Mosquitoes are responsible for more diseases than any other group of arthropods. Global health problems associated with mosquito-borne diseases put hundreds of millions of people who are living in tropical and subtropical regions and underdeveloped countries at risk. Currently, insecticides represent the major strategy for control of mosquitoes. Extensive use of synthetic insecticides, however, has led to the emergence of insecticide-resistant mosquitoes, environmental pollution, and rising prices for new synthetic insecticides (Rajkumar and Jebanesan 2009). These factors make vector control difficult in practice. Thus, it is crucial to search for new substances that are safe and ecologically friendlier.

Use of the entomopathogenic bacteria Bacillus thuringiensis (Bt) and B. sphaericus as biolarvicides is a viable alternative for insect control (Aronson 2010). Bt is a Gram-positive, rod-shaped, and sporulating bacterium that produces crystal proteins and has several unique features that include insecticidal, nematicidal, and anti-cancer activity. B. thuringiensis subsp. israelensis (Bti) is active against mosquitoes. Despite widespread application of Bti for more than 30 years, no resistant mosquitoes have been found in the field to date (Bravo and Soberon 2008, Sazhenskiy et al. 2010).

High cost, however, is becoming one of the most important difficulties in using Bt products. Finding more active strains against dipteran species could greatly impact the control of mosquitoes. The Bti strain LLP29 was first isolated from leaves of Magnolia denudata (Zhang et al. 2010). In the present study, its growth rate, flagellar antigen serology, biochemical profile, antibiotic sensitivity, and toxic activities against mosquitoes were investigated to learn about strain LLP29′s prospects for use in biological mosquito control. It would be of great value to develop a less costly and more efficient product.

For H-serotype identification of strain LLP29, a slide agglutination test was used, as described previously (Ohba and Aizawa 1978). Bti serotype H14 shows high toxicity against mosquito larvae, and it has been used for many decades in mosquito and black fly control programs worldwide (Bravo et al. 2007). Using H antisera against H14 serotypes of B. thuringiensis, it was determined that LLP29 is a novel Bti strain and that it is similar to IPS82 in its biochemical reactions and antibiotic sensitivity characteristics.

Thirty biochemical reactions and 29 tests of antibiotic sensitivity were conducted in this study using an HX-21A bacilli analyzer (Heng xing (Hefei) Co., Ltd., People's Republic of China). LLP29 and IPS82 were shown to have similar biochemical reactions in most of the reagents, except for fructose and sodium chloride LLP29 reacted positively for sodium chloride and negatively for fructose while IPS82 reacted just the opposite (Table 1). The two strains were also similar in many reagents in the antibiotic sensitivity screening, but there were also some differences. IPS82 was shown to be sensitive to cefoperazone, but LLP29 was resistant. LLP29 was shown to be sensitive to tobramycin, but IPS82 was resistant. They were also different for oxacillin, cefazolin, clindamycin, rifampin, and cefalotin (Table 2).

Table 1.  Biochemical profiles of IPS82 and LLP29.
Carbamide++Sodium chloride+
EsculinHydrogen sulfide
Xylose++Gelatin proteolysis++
MacConkey agar4-disulfonic acid
Voges ProskauerAnaerobic Glucose
Table 2.  Antibiotic sensitivity screening of IPS82 and LLP29.
  1. S = sensitive, R = resistant, I = intervenient, NA = no activity.

Amoxicillin/Clavulanic acidRNA   

Bioassays of Bt LLP29 cultures were compared using the standard Bti reference strain IPS82 against 3rd instar larvae of Ae. aegypti following WHO procedures (World Health Organization 2007). The results demonstrated that both strains were toxic to Ae. aegypti, but LLP29 had about a four-fold higher activity than did IPS82 with LC50 of 1.21 and 4.66 ng/ml, respectively. Thus, the toxic genes of the novel LLP29 strain must be examined further in order to determine the reasons for its high mosquitocidal activity. Meanwhile, characterization of the putative receptors would also be of great value in understanding these toxins’ mechanism of action. Explanations as to the mechanism of this novel isolate may lead to the development of new biological insecticides with high mosquitocidal activity.

To compare growth rates of the two tested strains, samples of LLP29 and IPS82 (2 ml) were taken from each culture medium at 2-h intervals from 0 to 72 h and cell growth was monitored by measuring optical density at 650 nm.

Culture samples at different growing times were examined microscopically. As an indicator of biomass production, the yield of bacterial cells/crystals was also estimated at different times during bacterial growth. It was shown that both LLP29 and IPS82 had three growth periods: an exponential phase, a stationary phase, and a lysis phase. LLP29 grew slightly faster than did IPS82. In the first period of 0–22 h, spores of LLP29 germinated and then cells divided constantly at a rapid rate of growth. The culture density showed a corresponding increase and reached a plateau at about 2.5 times that at the beginning. For IPS82, however, 0–24 h was the exponential phase, thus lagging LLP29 by 2 h.

When cell growth slowed, LLP29 came into its stationary phase. During 22–33 h, it was completely sporulated and spore/crystal toxins presented themselves. After 33 h, cells of LLP29 declined. Some of them partly lysed, releasing spores and parasporal crystals. However, IPS82′s stationary phase was during 24–36 h. Observed under the microscope, IPS82 was still at the beginning of its stationary phase when cultured for 28 h while LLP29 was well into its stationary phase. The lysis phase for LLP29 was during 33–38 h.

After 47 h, all spores had been released from cells. Meanwhile, 10–20% of spores had germinated in a new cycle (Figure 1). When LLP29 had released 20% of its spores after 36 h, IPS82 was just at the end of the stationary phase and had not yet begun to release spores. The lysis phase of IPS82 was during 36–45 h. After 47 h, all spores had been released from the cells. Meanwhile, 20% of spores had germinated in a new cycle. It was slower than LLP29, which had completed fermentation at 38 h (Figure 1).


Figure 1. Growth pattern of LL29 and IPS82 in LB culture media.

Download figure to PowerPoint

To date, Bt's use has been greatly limited by its high cost. In the present study, LLP29 was found to grow faster than IPS82. Loss of plasmids might be assumed to be one of the most important reasons for its shorter life cycle. According to the plasmid profile analysis in the preliminary study, there were fewer plasmids in LLP29 than in IPS82. It might be easier to reproduce LLP29 due to the absence of some unnecessary genes. Meanwhile, LLP29 was found to have high mosquitocidal activity (Zhang et al. 2010). Thus, LLP29 could have potential as a biocontrol agent in controlling mosquitoes and preventing mosquito-borne diseases.


  1. Top of page
  2. Acknowledgments

This study was supported by the National Natural Science Foundation of China (Grant No. 31071745), the Science Foundation of the Ministry of Education of China (Grants Nos. 20093515110010 and 20093515120010), Grant No. 2B08003 from the Ministry of Education, Youth, and Sport of the Czech Republic, and by Project No Z50070508 of the Institute of Entomology of the Academy of Sciences of the Czech Republic.


  1. Top of page
  2. Acknowledgments
  • Aronson, A.I. 2010. Bacillus thuringiensis and its use as a biological insecticide. Plant Breed. Rev. 12: 1945.
  • Bravo, A., S.S. Gill, and M. Soberón. 2007. Mode of action of Bacillus thuringiensis Cry and Cyt toxins and their potential for insect control. Toxicon 49: 423435.
  • Bravo, A. and M. Soberon. 2008. How to cope with insect resistance to Bt toxins. Trends Biotechnol. 26: 573579.
  • Krishnappa, K. and A. Anandan. 2011. Mosquito larvicidal, ovicidal, and repellent properties of botanical extracts against Anopheles stephensi, Aedes aegypti, and Culex quinquefasciatus (Diptera: Culicidae). Parasitol. Res. 108, doi: 10.1007s00436-011-2263-1.
  • Ohba, M. and K. Aizawa. 1978. Serological identification of Bacillus thuringiensisand related bacteria isolated in Japan. J. Invert. Pathol. 32: 303309.
  • Rajkumar, S. and A. Jebanesan. 2009. Larvicidal and oviposition activity of Cassia obtusifolia Linn (Family: Leguminosae) leaf extract against malarial vector, Anopheles stephensi Liston (Diptera: Culicidae). Parasitol. Res. 104: 337340.
  • Sazhenskiy, V., A. Zaritsky, and M. Itsko. 2010. Expression in Escherichia coli of the Native cyt1Aa from Bacillus thuringiensis subsp. israelensis. Appl. Environ. Microbiol. 73: 34093411.
  • World Health Organization. 2007. WHO specifications and evaluations for public health pesticides: Bacillus thuringiensis subspecies israelensis strain AM65–52. World Health Organization, Geneva .
  • Zhang L., E. Huang, J. Lin, I. Gelbič, Q. Zhang, Y. Guan, T. Huang, and X. Guana. 2010. A novel mosquitocidal Bacillus thuringiensis strain LLP29 isolated from the phylloplane of Magnolia denudata. Microbiol. Res. 165: 133141.