Venom Toxins of Fire Ants

Venom Genomics and Proteomics DOI 10.1007/978-94-007-6649-5_38-1 # Springer Science+Business Media Dordrecht 2014 Venom Toxins of Fire Ants Eduardo Gonçalves Paterson Fox* Département d’Ecologie et Evolution, UNIL-Sorge Le Biophore, Lausanne, VD, Switzerland Abstract Fire ants are aggressive invasive insects spread around the world via ship cargo, mainly originating from the United States. They can cause severe impacts on human activities and the environment. This chapter presents an overview of what is known about fire ant venom, ending with different open possibilities for investigation in this topic. For decades, studies on fire ant venoms have been limited in scope because of the technical difficulties in obtaining enough samples for analysis and bioassays; yet now there is one simple, effective, published method for extracting venom from whole colonies. Fire ant venom is mainly composed of a mixture of >95 % bioactive piperidine alkaloids and 0.01 % of proteins, which comprise mainly allergens, phospholipases, and neurotoxins. The alkaloids of fire ants, generally named solenopsins, are well known for their antifungal and insecticidal properties; however, many have also been suggested as promising alternatives for other biomedical applications, such as the treatment of parasitemia and various neurological diseases. The venom proteins of fire ants remain only superficially studied, as most published literature focuses on just four allergens. Crucially, others may contain compounds of interest to immunotherapy or even play a central role in aspects of social organization. Introduction Fire ants are a serious worldwide invasive pest originating from South America. Some of the first observations on the aggressiveness of fire ants were recorded by Henry Bates in his famous book The Naturalist on the River Amazon. As the author states, . . .Tapajos is nearly free from the insect pests [. . .] but the formiga de fogo is perhaps [the] greater. [. . .] They seem to attack persons out of sheer malice [. . .] whose sting is likened by the Brazilians to the puncture of a red-hot needle. (Bates 1856) Fire ants will typically attack in great numbers, often while foraging, but most markedly when their fragile nests are disturbed. Yet, fire ants will not only attack man and also not only when defending their nests. Indeed, fire ants have been observed sending out foragers to locate nests and burrows of other animals, including vertebrates, and then recruit worker raids to attack, kill, and consume the nestlings and their food (Chalcraft and Andrews 1999). This results in considerable ecological impact from a fire ant infestation on local avian (Drees 1994) and reptile fauna (Allen et al. 2001) and can likewise affect human activities, livestock, and domestic animals. This behavior can affect humans, where fire ants have been reported to recurrently enter homes and attack sleeping people of various ages in their beds both in residences and hospitals in infested areas (author’s observations in Brazil). Fire ant infestations have been a recurrent issue in small towns in the Amazon (reportedly from Novo Aripuanã, Envira, and Novo Airão), where people can be forced to *Email: [email protected] *Email: [email protected] Page 1 of 16 Venom Genomics and Proteomics DOI 10.1007/978-94-007-6649-5_38-1 # Springer Science+Business Media Dordrecht 2014 Fig. 1 A fire ant minor worker stinging the author (Picture courtesy of Roberto Eizemberg) abandon their homes until the infestation is controlled (Mendes 2008). Concerning other recurrent problems caused by fire ants in agriculture, they are known to attack several culture crops and some stored grains and can associate with sap-sucking pest insects, elevating their numbers, apart from being a constant nuisance to the exposed field worker. Moreover, they can frequently attack farm animals, especially young cubs. Some remarks on the stinging behavior of fire ants while attacking are as follows: they often take some time climbing on the victim prior to stinging; thus, it is common for prey and victims to realize the attack only when many ants are already stinging at roughly the same time. Given their small size, fire ants must hold with their mandibles in order to insert their sting (Fig. 1), and the injection is rather slow as the venom is poorly soluble in water and their venom apparatus lacks muscles around the venom reservoir (Fox et al. 2010). There is one erroneous description of their stinging behavior that has been repeated many times over in the literature: that “fire ants sting repeatedly, many times rotating in a circular pattern, while maintaining a grip with their mandibles” (e.g., Hoffman 1995) – this notion must be revisited, as logically an insect cannot rotate much while biting. Instead, a fire ant will quite often let go of the bite shortly after the sting was introduced into human skin and will remain waiting in place while venom is being injected; it generally will bite and sting again in the case of attempted removal or when eventually the grip of the inserted stinger comes lose. This pattern of behavior generally results in three or more different stings close together (usually not in circular pattern) delivered by each attacking fire ant while wandering, for instance, under the clothes of a human victim. On the other hand, during fights (usually against another insect), it will hold the bite hard and continuously with its mandibles (even postmortem) and will deliver venom whether or not the stinger is inserted, but this is generally like most other ants behave. Fire ants are among the most common ants to be found in South America, where they typically occur in the lawns around houses and public areas (only one species being recurrently found in woods). Given their prevalence, quite often local ant queens and small colonies can be accidentally transported elsewhere by sea among commercial goods and ship ballast, and this has been a major determinant for the spread of invasive fire ants around the world (Tschinkel 2006). In some invaded areas, fire ant colonies have become even more abundant than in their original habitats in South America, and local fauna and human residents must physiologically and behaviorally adapt to deal with the new aggressive ants. Thus, quite often the arrival of fire ants can prove disastrous to local populations. At some point in the middle of the twentieth century, two species of fire ants were introduced to the Southern United States, probably among shipped goods originating from Page 2 of 16 Venom Genomics and Proteomics DOI 10.1007/978-94-007-6649-5_38-1 # Springer Science+Business Media Dordrecht 2014 somewhere between Argentina and the south of Brazil (Tschinkel 2006). At least one other fire ant species may have arrived in the United States much earlier, perhaps coming from Central America (Wetterer 2011). Nowadays approximately 40 % of the territory in the United States is reported to contain fire ant colonies, and the estimated national spending surmounts over five billion dollars a year in losses and combative efforts, which are proving not quite effective (McDonald 2006). These figures are but a numeric indication of how fire ants can be dangerous to agriculture and human welfare when their numbers get out of hand. The world spread of fire ants among shipped commerce goods, mainly originating from the United States, has become a serious concern to specialists around the globe, and the potential damage to regions suitable for invasion (for an interesting predictive map, refer to Morrison et al. 2004) is impossible to precise. As a piece of irony, referring again to Bates (1856), who wrote about the problem with fire ants in the Brazilian Amazon, This species is exclusively found in sandy soils, in open semi-cultivated or neglected places [where] they increase only in the neighbourhood of deserted houses or unweeded plantations; consequently they are a scourge only to the lazy and worthless people that inhabit the shores of this magnificent river. As a result of the growing concern, fire ants are currently one of the most intensively studied insects in the world and as such have become a model species for studies of social insects. One of the most strongly emphasized aspects concerning fire ant biology is the effect of their stings and venom. This chapter aims to present a summarized overview of the state of knowledge on fire ant venom with special emphasis on some potential trends of future studies. Fire Ant Venom: A Dangerous Mystery As to illustrate how much of a threat fire ants can be to human populations, in some regions in the United States, over one fourth of the local residents may prove highly sensitive to fire ant stings (Ownby 2008). Quite often highly sensitive subjects live in heavily infested areas, where abundant fire ant nests can be found at house gardens and sidewalks where children and pets play. In such areas fire ants constitute a constant danger, especially to young toddlers and the elderly, who cannot react quickly and efficiently against aggressive insect attacks. The problem is fire ant stings can be often quite harmful, as will be explained in this section. Among the stinging ants that recurrently induce anaphylaxis – Hypoponera, Myrmecia, Odontomachus, Pogonomyrmex, Pachycondyla, Pseudomyrmex, Rhytidoponera, Solenopsis, and Tetramorium – registered allergic reactions following accidents with fire ant stings are by far the most frequent prevalent in a world scale, with an estimated 50 % of residents being stung in endemic regions (Kemp et al. 2000). The onset of a serious anaphylactic reaction to fire ant stings in sensitized victims can occur within minutes after few stings, often unnoticed stings, and this can rapidly escalate from systemic itching and swelling to tachycardia and difficulty breathing. In rare cases, such reactions may culminate in death (Stablein et al. 1985; Prahlow and Barnard 1989; More et al. 2008). As such, sensitive patients who reside in infested areas are advised to undergo immunotherapy as a security measure to increase their resistance to likely future encounters with fire ants (Stafford 1996). However, fire ant immunotherapy is even today done using whole-body extracts of workers, given the traditional impracticability in obtaining pure venom protein as delineated below. Most studies about fire ant venoms are actually based on a small amount of general published information about the venom composition. For over 30 years, the limited sensitivity of methods employed and difficulties in extracting venom in quantities sufficient for biochemical Page 3 of 16 Venom Genomics and Proteomics DOI 10.1007/978-94-007-6649-5_38-1 # Springer Science+Business Media Dordrecht 2014 Fig. 2 Traditional labor-intensive methods for collection of fire ant venom. (a) Many studies have reported milking individual ants using a capillary tube, inducing venom extrusion by different means (e.g., excision of gaster, contact with another ant, electric shock). (b) A depiction of an intricate method described in Hoffman (1995), where pure venom is amassed into a tube by sucking the gaster of individual ants with a capillary tube, followed by centrifugation for separation of alkaloidal phase and aqueous phase (Adapted from Hoffman (1995), not drawn to scale) characterization have hampered any ampler overview regarding the composition of fire ant venoms. Also, the unique composition of fire ant venom makes it hard to analyze: it is primarily composed (over 95 %) of a mixture of hydrophobic alkaloids somehow solubilized in conjunction with a comparatively minute amount of toxic proteins and peptides. Given the small mass of each individual ant (ca. 1 mg), venom extracted from just a few thousand fire ants will deliver quantities sufficient for only superficial analysis of the most abundant venom alkaloids. Traditional methods for extracting fire ant venom are remarkably laborious, based on dissecting or milking each individual ant for venom over several weeks and saving the samples in a freezer until use. A few examples of such methods are depicted in Fig. 2, using fine capillary tubes or tissue paper, or even some suction apparatus adapted to collect venom from individual workers. Haight and Tschinkel 2003 reported employing direct repetitive contact of fire ant workers with a larger carpenter ant so as to stimulate their releasing venom droplets to keep them alive after milking. However, one remarkable biochemical investigation from the late 1970s (Baer et al. 1979) reported obtaining as much as ~120 mg of pure venom, through milking individual amputated gasters (=posterior pedunculate body part of aculeate hymenopterans) of an estimated million ants – this feat having required three fully dedicated technicians over almost 3 years (personal communication by Murray S. Blum). This study by Baer et al. (1979) was the first to detect the existence and analyze proteins among the mixture of venom alkaloids, which were then partially separated by size-exclusion liquid chromatography and superficially described in terms of their amino acid composition. Unfortunately for scientific studies and immunotherapists, there is only one company from the United States selling pure venom extracts of fire ants for research, and they have not patented nor formally described their method of extraction. In some studies it was briefly stated that the ants can be milked by electrical stimulation (for a comprehensive overview on traditional methods for venom extraction and purification with fire ants, refer to Hoffman 1995), thus possibly done by placing a certain quantity of workers on tissue paper on an electric-pulse grid moistened with buffer solution and then recovering the extruded venom compounds from the tissue paper. Given such technical and practical impediments for collecting fire ant venom, research in this area has been delayed for decades, and as mentioned, even today immunotherapy with sensitive subjects is limited to the use of fire ant whole-body extracts. Page 4 of 16 Venom Genomics and Proteomics DOI 10.1007/978-94-007-6649-5_38-1 # Springer Science+Business Media Dordrecht 2014 Fig. 3 Chromatographic laboratory analyses of fire ant venom proteins extracted by the fast method described by Fox et al. (2013). (a) Liquid chromatography of aqueous phase of Solenopsis invicta venom through a Sephadex G-75 gel exclusion column, as tracked by absorption at 256 nm. (b) Bidimensional SDS-PAGE scanned result gel of lyophilized venom proteins of S. invicta, revealed by silver staining reaction. (c) Unidimensional SDS-PAGE scanned result gel of venom proteins of S. saevissima (middle lane) and S. invicta (right lane); on the left lane the molecular weight marker is presented with respective protein band masses Only recently a more practical method for extracting venom from large quantities of fire ants has been devised, enabling extraction of whole venom from fire ants in gram amounts within a few hours (Fox et al. 2013), based on a simple solvent extraction of live ants. This procedure has been shown to yield the very same results as obtained by the much more laborious methods employed by previous authors, as can be seen both by liquid chromatography and proteomic analysis. See Fig. 3 for a comparison of obtained results with previous studies employing extraction methods as shown in Fig. 2: compare 3A with similar profiles presented in Baer et al. (1979) and Hoffman et al. (1990), compare 3B with similar profiles presented in Pinto et al. (2012) and Sukprasert et al. (2012), and compare 3C with similar results shown in Hoffman et al. 1988, Hoffman et al. (1990), and Sukprasert et al. (2012). Basically, the fire ants are obtained from field nests (or cultured in the laboratory), separated from the soil by flotation, and then they can be allowed to clean/groom themselves for some time or can be directly immersed into a biphasic mixture of water and some strongly apolar solvent, such as chloroform or pentane. The venom alkaloids and cuticular hydrocarbons will be extracted into the upper (organic) phase, while the lower aqueous phase will prove rich in active venom proteins and Page 5 of 16 Venom Genomics and Proteomics DOI 10.1007/978-94-007-6649-5_38-1 # Springer Science+Business Media Dordrecht 2014 4 1 2 3 Organic phase with hydrocarbons and alkaloids (EVAPORATE) Water or buffer Add living ants Add apolar solvent Aqueous phase rich in proteins and peptides Fig. 4 A depiction of the fast method for massive venom extraction described by Fox et al. (2013) peptides (Fig. 4). Clean extracts of both fractions are obtained after centrifugation and evaporation of the solvents for further procedures or freeze storage. This new method has opened the possibility of obtaining fire ant venom in greater amounts, using minimum structure and enabling subsequent bioassays or purification steps. It has been hinted that the extraction method could be adapted to obtain venom from other aggressive animals, such as Pseudomyrmex ants or wasps and bees, but this still awaits direct experimentation (Fox et al. 2013). Some of the Most Toxic Known Proteins: A Brief Molecular Overview For decades, there were only a handful of proteins known from the venom of fire ants, given the general difficulty in obtaining enough samples for proper analysis. These few venom proteins were described for the first time in a comparative chromatographic study using purchased fire ant venom extract, and milked venom, shown in Hoffman et al. (1988). This study reported on four most abundant allergens, baptized Sol i 1–4. This pioneering study helped elucidate why the ant stings could often cause serious allergic reactions. Later, a series of studies focusing on each of these four allergens presented them in deeper detail (mainly Hoffman et al. 1990, 2005; Hoffman 1993; Hoffman 1995; Schmidt et al. 1996; Padavattan et al. 2008; Borer et al. 2012), for which the sequence, enzymatic activities, gene structure, and molecular structures were determined. This information will be briefly summarized below. Some insect allergens may also have enzymatic activity, as demonstrated here by Sol i 1, which is a phospholipase A1B – a member of the lipoprotein lipase family that has homology with other hymenopterans. The other three described allergens in fire ant venom have no apparent enzymatic activity (although Hoffman et al. (1988) stated on page 826 that “Sol i II is a phospholipase with A and some B activity,” as this activity was not mentioned in any subsequent papers except for Sol i 1 that was most probably a typographical mistake). Actually, venom allergen Sol i 2 was predicted as an odorant-binding protein (OBP) based on its sequence and modeled 3D structure, also suggesting that the similar protein Sol i 4 must be an OBP – OBPs are intermediary soluble molecules mediating the passage of pheromones or signal compounds between systems where they would be otherwise insoluble. The last described protein, venom allergen Sol i 3, has not yet demonstrated any alternate role, yet it belongs to one conserved class of known allergens known as cysteine-rich secretory proteins (CRISP), which is present in the venom of several aculeate hymenopterans, including the wasp antigen-5 and the ponerine ant allergen Pac c 3. The CRISP protein family also includes a series of cancer-related antigens and some inflammatory proteins known from sandfly saliva (Anderson et al. 2006). Page 6 of 16 Venom Genomics and Proteomics DOI 10.1007/978-94-007-6649-5_38-1 # Springer Science+Business Media Dordrecht 2014 Top-Hit species distribution BLAST Top-Hits Solenopsis invicta Camponotus floridanus Tribolium castaneum Acromyrmex echinatior Harpegnathos saltator Nasonia vitripennis Acyrthosiphon pisum Megachile rotundata Bombyx mori Aedes aegypti Drosophila melanogaster Hydra magnipapillata Trichinella spiralis Danaus plexippus Epicauta funebris Glyptapanteles flavicoxis Strongylocentrotus purpuratus Saccoglossus kowalevskii Candidatus Regieila Xenopus (Silurana) Drosophila ananassae Daphnia pulex Vitis vinifera Schistosoma mansoni Pediculus humanus Messor bouvieri Forficula auricularia Oryza sativa Mayetiola destructor others 25 50 75 100 125 150 175 200 225 250 275 300 325 350 375 400 425 450 475 Sequence similarity distribution Hits Species 0 500 450 400 350 300 250 200 150 100 50 0 0 10 20 30 40 50 60 70 #positives/alignment-length 80 90 100 Fig. 5 Preliminary BLAST analysis of the transcriptome of the venom gland of the Brazilian fire ant Solenopsis saevissima. Random PCR-amplified transcripts from a cDNA library were sequenced by 454 GS FLX Titanium chemistry, and the resulting trimmed reads mapped to the available genome version of the fire ant Solenopsis invicta. Resulting contigs were automatically compared using blastx algorithm to online NCBI nr database For decades, these four allergens were believed to be the only proteins present in fire ant venom; however, recently the first proteomics study focusing on the same commercial venom sample of previous studies revealed an unfathomed diversity of other venom proteins (Pinto et al. 2012). Fire ant venom proteins were shown to be mainly a mixture of toxins and allergens and also selfprotective components as to preserve the animal from its own venom, this way matching the typical pattern for other studied predator arthropods, such as scorpions and spiders, and in agreement with the habits of fire ants as active hunters. On this aspect it is worth stressing that some of the toxins reported by (Pinto et al. 2012) are unique among ants for specifically targeting vertebrates (e.g., U5-ctenotoxin Pk 1a and atrial neural peptide), illustrating why fire ants are capable of preying upon nests of alligators, lizards, birds, and even threaten humans and their livestock and brood. The most abundant protein in the fire ant venom – as based on the bidimensional gel chromatography of S. invicta of (Pinto et al. 2012) – was Sol i 3, followed by Sol i 2. A remarkable set of new identifications by (Pinto et al. 2012) mentions toxins involved in causing local necrosis and increased microvascular permeability and cytolysis: myotoxin 2-like toxin (similar to phospholipases from Crotalidae snake venoms) and disintegrins and metalloproteinases, as well as a cytolytic protein similar to PSTx-60 from the sea anemone. Among neurotoxins, it is worth mentioning a protein similar to U5-ctenotoxin Pk 1a, one similar to millipede Scolopendra toxin, and the paralytic alpha-toxin Tc48a-like, which is possibly a blocker of Na+ channels. Regarding self-protective factors, an identified vascular endothelial growth factor (VEGF) was also reported in honeybee and wasp venoms, as well as an inhibitor of phospholipases – it is believed that such factors are essential in keeping the venom apparatus integrity from the close contact with toxins. Some preliminary results of an ongoing transcriptomic investigation of the venom gland of the Brazilian fire ant Solenopsis saevissima (Smith) further reveal interesting information about the metabolic particularities of this organ (Fig. 5). There seem to be further undetected protein toxins that can be present in the venom in such minute amounts that they preclude direct analysis, or perhaps are produced only under particular conditions. Venom peptides appear to be poorly diverse, if even present at all. Some remarkable toxin transcripts identified were a series of peptidases (markedly dipeptidases) and proteases, including prophenoloxidase, which likely have both a Page 7 of 16 Venom Genomics and Proteomics DOI 10.1007/978-94-007-6649-5_38-1 # Springer Science+Business Media Dordrecht 2014 a c b N N CH3 N H (CH2)nCH3 N H Fig. 6 Examples of chemical structure of some well-studied piperidine alkaloids. (a) Nicotine, extracted from tobacco plants. (b) Solenopsin, extracted from fire ants (n = even number, varying according with solenopsin species). (c) Coniine, extracted from hemlock plants digestive function and a regulatory function in activating other venom secretions. Also identified were the paralyzing actin-depolymerizing factor cofilin, several mucin-like lubricant glycosylated proteins, the insecticidal protein mulatexin, several chitinases, and a number of potential new allergens, including one new Sol i 3-like allergen, and finally odorant-binding proteins such as the social regulator protein gp-9. While several of the results confirm the finds by (Pinto et al. 2012), the existence of newly identified compounds could be indicative of species-specific venom variation. Such diversity would indicate that studying the venom proteins of other fire ant species might reveal a yet unimagined source of new potent bioactive compounds. Apart from revealing new venom compounds, a transcriptome can provide insights into the biology and physiology of the targeted organ. In summary, preliminary data suggests that the venom gland of fire ants is a highly dynamic organ under strict physiological regulation that will immediately respond to metabolic and environmental cues. This is clearly illustrated by the fact that the most diverse of the expressed transcripts are related to transcription factors (mainly transmembrane receptors), followed by signalingpathway proteins. The observed proportions of classes of identified transcripts contrast with those described for venom glands of other organisms; thus, this is an interesting aspect of this organ to be further investigated. Indeed there are very few published studies assessing variation in venom composition resulting of different situations, yet one study has already demonstrated that fire ants will produce more venom during the flooding of their colonies (Haight 2006), reflecting a metabolic response of the venom gland to external stimuli. It would be interesting to check for the plasticity of venom profile variation within the same species and among individuals of different ages, as further insights into the venom nature and their pattern of synthesis could be drawn. Finally, while opening the topic of the next section, the preliminary results from the venom gland transcriptome have revealed potential candidates for the synthesis of venom toxins, including the synthesis of venom alkaloids. Among the transcripts of the fire ant venom gland, one can identify the presence of some enzymes related to the mevalonate pathway of the synthesis of polyketides, which are known only from some microorganisms and plants – such enzymes could be involved in the production of alkaloids, which have not been shown to be produced by other animals. Further knowledge on how such compounds are produced can be useful for their study. Piperidine alkaloids are of great biotechnological interest, as described in the next section. Worse than a Cigarette As previously mentioned, the venom of fire ants is almost completely composed of a mixture of hydrophobic alkaloids. These are essentially compounds with a piperidinic ring attached to a side hydrocarbon chain of variable length and with varied degrees of unsaturation (Fig. 6). These alkaloids in fire ants are hydrophobic dialkylpiperidines, generally called solenopsins (under Page 8 of 16 Venom Genomics and Proteomics DOI 10.1007/978-94-007-6649-5_38-1 # Springer Science+Business Media Dordrecht 2014 Fig. 7 Freshly extracted alkaloids from a whole colony of Solenopsis invicta, by the method described by Fox et al. (2013) terminologies as isosolenopsins, dehydrosolenopsins, or dehydroisosolenopsins according with their molecular conformation), similar in structure to coniine and nicotine. The solenopsins include ten different substances termed 2,6-methyl-6-alkylsolenopsins A-E and their unsaturated counterparts and isomers (for a general overview on their structures, refer to Chen and Fadamiro (2009)). Each of these compounds has slightly different chemical properties and biological activities. Given the fact that solenopsins are a class of alkaloids unique to fire ants and are thus compounds relatively easy to obtain and partially purify in endemic areas, there are numerous studies on their chemistry and physiological effects. Solenopsins are overall highly bioactive compounds, which have been demonstrated to be of interest to several biotechnological and biomedical applications. Working with solenopsins is much more straightforward than working with venom proteins: fire ant alkaloids can be easily obtained by immersing any amount of these ants in organic solvents, and from which they can be partially purified from contaminants by their relative affinity with silica powder, either by thin-layer chromatography or common resin columns. The mixture of purified venom alkaloids appears as a translucent yellowish oil (Fig. 7), and compounds are stable at room temperature. As much as 1.0 g can be obtained from one large nest within few hours of work. How can ants produce the venom alkaloids is presently a mystery. Several laboratory synthesis methods have been designed, and at least one biochemical pathway has been proposed (Leclercq et al. 1996a). When first discovered, it was supposed that the ants were acquiring these compounds from feeding on vegetable sources; however, it was soon made clear that they somehow synthesize alkaloids – given that venom alkaloids are species specific, general proportions and composition are maintained over the ant’s life and finally that ants can produce the compounds when cultured in a laboratory without any access to vegetable matter. Among solenopsins, there are also minor amounts of similar alkaloids named piperideines, which are currently regarded as unstable intermediates in the synthesis of alkaloids, but these are essentially unstudied compounds (Chen et al. 2009), except for two recent studies (Li et al. 2012; Rashid et al. 2013). Regarding their activities as antimicrobials in general, solenopsins have proved to be potent antifungals and insecticides after being tested against a range of microorganisms (Jouvenaz Page 9 of 16 Venom Genomics and Proteomics DOI 10.1007/978-94-007-6649-5_38-1 # Springer Science+Business Media Dordrecht 2014 Fig. 8 Bioassays with ethanolic suspensions of solenopsins at varied concentrations against different organisms. (a) Antimicrobial test against Micrococcus luteus by counting number of colony-forming units in a Petri dish. (b) Topical application/injection into hemolymph of Tenebrio molitor larvae et al. (1972), e.g., Candida albicans, Escherichia coli, and Pseudomonas aeruginosa) and several insects (mainly lepidopterans, see Blum et al. (1958) and Lai et al. (2010)). Some ongoing tests are presented in Fig. 8. As mentioned, some isomers prove more effective than others depending on the bioassay conditions, and this must correlate with the biological adaptations of the different fire ant species, given that they possess unique proportions of solenopsins (Fox et al. 2012). One aspect that should be emphasized is that by far the most intensively tested compound was solenopsin A, and much information is lacking about the other compounds and isomers, including one existing solenopsin still currently lacking a name with a side hydrocarbon chain shorter than solenopsin A, reported from poorly studied species of fire ants (e.g., Solenopsis virulens). As for reported physiological activities in mammals, solenopsins have been reported to trigger histamine production in mastocytes (Read et al. 1978), block neuromuscular junctions (Yeh et al. 1975), and inhibit ATP-dependent sodium-potassium pumps and respiratory chains (Koch et al. 1977; Lind 1982). Javors et al. (1993) also observed that solenopsins are capable of activating platelets and neutrophils, and Yi et al. (2001) and Yi et al. (2003) reported that they can inhibit three isoforms of neuronal nitric oxide synthases. One particular aspect that has made a greater impact in the mainstream media is that solenopsin A was demonstrated to be a potent inhibitor of class-1 phosphatidylinositol-3-kinase signaling and angiogenesis in embryonic fibroblasts of mice, and also angiogenesis in zebra fish (e.g., Arbiser et al. 2007), making it a promising anticancer candidate. Still on the physiological effects, yet concerning the toxicity of venom alkaloids towards mammals (for instance, if to be administrated as a drug), it was reported that intravenous application of solenopsins is capable of affecting the central nervous and cardiovascular system (Howell et al. 2005) of mice, as it is capable of crossing the hematoencephalic barrier. However, it becomes significantly toxic 3–30 mg/kg in a dose-dependent manner, causing dizziness, then cardiorespiratory complications, and death (Howell et al. 2005). The least toxic means of administration seems to be oral intake. In fact, some controversial applications for fire ant venoms have been proposed by recent patents deposited in the United States, for instance, one patent suggesting the oral administration of milligram amounts of venom to domestic cats and dogs and humans, for parasite control (Patent US 5098914 A); another patent recommends oral intake for humans (elderly people) as to revert aging-associated decline of motor skills, particularly in patients with Alzheimer’s disease (Patent EP 2043642 A2). Page 10 of 16 Venom Genomics and Proteomics DOI 10.1007/978-94-007-6649-5_38-1 # Springer Science+Business Media Dordrecht 2014 The wide diversity of existent alkaloids in fire ant venoms, and the present paucity of published investigations into most of them, is but emblematic of the poorly explored biotechnological potential of such compounds. However, as all existent compounds are chemically similar substances, greatly due to isomers, obtaining pure natural fractions of individual solenopsins is technically challenging. Obtaining pure compounds heavily relies upon finding the ant species in which they are prevalent and also perfecting the current purification methods, or else relying on laboratory synthesis of solenopsins. Unfortunately, currently available methods for synthesizing solenopsins are expensive and time-consuming and yield small amounts of product, even with racemic mixtures of product compounds (e.g., Leclercq et al. 1996b; Pianaro et al. 2012). Adjusting the synthesis to yield enantiomerically pure end products greatly increases the methodological challenge and associated costs (e.g., Pelletier et al. 2014). These difficulties have limited tests with these compounds to smallscale essays with mixtures of compounds and microscale in vitro tests with synthetic compounds. Given the ease of obtaining venom alkaloids from ants and the difficulties in synthesizing them, finding effective methods for their purification or practical biological synthesis will greatly speed up breakthroughs in this field. There is a complete lack of papers published towards this direction. Conclusion Taken overall, this chapter aimed at presenting an updated overview about the state of knowledge about fire ant venoms and indicated possibilities of research and developments on this topic. Recent finds and test results emphasize that ant venom secretions of ants can be rich in bioactive factors useful to many different fields of application and provide a yet essentially untapped rich source of knowledge about new biochemical pathways and potential new pharmacological compounds. Given the broad nature of possible applications and the necessary background knowledge to deal with the different chemical biochemical and physical aspects of the venom alkaloids and the immunological and metabolic questions about fire ant proteins and peptides, interdisciplinary research teams are necessary to advance in these topics. Many young scientists may find in this a hot topic for relevant discoveries. Some possible future directions are suggested below as a mind-provoking invitation to young researchers and investors to this field of research. Future Directions: Possible Uses for Fire Ant Venom A Social Role of Ant Venoms? There is some timid evidence suggesting that the venom may play an important central role in fire ant social organization. Not only are all ants in the nest covered with venom, which is recurrently sprayed by workers inside the colonies through a behavior known as gaster flagging, but the venoms of female ants of different castes are markedly different. Moreover, the venoms of fire ants are species specific, and workers have been seen to immediately present their gasters when introduced to other workers of a different colony (author’s personal observations). Given the abundance and relative volatility of fire ant alkaloids, it would be reasonable to assume that they may work as pheromones among the ants and towards other species. Additionally, the presence of abundant odorant-binding proteins in the venom as exemplified by some of the described allergens and also other molecules detected by proteomics (like the social pheromone protein gp-9) suggests a social role for venom secretions. Such odorant-binding proteins could be carrying a message among the Page 11 of 16 Venom Genomics and Proteomics DOI 10.1007/978-94-007-6649-5_38-1 # Springer Science+Business Media Dordrecht 2014 ants covered in secreted venom. Currently, studies with manipulation of fire ant venom in association with behavioral observations are still lacking, and this is still a field to be evaluated. Derivatives of Solenopsins to Prevent Biofilm Formation? Great industrial expenses are directed towards fighting off microbial biofilm formation, which cause continuous duct obstruction and corrosion and inutilization of surfaces in factories and refineries. In their attempt to prevent biofilm formation inside ducts, industries release highly toxic pollutants into the environment, resulting in the appearance of resistant microbial strains and causing massive environmental impact. There is thus considerable interest in the discovery of new (cheaper and less toxic) chemicals or surface materials that will delay or eliminate biofilm formation. This can be attained by preventing fixation of early-succession microbes or by disrupting biosignaling among the biofilm-secreting microbes. Treating exposed surfaces with several chemicals and paints – termed surface conditioning – has been proposed as an alternate solution. Park et al. (2008) have demonstrated that fire ant venom alkaloids can inhibit biofilm formation by gram-negative Pseudomonas aeruginosa, possibly from its structural resemblance of a quorumsensing molecule termed 3-oxo-C12-HSL. As mentioned, solenopsins strongly adsorbs to silica, and there are several reports of alkaloids adsorbing to surfaces (e.g., chinchonines adhering to metal layers) and plastics. Thus, maybe artificial solenopsin derivatives could be added to paints for surface conditioning as an alternative environmentally friendly measure against biofilm formation. This is an interesting aspect open for investigation. Venom Alkaloids as Sustainable Pesticides? As mentioned earlier in this chapter, solenopsins can be partially purified from contaminants based on their relative affinity to silica. During the process of solenopsin purification, some inevitably remain adsorbed to the silica phase employed (termed phase saturation of the system), which must be then replaced for continued use. However, as silica is an inert cheap material, one could picture practical utilities for alkaloid-saturated silica, given that it is impregnated with bioactive compounds. One idea, for instance, could be using solenopsin-saturated silica for delaying the growth of filamentous soil fungi on collected fruit. One of the greatest losses of commercial fruit is due to mold formation during its transport to points of sales or during its shelf life, and the usual method for preventing this is to apply toxic pesticides after harvesting fruits. Perhaps an inert thin layer of alkaloid-bound silica would provide good protection, while being less toxic and easier to wash away. How about adding alkaloid-bound minerals to the soil of crops in an attempt to make them obtain the insecticidal compounds? Such agricultural possibilities are worthy of experimentation. Fire Ant Venom for Immunotherapy As mentioned previously, immunotherapy of patients sensitive to fire ant stings is currently done with whole-body ant extracts. Logic entails that the new extraction method for pure venom protein could be used for immunotherapy, with the potential of more efficient and specific results. However, farming fire ants for venom extraction could prove impracticable and environmentally risky. Thus, further knowledge about the characteristics and diversity of the main allergens in fire ant venom could enable the recombinant production of antigen sites as to make immunotherapy less risky and also independent of biological samples. Combining recombinant antigens similar to wasp/bee/ant venoms could perhaps provide a broad-range immunizing therapy. Moreover, it has been demonstrated in some vertebrates (as illustrated by the use of bee or ant stings against arthritis) that adjuvant-mediated stimulation with hymenopteran venom phospholipases can induce interesting immunological reactions, such as anti-inflammatory effects, or acquired immunological resistance Page 12 of 16 Venom Genomics and Proteomics DOI 10.1007/978-94-007-6649-5_38-1 # Springer Science+Business Media Dordrecht 2014 to certain parasites. It seems reasonable that fire ant venom could prove to have the same properties given that it is rich in phospholipases. Alkaloids as Future Antibiotics? Previously in this chapter, it was mentioned that venom alkaloids were administered orally to mammals without any apparent significant side effects. Also, their potent activity as antifungals is widely reported in the scientific literature. Fungal infections are among the most difficult to treat clinically, as common fungi are naturally resistant to most known antibiotics, and most can easily spread to different tissues in the body. There is already some research on the use of microencapsulated preparations of solenopsins (as creams) for topical application as an alternative treatment against skin fungal infections. The reported possibility of oral administration is also suggestive that internal application in cases of respiratory or oral diseases should be feasible. Such alkaloids or artificial derivatives could be employed in association with other drugs as to maximize their effects and prevent the onset of resistant strains. As the antimicrobial effects of piperidinic alkaloids are yet unknown, it is hard to precise how enhanced drugs could be generated from simple modifications in the original molecules, and this is a hot topic in lack of further research. 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