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Knockout of Two Cry-Binding Aminopeptidase N Isoforms Does Not Change Susceptibility of Aedes aegypti Larvae to Bacillus thuringiensis subsp. israelensis Cry4Ba and Cry11Aa Toxins

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State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops, College of Plant Protection, Fujian Agriculture and Forestry University (FAFU), Fuzhou 350002, China
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College of Forestry, Fujian Agriculture and Forestry University (FAFU), Fuzhou 350002, China
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College of Life Science, Fujian Agriculture and Forestry University (FAFU), Fuzhou 350002, China
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Postdoctoral Station of Forestry, College of Forestry, Fujian Agriculture and Forestry University (FAFU), Fuzhou 350002, China
*
Authors to whom correspondence should be addressed.
Academic Editor: Kevin M. Myles
Insects 2021, 12(3), 223; https://doi.org/10.3390/insects12030223
Received: 10 February 2021 / Revised: 26 February 2021 / Accepted: 2 March 2021 / Published: 5 March 2021
(This article belongs to the Section Insect Pest and Vector Management)
The midgut aminopeptidase N (APN) isoforms have been identified as the binding receptor of insecticidal Cry toxins in numerous insects, including the major arbovirus vector Aedes aegypti (Ae. aegypti). However, whether the Cry-binding APN acts as an essential functional receptor to mediate Bacillus thuringiensis subsp. israelensis (Bti) toxicity in Ae. aegypti larvae remains to be determined. In this study, our results provide the direct molecular evidence demonstrating that two Cry-binding APN isoforms (AeAPN1 and AeAPN2) did not play a key role in mediating Bti Cry4Ba and Cry11Aa toxicity in Ae. aegypti larvae.
The insecticidal Cry4Ba and Cry11Aa crystal proteins from Bacillus thuringiensis subsp. israelensis (Bti) are highly toxic to Ae. aegypti larvae. The glycosylphosphatidylinositol (GPI)-anchored APN was identified as an important membrane-bound receptor for multiple Cry toxins in numerous Lepidoptera, Coleoptera, and Diptera insects. However, there is no direct molecular evidence to link APN of Ae. aegypti to Bti toxicity in vivo. In this study, two Cry4Ba/Cry11Aa-binding Ae. aegypti GPI-APN isoforms (AeAPN1 and AeAPN2) were individually knocked-out using CRISPR/Cas9 mutagenesis, and the AeAPN1/AeAPN2 double-mutant homozygous strain was generated using the reverse genetics approach. ELISA assays showed that the high binding affinity of Cry4Ba and Cry11Aa protoxins to the midgut brush border membrane vesicles (BBMVs) from these APN knockouts was similar to the background from the wild-type (WT) strain. Likewise, the bioassay results showed that neither the single knockout of AeAPN1 or AeAPN2, nor the simultaneous disruption of AeAPN1 and AeAPN2 resulted in significant changes in susceptibility of Ae. aegypti larvae to Cry4Ba and Cry11Aa toxins. Accordingly, our results suggest that AeAPN1 and AeAPN2 may not mediate Bti Cry4Ba and Cry11Aa toxicity in Ae. aegypti larvae as their binding proteins. View Full-Text
Keywords: Aedes aegypti; Cry4Ba; Cry11Aa; aminopeptidase N; CRISPR/Cas9 Aedes aegypti; Cry4Ba; Cry11Aa; aminopeptidase N; CRISPR/Cas9
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MDPI and ACS Style

Wang, J.; Yang, X.; He, H.; Chen, J.; Liu, Y.; Huang, W.; Ou, L.; Yang, Z.; Guan, X.; Zhang, L.; Wu, S. Knockout of Two Cry-Binding Aminopeptidase N Isoforms Does Not Change Susceptibility of Aedes aegypti Larvae to Bacillus thuringiensis subsp. israelensis Cry4Ba and Cry11Aa Toxins. Insects 2021, 12, 223. https://doi.org/10.3390/insects12030223

AMA Style

Wang J, Yang X, He H, Chen J, Liu Y, Huang W, Ou L, Yang Z, Guan X, Zhang L, Wu S. Knockout of Two Cry-Binding Aminopeptidase N Isoforms Does Not Change Susceptibility of Aedes aegypti Larvae to Bacillus thuringiensis subsp. israelensis Cry4Ba and Cry11Aa Toxins. Insects. 2021; 12(3):223. https://doi.org/10.3390/insects12030223

Chicago/Turabian Style

Wang, Junxiang, Xiaozhen Yang, Huan He, Jingru Chen, Yuanyuan Liu, Wanting Huang, Luru Ou, Zhaohui Yang, Xiong Guan, Lingling Zhang, and Songqing Wu. 2021. "Knockout of Two Cry-Binding Aminopeptidase N Isoforms Does Not Change Susceptibility of Aedes aegypti Larvae to Bacillus thuringiensis subsp. israelensis Cry4Ba and Cry11Aa Toxins" Insects 12, no. 3: 223. https://doi.org/10.3390/insects12030223

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