Bioinformatic analysis suggests potential mechanisms underlying parasitoid venom evolution and function.

Hymenopteran parasitoid wasps are a diverse collection of species that infect arthropod hosts and use factors found in their venoms to manipulate host immune responses, physiology, and behaviour. Whole parasitoid venoms have been profiled using proteomic approaches, and here we present a bioinformatic characterization of the venom protein content from Ganaspis sp. 1, a parasitoid that infects flies of the genus Drosophila. We find evidence that diverse evolutionary processes including multifunctionalization, co-option, gene duplication, and horizontal gene transfer may be acting in concert to drive venom gene evolution in Ganaspis sp.1. One major role of parasitoid wasp venom is host immune evasion. We previously demonstrated that Ganaspis sp. 1 venom inhibits immune cell activation in infected Drosophila melanogaster hosts, and our current analysis has uncovered additional predicted virulence functions. Overall, this analysis represents an important step towards understanding the composition and activity of parasitoid wasp venoms.

Outdated allergens are similar to unexpired extracts for the detection of allergen sensitization.

Background: Allergen extracts have relatively short shelf lives, which limits their use and increase financial loss and waste on unused extracts. It is thus important to determine if efficacy persists beyond the expiration date. Objective: To determine the in vivo efficacy and bioavailability of outdated allergen extracts for diagnosis of allergic sensitizations. Methods: We enrolled 34 participants with allergic rhinitis and 5 participants with Hymenoptera hypersensitivity. After confirming allergen sensitization with the unexpired extracts, each participant had a second skin test with the matched outdated one (up to 7 years after the expiration date). All pairs of extracts were from the same company, stored under identical conditions, and tested for microbiologic contamination. The results of 356 skin-prick tests between expired and 111 unexpired extracts were compared. Results: None of the extracts had bacterial or fungal contamination. All outdated extracts produced a positive wheal reaction, with an average of 9.4 mm, which was not significantly different than the unexpired allergens. Seven years outdated lyophilized Hymenoptera extracts showed no significant differences in the wheal's size for the intradermal test at 1 µg/mL, between 5 and 9 mm. Conclusion: Outdated allergen extracts were safe and did not seem to differ in potency and bioavailability from unexpired extracts for the detection of allergen sensitization by skin-prick testing. These results supported our hypothesis that allergen extracts have efficacy and bioavailability that extend beyond the expiry date provided by the manufacturer. For the diagnosis of aeroallergens and Hymenoptera sensitization, it seemed that allergens can be used beyond the expiration date.

Giant fish-killing water bug reveals ancient and dynamic venom evolution in Heteroptera.

True Bugs (Insecta: Heteroptera) produce venom or saliva with diverse bioactivities depending on their feeding strategies. However, little is known about the molecular evolution of the venom toxins underlying these biological activities. We examined venom of the giant fish-killing water bug Lethocerus distinctifemur (Insecta: Belostomatidae) using infrared spectroscopy, transcriptomics, and proteomics. We report 132 venom proteins including putative enzymes, cytolytic toxins, and antimicrobial peptides. Over 73% (96 proteins) showed homology to venom proteins from assassin bugs (Reduviidae), including 21% (28 proteins from seven families) not known from other sources. These data suggest that numerous protein families were recruited into venom and diversified rapidly following the switch from phytophagy to predation by ancestral heteropterans, and then were retained over > 200 my of evolution. In contrast, trophic switches to blood-feeding (e.g. in Triatominae and Cimicidae) or reversions to plant-feeding (e.g., in Pentatomomorpha) were accompanied by rapid changes in the composition of venom/saliva, including the loss of many protein families.

Melt With This Kiss: Paralyzing and Liquefying Venom of The Assassin Bug Pristhesancus plagipennis (Hemiptera: Reduviidae).

Assassin bugs (Hemiptera: Heteroptera: Reduviidae) are venomous insects, most of which prey on invertebrates. Assassin bug venom has features in common with venoms from other animals, such as paralyzing and lethal activity when injected, and a molecular composition that includes disulfide-rich peptide neurotoxins. Uniquely, this venom also has strong liquefying activity that has been hypothesized to facilitate feeding through the narrow channel of the proboscis-a structure inherited from sap- and phloem-feeding phytophagous hemipterans and adapted during the evolution of Heteroptera into a fang and feeding structure. However, further understanding of the function of assassin bug venom is impeded by the lack of proteomic studies detailing its molecular composition.By using a combined transcriptomic/proteomic approach, we show that the venom proteome of the harpactorine assassin bug Pristhesancus plagipennis includes a complex suite of >100 proteins comprising disulfide-rich peptides, CUB domain proteins, cystatins, putative cytolytic toxins, triabin-like protein, odorant-binding protein, S1 proteases, catabolic enzymes, putative nutrient-binding proteins, plus eight families of proteins without homology to characterized proteins. S1 proteases, CUB domain proteins, putative cytolytic toxins, and other novel proteins in the 10-16-kDa mass range, were the most abundant venom components. Thus, in addition to putative neurotoxins, assassin bug venom includes a high proportion of enzymatic and cytolytic venom components likely to be well suited to tissue liquefaction. Our results also provide insight into the trophic switch to blood-feeding by the kissing bugs (Reduviidae: Triatominae). Although some protein families such as triabins occur in the venoms of both predaceous and blood-feeding reduviids, the composition of venoms produced by these two groups is revealed to differ markedly. These results provide insights into the venom evolution in the insect suborder Heteroptera.

Dengue Virus Infection of Aedes aegypti Requires a Putative Cysteine Rich Venom Protein.

Dengue virus (DENV) is a mosquito-borne flavivirus that causes serious human disease and mortality worldwide. There is no specific antiviral therapy or vaccine for DENV infection. Alterations in gene expression during DENV infection of the mosquito and the impact of these changes on virus infection are important events to investigate in hopes of creating new treatments and vaccines. We previously identified 203 genes that were ≥5-fold differentially upregulated during flavivirus infection of the mosquito. Here, we examined the impact of silencing 100 of the most highly upregulated gene targets on DENV infection in its mosquito vector. We identified 20 genes that reduced DENV infection by at least 60% when silenced. We focused on one gene, a putative cysteine rich venom protein (SeqID AAEL000379; CRVP379), whose silencing significantly reduced DENV infection in Aedes aegypti cells. Here, we examine the requirement for CRVP379 during DENV infection of the mosquito and investigate the mechanisms surrounding this phenomenon. We also show that blocking CRVP379 protein with either RNAi or specific antisera inhibits DENV infection in Aedes aegypti. This work identifies a novel mosquito gene target for controlling DENV infection in mosquitoes that may also be used to develop broad preventative and therapeutic measures for multiple flaviviruses.

Clawing through evolution: toxin diversification and convergence in the ancient lineage Chilopoda (centipedes).

Despite the staggering diversity of venomous animals, there seems to be remarkable convergence in regard to the types of proteins used as toxin scaffolds. However, our understanding of this fascinating area of evolution has been hampered by the narrow taxonomical range studied, with entire groups of venomous animals remaining almost completely unstudied. One such group is centipedes, class Chilopoda, which emerged about 440 Ma and may represent the oldest terrestrial venomous lineage next to scorpions. Here, we provide the first comprehensive insight into the chilopod "venome" and its evolution, which has revealed novel and convergent toxin recruitments as well as entirely new toxin families among both high- and low molecular weight venom components. The ancient evolutionary history of centipedes is also apparent from the differences between the Scolopendromorpha and Scutigeromorpha venoms, which diverged over 430 Ma, and appear to employ substantially different venom strategies. The presence of a wide range of novel proteins and peptides in centipede venoms highlights these animals as a rich source of novel bioactive molecules. Understanding the evolutionary processes behind these ancient venom systems will not only broaden our understanding of which traits make proteins and peptides amenable to neofunctionalization but it may also aid in directing bioprospecting efforts.

Venom allergen 5 is Associated With Deltamethrin Resistance in Culex pipiens pallens (Diptera: Culicidae).

The mosquito, Culex pipiens pallens (L.), is an important vector of encephalitis and filariasis in northern China. The control of these mosquitoes occurs primarily via the use of pyrethroid insecticides, such as deltamethrin. The widespread and improper application of pyrethroid has resulted in the evolution of pyrethroid resistance amongst many mosquito populations, including Cx. pipiens pallens. Previous studies using high-throughput transcriptome sequencing have identified that the venom allergen 5 gene is differentially expressed between deltamethrin-susceptible and deltamethrin-resistant Cx. pipiens pallens. In this study, quantitative real-time polymerase chain reaction analyses revealed that venom allergen 5 was significantly overexpressed in adult females of both deltamethrin-resistant laboratory populations and two field populations. The transcriptional level of venom allergen 5 in the laboratory populations was elevated as the levels of deltamethrin resistance increased. Full-length cDNAs of the venom allergen 5 gene were cloned from Cx. pipiens pallens, and contained an open reading frame of 765 bp, encoding a protein with 254 amino acids. The deduced amino acid sequence shared 100% identity with the ortholog in Culex quinquefasciatus Say. The overexpression of venom allergen 5 decreased the susceptibility of mosquito cells to deltamethrin, while knockdown of this gene by RNAi increased the susceptibility of mosquitoes to deltamethrin. This study provides the first evidence of the association between the venom allergen 5 gene and deltamethrin resistance in mosquitoes.

Detection and Identification of the Invasive Sirex noctilio (Hymenoptera: Siricidae) Fungal Symbiont, Amylostereum areolatum (Russulales: Amylostereacea), in China and the Stimulating Effect of Insect Venom on Laccase Production by A. areolatum YQL03.

The Eurasian woodwasp Sirex noctilio F. was first detected in Daqing, Heilongjiang Province, in the northeast region of China in 2013. Here, we investigated the S. noctilio's fungal symbiont, Amylostereum areolatum, and insect venom produced in its acid (venom) gland. Overall, seven out of 10 fungal isolates obtained from the mycangia of 10 adult S. noctilio females in this study were identified as A. areolatum. The remaining three isolates were identified as Trichoderma viride, Verticillium dahlia, and Geosmithia pallida, which were probably contaminants that entered during the mycangia-spore extraction process. The enzyme activity bioassay showed that the level of laccase activity of A. areolatum YQL03 in liquid medium is prominently enhanced by insect venom, but was relatively low when venom was not available as an inducer. This study confirms the presence of A. areolatum in S. noctilio specimens from China. The putative heat-stable factors identified in S. noctilio venom may contribute novel information about the pathogenic mechanism of the S. noctilio-A. areolatum pine-killing pest complex on host trees.

Phylogeny, envenomation syndrome, and membrane permeabilising venom produced by Australia's electric caterpillar Comana monomorpha.

Zygaenoidea is a superfamily of lepidopterans containing many venomous species, including the Limacodidae (nettle caterpillars) and Megalopygidae (asp caterpillars). Venom proteomes have been recently documented for several species from each of these families, but further data are required to understand the evolution of venom in Zygaenoidea. In this study, we examined the 'electric' caterpillar from North-Eastern Australia, a limacodid caterpillar densely covered in venomous spines. We used DNA barcoding to identify this caterpillar as the larva of the moth Comana monomorpha (Turner, 1904). We report the clinical symptoms of C. monomorpha envenomation, which include acute pain, and erythema and oedema lasting for more than a week. Combining transcriptomics of venom spines with proteomics of venom harvested from the spine tips revealed a venom markedly different in composition from previously examined limacodid venoms that are rich in peptides. In contrast, the venom of C. monomorpha is rich in aerolysin-like proteins similar to those found in venoms of asp caterpillars (Megalopygidae). Consistent with this composition, the venom potently permeabilises sensory neurons and human neuroblastoma cells. This study highlights the diversity of venom composition in Limacodidae.

Divergent venom effectors correlate with ecological niche in neuropteran predators.

Neuropteran larvae are fierce predators that use venom to attack and feed on arthropod prey. Neuropterans have adapted to diverse and sometimes extreme habitats, suggesting their venom may have evolved accordingly, but the ecology and evolution of venom deployment in different families is poorly understood. We applied spatial transcriptomics, proteomics, morphological analysis, and bioassays to investigate the venom systems in the antlion Euroleon nostras and the lacewing Chrysoperla carnea, which occupy distinct niches. Although the venom system morphology was similar in both species, we observed remarkable differences at the molecular level. E. nostras produces particularly complex venom secreted from three different glands, indicating functional compartmentalization. Furthermore, E. nostras venom and digestive tissues were devoid of bacteria, strongly suggesting that all venom proteins are of insect origin rather than the products of bacterial symbionts. We identified several toxins exclusive to E. nostras venom, including phospholipase A2 and several undescribed proteins with no homologs in the C. carnea genome. The compositional differences have significant ecological implications because only antlion venom conferred insecticidal activity, indicating its use for the immobilization of large prey. Our results indicate that molecular venom evolution plays a role in the adaptation of antlions to their unique ecological niche.

Structure and bioactivity of an insecticidal trans-defensin from assassin bug venom.

Disulfide-rich peptides such as defensins play diverse roles in immunity and ion channel modulation, as well as constituting the bioactive components of many animal venoms. We investigated the structure and bioactivity of U-RDTX-Pp19, a peptide previously discovered in venom of the assassin bug Pristhesancus plagipennis. Recombinant Pp19 (rPp19) was found to possess insecticidal activity when injected into Drosophila melanogaster. A bioinformatic search revealed that domains homologous to Pp19 are produced by assassin bugs and diverse other arthropods. rPp19 co-eluted with native Pp19 isolated from P. plagipennis, which we found is more abundant in hemolymph than venom. We solved the three-dimensional structure of rPp19 using 2D 1H NMR spectroscopy, finding that it adopts a disulfide-stabilized structure highly similar to known trans-defensins, with the same cystine connectivity as human α-defensin (I-VI, II-IV, and III-V). The structure of Pp19 is unique among reported structures of arthropod peptides.

Construction and expression of an antimicrobial peptide scolopin 1 from the centipede venoms of Scolopendra subspinipes mutilans in Escherichia coli using SUMO fusion partner.

Antimicrobial peptide scolopin 1 (AMP-scolopin 1) is a small cationic peptide identified from centipede venoms of Scolopendra subspinipes mutilans. It has broad-spectrum activities against bacteria, fungi, and tumor cells, which may possibly be used as an antimicrobial agent. We first report here the application of small ubiquitin-related modifier (SUMO) fusion technology to the expression and purification of cationic antimicrobial peptide AMP-scolopin 1. The fusion protein expressed in a soluble form was purified to a purity of 95% by Ni-IDA chromatography. After the SUMO-scolopin 1 fusion protein was cleaved by the SUMO protease at 30°C for 1 h, the cleaved sample was reapplied to a Ni-IDA. The recombinant scolopin1 had similar antimicrobial properties to the synthetic scolopin 1. Thus, we successfully established a system for purifying peptide of centipede, which could be used for further research.

Molecular cloning and characterization of a new cDNA sequence encoding a venom peptide from the centipede Scolopendra subspinipes mutilans.

Many studies have been performed on venomous peptides derived from animals. However, little of this research has focused on peptides from centipede venoms. Here, a venom gland cDNA library was successfully constructed for the centipede Scolopendra subspinipes mutilans. A new cDNA encoding the precursor of a venom peptide, named SsmTx, was cloned from the venomous gland cDNA library of the centipede S. subspinipes mutilans. The full-length SsmTx cDNA sequence is 465 nt, including a 249 nt ORF, a 45 nt 5' UTR and a 171 nt 3' UTR. There is a signal tail AATAAA 31 nt upstream of the poly (A) tail. The precursor nucleotide sequence of SsmTx encodes a signal peptide of 25 residues and a mature peptide of 57 residues, which is bridged by two pairs of disulfide bonds. SsmTx displays a unique cysteine motif that is completely different from that of other venomous animal toxins. This is the first reported cDNA sequence encoding a venom peptide from the centipede S. subspinipes mutilans.

Venom resistance mechanisms in centipede show tissue specificity.

Venomous animals utilize venom glands to secrete and store powerful toxins for intraspecific and/or interspecific antagonistic interactions, implying that tissue-specific resistance is essential for venom glands to anatomically separate toxins from other tissues. Here, we show the mechanism of tissue-specific resistance in centipedes (Scolopendra subspinipes mutilans), where the splice variant of the receptor repels its own toxin. Unlike the well-known resistance mechanism by mutation in a given exon, we found that the KCNQ1 channel is highly expressed in the venom gland as a unique splice variant in which the pore domain and transmembrane domain six, partially encoded by exon 6 (rather than 7 as found in other tissues), contain eleven mutated residues. Such a splice variant is sufficient to gain resistance to SsTx (a lethal toxin for giant prey capture) in the venom gland due to a partially buried binding site. Therefore, the tissue-specific KCNQ1 modification confers resistance to the toxins, establishing a safe zone in the venom-storing/secreting environment.

Effects of sea-level rise on physiological ecology of populations of a ground-dwelling ant.

INTRODUCTION: Sea-level rise is a consequence of climate change that can impact the ecological and physiological changes of coastal, ground-dwelling species. Sea-level rise has a potential to inundate birds, rodents, spiders, and insects that live on the ground in coastal areas. Yet, there is still much to be learned concerning the specifics of these impacts. The red imported fire ant Solenopsis invicta (Buren) excavates soil for its home and is capable of surviving flooding. Because of their ground-dwelling life history and rapid reproduction, fire ants make an ideal model for discovery and prediction of changes that may be due to sea-level rise. There are up to 500,000 individuals in a colony, and these invasive ants naturally have a painful sting. However, observations suggest that colonies of fire ants that dwell in tidally-influenced areas are more aggressive with more frequent stings and more venom injected per sting (behavioral and physiological changes) than those located inland. This may be an adaption to sea-level rise. Therefore, the objective of this study is to elucidate differences in inland and coastal defensiveness via micro-dissection and comparison of head width, head length, stinger length, and venom sac volume. But first because fire ants' ability to raft on brackish tidal water is unknown, it had to be determined if fire ants could indeed raft in brackish water and examine the behavior differences between those flooded with freshwater vs. saltwater. METHODS: To test the coastal-aggression hypothesis, inland colonies and coastal colonies, which experience relatively greater amounts of flooding, specifically regular tidal and windblown water and oscillations (i.e. El Nino Southern Oscillation) from the Gulf of Mexico, were collected. To mimic sea-level rise, the colonies were flooded in salinities that correspond to both their collection site and conditions found in a variety of locales and situations (such as storm surge from a tropical storm). Individual ants were immediately taken from each colony for dissection before flooding, 1-hour into flooding, and 24-hours into flooding. RESULTS AND DISCUSSION: Fire ants use their venom to defend themselves and to communicate alarm or aggression. Dissections and measurement of heads, venom sacs, and stingers revealed both coastal and inland colonies experience an increase in venom sac volume after 24 hours; in fact coastal colonies increased their venom volume by 75% after 24 h of flooding Whether this venom sac enlargement is due to diffusion of water or venom sac production is unknown. These ground-dwelling ants exhibit physiological and behavioral adaptations to ongoing sea-level rise possibly indicating that they are responding to increased flooding. Fire ants will raft on high-salinity water; and sea-level rise may cause stings by flooded ants to be more severe because of increased venom volume.

Weaponisation 'on the fly': Convergent recruitment of knottin and defensin peptide scaffolds into the venom of predatory assassin flies.

Many arthropod venom peptides have potential as bioinsecticides, drug leads, and pharmacological tools due to their specific neuromodulatory functions. Assassin flies (Asilidae) are a family of predaceous dipterans that produce a unique and complex peptide-rich venom for killing insect prey and deterring predators. However, very little is known about the structure and function of their venom peptides. We therefore used an E. coli periplasmic expression system to express four disulfide-rich peptides that we previously reported to exist in venom of the giant assassin fly Dolopus genitalis. After purification, each recombinant peptide eluted from a C18 column at a position closely matching its natural counterpart, strongly suggesting adoption of the native tertiary fold. Injection of purified recombinant peptides into blowflies (Lucilia cuprina) and crickets (Acheta domestica) revealed that two of the four recombinant peptides, named rDg3b and rDg12, inhibited escape behaviour in a manner that was rapid in onset (<1 min) and reversible. Homonuclear NMR solution structures revealed that rDg3b and rDg12 adopt cystine-stabilised α/ß defensin and inhibitor cystine knot folds, respectively. Although the closest known homologues of rDg3b at the level of primary structure are dipteran antimicrobial peptides such as sapecin and lucifensin, a DALI search showed that the tertiary structure of rDg3b most closely resembles the KV11.1-specific α-potassium channel toxin CnErg1 from venom of the scorpion Centruroides noxius. This is mainly due to the deletion of a large, unstructured loop between the first and second cysteine residues present in Dg3b homologues from non-asiloid, but not existing in asiloid, species. Patch-clamp electrophysiology experiments revealed that rDg3b shifts the voltage-dependence of KV11.1 channel activation to more depolarised potentials, but has no effect on KV1.3, KV2.1, KV10.1, KCa1.1, or the Drosophila Shaker channel. Although rDg12 shares the inhibitor cystine knot structure of many gating modifier toxins, rDg12 did not affect any of these KV channel subtypes. Our results demonstrate that multiple disulfide-rich peptide scaffolds have been convergently recruited into asilid and other animal venoms, and they provide insight into the molecular evolution accompanying their weaponisation.

Human Chondrocyte Activation by Toxins From Premolis semirufa, an Amazon Rainforest Moth Caterpillar: Identifying an Osteoarthritis Signature.

Pararamosis is a disease that occurs due to contact with the hairs of the larval stage of the Brazilian moth Premolis semirufa. Envenomation induces osteoarticular alterations with cartilage impairment that resembles joint synovitis. Thus, the toxic venom present in the caterpillar hairs interferes with the phenotype of the cells present in the joints, resulting in inflammation and promoting tissue injury. Therefore, to address the inflammatory mechanisms triggered by envenomation, we studied the effects of P. semirufa hair extract on human chondrocytes. We have selected for the investigation, cytokines, chemokines, matrix metalloproteinases (MMPs), complement components, eicosanoids, and extracellular matrix (ECM) components related to OA and RA. In addition, for measuring protein-coding mRNAs of some molecules associated with osteoarthritis (OA) and rheumatoid arthritis (RA), reverse transcription (RT) was performed followed by quantitative real-time PCR (RT-qPCR) and we performed the RNA-sequencing (RNA-seq) analysis of the chondrocytes transcriptome. In the supernatant of cell cultures treated with the extract, we observed increased IL-6, IL-8, MCP-1, prostaglandin E2, metalloproteinases (MMP-1, MMP-2, MMP-3 and MMP-13), and complement system components (C3, C4, and C5). We noticed a significant decrease in both aggrecan and type II collagen and an increase in HMGB1 protein in chondrocytes after extract treatment. RNA-seq analysis of the chondrocyte transcriptome allowed us to identify important pathways related to the inflammatory process of the disease, such as the inflammatory response, chemotaxis of immune cells and extracellular matrix (ECM) remodeling. Thus, these results suggest that components of Premolis semirufa hair have strong inflammatory potential and are able to induce cartilage degradation and ECM remodeling, promoting a disease with an osteoarthritis signature. Modulation of the signaling pathways that were identified as being involved in this pathology may be a promising approach to develop new therapeutic strategies for the control of pararamosis and other inflammatory joint diseases.

Centipede Venom Peptides Acting on Ion Channels.

Centipedes are among the oldest venomous arthropods that use their venom to subdue the prey. The major components of centipede venom are a variety of low-molecular-weight peptide toxins that have evolved to target voltage-gated ion channels to interfere with the central system of prey and produce pain or paralysis for efficient hunting. Peptide toxins usually contain several intramolecular disulfide bonds, which confer chemical, thermal and biological stability. In addition, centipede peptides generally have novel structures and high potency and specificity and therefore hold great promise both as diagnostic tools and in the treatment of human disease. Here, we review the centipede peptide toxins with reported effects on ion channels, including Nav, Kv, Cav and the nonselective cation channel polymodal transient receptor potential vanilloid 1 (TRPV1).

Fire ant venom alkaloids act as key attractants for the parasitic phorid fly, Pseudacteon tricuspis (Diptera: Phoridae).

The phorid fly, Pseudacteon tricuspis Borgmeier, is an introduced parasitoid of imported fire ants, Solenopsis spp., in the USA. Although the assumption that phorid flies use fire ant alarm pheromones for host location is probably true, we demonstrated in a previous study the possible involvement of other ant semiochemicals in the response of P. tricuspis to fire ants. This study was conducted to determine the glandular sources and identity of the semiochemicals mediating this interaction. First, we tested the electroantennogram response of P. tricuspis to extracts of key body parts and glands of workers of the red imported fire ant, S. invicta Buren. The results confirm that the poison (venom) gland/sac is the key source of compounds which elicited strong antennal activity in P. tricuspis. Follow-up studies were conducted by using a combination of bioassay-guided fractionation and behavioral bioassays to test the hypothesis that attraction of this parasitoid to fire ants is mediated by venom alkaloids. The results confirm the response of P. tricuspis to physiologically relevant amounts of the two venom alkaloid fractions (cis and trans alkaloid fractions) of S. invicta. Further analysis by coupled gas chromatography-electroantennogram detection revealed nine venom alkaloid components including two novel 2,6-dialkylpiperideines that elicited significant antennal activity in P. tricuspis. This is the first demonstration of the role of venom alkaloids of ants as attractants for their natural enemies. We propose a semiochemical-mediated host location mechanism for P. tricuspis involving both alarm pheromones and venom alkaloids. The ecological significance of these findings, including the attraction of male P. tricuspis to fire ant venom alkaloids, possibly for mate location, is discussed.