The bacterium, Lysinibacillus sphaericus, as an insect pathogen

Since the first bacteria with insecticidal activity against mosquito larvae were reported in the 1960s, many have been described, with the most potent being isolates of Bacillus thuringiensis or Lysinibacillus sphaericus (formerly and best known as Bacillus sphaericus). Given environmental concerns over the use of broad spectrum synthetic chemical insecticides and the evolution of resistance to these, industry placed emphasis on the development of bacteria as alternative control agents. To date, numerous commercial formulations of B. thuringiensis subsp. israelensis (Bti) are available in many countries for control of nuisance and vector mosquitoes. Within the past few years, commercial formulations of L. sphaericus (Ls) have become available. Because Bti has been in use for more than 30 years, its properties are well know, more so than those of Ls. Thus, the purpose of this review is to summarise the most critical aspects of Ls and the various proteins that account for its insecticidal properties, especially the mosquitocidal activity of the most common isolates studied. Data are reviewed for the binary toxin, which accounts for the activity of sporulated cells, as well as for other toxins produced during vegetative growth, including sphaericolysin (active against cockroaches and caterpillars) and the different mosquitocidal Mtx and Cry toxins. Future studies of these could well lead to novel potent and environmentally compatible insecticidal products for controlling a range of insect pests and vectors of disease.     

Biologically based insecticides for the control of immature Australian mosquitoes: a review

Abstract  Our review outlines changes in insecticide usage for mosquito control against Australian mosquitoes, with a focus on biologically based insecticides. The most widely used microbial insecticide is Bacillus thuringiensis var. israelensis de Barjac ( Bti ) and it is regularly used in estuaries and freshwater. In comparison, the use of Bacillus sphaericus (Neide) is limited to freshwater. The only insect growth regulator registered for mosquito control in Australia is s -methoprene, and it is used in both estuaries and freshwater. Although biologically based insecticides have been used for more than 10 years, there remains knowledge gaps surrounding the use of specific formulations in specific habitats: for example, VectoBac WG ( Bti ) and VectoBac G ( Bti ) in containers containing freshwater, or Altosid pellets ( s -methoprene) in ground-pool freshwater habitats. Where broad-scale mosquito control programs have been implemented in Australia, a reduction in the incidence of mosquito-borne disease, mainly Ross River virus, has been recorded. Therefore, the future application of these products is supported. The use of insecticides should be integrated with public education, biological control, physical habitat modification and coupled with spatial risk assessment.     

Non-larvicidal effects of Bacillus thuringiensis israelensis and Bacillus sphaericus on oviposition and adult mortality of Culex quinquefasciatus Say (Diptera: Culicidae).

Two microbial mosquito larvicides, Bacillus thuringiensis ssp. israelensis (Bti) and Bacillus sphaericus (Bsph), have been shown to be highly effective in controlling mosquito larvae and have been used in larvicidal programs for many years. In exploring other modes of action of these agents, we studied the ovipositional response of Bsph susceptible and resistant Culex quinquefasciatus to aqueous suspensions of Bti and Bsph water dispersible granules (WDG). We quantified the level of mortality of adult mosquitoes caused by exposure to Bti and Bsph suspensions during oviposition. Significantly lower numbers of egg rafts were laid and collected from the treatments than the control regimen. There was an inverse relationship between Bacillus product concentrations and oviposition. As the concentration of Bti or Bsph increased from 0.0 to 2.0 mg/L, treated waters received progressively fewer egg rafts. In addition to the negative effects of Bacillus on oviposition, both male and female adult mosquitoes suffered high mortality on landing and imbibing on Bti and Bsph suspensions, the extent of mortality directly proportional to concentration. These two microbial agents used solely as mosquito larvicides thus have the additional benefits of reducing mosquito oviposition and killing adult mosquitoes, especially gravid females that come in contact with the treated water either for oviposition or drinking. Reducing the number of gravid females may also result in reduced transmission rates of pathogens. The combined effects of reduced oviposition and adult mortality could result in higher control potential of these microbial agents.     

Construction and characterization of a recombinant Bacillus thuringiensis subsp. israelensis strain that produces Cry11B

The mosquitocidal bacterium Bacillus thuringiensis subsp. israelensis (Bti) produces four major endotoxin proteins, Cry4A, Cry4B, Cry11A, and Cyt1A, and has toxicity in the range of many synthetic chemical insecticides. Cry11B, which occurs naturally in B. thuringiensis subsp. jegathesan, is a close relative of Cry11A, but is approximately 10-fold as toxic to Culex quinquefasciatus. To determine whether the addition of Cry11B to Bti would improve its toxicity, we produced this protein in Bti. High levels of Cry11B synthesis were obtained by expression of the cry11B gene under the control of cyt1A promoters and the STAB-SD sequence. This construct was cloned into the shuttle vector pHT3101, yielding the derivative plasmid pPFT11Bs, which was then transformed by electroporation into acrystalliferous (4Q7) and crystalliferous (IPS-82) strains of Bti. Synthesis of Cry11B in Bti 4Q7 produced crystals approximately 50% larger than those produced with its natural promoters without STAB-SD. However, less Cry11B was produced per unit culture medium with this construct than with the wild-type construct, apparently because the latter construct produced more cells per unit medium. Nevertheless, the Bti IPS-82 strain that produced Cry11B with pPFT11Bs was twice as toxic as the parental IPS-82 strain (LC(50) = 1.4 ng/ml versus 3.3 ng/ml, respectively) to fourth instars of C. quinquefasciatus. Against fourth instars of Aedes aegypti, no statistically significant difference between parental Bti IPS-82 (LC(50) = 4.7 ng/ml) and the Bti IPS-82 recombinant producing Cry11B (LC(50) = 3.5 ng/ml) was found in toxicity.     

Mosquito control actions affect chironomid diversity in temporary wetlands of the Upper Rhine Valley

The Upper Rhine Valley, a “hotspot of biodiversity” in Germany, has been treated with the biocide Bacillus thuringiensis var. israelensis (Bti) for mosquito control for decades. Previous studies discovered Bti nontarget effects in terms of severe chironomid abundance reductions. In this study, we investigated the impact of Bti on species level and addressed the community composition of the nontarget family Chironomidae by use of community metabarcoding. Chironomid emergence data were collected in three mosquito‐control relevant wetland types in the Upper Rhine Valley. For all three sites the chironomid species composition, based on operational taxonomic units (OTUs), was different to varying degrees in the Bti‐treated samples versus control samples, ranging from a significant 63% OTU reduction to an OTU replacement. We assumed that predatory chironomids are less prone to Bti than filter feeders, as the latter feed on floating particles leading to direct ingestion of Bti. However, a comparable percentage of predators and filter feeders (63% and 65%, respectively) was reduced in the Bti samples, suggesting that the feeding strategy is not the main driver for Bti sensitivity in chironomids. Finally, our data was compared to a three‐year‐old data set, indicating possible chironomid community recovery due to species recolonization a few years after the last Bti application. Considering the currently discussed worldwide insect decline we recommend a rethinking of the usage of the biocide Bti, and to prevent its ongoing application especially in nature protection reserves to enhance ecological resilience and to prevent boosting the current biodiversity loss.     

Differential effects of Bacillus sphaericus strain 2362 on Culex quinquefasciatus and its competitor Culex cinereus in West Africa

The larval susceptibility to Bacillus sphaericus strain 2362 of the non-man-biting mosquito Culex cinereus and the urban filariasis vector Culex quinquefasciatus, two competitor mosquitoes in polluted habitats, was compared. In the laboratory, both species ingested a similar amount of B. sphaericus spores when fed c. 2 x 10(5) spores per ml for 30 min. However, in the same experiment, third-instar larvae of Cx quinquefasciatus were reduced by 98% at 24 h exposure while Cx cinereus larvae were only reduced by 6% at 72 h. In the field, preimaginal populations of Cx cinereus ingested, within a week, more than 99% of the applied spores, but showed no significant decrease through 14 days in cesspools treated at 10 g/m2 of a flowable concentrate of B. sphaericus 2362, containing 2 x 10(10) spores/g. It is proposed that specific biological control of Cx quinquefasciatus could result from appropriate treatment of breeding-sites with larvicidal B. sphaericus and competitive displacement by Cx cinereus or other mosquitoes with larvae that are more tolerant of B. sphaericus.     

B.thuringiensis穿梭载体的构建及cry1C基因的表达

以ＵＣ１９为母体,克隆了Ｂｔ ｋｅｎ－Ａｇ（Ｂ。．ｔｈｒｕｉｎｇｉｅｎｓｉｓ ｓｕｂｓｐ．ｋｅｎｙａｅ Ａｇ）的复制起始区（～１．６ｋｂ）、ｐＵＣ４Ｋ的ａｐｈ１基因,构建成穿梭载体ｐＨＶ－１,ｐＨＶ－１在Ｅ．ｃｏｌｉ中经１００个世代,质粒保持率在８０％以Ｂｔｉ ４Ｑ８（Ｂ．ｔｈｕｒｉｎｇｉｅｎｓｉｓ ｓｕｂｓｐ．ｉｓｒａｅｌｅｎｓｉｓ ４Ｑ８）中经４０个世代,质粒保持率在８０％以上,将Ｂ．ｌｉｃｈ     

Cyt1A from Bacillus thuringiensis synergizes activity of Bacillus sphaericus against Aedes aegypti (Diptera: Culicidae)

Bacillus sphaericus is a mosquitocidal bacterium recently developed as a commercial larvicide that is used worldwide to control pestiferous and vector mosquitoes. Whereas B. sphaericus is highly active against larvae of Culex and Anopheles mosquitoes, it is virtually nontoxic to Aedes aegypti, an important vector species. In the present study, we evaluated the capacity of the cytolytic protein Cyt1A from Bacillus thuringiensis subsp. israelensis to enhance the toxicity of B. sphaericus toward A. aegypti. Various combinations of these two materials were evaluated, and all were highly toxic. A ratio of 10:1 of B. sphaericus to Cyt1A was 3, 600-fold more toxic to A. aegypti than B. sphaericus alone. Statistical analysis showed this high activity was due to synergism between the Cyt1A toxin and B. sphaericus. These results suggest that Cyt1A could be useful in expanding the host range of B. sphaericus.     

Sublethal effects of mosquito larvicides on swimming performance of larvivorous fish Melanotaenia duboulayi (Atheriniformes: Melanotaeniidae)

Laboratory studies were conducted to determine the sublethal effects of exposure to selected larvicides on the critical swimming speed (Ucrit) of crimson-spotted rainbowfish, Melanotaenia duboulayi (Castlenau). This native fish is common throughout southeastern Queensland, and it is increasingly being distributed as a biological control agent of mosquitoes. The selected larvicides included, two organophosphate (OP) compounds (temephos and pirimiphos-methyl), two microbial larvicides (Bacillus thuringiensis spp. israelensis [Bti] de Barjac and Bacillus sphaericus [Bs] Neide), and an insect growth regulator (IGR) (s-methoprene). Exposure to the OP temephos at 10 times the effective field concentration (EFC; 0.33 mg/liter), and OP pirimiphos-methyl at the EFC (0.50 mg/liter), resulted in a significant reduction in the Ucrit of M. duboulayi under controlled conditions. Conversely, exposure to the microbial (Bti and Bs) and IGR (s-methoprene) larvicides at 10 times the EFC had no effect on the Ucrit of M. duboulayi. Accordingly, these products are suitable for integrated pest management programs in Australia.     

Ingestion, dissolution, and proteolysis of the Bacillus sphaericus toxin by mosquito larvae

Larvae of Culex quinquefasciatus are much more susceptible to the toxin of Bacillus sphaericus than are larvae of Aedes aegypti. In the present study, the rate of ingestion, dissolution, and the cleavage by midgut proteases of the B. sphaericus toxin were compared in larvae of these species to determine whether these factors account for the differences in susceptibility. During filter feeding, larvae of both species removed significant quantities of B. sphaericus toxin from suspensions. Filtration rates for 1 hr, the time at which C. quinquefasciatus exhibited marked intoxication, were higher for A. aegypti (576-713 microliters/larva/hr) than for C. quinquefasciatus (446-544 microliters/larva/hr). Within 24 hr of exposure, A. aegypti larvae ingested 97-99% of the toxin particulates and suffered not more than 10% mortality in suspensions which induced complete mortality in C. quinquefasciatus within 2 hr of exposure. Quantification of the particulate toxin present in larvae after exposure to B. sphaericus suspensions revealed that larvae of both species contained only minor amounts of the toxin, suggesting the larvae had been able to solubilize the toxin after ingestion. Proteases recovered from the feces of larvae cleaved at 43-kDa protein isolated from B. sphaericus toxin extract to 40 kDa in both species. Thus, differences in susceptibility to the B. sphaericus toxin between A. aegypti and C. quinquefasciatus are not due to differences in rates of ingestion, dissolution, or the specificity of proteases.     

Ovipositional and ovicidal effects of the microbial agent Bacillus thuringiensis israelensis on Culex quinquefasciatus say (Diptera: Culicidae).

The magnitude of oviposition as well as the size, shape and the number of eggs per of egg rafts egg raft were determined after gravid Culex quinquefasciatus Say oviposited on water treated with water dispersible granules (WDG) of Bacillus thuringiensis ssp. israelensis (Bti) and on untreated water. The mean number of eggs/raft was lower in the treated than in the untreated water. Bti concentrations from 0.5 to 2.0mg/L affected the shape of egg rafts and number of eggs in each raft. As the concentration of Bti increased from 0.5 to 2.0 mg/L the shape of egg rafts became more irregular with fewer eggs in each raft. Exposure to Bti at 2- and 26-h reduced the hatching rates, and fewer eggs hatched at 26-h of exposure to Bti. As the concentration of Bti WDG increased from 0.5 to 2.0 mg/L, the hatching rate decreased. Eggs exposed for 2-h to 2.0mg/ L Bti had a hatch of 30% after 24 h, the rate increasing to 57% after 72 h. In contrast, in 26-h exposed eggs to 2.0 mg/L Bti, the hatching rate after 24 h was only 12% and this rate increased to 39% after 72 h. In larvae from eggs exposed for 2 h, the survival rate was 40% at 2.0 mg/L Bti and 87% in untreated controls. In contrast, the survival rates of larvae from 26-h exposed eggs was 91% in controls while it was 30% at 2.0 mg/L Bti. As the concentration of Bti increased from 0.5 to 2.0 mg/ 1 the survival rates of larvae decreased. The combined effects of reductions of egg rafts, low number of eggs per egg raft, and reduced hatching and survival rates could have significant cumulative effects on the yield of adult mosquitoes, and this could result in a greater control potential of this microbial agent.     

High-Level Field Resistance to Bacillus sphaericus C3-41 in Culex quinquefasciatus from Southern China

A flowable mosquito-larvicidal formulation of Bacillus sphaericus strain C3-41 has been continuously used for 8 years to control Culex quinquefasciatus larvae in Dongguan, Guangdong Province, China. This formulation had high efficacy against the target larvae during the first 6 years of treatment. However, under this high selection pressure, C. quinquefasciatus showed a significant level of resistance to C3-41 from years seven to eight. The resistance ratio of field-collected larvae at LC 50 was calculated to be 22 672-fold in comparison with the susceptible laboratory colony. Interestingly, no cross-resistance was observed to B. sphaericus strain LP1-G which had the same toxicity against both susceptible and resistant larvae, and B. thuringiensis subsp. israelensis was found to be more active to the latter than the former. After six months treatment with the B. thuringiensis subsp. israelensis formulation in the B. sphaericus resistant population area, the mosquito population recovered its susceptibility to B. sphaericus C3-41, with the resistance ratio of field-collected larvae dropping from 22 672- to 5.67-fold.     

Bacterial larvicides for vector control: mode of action of toxins and implications for resistance

Vector control can be an effective strategy to interrupt disease transmission and biolarvicides based on the entomopathogenic bacteria Bacillus sphaericus, and Bacillus thuringiensis serovar israelensis (Bti) have been successfully used to control species of public health relevance from the genera Aedes, Culex, Anopheles and Simulium. The most important feature of these agents is their ability to produce insecticidal proteins with selective action on the larval midgut. These protoxins are produced as crystals that, once ingested by larvae, are processed into active toxins, interact with receptors in the midgut epithelium and trigger cytopathological effects leading to larval death. B. sphaericus and Bti toxins share the initial steps of the mode of action; however, they interact with different midgut molecules. B. sphaericus presents a single larvicidal factor, the binary (Bin) toxin, whose action relies on the binding to one class of midgut receptors, while Bti crystals contain four protoxins (Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa), which display interactions with multiple midgut receptors. The mode of action of B. sphaericus displays a greater potential for resistance selection, compared to Bti, and, to date, there is no record of insect resistance to the latter, contrarily to B. sphaericus. The set of mosquitocidal toxins and their interaction with midgut target sites are described in this review, as well as the implications for the potential to select resistance amongst exposed populations. These biolarvicides have specific mode of action that rely on unique interactions and make them the most selective agents to control Diptera insects actually available.     

Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and their expression

The genes encoding the toxic determinants of Bacillus sphaericus have been expressed in a nontoxic and a toxic strain of Bacillus thuringiensis subsp. israelensis. In both cases, the B. sphaericus toxin proteins were produced at a high level during sporulation of B. thuringiensis and accumulated as crystalline structures. B. thuringiensis transformants expressing B. sphaericus and B. thuringiensis subsp. israelensis toxins did not show a significant enhancement of toxicity against Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae.     

Effects of Bacillus thuringiensis var. israelensis on Target and Nontarget Organisms: a Review of Laboratory and Field Experiments

Since the discovery of Bacillus thuringiensis var. israelensis (Bti) in 1976, extensive literature has proved its efficacy to control mosquitoes and black flies, of which many species are known as important vectors of diseases or simply as pests of humans and animals. Since 1978, Bti has been used in many countries on all continents and numerous studies have been made on target mosquitoes and black flies, as well as nontarget organisms (NTO). This review analyses the results of 75 studies on these organisms covering approximately 125 families, 300 genera and 400 species. Different factors such as species, instar, feeding behaviour and environmental parameters (larval density, water temperature, suspended matter etc.) may drastically affect the efficacy of the Bti products. This is addressed in detail by reviewing the main factors affecting mosquitoes as well as black flies. The results of a wide range of laboratory and field experiments using different target and nontarget species, various preparations and formulations of Bti and different biotic or abiotic factors are present in the literature, making the data difficult to compare on a common basis. Our analysis shows that, under different application conditions, the effects of Bti on target and nontarget organisms may be hard to predict. Although Bti has been proclaimed to be relatively highly specific, some studies show that some NTO are affected either by single or repeated Bti treatments. Present use against black flies seems ecologically acceptable. High frequencies of application and/or overdosages against mosquitoes may result in some persistence of the toxin crystals and ultimately this may have adverse effects on the food web. A long-term study (published in 1998) in mosquito habitats has shown that intensive Bti treatments over three years did in fact produce an impact on the food web in wetlands. This raises questions, for the first time, on Bti environmental specificity. The importance of this impact is discussed and the alternatives for practical pest control are considered. Some modifications of Bti use against mosquitoes, guided by research, is probably the best of these alternatives.     

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.     

Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and their expression.

The genes encoding the toxic determinants of Bacillus sphaericus have been expressed in a nontoxic and a toxic strain of Bacillus thuringiensis subsp. israelensis. In both cases, the B. sphaericus toxin proteins were produced at a high level during sporulation of B. thuringiensis and accumulated as crystalline structures. B. thuringiensis transformants expressing B. sphaericus and B. thuringiensis subsp. israelensis toxins did not show a significant enhancement of toxicity against Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae.     

Improvement of Bacillus sphaericus toxicity against dipteran larvae by integration, via homologous recombination, of the Cry11A toxin gene from Bacillus thuringiensis subsp. israelensis.

Integrative plasmids were constructed to enable integration of foreign DNA into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Integration of the aphA3 kanamycin resistance gene by a two-step procedure demonstrated that this strategy was applicable with antibiotic resistance selection. Hybridization experiments evidenced two copies of the operon encoding the binary toxin from B. sphaericus in the recipient strain. The Bacillus thuringiensis subsp. israelensis cry11Aal gene (referred to as cry11A), encoding a delta-endotoxin with toxicity against Culex, Aedes, and Anopheles larvae, was integrated either by a single crossover event [strain 2297 (::pHT5601), harboring the entire recombinant plasmid] or by two successive crossover events [strain 2297 (::cry11A)]. The level of the Cry11A production in B. sphaericus was high; two crystalline inclusions were produced in strain 2297 (::pHT5601). Synthesis of the Cry11A toxin conferred toxicity to the recombinant strains against Aedes aegypti larvae, for which the parental strain was not toxic. Interestingly, the level of larvicidal activity of strain 2297 (::pHT5601) against Anopheles stephensi was as high as that of B. thuringiensis subsp. israelensis and suggested synergy between the B. thuringiensis and B. sphaericus toxins. The toxicities of parental and recombinant B. sphaericus strains against Culex quinquefasciatus were similar, but the recombinant strains killed the larvae more rapidly. The production of the Cry11A toxin in B. sphaericus also partially restored toxicity for C. quinquefasciatus larvae from a population resistant to B. sphaericus 1593. In vivo recombination therefore appears to be a promising approach to the creation of new B. sphaericus strains for vector control.     

Evaluation of an rRNA-targeted oligonucleotide probe for the detection of mosquitocidal strains of Bacillus sphaericus in soils: characterization of novel strains lacking toxin genes

Abstract: Colonies of Bacillus sphaericus on primary isolation have been identified using an oligonucleotide probe targeted to a specific region of the 16S rRNA. Of 3440 colonies from soil samples from Brazil, 57 hybridized to the probe in colony blots but when purified DNA was used in slot-blots the probe was more specific and only 27 isolates hybridized. Of these, 20 strains were confirmed as members of DNA homology group IIA (potential mosquitocidal strains) by ribotyping and isoenzyme analysis. However, none of these strains was toxic to Anopheles or Culex larvae, nor did they contain recognized toxin genes. This is the first demonstration of such non-pathogenic strains of B. sphaericus DNA homology group IIA and their common occurrence suggests that pathogenicity is not an important contribution to the success of these bacteria in the environment. Similar screening of strains from Scottish soils indicated that B. sphaericus DNA homology group IIA strains were less common in this habitat and none were recovered on this occasion.