The genome of the ant Tetramorium bicarinatum reveals a tandem organization of venom peptides genes allowing the prediction of their regulatory and evolutionary profiles.

BACKGROUND: Venoms have evolved independently over a hundred times in the animal kingdom to deter predators and/or subdue prey. Venoms are cocktails of various secreted toxins, whose origin and diversification provide an appealing system for evolutionary researchers. Previous studies of the ant venom of Tetramorium bicarinatum revealed several Myrmicitoxin (MYRTX) peptides that gathered into seven precursor families suggesting different evolutionary origins. Analysis of the T. bicarinatum genome enabling further genomic approaches was necessary to understand the processes underlying the evolution of these myrmicitoxins. RESULTS: Here, we sequenced the genome of Tetramorium bicarinatum and reported the organisation of 44 venom peptide genes (vpg). Of the eleven chromosomes that make up the genome of T. bicarinatum, four carry the vpg which are organized in tandem repeats. This organisation together with the ML evolutionary analysis of vpg sequences, is consistent with evolution by local duplication of ancestral genes for each precursor family. The structure of the vpg into two or three exons is conserved after duplication events while the promoter regions are the least conserved parts of the vpg even for genes with highly identical sequences. This suggests that enhancer sequences were not involved in duplication events, but were recruited from surrounding regions. Expression level analysis revealed that most vpg are highly expressed in venom glands, although one gene or group of genes is much more highly expressed in each family. Finally, the examination of the genomic data revealed that several genes encoding transcription factors (TFs) are highly expressed in the venom glands. The search for binding sites (BS) of these TFs in the vpg promoters revealed hot spots of GATA sites in several vpg families. CONCLUSION: In this pioneering investigation on ant venom genes, we provide a high-quality assembly genome and the annotation of venom peptide genes that we think can fosters further genomic research to understand the evolutionary history of ant venom biochemistry.

Antimicrobial Peptide Arsenal Predicted from the Venom Gland Transcriptome of the Tropical Trap-Jaw Ant Odontomachus chelifer.

With about 13,000 known species, ants are the most abundant venomous insects. Their venom consists of polypeptides, enzymes, alkaloids, biogenic amines, formic acid, and hydrocarbons. In this study, we investigated, using in silico techniques, the peptides composing a putative antimicrobial arsenal from the venom gland of the neotropical trap-jaw ant Odontomachus chelifer. Focusing on transcripts from the body and venom gland of this insect, it was possible to determine the gland secretome, which contained about 1022 peptides with putative signal peptides. The majority of these peptides (75.5%) were unknown, not matching any reference database, motivating us to extract functional insights via machine learning-based techniques. With several complementary methodologies, we investigated the existence of antimicrobial peptides (AMPs) in the venom gland of O. chelifer, finding 112 non-redundant candidates. Candidate AMPs were predicted to be more globular and hemolytic than the remaining peptides in the secretome. There is evidence of transcription for 97% of AMP candidates across the same ant genus, with one of them also verified as translated, thus supporting our findings. Most of these potential antimicrobial sequences (94.8%) matched transcripts from the ant's body, indicating their role not solely as venom toxins.

Transcriptomic and biochemical analysis from the venom gland of the neotropical ant Odontomachus chelifer.

The genus Odontomachus is widely distributed in neotropical areas throughout Central and South America. It is a stinging ant that subdues its prey (insects) by injecting them a cocktail of toxic molecules (venom). Ant venoms are generally composed of formic acid, alkaloids, hydrocarbons, amines, peptides, and proteins. Odontomachus chelifer is an ant that inhabits neotropical regions from Mexico to Argentina. Unlike the venom of other animals such as scorpions, spiders and snakes, this ant venom has seldom been analyzed comprehensively, and their compositions are not yet completely known. In the present study, we performed a partial investigation of enzymatic and functional activities of O. chelifer ant venom, and we provide a global insight on the transcripts expressed in the venom gland to better understand their properties. The crude venom showed phospholipase A2 and antiparasitic activities. RNA sequencing (Illumina platform) of the venom gland of O. chelifer generated 61, 422, 898 reads and de novo assembly Trinity generated 50,220 contigs. BUSCO analysis against Arthropoda_db10 showed that 92.89% of the BUSCO groups have complete gene representation (single-copy or duplicated), while 4.05% are only partially recovered, and 3.06% are missing. The 30 most expressed genes in O. chelifer venom gland transcriptome included important transcripts involved in venom function such as U-poneritoxin (01)-Om1a-like (pilosulin), chitinase 2, venom allergen 3, chymotrypsin 1 and 2 and glutathione S-transferase. Analysis of the molecular function revealed that the largest number of transcripts were related to catalytic activity, including phospholipases. These data emphasize the potential of O. chelifer venom for prospection of molecules with biotechnological application.

Multipurpose peptides: The venoms of Amazonian stinging ants contain anthelmintic ponericins with diverse predatory and defensive activities.

In the face of increasing drug resistance, the development of new anthelmintics is critical for controlling nematodes that parasitise livestock. Although hymenopteran venom toxins have attracted attention for applications in agriculture and medicine, few studies have explored their potential as anthelmintics. Here we assessed hymenopteran venoms as a possible source of new anthelmintic compounds by screening a panel of ten hymenopteran venoms against Haemonchus contortus, a major pathogenic nematode of ruminants. Using bioassay-guided fractionation coupled with liquid chromatography-tandem mass spectrometry, we identified four novel anthelmintic peptides (ponericins) from the venom of the neotropical ant Neoponera commutata and the previously described ponericin M-PONTX-Na1b from Neoponera apicalis venom. These peptides inhibit H. contortus development with IC50 values of 2.8-5.6 µM. Circular dichroism spectropolarimetry indicated that the ponericins are unstructured in aqueous solution but adopt α-helical conformations in lipid mimetic environments. We show that the ponericins induce non-specific membrane perturbation, which confers broad-spectrum antimicrobial, insecticidal, cytotoxic, hemolytic, and algogenic activities, with activity across all assays typically correlated. We also show for the first time that ponericins induce spontaneous pain behaviour when injected in mice. We propose that the broad-spectrum activity of the ponericins enables them to play both a predatory and defensive role in neoponeran ants, consistent with their high abundance in venom. This study reveals a broader functionality for ponericins than previously assumed, and highlights both the opportunities and challenges in pursuing ant venom peptides as potential therapeutics.

An Integrated Proteomic and Transcriptomic Analysis Reveals the Venom Complexity of the Bullet Ant Paraponera clavata.

A critical hurdle in ant venom proteomic investigations is the lack of databases to comprehensively and specifically identify the sequence and function of venom proteins and peptides. To resolve this, we used venom gland transcriptomics to generate a sequence database that was used to assign the tandem mass spectrometry (MS) fragmentation spectra of venom peptides and proteins to specific transcripts. This was performed alongside a shotgun liquid chromatography-mass spectrometry (LC-MS/MS) analysis of the venom to confirm that these assigned transcripts were expressed as proteins. Through the combined transcriptomic and proteomic investigation of Paraponera clavata venom, we identified four times the number of proteins previously identified using 2D-PAGE alone. In addition to this, by mining the transcriptomic data, we identified several novel peptide sequences for future pharmacological investigations, some of which conform with inhibitor cysteine knot motifs. These types of peptides have the potential to be developed into pharmaceutical or bioinsecticide peptides.

A comprehensive portrait of the venom of the giant red bull ant, Myrmecia gulosa, reveals a hyperdiverse hymenopteran toxin gene family.

Ants (Hymenoptera: Formicidae) are diverse and ubiquitous, and their ability to sting is familiar to many of us. However, their venoms remain largely unstudied. We provide the first comprehensive characterization of a polypeptidic ant venom, that of the giant red bull ant, Myrmecia gulosa. We reveal a suite of novel peptides with a range of posttranslational modifications, including disulfide bond formation, dimerization, and glycosylation. One venom peptide has sequence features consistent with an epidermal growth factor fold, while the remaining peptides have features suggestive of a capacity to form amphipathic helices. We show that these peptides are derived from what appears to be a single, pharmacologically diverse, gene superfamily (aculeatoxins) that includes most venom peptides previously reported from the aculeate Hymenoptera. Two aculeatoxins purified from the venom were found to be capable of activating mammalian sensory neurons, consistent with the capacity to produce pain but via distinct mechanisms of action. Further investigation of the major venom peptide MIITX1-Mg1a revealed that it can also incapacitate arthropods, indicative of dual utility in both defense and predation. MIITX1-Mg1a accomplishes these functions by generating a leak in membrane ion conductance, which alters membrane potential and triggers neuronal depolarization. Our results provide the first insights into the evolution of the major toxin gene superfamily of the aculeate Hymenoptera and provide a new paradigm in the functional evolution of toxins from animal venoms.

Deciphering the Molecular Diversity of an Ant Venom Peptidome through a Venomics Approach.

The peptide toxins in the venoms of small invertebrates such as stinging ants have rarely been studied due to the limited amount of venom available per individual. We used a venomics strategy to identify the molecular diversity of the venom peptidome for the myrmicine ant Tetramorium bicarinatum. The methodology included (i) peptidomics, in which the venom peptides are sequenced through a de novo mass spectrometry approach or Edman degradation; (ii) transcriptomics, based on RT-PCR-cloning and DNA sequencing; and (iii) the data mining of the RNA-seq in the available transcriptome. Mass spectrometry analysis revealed about 2800 peptides in the venom. However, the de novo sequencing suggested that most of these peptides arose from processing or the artifactual fragmentations of full-length mature peptides. These peptides, called "myrmicitoxins", are produced by a limited number of genes. Thirty-seven peptide precursors were identified and classified into three superfamilies. These precursors are related to pilosulin, secapin or are new ant venom prepro-peptides. The mature myrmicitoxins display sequence homologies with antimicrobial, cytolytic and neurotoxic peptides. The venomics strategy enabled several post-translational modifications in some peptides such as O-glycosylation to be identified. This study provides novel insights into the molecular diversity and evolution of ant venoms.

Combined Venom Gland Transcriptomic and Venom Peptidomic Analysis of the Predatory Ant Odontomachus monticola.

Ants (hymenoptera: Formicidae) have adapted to many different environments and have become some of the most prolific and successful insects. To date, 13,258 ant species have been reported. They have been classified into 333 genera and 17 subfamilies. Except for a few Formicinae, Dolichoderinae, and members of other subfamilies, most ant species have a sting with venom. The venoms are composed of formic acid, alkaloids, hydrocarbons, amines, peptides, and proteins. Unlike the venoms of other animals such as snakes and spiders, ant venoms have seldom been analyzed comprehensively, and their compositions are not yet completely known. In this study, we used both transcriptomic and peptidomic analyses to study the composition of the venom produced by the predatory ant species Odontomachus monticola. The transcriptome analysis yielded 49,639 contigs, of which 92 encoded toxin-like peptides and proteins with 18,106,338 mapped reads. We identified six pilosulin-like peptides by transcriptomic analysis in the venom gland. Further, we found intact pilosulin-like peptide 1 and truncated pilosulin-like peptides 2 and 3 by peptidomic analysis in the venom. Our findings related to ant venom peptides and proteins may lead the way towards development and application of novel pharmaceutical and biopesticidal resources.

A reexamination of poneratoxin from the venom of the bullet ant Paraponera clavata.

In 1991, Piek et al. [45] described a voltage-gated sodium channel (VGSC) modifier from "bullet ant" (Paraponera clavata) venom they called poneratoxin (PoTx). Using UV chromatography and Edman degradation they showed two "identical peptides" of 25 residues. We reinvestigated PoTx using ultra performance liquid chromatography-tandem mass spectrometry (UPLC-TMS). De novo sequencing showed the two peptides were actually structurally different peptides: the originally described PoTx and a glycyl pro-peptide (glycyl-PoTx) that lacks C-terminus amidation. We examined P. clavata venom from different geographical locations and discovered two additional PoTx analogs: an A23E substitution analog and a D22N; A23V substitutions analog. We tested PoTx and these three natural analogs on the mammalian sensory voltage-gated sodium channel, Nav1.7, using whole cell voltage-clamp. PoTx and each analog induced slowly activating currents in response to small depolarizing steps and sustained currents due to blockade of channel inactivation, similar to that described previously in skeletal muscle [19]. Glycyl-PoTx had the same potency and efficacy as PoTx. A23E PoTx, with a decrease in both C-terminal net positive charge and hydrophobicity, had an eight-fold reduction in potency compared to PoTx. In contrast, the D22N; A23V PoTx, with an increase in both C-terminal net positive charge and hydrophobicity, had a nearly five-fold increase in potency compared to PoTx. We found that changes in PoTx C-terminus caused a significant change in PoTx potency.

Biochemical and biophysical combined study of bicarinalin, an ant venom antimicrobial peptide.

We have recently characterized bicarinalin as the most abundant peptide from the venom of the ant Tetramorium bicarinatum. This antimicrobial peptide is active against Staphylococcus and Enterobacteriaceae. To further investigate the antimicrobial properties of this cationic and cysteine-free peptide, we have studied its antibacterial, antifungal and antiparasitic activities on a large array of microorganisms. Bicarinalin was active against fifteen microorganisms with minimal inhibitory concentrations ranging from 2 and 25µmolL(-1). Cronobacter sakazakii, Salmonella enterica, Candida albicans, Aspergilus niger and Saccharomyces cerevisiae were particularly susceptible to this novel antimicrobial peptide. Resistant strains of Staphylococcus aureus, Pseudomonas aeruginosa and C. albicans were as susceptible as the canonical strains. Interestingly, bicarinalin was also active against the parasite Leishmania infantum with a minimal inhibitory concentrations of 2µmolL(-1). The bicarinalin pre-propeptide cDNA sequence has been determined using a combination of degenerated primers with RACE PCR strategy. Interestingly, the N-terminal domain of bicarinalin pre-propeptide exhibited sequence similarity with the pilosulin antimicrobial peptide family previously described in the Myrmecia venoms. Moreover, using SYTOX green uptake assay, we showed that, for all the tested microorganisms, bicarinalin acted through a membrane permeabilization mechanism. Two dimensional-NMR experiments showed that bicarinalin displayed a 10 residue-long α-helical structure flanked by two N- and C-terminal disordered regions. This partially amphipathic helix may explain the membrane permeabilization mechanism of bicarinalin observed in this study. Finally, therapeutic value of bicarinalin was highlighted by its low cytotoxicity against human lymphocytes at bactericidal concentrations and its long half-life in human serum which was around 15h.

Pilosulins: a review of the structure and mode of action of venom peptides from an Australian ant Myrmecia pilosula.

Myrmecia pilosula is an endemic Australian ant whose sting is a frequent cause of insect allergy in southeast Australia, and several deaths due to M. pilosula sting envenomation have been documented. In this review, we discuss the composition and bioactivity of M. pilosula venom. In addition to various enzymes and pharmacologically active constituents, the venom contains four families of highly basic low molecular weight peptides trivially named Pilosulins. These peptides are unique and have low structural homology to other Hymenoptera venom peptides. Moreover, M. pilosula venom is relatively simple in its composition with 5 predominant peptides making up about 90% by weight. These peptides display cytotoxic, hypotensive, histamine-releasing and antimicrobial activities. Within the M. pilosula venom, Pilosulin 3 has been classified as a major allergen and [Ile(5)]pilosulin 1 and Pilosulin 4.1 are classified as minor allergens. Several uncharacterised higher molecular weight components with allergenic activities have also been identified. The revised naming of M. pilosula venom peptides according to the International Union of Immunological Societies (IUIS) criteria for allergen nomenclature is discussed in this review.

De Novo sequencing and transcriptome analysis for Tetramorium bicarinatum: a comprehensive venom gland transcriptome analysis from an ant species.

BACKGROUND: Arthropod venoms are invaluable sources of bioactive substances with biotechnological application. The limited availability of some venoms, such as those from ants, has restricted the knowledge about the composition and the potential that these biomolecules could represent. In order to provide a global insight on the transcripts expressed in the venom gland of the Brazilian ant species Tetramorium bicarinatum and to unveil the potential of its products, high-throughput approach using Illumina technology has been applied to analyze the genes expressed in active venom glands of this ant species. RESULTS: A total of 212,371,758 pairs of quality-filtered, 100-base-pair Illumina reads were obtained. The de novo assemblies yielded 36,042 contigs for which 27,873 have at least one predicted ORF among which 59.77% produce significant hits in the available databases. The investigation of the reads mapping toxin class revealed a high diversification with the major part consistent with the classical hymenopteran venom protein signature represented by venom allergen (33.3%), followed by a diverse toxin-expression profile including several distinct isoforms of phospholipase A1 and A2, venom serine protease, hyaluronidase, protease inhibitor and secapin. Moreover, our results revealed for the first time the presence of toxin-like peptides that have been previously identified from unrelated venomous animals such as waprin-like (snakes) and agatoxins (spiders and conus).The non-toxin transcripts were mainly represented by contigs involved in protein folding and translation, consistent with the protein-secretory function of the venom gland tissue. Finally, about 40% of the generated contigs have no hits in the databases with 25% of the predicted peptides bearing signal peptide emphasizing the potential of the investigation of these sequences as source of new molecules. Among these contigs, six putative novel peptides that show homologies with previously identified antimicrobial peptides were identified. CONCLUSIONS: To the best of our knowledge, this work reports the first large-scale analysis of genes transcribed by the venomous gland of the ant species T. bicarinatum and helps with the identification of Hymenoptera toxin arsenal. In addition, results from this study demonstrate that de novo transcriptome assembly allows useful venom gene expression analysis in a species lacking a genome sequence database.

Transcriptome analysis in venom gland of the predatory giant ant Dinoponera quadriceps: insights into the polypeptide toxin arsenal of hymenopterans.

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.

Elucidation of the unexplored biodiversity of ant venom peptidomes via MALDI-TOF mass spectrometry and its application for chemotaxonomy.

The rise of integrative taxonomy, a multi-criteria approach used in characterizing species, fosters the development of new tools facilitating species delimitation. Mass spectrometric (MS) analysis of venom peptides from venomous animals has previously been demonstrated to be a valid method for identifying species. Here we aimed to develop a rapid chemotaxonomic tool for identifying ants based on venom peptide mass fingerprinting. The study focused on the biodiversity of ponerine ants (Hymenoptera: Formicidae: Ponerinae) in French Guiana. Initial experiments optimized the use of automated matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to determine variations in the mass profiles of ant venoms using several MALDI matrices and additives. Data were then analyzed via a hierarchical cluster analysis to classify the venoms of 17 ant species. In addition, phylogenetic relationships were assessed and were highly correlated with methods using DNA sequencing of the mitochondrial gene cytochrome c oxidase subunit 1. By combining a molecular genetics approach with this chemotaxonomic approach, we were able to improve the accuracy of the taxonomic findings to reveal cryptic ant species within species complexes. This chemotaxonomic tool can therefore contribute to more rapid species identification and more accurate taxonomies. BIOLOGICAL SIGNIFICANCE: This is the first extensive study concerning the peptide analysis of the venom of both Pachycondyla and Odontomachus ants. We studied the venoms of 17 ant species from French Guiana that permitted us to fine-tune the venom analysis of ponerine ants via MALDI-TOF mass spectrometry. We explored the peptidomes of crude ant venom and demonstrated that venom peptides can be used in the identification of ant species. In addition, the application of this novel chemotaxonomic method combined with a parallel genetic approach using COI sequencing permitted us to reveal the presence of cryptic ants within both the Pachycondyla apicalis and Pachycondyla stigma species complexes. This adds a new dimension to the search for means of exploiting the enormous biodiversity of venomous ants as a source for novel therapeutic drugs or biopesticides. This article is part of a Special Issue entitled: Proteomics of non-model organisms.

Identification, expression, and immuno-reactivity of Sol i 2 & Sol i 4 venom proteins of queen red imported fire ants, Solenopsis invicta Buren (Hymenoptera: Formicidae).

We report on two low-molecular weight proteins that are stored in the venom of queen red imported fire ants (Solenopsis invicta). Translated amino acid sequences identified one protein to have 74.8% identity with the Sol i 2w worker allergen, and the other protein was found to have 96/97% identity with Sol i 4.01w/4.02w worker allergens. Both Sol i 2 and Sol i 4 queen and worker proteins were expressed using pEXP1-DEST vector in SHuffle™ T7 Express lysY Escherichia coli. Proteins were expressed at significant concentrations, as opposed to the µg/ml amounts by our previous expression methods, enabling further study of these proteins. Sol i 2q protein bound weakly to human IgE, sera pooled from allergic patients, whereas Sol i 2w, Sol i 4.01w, and Sol i 4q proteins bound strongly. Despite Sol i 2w and Sol i 2q proteins having 74.8% identity, the queen protein is less immuno-reactive than the worker allergen. This finding is consistent with allergic individuals being less sensitive to queen than worker venom.

Similarity in venom alkaloid chemistry of alate queens of imported fire ants: implication for hybridization between Solenopsis richteri and S. invicta in the Southern United States.

Both cis- and trans-2-methyl-6-undecylpiperidines, MC11P, have been previously reported as the major components of the venom of alate queens of the imported fire ants, Solenopsis richteri (black) and S. invicta (red). To identify the minor components of venom alkaloids from alate queens and compare the venom alkaloid chemistry of alate queen of their hybrid (S. richteri×S. invicta) with that of the two parental fire ant species (S. richteri and S. invicta), silica-gel short-column chromatography was utilized for separating cis-stereoisomers of venom alkaloids from trans-stereoisomers. GC/MS Analyses of venom-alkaloid chemistry of alate queens demonstrated that fewer alkaloid peaks were detected in the chromatograms of the alate queens compared to those of workers. Three new compounds, 7, 12, and 13, were detected as minor components in the venom of alate queens of all three fire ant species. Alate queens of hybrid fire ants showed cis- and trans-alkaloid patterns similar to those of the parental species. Similarity in venom-alkaloid chemistry of alate queens of S. richteri and S. invicta, and their hybrid may indicate their reproductive compatibility in the hybrid zone in southern United States, where all three species occur sympatrically.

Interspecific variation of Delta1,6-piperideines in imported fire ants.

Delta(1,6)-piperideines have been recently reported in the venom of the red imported fire ants, Solenopsis invicta Buren and the black imported fire ants, Solenopsis richteri Forel. However, they have never been quantified in either species. Furthermore, there is no information available about those piperideines in the hybrid imported fire ants (S. invicta x S. richteri). The abundance of six Delta(1,6)-piperideines was investigated in both species and their hybrid using gas chromatography-mass spectrometer (GC-MS). They include 2-methyl-6-tridecenyl-6-piperideine, 2-methyl-6-pentadecenyl-6-piperideine, 2-methyl-6-heptadecenyl-6-piperideine, 2-methyl-6-tridecyl-6-piperideine, 2-methyl-6-pentadecyl-6-piperideine, and 2-methyl-6-heptadecyl-6-piperideine. S. invicta produced all six Delta(1,6)-piperideines, whereas, S. richteri did not produce 2-methyl-6-heptadecenyl-6-piperideine and 2-methyl-6-heptadecyl-6-piperideine. The Delta(1,6)-piperideine profiles of the hybrid was similar to that of S. richteri, except trace amounts of 2-methyl-6-heptadecenyl-6-piperideine and 2-methyl-6-heptadecyl-6-piperideine were found in some of the samples. The ratio of 2-methyl-6-pentadecenyl-6-piperideine to 2-methyl-6-pentadecyl-6-piperideine (C(15:1)/C(15:0)) was significantly different among two species and their hybrid. In addition to Delta(1,6)-piperideines, hybrid workers also contained significantly more piperidines than their parent species. This is the first evidence of heterosis of imported fire ants in venom production.

Crystal structure of the major allergen from fire ant venom, Sol i 3.

Fire ant venom is an extremely potent allergy-inducing agent containing four major allergens, Sol i 1 to Sol i 4, which are the most frequent cause of hypersensitivity reactions to hymenoptera in the southern USA. The crystal structure of recombinant (Baculovirus) major fire ant allergen Sol i 3 has been determined to a resolution of 3.1 A by the method of molecular replacement. The secondary-structure elements of Sol i 3 are arranged in an alpha-beta-alpha sandwich fold consisting of a central antiparallel beta-sheet surrounded on both sides by alpha helices. The overall structure is very similar to that of the homologous wasp venom allergen Ves v 5 with major differences occurring in the solvent-exposed loop regions that contain amino acid insertions. Consequently, the limited conservation of surface chemical properties and topology between Sol i 3 and Ves v 5 may explain the observed lack of relevant cross-reactivity. It is concluded that Sol i 3 recognizes immunoglobulin E antibodies with a distinct set of its own epitopes, which are different from those of Ves v 5. Indeed, the molecular area in Sol i 3 covered by non-conserved residues is large enough to accommodate four unique Sol i 3 epitopes.

Hymenoptera venom allergens.

Hymenoptera venoms each contain a variety of protein allergens. The major components have all been characterized, and most of the amino acid sequences are known. This article concentrates on the use of contemporary techniques including cloning, mass spectrometry and genomics in the characterization of venom allergens, and newer separation techniques for protein isolation. Examples of the use of these techniques with venom proteins are presented.

Molecular cloning and biological characterization of novel antimicrobial peptides, pilosulin 3 and pilosulin 4, from a species of the Australian ant genus Myrmecia.

Venom of an Australian ant species of the Myrmecia pilosula species complex (mss. name Myrmecia banksi Taylor) contains two major allergenic peptides, pilosulin 1 and pilosulin 2. To obtain novel cDNA clones that encode the pilosulin-related bioactive peptides, mRNA of another Myrmecia species was subjected to RT-PCR in which the forward primer corresponds to a nucleotide sequence in the leader sequences of pilosulin 1 and pilosulin 2. As a result, we isolated cDNA clones encoding the novel antimicrobial peptides pilosulin 3 and pilosulin 4. The nucleotide and the amino acid sequences of all four pilosulins have high homology except for the mature peptide coding regions. Synthetic pilosulin 3 and pilosulin 4 peptides displayed antimicrobial activity with histamine-releasing and low hemolytic activities.