Transcriptome Analysis in Venom Gland of the Predatory Giant Ant Dinoponera quadriceps: Insights into the Polypeptide Toxin Arsenal of Hymenopterans

Alba F. C. Torres; Chen Huang; Cheong-Meng Chong; Siu Wai Leung; Álvaro R. B. Prieto-da-Silva; Alexandre Havt; Yves P. Quinet; Alice M. C. Martins; Simon M. Y. Lee; Gandhi Rádis-Baptista

doi:10.1371/journal.pone.0087556

Abstract

Background

Dinoponera quadriceps is a predatory giant ant that inhabits the Neotropical region and subdues its prey (insects) with stings that deliver a toxic cocktail of molecules. Human accidents occasionally occur and cause local pain and systemic symptoms. A comprehensive study of the D. quadriceps venom gland transcriptome is required to advance our knowledge about the toxin repertoire of the giant ant venom and to understand the physiopathological basis of Hymenoptera envenomation.

Results

We conducted a transcriptome analysis of a cDNA library from the D. quadriceps venom gland with Sanger sequencing in combination with whole-transcriptome shotgun deep sequencing. From the cDNA library, a total of 420 independent clones were analyzed. Although the proportion of dinoponeratoxin isoform precursors was high, the first giant ant venom inhibitor cysteine-knot (ICK) toxin was found. The deep next generation sequencing yielded a total of 2,514,767 raw reads that were assembled into 18,546 contigs. A BLAST search of the assembled contigs against non-redundant and Swiss-Prot databases showed that 6,463 contigs corresponded to BLASTx hits and indicated an interesting diversity of transcripts related to venom gene expression. The majority of these venom-related sequences code for a major polypeptide core, which comprises venom allergens, lethal-like proteins and esterases, and a minor peptide framework composed of inter-specific structurally conserved cysteine-rich toxins. Both the cDNA library and deep sequencing yielded large proportions of contigs that showed no similarities with known sequences.

Conclusions

To our knowledge, this is the first report of the venom gland transcriptome of the New World giant ant D. quadriceps. The glandular venom system was dissected, and the toxin arsenal was revealed; this process brought to light novel sequences that included an ICK-folded toxins, allergen proteins, esterases (phospholipases and carboxylesterases), and lethal-like toxins. These findings contribute to the understanding of the ecology, behavior and venomics of hymenopterans.

Citation: Torres AFC, Huang C, Chong C-M, Leung SW, Prieto-da-Silva ÁRB, Havt A, et al. (2014) Transcriptome Analysis in Venom Gland of the Predatory Giant Ant Dinoponera quadriceps: Insights into the Polypeptide Toxin Arsenal of Hymenopterans. PLoS ONE 9(1): e87556. https://doi.org/10.1371/journal.pone.0087556

Editor: Paulo Lee Ho, Instituto Butantan, Brazil

Received: September 19, 2013; Accepted: December 23, 2013; Published: January 31, 2014

Copyright: © 2014 Torres et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: The giant ant project was sponsored by the program for funding new research groups of The National Council for Scientific and Technological Development (CNPq), Ministry of Science and Technology, Federal Government of Brazil (program PRONEM/FUNCAP/CNPq, Ref. numbers PRN-0040-00067.01.00/10 and SPU 10582724-0). This study was partly supported by grants from the Science and Technology Development Fund of Macau, China (Ref. no. from 045/2007 to 078/2011/A3). GR-B and ARBPS are grateful to CNPq and the Coordination for the Improvement of Higher Education Personnel (CAPES) Program on Toxinology (Issued 2010), Ministry of Education of the Federal Government of Brazil for partial financial support for this work. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

In recent years, the order Hymenoptera, which comprises numerous species of bees, wasps and ants, has received increasing attention because of their direct and indirect influences on human health, ecological balance, agriculture and the forestry economy. The importance of investigating hymenopterans is well-known; thus, information from diverse studies has culminated with the establishment of a genome database (http://hymenopteragenome.org). Presently, this database maintains scientific information about one species of bee (Apis mellifera), two species of bumblebees (Bombus terrestris and B. impatiens), a parasitoid wasp (Nasonia vitripennis), and seven species of ants [1]. The seven ant species detailed in this database include the fungus-growing (leaf cutter) ants Acromyrmex echinatior and Atta cephalotes, the Florida carpenter ant Camponotus floridanus, Jerdon's jumping ant Harpegnathos saltator, the Argentine ant Linepithema humile, the red harvester ant Pogonomyrmex barbatus, and the fire ant Solenopsis invicta (http://hymenopteragenome.org/ant_genomes/).

From the perspective of molecular toxinology and venomics, scientific interest and public concerns about animal venoms have sharply increased due the vast diversity of bioactive compounds, the safety hazards, and the potential therapeutic uses of isolated compounds [2]. Hymenopterans offer a myriad of biologically active molecules that incorporate the physiopathological bases of the causes of ant venom allergies and envenomation and the traditional and modern uses of ants for medicines and in programs of drug discovery. For example, in countries of East Asia, Africa and South America, ant venoms have been used to treat rheumatoid arthritis [3]. Badr and collaborators [4] demonstrated that the crude venom from Pachycondyla senaarensis induces death by apoptosis in breast carcinoma cells. The same venom has been proven to display anti-inflammatory and anti-oxidant properties [5]. Additionally, the venom of Chinese medicinal ant Polyrhachis lammellidens elicits analgesic and anti-inflammatory effects [6]. Peptides from Pachycondyla goeldii and Myrmecia sp. have been demonstrated to be effective against Gram-negative and Gram-positive bacteria, including the methicilin-resistant Staphyloccocus aureus and the pathogenic fungus Candida albicans [7], [8]. Moreover, the antimicrobial effects of crude venom from Crematogaster pygmaea, which is composed of electrophilic aldehydes, have recently been demonstrated [9].

Our group has been investigating the biological effects of D. quadriceps crude venom in different experimental models. In a system of isolated perfused rat kidney, D. quadriceps venom triggers increases in urinary flow, decreases in vascular resistance, and reductions in sodium tubular transport; these findings demonstrate the natriuretic and diuretic-induction effects of this crude venom [10]. In pentylenetetrazol-induced seizure models, this giant ant venom exhibits neuroprotective activities that depend on the route of administration [11], [12]. Additionally, models of antinociception, including those evoked by formalin, writhing, von Frey fibers and hot plates, the analgesic effects of D. quadriceps crude venom have been verified to be efficacious [13].

The genus Dinoponera (Hymenoptera: Vespoidea: Formicidae) belongs to the subfamily Ponerinae and comprises the following eight species with strict Neotropical distributions [14]–[16]: D. australis, D. gigantea, D. hispida, D. longipes, D. lucida, D. mutica, D. quadriceps, and D. snellingi. Collectively, these species form a group of primitive poneromorphs ants with body sizes ranging from 3 to 4 cm, which places them among the largest ants in the world [17], [18]. Along with some other ponerine genera, Dinoponera ants have queenless colonies, and reproductive functions are performed by one mated dominant worker called a gamergate [19]. In Brazil, Dinoponera ants, popularly called “falsas tocandiras”, exemplify the genera with strict New World distributions. The giant ant D. quadriceps preferentially inhabits the Brazilian northeast and can be found in a variety of vegetation types including caatinga (a xerophyllous vegetation), cerrado (a savannah-like vegetation), brejos de altitude (humid mountain forests) and Atlantic forest. D. quadriceps nests (Fig. 1-A) have an average population of 60 workers, one or two entrances, a vertical gallery that is between 30 to 120 cm deep, and up to 16 chambers [15], [20]. As in all Aculeata hymenopterans for which anatomical observations exist, the venom gland of Dinoponera is formed by free tubules, a convoluted gland and a reservoir (venom sac) (Fig. 1-B) [21], [22]. The free tubules and convoluted gland are involved in venom production, and the venom is a fluid that is rich in polypeptides and characteristic of the stinging ants that belong to the subfamilies Myrmicinae, Ponerinae and Pseudomyrmecinae [23].

Download:

Figure 1. Dinoponera quadriceps in the field and its dissected venom apparatus.

Part A - A single specimen of D. quadriceps protecting the nest's entrance. Part B - Dissected D. quadriceps venom apparatus (×40). Abbreviations: Dg - Doffur's gland; cv - convoluted gland (not observable); st - secretory tubule; vs- venom sac; sg – sting.

https://doi.org/10.1371/journal.pone.0087556.g001

Dinoponera ants preferentially prey on insects and small arthropods, but inevitable interactions with humans occur, and, upon venom injection, the location of the sting becomes extremely painful. Additionally, the stings may have systemic manifestations such as sweating, nausea, vomiting, fever, lymphadenopathy, cardiac arrhythmias and tissue damage [24]–[27]. These symptoms are mostly attributable to a cocktail of low molecular weight organic compounds and pharmacologically active polypeptides that is present in the venom. Indeed, regarding the polypeptide core of the venom, short peptides isolated from Paraponera clavata venom (i.e., poneratoxins (PoTx)) have been proven to prolong the action of sodium channels and interfere with neurotransmission [28], [29]. To identify individual venom peptides and proteins in the venom of Dinoponera australis, Johnson and colleagues [30] conducted a proteomic survey via high performance liquid chromatography coupled with mass-spectrometry (HPLC/DAD/MS). However, only a limited number of peptides and related partial protein fragments were fully characterized. Consequently, a comprehensive description of the toxic polypeptide repertoire of the venom of the Dinoponera giant ant does not yet exist at either the genus or species level. Moreover, despite the large amounts of information about the compositions of the venoms of some species of hymenopterans of the family Formicidae that are available, information about the effects of the components of the venoms on biological systems and the potential uses of the individual polypeptides to manipulate cell biology are still scarce and deserving of deeper investigation. Given this context and the ecological and pharmacological interest in D. quadriceps venomics, in this study we report on the transcriptome of the giant ant venom gland and present new data that should further our comprehension of the evolution of the toxin arsenal of Hymenoptera.

Results

Sanger sequencing and cluster assemblies

A full-length cDNA library was constructed with approximately 20 venom glands from female (wingless) D. quadriceps. A cloning efficiency of 1×10⁵ cfu/µg DNA was achieved, and the average insert size was 563 bp. A total of 420 individual randomly selected clones were sequenced by with the dideoxy chain-termination (Sanger) method. Nucleotide sequences with greater base calling values were processed via biocomputation (CLC Main Workbench v. 6.8.4, CLC Bio, U.S.A.). The high-quality trimmed ESTs generated an assemblage of 22 contigs and 44 singletons. As summarized in figure 2, the comparison of translated sequences with non-redundant protein databanks using BlastX analysis allowed for the classification and identification the following clusters (Fig. 2-A): (1) proteins involved in general metabolism (56% of contigs and singlets; e.g., transferases, ATP synthase, dehydrogenases, ribosomal proteins, and cytocrome c); (2) venom-linked polypeptide gene sequences (15%) that highlighted the predominant expression of dinoponeratoxins, allergen peptides, and an ICK motif-containing toxin; (3) cDNA precursor sequences that yielded no hits (20%), and (4) ESTs that represented hypothetical proteins with unknown function (5%). Comparisons of the amino acid and nucleotide sequences of the hypothetical proteins with a specific arthropod database revealed that the ESTs of D. quadriceps shared homology with polypeptides from scorpions and others ants, including H. saltator, S. invicta and S. saevissima (Fig. 2-B; Table S1).

Download:

Figure 2. Overall classification of ESTs from the D. quadriceps venom gland cDNA (ESTs) library based on function.

EST sequences were annotated by comparing contigs and singlets (E-values≤1.0E-5) with the non-redundant Genbank translated protein database using BLASTX. Search stringency was eventually adjusted (E-values lower than 1.0E-5) and database restricted to hymenopterans with the aim of finding relevant familial- and structural-related sequences of hymenopterans toxins. Part A – relative number of polypeptides which were counted as individual random sequenced clones. The group of <cellular process> refers to enzymes and polypeptides involved in the venom gland metabolism. Part B – relative numbers of venom-related sequences found in the overrepresented small toxin dataset. Under the term <toxin> is included dinoponeratoxins, venom allergens, and venom cysteine rich peptides (part B).

https://doi.org/10.1371/journal.pone.0087556.g002

Ion Torrent deep RNA sequencing and read assembly

Next generation RNA-sequencing of the same D. quadriceps venom gland starting material that was used to construct the cDNA library generated a total of 2,514,767 raw reads (average read length = 230 bps). The sequencing data were subjected in-depth bioinformatics analyses. Initially, the raw reads were filtered by an in-house Perl script to remove low quality reads, which resulted in a total of 18,546 assembled transcripts with contig sizes ranging from 101 to 8114 bp, and an average transcript length of 232.09 bp (Table S2). The size distribution of the assembled transcripts is shown in Figure S1. The processed contigs were annotated by querying the non-redundant (nr) protein database via transcript sequence comparison using the BLASTx algorithm, and 6429 contigs with high gene homology were retrieved (Table 1 and Fig. 3). The majority of D. quadriceps transcript sequences (3807 contigs) were matched to those of another ant species (H. saltator), which reflects their close phylogenetic relationship of these species. Interestingly, some contigs showed high sequence similarity with Hymenoptera species such as A. florae and Megachile rotundata (see Fig. 4 for details). Nearly all (∼98%) EST sequences retrieved from cDNA library matched and strongly supported the assembled contigs that were obtained by next generation RNA-sequencing. Based on non-redundant annotation information, these RNA-derived contigs were then categorized into protein families involved in, for example, cellular processes, Hymenoptera venom-related polypeptides and ribosomal proteins (Fig. 3). This Transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/GenBank under the accession GANS00000000. The version described in this paper is the first version, GANS01000000.

Download:

Figure 3. General overview of the transcripts in the D. quadriceps venom gland identified by deep RNA sequencing.

Annotation of contigs from the RNA-Seq assembly of the giant ant venom following the conventions of figure 2; i.e., the E-value cut-off was at least 1.0E-5 for BLASTX functional comparison.

https://doi.org/10.1371/journal.pone.0087556.g003

Download:

Figure 4. Distribution of the Hymenoptera species as determined by best protein hits.

The percentages of homologs from distinct hymenopterans species with which the 6,429 contigs of the D. quadriceps venom gland shared the high sequence similarities.

https://doi.org/10.1371/journal.pone.0087556.g004

Download:

Table 1. Summary of assembled contigs derived from RNA sequencing and comparison with the ESTs of the D. quadriceps venom gland transcriptome.

https://doi.org/10.1371/journal.pone.0087556.t001

Annotation of transcript clusters related to D. quadriceps venomics

From the RNA-sequencing data, Gene Ontology (GO) assignments were first used to classify the functions of these assembled transcripts. Based on sequence homology, 3024 transcript sequences were assigned at least one GO term, including 53 functional groups at the second level (Figure S2). Additionally, all assembled transcripts were compared against the Cluster of Orthologous Groups (COG) database to predict possible functions. The results revealed 1592 sequences with significant homology and assigned them into the appropriate clusters. These COG classifications were grouped into 23 functional categories (Figure S3, A to Z). Furthermore, according to the species and protein annotation information of the nr database, the transcripts related to toxins and venom components were grouped into the following four main categories: dinoponeratoxins, venom allergens, phospholipase-like toxin peptides and lethal-like proteins (Fig. 5). Again, as revealed by the cDNA library, there was a high preponderance of dinoponeratoxin transcripts. Venom phospholipase-related toxins and lethal-like proteins corresponded to the largest proportion of contigs. Specifically, the predominant encoded proteins found in the venom gland of D. quadriceps were the venom allergen 1 (Sol i 1) and venom allergen 3 (Sol i 3) antigens. Multiple alignments with sequences of other Hymenoptera species revealed several unique amino-acid sequence regions in Sol i 1 and Sol i 3 of D. quadriceps (Fig. 6, Fig. 7). The Sol i 3 protein sequences that was orthologous to those of other hymenoptera species, especially bees, were subjected to phylogenetic analyses (Fig. 8). As expected, the phylogenetic tree showed that D. australis was more closely related to S. invicta (the red imported fire ant) than other bee species. We found four contig sequences that coded for dinoponeratoxins and were homologous to the same toxin class of another ponerine ant species; i.e., D. australis. Multiple alignments of the peptide sequences from D. quadriceps and the D. australis dinoponeratoxins (Fig. 9) showed that these toxin peptides are highly conserved but also exhibited several amino acid substitutions that reflect the species-specific diversification of this toxin.

Download:

Figure 5. Proportions of transcript toxin and toxin-related components expressed in the venom gland of D. quadriceps.

Toxins and venom-related protein precursors represented less than 1% of all transcripts (18,524) in the giant ant venome. As shown, the major toxin core was composed of four groups of polypeptides.

https://doi.org/10.1371/journal.pone.0087556.g005

Download:

Figure 6. Multiple alignments of the deduced amino acid sequences of venom allergen I (Sol i 1) from D. quadriceps with known sequences from different ant and wasp species.

The identical and conserved amino acid residues of diverse ant and wasp hymenopterans are highlighted in black and gray, respectively. Dots represent gaps.

https://doi.org/10.1371/journal.pone.0087556.g006

Download:

Figure 7. Multiple alignments of the deduced amino acid sequences of the venom allergen antigen 5 (Sol i 3) of D. quadriceps with known sequences from different Hymenoptera species.

The conserved regions of the Sol i 3/allergen antigen 5 (Ag 5) polypeptide sequences of D. quadriceps and other hymenopterans are presented in black boxes. Conserved amino acid residue mutations are highlight in light gray boxes. Dots represent gaps. The unique amino acid sequences of the D. quadriceps allergen homologous to Sol i 3 protein are displayed in open boxes.

https://doi.org/10.1371/journal.pone.0087556.g007

Download:

Figure 8. Comparison of Sol i 3/Allergen Antigen 5 polypeptides from D. quadriceps with their hymenopteran orthologs.

Phylogenetic tree based on neighbor-joining analyses of a concatenated alignment of allergen 5/Sol i 3 and the orthology relationships of multiple insects. The scale bar indicates 0.05 substitutions per site. Vespula maculifrons, Vespula vulgaris, Vespula germanica, Vespula squamosal, Vespa crabro, Dolichovespula maculate, Polybia paulista, Polistes dominulus, Polistes annularis, and Solenopsis invicta.

https://doi.org/10.1371/journal.pone.0087556.g008

Download:

Figure 9. Multiple alignments of deduced amino acid sequences of different identified dinoponeratoxins from the D. quadriceps venom gland transcriptome and D. australis.

Deduced D. quadriceps dinoponeratoxin cDNA precursor sequences (contig_1 and contig_9). RNA deep sequencing contigs (contig_2 and contig_145) were compared to mature peptide sequences from D. australis (Da-3177 and Da-3105) (part A) and from another species of Dinoponera (Da-2501) (part B). ClustalW was used to multi-align the sequences. Identical amino acid residues are marked with asterisks. Stretches of deduced amino acid sequences supported by EST sequences are boxed. Signal peptides (pre-peptides) are doubled underlined, and pro-regions of pro-peptides are shown with a single line under the sequence. Contigs 1 and 9 were first identified in the EST library, and contigs 2 and 145 came primarily from RNA-Seq.

https://doi.org/10.1371/journal.pone.0087556.g009

Contigs that were not related to any known sequences in the nr database accounted for 65% of the total transcripts. This high percentage of “no match” contigs was not surprising because the E-value used in the BLAST search was stringent to ensure the reliability of the alignment results. Additionally, the numbers of annotated sequences related to venom from other ant species that are available in the nr database are limited. Moreover, de novo RNA-Seq assembly is a complicated process and probably resulted in some sequencing errors, repetitive sequences and aberrant amplicons that brought several “acceptable” incorrect assembled sequence fragments together. Nevertheless, these “no-match” contigs might represent mRNA transcripts that encode novel genes. To further identify and characterize the contigs that had no match in the nr database, these contigs were subjected to a blastx search against the UniProtKB database (2012). This analysis resulted in 34 best hit contigs. For the other “no match” contigs, we performed a BLASTn search against 411 ESTs obtained from the Sanger sequencing of the cDNA library of the venomous gland of D. quadriceps, and six contigs were well mapped by numerous ESTs from the venom gland of D. quadriceps's (Table S4). Additionally, ORF prediction was implemented with the OrfPredictor server, which was designed for ORF prediction and translation of batches of EST or cDNA sequences [31]; then, signal peptides were analyzed with PrediSi [32]. These seven contigs were novel candidate venom encoding transcripts in D. quadriceps because they were dissimilar proteins that did not match with any other sequences deposited the nr or UniProtKB databases.

Clusters related to proteins involved in venom gland physiology

The results of deep RNA-sequencing showed that the venom gland transcriptome was composed of a large proportion of housekeeping genes, such as ribosomal proteins, cellular process-related proteins and hypothetical proteins, but the proportion of venom proteins was small (Fig. 3). Additionally, we identified two sex determination proteins in the venom transcriptome that could be used to predict the phylogenetic relationships of selected ant and bee species. Our phylogenetic analyses indicated that H. saltator likely shares the closest parental species with the giant ant D. quadriceps and suggests these species might have evolved from a common ancestor (Fig. S4).

Inhibitor cystine knot- (ICK-) toxin transcripts in the venom gland of D. quadriceps

A deduced amino acid sequence encoded by contig 05 (Table S1) has a putative signal peptide of 23 amino acids followed by a mature core toxin of 30 amino acids that includes six cysteine residues. A pro-piece (pro-peptide) was absent. As was inferred by similarity comparisons, the cysteine residues impair the structural configuration of the so-called “disulfide through disulfide knot” and allow for the grouping of this peptide into the class of knottins (Fig. 10-A and -B). The Dinoponera ICK-like toxin precursor contains a cysteine framework that is typified by VI/VII; i.e., -C-C-CC-C-C-, which is characteristic of the ω-toxin super-family (PFAM Clan CL0083) that encompasses several type-1 conotoxins, funnel web spider toxins and assassin bug toxins, among others. In figure 10-C, ICK-like toxins from D. quadriceps, Conus species, and spiders are aligned for primary structure comparison. Another type of ICK-folded toxin transcript found in the venom of the giant ant was similar to the so-called cysteine-rich venom proteins that share structural similarities with the toxins in the venoms of distinct species of animals (e.g., contig 1144, table S3).

Download:

Figure 10. The Inhibitor cystine knot- (ICK-) toxin from D. quadriceps venom.

A) Dinoponera ICK-like tridimensional homology model from contig 05 (Table S1) mature peptide (yellow – β-sheet; green – cysteine; ted – 3/10-helix. N-terminus (N); C-terminus (C). The numbers indicate the cysteine positions in the Dinoponera sequence. The presence of a ‘disulfide through disulfide knot’ structurally defines this peptide as a knottin. B) Schematic representation of a knottin obtained when one disulfide bridge crosses the macrocycle formed by two other disulfides, and the interconnecting backbone (disulfide III–VI) goes through disulfides I–IV and II–V. C) Alignments of the Dinoponera ICK-like mature peptide with the tarantula and Conus snail sequences of the templates used in the homology model showing the highly conserved cysteine framework [C-C-CC-C-C] that is characteristic of the omega-toxin-like family. TXFK1_PSACA: psalmopeotoxin I (PcFK1) from the venom of the tarantula Psalmopoeus cambridgei (NMR structure 1X5V; UNIPROT Accession P0C201). CO16B_CONMR: Mu-Conotoxin MrVIB from conus snail Conus marmoreus (NMR structure 1RMK; UNIPROT Accession Q26443).

https://doi.org/10.1371/journal.pone.0087556.g010

Discussion

Ants play significant ecological roles not only in diverse terrestrial environments, particularly in the tropical regions of the planet, but also in extreme biomes. Ants are engaged in several beneficial interrelationships with other organisms that include pollination, microbial dispersal, insect predation, and symbiotic associations with microorganisms [33]. In agriculture, ants modulate and interfere with soil conditions and may cause economic losses due to several factors including damage to the foliage of commercial plants, direct and indirect reductions in crop yields and pest management-related increases in the costs production [34]. With over 12,000 known species, the ants are among the most successful insects in the animal kingdom, and many of these species depend on venom for defense against other organisms, including predators and other ant species [35].

The genus Dinoponera inhabits the tropical regions of the New World, and its nests are located in diverse Brazilian biomes including semi-arid and savannah-like vegetation and humid and rain forests. D. quadriceps preys exclusively on insects and is normally harmless to humans except when they are disturbed or inadvertently encountered; the resultant interactions may result in accidents of varying levels of severity. Therefore, knowledge of venom composition is desirable not only to comprehend the pathophysiological basis of the effects and symptoms elicited by the sting of D. quadriceps but also to characterize proteins and peptides that will potentially be useful in the diagnosis and prospective treatment of chronic human diseases [5], [36]. The biotechnological applications of ant venom include not only the development of pharmaceuticals but also the development of bio-insecticides; the latter benefit is exemplified by poneratoxin, a small 25-residue neuropeptide that is active at the Na+- and K⁺-channels of invertebrates and was originally isolated in the venom of Paroponera clavata [29].

A robust strategy for the study of the venom profiles of small poisonous creatures with tiny venom glands relies on molecular cloning and DNA/RNA sequencing [37]. Thus, we prepared a D. quadriceps venom gland cDNA library and investigated the toxin repertoire of cloned transcribed genes by Sanger and RNA deep sequencing. Hundreds randomly picked clones from the Sanger sequencing are presented in figure 2 and table S1. Regarding the contigs and singlets datasets, proteins classified as “no hits” accounted for ∼20% of all ESTs, and dinoponeratoxins accounted for about 5% of all sequences categorized as toxins (Fig. 2-A). In terms of the absolute numbers of individual clones, over 37% of all retrieved random picked sequences were dinoponeratoxins (130 clones out 420) and over 50% of all venom-related sequences (Fig. 2-B). Two novel full-length dinoponeratoxin isoform precursors were identified in EST library (discussed below). Additionally, representative sequences of allergens and other minor components were also annotated and catalogued. However, due to the steep rise in the redundancy in the number of transcript sequences rescued from the cDNA (ESTs) library, we decided to conduct a deep investigation using next generation RNA-sequencing (NG RNA-Seq) to characterize the entire transcriptome of the D. quadriceps venom gland. To describe the overall transcript profile, we obtained our sequencing dataset with the Ion Torrent Personal Genome Machine (PGM™) and the Trinity program, which is specially designed for RNA-seq de novo assembly. This strategy yielded over 4,000,000 bp, and over 2,000,000 reads with an average length of ∼230 nucleotides were deep sequenced (Table S2). These reads generated over 18,500 contigs, of which 6,463 contigs corresponded to BLASTx hits. Most of the expressed sequences in the venom gland encoded proteins related to biological processes, cellular architecture, and molecular functions (Fig. 3 and Fig. S2). Importantly, based on functional classification, the most abundant proteins (over 200) were related to ribosomal structure and biogenesis, translational and post-translational modification, protein turnover and chaperones (Fig. S3), as it would be expected for a specialized and dynamic metabolic active tissue that needs to synthesize venom components for ant defense and feeding. Another interesting finding from the giant ant transcriptome is related to the proportion of Hymenoptera species with which D. quadriceps sequences share high similarity based on BLASTx queries. Nearly 60% of all D. quadriceps transcript sequences had the greatest homology with sequences from H. saltator, followed by A. echinator (∼11%), S. invicta (∼7%) and M. rotundata (∼6%) (Fig. 4). As D. quadriceps and H. saltator belong to the same subfamily (Ponerini) and evolved from a common ancestor, it is reasonable to argue that they have a common framework of transcript sequences in their venom glands. Two groups of highly homologous proteins expressed by hymenopterans in the venom gland transcriptome of D. quadriceps that were disclosed here are related to “sex determination” and mixed-function “lethal-like” proteins.

The so-called “sex determination proteins” were first discovered in Drosophila [38] and thereafter shown to be present in ant species, such as A. echinatior, H. saltator and C. floridanus (Figure S4). This group of proteins belongs to the BTB-ZF family of transcriptional regulators, which are part of the somatic sex determination hierarchy [39], [40]. These proteins can switch fruitless gene (fru) splicing from the male-specific pattern to the female-specific pattern through the activation of the female-specific fru 5′-splice site [41]–[42]. Our analysis of the molecular phylogeny of sex determination proteins indicated that the amino acid sequences of the members of this class of proteins are phylogenetically related to the insect clade and, conversely, that the orthologous relationship is closely related to insect species (Figure S4), which suggests a common mechanism of sex determination in hymenopterans. Moreover, the sex determination proteins are usually expressed in specific regions of the adult male brain that are associated with the control of courtship songs and the steps of male courtship and are also expressed in the larval and pupal male mushroom bodies and optic lobes and thereby possess tissue-specific characteristics [38], [39]. Importantly, our results have revealed for the first time that this group of proteins is expressed in giant ant venom glands. Further investigation is required to understand the role of the orthologous sex determination proteins in the venom gland, although the presence of this group of proteins might suggest that they influence courtship behavior and social organization in the queenless nests of the giant ant species.

Several classes of polypeptides with multiple functions are grouped together under the term “lethal-like proteins”, despite the fact that these proteins have distinct structures and protein motifs (Fig. 5 and Table S2). For example, we found transcript that coded for protein lethal essential for life, multidrug resistance-associated protein lethal, lethal neighbor of tid protein, and lethal malignant brain tumor-like protein, among other lethal-like sequences. In the category of protein lethal essential for life, we found sequences that possessed the signatures of small heat shock proteins (sHSPs) and α-crystallins (αC); i.e., domain sHSP/αC. Polypeptides with these domains, apart from being responsive to a variety of stresses, are involved in a wide range of functions in multiple cell types and multiple organisms [43]. In the venom glands of ants, the protein lethal with domain sHSP/αC proteins might work as co-chaperones that assist in toxin folding and post-translational processing of key proteins. The other lethal-like or lethal-related proteins, namely, the lethal neighbor of tid proteins, contain the typical domain of ALG3 proteins; i.e., a structural motif dedicated to the enzymatic linkage of N-glycosidic groups to glycoproteins (pfam05208). The catalytic process of glycosilation post-translationally modifies numerous toxins including ant venom phospholipases [44]. Lethal-like proteins are also associated with multidrug resistance. A number of transcripts code for multidrug resistance-associated protein lethal proteins, which include the ABC transporter cassette motif in their structures. In ants, these polypeptides might be associated with the active transport of glycoproteins and xenobiotics to sub-cellular compartments like those that have been observed in several organisms and described in more detail in Drosophila Malpighian tubule physiology [45], [46]. Another lethal-related protein class in the giant ant transcriptome includes the lethal malignant brain tumor-like protein. The members in this class of proteins contain the domain of the MBT superfamily (smart00561) as exemplified by proteins of Drosophila and vertebrates that act in the process of transcriptional regulation. Additionally, proteins in this class possess the “sterile alpha motif (SAM)”, which interacts with diverse proteins, RNAs and membrane lipids, and plays a role in signal transduction and transcription. Thus, the categorization of lethal-like protein in the venomics of the giant ant relies on the fact that members of this group of polypeptides participate in critical biochemical events of venom gland physiology and might additionally induce disruption of the cellular circuitries of prey or victims after ant sting and envenomation.

Notably, less than 1% (exactly 0.72%) of all sequenced transcripts corresponded to toxins or venom-related peptides in hymenopterans (Fig. 3), and this percentage corresponded with hundreds of polypeptides. Accordingly, Johnson and colleagues [30] performed a proteomic-based investigation on the venom of D. australis and found over 75 polypeptides, but only described the six most abundant dinoponeratoxins and their congeners. In another recent study on the venom transcript profile of ants, Bouzid and collaborators [2] identified five major classes of toxins and toxin-related peptides (i.e., pilosulin 5-like, insect cytokine precursor, Sol I 3 antigen, secapins and allergen peptide) that corresponded to a total of 42 of the 85 ESTs that were Hymenoptera toxin-like candidates.

Despite of relative simplicity of ant venoms, which are comprised of a basic framework of allergen peptides, hyaluronidases, phospholipases and phosphatases, the small repertoire of venom-related components is highly effective in causing tissue permeability, edema, warmth of the local area, life-threatening systemic anaphylactic reactions, and, occasionally, delayed hypersensitivity; these reactions are observed after most Hymenoptera stings [47]. Moreover, due to the high levels of structural conservation and homology among hymenopterans allergens, venom cross-reactivity is one of the major risk factors related to insect stings [48].

In our transcriptome analysis, we determined that the giant ant venom gland possesses a relative simple toxin and venom-related peptide repertoire, the sequences of which predominantly fit into the following three major venomic classes: esterases (phospholipase-related polypeptides and carboxylesterases), venom allergens, and dinoponeratoxins (Fig. 2-B and Fig. 5; table S1 and S3). Minor venom components, which are not less toxic or harmless to human cells and tissue, included cysteine-rich peptides stabilized by disulfide bonds that are distributed across the venoms from animals of multiple phyla.

In table 2, the general venom polypeptide cores from D. quadriceps and several species of ants are shown and compared. There are venom components common to all species (e.g., phospholipases) and others, such as venom allergens and dinoponeratoxin, which are genus-specific. However, given that most of the common venom components share high levels of amino acid and epitope similarity, cross reactivity and anaphylaxis after Hymenoptera stings is a recurrent risk, as previously mentioned. This is a particular concern for phospholipases and other major venom allergens, which exhibit the highest levels of similarities in their primary structures and tri-dimensional topologies.

Download:

Table 2. Overview of major venom polypeptide components of D. quadriceps and other ant species.

https://doi.org/10.1371/journal.pone.0087556.t002

Notably, two of the most conserved venom allergens from fire ants (S. invicta) are also expressed in the venom gland of D. quadriceps: the homologous of Sol i 1 and Sol i 3 (Fig. 6 and Fig. 7, respectively). D. quadriceps venom allergen 1 belongs to the phospholipase AB₁ group and, consequently, displays a propensity for cross-reactivity with the Sol i 1 homologs of several hymenopteran-related sequences [49]. Allergen proteins are potent components of the Hymenoptera venom arsenal that induce hypersensitive allergic responses. As can be observed in figures 7 and S5, the structural conservation of the D. quadriceps Sol i 3/allergen 5-like transcript is not only related to sequences from other species of ants but also to sequences from wasp species. Indeed, these sequences are collectively called CAP proteins (cysteine-rich secretory proteins - CRISPs, insect venom allergen antigen 5, and pathogenesis-related 1 proteins), and they compose a superfamily of polypeptide sequences that are found in a wide range of organisms, including prokaryotes. In figure S5, the D. quadriceps CAP allergen Sol i 3/allergen 5-like is compared with homologous sequences from S. invicta and Vespula vulgaris, and its tri-dimensional model depicted. The spatial structure of D. quadriceps Sol i 3/allergen 5-like proteins extremely highly conserved and fits well with fire ant Sol i 3/Ag 5 experimental crystal structure data [50]. Interestingly, polypeptides in the mammalian male reproductive tract and in the venom secretory ducts (e.g., stecrisp, PSTx, triflin, helothermine) of several snake and lizard species are CAP proteins in the CRISP group. CRISPs have two domains: A CAP N-terminal domain followed by a C-terminal cysteine rich domain (CRD) containing 10 absolutely conserved cysteine residues. Importantly, from the perspective of venomics, the CRD possesses a fold in common with sea anemone ion channel inhibitors (Bgk- and Shk-type toxins), which are involved in the blocking of voltage gated Ca²⁺- and K⁺-channels and modulate ryanodine receptors. Interestingly, these biological activities are only observed when these polypeptides are associated with the CAP domain [51], [52]. Despite belonging to CAP superfamily, the Sol i 3/allergen antigen 5 (Sol i 3/Ag 5) homologs from Hymenoptera venom form a major and distinct clade. Sol i 3/Ag 5 homologs, besides having been initially characterized in the venoms of fire ants and several species of wasps, have also been identified in the midgut of Drosophila and in the saliva of ticks, sand flies, and mosquitoes. In the secretions of blood-feeding insects, the Ag5 proteins are part of a cocktail of salivary proteins that are believed to be active either in the suppression of the host immune system or in the prevention of clotting to prolong feeding [52]. Thus, the structural conservation of these allergens in the venom of D. quadriceps (Ponerinae), other ant species (e.g., Pachycondyla and S. invicta) and wasps is an interesting aspect of Hymenoptera venomics because these insect groups have a long natural history of divergence that dates to more than 150 million years ago [35].

In addition to the venom allergen homologous of Sol i 1, which are related to the group of phospholipase AB₁,esterases (phospholipases and carboxylesterases) comprises another class of predominantly expressed venom components in the transcriptome of the giant ant D. quadriceps. We found members and isoforms of PLA₁, PLA₂, PLB₁, PLD, which indicates that phospholipases compose an important part of the toxin core of giant ant venoms (Fig. 5, table S3). Broadly, the class of esterase-lipases comprises a superfamily of enzymes that includes diverse groups of phospholipases (A1, A2, B, C and D). The most well-known phospholipases, A₁, A₂ and C (PLA₁, PLA₂ and PLC), hydrolyze the ester bonds of phospholipids at the specific positions of sn-1, sn-2 and sn-3, respectively [53]–[55], and the resulting products are used as precursors in the synthetic pathways for pharmacological mediators such as leukotrienes and prostaglandins or play a role in signal transduction as secondary messengers [56]. PLA₁s can be classified into two groups based on their structures and cellular localizations. The first group encompasses intracellular enzymes that include phosphatidic acid-preferring phospholipase A₁, and the second group is composed of other extracellular enzymes that include phosphatidylserine-specific PLA₁ [57]. Members of the latter group of enzymes have been detected in hymenopterans, mainly in wasp species. The structures of these enzymes are conserved across a wide range of organisms, and, in humans, they induce platelet activation, allergic reactions and hemolysis [54], [58], [59]. Phospholipases A₂ is subdivided into more than a dozen subtypes (from I to XV, with subgroups) and is the most abundantly expressed type of phospholipase in the venom of hymenopterans. The venoms of bees and wasps have been shown to possess high levels of phospholipase activity; thus, this class of enzymes has been implicated in tissue damage and hypersensitivity reactions [60]–[62]. The pioneering report of Schmidt and Blum [63] detailed the high phospholipase A₂ and B contents of the venom of the Harvester ant (Pogonomyrmex badius). Since that early work, phospholipase activity has been described in the venoms of several ant species, including Neoponera apicalis, N. olivaceae, P. clavata, Pachycondyla cressiondes, Pogonomyrmex occidentalis and Dinoponera grandis [53]. These enzymes have been related to be the major allergen in hymenoptera venoms [49], [64], [65] and, notably, are responsible for the antigenic cross-reactivity between hymenoptera venoms [49], [60], [66], [67].

PLCs are ubiquitous enzymes that hydrolyze membrane lipid phosphoinositides to yield two important second messengers, inositol phosphates and diacylglycerol (DAG) (cl14615); however, despite finding numerous types of phospholipases, we did not find any PLC venom in our study of giant ant venomics, Two additional subfamilies of phospholipases should be mentioned: (1) phospholipase B, which has both esterase and phospholipase-A/lysophospholipase activity (cd01824) and is involved in the conversion of phosphatidylcholine to fatty acids and glycerophosphocholine (perhaps in the context of dietary lipid uptake); and (2) phospholipase D (PLD), which is a signal-activated enzyme that catalyzes the hydrolysis of the phosphodiester bond of glycerophospholipids to generate phosphatidic acid (PA); PA is an intracellular signaling lipid that has been implicated in vesicular trafficking [68], [69]. In the D. quadriceps venome, we found transcripts for the first giant ant PLD (sphingomyelinase-D, E.C. 3.1.4.41), a dermonecrotic toxin that, until now, had only been described venoms of spiders, particularly the brown spider Loxoceles gaucho. In spider venom, PLD is responsible for the necrotic arachnidism and massive inflammatory response observed in loxoscelism, which is the main effect of brown spider envenomation [70], [71]. Thus, the presence of PLD in the D. quadriceps venome is another remarkable finding that advances the comprehension of the pathophysiological effects of the venom of the giant ant. Moreover, the expression of several isoforms and subgroups of phospholipases in the giant ant venom gland indicate that lipid homeostasis and the lipid-mediated signaling circuitry are targets that mediate the tissue disruption of, and damage to, prey and/or victims.

We also identified transcripts for carboxylesterases in the venom of D. quadriceps; carboxylesterases are enzymes that hydrolyze carboxylic acid esters into their corresponding acids and alcohols (GO0004091). Enzymes in this class are considered to be protective molecules because promote cellular detoxification by inactivating toxicants and carcinogens. As described by Hatfield and Potter [72], diverse molecules such as drugs, pesticides and veterinary products contain ester moieties that are susceptible to catalytic conversion by these enzymes. Interestingly, carboxylesterases have been identified in diverse hymenoptera venoms, including the venoms of Apis mellifera [73], N. vitripennis [74], H. saltator, C. floridanus [75], A. echinatior (GI332028825). Thus, it is likely that such this class of venom components might be involved in the detoxification xenobiotics and confer resistance to insecticides [74], [75]. Despite this physiological function, the A. mellifera carboxylesterase has been shown to cause allergic reactions in humans [76], and this type of venom component might make a similar contribution to the symptoms of by giant ant stings.

Dinoponeratoxin transcripts were identified in high proportions in the transcriptome of D. quadriceps, and four novel dinoponeratoxin sequences were discovered (Fig. 9). As noted by Johnson and co-workers [30], dinoponeratoxins share variable primary sequence similarities with antimicrobial peptides from ponerine ants (i.e., ponericin G and W3) and other organisms; e.g., frogs (gaegurin-5 and brevinin-1 PTa). Additionally, it is notable that bioactive peptides seem to be encrypted into the dinoponeratoxin precursors, as numerous peptide fragments were detected in the venom proteomic of D. australis. The alignment of dinoponeratoxins with antimicrobial peptide sequences provides a clue regarding the functions of these peptides in the venom of D. quadriceps and other ponerine ants, although the actual biological activity of dinoponeratoxin - other than microbicide - deserves further investigation. It is known that many antimicrobial peptides cause cellular membrane damage via pore formation or micellization as a part of their modes of action [77], [78]; these processes might intensify toxin dispersal in the tissues of prey or victims and increase the efficacy of ant envenomation. Another interesting aspect of dinoponeratoxin transcripts is related to their cDNA precursors at the nucleotide level. We observed that the dinoponeratoxin (contig 9, table S1) precursor shares a certain degree of identity with the 5′-end of the pilosulin 5 cDNA precursor (not shown). First discovered in the venom of Australian jumper jack ant (Myrmecia pilosula), pilosulins comprise a family of short monomeric and dimeric peptides with antimicrobial activity that is mediated through histamine-releasing and hemolytic effects [79], [80]. In light of toxin evolution and diversification, the sharing of stretches of nucleotide sequences between dinoponeratoxin and pilosulin-5 is indicative of the conservation of parental ancestor genes and their recruitment as a template for the evolution of bioactive peptide variants in the venom that, in this case, cause the degranulation of mast cells and histamine release. Another pilosulin-related sequence of giant ant venom gland was observed to be encoded by the contig 6 (table S1).

Minor toxin-related components identified in the venom gland transcriptome of D. quadriceps include sequences with high homologies with the spider U8 agatoxin, the venom protein VN50, the cysteine-rich venom proteins of the endoparasitic wasp, and the ICK-like toxins from Conus and spiders. The U8-agatoxin-Ao1a-like isoform peptide precursor was found in an initiative to identify toxin-like structures based on new algorithms for the interrogation of the cDNA library of the spider Agelena orientalis and the data bank for neurotoxin sequences [81]. Based on this strategy, a contig (1144_B2-2, table S3) from the D quadriceps transcriptome was found to meet the criteria for the “extra structural motif” (ESM), in which a pair of CXC fragments in the C-region (or C1CZC1C, where Z is variable number of amino acid residues), code for a novel neurotoxin precursor that is homologous to spider toxin and diverse hymenopteran-related sequences (Fig. S6).

The venom protein VN50 is a serine protease homolog without enzymatic activity from the venom Cotesia rubecula that, once injected into the host, interferes with the immune defense mechanisms mediated by the prophenoloxidase (proPO) activation cascade [82]. The presence of a VN50-related sequence (contig 1219_B2-2) in the venom gland of D. quadriceps indicates that this venom component has the ability to disturb the homeostasis of invertebrates and augment the processes of their intoxication. Another less abundant, but not less important, transcript sequence in the D. quadriceps transcriptome (contig 23_B2-2) shares high similarity with the cysteine-rich venom proteins that make up a significant fraction of the polypeptides from the venom of the parasitoid wasp Pimpla hypochondriaca [83]. Venom peptides in this class include trypsin inhibitors (including the Kunitz type) and voltage-gated Ca²⁺-channel antagonists (ω-conotoxins) with analgesic properties from the gastropod mollusk sea snail Conus. A structural fold that is highly conserved across diverse bioactive venom cysteine-rich peptides (or disulfide-rich domains) is known as the inhibitor cysteine-knot (ICK) motif. The ICK motif is an evolutionarily conserved structure that is found in the venoms of a wide range of organisms of diverse phyla, including arthropods (spiders and scorpions), marine cnidarians (sea anemones), sea snails, fungi, plants, and vertebrates. The ICK motif is a chemically and thermally very stable protein fold that is characterized by a knot formed by segments (α-helix, β-sheet or random coil) connected by two disulfide bonds and interlaced with a third disulfide bond that forms up to six loops with variable sequences [84]. Venom from the marine mollusk Conus, spiders and scorpions include numerous toxins containing the ICK fold. In the transcriptome of D. quadriceps, we identified the first ponerine ant ICK-containing toxin (Fig. 10). The BLAST hits with the highest scores for this novel sequence were for the superfamily-A conotoxins and the scorpion like-toxins. The high scores between this novel sequence and members of these protein families were mainly due to similarities with the signal peptides of the Dinoponera ICK-like toxin precursors. However, the Dinoponera ICK-like toxin cysteine framework was proven to be VI/VII (C-C-CC-C-C), and this pattern of disulfide bonds would place this ant ICK-like toxin into the ω-toxin superfamily (PFAM Clan cl0083). This superfamily includes several type-1 conotoxins, funnel web spider toxins, and assassin bug toxins, all of which are characterized as neurotoxic antagonists of several subtypes of ion channel [85]. Curiously, the translated sequence of the Dinoponera ICK-like toxin also has structural homology with an uncharacterized protein family expressed that is in Schistosoma (result not shown). Moreover, ICK-type peptides with antimicrobial have recently been identified in the genomes of seven ant species; i.e., A. cephalotes, A. echinatior, L. humile, S. invicta, P. barbatus, C. floridanus, and H. saltator. Based on molecular phylogenetic analyses, these ant antimicrobial peptides with ICK motifs have been were categorized into three subtypes [86]. Thus far, transcripts encoding cysteine-rich peptides with variable numbers of disulfide bonds and cysteine stabilized ICK-folded peptides form a minor proportion of all expressed components of D. quadriceps venom.

Conclusions

In this study, we sequenced and characterized the transcriptome of the D. quadriceps venom gland and focused principally on the polypeptide core of the venom that is responsible for the symptoms caused by giant ant stings. With a combination of Sanger sequencing of the cDNA library and next generation deep RNA sequencing, a complete description of transcriptome of the venom gland was achieved. These experiments revealed the expression of coding sequences dedicated to specialized tissue physiology and toxin production. The polypeptide component framework of the venom is comprised of a major and a minor core of toxin classes, which, in combination, elicit local and systemic reactions to stings from this species of ponerine ant. The major toxin core is composed of venom allergens, phospholipases, carboxylesterases, lethal-like toxins, and dinoponeratoxins. With exception of dinoponeratoxins, all components of the major toxin core of the giant ant display high structural similarities with toxins of the same groups of proteins from different hymenopterans species (ants and wasps). This finding indicates that cross-reactivity is a recurrent phenomenon of hymenopteran envenomation and a property of the structures of venom components that has been maintained over the course of more than 100 million years of insect evolution and diversification. Moreover, the toxins that compose the major core may functionally interfere with lipid homeostasis and signaling, the trafficking of vesicles and protein-protein and protein-nucleic acid interactions. These toxins may also disrupt the biochemical circuitry of the tissues of the prey or victims. Dinoponeratoxins are another major toxic component of D. quadriceps venom and are exclusively expressed in the giant ant venom. This group of small disulfide-less toxins shares structural similarity with sequences from D. australis and, at the nucleotide level, shares an evolutionary relationship with pilosulins, which are antimicrobial and mast cell degranulating peptides from Myrmecia venom. Cysteine rich polypeptides that are shaped by disulfide bonds and folded into the typical ICK-motif comprise a fraction of the minor D. quadriceps venom protein core. The ICK-type peptides from the giant ant possess structural identity with peptides from scorpions, spiders and the marine venomous Conus gastropods. A component of the minor venom protein core of D. quadriceps is the homolog of a serine protease that is devoid of catalytic activity (i.e., the peptidase S1-derived scaffold venom protein of the solitary wasp and blood-feeding bat), which should interfere with innate immune responses and inhibit endogenous peptidase S1 homologs of the prey or victims. Taken together, the results of the transcriptome and venomic analysis of D. quadriceps provide an inestimable resource for the understanding of the molecular and physiopathological bases of giant ant envenomation and basic knowledge about the major and minor polypeptide venom components that may be useful in the future for medical biotechnology.

Methods

Dinoponera quadriceps sampling and processing

Specimens of the giant ant Dinoponera quadriceps were collected at twilight in the Serra de Maranguape (3°52′51″S and 38°42′39″W), which is a mountain range with peaks below 1000 m that is located about 40 km from the coast of the north-eastern state of Ceará, Brazil. A trap made with ground tuna meat was used to attract ants from inside their nests. The individuals were either kept in the laboratory in artificial nests or dissected with thin scissors and scalpels under a stereomicroscope (Nikon SMZ 800, Tokyo, Japan). The resting (not milked) venom gland was carefully separated from the other surrounding tissues, such as the Malpighi tubules and muscles, and quickly transferred to microtubes containing RNAlater solution (Life Technologies, U.S.A.). Authorization (no. 28794-1) to access Brazilian biodiversity was issued by the Brazilian Institute of the Environment and Renewable Natural Resources (Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis, IBAMA) of the Ministry of the Environment of the Federal Government of Brazil.

RNA isolation and venom gland cDNA library construction

Total RNA was prepared using the RNAeasy Mini Kit (Qiagen, U.S.A), which is based on a combination of guanidine-isothiocyanate lysis and silica-membrane purification in a mini-spin column. The procedures were performed according to the manufacturer's instructions. The quality of the RNA was analyzed with denaturating RNA electrophoresis in TAE agarose gel as described in [87] and quantified by UV absorption using NanoDrop (Thermo Scientific, Wilmington, DE-USA).

The cDNA library was constructed using a switching mechanism at the 5′ end of the RNA template to preferentially amplify the poly-A tail (IN-FUSION™ SMARTer™ cDNA Library Construction kit, Clontech, Mountain View, CA-USA). Briefly, the synthesis of single strand cDNA began with 1 µg of total RNA and 3′ In-fusion SMARTer CDS primers. The resulting first strands of cDNA were sequentially amplified with SMARTer V Primer. To synthesize double strand cDNA, the Advantage 2 Polymerase, the 5′ PCR primer IIA and 3′ In-fusion SMARTer PCR Primer with the lowest possible number of cycles (in this case 15) were used. The double stranded (ds-) cDNA was fractionated by size exclusion chromatography (CHROMA SPIN 400, Clontech, USA), and the fractions with specific molecular sizes (over 400 bp) were combined and precipitated with NaCl/ethanol. The cDNA was quantified by U.V. spectrophotometry and ligated into the plasmid vector (pSMART 2IFD). A commercial strain of E. Coli was used to as a host for the maintenance and propagation of the D. quadriceps venom gland cDNA library, which was then stored at −80°C.

Sanger sequencing of independent clones

The cDNA library was seeded in LB agar plates supplemented with appropriate antibiotics, and individual clones were selected and analyzed for insert size. To screen for positive clones, specific primers flanking the multi-cloning site of the vector were used in a PCR reaction. The amplified screening products were analyzed in 1% agarose gel and photodocumented. The PCR products of 420 independent clones were cleaned up by EXO/SAP treatment and sodium acetate/ethanol precipitation. The precipitated DNA were resuspended in deionized formamide and sequenced in a 3100 Avant Sequencer (Applied Biosystems, USA) using Prism Big Dye Terminator v3.0 Cycle Sequencing Ready Reactions (Applied Biosystems, USA).

Data analysis of Sanger sequencing

Sequences were analyzed with the CLC Bio Main Workbench v 6.8.3 Software. The sequences were trimmed to exclude vector contamination and the poly-A tail. The following parameters were selected for clustering and assembling: minimum aligned read length of 50 and medium (ambiguity level) alignment stringency. The resulting contigs (clusters and singlets) were compared against the non-redundant GenBank (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov) using BLASTx for all organisms and/or an arthropod databank (see below for deep sequencing analysis).

Sample preparation and deep Sequencing using Ion Torrent Technology

A fragment library was generated with an input of 100 ng DNA using the Ion Xpress Plus Fragment Library Kit (Life Technologies, Carlsbad, USA). Next, DNA fragmentation, adaptor ligation, fragment size selection and library amplification were carried out for a total of 8 cycles according to manufacturer's instructions. The targeting of fragments of approximately 330 bp was performed with 2% agarose gel cassettes for the Pippin Prep instrument (Sage Science, Beverly, USA). Prior to emulsion PCR, the size distribution and concentrations of the library were assessed with a Bioanalyzer 2100 using the DNA High Sensitivity Kit (Agilent, Santa Clara, CA). The fragment library was adjusted to approximately 26 pM and amplified with Ion Sphere particles™ (ISPs) by emulsion PCR using the Ion OneTouch™ Instrument with the Ion OneTouch™ 200 Template Kit (Life Technologies, Grand Island, NY) and template-positive ISP enrichment according to the manufacturer's protocol (Part Number 4472430 Rev. E, 06/2012). Over 50% of the ISPs were and then sequenced on an Ion 316™ chip using the Ion Torrent Personal Genome Machine (PGM™) (Life Technologies, San Francisco, CA, USA) for 130 cycles (520 flows) with the Ion PGM™ 200 Sequencing Kit (Part Number 4474246 Rev. D, 06/2012).

Sequence filtering and (de novo) assembly of deep sequencing data

Ion Torrent Personal Genome Machine (PGMTM) sequencing can generate 200–500 base pairs (bps) from each single end (SE) of a cDNA fragment. The raw reads were initially processed to obtain clean reads by removing the adapter sequences and removing low quality sequences (i.e., reads in which more than 50% of the bases had quality value s≤5). Next, de novo transcriptome assembly was carried out using the short-read assembling program Trinity [88]. Specifically the k-mer counting method was set to jellyfish, and the contigs that could not be extended on either end were defined as transcripts.

Gene ontology and functional annotation

All of the assembled transcripts were searched against the NCBI non-redundant protein sequence (nr) and COG databases using BLASTX to identify the proteins with the highest sequence similarities with the given transcripts to retrieve the function annotations of the transcripts, and standard cut-off E-value of <10⁻⁵ was used. Next, the species and function information from the nr database was used to perform taxonomic analyses with in-house Perl scripts. For the nr annotation, the in-house BLAST2GO embedded annotation pipeline was used to obtain the GO annotations of the assembled transcripts to describe the biological processes, molecular functions and cellular components of the transcripts [89], and the WEGO [90] software was used to conduct GO functional classification to understand the distribution of gene functions at the macroscopic level. Furthermore, the transcripts that had no matches in the nr database were subjected to another BLASTX search against the UniProtKB database.

Bioinformatic analyses of similarities and molecular phylogenies

https://doi.org/10.1371/journal.pone.0087556.s010

(TIF)

Author Contributions

Conceived and designed the experiments: AMCM ARBPS SMYL GR-B. Performed the experiments: AFCT CH AH C-MC YPQ. Analyzed the data: AFCT CH C-MC ARBPS SWL SMYL GR-B. Contributed reagents/materials/analysis tools: AMCM ARBPS SMYL GR-B. Wrote the paper: AFCT CH ARBPS SMYL GR-B. Field trip missions and rearing ants: AFCT YPQ.

References

1. Munoz-Torres MC, Reese JT, Childers CP, Bennett AK, Sundaram JP, et al. (2012) Hymenoptera Genome Database: integrated community resources for insect species of the order Hymenoptera. Nucleic Acids Research 39: 658–662.
- View Article
- Google Scholar
2. Bouzid W, Klopp C, Verdenaud M, Ducancel F, Vétillard A (2013) Profiling the venom gland transcriptome of Tetramorium bicarinatum (Hymenoptera: Formicidae): The first transcriptome analysis of an ant species. Toxicon 70: 70–81.
- View Article
- Google Scholar
3. Cherniak EP (2010) Bugs as drugs, Part 1: Insects: the new alternative medicine for the 21^st century? Altern Med Rev 15: 124–135.
- View Article
- Google Scholar
4. Badr G, Garraud O, Daghestani M, Al-Khalifa MS, Richard Y (2012) Human breast carcinoma cells are induced to apoptosis by samsum ant venom through an IGF-1-dependant pathway, PI3K/AKT and ERK signaling. Cel Immunol 273: 10–16.
- View Article
- Google Scholar
5. Ebaid H, Al-Khalifa M, Isa AM, Gadoa S (2012) Bioactivity of Samsum ant (Pachycondyla sennaarensis) venom against lipopolysaccharides through antioxidant and upregulation of Akt1 signaling in rats. Lipids in Health Disease 11: 93.
- View Article
- Google Scholar
6. Dkhil M, Abdel BA, Al Quraishi , Al Khalifa M (2010) Anti-inflammatory activities of the venom from samsumants Pachycondyla sennaarensis. Afr J Pharm Pharmacol 4: 115–118.
- View Article
- Google Scholar
7. Orivel J, Dejean A (2001) Comparative effect of the venoms of ants of the genus Pachycondyla (Hymenoptera: Ponerinae). Toxicon 39: 195–201.
- View Article
- Google Scholar
8. Zelezetsky I, Pag U, Antcheva N, Sahl HG, Tossi A (2005) Identification and optimization of an antimicrobial peptide from the ant venom toxin pilosulin. Biochem Biophys 434: 358–364.
- View Article
- Google Scholar
9. Quinet Y, Vieira RHSF, Sousa MR, Evangelista-Barreto NS, Carvalho FCT, et al. (2012) Antibacterial properties of contact defensive secretions in neotropical Crematogaster ants. JVAT 18: 441–445.
- View Article
- Google Scholar
10. Torres AFC, Quinet YP, Havt A, Rádis-Baptista G, Martins AMC (2013) Molecular pharmacology and toxynology of venom from ants.; Rádis-Baptista G, editor. An Integrated view of the molecular recognition and toxinology – From analytical procedures to biomedical applications. Croatia: InTech. pp. 207–222.
11. Lopes KS, Rios E, Dantas RT, Lima C, Linhares M, et al. (2011) Effect of Dinoponera quadriceps venom on chemical-induced seizures models in mice. Rencontres en Toxinologie – Meeting on Toxinology SFET Editions. Available: http://www.sfet.asso.fr. Accessed 20 August 2012.
12. Lopes KS, Rios ERV, Lima CNC, Linhares MI, Torres AFC, et al. (2013) The effects of the brazilian ant Dinoponera quadriceps venom on chemically induced seizure models. Neurochem Intern 63: 141–145.
- View Article
- Google Scholar
13. Sousa PL, Quinet Y, Ponte EL, do Vale JF, Torres AFC, et al. (2012) Venom's antinociceptive property in the primitive ant Dinoponera quadriceps. J Ethnopharmacol 144: 213–216.
- View Article
- Google Scholar
14. Kempf WW (1971) A preliminary review of the Ponerinae ant genus Dinoponera Roger (Hymenoptera: Formicidae). Studia Entomologica 14: 369–394.
- View Article
- Google Scholar
15. Paiva RVS, Brandão CRF (1995) Nests, worker population, and reproductive status of workers, in the giant queenless ponerinae ant Dinoponera Roger Hymenoptera Formicidae. Ethology Ecology and Evolution 7: 297–312.
- View Article
- Google Scholar
16. Lenhart PA, Dash ST, Mackay WP (2013) A revision of the giant Amazonian ants of the genus Dinoponera (Hymenoptera, Formicidae). J Hymenoptera 31: 119–164.
- View Article
- Google Scholar
17. Lattke, J. E. 2003. Subfamilia Ponerinae, p. 261–276 In: F. FERNÁNDEZ (ed.) Introducción a las hormigas de la región Neotropical. Bogotá, Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, 398 p.
18. Wilson EO, Hölldobler B (2005) Eusociality: origin and consequences. Proc Natl Acad Sci 102: 13367–13371.
- View Article
- Google Scholar
19. Monnin T, Peeters C (1998) Monogyny and regulation of worker mating in the queenless ant Dinoponera quadriceps. Animal Behaviour 55: 299–306.
- View Article
- Google Scholar
20. Vasconcellos A, Santana GG, Souza AK (2004) Nest spacing and architecture, and swarming of males of Dinoponera quadriceps (Hymenoptera, Formicidae) in a remnant of the Atlantic forest in Northeast Brazil. Braz J Biol 64: 357–362.
- View Article
- Google Scholar
21. Schoeters E, Billen J (1995) Morphology and ultra structure of the convoluted gland in the ant Dinoponera australis (Hymenoptera:Formicidae). Int J Insect Morphol Embryol 24: 323–332.
- View Article
- Google Scholar
22. Mathias MIC (2007) Glândulas de veneno em formigas. Biológico 69: 229–235.
- View Article
- Google Scholar
23. Holldobler B, Wilson EO (1990). The ants. Cambridge: Harvard University, Belknap Press. 746 p.
24. Haddad V Jr, Cardoso JLC, França FOS, Wen FH (1996) Acidentes por formigas: um problema dermatológico. An Bras Derm 71: 527–530.
- View Article
- Google Scholar
25. Haddad Jr V(2001) Acidentes por formigas. In: Manual de diagnóstico e tratamento de acidentes por animais peçonhentos. Brasília, FNS. pp.65–66
26. Haddad Jr V, Cardoso JLC, Moraes RHP (2005) Description of an injury in a human caused by a false tocandira (Dinoponera gigantea, Perty, 1833) with a revision on folkloric, pharmacological and clinical aspects of the giant ants of the genera Paraponera and Dinoponera (sub-family ponerinae). Rev Inst Med Trop 47: 235–238.
- View Article
- Google Scholar
27. Cruz Lopez L, Morgan ED (1997) Explanation of bitter taste of venom of ponerine ant Pachycondyla apicalis. J Chem Ecol 23: 705–712.
- View Article
- Google Scholar
28. Duval A, Malécot CO, Pelhate M, Piek T (1992) Poneratoxin, a new toxin from ant venom, reveals an interconversion between two gating modes of the Na channels in frog skeletal muscle fibres. Pflugers Arch 420: 239–247.
- View Article
- Google Scholar
29. Szolajska E, Poznanski J, Ferber ML, Michalik J, Gout E, et al. (2004) Poneratoxin, a neurotoxin from ant venom. Structure and expression in insect cells and construction of a bio-insecticide. Europ J Biochem 271: 2127–2136.
- View Article
- Google Scholar
30. Johnson SR, Copello JA, Evans MS, Suarez AV (2010) A biochemical characterization of the major peptides from the Venom of the giant Neotropical hunting ant Dinoponera australis. Toxicon 55: 702–710.
- View Article
- Google Scholar
31. Min XJ, Butler G, Storms R, Tsang A (2005) OrfPredictor: predicting protein-coding regions in EST-derived sequences. Nucleic Acids Res 33: 677–680.
- View Article
- Google Scholar
32. Menne KM, Hermjakob H, Apweiler R (2000) A comparison of signal sequence prediction methods using a test set of signal peptides. Bioinformatics 16: 741–742.
- View Article
- Google Scholar
33. Zientz E, Feldhaar H, Stoll S, Gross R (2005) Insights into the microbial world associated with ants. Arch Microbiol 184: 199–206.
- View Article
- Google Scholar
34. Boulogne I, Germosen-Robineau L, Ozier-Lafontaine H, Jacoby-Koaly C, Aurela L, Loranger-Merciris G (2012) Acromyrmex octospinosus (Hymenoptera: Formicidae) management. Part 1: Effects of TRAMIL's insecticidal plant extracts. Pest Manag Sci 68: 313–320.
- View Article
- Google Scholar
35. Ward PS (2006) Ants. Curr Biol 7:16: R152–155.
- View Article
- Google Scholar
36. Gruber CW, Muttenthaler M (2012) Discovery of Defense- and Neuropeptides in Social Ants by Genome-Mining. PLoS ONE 7 (3) e32559.
- View Article
- Google Scholar
37. Raìdis-Baptista G (2011). Molecular Toxinology – Cloning Toxin Genes for Addressing Functional Analysis and Disclosure Drug Leads.; Brown, G, editor. Molecular Cloning - Selected Applications in Medicine and Biology. Croatia: InTech. pp. 161–196.
38. Ryner LC, Goodwin SF, Castrillon DH, Anand A, Villella A, et al. (1996) Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene. Cell 87: 1079–1089.
- View Article
- Google Scholar
39. Ito H, Fujitani K, Usui K, Shimizu-Nishikawa K, Tanaka S, et al. (1996) Sexual orientation in Drosophila is altered by the satori mutation in the sex-determination gene fruitless that encodes a zinc finger protein with a BTB domain. Proc Natl Acad Sci U S A 93: 9687–9692.
- View Article
- Google Scholar
40. Zollman S, Godt D, Privé GG, Couderc JL, Laski FA (1994) The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila,. Proc Natl Acad Sci U S A 91: 10717–10721.
- View Article
- Google Scholar
41. Dauwalder B, Tsujimoto S, Moss J, Mattox W (2002) The Drosophila takeout gene is regulated by the somatic sex-determination pathway and affects male courtship behavior. Genes Dev 16: 2879–2892.
- View Article
- Google Scholar
42. Heinrichs V, Ryner LC, Baker BS (1998) Regulation of sex-specific selection of fruitless 5′ splice sites by transformer and transformer-2. Mol Cell Biol 18: 450–458.
- View Article
- Google Scholar
43. Basha E, O'Neill H, Vierling E (2012) Small heat shock proteins and α-crystallins: dynamic proteins with flexible functions. Trends Biochem Sci 37: 106–117.
- View Article
- Google Scholar
44. Hoffman DR (2010) Ant venoms. Curr Opin Allergy Clin Immunol 10: 342–346.
- View Article
- Google Scholar
45. Karnaky KJ Jr, Hazen-Martin D, Miller DS (2003) The xenobiotic transporter, MRP2, in epithelia from insects, sharks, and the human breast: implications for health and disease. J Exp Zool A Comp Exp Biol 300: 91–97.
- View Article
- Google Scholar
46. O'Donnell MJ (2009) Too much of a good thing: how insects cope with excess ions or toxins in the diet. J Exp Biol 212: 363–72.
- View Article
- Google Scholar
47. Fitzgerald KT, Flood AA (2006) Hymenoptera stings. Clin Tech Small Anim Pract 21: 194–204.
- View Article
- Google Scholar
48. Biló BM, Rueff F, Mosbech H, Bonifazi F, Oude-Elberink JN (2005) Diagnosis of Hymenoptera venom allergy. Allergy 60: 1339–1349.
- View Article
- Google Scholar
49. Hoffman DR, Sakell RH, Schmidt M (2005) Sol i 1, the phospholipase allergen of imported fire ant venom. J Allergy Clin Immunol 115: 611–616.
- View Article
- Google Scholar
50. Padavattan S, Schmidt M, Hoffman DR, Marković-Housley Z (2008) Crystal structure of the major allergen from fire ant venom, Sol i 3. J Mol Biol 383: 178–185.
- View Article
- Google Scholar
51. Gibbs GM, Scanlon MJ, Swarbrick J, Curtis S, Gallant E, Dulhunty AF, O'Bryan MK (2006) The cysteine-rich secretory protein domain of Tpx-1 is related to ion channel toxins and regulates ryanodine receptor Ca2+ signaling. J Biol Chem 281 (7) 4156–4163.
- View Article
- Google Scholar
52. Gibbs GM, Roelants K, O'Bryan MK (2008) The CAP Superfamily: Cysteine-Rich Secretory Proteins, Antigen 5, and Pathogenesis-Related 1 Proteins—Roles in Reproduction, Cancer, and Immune Defense. Endocrine Reviews 29 (7) 865–897.
- View Article
- Google Scholar
53. Zalat S, Schmidt J, Moawad TI (2003) Lipase and phospholipase activities of Hymenoptera venoms (wasps and ants). Egyptian J Biol 5: 138–147.
- View Article
- Google Scholar
54. Seismann H, Blank S, Cifuentes L, Braren I, Bredehorst R, Grunwald T, Ollert M, Spillner E (2010) Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity. Clin Mol Allergy 8: 7.
- View Article
- Google Scholar
55. Vines CM (2012) Phospholipase C. Adv Exp Med Biol 740: 235–254.
- View Article
- Google Scholar
56. Murakami M, Kudo I (2002) Phospholipase A2. JB Minireview-Lipid Signaling Biochem 131: 285–292.
- View Article
- Google Scholar
57. Richmond GS, Smith TK (2011) Phospholipases A1. Inter J Mol Sci 12: 588–612.
- View Article
- Google Scholar
58. Sonoda H, Aoki J, Hiramatsu T, Ishida M, Bandoh K, Nagai Y, Taguchi R, Inoue K, Arai H (2002) A Novel Phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic acid. J Biol Chem 277: 34254–34263.
- View Article
- Google Scholar
59. Aoki J, Nagai Y, Hosono H, Inoue K, Hiroyuki A (2002) Structure and function of phosphatidylserine-specific phospholipase A1. Bioch Biophys Acta 1582: 26–32.
- View Article
- Google Scholar
60. King TP, Spangfort MD (2000) Structure and biology of stinging insect venom allergens. Int Arch Allergy Immunol 123: 99–106.
- View Article
- Google Scholar
61. dos Santos Pinto JR, Fox EG, Saidemberg DM, Santos LD, da Silva Menegasso AR, et al. (2012) Proteomic view of the venom from the fire ant Solenopsis invicta Buren. J Proteome Res 11: 4643–4653.
- View Article
- Google Scholar
62. Huang WN, Chen YH, Chen CL, Wu W (2011) Surface pressure-dependent interactions of secretory phospholipase A2 with zwitterionic phospholipid membranes. Langmuir 27: 7034–7041.
- View Article
- Google Scholar
63. Schmidt JO, Blum MS (1978) A harvester ant venom: chemistry and pharmacology. Science 200: 1064–1066.
- View Article
- Google Scholar
64. Hoffman DR, Peroutka CM, Schmidt M (2008) Imported fire ant venom phospholipase, Sol I 1, is a protein allergen. J Allergy Clin Immunol 121: S30.
- View Article
- Google Scholar
65. Muller UR (2002) Recombinant Hymenoptera venom allergens. Allergy 57: 570–576.
- View Article
- Google Scholar
66. Hoffman DR, Dove DE, Moffitt JE, Stafford CT (1988) Allergens in hymenoptera venom. XXI. Cross-reactivity and multiple reactivity between fire ant venom and bee and wasps venoms. J Allergy Clin Immunol 82: 828–834.
- View Article
- Google Scholar
67. Sin BA, Akdis M, Zumkehr J, Bezzine S, Bekpen C, et al. (2011) T-cell and antibody responses to phospholipase A2 from different species show distinct cross-reactivity patterns. Allergy 66: 1513–1521.
- View Article
- Google Scholar
68. Liscovitch M, Czarny M, Fiucci G, Tang X (2000) Phospholipase D: molecular and cell biology of a novel gene family. Biochem J 3: 401–415.
- View Article
- Google Scholar
69. McDemott M, Wakelam MJ, Morris AJ (2004) Phospholipase D. Biochem Cell Biol 82: 225–253.
- View Article
- Google Scholar
70. Chaim OM, Trevisan-Silva D, Chaves-Moreira D, Wille AC, Ferrer VP, et al. (2011) Brown spider (Loxosceles genus) venom toxins: tools for biological purposes. Toxins 3: 309–344.
- View Article
- Google Scholar
71. Magalhães GS, Caporrino MC, Della-Casa MS, Kimura LF, Prezotto-Neto JP, et al. (2013) Cloning, expression and characterization of a phospholipase D from Loxosceles gaucho venom gland. Biochimie 9: 1773–1783.
- View Article
- Google Scholar
72. Hatfield MJ, Potter PM (2011) Carboxylesterase inhibitors. Expert Opin Ther 8: 1159–1171.
- View Article
- Google Scholar
73. Cui F, Lin Z, Wang H, Liu S, Chang H, et al. (2011) Two single mutations commonly cause qualitative change of nonspecific carboxylesterases in insects. Insect Biochem Mol Biol 41: 1–8.
- View Article
- Google Scholar
74. Werren JH, Richards S, Desjardins CA, Niehuis O, Gadau J, et al. (2010) Functional and evolutionary insights from the genomes of three parasitoid Nasonia species. Science 327: 343–348.
- View Article
- Google Scholar
75. Bonasio R, Zhang G, Ye C, Mutti NS, Fang X, et al. (2010) Genomic comparison of the ants Camponotus floridanus and Harpegnathos saltator. Science 329: 1068–1071.
- View Article
- Google Scholar
76. Blank S, Seismann H, Bockisch B, Braren I, Bredehorst R, et al. (2008) Identification and recombinant expression of a novel IgE-reactive 70 kDa carboxylesterase from Apis mellifera venom. Available: http://www.uniprot.org/uniprot/B2D0J5. Accessed 19 September 2013.
77. Shai Y (2002) Mode of action of membrane active antimicrobial peptides. Biopolymers 66: 236–248.
- View Article
- Google Scholar
78. Huang Y, Huang J, Chen Y (2010) Alpha-helical cationic antimicrobial peptides: relationships of structure and function. Protein Cell 2: 143–152.
- View Article
- Google Scholar
79. Inagaki H, Akagi M, Imai HT, Taylor RW, Kubo T (2004) Molecular cloning and biological characterization of novel antimicrobial peptides, pilosulin 3 and pilosulin 4, from a species of the Australian ant genus Myrmecia. Arch Biochem Biophys 428: 170–178.
- View Article
- Google Scholar
80. Inagaki H, Akagi M, Imai HT, Taylor RW, Wiese MD, et al. (2008) Pilosulin 5, a novel histamine-releasing peptide of the Australian ant, Myrmecia pilosula (Jack Jumper Ant). Arch Biochem Biophys 477: 411–416.
- View Article
- Google Scholar
81. Kozlov S, Malyavka A, McCutchen B, Lu A, Schepers E, et al. (2005) A novel strategy for the identification of toxin like structures in spider venom. Proteins 59: 131–140.
- View Article
- Google Scholar
82. Asgari S, Zhang G, Zareie R, Schmidt O (2003) A serine proteinase homolog venom protein from an endoparasitoid wasp inhibits melanization of the host hemolymph. Insect Biochem Mol Biol 33: 1017–1024.
- View Article
- Google Scholar
83. Parkinson NM, Conyers C, Keen J, MacNicoll A, Smith I, et al. (2004) Towards a comprehensive view of the primary structure of venom proteins from the parasitoid wasp Pimpla hypochondriaca. Insect Biochem Mol Biol 34: 565–571.
- View Article
- Google Scholar
84. Daly NL, Craik DJ (2011) Bioactive cystine knot proteins. Curr Opin Chem Biol 15: 362–368.
- View Article
- Google Scholar
85. Gracy J, Chiche L (2011) Structure and modeling of knottins, a promising molecular scaffold for drug discovery. Curr Pharm Des 17: 4337–4350.
- View Article
- Google Scholar
86. Zhang Z, Zhu S (2012) Comparative genomics analysis of five families of antimicrobial peptide-like genes in seven ant species. Dev Comp Immunol 38: 262–274.
- View Article
- Google Scholar
87. Masek T, Vopalensky V, Suchomelova P, Pospisek M (2005) Denaturing RNA electrophoresis in TAE agarose gels. Anal Biochem 336: 46–50.
- View Article
- Google Scholar
88. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, et al. (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29: 644–652.
- View Article
- Google Scholar
89. Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21: 3674–3676.
- View Article
- Google Scholar
90. Ye J, Fang L, Zheng H, Zhang Y, Chen J, et al. (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34: 293–297.
- View Article
- Google Scholar
91. Gelly JC, Gracy J, Kaas Q, Le-Nguyen D, Heitz A, Chiche L (2004) The KNOTTIN website and database: a new information system dedicated to the knottin scaffold. Nucleic Acids Res 32: 156–159.
- View Article
- Google Scholar
92. Matuszek MA, Hodgson WC, Sutherland SK, King RG (1992) Pharmacological studies of jumper ant (Myrmecia pilosula) venom: evidence for the presence of histamine, and haemolytic and eicosanoid-releasing factors. Toxicon 30: 1081–1091.
- View Article
- Google Scholar

[ref1] 1. Munoz-Torres MC, Reese JT, Childers CP, Bennett AK, Sundaram JP, et al. (2012) Hymenoptera Genome Database: integrated community resources for insect species of the order Hymenoptera. Nucleic Acids Research 39: 658–662.
View Article
Google Scholar

[2] View Article

[3] Google Scholar

[ref2] 2. Bouzid W, Klopp C, Verdenaud M, Ducancel F, Vétillard A (2013) Profiling the venom gland transcriptome of Tetramorium bicarinatum (Hymenoptera: Formicidae): The first transcriptome analysis of an ant species. Toxicon 70: 70–81.
View Article
Google Scholar

[5] View Article

[6] Google Scholar

[ref3] 3. Cherniak EP (2010) Bugs as drugs, Part 1: Insects: the new alternative medicine for the 21^st century? Altern Med Rev 15: 124–135.
View Article
Google Scholar

[8] View Article

[9] Google Scholar

[ref4] 4. Badr G, Garraud O, Daghestani M, Al-Khalifa MS, Richard Y (2012) Human breast carcinoma cells are induced to apoptosis by samsum ant venom through an IGF-1-dependant pathway, PI3K/AKT and ERK signaling. Cel Immunol 273: 10–16.
View Article
Google Scholar

[11] View Article

[12] Google Scholar

[ref5] 5. Ebaid H, Al-Khalifa M, Isa AM, Gadoa S (2012) Bioactivity of Samsum ant (Pachycondyla sennaarensis) venom against lipopolysaccharides through antioxidant and upregulation of Akt1 signaling in rats. Lipids in Health Disease 11: 93.
View Article
Google Scholar

[14] View Article

[15] Google Scholar

[ref6] 6. Dkhil M, Abdel BA, Al Quraishi , Al Khalifa M (2010) Anti-inflammatory activities of the venom from samsumants Pachycondyla sennaarensis. Afr J Pharm Pharmacol 4: 115–118.
View Article
Google Scholar

[17] View Article

[18] Google Scholar

[ref7] 7. Orivel J, Dejean A (2001) Comparative effect of the venoms of ants of the genus Pachycondyla (Hymenoptera: Ponerinae). Toxicon 39: 195–201.
View Article
Google Scholar

[20] View Article

[21] Google Scholar

[ref8] 8. Zelezetsky I, Pag U, Antcheva N, Sahl HG, Tossi A (2005) Identification and optimization of an antimicrobial peptide from the ant venom toxin pilosulin. Biochem Biophys 434: 358–364.
View Article
Google Scholar

[23] View Article

[24] Google Scholar

[ref9] 9. Quinet Y, Vieira RHSF, Sousa MR, Evangelista-Barreto NS, Carvalho FCT, et al. (2012) Antibacterial properties of contact defensive secretions in neotropical Crematogaster ants. JVAT 18: 441–445.
View Article
Google Scholar

[26] View Article

[27] Google Scholar

[ref10] 10. Torres AFC, Quinet YP, Havt A, Rádis-Baptista G, Martins AMC (2013) Molecular pharmacology and toxynology of venom from ants.; Rádis-Baptista G, editor. An Integrated view of the molecular recognition and toxinology – From analytical procedures to biomedical applications. Croatia: InTech. pp. 207–222.

[ref11] 11. Lopes KS, Rios E, Dantas RT, Lima C, Linhares M, et al. (2011) Effect of Dinoponera quadriceps venom on chemical-induced seizures models in mice. Rencontres en Toxinologie – Meeting on Toxinology SFET Editions. Available: http://www.sfet.asso.fr. Accessed 20 August 2012.

[ref12] 12. Lopes KS, Rios ERV, Lima CNC, Linhares MI, Torres AFC, et al. (2013) The effects of the brazilian ant Dinoponera quadriceps venom on chemically induced seizure models. Neurochem Intern 63: 141–145.
View Article
Google Scholar

[31] View Article

[32] Google Scholar

[ref13] 13. Sousa PL, Quinet Y, Ponte EL, do Vale JF, Torres AFC, et al. (2012) Venom's antinociceptive property in the primitive ant Dinoponera quadriceps. J Ethnopharmacol 144: 213–216.
View Article
Google Scholar

[34] View Article

[35] Google Scholar

[ref14] 14. Kempf WW (1971) A preliminary review of the Ponerinae ant genus Dinoponera Roger (Hymenoptera: Formicidae). Studia Entomologica 14: 369–394.
View Article
Google Scholar

[37] View Article

[38] Google Scholar

[ref15] 15. Paiva RVS, Brandão CRF (1995) Nests, worker population, and reproductive status of workers, in the giant queenless ponerinae ant Dinoponera Roger Hymenoptera Formicidae. Ethology Ecology and Evolution 7: 297–312.
View Article
Google Scholar

[40] View Article

[41] Google Scholar

[ref16] 16. Lenhart PA, Dash ST, Mackay WP (2013) A revision of the giant Amazonian ants of the genus Dinoponera (Hymenoptera, Formicidae). J Hymenoptera 31: 119–164.
View Article
Google Scholar

[43] View Article

[44] Google Scholar

[ref17] 17. Lattke, J. E. 2003. Subfamilia Ponerinae, p. 261–276 In: F. FERNÁNDEZ (ed.) Introducción a las hormigas de la región Neotropical. Bogotá, Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, 398 p.

[ref18] 18. Wilson EO, Hölldobler B (2005) Eusociality: origin and consequences. Proc Natl Acad Sci 102: 13367–13371.
View Article
Google Scholar

[47] View Article

[48] Google Scholar

[ref19] 19. Monnin T, Peeters C (1998) Monogyny and regulation of worker mating in the queenless ant Dinoponera quadriceps. Animal Behaviour 55: 299–306.
View Article
Google Scholar

[50] View Article

[51] Google Scholar

[ref20] 20. Vasconcellos A, Santana GG, Souza AK (2004) Nest spacing and architecture, and swarming of males of Dinoponera quadriceps (Hymenoptera, Formicidae) in a remnant of the Atlantic forest in Northeast Brazil. Braz J Biol 64: 357–362.
View Article
Google Scholar

[53] View Article

[54] Google Scholar

[ref21] 21. Schoeters E, Billen J (1995) Morphology and ultra structure of the convoluted gland in the ant Dinoponera australis (Hymenoptera:Formicidae). Int J Insect Morphol Embryol 24: 323–332.
View Article
Google Scholar

[56] View Article

[57] Google Scholar

[ref22] 22. Mathias MIC (2007) Glândulas de veneno em formigas. Biológico 69: 229–235.
View Article
Google Scholar

[59] View Article

[60] Google Scholar

[ref23] 23. Holldobler B, Wilson EO (1990). The ants. Cambridge: Harvard University, Belknap Press. 746 p.

[ref24] 24. Haddad V Jr, Cardoso JLC, França FOS, Wen FH (1996) Acidentes por formigas: um problema dermatológico. An Bras Derm 71: 527–530.
View Article
Google Scholar

[63] View Article

[64] Google Scholar

[ref25] 25. Haddad Jr V(2001) Acidentes por formigas. In: Manual de diagnóstico e tratamento de acidentes por animais peçonhentos. Brasília, FNS. pp.65–66

[ref26] 26. Haddad Jr V, Cardoso JLC, Moraes RHP (2005) Description of an injury in a human caused by a false tocandira (Dinoponera gigantea, Perty, 1833) with a revision on folkloric, pharmacological and clinical aspects of the giant ants of the genera Paraponera and Dinoponera (sub-family ponerinae). Rev Inst Med Trop 47: 235–238.
View Article
Google Scholar

[67] View Article

[68] Google Scholar

[ref27] 27. Cruz Lopez L, Morgan ED (1997) Explanation of bitter taste of venom of ponerine ant Pachycondyla apicalis. J Chem Ecol 23: 705–712.
View Article
Google Scholar

[70] View Article

[71] Google Scholar

[ref28] 28. Duval A, Malécot CO, Pelhate M, Piek T (1992) Poneratoxin, a new toxin from ant venom, reveals an interconversion between two gating modes of the Na channels in frog skeletal muscle fibres. Pflugers Arch 420: 239–247.
View Article
Google Scholar

[73] View Article

[74] Google Scholar

[ref29] 29. Szolajska E, Poznanski J, Ferber ML, Michalik J, Gout E, et al. (2004) Poneratoxin, a neurotoxin from ant venom. Structure and expression in insect cells and construction of a bio-insecticide. Europ J Biochem 271: 2127–2136.
View Article
Google Scholar

[76] View Article

[77] Google Scholar

[ref30] 30. Johnson SR, Copello JA, Evans MS, Suarez AV (2010) A biochemical characterization of the major peptides from the Venom of the giant Neotropical hunting ant Dinoponera australis. Toxicon 55: 702–710.
View Article
Google Scholar

[79] View Article

[80] Google Scholar

[ref31] 31. Min XJ, Butler G, Storms R, Tsang A (2005) OrfPredictor: predicting protein-coding regions in EST-derived sequences. Nucleic Acids Res 33: 677–680.
View Article
Google Scholar

[82] View Article

[83] Google Scholar

[ref32] 32. Menne KM, Hermjakob H, Apweiler R (2000) A comparison of signal sequence prediction methods using a test set of signal peptides. Bioinformatics 16: 741–742.
View Article
Google Scholar

[85] View Article

[86] Google Scholar

[ref33] 33. Zientz E, Feldhaar H, Stoll S, Gross R (2005) Insights into the microbial world associated with ants. Arch Microbiol 184: 199–206.
View Article
Google Scholar

[88] View Article

[89] Google Scholar

[ref34] 34. Boulogne I, Germosen-Robineau L, Ozier-Lafontaine H, Jacoby-Koaly C, Aurela L, Loranger-Merciris G (2012) Acromyrmex octospinosus (Hymenoptera: Formicidae) management. Part 1: Effects of TRAMIL's insecticidal plant extracts. Pest Manag Sci 68: 313–320.
View Article
Google Scholar

[91] View Article

[92] Google Scholar

[ref35] 35. Ward PS (2006) Ants. Curr Biol 7:16: R152–155.
View Article
Google Scholar

[94] View Article

[95] Google Scholar

[ref36] 36. Gruber CW, Muttenthaler M (2012) Discovery of Defense- and Neuropeptides in Social Ants by Genome-Mining. PLoS ONE 7 (3) e32559.
View Article
Google Scholar

[97] View Article

[98] Google Scholar

[ref37] 37. Raìdis-Baptista G (2011). Molecular Toxinology – Cloning Toxin Genes for Addressing Functional Analysis and Disclosure Drug Leads.; Brown, G, editor. Molecular Cloning - Selected Applications in Medicine and Biology. Croatia: InTech. pp. 161–196.

[ref38] 38. Ryner LC, Goodwin SF, Castrillon DH, Anand A, Villella A, et al. (1996) Control of male sexual behavior and sexual orientation in Drosophila by the fruitless gene. Cell 87: 1079–1089.
View Article
Google Scholar

[101] View Article

[102] Google Scholar

[ref39] 39. Ito H, Fujitani K, Usui K, Shimizu-Nishikawa K, Tanaka S, et al. (1996) Sexual orientation in Drosophila is altered by the satori mutation in the sex-determination gene fruitless that encodes a zinc finger protein with a BTB domain. Proc Natl Acad Sci U S A 93: 9687–9692.
View Article
Google Scholar

[104] View Article

[105] Google Scholar

[ref40] 40. Zollman S, Godt D, Privé GG, Couderc JL, Laski FA (1994) The BTB domain, found primarily in zinc finger proteins, defines an evolutionarily conserved family that includes several developmentally regulated genes in Drosophila,. Proc Natl Acad Sci U S A 91: 10717–10721.
View Article
Google Scholar

[107] View Article

[108] Google Scholar

[ref41] 41. Dauwalder B, Tsujimoto S, Moss J, Mattox W (2002) The Drosophila takeout gene is regulated by the somatic sex-determination pathway and affects male courtship behavior. Genes Dev 16: 2879–2892.
View Article
Google Scholar

[110] View Article

[111] Google Scholar

[ref42] 42. Heinrichs V, Ryner LC, Baker BS (1998) Regulation of sex-specific selection of fruitless 5′ splice sites by transformer and transformer-2. Mol Cell Biol 18: 450–458.
View Article
Google Scholar

[113] View Article

[114] Google Scholar

[ref43] 43. Basha E, O'Neill H, Vierling E (2012) Small heat shock proteins and α-crystallins: dynamic proteins with flexible functions. Trends Biochem Sci 37: 106–117.
View Article
Google Scholar

[116] View Article

[117] Google Scholar

[ref44] 44. Hoffman DR (2010) Ant venoms. Curr Opin Allergy Clin Immunol 10: 342–346.
View Article
Google Scholar

[119] View Article

[120] Google Scholar

[ref45] 45. Karnaky KJ Jr, Hazen-Martin D, Miller DS (2003) The xenobiotic transporter, MRP2, in epithelia from insects, sharks, and the human breast: implications for health and disease. J Exp Zool A Comp Exp Biol 300: 91–97.
View Article
Google Scholar

[122] View Article

[123] Google Scholar

[ref46] 46. O'Donnell MJ (2009) Too much of a good thing: how insects cope with excess ions or toxins in the diet. J Exp Biol 212: 363–72.
View Article
Google Scholar

[125] View Article

[126] Google Scholar

[ref47] 47. Fitzgerald KT, Flood AA (2006) Hymenoptera stings. Clin Tech Small Anim Pract 21: 194–204.
View Article
Google Scholar

[128] View Article

[129] Google Scholar

[ref48] 48. Biló BM, Rueff F, Mosbech H, Bonifazi F, Oude-Elberink JN (2005) Diagnosis of Hymenoptera venom allergy. Allergy 60: 1339–1349.
View Article
Google Scholar

[131] View Article

[132] Google Scholar

[ref49] 49. Hoffman DR, Sakell RH, Schmidt M (2005) Sol i 1, the phospholipase allergen of imported fire ant venom. J Allergy Clin Immunol 115: 611–616.
View Article
Google Scholar

[134] View Article

[135] Google Scholar

[ref50] 50. Padavattan S, Schmidt M, Hoffman DR, Marković-Housley Z (2008) Crystal structure of the major allergen from fire ant venom, Sol i 3. J Mol Biol 383: 178–185.
View Article
Google Scholar

[137] View Article

[138] Google Scholar

[ref51] 51. Gibbs GM, Scanlon MJ, Swarbrick J, Curtis S, Gallant E, Dulhunty AF, O'Bryan MK (2006) The cysteine-rich secretory protein domain of Tpx-1 is related to ion channel toxins and regulates ryanodine receptor Ca2+ signaling. J Biol Chem 281 (7) 4156–4163.
View Article
Google Scholar

[140] View Article

[141] Google Scholar

[ref52] 52. Gibbs GM, Roelants K, O'Bryan MK (2008) The CAP Superfamily: Cysteine-Rich Secretory Proteins, Antigen 5, and Pathogenesis-Related 1 Proteins—Roles in Reproduction, Cancer, and Immune Defense. Endocrine Reviews 29 (7) 865–897.
View Article
Google Scholar

[143] View Article

[144] Google Scholar

[ref53] 53. Zalat S, Schmidt J, Moawad TI (2003) Lipase and phospholipase activities of Hymenoptera venoms (wasps and ants). Egyptian J Biol 5: 138–147.
View Article
Google Scholar

[146] View Article

[147] Google Scholar

[ref54] 54. Seismann H, Blank S, Cifuentes L, Braren I, Bredehorst R, Grunwald T, Ollert M, Spillner E (2010) Recombinant phospholipase A1 (Ves v 1) from yellow jacket venom for improved diagnosis of hymenoptera venom hypersensitivity. Clin Mol Allergy 8: 7.
View Article
Google Scholar

[149] View Article

[150] Google Scholar

[ref55] 55. Vines CM (2012) Phospholipase C. Adv Exp Med Biol 740: 235–254.
View Article
Google Scholar

[152] View Article

[153] Google Scholar

[ref56] 56. Murakami M, Kudo I (2002) Phospholipase A2. JB Minireview-Lipid Signaling Biochem 131: 285–292.
View Article
Google Scholar

[155] View Article

[156] Google Scholar

[ref57] 57. Richmond GS, Smith TK (2011) Phospholipases A1. Inter J Mol Sci 12: 588–612.
View Article
Google Scholar

[158] View Article

[159] Google Scholar

[ref58] 58. Sonoda H, Aoki J, Hiramatsu T, Ishida M, Bandoh K, Nagai Y, Taguchi R, Inoue K, Arai H (2002) A Novel Phosphatidic acid-selective phospholipase A1 that produces lysophosphatidic acid. J Biol Chem 277: 34254–34263.
View Article
Google Scholar

[161] View Article

[162] Google Scholar

[ref59] 59. Aoki J, Nagai Y, Hosono H, Inoue K, Hiroyuki A (2002) Structure and function of phosphatidylserine-specific phospholipase A1. Bioch Biophys Acta 1582: 26–32.
View Article
Google Scholar

[164] View Article

[165] Google Scholar

[ref60] 60. King TP, Spangfort MD (2000) Structure and biology of stinging insect venom allergens. Int Arch Allergy Immunol 123: 99–106.
View Article
Google Scholar

[167] View Article

[168] Google Scholar

[ref61] 61. dos Santos Pinto JR, Fox EG, Saidemberg DM, Santos LD, da Silva Menegasso AR, et al. (2012) Proteomic view of the venom from the fire ant Solenopsis invicta Buren. J Proteome Res 11: 4643–4653.
View Article
Google Scholar

[170] View Article

[171] Google Scholar

[ref62] 62. Huang WN, Chen YH, Chen CL, Wu W (2011) Surface pressure-dependent interactions of secretory phospholipase A2 with zwitterionic phospholipid membranes. Langmuir 27: 7034–7041.
View Article
Google Scholar

[173] View Article

[174] Google Scholar

[ref63] 63. Schmidt JO, Blum MS (1978) A harvester ant venom: chemistry and pharmacology. Science 200: 1064–1066.
View Article
Google Scholar

[176] View Article

[177] Google Scholar

[ref64] 64. Hoffman DR, Peroutka CM, Schmidt M (2008) Imported fire ant venom phospholipase, Sol I 1, is a protein allergen. J Allergy Clin Immunol 121: S30.
View Article
Google Scholar

[179] View Article

[180] Google Scholar

[ref65] 65. Muller UR (2002) Recombinant Hymenoptera venom allergens. Allergy 57: 570–576.
View Article
Google Scholar

[182] View Article

[183] Google Scholar

[ref66] 66. Hoffman DR, Dove DE, Moffitt JE, Stafford CT (1988) Allergens in hymenoptera venom. XXI. Cross-reactivity and multiple reactivity between fire ant venom and bee and wasps venoms. J Allergy Clin Immunol 82: 828–834.
View Article
Google Scholar

[185] View Article

[186] Google Scholar

[ref67] 67. Sin BA, Akdis M, Zumkehr J, Bezzine S, Bekpen C, et al. (2011) T-cell and antibody responses to phospholipase A2 from different species show distinct cross-reactivity patterns. Allergy 66: 1513–1521.
View Article
Google Scholar

[188] View Article

[189] Google Scholar

[ref68] 68. Liscovitch M, Czarny M, Fiucci G, Tang X (2000) Phospholipase D: molecular and cell biology of a novel gene family. Biochem J 3: 401–415.
View Article
Google Scholar

[191] View Article

[192] Google Scholar

[ref69] 69. McDemott M, Wakelam MJ, Morris AJ (2004) Phospholipase D. Biochem Cell Biol 82: 225–253.
View Article
Google Scholar

[194] View Article

[195] Google Scholar

[ref70] 70. Chaim OM, Trevisan-Silva D, Chaves-Moreira D, Wille AC, Ferrer VP, et al. (2011) Brown spider (Loxosceles genus) venom toxins: tools for biological purposes. Toxins 3: 309–344.
View Article
Google Scholar

[197] View Article

[198] Google Scholar

[ref71] 71. Magalhães GS, Caporrino MC, Della-Casa MS, Kimura LF, Prezotto-Neto JP, et al. (2013) Cloning, expression and characterization of a phospholipase D from Loxosceles gaucho venom gland. Biochimie 9: 1773–1783.
View Article
Google Scholar

[200] View Article

[201] Google Scholar

[ref72] 72. Hatfield MJ, Potter PM (2011) Carboxylesterase inhibitors. Expert Opin Ther 8: 1159–1171.
View Article
Google Scholar

[203] View Article

[204] Google Scholar

[ref73] 73. Cui F, Lin Z, Wang H, Liu S, Chang H, et al. (2011) Two single mutations commonly cause qualitative change of nonspecific carboxylesterases in insects. Insect Biochem Mol Biol 41: 1–8.
View Article
Google Scholar

[206] View Article

[207] Google Scholar

[ref74] 74. Werren JH, Richards S, Desjardins CA, Niehuis O, Gadau J, et al. (2010) Functional and evolutionary insights from the genomes of three parasitoid Nasonia species. Science 327: 343–348.
View Article
Google Scholar

[209] View Article

[210] Google Scholar

[ref75] 75. Bonasio R, Zhang G, Ye C, Mutti NS, Fang X, et al. (2010) Genomic comparison of the ants Camponotus floridanus and Harpegnathos saltator. Science 329: 1068–1071.
View Article
Google Scholar

[212] View Article

[213] Google Scholar

[ref76] 76. Blank S, Seismann H, Bockisch B, Braren I, Bredehorst R, et al. (2008) Identification and recombinant expression of a novel IgE-reactive 70 kDa carboxylesterase from Apis mellifera venom. Available: http://www.uniprot.org/uniprot/B2D0J5. Accessed 19 September 2013.

[ref77] 77. Shai Y (2002) Mode of action of membrane active antimicrobial peptides. Biopolymers 66: 236–248.
View Article
Google Scholar

[216] View Article

[217] Google Scholar

[ref78] 78. Huang Y, Huang J, Chen Y (2010) Alpha-helical cationic antimicrobial peptides: relationships of structure and function. Protein Cell 2: 143–152.
View Article
Google Scholar

[219] View Article

[220] Google Scholar

[ref79] 79. Inagaki H, Akagi M, Imai HT, Taylor RW, Kubo T (2004) Molecular cloning and biological characterization of novel antimicrobial peptides, pilosulin 3 and pilosulin 4, from a species of the Australian ant genus Myrmecia. Arch Biochem Biophys 428: 170–178.
View Article
Google Scholar

[222] View Article

[223] Google Scholar

[ref80] 80. Inagaki H, Akagi M, Imai HT, Taylor RW, Wiese MD, et al. (2008) Pilosulin 5, a novel histamine-releasing peptide of the Australian ant, Myrmecia pilosula (Jack Jumper Ant). Arch Biochem Biophys 477: 411–416.
View Article
Google Scholar

[225] View Article

[226] Google Scholar

[ref81] 81. Kozlov S, Malyavka A, McCutchen B, Lu A, Schepers E, et al. (2005) A novel strategy for the identification of toxin like structures in spider venom. Proteins 59: 131–140.
View Article
Google Scholar

[228] View Article

[229] Google Scholar

[ref82] 82. Asgari S, Zhang G, Zareie R, Schmidt O (2003) A serine proteinase homolog venom protein from an endoparasitoid wasp inhibits melanization of the host hemolymph. Insect Biochem Mol Biol 33: 1017–1024.
View Article
Google Scholar

[231] View Article

[232] Google Scholar

[ref83] 83. Parkinson NM, Conyers C, Keen J, MacNicoll A, Smith I, et al. (2004) Towards a comprehensive view of the primary structure of venom proteins from the parasitoid wasp Pimpla hypochondriaca. Insect Biochem Mol Biol 34: 565–571.
View Article
Google Scholar

[234] View Article

[235] Google Scholar

[ref84] 84. Daly NL, Craik DJ (2011) Bioactive cystine knot proteins. Curr Opin Chem Biol 15: 362–368.
View Article
Google Scholar

[237] View Article

[238] Google Scholar

[ref85] 85. Gracy J, Chiche L (2011) Structure and modeling of knottins, a promising molecular scaffold for drug discovery. Curr Pharm Des 17: 4337–4350.
View Article
Google Scholar

[240] View Article

[241] Google Scholar

[ref86] 86. Zhang Z, Zhu S (2012) Comparative genomics analysis of five families of antimicrobial peptide-like genes in seven ant species. Dev Comp Immunol 38: 262–274.
View Article
Google Scholar

[243] View Article

[244] Google Scholar

[ref87] 87. Masek T, Vopalensky V, Suchomelova P, Pospisek M (2005) Denaturing RNA electrophoresis in TAE agarose gels. Anal Biochem 336: 46–50.
View Article
Google Scholar

[246] View Article

[247] Google Scholar

[ref88] 88. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, et al. (2011) Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol 29: 644–652.
View Article
Google Scholar

[249] View Article

[250] Google Scholar

[ref89] 89. Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21: 3674–3676.
View Article
Google Scholar

[252] View Article

[253] Google Scholar

[ref90] 90. Ye J, Fang L, Zheng H, Zhang Y, Chen J, et al. (2006) WEGO: a web tool for plotting GO annotations. Nucleic Acids Res 34: 293–297.
View Article
Google Scholar

[255] View Article

[256] Google Scholar

[ref91] 91. Gelly JC, Gracy J, Kaas Q, Le-Nguyen D, Heitz A, Chiche L (2004) The KNOTTIN website and database: a new information system dedicated to the knottin scaffold. Nucleic Acids Res 32: 156–159.
View Article
Google Scholar

[258] View Article

[259] Google Scholar

[ref92] 92. Matuszek MA, Hodgson WC, Sutherland SK, King RG (1992) Pharmacological studies of jumper ant (Myrmecia pilosula) venom: evidence for the presence of histamine, and haemolytic and eicosanoid-releasing factors. Toxicon 30: 1081–1091.
View Article
Google Scholar

[261] View Article

[262] Google Scholar

Figures

Abstract

Background

Results

Conclusions

Introduction

Results

Sanger sequencing and cluster assemblies

Ion Torrent deep RNA sequencing and read assembly

Annotation of transcript clusters related to D. quadriceps venomics

Clusters related to proteins involved in venom gland physiology

Inhibitor cystine knot- (ICK-) toxin transcripts in the venom gland of D. quadriceps

Discussion

Conclusions

Methods

Dinoponera quadriceps sampling and processing

RNA isolation and venom gland cDNA library construction

Sanger sequencing of independent clones

Data analysis of Sanger sequencing

Sample preparation and deep Sequencing using Ion Torrent Technology

Sequence filtering and (de novo) assembly of deep sequencing data

Gene ontology and functional annotation

Bioinformatic analyses of similarities and molecular phylogenies

Supporting Information

Author Contributions

References