Biological activity of Bacillus thuringiensis strains against larvae of the Blowfly Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae)

Atividade biológica de linhagens de Bacillus thuringiensis sobre larvas da mosca varejeira Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae)

PUBLIC HEALTH

Biological activity of Bacillus thuringiensis strains against larvae of the Blowfly Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae)

Atividade biológica de linhagens de Bacillus thuringiensis sobre larvas da mosca varejeira Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae)

Marcio S. Oliveira^I,II; Marcelo A. Nascimento^II; Clara F.G. Cavados^III; Jeane Q. Chaves^III; Leon Rabinovitch^III; Marli M. Lima^II; Margareth M.C. Queiroz^II

^ICoordenação de Vigilância Ambiental em Saúde/SES-RJ, Rua México, 128/413 - 200315-142, Rio de Janeiro, RJ [e-mail]

^IIDepto. Biologia. IOC/FIOCRUZ, Av. Brasil, 4365, 21045-900, Rio de Janeiro, RJ

^IIIDepto. Bacteriologia. IOC/FIOCRUZ, Av. Brasil, 4365, 21045-900, Rio de Janeiro, RJ

ABSTRACT

Different strains of Bacillus thuringiensis Berliner were proved to be a powerful biologic insecticide against larvae of several insect orders. Due to the epidemiological importance of blowflies of the Chrysomya Robineau-Desvoidy genus in the production of secondary cutaneous myiasis and mechanic transmission of pathogenic agents, the performance of two strains of B. thuringiensis (LFB-FIOCRUZ 907 and LFB-FIOCRUZ 856) was tested against larvae of Chrysomya putoria (Wiedemann). The LFB-FIOCRUZ 907 strain was tested in four different concentrations, added to the diet; the LFB-FIOCRUZ 856 strain was tested in three concentrations. C. putoria larvae showed sensibility to the treatment with the LFB-FIOCRUZ 907 strain at the tested concentrations. The higher concentration presented the best efficiency, causing higher mortality and reducing larval weight and adult emergence more intensely. The LFB-FIOCRUZ 856 strain showed low toxicity, sliglity reducing emergence time of adults at 326 mg/ 25 g concentration and larval weight at 326 mg/ 25 g and 86 mg/ 25 g concentrations.

Key words: Fly, entomopathogenic action, biological control

RESUMO

Diferentes linhagens de Bacillus thuringiensis Berliner têm provado ser poderoso inseticida biológico contra larvas de várias ordens de insetos. Dada a importância epidemiológica das moscas do gênero Chrysomya Robineau-Desvoidy na produção de miíases cutâneas secundárias e transmissão mecânica de agentes patogênicos, avaliou-se a atividade de duas linhagens de B. thuringiensis (LFB-FIOCRUZ 907 e LFB-FIOCRUZ 856) sobre larvas de Chrysomya putoria (Wiedemann). A linhagem LFB-FIOCRUZ 907 foi testada em quatro diferentes concentrações misturadas à dieta e a linhagem LFB-FIOCRUZ 856 em três concentrações. As larvas de C. putoria apresentaram sensibilidade ao tratamento com a linhagem LFB-FIOCRUZ 907, nas concentrações testadas, sendo a concentração mais alta a de maior eficiência, causando maior mortalidade e reduzindo mais intensamente o peso larval e a taxa de emergência dos adultos. A linhagem LFB-FIOCRUZ 856 apresentou toxicidade muito baixa, redizindo ligeiramente a emergência dos adultos na concentração de 326 mg/ 25 g e o peso larval nas concentrações de 326 mg/ 25 g e 86 mg/ 25 g.

Palavras-chave: Bacilo, ação entomopatogênica, controle biológico

The use of pathogens and their metabolic products for the control of vector insects have several advantages, such as specificity because they do not present resistance problems, do not pollute the environment, and are non toxic for humans (Alves 1998). The entomopathogenic action of Bacillus thuringiensis Berliner in some insect orders was already described (Feitelson et al. 1992, Alves 1998). The synthesis of toxins as intra-cytoplasmic proteic crystal occurs during B. thuringiensis esporulation (Höfter & Whiteley 1989); when ingested by susceptible insects, the pathogen causes serious lesions or death.

In this work, we observed the effects of the complex formed by the spore and the d-endotoxin pro-toxin of this bacterium in flies of the Chrysomya putoria (Wiedemann) (Diptera: Calliphoridae) species. The strains used were B. thuringiensis LFB-FIOCRUZ 907 and LFB-FIOCRUZ 856. The larvae produce secondary type cutaneous myiasis in vertebrates (Leclerq 1990) and the adults are mechanical vectors of pathogenic microorganisms (Furlanetto et al. 1984, Guimarães & Papavero 1999, Mariluis 1999). During the experiment under laboratory conditions, we tested the efficiency of bacillus strains for controlling blowflies. The results are an important contribution for further application to natural conditions.

Material and Methods

Bacterial strains. The strain B. thuringiensis LFB-FIOCRUZ 907, isolated from soil in Rio de Janeiro, Brazil, responded the same way as B. thuringiensis serovar israelensis IPS-82 flagelar serum type H-14. The latter, which is considered the standard strain by the Institut Pasteur of Paris, is currently used as an active source for commercial, bacterial and biological insecticides. The strain B. thuringiensis LFB-FIOCRUZ 856, which is identical to B. thuringiensis serovar oswaldocruzi was isolated from black pepper powder consumed in Rio de Janeiro (Cavados et al. 1998).

Preparing the bacterial biomass. The bacterial biomass was prepared according to Cavados et al. (1998). The samples of B. thuringiensis were grown in Nutrient Broth (Bacto Nutrient Broth, Difco Laboratories) supplemented with 5 g/l of glucose and metals, such as MgSO₄.7H₂O – 0.02 g/l, MnSO₄.7H₂O – 0.03 g/l, ZnSO₄.7H2O – 0.02 g/l, FeSO₄.7H₂O – 0.02 g/l, and CaCl₂ – 0.1 g/l, pH 7.0.

Growth started with a pre-inoculum, where samples were cultivated for environmental adaptation and shortening of the lag phase of bacterial growth. The samples were inoculated in 125 ml Erlenmeyer flasks containing 50 ml of the medium, and the flasks were incubated in a New Brunswick Scientific series 25D agitator, at 175 rpm and 30ºC for 6h. Afterwards, 3 ml were transferred to 500 ml Erlenmeyer flasks containing 150 ml of the medium and incubated as previously described, for more 72h.

Once sporulation reached 90% of free spores, each culture was centrifuged (6,000 g, 10ºC). Next, the biomasses were suspended in distilled water acidified with 0.7% propionic acid to pH 3.0 to keep the crystals active, and left to rest for 1h. For preservation, all samples were submitted to a second centrifugation and the biomasses kept in an amber container with pH adjusted with propionic acid to 5.0, in a refrigerator.

Determining the dry weight. Samples of 0.5 g biomass were weighed. Each sample was replicated three times and kept for 24h in a vacuum incubator at 70ºC and a negative pressure of 62 mm Hg. The dried biomasses were transferred to a desiccator and submitted to vacuum for 1h, to reach room temperature. The biomasses were weighed on an analytical balance up to the fourth decimal case, to determine dry biomass the mean weight and moisture content (Cavados et al. 1998).

Counting the number of viable cells. A sample corresponding to 25 mg (dry weight) of the biomass was transferred to a 50 ml volumetric flask. The volume was completed with distilled water and the solution was homogenized for 5 min. The mother suspension contained 0.5 mg/ml. For quantification of viable spores, 5 ml of the solution were subjected to a 80ºC water bath for 12 min. After cooling to room temperature, decimal dilutions (1 ml of suspension in 9 ml of sterile saline solution) were prepared in sterilized tubes, down to 1:105. The last three dilutions (1:103, 1:104, and 1:105) had 0.1 ml drawn for breeding, by spreading on the surface of Nutrient Agar poured on petri dishes. Three dishes were used for each dilution and incubation lasted 24h at 33ºC. The colonies were counted, the average determined, and the number of spores/mg calculated (Cavados et al. 1998).

Assessing the entomopathogenic activity in C. putoria. The bioassays were conducted in climatized chambers (B.O.D.) at 27 ± 1ºC, 60 ± 10% R.H., and 14h artificial photoperiod. The egg masses of C. putoria were obtained from adults kept under laboratory conditions, according to the methodology proposed by Queiroz & Milward-de-Azevedo (1991); these masses were transferred to petri dishes covered with paper filter dampened with distilled water. After hatching, 50 newly emerged larvae (first instar) were transferred to plastic recipients containing 25 g of diet (beef in early decomposition) mixed with the LFB-FIOCRUZ 907 strain, except for the control group, which had just received the diet. Four concentrations of bacillus (55 mg/25 g, 134 mg/25 g, 209 mg/25 g, and 326 mg/25 g) were used in three repetitions for each biomass preparated. The LFB-FIOCRUZ 856 strain was tested in 55 mg/25 g, 86 mg/25 g, and 326 mg/25 g concentrations, using the same procedures as for the first strain. The recipients were placed into bigger containers with vermiculite, covered with nylon mesh, and fixed around the edges with a rubberband.

The larvae and their relation to the development and ingestion of the spore-endotoxin complex were observed daily for number of dead larvae, weight of mature larvae, duration of larval and pupal stages, and average adult emergence. A slide rubbed with intestinal content of dead larvae and stained by the Gram method was used to observe vegetative forms of B. thuringiensis.

Statistical analysis. The biological activity (LC₅₀) was determined by the logarithmic probability analysis. The analysis of variance (ANOVA 1; P < 0.05) and Tukey (P < 0.05) tests were used.

Results and Discussion

Varieties of B. thuringiensis produce some toxins already well characterized and others still insufficiently known for many insects. The d-endotoxins of different subspecies of these bacilli can vary considerably in toxicity, what seems associated with differences in the amino acid sequences of toxins (Federici et al. 1990). These are codified by four types of Cry genes, which apparently share an evolutionary origin due to their important DNA homology (Gill et al. 1992). A different type of gene codifies a protein called Cyt, which differs from Cry proteins in structure and biological activity. This second type of gene is found in the parasporal bodies of israelensis subspecies (Gill et al. 1992). Heimpel & Angus (1959) were the first to report on the toxin mode of action and noted that the breached intestine membranes allowed for ionic flow to the haemolymph. Further studies showed that the toxin mode of action is related to formation of non-specific pores in the intestine epithelial cell membrane of insects (Knowles & Ellar 1987, Brousseau & Masson 1988, Gill et al.1992, Honée & Visser 1993).

Temeyer (1984) studied the larvicidal activity of B. thuringiensis serovar israelensis against Haematobia irritans L. larvae (Diptera: Muscidae), and reported that the d-endotoxine was only active during the larval, and not the pupal stage. Wilton & Klowden (1985) tested the effect of the preparation on Musca domestica L. adults (Diptera: Muscidae), Stomoxys calcitrans L. (Diptera: Muscidae), and Chrysomia carnea (Neuroptera: Chrysopidae), concluding that adults of M. domestica and C. carnea were not susceptible to quantity. However, Stomoxys was susceptible to dose.

In Table 1, the results of bioassays on the biological activity of LFB-FIOCRUZ 907 tested against C. putoria larvae are presented. The toxicity of LFB-FIOCRUZ 907 strain was high. The activity of LFB-FIOCRUZ 907 strain against larvae caused a depression effect on the average emergence percentage of C. putoria adults. The highest concentrations were the most efficient, affecting larval weight more intensely (Table 2). Variations in the duration of post-embryonic development are presented in Table 3. The LC₅₀ calculated for the larva- adult period was 7.2 mg/g.

The B. thuringiensis LFB-FIOCRUZ 907 strain was toxic in high doses for C. putoria larvae. Our results are comparable to Vankova’s (1981), who observed a small effect on larvae and pupae of M. domestica. The same has occurred with the strain of B. thuringiensis var kurstaki, which is present in a commercial product. However, when Vankova (1981) used a B. thuringiensis serovar thuringiensis (H-1) strain synthesizing b-exotoxina, the results were satisfactory (LC₅₀ = 65 mg/kg) because the strain inhibited larvae growth in animal excrements.

Cavados et al. (1998) evaluated the action of the LFB-FIOCRUZ 907 strain on larvae of Chrysomya megacephala (Fabricius) and observed toxicity in high doses (14.3 mg/g) and a reduction in average emergence. The LC₅₀ from larva to adult development was 6.1 mg/g. The authors also found vegetative forms in slides of the digestive tube of dead insects.

Lonc et al. (1991) used B. thuringiensis serovar morrisoni, B. thuringiensis serovar darmstadiensis, and two strains of B. sphaericus to evaluate toxicity in M. domestica in the laboratory. The activities performed by the tested strains were similar but the concentrations were high in relation to particle per volume unit (4 x 10⁷ - 4 x 10⁸ spores/ml).

Neither the viability percentage of larvae submitted to treatment with the LFB-FIOCRUZ 856 strain, nor the duration of larval and pupal stages differed among the tested concentrations. The only significant differences of the action of the LFB-FIOCRUZ 856 strain were in adult emergence percentage, at the concentration of 326 mg/25 g of diet (in relation to the control group) and in larval weight,at the concentrations of 326 mg/25 g and 86 mg/25 g of diet (Table 4).

An important difference among the tested strains was that the B. thuringiensis serovar oswaldocruzi, LFB-FIOCRUZ 856 was not significantly active on C. putoria in the concentrations used and did not affect the larvae, had null mortality, and affected adult emergence only at the highest diet concentration (326 mg/25 g).

The correlation between the LC₅₀ obtained with the strain LFB-FIOCRUZ 907 for larvae of C. putoria (382.82mg/g), and adult mortality rate is effective at a lower concentration (7.2 mg/g). This may be explained by the fact that death of the winged form is not due to the action of bacterial toxin but to the germination of bacterial spores in the blowfly haemolymph, as seen by microscopic examination of the slides displaying Gram positive vegetative forms of B. thuringiensis.

Received 29/XI/05.

Accepted 23/VI/06.

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Publication Dates

• Publication in this collection
  31 Jan 2007
• Date of issue
  Dec 2006

History

• Accepted
  23 June 2006
• Received
  29 Nov 2005