Research paper Receptors are affected by selection with each Bacillus thuringiensis israelensis Cry toxin but not with the full Bti mixture in Aedes aegypti Renaud Stalinski, Frederic Laporte, Guillaume Tetreau 1 , Laurence Després ⁎ a Université Grenoble Alpes, Laboratoire d'Ecologie Alpine UMR5553, F-38000 Grenoble, France b Centre National de la Recherche Scientifique (CNRS), Laboratoire d'Ecologie Alpine UMR5553, F-38000 Grenoble, France abstract article info Article history: Received 13 April 2016 Received in revised form 9 June 2016 Accepted 8 July 2016 Available online 11 July 2016 Bacillus thuringiensis israelensis (Bti) toxins are increasingly used for mosquito control, but little is known about the precise mode of action of each of these toxins, and how they interact to kill mosquito larvae. By using RNA sequencing, we investigated change in gene transcription level and polymorphism variations associated with re- sistance to each Bti Cry toxin and to the full Bti toxin mixture in the dengue vector Aedes aegypti. The up- regulation of genes related to chitin metabolism in all selected strain suggests a generalist, non-toxin-specific re- sponse to Bti selection in Aedes aegypti. Changes in the transcription level and/or protein sequences of several pu- tative Cry toxin receptors (APNs, ALPs, α-amylases, glucoside hydrolases, ABC transporters) were specific to each Cry toxin. Selective sweeps associated with Cry4Aa resistance were detected in 2 ALP and 1 APN genes. The lack of selection of toxin-specific receptors in the Bti-selected strain supports the hypothesis that Cyt toxin acts as a re- ceptor for Cry toxins in mosquitoes. © 2016 Elsevier B.V. All rights reserved. Keywords: RNA-seq Bacillus thuringiensis israelensis toxins Mosquito Bio-insecticide resistance Specific receptors Single Nucleotide Polymorphism 1. Introduction Biological control strategies involving Bacillus thuringiensis israelensis (Bti) are increasingly used as alternative to chemicals in order to limit the spreading of mosquito-borne pathogens transmission and nuisance. During sporulation, Bti produces a crystalline inclusion composed of four main toxins: Cry4Aa, Cry4Ba, Cry11Aa and Cyt1Aa. The classification of Cry toxins is based on amino-acid sequence homol- ogy: Cry4Aa and Cry4Ba share 60% homology while Cry11Aa share only 20% homology with Cry4Aa and Cry4Ba (Crickmore et al., 1998). Upon larval ingestion, the crystal is solubilized in the alkaline pH of the in- sect's gut, releasing Cry and Cyt protoxins that will be processed to ac- tive toxins by insect gut proteases (Ben-Dov, 2014; Lacey, 2007). Cyt and Cry toxins are known to act in synergy to kill mosquito larvae (Wirth et al., 2000), and evidence was provided that Cyt1Aa synergizes or suppresses resistance to Cry11Aa toxin by binding to the midgut membrane and functioning as a Cry receptor (Perez et al., 2005), but the precise mechanisms of their interaction is not fully understood. Other Bacillus thuringiensis subspecies produce Cry toxins that are specific to their target organism, and the selectivity of Cry toxins is mainly due to the interaction with specific receptors present at the sur- face of midgut epithelium cells of the larva. However, most research so far has been done on Cry1A toxin mode of action in lepidopterans (Ferré et al., 1995; Pigott and Ellar, 2007). The activated Cry toxins bind to spe- cific protein receptors, insert into the membrane and form pores, resulting in midgut disruption, bacterial infection and death of the in- sect. In the recent years, several models have been proposed to describe the precise mode of action of Bt Cry toxins (reviewed in (Soberon et al., 2009; Vachon et al., 2012)). In the most accepted model, binding to cadherin is required for the Cry proteins to oligomerize and then bind to other receptors, such as aminopeptidases N (APNs) and alkaline phosphatases (ALPs), mostly located in lipid rafts (reviewed in (Pigott and Ellar, 2007)) which favor toxin insertion into the membrane. More recently, the role of ABC transporters acting as Cry1A and/or Cry1C toxin receptors in several distant lineages of Lepidoptera has been demonstrated (Atsumi et al., 2012; Baxter et al., 2011; Heckel, 2012; Park et al., 2014; Xiao et al., 2014). In mosquitoes, besides cadherins, APNs and ALPs, α-amylases were also shown to bind to Cry4Ba and Cry11Aa toxins (Fernandez-Luna et al., 2010). As compared to what is known about the mode of action of Bt Cry toxins in lepidop- terans, the Bti mode of action in mosquito has been much less studied, mainly due to the lack of Bti-resistant field population of mosquito to dissect the mechanisms of resistance. Indeed, the cocktail of toxins pro- duced by Bti, and especially the presence of Cyt toxin, appears to hinder the evolution of resistance to Bti both in the laboratory and in the field (Wirth et al., 2005). Two main mechanisms of resistance to Cry toxins Infection, Genetics and Evolution 44 (2016) 218–227 Abbreviations: ALP, alkaline phosphatase; APN, aminopeptidase N; ABC transporters, ATP-binding cassette transporter; Bti, Bacillus thuringiensis israelensis; SNP, Single Nucleotide Polymorphism. ⁎ Corresponding author at: Laboratoire d'Ecologie Alpine, 2233 rue de la piscine, CS 40700 - 38058 Grenoble Cedex 9, France. E-mail address: Laurence.despres@ujf-grenoble.fr (L. Després). 1 Present address: Univ. Perpignan Via Domitia, IHPE UMR 5244, CNRS, IFREMER, Univ. Montpellier, F-66,860 Perpignan, France. http://dx.doi.org/10.1016/j.meegid.2016.07.009 1567-1348/© 2016 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Infection, Genetics and Evolution journal homepage: www.elsevier.com/locate/meegid