Mem Inst Oswaldo Cruz, Rio de Janeiro, 93(4) Jul/Ago 1998
Studies on the Bacillus sphaericus Larvicidal Activity against Malarial Vector Species in Amazonia
Instituto Nacional de Pesquisas da Amazônia, Caixa Postal 478, 69083-000 Manaus, AM, Brasilu00a0
*CENARGEN - EMBRAPA, Brasília, DF
In this work, bioassays were carried out in laboratory conditions (average temperature 26 ± 2u00baC) to test ten strains ofu00a0Bacillus sphaericus, isolated from Brazilian soils against third instar larvae from anopheline species recorded as malaria vectors in Amazonian -u00a0Anopheles nuneztovariu00a0andu00a0An. darlingi. With the former mosquito, three strains - S2, S20u00a0and S46u00a0showed relative activity, in 24 and 48 hr exposure to theu00a0B. spahericusu00a0strains. With the latter only the S2u00a0and S20u00a0were effective in the 48 hr reading. The studied strains that showed the most adequate response in the Amazonian region were S2u00a0and S20u00a0showing broader and more efficient results. Therefore, S2u00a0was the most effective when the 24 and 48 hr readings were considered, because it showed the greatest relative activity values.
In the Amazonian region there are great malaria transmission areas mainly those with recent disturbance caused by settling, mining camps and some native villages, where a large number of vectors exists. The methodology used for the control of these mosquitoes, in this region, has been based on intradomicilium chemical control, which has not accomplished the same results as observed in other regions in the southern and southeastern parts of the country.
The importance of new technologies in the seeking of intervention strategies, such as for planning and application of preventive measures and vector control in a selective way is being pointed out. The biological control is an important strategy when prioritizing the enviromental question. For malaria in Amazonian endemic areas, the application of this means of attack on immature forms is one of the most relevant methods for winged density control. Therefore, in order to be implemented, bioassays are needed to evaluate the Anopheles species response to the biological control agents.
This work presents the first data obtained on the susceptibility of anopheline larvae, which are pointed out as malaria vectors in Amazonia - Anopheles nuneztovari and An. darlingi, to ten strains of Bacillus sphaericus isolated from Brazilian soils.
MATERIALS AND METHODS
Anopheline females collected in the suburbs of Manaus, State of Amazonas and in the municipal district of Jaciparaná, State of Rondônia, Brazil, were used in bioassays. Mosquitoes were laid to oviposit one by one in the insectary at a temperature of 26 ± 2ºC, with relative humidity of 80-90%, and were maintained in the laboratory according to Santos et al. (1981).
Ten strains of B. sphaericus were utilized in this work. They were isolated from soils colected in the following localities: Brasília, Federal District - S2, S46; Corumbá, State of Mato Grosso - S7, S11, S15 , S17, S20, S21 and S24; Vitória, State of Espírito Santo - S32. These were taken from the Microbian Germoplasm Bank of Centro Nacional de Pesquisas de Recursos Genéticos e Biotec-nológicos (CENARGEN). All the strains belong to serotype H5a5b (Barjac 1990, Schenkel et al. 1992, Dias 1992). For comparing Brazilian strains, the B. sphaericus strain 2362, the World Health Organization (WHO) standard strain was utilized.
The bioassays were conducted with third instar larvae, with six or seven concentrations of lyophilized bacteria ranging from 0.5 ppb to 50 ppm. The lyophilized of each strain was prepared from cultures in a rotating incubator (30±0.5ºC, 200 rpm) for 48 hr, centrifuged and rinsed with destilled water, frozen and lyophilized from 12 to 15 hr. Same as Brazilian isolates, strain 2362 has been grown and prepared for assay in the same way.
Twenty larvae per concentration tested from each Anopheles species were used in one single bioassay. The method used in the observation of mortality and survival of the biossays was the one of Dulmage et al. (1990), with some changes.
The target population average for lethal concentration (LC50) in each period of observation (24 and 48 hr) was estimated through probits analysis (Finney 1981) using the POLO-PC program. The relative activity (RA) was calculated using B. sphaericus 2362 as a standard, acording to the following formula:
|RA =||LC50 standard
The values of RA were calculated for 48 hr results.
RESULTS AND DISCUSSION
The larvicidal activity of the ten Brazilian strains tested can be evaluated by the association of the LC50 values (Table I) with the RA (Table II). Regarding An. nuneztovari, it is observed that the S2, S20 and S46 are the most effective with LC50 values between 0.084-0.127 ppm in 48 hr reading. In this last reading, considering B. sphaericus 2362 as a standard, the three cited strains showed RA varying from 3.7 to 5.6 times. The RA shows values above four units, except for S2 in the 48 hr reading (Table II). Few strains were effective in the control ofAn. darlingi, when we associated LC50 to RA. The S2, S20 and S46 were again stressed (only in the 48 hr reading) with RA around four and five times.
Figure shows the graphic representation of the dosage response line for the S2, S20 and S46 strains to the two species. Figure also shows the values for the 2362 WHO standard strain. It is observed that for An. nuneztovari (Figs 1A, 1B) the S2, S20 and S46 strains were more effective than the 2362. It has also been shown that the linear regression straight lines of the 2362 strain, in both Figs, presents a relatively parallel pattern to the S2 and S20 straight lines. In An. darlingi (Figs 1C, 1D) the LC50 values of S2 and S20 are close to the 2362 strain, in the 24 hr reading. The S2 is more effective being close to the S20 and 2362 values, in 48 hr. The linear regression straight lines of S2, S20 and 2362 are divergent in the minor concentration doses (abscissa), for they show zero mortality (24 hr) or next to 10% (48 hr - S2 and S20). There are no studies aboutbiological activity of B. sphaericus against anophelines which are important in the Amazonian region. S2 and S20 straight lines cross with the one from 2362 in values near the LC50 from the three strains. The RA data (Table II) revealed that the S2 and S20 strains were effective for the two species either at 24 or at 48 hr readings. Considering the other tested strains, in a general way, the RA values were lower than when compared to standard B. sphaericus 2362 strain. Therefore, it is observed that the Brazilian strains that presented a potential for the control of the anopheline immature forms were S2, S20 and S46, at 24 or 48 hr readings.
The studies on new strains against anopheline immature forms have been carried out in different parts of the world. Mulla (1986) tested larvicidal activity of different B. sphaericus 2362 preparations, and of 1593 and 2297 strains against An. albimanus and An. quadrimaculatus. In relation to the two Anopheles species, an activity variation of mortality rate caused by B. sphaericus 2362 was observed An. albimanus was from 8 to 25 times more sensitive than An. quadrimaculatus. Formulations showed different levels of larvicidal activity, according to the 2297 AP and SD strain formulations and to the 1593 Sawdust formulation, which showed high larvicidal activity levels againstAn. albimanus and low activity against An. qua-drimaculatus. Studies with different B. sphaericus strains, distributed into the six serotypes, were made testing An.sthephensi third instar larvae. Comparatively, the average LC50 on An. stephensi larvae were around 10-5 FWC dilutions (Thiery & Barjac 1989).
Other strains from different places in Brazil, that were also isolated at CENARGEN, were used by Schenkel et al. (1992) in bioassays with An. albimanus. These authors tested S1, S2, S5 and L2 (all isolated from Brasília) against An. stephensi larvae, taking the mortality rate observed with B.sphaericus 2362 as a standard. They observed that only L2 did not show larvicidal activity similar to the 2362 isolated strain, against this mosquito species. The S2 strain showed a toxicity that also affects other genera and Anopheles sub-genera. B. sphaericus entomopatogenic strain was first reported in a soil sample from Distrito Federal, pointing to S2 (Schenkel et al. 1992). Later characterization and biological activity showing that S2 is equaly or even more toxic than 2362 for An. albimanus andAn. quadrimaculatus. Besides these, S2 was biochemicaly characterized and dif-ferenciated from 2362 through lipid gaseous chromagraphy of the cell wall (Vilarinhos 1991).
The continuos isolation of new strains from native ones has indicated that many of these isolates showed a greater larvicidal activity than the 2362 strain.
Concluding, it should be pointed out that in several experiments in which S2 was used, against several Anopheles species, this strain showed high larvicidal activity often greater than the standard 2362 strain.
Brazilian strains of B. sphaericus show a greater larvicidal activity against Anopheles species, the malarian vectors in Amazonia, in relation to the standard 2362 strain. An. nuneztovari third instar larvae were more susceptible to strains: S2, S20, and S46 in 24 and 48 hr exposure. InAn. darlingi only S2 was effective in the two readings. The S20 showed greater activity only in the 48 hr reading. These data revealed that S2, S20 and S46 strains were potentialy the most adequate for the Amazonian region, against the studied species immature forms. Therefore, S2can be pointed out as the most effective one, for it showed activity in both readings with higher RA values.
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