Immunolocalization of cuticular proteins in Johnston's organ and the corneal lens of Anopheles gambiae

Abstract

Previous work with EM immunolocalization examined the intracuticular placement of several antibodies directed against cuticular proteins (CPs) in various structures of Anopheles gambiae. Those structures had long stretches of fairly uniform cuticle. We have now used 19 antibodies directed against members of five CP families on two adult structures with considerable complexity, Johnston's organ and the corneal lens of the compound eye. We also localized chitin with colloidal-gold labeled wheat germ agglutinin. Twelve of these antibodies recognized structures in Johnston's organ. Only 6 were detected in the outer pedicel wall, but the internal structures were more complex with distinct distributions of members of the five CP families in six different structures. The corneal lens had four distinct regions of laminar cuticle. Thirteen of the 15 members of the CPR family were detected, none from the other CP families. Specific antibodies were localized to different regions and in different laminae within a region. The specificity of deployment of cuticular proteins revealed in this study is helping to explain why An. gambiae allocates about 2% of its protein coding genes to structural CPs.

Contributions of cuticle permeability and enzyme detoxification to pyrethroid resistance in the major malaria vector Anopheles gambiae

“…Fluorescence in situ hybridization would allow overexpressed proteins from cuticle genes to be localised to make sure they belonged to at least one of three cuticle layers. None of the cuticular proteins necessarily belongs to the cuticle since some of them have been found in Johnston’s organ and the corneal lens of Anopheles gambiae using EM immunolocalization 62 . In addition, some cuticle proteins with chitin-binding domain 2 belong to the procuticle and also to the midgut chitinous peritrophic matrix in Drosophila melanogaster 63 .…”

Culex pipiens pallens cuticular protein CPLCG5 participates in pyrethroid resistance by forming a rigid matrix

“…Sclerotisation of the cuticle is a primary consequence of the formation of cross-links between cuticle proteins, resulting in a rigid matrix in which chitin microfibrils can be embedded. Additionally, it has been demonstrated that cuticular proteins with low-complexity sequences play an integral role as cross-linked structural proteins in the formation of lightweight rigid cuticles [ 46 ]. The CPLCG family was first identified in A. gambiae by proteomic analysis of cuticle preparations, and members of the CPLCG family contain low-complexity regions (LCRs) [ 25 ].…”

Proteomics reveals localization of cuticular proteins in Anopheles gambiae

“…Those eliminated are shown with brackets around the U or S in the green colored cells on Table 1, leaving only 14 as larval-restricted. Furthermore, our EM immunolocalization studies revealed that antibodies raised against CPR22, CPR61, and CPR133/CPR153 reacted with proteins in adult structures (Vannini and Willis, 2016, 2017), revealing that they are not restricted to larvae, although this information was not used in our numerical summaries. We failed to detect any CPs that might be restricted to pupae.…”

“…One protein, CPR152, does appear to be unique in that by in situ hybridization it was restricted to Johnston’s organ and EM immunolocalization revealed it to be localized in several, but not all, discrete components of that structure (Vannini and Willis, 2016). We also recovered peptides for CPR152 from the rest of the antennae but nowhere else.…”

“…These brackets in larval columns indicate that the protein was not restricted to larvae based on Masson et al * Antenna includes Johnston’s organ (JO). They were analyzed separately in Zhou et al 2016 and JO not analyzed in Masson et al, 2018. Data for whole adults from Zhou et al., 2017 S6_Table. Gray highlighting in the” Up-to-date Summary” column indicates the 24 proteins identified by us for the first time in this study. Gray highlighting in cells in the “whole adults” column indicates that the protein was identified only in adults in this and other studies. Bolded “no” in Up-to-date summary column indicates that protein had no peptides in Mastrobuoni et al, 2013 or Masson et al, 2018 and/or on VectorBase as of5/16/18. V&W indicates that even though no peptides were detected, antibodies raised against a peptide in that protein or sequence cluster were found in that structure by EM immunolocalization (Vannini and Willis, 2016). A no in square brackets, means that trypsin peptides were unlikely.…”