Mosquito is at the top of the four pests and is carry of many diseases, and it poses a great threat to human survival and health (WHO, 2009; Staples et al., 2009). Aedes aegypti and Aedes albopictus is the main medium for the spread of dengue and Chikungunya, but the filariasis and West Nile virus mainly by the Culex transmit the disease to humans. At present, the most effective way to control the mosquito borne disease is to use the synthetic chemical pesticides (Baird, 2000), but the disadvantage is that the mosquito is easy to produce resistance to it. Such as, in the global scope of use the DDT, although with good effect, but the mosquitoes formed resistance to it within a short period of 8 years (Baird, 2000).

Bacillus thuringiensis (Bt) is a widely distributed gram positive bacteria with rod shape and is a kind of insect pathogenic microorganism which has strong toxicity to pest but no toxicity to its natural enemies. (Regis et al., 2001). B. thuringiensis subsp. israelensis (Bti for short), witch can form 4 kinds of Cry toxin (Cry4Aa, Cry4Ba, Cry10Aa and Cry11Aa) and 2 kinds of Cyt toxin (Cyt1Aa and Cyt2Ba), has a good ability to kill mosquitoes (Pigott and Ellar, 2007; Roh et al., 2007; Ohba et al.,2009). Bti has been used in actual production for more than thirty years, but so far the mosquitoes have not produced resistance to it (Bravo and Soberon, 2008).

The research of the effects of Bt sensitive strains on Diptera insects and mosquito control have great significance. This study through identified Bt BRC-ZLL13 (Zhang et al., 2010) which isolated from the leaves of Michelia alba to confirm the mosquito killing gene.

The bioassay results show that the toxicity against Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus of BRC-ZLL13 are higher than the international standard strain IPS82, and the growth rate of BRC-ZLL13 is faster than IPS82. The above characteristics indicate that this strain is potential for the development and application in the field of public health and biological control. This study provided a theoretical basis for the future research on the function of mosquito killing gene, the mechanism of action, and the generation of resistance.

1 Results and Analysis

1.1 Serotype identification, biochemical reactions, antibiotic sensitivity

Test results showed that BRC-ZLL13 Serotype is H14 and belong to B. thuringiensis subsp. Israelensis. The biochemical profiles of IPS82 and ZLL13 have similar biochemical reactions (Table 1). Two strains were capable of reacting with Arginine, Urea, Glucose, Xylose, Lysine, Ornithine and Amylase. However, there were no reaction with Nitrate, Esculin, Galactose, Maltase, Mannitol, Lactose, Macconkey Agar, Rhamnose, Gelatin, Sucrose, Trehalose, Vitamin and Auxin, and the reactions to Fructose and Sodium chloride were differently.

Table 1 The biochemical profile of IPS82 and ZLL13

Table 2 shows the sensitivity of the tested strains to 29 kinds of antibiotics. Two strains were sensitized to Piperacillin, Tazobactam Three, Gentamicin, Amikacin, Ciprofloxacin, Ofloxacin, Levofloxacin, Ciprofloxacin, Tetracycline, Erythromycin and Nitrofurantoin, resisted to Piperacillin, Mezlocillin, Ceftriaxone, Ceftazidime, Cefepime, Aztreonam, Cefotaxime, Cotrimoxazole. IPS82 sensitive to Cefoperazone and ZLL13 has resistance to it, ZLL13 sensitive to Cefoperazone and IPS82 has resistance to it, IPS82 and ZLL13 have different reactions to Oxacillin, Cefazolin, Clindamycin, Rifampicin and Cephalothin.

Table 2 Antibiotic sensitivity screening of IPS82 and ZLL13 Note: S = sensitive; R = resistant; I = intervenient

1.2 Growth rate

The growth rate of bacteria can be reflected by the total number of cells and the total cell number can be indicated by OD value under the same inoculation quantity and same culture condition. As shown in Figure 1, the two strains growth into the logarithmic period after 1 h stagnation period, the number of bacteria increased rapidly until reaching a stable level, the total number of cells in the two strains has no significant difference. By microscopic examination and comparison of both fermentation terminal (90% spore release), BRC-ZLL13 reached the end point after 38 h, and IPS82 reached the end point after 47 h, this indicates that the growth period of BRC-ZLL13 is shorter than that of IPS82, This means that the growth rate of BRC-ZLL13 is faster than IPS82.

Figure 1 Growth patternofZLL13 andIPS82inLB culturemedia

1.3 Insecticidal activity

The lethal concentrations of for test strains against different mosquito species were determined. The results showed that BRC-ZLL13 activity to Culex quinquefasciatus was 2 times higher than the IPS82, to Aedes aegypti was 4 times, to Aedes albopictus was 4 times. These suggest that BRC-ZLL13 is not only have toxic for the 3 test mosquito species, but also have higher toxicity than the standard strain IPS82 (Table 3).

Table 3 Toxicity of IPS82 and ZLL13 produced from LB against mosquito species

1.4 Plasmid profile analysis

Plasmid profiles of the tested strains were obtained by agarose gel electrophoresis. There are many plasmid bands on BRC-ZLL13, and there are 8 plasmid bands on the standard strain IPS82. In addition to 3 kb and 10 kb two strips with different bands, the rest of the bands of BRC-ZLL13 and IPS82 are basically similar (Figure 2).

Figure 2 Plasmid DNA from IPS82 and ZLL13 Note: M: Mark λDNA/ Hind Ⅲ ; 1: Bti ; 2: ZLL13

1.5 Mosquito killing gene

BRC-LL13 and ISP82 both contained the mosquito killing gene cyt1, cyt2, cry4A, cry4B, cry10 and cry11 (Figure 3; Figure 4).

Figure 3 Screening of cry gene in IPS82 and ZLL13 Note: M: 100 bp plus DNA ladder; 1: cry11 of ZLL13; 2: cry11 of IPS82; 3: cry10 of ZLL13; 4: cry10 of IPS82; 5: cry4B of ZLL13; 6: cry4B of IPS82; 7: cry4A of ZLL13; 8: cry4A of IPS82

Figure 4 Screening of cyt gene in IPS82 and ZLL13 Note: M: 100 bp plus DNA ladder; 1: cyt1 of ZLL13; 2: cyt1 of IPS82; 3: cyt2 of ZLL13; 4: cyt2 of IPS82

1.6 Analysis of the crystal protein map

Standard strain IPS82 expression of crystal protein of Cry4A, Cry4B, Cry10A, Cry11A, Cyt1 and Cyt2 (Guerchicoff et al., 1997), ZLL13 was also able to express the corresponding protein, and its expression was significantly higher than IPS82 (Figure 5).

Figure 5 SDS-PAGE pattern of crystal proteins which were separated from IPS82 and ZLL13 Note: M: Marker; 1: IPS82 with 5 μL sample; 2: ZLL13 with 5 μL sample; 3: IPS82 with 10 μL sample; 4: ZLL13 with 10 μL sample

2 Discussions

B. thuringiensis subsp. israelensis, serotype H14, is a strains with high activity of killing mosquito, the prepared products are used to control mosquitoes for decades (Bravo et al., 2007). Bt BRC-ZLL13, isolated from the leaves of Michelia alba, was not only had higher activity for killing mosquito than IPS82, but also had faster growth rate. The Serotype experiment confirmed that BRC-ZLL13 and IPS82 both belong to the Israelensis subspecies. Biochemical profile and antibiotic sensitivity test results show that there was a difference between the two strains.

Although there are many mosquito killing genes in the natural world, cry4, cry10 and cry11 are the most important genes. 14 pairs of primers for PCR amplification results showed that IPS82 and BRC-ZLL13 both contained cyt1, cyt2, cry4A, cry4B, cry10 and cry11 genes. In the spore capsule period, two strains were able to express the crystal protein of Cry4A, Cry4B, Cry10, Cry11, Cyt1 and Cyt2, which explained the high toxicity of the two strains, but the bioassay results showed that BRC-ZLL13 had higher toxicity than IPS82.

Plasmid is widely existed in Bt. The number of plasmids with different strains ranged from 1 to 16, and the molecular weight ranged from 1 MDa to 295 MDa (Yu, 1990), researchers have detected molecular weight of 3.3 MDa, 4.2 MDa, 4.9 MDa, 10.6 MDa, 68 MDa, 75 MDa, 105 MDa, and 135 Mda, respectively (Gonzdae and Carlton, 1984). In this study, 8 plasmid bands of ISP82 were detected, and the results were consistent with the above findings. ZLL13 contains 6 plasmids and those plasmids were same to IPS82 except the two plasmids at 3 kb and 10 kb (Figure 2).

In summary, Bt BRC-ZLL13 was similar to IPS82, But the growth rate of IPS82 was faster than BRC-ZLL13, the reason may be 2 lessened plasmids reduced the burden for BRC-ZLL13 protein expression. Although BRC-ZLL13 and IPS82 have the same insecticidal gene, but the expression efficiency of BRC-ZLL13 is higher, its reason waits for further studying.

3 Materials and Methods

3.1 Strains

Bt IPS82 was provided by the genetic stock center of Bacillus Ohio, BT BRC-ZLL13 was the Bt strain which were isolated and then preserved from Magnolia in our laboratory.

3.2 Culture medium

Liquid LB medium: 10 g/L of peptone, 5 g/L of yeast powder, 10 g/L of NaCl, pH 7.0. PM medium: 5 g/L of peptone, 1 g/L of yeast powder, 2.5 g/L of glucose, 1 g of KH₂PO₄, 0.02 g of ZnSO₄·7H₂O, 0.3 g of MgSO₄·7H₂O, 0.02 g of MnSO₄·7H₂O, 0.02 g of FeSO₄·7H₂O, pH 7.0.

3.3 Serotype

H serotype identification according to method in the literature (Ohba and Aizawa, 1978). Prepare H antigen of the test strains, intravenously inject rabbit. And then collect blood to prepare antiserum and determine titer, finally carry out agglutination test and cross absorption.

3.4 Growth rate

The same dose of IPS82 and BRC-ZLL13 were inoculated in 2 mL LB medium to actively culture 12 h under 30℃. 1 mL activated bacteria liquid was acquired to switch to 100 mL Erlenmeyer flask. Exact samples from the culture medium every 2 h (2 ml) to determine absorbance by UV spectrophotometry in 650 nm. Continuously measure72 h, observe under microscopic, when the 90% spore released, it deem to the end of culture.

3.5 Biological assay

The mosquito culture conditions: relative humidity was 80% and temperature was 25 (±3), daytime was 12 h. Culex quinquefasciatus was cultured more than 10 passages in the laboratory. Aedes aegypti and Aedes albopictus were provided by Dr. Huang Enjiong in Fujian Entry exit inspection and Quarantine Bureau. According to WHO (2007) bioassay standard, determine the biological toxicity of strains to Culex, Aedes aegypti and Aedes albopictus. Three mosquito species was repeated three times, each dose was repeated 3 times. The experiment was divided into 3 d, every 24 h to calculate mortality. The water in Institute of Biological assay was removed chlorine tap water, and the measured temperature was 28℃.

3.6 Statistical analysis

Excel Microsoft 2010 was used to carry on the variance analysis of the data that acquired in Institute of Biological assay, and then Windows SPSS 18.0 was used to calculate the median lethal concentration (LC50).

3.7 Physiological and biochemical reaction and drug sensitivity test

By using standard reagent plate of bacillus strain to test biochemical parameters and drug sensitivity. Concrete methods please read product specifications. The test results were analyzed by HX-21A bacteria analyzer.

3.8 Plasmid profiles

Plasmid extraction referred to molecular cloning experiments guidelines (Ibarra etal., 2003; Sambrook and Russell, 2001). Strains were inoculated to 5 mL liquid LB medium at 30℃, shaking culture overnight at 250 r/min, got the bacterial fluid, centrifuged for 2 min at 12000 r/min in 4℃, and when the centrifugation was end, then dry medium as far as possible. The precipitate resuspended on 100 μL pre cold alkaline lysis liquid I, after violent oscillation, added 5 μL lysozyme, put in a water bath for 30 min at 37℃.With the 200 μL of new configuration alkali liquor SⅡ, and quickly reversed the centrifugal tube several times, and then put centrifugal tube on ice for 5 min. With 150 mL of pre cold alkali liquor S, repeatedly reversed several times. The SⅢ distributed evenly, then put centrifuge tube on ice for 3~5 min. Centrifuged for 5 min at 12000 r/min in 4℃, the supernatant was transferred to another centrifuge tube, add an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), after intensive mixing, centrifuged for 5 min at 12000 r/min. Drew the upper aqueous phase, adding two times volume ethanol pre cooling, lay for 2 h at -20℃. Centrifuged, discarded supernatant, with 100 μL 70% of ethanol rinsed sediment, then removed ethanol, centrifugal tube placed 10 min at room temperature, after the evaporation of ethanol, with 50 μL of TE buffer which was containing pancreatic RNA enzyme (20 g/ml) to re dissolution precipitation, moderate oscillation in a few seconds, preserved at -20℃.

Mixed 5 μL plasmid solution and 1 μL 6 × sample buffer, according to the previous identification method, using IPS82 as reference, prepared agarose gel electrophoresis that concentration of 1% (buffer was 1 × TAE, 100 V, 1~2 h to detect(Reyes-Ramírez and Ibarra, 2008). After electrophoresis, the gel was stained with EB, and observed and photographed in Bio-Rad Gel Doc 1000 gel imager.

3.9 Detection of mosquito killing genes

3.9.1 Extraction of total DNA

The strains were cultured in 5 mL liquid LB culture medium and cultured overnight at 30℃. 1.5 mL bacteria was acquired, centrifuged for 2 min at 12000 r/min, precipitation was resuspended in 80 mL solutionⅠ (0.3 mol/L sucrose, 25 mmol/L EDTA), after violent oscillation added 5 μL lysozyme, mixing, lay for 30 min at 37℃. Added 420 μL of extraction buffer in 60℃ (100 mmol/L Tris-HCl, 25 mmol/L EDTA, 0.5 mol/L NaCl, 0.5% SDS), shaked up and down, with an equal volume of phenol: chloroform: isoamyl alcohol (25:24:1), centrifuged supernatant for 10 min at 12000 r/min. Supernatant was acquired to add 2 times ethanol, lay more than 2 h at -20℃. Centrifuged for 10 min at 12 000 r/min, discarded supernatant, dry for 5~10 min in a depurator, finally it was dissolved in TE buffer, preserved at -20℃.

3.9.2 PCR

According to the report of Ibarra et al (2003), 14 pairs of primers were synthesized to detect the following genes: cyt1, cyt2, cry2, cry4A, cry4B, cry10, cry11, cry17+27, cry19+29, cry24+40, cry25, cry29, cry 30 and cry32, the characteristics of each primer are shown in Table 4.

Table 4 Characteristics of general and specific primers used to amplify target genes

Using the total DNA as a template for PCR amplification, 25 μL of PCR reaction system was: 1.0 μL DNA template, 1.0 μL dNTP (each 200 mmol/L), 1.0 μL primers R (0.5 μmol/L), 1.0 μL primers F (0.5 μmol/L), 1.0 μL Taq enzyme (0.5~1 U/μL, 2.5 μL 10 × PCR Buffer, 17.5 μL ddH₂O). After reaction, 5 μL PCR product and 1 1 μL 6 × sample buffer were mixed, detected by agarose gel electrophoresis which the concentration of 1% (buffer of 1 × TAE, 100 V, 1~2 h). After electrophoresis, gel was stained with EB, and observed and photographed in Bio-Rad Gel Doc 1000 gel imager.

3.10 Mosquito killer crystal protein SDS-PAGE

SDS-PAGE referred to molecular cloning experiments guidelines. The strains were inoculated in 50 mL PM medium, cultures for 2 d in 30℃. Through the cultivation of microscopic observation of spores and crystal number, when the spore rate is 90%, removed bacteria, centrifuged for 15 min in 4℃ at 5000 r/min, precipitation resuspended in 1 mol/L NaCl solution, after vortex oscillation for 5 min, centrifuged for 15 min in 4℃ at 5000 r/min, repeated 3 times. The sediment resuspended in sterile water, after vortex oscillation for 5 min, centrifuged for 15 min in 4℃ at 5000 r/min, repeated 3 times. Precipitation resuspended in lysis buffer solution (1% Na₂CO₃, 0.08% DTT), oscillated for 30 min, in the constant temperature of 30, centrifuged for 15 min in 4℃ at 7000 r/min. Supernatant was acquired, adjusted to pH 4.6~4.8 by adding acetic acid, stored at 4℃ overnight. The supernatant was centrifuged for 15 min in 4℃ at 10 000 r/min. The precipitate resuspended in sterile water, after vortex oscillation for 5 min, centrifuged for 15 min in 4℃ at 10 000 r/min., repeated 3 times. The protein was dissolved in 0.05 mol/L NaOH, and preserved in -20℃.

Authors’ Contributions

Liu Zhaoxia, Li Mingwei and Wu Songqing were responsible for the implementation and application of data processing, and thesis writing; Ye Wenhui, Yu Xiongze and Lin Qiuqiu in charge of sampling, data analysis and the revision work in the study; Guan Xiong and Zhang Lingling were responsible for the guidance and verification of the modification.

Acknowledgements

The study was supported by the National 863 Project (2011AA10A203), Education Department University Leading Talent (k8012012a), National Health and Family Planning Commission Co Construction Science Research Fund--Fujian Provincial Health Joint Research Plan (WKJ-FJ-25), National Natural Science Foundation of China (31301724), Fujian Province Science and Technology Key Project (2013N0003), Natural Science Foundation of Fujian Province (2013J01079), University Distinguished Young Research Talent Training Program of Fujian Province (JA12092), and New Century Talent Support Program of Fujian Province (K80MKTO3a).

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