Potential of Cry10Aa and Cyt2Ba, Two Minority δ-endotoxins Produced by Bacillus thuringiensis ser. israelensis, for the Control of Aedes aegypti Larvae
Abstract
:1. Introduction
2. Results
2.1. Insecticidal Cry and Cyt Genes Identified in Bti Strain from VectoBac-12AS®
2.2. Cloning of Cyt2Ba, Cry10Aa and Cry11Aa
2.3. Characterization of Bt Recombinant Strains Expressing cyt2Ba, cry10Aa, cry4Aa, cry4Ba and cry11Aa
2.4. Mosquitocidal Activity of the δ-Endotoxins Produced by Bti
3. Discussion
4. Conclusions
5. Materials and Methods
5.1. Bacterial Strains and Plasmids
5.2. Insect Culture
5.3. Total DNA Extraction and Genomic Sequencing
5.4. Identification of Cry and Cyt Insecticidal Genes in VectoBac-12AS®
5.5. Amplification, Cloning and Sequencing of Cyt2Ba, Cry11Aa and Cry10Aa
5.6. Expression of Cyt2Ba, Cry10Aa, Cry4Aa, Cry4Ba and Cry11Aa Recombinant Proteins and SDS-PAGE Analysis
5.7. Mosquitocidal Activity of the δ-Endotoxins Produced by Bti
5.8. Statistical Analysis
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Treatment 1 | Concentration (ng/mL) | Mortality (% ± SD) |
---|---|---|
Cry10Aa | 40 | 26 ± 5 |
Cyt2Ba | 40 | 31 ± 8 |
Cry10Aa+Cyt2Ba (1:1) | 80 | 93 ± 6 |
Cry4Aa | 10 | 31 ± 10 |
Cyt2Ba | 10 | 28 ± 23 |
Cry4Aa+Cyt2Ba (1:1) | 20 | 100 ± 0 |
Cry4Ba | 0.02 | 32 ± 18 |
Cyt2Ba | 0.02 | 10 ± 9 |
Cry4Ba+Cyt2Ba (1:1) | 0.04 | 43 ± 19 |
Cry11Aa | 1.5 | 40 ± 16 |
Cyt2Ba | 1.5 | 21 ± 14 |
Cry11Aa+Cyt2Ba (1:1) | 3 | 46 ± 21 |
Cry10Aa | Cyt2Ba | Cry10Aa+Cyt2Ba | ||||||
---|---|---|---|---|---|---|---|---|
ng/mL | Dead/Total | Mortality (% ± SD) | ng/mL | Dead/Total | Mortality (% ± SD) | ng/mL | Dead/Total | Mortality (% ± SD) |
2000 | 39/50 | 78 ± 2% | 4000 | 84/92 | 91.3 ± 10% | 300 | 115/124 | 92.7 ± 9% |
666 | 30/50 | 60 ± 10% | 1333 | 64/85 | 75.3 ± 17% | 60 | 86/114 | 75.4 ± 9% |
222 | 23/41 | 56.1 ± 2% | 444 | 39/94 | 41.5 ± 9% | 12 | 75/135 | 55.6 ± 11% |
74 | 12/44 | 27.3 ± 12% | 148 | 28/78 | 35.9 ± 7% | 2.4 | 53/131 | 40.5 ± 6% |
24.7 | 6/46 | 13 ± 17% | 49.4 | 22/86 | 25.6 ± 6% | 0.48 | 34/116 | 29.3 ± 6% |
8.2 | 1/42 | 2.4 ± 3% | 16.4 | 21/84 | 25 ± 13% | 0.096 | 23/120 | 19.2 ± 14% |
Cry4Aa | Cyt2Ba | Cry4Aa+Cyt2Ba | ||||||
---|---|---|---|---|---|---|---|---|
ng/mL | Dead/Total | Mortality (% ± SD) | ng/mL | Dead/Total | Mortality (% ± SD) | ng/mL | Dead/Total | Mortality (% ± SD) |
486 | 63/70 | 90 ± 11% | 4000 | 84/92 | 91.3 ± 10% | 54 | 139/163 | 85.3 ± 13% |
162 | 53/71 | 74.6 ± 17% | 1333 | 64/85 | 75.3 ± 17% | 27 | 111/155 | 71.6 ± 17% |
54 | 42/70 | 60 ± 13% | 444 | 39/94 | 41.5 ± 9% | 13.5 | 87/200 | 43.5 ± 14% |
18 | 27/73 | 37 ± 11% | 148 | 28/78 | 35.9 ± 7% | 6.74 | 58/142 | 40.8 ± 24% |
6 | 13/64 | 20.3 ± 10% | 49.4 | 22/86 | 25.6 ± 6% | 3.36 | 11/102 | 10.8 ± 16% |
2 | 7/71 | 9.9 ± 7% | 16.4 | 21/84 | 25 ± 13% | 1.68 | 5/73 | 6.8 ± 8% |
Treatment (a) | Regression | LC50 Observed | FL (95%) (b) | LC50 Expected (c) | FL (95%) (b) | |||||
---|---|---|---|---|---|---|---|---|---|---|
Slope ± SE | Intercept ± SE | (ng/mL) | Lower | Upper | (ng/mL) | Synergistic Factor (d) | Potency | Lower | Upper | |
Cyt2Ba | 0.59 ± 0.13 | −3.33 ± 0.79 | 279.37 | 190.20 | 410.38 | - | - | 1 | - | - |
Cry10Aa | 0.74 ± 0.09 | −4.26 ± 0.53 | 299.62 | 245.06 | 366.34 | - | - | 0.93 | 0.78 | 1.12 |
Cry4Aa | 0.78 ± 0.02 | −2.77 ± 0.09 | 34.63 | 29.73 | 40.34 | - | - | 8.07 | 6.40 | 10.17 |
Cry10Aa+Cyt2Ba (e) | 0.45 ± 0.05 | −0.64 ± 0.14 | 4.22 | 3.25 | 5.50 | 289.27 | 68.55 | 66.20 | 58.52 | 74.61 |
Cry4Aa+Cyt2Ba (e) | 1.22 ± 0.17 | −3.17 ± 0.46 | 13.41 | 12.55 | 14.33 | 61.62 | 4.60 | 20.83 | 15.15 | 28.63 |
VectoBac-12AS® | 1.51 ± 0.29 | 3.44 ± 0.73 | 1.02 × 10−1 | 9.34 × 10−2 | 1.11 × 10−1 | - | - | 2.73 × 103 | 2.03 × 103 | 3.69 × 103 |
Primer Name | Primer Sequence | Reference |
---|---|---|
Cyt2B-Fw-XbaI | 5′-TTCTAGAGATAATGAAGGAGGGGAGTC-3′ | This study |
Cyt2B-Rv-PstI | 5′-CCTGCAGCAAAATTAAATTGCTGAGTTACTATAATAAC-3′ | This study |
Cry10A-Fw-SalI | 5′-ATGTCGACTTGCAACAGAAAAGAGTTGTGTC-3′ | [17] |
Cry10A-Rv-PaeI | 5′-GAGCATGCACATTTCCCCACAATTTTCA-3′ | [17] |
Cry10A-test-Fw | 5′-CGAAATTGTCAGACATAGAGAG-3′ | This study |
Cry10A-test-Rv | 5′-GAATTACCAAGTCTCCACCTG-3′ | This study |
p20-Fw-PstI | 5′-CCTGCAGGGATAAAATTGGAGGATAATTGATG-3′ | This study |
p20-Rv-PaeI | 5′-GGCATGCGTTTCCAGTGCATTCAATTTAC-3′ | This study |
p19-Fw-SalI | 5′-GTGTCGACGTTTTTTAAAATTGCATAGAAGGG-3′ | This study |
Cry11A-Rv-PstI | 5′-CTCTGCAGGTGCTAACATGACTTCTACTTTAG-3′ | This study |
Cry11A-test | 5′-GGTCATAATTTATGAATAAAAATATGAC-3′ | This study |
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Valtierra-de-Luis, D.; Villanueva, M.; Lai, L.; Williams, T.; Caballero, P. Potential of Cry10Aa and Cyt2Ba, Two Minority δ-endotoxins Produced by Bacillus thuringiensis ser. israelensis, for the Control of Aedes aegypti Larvae. Toxins 2020, 12, 355. https://doi.org/10.3390/toxins12060355
Valtierra-de-Luis D, Villanueva M, Lai L, Williams T, Caballero P. Potential of Cry10Aa and Cyt2Ba, Two Minority δ-endotoxins Produced by Bacillus thuringiensis ser. israelensis, for the Control of Aedes aegypti Larvae. Toxins. 2020; 12(6):355. https://doi.org/10.3390/toxins12060355
Chicago/Turabian StyleValtierra-de-Luis, Daniel, Maite Villanueva, Liliana Lai, Trevor Williams, and Primitivo Caballero. 2020. "Potential of Cry10Aa and Cyt2Ba, Two Minority δ-endotoxins Produced by Bacillus thuringiensis ser. israelensis, for the Control of Aedes aegypti Larvae" Toxins 12, no. 6: 355. https://doi.org/10.3390/toxins12060355