Summary
The present study was carried out to partially characterize the venom and hemolymph of spider Tetragnatha javana (Araneae: Tetragnathidae). Live spiders were collected by using hand picking method from the rice field of Kolowal Nangiana, (32.13° N, 72.45° E), Sargodha, Punjab, Pakistan. Spiders were kept hungry for two days to extract maximum amount of venom. After extraction, venom and hemolymph were transferred into separate eppendrofs containing 0.5ml tris HCl buffer.
By using SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis), protein fraction of venom and hemolymph of T. javana was determined. The approximate molecular weight of proteins was determined by comparing them with protein marker of known values. Concentration of free ions (Na+ and K+) was determined by using flame photometer, against the concentration of Na+ and K+ ions in the standard solutions.
The result of present work indicated that the venom of T. javana contained protein bands from 15kDa to 70kDa, while the hemolymph contained protein bands from 10kDa to 68kDa. Band of 70kDa was most intense in venom and band of 15kDa was least intense.In case of hemolymph band of 68kDa was most intense and band of 10 kDa was least intense.
The analysis of free ions concentration showed that venom of T. javana contained higher concentration of Na+ (4.40ppm) as compared to K+ ( 1.54ppm). Hemolymph of T. javana also contained more concentration of Na+ (3.16) than K+ (0.88ppm).
One tailed unpaired t test was used for comparative analysis of concentration of free ions and it indicated that there is non-significant difference between Na+ and K+ ions concentrations in venom (P=0.0015, t=6.50, df=4),while there is significant difference in case of hemolymph ( P>0.001, t=18.36, df=4). There was non-significant difference in Na+ (P=0.24, t=2.81, df=4) and K+ (P=0.003, t=5.25, df=4) ions concentrations between venom and hemolymph.
- javana contains peptides that might play role in manufacturing of novel selective insecticides. The venom and hemolymph components might be of help in future in pharmacological industry, but further work is required to establish this.
INTRODUCTION
Spiders are considered as one of the most successful arthropods due to their ability to build web and evolution of complex pharmacological venom that helps them in successful predation (King & Hardy, 2013). Spiders are a diverse group of arachnids having 48,688 described species (World Spider Catalogue, 2020) but only 1,576 toxins have been characterized and registered from 100 spider species (Arachno Server, 2020).
Conventional insecticides used to control insect pests have problems like rapid resistance development, hazardous to non-target species, inclining pollinators and natural enemies of insect pests and unpleasant side effects on environment. Recently, the attention towards insecticidal peptides obtained from venom of spiders is increasing. The major advantage of spider venom derived insecticides over other chemical insecticides is that chance of resistance development is less. They are selective and safe for non-target species. Furthermore, the bioinsecticides containing spider venom’s disulfide rich peptides have fast neurotoxic activity as compared with other insecticides and they can act on wide series of ion channels and receptors in their prey’s nervous system. The ions present in venom of spiders also participate in immobilizing their prey (Bloom, 2011; Carneiro et al., 2003; Hardy, 2014; King & Hardy, 2013; King, 2019; Nakasu et al., 2014; Oerke, 2006; Saez & Herzig, 2019; Tedford et al., 2004; Windley et al., 2012).
Spider venom peptides have potential for future use to treat various health issues related to heart arhythmicity and dystrophy of muscles etc. Recent researches have confirmed that spider venom peptides have the characteristics to act as effective antinociceptive drugs as they can act on a broad range of ion channels. Some peptides from spider venom have been proved to have anti-tumor activities, by inducing apoptosis of cancerous cells. Some spiders have toxins which can be useful for future development of novel drugs for treating mental disorders. Antimicrobial peptides present in spiders have the ability to stop bacterial growth. Some spider species have been confirmed to be effective against malaria (Benli & Yigit, 2008; Estrada et al., 2007; Saez et al., 2010; Santos et al., 2019; Saez & Herzig, 2019; Wu et al., 2019).
Venom of spiders is constituted by amino acids, neurotoxins, enzymes, proteins, nucleic acids, ion channel toxins and polypeptides. The hemolymph of spiders consists of amino acids, sodium and chloride ions, inorganic phosphates, lipids, fatty acids, carbohydrates, peptides and proteins. The components of hemolymph have been confirmed to possess anti-bacterial and antifungal activity (Heinen & Veiga, 2011; Punzo, 1982; Silva et al., 2000; Yigit & Benli, 2008).
In Pakistan, no work has been done on the venom and hemolymph of Tetragnatha javana. Therefore, the aim of the present study was to partially characterize the venom and hemolymph of T. javana.
The objectives of the current work were:
- Extraction of venom and hemolymph of javana
- Identification of different peptides present in venom and hemolymph of javana, by using SDS-PAGE
- Estimation of different ions in venom and hemolymph using flamephotometer
LITERATURE REVIEW
Stapleton et al. (1990) separated three peptides from Hololena curta’s venom. These curtatoxins are cystine rich polypeptides having 36-38 residues of amino acids. These peptides caused instant paralysis and eventually death in Acheta domestica. Their effective paralysis activity makes them a suitable candidate for development of insecticidal compounds.
Deng et al. (2008) purified neurotoxin (huwentoxin-V) from venom of Ornithoctonus huwena and investigated its activity against different voltage gated channels in cockroaches. This insecticidal peptide expressed inhibitory effect against voltage gated calcium channels and did not affect voltage gated sodium and voltage gated potassium channels. This neurotoxin’s specificity makes it a suitable tool for manufacturing novel insecticides.
Corzo et al. (2009) isolated two insect-killing peptides (Ba1 and Ba2) from Brachypelma albiceps’s venom, having 39 amino acids residues and molecular weight 4.40kDa and 4.44kDa, respectively. These peptides proved to be effective against crickets and were not associated with inhibition of voltage gated sodium channels in other insects.
Gao et al. (2007) concluded that the venom derived from Macrothele raveni showed anti-cancer activity in human breast cancer cell line MCF7 by stopping DNA synthesis and starting necrosis and arresting the cell cycle G2 and M phase. These findings make it obvious that spider venom peptides are one of the best suitable options for the development of anti-tumor drugs.
Vorontsova et al. (2011) reported that Lachesana tarabaevi has Latarcin 2a in its venom which exhibits cytotoxic effect on erythroleukemia K562 cells in humans. This effect includes destabilization of plasma membrane and formation of small pores which is followed by swelling and causes death of cell.
Liu et al. (2012) worked on venom of M. raveni and reported that it reduced the growth of myeloid leukaemia K562 cells through triggering apoptosis by altering the signaling pathways associated with caspases.
Liu et al. (2012) investigated the effect of Lycosin-1 peptide derived from venom of Lycosa singoriensis and concluded that it has the potential to stop the growth of tumor cells by triggering apoptosis and stopping the proliferation of tumor cells and showing less toxic effects on normal cells. He suggested that Lycosin-1 might be used as structural lead for preparation of new anti-tumor dugs.
Grishin et al. (2010) isolated Purotoxin-1(PT1) from venom of Geolycosa vultuosa and investigated its action on purinoceptors. PT1 is a single chain peptide made up of 35 amino acid residues with molecular weight 3.83kDa. PT1 exhibited antinociceptive activity against P2X3 receptors in rats. Therefore, PT1 seems to be an appropriate peptide for manufacturing of novel antinociceptive drugs.
Dalmolin et al. (2011) isolated Tx3-3 peptide from the venom of Phoneutria nigriventer and checked it effects on different models keeping in view the type of pain it relieved. Tx3-3 exhibited a long term effect in case of neuropathic pain while it did not play any role in decreasing pain related with swelling. This peptide can be used for development of novel drug for controlling neuropathic pain.
Revell et al. (2013) found Huwentoxin-IV from venom of Selenocosmia huwena that inhibits Nav1.7 channel to produce analgesic effect.
Zhang et al. (2019) recognized a new peptide, µ-TRTX-Ca1a, from the venom of tarantula spider (Cyriopagopus albostriatus) that contains 38 residues, six of which are cysteines. This toxin showed high inhibiting effect on voltage gated sodium channels in mammalian cells, which makes it suitable for development of a novel antinociceptive drug.
Wu et al. (2019) concluded that peptides obtained from spider venom are good candidate for development of drugs which can be used for relieving pain. These peptides act on ion channels and other receptors for inducing the pain relieving effect. Spider Phoneutria nigriventer‘s venom contains number of peptides having pain relieving effect.
Suchyna et al. (2000) separated GsMTx-4, a toxin having 35 amino acid residues. This toxin has the ability to inhibit the activity of stretch-activated channels (SACs),that will be helpful in identifying the properties of SACs under different situations.
Escoubas et al. (2000) reported that PcTx1, a toxin present in Psalmopoeus cambridgei’ s venom plays its role by blocking the novel type of ion channels called as acid sensing ion channels (ASICs). The gating activity of these ASICs depends on pH and they are related with pain receptors. PcTx1 is the first toxin found to act against these channels and can play role in understanding the structure and characteristics of these channels.
Xiao et al. (2004) separated the Jingzhaotoxin –III from the Chilobrachys jingzhao’s venom, and tested its activity in rat models. This toxin contains 36 residues and did not affect voltage gated sodium channels (VGSCs) in neurons but inhibited the cardiac subtype VGSCc. This proved that Jingzhaotoxin –III is a cardiotoxin and has specificity.
Yuan et al. (2007) worked on venom of C. jingzhao and separated three peptides that exhibit gate modulating activity on voltage gated potassium ion channels by changing the channel activation to depolarized stages, decreasing the channel activation rate. 29-36 residue constitute these neurotoxin peptides namely Jingzhaotoxin-I, III and V which inhibit Kv4.1, Kv2.1 and Kv4.2 channel, respectively.
Yigit and Benli (2008) worked on hemolymph of Agelena labyrinthica to check the antimicrobial activity. 5 out of 10 bacterial strains used in study, expressed sensitivity to its hemolypmh. This exploration gives high hopes about usage of spider hemolymph in development of antimicrobial drugs in future against microbes which show antibiotic resistance.
Benli and Yigit (2008) worked on venom of Agelena labyrinthica to check its antimicrobial activity. Venom exhibited antimicrobial activity against 6 of 10 bacterial stains studied. Cell wall depression was the result showed by venom-treated cells as they lost their cytoplasm. Staphylococcus aureus,a drug resistant and fatal bacteria, did not show resistance to venom of A. labyrinthica.
Silva et al. (2000) isolated gomesin, a mini peptide having molecular weight of 2.27 kDa and having eighteen amino acids from the homocytes of Acanthoscurria gomesiana. Gomesin exhibited antibacterial activity against a large number of bacterial strains of total strains tested. Gomesin also inhibits the growth of fungi.
Zhao et al. (2011) purified an antimicrobial peptide, OH-denfensin from the venom of Ornithoctonus hainana that played antimicrobial role against fungi and both gram negative and positive bacteria.
Riciluca et al. (2012) purified Rondonin, a small peptide of 1.24 kDa, from hemolypmh of Acanthoscurria rondoniae and reported that it showed antimicrobial activity against fungi. Yeast can be killed by rondonin in 10 minutes. The future work can be done by synthesizing rondonin commercially to obtain antimicrobial drugs, as it is a small peptide and easy to produce.
Ayroza et al. (2012) worked on venom of Avicularia juruensis and separated antimicrobial peptides which can be useful in future as template for development of drugs. Antifungal peptide, juruin having thirty eight amino acids, molecular weight between 3.5-4.5 kDa and three disulfide bonds exhibits resistant against proteases and lacks hemolytic activity against erythrocytes in humans. All these properties make it a good candidate for efficient antifungal drug development.
Ji et al. (2019) reported that venom of spider, Alopecosa nagpag, contains Antiviral-Lycotoxin-An1a, which restricts Zika virus infection.This study suggested An1a as future candidate for drug development against flavivirus.
Schartau and Leidescher (1983) studied hemolymph compostion of Eurypelma californicum. The dominant amino acid present in hemolymph was proline. Stearic acid and palmitic acid are the major lipids present in hemolymph. Hemolymph of E.californicum exhibited highest concentrations of sodium and chloride ions among all cations and anions present, respectively.
Bednaski et al. (2015) worked on hemolymph of Loxosceles intermedia and identified homocytes of four different kinds including adipohemocyte, granulocyte, prohemocyte and plasmatocyte.
Jalal et al. (2010) worked on hemolymph profiles of various spider species and found that Phintella castriesian and spiders belonging to family Sparassidae showed higher concentration of potassium, sodium, calcium and chloride ions in comparison of other studied species. The hemolymph contains both high and low molecular weight polypeptides. Protein having molecular weight of 200 kDa was present in all studied spider species. Glutamine is the most abundant amino acid present in hemolymph of spiders. The presence of useful components in spider hemolymph is promising and it can lead to new discoveries in field of agriculture and medicine.
Wullschleger et al. (2004) worked on the neurotoxin, CSTX-13 present in the venom of Cupiennius salei spider and concluded that it helps in paralyzing the prey only when it is applied in high concentration. When CSTX-13 works synergistically with the major neurotoxin that is CSTX-1, it increases the paralytic effect venom at lower concentration.
Wullschleger et al. (2005) concluded that cytolytic peptides such as taurine and histamine augment the activity of CSTX-1 and high concentration of potassium ions in the venom helps in facilitating the activity of CSTX-1.
Nentwig et al. (2012) reported that a neurotoxic peptide, CSTX-1 obtained from the venom of C. salei containing 74 amino acid residues and ICK motif exhibits insecticidal activity against Drosophila flies by inhibiting the calcium ion channels. It can also kill Escherichia coli cells by rupturing their cell membranes which proves its antibacterial property as well.
Carneiro et al. (2003) made a recombinant PnTx3-1 toxin, using bacterial expression system and the activity of recombinant toxin was the same as of the original toxin present in venom of C. Salei. This successful work highlights the future aspect of using the cDNA methods to obtain functional spider toxins.
Gunning et al. (2008) concluded that Janus-faced atracotoxin (J-ACTX)-Hv1c, present in venom of Australian funnel web spiders play its role by blocking the pores in Ca2+-activated K+ (KCa) channels of cockroach and does not exhibit any activity in mouse, which confirmed its selective nature.
Gremski et al. (2010) obtained an overall glimpse of expression profile of venom of Loxosceles intermedia, with the use of expressed sequence tag method. Notorious toxins such as metalloproteases and phospholipases were present in its venom. Hyaluronidases, translationally controlled tumor protein (TCTP) and venom allergins were also found in its venom. These findings prove that the venom of L. intermedia contains a number of novel components which can be used in pharmacological and agri-chemical industry.
Calabria et al. (2019) constructed a recombinant toxin by using hydrophilic parts of Astacin-like metalloproteases and phospholipases D from venom of Loxosceles gaucho, named as LgRec1ALP1.The toxin showed effective results in mice leading to production of effective antibodies against L. gaucho, L. laeta, and L. intermedia venoms.
MATERIALS AND METHODS
Collecting Spiders:
The long jawed spiders (Tetragnatha javana) were collected from rice fields of Kolowal Nangiana, (32.13° N, 72.45° E), Sargodha, Punjab, Pakistan. Live Spiders were collected by hand picking method and were kept in plastic containers. These containers were covered by thin cloth so that air could pass through. They were kept hungry for two days to get maximum quantity of venom (Nagaraju et al., 2007).
Extracting venom:
Spiders were made motionless by putting them in low temperature of 4 C in refrigerator for 4-5 minutes. The chelicerae containing the venom glands were isolated with the help of needles and forceps. Venom glands were transferred into 1.5 ml eppendorfs having 0.5ml Tris buffer (pH 8.2) for analyzing the protein content. For estimation of ions, the venom glands were transferred into 0.5ml deionized water contained in 1.5ml eppendorfs. Twenty venom glands were transferred in each eppendorf. Venom was homogenized using small plastic rod and was centrifuged at 20,000 G for twenty minutes at 4 C (Centrifuge MPW-352R). After that, the supernatant was placed in ultra freezer at -20 C until further use (Guerrero et al., 2010: Rates et al., 2013).
Extracting hemolymph:
Using micropipettes, hemolymoph of spider was extracted after removing the legs from coaxal parts of spider. For analysis of proteins, hemolymoh was transferred into 1.5 ml eppendorfs having 0.5ml Tris buffer (pH 8.2). For estimation of ions, the hemolypmh was shifted into 0.5ml deionized water contained in 1.5ml eppendorf. In each eppendrof, hemolypmh of twenty spiders was collected. The hemolymph was centrifuged at 20,000 G for twenty minutes at 4 C (Centrifuge MPW-35-R). After that, the supernatant was placed in ultra freezer at -20 C until further use (Moreira et al., 2014).
Partial characterization of venom and hemolymph:
Gel Electrophoresis:
Protein profile of venom and hemolypmh was characterized by Sodium Dodecyl Sulfate gel electrophoresis (SDS-PAGE). Sambrook and Russle (2011) protocol was followed with some modification for analyzing the protein fractions. Different peptides in venom and hemolypmh were differentiated depending on their molecular weight.
Solution used in SDS-PAGE:
- Solution A:
Solution A was prepared by mixing 30g Acrylamide and 0.8g Methylenebisacrylamide in 100ml of distilled water. It can be stored at -20°C in ultra freezer.
- Solution B:
Solution B was prepared by mixing Tris base (18.2 g), 0.4g of SDS (Sodium Dodecyl Sulfate) in distilled water (100ml).Few drops of 5N HCL were added to the solution to set the pH to 8.8.It can be saved in ultra freezer at -20°C.
- Solution C:
Solution C was prepared by mixing 6.06 g of Tris base, 0.4 g of SDS in distilled water (100ml). The pH was adjusted to 6.8.It can be saved at -20° C in ultra freezer.
- 10 % Ammonium Persulfate (APS):
10 g Ammonium persulfate was mixed in distilled water (10ml) to prepare 10% Ammonium Persulfate (APS).It can be stored at -20°C in ultra freezer.
- Sample Buffer (2X):
1.25ml Sol.C , 5ml Mercaptoethanol , 11.5ml Glycerol,2g SDS, 5mg Bromophenol blue and 50ml of distilled water were mixed for preparation of sample buffer (2X).
- Running Buffer (4X):
12g Tris base, 57.6g Glycine, 4g SDS and 1.5 liter of distilled water were mixed to prepare Running buffer (4X).
- Staining Solution:
Staining solution was prepared by mixing 0.25 g Coomassie blue, 40ml Methanol, 10ml glacial acetic acid and distilled water (100ml).It was stored in ultra freezer at -20°C.
- De-staining Solution:
De-stainimng solution was prepared by 40ml Ethanol, 10ml glacial Acetic acid and distilled water (60ml).
Gel Preparation:
Gel plates were put together in a sealed gel apparatus (MSmidi10), after a thorough cleanup. They were positioned properly by placing 1mm spacer. Separating gel (10%) and staking gel (5%) were prepared.
- Separating Gel (10%):
3.3ml sol.A ,4ml distilled water, 2.5 ml sol.B, 40μl APS(10%) and 20μl TEMED were mixed in a 50ml falcon tube in order to prepare 10ml separating gel(10%).
- Staking Gel (5%):
0.7ml sol.A , 3.05ml distilled water, 1.25ml sol.C, 25μl APS(10%) and 15 TEMED were added together in a 50ml falcon tube for preparation of 3ml staking gel solution(5%).
Separating gel solution was poured between the glass plates with the help of micropipette up till the lower edge of comb, keeping the place for staking gel. It was allowed to polymerize for twenty minutes. Then, staking gel was poured over the separating gel between the plates using micropipette. The comb was placed into the staking gel for formation of wells and was removed after complete polymerization of gel.
Sample Preparation:
Eppendorfs containing supernatant (venom and hemolymph sample) were taken out from freezer and allowed to melt at room temperature. Samples (60μl) were mixed with sample buffer (2X) in 1:1 ratio and were thawed using dry heat chamber for thirty minutes in order to denature the proteins present in samples. Same procedure was followed for preparation of hemolymph sample.
Assembly of SDS gel electrophoresis apparatus:
The gel apparatus was set vertically in the electrophoresis tank. Then running buffer was poured into the tank up to the mark on the tank. Samples (20μl) were then loaded into the wells with the help of micropipette. One well was loaded with protein marker (thermoscientific- PageRuler Prestained Protein Ladder) having known molecular weight (10-180kDa) as standard scale. Then the tank was closed with its lid and was connected to power supply. The gel was allowed to run at eighty volts until it reached the separating gel then the voltage was increased to hundred volts. After 3-4 hours, when the samples reached the bottom of gel, power supply was turned off.
Staining and de-staining of gel:
Glass plates were taken out from gel apparatus and gel was isolated by separating the plates carefully. Gel was placed in staining solution for about one hour and then was placed into de-staining solution for eight to ten hours and was photographed. The molecular weights of bands of different fractions were determined by comparing them with standard reference proteins.
Free ion determination:
Eppendorfs containing venom and hemolymph samples were taken out of freezer and were allowed to melt at room temperature. Ion concentrations in samples were determined with the help of flame photometer (Jenway PFP 7).
Sample Digestion:
Concentrated H2SO4 (5ml) and 0.4ml of venom sample were mixed in a beaker and solution was heated over Bunsen burner for 5-10 minutes. Then, H2SO4 (5ml) and HNO3 (3ml) were added into the solution and the mixture was further heated for 20-25 minutes. After emission of black fumes, water was added into the solution. After heating the solution for five more minutes, it was removed from burner and was filtered with the help of Whatman filter paper (Sharma & Tyagi, 2013). Same directions were followed for the digestion of hemolymph samples.
Preparation of solutions used in determination of free ions:
- Stock NaCl Solution (1000 ppm):
1.27g of NaCl was added into 500 ml of distilled water in order to prepare the stock NaCl solution (1000 ppm) in a volumetric flask.
- Stock NaCl Solution (100 ppm):
10 ml of stock NaCl solution (1000 ppm) was mixed in 90 ml for preparation of Stock NaCl solution (100 ppm) in a volumetric flask.
- Standard Solutions (Na+) :
Serial dilution of stock NaCl solution (100 ppm) was performed in order to prepare the standard solutions of Na+. Eight test tubes were used for preparation of eight different concentrations of standard solution (10, 20, 30, 40, 50, 60, 70, 80 ppm). In one test tube, 1 ml of stock NaCl solution (100 ppm) was mixed with 9 ml of
distilled water for preparation of 10 ppm standard solution. Similarly concentrations of 20, 30, 40, 50, 60, 70 and 80 ppm standard solutions were prepared.
- Stock KCl Solution (1000 ppm):
1.91 g KCL was mixed in 500 ml distilled water in order to make stock KCL solution (1000 ppm) in a volumetric flask.
- Stock KCL Solution (100 ppm):
10 ml of stock KCL solution (1000 ppm) was added into 90 ml distilled water for preparation of stock KCL solution (100 ppm) in a volumetric flask.
- Standard Solutions (K+):
Volumetric dilution of stock KCL solution (100 ppm) was performed in order to prepare the standard solutions of K+. Concentrations of 10, 20, 30, 40, 50, 60, 70 and 80 ppm standard solutions were prepared, following the same procedures as standard solution Na+.
Determination of Ion Concentration:
Diluents were added to the instrument after warming the instrument for 5-10 minutes. Distilled water was used as aspirate for cleaning the system. After water aspiration, standard solutions were aspirated in ascending order and steady display readings were recorded. Then the digested samples were run through the photometer and their readings were noted.
Data analysis:
One tailed unpaired t test was used for comparison of free ion concentrations in venom and hemolymph. Microsoft Office Excel (2007) was used for graphic interpretation of data.
| Elements | Sodium | Potassium |
| Wavelength | 589 | 766 |
| Flame Color | Yellow | Violet |
Table1.The wavelength and color of light emmited by Na+ and K+
RESULTS
Electrophoretic analysis of venom of Tetragnatha javana:
The bands of venom samples were compared with the protein ladder of known value (10-180kDa) and results showed that venom of Tetragnatha javana contained three different protein fractions, i.e. 15kDa, 43kDa and 70 kDa. Band of 70 kDa was most intense followed by 43 kDa. Band of 15 kDa was least intense. (Figure 6)
Electrophoretic analysis of hemolymph of Tetragnatha javana:
The bands of hemolymph sample were compared with the protein ladder of known value (10-180kDa) and result showed that hemolymph of T. javana contained three different protein fractions, i.e. 10kDa, 43kDa and 68 kDa. Band of 68 kDa was most intense followed by 43kDa, whereas band of 10 kDa was least intense.(Figure 7)
4.3. Estimation of free ions in venom and hemolymph of T. javana:
Against the standard solutions of Na and K, concentrations of Na+ and K+ ions in venom and hemolymph of T. javana were estimated with the help of flame photometer (Jenway PFP7) and there was direct relation between emission concentration of ions and concentration of standard solutions.
The concentration of Na+ and K+ ions in venom and hemolymph was estimated in multiple samples and their mean±SE was calculated. It was found that venom of T. javana contained higher concentration of Na+ (4.40ppm) than K+ (1.54ppm). In case of hemolymph, same trend was observed as concentration of Na+ (3.16ppm) was higher than than K+ (0.88ppm).(Figure 10 & 11)
Table 2. Absorbance of Na+ ions against different standard solutions
|
No. |
Conc. Of Standard Solution (ppm) |
Absorbance (ppm) |
| 1 | 00 | 00 |
| 2 | 10 | 11.1 |
| 3 | 20 | 22.4 |
| 4 | 30 | 31.3 |
| 5 | 40 | 43.6 |
| 6 | 50 | 53.2 |
| 7 | 60 | 61.4 |
| 8 | 70 | 72.7 |
| 9 | 80 | 83.5 |
Table 3. Absorbance of K+ ions against different standard solutions
|
No. |
Conc. Of Standard Solution (ppm)
|
Absorbance (ppm) |
| 1 | 00 | 00 |
| 2 | 10 | 14.1 |
| 3 | 20 | 24.4 |
| 4 | 30 | 37.3 |
| 5 | 40 | 48.6 |
| 6 | 50 | 52.3 |
| 7 | 60 | 63.4 |
| 8 | 70 | 74.1 |
| 9 | 80 | 81.2 |
The result showed that there is non-significant difference between Na+ and K+ ions concentrations in venom (P=0.0015, t=6.50, df=4),while there is significant difference in case of hemolymph( P>0.001, t=18.36, df=4). There was non-significant difference in Na+ (P=0.24, t=2.81, df=4) and K+ (P=0.003, t=5.25, df=4) ions concentrations between venom and hemolymph.
Calibration curves for Na+ and K+ were drawn by using data of table 2 and 3 respectively as shown in figure 8 and 9.
Table 4. Mean Concentration of Na+ and K+ ions in venom and hemolymph of T. javana
| NO. | Na+ Content | K+ Content | ||
| 1. | Hemolymph | Venom | Hemolymph | Venom |
| 2. | 3.16±0.120 | 4.40±0.422 | 1.54±0.121 | 0.88±0.032 |
DISCUSSION
In the present study, venom and hemolymph of Tetragnatha javana was partially characterized. The results showed that venom of T. javana contains protein with molecular weight, greater than 10kDa. The venom of T. javana contained bands of 15kDa, 43kDa and 70 kDa. Band of 70kDa was most intense whereas band of 15kDa was least intense. The hemolymph contained protein bands of 10kDa, 43kDa and 68 kDa. Band of 68 kDa was most intense followed by 43kDa and 10 kDa band. High and low intensity of protein bands directly correlates with concentration of proteins in the venom and hemolymph.
Venom of spiders contains amino acids, neurotoxins, enzymes, proteins, nucleic acids, ion channel toxins and polypeptides. On the basis of molecular weight, components of spider venom can be categorized into three major classes. First class consists of compounds having less than 1kDa molecular mass such as organic acids, amino acids, ions, nucleosides, polyamines, neurotransmitters and nucleotides etc. Second class comprises of peptides having molecular mass between 1 to t10 kDa, which are further classified in two main groups on the basis of their function viz. linear peptides (having cytolytic activity) and disulfide peptides (having neurotoxic activity), while the third class contains substances having molecular mass greater than 10 kDa e.g. enzymes, neurotoxins and proteins (Heinen & Veiga, 2011; Vassilevski et al., 2009).
Venom of T. javana contains peptides belonging to third category having molecular mass greater than 10kDa that may contain enzymes, proteins and neurotoxins. In Hawaiian Tetragnatha spiders (both orb-weaver and wandering), bands of 25 kDa, 43 kDa and bands between 40kDa to 90 kDa were observed (Binford, 2001). Venom of T. javana also contained peptide bands of 43kDa and 70kDa.
Venom of Araneus ventricosus contains peptides ranging in molecular weight from 2 to 70 kDa and they can block the VGSCs in cockroach (Periplanta americana), but not harmful to vertebrate (Liu et al., 2016). Peptides of 24kDa and 70kDa were present in 22 species of prey-specialized arachnids and they all showed toxin specificity for their respective prey, and it can be assumed that they have prey specific toxins in their venom (Pekar et al., 2018). Venom of T. javana also contains peptides between 15kDa to 70 kDa that may be used for development of novel selective insecticides.
Loxosceles intermedia contained hylauronidases of moleculer weight 41 and 43 kDa. These enzymes have the ability to disintegrate the contents of extracelluar membrane and enhance the permeability of membrane. Hylauronidase can be used to treat many health issues related to surgery, gynaecology, dermatology and orthopaedics etc (Chaim et al., 2011; Sneff-ribeiro et al., 2008). Venom of Agelena labyrinthica venom contained peptides ranging between 10 and 40 kDa. Its venom showed antimicrobial activity (Benli & Yigit, 2008; Yigit & Guven, 2006).
In the present work, the analysis of free ions concentration showed that venom of T. javana contains higher concentration of Na+ ions (4.40 ppm) as compared to K+ ions (1.54 ppm). The hemolypmh of T. javana also have higher concentration of Na+ ions (3.16 ppm) as compared to K+ ions (0.88 ppm). The comparison of free ions indicated that venom contained higher concentration of free ions (Na+ and K+), as compared to hemolymph of T. javana.
The concentration of free ions in Arachnids varies from species to species. Factors such as sex, nutrition and age play role in concentration of free ions in venom and hemolymph (Vassilevski et al., 2009). Several spiders belonging to different families viz. Sparassidae, Salticidae (Thyene imperialis, Phidippus workmani, Plexippus paykulli, Phintella castriesiana, Pelegrina verecunda), Miturgidae (Cheiracanthium mildei), Eresidae (Stegodyphus sarasinorium) and Clubionidae (Elaver spp.) showed higher concentrations of Na+ ions in hemolymph as compared to K+ ions ( Jalal et al., 2010). Hemolymph of lycosid spiders also showed higher concentration of Na+ ions ranging between 35-41% of hemolymph osmolytes as compared to K+ ions (Punzo, 1982).
Efficiency of venom is enhanced by Na+ ions and helps the spider in suceesful predation. High concentration of K+ ions causes short term paralysis in prey, immediately by depolarizing the excitable cell membrane. K+ ions play role in enhancing the insecticidal potential of spider peptides (Wullschleger er al., 2004). There is very little research regarding free ions concentration of venom and hemolymph.
Tiger wandering spider Cupiennius salei contains higher concentration of K+ ions in venom and low in hemolymph (Wullschleger er al., 2004), which is contradictory to the concentration of K+ ions in venom and hemolymph of T. javana.
From above discussion it is concluded that venom of T. javana contains proteins ranging from 15kDa to 70 kDa and hemolymph contains proteins ranging from 10kDa to 68kDa. These peptides could have insecticidal potential and might have neurotoxic role and antimicrobial potential and might be used in future pharmacological applications, but further research is needed to establish this.
CONCLUSION
It is concluded from above discussion that the venom of Tetraganatha javana contains proteins of size ranging from 15kDa to 70 kDa and the hemolymph has proteins of size ranging from 10kDa to 68kDa. These could have insecticidal potential and antimicrobial potential.
In present work, the comparison of concentration of free ions in venom and hemolymph indicates that venom of T. javana has higher concentration of Na+ ions (4.40ppm) as compared to K+(1.54 ppm).Same trend is followed by hemolymph as it contains lower concentration of K+ ions (0.88ppm) as compared to Na+(3.16ppm). The comparison between venom and hemolymph shows that venom contains higher concentration of free ions as compared to hemolymph. The venom and hemolymph may have important role in agriculture and pharmacological industries but more research is required to establish this.