Skip to main content
SpringerLink
Log in

Genotoxic evaluation in Oreochromis niloticus (Fish: Characidae) of recombinant spore–crystal complexes Cry1Ia, Cry10Aa and Cry1Ba6 from Bacillus thuringiensis

  • Published:
Ecotoxicology Aims and scope Submit manuscript

Abstract

Bioinsecticides from Bacillus thuringiensis (Bt) are widely used around the world in biological control against larval stages of many insect species. Bt has been considered a biopesticide that is highly specific to different orders of insects, non-polluting and harmless to humans and other vertebrates, thus becoming a viable alternative for combating agricultural pests and insect vectors of diseases. The family of Bt δ-endotoxins are crystal-protein inclusions showing toxicity to insects’ midgut, causing cell lysis leading to starvation, septicemia and death. The aim of this study is to evaluate the genotoxic potential of recombinant Bt spore–crystals expressing Cry1Ia, Cry10Aa and Cry1Ba6 on peripheral erythrocyte cells of Oreochromis niloticus, through comet assay, micronucleus (MN) test and nuclear abnormalities (NA) analysis. Fish (n = 10/group) were exposed for 96 h at 107 spores 30 l−1, 108 spores 30 l−1 or 109 spores 30 l−1 of Bt spore–crystals. Cry1Ia showed a significant increase in comet cells at levels 1 and 2, but not at levels 3 and 4, so it was not mutagenic nor did it induce MN or NA. These three spore–crystals showed some fish toxicity at only the highest exposure level, which normally does not occur in the field.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Aguiar RWS et al (2012) Cry10Aa protein is highly toxic to Anthonomus grandis Boheman (Coleoptera: Curculionidae), an important insect pest in Brazilian cotton crop fields. Bt Res 3:20–28

    Google Scholar 

  • Ali FK, El-Shehawi AM, Seehy MA (2008) Micronucleus test in the fish genome: a sensitive monitor for aquatic pollution. Afr J Biotechnol 7:606–612

    CAS  Google Scholar 

  • Alves SB, Moraes SB (1998) Controle microbiano de insetos, 2nd edn. Piracicaba, São Paulo, p 329

    Google Scholar 

  • Ayllon F, Garcia-Vazquez E (2000) Induction of micronuclei and other nuclear abnormalities in European minnow Phoxinus phoxinus and mollie Poecilia latipinna: an assessment of the fish micronucleus test. Mutat Res 467:177–186

    Article  CAS  Google Scholar 

  • Betz FS, Hammond BG, Fuchs RL (2000) Safety and advantages of Bacillus thuringiensis protected plants to control insect pests. Regul Toxicol Pharmacol 32:156–173

    Article  CAS  Google Scholar 

  • Bolognesi C, Hayashi M (2011) Micronucleus assay in aquatic animals. Mutagen 26:205–213

    CAS  Google Scholar 

  • Cantón PE et al (2013) Membrane binding and oligomer membrane insertion are necessary but insufficient for Bacillus thuringiensis Cyt1Aa toxicity. Peptides. doi:10.1016/j.peptides.2013.10.011

    Google Scholar 

  • Cao S et al (2010) Safety assessment of Cry1C protein from genetically modified rice according to the standards of PR China for new food resource. Regul Toxicol Pharmacol 58:474–481

    Article  CAS  Google Scholar 

  • Carlberg G, Tikkanen L, Abel-Hameed A (1995) Safety testing of Bacillus thuringiensis preparations, including thuringiensin, using the Salmonella Assay. J Invertebr Pathol 66:68–71

    Article  CAS  Google Scholar 

  • Carvalho JV et al (2011) Desempenho zootécnico e morfometria intestinal de alevinos de tilápia-do-nilo alimentados com Bacillus subtilis ou mananoligossacarídeo. Revista Brasileira Saúde e Produção Animal, p 121

  • Chen J et al (2013) A 104 kDa Aedes aegypti aminopeptidase N is a putative receptor for the Cry11Aa toxin from Bacillus thuringiensis subsp. israelensis. Insect Biochem Mol Biol 43(12):1201–1208. doi:10.1016/j.ibmb.2013.09.007

    Article  CAS  Google Scholar 

  • Collins AR, Ai-Guo S, Duthie SJ (1995) The kinetics of repair of oxidative DNA damage (strand breaks and oxidised pyrimidines) in human cells. Mutat Res 336:69–77

    Article  CAS  Google Scholar 

  • Cuong DV et al (2006) Nitric oxide-cGMP-protein kinase G signaling pathway induces anoxic preconditioning through activation of ATP-sensitive K+ channels in rat hearts. Am J Physiol Heart Circ Physiol 290(5):H1808–H1817

    Article  Google Scholar 

  • Douville M et al (2005) Tracking the source of Bacillus thuringiensis Cry1Ab endotoxin in the environment. Biochem Syst Ecol 33:219–232

    Article  CAS  Google Scholar 

  • Douville M, Gagné F, André C, Blaise C (2009) Occurrence of the transgenic corn Cry1Ab gene in freshwater mussels (Elliptio complanata) near corn fields: evidence of exposure by bacterial ingestion. Ecotoxicol Environ Saf 72:17–25

    Article  CAS  Google Scholar 

  • EPA Microbial Pesticide Tests Guidelines. (1996) Freshwater Fish Testing number 885.4200/1996

  • Escudero IR et al (2006) Molecular and insecticidal characterization of a Cry1I protein toxic to insects of the families Noctuidae, Tortricidae, Plutellidae, and Chrysomelidae. Appl Environ Microbiol 72:4796–4804

    Article  Google Scholar 

  • Gallo D et al (2002) Entomologia agrícola. Piracicaba, São Paulo, p 586

    Google Scholar 

  • Grisolia CK et al (2009a) Genotoxic evaluation of different delta-endotoxins from Bacillus thuringiensis on zebrafish adults and development in early life stages. Mutat Res 672:119–123

    Article  CAS  Google Scholar 

  • Grisolia CK et al (2009b) Acute toxicity and cytotoxicity of Bacillus thuringiensis and Bacillus sphaericus strains on fish and mouse bone marrow. Ecotoxicology 8:22–26. doi:10.1007/s10646-008-0252-7

    Article  Google Scholar 

  • Günther J, Jimenez-Montealegre R (2004) Efeito del probiótico Bacillus subtilis sobre el crescimento y alimentación de tilapia (Oreochromis niloticus) e langostino (Macrobrachium rosenbergii) em laboratório. Revista de Biología Tropical 52:937–943

    Google Scholar 

  • Heddle JA, Carrano AV (1977) The DNA content of micronuclei induced in mouse bone marrow by gamma-irradiation: evidence that micronuclei arise from acentric chromosomal fragments. Mutat Res 44:63–69

    Article  CAS  Google Scholar 

  • Jaloszynski P et al (1997) Bleomycin-induced DNA damage and its removal in lymphocytes of breast cancer patients studied by comet assay. Mutat Res 385:223–233

    Article  Google Scholar 

  • Johnson DE, McGaughey WH (1996) Contribution of Bacillus thuringiensis spore to toxicity of purified cry protein towards indianmeal moth larvae. Curr Microbiol 33:54–59

    Article  CAS  Google Scholar 

  • Jonsson CM, Maia AHN, Capalbo D, Carballo-Hondal O (2009) Avaliação de Risco do Biopesticida Bacillus thuringiensis (Cepa 344) em Organismos Bioindicadores Presentes em Sistemas de Reprodução Aquicola. In: Anais Zootec, Águas de Lindóia, São Paulo, p 988

  • Leite EMA, Amorim LCA (2001) Toxicologia Geral. Departamento de Análises Clínicas e Toxicologia Universidade Federal de Minas Gerais, p 695

  • Marec F, Matha V, Weiser J (1989) Analysis of genotoxic activity of Bacillus thuringiensis β-exotoxin by means of the Drosophila wing test. J Invertebr Pathol 53:347–353

    Article  CAS  Google Scholar 

  • Martins ES et al (2008) Recombinant Cry1Ia protein is highly toxic to cotton boll weevil (Anthonomus grandis Boheman) and fall armyworm (Spodoptera frugiperda). J Appl Microbiol 104:1363–1371

    Article  CAS  Google Scholar 

  • Martins ES et al (2010) Midgut GPI-anchored proteins with alkaline phosphatase activity from the cotton boll weevil (Anthonomus grandis) are putatite receptors for the Cry1B protein of Bacillus thurigiensis. Insect Biochem Mol Bio 40:138–145

    Article  CAS  Google Scholar 

  • Meher SM et al (2002) Toxicity studies of microbial insecticide Bacillus thuringiensis var. kenyae in rats, rabbits, and fish. Int J Toxicol 21:99–105

    Article  CAS  Google Scholar 

  • Mezzomo BP et al (2013) Hematotoxicity of Bacillus thuringiensis as spore–crystal strains Cry1Aa, Cry1Ab, Cry1Ac or Cry2Aa in Swiss Albino Mice. J Hematol Thromb Dis 1:1–9

    Article  Google Scholar 

  • Miranda-Vilela AL et al (2010) Gene polymorphisms against DNA damage induced by hydrogen peroxide in leukocytes of healthy humans through comet assay: a quasi-experimental study. Environ Health. doi:10.1186/1476-069X-9-21

    Google Scholar 

  • Monnerat RG, Praça LBB (2006) Bacillus thuringiensis and Bacillus sphaericus. In: Oliveira-Filho EC, Monnerat RG (eds) Fundamento para a Regulação de Semioquímicos. Inimigos Naturais e Agentes Microbiológicos de Controle de Pragas. E-Publising Inc., Planaltina, pp 121–155

    Google Scholar 

  • Monnerat RG et al (2007) Characterization of Brazilian Bacillus thuringiensis strains active against Spodoptera frugiperda, Plutella xylostella and Anticarsia gemmatalis. Biol Control 41:291–295

    Article  Google Scholar 

  • OECD Guideline for testing of chemicals (1992) Fish, Acute Toxicity Test number 203/1992

  • Oliveira-Filho EC et al (2011) Susceptibility of non target invertebrates to brazilian microbial pest control agents. Ecotoxicology 20:1354–1360

    Article  CAS  Google Scholar 

  • Patetsini E, Dimitriadis VK, Kaloyianni M (2013) Biomarkers in marine mussels, Mytilus galloprovincialis, exposed to environmentally relevant levels of the pesticides, chlorpyrifos and penoxsulam. Aquat Toxicol 126:338–345

    Article  CAS  Google Scholar 

  • Reardon R, Dubois N, McLane W (1994) Bacillus thuringiensis for manaing gypsy moth: a review. National Center of Forest Health Management, Morgantown, p 854

    Google Scholar 

  • Ren Z, Ma E, Guo Y (2002) Chromosome aberration assays for the study of cyclophosphamide and Bacillus thuringiensis in Oxya chinensis (Orthoptera: Acrididae). Mutat Res 520:141–150

    Article  CAS  Google Scholar 

  • Rivero CL et al (2008) Evaluation of genotoxicity and effects on reproduction of nonylphenol in Oreochromis niloticus (Pisces: Cichlidae). Ecotoxicology 17:732–737

    Article  CAS  Google Scholar 

  • Rivero-Wendt CL et al (2013) Cytogenetic toxicity and gonadal effects of 17 α-methyltestosterone in Astyanax bimaculatus (Characidae) and Oreochromis niloticus (Cichlidae). Genet Mol Res 12:3862–3870

    Article  CAS  Google Scholar 

  • Sanches V et al (1999) Development and field performance of a broad-sprctrum nonviable asporogenic recombinant strain of Bacillus thuringiensis with greater potency and UV resistance. Appl Environ Microbiol 65:4032–4039

    Google Scholar 

  • Schnepf E et al (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:774–806

    Google Scholar 

  • Silva-Filho MC, Falco MC (2000) Interação planta-inseto: adaptação dos insetos aos inibidores de proteinase produzidos pelas plantas. Biotecnologia Ciência Desenvolvimento 2:38–42

    Google Scholar 

  • Silva-Werneck JO, Ellar DJ (2008) Characterization of a novel Cry9Bb δ-endotoxin from Bacillus thuringiensis. J Invertebr Pathol 98:320–328

    Article  CAS  Google Scholar 

  • Singh NP et al (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp Cell Res 175:184–191

    Article  CAS  Google Scholar 

  • Souza TS, Fontanetti CS (2006) Micronucleus test and observation of nuclear alterations in erythrocytes of Nile tilapia exposed to waters affected by refinery effluent. Mutat Res 605:87–93

    Article  CAS  Google Scholar 

  • Yang W et al (2012) pH-controlled Bacillus thuringiensis Cry1Ac protoxin loading and release from polyelectrolyte microcapsules. PLoS One 9:1–7

    CAS  Google Scholar 

  • Zhou G et al (2008) The residual occurrences of Bacillus thuringiensis biopesticides in food and beverages. Int J Food Microbiol 127:68–72

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Research project supported by the Brazilian National Council for Research and Biotechnology (CNPq), Grant 552113/2011-5. Ingrid S. Freire had a fellowship from the Brazilian Ministry of Education.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Genetics and Morphology, Institute of Biological Sciences, University of Brasilia, Brasília, DF, Brazil

    I. S. Freire, A. L. Miranda-Vilela, M. L. Fascineli & C. K. Grisolia

  2. Faculty of Medicine, Faciplac, Campus Gama, Gama, DF, Brazil

    A. L. Miranda-Vilela

  3. Laboratory of Ecotoxicology, EMBRAPA-CERRADOS, Planaltina, DF, Brazil

    E. C. Oliveira-Filho

  4. University Center of Brasilia, UniCEUB, Brasília, DF, Brazil

    E. C. Oliveira-Filho

  5. Laboratory of Bacteriology, EMBRAPA-CENARGEN, Brasília, DF, Brazil

    E. S. Martins & R. G. Monnerat

Authors
  1. I. S. Freire
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. L. Miranda-Vilela
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. M. L. Fascineli
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. E. C. Oliveira-Filho
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. E. S. Martins
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. R. G. Monnerat
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. C. K. Grisolia
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to C. K. Grisolia.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Freire, I.S., Miranda-Vilela, A.L., Fascineli, M.L. et al. Genotoxic evaluation in Oreochromis niloticus (Fish: Characidae) of recombinant spore–crystal complexes Cry1Ia, Cry10Aa and Cry1Ba6 from Bacillus thuringiensis . Ecotoxicology 23, 267–272 (2014). https://doi.org/10.1007/s10646-013-1170-x

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10646-013-1170-x

Keywords

Search

Navigation