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Assessing the Proteomic Activity of the Venom of the Ant Ectatomma tuberculatum (Hymenoptera: Formicidae: Ectatomminae).

1. Introduction

The biotechnological potential of venoms to produce drugs with different applications such as antimicrobials, analgesics, antihypertensives, and insecticides has motivated studies of this kind of products [1]. The use of substances obtained from natural products such as animal venoms, including those from insects, is a practice adopted in folk medicine by numerous native populations since ancient times. They would find the raw materials for these therapeutic resources in nature [2,3] for the treatment of the most varied diseases [4]. This is due to the immunological, analgesic, antimicrobial, diuretic, anesthetic, and antirheumatic properties of these products [3], which also include several substances with potential in the treatment of cancer, microbial, and viral infections, as well as of possible biocide properties such as those of insecticides [5, 6].

Therefore, the search for substances with biological activity obtained from natural products has been an increasingly valuable alternative to find and produce bioactive compounds with practical application [1, 9, 10]. This has motivated numerous recent scientific studies supported by the pharmaceutical industry to search for new bioactive molecules [6,11].

A series of bioactive molecules that are important for several fields such as allergy, immunology, microbiology, biotechnology have already been identified in the venoms of Hymenoptera. Proteins like phospholipases, hyaluronidases, and proteinases have been observed among the identified components, and peptides with diverse activities have already been found in ants, wasps, and bees [1,12].

Thanks to the richness and diversity of bioactive compounds in their venom, Hymenoptera have been gaining notoriety over the last decades, through research studies that use diverse techniques of characterization and biochemical analysis [1]. Many substances of pharmacological interest have been discovered, along with information regarding their structures, biological activities, and action mechanisms. These substances, which vary in diversity and complexity, include pheromones, hormones, defense substances, enzymes, and peptides [1,12,13].

Nevertheless, these insects can cause physiological damage and serious pathologies such as allergies, intense pain, intense sweating, nausea, tachycardia, among other manifestations [14, 15], which in some cases may lead to death of the affected individual [16]. Although the amount of ant venoms produced is small, when compared to that produced by bees and wasps, there is a much larger structural diversity of its components [14, 17]. In light of this, an in depth study of the venoms of Hymenoptera is fundamental as their complex composition and diversity of functions suggest an enormous biotechnological potential for the synthesis of new macromolecules that canbe used for therapeutic purposes or even pest control [18].

Among the studies on ants, [19] pointed out the presence of protein allergens with posttranslational modifications in the venom of the Australian ant Myrmecia pilosula. Touchard and collaborators [20,21] have revealed the profiles of venom proteins and peptides of different species of ants through mass spectrometry and high performance liquid chromatography (HPLC), thus reinforcing the thesis that this group of Hymenoptera is promising in the search for molecules to produce new drugs and bioinsecticides. According to Schmidt, Blum, and Overal [22], E. tuberculatum has one of the most toxic venoms among ants. Pluzhnikov and collaborators and Arseniev [23, 24] have confirmed the high toxicity of its raw venom and have studied the action of the isolated peptide ectatomin. In order to identify the chemical compositionofthe venomof E. tuberculatum in greater detail, it is moreover extremely important to obtain information on the characteristics of its components and their mechanism of action.

In the present study, proteins of the Ectatomma tuberculatum venom have been identified by means of 2D PAGE, followed by tandem nanochromatography with mass spectrometry. A total of 48 proteins have been identified and are discussed for their mechanisms of action. This may help to point out some of these components as a natural prototype of bioactive molecule worthy of synthesizing for pharmaceutical application.

2. Material and Methods

2.1. Protein Extraction from the Ant Glands. Workers of the Ectatomminae ant E. tuberculatum have been collected in the experimental fields of the Cacao Research Center (CEPLAC) at Ilheus, Bahia, Brazil. Additional information on the biology of this species in cacao plantations can be found in Delabie and Hora and collaborators [26, 27]. In the laboratory, 100 workers were anesthetized with C[O.sub.2] and dissected for extraction of venom glands.

The reservoirs of these venom containing glands were removed from the sting apparatus by pulling them out of bodies with forceps and microscissors. The venom reservoirs were then carefully washed and suspended in small volumes of a phosphate buffered saline solution with pH 7.8, punctured, and centrifuged at 10.000x g for 10 min at 4[degrees]C. The supernatants were collected and maintained at -20[degrees]C until use. Venom proteins were extracted from the supernatants according to Rocha and collaborators [28] and the 2D Quant Kit (GE Healthcare) was used to quantify the proteins. Bovine serum albumin (BSA) was used as standard.

2.2. Two-Dimensional Electrophoresis (2D PAGE). For each sample, 350 [micro]g of venom proteins homogenized in rehydration buffer (7 molx[L.sup.-1] urea, 2 molx[L.sup.-1] thiourea, 1% CHAPS, and traces of bromophenol blue) have been used with DTT 40 mmolx[L.sup.-1] and 0.5% ampholytes at pH 3-10NL with a final volume of 250 [micro]L.

The samples were subjected to IEF in 13 cm gel strips, pH gradient pH 3-10NL (Amersham Biosciences, Immobiline[TM] Dry-Strip). The IEF in the Ettan IPGphor 3 (GE Healthcare) system was carried out according to the manufacturer's manual. The 2D Electrophoresis was performed on 12.5% gel in a SE600 Ruby Vertical Electrophoresis System (Hoefer). The running conditions were similar to those used by Mares and collaborators [29], staining in 0.08% colloidal Coomassie [30] for 5 days, followed by destaining in distilled water for 5 days with daily water replacements to remove excess of colloidal Coomassie.

The gels were scanned on ImageScanner II (GE Healthcare) for imaging. Afterwards, it was analyzed using Image Master 2D Platinum 7.0 (GE Healthcare) software to evaluate the number of spots, isoelectric point (pI), molecular mass (MM), and relative abundance of spots on the gels.

2.3. Spot Preparation and MS/MS Analysis. All spots on the gels were excised with the aid of a scalpel, inserted separately into microtubes, and processed according to Silva and collaborators [31].

After peptide digestion and extraction, 4 [micro]L of the sample corresponding to each spot was applied to the nanoAcquity UPLC (WATERS[R]) and subjected to a C18 "trapping" column of 5 [micro]m, 180 [micro]m x 20 mm, followed by the analytical column of 1.7 [micro]m, 100 [micro]m x 100 mm, under a flow of 0.6 [micro]L/min in a 50 minute run. The peptides were separated according to a gradient of 1% acetonitrile for 1 minute, from 1% to 50% for 40 minutes, from 50% to 85% for 5 minutes, remaining at that concentration for another 2 minutes, then returning to a concentration of 1% in one minute, and remaining in this condition for 2 minutes, totaling 5 minutes of run.

Separated peptides were transferred to the mass spectrometer (Micromass Q-TOF micro), and ionized in a capillary under 3000 V voltage. Next, they were fragmented in positive mode with selection of the minimum intensity of 10 counts, and the 3 of the most intense ions were analyzed by each scan of 1 second, with collision energy ranging from 20 to 95 eV according to the mass/charge (m/z) ratio of the peptides.

The obtained spectra were analyzed by ProteinLynx Global Server 4.2 (WATERS), as well as by MASCOT (Matrix Science), and compared with the SWISSPROT and NCBI databases, respectively. In trypsin hydrolysis, the possible loss of a cleavage site was considered. The tolerance of the peptide masses was [+ or -] 0.3 Da, and the mass tolerance of the fragments was [+ or -] 0.1 Da.

The peptides identified through the NCBI database had their complete sequence located on the same bank and analyzed with NCBI Blast2GO using the BlastP algorithm for protein identification.

Since the E. tuberculatum ant genome has not yet been sequenced, the identification ofthe proteins was based on the complete genome sequences of organisms phylogenetically close to the species.

3. Results

The 350 [micro]g mass of E. tuberculatum venom protein resolved on 2D PAGE resulted in a well-resolved, low-drag spot profile in the range of 14 to 97 kDa and pH of 4 to 9. A higher density of spots appears for the pH range of 5 to 8.5 and molecular weight of 25 to 93 kDa (Figure 1).

Gels analysis in the ImageMaster 2D Platinum 7.0 software showed the number of spots, the pI and molecular mass values, and the spots' expression levels in each replicate. A total of 244 spots were detected in the gels.

From a total of 129 spots treated with trypsin and analyzed by mass spectrometry, 48 proteins have been identified in the venom of E. tuberculatum. Among these, six showed similarity with not yet characterized proteins.

Table 1 shows the proteins identified with their respective access codes in the NCBI, molecular mass (MM), isoelectric point (pI), score, and peptide sequence for each spot. E. tuberculatum proteins showed sequences similar to the proteins of other Hymenoptera, including wasps, bees, and ants.

4. Discussion

According to the Blast2Go software, 42 proteins identified in E. tuberculatum venom can be grouped basically into proteins involved in insect metabolism (annexin B9, nucleoside diphosphate kinase; aconitate hydratase; superoxide dismutase; triose phosphate isomerase; glyceraldehyde-3phosphate dehydrogenase; succinyl-CoA ligase; fructosebisphosphate aldolase; arginine kinase; citrate synthase; enolase; ATP synthase; 4-hydroxybutyrate coenzyme A transferase; medium-chain specific acyl-CoA dehydrogenase, and isocitrate dehydrogenase); transport protein (transferrin); and structural proteins (cyclophilin, actin, and chitinase).

Three proteins found in the venom ofE. tuberculatum are part of a group of compounds with known allergenic effects: triose phosphate isomerase, arginine kinase, and cyclophilinlike protein. Through various techniques of molecular biology, including proteomics studies, allergenic proteins present in Hymenoptera venoms have been identified, as well as their respective action mechanisms.

The triose phosphate isomerase protein, identified in spot 9, relates to the metabolism of carbohydrates, acting as an important enzyme of the glycolytic pathway. It has been studied and classified by [32] as allergenic because it appeared in the serum of patients presenting allergy to bites of the midge Forcipomyia taiwana (Ceratopogonidae). Also Hoppe, Steinhart, and Paschke [33] found allergenic enzymes in the protein extract of lychee fruit (Litchi chinensis, Sapindaceae) with high homology with triose phosphate isomerase.

Arginine kinase, expressed in spots 21, 22, 23, 24, and 25 as isoforms of the same protein, is an enzyme that catalyzes the reversible transfer of the phosphate group from ATP to supply the need for energy-requiring cells. It was found in the venom of solitarywasps Eumenidae Eumenes pomiformis and Orancistrocerus drewseni [34], in the venom of social wasps Polybia paulista (Vespidae), with evidence of its allergenic action in humans [35] as well as in various other invertebrates such as Dermatophagoides pteronyssinus (Acari: Pyroglyphidae), Blatella germanica (Insecta: Blattodea: Blattellidae), Penaeus monodon (Crustacea: Penaeidae), Homarus gammarus (Crustacea: Nephropidae), Mytilus edulis (Mollusca: Bivalvia: Mytilidae), and Plodia interpunctella (Insecta: Lepidoptera: Pyralidae), a new class of allergens [36]. Yamamoto and collaborators [37] have isolated arginine kinase from the venom of solitary wasp Pompilidae Cyphononyx dorsalis and confirmed its paralytic activity in spiders, their natural prey. According to Hofling and Rocha [18], Hymenoptera use their venoms to paralyze or kill their prey, as well as to defend themselves when they feel threatened by vertebrates, including humans. According to [38], E. tuberculatum is an active predator of cacao pests, feeding on Chrysomelidae the conserved domains to which these proteins belong have been identified, classifying them into specific superfamilies. Spots 6 (SINV.05483), 7 (EAL1007), 8 (L0C.100743840), 64 (L0C_100743840), and 73 (EAL10007) belong to the superfamily LPM010 (Lytic polysaccharide monooxygenase, cellulose degrading). They form a recently discovered group of oxidative enzymes that carries the important role of degrading polysaccharides such as chitin and cellulose, cleaving the glycoside bonds with the oxidation of the C-1, C4 carbons, or both [56, 57]. Spot 77 (SINV.07147) belongs to the superfamily GMC-OXRED-C (glucose-methanolcholine oxidoreductase). They aggregate homologous proteins with oxidation-reduction enzymatic activity, which uses FAD as a cofactor [58, 59].

Some proteins found in the venom ofE. tuberculatum are not part of the protein composition generally found in the venoms of Hymenoptera. It is likely that these proteins are components of the membrane cells of the venom reservoir. Table 2 summarizes the situation for these proteins and their corresponding functions.

The results of the proteomic analysis of the venom of E. tuberculatum allowed the identification of nine proteins related to the process of attack/defense and maintenance of the colony. The active components of the venom have been classified into five subgroups:

(1) Colony asepsis--transferrin and annexin B9: they have antioxidant and antimicrobial activity and are used by the insects to prevent infections of stored food and colony members themselves [34, 53, 54].

(2) Protein of the venom constituents--superoxide dismutase and cyclophilin-like protein: they relate to the preservation of the functional integrity of cellular/glandular constituents, preventing oxidation and damage to the cellular structure of the venom bag and its reservoir [35, 40, 41,44].

(3) Diffusion of the venom in the prey--chitinase, enolase, annexin B9, and actin: the process of diffusion of the constituents of the venom [46].

(4) Prey paralysis--arginine kinase: it causes neurotoxicity, leading to paralysis and subsequent death of prey [38].

(5) Alteration of homeostasis and cellular toxicity--actin, enolase, glyceraldehyde-3-phosphate dehydrogenase: they cause damage to different tissue types and alteration of cellular homeostasis and maybe neurotoxic [34].

Figure 2 resumes the scheme for the aforementioned functions.

The identification and characterization of the proteins present in the venom of E. tuberculatum can aid in the better understanding of how the ants use the protein constituents for different behaviors such as defense against natural and pathogenic enemies, attack on prey, and maintenance of the colony, in addition to correlating the presence of certain allergic components in the venom with effects occurring in humans after inoculation. Furthermore, for providing knowledge as explained above, studies like this one also contribute to the identification of substances as possible prototypes for synthesis of macromolecules for new purposes, from their use as molecular markers to the manufacture of drugs and insecticides. 10.1155/2018/7915464

Conflicts of Interest

The authors declare that they have no conflicts of interest.


Special thanks are due to Martin Brendel for reviewing the English manuscript. This research received support from the PRONEX Program [SECTI-FAPESB & CNPq, project 011/2009]. Finally JRS acknowledges her study (MS) grant from CAPES and JHCD acknowledges his research grant from CNPq.


[1] R. Fontana, C. P. Pirovani, H. Costa et al., "Complexidade e atividade biologica das peconhas de formigas, em particular de poneromorfas," in As formigas Poneromorfas do Brasil, J. H. C. Delabie, R. M. Feitosa, J. E. Serrao, C. S. F. Mariano, and J. D. Majer, Eds., pp. 271-284, Editus, Ilheus, Brasil, 2015.

[2] H. B. Weiss, "Entomological medicaments of the past," Journal of the New York Entomological Society, vol. 55, pp. 155-168,1947.

[3] E. M. Costa-Neto, "Entomotherapy, or the medicinal use of insects," Journal ofEthnobiology, vol. 25, no. 1, pp. 93-114, 2005.

[4] E. M. Costa Neto and J. M. Pacheco, "Utilizacao medicinal de insetos no povoado de Pedra Branca, Santa Terezinha, Bahia, Brasil," Biotemas, vol. 18, no. 1, pp. 113-133, 2005.

[5] A. T. Dossey, "Insects and their chemical weaponry: new potential for drug discovery," Natural Product Reports, vol. 27, no. 12, pp. 1737-1757, 2010.

[6] N. A. Ratcliffe, C. B. Mello, E. S. Garcia, T. M. Butt, and P. Azambuja, "Insect natural products and processes: New treatments for human disease," Insect Biochemistry and Molecular Biology, vol. 41, no. 10, pp. 747-769, 2011.

[7] A. W. Fenton, P. R. West, G. V. Odell et al., "Arthropod venom citrate inhibits phospholipase A2," Toxicon, vol. 33, no. 6, pp. 763-770, 1995.

[8] P. Yang, J. Zhu, M. Li, Li J., and X. Chen, "Soluble proteome analysis of male Ericerus pela Chavannes cuticle at the stage of the second instar larva," African Journal of Microbiology Researc, vol. 5, no. 9, pp. 1108-1118, 2011.

[9] A. C. Pinto, D. H. Silva, V d. Bolzani, N. P. Lopes, and R. d. Epifanio, "Produtos naturais: atualidade, desafios e perspectivas," Quimica Nova, vol. 25, pp. 45-61, 2002.

[10] B. Graz, J. Falquet, and E. Elisabetsky, "Ethnopharmacology, sustainable development and cooperation: The importance of gathering clinical data during field surveys," Journal of Ethnopharmacology, vol. 130, no. 3, pp. 635-638, 2010.

[11] P. Bulet and R. Stocklin, "Insect antimicrobial peptides: Structures, properties and gene regulation," Protein and Peptide Letters, vol. 12, no. 1, pp. 3-11, 2005.

[12] B. E. C. Banks and R. A. Shipolini, "Chemistry and pharmacology of honey-bee venom," in Venoms of the Hymenoptera: Biochemical, Pharmacological and Behavioural Aspects, T. Piek, Ed., pp. 330-416, Academic Press, London, UK, 1986.

[13] A. F. C. Torres, Y. P. Quinet, A. Havt, G. Radis-Baptista, and A. M. C. Martins, "Molecular pharmacology and toxinology of venom from ants. Hymenopteran integrated view of the molecular recognition and toxinology," Analytical Procedures to Biomedical Applications, vol. 8, pp. 207-222, 2013.

[14] M. S. Blum, "Ant venoms: Chemical and pharmacological properties," Toxin Reviews, vol. 11, no. 2, pp. 115-164,1992.

[15] V. Haddad Jr., J. L. C. Cardoso, and R. H. P. Moraes, "Description of an injury in ahuman caused by a false tocandira (Dinoponera gigantea, perty, 1833) with a revision on folkloric, pharmacological and clinical aspects of the giant ants of the genera Paraponera and Dinoponera (sub-family Ponerinae)," Revista do Instituto de Medicina Tropical de Sao Paulo, vol. 47, no. 4, pp. 235-238, 2005.

[16] C. J. Steen, C. K. Janniger, S. E. Schutzer, and R. A. Schwartz, "Insect sting reactions to bees, wasps, and ants," International Journal of Dermatology, vol. 44, no. 2, pp. 91-94, 2005.

[17] J. Orivel and A. Dejean, "Comparative effect of the venoms of ants of the genus Pachycondyla (Hymenoptera: Ponerinae)," Toxicon, vol. 39, no. 2-3, pp. 195-201, 2000.

[18] M. A. C. Hofling and T. Rocha, "Aspectos da Miotoxicidade e Neurotoxicidade de Venenos de Hymenoptera," in Alergia a Venenos de Insetos, F. F. M. Castro and M. S. Palma, Eds., p. 231, Manole, Barueri, Brazil, 2009.

[19] M. D. Wiese, S. G. A. Brown, T. K. Chataway et al., "Myrmecia pilosula (Jack Jumper) ant venom: Identification of allergens and revised nomenclature," Allergy: European Journal of Allergy and Clinical Immunology, vol. 62, no. 4, pp. 437-443, 2007.

[20] A. Touchard, J. M. S. Koh, S. R. Aili et al., "The complexity and structural diversity of ant venom peptidomes is revealed by mass spectrometry profiling," Rapid Communications in Mass Spectrometry, vol. 29, no. 5, pp. 385-396, 2015.

[21] S. R. Aili, A. Touchard, J. M. S. Koh et al., "Comparisons of Protein and Peptide Complexity in Poneroid and Formicoid Ant Venoms," Journal of Proteome Research, vol. 15, no. 9, pp. 3039-3054, 2016.

[22] J. O. Schmidt, M. S. Blum, and W. L. Overal, "Comparative enzymology of venoms from stinging Hymenoptera," Toxicon, vol. 24, no. 9, pp. 907-921,1986.

[23] K. Pluzhnikov, E. Nosyreva, L. Shevchenko et al., "Analysis of ectatomin action on cell membranes," European Journal of Biochemistry, vol. 262, no. 2, pp. 501-506,1999.

[24] A. S. Arseniev, K. A. Pluzhnikov, D. E. Nolde et al., "Toxic principle of selva ant venom is a pore-forming protein transformer," FEBS Letters, vol. 347, no. 2-3, pp. 112-116,1994.

[25] D. E. Nolde, A. G. Sobol, K. A. Pluzhnikov, E. V. Grishin, and A. S. Arseniev, "Three-dimensional structure of ectatomin from Ectatomma tuberculatum ant venom," Journal of Biomolecular NMR, vol. 5, no. 1, pp. 1-13,1995.

[26] J. H. C. Delabie, "The ant problems of cocoa farms in Brazil," in Applied Myrmecology: A World Perspective, R. K. Vander Meer, K. Jaffe, and A. Cedeno, Eds., pp. 555-569, Westview Press, Boulder, CO, USA, 1990.

[27] R. R. Hora, E. Vilela, R. Feneron, A. Pezon, D. Fresneau, and J. Delabie, "Facultative polygyny in Ectatomma tuberculatum (Formicidae, Ectatomminae)," Insectes Sociaux, vol. 52, no. 2, pp. 194-200, 2005.

[28] T. L. Rocha, P. H. A. Costa, and J. C. C. Magalhaes, Comunicado Tocnico 136: Eletroforese Bidimensional e Analise de Proteomas, EMBRAPA, Brasilia, Brazil, 2005.

[29] J. H. Mares, K. P. Gramacho, E. C. Dos Santos et al., "Protein profile and protein interaction network of Moniliophthora perniciosa basidiospores," BMC Microbiology, vol. 16, no. 1, article no. 120, 2016.

[30] V. Neuhoff, N. Arold, D. Taube, and W. Ehrhardt, "Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250," Electrophoresis, vol. 9, no. 6, pp. 255-262, 1988.

[31] F. A. C. Silva, C. P. Pirovani, S. Menezes et al., "Proteomic response of Moniliophthora perniciosa exposed to pathogenesis-related protein-10 from Theobroma cacao," Genetics and Molecular Research, vol. 12, no. 4, pp. 4855-4868, 2013.

[32] Y.-H. Chen, M.-F. Lee, J.-L. Lan et al., "Hypersensitivity to Forcipomyia taiwana (biting midge): Clinical analysis and identification of major For t 1, For t 2 and For t 3 allergens," Allergy: European Journal of Allergy and Clinical Immunology, vol. 60, no. 12, pp. 1518-1523, 2005.

[33] S. Hoppe, H. Steinhart, and A. Paschke, "Identification of a 28 kDa lychee allergen as a triose-phosphate isomerase," Food and Agricultural Immunology, vol. 17, no. 1, pp. 9-19, 2006.

[34] J. H. Baek and S. H. Lee, "Isolation and molecular cloning of venom peptides from Orancistrocerus drewseni (Hymenoptera: Eumenidae)," Toxicon, vol. 55, no. 4, pp. 711-718, 2010.

[35] L. D. Dos Santos, K. S. Santos, J. R. A. Pinto et al., "Profilingthe proteome of the venom from the social wasp polybia paulista: A clue to understand the envenoming mechanism," Journal of Proteome Research, vol. 9, no. 8, pp. 3867-3877, 2010.

[36] M. Binder, V. Mahler, B. Hayek et al., "Molecular and immunological characterization of arginine kinase from the indianmeal moth, Plodia interpunctella, a novel cross-reactive invertebrate pan-allergen," The Journal of Immunology, vol. 167, no. 9, pp. 5470-5477, 2001.

[37] T. Yamamoto, H. Arimoto, T. Kinumi, Y. Oba, and D. Uemura, "Identification of proteins from venom of the paralytic spider wasp, Cyphononyx dorsalis," Insect Biochemistry and Molecular Biology, vol. 37, no. 3, pp. 278-286, 2007.

[38] J. H. C. Delabie, H. S. R. Alves, V. C. Franca, P. T. A. Martins, and I. C. Nascimento, "Biogeografia das formigas predadoras do genero Ectatomma (Hymenoptera: Formicidae: Ectatomminae) no leste da Bahia e regioes vizinhas," Agrotropica, vol. 19, pp. 1320, 2007.

[39] S. Kumari, S. Roy, P. Singh, S. L. Singla-Pareek, and A. Pareek, "Cyclophilins: Proteins in search of function," Plant Signaling and Behavior, vol. 8, no. 1, pp. 25-32, 2013.

[40] R. Weisman, J. Creanor, and P. Fantes, "A multicopy suppressor of a cell cycle defect in S.pombe encodes a heat shock-inducible 40 kDa cyclophilin-like protein," EMBO Journal, vol. 15, no. 3, pp. 447-456,1996.

[41] P. Wang and J. Heitman, "The cyclophilins," Genome Biology, vol. 6, no. 7, article no. 226, 2005.

[42] S. Fluckiger, H. Fijten, P. Whitley, K. Blaser, and R. Crameri, "Cyclophilins, a new family of cross-reactive allergens," European Journal of Immunology, vol. 32, no. 1, pp. 10-17, 2002.

[43] J. M. McCord and I. Fridovich, "Superoxide dismutase. An enzymic function forerythrocuprein (hemocuprein)," The Journal ofBiological Chemistry, vol. 244, no. 22, pp. 6049-6055,1969.

[44] N. Peiren, D. C. de Graaf, F. Vanrobaeys et al., "Proteomic analysis of the honey bee worker venom gland focusing on the mechanisms of protection against tissue damage," Toxicon, vol. 52, no. 1, pp. 72-83, 2008.

[45] P. Yang, J. Zhu, M. Li, J. Li, and X. Chen, "Soluble proteome analysis of male Ericerus pela Chavannes cuticle at the stage of the second instar larva," African Journal of Microbiology Research, vol. 5, no. 9, pp. 1108-1118, 2011.

[46] J. R. Edgar, "Q&A: What are exosomes, exactly?" BMC Biology, vol. 14, p. 46, 2016.

[47] M. Van Vaerenbergh, G. Debyser, G. Smagghe, B. Devreese, and D. C. De Graaf, "Unraveling the venom proteome of the bumblebee (Bombus terrestris) by integrating a combinatorial peptide ligand library approach with FT-ICR MS," Toxicon, vol. 102, article no. 4698, pp. 81-88, 2013.

[48] A. Nakabachi, S. Shigenobu, and S. Miyagishima, "Chitinaselike proteins encoded in the genome of the pea aphid, Acyrthosiphon pisum," Insect Molecular Biology, vol. 19, no. 2, pp. 175-185, 2010.

[49] K. J. Kramer and S. Muthukrishnan, "Insect chitinases: Molecular biology and potential use as biopesticides," Insect Biochemistry and Molecular Biology, vol. 27, no. 11, pp. 887-900,1997.

[50] H. Merzendorfer and L. Zimoch, "Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases," Journal ofExperimental Biology, vol. 206, no. 24, pp. 4393-4412, 2003.

[51] A. Krishnan, P. N. Nair, and D. Jones, "Isolation, cloning, and characterization of new chitinase stored in active form in chitinlined venom reservoir," The Journal of Biological Chemistry, vol. 269, no. 33, pp. 20971-20976, 1994.

[52] K. Y. Jeong, C.-S. Hong, J.-S. Lee, and J.-W. Park, "Optimization of allergen standardization," Yonsei Medical Journal, vol. 52, no. 3, pp. 393-400, 2011.

[53] S. M. Valles and R. M. Pereira, "Solenopsis invicta transferrin: cDNA cloning, gene architecture, and up-regulation in response to Beauveria bassiana infection," Gene, vol. 358, no. 1-2, pp. 60-66, 2005.

[54] E. A. B. Undheim, B. G. Fry, and G. F. King, "Centipede venom: Recent discoveries and current state of knowledge," Toxins, vol. 7, no. 3, pp. 679-704, 2015.

[55] E. De Gregorio, P. T. Spellman, G. M. Rubin, and B. Lemaitre, "Genome-wide analysis of the Drosophila immune response by using oligonucleotide microarrays," Proceedings of the National Acadamy of Sciences of the United States of America, vol. 98, no. 22, pp. 12590-12595, 2001.

[56] Z. Forsberg, A. K. Mackenzie, M. Sorlie et al., "Structural and functional characterization of a conserved pair of bacterial cellulose-oxidizing lytic polysaccharide monooxygenases," Proceedings of the National Acadamy of Sciences of the United States ofAmerica, vol. 111, no. 23, pp. 8446-8451, 2014.

[57] G. R. Hemsworth, E. J. Taylor, R. Q. Kim et al., "The Copper Active Site of CBM33 Polysaccharide Oxygenases," Journal of the American Chemical Society, vol. 135, no. 16, pp. 6069-6077, 2013.

[58] D. R. Cavener and P. A. Krasney, "Drosophila glucose dehydrogenase and yeast alcohol oxidase are homologous and share N-terminal homology with other flavoenzymes," Molecular Biology and Evolution, vol. 8, no. 1, pp. 144-150,1991.

[59] K. Iida, D. L. Cox-Foster, X. Yang, W.-Y. Ko, and D. R. Cavener, "Expansion and evolution of insect GMC oxidoreductases," BMC Evolutionary Biology, vol. 7, article no. 75, 2007.

Juliana Rocha da Silva, (1) Aline Zanotelli de Souza, (2) Carlos Priminho Pirovani, (2) Helena Costa, (2) Aline Silva, (2) Joao Carlos Teixeira Dias, (2) Jacques Hubert Charles Delabie, [ID](2,3) and Renato Fontana [ID] (2)

(1) Universidade Estadual de Santa Cruz (UESC), Programa de Pos-Graduacao em Biologia e Biotecnologia de Microrganismos (PPGBBM), 45.662-900 Ilhous, BA, Brazil

(2) Universidade Estadual de Santa Cruz (UESC), Ilheus, BA, Brazil

(3) Laboratorio de Mirmecologia, Centro de Pesquisas do Cacau (CEPLAC), Itabuna, BA, Brazil

Correspondence should be addressed to Renato Fontana;

Received 22 November 2017; Revised 20 March 2018; Accepted 29 May 2018; Published 21 June 2018

Academic Editor: Leonardo Dapporto

Caption: Figure 1: Spots corresponding to the proteins identified in the bidimensional gel of Ectatomma tuberculatum venom. Isoelectric point (pI) of 3 to 10, molecular weight (MW) of 14.4 to 97.0 kDa. The numbered spots correspond to proteins identified by mass spectrometry.

Caption: Figure 2: Representation of the action mechanisms proposed for the proteins identified in the venom of E. tuberculatum; 'peptide ectatomin [23, 25] not identified in the present study.
Table 1: Proteins identified in the Ectatomma tuberculatum venom
with their respective access codes in NCBI, molecular mass (MM),
isoelectric point (pI), score, and peptide sequence of each spot
were analyzed in the Mascot program (Matrix Science).

Spot         Protein/Source         Accession code NCBI

01       Annexin B9/Harpegnathos    gi|307194598
          saltator (Formicidae)

76       Annexin B9/Harpegnathos    gi|307194598
          saltator (Formicidae)

02       Nucleoside diphosphate     gi|307173082
         floridanus (Formicidae)
          Aconitate hydratase,

03        Acromyrmex echinatior     gi|332018721
          Aconitate hydratase,

51        Acromyrmex echinatior     gi|332018721

52        Aconitate hydratase,      gi|332018721
          Acromyrmex echinatior

04        Superoxide dismutase      gi|307204104
          saltator (Formicidae)

05      Cyclophilin-like protein-   gi|292397870
        Nylanderia nr. pubens LZ-
            2010 (Formicidae)

09           Triosephosphate        gi|332024520
         echinatior (Formicidae)

12       Phosphoglycerate mutase    gi|340726229
        2-like/Bombus terrestris

16          Glyceraldehyde-3-       gi|307181618
        phosphate dehydrogenase/
          Camponotus floridanus

17          Glyceraldehyde-3-       gi|307181618
        phosphate dehydrogenase/
         Camponotus floridanus
18          Glyceraldehyde-3-       gi|307181618
        phosphate dehydrogenase-
          Camponotus floridanus

69          Glyceraldehyde-3-       gi|332026368
        phosphate dehydrogenase
        2/ Acromyrmex echinatior

19      Succinyl/CoA ligase [GDP/   gi|307170861
         forming] subunit alpha,
         floridanus (Formicidae)

26      Succinyl/CoA ligase [ADP/   gi|307178345
         forming] subunit beta,
             mitochondrial /
          Camponotus floridanus

57      Succinyl/CoA ligase [GDP/   NCBI gi|307170861
         forming] subunit alpha,
         floridanus (Formicidae)

20        Fructose/bisphosphate     gi|110748949
           mellifera (Apidae)

58        Fructose/bisphosphate     gi|110748949
           aldolase/like /Apis
           mellifera (Apidae)

75        Fructose-bisphosphate     gi|307206615
         aldolase/ Harpegnathos
          saltator (Formicidae)

78        Fructose-bisphosphate     gi|307206615
         aldolase/ Harpegnathos
          saltator (Formicidae)

21          Arginine kinase /       gi|332018357
          Acromyrmex echinatior

22          Arginine kinase /       gi|332018357
          Acromyrmex echinatior

23          Arginine kinase /       gi|307197996
          Harpegnathos saltator

24          Arginine kinase /       gi|332018357
          Acromyrmex echinatior
          (Formicidae) Arginine
                kinase /

25        Acromyrmex echinatior     gi|332018357

27      Actin-5, muscle-specific/   gi|307197034
          Harpegnathos saltator

29      Probable citrate synthase   gi|307202019
            1, mitochondrial/
          Harpegnathos saltator

30      Probable citrate synthase   gi|350425352
        2, mitochondrial/ Bombus
           impatiens (Apidae)

34      ATP synthase subunit beta   gi|307195440
          Harpegnathos saltator

32        ATP synthase subunit      gi|269784695
          alpha, mitochondrial
           precursor/ Nasonia

31        Enolase/ Harpegnathos     gi|307211488
          saltator (Formicidae)

33        Enolase/ Harpegnathos     gi|307211488
          saltator (Formicidae)

61        Enolase/ Harpegnathos     gi|307211488
          saltator (Formicidae)

35          4-hydroxybutyrate       gi|307174077
         coenzyme A transferase/
          Camponotus floridanus

37       Chitinase-like protein     gi|307207611
          saltator (Formicidae)

60        Probable medium-chain     gi|156553409
            specific acyl-CoA
           Nasonia vitripennis

66      Transferrin/ Harpegnathos   gi|307215135
          saltator (Formicidae)

71       Transferrin/ Acromyrmex    gi|332029256
         echinatior (Formicidae)

72       Transferrin/ Acromyrmex    gi|332029256
         echinatior (Formicidae)

74       Transferrin/Acromyrmex     gi|332029256
         echinatior (Formicidae)
           Probable isocitrate
           dehydrogenase [NAD]
             subunit alpha,

70             Camponotus           gi|307166568
         floridanus (Formicidae)

06       Uncharacterized protein    gi|322778685
         SINV.05483/ Solenopsis
          invicta (Formicidae)
         Uncharacterized protein

07       EAL10007/ Harpegnathos     gi|307206139
          saltator (Formicidae)
         Uncharacterized protein

73       EAL10007/ Harpegnathos     gi|307206139
          saltator (Formicidae)

08       Uncharacterized protein    gi|350427541
          LOC100743840/ Bombus
           impatiens (Apidae)

64       Uncharacterized protein    gi|350427541
           impatiens (Apidae)
         Uncharacterized protein

77        SINV_07147/Solenopsis     gi|322796407
          invicta (Formicidae)

Spot    MM/pI         Score

01      35836/4.93    68

76      35836/4.93    106

02      19547/8.41    68

03      92836/8.65    101

51      92836/8.65    371

52      92836/8.65    406

04      14008/6.18    218

05      18008/8.89    162

09      26935/7.71    129

12      35071/7.17    90

16      35815/7.71    133

17      35815/7.71    194

18      35815/7.71    174

69      37576/8.15    81

19      35682/8.93    108

26      48972/6.63    125

57      35682/8.93      158

20      39975/7.57    85

58      39975/7.57    85

75      40185/8.31    264

78      40185/8.31    338

21      40032/5.86    370

22      40032/5.86    238

23      39996/5.75    447

24      40032/5.86    333

25      40032/5.86    333

27      42098/5.30    114

29      49316/8.94    85

30      51596/9.03    72

34      55223/5.32    397

32      59247/9.18      131

31      47379/5.79      276

33      47379/5.79    230

61      47379/5.79    400

35      53245/7.99    104

37      55766/8.45    56

60      45544/8.37    67

66      82014/5.66    87

71      79357/5.47    145

72      79357/5.47    431

74      79357/5.47    84

70      29723/6.01    74

06      17131/6.70    123

07      27511/6.70    74

73      27511/6.70    85

08      24614/6.41    72

64      24614/6.41    78

77      68188/6.51    97

Spot                        Peptide sequences

01                             GIGTTDSTLIR

76                             GIGTTDSTLIR







09                     IIYGGSVTAANAK, VIACIGEKLEER





69                              IAVFSEREPK



57                           LIGPNCPGIIAPEQCK

20                             LAILENANVLAR

58                             LAILENANVLAR



                         LIDDHFLFK, EMNDGIAELIK,







30                     VVPPILLETGK, VGEVTVDMMYGGMR







37                            EADYPAPIYGSYGR

60                             TNPDPKAPASK

66                             DLDINNVQGLR



74                           DNGADITIIDGGSVK

70                            VAEFAFKYATDNNR


07                             GAMGCGPQETFR

73                             GAMGCGPQETFR

08                              YGTGVIVQR

64                              YGTGVIVQR

77                              VYKDLPVGK

Table 2: Proteins found in the venom of E. tuberculatum with
unknown function in the venoms of Hymenoptera.

Protein                                     Spot

Medium-chain specific acyl-CoA               60
dehydrogenase, mitochondrial-like
(E. C.

Aconitate hydratase mitochondrial       3, 51 and 52
(E.C.                           (isoforms)

ATP Synthase (E.C.                 32

Citrate Synthase (E.C.         29, 30 and 34

Fructose-bisphosphate aldolase        20, 58, 75 and 78

Glyceraldehyde-3-phosphate             16,17,18 and 69
dehydrogenase (E.C.,

Isocitrate dehydrogenase (E.C.               70

Nucleoside diphosphate kinase                 2

Phosphoglycerate mutase 2-like               12

Succinyl-CoA ligase (E.C.      19, 26 and 57

4-Hydroxybutyrate coenzyme A                 35
transferase (E.C.

Protein                                       Action Mechanism

Medium-chain specific acyl-CoA        Enzyme that acts on the [beta]-
dehydrogenase, mitochondrial-like       oxidation of fatty acids in
(E. C.                               mitochondria.

Aconitate hydratase mitochondrial      Catalyzes the isomerization of
(E.C.                          citrate in isocitrate in the
                                      citric acid cycle. Some studies
                                       have revealed the presence of
                                       this protein in the venoms of
                                        several arthropods, such as

                                      Vespula maculifrons, Sicariidae
                                       Loxosceles reclusa spider, A.
                                      mellifera, among others, as well
                                       as in the cuticle of Hemiptera
                                       Coccidae Ericeruspela [7, 8].

ATP Synthase (E.C.          ATP-forming enzyme complex from
                                        ADP and inorganic phosphate
                                      during oxidative phosphorylation
                                             in respiration and
                                         photosynthesis processes.

Citrate Synthase (E.C.        Participates in the first step
                                        of the citric acid cycle by
                                        catalyzing the condensation
                                       reaction of an acetate residue
                                        containing two carbons of an
                                         acetyl coenzyme A with an
                                      oxaloacetate molecule containing
                                        four carbons to form a six-
                                              carbon citrate.

Fructose-bisphosphate aldolase            Involved in carbohydrate
(E.C.                       metabolism, and responsible for
                                       the cleavage of fructose 1,6-
                                        bisphosphate in two trioses:
                                       glyceraldehyde 3-phosphate and
                                       dihydroxyacetone phosphate in

Glyceraldehyde-3-phosphate                Participates in glucose
dehydrogenase (E.C.,           metabolism by the Embden-
                                       Meyerhof route, catalyzing the
                                      conversion of glyceraldehyde 3-
                                             phosphate to 1,3-

Isocitrate dehydrogenase (E.C.        Enzyme that catalyzes oxidative                             decarboxylation of isocitrate to
                                        form a-ketoglutarate in the
                                             citric acid cycle.

Nucleoside diphosphate kinase            Participates in nucleotide
(E.C.                          biosynthesis, catalyzing the
                                        conversion of the nucleoside
                                        diphosphate into nucleoside

Phosphoglycerate mutase 2-like            Glycolytic enzyme which
(E.C.                        catalyzes the conversion of 3-
                                           phosphoglycerate to 2-

Succinyl-CoA ligase (E.C.    Enzyme that participates in the
                                       citric acid cycle, catalyzing
                                       the conversion of succinyl-CoA
                                               to succinate.

4-Hydroxybutyrate coenzyme A          Enzyme that participates in the
transferase (E.C.              metabolic process involving
                                          acetyl Co-A, exhibiting
                                      transferase activity. It is well
                                          studied in bacteria and
                                      participates in the fermentation
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Title Annotation:Research Article
Author:da Silva, Juliana Rocha; de Souza, Aline Zanotelli; Pirovani, Carlos Priminho; Costa, Helena; Silva,
Publication:Psyche (Cambridge, 1874)
Date:Jan 1, 2018
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