Integrated proteomic and transcriptomic analysis of the Aedes aegypti eggshell

Abstract

Background: Mosquito eggshells show remarkable diversity in physical properties and structure consistent with adaptations to the wide variety of environments exploited by these insects. We applied proteomic, transcriptomic, and hybridization in situ techniques to identify gene products and pathways that participate in the assembly of the Aedes aegypti eggshell. Aedes aegypti population density is low during cold and dry seasons and increases immediately after rainfall. The survival of embryos through unfavorable periods is a key factor in the persistence of their populations. The work described here supports integrated vector control approaches that target eggshell formation and result in Ae. aegypti drought-intolerant phenotypes for public health initiatives directed to reduce mosquito-borne diseases.

Results: A total of 130 proteins were identified from the combined mass spectrometric analyses of eggshell preparations.

Conclusions: Classification of proteins according to their known and putative functions revealed the complexity of the eggshell structure. Three novel Ae. aegypti vitelline membrane proteins were discovered. Odorant-binding and cysteine-rich proteins that may be structural components of the eggshell were identified. Enzymes with peroxidase, laccase and phenoloxidase activities also were identified, and their likely involvements in cross-linking reactions that stabilize the eggshell structure are discussed.

Figure 1

Aedes aegypti eggshell purification. A) Eggshells purified by differential sedimentation from ovaries dissected at 72 hPBM and visualized by light microscopy. Top image, ovary dissected at 72 hPBM. Bottom images, eggshells purified by differential sedimentation. B) Ovary (Ov) and eggshell (ES) protein preparations dissolved in SDS/β-mercaptoethanol buffer resolved using 4–15% SDS-PAGE and stained with Coomassie blue R. The electrophoretic migrations of molecular weight markers (M - Precision Plus Protein Kaleidoscope, Bio-Rad Laboratories) and their corresponding molecular weights are indicated at the left of the figure. The excised gel slices submitted to mass spectrometry are indicated by letters A-J, and the identified proteins are listed in Additional file 1. Vitellin subunits are indicated with asterisks.

Figure 2

Hybridizations in situ of four transcripts encoding Ae. aegypti eggshell proteins. AaegOBP45 (AAEL01714-RA), AaegOBP31 (AAEL006396-RA), AaegOBP29 (AAEL006387-RA), chitin-binding protein (AAEL007112-RA). All anti-sense probes hybridize to the follicle cells while the sense probe does not. The clear, stain-free, elliptical shapes indicate the positions of the follicular cell nuclei.

Figure 3

Functional annotations and expression profiles of Aedes aegypti genes associated with eggshell formation. Expression profiles are derived from qRT-PCR (this publication), microarray [25] and RNAseq data [26]. A) Schematic representation of the sequential expression of genes belonging to distinct functional categories: VMPs, vitelline membrane proteins; CBPs, chitin binding proteins/chitinases; OBP, odorant binding proteins; CRP, cysteine-rich proteins. Panels B, C, D, E and F display lists of annotations and expression profiles for VMPs, CBPs, OBPs, CRPs and enzymes involved in eggshell formation. Selected genes (underlined) had their spatial and temporal expression profiles validated by hybridization in situ (indicated by Δ; Figure 2) or quantitative RT-PCR (indicated by ●; Additional file 5). Expression values indicated by ‘++’ and ‘+’ refer to higher and lower relative abundance, respectively, with blanks representing negligible or undetectable levels.

Figure 4

Conservation of a hydrophobic amino acid sequence motif among Aedes aegypti vitelline membrane proteins. The conserved motif is composed of a sequence 30 amino acids in length. Conserved motif discovery was performed using MEME [65]http://meme.nbcr.net/meme/cgi-bin/meme.cgi.

Figure 5

Identification and alignment and of a motif 59 amino acids in length that is conserved among the Aedes aegypti eggshell cysteine-rich proteins. Conserved motif discovery was performed using MEME [65]http://meme.nbcr.net/meme/cgi-bin/meme.cgi.