Biosynthesis of 130-kilodalton mosquito larvicide in the cyanobacterium Agmenellum quadruplicatum PR-6

The 130-kilodalton mosquito larvicidal gene, cloned from Bacillus thuringiensis var. israelensis, was introduced into the cyanobacterium Agmenellum quadruplicatum PR-6 by plasmid transformation. Transformed cells synthesized 130-kilodalton delta-endotoxin protein and showed mosquito larvicidal activity. Results demonstrate a potential use of a cyanobacterium for biological control of mosquitoes.     

Immunological relationships among proteins making up the Bacillus thuringiensis subsp. israelensis crystalline toxin.

The immunological relationships among the proteins of the mosquito larvicidal toxin produced by Bacillus thuringiensis subsp. israelensis have been investigated by using polyclonal antisera specific for the 28-, 70-, and 135-kilodalton proteins. Each of these proteins was immunologically distinct. There was no cross-reaction among the three proteins and the two non-homologous antisera. Treatment of toxin proteins with larval gut enzymes for 20 h identified protease-resistant domains at approximately 65, 38, and 22 kilodaltons. Similar domains were generated by treatment with trypsin and chymotrypsin. Our immunological and kinetic data indicate that the 28-kilodalton protein is degraded successively to protein bands at 26, 25, 23, and 22 kilodaltons, the 70-kilodalton protein is degraded to a protein at 38 kilodaltons, and the 135-kilodalton protein is degraded successively to protein bands at 94, 72, and, probably, 65 kilodaltons. Solubilized toxin possesses two biological activities, larvicidal and general cytolytic (hemolytic). We used nondenaturing gel electrophoresis to show that the hemolytic activity resides in the 28-kilodalton protein. However, higher-molecular-weight proteins are required to achieve the level of toxicity observed in intact toxin.     

Immunological relationships among proteins making up the Bacillus thuringiensis subsp. israelensis crystalline toxin

The immunological relationships among the proteins of the mosquito larvicidal toxin produced by Bacillus thuringiensis subsp. israelensis have been investigated by using polyclonal antisera specific for the 28-, 70-, and 135-kilodalton proteins. Each of these proteins was immunologically distinct. There was no cross-reaction among the three proteins and the two non-homologous antisera. Treatment of toxin proteins with larval gut enzymes for 20 h identified protease-resistant domains at approximately 65, 38, and 22 kilodaltons. Similar domains were generated by treatment with trypsin and chymotrypsin. Our immunological and kinetic data indicate that the 28-kilodalton protein is degraded successively to protein bands at 26, 25, 23, and 22 kilodaltons, the 70-kilodalton protein is degraded to a protein at 38 kilodaltons, and the 135-kilodalton protein is degraded successively to protein bands at 94, 72, and, probably, 65 kilodaltons. Solubilized toxin possesses two biological activities, larvicidal and general cytolytic (hemolytic). We used nondenaturing gel electrophoresis to show that the hemolytic activity resides in the 28-kilodalton protein. However, higher-molecular-weight proteins are required to achieve the level of toxicity observed in intact toxin.     

Antitumor Activity of an Alkali Extract of Clostridium saccharoperbutylacetonicum Cells

Bacillus thuringiensis var. israelensis produces 130-kDa proteins which are toxic to mosquito larvae. The ISRH4 gene encoding 1,180 amino acids of the 130-kDa insecticidal protein was fused with lac Z′ on a plasmid, pUC19, and sequentially deleted from the C-terminus to construct a series of deletion mutants. All the deletion mutant genes directed the production of truncated ISRH4 proteins fused with the α-complementing fragment of β-galactosidase in Escherichia coli cells in the presence of isopropyl β-d-thiogalactopyranoside. Analysis of the mosquito larvicidal activity of deletion mutant proteins revealed that the N-terminal 29 amino acids and the C-terminal 485 amino acids could be removed without loss of the activity.     

Insecticidal activities of leaf essential oils from Cinnamomum osmophloeum against three mosquito species

The larvicidal activities of leaf essential oils and their constituents from six chemotypes of indigenous cinnamon (Cinnamomum osmophloeum Kaneh.) trees were evaluated against three mosquito species. Results of larvicidal tests demonstrated that the leaf essential oils of cinnamaldehyde type and cinnamaldehyde/cinnamyl acetate type had an excellent inhibitory effect against Aedes albopictus larvae, and their LC(50) values in 24h were 40.8 microg/ml (LC(90)=81.7 microg/ml) and 46.5 microg/ml (LC(90)=83.3 microg/ml), respectively. Results of the 24-h mosquito larvicidal assays also showed that the effective constituents in leaf essential oils were trans-cinnamaldehyde and benzaldehyde and that the LC(50) values of these constituents against A. albopictus larvae were below 50 mug/ml. In addition, cinnamaldehyde type leaf essential oil and trans-cinnamaldehyde have also exhibited great larvicidal performance against Culex quinquefasciatus and Armigeres subalbatus larvae. Comparisons of mosquito larvicidal activity of trans-cinnamaldehyde congeners revealed that alpha-methyl cinnamaldehyde, benzaldehyde, and trans-cinnamaldehyde exhibited strong mosquito larvicidal activity.     

Larvicidal activity of tectoquinone isolated from red heartwood-type Cryptomeria japonica against two mosquito species

Mosquito larvicidal activities of methanolic extracts from different plant parts of red heartwood-type Cryptomeria japonica D. Don against the fourth-instar larvae of Aedes aegypti and Aedes albopictus were examined. Results of mosquito larvicidal tests demonstrated that the n-hexane fraction of C. japonica sapwood methanolic extract had an excellent inhibitory effect against the larvae of A. aegypti and A. albopictus and its LC50 values were 2.4 and 3.3 microg/ml, respectively, in 24h. Following the bioactivity-guided fractionation procedure, the active constituent isolated from C. japonica sapwood was characterized as tectoquinone by spectroscopic analyses. The LC50 values of tectoquinone against A. aegypti and A. albopictus in 24h were 3.3 and 5.4 microg/ml, respectively. In addition, comparisons of mosquito larvicidal activity of anthraquinone congeners demonstrated that anthraquinone skeleton with a methyl group at C-2 position, such as tectoquinone, exhibited the strongest mosquito larvicidal activity. Results of this study show that the methanolic extract of C. japonica sapwood may be considered as a potent source and tectoquinone as a new natural mosquito larvicidal agent.     

Variations in insecticidal activity and chemical compositions of leaf essential oils from Cryptomeria japonica at different ages

The larvicidal effects of the essential oils extracted from the leaves of Cryptomeria japonica at different ages (58, 42, and 26 years old) against 2 mosquito species, Aedes aegypti and Aedes albopictus, were studied. The analysis of major constituents of these essential oils was also investigated. Results obtained from the larvicidal tests, using essential oil from the leaves of 58-year-old C. japonica was found to be most effective against both A. aegypti and A. albopictus larvae, indicating tree age has significant influence on mosquito larvicidal activity. In addition, the eleven pure constituents from C. japonica leaf essential oil were also tested individually against the two mosquito larvae. Among them, alpha-terpinene, gamma-terpinene, p-cymene, 3-carene, terpinolene, and beta-myrcene shows strong larvicidal effect against the two mosquito larvae. Among these pure constituents, 3-carene exhibits the best larvicidal effect against A. aegypti and terpinolene shows an excellent inhibitory action against A. albopictus larvae. The results of this study show that the leaf essential oil and its effective constituents might be considered as a potent source for the production of fine natural larvicides.     

Enhanced expression of a second mosquito larvicidal gene fromB.sphaericus 1593M inE.coli

Summary Enhanced expression of a second mosquito larvicidal gene fromB.sphaericus 1593M inE.coli has been achieved by the recloning of the DNA fragment encoding for larvicidal activity previously reported by us, in a pMal vector system. The potency of this recombinant strain was only 10 fold lower than the parentalB.sphaericus 1593M strain. The protein encoded was different from the previously reported larvicidal gene products ofB.sphaericus. Neverthelesss, this protein is recognized by the antiserum raised against crystal proteins. This result has indicated the presence of multiple mosquito larvicidal genes inB.sphaericus, a situation similar to that encountered withB.thuringiensis toxins.     

Chemical compositions and larvicidal activities of leaf essential oils from two eucalyptus species

In the current study, the mosquito larvicidal activity of leaf essential oils and their constituents from two eucalyptus species (Eucalyptus camaldulensis and Eucalyptus urophylla) against two mosquito species, Aedes aegypti and Aedes albopictus, was investigated. In addition, the chemical compositions of the leaf essential oils were analyzed using gas chromatography-mass spectrometry. Results from the larvicidal tests revealed that essential oil from the leaves of E. camaldulensis had an excellent inhibitory effect against both A. aegypti and A. albopictus larvae. The 12 pure constituents extracted from the two eucalyptus leaf essential oils were also tested individually against two mosquito larvae. Among the six effective constituents, alpha-terpinene exhibits the best larvicidal effect against both A. aegypti and A. albopictus larvae. Results of this study show that the leaf essential oil of E. camaldulensis and its effective constituents might be considered as a potent source for the production of fine natural larvicides.     

Insect growth regulatory and larvicidal activity of chalcones against Aedes albopictus

Widespread use of chemical insecticides has resulted in development of insect resistance and natural products with biological activities could become an attractive alternative to control insect pests. In order to find more effective insecticides for controlling mosquito, various mosquitocidal compounds are studied. Recently, juvenile hormone antagonists (JHANs) have been found to be to safe and effective insecticides for control of mosquito. In order to identify novel insecticidal compounds with JHAN activity, several chalcones were surveyed on their JHAN activities and larvicidal activities against Aedes albopictus larvae. Among them, 2′‐hydroxychalcone and cardamonin showed high levels of JHAN and mosquito larvicidal activities. These results suggested that chalcones with JHAN activity could be useful for control of mosquito larvae.     

Synthesis,larvicidal activity,and SAR studies of new benzoylphenylureas containing oxime ether and oxime ester group

A series of new structural benzoylphenylureas (BPUs) containing oxime ether and oxime ester group were designed and synthesized. The larvicidal activities against Oriental armyworm and mosquito of these benzoylphenylureas were evaluated and the result of bioassay displayed specific structure–activity relationship (SAR). Most of the compounds exhibited excellent larvicidal activities against Oriental armyworm and mosquito. Interestingly, some compounds showed different structure–activity relationship towards diamondback moth, beet armyworm, and corn borer although three tested insects all belong to the same insect order.     

Affinity purification of a 65-kilodalton parasporal protein from Bacillus thuringiensis PG-14 that shows mosquitocidal activity

By using antibody-mediated affinity chromatography, a highly mosquito larvicidal but nonhemolytic fraction was obtained from alkali-solubilized, silkworm (Bombyx mori) larval gut juice-treated parasporal inclusions of Bacillus thuringiensis strain PG-14 (serotype 8a : 8b). This fraction contained a 65-kDa protein only but not a 25-kDa protein, the main component in the flow through fraction unbound to the affinity column. The 25-kDa protein purified from the unbound fraction by CM-cellulose chromatography demonstrated a high hemolytic activity against sheep red blood cells but very low mosquito larvicidal activity.     

Enhanced and constitutive expression of mosquito larvicidal protein genes of Bacillus sphaericus under leaky regulation of T7 expression system in Escherichia coli

Expression of the mosquito larvicidal genes coding for 51 and 42kDa proteins from Bacillus sphaericus cloned into E. coli, under the control of T7RNA polymerase promoter system, resulted in a high level of larvicidal activity of the recombinant. The clone MS16 expressed the binary toxins at high level even under uninduced conditions. This indicates that the leaky regulation of the T7RNA polymerase gene was responsible for the constitutive expression of mosquito larvicidal proteins (MLP). The economic advantage of producing the MLP in a rapidly-growing and sugar-utilising organism such as E. coli was demonstrated in a batch cultivation where production of the MLP was growth-associated.     

Mosquito larvicidal activity of transgenic Anabaena PCC 7120 expressing toxin genes from Bacillus thuringiensis subsp. israelensis

Genes encoding the mosquito larvicidal toxins Cry4Aa, Cry11Aa, Cyt1Aa and the regulatory P20 from Bacillus thuringiensis subsp. israelensis were introduced into the nitrogen-fixing, filamentous cyanobacterium Anabaena PCC 7120 for expression under control of two strong promoters P(psbA) and P(A1). The clone pRVE4-ADRC displayed toxicity against fourth-instar larvae of Aedes aegypti, the highest ever achieved in cyanobacteria. It was about 2.5-fold more toxic than the respective clone without cyt1Aa [Wu et al., Appl. Environ. Microbiol. 63 (1997) 4971-4975]. Cyt1Aa synergized the combination of Crys by about five-fold. Consistently, the lethal times exerted by pRVE4-ADRC were also reduced (it killed exposed larvae more quickly). This clone may become a useful biological control agent which reduces the probability of resistance development in the target organisms [Wirth et al., Proc. Natl. Acad. Sci. USA 94 (1997) 10536-10540].     

The 23-kilodalton CytB protein is solely responsible for mosquito larvicidal activity ofBacillus thuringiensis serovarkyushuensis

To identify the actual mosquito larvicidal toxin(s) inBacillus thuringiensis serovarkyushuensis, we conducted a comparative toxicity study by a combination of ion-exchange chromatographies and SDS-PAGE. Among the proteins contained in solubilized and protease-treated inclusions, only the 23-kDa protein, a cytolysin referable to the CytB protein, exhibited mosquitocidal activity. The LC₅₀ value of this protein was 51.4 g/ml for the third-instar larvae of the mosquito,Aedes aegypti. The fractions of other inclusion proteins showed no mosquito larvicidal activity at protein concentrations up to 1000 g/ml.     

Larvicidal activity of saponins from Balanites aegyptiaca callus against Aedes aegypti mosquito

Seeking an alternative approach for producing a larvicidal product from Balanites aegyptiaca plants, callus was produced from in vitro cultures of root explants and its larvicidal activity against Aedes aegypti mosquito larvae was evaluated. Concentrations of 0, 50, 100, 500, 1000, and 1500 ppm of saponins from the root-derived callus of B. aegyptiaca were used to determine larvicidal effects and consequent effect on adult emergence. A dose-dependent effect was observed. In a chronic mortality assessment (after 7 days of exposure), concentrations of 500 ppm or greater killed 100% of the test larvae population. Fifty parts per million showed no difference in larval mortality compared to the control (0 ppm); however, this concentration allowed one-fourth of the adult emergence of the control treatment. These results suggest that saponins from in vitro cultures of the root explant of B. aegyptiaca can be used as a larvicidal agent against A. aegypti larvae.     

Insecticidal, repellent and oviposition-deterrent activity of selected essential oils against Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus

Essential oils extracted from 10 medicinal plants were evaluated for larvicidal, adulticidal, ovicidal, oviposition-deterrent and repellent activities towards three mosquito species; Anopheles stephensi, Aedes aegypti and Culex quinquefasciatus. The essential oils of Juniperus macropoda and Pimpinella anisum were highly effective as both larvicidal and ovicidal. The essential oil of P. anisum showed toxicity against 4th instar larvae of A. stephensi and A. aegypti with equivalent LD95 values of 115.7 microg/ml, whereas it was 149.7 microg/ml against C. quinquefasciatus larvae. Essential oils of Zingiber officinale and Rosmarinus officinalis were found to be ovicidal and repellent, respectively towards the three mosquito species. The essential oil of Cinnamomum zeylanicum resulted into highest repellent (RD95) values of 49.6, 53.9 and 44.2 mg/mat against A. stephensi, A. aegypti and C. quinquefasciatus, respectively apart from oviposition-deterrent potential.     

Chicory (Cichorium intybus) and wormwood (Artemisia absinthium) extracts exhibit strong larvicidal activity against mosquito vectors of malaria,dengue fever,and filariasis

Vector-borne diseases transmitted by mosquitoes cause globally important diseases such as malaria, dengue fever, and filariasis. The incidence of these diseases can be reduced through mosquito control programs but these control programs currently rely on synthetic insecticides that can impact the environment, and has selected widespread mosquito resistance. Environment friendly and biodegradable natural insecticides discovered in plants offer an alternative approach to mosquito control. Here, we investigated extracts from root or aerial parts of Chicory (Cichorium intybus) and wormwood (Artemisia absinthium) against the early 4th instar larvae of Anopheles stephensi (malaria vector), Aedes aegypti (dengue fever vector), and Culex quinquefasciatus (filariasis vector). The root and aerial parts extracts of A. absinthium and C. intybus at 200, 100, 50, 25 and 12.5?ppm caused significant mortality of the tested mosquito species. Root extracts exhibited higher larvicidal activity that aerial part extracts. The highest larvicidal activity was recorded in methanol extract of roots of C. intybus with LC50?=?66.16, 18.88 and LC¬90?=?197.56, 107.16?ppm for An. stephensi; LC50?=?78.51, 40.15 and LC90?=?277.31, 231.28?ppm for Ae. aegypti and LC50?=?103.99, 64.56 and LC¬90?=?314.04, 247.54?ppm for Cx. quinquefasciatus. These results reveal potent mosquito larvicidal activity against vectors of malaria, dengue fever, and filariasis is present in extracts of chicory and wormwood.     

Growth,sporulation and larvicidal activity of Bacillus sphaericus

Summary A new medium (MBS) for optimal sporulation of Bacillus sphaericus was defined. With the two main mosquito pathogenic strains grown in this medium, 1593-4 and 2297, highest cell and spore yields were obtained, concomitantly with an highest larvicidal activity against Culex pipiens. Study of both strains asporulated mutants showed a decrease in larvicidal power. After plasmid curing treatments, toxicity of strain 1593-4 did not decrease, neither toxic parasporal inclusion bodies of strain 2297 disappear.     

Identification of the peptides of the crystals of Bacillus thuringiensis var israelensis involved in the mosquito larvicidal activity

Tryptic digestion of the proteins from the purified crystals of B.thuringiensis var israelensis resulted in the decline of high molecular weight peptides without the loss of mosquito larvicidal activity, measured after immobilization of the digests with DEAE- Sephadex A 50 beads. Amongst the peptides generated (less than 44 kDa), a 21 kDa peptide was immunoreactive to the crystal antiserum. Analysis of the peptides released from spores of the toxic (Cry+) and non-toxic (Cry-) strains has revealed a pattern in which only the 26kDa peptide was missing in the Cry-strain. Sporulation and crystal formation were dissociated by the addition of the antibiotic netropsin, which could also inhibit the crystal assembly, without considerable decrease of the larvicidal activity and retention of the 26kDa peptide. These results implicate the 26kDa peptide in the larvicidal action.