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Isolation of novel Bacillus species showing high mosquitocidal activity against several mosquito species
Journal of Invertebrate Pathology 107 (2011) 79–81
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Isolation of novel Bacillus species showing high mosquitocidal activity against several mosquito species Sabrina R. Hayes a, Michael Hudon b, Hyun-Woo Park a,c,d,⇑ a
John A. Mulrennan, Sr., Public Health Entomology Research & Education Center, Florida A&M University, Panama City, FL 32405, USA Indian River Mosquito Control District, 5655 41st Street, Vero Beach, FL 32967, USA c Department of Natural and Mathematical Sciences, California Baptist University, Riverside, CA 92504, USA d Department of Entomology, University of California Riverside, Riverside, CA 92521, USA b
a r t i c l e
i n f o
Article history: Received 3 September 2010 Accepted 21 January 2011 Available online 27 January 2011 Keywords: Bacillus sp. Mosquitocidal GC-FAME LC–MS/MS
a b s t r a c t Two novel mosquitocidal bacteria, VB17 and VB24, identified as new Bacillus species were isolated from dead mosquito larvae obtained in Florida aquatic habitats. Gas chromatographic analysis of fatty acid methyl esters (GC-FAME) and 16S rRNA sequencing indicated that VB24 is closely related to Bacillus sphaericus whereas VB17 does not have a close relationship with either Bacillus thuringiensis or B. sphaericus. Both isolates were significantly more active than B. sphaericus 2362 against Aedes taeniorhynchus, Anopheles quadrimaculatus, Culex quinquefasciatus larvae, and as active as B. sphaericus 2362 against Anopheles gambiae. Interestingly, however, both were not active against Aedes aegypti larvae, indicating some level of insecticidal specificity. Ó 2011 Elsevier Inc. All rights reserved.
Although the use of chemical insecticides proved successful for many decades, the emergence of resistant mosquitoes, coupled with an appreciation of the long-term detrimental effects of chemicals to non-target organisms and concern about accumulation of chemicals in the environment have accelerated the need to develop alternatives. Using entomopathogenic bacteria to control mosquitoes is a promising environmentally friendly alternative to chemical insecticides (Park and Federici, 2009). The most widely used alternative control agents for mosquitoes are the insecticidal spore-forming bacteria, Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus (Federici et al., 2006; Park et al., 2010). These bacteria produce parasporal endotoxin crystals during sporulation and these crystal proteins are responsible for their mosquitocidal activity. However, mosquito colonies develop resistance to B. sphaericus over a period of time where it is used extensively (Rao et al., 1995; Silva-Filha et al., 1995; Yuan et al., 2000). Although no resistance to B. thuringiensis subsp. israelensis has been reported in the field yet, laboratory selection of mosquitoes with B. thuringiensis subsp. israelensis proteins results in high levels of resistance (Georghiou and Wirth, 1997; Wirth et al., 1997). Furthermore, mosquito resistance to any of these proteins results in significant cross-resistance to the others (Wirth et al., 2007). Consequently, there is an urgent need to search for and isolate new indigenous ⇑ Corresponding author at: Department of Natural and Mathematical Sciences, California Baptist University, 8432 Magnolia Avenue, Riverside, CA 92504, USA. Fax: +1 951 343 4584. E-mail address: [email protected] (H.-W. Park). 0022-2011/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2011.01.005
and more effective mosquitocidal bacterial strains. As part of this effort, recently we have identified two Bacillus cereus strains that show moderate toxicity against several mosquito species (Park et al., 2009). VB17 and VB24, two spore-forming rod-shaped bacteria, were isolated from dead mosquito larvae collected from an untreated area in Vero Beach, Florida using the method established (Park et al., 2008, 2009). Mosquitocidal activities of VB17 and VB24 were determined in two steps. Preliminary mosquito bioassays were performed by growing both isolates in 50 ml of MBS medium (Park et al., 2007) for 3 days at 30 °C and by using early 4th instar Aedes taeniorhynchus and Culex quinquefasciatus maintained at John A. Mulrennan, Sr., Public Health Entomology Research & Education Center, Florida A&M University. Growth and sporulation of both isolates were poor when other sporulation media such as GYS and NBG (Park et al., 2008) were used and, therefore, MBS was used for the rest of the study. Ten milliliter of undiluted bacterial culture was transferred to a 15 ml conical tube, five mosquito larvae were added and tubes were placed at 28 °C. Ten milliliter of tap water with five larvae was used as a control. Both isolates showed 100% mortality against these mosquito species after 24 h whereas control did not show any mortality. Therefore, advanced bioassay was performed to calculate median lethal concentrations (LC50s) and to determine the host range of these isolates using the method previously established (Park et al., 2007, 2009). Lyophilized powder of sporulated cultures of VB17, VB24 and B. sphaericus 2362, active ingredient of VectoLexÒ (Valent BioSciences, Libertyville, IL) were prepared using the same procedure described above and tested against early 4th
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S.R. Hayes et al. / Journal of Invertebrate Pathology 107 (2011) 79–81
instars of Aedes aegypti, Ae. taeniorhynchus, and Cx. quinquefasciatus and 3rd instars of Anopheles gambiae and Anopheles quadrimaculatus. Ae. aegypti eggs were purchased from Benzon Research (Carlisle, PA), and An. gambiae and An. quadrimaculatus eggs were kindly provided by Dr. Jiannong Xu, Department of Biology, New Mexico State University (Las Cruces, NM) and Dr. James Becnel, USDA/ARS/ CMAVE (Gainesville, FL), respectively. The results showed that both isolates are highly toxic to Ae. taeniorhynchus, An. gambiae, An. quadrimaculatus and Cx. quinquefasicatus (Table 1). Among these mosquito species, both isolates are most active against Ae. taeniorhynchus (LC50 = 1.3 and 4.6 ng/ml for VB17 and VB24, respectively), and least active against An. gambiae (LC50 = 16.6 and 19.5 ng/ml for VB17 and VB24, respectively). Ae. taeniorhynchus is the most problematic mosquito species in Florida and B. sphaericus 2362 used as a control did not show any activity against this mosquito species. In addition, both showed similar toxicity against An. quadrimaculatus and Cx. quinquefasciatus. Interestingly, none of these isolates were active against Ae. aegypti, although they showed high activities against Ae. taeniorhynchus, a closely related species. This may be explained by different feeding behavior between the two species (Khawaled et al., 1988). Both isolates showed significantly higher toxicity against Ae. taeniorhynchus, An. quadrimaculatus and Cx. quinquefasicatus and compared with B. sphaericus 2362. For identification of VB17 and VB24, gas chromatographic analysis of fatty acids methyl esters (GC-FAME) (Osterhout et al., 1991; Kaufman et al., 1999) and 16S rRNA gene sequence alignment (Lane, 1991; Kolbert and Pershing, 1999) were performed as described previously (Park et al., 2009). The results showed that VB17 has 4.72% difference in 16S rRNA gene sequence and the FAME Similarity Index (SI) of 0.042 with Bacillus badius, indicating that it is a new Bacillus species. Furthermore, a neighbor-joining tree of VB17 indicated it does not have close relationship to either B. sphaericus or B. thuringiensis (Saitou and Nei, 1987) (data not shown). For VB24, it has 2.23% and 2.42% differences in 16S rRNA gene sequence with Bacillus fusiformis and B. sphaericus, respectively. However, it showed high FAME SI value (0.926) with B. sphaericus. However, despite this high FAME SI value, VB24 cannot be B. sphaericus because it should have less than 1% difference in 16S rRNA gene sequence with B. sphaericus to be identified as B. sphaericus. Therefore, we concluded that VB24 is also a new Bacillus species that has close relationship to B. spahericus. In order to determine whether these isolates produce toxin crystals like B. thuringiensis or B. sphaericus, transmission electron microscopy was performed at the Electron Microscopy Core Laboratory, Interdisciplinary Center for Biotechnology Research, University of Florida (Gainesville, FL). Cultures were processed using a laboratory microwave PELCOÒ BioWave with ColdSpot (Ted Pella, Inc., Redding CA). Microwave settings were as follows: temperature probe in ColdSpot port, restricted temperature 37 °C, vacuum 22 bars, 180 W unless otherwise stated. Cells were
immersed into 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, under vacuum, buffer washed three times for 45 s, post-fixed with 1% buffered OsO4 under vacuum, water washed twice for 45 s, dehydrated in a graded ethanol series once for 45 s per sample, and followed by 100% acetone twice for 45 s per sample. Dehydrated samples were then infiltrated in a graded acetone/Spurrs resin series, 250 W, under vacuum and embedded in 100% Spurrs resin (Ellis, 2006), polymerized in laboratory oven at 60 °C for 2 days. Cured sample resin blocks were trimmed with Leica EM Trim (Leica Microsystems, Inc., Bannockburn, IL). Sections were cut with a Leica UltraCut R and ultrathin sections were collected on 200 mesh Cu grids. Ultrathin sections were post-stained with 2% aq. Uranyl acetate and Reynold’s lead citrate, examined in a Hitachi H-7000 (Hitachi High Technologies America, Inc., Pleasanton, CA) operated at 75 kV and digital images acquired with MegaViewIII camera (Soft Imaging Solutions Corp, Lakewood, CO). Electron Micrographs showed that some of the sporulated cells of VB17 produce oval inclusions (Fig. 1A) that were separated from spores after cell lysis. In addition, VB17 has hair-like projectiles coming off the spores. The role of these projectiles needs to be determined. In contrast, VB24 neither forms inclusions nor has projectiles observed in VB17 (Fig. 1B). Spores of both isolates are oval and they are not deforming as head-localized spherical B. sphaericus spores are. Other mosquitocidal bacterial isolates (e.g. Brevibacillus laterosporus), that are known for their potential against mosquitoes, produce either flat square crystals or no crystals, suggesting inclusions produced by VB17 may or may not be responsible for mosquitocidal activities (Rivers et al., 1991; Orlova et al., 1998). SDS–polyacrylamide gel electrophoresis (PAGE) was performed using the fully-sporulated cultures of the Bacillus isolates grown in MBS medium using established methods (Laemmli, 1970; Park et al., 2007, 2009). Bacterial preparations used for protein analyses were the same as those for mosquito bioassay. VB17 produced two major proteins bands of about 180 and 160 kDa whereas VB24 produced a thick protein band of about 130 kDa (data not shown). To determine the protein composition in the sporulated cultures of these Bacillus isolates, the solubilized protein mixture from each isolate was analyzed by use of polyacrylamide gel block coupled with LC–MS/MS as described previously with minor modifications at the Protein Chemistry Core Laboratory, Interdisciplinary Center for Biotechnology Research, University of Florida (Gainesville, FL) (Fu et al., 2008; Golovko and Murphy, 2008). The proteins of VB17 and VB24 showed 100% sequence identity with spore coat protein,
A
Table 1 Mosquitocidal activity of the Bacillus isolates in comparison with Bacillus sphaericus 2362. Mosquito species
LC50 (fiducial limits)a VB17
VB24
B. sphaericus 2362
Aedes aegypti Aedes taeniorhynchus Anopheles gambiae Anopheles quadrimaculatus Culex quinquefasciatus
N/Tb 1.3 (0.1–3.7) 16.6 (12.3–24.7) 6.4 (3.9–9.0)
N/T 4.6 (2.4–7.0) 19.5 (14.6–25.8) 6.0 (3.5–8.6)
N/T N/T 13.7 (9.7–18.3) 50.7 (37.8–70.1)
6.1 (4.6–7.6)
6.7 (4.5–8.9)
12.6 (8.7–17.5)
a 24-h Mortality, in nanograms of lyophilized sporulated whole culture per milliliter of larval assay water. b Not toxic (not able to determine LC50 using 1 lg/ml as the highest concentration).
B
Fig. 1. Transmission electron micrographs of fully sporulated Bacillus sp. VB17 (A) and VB24 (B). S, spore; I, inclusion; P, hair-like projections.
S.R. Hayes et al. / Journal of Invertebrate Pathology 107 (2011) 79–81
CotZ of a marine Bacillus sp. B14905 (molecular weight = 19 kDa; GenBank accession number 126649930) (data not shown). It has been reported that the toxins responsible for insecticidal and nematocidal activities were isolated from the spores of certain B. thuringiensis and B. laterosporus strains, respectively (Du and Nickerson, 1996; Ruiu et al., 2007). Therefore, the possibility of presence of mosquitocidal toxin cannot be ruled out. Also, they showed 95% sequence identities with DNA-directed RNA polymerase subunit beta (molecular weight = 134 kDa; GenBank accession number 126654238) and serine protein kinase (molecular weight = 75 kDa; GenBank accession number 126649571). Interestingly, proteins of VB17 showed 95% sequence identity with outer membrane protein A of Acinetobacter baumannii (molecular weight = 26 kDa; GenBank accession number 109675218), a major envelope protein known to be responsible for adherence to and invasion of bacteria in epithelial cells (Choi et al., 2008; Lee et al., 2010). However, they did not show any identity with known insecticidal proteins, and DNA-directed RNA polymerase subunit beta was the only one that matches with the major protein bands appeared in the SDS–PAGE. Therefore, in order to determine whether their mosquitocidal activities come from other unknown metabolites and/or compounds, both isolates were cultured using the same procedure described above and the cultures were treated at 80, 90 and 100 °C for 15 min. All three treatments for both isolates showed 100% mortality against Ae. taeniorhynchus and Cx. quinquefasciatus 4th instars when the undiluted cultures were used for bioassay, suggesting that factors other than proteins such as a heat-stable insecticidal adenine-nucleotide analog, b-exotoxin play a key role in mosquitocidal activities. Further study to determine the mosquitocidal factor(s) for these bacteria is necessary. VB17 and VB24 have been deposited as accession numbers, respectively, PW1 and PW2 at the Bacillus Genetic Stock Center, Ohio State University, Columbus, OH. In summary, VB17 and VB24 both showed significantly higher toxicity against several mosquito species compared with B. sphaericus 2362. Some specificity may exist since both isolates showed no activity against Ae. aegypti. They may eventually be used to control mosquitoes that transmit many medically important diseases because they are novel insecticidal bacteria. A possible effect of these isolates on non-target organisms is being studied for further characterization. Depending on the results obtained from the nontarget studies, these isolates could be used in either as a replacement for or in conjunction with B. sphaericus and B. thuringiensis subsp. israelensis. Acknowledgments We thank Karen Kelley and Byung-Ho Kang for technical assistance. This research was supported by mosquito research grants to H.-W.P. from the Florida Department of Agriculture and Consumer Services (Award No. 012966). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jip.2011.01.005. References Choi, C.H., Hyun, S.H., Lee, J.Y., Lee, J.S., Lee, Y.S., Kim, S.A., Chae, J.P., Yoo, S.M., Lee, J.C., 2008. Acinetobacter baumannii outer membrane protein A targets the nucleus and induces cytotoxicity. Cell. Microbiol. 10, 309–319.
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Du, C., Nickerson, K.W., 1996. Bacillus thuringiensis HD-73 spores have surfacelocalized Cry1Ac toxin: physiological and pathological consequences. Appl. Environ. Microbiol. 62, 3722–3726. Ellis, E.A., 2006. Solutions to the problem of substitution of ERL 4221 for vinyl cyclohexen dioxide in Spurr low viscosity embedding formulations. Microsc. Today 14, 32–33. Federici, B.A., Park, H.-W., Sakano, Y., 2006. Insecticidal protein crystals of Bacillus thuringiensis. In: Shively, J.M. (Ed.), Inclusions in Prokaryotes. Springer-Verlag, Berlin, Heidelberg, Germany, pp. 195–236. Fu, Z., Sun, Y., Xia, L., Ding, X., Mo, X., Li, X., Huang, K., Zhang, Y., 2008. Assessment of protoxin composition of Bacillus thuringiensis strains by use of polyacrylamide gel block and mass spectrometry. Appl. Microbiol. Biotechnol. 79, 875–880. Georghiou, G., Wirth, M.C., 1997. Influence of exposure to single versus multiple toxins of Bacillus thuringiensis subsp. israelensis on development of resistance in the mosquito Culex quinquefasciatus (Diptera: Culicidae). Appl. Environ. Microbiol. 63, 1095–1101. Golovko, M., Murphy, E.J., 2008. An improved LC–MS/MS procedure for brain prostanoid analysis using brain fixation with head-focused microwave irradiation and liquid–liquid extraction. J. Lipid Res. 49, 893–902. Kaufman, M.G., Walker, E.D., Smith, T.W., Merritt, R.W., Klug, M.J., 1999. Effects of larval mosquitoes (Aedes triseriatus) and stemflow on microbial community dynamics in container habitats. Appl. Environ. Microbiol. 65, 2661–2673. Khawaled, K., Barak, Z., Zaritsky, A., 1988. Feeding behavior of Aedes aegypti larvae and toxicity of dispersed and of natural encapsulated Bacillus thuringiensis var. israelensis. J. Invertebr. Pathol. 52, 419–426. Kolbert, C.P., Pershing, D.H., 1999. Ribosomal DNA sequencing as a tool for identification of bacterial pathogens. Curr. Opin. Microbiol. 2, 299–305. Laemmli, U.K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685. Lane, D.J., 1991. 16S/23S rRNA sequencing. In: Stackebrandt, E., Goodfellow, M. (Eds.), Nucleic Acid Techniques in Bacterial Systematics. John Wiley and Sons, New York, USA, pp. 115–175. Lee, J.S., Choi, C.H., Kim, J.W., Lee, J.C., 2010. Acinetobacter baumannii outer membrane protein A induces dendritic cell death through mitochondrial targeting. J. Microbiol. 48, 387–392. Orlova, M.V., Smirnova, T.A., Ganushkina, L.A., Yacubovich, V.Y., Azizbekyan, R.R., 1998. Insecticidal activity of Bacillus laterosporus. Appl. Environ. Microbiol. 64, 2723–2725. Osterhout, G.J., Shull, V.H., Dick, J.D., 1991. Identification of clinical isolates of Gram negative nonfermentative bacteria by an automated cellular fatty acid identification system. J. Clin. Microbiol. 29, 1822–1830. Park, H.-W., Bideshi, D.K., Federici, B.A., 2010. Properties and applied use of the mosquitocidal bacterium, Bacillus sphaericus. J. Asia–Pac. Entomol. 13, 159–168. Park, H.-W., Federici, B.A., 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., Hayes, S.R., Mangum, C.M., 2008. Distribution of mosquitocidal Bacillus thuringiensis and Bacillus sphaericus from sediment samples in Florida. J. Asia– Pac. Entomol. 11, 217–220. Park, H.-W., Hayes, S.R., Stout, G.M., Day-Hall, G., Latham, M.D., Hunter, J.P., 2009. Identification of two mosquitocidal Bacillus cereus strains showing different host ranges. J. Invertebr. Pathol. 100, 54–56. Park, H.-W., Mangum, C.M., Zhong, H., Hayes, S.R., 2007. Isolation of Bacillus sphaericus with improved efficacy against Culex quinquefasciatus. J. Am. Mosq. Control Assoc. 23, 478–480. Rao, D.R., Mani, T.R., Rajendran, 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. Control Assoc. 11, 1–5. Rivers, D.B., Vann, C.N., Zimmack, H.L., Dean, D.H., 1991. Mosquitocidal activity of Bacillus laterosporus. J. Invertebr. Pathol. 58, 444–447. Ruiu, L., Floris, I., Satta, A., Ellar, D.J., 2007. Toxicity of a Brevibacillus laterosporus strain lacking parasporal crystals against Musca domestica and Aedes aegypti. Biol. Control 43, 136–143. Saitou, N., Nei, M., 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Silva-Filha, M.H., Regis, L., Nielsen-LeRoux, C., 1995. Low-level resistance to Bacillus sphaericus in a field-treated population of Culex quinquefasciatus (Diptera: Culicidae). J. Econ. Entomol. 88, 525–530. Wirth, M.C., Georghiou, G.P., Federici, B.A., 1997. CytA enables CryIV endotoxins of Bacillus thuringiensis to overcome high levels of CryIV resistance in the mosquito, Culex quinquefasciatus. Proc. Natl. Acad. Sci. USA 94, 10536–10540. Wirth, M.C., Zaritsky, A., Ben-Dov, E., Manasheron, R., Khasdan, V., Boussiba, S., Walton, W.E., 2007. Cross-resistance spectra of Culex quinquefasciatus resistant to mosquitocidal toxins of Bacillus thuringiensis towards recombinant Escherichia coli expressing genes from B. thuringiensis ssp. israelensis. Environ. Microbiol. 9, 1393–1401. Yuan, Z., Zhang, Y.M., Cai, Q., Liu, E.Y., 2000. High-level field resistance to Bacillus sphaericus C3–41 in Culex quinquefasciatus from southern China. Biocontrol Sci. Technol. 10, 41–49.
Contents lists available at ScienceDirect
Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip
Short Communication
Isolation of novel Bacillus species showing high mosquitocidal activity against several mosquito species Sabrina R. Hayes a, Michael Hudon b, Hyun-Woo Park a,c,d,⇑ a
John A. Mulrennan, Sr., Public Health Entomology Research & Education Center, Florida A&M University, Panama City, FL 32405, USA Indian River Mosquito Control District, 5655 41st Street, Vero Beach, FL 32967, USA c Department of Natural and Mathematical Sciences, California Baptist University, Riverside, CA 92504, USA d Department of Entomology, University of California Riverside, Riverside, CA 92521, USA b
a r t i c l e
i n f o
Article history: Received 3 September 2010 Accepted 21 January 2011 Available online 27 January 2011 Keywords: Bacillus sp. Mosquitocidal GC-FAME LC–MS/MS
a b s t r a c t Two novel mosquitocidal bacteria, VB17 and VB24, identified as new Bacillus species were isolated from dead mosquito larvae obtained in Florida aquatic habitats. Gas chromatographic analysis of fatty acid methyl esters (GC-FAME) and 16S rRNA sequencing indicated that VB24 is closely related to Bacillus sphaericus whereas VB17 does not have a close relationship with either Bacillus thuringiensis or B. sphaericus. Both isolates were significantly more active than B. sphaericus 2362 against Aedes taeniorhynchus, Anopheles quadrimaculatus, Culex quinquefasciatus larvae, and as active as B. sphaericus 2362 against Anopheles gambiae. Interestingly, however, both were not active against Aedes aegypti larvae, indicating some level of insecticidal specificity. Ó 2011 Elsevier Inc. All rights reserved.
Although the use of chemical insecticides proved successful for many decades, the emergence of resistant mosquitoes, coupled with an appreciation of the long-term detrimental effects of chemicals to non-target organisms and concern about accumulation of chemicals in the environment have accelerated the need to develop alternatives. Using entomopathogenic bacteria to control mosquitoes is a promising environmentally friendly alternative to chemical insecticides (Park and Federici, 2009). The most widely used alternative control agents for mosquitoes are the insecticidal spore-forming bacteria, Bacillus thuringiensis subsp. israelensis and Bacillus sphaericus (Federici et al., 2006; Park et al., 2010). These bacteria produce parasporal endotoxin crystals during sporulation and these crystal proteins are responsible for their mosquitocidal activity. However, mosquito colonies develop resistance to B. sphaericus over a period of time where it is used extensively (Rao et al., 1995; Silva-Filha et al., 1995; Yuan et al., 2000). Although no resistance to B. thuringiensis subsp. israelensis has been reported in the field yet, laboratory selection of mosquitoes with B. thuringiensis subsp. israelensis proteins results in high levels of resistance (Georghiou and Wirth, 1997; Wirth et al., 1997). Furthermore, mosquito resistance to any of these proteins results in significant cross-resistance to the others (Wirth et al., 2007). Consequently, there is an urgent need to search for and isolate new indigenous ⇑ Corresponding author at: Department of Natural and Mathematical Sciences, California Baptist University, 8432 Magnolia Avenue, Riverside, CA 92504, USA. Fax: +1 951 343 4584. E-mail address: [email protected] (H.-W. Park). 0022-2011/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jip.2011.01.005
and more effective mosquitocidal bacterial strains. As part of this effort, recently we have identified two Bacillus cereus strains that show moderate toxicity against several mosquito species (Park et al., 2009). VB17 and VB24, two spore-forming rod-shaped bacteria, were isolated from dead mosquito larvae collected from an untreated area in Vero Beach, Florida using the method established (Park et al., 2008, 2009). Mosquitocidal activities of VB17 and VB24 were determined in two steps. Preliminary mosquito bioassays were performed by growing both isolates in 50 ml of MBS medium (Park et al., 2007) for 3 days at 30 °C and by using early 4th instar Aedes taeniorhynchus and Culex quinquefasciatus maintained at John A. Mulrennan, Sr., Public Health Entomology Research & Education Center, Florida A&M University. Growth and sporulation of both isolates were poor when other sporulation media such as GYS and NBG (Park et al., 2008) were used and, therefore, MBS was used for the rest of the study. Ten milliliter of undiluted bacterial culture was transferred to a 15 ml conical tube, five mosquito larvae were added and tubes were placed at 28 °C. Ten milliliter of tap water with five larvae was used as a control. Both isolates showed 100% mortality against these mosquito species after 24 h whereas control did not show any mortality. Therefore, advanced bioassay was performed to calculate median lethal concentrations (LC50s) and to determine the host range of these isolates using the method previously established (Park et al., 2007, 2009). Lyophilized powder of sporulated cultures of VB17, VB24 and B. sphaericus 2362, active ingredient of VectoLexÒ (Valent BioSciences, Libertyville, IL) were prepared using the same procedure described above and tested against early 4th
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instars of Aedes aegypti, Ae. taeniorhynchus, and Cx. quinquefasciatus and 3rd instars of Anopheles gambiae and Anopheles quadrimaculatus. Ae. aegypti eggs were purchased from Benzon Research (Carlisle, PA), and An. gambiae and An. quadrimaculatus eggs were kindly provided by Dr. Jiannong Xu, Department of Biology, New Mexico State University (Las Cruces, NM) and Dr. James Becnel, USDA/ARS/ CMAVE (Gainesville, FL), respectively. The results showed that both isolates are highly toxic to Ae. taeniorhynchus, An. gambiae, An. quadrimaculatus and Cx. quinquefasicatus (Table 1). Among these mosquito species, both isolates are most active against Ae. taeniorhynchus (LC50 = 1.3 and 4.6 ng/ml for VB17 and VB24, respectively), and least active against An. gambiae (LC50 = 16.6 and 19.5 ng/ml for VB17 and VB24, respectively). Ae. taeniorhynchus is the most problematic mosquito species in Florida and B. sphaericus 2362 used as a control did not show any activity against this mosquito species. In addition, both showed similar toxicity against An. quadrimaculatus and Cx. quinquefasciatus. Interestingly, none of these isolates were active against Ae. aegypti, although they showed high activities against Ae. taeniorhynchus, a closely related species. This may be explained by different feeding behavior between the two species (Khawaled et al., 1988). Both isolates showed significantly higher toxicity against Ae. taeniorhynchus, An. quadrimaculatus and Cx. quinquefasicatus and compared with B. sphaericus 2362. For identification of VB17 and VB24, gas chromatographic analysis of fatty acids methyl esters (GC-FAME) (Osterhout et al., 1991; Kaufman et al., 1999) and 16S rRNA gene sequence alignment (Lane, 1991; Kolbert and Pershing, 1999) were performed as described previously (Park et al., 2009). The results showed that VB17 has 4.72% difference in 16S rRNA gene sequence and the FAME Similarity Index (SI) of 0.042 with Bacillus badius, indicating that it is a new Bacillus species. Furthermore, a neighbor-joining tree of VB17 indicated it does not have close relationship to either B. sphaericus or B. thuringiensis (Saitou and Nei, 1987) (data not shown). For VB24, it has 2.23% and 2.42% differences in 16S rRNA gene sequence with Bacillus fusiformis and B. sphaericus, respectively. However, it showed high FAME SI value (0.926) with B. sphaericus. However, despite this high FAME SI value, VB24 cannot be B. sphaericus because it should have less than 1% difference in 16S rRNA gene sequence with B. sphaericus to be identified as B. sphaericus. Therefore, we concluded that VB24 is also a new Bacillus species that has close relationship to B. spahericus. In order to determine whether these isolates produce toxin crystals like B. thuringiensis or B. sphaericus, transmission electron microscopy was performed at the Electron Microscopy Core Laboratory, Interdisciplinary Center for Biotechnology Research, University of Florida (Gainesville, FL). Cultures were processed using a laboratory microwave PELCOÒ BioWave with ColdSpot (Ted Pella, Inc., Redding CA). Microwave settings were as follows: temperature probe in ColdSpot port, restricted temperature 37 °C, vacuum 22 bars, 180 W unless otherwise stated. Cells were
immersed into 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.2, under vacuum, buffer washed three times for 45 s, post-fixed with 1% buffered OsO4 under vacuum, water washed twice for 45 s, dehydrated in a graded ethanol series once for 45 s per sample, and followed by 100% acetone twice for 45 s per sample. Dehydrated samples were then infiltrated in a graded acetone/Spurrs resin series, 250 W, under vacuum and embedded in 100% Spurrs resin (Ellis, 2006), polymerized in laboratory oven at 60 °C for 2 days. Cured sample resin blocks were trimmed with Leica EM Trim (Leica Microsystems, Inc., Bannockburn, IL). Sections were cut with a Leica UltraCut R and ultrathin sections were collected on 200 mesh Cu grids. Ultrathin sections were post-stained with 2% aq. Uranyl acetate and Reynold’s lead citrate, examined in a Hitachi H-7000 (Hitachi High Technologies America, Inc., Pleasanton, CA) operated at 75 kV and digital images acquired with MegaViewIII camera (Soft Imaging Solutions Corp, Lakewood, CO). Electron Micrographs showed that some of the sporulated cells of VB17 produce oval inclusions (Fig. 1A) that were separated from spores after cell lysis. In addition, VB17 has hair-like projectiles coming off the spores. The role of these projectiles needs to be determined. In contrast, VB24 neither forms inclusions nor has projectiles observed in VB17 (Fig. 1B). Spores of both isolates are oval and they are not deforming as head-localized spherical B. sphaericus spores are. Other mosquitocidal bacterial isolates (e.g. Brevibacillus laterosporus), that are known for their potential against mosquitoes, produce either flat square crystals or no crystals, suggesting inclusions produced by VB17 may or may not be responsible for mosquitocidal activities (Rivers et al., 1991; Orlova et al., 1998). SDS–polyacrylamide gel electrophoresis (PAGE) was performed using the fully-sporulated cultures of the Bacillus isolates grown in MBS medium using established methods (Laemmli, 1970; Park et al., 2007, 2009). Bacterial preparations used for protein analyses were the same as those for mosquito bioassay. VB17 produced two major proteins bands of about 180 and 160 kDa whereas VB24 produced a thick protein band of about 130 kDa (data not shown). To determine the protein composition in the sporulated cultures of these Bacillus isolates, the solubilized protein mixture from each isolate was analyzed by use of polyacrylamide gel block coupled with LC–MS/MS as described previously with minor modifications at the Protein Chemistry Core Laboratory, Interdisciplinary Center for Biotechnology Research, University of Florida (Gainesville, FL) (Fu et al., 2008; Golovko and Murphy, 2008). The proteins of VB17 and VB24 showed 100% sequence identity with spore coat protein,
A
Table 1 Mosquitocidal activity of the Bacillus isolates in comparison with Bacillus sphaericus 2362. Mosquito species
LC50 (fiducial limits)a VB17
VB24
B. sphaericus 2362
Aedes aegypti Aedes taeniorhynchus Anopheles gambiae Anopheles quadrimaculatus Culex quinquefasciatus
N/Tb 1.3 (0.1–3.7) 16.6 (12.3–24.7) 6.4 (3.9–9.0)
N/T 4.6 (2.4–7.0) 19.5 (14.6–25.8) 6.0 (3.5–8.6)
N/T N/T 13.7 (9.7–18.3) 50.7 (37.8–70.1)
6.1 (4.6–7.6)
6.7 (4.5–8.9)
12.6 (8.7–17.5)
a 24-h Mortality, in nanograms of lyophilized sporulated whole culture per milliliter of larval assay water. b Not toxic (not able to determine LC50 using 1 lg/ml as the highest concentration).
B
Fig. 1. Transmission electron micrographs of fully sporulated Bacillus sp. VB17 (A) and VB24 (B). S, spore; I, inclusion; P, hair-like projections.
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CotZ of a marine Bacillus sp. B14905 (molecular weight = 19 kDa; GenBank accession number 126649930) (data not shown). It has been reported that the toxins responsible for insecticidal and nematocidal activities were isolated from the spores of certain B. thuringiensis and B. laterosporus strains, respectively (Du and Nickerson, 1996; Ruiu et al., 2007). Therefore, the possibility of presence of mosquitocidal toxin cannot be ruled out. Also, they showed 95% sequence identities with DNA-directed RNA polymerase subunit beta (molecular weight = 134 kDa; GenBank accession number 126654238) and serine protein kinase (molecular weight = 75 kDa; GenBank accession number 126649571). Interestingly, proteins of VB17 showed 95% sequence identity with outer membrane protein A of Acinetobacter baumannii (molecular weight = 26 kDa; GenBank accession number 109675218), a major envelope protein known to be responsible for adherence to and invasion of bacteria in epithelial cells (Choi et al., 2008; Lee et al., 2010). However, they did not show any identity with known insecticidal proteins, and DNA-directed RNA polymerase subunit beta was the only one that matches with the major protein bands appeared in the SDS–PAGE. Therefore, in order to determine whether their mosquitocidal activities come from other unknown metabolites and/or compounds, both isolates were cultured using the same procedure described above and the cultures were treated at 80, 90 and 100 °C for 15 min. All three treatments for both isolates showed 100% mortality against Ae. taeniorhynchus and Cx. quinquefasciatus 4th instars when the undiluted cultures were used for bioassay, suggesting that factors other than proteins such as a heat-stable insecticidal adenine-nucleotide analog, b-exotoxin play a key role in mosquitocidal activities. Further study to determine the mosquitocidal factor(s) for these bacteria is necessary. VB17 and VB24 have been deposited as accession numbers, respectively, PW1 and PW2 at the Bacillus Genetic Stock Center, Ohio State University, Columbus, OH. In summary, VB17 and VB24 both showed significantly higher toxicity against several mosquito species compared with B. sphaericus 2362. Some specificity may exist since both isolates showed no activity against Ae. aegypti. They may eventually be used to control mosquitoes that transmit many medically important diseases because they are novel insecticidal bacteria. A possible effect of these isolates on non-target organisms is being studied for further characterization. Depending on the results obtained from the nontarget studies, these isolates could be used in either as a replacement for or in conjunction with B. sphaericus and B. thuringiensis subsp. israelensis. Acknowledgments We thank Karen Kelley and Byung-Ho Kang for technical assistance. This research was supported by mosquito research grants to H.-W.P. from the Florida Department of Agriculture and Consumer Services (Award No. 012966). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jip.2011.01.005. References Choi, C.H., Hyun, S.H., Lee, J.Y., Lee, J.S., Lee, Y.S., Kim, S.A., Chae, J.P., Yoo, S.M., Lee, J.C., 2008. Acinetobacter baumannii outer membrane protein A targets the nucleus and induces cytotoxicity. Cell. Microbiol. 10, 309–319.
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