e at SciVerse ScienceDirect Toxicon 65 (2013) 5–8 Contents lists availabl Toxicon journal homepage: www.elsevier .com/locate/ toxicon Short communication A simple, rapid method for the extraction of whole fire ant venom (Insecta: Formicidae: Solenopsis) Eduardo Gonçalves Paterson Fox a,*, Daniel Russ Solis b, Lucilene Delazari dos Santos c, Jose Roberto Aparecido dos Santos Pinto b, Anally Ribeiro da Silva Menegasso b, Rafael Cardoso Maciel Costa Silva a, Mario Sergio Palma b, Odair Correa Bueno b, Ednildo de Alcântara Machado a a Laboratório de Entomologia Médica e Molecular, Instituto de Biofísica Carlos Chagas Filho, Federal University of Rio de Janeiro (IBCCF/UFRJ), Rio de Janeiro, Brazil b Instituto de Biociências, UNESP – Universidade Estadual Paulista, Campus de Rio Claro, Departamento de Biologia – Centro de Estudos de Insetos Sociais, SP, Brazil cCenter of Studies of Venoms and Venomous Animals (CEVAP), University of São Paulo State (UNESP), Botucatu, SP, Brazil a r t i c l e i n f o Article history: Received 12 June 2012 Received in revised form 19 October 2012 Accepted 13 December 2012 Available online 18 January 2013 Keywords: Hymenoptera Allergen Toxin Myrmecology Chromatography * Corresponding author. Tel.: þ55 21 2205 3314. E-mail addresses: ofox@biof.ufrj.br, ofoxofox@gm Paterson Fox). 0041-0101� 2013 Elsevier Ltd. http://dx.doi.org/10.1016/j.toxicon.2012.12.009 Open access under the E a b s t r a c t The invasive fire ant Solenopsis invicta is medically important because its venom is highly potent. However, almost nothing is known about fire ant venom proteins because obtaining even milligram-amounts of these proteins has been prohibitively challenging. We present a simple and fast method of obtaining whole venom compounds from large quantities of fire ants. For this, we separate the ants are from the nest soil, immerse them in dual-phase mixture of apolar organic solvent and water, and evaporate each solvent phase in separate. The remaining extract from the aqueous phase is largely made up of ant venom proteins. We confirmed this by using 2D gel electrophoresis while also demon- strating that our new approach yields the same proteins obtained by other authors using less efficient traditional methods. � 2013 Elsevier Ltd. Open access under the Elsevier OA license. Solenopsis fire ants are native to the Americas, withmost of the species occurring in lower regions of South America (Tschinkel, 2006). The most notorious of these is Solenopsis invicta Buren which was introduced to Alabama, US in the early 20th century and has since then successfully invaded warm regions around theworld including in the Galapagos, China, and Vietnam (Lofgren, 1986; Luo, 2005; Ascunce 2011) via commercial ships. This species is characterized by high population densities, aggressive behavior and a very potent sting. In the United States alone, more than 14 million people per year are stung by fire ants, as many as 100,000 of them seek medical attention (Apperson and Adams, 1983) and more than 80 people have died ail.com (E. Gonçalves lsevier OA license. because of high sensitivity to compoundswithin the venom (deShazo et al., 1990; Stablein et al., 1985; Rhoades et al., 1989; Stafford, 1996; Prahlow and Barnard, 1989). Fire ant venom includes a major (more than 95%) frac- tion of piperidinic alkaloids and a minor (less than 5%) aqueous fraction of allergenic proteins (e.g., Baer et al., 1979; Hoffman et al., 1988, 1990, 2005; Hoffman, 1993). The composition and toxic and antimicrobial properties of the piperidinic alkaloids are well described (Blum et al., 1958; Storey et al., 1991; Jouvenaz et al., 1972; Howell et al., 2005). However, almost nothing is known about the proteins. Indeed, only four proteins have been described in any detail (Hoffman, 1993; Tschinkel, 2006; King and Spangfort, 2000) out of an estimated total of over 40 fire ant venom proteins (Pinto et al., 2012). This is due to the difficulty of extracting amounts of proteins sufficient for proper purification and extensive characterization. Indeed, mailto:ofox@biof.ufrj.br mailto:ofoxofox@gmail.com http://crossmark.dyndns.org/dialog/?doi=10.1016/j.toxicon.2012.12.009&domain=pdf www.sciencedirect.com/science/journal/00410101 http://www.elsevier.com/locate/toxicon http://dx.doi.org/10.1016/j.toxicon.2012.12.009 http://dx.doi.org/10.1016/j.toxicon.2012.12.009 http://www.elsevier.com/open-access/userlicense/1.0/ http://www.elsevier.com/open-access/userlicense/1.0/ Fig. 1. Fluxogram illustrating the venom extraction steps described. E. Gonçalves Paterson Fox et al. / Toxicon 65 (2013) 5–86 methods of fire ant venom extraction described in the lit- erature are extremely inefficient because they are based on “milking” the venom from individual ants (e.g., Padavattan et al., 2008). We propose here a novel venom protein extraction method that is simpler, faster and provides extraction yields orders of magnitude higher. 1. Ant collection & separation from nest soil First, locate a fire ant nest in the field and shovel the upper portion of the mound into a bucket that was rimmed with Teflon paint. Following the methods described in Banks et al. (1981), separate the ants from the nest earth by slowly flooding the bucket with water (one drop every w2 seconds). This takes several hours thus the extraction so- lution can be prepared in the mean time (below). Once completely flooded, the ants form a raft at the surface of the water (Banks et al., 1981). 2. Preparing the extraction solution Obtain a clean glass recipient of appropriate size (e.g., 500 mL, depending on the amount of obtained ants). We recommend using a wide-mouth recipient (e.g., beaker or glass tumbler) rather than a narrow-mouth recipient (e.g., Erlenmeyer) because it is easier to put the ants inside in a single move. Add into the glass recipient a small quantity (ca. 1 mL per gram of ant) of distilled water or preferred buffer solution and a larger amount (ca. 5 mL per gram of ant) of a strong apolar solvent such as hexane (hexane was preferred because it is less volatile than ether or chloro- form). The extraction mixture should clearly separate into two phases, and the volume of organic solvent should be enough to completely immerse the ants. 3. Venom extraction Wearing protective rubber gloves, transfer the raft of floating ants into the extraction solution. Alternatively a cleaner extract can be obtained if the ants are first transferred into another recipient for several hours during which they dry and clean themselves. Transferring the ants requires utmost care, because accidents can result in escaped ants, stings and solvent spillage. When the ants enter the organic solvent, they instinctively discharge their venomwhile sinking – perhaps because of their aggressive nature – and rapidly die. These two phases are easily sep- arated into individual tubes using pipettes or a separatory funnel (mind to use glass tubes for organic solvent). The tubes can be frozen at �20 �C for long-term storage. The upper, organic phase contains venom alkaloids and cutic- ular hydrocarbons. Venom alkaloids can be separated from the cuticular hydrocarbons by washing this organic phase with additional hexane through a silica column and then eluting the alkaloids with acetone (further described in Chen and Fadamiro 2009). The lower, aqueous phase con- tains water-soluble proteins. These proteins can be extracted by either precipitation, or lyophilizing this phase and resuspending it in a solution of preference. A video was produced illustrating the extraction procedure http:// youtu.be/dWo-4uxpZK4; all steps are summarized in Fig. 1. Supplementary video related to this article can be found at http://dx.doi.org/10.1016/j.toxicon.2012.12.009. We performed the described extraction procedures on whole nests of S. invicta collected on the campus of the Federal University of Rio de Janeiro. Species identification followed Pitts et al. (2005) using the following diagnostic characters: absence of post-petiolar process, complete mandibular costulae, presence of a frontal medial streak, well developed median clypeal tooth, and males being distinctly black. Voucher specimens are deposited in the Adolph Hempel Entomological Collection of Instituto Bio- lógico de Sao Paulo, SP, Brazil. Hexane was purchased from Merck. Protein quantification was made by the method of Bradford (1976), using bovine serum albumin as standard. The extracted venom alkaloids were air-dried and weighed using a digital precision scale (Bioprecisa FA – 2104N TDS Instrumental Tecnológico). We estimated the number ants used based on their total wet weight (each fire ant weights on average 0.8 mg). We thus deduced that each ant yields approximately 10 mg of alkaloids and 50–100 ng of protein. To compare the quality of extracted proteins with pro- teins obtained by other venom extraction methods, we prepared a bidimensional gel electrophoresis (2DE gel) using about 300 mg of putative protein from an aqueous phase extraction from S. invicta, and a 2DE gel of pure venom protein extract purchased from Vespa Labs Inc. (Spring Mills, PA, USA) (Fig. 2; also refer to Pinto et al., 2012). Gels were digitalized with a table scanner, and the software Adobe Photoshop CS was used to discard color information, normalize contrast between images, and number the obtained spots. The general patterns of the two http://youtu.be/dWo-4uxpZK4 http://youtu.be/dWo-4uxpZK4 http://dx.doi.org/10.1016/j.toxicon.2012.12.009 Fig. 2. Bidimensional gel electrophoresis of fire ant venom from Solenopsis invicta, as obtained by the methods herein described (left), and from the commercial extract of Vespa Labs, US (right). E. Gonçalves Paterson Fox et al. / Toxicon 65 (2013) 5–8 7 2DE gels are clearly similar. Indeed, most proteins are found at similar isoelectric points vs. molecular size positions, and the number of obtained proteins was almost identical. Furthermore, preliminary results of mass spectrometry analysis of individual protein spots in similar positions showed no difference in protein content; the individual proteomic identifications of fire ant venom are dealt within a separate publication (Pinto et al., 2012). The described method of venom extraction is rapid and inexpensive, and depends only on the ability of locating and handling fire ants and the necessary solvents. This method can likely be adapted for venom extraction from other aggressive hymenopterans (e.g., other ants, or cold- anesthetized bees and wasps). Furthermore, the protocol may be further revisited and optimized to increase the purity of each fraction and possibly replace the used sol- vents with environment-friendly alternatives (e.g., using ethanol or cold acetone). We hope that the presented method will encourage investigators to advance the study of venom proteins and peptides of fire ants and other venomous insects. Acknowledgments The present investigation was funded by grants from FAPESP, CNPq, and FAPERJ. We thank Miles Guralnik for technical information on the purchased venom sample, Sandra Fox Lloyd for assistance in obtaining and extracting fire ant colonies, and Daniela R. P. Fernandes, Diogo Gama dos Santos and Willy Jablonka for help making the accompanying video.We are indebted to YannickWurm for revising and proofreading the manuscript. Conflict of interest The authors declare that there are no conflicts of interest. References Apperson, C.S., Adams, C.T., 1983. Medical and agricultural importance of red imported fire ant. Florida Entomologist 66, 121–126. Ascunce, M.S., Yang, C.-C., Oakey, J., Calcaterra, L., Wu, W.-J., Shih, C.-J., Goudet, J., Ross, K.J., Shoemaker, D.D., 2011. Global invasion history of the fire ant Solenopsis invicta. Science 331, 1066–1068. Baer, H., Liu, T.Y., Anderson, M.C., Blum, M., Schmidt, W.H., James, F.J., 1979. Protein components of fire ant venom. 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