Hymenopterans insects mainly wasps, honey bees, hornets inflict venom to maintain self-defense for protection of territory. Hymenoptera venom is a secreted from social poison glands of insects attached to a sting apparatus present in last segment of the body. It serves both as defensive substances against aggressors as well as weapon used to paralyze the victim during gaining food. After sensing little disturbance in the territory, wasps make lethal attack in large numbers and they inflict venom into the body of mammals mainly man and his pets [1] . Wasps react and respond very fast to make an attack on predators and mammals. It is also highly toxic to microorganisms. These inflict venom into the body of enemy within no time by opening venom apparatus and charge upon heavily for making self defense. Venom toxins have been evolved to capture prey and make defense against predators and/or microorganisms. Generally wasp envenomation occurs after little disturbance occurred in near vicinity of their hive. Chemically, the venom is a mixture of biologically active substances of high- medium, and small molecular weight with a variety of physiological function [2] . Wasp venom is a complex cocktail of several hundreds of different components and lethal toxins. It is a complex mixture of active amines (serotonin, histamine, tyramine, dopamine, noradrenaline and adrenaline), peptides (pain producing peptides such as kinins and chemotactic peptides like mastoparans or crabrolin) and proteins including many types of enzymes such as hydralases (proteases, hyaluronidase, phosphatases, nucleotidases, phospholypases A) as well as allergens [3] and toxins [4] . Wasp venom toxins possess a number of charged amino acids which are highly critical for their biological activity. The wasp venom is highly toxic to small mammals in which it causes tissue irritation, swelling and inflammation and pain. Venom toxins induce severe anaphylaxis in animals and man soon after stinging [5] and cause early and delayed hypersensitivity, inflammatory reaction, necrosis and toxic complications in mammals and even invertebrates animals [6] . Venom toxins of Polybia paulista showed genotoxic and mutagenic effects. Polyamine toxin from yellow wasp is potent open-channel blockers of ionotropic glutamate (IGlu) receptors that show selective ligand binding [7] .

Wasp stinging impose multisystem changes and show wide range of biological effects such as intravascular hemolysis, rhabdomylysis, acute renal failure, cardiac involvement, hepatic dysfunction, thrombocytopenia and coagulopathy. Wasp venom also causes scanty micturation, generalization, swelling, and respiratory distress [8] . Wasp venom toxins cause significant serological changes [9] and impose multisystem changes and show wide range of activities such as intravascular hemolysis, rhabdomylysis. Wasp venom toxins cause acute renal failure, hepatic dysfunction and occasionally thrombocytopenia and coagulopathy. At cellular level a large group of toxins breach the normal barrier to free the movement of molecules across cell membrane. More specifically cytotoxins in large amount cause cytolysis, by which soluble molecules leak out of the cell. Wasp toxins cause hemolytic activity and damage nerve and blood cells [10] . Its low concentration causes mild damage to plasma membrane. Besides these toxins act as enzymes and hydrolyze membrane phospholipids [11] and insert themselves into membrane and form channel blockage by binding through which small molecules may pass [12] .

The wasp venom is a toxic substance that causes severe inflammation, pain and allergic reactions after infliction in farmers, researchers, free dwellers, rural and urban people. After an intense attack in large numbers with multiple stings, patient generates fast immediate (anaphylactic) responses that show severe or even fatal illness. Wasp venom toxins generate toxic effects with multiple organ dysfunction followed by anaphylactic reaction [13] . It causes significant serological changes [9] and severely effect blood biochemical parameters and generates toxic effects in man [14] . Patients face serious physiological effects like rhabdomyolysis, hemolysis, cerebral disturbances, hepatic and renal dysfunctions after multiple stings. Although allergic reactions to hymenoptera stings are often considered as general model for the underlying principles of the allergic diseases, hence venom immunotherapy is highly applicable to release or cut down systemic side effects of allergens [15] .

Hymenopteran insect venom is a rich source of bioactive compounds that are highly useful tools in neuro- science and pharmacological investigations. Components of wasp venom can be used as important pharmacological tools or powerful probes or diagnostic techniques for the elucidation of complex biological processes of pharmaceutical importance. Currently, various toxin structures are used to drug design suitable three-dimen- sional templates for creating small molecules, which might mimic interesting pharmacological properties. Wasp envenomation is a worldwide public health problem that has economic and social implications. Wasp venom also contains some peptides, which are antimicrobial [12] [16] and anticancer in nature [17] and may act like antibiotics [18] [19] . In the present study antibacterial activity of purified yellow wasp toxins were evaluated against seven bacteria by MIC, MBC and zone inhibition. No doubt wasp venom toxins are of very high pharmacological importance. Several insecticidal compounds belonging to the class of peptides or polyamines like compounds have been purified and characterized from the venom of arachnids and hymenoptera. Few invertebrates’ specific peptides neurotoxins that have been isolated from the venom of wasp which are used as good pest control agents and are also used as invaluable tools in neuropharmacology [20] . Toxin peptides can also become a good source of strong antimicrobials and can replace broad spectrum antibiotics which are highly toxic and show multiple biological effects and responsible for drug resistance in microbes [21] .

Triton X-100 (0.1%) proved to be a good solubilizing agent for toxins/proteins. Higher protein solubilization was observed in the supernatant than in the residue, except TCA (Figure 1). The elution pattern of purified and homogenized sting glands exhibited two major peaks at 280 nm in fraction no. 41 - 61 and 81 - 101 (Figure 2(a)). Further concentration and fractionation of venom proteins again revealed two peaks at 640 nm, a minor one between fractions 46 - 51 and a major peak between fractions 60 - 101 (Figure 2(b)). Both peaks were eluted with 0.13 M NaCl PBS buffer (pH 6.9) and protein estimation was done for each fraction by using Lowry’s method (Figure 2(c)). The total yield of protein was 69.21% and specific activity was determined in each fraction (Figure 2(d)). Molecular weights in protein fractions were ranging from 6 - 70 kD (Figure 2(e)).

MIC values were 12.3 μg/ml against K. pneumoniae 12.3 μg/ml against E. coli and L. acidophilus, 24.6 μg/ml against B. cereus; 49.24 μg/ml against S. aureus and M. luteus. The results suggest that E. coli and B. cereus were most susceptible to yellow wasp toxins while K. pneumoniae was least susceptible. MIC values for tetracycline and ampicillin were 14 μg/ml and for ciprofloxin 28 μg/ml against K. pneumoniae (Table 1). Salmonella typhi 24.69 μg/ml, Vibrio cholera 24.69 μg/ml, Pseudomonas aeruginosa 12.3 μg/ml respectively, while MIC value obtained against fungal strains i.e. Aspergillus niger 49.2 μg/ml, Candida albicans 12.3 μg/ml, Rhizopus stolonifer 6.9 μg/ml (Table 1).

By agar disc diffusion method the diameter of inhibition zones in mm in presence of yellow wasp toxins at a concentration range of 98.56 - 6.9 μg/ml E. coli 18.36 ± 0.14, Bacillus cereus 14.566 ± 0.21, L. acidophilus 18.10 ± 0.21, Micrococcus luteus 18.76 ± 0.19, S. aeurus 17.36 ± 0.43, Klebsiella pneuminiae 19.56 ± 0.21,

Salmonella typhi 19.96 ± 0.31, Vibrio cholera 21.83 ± 0.22, Pseudomonas aeruginosa 22.90 ± 0.09, Aspergillus niger 21.66 ± 0.16, Candida albicans 23.33 ± 0.26, Rhizopus stolonifer 24.96 ± 0.16 (Table 2).

In the present study, antimicrobial activity of purified yellow wasp toxins was determined in vitro and compared with broad-spectrum antibiotics. From antimicrobial susceptibility tests it was found that antibiotics were found

^*Positive controls are 1) tetracycline, 2) ampicillin & 3) ciprofloxacin and T represents wasp toxins, negative control is DMSO, Gsf Griseofulvin, ND = not done.

^*Values are expressed as mean ± SD (N = 3) and values followed by same letter are not significantly different at the p < 0.05 determined by Duncan’s Multiple Range Test. Negative control is DMSO, Positive control are 1) tetracycline, 2) ampicillin & 3) ciprofloxacin and T represents wasp toxins.

less active than the wasp venom toxins against the test bacteria and fungi. From the inhibition zone diameter assays the IZDs obtained were larger in case of wasp venom than antibiotics. Yellow wasp venom toxin (16.4 mg/ml) gave inhibition zone diameters of 18 - 23 mm while tetracycline, ampicillin, and ciprofloxacin gave inhibition zone diameters in the range of 15 - 26 mm at a concentration of 8 mg/disc (Table 1). By agar disc diffusion method the diameter of inhibition zones in mm in presence of yellow wasp toxins at a concentration range of 6.9 - 49.24 μg/ml were 17.36 ± 0.43, 19.56 ± 0.21, 14.566 ± 0.21, 18.36 ± 0.14, 18.10 ± 0.21, 18.76 ± 0.19 for S. aureus, K. pneumoniae, B. cereus, E. coli, L. acidophilus and M. luteus respectively. Inhibition zone diameters in mm for tetracycline, ampicillin and ciprofloxacin against S. aureus, K. pneumoniae, E. coli, L. acidophilus and M. luteus, Streptococcus pneumoniae and Bacillus cereus were 15.23 ± 0.38, 10.63 ± 0.25, 11.16 ± 0.21; 17.06 ± 0.21, 14.23 ± 0.21, 9.80 ± 0.18; 15.66 ± 0.22, 16.43 ± 0.28, 9.86 ± 0.17; 16.00 ± 0.29, 10.30 ± 0.21, 12.83 ± 0.26; 17.66 ± 0.24, 10.23 ± 0.19, 14.13 ± 0.19 respectively (Table 2). In all the cases venom toxins displayed much better antimicrobial action against bacterial and fungal strains. The results suggest that 22.90 ± 0.09 was most susceptible to yellow wasp toxins while B. cereus and S. aeurus least susceptible (Table 2). Similar results were also obtained in Egyptian yellow wasp on the growth of Aspergillus versicolor [26] .

Melittin, a highly toxic and immunogenic peptide isolated from honey bee, shows an MIC value between 0.5 - 0.03125 mg/ml against B-pseudomallei [27] [28] . It also exhibits powerful in vitro inhibitory activity against spirochetes [29] . Yellow wasp venom also contains few other peptides such as apamin, procamine, abcaecin and secapin [17] [30] [31] . Apamin constitutes less than 2% of venom dry weight; has few amino acid residues and shows neurotoxic properties. It acts as an inductor of convulsion in mice [32] but does not exert any influences on mammalian cells. Apamin also affects sodium and calcium channels in cells and influence nerve transmission [10] [33] . It also blocks the postsynaptic ion channels present in neurons of both vertebrates and invertebrates [34] . Most of the peptides isolated from yellow wasp venom strongly act upon bacterial cell membrane. These toxin peptides interact with lipid membrane, disrupt its integrity [10] [35] [36] and increase the diffusion of molecules from inside to outside of the cell [32] [37] . Few venom peptides which occur as monomer but in presence of salt these transform into tetramer and form helical structure [38] [39] . This tetramer structure forms trans-membrane pores in lipid bilayers and in lipid vesicles [40] [41] . It also acts like a detergent [42] [43] and results in heavy cell lyses [44] , and disruption of membrane integrity. This might be due to hydrophilic nature of toxin peptides, and presence of basic amino acids in their active site region [35] .

Similar antimicrobial activity is also reported in cationic peptides polybia-MPI [35] , anoplin [35] [45] and their synthetic analogues [35] [46] . Anoplin shows membrane anchoring through a lipophilic amino acid derivative and slight changes in its structure shows improvement in biological activity [47] . Wasp venom toxins interact with voltage-sensitive sodium channels and generate action potentials in most of excitable tissues. Membrane depolarization causes voltage-dependent conformational changes that increase the permeability of sodium ions and can work as local anesthetics [48] . α-Pompilidotoxin (α-PMTX) isolated from the venom of solitary wasp Anopolis samariensis [49] greatly facilitates both excitatory and inhibitory synaptic transmission in the lobster neuromuscular synapse [49] as well as disrupts synchronous firing in rat cortical neurons [50] . α-PMTX affects presynaptic neurons while carybdotoxins or apamin affects calcium ions activated potassium ion channels [49] . From the results it can be concluded that the purified yellow wasp toxin has displayed similar, better or worse anti bacterial activity against different bacterial and fungal strains in comparison to the three antibiotics tested. These could replace highly toxic antibiotics which can solve the problem of drug resistance.

The authors would like to thanks the D. D. U. Gorakhpur University, Gorakhpur U.P. for providing research opportunity and for the use of laboratory facilities to carry out this study.

Authors have no conflict of interest. Authors are responsible for the content and writing of the paper.

Krishna Kumar Prajapati,Ravi Kant Upadhyay, (2016) Antimicrobial Activity of Purified Toxins from Yellow Wasp Polistes flavus (Vespidae) against Certain Bacteria and Fungi. Journal of Biosciences and Medicines,04,95-104. doi: 10.4236/jbm.2016.47010