Cadherin binding is not a limiting step for Bacillus thuringiensis subsp. israelensis Cry4Ba toxicity to Aedes aegypti larvae.

Bacillus thuringiensis subsp. israelensis produces three Cry toxins (Cry4Aa, Cry4Ba and Cry11Aa) that are active against Aedes aegypti larvae. The identification of the rate-limiting binding steps of Cry toxins that are used for insect control in the field, such as those of B. thuringiensis subsp. israelensis, should provide targets for improving insecticides against important insect pests. Previous studies showed that Cry11Aa binds to cadherin receptor fragment CR7-11 (cadherin repeats 7-11) with high affinity. Binding to cadherin has been proposed to facilitate Cry toxin oligomer formation. In the present study, we show that Cry4Ba binds to CR7-11 with 9-fold lower binding affinity compared with Cry11Aa. Oligomerization assays showed that Cry4Ba is capable of forming oligomers when proteolytically activated in vitro in the absence of the CR7-11 fragment in contrast with Cry11Aa that formed oligomers only in the presence of CR7-11. Pore-formation assays in planar lipid bilayers showed that Cry4Ba oligomers were proficient in opening ion channels. Finally, silencing the cadherin gene by dsRNA (double-stranded RNA) showed that silenced larvae were more tolerant to Cry11Aa in contrast with Cry4Ba, which showed similar toxic levels to those of control larvae. These findings show that cadherin binding is not a limiting step for Cry4Ba toxicity to A. aegypti larvae.

Multiple receptors as targets of Cry toxins in mosquitoes.

Bacillus thuringiensis (Bt) produces inclusions that are composed of proteins known as crystal proteins or Cry toxins. Due to their high specificity and their safety to humans and the environment, these Cry toxins are considered to be valuable alternatives to chemical pesticides in insect control programs. It is believed that Cry toxin-induced membrane pore formation is responsible for insect toxicity. The molecular mechanism of pore formation involves recognition and subsequent binding of the toxin to membrane receptors. This binding is accompanied by toxin oligomerization and transfer of domain I helices of the toxin to the lipid-water interface. This toxin insertion creates pores that lyse the cells. Several receptors from lepidopteran, coleopteran, and dipteran insects have been well characterized. This paper provides an overview of the understanding of the interactions between Cry toxin and multiple receptors in mosquitoes, in particular Aedes aegypti and reviews the manner by which the receptors were identified and characterized, with a focus on three proteins, cadherin, alkaline phosphatase, and aminopeptidase-N.

Characterization of a blood-meal-responsive proton-dependent amino acid transporter in the disease vector, Aedes aegypti.

After anautogenous mosquitoes ingest the required blood meal, proteins in it are rapidly cleaved, yielding a large pool of amino acids. Transport of these amino acids into gut epithelial cells and their subsequent translocation into other tissues is critical for oogenesis and other physiological processes. We have identified a proton amino acid transporter (PAT) in Aedes aegypti (AaePAT1, AAEL007191) which facilitates this transport and is expressed in epithelial cell membranes of larval caecae and the adult midgut. AaePAT1 encodes a 475 amino acid protein showing high similarity to Anopheles gambiae AGAP009896, Culex pipiens CPIJ011438 and Drosophila melanogaster CG7888. When expressed in Xenopus oocytes the transport kinetics showed AaePAT1 is a low affinity transporter with low substrate specificity, having Km and Vmax values of about 7.2 mmol l(-1) and 69 pmol oocyte(-1) min(-1), respectively, for glutamine. A number of other amino acids are also transported by this PAT. In female adult midgut, AaePAT1 transcript levels were induced after ingestion of a blood meal.

Sindbis virus induces transport processes and alters expression of innate immunity pathway genes in the midgut of the disease vector, Aedes aegypti.

The midgut of hematophagous insects is the initial site of infection by arthropod-borne viruses (arboviruses) and plays a crucial role in vector competence. To further understand processes that occur in the midgut in response to infection by an arbovirus, DNA microarrays were used to analyze gene expression changes following infection by the alphavirus, Sindbis (MRE16 Malaysian strain). Midgut transcription profiles from mosquitoes fed blood containing 10(8)pfu/ml of virus were compared with those from mosquitoes ingesting blood meals having no virus. Transcription profiles from both experimental groups were analyzed at 1, 4, and 8 days post-feeding. Among the many transcription changes observed by microarray analysis, the most dramatic involved three genes that had 25-40-fold increases in transcript levels in virus infected mosquitoes at 4 days post-infection. These genes were synaptic vesicle protein-2 (SV2), potassium-dependent sodium/calcium exchanger (NCKX), and a homologue of Caenorhabditis elegans Unc-93, a putative component of a two-pore potassium channel. We speculate that these changes represent changes in vesicle transport processes. In addition to these observations, transcript changes were observed in infected mosquitoes that suggested involvement of Toll and c-jun amino terminal kinase immune cascades as a response to viral infection.

Blood meal induces global changes in midgut gene expression in the disease vector, Aedes aegypti.

Blood feeding is an essential developmental process for many arthropods and plays a significant role in disease transmission. Understanding physiological responses in the midgut is important because it is the primary site of blood meal digestion and pathogenic infection. Processes that occur in the midgut in response to a blood meal have been studied but are poorly understood. Here, we use cDNA microarrays to examine midgut gene expression on a global level in response to blood feeding to assist in unraveling these processes. We have developed Aedes aegypti microarrays consisting of clones obtained from an expressed sequence tag project. Individual clones were amplified by polymerase chain reaction and printed onto glass slides. These microarrays were used to study the effects of a blood meal on midgut gene expression over a 72-h time course. As a result, a number of genes involved in processes such as nutrient uptake and metabolism, cellular stress responses, ion balance, and PM formation, as well as a number of unknown genes were induced or repressed in response to a blood meal based on this microarray data.