Mosquitocidal Activity Research Articles

Bacillus thuringiensis Cry2Ab is active on Anopheles mosquitoes: single D block exchanges reveal critical residues involved in activity

Cry2Aa exhibits dual activity to Lepidoptera and Diptera. Cry2Ab differs in amino acid sequence from Cry2Aa by 13% and has shown significant lepidopteran activity, but no mosquitocidal activity. Previous studies implicate 23 Cry2Aa specificity-conferring residues of domain II, which differ in Cry2Ab. Nine residues are putatively involved in conferring Cry2Aa dipteran specificity. To explore Cry2Ab dipteran toxicity, site-directed mutagenesis was employed to exchange Cry2Ab residues with Cry2Aa D (dipteran) block residues. Cry2Ab wild type demonstrated high toxicity (LC(50) of 540 ng mL(-1)) to Anopheles gambiae, but not to Aedes or Culex, within a 24-h time period. Cry2Ab should be reclassified as a dual active Cry toxin. Cry2Ab mutagenesis revealed critical residues for Cry2Ab protein function, as well as enhanced activity against the malarial mosquito, An. gambiae.

Chitinolytic enzymes from the newly isolated Aeromonas hydrophila SBK1: study of the mosquitocidal activity

Aeromonas hydrophila SBK1 (GenBank accession no. HM802878.1), a potent chitinolytic bacterium, was isolated from a pool of 30 chitinolytic isolates. The isolate showed higher chitinolytic activity in respect to clear zone to colony size ratio of 2.15. Maximum production of chitinolytic enzymes, viz., β-N-acetyl-glucosaminidase and chitinase (specific activity 655.3 and 71.6 U mg−1, respectively) by A. hydrophila SBK1 was observed in the synthetic media, containing (w/v)-colloidal chitin, 4.0%; peptone, 0.3%; phosphate, 0.3% (0.15% of each KH2PO4 and K2HPO4); NaCl, 0.25%; MgSO4, 0.05%; KCl, 0.05%; pH 7.0 and at 35°C after 72 h of incubation. Both carbon-to-nitrogen (C/N) and carbon-to-phosphate (C/P) ratio of 13.33 were found optimum for chitinase production. Enzyme productivity increased about twofold in optimized culture condition in respect to its un-optimized state. The crude enzyme showed optimum activity against Culex quinquefasciatus larvae in native water at pH 7.0 and 35°C (LD50 0.60 U ml−1 at 48 h). Therefore, the studied chitinases can be used as an effective mosquitocidal agent.

Screening for adulticidal bioactivity of South African plants against Anopheles arabiensis

BackgroundThis study was conducted to evaluate whether a selection of South African ethnomedicinal plants included in this study displayed insecticidal properties when screened against adult stages of the mosquito.Methods381 crude extracts of 80 plant taxa in 42 families were sprayed onto ceramic tiles and screened using the cone bio-assay method for insecticide efficacy testing. Blood-fed, female Anopheles arabiensis mosquitoes were exposed to the treated tiles for a period of sixty minutes. Mosquito mortality was monitored for twenty-four hours.ResultsOf all the extracts analysed, the highest activity was observed in Ptaeroxylon obliquum (Ptaeroxylaceae) and Pittosporum viridiflorum (Pittosporaceae), a single extract from each, exhibiting more than 50% mortality. A large proportion (81.63%) of the extracts tested displayed low levels of mosquitocidal activity. The remainder of the extracts (17.85%) exhibited no bioactivity (0% mortality).ConclusionsThe screening results have shown that in accordance with WHO standards, none of the crude extracts tested had exhibited greater than 60% mortality against the adult stages of the malaria vector Anopheles arabiensis.

Insecticidal activity of different extracts from Scrophularia canina L. against Culex pipiens molestus Forskal (Diptera, Culicidae)

The insecticidal activity of different extracts (aqueous, methyl alcohol, hexane and petroleum ether) of the aerial part of Scrophularia canina against the second and fourth-instar larvae and adult females of Culex pipiens molestus was investigated. The larvicidal activity of all the extracts was tested in the dose range from 7.8 to 1000 ppm. The highest toxicity was exhibited by the petroleum ether extract against second-instar larvae (48 h LC50 = 23.5 ppm) and by the hexane extract against fourth-instar larvae (48 h LC50 = 23.6 ppm). Methyl alcohol and aqueous extracts did not show any larvicidal activity. Sub-lethal doses of petroleum ether and hexane extracts induced increasing mortalities during 13 days after treatment but did not affect the duration of larval and pupal stages. In persistence tests, the hexane extract retained a satisfactory larvicidal activity after a 10-day period of test solutions incubation. Topical treatment of adult females with doses from 0.25 to 8 μg per mg of female body weight of different extracts showed a similar toxicity for the hexane (24 h LD50 = 1.7 μg mg−1) and petroleum ether (24 h LD50 = 1.8 μg mg−1) extracts which were significantly more toxic than methyl alcohol extract (4.2 μg mg−1). Aqueous extract did not induce adult mortality. The marked mosquitocidal activity of petroleum ether and hexane extracts of the aerial part of S. canina against different life stages of C. pipiens molestus is promising to develop effective alternatives to synthetic insecticides.

Bacillus amyloliquefaciens: A mosquitocidal bacterium from mangrove forests of Andaman & Nicobar islands, India

Samples collected from the mangrove forests of Andaman & Nicobar islands yielded a mosquitocidal bacterium, whose extracellular metabolite(s) exhibited mosquito larvicidal and pupicidal activity. The bacterium was isolated using standard microbiological methods and identified using classical biochemical tests and rpoB gene sequences. The mosquitocidal bacterium was identified as Bacillus amyloliquefaciens. Mosquitocidal metabolite(s) was separated from the culture supernatant of the bacterium and its efficacy against the larval and pupal stages of different species of mosquitoes was determined in terms of LC50 and LC90. Mosquito larvicidal activity in terms of LC50 against Anopheles stephensi, Culex quinquefasciatus and Aedes aegypti was respectively, 26.4μg, 22.2μg and 20.5μg/ml and its pupicidal activity was 4.4μg, 8.2μg and 14.5μg/ml respectively. The mosquitocidal metabolite(s) was found to be a biosurfactant. This is the first report of the mosquitocidal activity of B. amyloliquefaciens and it is a new weapon which can be added to the array of microbial agents for use against mosquitoes.

Mosquitocidal activity of two Bacillus bacterial endotoxins combined with plant oils and conventional insecticides

The full whole culture (FWC), containing parasporal protein toxins of Bacillus thuringiensis israelinsis (Bti) and Bacillus sphaericus 2362 (Bs), either singly or in combination with plant oils and commercial insecticides, was tested against larval and adult stages of Culex pipiens mosquitoes under controlled laboratory conditions. In terms of LC 50 values recorded after 24, 48, 72 and 96 h, the bacterial toxins showed high potency towards both larvae and adults of mosquitoes in a dose-dependent manner. Generally, the Bti toxin seemed to be more potent than the Bs toxin. For example, the Bti toxin showed a 24 h LC 50 of 8.2 ppm against mosquito larvae compared to 13.6 ppm for the Bs toxin. In the adult bioassay, the obtained 24 h LC 50 values were 0.064 and 0.085 mg/cm 2, respectively for the two bacterial toxins. The bacterial toxins mixed with plant oils or insecticides at equitoxic doses (e.g., LC 25 values) mostly showed potentiation effects, either against larvae or adults of the tested insect. Among a total of 14 paired mixtures, only the joint action estimated for the mixture of malathion + Bti or Bs was accounted as additively. Combining Bti or Bs endotoxins at LC 0 with different plant oils and insecticides at LC 50 concentration levels each, has resulted in considerable synergism against either larvae or adults. In the case of larval bioassays, the maximum synergistic factor (SF) obtained (ca. 2.0) was entitled to the mixture of Bti + spinosad. In the adult bioassays, the mixtures containing Curcuma longa or Melia azedarach oil extracts with Bti or Bs toxins achieved a SF accounted to 2.0. The results of the present study may be considered as an additional contribution to the area of joint toxicity of biocidal agents combining bacterial toxins, plant oils and traditional insecticides. The reached findings may encourage future research to elucidate its performance under practical field conditions.

Insecticidal activity of the essential oil from fruits and seeds of Schinus terebinthifolia Raddi against African malaria vectors.

BackgroundAlternative insecticides for the control of malaria and filarial vectors are of paramount need as resistance is increasing among classes of insecticides currently in use in the public health sector. In this study, mosquitocidal activity of Schinus terebinthifolia essential oil against Anopheles gambiae s.s., An. arabiensis and Culex quinquefasciatus was assessed in laboratory, semi- field and full- field conditionsMethodTwenty third instar larvae of both Anopheles gambiae s.s. and Cx. quinquefasciatus were exposed to different dosages of plant extract in both laboratory and semi- field environments. Observation of the mortality response was assessed at intervals of 12, 24, 48 and 72 hours. Adult semi- gravid female mosquitoes were exposed to papers treated with S. terebinthifolia and compared with WHO standard paper treated with alphacypermethrin (0.05%).ResultsGas chromatography, coupled to mass spectrometry, identified 15 compounds from S. terebinthifolia extracts, the most abundant identified compound was δ-3-carene (55.36%) and the least was γ-elemene (0.41%). The density of the oil was found to be 0.8086 g/ml. The effective dosages in the insectary ranged from 202.15 to 2625.20 ppm and were further evaluated in the semi- field situation. In the laboratory, the mortality of Cx. quinquefasciatus ranged from 0.5 to 96.75% while for An. gambiae s.s it was from 13.75 to 97.91%. In the semi- field experiments, the mortality rates observed varied for both species with time and concentrations. The LC50 and LC95 value in the laboratory was similar for both species while in the semi- field they were different for each. In wild, adult mosquitoes, the KT50 for S. terebinthifolia was 11.29 minutes while for alphacypermethrin was 19.34 minutes. The 24 hour mortality was found to be 100.0% for S. terebinthifolia and 75.0% for alphacypermethrin which was statistically significant (P < 0.001).ConclusionThe efficacy shown by essential oils of fruits and seeds of S. terebinthifolia has given an opportunity for further investigation of individual components of these plant extracts and to evaluate them in small- scale field trials.

Enhanced activity of an insecticidal protein, trypsin modulating oostatic factor (TMOF), through conjugation with aliphatic polyethylene glycol

Trypsin modulating oostatic factor (TMOF), a decapeptide (Tyr-Asp-Pro-Ala-Pro(6)) isolated from the ovaries of the adult yellow fever mosquito, Aedes aegypti, regulates trypsin biosynthesis. TMOF per os is insecticidal to larval mosquitoes and a good model for the development of technologies to enhance protein insecticide activity by reduced catabolism and/or enhanced delivery to the target. TFA-TMOF-K (TFA = trifluoro acetyl) allowed the specific conjugation of monodispersed, aliphatic polyethylene glycol (PEG) to the amino group of lysine-producing TMOF-K-methyl(ethyleneglycol)(7)-O-propionyl (TMOF-K-PEG(7) P). The addition of lysine to TMOF reduced its per os larval mosquitocidal activity relative to the parent TMOF, but conjugation of TMOF-K with methyl(ethyleneglycol)(7)-O-propionyl increased its toxicity 5.8- and 10.1-fold above that of TMOF and TMOF-K for Ae. aegypti. Enhanced insecticidal activity was also found for larval Ae. albopictus and for neonates of Heliothis virescens and Heliocoverpa zea. Only TMOF-K was found by MS/MS in the hemolymph for H. virescens fed on TMOF-K-PEG(7) P. No TMOF, TMOF-K or PEGylated TMOF-K was detected in the hemolymph after topical applications. This research suggests that aliphatic PEG polymers can be used as a new method for increasing the activity of insecticidal proteins.

The dual-activity insecticidal protein, Cry2Aa, does not enhance the mosquitocidal activity of Bacillus thuringiensis subsp. israelensis

Cry2Aa, one of the major insecticidal proteins produced by Bacillus thuringiensis subsp. kurstaki HD1, is known to be active against both lepidopteran and dipteran larvae. In order to determine whether Cry2Aa could enhance or synergize the mosquitocidal activity of B. thuringiensis subsp . israelensis, we constructed a plasmid vector that harbored the cry2Aa operon and transformed crystalliferous and acrystalliferous strains of this bacterium. The wild-type B. thuringiensis subsp . israelensis, a recombinant B. thuringiensis subsp . israelensis producing Cry2A along with its native major mosquitocidal proteins, and a recombinant B. thuringiensis subsp . israelensis producing Cry2Aa alone were tested against three major mosquito species — Aedes aegypti, Anopheles gambiae and Culex quinquefasciatus. Our results demonstrated that Cry2Aa does not synergize or enhance the mosquitocidal activity of B. thuringiensis subsp . israelensis against these important vectors of disease.

Potent Insecticidal Secondary Metabolites Isolated from Acanthus montanus

Acanthus montanus (Nees) T. Anders. (Family: Acanthaceae) is a small shrub with sparse branches and soft stems, widespread in Africa, the Balkans, Romania, Greece and Eastern Mediterranean [1]. Documented evidence showed that the leaves of the plant possess spasmolytic, analgesic, anti-inflammatory and anti-pyretic [2, 3] activities. In our ongoing research project, aimed at identifying new natural compounds with insecticidal activity, the alcohol extract of the aerial parts of A. montanus were the most active. Phytochemical study of the aerial parts of A. montanus has resulted in isolation of nine compounds, eight of which exhibit variable degrees of insecticidal activity. β-sitosterol-3-O-β-D-glucoside 2 exhibited potent mosquitocidal activity (100% mortality) against adult Aedes aegypti at 3.1ppm concentration, followed by palmitic acid 1 (90%), linaroside 3 (80%), and acetoside 9 (70%) respectively. It is noteworthy that this is the first report of insecticidal activity of β-sitosterol-3-O-β-D-glucoside, linaroside and acetoside.

Isolation of novel Bacillus species showing high mosquitocidal activity against several mosquito species

Two novel mosquitocidal bacteria, VB17 and VB24, identified as new Bacillus species were isolated from dead mosquito larvae obtained in Florida aquatic habitats. Gas chromatographic analysis of fatty acid methyl esters (GC-FAME) and 16S rRNA sequencing indicated that VB24 is closely related to Bacillus sphaericus whereas VB17 does not have a close relationship with either Bacillus thuringiensis or B. sphaericus. Both isolates were significantly more active than B. sphaericus 2362 against Aedes taeniorhynchus, Anopheles quadrimaculatus, Culex quinquefasciatus larvae, and as active as B. sphaericus 2362 against Anopheles gambiae. Interestingly, however, both were not active against Aedes aegypti larvae, indicating some level of insecticidal specificity.

Sustained-release biopolymer based formulations for Bacillus sphaericus Neide ISPC-8

Sustained-release biopolymer based formulations for an indigenous Bacillus sphaericus Neide ISPC-8 strain were developed and evaluated under laboratory conditions. These formulations were assessed for residual activity, response to UV-B radiation and shelf-life. The mosquitocidal activity of tested formulations showed a varied residual activity pattern against Culex quinquefasciatus Say larvae. The residual activity of unformulated spore-crystal suspension drastically decreased after 4 days whereas the formulated preparations persisted until 28 days. All the formulations except sodium alginate-based formulation showed more than 95% mortality within 48 h. Although the sodium alginate-based formulation showed delayed initial mortality, it persisted for a longer period of time (42 days). Based on the persistence studies, promising formulations based on sodium alginate, gelatin and acacia gum were selected for further evaluation. The respective LC50 values of these three formulations were 0.085, 0.117, and 0.196 mg/l against C. quinquefasciatus after 48 h. The biopolymers combined with Congo red protected the spore-crystal moiety from UV-B radiation. These formulations were found to be stable up to 18 months when stored at room temperature. Thus, these biopolymers could serve as suitable ingredients for development of biopesticide based on B. sphaericus ISPC-8.

The gene expression of caspasses is up-regulated during the signaling response of Aedes caspius against larvicidal bacteria

Our current knowledge on the key molecular mechanisms and cognate signaling transduction, by the δ -endotoxin-mediated mosquitoticidal effects, associated with exposure to Bacillus thuringiensis ( Bt ) and Bacillus sphaericus ( Bs ), is limited. Moreover, this observed mosquitocidal activity that is related to program cell death is largely unknown. Therefore, in an attempt to answer this question, the current study was primarily sought to provide evidence as to the molecular mechanism of mortality in Bt/B s infected Aedes caspius mosquito larvae. Thus, the impact of Bt and Bs treatment on the expression of some selected apoptosis related caspase genes in A. caspius mosquito larvae was investigated, via quantitative reverse-transcriptase PCR (qRT-PCR). Mosquito larvae were collected from natural water niches. Larvae were grown to adult stage and were subsequently identified as A. caspius at Natural History Museum, London, UK. Remarkably, light and transmission electron microscopy studies of the midgut epithelial tissues revealed that both Bt and Bs brought about significant histopathological effects. Moreover, this treatment resulted in severe destruction at the sub-cellular organelle level for the mitochondria. Interestingly, qRT-PCR studies revealed that the treatment of A. caspius mosquito larvae with both Bt and Bs caused a significant up-regulation in the transcription level of all caspase genes under study, namely: CASPS17 , CASPS18 , CASPS19, CASPS20 and CASPS21 . The results are discussed in the light of our current understanding of the signaling transduction pathway of apoptosis in insects and mosquitoes and the putative role of caspases gene expression in response to the treatment of A. caspius mosquito larvae with larvicidal bacteria. Keywords: Aedes caspius, Bacillus thuringenesis, Bacillus sphaericus , apoptosis, caspase, larvicidal bacteria

Isolation, characterization and evaluation of mosquitocidal activity of Lysinibacillus strains obtained from Culex pipiens larvae

Three strains—C107, C207, and C307—of spore-forming Gram-positive motile rod-shaped bacteria were isolated from dead larvae of Culex pipiens in La Plata city, Argentina. The three bacterial strains have different phenotypic and molecular characteristics. A comparative analysis of their 16S rRNA gene sequences and phylogenetic analysis demonstrated that isolates C107 and C207 were related to reference strains of Lysinibacillus sphaericus, while C307 was related to Bacillus licheniformis. The cytomorphology, biochemical characterization, and phylogenetic relatedness corroborated these respective group assignments. The isolated bacterial strain exhibited the same PCR amplified pattern for the binA, binB, and mtx genes as did the reference strains used. These bacterial strains presented different pathogenic actions among the following mosquito species tested: Culex pipiens, Aedes aegypti, Culex dolosus, Culex apicinus, Ochlerotatus albifasciatus, and Anopheles albitarsis. Only isolates C107 and C207 exhibited mosquitocidal activity. Culex pipiens was the species most susceptible to C107 (LC50, 4 × 104 spores/ml), while O. albifasciatus was most susceptible to C207 (LC50, 3.4 × 106 spores/ml).

Triterpenoidal Saponins: Bioactive Secondary Metabolites fromZygophyllum coccineum

Phytochemical investigation of the aerial parts of Zygophyllum coccineum L. led to the isolation of nine ursane-type triterpene saponins (1- 9), including the new one; zygophylloside S (1), together with a known flavonoid glycoside (10) and a sterol glycoside (11). The isolated compounds were tested for antifungal activity against several important plant pathogens and for insecticidal activity against two important mosquito species. Among the isolated compounds 1, 3, 5, 6, and 9 showed 32-77 % fungal growth inhibition at a concentration of 30 µM against Phomopsis viticola. Compound 9 showed 90 % and 80 % mosquitocidal activity at 3.1 µg/0.5 µL against Aedes aegypti and Culex quinquefasciatus, respectively.

ChemInform Abstract: Bioactivity‐Guided Isolation of Mosquitocidal Constituents from the Rhizomes of Plumbago capensis Thunb

Abstractisolation and structure elucidation of two new naphthoquinone derivatives with moderate mosquitocidal activity, isoplumbagolone (I) and chitranane (II), along with other known compounds possessing potent larvicidal potentials

ChemInform Abstract: The First Isolation and Crystal Structure of a Boron Difluoro Complex (Isoflavone Yellow). Biologically Active Intermediates Produced During Isoflavone Synthesis.

The yellow intermediate 4f produced during the reaction of the deoxybenzoin 3f with N,N′-dimethyl(chloromethylene)ammonium chloride in the presence of BF3·Et2O prior to the formation of the corresponding isoflavone was isolated for the first time and characterized by NMR, MS and single-crystal X-ray diffraction techniques. These yellow intermediates showed anticancer, nematicidal and mosquitocidal activities.

Characterization of highly toxic indigenous strains of mosquitocidal organismBacillus sphaericus

Three indigenous isolates of Bacillus sphaericus (ISPC-5, ISPC-6 and ISPC-8), along with standard 2362 and 1593 strains, were evaluated for spore viability and mosquitocidal activity. Among these, ISPC-8 was the most viable and virulent isolate, exhibiting a significantly higher total viability count (TVC) and lower LC(50) values. The TVC of the standard strains ranged from 4.0 to 9.2 x 10(8) spores mL(-1), whereas it was 1.3 x 10(9) spores mL(-1) for ISPC-8. The LC(50) values of ISPC-8, 2362 and 1593 against Culex quinquefasciatus were 0.68 x 10(3), 1.22 x 10(3) and 1.85 x 10(3) spores mL(-1), respectively. The ISPC-8 was further assessed for host spectrum and found to be more active against C. quinquefasciatus, followed by Culex tritaeniorhynchus, Aedes albopictus and Aedes aegypti. The ISPC-8 strain was thus found to be a promising isolate for developing biopesticides. Among the indigenous strains, only ISPC-8 was found to have binary toxin genes (binA and binB). Comparative sequence analysis revealed that the BinA (41.9 kDa) protein of ISPC-8 differs by one amino acid (R197M), whereas BinB (51.4 kDa) differs by two amino acids (H99P, P174S) as compared with 1593 and 2362 strains. The purified binary proteins of ISPC-8 showed an LC(50) value of 6.32 ng mL(-1) against C. quinquefasciatus larvae after 48 h.

Alanine Scanning Analyses of the Three Major Loops in Domain II of Bacillus thuringiensis Mosquitocidal Toxin Cry4Aa

Cry4Aa produced by Bacillus thuringiensis is a dipteran-specific toxin and is of great interest for developing a bioinsecticide to control mosquitoes. Therefore, it is very important to characterize the functional motif of Cry4Aa that is responsible for its mosquitocidal activity. In this study, to characterize a potential receptor binding site, namely, loops 1, 2, and 3 in domain II, we constructed a series of Cry4Aa mutants in which a residue in these three loops was replaced with alanine. A bioassay using Culex pipiens larvae revealed that replacement of some residues affected the mosquitocidal activity of Cry4Aa, but the effect was limited. This finding was partially inconsistent with previous results which suggested that replacement of the Cry4Aa loop 2 results in a significant loss of mosquitocidal activity. Therefore, we constructed additional mutants in which multiple (five or six) residues in loop 2 were replaced with alanine. Although the replacement of multiple residues also resulted in some decrease in mosquitocidal activity, the mutants still showed relatively high activity. Since the insecticidal spectrum of Cry4Aa is specific, Cry4Aa must have a specific receptor on the surface of the target tissue, and loss of binding to the receptor should result in a complete loss of mosquitocidal activity. Our results suggested that, unlike the receptor binding site of the well-characterized molecule Cry1, the receptor binding site of Cry4Aa is different from loops 1, 2, and 3 or that there are multiple binding sites that work cooperatively for receptor binding.

Bacillus thuringiensis serovar mogi (flagellar serotype 3a3b3d), a novel serogroup with a mosquitocidal activity

The flagellated vegetative cells of the Bacillus thuringiensis strain K4 were agglutinated with the H3 reference antiserum and further, agglutinated with 3b and 3d monospecific factor sera but non-reactive for 3c and 3e factor sera. This creates a new serogroup with flagellar antigenic structure of 3a3b3d: B. thuringiensis serovar mogi. The strain K4 showed high activity against dipteran larvae, Anopheles sinensis and Culex pipiens pallens while no lepidopteran toxicity. It produced ovoidal parasporal inclusions (crystals) whose SDS–PAGE protein profile consisted of several bands ranging from 75 to 30 kDa. Through the protein identification by nano-LC-ESI-IT MS analysis, the putative peptides of Cry19Ba, Cry40ORF2, Cry27Aa and Cry20Aa were detected.