New mosquitocidal strains from Ghana belonging to serotypes H3, H6 and H48 of Bacillus sphaericus

Summary Seven bacterial isolates from Ghana, IAB 763, IAB 769-1, IAB 769-2, IAB 774, IAB 871, IAB 872, IAB 881, are characterized as Bacillus sphaericus strains highly toxic to mosquito larvae. Most of them belong to serotype H6, except for IAB 881 and IAB 872, which belong pesrespectively to serotypes H3 and H48. Phenotypic characters of all these strains are identical to those of strains 2362 (serotype H5) and IAB 59 (serotype H6), used for comparison. Five strains out of seven produce final whole cultures and alkali-solubilized toxins, which have very high potency against Culex pipiens larvae. Their larvicidal power is similar to that of strains 2362 and IAB 59. By using polyclonal antibodies raised against 42- and 56-kDa toxic polypeptides of strain 2362, Western-blot of the alkali-solubilized toxins of these new five strains showed homologies. It is the first time that strains belonging to serotypes H3 and H48 have been found pathogenic to mosquito larvae, thus increasing to eight the number of toxic serotypes of B. sphaericus. Correspondence to: I. Thiery     

Activité comparée de 22 variétés deBacillus thuringiensis sur 3 espèces deCulicidae

Résumé Le pouvoir larvicide des cultures totales de 22 variétés deBacillus thuringiensis Berliner représentant 15 sérotypes H a été testé sur larves L4 d'Aedes aegypti (L.)Culex pipiens pipiens (L.) etAnopheles stephensi (Liston). Seul le sérotype H 14, variétéisraelensis, est réellement actif, provoquant 100% de mortalité à la dilution 10⁻⁵. Avec des doses beaucoup plus fortes, 10⁻², une certaine toxicité peut être manifestée par les variétésentomocidus, galleriae etkyushuensis en ce qui concerneAe. aegypti etC. pipiens pipiens, ou par les variétésentomocidus, tolworthi, kyushuensis etaizawai, pourAn. stephensi. Cependant cette activité n'a rien de comparable avec celle de la variétéisraelensis.

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Summary We have studied the 15 H serotypes ofBacillus thuringiensis Berliner including 22 varieties. The larvicidal potency of the whole cultures of these varieties is evaluated on 4th instar larvae ofAedes aegypti (L.),Culex pipiens pipiens (L.) andAnopheles stephensi (Liston). The H-14 serotype, varietyisraelensis is the only one to show a true toxicity at 10⁻⁵ dilution on larvae of the 3 mosquito species. A low mortality at 10⁻² dilution is observed onAe. aegypti andCx. pipiens pipiens larvae withentomocidus, galleriae andkyushuensis varieties; onAn. stephensi withentomocidus, tolworthi, kyushuensis andaizawa? varieties. Nevertheless, this activity cannot be compared to the extremely high toxicity of theisraelensis variety.

     

Characterization of a Bacillus thuringiensis strain with a broad spectrum of activity against lepidopteran insects

The insect pathogen Bacillus thuringiensis is suitable for use in biological control, and certain strains have been developed as commercial bioinsecticides. The molecular and biological characterization of a Bacillus thuringiensis subsp. aizawai strain, named HU4‐2, revealed its potential as a bioinsecticide. The strain was found to contain eight different cry genes: cry1Ab, cry1Ad, cry1C, cry1D, cry1F, cry2, cry9Ea1, and a novel cry1I‐type gene. Purified parasporal crystals from strain HU4‐2 comprised three major proteins of 130–145 kDa, which were tested for their insecticidal potency to four species of Lepidoptera (Helicoverpa armigera, Spodoptera exigua, S. littoralis, and S. frugiperda) and three species of mosquito (Culex pipiens pipiens, Aedes aegypti, and Anopheles stephensi). The crystal proteins were highly toxic against all the species of Lepidoptera tested, moderately toxic against two of the mosquito species (C. pipiens and Ae. aegypti), but no toxicity was observed against a third species of mosquito (An. stephensi) at the concentrations used in our study. The LC₅₀ values of the HU4‐2 Bt strain against H. armigera larvae (5.11 µg/ml) was similar to that of HD‐1 Bt strain (2.35 µg/ml), the active ingredient of the commercial product Dipel®. Additionally, the LC₅₀ values of the HU4‐2 Bt strain against S. littoralis, S. frugiperda, and S. exigua (2.64, 2.22, and 3.38 µg/ml, respectively) were also similar to that of the Bt strain isolated from the commercial product Xentari® for the same three species of Spodoptera (1.94, 1.34, and 2.19 µg/ml, respectively). Since Xentari® is significantly more toxic to Spodoptera spp. than Dipel® and, reciprocally, Dipel® is significantly more toxic against H. armigera than Xentari®, we discuss the potential of the HU4‐2 strain to control all these important lepidopteran pests.     

Specificity of action on mosquito larvae of Bacillus thuringiensis israelensis toxins encoded by two different genes

Summary A 135 kDa protein gene and two open reading frames (ORF1 and ORF2) have been cloned from a large plasmid of Bacillus thuringiensis israelensis (Bourgouin et al. 1986). The Escherichia coli recombinant clones containing these genes were highly toxic to larvae of Aedes aegypti, Anopheles stephensi and Culex pipiens. From subcloning experiments it was deduced that the 135 kDa polypeptide alone was responsible for the toxic activity on both A. aegypti and An. stephensi larvae. In contrast, the presence of two polypeptides, the 135 kDa protein and the ORF1 product was required for toxicity to C. pipiens larvae. The minimal toxic fragment of the 135 kDa polypeptide has been delineated. The results indicate that a polypeptide of about 65 kDa, corresponding to an amino-terminal part of the 135 kDa protein is sufficient for toxicity. Sequence comparisons indicate that the ORF1 product may correspond to an N-terminal part of a rearranged 130 kDa protein.     

Efficacy of dry powders from Bacillus sphaericus: RB 80, a potent reference preparation for biological titration

Larvicidal potency of three primary powders based on Bacillus sphaericus strains 1593 and 1881 was studied on mosquito larvae. Two acetone powders, P 1593 and P 1881, were very toxic for Anopheles stephensi larvae. The potency of a third lyophilized powder RB 80 made from 1593 strain compared even better when tested against Anopheles stephensi and Culex pipiens pipiens larvae. LC₅₀'s after 48 hr were 0.15 and 0.003 mg/ml, respectively. After storage of RB 80 aqueous suspensions over 2 years or after heat exposure of RB 80 powder, larvicidal potency was still high, indicating an excellent stability. The use of RB 80, because of all its qualities, is suggested as a first experimental standard for titration of B. sphaericus preparations.     

Culex torrentium mosquitoes from Germany are negative for Wolbachia

Wolbachia (Rickettsiales: Anaplasmataceae) infects a wide range of arthropods, including several mosquito species. The bacterium is known to induce a plethora of phenotypes in its host, examples being the reproductive phenotype cytoplasmic incompatibility or resistance against infection with arboviruses. The latter is especially relevant when assessing the vector competence of mosquito species for emerging arboviruses. Thus, knowledge of Wolbachia infection status is important for the assessment of vector competence. To facilitate Wolbachia screening in mosquito populations, a quantitative polymerase chain reaction (qPCR) assay was developed to enable high‐throughput analysis of mosquito samples. Using this assay, the Wolbachia infection status of the two most common Culex mosquito species in Germany, Culex pipiens biotype pipiens Linnaeus (Diptera: Culicidae) and Culex torrentium Martini (Diptera: Culicidae), was assessed. About 93% of all tested C. pipiens biotype pipiens individuals were positive for Wolbachia, whereas none of the C. torrentium samples was found to be infected. Furthermore, other applications of the qPCR assay were explored by assessing a potential link between the levels of Wolbachia and West Nile virus (WNV) infections in German C. pipiens biotype pipiens mosquitoes. No relationship was found between the two variables, indicating that a Wolbachia‐induced antiviral phenotype in this mosquito population is not exclusively attributable to the general level of bacterial infection.     

Cloning and expression of <Emphasis Type="Italic">Bacillus thuringiensis cry</Emphasis>11 crystal protein gene in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The six most toxic Pakistani isolates of Bacillus thuringiensis (SBS Bt-23, 29, 34, 37, 45 and 47), which were previously characterized for their toxicity against larvae of mosquito, Anopheles stephensi, and the presence of cry4 gene, were used for cry11 (cry4D) gene amplification. A 1.9-kb DNA fragment of cry11 gene was PCR-amplified, cloned in expression vector pT7-7, and then used for transformation of E. coli BL21C. The optimum expression was obtained with 1 mM IPTG at 37°C for 3 h. This gene showed different percentage homologies at protein level with scattered mutations in the toxic region. Biotoxicity assay of recombinant protein showed that Cry11 of SBS Bt 45 (DAB Bt 5) was the most toxic protein against third instar larvae of mosquito, A. stephensi, and has potentiality of a bioinsecticide against mosquitoes.     

Characterization of Six Highly Mosquitocidal Bacillus thuringiensis Strains That Do Not Belong to H-14 Serotype

Four strains belonging to Bacillus thuringiensis serovars thompsoni, malaysiensis, canadensis, jegathesan and two auto-agglutinating B.t. strains were identified as being highly toxic to the mosquito larvae of the species Aedes aegypti, Anopheles stephensi, and Culex pipiens. Their larvicidal and hemolytic activities were determined and compared with those of strains known to be highly mosquitocidal and/or cytolytic from serovars of B.t. israelensis, morrisoni, darmstadiensis, medellin, kyushuensis, and fukuokaensis. The electrophoretic protein profiles of purified crystals and immunological relationships with B.t.i. polypeptides were studied. Five out of the six new strains showed the same larvicidal and hemolytic activities and the same crystal proteins and toxin genes as B.t.i. One strain, B.t. jegathesan 367, presented a novel pattern of larvicidal activity and a protein profile different from those of other strains.     

Virulence of natural and insect-passaged strains of Metarhizium anisopliae to mosquito larvae

Forty-seven isolates of Metarhizium anisopliae var. anisopliae (small-spored form) and five isolates of M. anisopliae var. major (large-spored form) obtained from widely separated geographical regions from various insect hosts were screened for virulence against Culex pipiens pipiens larvae. Pathogenesis was variable with mortalities ranging from 0 to 100%. However, much of the variation in mortality among small-spored isolates was due to lowered natural viabilities. The most virulent isolates were from Austria, Australia, and Brazil from insect species in three different orders. Isolates from the major strain were generally avirulent. There was no correlation of strain morphology, geographical region of isolation, or original host species with strain virulence. The strains most virulent to C. pipiens larvae were also highly infective to Aedes aegypti and Anopheles stephensi larvae. Virulence of two strains (E₆ and E₉) to C. pipiens larvae was significantly enhanced by one passage through a C. pipiens larval siphon. Relative potencies increased approximately 1.63 to 2.45 times. A smaller increase in virulence, depending upon the isolate, was also shown when these same strains were tested against A. aegypti and A. stephensi. Virulence of strain E₉ was also increased significantly by passage through an alternate host, Nilaparvata lugens.     

In vivo germination and host specificity of Nosema algerae in mosquitoes.

Of five species of mosquitoes tested, Anopheles stephensi and A. albimanus were the most susceptible, A. quadrimaculatus and Culex pipiens were relatively unsusceptible and A. atroparvus was almost completely refractory to Nosema algerae. Spore germination percentages differed in the larval midguts of the five species of mosquito but not in the order which would explain the susceptibility differences. Neither midgut pH difference nor differences in passage rate through the midgut were sufficient to explain the observed differences in spore germination percentages.     

Distribution, Serological Identification, and PCR Analysis of Bacillus thuringiensis Isolated from Soils of Korea

A total, 58 strains of Bacillus thuringiensis were isolated from soils of various regions in Korea. Serological tests showed that B. thuringiensis isolates represented 10 H serotypes, indicating a varied flora of B. thuringiensis. But the H serotypes did not have a significantly uneven distribution, ranging from 1 to 11 isolates. In toxicity tests, 35% of all isolates were toxic to lepidoptera, 20% were toxic to diptera, and 9% were non-toxic isolates. Especially, a large number of lepidopteran/dipteran-active isolates (36%) were found. Forty all lepidopteran-active isolates produced typical rhomboidial inclusions, and the remainder, which belong to dipteran-active and non-toxic isolates, were spherical in shape. In addition, lepidopteran/dipteran-active isolates produced rhomboidal or spherical inclusions. PCR analysis using cryI, II, III, IV, and V gene-specific primers showed that the frequency of the cryIC gene (57%) predominated, followed by the cryIA(b) (45%) and cryIIA genes (34%). But, the cryIE, cryIF, cryIII, cryIVC and cryV genes were not reactive. Several isolates had unusual PCR products and multiple insecticidal crystal protein genes. PCR results showed varied distribution of the cry-type gene. Seven isolates were selected for evaluation of novel activity according to the following criteria: flagellar serotypes, parasporal inclusion morphology, SDS-PAGE, plasmid DNA patterns, toxicity, and the cry-type gene in PCR analysis. Two isolates, named S333 (H7) and S225 (H7), among them synthesized PCR products of the cryIC gene, but the S333 isolate producing rhomboidal inclusion was toxic to both Plutella xylostella and Culex pipiens, whereas the S225 isolate having toxicity to only C. pipiens produced spherical inclusion. Received: 13 March 1998 / Accepted: 14 April 1998     

Aquatain AMF efficacy on juvenile mosquito stages in control of Culex pipiens complex and Aedes albopictus

Aquatain^® mosquito formulation (AMF) is a silicone-based monomolecular film, which has recently been approved for use in the European Union. The physical mode of action based on lowering water surface tension prevents mosquito larvae/pupae respiration. Additionally, AMF disables gravid females from landing on the water surface and obstructs the natural oviposition process. Due to multistage effects on mosquitoes, AMF could be a product of choice for defined water body and container breeders such as Culex pipiens L. complex, principal vector of West Nile virus in Europe, and the invasive Aedes albopictus (Skuse) (both Diptera: Culicidae), vector of dengue and chikungunya viruses. The primary objectives of this study were to evaluate the efficacy of AMF, to determine the susceptibility of the immature forms of C. pipiens and A. albopictus, and the persistence/longevity of the product to suppress the eclosion of adults. AMF achieved high mortality rates of juvenile A. albopictus and C. pipiens under laboratory conditions. However, in the field C. pipiens larvae showed higher susceptibility to AMF than A. albopictus. Pupae of the two mosquito species were highly susceptible to the presence of AMF. When C. pipiens juveniles were exposed to AMF in the wild, effects lasted for 21 days in densely covered water bodies and 56 days in water recipients with less vegetation. In both breeding sites, natural habitat and artificial water recipient, the two mosquito species with high impact on public health in Europe could successfully be suppressed by application of AMF (1 ml m⁻²).     

Insecticidal Activity of Bacillus laterosporus

The Bacillus laterosporus strains 921 and 615 were shown to have toxicity for larvae of the mosquitoes Aedes aegypti, Anopheles stephensi, and Culex pipiens. The larvicidal activity of B. laterosporus was associated with spores and crystalline inclusions. Purified B. laterosporus 615 crystals were highly toxic for Aedes aegypti and Anopheles stephensi.     

Various Levels of Cross-Resistance to Bacillus sphaericus Strains in Culex pipiens (Diptera: Culicidae) Colonies Resistant to B. sphaericus Strain 2362

We studied the cross-resistance to three highly toxic Bacillus sphaericus strains, IAB-59 (serotype H6), IAB-881 (serotype H3), and IAB-872 (serotype H48), of four colonies of the Culex pipiens complex resistant to B. sphaericus 2362 and 1593, both of which are serotype H5a5b strains. Two field-selected highly resistant colonies originating from India (KOCHI, 17,000-fold resistance) and France (SPHAE, 23,000-fold resistance) and a highly resistant laboratory-selected colony from California (GeoR, 36,000-fold resistance) showed strong cross-resistance to strains IAB-881 and IAB-872 but significantly weaker cross-resistance to IAB-59 (3- to 43-fold resistance). In contrast, a laboratory-selected California colony with low-level resistance (JRMM-R, 5-fold resistance) displayed similar levels of resistance (5- to 10-fold) to all of the B. sphaericus strains tested. Thus, among the mosquitocidal strains of B. sphaericus we identified a strain, IAB-59, which was toxic to several Culex colonies that were highly resistant to commercial strains 2362 and 1593. Our analysis also indicated that strain IAB-59 may possess other larvicidal factors. These results could have important implications for the development of resistance management strategies for area-wide mosquito control programs based on the use of B. sphaericus preparations.     

Single amino acids set apparent temperature thresholds for heat-evoked activation of mosquito transient receptor potential channel TRPA1

Animals detect heat using thermosensitive transient receptor potential (TRP) channels. In insects, these include TRP ankyrin 1 (TRPA1), which in mosquitoes is crucial for noxious heat avoidance and thus is an appealing pest control target. However, the molecular basis for heat-evoked activation has not been fully elucidated, impeding both studies of the molecular evolution of temperature sensitivity and rational design of inhibitors. In TRPA1 and other thermosensitive TRPs, the N-terminal cytoplasmic ankyrin repeat (AR) domain has been suggested to participate in heat-evoked activation, but the lack of a structure containing the full AR domain has hindered our mechanistic understanding of its role. Here, we focused on elucidating the structural basis of apparent temperature threshold determination by taking advantage of two closely related mosquito TRPA1s from Aedes aegypti and Culex pipiens pallens with 86.9% protein sequence identity but a 10 °C difference in apparent temperature threshold. We identified two positions in the N-terminal cytoplasmic AR domain of these proteins, E417 (A. aegypti)/Q414 (C. pipiens) and R459 (A. aegypti)/Q456 (C. pipiens), at which a single exchange of amino acid identity was sufficient to change apparent thresholds by 5 to 7 °C. We further found that the role of these positions is conserved in TRPA1 of a third related species, Anopheles stephensi. Our results suggest a structural basis for temperature threshold determination as well as for the evolutionary adaptation of mosquito TRPA1 to the wide range of climates inhabited by mosquitoes.     

Duplex real-time PCR detection of type III effector tccP and tccP2 genes in pathogenic Escherichia coli and prevalence in raw milk cheeses

Aims: To develop a duplex real‐time PCR assay targeting enterohaemorrhagic Escherichia coli (EHEC) type III effector TccP/TccP2‐encoding genes which are pivotal to EHEC‐mediated actin cytoskeleton reorganization in human intestinal epithelial cells. Methods and Results: The specificity of the assay was demonstrated with DNA from EHEC reference strains and non‐E. coli bacterial species. The detection limit was determined as five tccP or tccP2 copies per reaction. The assay was then evaluated on a large collection of 526 E. coli strains of human, animal, food and environmental origins. The results showed that tccP was restricted to a limited number of serotypes (i.e. O5:H^?, O55:H7, O125:H6 and O157:H7). The tccP2 gene was present in a higher number of serotypes including the five most frequent EHEC serotypes (i.e. O26:H11, O103:H2, O111:H8, O145:H28 and O157:H7), and a few other serotypes that caused human infections (i.e. O4:H^?, O45:H2 and O55:H7). A minority of O26:H11 and O103:H2 strains however tested negative for tccP2, though it is not known whether the lack of tccP2 affected their pathogenic potential. Real‐time PCR analysis of 400 raw milk cheeses revealed the presence of tccP and/or tccP2 genes in 19·75% of the cheese enrichment suspensions. Conclusions: A highly specific and sensitive duplex real‐time PCR method was developed for rapid and simultaneous detection of tccP and tccP2. Unpasteurized dairy products may be contaminated with E. coli strains carrying tccP and/or tccP2. Significance and Impact of the Study: The developed real‐time PCR assay represents a valuable alternative to conventional PCR tests and should be useful for characterization of the virulome of pathogenic E. coli strains.     

Novel spirochetes isolated from mosquitoes and black flies in the Czech Republic

During the years 1999–2002, a total of 4,898 individuals of 26 species of hematophagous insects (4,149 mosquitoes, 583 black flies, and 166 tabanid flies) was examined for the presence of spirochetes using dark‐field microscopy. There was an overall recovery of spirochetes from the midguts of Culicidae and Simuliidae of 23.5% and 11.4%, respectively. Spirochetes were not detected in Tabanidae. Seven spirochetal strains have been successfully recovered from mosquitoes and black flies: BR149 (Culex pipiens), BR151 (Cx. pipiens), BR173 (Cx. pipiens), BR177 (Cx. pipiens), BR193 (Aedes cinereus), BR208 (Cx. pipiens), and BR231 (Simulium noelleri). The strains have been adapted to laboratory conditions (BSK‐H Complete medium). Their preliminary determination based on 16S rRNA gene sequencing has shown that they differ from the Lyme disease spirochete Borrelia burgdorferi sensu lato as well as other members of the Order Spirochaetales indicating novel bacterial species in the Family Spirochaetaceae.     

Growth, Sporulation, δ-Endotoxins Synthesis, and Toxicity During Culture of Bacillus thuringiensis H14

Growth, sporulation, synthesis of δ-endotoxins, and toxicity against the larvae of Aedes aegypti and Culex pipiens were studied during fermentation of Bacillus thuringiensis H14 in a 20-L fermentor. Measurements of optical density and dielectric permittivity for biomass determination suggest a highly promising technique for on-line evaluation of sporulation. The synthesis of 65-, 25- and 130-kDa proteins started at 16, 18, and 23 h, respectively. These proteins were enriched in different ways until the end of culture (48 h). Toxicity in the course of sporulation was significantly different for the larvae of both mosquito species. Maximal activity against Ae. aegypti was obtained at the end of culture, whereas for Cx. pipiens, the sample at 38 h was the most active.     

Acid production by Metarhizium anisopliae: Effects on virulence against mosquitoes and on detection of in vitro amylase,protease, and lipase activity

The imperfect fungus Metarhizium anisopliae infects and kills larvae of many insect species, including the mosquito Culex pipiens. Mutants of M. anisopliae selected for enhanced production of amylase have been found to have simultaneously acquired hypervirulence against C. pipiens larvae. In the present work, wild-type and some mutant strains of M. anisopliae were found to excrete an acid or acids which alter the pH of fungal cultures below that permissible for amylase activity. The amylase-enhanced hypervirulence mutants did not excrete acid. Detection of protease and lipase activities was complicated by the acid excretion. When this was taken into account, mutants having altered lipase or protease production were found to have no alteration in virulence against mosquito larvae. The link between acid excretion, amylase activity, and hypervirulence is discussed.     

Predation on Mosquito Larvae by Mesocyclops thermocyclopoides (Copepoda: Cyclopoida) in the Presence of Alternate Prey

The cyclopoid copepod Mesocyclops thermocyclopoides, a dominant invertebrate predator in many shallow ponds and temporary water bodies in northern India, feeds on cladocerans, rotifers, ciliates and when present, on mosquito larvae also. We studied in the laboratory the prey consumption rates of the copepod on first and fourth instar larvae of two species of mosquito (Anopheles stephensi and Culex quinquefasciatus) in relation to their density. We also studied its prey selectivity with mosquito larvae in the presence of an alternate prey (the cladocerans‐either Moina macrocopa or Ceriodaphnia cornuta) in different proportions. With either mosquito species, the copepod actively selected Instar‐I larvae, avoiding the Instar‐IV larvae, and with either instar, selected Anopheles stephensi over Culex quinquefasciatus. When prey choice included the cladoceran as an alternate prey, the copepod selected the cladoceran only when the other prey was Instar‐IV mosquito larvae. Our results point to the potential and promise of M. thermocyclopoides as a biological agent for controlling larval populations of vectorially important mosquito species.