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On-line version ISSN 1981-5328
An. Soc. Entomol. Bras. vol.29 no.3 Londrina Sept. 2000
Toxicity of Bacillus species to larvae of Lutzomyia longipalpis (L. & N.) (Diptera: Psychodidae: Phlebotominae)
Toxicidade de espécies de Bacillus a larvas de Lutzomyia longipalpis (L. & N.) (Diptera: Psychodidae: Phlebotominae)
Eduardo D. WermelingerRI, II; José C. ZanuncioII; Elizabeth F. RangelIII; Paulo R. CeconIV; Leon RabinovitchV
IDepartamento de Ciências Biológicas, Escola Nacional de Saúde Pública/FIOCRUZ, Caixa postal 931, 21041-210, Rio de Janeiro, RJ, Brasil
IIDepartamento de Biologia Animal, Universidade Federal de Viçosa, 36571-000,Viçosa, MG, Brasil
IIIDepartamento de Entomologia, Instituto Oswaldo Cruz/FIOCRUZ, Caixa postal 926, 21045-900, Rio de Janeiro, RJ, Brasil
IVDepartamento de Informática, Universidade Federal de Viçosa, 36571-000, Viçosa, MG, Brasil
VDepartamento de Bacteriologia, Instituto Oswaldo Cruz/FIOCRUZ, Caixa postal 926, 21045-900, Rio de Janeiro, RJ, Brasil
A study was conducted to compare the susceptibility of third instar larvae of Lutzomyia longipalpis (L. & N.) (Diptera: Psychodidae: Phlebotominae), the vector of the American visceral leishmaniasis to two strains of Bacillus thuringiensis serovar israelensis, one strain of Bacillus sphaericus (all pathogenic to Diptera Culicidae) and a strain of B. thuringiensis ser. morrisoni (pathogenic to larvae of Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae)). Larvae of L. longipalpis showed similar susceptibility to the two strains of B. thruringiensis ser. israelensis, while B. sphaericus and B. thuringiensis ser. morrisoni showed low and no larvicidal effect to this vector, respectively.
Key words: Insecta, microbial control, leishmaniasis.
Foi feito um estudo comparativo da susceptibilidade de larvas de terceiro estádio de Lutzomyia longipalpis (L. & N.) (Diptera: Psychodidae: Phlebotominae), vetor da leishmaniose visceral americana, a duas estirpes de Bacillus thuringiensis sorovar israelensis e uma de Bacillus sphaericus, todas patogênicas a dípteros Culicidae, além de uma de B. thuringiensis sorovar morrisoni, patogênica a larvas de Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae). Larvas de L. longipalpis mostraram-se susceptíveis às duas amostras de B. thuringiensis sorovar israelensis, mas com baixa e nenhuma susceptibilidade às estirpes de B. sphaericus e de B. thuringiensis sorovar morrisoni, respectivamente.
Palavras-chave: Insecta, controle microbiológico, leishmaniose.
Control programs against phlebotomine sandfly vectors of leishmaniasis depend largely on household application of conventional insecticides (Gratz & Jany 1994, Perich et al. 1995, Service 1996). These applications target only adult populations since no practical means to control the immature stages of this pest are available, due to limited knowledge of their breeding sites in nature.
Diptera Nematocera larvae, including phlebotomine sandflies have a highly alkaline midgut, live in humid habitats and feed on decaying matter or associated fungi (Panizzi & Parra 1991). Although information regarding their breeding sites is scarce, it is likely that phlebotomine larvae are detritivores, similar to the majority of soil inhabiting invertebrates which consum dead organic matter in various decomposition stages. For this reason they can live in many microhabitats, such as fallen tree trunks, rock holes and cracks, humus, and so on. Such conditions are suitable for the development and long-term persistence of many entomopathogens, which can be used to control phlebotomine larvae (Warburg et al. 1991). This presents an opportunity, alone or in combination with other methods to control vectors of the leishmaniasis.
The spore forming bacteria of the genus Bacillus is efficient against important Diptera vectors of diseases, particularly Culicidae and Simulidae (Barjac & Sutherland 1990, Priest 1992, Rozendaal 1997, Vilarinhos et al. 1998). The susceptibility of sandfly larvae to Bacillus thuringiensis serovar israelensis was previously demonstrated by Barjac et al. (1981) against Lutzomyia longipalpis and Phlebotomus papatasi. Pener & Wilamowski (1996) showed that P. papatasi is susceptible to B. sphaericus. Such studies do not clarify the pathogenicity of Bacillus to sandfly larvae and no study has compared toxicity of entomopathogenic Bacillus species to sandfly larvae.
The objective of this study was to compare the susceptibility of L. longipalpis, the vector of the American visceral leishmaniasis, to four strains of entomopathogenic Bacillus. The Bacillus strains used were: (1) B. thuringiensis ser. israelensis LFB/FIOCRUZ 584, from the standard powder IPS-82; (2) B. thuringiensis ser. israelensis LFB/FIOCRUZ 710, isolated from soil of the Monte Paschoal National Park, Bahia, Brazil, and serotyped by Dr. H. de Barjac; (3) B. thuringiensis ser. morrisoni, LFB/FIOCRUZ 756, isolated from Brazilian soil of João Pessoa, Paraíba, Brazil, which was also serotyped by Dr. H. de Barjac; and (4) a standard strain of B. sphaericus ATCC 2362. All strains were supplied by the Collection Culture of the Genus Bacillus (CCGB), of the Department of Bacteriology of the "Instituto Oswaldo Cruz." The IPS 82 was originally supplied by Dr. H. de Barjac from the Pasteur Institute, Paris. B. thuringiensis ser. morrisoni proved pathogenic to larvae of Anticarsia gemmatalis (Hübner) (Lep. Noctuidae), while the other three strains have previously shown larvicidal action against dipterous Culicidae.
L. longipalpis larvae were obtained from a colony maintained through 78 generations in the insectary of the Leishmaniasis and Onchocerciasis Vectors Laboratory of the Entomology Department of the Instituto Oswaldo Cruz (Wermelinger et al. 1987).
Two concentrations of each bacteria were tested (10 mg and 20 mg of dry weight) of biomass bearing delta-endotoxin crystals and spores of Bacillus, obtained by bacterial growth in a New Brunswick 14 liter capacity fermentator loaded with a soya flour, divalent metals and yeast extract liquid culture medium, for 22 hours at 33ºC. This biomass was harvested by continuous centrifugation (3500 rpm). Samples of equivalent biomass to dry weigh material were mixed and homogenized with 1 g of autoclaved larval food, and used during seven days in each experimental lot. After this period, all lots received normal food, a commercial aquarium fish food "Vitormonio", first used by Rangel et al. (1985).
Each experimental unit was represented by 20 third instar L. longipalpis larvae, kept in 150 x 20 mm petri dishes with a 6 mm thick plaster of Paris in its bottom. Each dish was placed in a cylindrical plastic pot (170 mm diameter by 70 mm height), and covered with a filter paper. The experiment was developed at 25ºC in a B.O.D. incubator with relative humidity above 90%. Each bacterium concentration had four replications, and the control had at least five replications.
The experiment was carried out in three phases: the first with B. sphaericus ATCC 2362 and B. thuringiensis ser. israelensis LFB 584; the second with B. thuringiensis ser. israelensis LFB 584 and B. thruringiensis ser. israelensis LFB 710; and the third with B. thuringiensis ser. israelensis LFB 710 and B. thuringiensis ser. morrisoni LFB 756. Readings (three per week) were performed until the total number of surviving pupae in all dishes was recorded. Larval food was changed during each observation in order to avoid excessive fungal growth. Mortality rates of L. longipalpis larvae were based on total number of surviving pupae. Average mortality rates in the three phases, including the controls, were compared using Tukey's test (Pimentel Gomes 1990) at 5% probability level.
Data on the results of the tree phases are presented on Table 1. In the first phase, B. sphaericus was slightly virulent to L. longipalpis larvae with mortalities of 35% and 28,7% while B. thuringiensis ser. israelensis LFB 584 showed mortality above 70%. Mortalities atributed to B. sphaericus were not significantly different from the control while B. thuringienis ser. israelensis showed higher mortality than both B. sphaericus and the control. In the second phase, no significant differences were observed between three of the four mortality attributed to two strains of B. thuringiensis ser. israelensis. The mortality of 36.2% obtained for the LFB 584 strain in a concentration of 20 mg/g was statistically different from the 60% obtained for the concentration of 10 mg/g of this strain, and from averages of both concentrations (61.2%) of the strain LFB 710, and from mortality of 9.1% in the control. In the third phase, B. thuringiensis ser. morrisoni seemed to be non-pathogenic to L. longipalpis larvae, with mortality rates of 23.7% and 21.1% for the concentration of 10 mg/g and 20 mg/g, respectively. These results were lower than the mortality rate of 25% obtained in the control (Table 3). The LFB 710 strain of B. thuringiensis ser. israelensis showed 77.5% and 81.2% mortality rates with 10 mg/g and 20 mg/g respectively, which were significantly different from the 25% obtained in the control.
L. longipalpis larvae proved to be more susceptible to B. thuringiensis ser. israelensis than B. sphaericus and B. thuringiensis ser. morrisoni. B. thuringiensis ser. israelensis is pathogenic to sandfly larvae (Barjac et al. 1981), to mosquitoes of the genus Culex, Anopheles, Aedes and Mansonia (Nicolas 1992) and also to larvae of other Diptera in the Blephariceridae (Boisvert et al. 1985), Chironomidae (Ali et al. 1981, Boisvert et al. 1985, Mulla et al. 1990, Molloy 1992), Ceratopogonidae (Lacey & Kline 1983), Muscidae (Temeyer 1984), Sciaridae (Osborne et al. 1985, Keil 1991) and Tipulidae (Waalwijk et al. 1992, Smiths et al. 1993) which indicates that this bacteria is pathogenic to a wide spectrum of Diptera larvae species.
Larvicidal activity of B. thuringiensis ser. israelensis to L. longipalpis or to other species of sandfly vectors is poorly studied and needs to be more carefully evaluated because larval susceptibility to bacterial toxins varies considerably, even between closely related taxa. It may also vary with different conditions such as temperature, nutrition and larval stage. For example, in our results B. sphaericus showed slight pathogenicity to L. longipalpis larvae, but under different conditions, Pener and Wilamowski (1996) observed high susceptibility of 13 + 2 and 20 + 2 days old larvae of P. papatasi to B. sphaericus.
Mortalities in the control (first and third phases) were higher than 10%, which must be discarded according to Alves et al. (1998). However, normal mortality in experimental conditions and insetary, from third instar to pupae ranged from 10% to 30%. For these reason mortalities in the control groups can be accepted, although these rates should be kept as low as possible.
We conclude that L. longipalpis larvae are more susceptible to Bacillus thuringiensis ser. israelensis than B. sphaericus and B. thuringiensis ser. morrisoni. In comparison with B. t. israelensis, the other two strains proved to have low or none larvicidal activity.
To the staff of the Laboratory of Bacterial Physiology of the Dept. of Bacteriology and to the Sandfly Insectary, of the Leishmaniasis and Onchocerciasis Vector Laboratory, of the Dept. of Entomology of Oswaldo Cruz Institute for technical support. To Prof. Raul Guedes for suggestions on the manuscript. To CNPq and FAPEMIG for support.
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