SUMMARY
The aim of present study was to conduct partial characterization of venom and hemolymph of Cheiracanthium spp (Eutichuridae : Araneae). Hand picking method was used to collect live spiders from citrus plants of Khayaban e Naveed, Sargodha, Punjab, Pakistan and the collected specimens were shifted in plastic containers covered with mesh cloth for ventilation. After collection, spiders were kept without food for two days to obtain maximum amount of venom. Venom glands were removed from cephalothorax region of spider while hemolymph was collected from the coxal region. After that, the venom glands and hemolymph were shifted into separate eppendrof containing 0.5 ml tris HCl buffer of 8.2 pH.
The protein fraction of the venom and hemolymph was determined by using SDS-PAGE (Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis) following standard protocol. The fairly accurate molecular weight of protein fractions was determined by comparing them with the standard protein markers. The comparative free ions concentration in digested sample of venom and hemolymph was determined by flame photometer. The concentration of sodium and potassium ions was determined against the concentration of Na+ and K+ ions in standard. The emission intensity of free ions was in direct relation with concentration.
The results of current study indicated that the venom of the selected common spider Cheiracanthium spp (Eutichuridae : Araneae) contained protein bands ranged from 15 to160 kDa. Among all the bands, 70 kDa band was the densest, while the protein bands of hemolymph ranged from 27-150 kDa and the 70 kDa bands were the densest followed by 35 kDa and 27 kDa. The band of 150 kDa and 45 kDa were less intense followed by bands of 35 kDa and 90 kDa.
The one tailed unpaired t test was used to compare the concentration of Na+ and K+ ions in venom and hemolymph composed for multiple samples. The proportional molecular characterization of these free ions of venom and hemolymph indicates that venom of common spider Cheiracanthium spp contains high concentration of Na+ ions (4.98 ppm) as compared to K+ ions (0.96 ppm). The hemolymph of Cheiracanthium spp also shows same trend of Na+ ions (3.54 ppm) and K+ ions (0.65 ppm).
The comparison of Na+ and K+ and in venom, and comparison of both ions in hemolymph showed significant difference (P = 0.33, t = 2.51, df = 4) and (P = 0.155, t = 3.27, df = 4), respectively (Table.3). The results showed non-significant difference between Na+ ions concentration in venom and hemolymph (P = 0.33, t =2.51, df = 4) and non-significant difference in K+ ions concentration in both fluids (P = 0.155, t =3.27, df = 4).
INTRODUCTION
Spiders (Araneae: Arachnida) are the most miscellaneous and dominant group among all organisms which produce venom for their success and survival. Spider’s are abundant terrestrial cosmopolitan predators having broad range of distribution throughout globe (Windley, 2012) and have currently 48,643 described species (World spider catalog, 2020).
Spiders venom is a rich source of active biological compounds having different chemical nature and is a complex mixture of proteins (enzymatic and non-enzymatic) and peptide toxins as well as polyamine neurotoxins, monoamines, free amino acids, nucleic acids, and inorganic salts such as Mg++, Ca++, Na+, K+, Cl- (Baron et al., 2013; Gremski et al., 2015; Vassilevski et al., 2009).The important function of the venom is protection from predators and prey acquisition. All spiders paired venom glands except hackled orb weaver (King & Hardy, 2013).
The overall achievement of venom is its chemical composition (Pineda, 2014). On the basis of molecular mass, the venom components are classified into three categories. The lowest category having molecular weight (<1 kilo Dalton) contains acyl polyamines, organic acids, amines and nucleotides as well as non peptidic molecules and the middle category ranging from (1–10 kilo Dalton) contains neurotoxins having huge disulphide bonds and linear cytolytic peptides, and the highest categoryhaving molecular mass >30 kilo Dalton contains enzymes along with neurotoxins (Windley, 2012). The factors that influence venom composition are sex, natural habitat and nutrition and climate (Vassilevski, 2009).Venom is classified into two groups on the basis of mode of action i.e necrotic and neurotoxic. Necrotic venom leads to tissue damage such as lesions and blisters while neurotoxic venom has unpleasant effect on nervous system of insects.
Spider’s venom contains peptides with anti-parasitic, cytolytic, pain-relieving and enzymes inhibitory activities. Spider venom peptides can be used as healing agents like fungal and bacterial infections, cardiovascular disorders and chronic neurological disorders. Venom of a variety of spiders contains several groups of cytolytic and antimicrobial peptide that performed different functions. After prey ingestion, the risk of infection for spider enhances but these cytolytic and antimicrobial peptides protect spider from infection (Saez, 2010).
Spiders have highly precise substance present in venom that affect different receptors and ion channels and also treat diseases that affect the functioning of membrane transport system due to which they have definite task in modern neurobiology (Vassilevski, 2009). From the venom of Brown spiders, some toxins such as Phospholipase-D have been cut off and then purified can be used as an anti cancerous phenomenon. Some spider’s venom contains a toxin compound hyaluronidase, which increases the permeability of tissue and then any drug can easily enter into that tissue (Silva and Bulet, 2004).
Spider venom peptides also have potential to treat abnormal heart rhythm in humans and also have capability to kill or diminish the microorganism that affects the behavior of humans and their fitness. The venom of lynx spider (Oxyopes kitabensis) has peptides oxyopinins that act as important tools for investigating and discovering the operation of ion channels in the body (Heinen & Veiga, 2011). Spiders venom toxin can also be used as an insecticides because their natural targets are insects and are species specific, shows that their venom are highly toxic for insects and harmless for some members of other taxon.
The hemolymph and venom gland of spiders are in direct contact with each other, which shows that hemolymph composition is very precious and valuable for investigation. Like venom, hemolymph has vast pharmaceutical potential. They contain different peptides which reduce the growth of bacteria, yeast and fungi. These antibacterial peptides will provide new approach for the development of biologically active compounds, which may be used as a substitute to antibiotics. The hemolymph of spiders also contains toxins with anti-melanoma activity (Benli & Yigit, 2008).
Besides huge importance of spider venom and hemolymph in medical and agriculture, little work has been done on the characterization of venom of spiders. Out of 10 million venom peptides, only 0.01% have been explored yet (King, 2012). The hemolymph of arachnids has been studied even less than venom (Bednaski et al., 2015). However advances in molecular biology techniques in recent years has led to investigation of venom and hemolymph peptide toxins, which created new pathways to explore these accessible integrative libraries (Escoubas & Bosman, 2007).
In Pakistan, very little work has been done in this regard and the venom of genus Chieracanthium has not been explored yet. The aim of this study was partial characterization of venom and hemolymph extracted from a common spider of genus Chieracanthium and checks the different components present in its venom. Therefore, the objectives of the present study were as follows:
- Collection of a common spider of genus Chieracanthium for the extraction of venom and hemolymph.
- To partially characterized different protein fractions present in venom and hemolymph of spider by using SDS page.
- Determination of free ions concentration in the venom and hemolymph by using Flame photometer.
REVIEW OF LITERATURE
Akef (2018) stated that spider venom is multifaceted mixture of a variety of compounds that mainly includes salts, polypeptides, enzymes, small organic molecules and proteins. Most spiders’ venom contains diversity of compounds that target far ranging insect prey due to which they are polyphagous. The μ-Agatoxin-1 family toxins mainly effect on insects and paralyze them. These toxins are specific for certain orders of insects.
Adams (2004) stated that there are approximately 50,000 species of spiders present throughout the world but only 100 of them are venomous for human beings and other vertebrates. Jalal et al. (2010) reported spider venom as a most significant and unique group because of their necroticn neurotoxic and cytotoxic effects and having a lot of interest due to their potential applications in biological, pharmaceutical and toxicological aspects. Necrotoxic venom effects on tissues of the body and neurotoxins venom effects on nervous system. After venom attack, the symptoms are difficulty in breathing and eventually death occurs.
Jalal et al. (2010) estimated mineral composition in venom and hemolymph of five common spider families. They found major differences in ionic content of venom in different families and concluded that potassium ion concentration was higher in venom than calcium and sodium as compared to other body fluids. But in several cases, like in females of genus Myrmarachne had lower concentration of sodium and potassium in venom than hemolymph and chloride was higher in hemolymph than that in venoms while Sparassidae specie had higher concentration of K+ ions as compared to their males. These variations and differences were mainly species specific.
Kuhn-Nentwig et al. (2013) evaluated the venom of neotropical spider Cupiennius salei classified into three groups of molecules. The first group contains low molecular mass substances such as ions, polyamines and free amino acids. The second group contains peptides containing ICK motif, disulphide brigdes and peptide lacking cysteines. The third group includes proteins such as hyaluronidase which is highly active and accelerates the transfer of neurotoxins. Cupiennin 1 and cupiennin 2 belonging to these groups have low molecular mass mechanism in spider venom. These peptides showed great interest because of their antimicrobial activities and cytolytic variability as well as paralysis of prey.
Khan et al. (2006) determined insecticidal function of spider venom by the segregation of three polypeptide neurotoxin (curtatoxin) from the funnel web spider (Hololena curta) venom. The curtatoxin is rich in cysteine which consists of 36 to 38 amino acid residues. In terms of specificity of action, the folded structure of curtatoxin as compared to other cysteine rich venom proteins is more significant. The purified toxin induces a quick paralysis followed by unexpected death in the cricket ,Acheta domestica, within few hours. Gupta et al. (2016) found Omega-ACTH-HV toxin in the venom of Blue mountain funnel web spider (Hadrony cheversuta), that has insecticidal potential against insect pests of tobacco plants. This toxin is insect specific and provides no damage to humans.
Vassilevskiet al. (2013) reported that lynx spider (Oxyopes takobius) venom have two toxins named as spiderines OtTx 1a and 1b, 2a and 2b. These toxins illustrate both successful antimicrobial effects and insecticidal activity.
Tahir et al. (2018) studied the venom removal and characterization of two dominant wolf spiders, Pardosa birmanica, Simon and, Pardosa sumatrana, Thorell. Rhopalosiphum padi (Linnaeus) (Homoptera: Aphididae) used as a representation pest in laboratory for the assessment of insecticidal potential of basic venom and preferred peptide fractions i.e., 35-kDa fraction of both spiders were used. Results of the study showed that, both the protein fractions and crude venom causing considerably higher mortality rate in treated aphids as compared to the control. So, it is concluded that both the protein fractions and crude venom possess immense insecticidal potential.
Ferrer et al. (2013) recognized metalloproteases in the venom of (loxosceles intermedia). They found that Astacin like metalloproteases are organically energetic molecules that could be used as a tool for research protocol and have most important role in pharmaceutical studies. The Astacin act on different proteins and these molecules used in the study of extracellular matrix deprivation and protein degradation.
Saez et al. (2010) investigated that in the venom of the (Chevron tarantula), remote ICK peptide toxins U1-TRTX-Pc1a and U2-TRTX-Pc1a were used as an anti-malarial drug which are competent against the intra erythrocyte developmental stage of ,Plasmodium falciparum, (most dangerous cause of malaria).
Pringos et al. (2011) isolated a peptide neurotoxin SNX-482 from African tarantula (Hysterocrates gigas) venom. SNX-482 is 41 residues acidic peptide containing 3 disulfide bonds. This toxin stops premature labor pain during pregnancy and also acts as an inhibiting oxytocin release in vertebrates.
Varl et al. (2017) described that, (Cheiracanthium punctorium) bite comprise pain, burning, localized swelling, itching close to the bite area and erythema. The orderly symptoms which may be linked with yellow sac spider bites are shivers, nausea, sweat, vomiting, headache, tachycardia, abdominal cramps, mild to high fever, widespread itching, hypotension, neutrophilia, bilirubinemia, as well as respiratory complexity and even circulatory breakdown happen.
Gao et al. (2005) studied the effect of spider (Macrothele raven) venom on cell propagation & cytotoxicity in HeLa cells1. By using (AO/EB) staining, signs of apoptosis appeared biochemically and morphologically. After whole phenomenon of treatments, it concluded that the inhibition of HeLa cells in the spider (Macrothele raveni) venom was completed in 3 steps including induction of apoptosis, nonstop lysis and necrosis of toxicity injure which shows that spiders venom act as an anti-tumor material.
Chaves-Moreira et al. (2017) reported that the venom of Brown spider is a multifaceted combination of toxins containing low molecular mass proteins of 4–40 kDa. They originate that extremely expressed proteins comprise phospholipases D, insecticidal peptides (knottins) and metalloproteases (astacins). Whereas in Loxosceles venom, the toxins with low level of expressed toxins were protease inhibitors (serpins), allergen-like toxins, hyaluronidases, serine proteases, and histamine-releasing factors. Phospholipases D family of toxins individually can persuade dermonecrosis, inflammatory response, thrombocytopenia, hemolysis, and renal failure.
Windley et al. (2012) reported that, from the venom of the Western grass spider (Agelenopsis aperta) isolated μ-agatoxin-Aa1 toxins having 36–38 acid residue peptides. µ-Agatoxin-1 toxins are insects selective neurotoxins resulted in paralysis in insects. NaV channel inactivation also occurs in insect motor neurons due to µ-AGTX-1 toxins.
Sannaningaiah et al. (2014) determined the pharmacology of spiders venom containing a mixture of target specific enzymatic toxins (Sphingomyelinase, Phospholipase, Hyaluronidase, Collagenase, Protease, ATPases, Alkaline phosphatase, Phosphodiesterase, and Peptide isomerases) andnon-enzymatic toxins (serine protease inhibitors andtranslationally controlled tumor proteins). In addition, the venom also contains polyamine neurotoxins, AMP, ADP, ATP, c-aminobutyric acid, guanosine, glutamic acid,2,4,6-trihydroxy purine, aspartic acid, histamine, taurine, serotonin, octomine, nor-adrenaline, tyramine, and inorganic salts.
The extensively characterized enzymes from spider venoms are hyaluronidase, phospholipase D, protease, neurotoxic peptidesand sphingomyelinase. Spider bite is an unintentional event, can cause both systemic toxicity (neurotoxicity, cytotoxicity, myotoxicity, and hemostatic alterations) and local toxicity (hemorrhage, edema and myo/dermonecrosis). As an entire, spider venom components possess huge potential for therapeutic and biotechnological applications.
Sharma and Tyagi (2013) studied the partial characterization of ,Grammostola rosea,(Rose hair tarantula) venom. From characterization, different peptides are isolated from its venom and they also ensure their effect on membrane receptor system. Their results showed changes in voltage activated Na+, K+ and Ca+ channels. The Huwentoxin 1Vobtained from the venom of the spider (Heplo pelmaschmidti) is a pure blocker of Nav ion channel. Vassilevski et al. (2009) acknowledged that Atracotoxin obtained from ACTX-1 family are present in venom of the Sydney funnel-web spiders, (Altrax & Hadronyche) spider genera and selectively reduce insect central neuron Cav ion channel.
King et al. (2008) revealed the characterized proteins from venom of (Loxosceles gaucho) species. They used electrospray mass spectrometry, for the motive of characterization to determine the isoforms of toxin and to develop internal amino acid sequences. L. gaucho venom induces P-selectin, aggregation of platelets and role of platelets in the development of dermonecrosis. Kuhn-Nentwig et al. (2012) studied on (Nephila clavata) and isolated neurotoxin JSTX from venom of spider, this neurotoxin JSTX infertile the potential of glutamate and postsynaptic receptors.
The w-Agatoxin is the Cav ion channel inhibitor in the venom of (Agelenopsis aperta). The w-Agatoxin is a peptide toxin having four families which particularly acts on a variety of mammalian calcium channels. The first family w-Agatoxin IA and IB are peptides consisting two chains and they affect L type Ca channels. The second family w-Agatoxin IIA and IIB effects on the functioning of N type Ca channesl. The third family w-Agatoxin IIIA-IIID inhibits the working of all Ca channel except T type Ca channel. The fourth family of w-Agatoxin IVA and IVB specifically influence Cav 2.1 channels (Vassilevski et al., 2009).
Dutertre et al. (2010) reported that little studies were imparted with Tarantula toxins, particularly on Brazillian species. AP1a is a first peptide toxin which is isolated from (Acanthoscurri apaulensis) venom was characterized chemically as well as biologically. They found that it has molecular mass of 5457.79Da having 48 amino acids residues. Escoubas et al. (2006) characterized venom of (Selenocosmi ajiafu) by using RP-HPLC and MALDI-TOF-MS techniques. The molecular masses range from 1KDa to 10KDa of different venom peptides of this specie, in which most peptides mass range of 3 to 4.5KDa.
Ecoubas and Bosmans (2007) observed that acid sensing ion channels which are present in various neurons of PNS and CNS and are activated by changes in external pH. These acid sensing ion channels act as analgesics due to novel pain receptors. The venom of the tarantula spider contains Psalmotoxin1 (PcTx1). It acts as a gating modifier and selectively restrain acid sensing ion channel. This PcTx1 shifts the channel from resting to inactivated condition.
The American funnel web spider (Agelenospsis aperta) has insecticidal potential and it contains venom having rich source of toxins that act particularly on Na+ channel (Escoubas & Bosmans, 2007). The Japanese funnel web spider (Macrothele gigas) contains a toxin (µ-hexatoxin-Mg1a) in its venom that causes flaccid paralysis of insects larvae by blocking Nav channel (Windly et al., 2012).
MATERIALS AND METHOD
Collection of spiders
Spiders of Cheiracanthium spp (Family: Eutichuridae) were collected by jerking method and hand picking from citrus plants of Khayaban e Naveed, Sargodha, Punjab, Pakistan.The sampling was completed mainly from March through September 2019. Almost 200 spiders were collected that were shifted in separate plastic bottles to stay away from cannibalism. The bottles covered with mesh cloth to give proper ventilation. After collection, spiders were kept hungry for two days to get maximum amount of venom and hemolymph (Nagaraju et al., 2007).
Extraction of Venom
Spiders were immobilized by placing them in freezer at low temperature of 4 o C for 3-4 minutes. After that spiders were dissected under stereomicroscope and their chelicerae and venom glands were separated with the help of forceps and needles. For protein analysis, venom glands of spiders were instantly put into 1.5 ml of eppendorfs, containing 0.5 ml of tris-HCl buffer having pH of 8.2 (Guerrero et al., 2010).
For ions estimation, the venom glands were transferred into 1.5 ml of eppendorfs containing 0.5 ml of deionized water. In each eppendorff, approximately 20 venom glands were present. By using small plastic rods, the venom glands were macerated manually until they were homogenized. The homogenized mixture in eppendorfs was centrifuged by using refrigerated centrifuge at 20, 000 G for 20 minutes at 4 o C (MPW-352R). The supernatant which was obtained after centrifugation was separated from residue and was kept at -20 o C in ultra freezer until use (Rates et al., 2013).
Extraction of Hemolymph
Hemolymph was extracted after two days of spider collection. After Immobilization, the legs of spiders were removed from the coxal region of spiders. The hemolymph was collected with the help of micropipette. For protein analysis, the hemolymph was shifted into 1.5 ml eppendorfs containing Tris HCl buffer of 0.5 ml having pH 8.2. For ions estimation, the hemolymph of spiders was shifted into 1.5 ml of eppendorfs containing 0.5 ml of deionize water. In one eppendorf, hemolymph of 20 spiders was collected. Venom glands were homogenized by using small plastic rods. The homogenate was centrifuged in refrigerated centrifuge at 20, 000 G for 20 minutes at 4 o C (MPW-352R). After centrifugation, supernatant was kept at -20 o C in Ultra freezer until use (Moreira et al., 2014).
Characterization of Spider Venom and Hemolymph
Characterization of Venom and Hemolymph by Sodium Dodecyl Sulphate Gel Electrophoresis
Preparation of solutions Used in Gel preparation
- Solution B
Solution B was prepared by mixing 18.2 g of Tris base, 0.4 g SDS (Sodium Dodecyl sulphate) in 100 ml of distilled water. The pH was adjusted upto 8.8 by adding few drops of 5N HCl into the solution. It can be stored at -20 o C in Ultra freezer.
- Solution C
Solution C was used in stacking gel and was prepared by mixing 6.06 g of Tris base, 0.4 g of SDS in 100 ml of distilled water. The pH was adjusted upto 6.8. It can be stored at -20 o C in Ultra freezer.
- Solution A (30% acrylamide-bisacrylamide solution)
100ml of solution was prepared by dissolving 9g acrylamide and 1g bisacrylamide in deionized water.
- Tris-HCl buffer (0.05M, 8.2 pH)
6.05 g of Tris base was dissolved in 800ml of water and pH of 8.2 was adjusted by adding 1MNaOH solution. Distilled Water was added to make a concluding volume upto1 liter.
- Tris-HCl buffer (1M, 8.8 pH)
In 800ml of water, Tris base of 121.1 g was dissolved and pH was adjusted to 8.8. Water was added to make a finishing volume upto1000ml.
- Tris-HCl buffer (1M, 6.8 pH)
121.1 g of Tris base was dissolved in 800ml of water and pH was adjusted to 6.8 by adding 1M HCL solution. Water was added to make an ultimate volume upto1000ml.
- 10% Ammonium persulphate (APS) solution
10% Ammonium persulphate solution was prepared by mixing 10 g ammonium persulphate in 10ml of water. It can be stored at -20 o C in Ultra freezer.
- 4X Tris-glycine running buffer
Running buffer was prepared by mixing 12 g Tris base, 57.6 g Glycine and 4 g SDS in 1.5 liter of distilled water. The running buffer can be stored in -20 o C in Ultra freezer to use it again.
- 2X SDS gel Sample buffer
2X SDS Loading buffer was prepared by mixing 12.5ml of Solution C, 11.5ml glycerol, 2g SDS, 5mg bromophenol blue and 5ml of β-mercaptoethanol were added to 50ml of water. Mixture was stirred at 4°C.
- Coomassie blue staining solution
For the preparation of Coomassie blue staining solution, 0.25g Coomassie brilliant blue was dissolved in 40ml methanol, then 10ml glacial acetic acid and water were added up to 100ml. It can was stored at -20 o C in ultra freezer to use again.
- Destaining solution
To prepare destaining solution 40ml of ethanol and 10ml of Glacial acetic acid were mixed in 60ml of water.
Gel preparation
Glass plates were cleaned carefully and then assembled in a sealed gel apparatus by following the commands of manufacturer. Gel plates were assembled appropriately by placing 1mm spacer. The two gels of different percentages (5% stacking gel and 10% separating gel) were prepared.
10% separating gel (10ml) was prepared in 50ml Falcon Tube by mixing 3.3 ml of solution A (30g acryl amide + 0.8g methylen bisacrylamide, water up to 100 ml), 4.1 ml of distilled water, 2.5 ml of solution B (18.2 g tris base + 0.4g SDS, adjust Ph to 8.8 with 5N HCL, water up to 100ml), 20 μl TEMED, 40μl 10 % APS.The solution was shifted between two glass plates with the help of micropipette beneath the edges of comb and then polymerizes it for 30 minutes and poured below the edges of comb sparing space for stacking gel.
Stacking gel (3 ml, 5%) was prepared by adding 0.7ml of solution A (30g acryl amide +0.8 g methylen bisacrylamide solution, water up to 100ml) 3.05 ml distilled water, 1.25 ml of solution C (6.06 g tris base + 0.4 g SDS; and adjust Ph to 6.8; water up to 100 ml: add 100 μl Phenol Red concentrate) 15 μl TEMED, 25 μl APS in separate Falcon tube. After suitable mixing, the staking gel was poured over separating gel and comb was inserted into stacking gel to make the wells. Comb was removed after 30 minutes until the gel polymerize properly.
Sample preparation
Supernatant which contains venom sample was removed from freezer and thawed at
room temperature. Supernatant 60 µl was mixed with 2X SDS Sample buffer (100 mMtrisCl pH 6.8) in an eppendorfs in equal ratio 1: 1 and heated in dry heating chamber at 100°C for 30 minutes to denature the proteins. Same protocol was applied for the sample preparation of hemolymph.
Assembly of apparatus, sample loading and electrophoresis
Gel apparatus before its use was cleaned accurately and gel plates were adjusted vertically into the gel tank, after that running buffer was added in both upper and lower tanks of gel apparatus and fill tank upto the mark. A comb was inserted into gel for making wells. The samples (17µl each) were loaded into wells by using micropipette.The protein marker was of known molecular weight used as standard reference having range of 12 to 160 kDa (EZTM-Prestained protein Ladder Marker).
After loading of all samples in wells, the tank lid was positioned on tank and after proper arrangement, the gel apparatus was connected to power supply. First 30 minutes, the gel was run up to voltage of 80 after reaching resolving gel the voltage was increased to about 105 volts until the dye reached at the bottom of resolving gel. After 3-4 hours power supply was detached until the samples reached at the bottom of gel and gel was taken out of the plates.
Gel staining and De staining
The polymerized gel was separated cautiously from both gel plates with the help of forceps. Proteins were stained with Coomassie brillient blue stain (Wray et al., 1981). Gel was kept in staining solution (1% Coomassi brilliant blue R 250, 10% ethanol, 40% glacial acetic acid) for about 1 hour and then in destaining solution (40% methanol and 30% acetic acid) for 6-8 hours and photographed.The bands on gel were clearly seen and molecular weight of different fractions was determined by comparing it with reference protein standards.
Free ions determination
The ions concentration was detected by using flame photometer (JENWAY, PFP 7). The supernatant which contains venom sample was removed from freezer and thawed at room temperature.
Digestion of samples
For digestionof samples, 0.4 ml of venom was taken and mixed with 5 ml of concentrated H2SO4 in a beaker and then the mixture was boiled for 5-10 minutes on Bunsen burner. After heating of 5-10 minutes, 2 ml of H2SO4 and 3 ml of HNO3 was added into the mixture and further heated it for 20-25 minutes. When black fumes came out from mixture, 2-3 drops of water was added in it. After 5 minutes, the mixture was chilled when it removed from burner and filtered with Whatman filter paper (Sharma & Tyagi, 2013). For digestion of hemolymph, same procedure was repeated.
Solutions preparation (Stock’s and Standard)
The preparation of the solutions (Stock’s and Standard) is given the following section.
Stock’s Solution
It is a solution of relatively high concentration of 1000 ppm used to prepare lower concentration solutions (Standard Solutions). These solutions are accessible in the lab with instrument (Flame Photometer) by the company i.e., Merk etc. However, these can be prepared by using some salt of the metal like for preparation of sodium Stock’s solution NaCl, NaI, NaBr etc. can be used.
Preparation of Stock’s and Standard Solutions of ‘Na+ and K+’
Stock NaCl solution (1000 ppm)
1000 ppm stock solution of Na+ was prepared by mixing1.27 g of NaCl in 500 ml of water in a volumetric flask.
Stock NaCl solution (100ppm)
This stock solution was prepared by mixing 10 ml of stock solution of Na+ (1000 ppm) in 90 ml of distilled water in volumetric flask.
Standard Solution Preparation
The standard solutions of Na+ionwere prepared by volumetric dilution of the stock solution. For determination of Na+ ion concentration 10 ppm, 20, 30, 40, 50, 60, 70, 80 ppm was taken as standard solutions.
The standard solutions were prepared by serial dilution of stock solution. The 100 ppm stock solution was more diluted to form these eight standard solutions. Eight test tubes were used for making standard solution. Each tube was labeled in series. In first tube, 1ml of 100 ppm stock solution was added in 9 ml of distilled water to form 10 ppm standard solution. In the same way different concentration of 20 ppm, 30, 40, 50, 60, 70 and 80 ppm was prepared for analysis.
Stock KCl solution (1000ppm):
The 1000 ppm stock solution of K+ was prepared by mixing 0.955 g of KCl in 500 ml of water in a volumetric flask.
Stock KCl solution (100ppm):
This stock solution was prepared by mixing 10 ml of stock solution of K+ (1000 ppm) in 90 ml of distilled water in volumetric flask.
Standard solution Preparation:
The standard solution of K+ion was prepared by volumetric dilution of the stock solution. For determination of K+ion concentration 10 ppm, 20, 30, 40, 50, 60, 70, 80 ppm was taken as standard solutions. The procedure for manufacturing standard solution for K+ was similar to Na+.
Detection of ions concentration by flame photometer
Instrument was warmed up for 5-10 minutes before use. After warming, the instrument was fed with the diluents. The instrument contains tubing which was of small diameter attached to aspiration needle on one end and the other end is positioned in sample cup. To fresh the system, distilled water was used as aspirate.
The aspiration was take place for at least 5 minutes, and then the prepared standards solution was aspirated orderly and their stable display readings were recorded. After recording the readings of standard the digested sample were run through the flame photometer and their stable display reading was recorded. Light emitted from the flame was measured on the instrument transmission scale. Ions are produced by a flame that absorb light of a specific wavelength and then emit light of the same wavelength. The emission spectra of sample became visible. The flame color is prejudiced by the intensity of the sample (Hald, 1947).
Table 1: The wavelength of light emitted by cations (K+and Na+ ) along with flame color
| Cations | Sodium | Potassium |
| Wavelength | 589 | 766 |
| Color of flame | Yellow | Violet |
Analysis of data:
For data explanation graph pad prism version eight was used. To compare ions concentration in venom and hemolymph one tailed unpaired t test was used.
RESULTS
4.1. Description of spider venom electrophoretic pattern:
The electrophoretic analysis revealed that venom extracted from Cheiracanthium spp contains eight different proteins bands. The venom of this spider species contained bands of 15 kDa, 25 kDa, 35 kDa, 50 kDa, 70 kDa, 100 kDa, 110 kDa and 160 kDa. Among all the bands, 70 KDa band was the densest followed by bands of 35 kDa and 25 kDa. The band of 50 kDa was less intense followed by band of 35 kDa
4.2. Description of spider hemolymph electrophoretic pattern:
During gel electrophoresis, the bands of unknown resolved protein were compared to the protein bands of known molecular weight and aligned on the same gel. The electrophoretic analysis revealed that hemolymph extracted from Cheiracanthium spp contains eight different proteins bands. The hemolymph of this spider species contained bands of 27 kDa, 35 kDa, 45 kDa, 55 kDa, 70 kDa, 90 kDa, 100 kDa and 150 kDa. Among all the bands, 70 KDa band was the densest followed by bands of 35 kDa and 27 kDa. The band of 150 kDa and 45 kDa were less intense followed by bands of 35 kDa and 90 kDa
4.3. Estimation of free ions (Na+ and K+) in venom and hemolymph of spider:
The absorption of sodium and potassium ions was determined by flame photometer (JENWAY, PFP7) adjacent to the concentration of sodium and potassium ion in standard. The emission intensity of free ions was in undeviating relation with concentration shown in table 2.
The Na+ and K+ ions concentration in venom and hemolymph was calculated with their mean and standard error. The relative molecular characterization of these free ions of venom and hemolymph indicates that Cheiracanthium spp venom contains high concentration of Na+ ions (4.98 ppm) as compared to K+ ions (0.96 ppm). The hemolymph of Cheiracanthium spp also shows same trend of Na+ ions (3.54 ppm) and K+ ions (0.65 ppm). (Table.3)
Table 2: Concentration of different standard samples of “Na” and “K+” and their absorbance is shown in the table
|
No |
Standard conc for Na+ and K+ (ppm) | Absorbance of
Na+ |
Absorbance of
K+ |
| 1 | 10 | 11.1 | 14.1 |
| 2 | 20 | 22.4 | 24.4 |
| 3 | 30 | 31.3 | 37.3 |
| 4 | 40 | 43.6 | 48.6 |
| 5 | 50 | 53.2 | 52.3 |
| 6 | 60 | 61.4 | 63.4 |
| 7 | 70 | 72.7 | 74.1 |
| 8 | 80 | 83.5 | 81.2 |
The one tailed unpaired t test was used to compare the concentration of Na+ and K+ ions in venom and hemolymph composed for multiple samples. The proportional molecular characterization of these free ions of venom and hemolymph indicates that venom of Cheiracanthium spp contains high concentration of Na+ ions (4.98 ppm) as compared to K+ ions (0.96 ppm). The hemolymph of Cheiracanthium spp also shows same trend of Na+ ions (3.54 ppm) and K+ ions (0.65 ppm).
The comparison of Na+ and K+ in venom, and comparison of both ions in hemolymph showed significant difference (P = 0.33, t = 2.51, df = 4) and (P = 0.155, t = 3.27, df = 4), respectively (Table.3). The results showed non-significant difference between Na+ ions concentration in venom and hemolymph (P = 0.33, t =2.51, df = 4) and non-significant difference in K+ ions concentration in both fluids (P = 0.155, t =3.27, df = 4)
Using data of above table, calibration cures were drawn for “Na+” and “K+” respectively as shown in figure 01 and 02.
Table.3. Mean concentration of Na+ and K+ content in venom and hemolymph of Cheiracanthium spp.
| Na+ content | K+ content | ||
| Venom | Hemolymph | Venom | Hemolymph |
| 4.98±0.422 | 3.54±0.389 | 0.96±0.78 | 0.65±0.52 |
DISCUSSION
In the present study, partial characterization of venom and hemolymph of Cheiracanthium spp was done. The venom of the selected spider species contained protein bands of molecular mass (>10 kDa). The venom and hemolymph of different spider species contained several components that have important role in pharmacology and agriculture (Tahir et al., 2016). On the basis of the molecular mass, spider venom components are classified into three categories. The compounds having molecular mass less than 1 kDa are named as acylpolyamines.
The compounds having molecular mass ranged from 1-10 kDa are named as peptides and the compounds having molecular mass greater than 10 kDa are named as structural and functional proteins, playing vital role as enzymes and toxins ,neuro- and necrotoxin,. (Vassilevski et al., 2009). The venom of this spider species contained bands of 15 kDa, 25 kDa, 35 kDa, 50 kDa, 70 kDa, 100 kDa, 110 kDa and 160 kDa. Among all the bands, 70 kDa band was the densest followed by bands of 35 kDa and 25 kDa. The band of 50 kDa was less intense followed by band of 35 kDa. The hemolymph contained bands of 27 kDa, 35 kDa, 45 kDa, 55 kDa, 70 kDa, 90 kDa, 100 kDa and 150 kDa. Among all the bands, 70 kDa band was the densest followed by bands of 35 kDa and 27 kDa. The band of 150 kDa and 45 kDa were less intense followed by bands of 35 kDa and 90 kDa. The less intensity of these bands is due to small concentration of proteins expressed in these bands.
The protein fraction of Cheiracanthium spp showed comparable trend with other spider species such as the black widow spider of theridiidae family that contains a neurotoxin named as latrotoxin having molecular weight of about 150 kDa (Grishin, 1999). The Cheiracanthium spp hemolymph also contains 150 kDa protein bands.
The genus Loxosceles of brown spiders venom contains huge number of low molecular mass proteins ranging from 5–40 kDa. The serine proteases of brown spider having elevated molecular mass ranging from 85-95 kDa. This enzyme has a massive consequence on hemostatic system. The lycosids spiders (Hippasa agelenoides) include two serine proteases having range of 16.3- 28.7 kDa that hinder with hemostasis.
The Loxosceles spiders also surround metalloproteases in their venom. These metalloproteases have broad range of proteolytic activity (Chaim et al., 2011). The genus Loxosceles have two different type of metalloproteases, one is Loxolisin A having molecular mass ranging between 20–28 kDa, and other one is Loxolisin B having molecular mass ranging between 32–35 kDa (Feitosa et al., 2007). The venom of Cheiracanthium spp also shows bands of 25 kDa, 27 kDa and 35 kDa. Cheiracanthium spp might have important role in pharmacology.
The loxosceles spiders also contain hyaluronidase enzymes in their venom. The hyaluronidase activity in the venom was first described by Wray et al. in 1981 in Loxosceles genus. The venom of Loxosceles intermedia contains hyaluronidase enzymes of molecular weight of about 41–43 kDa. Hyaluronidases are referred as spreading factor due to their enzymatic activity upon extracelluar components (Chaim et al., 2011). In according with this information, venom of Cheiracanthium spp analyzed in this study by SDS-PAGE shows prominent bands at the molecular weight region corresponds to hyaluronidase.
In the current study, the comparison of free ions indicates that venom of Cheiracanthium spp has higher concentration of Na+ ions (4.98 ppm) than K+ ion (0.96 ppm). The hemolymph of Cheiracanthium spp also has higher concentration of Na+ ions (3.54 ppm) as compared to K+ ions (0.65 ppm). The results indicate that venom has high concentration of both ions as compared to hemolymph. In different spider species, the ion concentration in the body fluid varies depends upon sex, habitat and nutition (Vassilervski et al, 2009).
The Cupiennius salei contains high concentration of K+ ion in venom as compared to other cations. These results contradicted with our findings because Cheiracanthium spp contains high concentration of Na+ ions. The high concentration of Na+ ions increases the competence and effectiveness of venom and helps the spider to dominate. Like Cheiracanthium spp the hemolymph of Cupiennius salei spiders have high concentration of Na+ as compared to other cations (Wullschleger et al., 2005).
The outcomes of our study showed similarity with the results of many other researchers. The hemolymph of lycosid spiders contain 35 to 45% of Na+ while the other cations such as K+ , Ca2+ , Mg2+ were present in little concentration (Punzo, 1989). Similar to Cheiracanthium spp, the hemolymph of citrus spider species (Picnus versicunda) contain higher concentration of Na+ as compared to K+ ions in venom (Jalal et al., 2010). Unlike Cheiracanthium spp, the tatantula spider’s venom contains elevated concentration of K+ as compared to other cations (Tahir et al., 2016). The high concentration K+ might be capable to bring paralysis in prey by depolarizing highly strung cell membrane. In Cupiennius salei K+ ion works synergistically with other neurotoxin and enhances their efficiency and effectiveness (Wullschleger et al., 2005).
In different spider species, the free ions concentration of venom and hemolymph showed same trend like Cheiracanthium spp such as the gaint crab spider belonging to family Sparassidae and the jumping spider species (Thyene imperialis) in the genus Thyene belonging to salticidae family contains high concentration of Na+ in the venom and hemolymph (Jalal et al., 2010). From above discussion, it is concluded that the venom of Cheiracanthium spp contains comparatively high molecular weight proteins ranging from 15 to 160 kDa and the hemolymph also contain high molecular mass proteins ranging from 27 to 150 kDa. These proteins probably have enzymatic or neurotoxic role or might work as membrane disrupting agents.
CONCLUSION
From above discussion, it is concluded that the venom of Cheiracanthium spp contains relatively high molecular weight proteins ranging from 15 to 160 kDa and the hemolymph contains high molecular mass proteins ranging from 27 to 150 kDa. These proteins have probably enzymatic or neurotoxic role or could act as membrane disrupting agents. This spider species perhaps does not contain any cytolytic property.
In current study the relative molecular characterization of free ions indicates that venom samples of Cheiracanthium spp contains high concentration of Na+ ions (4.98 ppm ) than K+ ion (0.96 ppm). The hemolymph of Cheiracanthium spp also has high concentration of Na+ ions (3.54 ppm) as compared to K+ ions (0.65 ppm).The comparison between venom and hemolymph also indicates that venom of Cheiracanthium spp contains high concentration of cations than other body fluids. The venom and hemolymph of Cheiracanthium spp might have necessary function in pharmacology and agriculture but further studies are required to establish this fact. But such kinds of studies are more valuable for extraction of venom from different species of spiders and use them for enhancing anti-venomus drugs and for therapeutics against the injurious and damaging effects of venom.