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Elimination of Gut Microbes with Antibiotics Confers Resistance to Bacillus thuringiensis Toxin Proteins in Helicoverpa armigera (Hubner)

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Abstract

Helicoverpa armigera is one of the most important pests worldwide. Transgenic crops with toxin genes from Bacillus thuringiensis (Bt) have been deployed on a large scale to control this pest. The insecticidal activity of Bt is probably influenced by the insect midgut microbes, which vary across crop hosts and locations. Therefore, we examined the role of gut microbes in pathogenicity of Bt toxins in the H. armigera. Antibiotic cocktail was used for the complete elimination of the H. armigera gut microbes. Activated Cry1Ac, Bt formulation, and transgenic cotton resulted in larval weight loss and increase in mortality, but pretreatment of larvae with antibiotic cocktail significantly decreased larval mortality and increased the larval weight gain. Activated Cry1Ac and Bt formulation inhibited the activity of proteases in midgut of H. armigera larvae but showed no such effect in the larvae pretreated with antibiotic cocktail. Five protease bands in activated Cry1Ac and two in Bt formulation-treated larvae were inhibited but no such effect in the larvae pretreated with antibiotic cocktail. Cry1Ac protein was detected in Bt/Cry1Ac protoxin-fed larval gut extract in the absence of antibiotic cocktail, but fewer in larvae pretreated with antibiotic cocktail. The activity of antioxidant enzymes and aminopeptidases increased in larvae fed on Bt toxin, but there was no significant increase in antioxidant enzymes in larvae reared on toxin protein in combination with antibiotic cocktail. The results suggest that gut microbes exercise a significant influence on the toxicity of Cry1Ac and Bt formulation in H. armigera larvae. The implications of these results have been discussed in relation to development of insect resistance to Bt transgenic crops deployed for pest management.

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References

  1. Sharma, H. C. (2005). Heliothis/Helicoverpa management: emerging trends and strategies for future research. New Delhi: Oxford and IBH Publishing Co.

    Google Scholar 

  2. Konecka, E., Kaznowski, A., Ziemnicka, J., & Ziemnicki, K. (2007). Molecular and phenotypic characterization of Bacillus thuringiensis isolated during epizootics in Cydia pomonella L. Journal of Invertebrate Pathology, 94, 56–63.

    Article  CAS  Google Scholar 

  3. Brar, S., Verma, M., Tyagi, R., Surampalli, R., Bernabe, S., & Valero, J. (2007). Bacillus thuringiensis proteases: production and role in growth, sporulation and synergism. Process Biochemistry, 42, 773–790.

    Article  CAS  Google Scholar 

  4. Bravo, A., Gomez, I., Conde, J., Garay, C. M., Sanchez, J., & Miranda, R. (2004). Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane micro domains. Biochimica et Biophysica Acta, 1667, 38–46.

    Article  CAS  Google Scholar 

  5. Chen, D. Q., & Purcell, A. H. (1997). Occurrence and transmission of facultative endosymbionts in aphids. Current Microbiology, 34, 220–225.

    Article  CAS  Google Scholar 

  6. Wilkinson, T. L. (1998). The elimination of intracellular microorganisms from insects: an analysis of antibiotic-treatment in the pea aphid (Acyrthosiphon pisum). Comparative Biochemistry and Physiology Part A, 119, 871–881.

    Article  Google Scholar 

  7. Indiragandhi, P., Anandha, R., Madhaiyan, M., & Sa, T. M. (2008). Characterization of plant growth-promoting traits of bacteria isolated from larval guts of diamondback moth Plutella xylostella (Lepidoptera: Plutellidae). Current Microbiology, 56, 327–333.

    Article  CAS  Google Scholar 

  8. Pardo, S. S., & Almeida, R. P. P. (2009). Role of symbiotic gut bacteria in the development of Acrosternum hilare and Murgantia histrionica (Hemiptera: Pentatomidae). Entomological Experimental et Applica, 132, 21–29.

    Article  Google Scholar 

  9. Visotto, L. E., Oliveira, M. G. A., Guedes, R. N. C., Ribon, A. O. B., & Good-God, P. I. V. (2009). Contribution of gut bacteria to digestion and development of the velvetbean caterpillar, Anticarsia gemmatalis. Journal of Insect Physiology, 55, 185–191.

    Article  CAS  Google Scholar 

  10. Paramasiva, I., Souche, Y., Kulakarni, G. J., Akbar, S. M. D., Sharma, H. C., & Krishnayya, P. V. (2014). Diversity in gut microflora of Helicoverpa armigera populations from different regions in relation to biological activity of Bacillus thuringiensis δ-endotoxin Cry1Ac. Archives of Insect Biochemistry and Physiology, 87, 201–213.

    Article  CAS  Google Scholar 

  11. Broderick, N. A., Raffa, K. F., & Handelsman, J. (2006). Midgut bacteria required for Bacillus thuringiensis insecticidal activity. Proceedings of the National Academy of Sciences of the United States of America, 103, 15196–15199.

    Article  CAS  Google Scholar 

  12. Broderick, N. A., Robinson, C. J., McMahon, M. D., Holt, J., Handelsman, J., & Raffa, K. F. (2009). Contributions of gut bacteria to Bacillus thuringiensis-induced mortality vary across a range of Lepidoptera. BMC Biology, 7, 11.

    Article  Google Scholar 

  13. Johnston, P. R., & Crickmore, N. (2009). Gut bacteria are not required for the insecticidal activity of Bacillus thuringiensis toward the Tobacco Horn worm, Manduca sexta. Applied and Environmental Microbiology, 75, 5094–5099.

    Article  CAS  Google Scholar 

  14. Raymond, B., Johnston, P. R., Wright, D. J., Ellis, R. J., Crickmore, N., & Bonsall, M. B. (2009). A midgut microbiota is not required for the pathogenicity of Bacillus thuringiensis to diamondback moth larvae. Environmental Microbiology, 11, 2556–2563.

    Article  CAS  Google Scholar 

  15. Babu, G. C., Sharma, H. C., Madhumati, T., Raghavaiah, G., Murthy, K. V. M. K., & Rao, V. S. (2014). A semi-synthetic chickpea flour based diet for long-term maintenance of laboratory culture of Helicoverpa armigera. Indian Journal of Entomology, 76, 336–340.

    Google Scholar 

  16. Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265–275.

    CAS  Google Scholar 

  17. Maa, X., Liu, X., Ning, X., Zhang, B., Han, F., Guan, X., Tan, Y., & Zhang, Q. (2008). Effects of Bacillus thuringiensis toxin Cry1Ac and Beauveria bassiana on Asiatic corn borer (Lepidoptera: Crambidae). Journal of Invertebrate Pathology, 99, 123–128.

    Article  Google Scholar 

  18. Laemmli, U. K. (1970). Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  19. Vinod, D. P., Sharma, H. C., & Kochole, M. S. (2010). In vivo inhibition of Helicoverpa armigera gut pro-proteinase activation by non-host plant protease inhibitors. Journal of Insect Physiology, 56, 1315–1324.

    Article  Google Scholar 

  20. Purushottam, R. L., & Vandana, K. H. (2013). Effect Bacillus thuringiensis (Bt) Cry1Ac toxin and protease inhibitor on growth and development of Helicoverpa armigera (Hubner). Pesticide Biochemistry and Physiology, 105, 77–83.

    Article  Google Scholar 

  21. Li, H., Oppert, B., Higgins, R. A., Huang, F., Zhu, K. Y., & Buschman, L. L. (2004). Comparative analysis of proteinase activities of Bacillus thuringiensis-resistant and -susceptible Ostrinia nubilalis (Lepidoptera: Crambidae). Insect Biochemistry and Molecular Biology, 34, 753–762.

    Article  Google Scholar 

  22. Garcia-Carreno, F. L., Dimes, L. E., & Haard, N. F. (1993). Substrate-gel electrophoresis for composition and molecular weight of proteinases or proteinaceous proteinase inhibitors. Analytical Biochemistry, 214, 65–69.

    Article  CAS  Google Scholar 

  23. Wang, Y., Oberley, L. W., & Murhammer, D. W. (2001). Evidence of oxidative stress following the viral infection of two Lepidopteran insect cell lines. Free Radical Biology and Medicine, 31, 1448–1455.

    Article  CAS  Google Scholar 

  24. McCord, J. M., & Fridovich, I. (1969). Superoxide dismutase: an enzymic function for erythro-cuprein (hemocuprein). Journal of Biological Chemistry, 244, 6049–6055.

    CAS  Google Scholar 

  25. Habig, W. H., Pabst, M. J., & Jakoby, W. B. (1974). Glutathione-S-transferases. Journal of Biological Chemistry, 249, 7130–7139.

    CAS  Google Scholar 

  26. Janero, D. R. (1990). Malondialdehyde and thiobarbituric acid-reactivity as diagnostic indices of lipid peroxidation and peroxidative tissue injury. Free Radical Biology and Medicine, 9, 515–540.

    Article  CAS  Google Scholar 

  27. Wolfersberger, M. G., Luethy, P., Maurer, A., Parenti, P., Sacchi, F. V., Giordana, B., & Hanozet, G. M. (1987). Preparation and partial characterization of amino acid transporting brush bordered membrane vesicles from larval midgut of the cabbage butterfly (Pieris brassicae). Comparative Biochemistry and Physiology, 86, 301–308.

    Article  Google Scholar 

  28. Nair, R., Kalia, V., Aggarwal, K. K., & Gujar, G. T. (2013). Variation in the cadherin gene sequence of Cry1Ac susceptible and resistant Helicoverpa armigera (Lepidoptera: Noctuidae) and the identification of mutant alleles in resistant strains. Current Science, 104, 215–223.

    CAS  Google Scholar 

  29. Wang, P., Zhao, J. Z., Simon, A. R., Kain, W., Janmaat, A. F., Shelton, A. M., Ferre, J., & Myers, J. (2007). Mechanism of resistance to Bacillus thuringiensis toxin Cry1Ac in a greenhouse population of the cabbage looper, Trichoplusia ni. Applied and Environmental Microbiology, 73, 1199–1207.

    Article  CAS  Google Scholar 

  30. Keller, M., Sneh, B., Strizhov, N., Prudovsky, E., Regev, A., Koncz, C., Schell, J., & Zilberstein, A. (1996). Digestion of δ-endotoxin by gut proteases may explain reduced sensitivity of advanced instar larvae of Spodoptera littoralis to Cry1C. Insect Biochemistry and Molecular Biology, 26, 365–373.

    Article  CAS  Google Scholar 

  31. Ogiwara, K., Indrasith, L. S., Asano, S., & Hori, H. (1992). Processing of δ-endotoxin from Bacillus thuringiensis subsp. Kurstaki HD-1 and HD-73 by gut juices of various insect larvae. Journal of Invertebrate Pathology, 60, 121–126.

    Article  CAS  Google Scholar 

  32. Rajagopal, R., Arora, N., Sivakumar, S., Rao, N. G. V., Nimbalkar, S. A., & Bhatnagar, R. K. (2009). Resistance of Helicoverpa armigera to Cry1Ac toxin from Bacillus thuringiensis is due to improper processing of the protoxin. Biochemical Journal, 419, 309–316.

    Article  CAS  Google Scholar 

  33. Oppert, B., Kramer, K. J., Johnson, D., Upton, S. J., & McGaughey, W. H. (1996). Proteinase-mediated insect resistance to Bacillus thuringiensis toxin. Insect Biochemistry and Molecular Biology, 26, 571–583.

    Article  CAS  Google Scholar 

  34. Gunning, R. V., Dang, H. T., Kemp, F. C., Nicholson, I. C., & Graham, D. M. (2005). New resistance mechanism in Helicoverpa armigera threatens transgenic crops expressing Bacillus thuringiensis Cry1Ac toxin. Applied and Environmental Microbiology, 71, 2558–2563.

    Article  CAS  Google Scholar 

  35. Frankenhuyzen, K. V., Liu, Y., & Amanda Tonon, A. (2010). Interactions between Bacillus thuringiensis subsp. kurstaki HD-1 and midgut bacteria in larvae of gypsy moth and spruce budworm. Journal of Invertebrate Pathology, 103, 124–131.

    Article  Google Scholar 

  36. Paramasiva, I., Sharma, H. C., & Krishnayya, P. V. (2014). Antibiotics influence the toxicity of the delta endotoxins of Bacillus thuringiensis towards the cotton bollworm, Helicoverpa armigera. BMC Microbiology, 14, 200.

    Article  Google Scholar 

  37. Hoeven, R. V., Betrabet, G., & Forst, S. (2008). Characterization of the gut bacterial community in Manduca sexta and effect of antibiotics on bacterial diversity and nematode reproduction. FEMS Microbiology Letters, 286, 249–256.

    Article  Google Scholar 

  38. Somerville, H. J., Tanada, Y., & Omi, E. M. (1970). Lethal effect of purified spore and crystalline endotoxin preparations of Bacillus thuringiensis on several lepidopterous insects. Journal of Invertebrate Pathology, 16, 241–248.

    Article  CAS  Google Scholar 

  39. Mohan, M., & Gujar, G. T. (2003). Characterization and comparison of midgut proteases of Bacillus thuringiensis susceptible and resistant diamondback moth (Plutellidae: Lepidoptera). Journal of Invertebrate Pathology, 82, 1–11.

    Article  CAS  Google Scholar 

  40. Siqueira, H. A. A., Nickerson, K. W., Moellenbeck, D., & Siegfried, B. D. (2004). Activity of gut proteinases from Cry1Ab-selected colonies of the European corn borer, Ostrinia nubilalis (Lepidoptera: Crambidae). Pest Management Science, 60, 1189–1196.

    Article  CAS  Google Scholar 

  41. Dubovskii, I. M., Olifirenko, O. A., & Glupov, V. V. (2005). Level and activities of antioxidants in intestine of larvae Galleria mellonella L. (Lepidoptera, Pyralidae) at peroral infestation by bacteria Bacillus thuringiensis ssp. galleriae. Journal of Evolutionary Biochemistry and Physiology, 41, 20–25.

    Article  CAS  Google Scholar 

  42. Dubovskiy, I. M., Martemyanov, V. V., Vorontsova, Y. L., Rantala, M. J., Gryzanova, E. V., & Glupov, V. V. (2008). Effect of bacterial infection on antioxidant activity and lipid peroxidation in the midgut of Galleria mellonella L. larvae (Lepidoptera: Pyralidae). Comparative Biochemistry and Physiology - Part C, 148, 1–5.

    CAS  Google Scholar 

  43. Guo, J. Y., Wu, G., & Wan, F. H. (2010). Activities of digestive and detoxification enzymes in multiple generations of beet armyworm, Spodoptera exigua (Hubner), in response to transgenic Bt cotton. Journal of Pest Science, 83, 453–460.

    Article  Google Scholar 

  44. Liao, C. Y., Trowell, S. C., & Akhuist, R. (2005). Purification and characterization of Cry1Ac toxin binding proteins from the brush border membrane of Helicoverpa armigera midgut. Current Microbiology, 51, 367–371.

    Article  CAS  Google Scholar 

  45. Ingle, S. S., Trivedi, N., Prasad, R., Kuruvilla, J., Rao, K. K., & Chhatpar, H. S. (2001). Aminopeptidase-N from the Helicoverpa armigera (Hubner) brush border membrane vesicles as a receptor of Bacillus thuringiensis Cry1Ac δ-endotoxin. Current Microbiology, 43, 255–259.

    Article  CAS  Google Scholar 

  46. Mason, K. L., Stepien, T. A., Blum, J. E., Labbe, N. H., Rush, J. S., Raffa, K. F., & Handelsman, J. (2011). From commensal to pathogen: translocation of Enterococcus faecalis from the midgut to the hemocoel of Manduca sexta. MBio, 2, 1–7.

    Article  Google Scholar 

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Acknowledgments

We thank the staff, Insect Rearing Laboratory, Entomology, for providing the insects throughout the study.

Funding

This work was supported by UGC under UGC-SAP (DSR-I) and UGC-MRP scheme.

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Authors and Affiliations

  1. Department of Biochemistry, Gulbarga University, Kalaburagi, Karnataka, 585 106, India

    R. Visweshwar & K. Sreeramulu

  2. Department of Entomology, International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, Telangana, 502324, India

    R. Visweshwar, H. C. Sharma & S. M. D. Akbar

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  1. R. Visweshwar
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  2. H. C. Sharma
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  3. S. M. D. Akbar
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Correspondence to K. Sreeramulu.

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Visweshwar, R., Sharma, H.C., Akbar, S.M.D. et al. Elimination of Gut Microbes with Antibiotics Confers Resistance to Bacillus thuringiensis Toxin Proteins in Helicoverpa armigera (Hubner). Appl Biochem Biotechnol 177, 1621–1637 (2015). https://doi.org/10.1007/s12010-015-1841-6

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