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.

Testing the Novel Weapons Hypothesis of the Argentine Ant Venom on Amphibians.

The globally invasive Argentine ant (Linepithema humile) possesses a venom lethal to some amphibian species in the invaded range. To test the novel weapons hypothesis (NWH), the effects of the toxin on the cohabiting amphibian species in the ant's native range need to be investigated. The invader should benefit from the novel chemical in the invaded range, because the species are not adapted, but the venom should not be effective in the native range. We explore the venom effects on juveniles of three amphibian species with different degrees of myrmecophagy inhabiting the ant's native range: Rhinella arenarum, Odontophrynus americanus, and Boana pulchella. We exposed the amphibians to the ant venom, determined the toxic dose, and evaluated the short- (10 min to 24 h) and medium-term (14 days) effects. All amphibian species were affected by the venom independently of myrmecophagy. In addition to amphibian sensitivity, we discuss how the differential Argentine ant abundance and density in the two ranges could be the key to the susceptibility of amphibians to the venom, resulting in the possibility of NWH. Our results confirm the potential magnitude of the impact of the Argentine ant in successfully invaded areas for the conservation of already threatened amphibians.

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.

Shedding Lights on Crude Venom from Solitary Foraging Predatory Ant Ectatomma opaciventre: Initial Toxinological Investigation.

Some species of primitive predatory ants, despite living in a colony, exercise their hunting collection strategy individually; their venom is painful, paralyzing, digestive, and lethal for their prey, yet the toxins responsible for these effects are poorly known. Ectatomma opaciventre is a previously unrecorded solitary hunting ant from the Brazilian Cerrado. To overcome this hindrance, the present study performed the in vitro enzymatic, biochemical, and biological activities of E. opaciventre to better understand the properties of this venom. Its venom showed several proteins with masses ranging from 1-116 kDa, highlighting the complexity of this venom. Compounds with high enzymatic activity were described, elucidating different enzyme classes present in the venom, with the presence of the first L-amino acid oxidase in Hymenoptera venoms being reported. Its crude venom contributes to a state of blood incoagulability, acting on primary hemostasis, inhibiting collagen-induced platelet aggregation, and operating on the fibrinolysis of loose red clots. Furthermore, the E. opaciventre venom preferentially induced cytotoxic effects on lung cancer cell lines and three different species of Leishmania. These data shed a comprehensive portrait of enzymatic components, biochemical and biological effects in vitro, opening perspectives for bio-pharmacological application of E. opaciventre venom molecules.

Venenos animais como possíveis ferramentas terapéuticas contra o estresse oxidativo em diversas doengas: Revisao da Literatura / Animal venoms as possible therapeutic tools against oxidative stress in several diseases: Literature Review

Resumo Venenos sao uma substancia tóxica (composta por uma ou mais toxinas) que podem causando lesao fisiológica dependente da dose. As toxinas sao moléculas bioativas formadas principalmente por compostos enzimáticos e nao enzimático que porque provocam consequéncias indesejáveis nas presas, além disso, exibem atividades biológicas únicas, diversas e específicas que perturbam os processos fisiológicos normais. Entretanto, muitas toxinas, de diferentes animais, tém sido isoladas e muitas delas sao consideradas ótimas ferramentas para pesquisa básica e alvos terapéuticos. Foi relatado que o estresse oxidativo desempenha um papel fundamental na patogénese de várias doengas, como distúrbios neurodegenerativos, distúrbios cardiovasculares e cáncer. O mecanismo pelo qual as toxinas animais atuam nos parametros de estresse oxidativo em várias doengas, ainda nao está estabelecido. O foco principal desta revisao é destacar os principais estudos com toxinas animais como ferramenta terapéutica em diversas doengas, atuando no balango redox do organismo.

Abstract Venoms are a toxic substance (comprised of one or more toxins) that can cause dose-dependent physiological injury. Toxins are bioactive molecules formed primarily by enzymatic and non-enzymatic compounds that cause undesirable conse-quences in prey, in addition, exhibit unique, diverse and specific biological activities that disrupt normal physiological processes. However, many toxins, from different animals, have been isolated and many of them are considered great tools for basic research and therapeutic targets. Oxidative stress has been reported to play a key role in the pathogenesis of various diseases such as neurodegenerative disorders, cardiovascular disorders and cancer. How animal toxins act on oxidative stress parameters in several diseases is not yet established. The main focus of this review is to highlight the main studies with animal toxins as a therapeutic tool in several diseases, acting on the organism's redox balance.

Resumen Los venenos son sustancias tóxicas (compuestas por una o más toxinas) que pueden causar daño fisiológico dependiente de la dosis. Las toxinas son moléculas bioactivas formadas principalmente por compuestos enzimáticos y no enzimáticos que debido a que causan consecuencias indeseables en las presas, además, exhiben actividades biológicas únicas, diversas y específicas que alteran los procesos fisiológicos normales. Sin embargo, se han aislado muchas toxinas de diferentes animales, y muchos de ellos se consideran grandes herramientas para la investigación básica y dianas terapéuticas. Se ha informado que el estrés oxidativo juega un papel clave en la patogenia de diversas enfermedades, como los trastornos neurodegenerativos, enfermedades cardiovasculares y cáncer. El mecanismo por el cual las toxinas animales actúan sobre los parámetros de estrés oxidativo en vários enfermedades, aún no está establecido. El enfoque principal de esta revisión es resaltar los principales estudios con toxinas animales como herramienta terapéutica en diversas enfermedades, actuando en el equilibrio redox del organismo.

Mutualistic interaction of aphids and ants in pepper, Capsicum annuum and Capsicum frutenscens (Solanaceae) / Interacción mutualista de pulgones y hormigas en pimiento, Capsicum annuum y Capsicum frutenscens (Solanaceae)

Abstract Introduction: Adequate biological identification is fundamental for establishing integrated pest management programs and identifying the trophic and mutualist relationships that can affect pest population dynamics. Aphids are the main pest of pepper Capsicum spp. (Solanaceae) crops in Southwestern Colombia, due to their role as vectors of viruses. However, the identification of aphid species is complex, limiting the investigations performed to address their interactions with other organisms. Ants and aphids present a facultative mutualistic relationship, that promotes the growth of hemipteran colonies, for this reason, the study of the ecological mutualistic association between aphids and ants is important. Objective: The main objective was to discriminate the aphid species present in commercial crops of Capsicum spp., and to identify the ant community that attends the aphid colonies and its effects on the size of the aphid colonies. Methods: Aphid species, and their ant mutualist, were collected from Capsicum annuum and Capsicum frutescens, in the Cauca valley, Southwestern Colombia. We used the DNA barcoding approach to identify aphid species, and the ants were identified by morphology-based taxonomy. To evaluate the effect of ant care on the size and structure of aphid colonies, generalized linear models were calculated using as the response variables the total number of aphids for each colony and the proportion of nymphs. Results: The aphid species that attack pepper crops, are: Aphis gossypii and Myzus persicae (Hemiptera: Aphididae), with A. gossypii being the species that interacts with ants (19 ant species). A. gossypii colonies attended by ants had larger sizes and more nymphs per colony, than those not attended. Conclusions: Although the aphid-ant interaction is not species-specific, it is necessary to consider its role in the propagation of viral diseases in peppers and to determine how this interaction may affect regional biological control strategies.

Resumen Introducción: La adecuada identificación biológica es fundamental para establecer programas de manejo integrado de plagas e identificar las relaciones tróficas y mutualistas que pueden afectar la dinámica poblacional de insectos plaga. Los áfidos son las principales plagas del ají Capsicum spp. (Solanaceae) en el suroccidente colombiano, debido a su rol como vectores de virus. Sin embargo, su identificación es compleja, y limita las investigaciones que intentan revelar sus interacciones con otros organismos. Las hormigas y los áfidos presentan una relación mutualista facultativa, que promueve el crecimiento de las colonias de los hemípteros, por esta razón, el estudio de la asociación ecológica y mutualista entre áfidos y hormigas es importante. Objetivo: El principal objetivo de esta investigación fue discriminar las especies de áfidos presentes en cultivos comerciales de Capsicum spp., e identificar la comunidad de hormigas que atiende las colonias de áfidos y su efecto en el tamaño de las colonias de áfidos. Métodos: Los áfidos, y las hormigas mutualistas de estos áfidos, se recolectaron de Capsicum annuum y Capsicum frutescens, en el valle del rio Cauca, en el suroccidente colombiano. Se empleó el Código de barras del ADN para identificar las especies de áfidos, y las hormigas se identificaron empleando taxonomía basada en morfología. Para evaluar el efecto que tiene el cuidado de las hormigas sobre el tamaño de las colonias de áfidos, se empleó un modelo lineal generalizado, utilizando como variables de respuesta el número total de áfidos por cada colonia y la proporción de ninfas por colonia. Resultados: Las especies de áfidos que atacan los cultivos de ají, son: Aphis gossypii y Myzus persicae (Hemiptera: Aphididae), siendo A. gossypii la especie que interactúa con hormigas (19 especies). Las colonias de A. gossypii atendidas por hormigas presentan mayor tamaño y número de ninfas, que aquellas desatendidas. Conclusiones: Aunque la interacción áfido-hormiga no es especie específica, es necesario considerar su rol en la propagación de enfermedades virales en plantas cultivadas y determinar cómo esta interacción puede afectar la implementación de estrategias de control biológico.

Effects of the Argentine ant venom on terrestrial amphibians.

Invasive species have major impacts on biodiversity and are one of the primary causes of amphibian decline and extinction. Unlike other top ant invaders that negatively affect larger fauna via chemical defensive compounds, the Argentine ant (Linepithema humile) does not have a functional sting. Nonetheless, it deploys defensive compounds against competitors and adversaries. We estimated levels of ant aggression toward 3 native terrestrial amphibians by challenging juveniles in field ant trails and in lab ant foraging arenas. We measured the composition and quantities of toxin in L. humile by analyzing pygidial glands and whole-body contents. We examined the mechanisms of toxicity in juvenile amphibians by quantifying the toxin in amphibian tissues, searching for histological damages, and calculating toxic doses for each amphibian species. To determine the potential scope of the threat to amphibians, we used global databases to estimate the number, ranges, and conservation status of terrestrial amphibian species with ranges that overlap those of L. humile. Juvenile amphibians co-occurring spatially and temporally with L. humile die when they encounter L. humile on an ant trail. In the lab, when a juvenile amphibian came in contact with L. humile the ants reacted quickly to spray pygidial-gland venom onto the juveniles. Iridomyrmecin was the toxic compound in the spray. Following absorption, it accumulated in brain, kidney, and liver tissue. Toxic dose for amphibian was species dependent. Worldwide, an estimated 817 terrestrial amphibian species overlap in range with L. humile, and 6.2% of them are classified as threatened. Our findings highlight the high potential of L. humile venom to negatively affect amphibian juveniles and provide a basis for exploring the largely overlooked impacts this ant has in its wide invasive range.

Efectos del Veneno de la Hormiga Argentina sobre los Anfibios Terrestres Resumen Las especies invasoras tienen un impacto importante sobre la biodiversidad y son una de las causas principales del declive y extinción de los anfibios. A diferencia de otras hormigas super-invasoras que afectan negativamente a animales más grandes por medio de compuestos químicos de defensa, la hormiga argentina (Linepithema humile) no tiene unaguijón funcional. Sin embargo, esta hormiga despliega compuestos defensivos contra sus competidores y adversarios. Estimamos los niveles de agresión de las hormigas hacia tres anfibios terrestres nativos exponiendo a los anfibios juveniles en pistas de hormigas en el campo y en las arenas de forrajeo de las hormigas en el laboratorio. Medimos la composición y las cantidades de toxina que presenta L. humile por medio del análisis de las glándulas pigidiales y el contenido en el cuerpo completo. Examinamos los mecanismos de la toxicidad en los anfibios juveniles cuantificando la toxina en el tejido del anfibio, buscando daños histológicos y calculando las dosis tóxicas para cada especie de anfibio. Para determinar el alcance potencial de la amenaza para los anfibios usamos bases de datos mundiales para estimar el número, distribución y estado de conservación de las especies terrestres de anfibios con distribuciones que se solapan con la de L. humile. Los anfibios juveniles que co-ocurren temporal y espacialmente con L. humile mueren al encontrarse con esta especie de hormiga en sus pistas. En el laboratorio, cuando un anfibio juvenil entró en contacto con L. humile, las hormigas reaccionaron rápidamente rociando a estos juveniles con veneno proveniente de las glándulas pigidiales. La iridomyrmecina fue el compuesto tóxico que encontramos en las glándulas pigidiales. Después de ser absorbida por la piel del anfibio, se acumuló en el cerebro, los riñones y el hígado. La dosis tóxica para los anfibios depende de la especie. A nivel mundial, se estima que 817 especies de anfibios terrestres tienen una distribución que se solapa con la de L. humile, y el 6.2% de estas especies se encuentran clasificadas como amenazadas. Nuestros hallazgos resaltan el potencial alto del veneno de L. humile para tener efectos negativos sobre los anfibios juveniles y también proporcionan una base para la exploración de los impactos de esta hormiga en su amplio rango invasivo, los cuales generalmente son ignorados.

Dinoponera quadriceps venom as a source of active agents against Staphylococcus aureus.

Staphylococcus aureus is a highly virulent pathogen, capable of biofilm formation and responsible for thousands of deaths each year. The prevalence of Methicillin-Resistant S. aureus (MRSA) strains has increased in recent years and thus, the development of new antibiotics has become necessary. Antimicrobial Peptides (AMPs) are effective against a variety of multidrug-resistant bacteria and low levels of resistance have been reported regarding these molecules. Dinoponera quadriceps ant venom (DqV) has been described regarding its effect against S. aureus. In this study, we have evaluated the antibacterial effect of DqV-AMPs, the dinoponeratoxins (DNTxs), against Methicillin-Sensitive and a Methicillin-Resistant S. aureus strains. Our results show DNTx M-PONTX-Dq3a as a potent inhibitor of both strains, being able to prevent biofilm formation at low micromolar range (0.78-3.12 µM). It also showed a short-time effect through membrane disruption. M-PONTX-Dq3a opens up new perspectives for the prevention of biofilm formation through the development of anti-adhesive surface coatings on medical devices, as well as the treatment of resistant strains in skin or soft tissue infections.

Cross-Reactive Carbohydrate Determinant in Apis mellifera, Solenopsis invicta and Polybia paulista Venoms: Identification of Allergic Sensitization and Cross-Reactivity.

Allergic reactions to Hymenoptera venom, which could lead to systemic and even fatal symptoms, is characterized by hypersensitivity reactions mediated by specific IgE (sIgE) driven to venom allergens. Patients multisensitized to sIgE usually recognize more than one allergen in different Hymenoptera species. However, the presence of sIgE directed against Cross-Reactive Carbohydrate Determinant (CCD), which occurs in some allergens from Hymenoptera venom, hampers the identification of the culprit insects. CCD is also present in plants, pollen, fruits, but not in mammals. Bromelain (Brl) extracted from pineapples is a glycoprotein commonly used for reference to sIgE-CCD detection and analysis. In sera of fifty-one Hymenoptera allergic patients with specific IgE ≥ 1.0 KU/L, we assessed by immunoblotting the reactivity of sIgE to the major allergens of Apis mellifera, Polybia paulista and Solenopsis invicta venoms. We also distinguished, using sera adsorption procedures, the cases of CCD cross-reaction using Brl as a marker and inhibitor of CCD epitopes. The presence of reactivity for bromelain (24-28 kDa) was obtained in 43% of the patients, in which 64% presented reactivity for more than one Hymenoptera venom in radioallergosorbent (RAST) tests, and 90% showed reactivity in immunoblot analysis to the major allergens of Apis mellifera, Polybia paulista and Solenopsis invicta venoms. Sera adsorption procedures with Brl lead to a significant reduction in patients' sera reactivity to the Hymenoptera allergens. Immunoblotting assay using pre- and post-Brl adsorption sera from wasp-allergic patients blotted with non-glycosylated recombinant antigens (rPoly p1, rPoly p5) from Polybia paulista wasp venom showed no change in reactivity pattern of sIgE that recognize allergen peptide epitopes. Our results, using Brl as a marker and CCD inhibitor to test sIgE reactivity, suggest that it could complement diagnostic methods and help to differentiate specific reactivity to allergens' peptide epitopes from cross-reactivity caused by CCD, which is extremely useful in clinical practice.

Antimicrobial activity of synthetic Dq-3162, a 28-residue ponericin G-like dinoponeratoxin from the giant ant Dinoponera quadriceps venom, against carbapenem-resistant bacteria.

The predatory giant ant Dinoponera quadriceps is one of the largest venomous ants on Earth. The venom of D. quadriceps comprises a rich blend of bioactive peptides that includes structures related to at least five classes of antimicrobial peptides. In the present study, two representative synthetic peptides, sDq-2562 and sDq-3162, belonging to the ponericin-like dinoponeratoxin family, were evaluated for their microbicide activity against antibiotic-resistant bacteria. The most effective peptide, the 28-residue sDq-3162 displayed a significant bacteriostatic and bactericidal effect with minimal inhibitory concentrations (MICs) between 5 µM and 10 µM (15.6 µg mL-1 and 31.2 µg mL-1), according to the strain of drug-resistant bacteria tested. In combination with conventional antibiotics, sDq-3162 displayed in vitro synergistic effects, reducing the MICs of antibiotics for more than 2-log against clinical isolates of carbapenem-resistant Acinetobacter baumannii, Klebsiella pneumoniae and Pseudomonas aeruginosa, with low cytotoxicity to human erythrocytes, in vitro. Since the development of molecules to circumvent the spread of antibiotic-resistant bacteria is demanding, ant venom peptides arise as useful molecular resources to contribute with the antimicrobial arsenal and therapeutic strategies to fight clinically relevant microbial infections.

Comprehensive analysis of peptides and low molecular weight components of the giant ant Dinoponera quadriceps venom.

Ants (Hymenoptera, Apocrita, Aculeata, Formicoidea) comprise a well-succeeded group of animals. Like bees and wasps, ants are mostly venomous, having a sting system to deliver a mixture of bioactive organic compounds and peptides. The predatory giant ant Dinoponera quadriceps belongs to the subfamily Ponerinae that includes one of the largest known ant species in the world. In the present study, low molecular weight compounds and peptides were identified by online peptide mass fingerprint. These include neuroactive biogenic amines (histamine, tyramine, and dopamine), monoamine alkaloid (phenethylamine), free amino acids (e.g. glutamic acid and proline), free thymidine, and cytosine. To the best of our knowledge, most of these components are described for the first time in an ant venom. Multifunctional dinoponeratoxin peptide variants (pilosulin- and ponericin-like peptides) were characterized that possess antimicrobial, hemolytic, and histamine-releasing properties. These venom components, particularly peptides, might synergistically contribute to the overall venom activity and toxicity, for immobilizing live prey, and for defending D. quadriceps against aggressors, predators, and potential microbial infection.

Bottom-Up Proteomic Analysis of Polypeptide Venom Components of the Giant Ant Dinoponera Quadriceps.

Ant species have specialized venom systems developed to sting and inoculate a biological cocktail of organic compounds, including peptide and polypeptide toxins, for the purpose of predation and defense. The genus Dinoponera comprises predatory giant ants that inoculate venom capable of causing long-lasting local pain, involuntary shaking, lymphadenopathy, and cardiac arrhythmias, among other symptoms. To deepen our knowledge about venom composition with regard to protein toxins and their roles in the chemical-ecological relationship and human health, we performed a bottom-up proteomics analysis of the crude venom of the giant ant D. quadriceps, popularly known as the "false" tocandiras. For this purpose, we used two different analytical approaches: (i) gel-based proteomics approach, wherein the crude venom was resolved by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and all protein bands were excised for analysis; (ii) solution-based proteomics approach, wherein the crude venom protein components were directly fragmented into tryptic peptides in solution for analysis. The proteomic data that resulted from these two methodologies were compared against a previously annotated transcriptomic database of D. quadriceps, and subsequently, a homology search was performed for all identified transcript products. The gel-based proteomics approach unequivocally identified nine toxins of high molecular mass in the venom, as for example, enzymes [hyaluronidase, phospholipase A1, dipeptidyl peptidase and glucose dehydrogenase/flavin adenine dinucleotide (FAD) quinone] and diverse venom allergens (homologous of the red fire ant Selenopsis invicta) and venom-related proteins (major royal jelly-like). Moreover, the solution-based proteomics revealed and confirmed the presence of several hydrolases, oxidoreductases, proteases, Kunitz-like polypeptides, and the less abundant inhibitor cysteine knot (ICK)-like (knottin) neurotoxins and insect defensin. Our results showed that the major components of the D. quadriceps venom are toxins that are highly likely to damage cell membranes and tissue, to cause neurotoxicity, and to induce allergic reactions, thus, expanding the knowledge about D. quadriceps venom composition and its potential biological effects on prey and victims.

Fire Ant Venom Alkaloids Inhibit Biofilm Formation.

Biofilm formation on exposed surfaces is a serious issue for the food industry and medical health facilities. There are many proposed strategies to delay, reduce, or even eliminate biofilm formation on surfaces. The present study focuses on the applicability of fire ant venom alkaloids (aka 'solenopsins', from Solenopsis invicta) tested on polystyrene and stainless steel surfaces relative to the adhesion and biofilm-formation by the bacterium Pseudomonas fluorescens. Conditioning with solenopsins demonstrates significant reduction of bacterial adhesion. Inhibition rates were 62.7% on polystyrene and 59.0% on stainless steel surfaces. In addition, solenopsins drastically reduced cell populations already growing on conditioned surfaces. Contrary to assumptions by previous authors, solenopsins tested negative for amphipathic properties, thus understanding the mechanisms behind the observed effects still relies on further investigation.

The allergic response mediated by fire ant venom proteins.

Fire ants are widely studied, invasive and venomous arthropod pests. There is significant biomedical interest in immunotherapy against fire ant stings. However, mainly due to practical reasons, the physiological effects of envenomation has remained poorly characterized. The present study takes advantage of a recently-described venom protein extract to delineate the immunological pathways underlying the allergic reaction to fire ant venom toxins. Mice were injected with controlled doses of venom protein extract. Following sensitization and a second exposure, a marked footpad swelling was observed. Based on eosinophil recruitment and production of Th2 cytokines, we hereby establish that fire ant proteins per se can lead to an allergic response, which casts a new light into the mechanism of action of these toxins.

Phospholipase A1-based cross-reactivity among venoms of clinically relevant Hymenoptera from Neotropical and temperate regions.

Molecular cross-reactivity caused by allergen homology or cross-reactive carbohydrate determinants (CCDs) is a major challenge for diagnosis and immunotherapy of insect venom allergy. Venom phospholipases A1 (PLA1s) are classical, mostly non-glycosylated wasp and ant allergens that provide diagnostic benefit for differentiation of genuine sensitizations from cross-reactivity. As CCD-free molecules, venom PLA1s are not causative for CCD-based cross-reactivity. Little is known however about the protein-based cross-reactivity of PLA1 within vespid species. Here, we address PLA1-based cross-reactivity among ten clinically relevant Hymenoptera venoms from Neotropical and temperate regions including Polybia paulista (paulistinha) venom and Vespula vulgaris (yellow jacket) venom. In order to evaluate cross-reactivity, sera of mice sensitized with recombinant PLA1 (rPoly p 1) from P. paulista wasp venom were used. Pronounced IgE and IgG based cross-reactivity was detected for wasp venoms regardless the geographical region of origin. The cross-reactivity correlated well with the identity of the primary sequence and 3-D models of PLA1 proteins. In contrast, these mice sera showed no reaction with honeybee (HBV) and fire ant venom. Furthermore, sera from patients monosensitized to HBV and fire ants did not recognize the rPoly p 1 in immunoblotting. Our findings reveal the presence of conserved epitopes in the PLA1s from several clinically relevant wasps as major cause of PLA1-based in vitro cross-reactivity. These findings emphasize the limitations but also the potential of PLA1-based HVA diagnostics.

Peptidomic investigation of Neoponera villosa venom by high-resolution mass spectrometry: seasonal and nesting habitat variations

Advancements in proteomics, including the technological improvement in instrumentation, have turned mass spectrometry into an indispensable tool in the study of venoms and toxins. In addition, the advance of nanoscale liquid chromatography coupled to nanoelectrospray mass spectrometry allows, due to its high sensitivity, the study of venoms from species previously left aside, such as ants. Ant venoms are a complex mixture of compounds used for defense, predation or communication purposes. The venom from Neoponera ants, a genus restricted to Neotropical regions, is known to have cytolytic, hemolytic, antimicrobial and insecticidal activities. Moreover, venoms from several Neoponera species have been compared and differences in their toxicity related to nesting habitat variation were reported. Therefore, the present study aimed to perform a deep peptidomic analysis of Neoponera villosa venom and a comparison of seasonal and nesting habitat variations using high-resolution mass spectrometry. Methods: Specimens of N. villosa ants were captured in Panga Natural Reserve (Uberlândia, MG, Brazil) from arboreal and ground-dwelling nests during summer and winter time. The venom glands were dissected, pooled and disrupted by ultra-sonic waves. The venom collected from different habitats (arboreal and ground-dwelling) and different seasons (summer and winter) was injected into a nanoACQUITY ULPC hyphened to a Q-Exactive Orbitrap mass spectrometer. The raw data were analyzed using PEAKS 7. Results: The results showed a molecular diversity of more than 500 peptides among these venoms, mostly in the mass range of 800-4000 Da. Mutations and post-translational modifications were described and differences among the venoms were observed. Part of the peptides matched with ponericins, a well-known antimicrobial peptide family. In addition, smaller fragments related to ponericins were also identified, suggesting that this class of antimicrobial peptide might undergo enzymatic cleavages. Conclusion: There are substantial differences among the venom of N. villosa ants collected in different seasons and from different nest habitats. The venom composition is affected by climate changes that influence prey availability and predator presence. Clearly, nano-LC-MS boosted the knowledge about ant venom, a rich source of unexplored and promising bioactive compounds.(AU)

Identification, expression and characterization of the recombinant Sol g 4.1 protein from the venom of the tropical fire ant Solenopsis geminata

Background: Fire ant venom is a complex mixture consisting of basic piperidine alkaloids, various biologically active peptides and protein components, including a variety of major allergenic proteins. Tropical fire ant Solenopsis geminata is an important stinging ant species that causes anaphylaxis and serious medical problems. Although the biological activities of allergenic venom proteins that are unique to ant venom, particularly Solenopsis 2 and 4, are still unknown, these proteins are believed to play important roles in mediating the effects of the piperidine derivatives in the venom. Methods: In the present study, the cDNA cloning, sequencing and three-dimensional structure of Sol g 4.1 venom protein are described. The recombinant Sol g 4.1 protein (rSol g 4.1) was produced in E. coli , and its possible function as a hydrophobic binding protein was characterized by paralyzing crickets using the 50% piperidine dose (PD50). Moreover, an antiserum was produced in mice to determine the allergenic properties of Sol g 4.1, and the antiserum was capable of binding to Sol g 4.1, as determined by Western blotting. Results: The molecular weight of Sol g 4.1 protein is 16 kDa, as determined by SDS-PAGE. The complete cDNA is 414 bp in length and contains a leader sequence of 19 amino acids. The protein consists of six cysteines that presumably form three disulfide bonds, based on a predicted three-dimensional model, creating the interior hydrophobic pocket and stabilizing the structure. The rSol g 4.1 protein was expressed in inclusion bodies, as determined by SDS-PAGE. Dialysis techniques were used to refold the recombinant protein into the native form. Its secondary structure, which primarily consists of -helices, was confirmed by circular dichroism analysis, and the three-dimensional model was also verified. The results of allergenic analysis performed on mice showed that the...(AU)

Peptidomic investigation of Neoponera villosa venom by high-resolution mass spectrometry: seasonal and nesting habitat variations

Background: Advancements in proteomics, including the technological improvement in instrumentation, have turned mass spectrometry into an indispensable tool in the study of venoms and toxins. In addition, the advance of nanoscale liquid chromatography coupled to nanoelectrospray mass spectrometry allows, due to its high sensitivity, the study of venoms from species previously left aside, such as ants. Ant venoms are a complex mixture of compounds used for defense, predation or communication purposes. The venom from Neoponera ants, a genus restricted to Neotropical regions, is known to have cytolytic, hemolytic, antimicrobial and insecticidal activities. Moreover, venoms from several Neoponera species have been compared and differences in their toxicity related to nesting habitat variation were reported. Therefore, the present study aimed to perform a deep peptidomic analysis of Neoponera villosa venom and a comparison of seasonal and nesting habitat variations using high-resolution mass spectrometry. Methods: Specimens of N. villosa ants were captured in Panga Natural Reserve (Uberlândia, MG, Brazil) from arboreal and ground-dwelling nests during summer and winter time. The venom glands were dissected, pooled and disrupted by ultra-sonic waves. The venom collected from different habitats (arboreal and ground-dwelling) and different seasons (summer and winter) was injected into a nanoACQUITY ULPC hyphened to a Q-Exactive Orbitrap mass spectrometer. The raw data were analyzed using PEAKS 7. Results: The results showed a molecular diversity of more than 500 peptides among these venoms, mostly in the mass range of 8004000 Da. Mutations and post-translational modifications were described and differences among the venoms were observed. Part of the peptides matched with ponericins, a well-known antimicrobial peptide family...(AU)

Peptidomic investigation of Neoponera villosa venom by high-resolution mass spectrometry: seasonal and nesting habitat variations

Background: Advancements in proteomics, including the technological improvement in instrumentation, have turned mass spectrometry into an indispensable tool in the study of venoms and toxins. In addition, the advance of nanoscale liquid chromatography coupled to nanoelectrospray mass spectrometry allows, due to its high sensitivity, the study of venoms from species previously left aside, such as ants. Ant venoms are a complex mixture of compounds used for defense, predation or communication purposes. The venom from Neoponera ants, a genus restricted to Neotropical regions, is known to have cytolytic, hemolytic, antimicrobial and insecticidal activities. Moreover, venoms from several Neoponera species have been compared and differences in their toxicity related to nesting habitat variation were reported. Therefore, the present study aimed to perform a deep peptidomic analysis of Neoponera villosa venom and a comparison of seasonal and nesting habitat variations using high-resolution mass spectrometry. Methods: Specimens of N. villosa ants were captured in Panga Natural Reserve (Uberlândia, MG, Brazil) from arboreal and ground-dwelling nests during summer and winter time. The venom glands were dissected, pooled and disrupted by ultra-sonic waves. The venom collected from different habitats (arboreal and ground-dwelling) and different seasons (summer and winter) was injected into a nanoACQUITY ULPC hyphened to a Q-Exactive Orbitrap mass spectrometer. The raw data were analyzed using PEAKS 7. Results: The results showed a molecular diversity of more than 500 peptides among these venoms, mostly in the mass range of 8004000 Da. Mutations and post-translational modifications were described and differences among the venoms were observed. Part of the peptides matched with ponericins, a well-known antimicrobial peptide family...

Identification, expression and characterization of the recombinant Sol g 4.1 protein from the venom of the tropical fire ant Solenopsis geminata

Background: Fire ant venom is a complex mixture consisting of basic piperidine alkaloids, various biologically active peptides and protein components, including a variety of major allergenic proteins. Tropical fire ant Solenopsis geminata is an important stinging ant species that causes anaphylaxis and serious medical problems. Although the biological activities of allergenic venom proteins that are unique to ant venom, particularly Solenopsis 2 and 4, are still unknown, these proteins are believed to play important roles in mediating the effects of the piperidine derivatives in the venom. Methods: In the present study, the cDNA cloning, sequencing and three-dimensional structure of Sol g 4.1 venom protein are described. The recombinant Sol g 4.1 protein (rSol g 4.1) was produced in E. coli , and its possible function as a hydrophobic binding protein was characterized by paralyzing crickets using the 50% piperidine dose (PD50). Moreover, an antiserum was produced in mice to determine the allergenic properties of Sol g 4.1, and the antiserum was capable of binding to Sol g 4.1, as determined by Western blotting. Results: The molecular weight of Sol g 4.1 protein is 16 kDa, as determined by SDS-PAGE. The complete cDNA is 414 bp in length and contains a leader sequence of 19 amino acids. The protein consists of six cysteines that presumably form three disulfide bonds, based on a predicted three-dimensional model, creating the interior hydrophobic pocket and stabilizing the structure. The rSol g 4.1 protein was expressed in inclusion bodies, as determined by SDS-PAGE. Dialysis techniques were used to refold the recombinant protein into the native form. Its secondary structure, which primarily consists of -helices, was confirmed by circular dichroism analysis, and the three-dimensional model was also verified. The results of allergenic analysis performed on mice showed that the...