Proof

1.Introduction

In our project, we transformed the expression vector of Cry and Cyt toxins. Based on the toxin groups, we constructed six toxins express devices as Figure1. To compare the toxicities, we also tested the toxicity of Bacillus thuringiensis strain LLP229.

2.Material, Condition and Method

The larvae of Ae. albopictus are fed in the Key Laboratory of Biopesticide and Chemical Biology. The generation of Ae. albopictus is 118. The LLP229 strain of Bacillus thuringiensis is provided by Key Laboratory of Biopesticide and Chemical Biology. The concentration of LLP229 is listed in each table.

The CC530 and FA479 strains of Chlamydomonas reinhardtii and TAP medium are bought from the Aquatic Species Research Institute in Wuhan, Hubei. The concentration of Chlamydomonas reinhardtii is recorded in each table.

We contrasted various toxins group expression vectors and transform them into Chlamydomonas reinhardtii as following groups: Cry4Aa+2A+Cyt1, Cry4Aa+2A+Cyt2, Cry10Aa+2A+Cyt1, Cry10Aa+2A+Cyt2, Cry11Aa+2A+Cyt1, Cry11Aa+2A+Cyt2.

The toxicity analysis condition is in an environmentally controlled room (T=28±1 °C, RH=85%±5%, L: D=14:10).

Bioassay of various concentrations was assayed against 30 third-instar larvae in 20 ml of dechlorinated water. Each group was replicated 3 times. The level of mortality was recorded after 48 hours. Then calculated the corrected mortality. The bioassay was then repeated with such that a concentration range yielding 10–90% mortality to obtain and the 50% lethal concentration (LC₅₀) was calculated.

Table 1.1 Toxicity of LLP29 to A. albopictus

Repeating group	Number of insects	Number of mortality	Corrected mortality (%)
1	30	30	100
2	30	30	100
3	30	30	100
Control	30	0	0

Note. Then concentration of LLP29 is 2.16 x 10⁹ (OD₆₀₀ of 1.0 = 8 x 10⁸ cells/ml, OD₆₀₀ =2.7A.)

 

Table 1.2 Toxicity of LLP29 to Culex quinquefasciatus

Repeating group	Number of insects	Number of mortality	Corrected mortality (%)
1	30	30	100
2	30	30	100
3	30	30	100
Control	30	0	0

Note. Then concentration of LLP29 is 2.16 x 10⁹ (OD₆₀₀ of 1.0 = 8 x 10⁸ cells/ml, OD₆₀₀ =2.7A)

 

Table 1.3 Toxicity of LLP29 to Culex quinquefasciatus

Repeating group	Number of insects	Number of mortality	Corrected mortality (%)
1	30	30	100
2	30	30	100
3	30	30	100
Control	30	0	0

Note. Then concentration of LLP29 is 2.16 x 10⁹ (OD₆₀₀ of 1.0 = 8 x 10⁸ cells/ml, OD₆₀₀ =2.7A)

 

Table 2.1 Toxicity of Chlamydomonas reinhardtii (Cry4Aa+2A+Cyt1) to A. albopictus

Repeating group	Number of insects	Number of mortality	Corrected mortality (%)
1
2
3
Control 1 Control 2

Note.1. Then concentration of Chlamydomonas reinhardtii is xxx (OD₆₀₀ of 1.0 = 8 x 10⁸ cells/ml, OD₆₀₀ =xxxx)

         2. The Control 1 group is Chlamydomonas reinhardtii (no transformation, OD=?)

         3. The Control 2 group is TAP medium (Concentration =?)

 

Table 2.2 Toxicity of Chlamydomonas reinhardtii (Cry4Aa+2A+Cyt2) to A. albopictus

Repeating group	Number of insects	Number of mortality	Corrected mortality (%)
1
2
3
Control 1 Control 2

Note.1. Then concentration of Chlamydomonas reinhardtii is xxx (OD₆₀₀ of 1.0 = 8 x 10⁸ cells/ml, OD₆₀₀ =xxxx)

         2. The Control 1 group is Chlamydomonas reinhardtii (no transformation, OD=?)

         3. The Control 2 group is TAP medium (Concentration =?)

 

Table 2.3 Toxicity of Chlamydomonas reinhardtii (Cry10Aa+2A+Cyt1) to A. albopictus

Repeating group	Number of insects	Number of mortality	Corrected mortality (%)
1
2
3
Control 1 Control 2

Note.1. Then concentration of Chlamydomonas reinhardtii is xxx (OD₆₀₀ of 1.0 = 8 x 10⁸ cells/ml, OD₆₀₀ =xxxx)

         2. The Control 1 group is Chlamydomonas reinhardtii (no transformation, OD=?)

         3. The Control 2 group is TAP medium (Concentration =?)

 

Table 2.4 Toxicity of Chlamydomonas reinhardtii (Cry10Aa+2A+Cyt2) to A. albopictus

Repeating group	Number of insects	Number of mortality	Corrected mortality (%)
1
2
3
Control 1 Control 2

Note.1. Then concentration of Chlamydomonas reinhardtii is xxx (OD₆₀₀ of 1.0 = 8 x 10⁸ cells/ml, OD₆₀₀ =xxxx)

         2. The Control 1 group is Chlamydomonas reinhardtii (no transformation, OD=?)

         3. The Control 2 group is TAP medium (Concentration =?)

 

Table 2.5 Toxicity of Chlamydomonas reinhardtii (Cry11Aa+2A+Cyt1) to A. albopictus

Repeating group	Number of insects	Number of mortality	Corrected mortality (%)
1
2
3
Control 1 Control 2

Note.1. Then concentration of Chlamydomonas reinhardtii is xxx (OD₆₀₀ of 1.0 = 8 x 10⁸ cells/ml, OD₆₀₀ =xxxx)

         2. The Control 1 group is Chlamydomonas reinhardtii (no transformation, OD=?)

         3. The Control 2 group is TAP medium (Concentration =?)

 

Table 2.5 Toxicity of Chlamydomonas reinhardtii (Cry11Aa+2A+Cyt2) to A. albopictus

Repeating group	Number of insects	Number of mortality	Corrected mortality (%)
1
2
3
Control 1 Control 2

Note.1. Then concentration of Chlamydomonas reinhardtii is xxx (OD₆₀₀ of 1.0 = 8 x 10⁸ cells/ml, OD₆₀₀ =xxxx)

         2. The Control 1 group is Chlamydomonas reinhardtii (no transformation, OD=?)

         3. The Control 2 group is TAP medium (Concentration =?)

 

 

 

 

 

 

 

 

Reference

1.Zhang, L., Huang, E., Lin, J., Gelbič, I., Zhang, Q., Guan, Y., … Guan, X. (2010). A novel mosquitocidal strain LLP29 isolated from the phylloplane of. Microbiological Research, 165(2), 133–141. doi: 10.1016/j.micres.2009.03.002