On-line version ISSN 1678-9946
Rev. Inst. Med. trop. S. Paulo vol.41 n.2 São Paulo Mar./Apr. 1999
In this present study, preliminary data was obtained regarding the mortality rate of the Amazonian anophelines, Anopheles nuneztovari, Anopheles darlingi and Anopheles braziliensis when subjected to treatment with Bacillus sphaericus strain 2362, the WHO standard strain. Initially, experiments were conducted to test the mortality rate of the three species of anopheline larvae. The third larval instar of An. nuneztovari and the second and third larval instars of An. darlingi proved to be the least susceptible. In other experiments, the same three mosquito species were tested with the standard strain 2362, An. nuneztovari was the least susceptible to this insect pathogen, while An. braziliensis was the most susceptible. This latter species showed a difference in the level of LC50 concentration, when compared to the former, of 2.4, 2.5 and 1.8 in readings taken 24, 48 and 72 hours after exposure to the bacillus.
KEYWORDS: Malaria; Biological control; Anopheline; B. sphaericus.
The Amazon is considered to be a region of high malaria transmission. This is particularly so in areas recently cleared of forest, areas of colonization and mining, and some indigenous settlements. In Manaus, the capital of Amazonas, 587 people out of 100,000 inhabitants contracted malaria in 199520. These figures are alarming because this is an area where vector control with chemical insecticides and treatment of the Plasmodium bearing patients are routinely carried out.
Important aspects of malaria were discussed at the Ministerial meeting on malaria in Amsterdam (1992)22. Emphasis was placed on the necessity of launching a search for more selective, alternative and/or complementary methods of vector control, while taking into account man's relationship with the environment.
The incidence of human malaria cases in the forest edges around Manaus is very high. An. darlingi is the main human malaria vector in Amazonia and most of Brazil2,19,21. Other species were also shown to be malaria vectors. An. nuneztovari has been found to be infected with Plasmodium vivax and Plasmodium falciparum1,13,18 and An. braziliensis with P. falciparum3.
Biological control is a method which uses biotic agents that are toxic, antagonostic or lethal to a target insect. Available literature cites several bacteria that, according to laboratory and field trials could be used to control the vector at breeding sites, however, few deal with B. sphaericus against anophelines.
In view of the increasing resistance of mosquitoes to chemical insecticides and the lack of new alternative and/or complimentary methods to control these insects, Bacillus sphaericus is being considered as a possible control measure against malaria vectors in Brazil.
LACEY & ORR (1994)9 report differences in the mortality rate to B. sphaericus strain 2297 (H25) between An. albimanus and An. quadrimaculatus. Differences were also reported between An. culicifacies and An. stephensis10. In field trials, using a concentrated spray of the B. sphaericus 2362 against An. gambiae, to obtain complete population control, 1x102 to 2x103 viable spores were necessary in the feeding zone12. KARCH et al. (1991)7 treated a natural breeding site with a 2362 strain showing larvicidal activity in high dosages against An. stephensis, An. subpictus and An. culicifacies.
This first report on the activity of B. sphaericus 2362 against Anopheles species in Amazonia constitutes the basis of this paper. The mortality rate of various larval instars and the dosage response line of B. sphaericus strain 2362 against larvae of An. nuneztovari, An. darlingi and An. braziliensis was studied under laboratory conditions.
MATERIAL AND METHODS
Adult females anopheline were collected around Manaus (AM), Jaciparaná (RO) and Maruanum, Macapá (AP). They were individualy isolated to ovulate. After emerging from the eggs, the larvae were transfered to enamel pans each containing 500 ml of potable water from an artesian well and 2ml of food. The previously prepared food, contained 1g of liver powder and 8g of fish flour, dissolved in 500 ml of water. The water in the enamel pans was changed every two days, until the larvae reached the appropriate larval developmental stage to be used in the bioassays, taking from three to five days. The insectary was maintained at a temperature of 26 ± 2ºC with relative humidity of between 80% to 90%. The standard methodology for maintaining anopheline larvae is consistent with methods of the Malaria Vector Laboratory (INPA)15,16.
Bacillus sphaericus 2362 samples
The bacillus used was the WHO standard strain 2362 obtained from the Bacterial Entomopathogens Unit of the Pasteur Institute (France). The bacteria were grown in a nutritive broth in a rotating incubator (30±0.5ºC, 200 rpm) for 48 - 72 hours, centrifuged at 10,000 rpm for 20 minutes and afterwards the pellets were lyophilized for 12 to 15 hours.
The bioassays were carrried out at a mean temperature of 26±2ºC, using plastic cups containing 100 ml of drinking water, 20 anopheline larva, 1 ml of food for the larvae and a specific volume of bacillar suspension (mother-suspension) to attain the previously defined concentrations.The method used in the observation of mortality and survival of the bioassays was that described by DULMAGE et al. (1990)4, with some modifications.
The mortality rate in the control group could not exceed 20% of the larval population as this was considered the demarcation point for acceptability of the bioassay.
Suspensions formulated for bioassays
Mother suspensions were formed by dissolving 50mg of lyophilized bacteria in 10 ml of drinking water. The test tube was shaken in vortex for two minutes to facilitate dissolution. Starting from the mother suspension, other suspensions were prepared for decimal dilution.
Bioassays for evaluating susceptibility of different instars
Bioassays with anopheline larvae that were in one of the four developing larval stages were carried out using the following bacillus concentrations: 5 ppm, 5x10-3 ppm and 5x10-6 ppm. In each concentration, 60 larvae were tested for each of the three species, totalling 180 for each instar not including the control treatment. The readings were made after 24, 48 and 72 hours of exposure to the bacillus. The first set of experiments aimed to evaluate the larvicidal activity of B. sphaericus 2362 strain against larvae in different instars of An. nuneztovari and An. darlingi.
Bioassays for calculating LC50 against An. darlingi, An. nuneztovari and An. braziliensis.
The methods used were those described by WHO (1985)23, using anopheline larvae in the third instar with concentrations of B. sphaericus 2362 of 1ppm, 0.5 ppm, 0.25 ppm, 0.12 ppm, 0.06 ppm, 0.02 ppm, 0.01 ppm. Three trials were conducted for each concentration. Mortality rate readings were made at 24, 48 and 72 hs of exposure.
Corrected mortality calculations were made using Abbott's formula as modified by Finney (1981)5. The LC50 calculations of the target population in each reading were performed by the probit analysis using the POLO PC program14. There was a confidence limit of 95% in all tests.
RESULTS AND DISCUSSION
Larvicidal Activity Against Various Instars
The results of the tests carried out on the four larval instars, and using different concentrations of bacteria (5 ppm, 5x10-3 and 5x10-6 ppm) showed that the third larval instar of An. nuneztovari was the least susceptible. In all situations 100% mortality was observed at 48 h using the highest concentration (Fig.1). Anopheles darlingi showed the lowest mortality rate when in it's third larval instar. The second instar also showed low corrected mortality rates at the three concentrations used, especially at 48 hour exposure (Fig. 2).
Fig. 1 - Corrected mortality rates of B. sphaericus against 3rd. larvae of An. nuneztovari, using 3 concentrations.
Fig. 2 - Corrected mortality rates of B. sphaericus 2362 against 3rd. larvae of An. darlingi, using 3 concentrations.
The results showed that to standardize the bioassays, third instar larvae must be used. These results are in accordance with observations made by KARCH (1984)6 who tested the sensitivity of the last three larval instars of An. stephensi to B. sphaericus 1593-4. His results showed that third and fourth larval instars were less affected than were earlier instars. MULLA (1990)11 reported that first larval instars were difficult to handle, which might have caused high mortality rates due to the handling procedures. The fourth instar larvae that feed very little or have ceased to feed are less susceptible, since their ingestion of the toxin is minimal during this short period11.
Mortality Rate of Anopheles Species Mosquitoes When Subjected to B. sphaericus 2362
Figure 3 represents the dosage response line of B. sphaericus 2362 against third instar larvae of An. nuneztovari, An. darlingi and An. braziliensis at 24, 48 and 72 hs of exposure. These results were used to calculate the LC50 values presented in Table 1. Considering the mortality rates of An. nuneztovari, An. darlingi and An. braziliensis to B. sphaericus 2362, the results of the different concentration tests (1 ppm to 0.1 ppm ) showed that the first species is only a little less sensitive than the second, with values very close to LC50 (0.471 ppm and 0.457 ppm respectively). Anopheles braziliensis is the species that showed the greatest mortality rate when exposed to the bacillus for 24, 48 and 72 hs, as can be seen in Table 1. In the case of the other two anopheline species, very close values of LC50 in the three readings were observed. For 24 and 48 h, the LC50 values for An. darlingi are slightly lower than those calculated for An. nuneztovari. In the 72-h exposure, the reverse is seen: An. nuneztovari is slighty more susceptible than An. darlingi to B. sphaericus 2362.
Fig. 3 - Dosage response line of Bacillus sphaericus 2362 instar larvae of An. nuneztovari and An. darlingi at 24 (A), 48 (B) and 72 (C) hours.
The probit analysis showed that An. darlingi became more sensitive than An. nuneztovari to the bacillus when the concentration was higher than 0.12 ppm, causing a reversal in mortality rate. A similar reversal in mortality rate occurs when comparing An. braziliensis and An. nuneztovari. In comparing the sensitivity of An. braziliensis and An. darlingi, the results showed that the bacillus caused a higher mortality rate in the first species at all concentrations and exposure times (Fig. 3).
In order to obtain values that can indicate the anopheline mortality rate differences when exposed to B. sphaericus 2362, the relative mortality rate index was calculated by relative susceptibility (RS):
In the present, study An. nuneztovari was chosen as the standard because the highest LC50 values for 24 and 48 hs were obtained in bioassays with this mosquito. The relative susceptibility values (LC50 standard / LC50 anopheline) of B. sphaericus 2362, in relation to An. darlingi and An. braziliensis, considering An. nuneztovari as standard are shown in Table 2. The data in this table show that the values are much lower for An. darlingi in relation to An. braziliensis. For the 48-hr reading of An. darlingi, the susceptibility of the two species is very close (RS=1.30), with a reversal occurring in the 72-h reading (RS=0.90). The high mortality rate of An. braziliensis remains evident, especially in the 24-hs reading, where it was 189% more sensitive than An. nuneztovari.
Therefore, it can be expected that different mosquitoes will have a different susceptibility to B. sphaericus toxins. This fact has been showed by LACEY & SINGER (1982)8, who found different LC50 values for An. albimanus and An. quadrimaculatus. Testing B. sphaericus strains 2013-4, they obtained LC50 values of 0.0187 and 0.0527 ppm, respectively. Using B. sphaericus strains 2013-6, the LC50 values were 0.0168 and 0.0558 ppm.
Little information exists regarding susceptibility in the laboratory of anopheline species to B. sphaericus strain 2362.
Bacillus sphaericus 2362 strain proved to be effective against all larval instars of An. nuneztovari, An. darlingi and An. braziliensis. It was shown that the third instar larvae of An. nuneztovari are less susceptible than the larvae in other instars. In An. darlingi, the second and third instars were equally less susceptible. When comparing susceptibility of the three Anopheles species to B. sphaericus 2362, An. braziliensis is the most susceptible, followed by An. darlingi and An. nuneztovari.
In field trials, it has been observed that An. nuneztovari and An. darlingi and, to a lesser extent, An. braziliensis, may coexist in breeding sites18,20. These ecological facts are relevant when considering the possibility of using B. sphaericus for the biological control of these three anopheline species. The LC50 values reported here show that, in habitats supporting the coexistence of all three species, a program of B. sphaericus application will affect even the species with the lowest susceptibly, An. nuneztovari.
Atividade larvicida do Bacillus sphaericus 2362 contra Anopheles nuneztovari, Anopheles darlingi e Anopheles braziliensis (Diptera, Culicidae)
Foram obtidos neste trabalho, os primeiros dados relativos a sensibilidade de anofelinos vetores da malária na Amazônia - An. nuneztovari, An. darlingi e An. braziliensis, a B. sphaericus 2362 estirpe padrão da OMS. Inicialmente, foram realizados bioensaios para verificar a susceptibilidade dos estádios larvais. Para as duas primeiras espécies, os dados evidenciaram que o terceiro estádio é o menos sensível e que, para An. darlingi, além deste, o segundo estádio mostrou baixa mortalidade. Na continuidade, a estirpe padrão foi testada contra as espécies de Anopheles e verificou-se que An. nuneztovari foi a menos sensível a estirpe 2362 quando comparada as outras espécies, mostrando reduções nas concentrações da CL50, em relação à primeira variando de 2,4 a 1,8, nas leituras 24, 48 e 72 horas de exposição ao bacilo.
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Correspondence to: I.B. RODRIGUES, Caixa Postal 478, 69083-000 Manaus, AM, Brasil. e-mail: firstname.lastname@example.org
Received: 31 August 1998
Accepted: 30 November 1998