Bacteriological larvicides of dipteran disease vectors.

The apparent success in vector control observed between 1950 and 1970 was followed by worldwide resistance to organosynthetic insecticides wherever they were used intensively. Insect resistance to one or more categories of insecticides has limited the effectiveness of these compounds, and their non-selective mode of action adversely affects non-target organisms. This scenario highlights the need for selective agents in integrated vector control programs. This article gives an overview of the main fundamental and applied research topics on entomopathogenic bacteria in relation to their role in vector control.

Impact of a 26-month Bacillus sphaericus trial on the preimaginal density of Culex quinquefasciatus in an urban area of Recife, Brazil.

A field trial using the entomopathogen Bacillus sphaericus against Culex quinquefasciatus was conducted in a district of Recife, Brazil, an area with a high prevalence of lymphatic filariasis. In this urban area more than 2,500 Cx. quinquefasciatus breeding sites within a 1.2-km2 area were found and subsequently submitted to a long-term treatment with B. sphaericus. To evaluate the impact of treatment on the densities of Cx. quinquefasciatus preimaginal forms, 26 breeding sites, representative of the major site types found in that area, were monitored for 3 years. Parameters such as mean and maximum densities of larval population as well as the frequency of infested sites were recorded before, during, and after the trial. The level of each parameter was greatly reduced during the treatment period, particularly in the 2nd treatment year. A low density of Cx. quinquefasciatus was also maintained throughout the last year, in the absence of treatment. Analysis of the data reported here confirms B. sphaericus as a very effective larvicide against Cx. quinquefasciatus despite the optimal environmental conditions for mosquito proliferation in the urban area of Recife.

Efficacy of Bacillus sphaericus in control of the filariasis vector Culex quinquefasciatus in an urban area of Olinda, Brazil.

The efficacy of Bacillus sphaericus 2362 against Culex quinquefasciatus was tested in 1991-94 in a major Brazilian endemic zone for bancroftian filariasis. Continuous selection pressure against the mosquito population was sustained for 18 months through treatment of 2500 potential breeding sites occurring within a 5.7-km2 urban area in the Metropolitan Region of Recife. The impact of this control intervention was evaluated by comparing entomological indices with those from an untreated area. Application of the larvicide kept the Cx. quinquefasciatus population density significantly lower when compared to the untreated area, despite some operational difficulties. Adult densities remained lower for at least 5 months after spraying ceased. Pre-trial microfilaria rates from the untreated and operational area were 13.1% and 7.2%, respectively. A 60% reduction in human exposure to infective bites was recorded as a consequence of this vector population control.

Identification of the receptor for Bacillus sphaericus crystal toxin in the brush border membrane of the mosquito Culex pipiens (Diptera: Culicidae).

The binary toxin (Bin) from Bacillus sphaericus crystals specifically binds to soluble midgut brush border membrane proteins from Culex pipiens larvae. A single 60 kDa midgut membrane protein is identified as the binding protein. This protein is anchored in the mosquito midgut membrane via a glycosyl-phosphatidylinositol (GPI) anchor, and is partially released by phosphatidylinositol specific-phospholipase C (PI-PLC). Fractionation of soluble proteins by anion exchange chromatography indicates that the binding protein does not co-elute with leucine aminopeptidase activity. After partial purification, the sequences of internal amino acid fragments of the 60 kDa protein were determined. The peptide sequences were compared with data in GenBank, and showed a very high degree of similarity with enzymes belonging to the alpha-amylase family. Further enzymatic investigation showed that the receptor of the Bin toxin in C. pipiens larval midgut may be an alpha-glucosidase.

Binding of the 51- and 42-kDa individual components from the Bacillus sphaericus crystal toxin to mosquito larval midgut membranes from Culex and Anopheles sp. (Diptera: Culicidae).

Individual components (P51 and P42) from the crystal toxin (Bin) of Bacillus sphaericus were used for in vitro binding competition experiments with brush border membranes (BBMFs) from Culex pipiens and Anopheles gambiae larval midguts. P51 competed for the Bin binding site with a similar affinity to the Bin toxin, on both BBMFs. For C. pipiens, P42 bound non-specifically until P51 was added with maximum binding of P42 at a molar ratio of each component. The binding of P42 was much greater on A. gambiae BBMFs and the presence of either P51 or P42 enhanced the binding of the other component, with highest binding when a mole ratio of each protein was supplied.

Binding kinetics of Bacillus sphaericus binary toxin to midgut brush-border membranes of Anopheles and Culex sp. mosquito larvae.

Direct-binding assays and homologous-competition assays were used to identify specific binding between the radiolabelled toxin of Bacillus sphaericus and brush-border membrane fractions (BBMF) from Anopheles gambiae and Anopheles stephensi, obtained from whole larvae preparations. In both species, the toxin bound to a single class of receptors. BBMF of A. gambiae had the highest binding affinity for the toxin of the species tested, with a dissociation constant (Kd) of 30 +/- 15 nM and a maximum receptor concentration of 5 +/- 1 pmol/mg. Toxin binding to A. gambiae BBMF was compared with that to BBMF from B. sphaericus-susceptible (IP) and B. sphaericus-resistant (SPHAE) Culex pipiens populations. BBMF toxin binding was slower in A. gambiae than in the C. pipiens populations. The BBMF of the B. sphaericus-resistant population of C. pipiens had an association profile that was similar to the susceptible population, despite of the lack of susceptibility in vivo. No relationship between toxicity and irreversibility of toxin binding was detected. On the contrary, toxin dissociation from BBMF was fast and almost complete in BBMF of all species studied.

Reversal of low-level resistance to Bacillus sphaericus in a field population of the southern house mosquito (Diptera:Culicidae) from an urban area of Recife, Brazil.

A larval population of Culex quinquefasciatus Say from an urban area (Coque) of Recife, Brazil, submitted to selection with Bacillus sphaericus Neide for a 26-mo trial, was 10 times less susceptible to B. sphaericus and slightly more susceptible to Bacillus thuringiensis israelensis, compared with untreated populations. The low-level resistance to B. sphaericus was unstable in the absence of selection pressure. The LC50 of B. sphaericus to the Coque population declined gradually and attained a susceptibility level similar to that observed in a laboratory control colony 16 mo after the treatment period was interrupted. In parallel to the recovery of B. sphaericus susceptibility. Coque larvae also were as susceptible as laboratory larvae to B. thuringiensis israelensis.

Integrated control measures against Culex quinquefasciatus, the vector of filariasis in Recife.

Integrated control measures against Culex quinquefasciastus have been implemented in a pilot urban area in Recife, Brazil. About 3,000 breeding sites found within the operational area were responsible for very high mosquito densities recorded during the pretrial period. Physical control measures have been applied to cess pits before starting a series of 37 treatments of the other sites with Bacillus sphaericus strain 2362, over 27 months. In spite of the difficulties due to environmental conditions, very significant reductions in preimaginal population of C. quinquefasciatus were achieved and, as a consequence, low adult mosquito densities were maintained for a relatively long period of time. Entomological and environmental data gathered in this pilot project can contribute to design an integrated mosquito control program in Recife city.