open reading frame translating to a 259-amino acid sequence. The cloned gene was designated cyt2Aa2. This gene was highly expressed in Escherichia coli as inclusion bodies that could be solubilized in 50 m M Na(2)CO(3), pH 10.5. Activation of the solubilized protoxin by proteinase K (1% wt/wt, proteinase K/protoxin) yielded the active fragment of about 23 kDa. Cyt2Aa2 showed high hemolytic activity against sheep erythrocytes (hemolytic end- point 0.25 microgram/ml) and was toxic to Culex quinquefasciatus and Aedes aegypti larvae (LC(50) 0.5-1.0 microgram/ml).     

Larvicidal activity of essential oils from Brazilian plants against Aedes aegypti L

Aedes aegypti L. is the major vector of dengue fever, an endemic disease in Brazil. In an effort to find effective and affordable ways to control this mosquito, the larvicidal activities of essential oils from nine plants widely found in the Northeast of Brazil were analyzed by measurement of their LC50. The essential oils were extracted by steam distillation and their chemical composition determined by GL-chromatography coupled to mass spectroscopy. The essential oils from Cymbopogon citratus and Lippia sidoides, reported in the literature to have larvicidal properties against A. aegypti, were used for activity comparison. The results show that Ocimum americanum and Ocimum gratissimum have LC50 of 67 ppm and 60 ppm respectively, compared to 63 ppm for L. sidoides and 69 ppm for C. citratus. These results suggest a potential utilization of the essential oil of these two Ocimum species for the control of A. aegypti.     

A new black fly isolate of Bacillus thuringiensis autoagglutinating strain highly toxic to Simulium pertinax (Kollar) (Diptera, Simuliidae) larvae

Formulations containing the entomopathogenic Bacillus thuringiensis serovar israelensis strain IPS-82 has been widely applied for mosquito control around the world. Strain IPS-82 is highly active against Aedes aegypti but less active against other well-known vectors such as Culex quinquefasciatus and Simulium spp. larvae. Eighteen strains of B. thuringiensis were isolated from Simulium pertinax larvae naturally occurring in rivers of Southeast Brazil with one demonstrating special toxic effects. Simulated field tests against S. pertinax larvae showed that the native Brazilian autoagglutinating B. thuringiensis (LFB-FIOCRUZ 1035) has an LC50 at least 25 times lower than the standard IPS-82 strain. The same bacterial preparation was also tested against Ae. aegypti larvae in laboratory trials and the LC50 values obtained with LFB-FIOCRUZ 1035 were at least three times lower than the one for the IPS 82 strain. The results indicate that this strain is more toxic than the standard B. thuringiensis serovar israelensis (H14) in the two Dipteran species tested. It is noteworthy that differences between LC50 values were more pronounced in S. pertinax larvae, the source of the original isolation.