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Identification and characterization of a novel marine Bacillus cereus for mosquito control

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Abstract

Entomopathogenic bacteria to control mosquitoes are a promising environmentally friendly alternative to synthetic pesticides. In the present study, a novel mosquitocidal bacterium was isolated from marine soil collected from east coastal areas at Pondicherry (India). 16S rRNA gene sequence alignment depicted that this isolate belonged to Bacillus cereus VCRC-B520 (NCBI: KC-119192). Biochemical studies on bacterial growth, biomass, and toxin production have revealed that this strain could possibly be helpful in the production of a biopesticide in mosquito control. Toxicity assay with B. cereus against mosquito larvae has shown that the filariasis vector, Culex quinquefasciatus, is more susceptible than the other two species (Anopheles stephensi and Aedes aegypti). The LC50 and LC90 values for C. quinquefasciatus were 0.30 and 2.21 mg/L, respectively. No effect of B. cereus was found on nontargeted organisms. SDS-PAGE analysis and protein purification result from the cell mass of B. cereus have shown that a well-perceptible polypeptide was the dependable factor (85 kDa) for mosquitocidal action. Protein characterization (M/S MALDI-TOF) has shown that it is an endotoxin-specific insecticidal protein, namely “Cry4Aa”. Phylogenetic analyses of 16S rDNA gene sequence from this marine isolate have revealed the presence of homology among closely related Bacillus strains. Therefore, considerable interest has been shown on the identification of a potential mosquitocidal bacterium from marine environment (B. cereus), which was not reported earlier in view of the current scenario of the rapid development of resistance to Bacillus sphaericus in mosquito vector control program.

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References

  • Abbott WS (1925) A method of computing the effectiveness of an insecticide. J Econom Entom 18:256–267

    Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analit Biochem 72:248–254

    Google Scholar 

  • Cadavid-Restrepo G, Sahaza J, Orduz S (2012) Treatment of an Aedes aegypti colony with the Cry11Aa toxin for 54 generations results in the development of resistance. Mem Inst Oswaldo Cruz 107:74–79

    Article  CAS  PubMed  Google Scholar 

  • Charles J-F, Silva-Filha MH, Nielsen-LeRoux C, Humphreys M, Berry C (1997) Binding of the 51 and 42 kDa individual components from the Bacillus sphaericus crystal toxin on mosquito larval midgut membranes from Culex and Anopheles sp. (Diptera: Culicidae). FEMS Microbiol 63:3254–3260

    Google Scholar 

  • Chatterjee S, Subhra Ghosh T, Das S (2010) Virulence of Bacillus cereus as natural facultative pathogen of Anopheles subpictus Grassi (Diptera: Culicidae) larvae in submerged rice-fields and shallow ponds. Afr J Biotechnol 9:6983–6987

    Google Scholar 

  • Chen ML, Tsen HY (2002) Discrimination of Bacillus cereus and Bacillus thuringiensis with 16S rRNA and gyrB gene based PCR primers and sequencing of their annealing sites. J Appl Microbiol 92:912–919

    Google Scholar 

  • Chenniappan K, Ayyadurai N (2012) Synergistic activity of Cyt1A from Bacillus thuringiensis subsp. israelensis with Bacillus sphaericus B101 H5a5b against Bacillus sphaericus B101 H5a5b-resistant strains of Anopheles stephensi Liston (Diptera: Culicidae). Parasitol Res 110:381–388

    Article  PubMed  Google Scholar 

  • de Maagd RA, Bravo A, Crickmore N (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet 17:193–199

    Article  PubMed  Google Scholar 

  • Federici BA, Park HW, Sakano Y (2006) Insecticidal protein crystals of Bacillus thurigiensis. In: Shively JM (ed) Inclusions in prokaryotes. Springer, Berlin, pp 195–236

    Chapter  Google Scholar 

  • Freitas DB, Reis MP, Lima-Bittencourt LI, Costa PS (2008) BMC Research Notes 1:1–11

  • Girisch M, Ries M, Zenker M, Carbon R, Rauch R Hofbeck M (2003) Intestinal perforations in a premature infant caused by Bacillus cereus infection. 31:192–203

  • Hayes SR, Hudson M, Park HW (2011) Isolation of novel Bacillus species showing high mosquitocidal activity against several mosquito species. J Invertebr Patho 107:79–81

    Article  CAS  Google Scholar 

  • Hemingway J (2000) The molecular basis of two contrasting metabolic mechanisms of insecticide resistance. Insect Biochem Mol Bio 30:1009–1015

    Article  CAS  Google Scholar 

  • Henriques AO, Moran CP (2007) Structure, assembly, and function of the spore surface layers. Annu Rev Microbiol 61:555–588

    Article  CAS  PubMed  Google Scholar 

  • Jahan N, Shahid A (2012) Evaluation of resistance against Bacillus thuringiensis israelensis (Bti) in dengue vector from Lahore, Pakistan. Pak J Zool 44:945–949

    Google Scholar 

  • Jalalpour SH (2011) Frequency of beta lactamase enzyme in isolated pathogen bacteria from hospital in-vivo and in-vitro condition. J Isfah Med School 29:1–9

    Google Scholar 

  • Johnson DA, Aulicino PL, Newby JG (1984) Bacillus cereus-induced myonecrosis. J Trauma 24:267–270

    Article  CAS  PubMed  Google Scholar 

  • Joseph MM, Kahindi SC, Kibe LW, Nzovu JG, Luethy P, Githure JI, Mbogo CM (2011) Wide-scale application of Bti/Bs biolarvicide in different aquatic habitat types in urban and peri-urban Malindi, Kenya. Parasil Res 108:1355–1363

    Google Scholar 

  • Jung HJG, Samac DA, Sarath G (2012) Modifying crops to increase cell wall digestibility. J Plant Sci 185–186:65–77

    Google Scholar 

  • Karch S, Monteny N, Jullien JL, Sinegre G, Coz J (1990) Control of Culex pipiens by Bacillus sphaericus and role of non-target arthropods in its recycling. J Am Mosq Control Assoc 6:47–54

    CAS  PubMed  Google Scholar 

  • Krieg A (1971) Concerning a-exotoxin produced by vegetative cells of Bacillus thuringiensis and Bacillus cereus. J Invertebr Pathol 17:134–135

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during assembly of head bacteriophage T4. Nature 227:680–685

    Article  CAS  PubMed  Google Scholar 

  • Mulla MS, Federici BA, Darwazeh HA (1982) Larvicidal efficacy of Bt serotype H-14 against stagnant water mosquitoes and its effects on non-target organisms. Environ Entomol 11:788–795

    Google Scholar 

  • Mwangangi JM, Kahindi SC, Kibe LW, Nzovu JG, Luethy P, Githure JI, Mbogo CM (2011) Wide-scale application of Bti/Bs biolarvicide in different aquatic habitat types in urban and peri-urban Malindi, Kenya. Parasitol Res 108:1355–1363

    Article  PubMed  Google Scholar 

  • Nadarajah VD, Ting D, Chan KK, Mohamed SM, Kankeswary K, Lee HL (2008) Selective cytotoxic activity against leukemic cell lines from mosquitocidal Bacillus thuringiensis parasporal inclusions. Southeast Asian J Trop Med Public Health 39:235–245

    CAS  PubMed  Google Scholar 

  • Nielsen-LeRoux C, Pasquier F, Charles J-F, Sinegre G, Gaven B, Pasteur N (1997) Resistance to Bacillus sphaericus involves different mechanisms in Culex pipiens (Diptera: Culicidae) larvae. J Med Entomol 34:321–327

    CAS  PubMed  Google Scholar 

  • Ohba M, Tantichodok A, Aizawai K (1981) Production of heat-stable exotoxin by Bacillus thuringiensis and related bacteria. J Invertebr Pathol 38:26–32

    Article  Google Scholar 

  • Oren A (2008) Microbial life at high salt concentration: phylogenetic and metabolic diversity. Saline Syst 4:2. doi:10.1186/1746-1448-4-2

    Article  PubMed Central  PubMed  Google Scholar 

  • Orlova MV, Smirnova TA, Ganushkina LA, Yacubovich VY, Arizbekyan RR (1998) Insecticidal activity of Bacillus laterosporus. Appl Environ Microbiol 64:272–275

    Google Scholar 

  • Ouled-Haddar H, Zaghloul TI, Saeed HM (2010) Expression of alkaline proteinase gene in two recombinant Bacillus cereus feather-degrading strains. Folia Microbiol (Praha) 55:23–27

    Article  CAS  Google Scholar 

  • Paris M, Tetreau G, Laurent F, Lelu M et al (2011) Persistence of Bacillus thuringiensis israelensis (Bti) in the environment induces resistance to multiple Bti toxins in mosquitoes. Pest Manag Sci 67:122–128

    Article  CAS  PubMed  Google Scholar 

  • Park HW, Federici BA (2009) Genetic engineering of bacteria to improve efficacy using the insecticidal proteins of Bacillus species. In: Stock, S.P. (Ed), Insect pathogens, molecular approaches and techniques. CABI International, pp. 275–305

  • Park H-W, Federici BA, Sakano Y (2006) Inclusion proteins from other insecticidal bacteria. In: Shively JM (ed) Inclusions in prokaryotes. Springer, Berlin, pp 321–330

    Chapter  Google Scholar 

  • Park HW, Mangum CM, Zhong H, Sabrina SR (2007) Isolation of Bacillus sphaericus with improved efficacy against Culex quinquefasciatus. J Am Mosq Control Assoc 23:478–480

    Article  CAS  PubMed  Google Scholar 

  • Patil CD, Borase HP, Salunke BK, Patil SV (2013) Alteration in Bacillus thuringiensis toxicity by curing gut flora: novel approach for mosquito resistance management. Parasitol Res 112:3283–3288

    Article  PubMed  Google Scholar 

  • Perchat S, Buisson C, Chaufaux J, Sanchis V, Lereclus D, Gohar M (2005) Bacillus cereus produces several nonproteinaceous insecticidal exotoxins. J Invertebr Pathol 90:131–133

    Article  CAS  PubMed  Google Scholar 

  • Peruca AP, Vilas-Bôas GT, Arantes OM (2008) Genetic relationships between sympatric populations of Bacillus cereus and Bacillus thuringiensis, as revealed by rep-PCR genomic fingerprinting. Mem Inst Oswaldo Cruz 103:497–500

    Article  CAS  PubMed  Google Scholar 

  • Poopathi S (2012) Current trends in the control of mosquito vectors by means of biological larvicides. J Biofert Biopest 3:1–14

    Article  Google Scholar 

  • Poopathi S, Tyagi BK (2004) Mosquitocidal toxins of spore forming bacteria: recent advancement. Afr J Biotechnol 3:643–650

    CAS  Google Scholar 

  • Poopathi S, Tyagi BK (2006) The challenge of mosquito control strategies: from primordial to molecular approaches. Biotechnol Mol Bio Rev 1:51–65

    Google Scholar 

  • Poopathi S, Nielsen-LeRoux C, Charles J-F (2002) Alternative methods for preservation of mosquito larvae to study binding mechanism of Bacillus sphaericus toxin. J Invertebr Pathol 79:132–134

    Article  CAS  PubMed  Google Scholar 

  • Priest FG (1993) Systematic and ecology of Bacillus. In: Sonenshein AL, Hoch JA, Losick R (eds) Bacillus subtilis and other gram-positive bacteria: biochemistry, physiology, and molecular genetics. American Society for Microbiology, Washington, pp 369–373

    Google Scholar 

  • Raffel SJ, Stabb EV, Milner JL, Handelsman JO (1996) Genotypic and phenotypic analysis of zwittermicin A-producing strains of Bacillus cereus. Microbiol 142:3425–3436

    Google Scholar 

  • Raghavendra K, Barik TK, Niranjan BP, Sharma P, Dash AP (2011) Malaria vector control: from past to future. Parasitol Res 108:757–779

    Article  PubMed  Google Scholar 

  • Rao DR, Mani TR, Rajendran R, Joseph ASJ, Gajanana A, Reuben R (1995) Development of a high level of resistance to Bacillus sphaericus in a field population of Culex quinquefasciatus from Kochi, India. J Am Mosq Contol Assoc 11:1–5

    CAS  Google Scholar 

  • Read TD, Peterson SN, Tourasse N, Baillie LW, Paulsen IT, Nelson KE, Tettelin H, Fouts DE, Eisen JA, Gill SR, Holtzapple EK et al (2003) The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature 423:81–86

    Article  CAS  PubMed  Google Scholar 

  • Rusul G, Yaacob NH (1995) Prevalence of Bacillus cereus in selected foods and detection of enterotoxin using TECRA-VIA and BCET-RPLA. Int J Food Microbiol 25:131–139

    Article  CAS  PubMed  Google Scholar 

  • Saleem M, Rehman A, Yasmin R, Munir B (2012) Biochemical analysis and investigation on the prospective applications of alkaline protease from a Bacillus cereus strain. Mol Biol Rep 39:6399–6408

    Article  CAS  PubMed  Google Scholar 

  • Sambrook J, Fritsch E, Maniatis A (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbour Laboratory, Cold Spring Harbor

    Google Scholar 

  • Sánchez B, Arias S, Chaignepain S, Denayrolles M, Schmitter JM, Bressollier P, Urdaci MC (2009) Identification of surface proteins involved in the adhesion of a probiotic Bacillus cereus strain to mucin and fibronectin. Microbiology 155:1708–1716

    Article  PubMed  Google Scholar 

  • Saurav K, Rajakumar G, Kannabiran K, Rahuman AA, Velayutham K, Elango G, Kamaraj C, Zahir AA (2013) Larvicidal activity of isolated compound 5-(2,4-dimethylbenzyl) pyrrolidin-2-one from marine Streptomyces VITSVK5 sp. against Rhipicephalus (Boophilus) microplus, Anopheles stephensi, and culex tritaeniorhynchus. Parasitol Res 112(1):215–226

    Google Scholar 

  • Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH (1998) Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol Mol Biol Rev 62:775–806

    CAS  PubMed Central  PubMed  Google Scholar 

  • Silva-Filha MH, Regis L, Nielsen-LeRoux C, Charles JF (1995) Low-level resistance to Bacillus sphaericus in a field-treated population of Culex quiquefasciatus (Diptera:Culicidae). J Econ Entomol 88:525–530

    Google Scholar 

  • Simonsen PE, Mwakitalu ME (2013) Urban lymphatic filariasis. Parasitol Res 112:35–44

    Article  PubMed Central  PubMed  Google Scholar 

  • Singh G, Prakash S (2009) Efficacy of Bacillus sphaericus against larvae of malaria and filarial vectors: an analysis of early resistance detection. Parasitol Res 104:763–766

    Article  PubMed  Google Scholar 

  • Sneath IHA, Sokal RR (1973) Numerical taxonomy: The principles and practice of numerical classification. W. H. Freeman and Company, San Francisco. pp 400-408

  • Tamura K, Nei M, Kumar S (2004) Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci USA 101:11030–11035

    Google Scholar 

  • Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol Biol Evol 24:1596–1599

    Article  CAS  PubMed  Google Scholar 

  • Tetraeu G, Stalinski R, David J-P, Despres L (2013) Monitoring resistance to Bacillus thuringiensis israelensis (Bti) in the field by performing bioassays with each Cry toxin superlatively. Mem Inst Oswaldo Cruz 108:000–000

    Article  Google Scholar 

  • Thenmozhi M, Gopal JV, Kannabiran K, Rajakumar G, Velayutham K, Rahuman AA (2013) Eco-friendly approach using marine actinobacteria and its compounds to control ticks and mosquitoes. Parasitol Res 112:719–729

    Article  PubMed  Google Scholar 

  • Thiery I, Hamon S, Gaven B, de Barjac H (1992) Host range of Clostridium bifermentans serovar Malaysia, a mosquitocidal anaerobic bacterium. J Am Mosq Control Assoc 8:272–277

    CAS  PubMed  Google Scholar 

  • Tokieda K, Morikawa Y, Maeyama K, Mori K, Ikeda K (1999) Clinical manifestations of Bacillus cereus meningitis in newborn infants. J Paediatric Child Health 35:582–584

    Article  CAS  Google Scholar 

  • Vilas-Boas GT, Peruca APS, Arantes OMN (2007) Biology and taxonomy of Bacillus cereus, Bacillus anthracis, and Bacillus thuringiensis. Can J Microbiol 53:673–687

    Article  CAS  PubMed  Google Scholar 

  • WHO (1982) Report of informal consultation on standardization of Bacillus thuringiensis H-14. TDR/BVC/BTH14/811, WHO/VBC/81-82

  • WHO (1985) Informal consultation on the development of Bacillus sphaericus as a microbial larvicide, TDR/BVC/sphaericus/853/WHO/VBC/1-24

  • Wipfli MS, Merritt RW (1994) Effect of Bacillus thuringiensis var israelensis on non-target benthic insects through direct and indirect exposure. J N Am Benthol Soc 13:190–205

    Article  Google Scholar 

  • Wirth MS, Walton WE, Federici BA (2010) Evolution of resistance to the Bacillus sphaericus Bin toxin is phenotypically masked by combination with the mosquitocidal proteins of Bacillus thuringiensis subspecies israelensis. Environ Microbiol 12:1154–1160

    Article  CAS  PubMed  Google Scholar 

  • Wirth MC, Walton WE, Federici BA (2012) Inheritance, stability, and dominance of Cry resistance in Culex quinquefasciatus (Diptera: Culicidae) selected with the three Cry toxins of Bacillus thuringiensis subsp. israelensis. J Med Entomol 49:886–894

    Article  CAS  PubMed  Google Scholar 

  • Zahner V, Rabinovitch L, Suffys P, Momen H (1999) Genotypic diversity among Brevibacillus laterosporus strains. Applied, Environ Microbiol 65:5182–5185

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Acknowledgments

The authors thank Dr. P. Jambulingam, the Director, VCRC, Pondicherry 605006, India, for the permission; the Department of Biotechnology (DBT), New Delhi, (project ID: BT/PR13776/PID/06/527/2010) for the funding, and to Smt. R. Sundarammal, Sr. Library Information Officer, VCRC, Pondicherry, for updating the scientific information.

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Correspondence to Subbiah Poopathi.

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Poopathi, S., Mani, C., Thirugnanasambantham, K. et al. Identification and characterization of a novel marine Bacillus cereus for mosquito control. Parasitol Res 113, 323–332 (2014). https://doi.org/10.1007/s00436-013-3658-y

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