The Peptide Venom Composition of the Fierce Stinging Ant Tetraponera aethiops (Formicidae: Pseudomyrmecinae)

Abstract

In the mutualisms involving certain pseudomyrmicine ants and different myrmecophytes (i.e., plants sheltering colonies of specialized "plant-ant" species in hollow structures), the ant venom contributes to the host plant biotic defenses by inducing the rapid paralysis of defoliating insects and causing intense pain to browsing mammals. Using integrated transcriptomic and proteomic approaches, we identified the venom peptidome of the plant-ant Tetraponera aethiops (Pseudomyrmecinae). The transcriptomic analysis of its venom glands revealed that 40% of the expressed contigs encoded only seven peptide precursors related to the ant venom peptides from the A-superfamily. Among the 12 peptide masses detected by liquid chromatography-mass spectrometry (LC-MS), nine mature peptide sequences were characterized and confirmed through proteomic analysis. These venom peptides, called pseudomyrmecitoxins (PSDTX), share amino acid sequence identities with myrmeciitoxins known for their dual offensive and defensive functions on both insects and mammals. Furthermore, we demonstrated through reduction/alkylation of the crude venom that four PSDTXs were homo- and heterodimeric. Thus, we provide the first insights into the defensive venom composition of the ant genus Tetraponera indicative of a streamlined peptidome.

Keywords: Tetraponera aethiops; defensive venom; dimeric peptides; peptidome.

Figure 1

Positive mode total ion chromatogram (TIC) of T. aethiops venom using LCQ Advantage ESI mass spectrometer. Crude venom was separated by C₁₈ RP-HPLC using an ACN gradient. The mobile phase was 0.1% aqueous formic acid (solvent A) and 0.1% formic acid in acetonitrile (solvent B). The peptides were eluted using a linear gradient from 0 to 50% of solvent B during 45 min, then from 50 to 100% during 10 min, and finally held for 5 min at a 250 µL min⁻¹ flow rate. Note that ‘U₄-PSDTX-Ta1a’ accounts for 66.41% of the venom peptide content.

Figure 2

Proportions of addressed functions of the 230 most expressed transcripts (≥100,000 hits) from T. aethiops venom glands. Functional annotations were made with the NCBI blastp program. The category “Others” groups functions involved in cellular metabolism. Contigs coding for venom peptides accounted for 40% of the transcripts expressed by the venom glands. Seventeen percent of the transcripts coded for venom proteins such as venom allergens and phospholipases, and 3% of the transcripts were dedicated to protein maturation. The contigs names and functions are presented in detail in Supplementary Data (Table S1).

Figure 3

Alignment of consensus prepro sequences from pilosulin-like ant venom peptides. The alignment was generated with the Muscle program in Seaview version 4.6.1 and edited using BOXSHADE version 3.2. Identical residues are highlighted in magenta. Similar residues are highlighted in blue. The consensus sequence of the prepro-region of Tetraponera aethiops venom peptides shared 43%, 35%, 52%, and 48% with the consensus prepro-regions sequences obtained from previous studies on Odontomachus monticola [26], Myrmecia pilosula [27], Myrmecia gulosa [28], and Tetramorium bicarinatum (Superfamily A) [29], respectively.

Figure 4

Alignments of T. aethiops venom peptide precursors with pilosuline-like peptides from Odontomachus monticola [26], Myrmecia gulosa [28], Myrmecia pilosula [27], and Tetramorium bicarinatum (Superfamily A) [29] venoms. Alignments were generated with the Muscle program in Seaview version 4.6.1 and edited using BOXSHADE version 3.2. The prepro- and mature regions were aligned separately. Identical residues are highlighted in magenta and similar residues are highlighted in blue. The black triangle indicates the cleavage site between the prepro-regions and the mature peptides. The black line marks the signal regions. Post-translational modifications are not shown. The prepro-regions showed themselves to be conserved, whereas mature peptide sequences were highly variable. Tetraponera aethiops venom peptide precursor cDNA sequences were submitted to GenBank, with the following accession numbers: U₁-PSDTX-Ta1a (MN607166), U₂-PSDTX-Ta1a (MN607169), U₂-PSDTX-Ta1b (MN607170), U₂-PSDTX-Ta1c (MN607168), U₃-PSDTX-Ta1a (MN607165), U₄-PSDTX-Ta1a (MN607167), and U₅-PSDTX-Ta1a (MN607171).

Figure 5

Identification of the dimeric features of the Ta-5680 peptides in T. aethiops venom. (A) Extracted-ion chromatogram and MS spectrum of the peptide Ta-5680 from the LC–MS analysis of T. aethiops venom before reduction/alkylation. We hypothesized that Ta-5680 is a heterodimeric peptide having the hypothetic sequence shown on the right. The distinctive residues between both monomers are highlighted in grey and the red bar represents the disulfide bond. (B) Comparison of chromatograms and spectra before and after reduction with DTT (Dithiothreitol) revealed the presence of two novel masses (2875.47 and 2805.39 Da) corresponding to both A and B chains while the Ta-5680 mass disappeared. (C) Alkylation experiment using IA (iodoacetamide) confirmed the presence of one cysteine on each alkylated monomer.

Figure 6

Of Tetraponera aethiops mature venom peptides with similar venom peptides from ant venoms [28,29,34,36]. Alignments were generated with the Muscle program in Seaview version 4.6.1 and edited using BOXSHADE version 3.2. Conserved residues are highlighted in cyan, identical residues are highlighted in magenta and similar residues are highlighted in blue.