SUMMARY
The aim of study to partially characterize, venom and hemolymph of spider Marpissa tigrina (Araneae; Salticidae). Hand picking method was used to collect live spiders from the different trees like Mangifera indica, Dalbergia sisso, Morus alba, Saccharium officinarum from 10 Chak NB Bhalwal, district Sargodha, Punjab, Pakistan. The collected specimens were shifted in plastic containers covered with mesh cloth for aeration. 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 were collected from the coxal region and then the venom glands and hemolymph were shifted into separate eppendrof containing 0.5 ml of 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. By comparing with standard protein markers the approximate molecular weight of protein fraction was determined. The comparative free ions concentration in digested sample of venom and hemolymph was determined by flame photometer. The concentration of potassium, calcium and sodium ions was determined against the concentration of potassium, calcium and sodium ion in standard. The emission intensity of free ions was in direct relation with concentration.
The results of current study indicated that the venom of selected spider species (M. tigrina) contained protein bands ranged from 35 to 125 KDa. Bands of 80 KDa was most dense and broad among all bands, while the protein bands of hemolymph ranged from 43 to 155 KDa and bands of 43 KDa was most dense among all other bands followed by 100 KDa, 70 KDa and 155 KDa. The bands of 140 KDa was less intense.
The one tailed unpaired t test was used to compare the concentration of three ions Na+, K+ and Ca+ ions in the venom and hemolymph collected for multiple samples. The comparative molecular characterization of these free ions of venom and hemolymph indicates that M. tigrina venom contains high concentration of Na+ ions (33.70 ppm) as compared to K+ ions (14.75 ppm) and Ca+ ions (23.57 ppm). The hemolymph of M. tigrina also shows same trend for Na+ ions (43.08 ppm) as compared to K+ ions (32.50 ppm) and Ca+ ions (22.33 ppm).
The results showed that there is significant difference between Na+ ions concentration in venom and hemolymph (P=0.0005). The K+ ions concentration in both fluids also showed significant difference (P=0.0007). The Ca+ ions concentration showed non-significant difference (P=0.1065). The comparison of these three ions in venom and hemolymph indicates that there is significant difference between these ions in both fluids.
INTRODUCTION
Spiders (Araneae: Arachnida) which produce venom and silk and these two important properties contribute the success and survival of spider’s diversity. They are present in all terrestrial habitats (Coddington & Levi, 1999; Wolf, 1990). Spider’s venom is a rich source of active biological compounds that have different chemical nature. All spiders produce venom except hackled orb weaver (King & Hardy, 2012). The total described species of spiders are 48, 321 (World spider catalog, 2020). The venom of spiders is classified into three categories from which lowest category contains polyamines having molecular weight (<1 kDa) middle category contains neurotoxins having huge disulphide bonds and linear cytolytic peptides ranging from (1–10 kDa) and higher category contains enzymes along with neurotoxins having molecular mass >30 kDa (Windley, 2012). In previous times Spiders activities causes many bad problems for humans like Brown reculse and Black widow spiders their venom even causes death of humans and hence these spiders got attention for study and for the estimation of different components which are present in their venom (Greenstone et al., 2002).
Araneae is the largest order of Arachnid and its ranks seventh among all other orders on the basis of species diversity. The venom of spiders is rich source of different kinds of peptides which are very effective for blocking ion channels of its prey (Turnbull, 1973). Major constituents of spider venom are proteins, polypeptide and polyamine, enzymes, neurotoxins, nucleic acids, monoamines and inorganic salts such as Mg++, Ca++, Na+, K+, Cl– and neurotransmitters that play central role in immobilizing prey (craig et al., 1999).
There are 10,000 species of arthropods throughout the world which acts as pest (Windley et al., 2012). Due to which 14% of world crops and stored food grains were destroyed, also many of them transmits diseases (Nauen., 2007). The venom of spider receiving attention as it comprises of potent neurotoxic peptides and stable mini proteins which having insecticidal effects and induces lethality in insects by blocking receptors, enzymes and ion channels (Herzig et al., 2012).
Venom of spiders has anti-cancerous potential. According to the world health organization, 14.1 million cases of cancer reported throughout the world in year 2012. There are different treatments available for cancer such as radiotherapy, chemotherapy, separation of tumors and therapy through hormones etc. But all of these treatments have limited cure because toxicity spread towards other tissues. Therefore, its substitutive medicines used for the treatment of cancer. The brown reculse spider’s venom consists of a poisonious, substance which named as phospholipase-D. it exhibits both hemolytic and anti-cancerous effect. In the venom of various spiders a compound hyaluronidase is present which enhance the permeability of tissue and hence any drug easily penetrated into that tissue. So it is used as anti-cancer agent (Silva., 2004). Venom of spiders also have antifungal potential, which imposes a great threat for the person suffering from cancer, organ transplantation or AIDS. So an alternative medication is required for the cure of such fungal infections. The venom of spiders contains these antifungal peptides. Due to this, the toxins of spiders used for the development of antifungal drugs (Venkatesan et al., 2005).
Hemolymph of spiders have direct contact with venom glands so the composition of hemolymph is very useful for investigation (Bednaski et al., 2015). Hemolymph has pharmaceutical potential as it contains peptides that inhibit the growth of bacteria and fungi. These antibacterial peptides will provide new insight 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 immense importance of spider venom and hemolymph in medical and agriculture, the venom of spider characterized very little. Out of 10 million spider venom peptides, only 0.01 percent has been explored yet (Klint, 2012). The hemolymph of arachnid has been studied even less than venom (Bednaski et al., 2015). However, advances in molecular biology techniques in few years led to exploration of venom and hemolymph peptide toxins, which create a new pathway to explore these existing integrative libraries (Escoubas & Bosman, 2007). In Pakistan, very little work has been done in this regard. The importance of this topic to partial characterize the venom of Marpissa tigrina and check the different components present in its venom by the help of SDS-PAGE. There is no work done on the venom of Marpissa tigrina in Pakistan. It is very crucial to characterize its venom and estimate different components in venom.
Objectives:
The present study was designed to investigate the following concern:
- To extract venom and hemolymph from a jumping spider: Marpissa tigrina.
- To partially characterize different proteins on the basis of molecular weight by SDS-PAGE.
- To estimate ions concentration in venom and hemolymph of spiders using flamephotometer.
REVIEW OF LITERATURE
Geren et al. (1973) extracted venom of fiddleback spider, Loxosceles reculsa and prepared a lethal abstract. They used column chromatography techniques for fractionation, extract into several components. Some of the isolated components produced wounds and causes death in tested animals. Budd et al. (1988) isolated toxins from the venom of specie Argiope trifasciata which is an orb-web spider. They purified the toxins after that characterized by using different techniques like spectroscopic, micro chemical analysis and mass spectrometric method. The result of experiments showed that toxins act as non-competitive inhibitors on receptors.
Kawai et al., (1991) studied on Nephila clavata and isolated neurotoxin JSTX from venom of spider, this JSTX blocked the potential of glutamate and postsynaptic receptors. Cunha et al. (2003) characterized proteins from venom of Loxosceles gaucho species. For the purpose of characterization they used electrospray mass spectrometry for the determination of isoforms of toxin and to develop internal amino acids sequence.
Silva et al. (2004) found venom of spiders having anti-cancerous potential. Venom of spiders has anti-cancerous potential. According to the world health organization, 14.1 million cases of cancer reported throughout the world in year 2012. There are different treatments available for cancer such as radiotherapy, chemotherapy, separation of tumors and therapy through hormones etc. But all of these treatments have limited cure because toxicity spread towards other tissues. Therefore, its substitutive medicines used for the treatment of cancer. The brown reculse spider’s venom consists of a poisonious, substance which named as phospholipase-D. it exhibits both hemolytic and anti-cancerous effect. In the venom of various spiders a compound hyaluronidase is present which enhance the permeability of tissue and hence any drug easily penetrated into that tissue effect. So it is used as anti-cancer agent.
Nentwig et al. (2004) studied on venom of Cupiennius salei and determined Ca++, Na+, K+ and Cl– ions concentration in its venom. The results of experiment revealed that Na+, Ca++ and Cl– ions concentration is lower in venom as compared to potassium ions. Duan et al. (2006) evaluated after collecting venom from Latrodactus tredecimguttatis by extraction of separated venom glands. To evaluate protein constituents they performed combinative proteomic strategies. Results revealed that venom contains acidic proteins of molecular mass greater than 15KDa in abundance while proteins and peptides with molecular masses below 15KDa are less.
Gao et al. (2005) found that spider venom full of neurotoxic peptides but it also consists of peptide toxins having anti-microbial, anti-malarial, anti-cancerous, hemolytic and enzyme inhibitory activity. Now-a-days spiders venom peptides are used for the development of new therapeutics agents against a number of diseases like chronic pain, bacterial and fungal infections, chronic neurological diseases and cardiovascular disorders. They also possess selective insecticidal potential.
Venkatesan et al. (2005) found that venom of spiders have antifungal potential, which imposes a great threat for the person suffering from cancer, organ transplantation or AIDS. So an alternative medication is required for the cure of such fungal infections. The venom of spiders contains these antifungal peptides. Due to this, the toxins of spiders used for the development of antifungal drugs. Due to attack of mycosis the percentage of mortality had been increased over the last 20 years. In past few years, azoles and Amphotericin B were used as a fungicidal pharmacological drugs but their often use develop resistance. So an alternative medication is required for the curing fungal infections.
Abdullah et al. (2007) 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. Latrodectus atrax and phoneutria are venomous species which are present in toilets, workshops, living rooms, huts and walls etc. spider venom is of two categories necrotoxic and neurotoxic. Neurotoxins venom attacks on nervous system and necrotoxic attacks on tissues of the body. After venom attack the symptoms are difficulty in breathing takes place and ultimately death occurs. Chemically venom is a heterogeneous compound which contains polypeptides, nucleic acids, amino acids, enzymes, inorganic ions among enzymes protease, hyaluronidase, isomerase form necrosis.
Vassilevski et al. (2009) have reported that there is no significant work have been done on the peptides which were separated from spider’s venom. Therefore, they noted less number of peptides in the spider venom. There is less study on its venom in spite of this, spiders venom consists of large number of applications in different fields.
Jalal et al. (2010) revealed that mineral composition in venom and hemolymph or body fluid of five common spider families. They identified and compared ions concentration in males and females spiders, in venom and other body fluid and concluded that potassium ions concentration is higher in venom than sodium, calcium and chloride. Sodium is most abundant cation and chloride is most abundant anion in spiders.
Jalal et al. (2010) reported that in the hemolymph of spiders a toxic protein is present which is erythrocyanin. Hemolymph consists of small proportion of toxins while it contains low and high molecular weight components in large amount. There are different spiders species which are involved in this study and their biochemical properties as well as free ions were studied. SDS-PAGE exposed that in all spider species 200KDa and 60KDa bands were present.
Monique et al. (2012) observed that venom of spiders is very important. Its play major role to control pests and hence avoids the crops from the attack of pests hence its act as potential source of bioinsecticides. Spider is consider as major successful predator of arthropods. Its venom consists of hyper stable insecticidal mini-proteins which is responsible for lethality or insect paralysis by modification of ion channels, receptors and enzymes. There are many toxins of spider venoms which are characterized newly which target on insects new sites and paralyze them.
Nicholson et al. (2012) reported that spiders are evolved around 300 million years ago from arachnid ancestors in carboniferous period that’s why they are ancient creatures. This express the long ancient time scale from which spiders evolved their complex venom. Spiders along with beetles consider as most successful terrestrial predator. One major reason reason which contributes the success of spiders is the production of toxic venom from their venomous glands, which act as a tool for capturing prey and avoid predators. Their venom contains enzymes and neurotoxins which paralyze the prey. Recent research shows that spider venom is very complex, than previously described studies. Their venom contains more than 1000 peptides in some species.
Mourao et al. (2013) little studies were imparted with Tarantula toxins, particularly on Brazillian species. AP1a is a first peptide toxin which is isolated from Acanthoscurria paulensis venom was characterized biologically as well as chemically. They find that it has molecular mass of 5457.79Da and It consists of 48 amino acids residues. Hu et al. (2014) characterized venom of S. jiafu by using RP-HPLC and MALDI-TOF-MS techniques. The molecular masses range from 1KDa to 10KDa of different venom peptides of this species, in which most peptides mass range of 3 to 4.5KDa.
Pamela et al. (2014) characterized the venom of Plectreurys tristis by using proteomic and transcriptomic methods. They concluded that new group of venom U1 and ɷ-PLTX, peptides and proteins are present in their venom.
Shi et al. (2014) studied on Grammostola rosea (Rose hair tarantula) venom and partially characterized its venom, different peptides are isolated from its venom and they also check their effect on membrane receptor system. Their results revealed changes in voltage activated K+, Na+ and Ca+ channels.
Garcia et al. (2015) noted that venom of three different therapsid spiders. They compared the pharmacological activity of venom. P. regalis and B. epicureanum venom caused deadly effect on cricket while C. darling shows less lethality. Sebastian et al. (2015) studied characterization of venom Phoneutria boliviensis (Aranae) done with colombian species its venom expressed physicochemical properties, liquid having 0.86 mg/ml density when electrostimulation extraction process was applied. This specie venom showed the hemolytic activity it hydrolyzed the synthetic substrate which indicates the presence of phospholipases A2 enzymes. The electrophoretic analysis of this protein content reveals that it has molecular masses below 14KDa and it also expose the difference of protein content between male and female. Mass analysis shows that presence of peptides in proteins ranging from 1047.71 to 3278.07 Da.
Tahir et al. (2016) studied that in the venom of spiders there are 10 million bioactive peptides, but from such a huge amount only 800 peptides from them are pharmacologically characterized. These peptides perform a great role in biological activities. The venom of spiders is powerfully block a variety of receptors, channels, enzymes other various target sites. Therefore, peptides which are isolated from venom of spiders posses significant ability for therapeutic applications.
Changxiao et al. (2016) noted that there are 40,000 species of spiders on this planet, they are ecologically very diverse most of them are harmless for humans. All of spiders kill their prey by their venom which is realized from venomous apparatus of spiders. Their venom have complex toxins consists of small peptides, amino acids, glucose, salts and ions, proteins and neurotransmitters. Recently, the peptide toxins which is present in the venom of spiders uses as a tool in pharmaceuticals industry. In most of spiders their peptide toxins consists of disulphide bonds which directly affects the nervous system. Some spiders toxins contains cysteine residues amino acids deficiency, it has great importance due to its deficiency the toxins positively charged and shows strong antimicrobial activity. The first reported peptides are Lycotoxin 1 amd Lycotoxin 2 which are separated from the venom of wolf spider both of these involved in prey capturing and protects them if they ingest infectious prey.
Yazho et al. (2016) observed that a large number of organisms produce venom naturally for capturing of prey or defense from their predators. Generally venom contains many bioactive chemical substances, such as proteins, peptides and enzymes. From all of molecules peptide toxins have particular importance. The toxins which is separated from the venom of arthropods/spiders include acetylcholine receptors, ion channels, plasma membrane and acetyl cholinesterase etc. generally, animals like snakes, spiders, ants, wasps and bees the toxins is synthesized in their venomous ducts. A small peptide toxins consists of only few amino acids possess great activity for treatment of various diseases like cardiovascular disorders, diabetes, multiple sclerosis and neurological disorders etc.
Kairong et al. (2016) reported that infectious diseases are caused by microbes but the antibiotics which are used against these microbes they quickly acquired resistance to the antibiotics. Therefore, new varities of antimicrobial agents are required to control the resistance issues. Many efforts have been done to discover new antimicrobial agents from natural resources. Peptide toxins which are present in the venom of spiders or other arthropods demonstrated great importance in this aspect. Peptide toxins performs a great antimicrobial activity because of its particular structures, and it has potential to create novel antimicrobial agents or used as a template for new drugs formation.
Huge number of toxins obtained from spider’s venom contains an unusual structural motif named inhibitory cysteine knot motif (ICK). The ICK is a three disulfide bridge containing structure surrounding by protein backbone and has high resistance to proteases, acidic pH, high temperature and organic solvents. The peptide toxin hexatoxin-Hv1a obtained from Australian funnels-web spider’s venom contains inhibitory cysteine knot motif structure. This ICK containing peptide enhances insecticidal ability of Australian funnel-web spider to attack prey that lives in a hot climate and also protect spider from proteinase K activity (King and Hardy, 2012). The CSTX-1 is a venom toxin which is present in Cuplennius salei. It contains an inhibitor cysteine knot motif that acts as ion channel blocker Nentwig et al. (2012)
MATERIALS AND METHOD
- Spider collection:
The jumping spiders (Marpissa tigrina) were collected from the leaves and trunks of different trees like Morus alba, Dalbergia sisoo, zea mays, Citrus limon, Ficus religiosa, Oryza sativa, Morus nigra, Citrus sinensis from different places of Sargodha, Bhalwal. A total of 200 spiders were collected by hand picking method. Spiders were placed in separate bottles to avoid cannibalism. The bottles covered with having pores to provide proper ventilation. Spiders were kept hungry for two days to ensure the presence of maximum venom.
- 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 (Labomed CSM 2). Their chelicerae and venom glands were separated with the help of forceps and needles. For protein analysis venom glands were immediately transferred in 1.5 ml of eppendorfs, containing 0.5 ml of tris-HCl buffer of pH 8.2 and 0.5 molarity (Guerrero et al., 2010). For ions estimation the venom glands were transferred into 1.5 ml eppendorfs containing 0.5 ml of deionized water. Each Eppendorff consists of 20 venom glands on average. The venom glands were then macerated manually by using small plastic rod untill they were homogenized. Eppendorfs were centrifuged by using refrigerated centrifuge at 15000 G for 20 minutes at 4 oC (MPW-352R). The supernatant which was obtained kept at -20 o C in ultra freezer until use (Rates et al., 2013).
- Hemolymph extraction:
Hemolymph was extracted after two days of spider collection. After immobilization of spiders, the legs of spiders were removed from the coxal region. The hemolymph was collected with the help of micropipette. For the analysis of protein, the hemolymph was shifted into 1.5 ml eppendorfs containing Tris HCl buffer (0.5 ml) of 8.2 pH. For ions estimation the hemolymph of spiders was shifted into 1.5 ml of eppendorfs containing 0.5 ml of deionize water. In one eppendorfs hemolymph of 20 spiders was collected. The homogenate was centrifuged in refrigerated centrifuge at 20, 000 G for 20 minutes at 4oC (MPW-352R). After centrifugation supernatant was kept at -20 oC 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:
For the analysis of various proteins fractions which were present in the venom and hemolymph of spiders SDS polyacrylamide gel electrophoresis was performed by following the protocol of Sambrook and Russle (2003) with slight adjustment. Venom and Hemolymph also contains peptides which were separated according to the molecular weight.
- 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 to 8.8 by adding few drops of 5N HCl into the solution. It can be stored at -20 oC 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 set to 6.8. it can be stored at -20 oC in Ultra freezer.
- 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 to 8.2 was adjusted by adding 1M NaOH solution. Distilled Water was added to make a final volume upto1 liter.
- Tris-HCl buffer (1M, 8.8 pH)
121.1 g of tris base was dissolved in 800ml of water and pH was adjusted to 8.8. Water was added to make a final volume upto 1000ml.
- 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 a final volume upto1000ml.
- 10% sodium dodecyl sulfate (SDS) solution:
To prepare 10 % (w/v) solution 10g of SDS was dissolved in 80ml of water and then water was added to make a final volume upto100ml.
- 10% Ammonium persulphate (APS) solution:
To prepare a 10% (w/v) solution 1 g ammonium per sulfate was dissolved in 8ml of water and then water was added to make a final volume upto 10ml.
- 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 oC in ultra freezer to use it again.
- 2X SDS gel loading buffer:
12.5ml of 1M Tris-cl buffer (pH 7), 15ml glycerol, 4g SDS, 4mg bromophenol blue and 5ml of β-mercaptoethanol were added to 17.5ml of water. Mixture was stirred at 4°C.
- Coomassie blue staining solution:
To prepare Coomassie blue staining solution, 0.25g Coomassie brilliant blue (G-250) was dissolved in 40ml methanol, then 10ml glacial acetic acid and water were added up to 100ml.
- Destaining solution:
To prepare destaining solution 40ml ethanol and 10ml Glacial acetic acid were mixed in 60ml of water.
- Gel preparation:
Glass plates were cleaned properly and then gather together in a sealed gel apparatus following the instructions of manufacturer. Gel plates were assembled properly by placing 1mm spacer. The gel of different percentages (5% stacking gel and 10% separating gel) prepared.
10% separating gel was prepared in 3.3 ml of solution A (30g acryl amide + 0.8g methylenbisacrylamide, water up to 100 ml) was added in 4.1 ml of 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, 40 μl 10 % APS.
This solution was poured in a space between two glass plates sparing the space for stacking gel 0.5 inch below the edges of comb and was allowed to polymerize for 30 minutes.
Stacking gel was prepared by 0.8 ml of solution A (30g acryl amide +0.8 g methylenbisacrylamide, water up to 100ml) 3.05 ml 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) 1.5 μl Temed, 25 μl APS.
Stacking gel was poured over separating gel and wells were made by inserting comb into the stacking gel, comb was removed after polymerization of gel.
- Sample preparation:
Eppendorff containing venom sample was thawed at room temperature of equal ratio 1:1 of supernatant 60 µl and 2×SDS loading buffer (100 mM tris-Cl pH 6.8) were mixed in an eppendorff and heated at 100 °C for 10 minutes in dry heating chamber to denature the proteins.
- Assembly of apparatus, sample loading and electrophoresis:
Gel apparatus was cleaned properly before use firstly, gel plates were placed vertically into the gel and running buffer was added in both upper and lower tanks of gel apparatus. A comb was placed into gel for making wells. Samples (17µl each) were loaded into wells by using micropipettes.
For standard reference one well was loaded with protein ladders of known molecular weight ranging 12 to 160 KDa. After loading of all samples, the lid was placed and units were attached to power supply. First 15 minutes gel was run at 80 volts after reaching resolving gel the voltage was increased to about 105 volts until the dye reached at the bottom of resolving gel. After 4 hours power supply was disconnected and gel was taken out of the plates.
- Gel staining and determination of molecular weight:
Plates were removed from gel apparatus and gel was obtained by placing apart both plates. Comassi brilliant blue staining of proteins was performed. Gel was kept in staining solution (1% Coomassi brilliant blue R 250, 10% ethanol, 40% glacial acetic acid) for 1 hour and then in destaining solution for 6-8 hours. Destaining solution was a mixture of 40% methanol and 30% acetic acid. Stained gels were photographed and approximate molecular weight of different fractions was determined by comparing with reference protein standards.
- Free ions determination:
The ions concentration was detected by using flame photometer (BWB-XP). The supernatant which contains venom sample was removed from freezer and thawed at room temperature.
- Quantitative procedure of sample digestion:
For sample digestion 0.4 ml of venom was taken and was mixed with 5 ml of conc. H2SO4 in a beaker and then the mixture was boiled for 5-10 minutes on Bunsen burner. After heating 2 ml of H2SO4 and 3 ml of HNO3 was added into the mixture and further heated for 20-25 minutes. when black fumes appeared 2-3 drops of water was added in it, after sometimes the mixture was cooled and it was filtered with Whatman filter paper (Sharma & Tyagi, 2013). Same procedure was applied for the digestion of hemolymph.
- Analysis of Venom & Hemolymph of Spider Marpissa tigrina
The analysis of the samples was performed on Flame Photometer by using standard samples. Calibration curve was drawn between concentration of standards and detector (flame photometer) response (absorbance). Standard samples of different concentration were prepared by using Stock’s solution (1000 pmm) with the help of dilution law (. The preparation of the solutions (Stock’s and Standard) is given the following section.
Preparation of the Solutions (Stock’s and Standard)
- Stock’s Solution
It is a solution of relatively high concentration (1000 ppm) used to prepare lower concentration solutions (Standard Solutions). These solutions are available 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 potassium Stock’s solution KCl, KI, KBr etc. can be used.
- Preparation of Stock’s and Standard Solutions of ‘Na+, K+, Ca+2’
- Stock’s Solution of Potassium “K”
Stock’s solution of potassium was prepared by using KCl salt. 1.91g KCl was dissolved in 1000 ml (1.0 litter) of distilled water in a volumetric flask of 1000 ml volume.
- Preparation of Standard Solutions of Potassium ‘K’
Standard solutions of K were prepared by diluting Stock’s solution (1000 ppm) of K with the help of dilution law (. 1.0, 2.0, 3.0, 4.0 ppm standard solution were prepared by using following calculations.
Dilution Law:
Where,
For 1.0 ppm standard solution,
100 of Stock’s solution was taken with the help of micropipette and diluted up to the mark with distilled water in 100 ml volumetric flask. Similarly for 2, 3 and 4ppm standard solution 200, 300 and 400 were taken and diluted up to mark in 100 ml volumetric flask respectively.
- Stock’s Solution of Calcium “Ca”
Stock’s solution of potassium was prepared by using CaCl2 salt. 2.769g CaCl2 was dissolved in 1000 ml (1.0 litter) of distilled water in a volumetric flask of 1000 ml volume.
- Preparation of Standard Solutions of Calcium ‘Ca’
These solutions (0.5, 1.0, 1.5, 2.0) were prepared in similar with that of potassium by using dilution law.
These calibration curves along with data used to draw them are shown below in graphs and tables respectively.
- 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 placed in sample cup. To clean 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 influenced by the intensity of the sample (Haid, 1946).
Table 1: The wavelength of light emitted by cations along with flame color
| Cation | Sodium | Potassium | Calcium |
| Wavelength | 589 | 766 | 622 |
| Color of flame | Yellow | Violet | Orange |
- Analysis of data:
For graph interpretation graph pad prism version seven was used. To compare ions concentration in venom and hemolymph one tailed unpaired t test was used.
RESULTS
- Characterization of venom and molecular weight determination of peptide fractions
The resolved venom protein bands were compared to protein bands of known molecular weight separated on the same gel. Electrophoretic analysis of venom extracted from Marpissa tigrina revealed presence of six different protein bands. The venom of this spider specie contained bands of 125kDa, 120kDa, 80kDa, 42kDa, 38kDa and 35kDa. Band of 80kDa was most dense and broad among all bands followed by 120kDa, 38kDa and 35kDa.The band of 125kDa was less dense.
Lane 1 was loaded with protein ladder. While Lane 2, 3, 4 & 5 contained venom of Marpissa tigrina.
- Partial characterization of Spider hemolymph electrophoretic pattern
During gel electrophoresis the resolved unknown protein bands were compared to protein bands of known molecular weight, alienated on the same gel. The results indicated that hemolymph of Marpissa tigrina comprised of five different protein bands viz, 155kDa, 140kDa, 100kDa, 65kDa and 43kDa. Band of 43KDa was the most intense and broad among all bands followed by 100kDa, 70kDa and 155KDa. Whereas band of 140kDa was less intense.
Lane 1 was loaded with protein ladder. While Lane 2, 3, and 4 contained Haemolymph of Marpissa tigrina.
- Estimation of free cations (Na+, K+ and Ca+) in venom and hemolymph of spider:
The concentration of Na+, K+ and Ca+ ions was determined by flame photometer (BWB-XP) against the concentration of Na+, K+ and Ca+ ions in standard. Emission concentration of free ions was in direct relation with concentration of standard.
The sodium, potassium and calcium ions concentration in venom and hemolymph was measured in multiple samples and results were expressed as mean±SE. The comparison of these free ions in venom and hemolymph indicates that Marpissa tigrina venom contains high concentration of Na+ ions (33.70 ppm) as compared to K+ (14.75 ppm) and Ca+ (23.57 ppm) ions. Whereas the hemolymph of Marpissa tigrina also shows same trend for Na+ ions (43.08 ppm) as compared to K+ (32.50 ppm) and Ca+ (22.33 ppm) ions.
Table 2: Concentration of different standard samples of “Na” and their absorbance is shown in the table
| NO. | Conc. (ppb) | Absorbance |
| 1 | 0 (Blank) | 0 |
| 2 | 100 | 0.144 |
| 3 | 200 | 0.232 |
| 4 | 500 | 0.543 |
| 5 | 1000 | 1.0 |
Table 3: Concentration of different standard samples 0f “K” and their absorbance is shown in the table
| NO. | Conc. (ppm) | Absorbance |
| 1 | 0 | 0 |
| 2 | 1.0 | 0.202 |
| 3 | 2.0 | 0.491 |
| 4 | 3.0 | 0.791 |
| 5 | 4.0 | 1.0 |
Table 4: Concentration of different standard samples of “Ca” and their absorbance is shown in the table
| No. | Conc. (ppm) | Abosrbance |
| 1 | 0 | 0 |
| 2 | 0.5 | 0.276 |
| 3 | 1.0 | 0.538 |
| 4 | 1.5 | 0.799 |
| 5 | 2.0 | 1.0 |
The results showed that there is significant difference between Na+ ions concentration in venom and hemolymph (P=0.0005). The K+ ions concentration in both fluids also showed significant difference (P=0.0007). The Ca+ ions concentration showed non-significant difference (P=0.1065 ). The comparison of these three ions in venom and hemolymph indicates that there is significant difference between these ions in both fluids.
Using data of table 01,02 & 03 calibration cures were drawn for “Na”, “K” & “Ca” respectively as shown in figure 01,02 & 03.
Figure 7 : Calibration curve for “Na”
Figure 8: Calibration Curve for Potassium “K”
Figure 9 : Calibration Curve for calcium “Ca”
Table 5: Mean concentration of Na+, K+ and Ca+ in venom and hemolymph of M. tigrina
| NO. | Na+ Content | K+ Content | Ca2+ Content | |||
| 1. | Hemolymph | Venom | Hemolymph | Venom | Hemolymph | Venom |
| 2. | 43.08±3.96 | 33.70±0.7041 | 32.50±7.10 | 14.75±2.38 | 22.33±0.08 | 23.57±3.040 |
DISCUSSION
In the present study partial characterization of venom and hemolymph of Marpissa tigrina spider was done. The venom and hemolymph of different spider species contained multiple components that have essential role in pharmacology and agriculture (Tahir et al., 2016). On the basis of molecular mass spider venom components are classified into three categories. The compounds having low molecular mass molecular weight (<1 kDa) are named as acyl polyamines. 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 most probably playing vital role as enzymes, toxins such as neuro and necrotoxin (Vassilevski et al., 2009).
The present study indicates that venom of selected spider species contained protein bands of molecular mass (>10 KDa). The venom of this spider species contained six different protein bands of approximately 125 KDa, 120 KDa, 80 KDa, 42 KDa, 38 KDa and 35 KDa. Among all the bands, bands of 80 KDa was most dense and broad followed by 120 KDa, 38 KDa and 35 KDa. The band of 125 KDa was less dense followed by band of 42 KDa. While the hemolymph contained bands of approximately 155 KDa, 140 KDa, 100 KDa, 65 KDa and 43 KDa. Among all bands, 43 KDa was densest followed by 100 KDa, 70 KDa and 155 KDa. The bands of 140 KDa was less intense. The less intensity of these bands is due to small concentration of proteins expressed in these bands.
The protein fraction of Marpissa tigrina showed similar trend with other species of spider such as the black widow spider (Latrodectus; Theridiidae) contains neurotoxin having molecular weight >125 kDa (Hayashi, 2013) the venom of M. tigrina also contain 125 KDa protein band. The Brown reculse spider belonging to Sicariidae family contain a wide range of peptides having molecular range 5-40 KDa (Chaim, 2011). Likewise, the venom of Loxosceles intermedia contains peptides having molecular weights 55 KDa, 42 KDa and 38 KDa respectively (de Castro, 2004). In the venom of Central Asian spiders (Pholcoides seclusa) a venomous proteins of molecular weight ranging from 40 to 80 KDa molecular weight were reported (Futrell, 1992). Australian funnel-web spiders Hadronyche versuta contain many hundreds of venom proteins with about 75% of the peptides ranging from 35 to 80KDa. Another Australian funnel-web spider Hadronyche infensa is also found to be rich in toxic peptides with masses ranged from approximately 42 to 100 KDa with 85% of the peptides in the 42 to 80 KDa range (Gimenez, 2002). Like other species of spider Marpissa tigrina also contains wide range of protein bands.
The venom of Lycosa terrestris was contained six different proteins bands of range 35-125 KDa Band of 80kDa was denser and broader among all bands indicating that higher concentration of this peptide is present in this spider venom, the venom peptides of more than 10 KDa molecular weight acts as toxin these species are neither cytolytic nor neurotoxic in action. The cytolytic peptides are typically rather small (∼30 kDa) and the size of spider neurotoxins ranges from 30–70 KDa (Nentwig, 2003). The venom of Hippasa partita consists of a low molecular weight peptide 35 KDa having non-enzymetic neurotoxin effect ( Nagarajju, 2013). The funnel web spider, Agelenopsis aperta venom having molecular masses 45, 48, 50, and 55 KDa these were paralytic agents and produced immediate but reversible paralysis in Manduca sexta, the funnel web spiders also contains peptides in range 43-50 KDa these peptides contributed beneficial role in pharmacology. Similarly, a range of polypeptides in Australian funnel web spiders 50 % in range of 38-52 KDa and almost 50 % in range of 38-45 KDa these peptides provides antimicrobial activity. The venom of Loxosceles contain enzymes of 85-95 KDa molecular weigh that digest serine amino acid (Veiga, 2000). These enzymes disturb normal physiological processes by altering body homeostasis. Similarly three new potential insecticidal toxins named LiTx1, LiTx2 and LiTx3 of approximately 60, 65 and 80 KDa has been reported from the venom of Loxosceles. A poison (latrotoxin) of >130 KDa molecular weight is present in black widow spiders venom (Latrodectus; Theridiidae) which disrupts nervous system. In accordance with this information, venom of Marpissa tigrina analyzed in this study, also shows prominent bands at the molecular weight region, so we can say that possibly M. tigrina have important role in pharmacology.
In the present study, the comparison of free ions indicates that venom samples of Marpissa tigrina contain higher concentration of Na+ ions (33.70 ppm) than Ca+ ion (23.57 ppm) and K+ ions (14.75 ppm). The hemolymph of Marpissa tigrina also has higher concentration of Na+ ions (43.08 ppm) as compared to K+ ions (32.50 ppm) and Ca+ ions (22.33 ppm). The characterization of free ions indicates that hemolymph has high concentration of two ions like Na+ and K+ as compared to venom of the spider. In different spider species the ion concentration in the body fluid varies depends upon sex, nutrition and habitat (Vassilervski et al, 2009). The Cupiensis salei contains high concentration of K+ ions in venom as compared to other cations. These results contradicted with our findings because Marpissa tigrina contains high concentration of Na+ ions. The high concentration of Na+ ions enhances the efficiency of venom and helps the spider to dominate. Like Marpissa tigrina the hemolymph of Cupiennius salei spiders have high concentration of Na+ as compared to other cations.
The results of our study showed similarity with the results of many other researchers. The hemolymph of orb web spiders contain 30 to 45 % of Na+ while the other cations such as K+, Ca+ and Mg+ were present in low concentration (Binford, 2001). Like M. tigrina the hemolymph of citrus spider species (Erovixia excelsa) contains lower concentration of potassium ions as compared to the venom (Jalal et al., 2010). Unlike, M. tigrina the tarantula spider’s venom contains high concentration of K+ as compared to other cations (Tahir et al., 2016). The high concentration of K+ might be able to induce paralysis in prey by depolarizing excitable cell membrane.
In different spider species, the free ions concentration of venom and hemolymph showed same trend like M. tigrina such as the giant crab spider 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). There are very few studies conducted on the mineral component of spider. This study indicated that spider minerals components are also of much importance and further study is required to explore the exact role of these inorganic ions to get more benefits in the field of health and medicine. From above discussion it is concluded that the venom of Marpissa tigrina contains relatively high molecular proteins ranging from 35 to 125 KDa and the hemolymph also contain high molecular mass proteins ranging from 43 to 155 KDa. These proteins have possibly enzymatic or neurotoxic role.
CONCLUSION
From above discussion it is concluded that the venom of Marpissa tigrina contains relatively high molecular weight proteins 35 to 125 KDa and the hemolymph contains high molecular mass proteins ranging from 43 to 155 KDa. These proteins have possibly enzymetic or neurotoxic role or could act as membrane disrupting agents. This spider species possibly does not contain any cytolytic activity.
The ion concentration in different spiders varies from species to species. There are various factors which act on the ion concentration of venom and hemolymph such as age, sex and nutrition. In present study the comparative molecular characterization of free ions indicates that venom samples of M. tigrina contains high concentration of Na+ ions (33.70 ppm) as compared to K+ ions (14.75 ppm) and Ca+ ions (23.57 ppm). The hemolymph of M. tigrina also shows same trend for Na+ ions (43.08 ppm) as compared to K+ ions (32.50 ppm) and Ca+ ions (22.33 ppm). The comparison between venom and hemolymph also indicates that hemolymph of M. tigrina contains high concentration of cations than other body fluids. The venom and hemolymph of M. tigrina might have essential role in pharmacology and agriculture but further studies are required to establish this fact.