Myrmecia Pilosula Research Articles

Stinging Ant Anaphylaxis: Advances in Diagnosis and Treatment

Stinging ants represent a wide range of over 200 different species across the world, of which Solenopsis, Myrmecia, Pogonomyrmex, and Brachyponera genera, account for a substantial economic and healthcare burden. S. invicta (red imported fire ant; IFA) and M. pilosula (jack jumper ant; JJA) are 2 species of high clinical importance, known to cause anaphylaxis in humans, with numerous reported fatalities. Diagnostic testing should be performed in patients with a history of a systemic reaction with skin testing and/or in vitro specific-IgE testing. In vitro testing is commercially available for IFA through whole-body extract (WBE) specific-IgE and JJA venom specific-IgE, but not widely available for other stinging ant species. Commercial venom component testing for IFA and JJA is currently not available. Patients with a clinical history and positive specific-IgE testing, should undergo treatment with specific immunotherapy, which is currently available for IFA and JJA. Build-up may be performed using conventional, semi-rush, rush, or ultra-rush schedules with similar risk profiles for IFA. Optimal duration for WBE immunotherapy for IFA and specific JJA venom immunotherapy is not well-studied, but generally recommended for at least 3-5 years. Sting challenges are used in research settings, primarily to assess treatment efficacy of immunotherapy.

Structure-based epitope prediction and assessment of cross-reactivity of Myrmecia pilosula venom-specific IgE and recombinant Sol g proteins (Solenopsis geminata)

The global distribution of tropical fire ants (Solenopsis geminata) raises concerns about anaphylaxis and serious medical issues in numerous countries. This investigation focused on the cross-reactivity of allergen-specific IgE antibodies between S. geminata and Myrmecia pilosula (Jack Jumper ant) venom proteins due to the potential emergence of cross-reactive allergies in the future. Antibody epitope analysis unveiled one predominant conformational epitope on Sol g 1.1 (PI score of 0.989), followed by Sol g 2.2, Sol g 4.1, and Sol g 3.1. Additionally, Pilosulin 1 showed high allergenic potential (PI score of 0.94), with Pilosulin 5a (PI score of 0.797) leading in B-cell epitopes. The sequence analysis indicated that Sol g 2.2 and Sol g 4.1 pose a high risk of cross-reactivity with Pilosulins 4.1a and 5a. Furthermore, the cross-reactivity of recombinant Sol g proteins with M. pilosula-specific IgE antibodies from 41 patients revealed high cross-reactivity for r-Sol g 3.1 (58.53%) and r-Sol g 4.1 (43.90%), followed by r-Sol g 2.2 (26.82%), and r-Sol g 1.1 (9.75%). Therefore, this study demonstrates cross-reactivity (85.36%) between S. geminata and M. pilosula, highlighting the allergenic risk. Understanding these reactions is vital for the prevention of severe allergic reactions, especially in individuals with pre-existing Jumper Jack ant allergy, informing future management strategies.

Ultrarush versus semirush venom immunotherapy: Outcome observations in real-world jack jumper ant cohort

Ultrarush versus semirush venom immunotherapy: Outcome observations in real-world jack jumper ant cohort

Cytotoxicity and Radiosensitizing Potentials of Pilosulin-3, a Recombinant Ant Venom, in Breast Cancer Cells.

Venom peptides are promising agents in the development of unconventional anticancer therapeutic agents. This study explored the potential of Pilosulin-3, a recombinant peptide from the venom of the Australian jack jumper ant "Myrmecia pilosula", as a cytotoxic and radiosensitizing agent in MCF-7 and MDA-MB-231 breast cancer (BC) cell lines. Pilosulin-3's cytotoxicity was evaluated across a wide range of concentrations using a proliferation assay. Cell cycle progression and apoptosis were examined at the inhibitory concentration 25% (IC25) and IC50 of Pilosulin-3, both with and without a 4Gy X-ray irradiation dose. Radiosensitivity was assessed at IC25 using the clonogenic survival assay. The study revealed that Pilosulin-3 exerted a concentration-dependent cytotoxic effect, with IC25 and IC50 values of 0.01 and 0.5 µM, respectively. In silico screening indicated high selectivity of Pilosulin-3 peptide, which was predicted to be the most likely anticancer agent (PROB = 0.997) with low hemolytic activity (PROP = 0.176). Although Pilosulin-3 exhibited a significant (p < 0.05) G2/M cell cycle arrest in combination with radiation, there was no discernible effect on apoptosis induction or cell survival following irradiation. In conclusion, Pilosulin-3 proved to be cytotoxic to BC cells and induced a cytostatic effect (G2/M arrest) when combined with radiation. However, it did not enhance the efficacy of cell killing by irradiation. While it holds potential as a cytotoxic agent in breast cancer treatment, its application as a radiosensitizer does not find support in these results.

The development of Jack Jumper ant venom immunotherapy: our 25 years' experience.

Jack Jumper ant venom allergy is a uniquely Australian medical issue. The stinging ant is a leading cause of insect venom allergy in south-eastern Australia. An effective venom immunotherapy-based treatment was successfully developed by the Tasmanian Jack Jumper Allergy Research group. This paper provides a synopsis of our 25 years' research journey in developing this evidence-based treatment modality.

It Takes Two: Dimerization Is Essential for the Broad-Spectrum Predatory and Defensive Activities of the Venom Peptide Mp1a from the Jack Jumper Ant Myrmecia pilosula.

Ant venoms have recently attracted increased attention due to their chemical complexity, novel molecular frameworks, and diverse biological activities. The heterodimeric peptide ∆-myrtoxin-Mp1a (Mp1a) from the venom of the Australian jack jumper ant, Myrmecia pilosula, exhibits antimicrobial, membrane-disrupting, and pain-inducing activities. In the present study, we examined the activity of Mp1a and a panel of synthetic analogues against the gastrointestinal parasitic nematode Haemonchus contortus, the fruit fly Drosophila melanogaster, and for their ability to stimulate pain-sensing neurons. Mp1a was found to be both insecticidal and anthelmintic, and it robustly activated mammalian sensory neurons at concentrations similar to those reported to elicit antimicrobial and cytotoxic activity. The native antiparallel Mp1a heterodimer was more potent than heterodimers with alternative disulfide connectivity, as well as monomeric analogues. We conclude that the membrane-disrupting effects of Mp1a confer broad-spectrum biological activities that facilitate both predation and defense for the ant. Our structure–activity data also provide a foundation for the rational engineering of analogues with selectivity for particular cell types.

Australian jack jumper ant foragers have no need for mental map

![Figure][1] An Australian jack jumper ant ( Myrmecia croslandi ). Photo credit Ajay Narendra. Before embarking on their careers as fully fledged foragers, novice ants take a few tours of their neighbourhood to familiarise themselves with the lay of the land. ‘Much evidence has

Pharmaceutical and preclinical evaluation of Advax adjuvant as a dose-sparing strategy for ant venom immunotherapy

A major challenge in broader clinical application of Jack Jumper ant venom immunotherapy (JJA VIT) is the scarcity of ant venom which needs to be manually harvested from wild ants. Adjuvants are commonly used for antigen sparing in other vaccines, and thereby could potentially have major benefits to extend JJA supplies if they were to similarly enhance JJA VIT immunogenicity. The purpose of this study was to evaluate the physicochemical and microbiological stability and murine immunogenicity of low-dose JJA VIT formulated with a novel polysaccharide adjuvant referred to as delta inulin or Advax™. Jack Jumper ant venom (JJAV) protein stability was assessed by UPLC-UV, SDS-PAGE, SDS-PAGE immunoblot, and ELISA inhibition. Diffraction light scattering was used to assess particle size distribution of Advax; pH and benzyl alcohol quantification by UPLC-UV were used to assess the physicochemical stability of JJAV diluent, and endotoxin content and preservative efficacy test was used to investigate the microbiological properties of the adjuvanted VIT formulation. To assess the effect of adjuvant on JJA venom immunogenicity, mice were immunised four times with JJAV alone or formulated with Advax adjuvant. JJA VIT formulated with Advax was found to be physicochemically and microbiologically stable for at least 2 days when stored at 4 and 25 °C with a trend for an increase in allergenic potency observed beyond 2 days of storage. Low-dose JJAV formulated with Advax adjuvant induced significantly higher JJAV-specific IgG than a 5-fold higher dose of JJAV alone, consistent with a powerful allergen-sparing effect. The pharmaceutical data provides important guidance on the formulation, storage and use of JJA VIT formulated with Advax adjuvant, with the murine immunogenicity studies providing a strong rationale for a planned clinical trial to test the ability of Advax adjuvant to achieve 4-fold JJAV dose sparing in JJA-allergic human patients.

P31: SEVERE SYSTEMIC ALLERGIC REACTIONS TO JACK JUMPER ANT VENOM IMMUNOTHERAPY: BASOPHIL RESPONSE DURING TREATMENT WITH OMALIZUMAB

Background Recurrent severe systemic reactions to Jack Jumper Ant (JJA) Venom Immunotherapy (VIT) although rare, hinder a patient's ability to achieve protective maintenance doses. Patients who discontinue treatment remain at high risk of potentially life threatening reactions. The administration of concurrent Omalizumab has been reported to reduce the rate of such reactions. We sought to evaluate the basophil response during treatment with Omalizumab in this setting. Case History We describe the case of a 50 year old female who has suffered multiple episodes of severe anaphylaxis following JJA stings. She commenced JJA VIT by conventional semirush protocol complicated by a severe (Brown grade III) anaphylaxis following a 10 mcg JJA venom (JJAV) dose. She suffered a further moderate (Brown grade II) severity anaphylaxis three weeks later. Geographic considerations were significant and treatment proceeded with a modified ultrarush protocol. A cumulative total of 45mcg of JJAV was achieved before she experienced another severe (Brown grade III) anaphylaxis. A subsequent basophil activation test (BAT) demonstrated 44.4%, reactive, CD63 positive basophils at a dilution of 0.1 mcg/ml JJAV. Immunotherapy was recommenced with the addition of Omalizumab 150 mcg 30 minutes before JJA VIT every fortnight. She achieved a maintenance dose of 50mcg JJAV 10 weeks later. A BAT was repeated 4 weeks following her first maintenance dose demonstrating no JJA venom basophil reactivity. Omalizumab was continued for 6 months. Three months after discontinuing Omalizumab our patient experienced a mild (Brown grade I) systemic reaction. Repeat BAT demonstrated reduction of CD63 positive, reactive, basophils from 44.4% to 18.9% at a dilution of 0.1mcg/ml JJAV. Conclusion Recurrent systemic VIT allergic reactions may occur after Omalizumab has been discontinued or reduced. The potential role of BAT for therapeutic monitoring in this setting is promising and requires further investigation.

Towards complete identification of allergens in Jack Jumper (Myrmecia pilosula) ant venom and their clinical relevance: An immunoproteomic approach.

The venomous stings of Jack Jumper ant (JJA; species of the Myrmecia pilosula taxonomic group) are a significant public health issue in parts of south-eastern and south-western Australia, causing anaphylaxis in approximately 3% of the population. Three allergenic peptides, Myr p 1, Myr p 2 and Myr p 3, and one histamine-releasing peptide, pilosulin 5, have been fully described, but there are at least 5 additional high molecular weight IgE-binding components that have not been identified. To identify IgE-binding components in JJA venom (JJAV) and to relate the IgE recognition of these components to relevant clinical parameters. Identification of IgE-binding components and determination of their sensitizing prevalence was performed using SDS-PAGE immunoblot assay and sera from 90 patients with confirmed allergy to JJAV. Tandem mass spectrometry was used for identification of novel JJAV components fractionated by size exclusion chromatography (SEC) and SDS-PAGE. Using SDS-PAGE immunoblot, 10 IgE-binding bands were identified in JJAV, two of which were recognized by 81% and 47% of the population studied. Mass spectrometry identified 17 novel JJAV proteins, including 2 glycoproteins, and confirmed the presence of 4 known Myr p and pilosulin peptides in JJAV. Most of the newly identified IgE-binding proteins were enzymes, including phospholipase A2 , hyaluronidase, arginine kinase and dipeptidyl peptidase IV. Correlations were found between recognition of certain IgE-binding bands with JJAV-specific IgE titre by ImmunoCAP, intradermal test threshold and treatment-related issues. This study has for the first time revealed the identity of various proteins with IgE-binding capacity in the venom of JJA and demonstrated their clinical relevance in the diagnosis and treatment of JJAV allergy.

The choreography of learning walks in the Australian jack jumper ant Myrmecia croslandi.

We provide a detailed analysis of the learning walks performed by Myrmecia croslandi ants at the nest during which they acquire visual information on its location. Most learning walks of 12 individually marked naïve ants took place in the morning with a narrow time window separating the first two learning walks, which most often occurred on the same day. Naïve ants performed between two and seven walks over up to four consecutive days before heading out to forage. On subsequent walks, naïve ants tend to explore the area around the nest in new compass directions. During learning walks, ants move along arcs around the nest while performing oscillating scanning movements. In a regular temporal sequence, the ants' gaze oscillates between the nest direction and the direction pointing away from the nest. Ants thus experience a sequence of views roughly across the nest and away from the nest from systematically spaced vantage points around the nest. Further, we show that ants leaving the nest for a foraging trip often walk in an arc around the nest on the opposite side to the intended foraging direction, performing a scanning routine indistinguishable from that of a learning walk. These partial learning walks are triggered by disturbance around the nest and may help returning ants with reorienting when overshooting the nest, which they frequently do. We discuss what is known about learning walks in different ant species and their adaptive significance for acquiring robust navigational memories.

Factors influencing the quality of Myrmecia pilosula (Jack Jumper) ant venom for use in invitro and invivo diagnoses of allergen sensitization and in allergen immunotherapy.

Allergen immunotherapy uses pharmaceutical preparations derived from naturally occurring source materials, which contain water-soluble allergenic components responsible for allergic reactions. The success of invivo and invitro diagnoses in allergen sensitization and allergen immunotherapy largely depends on the quality, composition and uniformity of allergenic materials used to produce the active ingredients, and the formulation employed to prepare finished products. We aimed to examine the factors influencing batch-to-batch consistency of Jack Jumper (Myrmecia pilosula) ant venom (JJAV) in the form of active pharmaceutical ingredient (AI) and informed whether factors such as temperature, artificial light and container materials influence the quality of JJAV AIs. We also aimed to establish handling and storage requirements of JJAV AIs to ensure preservation of allergenic activities during usage in the diagnosis of allergen sensitization and in allergen immunotherapy. The quality and consistency of JJAV AIs were analysed using a combination of bicinchoninic acid assay for total protein quantification, HPLC-UV for JJAV allergen peptides quantification, ELISA inhibition for total allergenic potency, SDS-PAGE, AU-PAGE and immunoblot for qualitative assessment of JJAV components, and Limulus Amebocyte Lysate assay for the quantification of endotoxin concentration. API-ZYM and Zymogram assays were used to probe the presence of enzymatic activities in JJAV. Pharmaceutical-grade JJAV for allergen immunotherapy has good batch-to-batch consistency. Temporary storage at 4°C and light exposure do not affect the quality of JJAV. Exposure to temperature above 40°C degrades high MW allergens in JJAV. Vials containing JJAV must be stored frozen and in upright position during long-term storage. We have identified factors, which can influence the quality and consistency of JJAV AIs, and provided a framework for appropriate handling, transporting and storage of JJAV to be used for the diagnosis of allergen sensitization and in AIT.

Δ-Myrtoxin-Mp1a is a Helical Heterodimer from the Venom of the Jack Jumper Ant that has Antimicrobial, Membrane-Disrupting, and Nociceptive Activities.

Δ-Myrtoxin-Mp1a (Mp1a), a 49-residue heterodimeric peptide from the venom of Myrmecia pilosula, comprises a 26-mer A chain and a 23-mer B chain connected by two disulfide bonds in an antiparallel arrangement. Combination of the individual synthetic chains through aerial oxidation remarkably resulted in the self-assembly of Mp1a as a homogenous product without the need for directed disulfide-bond formation. NMR analysis revealed a well-defined, unique structure containing an antiparallel α-helix pair. Dual polarization interferometry (DPI) analysis showed strong interaction with supported lipid bilayers and insertion within the bilayers. Mp1a caused non-specific Ca2+ influx in SH-SY5Y cells with a half maximal effective concentration (EC50 ) of 4.3 μm. Mp1a also displayed broad-spectrum antimicrobial activity, with the highest potency against Gram-negative Acinetobacter baumannii (MIC 25 nm). Intraplantar injection (10 μm) in mice elicited spontaneous pain and mechanical allodynia. Single- and two-chain mimetics of Mp1a revealed functional selectivity.

Δ‐Myrtoxin‐Mp1a is a Helical Heterodimer from the Venom of the Jack Jumper Ant that has Antimicrobial, Membrane‐Disrupting, and Nociceptive Activities

AbstractΔ‐Myrtoxin‐Mp1a (Mp1a), a 49‐residue heterodimeric peptide from the venom of Myrmecia pilosula, comprises a 26‐mer A chain and a 23‐mer B chain connected by two disulfide bonds in an antiparallel arrangement. Combination of the individual synthetic chains through aerial oxidation remarkably resulted in the self‐assembly of Mp1a as a homogenous product without the need for directed disulfide‐bond formation. NMR analysis revealed a well‐defined, unique structure containing an antiparallel α‐helix pair. Dual polarization interferometry (DPI) analysis showed strong interaction with supported lipid bilayers and insertion within the bilayers. Mp1a caused non‐specific Ca2+ influx in SH‐SY5Y cells with a half maximal effective concentration (EC50) of 4.3 μm. Mp1a also displayed broad‐spectrum antimicrobial activity, with the highest potency against Gram‐negative Acinetobacter baumannii (MIC 25 nm). Intraplantar injection (10 μm) in mice elicited spontaneous pain and mechanical allodynia. Single‐ and two‐chain mimetics of Mp1a revealed functional selectivity.

Characterizing the structure-function relationship reveals the mode of action of a novel antimicrobial peptide, P1, from jumper ant Myrmecia pilosula.

Microbial infections of antibiotic-resistant strains cause serious diseases and have a significant impact on public health worldwide, so novel antimicrobial drugs are urgently needed. Insect venoms, a rich source of bioactive components containing antimicrobial peptides (AMPs), are attractive candidates for new therapeutic agents against microbes. Recently, a novel peptide, P1, identified from the venom of the Australian jumper ant Myrmecia pilosula, showed potent antimicrobial activities against both Gram-negative and Gram-positive bacteria, but its structure-function relationship is unknown. Here, we used biochemical and biophysical techniques coupled with computational simulations to explore the mode of action of P1 interaction with dodecylphosphocholine (DPC) micelles as a model membrane system. Our circular dichroism (CD) and NMR studies revealed an amphipathic α-helical structure for P1 upon interaction with DPC micelles. A paramagnetic relaxation enhancement approach revealed that P1 orients its α-helix segment (F6-G14) into DPC micelles. In addition, the α-helix segment could be essential for membrane permeabilization and antimicrobial activity. Moreover, the arginine residues R8, R11, and R15 significantly contribute to helix formation and membrane-binding affinity. The lysine residue K19 of the C-terminus functionally guides P1 to interact with DPC micelles in the early interaction stage. Our study provides insights into the mode of action of P1, which is valuable in modifying and developing potent AMPs as antibiotic drugs.

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.

Incidence and Patient Demographics of Pre-Hospital Anaphylaxis in Tasmania

Introduction Anaphylaxis is widely accepted to be an increasing problem worldwide. This inquiry into pre-hospital incidence and patient demographics is useful to determine status of anaphylaxis in Tasmania. Our main goal was to determine the incidence of pre-hospital anaphylaxis in Tasmania and identify trends in characteristics of affected patients. Methods Raw data was searched and extracted from Ambulance Tasmania electronic recording system and case records for the period 1st January 2007 to 31st December 2011. This involved data mining 279,482 cases with the search parameters of anaphylaxis and allergy/ allergic reaction. Results There were 1,570 patients were classified as having allergic reaction (including the most severe form of anaphylaxis). 379 (24.1%) of the atopic group were given a final primary diagnosis of anaphylaxis. The adult cases distribution was female at 219 (57.8%) versus males at 160 cases (42.2%). Interestingly 21.1% of the total anaphylaxis cases were paediatric (n=80) with a greater percentage of male (n=46) to female (n=34) paediatric patients. Aetiology was identified in 85.5% of the cases accordingly: envenomation (insects) 141 (37.2%), food 118 (31.1%), medication 58 (15.3%), known other 4 (1.1%), exercise 3 ( Conclusion These findings suggest that almost a quarter of all calls for allergy or allergic reaction are of a severe potentially life-threatening nature, with the most common aetiology envenomation from insects (jack jumper bites).

Ant venom immunotherapy in Australia: the unmet need.

Jack jumper ant (JJA) venom allergy is an important cause of anaphylaxis in south-eastern Australia. The efficacy and real-world effectiveness of JJA venom immunotherapy (VIT) to prevent anaphylaxis in allergic patients are now well established, with an evidence base that is at least equivalent to that supporting VIT for allergy to other insect species. The tolerability and safety of JJA VIT are comparable with those of honeybee VIT.

Looking and homing: how displaced ants decide where to go

We caught solitary foragers of the Australian Jack Jumper ant, Myrmecia croslandi, and released them in three compass directions at distances of 10 and 15 m from the nest at locations they have never been before. We recorded the head orientation and the movements of ants within a radius of 20 cm from the release point and, in some cases, tracked their subsequent paths with a differential GPS. We find that upon surfacing from their transport vials onto a release platform, most ants move into the home direction after looking around briefly. The ants use a systematic scanning procedure, consisting of saccadic head and body rotations that sweep gaze across the scene with an average angular velocity of 90° s(-1) and intermittent changes in turning direction. By mapping the ants' gaze directions onto the local panorama, we find that neither the ants' gaze nor their decisions to change turning direction are clearly associated with salient or significant features in the scene. Instead, the ants look most frequently in the home direction and start walking fast when doing so. Displaced ants can thus identify home direction with little translation, but exclusively through rotational scanning. We discuss the navigational information content of the ants' habitat and how the insects' behaviour informs us about how they may acquire and retrieve that information.

Mapping the navigational knowledge of individually foraging ants,Myrmecia croslandi

Ants are efficient navigators, guided by path integration and visual landmarks. Path integration is the primary strategy in landmark-poor habitats, but landmarks are readily used when available. The landmark panorama provides reliable information about heading direction, routes and specific location. Visual memories for guidance are often acquired along routes or near to significant places. Over what area can such locally acquired memories provide information for reaching a place? This question is unusually approachable in the solitary foraging Australian jack jumper ant, since individual foragers typically travel to one or two nest-specific foraging trees. We find that within 10 m from the nest, ants both with and without home vector information available from path integration return directly to the nest from all compass directions, after briefly scanning the panorama. By reconstructing panoramic views within the successful homing range, we show that in the open woodland habitat of these ants, snapshot memories acquired close to the nest provide sufficient navigational information to determine nest-directed heading direction over a surprisingly large area, including areas that animals may have not visited previously.