Entomopathogenic bacteria isolated fromSimuliids are hematophagus insects naturally occurring in waterways throughout Brazil. Although some species have been involved in the transmission of human onchocerciasis in other regions (Gerais & Ribeiro 1986, Maia-Herzog et al. 1999) the main importance of these insects in the southeast of Brazil is socio-economic. The frequent and intensive attacks by simuliids on the transitory population in this area can reduce tourism in summer months, a fact that could negatively impact the economies of affected municipalities. To decrease the presence of these insects in these areas, a full-scale programme, based on Bacillus thuringiensis serovar israelensis, has been carried out in the north littoral zone of the State of São Paulo since 1990 (Araújo-Coutinho 1995).Recent findings of B. thuringiensis serovar oswaldocruzi and B. thuringiensis serovar braziliensis from unusual habitats (Rabinovitch et al. 1995) encourage the continued search for other endemic entomopathogens. For that purpose, Simulium larvae and adults from the north littoral zone of São Paulo and some other rivers in the State of Rio de Janeiro were evaluated for the presence of entomopathogenic bacteria.
MATERIALS AND METHODSCollection of simuliids -Black fly larvae and adults were collected from breeding sites in Pau d'Alho river, State of São Paulo. This river was divided into two regions, one downstream of B. thuringiensis serovar israelensis applications and another, upstream. Another collection point was the Soberbo river, Guapimirim, State of Rio de Janeiro (Fonseca et al. 1998). This river has never been exposed to commercial formulations based on B. thuringiensis serovar israelensis. Only larvae were collected in this waterway. The third point where larvae were collected was in Rio das Pedras, Mangaratiba, State of Rio de Janeiro.Isolation of Bacillus -To eliminate external contamination, the insects were sterilized following the methodology described by Alves (1986) with a slight modification; "Superbonder" glue (cyanoacrilate ester) was used to close both oral and anal cavities. This modification was adopted because Simulium larvae are very small and the dental floss used in the original technique was not applicable. After this step, the original method was performed passing the larvae through three solutions, first in 70% alcohol for 2 sec, second in 5% sodium hypoclorite for 3 min and finally in sterile 10% sodium thiossulfate for 5 min. The specimens were then washed three times in sterile distilled water.Two different methods were used to isolate B. thuringiensis samples. In both, Simulium larvae were transferred aseptically into a sterile mortar and macerated with a sterile pestle. In the first method, the macerate was placed in tubes containing distilled water (10 ml). The suspension was heated (65ºC) for 12 min, and immediately diluted (1x10 -2