To Study the Antibacterial Potential of Scorpion Venom and Hemolymph

by

Introduction

            Microbes are one of the major threat to human health. They cause mild to severe infections and are sometimes fatal for humans. In recent years, microbes have developed resistance against commonly used drugs and this is a global problem (Allen et al, 2010; Andersson & Hughes, 2010; Nhung et al, 2016). There is a dire demand for the production of new antibiotics with new approaches to resolve the issue (Samy et al, 2006). Now a days, scientist have switched to natural products for the production of new antibiotics (Newman & Cragg, 2007; Wang et al, 2016).

            Many researchers have reported the antibacterial potential of different plants and animals (Juneja et al, 2012). Scorpion venom is also important in this regard (Tarazi, 2015). The scorpions are among the earliest arthropods living on the earth. These belong to class Arachnida and feed on small arthropods mostly insects (Luna-Ramirez et al, 2017). These are present in almost all regions of the world excluding Antarctica. Aproximately 1,500 species and subspecies of scorpions have been reported till now which are classified into 18 families (H. S. Bawaskar & H. P. Bawaskar, 2012; Chaubey, 2017; Prendini & Wheeler, 2005). Among these approximately 50 species have very strong venom (Isbister et al, 2003; Osnaya-Romero et al, 2001). Possani et al. (2000) reported that from 1,500 diverse species of scorpions 100,000 different peptides have been extracted throughout the world, and only 0.02% among them are identified for their interaction with ion channels. They use their venom as a strong weapon against predators. The scorpion species with medical importance are from Buthidae family and genera Buthotus, Androctonus, Parabuthus, Leiurus, Buthus and Mesobuthus. These are placed in topographical areas i.e., middle East and Asia (Attarde & Pandit, 2016; Chaubey, 2017).

            Scorpion venom is mainly composed of proteinaceous substances (Nisani et al, 2007). It contains a mixture of neurotoxins and bioactive compounds (Luna-Ramirez et al, 2017). It is enriched with nucleotides, enzymes, mucoproteins, mixture of salts, peptides and proteins. Venom of scorpions also include different enzymes i.e., lipase, hyaluronidase, phospholipase (Park et al, 2007; Zouari et al, 2005) proteolytic enzymes and alkalic phosphatases (Incesu et al, 2005). It is being used in medicines for over thousands of years (Attarde & Pandit, 2016; Wang et al, 2016). The venom of scorpions and spiders contain many different chemical compounds, from these organisms antimicrobial peptides were first discovered in 1930 (Wang, X & Wang, G 2016). Two venom glands in the tail of scorpion produce venom and kept in two venom sacs in the telson of scorpions. Both these functional structures are very important for the existence of scorpions because these help in prey capturing as well as for defensive purpose (Elgar et al, 2006).

            Scorpion venom is a diverse combination of different peptides and protein toxins. These special characteristics makes the venom of this animal a precious source for new complexes in the development of new medicines as well as in basic research (Billen et al, 2008). Scorpion venom contains toxins which can be used for cancer treatment (Incesu et al, 2005) and the manufacturing of eco-friendly insecticides (Leng et al, 2011). Venom of scorpions is a reservoir of tremendously diverse active peptides that induce immunological and toxicological responses. And it also contains low molecular peptides that proposed a brilliant resource for drug development (Zafar et al, 2013). Venom of scorpions has also been used for the manufacturing of different vaccines and painkillers (Fabiano et al, 2008).

            Antimicrobial peptides (AMPs) are short length peptides which are found in blood, venoms of animals and other body secretions (Dang & Van-Damme, 2015; Harrison et al, 2014; Wang et al, 2015). Their structure shows great amphiphilic topologies (Godballe et al, 2011; Zasloff, 2002). These AMPs are cationic, low molecular with mass (2–5 kD), α- helical peptides which exhibits an expansive potential against fungi, G positive and negative bacteria through the lysis of membrane (Arpornsuwan et al, 2014; Dai et al, 2001). Some species of scorpions also use their venom as a shield against the microbes of skin (Torres-Larios et al, 2000). Host defense cationic peptides have been produced by many organisms (Hancock & Leher, 1998; Hancock & Sahl, 2006; Wang et al, 2006). These peptides are considered to be very effective against microorganisms and are called as antimicrobial peptides (Brogden et al, 2003; Cahalan, 1975). The charge on AMPs also play an important role. Positive charge of cationic AMPs plays an effective role in the electrostatic attachment to the negatively charged targeted membrane components like acidic and phospholipids of bacteria (Hancock & Sahl, 2007).

            Antimicrobial peptides (AMPs) isolated from venom of scorpions show an extensive activity against parasites microorganisms, and fungi (Boman, 2000). The antimicrobial toxins in venom of scorpions exhibit their bioactivities by blocking ion channels or by modifying its functions (Couraud et al, 1982). Their therapeutic potentials include treatment of autoimmune diseases, antimicrobial activity, cardiovascular effects and others (Attarde & Pandit, 2016). Antimicrobial peptides also known to have crucial first line defense against microbes (Ganz & Lehrer, 1997).

            AMPs can form three dimensional structures at sufficient concentrations when these are in contact with mammalian or bacterial membranes (Elgar et al, 2006). Peptide toxins extracted from scorpion venom also exhibit their antimicrobial activity by acting upon muscle transmembranes and nerve membranes (Possani et al, 2000). There are different ways to categorize AMPs created on different standards like peptides precursors, peptide source and peptides enrichment in one or more amino acids and based on other parameters (Palffy et al, 2002). For classification of AMPs one of the most used techniques is based on structural motifs homology (Palffy et al, 2002). According to the structural motifs homology, AMPs have been grouped into four main groups. Among these α-helical peptides and β-sheet peptides are common while extended peptides and loop peptides are less common. Moreover α-helical peptides are the most studied group till now (Powers & Hancock, 2003).

Many AMPs have been reported with positive charge ranged in between +2 to +9. There is a direct relationship between peptides net positive charge and its activity within limited range, which differs in different peptides. An increase or decrease in the charge beyond its limit results in the loss of optimum activity (Dathe et al, 2001). These peptides are different widely in structure and sequence, and kill the microbes by two ways i.e., thinning and disruption of bacterial membrane (Hancock & Scott, 2000), and as a result these peptides play significant role to produce the new medicinal agents (Silphaduang & Noga, 2001). Possani et al. (1998) identified some antibacterial peptides defensin and cecropins in scorpion’s hemolymph.  These peptides cause the killing of microbes by lysis of their cells.

Scorpion venom also shows its tremendous activity in the treatment of other disorders. Cancer is one of the serious health issue of this developed world. Scorpion venom has also shown tremendous activity against different types of cancers (Gomes et al, 2009). Scorpion peptides can also be used alternatively in cardiovascular therapy to regulate blood vasomotion (Hmed et al, 2013). It has been reported that many AMPs, originating from scorpions also possess antiviral activities against H5N1, SARS-Cov (Li et al, 2011), measles, hepatitis B (Zhao et al, 2012), and hepatitis C (Yan et al, 2011), and HIV-1 (Cao et al, 2012; Chen et al, 2012) viruses.

Scorpion peptides showing cytotoxic and anti-proliferative effect, and new components are being continuously added (Gupta et al, 2007; Salarian et al, 2012; Song et al, 2012; Wang & Ji, 2005; Xiao et al, 2012). The most remarkable use of scorpion venom have been seen in Chinese history where it was used in ethnopharmacy for the improvement of blood rheology and homeostasis (Song et al, 2002). Buthus martensii Karsch scorpion is extensively used in Chinese ethno medicine with the handling of certain nervous disorders i.e., cerebral palsy, apoplexy and epilepsy (Liu et al, 2003).

Objectives of Study:

Objectives of the study include.

  • Milking scorpion venom
  • Collection of hemolymph
  • Check the potential of scorpion venom against bacterial strains
  • Check the potential of scorpion hemolymph against bacterial strains

Scope and Significance of Study:

Now a days, no one can deny from the dire need of efficient antibiotics. Drug resistance is the major issue. This research will be an important contribution for the development of new antibiotics with better efficacy.

Literature Review

Infectious diseases cause millions of deaths annually, even with advances in antimicrobial therapy. The main reason is rapid emergence of antibiotic resistance in microbes so decreasing the efficacy of antibiotics (Alanis 2005; Nordmann et al, 2007). In such scenario there is a dire need of new researches for serious management (Gould & Bal, 2013; Spellberg & Gilbert, 2014). The issue has also drawn the attention of scientist towards antimicrobial peptides (AMPs) (Luna-Ramirez et al, 2016). These are short length peptides which have a reputed antibacterial potential against resistant microbes (Fan et al, 2011). Venom of animals are brilliant source of AMPs (Bouzid et al, 2014; Konig et al, 2012). Venom of scorpions is enriched with novel and effective AMPs (Ramirez et al, 2017).

A large quantity of peptides have been purified and categorized from scorpion venom. Approximately 100,000 different peptides are reported and only 1% are known among them (Possani et al, 1999). Venom and hemolymph of scorpions possess a diverse group of many bioactive compounds (Hmed et al, 2013). Scorpion venom is an effective self-protective device. Venom of scorpions contain some distinctive characteristics which have been proved to be very beneficial for mankind (Chaubey, 2017).

Venom of scorpion is a therapeutic tool against a variety of diseases. It comprises of salts, neurotoxins, enzymes with high molecular weight and other low molecular peptides. Venom of scorpion had been used in medicine since medieval times in China (Mishal et al, 2013). Scorpions belong to family buthidae are medically important. Scorpion venom is water soluble and it is a blend of many compounds and enzymes i.e., phospholipases, alkaline phosphatases and proteolytic enzymes (Possani et al, 1999; Incesu et al, 2005; Zouari et al, 2005; Park et al., 2007).

Scorpion venom possess a variety of bioactive compounds that are remarkable source for the development of new drugs.  AMPs were first revealed in 1990s. According to antimicrobial peptide database of May 2015 63 scorpion AMPs and 42 spider AMPs were reported. These AMPs have shown a wide ranged activity against microbes (Wang, G & Wang, X 2016). AMPs are short length peptides (less than 100 amino acids). These have been derived from an extensive variety of natural resources i.e., animals and plants. AMPs are cationic amphipathic peptides. Generally AMPs acts on different ion channels and block their activity. The scorpion peptides are highly specific to their targeted sites (Hmed et al, 2013).

The first scorpion AMP was reported in 1993.  Then in 1996, three more scorpion peptides were reported (Ehret-Sabatier et al, 1996). Buthinin and androctonus are three disulfide bond peptides and androctonin is a two disulfide bond peptide toxin. After this from 2004 to 2007, further three disulfide-linked AMPs were discovered. These comprise of opiscorpine, charybdotoxin, and heteroscorpine (Uawonggul et al, 2007; Yount & Yeaman, 2004; Zhu & Tytgat, 2004). In 2008, one more peptide BmK AS with four disulfide bonds was reported (Ramírez-Carreto, 2015). After that two more peptides with four disulfide bonds were successively identified. Amongst these, bactridines 1 and 2 were proven to be antimicrobial (Diaz et al, 2009).

 In APD database (Antimicrobial Peptide Database) from total 63 AMP enteries, AMPs of 56 scorpion are antibacterial. Moreover, 22 are antifungal, five are antiviral and three are antiparasitic. In addition six are anticancer peptides (Wang, G & Wang, X 2016). In the APD database of May 2015, 21 peptides of scorpion are labbled as helix (14 determined by CD and seven discovered by NMR). Many antibacterial peptides are short in length (13–19 residues). These consist of BmKn2, meucin-13, IsCT, IsCT2, , meucin-18, StCT2, VmCT1, VmCT2, , hp1404, ctriposin, stigmurin, and so on (Moerman et al, 2002). IsCT is a short 13 amino acids cysteine free peptide with helical structure (Lee et al, 2004). Its net charge is +2. It shows antimicrobial activity against G+ and G– bacteria (Dai et al, 2001).

IsCT 2 shows great potential against bacteria as well as fungi (Dai et al, 2002). In addition of it two peptides Meucin-13 and meucin-18 were separated from Mesobuthus eupeus (Gao et al, 2009). They have similar sequence to amphibian 13-residue temporins and 18-residue brevinins. These peptides have great potential against gram positive and negative bactreria. Meucin 18 is more powerful and hemolytic than Meucin 13. Three more peptides Pantinin-1, pantinin-2 and pantinin-3 were derived from Pandinus imperator (Zeng et al, 2013). All of these three peptides have more antibacterial potential against Gram +ve bacteria than gram –ve bacteria.

In the venom of Odontobuthus odontrous Zafar et al. (2013) evaluate the presence of low molecular weight peptides. They confirmed the presence of at least five low molecular weight peptides. Peptide toxins of scorpions target at different channels of ions i.e., Na+, K+, Ca++, Cl– and block their activity (Possani et al, 1999; Becceril et al, 1997). In scorpion haemolymph Leiurus quinquestriatus hebraeus and Androctonus australis several cysteine-containing defensin-type peptides have been derived (Cociancich et al, 1993; Sabatier et al, 1996). Buthinin is a bactericidal and fungicidal peptide with three disulphide-bridges, and from the venom of Androctonus australis androctonin  peptide is with two disulphide bridges (Sabatier et al, 1996).

Scorpine is a 75-residue antimalarial peptide separated from venom of Pandinus imperator (Conde et al, 2000). Scorpine-like peptides show great activity against potassium channel blockers, fungi and bacteria (i.e., K. pneumoniae, B. subtilis and P. aeruginosa). Scorpine also show antiparasitic activities (Luna-Ramirez et al, 2016). Scorpine-like peptides have also been exposed in other scorpion species, i.e., in Opisthacanthus cayaporum (Silva et al, 2009),  Hadrurus gertschi (Diego-García et al, 2007; Schwartz et al, 2007) Tityus costatus (Diego-García et al, 2005) Opistophthalmus carinatus (Zhu et al, 2004), Pandinus cavimanus (Diego-García et al, 2012), Vaejovis species (Quintero-Hernández et al, 2015) Euscorpiops validus (Feng et al, 2013), Urodacus yaschenkoi (Luna-Ramirez et al, 2013), and Heterometrus laoticus (Uawonggul et al, 2007).

 From venom of Hadrurus aztecus (hadrurin) and Parabuthus schlechteri (parabutoporin) α-Helical peptides have been reported (Torres-Larios et al, 2000; Verdonck et al, 2000). From scorpion venom of different geographical regions many insect selective toxins were identified (Nakagawa et al, 1997; Becceril et al, 1997; Bontems et al, 1991). From venom of Androctonus australis three peptides toxins (AaHIT1, AaHIT2, and AaHIT3) have been isolated which are used as potential anti insect agents (Higgs et al, 1995).

The most primitive peptide toxin was androctonin which isolated from the venom of A. australis. Androtonin possess strong antibacterial activity (Hetru et al, 2000). A peptide toxin opistoporins from Opistophthalmus carinatus and parbutoporin from P. schlechteri also showed antibacterial activity. These toxins target G-proteins for their antibacterial activity (Powers & Hancock, 2003).

 Some peptide toxins have also been taken from Vaejovis punctatus that obstructs the growth of G-positive (Streptococcus agalactiae and Staphylococcus aureus) and G-negative (E.coli and P.aeruginosa) bacteria. It also inhibits yeasts (Candida albicans and Candida glabrata) and two strains of Mycobacterium tuberculosis (Ramirez-Carreto et al, 2015). Opisin is cationic, amphipathic peptide with 19 amino acids. These peptides targets the Gram positive bacteria (Bao et al, 2015). Brazilian yellow scorpion Tityus stigmurus also represents antifungal and antibacterial activity, a peptide toxin has been derived from yellow scorpion named stigmurin (Melo et al, 2015). Ctriporin is a peptide toxin isolated from Chaerilus tricostatus. It exhibits a wide range of activity against microbes and also have capability against antibiotic resistant pathogens (Bandyopadhyay et al, 2014).  Li et al, 2014 has reported another peptide toxin Hp1404 extracted from Heterometrus petersii. Its an amphipathic α-helical peptide effective against G positive bacteria (Li et al, 2014).

Scorpion venom also possess anticancer activity. The venom of Hadrurus aztecus and P. schlechteri also contain Alpha helical properties which possess antimicrobial activity (Torres et al, 2000; Verdonck et al, 2000). Pandinin 1 and Pandinin 2 peptides have been isolated from P. impretor also possess antimicrobial activity (Corzo et al, 2001). From Mesobuthus martensii Karsch BmKn2 antimicrobial peptide was derived. Afterwards one more peptide Kn2-7 was separated from BmKn2 to enhance its antibacterial activity. Kn2-7 showed great inhibitory effects against gram +ve and gram -ve bacteria (Cao et al, 2012).

Venom of scorpions is a plentiful source of toxins which also acts on the potassium ion channels. By using fluorescent test system a research was designed to explore the new toxins from the venom of Mesobuthu seupeus (Buthidae). In this research they found five new significant toxins which showed great potential for the blockage of potassium channels (Kuzmenkov et al, 2015). A combinational research was designed to explore venom peptides from Mesobuthus martensii. This research was based on liquid chromatography, mass spectrometry and RNA sequencing. Total 153 proteins were identified from the venom of M. martensii, 26 previously known and 30 was newly explored. This research was proved significant, because many innovative venom peptides were discovered and zoologist gained more knowledge about diversity of venom (Luan et al, 2016).

On the basis of molecular weight 40 peptides were discovered and categorized from venom of Buthus martensi Karsch Among these 29 long chain peptides were discovered. A novel anti-insect toxin BmKaIT1 and a new anti-mammal toxin BmK IV were also discovered. Three potassium channel inhibitors were also identified. NMR spectroscopy and EDMAN degradation technique was used (Wu et al, 1999). Antimicrobial peptides (AMP’s) from venom of scorpions have also been used in agricultural department. AMP’s of scorpions proved very beneficial against aphids (Ramirez et al, 2017).

Some scorpions derived AMP’s suppressed the growth of microbes by their phospholipase activity (Guillaume et al, 2014; Incomnoi et al, 2013). A 21 amino acid peptide was derived from North African Buthus occitanus scorpion’s venom (Meki et al, 1995). From crude venom of Buthus sindicus B10 peptide was derived but its function is still unknown (Ali et al, 1998; Zeng et al, 2004).  A new antimicrobial peptide Vejovine was islolated from Mexican scorpion Vaejovis mexicanus. Its a 47 A.A peptide that hinders the growth of multidrug resistant G-negative bacteria (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloacae, Klebsiella pneumoniae and Acinetobacter baumanii) (Hernández-Aponte et al, 2011).

Borges et al. (2000) reported that venom of Brazilian scorpion Tityus serrulatus regulates the blood clot formations. It was reported in 1980 that the hemolymph of native Androctonus australis and its partial fraction stimulates the rabbit, human and mouse lymphocytes mitogenesis in in vitro studies (Brahmi & Cooper, 1980). Two more peptides VmCT1 and VmCT2 were isolated from scorpion Vaejovis mexicanus. These peptides have great structural similarity among them but their biological activities are very much different from each other. They have great antibacterial potential (Ramírez-Carreto et al, 2012).

In 2012, Eauclaire & Bougis has reported many high potential peptide toxins against K+ channels from Moroccan scorpion Androctonus mauretanicus mauretanicus. They have characterized and isolated various structurally different families of scorpion peptide toxins which exhibit activity against K+ ion channels. One more new AMP StCT2 has been derived from the venomous gland cDNA library of the Scorpiops tibetanus. It’s a 369 nucleotides peptide. It contains a presumed 14 residues mature peptide, a putative 24 residues signal peptide, and a putative 37 residues acidic propeptide at the C-terminus. It has shown high potential activity against S. aureus (Cao et al, 2012).

From the venom of Androctonus aeneas North African scorpion two non-disulphide bridged antimicrobial peptides have been reported. Broad spectrum antimicrobial activity shown by the synthetic version of natural peptides. But after modulations they shown high antimicrobial activity and antiproliferative activity (Du et al, 2015). Baradaran et al, 2017 have reported three new cationic AMPs, i.e., meuVAP-6, meuAP-18-1, and meuPep34 from the venom gland of the Iranian scorpion, Mesobuthus eupeus. Two of these peptides MeuVAP-6 and meuAP-18-1 are non-disulphide-bridged antimicrobial peptides. And meuPep34 is a cysteine-rich defensin-like peptide. All three AMPs contain high amount of tryptophan and arginine. Among all three AMPs uPep34 shown high potential activity against biofilm inhibition.

Another research was conducted by Salama & Geasa in 2014 on the antimicrobial activity of venom of  Egyptian scorpions Androctonus amoreuxi, Leuirus quinquestriatus, and Androctonus australis. Four Gram-positive and negative bacterial strains (Bacillus cereus, Citrobacter freundi, Bacillus subtillis and Klibsella pneumonia) and a fungus specie Candida albicans were used. The venom of Leuirus quinquestriatus showed great antibacterial potential against C. freundi. and B. subtillis. While A. australis and A. amoreuxi venoms did not show antibacterial potential against selected microbes. Ctriporin is another new antimicrobial peptide extracted from scorpion Chaerilus tricostatus. It exhibits great potential against microbes (Fan et al, 2011).

Venom of scorpion Tityus serrulatus results in relaxation of human and rabbit cavernosal smooth muscle in vitro (Teixeira et al, 2001 & Teixeira et al, 1998). Another research has been reported in which a polypeptide toxin OSK1 was derived from scorpion Orthochirus scrobiculosus. It works against voltage-gated potassium channels, Kv1.3 (Chen & Chung et al, 2012). Two more peptides Meucin-24 and Meucin-25 were discovered from Mesobuthus eupeus. They work as effective anti-malarial drugs (Gao et al, 2010). Thus this review shows the unique nature of scorpion venom and its peptides which shows their great effectiveness against microbes and other different type of diseases.

Materials and Methods

Collection of Scorpions

            Scorpions were collected by digging from burrows, under the soil, logs, dry places in the different areas of Dera Gazi Khan (Pakistan). Hottentota tamulus specie was used. Scorpions are nocturnal and they were coptured at dark nights by using the portable UV lamp (Ahsan et al, 2016; Skutelsky, 1996). Thess were handled by using 12 inches long forceps and were kept in transparent boxes with perforated lids to monitor them easily. Floor of the boxes was covered up by soil and sand to provide them their natural habitat. They were provided with insects like grasshoppers, cockroaches, flies and other small arthropods. Water was also provided by spraying the soil at different intervals. These boxes were kept at room temperature.

Milking Scorpion Venom

            Two methods were used for milking purpose. One was electricity controlled method (Ozkan & Filazi, 2004) and the second was maceration method of venom gland (telsons) (Ozkan & Filazi, 2004; Ozkan et al, 2006).

Electricity Controlled Method:

            The scorpions were kept hungry for five days prior to venom extraction. Electricity controlled method was used for venom extraction. Few droplets of saline solution (10%) were placed on the base of telson of scorpion for better conduction of current and the shock was given with two electrodes. Electric shock of 25 V was applied at the base of telson. Venom was obtained from the telson with the help of microtip and was transferred to eppendorf. It was stored at -180C until use (Ozkan & Filazi, 2004).

Maceration Method:

          Telsons from all scorpions were removed and taken in eppendorf. Its homogenized solution was prepared by adding distilled water in it. Then it was centrifuged at 7000 rpm in mini-centrifuge machine model (D1008).

Collection of Hemolymph:

            Scorpions were kept hungry for five days before the collection of hemolymph (Geethabali & Rajashekhar, 1988). Hemolymph was collected by cutting the different body segments (i.e., coxae and keels) of scorpions by using sterile scissor. It was recovered in eppendorf and was stored at -180C.

Dilutions of Venom:

            Venom in crude and diluted form (1/1 & 1/10) was applied on bacterial culture. Ratio of 1/1 venom was prepared by mixing equal amount of venom and water. 1/10 was prepared by mixing 1ul venom and 9ul water (Ahmad et al, 2012).

Dilutions of Hemolymph:

            Hemolymph was also applied in crude and diluted form (1/1 & 1/10) on bacterial culture. Ratio of 1/1 hemolymph was prepared by mixing equal amount of hemolymph and water. 1/10 was prepared by mixing 1ul hemolymph and 9ul water (Ahmad et al, 2012). Antibiotic Ampicillin (1%) was taken as +ve control and distilled water as -ve control.

Microbial Strains:

            Antibacterial activity was determined against four strains of pathogenic bacteria i.e., Staphylococcus aureus (Gram positive), Escherichia coli (Gram negative), Pseudomonas aeruginosa (Gram negative) and Klebisella pneumoniae (Gram negative).

Evaluation of Antibacterial Activity:

            Agar disc diffusion method was used for evaluation of antibacterial activity (Tendencia, 2004).

Preparation of Nutrient Broth Medium:

            Broth medium was prepared by adding 0.3g yeast extract & 0.5 g peptone in 100 ml distilled water. This media was prepared in flask, then poured into test tubes and later on it was autoclaved. Afterwards bacteria was taken from bacterial culture and applied in test tubes. Then those test tubes were kept in incubator model (DNP-9052A) at 370C for 24 hours. After that test tubes were kept in freezer until use.

Preparation of Nutrient Agar Medium:

            Nutrient agar medium was prepared by mixing 0.3g yeast extract, 0.5 g peptone & 2g agar in 100 ml distilled water. This mixture was prepared in flask and then autoclaved. After autoclaving media was poured in petri plates and was kept in incubator at 370C for 24 hours.

Inoculation of plates:

          After 24 hours contamination free agar plates were inoculated with the bacterial strains (50ul) obtained from nutrient broth medium with the help of micropipette. Glass spreader was used to make sure the proper spreading of the bacteria on agar plates.

Disc Diffusion method:

            Each agar plate was divided into three segments by using marker. Afterwards filter paper discs were impregnated with ampicillin, distilled water, and venom. Then the petri dishes were incubated at 370C for 24 hours. Same procedure was performed for hemolymph and different dilutions of both. This whole procedure was conducted in Laminar air flow.

Observations:

            Inhibitory zones made by control groups and test compounds were observed for all treatments and results were recorded.

Results

This research was designed to study the antibacterial potential of scorpion venom and hemolymph. Hottentota tamulus specie of scorpion was used to study the antibacterial effects of scorpion venom and hemolymph. Four bacterial strains gram positive and negative were used. A standard antibiotic ampicillin (1%) was used as positive control group. Distilled water was taken as negative control group.

Antibacterial Activity of venom:

Venom of Hottentota tamulus was not effective against any bacterial strain. Venom in crude form was applied on all four strains i.e., Staphylococcus aureus (Gram positive), Pseudomonas aeruginosa (Gram negative), Escherichia coli (Gram negative) and Klebsiella pneumoniae (Gram negative), but it did not show antibacterial effects against any strain. Venom in diluted form (1/1 & 1/10) was also tested but it also failed to inhibit the growth of bacterial colonies. On the other hand a wide inhibition zone was seen around the disk that was impregnated with 1% amplicin (positive control). Negative control group (distilled water) was also unsuccessful to hinder the growth of bacteria.

Antibacterial Activity of Hemolymph:

Hemolymph of Hottentota tamulus showed negative results against all four bacterial strains. Pure hemolymph was applied on all four strains i.e., Staphylococcus aureus (G positive), Escherichia coli (G negative), Pseudomonas aeruginosa (G -ve) and Klebsiella pneumoniae (Gram negative), but it did not produce any noticeable antibacterial effects against any strain. Hemolymph was also diluted in two ratios i.e., 1/1 and 1/10 but failed to make inhibition zones. While positive control group successfully make a clear inhibition zone against all strains and control group remain ineffective to stop the growth of bacteria.

Discussion

            Due to the rapid emergence of antibiotic resistance in microbes, health issues are increasing day by day. So, we terribly need some new drugs with better mode of action. Researchers are taking much interest in naturally occurring compounds which have always been helpful to mankind. Antimicrobial peptides are found in animal fluids, venom and hemolymph which can effectively kill microbes. Present work was designed to check out the antibacterial potential of scorpion (Hottetntota tamulus) venom and hemolymph, both in crude and diluted form (1/1 and 1/10). Their activity was evaluated against four strains of pathogenic bacteria i.e., Staphlococcus aureus (Gram positive), Escherichia coli (Gram negative), Pseudomonas aeruginosa (Gram negative) and Klebsiella pneumoniae (Gram negative).

Results of present research revealed that crude venom of Hottentota tamulus and its different dilutions didn’t stop the growth of Staphylococcus aureus. These results are in accordance to Ahmad et al. (2012). They reported that the growth of Staphylococcus aureus was not inhibited by the crude venom of Heterometrus xanthopus, but its crude venom was effective against Bacillus subtilis and Enterococcus faecalis. They also reported that different dilutions (1/1 and 1/10) of venom used couldn’t inhibit growth of all these strains.  The possible reason might be too much dilutions of venom (Benli & Yigit, 2000; Tatu, 1989). However, Benli & Yigit. (2008) and Haeberli et al. (2000) reported that Staphylococcus aureus growth was inhibited by spider venom. Salama & Geasa (2014) also studied the antibacterial potential of three Egyptian scorpions Leuirus quinquestriatus, Androctonus amoreuxi and Androctonus australis. They use two dilutions (20 and 10 mg/ml) and confirmed the antibacterial potential against Gram-positive bacteria (B. subtillis and B. cereus). B. subtillis showed excellent results with both concentrations of L. quinquestriatus venom. The venom of other two scorpions did not show any results against any strain.

Ctriporin is a new antimicrobial peptide extracted from Chaerilus tricostatus. It produce effective results against microbes. 5g/ml dose of ctriporin can totally stop the growth of Staphylococcus aureus as efficiently as 5g/ml dose of cefotaxime or 5g/ml dose of penicillin. While ampicillin can’t inhibit its growth at the same concentration even after 7 hours of test. But when the ctriporin concentration was decreased to 3.5g/ml, its growth was inhibited for the first 7 hours. After reducing ctriporin concentration to 2.5g/ml, its growth was stopped in the first 4 hours of treatment. Their results recommended that growth inhibitory effects of ctriporin on S. aureus were concentration dependent. They also used one more gram positive bacteria C. albicans which showed similar results to S. aureus (Fan et al, 2011).

In present findings the growth of all gram negative strains was not inhibited by the application of scorpion (Hottentota tamulus) venom. While Hernández-Aponte et al (2011) reported the antibacterial activity of a new peptide vejovine extracted from scorpion Vaejovis mexicanus. They checked its antibacterial activity against gram negative strains (Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterobacter cloacae, Escherichia coli, & Acinetobacter baumanii) and they all showed minimum inhibitory concentrations from a range of 4.4 μM up to 50 μM. I used crude venom and hemolymph which may contain many undesired substances and the activity of venom get reduced. Many researchers have worked on specific peptides and their application on microbes. Two gram negative strains (C. freundi and K. pneumoniae) was also studied by Salama & Geasa, 2014. They checked the antibacterial potential of three Egyptian scorpions Androctonus amoreuxi, Leuirus quinquestriatus, and Androctonus australis. Only venom of L. quinquestriatus showed zone of inhibition against C. freundi. The venom of Androctonus amoreuxi and Androctonus australis failed to produce results against any strain.

Ahamd et al. (2006) have reported the antibacterial activity of two scorpions against six microbial strains Enterobacter aerogenes, Proteus vulgaris, Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis and Staphylococcus aureus. One scorpion was Buthotus hottentota hottentota which showed antibacterial potential only against S. aureus. And the second was Chinese red scorpion Buthus martensi karsch showed antibacterial potential against Enterobacter aerogenes. Remaninig strains were not affected by venom of both scorpions.

 Conclusion and Recommendations:

It is concluded from present research that venom and hemolymph of Hottentota tamulus failed to inhibit the growth of studied pathogenic bacteria. However, scorpion venom is a mixture of many bioactive compounds. So, it is recommended that further researches should be conducted to characterize the components of scorpion venom and hemolymph. These components should also be evaluate to determine potent peptides.

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