Analysis of toxicity on Bacillus sphaericus from amazonian soils to Anopheles darlingi and Culex quinquefasciatus larvae

Análise da toxicidade em Bacillus sphaericus de solos da Amazônia em larvas de Anopheles darlingi e Culex quinquefasciatus

Analysis of toxicity on Bacillus sphaericus from amazonian soils to Anopheles darlingi and Culex quinquefasciatus larvae

Análise da toxicidade em Bacillus sphaericus de solos da Amazônia em larvas de Anopheles darlingi e Culex quinquefasciatus

Eleilza de Castro Litaiff^I; Wanderli Pedro Tadei^II; Jorge Ivan Rebelo Porto^III; Ila Maria de Aguiar Oliveira^IV

^IInstituto Nacional de Pesquisas da Amazônia, Coordenação de Ciências da Saúde, Laboratório de Vetores de Malária e Dengue. Av. André Araújo, 2936 - Aleixo CEP 69060-001. Manaus - AM - Brasil. Telefone: 55 92 3643 3223. e-mail: [e-mail] ^IIInstituto Nacional de Pesquisas da Amazônia, Coordenação de Ciências da Saúde, Laboratório de Vetores de Malária e Dengue (Casa 15). Av. André Araújo, 2936 - Aleixo CEP 69060-001. Manaus - AM - Brasil. Telefone: 55 92 3642 3435. e-mail: [e-mail] ^IIIInstituto Nacional de Pesquisas da Amazônia, Coordenação de Pesquisa em Biologia de Água Doce. Av. André Araújo, 2936 - Aleixo CEP 69060-001. Manaus - AM - Brasil. e-mail: [e-mail]

^IVUniversidade Federal do Amazonas/Faculdade de Farmácia. Rua. Alexandre Amorim 330, Aparecida. CEP 69010-300. Manaus - AM - Brasil. e-mail: [e-mail]

ABSTRACT

Bioassays under laboratory conditions aiming to determine the larvicidal activity of Bacillus sphaericus were carried out on Anopheles darlingi and Culex quinquefasciatus. In order to estimate the toxicity through median lethal concentration (LC₅₀) and the relative potency of the strains to B. sphaericus standard strain 2362, probit analysis was performed utilizing the POLO-PC program. The findings of LC₅₀ pointed out high effectiveness on strains IB15 (0.040 ppm), IB19 and S1116 (0.048 ppm), IB16 (0.052 ppm) and S265 (0.057 ppm). Strain IB15 presented nearly 50% more potency than strain 2362 in bioassays conducted on A. darlingi. It was observed that IB16 and S1116 strains were the most powerful against C. quinquefasciatus, showing to be about 300-400% stronger than 2362 strain. The results show that laboratory conditioned evaluation can be an important way to select promising bacteria with entomopathogenic action on biolarvicides production for use on mosquitoes breeding sites.

Keywords: Vector control, Bioassays, Biolarvicides, Amazonian, Entomopathogens.

RESUMO

Bioensaios sob condições de laboratório foram realizados em larvas de Anopheles darlingi e Culex quinquefasciatus, visando determinar a atividade larvicida de Bacillus sphaericus. Para estimar a toxicidade através da concentração letal mediana (CL₅₀) e a potência das estirpes em relação à estirpe padrão 2362, foi realizada a análise de probit utilizando o programa POLO-PC. Os resultados da CL₅₀ apontaram alta efetividade para as estirpes IB15 (0,040 ppm), IB19 e S1116 (0,048 ppm), IB16 (0,052 ppm) e S265 (0,057 ppm). A estirpe IB15 apresentou potência cerca de 50% maior que a estirpe 2362 nos bioensaios realizados com A. darlingi. Foi observado que as estirpes IB16 e S1116 foram as mais tóxicas para controle de C. quinquefasciatus, mostrando-se cerca de 300-400% mais potente. Os resultados mostram que a avaliação em laboratório é uma importante etapa para selecionar bactérias com ação entomopatogênica a serem usadas na para a produção de biolarvicidas para uso nos criadouros das larvas de mosquitos.

Palavras-chave: Controle de vetores, Bioesnasios, Biolarvicidas, Amazônia, Entomopatógenos.

INTRODUCTION

The intensive use of chemical products for controlling insects down through the years has confirmed their negative impact on the environment. This can be observed by the seriously damaged natural conditions as well as by the increased resistance to these products by insect populations (Tadei, 2001). In compensation, the use of entomopathogenic bacteria has been consolidating itself as a feasible alternative for the integrated control of vectors (Becker, 2003; Tadei & Rodrigues, 2002).

A program on biological control of mosquitoes, virulence prospecting and evaluation of new isolates around the world is one of the most important steps taken to determine their effect on target populations, and thereby selecting the most promising ones for producing biological insecticides.

In Brazil, investigating activities carried out with Bacillus sphaericus in several regions of the country has made the discovery of high toxicity bearing strains possible (Schenkel et al., 1992; Vilarinhos et al., 1996; Rodrigues et al., 1999; Litaiff, 2002; Silva et al., 2002). Nevertheless, there is little information regarding the effects of Amazonian strains on vector-borne diseases, such as malaria which represents a severe problem in the region, with a yearly average of about 500,000 cases, accounting for 99.7% of those registered in Brazil (FUNASA/DIVEP/SISMAL). Hence, this study aims to evaluate the toxicity of B. sphaericus in several Amazonian locations on Anopheles darlingi and Culex quinquefasciatus larvae, to establish dose- response lines against susceptible vector species, to select the most powerful ones as biological control agents, and to contribute towards implementing a strategy for controlling vectors in the region.

MATERIAL AND METHODS

The isolation of B. sphaericus was performed according to the World Health Organization (2005) from soil samples collected in different localities in Amazonia (Table 1). Soil samples were mixed in NaCl (0.85%) solution and submitted to thermal shock (80ºC, 12 min; ice, 5 min). Aliquots of the solution were placed on plates in a nutrient agar medium (meat extract 3 g.l^-1, peptone 5 g.l^-1, and agar 15 g.l^-1) and incubated at 30ºC for 48 h. Colonies were identified by morphology of spores and by observation on a phase contrast light microscope. Later, the cultures of 108 spores.ml^-1, obtained on standardized growth on NYSM medium (Myers & Yousten, 1978), were lyophilised before use in the bioassays. Strains from the remaining states in Northern Brazil were provided by the CENARGEN/EMBRAPA culture collection, including the 2362 strain (Weiser, 1984) which was used as standard.

Twenty strains of B. sphaericus was tested on the third instar A. darlingi and C. quinquefasciatus larvae cultivated at 26 ± 2ºC, relative humidity above 85% and photoperiod of 12L:12D according to Scarpassa & Tadei (1990).

Bioassays were carried out by testing seven doses: 1.00 ppm, 0.50 ppm 0.25 ppm, 0.12 ppm, 0.06 ppm 0,02 ppm and 0.01 ppm, obtained from successive dilutions of stock solution of lyophilised Bacillus culture according WHO guidelines (WHO, 2005). In each dose, five replicates of plastic cups were set up containing distilled water, 20 late third instar larvae and bacteria doses. In each cup was added 1 ml of food (1 g of fish flour and 8 g of liver flour diluted on 1000 ml of destiled water). The final volume in each cup was 100 ml.

Control groups were set up under the same conditions, but without spores application. Bioassays were performed in three replications on different days, totalling 2,100 larvae per strain. Monitoring was conducted at 24 and 48 h intervals following the Bacillus application, when readings of live and dead larvae were made. The bioassays were held at 26 ± 2ºC, and a photoperiod of 12L:12D (Dulmage et al., 1990). Bioassays, where the control group showed mortality between 5-10%, were corrected by the Abbott formula (Finney, 1971)

Mortality data was analysed if variance (ANOVA) and average mortality rates in the three bioassays, including the control group, were compared by Tukey's test at 0.05 probability level. Mean lethal concentrations (LC50) at 95% confidence intervals and relative potency to standard strain 2362 were obtained through the Probit analysis (Finney, 1971), utilising the POLO-PC program (LeOra Software), which tests the linearity of dose responses and estimates slopes.

RESULTS

Twenty B. sphaericus strains were studied: twelve strains isolated in soil samples from the state of Amazonas, eight strains were provided by the CENARGEN/EMBRAPA from the entomopathogenic collection. As standard strain, was used the B. sphaericus 2362.

Overall, 100,800 larvae were used in bioassays with A. darlingi and C. quinquefasciatus, with nearly 90% of the larvae mortality occurring within 24 h. The highest susceptibility was found in C. quinquefasciatus, with larvae mortality variance at 24 h (F = 316.47; P < 0.001) and 48 h (F = 299.58; P < 0.001). Strains IB15, S1116, IB19 (average of 65.8 %) presented the highest mortality percentiles in A. darlingi larvae and strains IB16, S265, S1116, IB10, IB15, IB12, S594, IB19, S580, and IB08 (74.4 %) in C. quinquefasciatus, considering the 24 h reading. In 48 h, IB15, IB19, and S1116 (69.0 %) strains were the most powerful in the bioassays with A. darlingi and IB16, S1116, S265, and IB10 (82.8 %) in the bioassays with C. quinquefasciatus (Table 2).

These findings are reflected in the LC₅₀ values with confidence interval at 95% (Table 3). The 24 h reading was considered for the analysis on account of its high mortality index (>90%). With A. darlingi the greatest effectiveness was found in IB15 with 0.040 ppm (0.034-0.047), which was statically significant; S1116 and IB19, 0.048 ppm (0.039-0.069); in IB16, 0.052 ppm (0.045-0.060); and S265, 0.057 ppm (0.051-0.064), however, with no statistically significant difference for the findings with 2362, 0.057 ppm (0.047-0.069). Lower effect was found in IB18, 0.864 ppm (0.625-1.312); IB12, 0.617 ppm (0.527-0.737); and S579, 0.524ppm (0.453-0.616). In the bioassays with C. quinquefasciatus, the most effective strains were IB16, 0.014 ppm (0.012-0.016); S1116, 0.016 ppm (0.014-0.018); S265, 0.017 ppm (0.014-0.019) IB10 and IB19, 0.018 ppm (0.014-0.022); IB12, 0.024 ppm (0.021-0.072); and IB15, 0.025 ppm (0.020-0.030). We were able to reach LC50 of 0.065 ppm (0.059-0.072) with the standard strain 2362. this comparison among larvae mortality relative to 2362 strain to A. darlingi and LC₅₀ values is summarized on Figure 1.

As for standard strain 2362, four of the examined isolates presented greater relative potency in bioassays with A. darlingi: IB15 (1.515), S1116 (1.244) and IB19 (1.238), IB16 (1.156). With C. quinquefasciatus, higher potency was ascertained in 17 isolates, while IB16, S1116, S265, and IB19 were about three to four times superior (Table 4).

Isolates IB15, IB19 and S1116 were compared for their pathogenicity to 2362 strain, and the findings graphically represented in Figure 2 (A-F). Stains IB15, IB16, S1116 show themselves to be more efficient than standard strains, and even more effective than IB19 and S1116. These last two presented the same toxicity as confirmed in the coinciding straight lines.

DISCUSSION

The high mortality rate of larvae within a time interval lower than 24 h observed on the tests with entomopathogenic bacteria, which is mainly observed in dipterous aquatic larvae, confirms one advantage from use of this bacteria in fast response time, namely when compared with terrestrial injurious insects.

The Bacillus that present entomopathogenic activity brings about a collapse in the nervous and muscle systems, resulting in the loss of ability to fluctuate, and, consequently, asphyxia by drowning becomes the main cause of death (Habib, 1983). Oliveira & Tadei (2005) described the body paralysis as the initial disturbance of larval behaviour of A. albitarsis, C. quinquefasciatus and A. aegypti, after being treated for 30 minutes with 0.01, 0.1 and 1.0 mg/l of B. sphaericus of 2362 and S1116 strains. Changes on internal and external morphology were observed after 15 minutes with evident structural disorganisation of the intestinal epithelium, showing most of the cells to be swollen, vacuolated, with an increased number of secretion vesicles and an irregularly disposed brush border (Oliveira et al., 2005).

In this study, all tested strains presented toxicity, but in differentiated levels in both target-species. Only the IB15 strain showed high toxicity in tests with A. darlingi, (larvae mortality above 70%), twelve isolates showed mean toxicity (30 and 70%) and seven showed low toxicity (under 30%). In C. quinquefasciatus, ten, seven, and three isolates presented high, mean, and low toxicity respectively. C. quinquefasciatus was more susceptible, reaching 66.8% versus 51.5% in A. darlingi. These findings agree with those from earlier studies pointing out the high susceptibility of Culex sp. to B. sphaericus (Singer, 1980; Yousten, 1984; Mulla et al., 1986).

Gujar (2001) had considered that the difference among observed effects in distinct larvae instars treated with B. thuringiensis may be explained by less food consumed by later instar larvae, resulting in less absorption efficiency in digesting food, but compensated by an increase in the utilisation of ingested and digested food into body substance. According to Nielsen-LeRoux (1992) and Silva-Filha (2005), the differentiated activity of the toxin in insects may be also attributed to the affinity of the receptors present in the intestinal epithelium with toxin among some mosquito species. The description of membrane receptors and their interaction with the toxin contribute to elucidate how the B. sphaericus and resistance mechanisms act.

Pathogenicity tests among new isolates are essential in order to select strains for the production of biolarvicides as well as to estimate the virulence in commercial products. Laboratory bioassays determine the Bacillus minimum effective dose, as a parameter for use in the field (Becker, 2003). In the assays carried out with C. quinquefasciatus, eleven strains presenting LC₅₀ between 0.014 and 0.038 ppm were significantly more efficient than the results obtained from strain 2362 (0.065 ppm). On A. darlingi only IB15 (0.040 ppm) was more effective than the standard strain (0,057 ppm). Similar findings were obtained by Lacey & Singer (1982) in their tests with B. sphaericus 2013-4 and 2013-6. These authors obtained LC₅₀ equal to 0.0187 and 0.0168 ppm on A. albimanus and LC₅₀ of 0.0527 and 0.0558 ppm on A. quadrimaculatus larvae.

In relation to the standard strain, IB15 was nearly 50% more efficient, followed by S1116 and IB19 (24%). On C. quinquefasciatus, isolates IB16, S1116, S265, and IB19 were 300-400% more powerful. Other works with B. sphaericus indicated strains with greater potency than the 2362 in bioassays with A. nuneztovari and A. darlingi, S20, S46, S2, and S4, with a potency four and five times greater (Rodrigues et al., 1998).

In Brazil, several isolates from all regions of the country have shown great potential for use in biolarvicides, but due to differences in methodology employed in bioassays, these findings cannot be compared with those found in the present study (Silva et al., 2002; Monnerat et al., 2004).

Comparison between IB15, IB19, and S1116 Probit lines, showed parallel lines, indicating qualitative similarity; however, it was necessary to use a smaller dose of the Bacillus to kill 50% of the target population in the three Amazonian strains. In one to one comparisons, IB15 presented a greater effectiveness than IB19 and S1116; the two were similar as indicated by the coinciding straight-lines.

Larvicidal activity was observed in all strains of B. sphaericus from Amazonia in differentiated toxicity levels, while the C. quinquefasciatus larvae was more susceptible than A. darlingi. Strains IB15, IB19, and S1116 showed greater relative potency to the standard strain 2362, and are recommended as potential agents for the biological control of mosquitoes. In field trials, the diversity in larvicidal activity plus the ecological effects are relevant when considering the possibilities of using B. sphaericus for the biological control of mosquito target-species that coexist in breeding sites, and the laboratory bioassays are a good tool for screening entomopathogenic microrganisms.

ACKNOWLEDGEMENTS

The authors thank FAPEAM, CTPETRO and PIATAM for financial support on this study; Dr Rose Gomes Monnerat (CENARGEN/EMBRAPA) for providing some B. sphaericus strains; and the Malaria Dengue Laboratory' technicians for collecting and identifying the mosquitoes species.

LITERATURE CITED

Recebido em 10/05/2007

Aceito em 19/03/2008

• Becker, N. 2003. Implementation and integration of mosquito control measures into routine treatments and resistence management.1o Simpósio - Mosquito vetores de doenças tropicais e controle biológico. Procedings -3ş Curso: Implementação do controle biológico mosquitos usando bioinseticidas bacterianos, Manaus. p. 1-25.
• Dulmage, H.T.; Yousten, A.A.; Singer, S.; Lacey, L.A. 1990. Guidelines for Production of Bacillus thuringiensis H-14 and Bacillus sphaericus, UNDP/World Bank/WHO, Steering Committee to Biological Control of Vetores, Geneva. 59pp.
• Finney, D.J. 1971. Probit Analysis, 3rd., S. Chand & Company Ltd, New Delhi, 333pp.
• Gujar, G.T.; Kalia, V.; Kumari, A. 2001. Effect of sublethal concentration of Bacillus thuringiensis var. kurstaki on food and developmental needs of the american bollworm, Helicoverpa armigera (Hubner). Indian J Exp Biol, 39: 1130-1135.
• Habib, M.E.M. 1983. Potency of Bacillus thuringiensis var. israelensis (H14) against some aquatic dipterous insects. Z. Ang. Ent, 95: 368 - 376.
• Lacey, L.A.; Singer, S. 1982. Larvicidal activity of new isolates of Bacillus sphaericus and Bacillus thuringiensis H-14 against Anopheline and Culicine mosquitoes. Mosq News, 42: 537-543.
• Litaiff, E.C. 2002. Controle da Malária: Isolamento, Caracterização Molecular e Avaliação da Atividade larvicida contra Anopheles darlingi e Anopheles nuneztovari, de Estirpes de Bacillus sphaericus Dissertação de Mestrado. Instituto Nacional de Pesquisas da Amazônia/Universidade Federal do Amazonas. Manaus, AM. 89pp.
• Monnerat, R.G.; Silva, S.F.; Silva-Werneck, J.; Dias, J.M.C.S. 2001. Métodos de coleta, isolamento, caracterização e armazenamento de estirpes de Bacillus sphaericus. Brasília: EMBRAPA Recursos Genéticos e Biotecnologia, Circular Técnica 9: 4.
• Monnerat, R.G.; Silva, S.F.; Dias, D.S.; Martins, É.S.; Praça, L.B.; Jones, G.W.; Soares, C.M.; Dias, J.M.C.S.; Berry, C. 2004. Screening of Bacillus sphaericus strains for high toxicity against Culex quinquefasciatus and Aedes aegypti JEN,128: 469-473.
• Mulla, M.S. 1986. Efficacy of the microbial agent Bacillus sphaericus Neide against mosquitoes (Diptera: Culicidae) in southern California.Bull Soc Vector Ecol, 11: 247-254.
• Myers, P.; Yousten, A.A. 1978. Toxic activity of Bacillus sphaericus SSII-I for mosquito larvae. Infect Immun, 19: 1047-1053.
• Nielsen-LeRoux, C.; Charles, J.F. 1992. Binding of Bacillus sphaericus binary toxin to a especific receptor on midgut brush-border membranes from mosquito larvae. Eur J Biochem, 210: 585-590.
• Oliveira, C.D.; Tadei, W.P.; Abdalla, F.C. 2005. Estudo histopatológicos de larvas de Anopheles albitarsis (Lynch-Arribalzaga, 1878), Culex quinquefasciatus (Say, 1823) e Aedes aegypti (Linnaeus, 1762) em contato com Bacillus sphaerius (Neide, 1904). In: 1ş Congresso Internacional do Piatam: Ambiente, Homem, Gás e Petróleo, EDUA. Manaus, AM. p. 268.
• Oliveira, C.D.; Tadei, W.P. 2005. Observação da sintomatologia em larvas de Anopheles albitarsis (Lynch- Arribalzaga, 1878), Culex quinquefasciatus (Say, 1823) e Aedes aegypti (Linnaeus, 1762), em contato com Bacillus sphaericus (Neide, 1904). 1ş Congresso Internacional do Piatam: Ambiente, Homem, Gás e Petróleo, EDUA, Manaus, AM. p. 275.
• Rodrigues, I.B.; Tadei, W.P.; Dias, J.M.C.S. 1998. Studies on the Bacillus sphaericus larvicidal activity against malariol vetor species in Amazonia. Mem Inst Oswaldo Cruz, 93: 441-444.
• Rodrigues, I.B.; Tadei, W.P.; Dias, J.M.C.S. 1999. Larvicidal activity of Bacillus sphaericus 2362 against Anopheles nuneztovari, Anopheles darlingi and Anopheles braziliensis (Diptera, Culicidae). Rev Inst Med Trop São Paulo, 4: 101-105.
• Scarpassa, V.M.; Tadei, W.P. 1990. Biologia de anofelinos amazônicos. XIII. Estudo do ciclo biológico de A. nuneztovari Acta Amazonica, 20: 95-118.
• Schenkel, R.G.M.; Nicolas, L.; Frachon, E.; Hamon, S. 1992. Characterization and toxicity to Mosquito larvae of four Bacillus sphaericus strains Isolated from brazilian soils. J Invert Pathol, 60: 10-14.
• Silva, S.F.; Dias, D.G.S.; Martins, E.S.M.; Soares, C.M.S.; Dias, J.M.C.S.; Monnerat, R.G. 2002. Prospecção de estirpes de Bacillus sphaericus tóxicas contra Aedes aegypti e Culex quinquefasciatus. Boletim de Pesquisa e Desenvolvimento/EMBRAPA. p. 4- 23.
• Silva-Filha, M.H. 2005. Bactérias patógenas de vetores: modo de ação e resistência, vantagens e limites. 9ş Simpósio de Controle Biológico, Recife, p.70.
• Singer, S. 1980. Bacillus sphaericus for the control of mosquitoes. Biotechnol Bioeng, 22: 1335-1355.
• Tadei, W.P. 2001. Controle da malária e dinâmica de vetores na Amazônia. 7a Reunião especial da SBPC (CD-ROM). Sociedade Brasileira para o Progresso da Ciência. p. 1-6.
• Tadei, W.P.; Rodrigues, IB 2002. O controle biológico para anofelinos na Amazônia. Anais do XIX Congresso Brasileiro de Entomologia (CD-ROM). Manaus.
• Vilarinhos, P.T.R.; Maruniak, J.E.; Hall, D.W. 1996. Characterization and biological activity of a brazilian isolate ofBacillus sphaericus (Neide) highly toxic to mosquito larvae. Mem Inst Oswaldo Cruz, 91: 771 - 776.
• Weiser, J. 1984. A mosquito-vilulent Bacillus sphaericus in adult Simulium damnosum from Northern Nigeria. Zbl Microbiol, 139: 57-60.
• WHO. 1985. Informal consultation on the development of Bacillus sphaericus as a microbial larvicide. Geneva: UNDP/World Bank/WHO special programme for rearch and training in tropical diseases (TDR).
• WHO. 2005. Guidelines for laboratory and field testing of mosquito larvicides. Geneva, World Health Organization (WHO/CDS/WHOPES/GCDPP/2005.13), 39 pp.
• Yousten, A. 1984. Bacillus sphaericus: microbiological factors related to its potential as a mosquito larvicide. In: Mizahi A, Van Wezel AL Advances in biotechnology processes, v.3, AR Liss. New York. p. 315-344

Publication Dates

• Publication in this collection
  04 Aug 2008
• Date of issue
  2008

History

• Accepted
  19 Mar 2008
• Received
  10 May 2007