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A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors
Published on: April 20, 2011
Aedes cadherin receptor that mediates Bacillus thuringiensis Cry11A toxicity is essential for mosquito development
Jianwu Chen1, Karly G Aimanova1, Sarjeet S Gill1
1Department of Molecular, Cell and Systems Biology, University of California, Riverside, California, United States of America.
Abstract:
Aedes cadherin (AaeCad, AAEL024535) has been characterized as a receptor for Bacillus thuringiensis subsp. israelensis (Bti) Cry11A toxins. However, its role in development is still unknown. In this study, we modified the cadherin gene using ZFN and TALEN. Even though we obtained heterozygous deletions, no homozygous mutants were viable. Because ZFN and TALEN have lower off-targets than CRISPR/Cas9, we conclude the cadherin gene is essential for Aedes development. In contrast, in lepidopteran insects loss of a homologous cadherin does not appear to be lethal, since homozygous mutants are viable. To analyze the role of AaeCad in vivo, we tagged this protein with EGFP using CRISPR-Cas9-mediated homologous recombination and obtained a homozygous AaeCad-EGFP line. Addition of Aedes Rad51 mRNA enhanced the rate of recombination. We then examined AaeCad protein expression in most tissues and protein dynamics during mosquito development. We observe that AaeCad is expressed in larval and adult midgut-specific manner and its expression pattern changed during the mosquito development. Confocal images showed AaeCad has high expression in larval caecae and posterior midgut, and also in adult midgut. Expression of AaeCad is observed primarily in the apical membranes of epithelial cells, and not in cell-cell junctions. The expression pattern observed suggests AaeCad does not appear to play a role in these junctions. However, we cannot exclude its role beyond cell-cell adhesion in the midgut. We also observed that Cry11A bound to the apical side of larval gastric caecae and posterior midgut cells exactly where AaeCad-EGFP was expressed. Their co-localization suggests that AaeCad is indeed a receptor for the Cry11A toxin. Using this mosquito line we also observed that low doses of Cry11A toxin caused the cells to slough off membranes, which likely represents a defense mechanism, to limit cell damage from Cry11A toxin pores formed in the cell membrane.
Insights
Aedes cadherin (AaeCad) is essential for mosquito development and acts as a receptor for Bacillus thuringiensis israelensis (Bti) Cry11A toxins. Its disruption is lethal, indicating a critical role in mosquito biology.
Area of Science:
- Molecular Biology
- Entomology
- Toxicology
Background:
- Aedes cadherin (AaeCad) is identified as a receptor for Bacillus thuringiensis subsp. israelensis (Bti) Cry11A toxins.
- The developmental role of AaeCad in Aedes mosquitoes remains largely uncharacterized.
Purpose of the Study:
- To investigate the essentiality of AaeCad during Aedes mosquito development.
- To elucidate the in vivo function and expression patterns of AaeCad.
- To confirm AaeCad's role as a Cry11A toxin receptor.
Main Methods:
- Gene editing using ZFN, TALEN, and CRISPR-Cas9-mediated homologous recombination to create AaeCad mutants and an AaeCad-EGFP tagged line.
- Analysis of mutant viability and protein expression patterns via confocal microscopy.
- Co-localization studies of AaeCad-EGFP and Cry11A toxin.
Main Results:
- Homozygous AaeCad deletion mutants were non-viable, indicating essentiality for Aedes development.
- AaeCad is expressed in a midgut-specific manner in larvae and adults, particularly in gastric caeca and posterior midgut.
- AaeCad localizes to the apical membrane of epithelial cells and co-localizes with Cry11A toxin, confirming its role as a receptor.
- Cry11A toxin exposure induced cell membrane sloughing, suggesting a defense mechanism.
Conclusions:
- AaeCad is indispensable for Aedes mosquito development.
- AaeCad functions as a specific receptor for Bti Cry11A toxins on the apical surface of midgut epithelial cells.
- The observed cellular response to Cry11A suggests a novel host defense mechanism in mosquitoes.
