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.

Peptide toxins that target vertebrate voltage-gated sodium channels underly the painful stings of harvester ants.

Harvester ants (genus Pogonomyrmex) are renowned for their stings which cause intense, long-lasting pain, and other neurotoxic symptoms in vertebrates. Here, we show that harvester ant venoms are relatively simple and composed largely of peptide toxins. One class of peptides is primarily responsible for the long-lasting local pain of envenomation via activation of peripheral sensory neurons. These hydrophobic, cysteine-free peptides potently modulate mammalian voltage-gated sodium (NaV) channels, reducing the voltage threshold for activation and inhibiting channel inactivation. These toxins appear to have evolved specifically to deter vertebrates.

Discovery of an Insect Neuroactive Helix Ring Peptide from Ant Venom.

Ants are among the most abundant terrestrial invertebrate predators on Earth. To overwhelm their prey, they employ several remarkable behavioral, physiological, and biochemical innovations, including an effective paralytic venom. Ant venoms are thus cocktails of toxins finely tuned to disrupt the physiological systems of insect prey. They have received little attention yet hold great promise for the discovery of novel insecticidal molecules. To identify insect-neurotoxins from ant venoms, we screened the paralytic activity on blowflies of nine synthetic peptides previously characterized in the venom of Tetramorium bicarinatum. We selected peptide U11, a 34-amino acid peptide, for further insecticidal, structural, and pharmacological experiments. Insecticidal assays revealed that U11 is one of the most paralytic peptides ever reported from ant venoms against blowflies and is also capable of paralyzing honeybees. An NMR spectroscopy of U11 uncovered a unique scaffold, featuring a compact triangular ring helix structure stabilized by a single disulfide bond. Pharmacological assays using Drosophila S2 cells demonstrated that U11 is not cytotoxic, but suggest that it may modulate potassium conductance, which structural data seem to corroborate and will be confirmed in a future extended pharmacological investigation. The results described in this paper demonstrate that ant venom is a promising reservoir for the discovery of neuroactive insecticidal peptides.

Ant venoms contain vertebrate-selective pain-causing sodium channel toxins.

Stings of certain ant species (Hymenoptera: Formicidae) can cause intense, long-lasting nociception. Here we show that the major contributors to these symptoms are venom peptides that modulate the activity of voltage-gated sodium (NaV) channels, reducing their voltage threshold for activation and inhibiting channel inactivation. These peptide toxins are likely vertebrate-selective, consistent with a primarily defensive function. They emerged early in the Formicidae lineage and may have been a pivotal factor in the expansion of ants.

The mechanism underlying toxicity of a venom peptide against insects reveals how ants are master at disrupting membranes.

Hymenopterans represent one of the most abundant groups of venomous organisms but remain little explored due to the difficult access to their venom. The development of proteo-transcriptomic allowed us to explore diversity of their toxins offering interesting perspectives to identify new biological active peptides. This study focuses on U9 function, a linear, amphiphilic and polycationic peptide isolated from ant Tetramorium bicarinatum venom. It shares physicochemical properties with M-Tb1a, exhibiting cytotoxic effects through membrane permeabilization. In the present study, we conducted a comparative functional investigation of U9 and M-Tb1a and explored the mechanisms underlying their cytotoxicity against insect cells. After showing that both peptides induced the formation of pores in cell membrane, we demonstrated that U9 induced mitochondrial damage and, at high concentrations, localized into cells and induced caspase activation. This functional investigation highlighted an original mechanism of U9 questioning on potential valorization and endogen activity in T. bicarinatum venom.

Venomics survey of six myrmicine ants provides insights into the molecular and structural diversity of their peptide toxins.

Among ants, Myrmicinae represents the most speciose subfamily. The venom composition previously described for these social insects is extremely variable, with alkaloids predominant in some genera while, conversely, proteomics studies have revealed that some myrmicine ant venoms are peptide-rich. Using integrated transcriptomic and proteomic approaches, we characterized the venom peptidomes of six ants belonging to the different tribes of Myrmicinae. We identified a total of 79 myrmicitoxins precursors which can be classified into 38 peptide families according to their mature sequences. Myrmicine ant venom peptidomes showed heterogeneous compositions, with linear and disulfide-bonded monomers as well as dimeric toxins. Several peptide families were exclusive to a single venom whereas some were retrieved in multiple species. A hierarchical clustering analysis of precursor signal sequences led us to divide the myrmicitoxins precursors into eight families, including some that have already been described in other aculeate hymenoptera such as secapin-like peptides and voltage-gated sodium channel (NaV) toxins. Evolutionary and structural analyses of two representatives of these families highlighted variation and conserved patterns that might be crucial to explain myrmicine venom peptide functional adaptations to biological targets.

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.

Heterodimeric Insecticidal Peptide Provides New Insights into the Molecular and Functional Diversity of Ant Venoms.

Ants use venom for predation, defense, and communication; however, the molecular diversity, function, and potential applications of ant venom remains understudied compared to other venomous lineages such as arachnids, snakes and cone snails. In this work, we used a multidisciplinary approach that encompassed field work, proteomics, sequencing, chemical synthesis, structural analysis, molecular modeling, stability studies, and in vitro and in vivo bioassays to investigate the molecular diversity of the venom of the Amazonian Pseudomyrmex penetrator ants. We isolated a potent insecticidal heterodimeric peptide Δ-pseudomyrmecitoxin-Pp1a (Δ-PSDTX-Pp1a) composed of a 27-residue long A-chain and a 33-residue long B-chain cross-linked by two disulfide bonds in an antiparallel orientation. We chemically synthesized Δ-PSDTX-Pp1a, its corresponding parallel AA and BB homodimers, and its monomeric chains and demonstrated that Δ-PSDTX-Pp1a had the most potent insecticidal effects in blowfly assays (LD50 = 3 nmol/g). Molecular modeling and circular dichroism studies revealed strong α-helical features, indicating its cytotoxic effects could derive from cell membrane pore formation or disruption. The native heterodimer was substantially more stable against proteolytic degradation (t 1/2 = 13 h) than its homodimers or monomers (t 1/2 < 20 min), indicating an evolutionary advantage of the more complex structure. The proteomic analysis of Pseudomyrmex penetrator venom and in-depth characterization of Δ-PSDTX-Pp1a provide novel insights in the structural complexity of ant venom and further exemplifies how nature exploits disulfide-bond formation and dimerization to gain an evolutionary advantage via improved stability, a concept that is highly relevant for the design and development of peptide therapeutics, molecular probes, and bioinsecticides.

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.

Venom Peptide Repertoire of the European Myrmicine Ant Manica rubida: Identification of Insecticidal Toxins.

Using an integrated transcriptomic and proteomic approach, we characterized the venom peptidome of the European red ant, Manica rubida. We identified 13 "myrmicitoxins" that share sequence similarities with previously identified ant venom peptides, one of them being identified as an EGF-like toxin likely resulting from a threonine residue modified by O-fucosylation. Furthermore, we conducted insecticidal assays of reversed-phase HPLC venom fractions on the blowfly Lucilia caesar, permitting us to identify six myrmicitoxins (i.e., U3-, U10-, U13-, U20-MYRTX-Mri1a, U10-MYRTX-Mri1b, and U10-MYRTX-Mri1c) with an insecticidal activity. Chemically synthesized U10-MYRTX-Mri1a, -Mri1b, -Mri1c, and U20-MYRTX-Mri1a irreversibly paralyzed blowflies at the highest doses tested (30-125 nmol·g-1). U13-MYRTX-Mri1a, the most potent neurotoxic peptide at 1 h, had reversible effects after 24 h (150 nmol·g-1). Finally, U3-MYRTX-Mri1a has no insecticidal activity, even at up to 55 nmol·g-1. Thus, M. rubida employs a paralytic venom rich in linear insecticidal peptides, which likely act by disrupting cell membranes.

Label-Free, Real-Time Phospholipase-A Isoform Assay.

Phospholipase-A (PLA) enzymes catalyze the hydrolysis of ester bonds in select glycerophospholipids. Sensors for rapidly measuring the PLA activity in biological samples have relevance in the study of venom compositions and in medical diagnostics for the diagnosis of diseases such as acute pancreatitis. Current PLA sensor technologies are often restricted by the time it takes to prepare an assay, the necessity of using fluorescent labels, or the fact they might require strict pH control of the buffer vehicles used. Here we present a tethered bilayer lipid membrane (tBLM) impedance sensor array for the rapid and real-time detection of PLA, which includes the ability to selectively detect phospholipase-A2 (PLA2) from phospholipase-A1 (PLA1) isoforms. Comparing the activity of PLA1 and PLA2 in an array of tBLMs composed of ether phospholipids, ester phospholipids or ether-ester phospholipids allows for the rapid and reliable distinction between the isoforms, as measured using swept-frequency electrical impedance spectroscopy. After testing the assay using pure enzymes, we demonstrate the capacity of the sensor to identify specific PLA2-type, calcium-dependent activity from the venom of the South American bullet ant, Paraponera clavata, at a concentration of 1 µg/mL. The specificity of the phospholipase activity was corroborated using matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry. As further validation, we tested the activities of a PLA1 isoform in the presence of different buffers commonly used in biology and biochemistry experiments. Sensitivity testing shows that PLA1 can be detected at an activity as low as 0.06 U/mL. The rapid and reliable detection of phospholipases presented in this study has potential applications in the study of animal venoms as well as in lipase bioreactors and point-of-care devices.

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

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

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.

Anti-Helicobacter pylori Properties of the Ant-Venom Peptide Bicarinalin.

The venom peptide bicarinalin, previously isolated from the ant Tetramorium bicarinatum, is an antimicrobial agent with a broad spectrum of activity. In this study, we investigate the potential of bicarinalin as a novel agent against Helicobacter pylori, which causes several gastric diseases. First, the effects of synthetic bicarinalin have been tested against Helicobacter pylori: one ATCC strain, and forty-four isolated from stomach ulcer biopsies of Peruvian patients. Then the cytoxicity of bicarinalin on human gastric cells and murine peritoneal macrophages was measured using XTT and MTT assays, respectively. Finally, the preventive effect of bicarinalin was evaluated by scanning electron microscopy using an adherence assay of H. pylori on human gastric cells treated with bicarinalin. This peptide has a potent antibacterial activity at the same magnitude as four antibiotics currently used in therapies against H. pylori. Bicarinalin also inhibited adherence of H. pylori to gastric cells with an IC50 of 0.12 µg·mL-1 and had low toxicity for human cells. Scanning electron microscopy confirmed that bicarinalin can significantly decrease the density of H. pylori on gastric cells. We conclude that Bicarinalin is a promising compound for the development of a novel and effective anti-H. pylori agent for both curative and preventive use.

Combined Peptidomic and Proteomic Analysis of Electrically Stimulated and Manually Dissected Venom from the South American Bullet Ant Paraponera clavata.

Ants have evolved venoms rich in peptides and proteins used for predation, defense, and communication. However, they remain extremely understudied due to the minimal amount of venom secreted by each ant. The present study investigated the differences in the proteome and peptidome of the venom from the bullet ant, Paraponera clavata. Venom samples were collected from a single colony either by manual venom gland dissection or by electrical stimulation and were compared using proteomic methods. Venom proteins were separated by 2D-PAGE and identified by nanoLC-ESI-QTOF MS/MS. Venom peptides were initially separated using C18 reversed-phase high-performance liquid chromatography, then analyzed by MALDI-TOF MS. The proteomic analysis revealed numerous proteins that could be assigned a biological function (total 94), mainly as toxins, or roles in cell regulation and transport. This investigation found that ca. 73% of the proteins were common to venoms collected by the two methods. The peptidomic analysis revealed a large number of peptides (total 309) but with <20% shared by the two collection methods. There was also a marked difference between venoms obtained by venom gland dissection from different ant colonies. These findings demonstrate the rich composition and variability of P. clavata venom.

Isolation and characterization of a structurally unique ß-hairpin venom peptide from the predatory ant Anochetus emarginatus.

BACKGROUND: Most ant venoms consist predominantly of small linear peptides, although some contain disulfide-linked peptides as minor components. However, in striking contrast to other ant species, some Anochetus venoms are composed primarily of disulfide-rich peptides. In this study, we investigated the venom of the ant Anochetus emarginatus with the aim of exploring these novel disulfide-rich peptides. METHODS: The venom peptidome was initially investigated using a combination of reversed-phase HPLC and mass spectrometry, then the amino acid sequences of the major peptides were determined using a combination of Edman degradation and de novo MS/MS sequencing. We focused on one of these peptides, U1-PONTX-Ae1a (Ae1a), because of its novel sequence, which we predicted would form a novel 3D fold. Ae1a was chemically synthesized using Fmoc chemistry and its 3D structure was elucidated using NMR spectroscopy. The peptide was then tested for insecticidal activity and its effect on a range of human ion channels. RESULTS: Seven peptides named poneritoxins (PONTXs) were isolated and sequenced. The three-dimensional structure of synthetic Ae1a revealed a novel, compact scaffold in which a C-terminal ß-hairpin is connected to the N-terminal region via two disulfide bonds. Synthetic Ae1a reversibly paralyzed blowflies and inhibited human L-type voltage-gated calcium channels (CaV1). CONCLUSIONS: Poneritoxins from Anochetus emarginatus venom are a novel class of toxins that are structurally unique among animal venoms. GENERAL SIGNIFICANCE: This study demonstrates that Anochetus ant venoms are a rich source of novel ion channel modulating peptides, some of which might be useful leads for the development of biopesticides.

Phthalate pollution in an Amazonian rainforest.

Phthalates are ubiquitous contaminants and endocrine-disrupting chemicals that can become trapped in the cuticles of insects, including ants which were recognized as good bioindicators for such pollution. Because phthalates have been noted in developed countries and because they also have been found in the Arctic, a region isolated from direct anthropogenic influence, we hypothesized that they are widespread. So, we looked for their presence on the cuticle of ants gathered from isolated areas of the Amazonian rainforest and along an anthropogenic gradient of pollution (rainforest vs. road sides vs. cities in French Guiana). Phthalate pollution (mainly di(2-ethylhexyl) phthalate (DEHP)) was higher on ants gathered in cities and along road sides than on those collected in the pristine rainforest, indicating that it follows a human-mediated gradient of disturbance related to the use of plastics and many other products that contain phthalates in urban zones. Their presence varied with the ant species; the cuticle of Solenopsis saevissima traps higher amount of phthalates than that of compared species. However, the presence of phthalates in isolated areas of pristine rainforests suggests that they are associated both with atmospheric particles and in gaseous form and are transported over long distances by wind, resulting in a worldwide diffusion. These findings suggest that there is no such thing as a "pristine" zone.

Comparisons of Protein and Peptide Complexity in Poneroid and Formicoid Ant Venoms.

Animal venom peptides are currently being developed as novel drugs and bioinsecticides. Because ants use venoms for defense and predation, venomous ants represent an untapped source of potential bioactive toxins. This study compared the protein and peptide components of the poneroid ants Neoponera commutata, Neoponera apicalis, and Odontomachus hastatus and the formicoid ants Ectatomma tuberculatum, Ectatomma brunneum, and Myrmecia gulosa. 1D and 2D PAGE revealed venom proteins in the mass range <10 to >250 kDa. NanoLC-ESI-QTOF MS/MS analysis of tryptic peptides revealed the presence of common venom proteins and also many undescribed proteins. RP-HPLC separation followed by MALDI-TOF MS of the venom peptides also revealed considerable heterogeneity. It was found that the venoms contained between 144 and 1032 peptides with 5-95% of peptides in the ranges 1-4 and 1-8 kDa for poneroid and formicoid ants, respectively. By employing the reducing MALDI matrix 1,5-diaminonapthalene, up to 28 disulfide-bonded peptides were also identified in each of the venoms. In particular, the mass range of peptides from poneroid ants is lower than peptides from other venoms, indicating possible novel structures and pharmacologies. These results indicate that ant venoms represent an enormous, untapped source of novel therapeutic and bioinsecticide leads.

The Biochemical Toxin Arsenal from Ant Venoms.

Ants (Formicidae) represent a taxonomically diverse group of hymenopterans with over 13,000 extant species, the majority of which inject or spray secretions from a venom gland. The evolutionary success of ants is mostly due to their unique eusociality that has permitted them to develop complex collaborative strategies, partly involving their venom secretions, to defend their nest against predators, microbial pathogens, ant competitors, and to hunt prey. Activities of ant venom include paralytic, cytolytic, haemolytic, allergenic, pro-inflammatory, insecticidal, antimicrobial, and pain-producing pharmacologic activities, while non-toxic functions include roles in chemical communication involving trail and sex pheromones, deterrents, and aggregators. While these diverse activities in ant venoms have until now been largely understudied due to the small venom yield from ants, modern analytical and venomic techniques are beginning to reveal the diversity of toxin structure and function. As such, ant venoms are distinct from other venomous animals, not only rich in linear, dimeric and disulfide-bonded peptides and bioactive proteins, but also other volatile and non-volatile compounds such as alkaloids and hydrocarbons. The present review details the unique structures and pharmacologies of known ant venom proteinaceous and alkaloidal toxins and their potential as a source of novel bioinsecticides and therapeutic agents.

The Guianese population of the fire ant Solenopsis saevissima is unicolonial.

In this study, conducted in French Guiana, a part of the native range of the fire ant Solenopsis saevissima, we compared the cuticular hydrocarbon profiles of media workers with previous results based on intraspecific aggressiveness tests. We noted a strong congruence between the two studies permitting us to delimit 2 supercolonies extending over large distances (up to 54 km), a phenomenon known as unicoloniality. Solenopsis geminata workers, taken as an out-group for cluster analyses, have a very different cuticular hydrocarbon profile. Because S. saevissima has been reported outside its native range, our conclusion is that this species has the potential to become invasive because unicoloniality (i.e., the main attribute for ants to become invasive) was shown at least for the Guianese population.