A Strain of Bacillus sphaericus Causes Slower Development of Resistance in Culex quinquefasciatus

Two field-collected Culex quinquefasciatus colonies were subjected to selection pressure by three strains of Bacillus sphaericus, C3-41, 2362, and IAB59, under laboratory conditions. After 13 and 18 generations of exposure to high concentrations of C3-41 and IAB59, a field-collected low-level-resistant colony developed >144,000- and 46.3-fold resistance to strains C3-41 and IAB59, respectively. A field-collected susceptible colony was selected with 2362 and IAB59 for 46 and 12 generations and attained >162,000- and 5.7-fold resistance to the two agents, respectively. The pattern of resistance evolution in mosquitoes depended on continuous selection pressure, and the stronger the selection pressure, the more quickly resistance developed. The resistant colonies obtained after selection with B. sphaericus C3-41 and 2362 showed very high levels of cross-resistance to B. sphaericus 2362 and C3-41, respectively, but they displayed only low-level cross-resistance to IAB59. On the other hand, the IAB59-selected colonies had high cross-resistance to both strains C3-41 and 2362. Additionally, the slower evolution of resistance against strain IAB59 may be explained by the presence of another larvicidal factor. This is in agreement with the nontoxicity of the cloned and purified binary toxin (Bin1) of IAB59 for 2362-resistant larvae. We also verified that all the B. sphaericus-selected colonies showed no cross-resistance to Bacillus thuringiensis subsp. israelensis, suggesting that it would be a promising alternative in managing resistance to B. sphaericus in C. quinquefasciatus larvae.     

Cytopathological Effects of Bacillus sphaericus Cry48Aa/Cry49Aa Toxin on Binary Toxin-Susceptible and -Resistant Culex quinquefasciatus Larvae

The Cry48Aa/Cry49Aa mosquitocidal two-component toxin was recently characterized from Bacillus sphaericus strain IAB59 and is uniquely composed of a three-domain Cry protein toxin (Cry48Aa) and a binary (Bin) toxin-like protein (Cry49Aa). Its mode of action has not been elucidated, but a remarkable feature of this protein is the high toxicity against species from the Culex complex, besides its capacity to overcome Culex resistance to the Bin toxin, the major insecticidal factor in B. sphaericus-based larvicides. The goal of this work was to investigate the ultrastructural effects of Cry48Aa/Cry49Aa on midgut cells of Bin-toxin-susceptible and -resistant Culex quinquefasciatus larvae. The major cytopathological effects observed after Cry48Aa/Cry49Aa treatment were intense mitochondrial vacuolation, breakdown of endoplasmic reticulum, production of cytoplasmic vacuoles, and microvillus disruption. These effects were similar in Bin-toxin-susceptible and -resistant larvae and demonstrated that Cry48Aa/Cry49Aa toxin interacts with and displays toxic effects on cells lacking receptors for the Bin toxin, while B. sphaericus IAB59-resistant larvae did not show mortality after treatment with Cry48Aa/Cry49Aa toxin. The cytopathological alterations in Bin-toxin-resistant larvae provoked by Cry48Aa/Cry49Aa treatment were similar to those observed when larvae were exposed to a synergistic mixture of Bin/Cry11Aa toxins. Such effects seemed to result from a combined action of Cry-like and Bin-like toxins. The complex effects caused by Cry48Aa/Cry49Aa provide evidence for the potential of these toxins as active ingredients of a new generation of biolarvicides that conjugate insecticidal factors with distinct sites of action, in order to manage mosquito resistance.Bacillus sphaericus is considered an important entomopathogen due to its capacity to produce insecticidal proteins with specific action against mosquitoes (Diptera: Culicidae). The binary (Bin) toxin, which is produced during bacterial sporulation and deposited in parasporal crystalline inclusions, is the most important larvicidal factor. Other proteins characterized, such as mosquitocidal toxins (Mtx proteins), can be produced during vegetative growth, and although these proteins may have larvicidal potential, they play a minor role in the toxicity of the native strains since they are produced by vegetative cells and are degraded by B. sphaericus proteinases (20, 30), and do not form components of the spore-crystal preparations that are used in control programs. Recently, a new two-component toxin was characterized from B. sphaericus strain IAB59. This is formed by the proteins Cry48Aa (135 kDa) and Cry49Aa (53 kDa), which are produced as crystalline inclusions (13). The toxin has a unique composition since the Cry48Aa component belongs to the three-domain family of Cry proteins with 30% similarity to the mosquitocidal Cry4Aa protein from Bacillus thuringiensis serovar israelensis, while Cry49Aa is one of the Bin-toxin-like proteins, a family that comprises the Bin toxin from B. sphaericus, in addition to the Cry36 and Cry35 proteins from B. thuringiensis (9, 13).Cry48Aa/Cry49Aa is considered a two-component toxin because neither component shows toxicity alone, whereas both can act in synergy and the optimum level of toxicity to Culex species is achieved when the two are present at an equimolar ratio. The 50% lethal concentration for third-instar larvae equates to 15.9 ng/ml Cry48Aa and 6.3 ng/ml Cry49Aa of purified toxins, which is a level of toxicity comparable to that of the Bin toxin (13). However, in contrast to the Bin toxin, which is naturally produced in an equimolar ratio, Cry48Aa production is low in native strains and does not confer high toxicity (13). The initial steps of the mode of action of Bin and Cry48Aa/Cry49Aa crystals are similar and comprise the ingestion of crystals, solubilization under alkaline pH, and activation of protoxins into toxins by midgut proteases. After processing, Bin toxin recognizes and binds to specific receptors in the midgut of Bin-toxin-susceptible species through its subunit BinB (51 kDa), while the component BinA (42 kDa) confers toxicity and is likely to form pores in the cell membrane (7, 25). The membrane-bound receptors of Bin toxin on the midgut of Culex quinquefasciatus larvae, Cqm1, were characterized as 60-kDa α-glucosidases (24). The mode of action of Cry48Aa/Cry49Aa is still unknown, but a remarkable feature of this new two-component toxin is the capacity to overcome C. quinquefasciatus resistance to the Bin toxin (13, 19, 21). Resistance of Culex larvae to the Bin-toxin-based larvicides often relies on the absence of functional Cqm1 receptors in the midgut (19, 24, 26). As a consequence, toxins with a distinct mode of action, such as Cry48Aa/Cry49Aa as well as B. thuringiensis serovar israelensis toxins (Cry11Aa, Cry4Aa, Cry4Ba, and Cyt1Aa), do not experience cross-resistance in the Bin-toxin-resistant larvae (12, 21, 32). Such toxins can play a strategic role in the management of resistance, and the major goal of this study was to investigate the ultrastructural effects of the Cry48Aa/Cry49Aa toxin on Bin-toxin-susceptible and -resistant C. quinquefasciatus larvae and to compare these with the effects of a synergistic mixture of Bin/Cry11Aa used to overcome Bin toxin resistance.     

球形芽孢杆菌对致倦库蚊的后致死作用

研究了球形芽孢杆菌Bacillus sphaericus C3-41菌株对致倦库蚊Culex quinquefasciatus幼虫的毒力及其后致死作用。生物测定表明,该菌株对目标蚊幼虫具有很高的毒力,其丙酮粉剂对3～4龄幼虫48 h的半致死浓度(LC₅₀)为(6.92±0.22) μg/L。用不同亚致死浓度处理2～3龄致倦库蚊幼虫,存活幼虫在后期发育中存在明显的延续死亡和损伤现象,经LC₃₀、LC₅₀、LC₇₀、LC₉₀和LC₉₈剂量的C3.41粉剂处理的致倦库蚊羽化前的总死亡率分别为84%、91%、95%、97%和100%,同时存活的幼虫、蛹和成蚊的发育和行为也受到一定的影响。这种后致死作用随处理浓度的升高而增强,可能同球形芽孢杆菌毒素蛋白对处理期间蚊幼虫中肠上皮细胞造成的损伤相关。     

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.     

Screening of Brazilian Bacillus sphaericus strains for high toxicity against Culex quinquefasciatus and Aedes aegypti

Abstract:  In this work, 246 Bacillus sphaericus strains were evaluated against Aedes aegypti and Culex quinquefasciatus larvae to select the most effective ones to be used as the basis of a national product. All strains were isolated from different regions of Brazil and they are stored in a Bacillus spp. collection at Embrapa Genetic Resources and Biotechnology. The selected strains were characterized by biochemical and molecular methods. Based on selective bioassays, 87 strains were identified as toxic to one or both target species. All of these strains contain genes that encode the 42, 51 kDa proteins that constitute the binary toxin and the 100 kDa Mtx1 toxin. All toxic strains presented a very high LC₅₀ against A. aegypti , so, a product based on any of these B. sphaericus strains would not be recommended for use in programmes to control A. aegypti . S201 had highest activity against C. quinquefasciatus , presenting the lowest LC₅₀ and LC₉₀ in bioassays.     

A strain of Bacillus sphaericus causes slower development of resistance in Culex quinquefasciatus

Two field-collected Culex quinquefasciatus colonies were subjected to selection pressure by three strains of Bacillus sphaericus, C3-41, 2362, and IAB59, under laboratory conditions. After 13 and 18 generations of exposure to high concentrations of C3-41 and IAB59, a field-collected low-level-resistant colony developed >144,000- and 46.3-fold resistance to strains C3-41 and IAB59, respectively. A field-collected susceptible colony was selected with 2362 and IAB59 for 46 and 12 generations and attained >162,000- and 5.7-fold resistance to the two agents, respectively. The pattern of resistance evolution in mosquitoes depended on continuous selection pressure, and the stronger the selection pressure, the more quickly resistance developed. The resistant colonies obtained after selection with B. sphaericus C3-41 and 2362 showed very high levels of cross-resistance to B. sphaericus 2362 and C3-41, respectively, but they displayed only low-level cross-resistance to IAB59. On the other hand, the IAB59-selected colonies had high cross-resistance to both strains C3-41 and 2362. Additionally, the slower evolution of resistance against strain IAB59 may be explained by the presence of another larvicidal factor. This is in agreement with the nontoxicity of the cloned and purified binary toxin (Bin1) of IAB59 for 2362-resistant larvae. We also verified that all the B. sphaericus-selected colonies showed no cross-resistance to Bacillus thuringiensis subsp. israelensis, suggesting that it would be a promising alternative in managing resistance to B. sphaericus in C. quinquefasciatus larvae.     

Tightly Bound Binary Toxin in the Cell Wall of Bacillus sphaericus

We have shown that urea-extracted cell wall of entomopathogenic Bacillus sphaericus 2297 and some other strains is a potent larvicide against Culex pipiens mosquitoes, with 50% lethal concentrations comparable to that of the well-known B. sphaericus binary toxin, with which it acts synergistically. The wall toxicity develops in B. sphaericus 2297 cultures during the late logarithmic stage, earlier than the appearance of the binary toxin crystal. It disappears with sporulation when the binary toxin activity reaches its peak. Disruption of the gene for the 42-kDa protein (P42) of the binary toxin abolishes both cell wall toxicity and crystal formation. However, the cell wall of B. sphaericus 2297, lacking P42, kills C. pipiens larvae when mixed with Escherichia coli cells expressing P42. Thus, the cell wall toxicity in strongly toxic B. sphaericus strains must be attributed to the presence in the cell wall of tightly bound 51-kDa (P51) and P42 binary toxin proteins. The synergism between binary toxin crystals and urea-treated cell wall preparations reflects suboptimal distribution of binary toxin subunits in both compartments. Binary toxin crystal is slightly deficient in P51, while cell wall is lacking in P42.     

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.     

A second independent resistance mechanism to Bacillus sphaericus binary toxin targets its alpha-glucosidase receptor in Culex quinquefasciatus

The entomopathogen Bacillus sphaericus is an important tool for the vector control of Culex sp., and its effectiveness has been validated in field trials. The appearance of resistance to this bacterium, however, remains a threat to its use, and attempts have been made to understand the resistance mechanisms. Previous work showed that the resistance to B. sphaericus in a Culex quinquefasciatus colony is associated with the absence of the approximately 60-kDa binary toxin receptor in larvae midgut microvilli. Here, the gene encoding the C. quinquefasciatus toxin receptor, Cqm1, was cloned and sequenced from a susceptible colony. The deduced amino-acid sequence confirmed its identity as an alpha-glucosidase, and analysis of the corresponding gene sequence from resistant larvae implicated a 19-nucleotide deletion as the basis for resistance. This deletion changes the ORF and originates a premature stop codon, which prevents the synthesis of the full-length Cqm1. Expression of the truncated protein, however, was not detected when whole larvae extracts were probed with antibodies raised against an N-terminal 45-kDa recombinant fragment of Cqm1. It seems that the premature stop codon directs the mutated cqm1 to the nonsense-mediated decay pathway of mRNA degradation. In-gel assays confirmed that a single alpha-glucosidase protein is missing from the resistant colony. Further in vitro affinity assays showed that the recombinant fragment binds to the toxin, and mapped the binding site to the N-terminus of the receptor.     

Persistence and recycling of Bacillus sphaericus 2362 spores in Culex quinquefasciatus breeding sites in West Africa

Summary A flowable concentrate of Bacillus sphaericus strain 2362 was applied at 10 g/m² against Culex quinquefasciatus mosquito larvae in cesspools. Complete control of larvae was maintained during 5 to 6 weeks, due to a very low settling of B. sphaericus spores, and was related to the presence of at least 100 to 500 spores per ml in upper water layers. Larval cadavers sedimented within 48 h after treatment. B. sphaericus was shown to recycle in dead larvae but not in mud. Spore persistence exceeded 5 months in bottom mud and the concentration of persistent spores was higher in cesspools, the bottom of which was cemented. Depth, temperature, pH, dissolved oxygen and suspended matter content of the water remained relatively constant throughout 4 months. In laboratory experiments, the final amounts of spores recycled in larvae was not influenced by spore concentration in water or by detergent, but it was affected by organic matter. Projected costs of B. sphaericus formulation indicates that its use even at high dosages, would be more cost effective than the use of chemical insecticides, especially where c. quinquefasciatus is resistant to these latter. A new strategy for controlling this vector could be deployed, using B. sphaericus and insect growth regulators in alternation.     

Improving the Insecticidal Activity against Resistant Culex quinquefasciatus Mosquitoes by Expression of Chitinase Gene chiAC in Bacillus sphaericus

Expression of a chitinase gene, chiAC, from Bacillus thuringiensis in B. sphaericus 2297 using the binary toxin promoter yielded a recombinant strain that was 4,297-fold more toxic than strain 2297 against resistant Culex quinquefasciatus. These results show that this chitinase can synergize the toxicity of the binary toxin against mosquitoes and thus may be useful in managing mosquito resistance to B. sphaericus.     

Permeabilization of Model Lipid Membranes by Bacillus sphaericus Mosquitocidal Binary Toxin and its Individual Components

The high larvicidal effect of Bacillus sphaericus (Bs), a mosquito control agent, originates from the presence of a binary toxin (Bs Bin) composed of two proteins (BinA and BinB) that work together to lyse gut cells of susceptible larvae. We demonstrate for the first time that the binary toxin and its individual components permeabilize receptor-free large unilamellar phospholipid vesicles (LUVs) and planar lipid bilayers (PLBs) by a mechanism of pore formation. Calcein-release experiments showed that LUV permeabilization was optimally achieved at alkaline pH and in the presence of acidic lipids. BinA was more efficient than BinB, BinB facilitated the BinA effect, and their stoichiometric mixture was more effective than the full Bin toxin. In PLBs, BinA formed voltage-dependent channels of ≈100–200 pS with long open times and a high open probability. Larger channels (≥400 pS) were also observed. BinB, which inserted less easily, formed smaller channels (≤100 pS) with shorter mean open times. Channels observed after sequential addition of the two components, or formed by their 1:1 mixture (w/w), displayed BinA-like activity. Bs Bin toxin was less efficient at forming channels than the BinA/BinB mixture, with channels displaying the BinA channel behavior. Our data support the concept of BinA being principally responsible for pore formation in lipid membranes with BinB, the binding component of the toxin, playing a role in promoting channel activity. Received: 29 March 2001/Revised: 20 July 2001     

Cross-resistance between strains of Bacillus sphaericus but not B. thuringiensis israelensis in colonies of the mosquito Culex quinquefasciatus

Two colonies of Culex quinquefasciatus Say (Diptera: Culicidae) were selected with Bacillus sphaericus strains C3-41 and IAB59 in the laboratory for 13 and 18 generations; they attained 145,000- and 48.3-fold resistance, respectively, in comparison with a susceptible laboratory colony (SLCq) and showed very high levels of cross-resistance (8500- to 145,000-fold) to B. sphaericus strains C3-41, 1593, 2297 and 2362. They were relatively susceptible to B. sphaericus strains LP1-G and 47-6B (only 0.8- to 2.8-fold tolerance), with 24.8- to 48.3-fold cross-resistance to strain IAB59. B. sphaericus-resistant mosquito colonies remained highly susceptible to B. thuringiensis israelensis, suggesting that B.t.i. would be of value in the management of B. sphaericus-resistant Cx. quinquefasciatus colonies. The demonstration of low or no cross-resistance of two selected resistant Cx. quinquefasciatus colonies to IAB59, LP1-G and 47-6B strains of B. sphaericus and the finding of a major 49 kDa protein in these strains suggest that there is likely to be another mosquitocidal factor in the three strains.     

Development of a Bacillus sphaericus tablet formulation and its evaluation as a larvicide in the biological control of Culex quinquefasciatus

This study aimed to analyze the final fermentation culture of Bacillus sphaericus 2362, standardize it and develop an active tablet formulation for use in urban mosquito breeding sites. It was performed in three phases: analysis and standardization of a B. sphaericus fermented culture; physical, chemical, and biological analysis of the active powder (solubility, residual humidity, particle size, resting angle, flowing off time, compacted density, and biological activity against Culex quinquefasciatus larvae); and the development of fast-disintegrating tablets. Five formulations with differing compositions were developed and a UV protector was added to the selected formulation. The formulation products with or without UV protector, as well as the active powder caused 100% larval mortality from 1 day to 2 months after a single treatment under simulated field conditions. These results show that the UV protector does not affect the initial larvicide activity of B. sphaericus, nor its persistence over a period of two months.     

Ultrastructure of midgut events in the pathogenesis of Bacillus sphaericus strain SSII-1 infections of Culex pipiens quinquefasciatus larvae.

The fate of Bacillus sphaericus strain SSII-1 cells ingested by Culex pipiens quinquefasciatus (= C. pipiens fatigans, C. fatigans, C. quinquefasciatus of authors; Diptera: Culicidae) larvae and the cytological events preceding death of the host were observed using electron microscopy. Bacillus sphaericus cells were digested rapidly in the anterior and central midgut. The outer cell wall layer and cytoplasmic ground substance disappeared soon after ingestion. Cytolysosomes became prominent in midgut cells as these cells gradually separated from one another. All bacteria, including B. sphaericus, were confined within the peritrophic membrane until after death of the host. Digestion by the larval host is confirmed as a possible mechanism for release of B. sphaericus toxin from the bacterial cells.