Introduction
Human population is developing at a rate of 80 million every year and is anticipated to be even more in coming years (Bloom et al., 2011). This population explosion will result in difficulties in food supply (Oerke et al., 2006). The vast majority of food resources are possible from enhanced harvest yields in light of the fact that there are less possibilities to increase the area of crop fields. Pests are the most significant reason for the low crop production (Oerke et al., 2006). About 1,000 insect species are viewed as major pests, they are considered to be the largest factor in reducing yields by 10– 14% regardless of serious control measures (Pimental et al., 2009).
From twentieth century and onward, scientists are concerning for the increase food security, this may be possible by increasing the crop production, this process is called green revolution or agricultural revolution. Different agro-techniques were applied, which includes pest and soil management techniques (Raj et al., 2003). Because of the effective control by agrochemicals such as DDT, carbamate and organophosphates in 1940’s (Casida et al., 2009). In 1960’s the use of synthetic pesticides has been increased to control pest (Nicholson et al., 2007), this technology of green revolution seemed to be helpful in increasing food production, but produced adverse effects on soil health, water and produce mutations in plants (Alzaidi et al., 2011). Agrochemicals also have adverse effect on mother milk and damage the infant health, that’s why scientists are interested in organic farming because it inhibits the plant interaction with chemicals (Berg et al., 2009). To avoid these harmful aspects of agrochemicals the best option is integrated pest management.
Integrated pest management (IPM) is the most ideal path, and the European Union has set it as most directive biocontrol program (Chandler et al., 2011). IPM is a framework that approaches the most distinctive harvest security practices with checking of pest and their enemies (Bajwa et al., 2002 & Flint et al., 1981). IPM uses various natural and synthetic techniques for effective control of the pest while bringing down reliance on chemicals. The thought behind IPM is that application of different strategies overcome the weaknesses of single strategy. The point is not to destroy all insect populations but to those that are responsible for economic losses. Following these characteristics and control measures, bio- pesticides considered to be the most important component of IPM (Thomas et al., 1996).
Bio-pesticides are considered as safer alternative rather than chemical pesticides as they do not cause any severe damage nor create any environmental pollution (Leng et al., 2011). The commercial status of bio-pesticide is increasing at 10-25% per year, while of the agrochemical is decreasing with the same pace. In year 2008, hundreds of bio pesticidal products were in global market and their number is increasing day by day (Kumar et al., 2008). As per record of 2013 there are 400 component of bio pesticides and total 1250 bio-pesticide products that are registered (Villaverde et al., 2014). Environmental protection agency is working for the registration of bio-pesticides and the non-toxic pesticides.
Bio-pesticides when utilized as a part of IPM, can significantly reduce the utilization of chemical pesticides. They are very potent in little amounts and decayed rapidly. This can bring about lower exposures and have no contamination issues, they are target specific organism rather than pesticide which affect many other organisms (IUPAC 1994).
Various animal toxin based bio insecticides were the main focus of biologists in the field of bio agriculture. Researchers have isolated the peptide neurotoxins from the venom of different organisms such as spiders, (Nicholson et al., 2007), scorpions (Froy et al., 2000), parasitic wasp (Quistad et al., 1994) and straw itch mite (Tomalski et al., 1994). There venom is beneficial because it contain toxins that can block the membrane ion channels and also affect enzymes of insects (Saez et al., 2010) Spiders are most intense predator of these pest (Clausen et al., 1986). Member of tetragnatha are dominant hunter of pest in citrus fields (Yan et al., 1994), they are the best predator of lepidopteron species (Hendawy et al., 2015) and also the most active predator of other crops e.g. rice and crucifer. They are potentially more efficient in organic food farming (Rosa et al., 2009).
Pakistan is an agricultural country. Major part of our economy is based on agriculture. There is an intense need to introduce bio-pesticides in order to save our crops. In Pakistan, spider venom has not yet been used as bio-pesticide. Very little research has been done on spider venom in Pakistan. To cope with current challenges, there is need to conduct research on spider venom bio pesticidal potentials. In the present study bio pesticidal potential of venom of species of spider Tetragnatha guatemalensis and Tetragnatha javana was evaluated against armyworm (Spodoptera exigua) which was used as model pest. The outcomes of this study will be helpful to generate very useful information regarding the use of spider venom in the field of agriculture. The results of this research will also be very beneficial for farmers
Objectives:
Objectives of the study are as under:
- To extract venom from selected spider species (Tetragnatha guatemalensis and Tetragnatha javana).
- To evaluate bio-pesticidal potential of crude venom of selected species against armyworm (Spodoptera exigua) and compare their effects from insecticide (Lufenuron).
Review of Literature
Agribusiness is a vital component in the economic status of Pakistan and it plays a fundamental role in the development of its identity, about 21% of the revenue is generated by this sector. (Talib et al., 2016). Agricultural industry is meeting the demand of food, as human population is increasing day by day. This population increase will exhibit huge difficulties in the food supply (Oerke et al., 2006). So the requirement of crops should be doubled, the production of more food from a same region of land is called ” sustainable intensification” (Godfray et al., 2010).
Guaranteeing for the security and quality of crops, the farmers were recommended for the use of pesticides. The yearly worldwide use of synthetic pesticide is around 3 million ton (Popp et al., 2011). The developing reliance on pesticides had been known as the “pesticide treadmill” by entomologists (Bosch et al., 1976). Since 1940s, protection from pest has been dependent only on chemical insecticides for example, DDT and organophosphates. These chemicals enhanced yields, however have negative outcomes on non-target organisms. (Carson et al., 1962). Pyrethroids, carbamates, organophosphates, sulphur and carbaryl compounds decrease the population of many useful organisms such as spider from fields. Neonicotoids were also successful due to their high insect toxicity, as they are strong antagonist of acetylcholine receptor, but they had adverse effects on pollination of insects (Glare et al., 2012). Scientists noticed that more prominent the effect of control measures on pest, the more outrageous are their responses, because the pest develop resistance against these chemicals by metabolizing the chemical to non-toxic, or by modifying its own target site (Elbert et al., 2008). Seeing these side effects scientists focused to use alternatives for these chemicals, the best option is integrated pest management.
Integrated pest management (IPM) emphasizes not only on the use of other environment friendly ways of controlling pests but also on even more intelligent use of those pesticides which are selective in action (Benamu et al., 2007). This is the most feasible strategy, because it limits negative ecological effect of pesticides and other chemical sprays on human and other organisms (Herzog et al., 1985). IPM uses various natural and synthetic techniques for effective control the pest while bringing down reliance on chemicals. Through its multi-strategic approach, it lessens pesticide resistance in pest by allowing moderate and appropriate use of pesticides, and by using chemical sprays only at particular stages in the life cycle of pest. It also decreases chemical expenses, and minimizes human contact with pesticide sprays (through blending, splashing, and so forth (Luna et al., 1990 & McDonald et al., 1993). The use of microbial pesticides in IPM requires high investigation, for example, the properties, mode of action, and pathogenicity of these pesticides should be clear. Ecological investigations are important because the ecological factors also play an important role to control pest (Mills et al., 2010).
Bioinsecticides are the most suitable component of IPM and INM (integrated Nutrient Management), they are ecofriendly substances, they are very safe, and are biodegradable, (Balasubramanian e al., 2008). Hubbard et al., 2014 described that their mechanism of action is nontoxic, they are going their best to meet the demands of organic food. They are grown in awesome sum and use against pest in crop protection on seasonal basis (Rodger et al., 1993). It is estimated that over 3000 tons per year of bio pesticides are producing and its coverage is 2% of all the protectants (Hubbard et al 2014). Of all the agrochemical, bio pesticides constitute 1% in international market, the use of bio pesticides has improved the pest management and also improved already existing pest management techniques (Wratten et al., 2002) Scientists are interested to shift chemical based management to integrated pest management (Frank et al., 1996). Integrated crop management is practicing the use of different methods to improve the cultivation either by traditional, physical ways or by using biological methods to control pest (Kumar et al., 2013).
Spider along with the beetle are most intense land predator and are the best indicator to maintain equilibrium in a terrestrial ecosystem. (Clauses et al., 1986). They are very alert and have broad host range (Riechert et al., 1984). They can kill large number of pest in a very short span from massacres, the high specificity and power of spider poisons prescribe them as lead chemical for bio insecticides (Platnick et al., 2012).
Spiders evolved around 300 million years back in the Carboniferous period. This features the long developmental timescale over which they have advanced their venom, they are the most venomous creatures (Roy et al., 2005). They have high pest specificity, however is not lethal in non-target life forms, they stay in the earth for sufficiently long time, and their formulation is not expensive (whetstone et al., 2007). In agrochemicals, some of these objectives can’t seem to be completely accomplished. One of the distinguishing feature to the general accomplishment of spiders is the generation of exceptionally lethal venom, because they are the natural way and their metabolic products are used to control pests (Nauen et al., 2002).
Various toxin based bio insecticides were the main focus of biologists, they isolated the peptide neurotoxins from the venom of different organisms such as spiders (Nicholson et al., 2007), scorpions (Froy et al., 2000), parasitic wasp (Quistad et al., 1994) and straw itch mite (Tomalski et al., 1988). Spider uses their venom to kill prey or predators as quickly as possible. The venom is especially rich in neurotoxins that quickly alter ion conductance, and influence neurotransmitter exocytosis, some of the toxins that target the voltage-gated calcium channels (CaV) are exceptionally intense and specific. Since CaV channels are not conserved in pests, this makes them best option for the activity of chemical pesticides (Tedford et al., 2004).
Spider and scorpion, both contain toxins with varying range (2– 12 kDa) of atomic masses, however spider venoms clearly have considerable variety of channel and other cell receptor than scorpion venoms (Escoubas et al., 2000) .Spider peptides are getting to be noticeably perceived as fundamental for the investigation of cellular receptors. Acylpolyamines seem to have basic insecticidal chemical and it cause severe paralysis of pest by means of blocking neuromuscular junction. Polypeptide poisons incorporate the most important channel blockers, and they are fundamental compounds in the venom of spider.
Other components of spider venom include toxins that can block the membrane ion channels. (Saez et al., 2010). Chemical nature of spider venom is basic or neutral, a very few have acidic venom (wiener et al., 1961). The venom in its major composition contain toxic peptides and acyl polyamines (Escoubas et al., 2006). They are the most important toxins in venom, acyl polyamines impede glutamate receptor (Mc cormick et al., 1993), and these neurotransmitters release action potential (Usherwood et al., 1994), and are supposed to be helpful in the field of neuropathology (Quistad et al., 1991). Other organic components may include amino acids, nucleic acids, polyamines and some inorganic salts (Jackson et al., 1989). These toxins have biological, biochemical, immunological, and cellular applications.
Toxins are studied by crystallographic techniques (Mirshafiey et al., 2007). The neurotoxins target both the basic functions i.e. irritability and conductance of nerve impulse in the CNS either by blocking the ions channels (Na+, K+ Ca+2, Cl-1) or by disrupting the receptors such as choline esterase receptor (Escoubas et al., 2000). The venom of the funnel web spider (Agelenopsis aperta), was the first of peptide blockers of non-L voltage sensitive calcium channels (VSCCs). The peptide poisons effect the neuromuscular structure of insects and the relocation of the calcium channel blockers, for example, the conotoxins in chick synaptosomes. Four kinds of polypeptide presynaptic opponents of VSCCs (w-agatoxins) were explained by toxicologist. The most effective toxins in venom of arachnids are the atracotoxins.
They come from the hadronyche and atrax (Nicholson et al., 2007). Other toxins include acyl polyamine and conotoxin. A- aperta toxins from venom of spider cause the paralysis of insects in housefly in 3rd instar (Adam et al., 1989) Alpha- agatoxins have less molar mass, they are the most primitive toxins in venom, they act on neuromuscular junction and cause paralysis which is reversible (Adam et al., 1989), they have the ability to reduce the action potentials. Mew agatoxins are toxins that have Disulphide Bridge in their structure, the peptide contain 36-37 amino acids (Skinner et al., 1989), they cause multiple action potential at neuromuscular junction (Adam et al., 1989), it also causes the depolarization by modification of sodium ion channels. This toxin only effect the insect on their nerve junction (Bindokas e al., 1989). ω–agaIA and ω-AgaIIA cause insect CNS impairment (Bindokas et al., 1991). Plectreurys poison II (PLTX-II) is a 44- molecule peptide poison obtained from the venom of the spider Plectreurys tristis (Branton et al., 1987).
It contains a novel post translational character i.e a C-terminal O-palmitoyl threonine amide, which is fundamental in poisoning (Branton et al., 1993). It was indicated approximately 20 years ago that PLTX-II poison targets the Dmca1A CaV channel in Drosophila (Leung et al., 1989). Spider venoms contain in excess of a thousand peptide poisons (Escoubas et al., 2006). Spider inject their venom through needle like structure called fangs. Throughout evolution spider didn’t possess oral activity to inject these toxins. However it was observed that some of the spider toxins have this property of being orally active (Mukherjee et al., 2006)
Lepidopteran species (moth armyworm etc.) are regular pests in agriculture, the utilization of this pest in the research of bio pesticide give an understanding that pest develop resistance against insecticides and can’t protect themselves from lethal impacts of spider venoms. This shows that Lepidoptera is the best objective for the improvement of bio insecticide. By the advancement of new bio pesticide items, the scientists are testing them to distinguish dangers before their usage in the field.
Being an agricultural country, we must have to enhance the profitability of the agribusiness through these resources of bio pesticides, A great work in the most recent decade has tried to overcome the constraints of bio pesticide activity, host specificity, and to improve the transport of the bio pesticide. Most of these issues have been solved through a blend of different poisons inside single frameworks (Ferre et al., 2002). Spiders are the best natural resource and their bio-pesticidal potentials are the main focus of present study.
Materials and Methods
Collection of spiders:
Study was conducted on two spider species i.e.,Tetragnatha javana and Tetragnatha guatemalensis. Spiders were collected by hand-picking method along the pond sides in the month of April in district Sargodha. Live spiders were kept at 25°C.
Venom extraction:
To ensure the presence of venom in the venom gland of spiders, they were kept in separate containers (8.5cm diameter ×7cm height and 250cm3 volume) without food for 2-3 days. Spiders were anaesthetized by keeping them in the freezer at 3°C for 5-7 minutes. Adult spiders were dissected and their cephalothoraxes were separated (Frontali et al., 1976) and venom glands were extracted.
Manual homogenization of venom glands from 250 spiders in 4-4.5 ml cold 0.05 M Tris-Cl buffer, pH 8.8 was done to get the venom from each spider species (Frontali et al., 1976).Two Eppendorf were made each with venom of 70 spiders, for both species two dilutions were prepared, one Eppendorf with 70 venom glands in 1ml of Tris-Cl buffer. and other with 70 venom glands in 0.5ml of Tris-Cl buffer.The homogenate was centrifuged at 16,000G in a refrigerated centrifuge at -5°C for 30 min The supernatant was frozen at -20°C and used for fractionation (Frontali et al.,. 1976)
The subject pest is armyworm Spodoptera exigua (lepidoptera: Noctuidae). Pest are collected from fodder research institute Sargodha. Armyworm has 6 instar stages, our concern was on 2nd instar stage, and it is recognized by orangish head green body and without patches. The pest was in masses on leaves in summer
Mortality tests
Mortality test was taken for 24 hours for venom of both spider species and insecticide against armyworm pest. Mortality observation was taken after every hour and their results were noted and compiled by one way anova.
Mortality test with venom:
Two species of spider were collected and their venom was homogenized and stored, each specie with two dilutions,The volume of each venom gland was calculated, 1ml solution of venom of T. javana in Tris-HCl buffer, and 0.5 ml soln of venom of T. javana Tris-HCl buffer, this is termed bioassay same is done with the T. guatemalensis. Ten armyworms were taken in each Petri dish, and four groups were arranged.
Mortality test with Insecticide (Lufenuron):
The mortality test with insecticide was arranged in four groups based on its four different dilutions of Lufenuron (Marshall 5 EC) i.e. field dose ( L1), half field dose (L2), quarter field dose (L3) and 1/10th field dose (L4). Ten armyworms were taken in each group, and 5µl of insecticide was poured on it.
Preparation of Solutions Used:
Tris-HCl buffer (1M, 8.8 pH)
Dissolve 121.1 g of tris base in 800ml of water and adjust pH to 8.8 by adding 1M NaOH solution. Water was added to make a final volume upto1000ml
- Control Group:
First two groups were control contain distilled water, and Tris-HCl buffer.
- Venom Dilution of javana:
Each venom gland of T. javana contain approximately 0.25 µL of venom. Based on this two dilutions of it were formed in third group i.e. 35µL/ml solution of venom in Tris-HCl buffer, and 70µL/ml solution of venom in Tris-HCl buffer.
- Venom Dilutionof guatemalensis:
Fourth group also has two dilutions i.e. 140µL/ml soln of venom in Tris-HCl buffer and 70 µL/ml solution of venom in Tris-HCl buffer of T. guatemalensis.The mortality test was taken for 24 hours and the readings were noted.
- Dilutions of Insecticides:
Different insecticides such lufenuron (marshal 5 EC,) used to see their comparison in effect with themselves and with the venom on armyworm Spodoptera exigua.
Field Dose Lufenuron:
Field dose of insecticide lufenuron is 200ml/hectare. On laboratory scale we take 2ml of lufenuron and mixing it in one liter of distil water. This is 2 µL/ml solution of lufenuron.
- Half field dose Lufenuron:
Half field dose is prepared by taking 100 ml solution from field dose and mixing it in 1 litre of distil water. This is 1µL/ml solution of lufenuron.
- Quarter field dose:
Quarter field dose is prepared by taking 50ml from field dose and mixing it in 1 litre of distil water. This is 0.5 µL/ml solution of lufenuron.
One-tenth field dose:
One-tenth field dose is prepared by mixing 20ml from field dose and mixing it in 100 litre 0f distil water or we can say 0.2µL/ml solution of lufenuron
Results
The purpose of the work was to observe the bio pesticidal potentials of venom against armyworm. Bioassays were made by using venom from two species of spiders i.e. Tetragnatha javana and Tetragnatha guatemelensis. Five microliters of the crude venom and its dilutions were applied topically on each pest. The bio pesticidal potential of venom was compared with insecticide (Lufenuron). The mortalities were observed for 24 hours. The results of bioassay showed that the experimental group that was applied with insecticide lufenuron showed 100% mortality at relatively shorter time as compared to venom.
Table 1.Percentage mortality in armyworm (Spodoptera exigua) treated with different Concentrations of Venom Tetragnatha guatemelensis and tetragnatha javana.
| Treatment | Time intervals | |||
| Percentage mortality | ||||
| 6 | 12 | 18 | 24 | |
| control (Tris HCl) | 3 | 7 | 7 | 7 |
| Venom TG1 (70 µL/ml) | 70 | 93 | 100 | 100 |
| Venom TG2 (35 µL/ml) | 27 | 47 | 63 | 80 |
| Venom TJ1(35 µL/ml) | 17 | 37 | 43 | 43 |
| Venom TJ2(17.5 µL/ml) | 20 | 40 | 43 | 43 |
When armyworm (S.exigua) were treated with different concentrations of Lufenuron i.e field dose half field dose quarter field dose and 1/10th field dose. High mortality was observed in (L1) treated venoms . Observations were made in different hours such as 6h, 12h, 18h, 24h. 100% mortality was recorded with field dose (L1) after 12 h of post treatment , 100% with half field dose after 18h, 100% with quarter field dose, and 97% in 1/10th field dose after 24h of post treatment, there is no significant mortality 33% in the control group after 24h of post treatment ( Table 2).
| Treatment | Time intervals | |||
| Percentage mortality | ||||
| 6hr | 12 | 18 | 24 | |
| Control (0 µL/ml) | 0 | 0 | 33 | 33 |
| L4 (0.2 µL/ml) | 13 | 46 | 70 | 97 |
| L3 (0.5 µL/ml) | 30 | 40 | 87 | 100 |
| L2 (1 µL/ml) | 30 | 73 | 100 | 100 |
| L1 (2 µL/ml) | 50 | 100 | 100 | 100 |
Table2. Percentage mortality in armyworm treated with different concentrations of Insecticide (Lufenuron)
Toxicity of T. guatemalensis and T. javana on armyworm.
Lethal doses i.e. LD50 and LD95 values for T. guatemalensis and T. javana venom applied on armyworm were calculated by using probit analysis. It is depicted in Figure 6 that +23.94 ± 5996.23 µl/ml of crude venom of T. guatemalensis was sufficient to kill the 50% population (LD50) of armyworm. While LD95 recorded from probit analysis was (35.785 ± 722.72) which showes that 95% population was died at this dose (70µl/ml) venom of T. guatemalensis. The values of LD50 and LD95 for insecticide Lufenuron applied against armyworm were also recorded from probit analysis.
The values of LT50 and LT95 for different dilutions of T. guatemalensis and T. javana on armyworm venom i.e. 70 µl/ml (TG1), 35µl/ml (TG2), 35µl/ml (TJ1) and 17.5µl/ml (TJ2) were also recorded from probit analysis. The LT50 and LT95 for 70 µl/ml (TG1) showed lowest value (3.01 ±1.27, 11.48±1.58) (Fig: 7) as compared to 35µl/ml (TG2) (13.59±1.09, 27.71±2.79). Furthermore the values of LT50 and LT95 for different dilutions of venom of TJ1 and TJ2 and insecticide Lufenuron i.e., L1, L2, L3, and L4 are summarized in the Table: 3.
Table 3. Calculated LT50 & LT95 of Venom Tetragnatha guatemelensis, Tetragnatha javana and insecticides.
| Treatments | LT50 | LT95 |
| TG1 (70 µL/ml)
|
3.01 ±1.27 | 11.48±1.58 |
| TG2 (35µL/ml)
|
13.59±1.09 | 27.71±2.79 |
| TJ1 (35µL/ml)
|
23.07±3.892 | 53.85±14.11 |
| TJ2 (17.5 µL/ml)
|
25.72±7.03 | 72.82±30.20 |
| L1 | 5.38 ±0.69 | 11.21±1.25 |
| L2 | 8.25±0.73 | 15.58±1.36
|
| L3 | 12.68±0.86 | 22.98±1.82 |
| L4 | 13.05 ±0.90 | 23.96±1.94 |
The graphs after minitab analysis are shown:
Figure No 6. LD50 and LD95 of T.guatamelensis at 24hrs
LD50= +23.94± 5996.23
LD95= 35.78 5±722.72
Graph No 7. LT50 & LT95 of Tetragnatha guatemalensis (70µl/ml)
LT50=3.01 ±1.27
LT95=11.48±1.58
Graph No 8. LT50 & LT95 of Tetragnatha guatemalensis (35µl/ml).
LT50= 13.59±1.09
LT95= 27.71±2.79
Graph No 9. LT50 & LT95 of Tetragnatha javana (35µl/ml)
LT50= 23.07±3.892
LT95= 53.85±14.11
Graph No 10. LT50 & LT95 of Tetragnatha javana (17.5µl/ml)
LT50= 25.72±7.03
LT95= 72.82±30.20
Graph No 11. LT50 & LT95 of insecticide Lufenuron field dose (2µl/ml)
LT50=5.38 ±0.69
LT95= 11.21±1.25
Graph No 12. LT50 & LT95 of insecticide Lufenuron Half field dose (1µl/ml)
LT50= 8.25±0.73
LT95= 15.58±1.36
Graph No 13. LT50 & LT95 of insecticide Lufenuron Quarter field dose (0.5µl/ml)
LT50= 12.68±0.86
LT95= 22.98±1.82
Graph No 14. LT50 & LT95 of insecticide Lufenuron 1/10th field dose (0.2µl/ml)
LT50=13.05 ±0.90.
LT95= 23.96±1.94.
Statistical analysis:
Analysis for variance followed by Tukey test was applied to compare the efficacy of venom T. guatemalensis and T. javana and insecticide in treated groups by using SPSS. The analysis Shows maximum mortalities (100±0.00) at post 18hours treatment & (80.00±5.77) at post 24h treatment by 70µl/ml & 35µl/ml dilution, respectively. While T. javana (35µl/ml) shows mortality (36.66±5.77) at post 12h, and 17.5µl/ml mortality (43.33±3.33) at post 24h of treatment (Fig) The mortality in the control group was significantly lower than experimental groups. The degree of freedom and significant values are shown in table.
The statistical analysis of each dilution of venom of T. guatemalensis and T. javana and insecticide was observed for different hours i.e. 2h ,6h 12h 18h and 24h .It is clear from the Table: 4 that there was significant difference of different hours with different dilutions . The values for p=0.00, DF = 9, 20 were same for 2, 4, 12, 18, and 24 hours, for 8th hour the DF = 9, 19.
Table 4. Calculated mortalities in armyworm (Spodoptera exigua), by crude venom of Tetragnatha guatemelensis Tetragnatha javana & insecticide (Lufenuron) are given:
| Treatments | Time (Hours) | |||||
| 2 | 4 | 8 | 12 | 18 | 24 | |
| Control (Buffer) Mean ±SE | 0±0a | 0.00±0.00 a | 5.00±5.00 a | 6.66 ±6.66 a | 6.66±6.66 a | 6.66±6.66 a |
| TG1 (70 µL/ml) | 36.66 ±3.33c | 6.33±1.15 ab | 90.00±0.00f | 93.33 ±3.33 d | 100±0.00 d | 100.0±0.00 d |
| TG2 (35µL/ml) | 0±0 a | 0.00±0.00 a | 30.00±0.00 a | 46.66± 3.33b | 63.33±3.33 bc | 80.00±5.77bcd |
| TJ1 (35µL/ml)
mean ±SE |
0±0 a | 0.00±0.00 a | 30.00±0.00 a | 36.66± 5.77 b | 43.33±3.33ab | 43.33±3.33 ab |
| TJ2 (17.5µL/ml) mean ±SE | 13.33 ±11.54 a | 16.6±5.77 c | 26.66±3.33 a | 40.00± 0.00 b | 33.33±33.33 ab | 43.33±3.33 ab |
| Control Mean±SE | 0±0 a | 0.00±0.00 a | 0.00±0.00 a | 0.00 ±0.00 a | 100±0.00 ab | 33.33±33.33 ab |
| L1 Mean±SE | 16.66 ±5.77 b | 40.00±0.00d | 70.00±5.77 d | 100 ±0.00 d | 100±0.00 d | 100±0.00 d |
| L2Mean±SE | 6.66 ±5.77 a | 20.00±0.00 c | 46.66±3.33 c | 73.33± 3.33 c | 86.66±6.66 d | 100±0.00 d |
| L3 Mean±SE | 0±0 a | 13.33±5.77 bc | 30.00±0.00 b | 40.00± 0.00 b | 70.00±5.77bc | 96.66±3.33 d |
| L4 Mean±SE | 7.66± 12.2 a | 3.33± 5.77 a | 36.58 ± 3.33bc | 46.66± 3.33 b | 64.66± 6.44 bc | 70.3± 6.85cd |
| DF | DF9,20 | DF9,20 | DF9,19 | DF9,20 | DF9,20 | DF9,20 |
| SG | 0 | 0 | 0 | 0 | 0 | 0 |
The biopesticidal potential of venom extracted from Tetragnatha guatemelensis against armyworms was evaluated in table 4. Maximum mortality was observed in T.gutamulensis treated groups (70.00 ±11.54) after 6 hours of exposure. While T.javana shower least mortality at same time interval in the table no 5 is showing there is maximum mortality (70.00± 11.54) of subject pest armyworm (Spodoptera exigua) at 6th hour of post treatment by T.guatamelensis. While T. javana showed little mortality (16.66± 3.33).
Table 5. Biopesticidal potentials of different dilutions of Tetragnatha guatemelensis Tetragnatha javana venom and compatible insecticide.
| Treatment | Time interval (hour) | |||
| 6 | 12 | 18 | 24 | |
| control Tris HCl | 3.33a± 3.33 | 6.66 a ±6.66 | 6.66 a ± 6.66 | 6.66 a ± 6.66 |
| Venom T. G1 (70 µL/ml) | 70.00 c ±11.54 | 93.33 d ±3.33 | 100 d ± 0.00 | 100 d ± 0.00 |
| Venom T.G2 (35 µL/ml) | 26.66ab ±3.33 | 46.66b ±3.33 | 63.33 c ± 5.77 | 80 c ± 5.77 |
| Venom T.J1(35 µL/ml) | 16.66ab± 3.33 | 36.66 b ± 3.33 | 43.33 b ± 3.33 | 43.33 b ± 3.33 |
| Venom T.J2(17.5 µL/ml) | 20ab ±0.00 | 40.00 b ± 0.00 | 43.33 b ± 3.33 | 43.33 b ± 3.33 |
| Lufenuron (1µl/ml) | 30.0c± 0.00 | 73.33 c ± 3.33 | 100 d ±0.00 | 100 d ± 0.00 |
| Df | 5,12 | 5,12 | 5,12 | 5,12 |
| F | 18.5 | 61.92 | 101.97 | 83.64 |
| P | 0 | 0 | 0 | 0 |
Discussion
There is great diversity of organism that is damaging crops and reducing the rate of annual production in the whole world (Bergvinson et al., 2004). Various chemicals are used to control them, but unfortunately the pest may develop a resistance to these insecticides (Schumann et al., 1991). In searching for more organic and sustainable alternatives to those agrochemicals, a new variety of insecticides has been found in some organisms. Among these bio insecticides, spiders are the most significant (Platnick et al., 2012). Spiders are one of the most abundant natural enemies of pests which play a significant role in controlling pest population in different agro-ecosystems. They are the predator of lepidopteron pests (armyworm) (Hendawy et al., 2015).
Various studies have been conducted on spider venom of different species. Many researchers have accounted that spider venom have bio-pesticidal potential (Tedford et al., 2004). It is reported by (Zlotkin et al., 2000) that the peptide toxins present in spider venom have selective insecticidal effect. This biological activity of spider venom is a consequence of its main components: proteins, polypeptides, enzymes, nucleic acids, neurotransmitters, amino acids, and inorganic salts (Frew et al., 1994). In order to control insect pests specifically insect selective toxins are very effective to use them as bio insecticides.
In the present study bioassay tests were performed in order to assess bio-pesticidal potential of spider venom of Tetragnatha javana and Tetragnatha guatemelensis (Family Tetragnathidae, Order Araneae) has been assessed against armyworm (Spodoptera exigua). Different venom dilutions were applied topically on dorsal side of armyworm (Spodoptera exigua) and percent mortality was observed for 24 hours. The results of present study showed that the percent mortality of armyworm (100±0.00) was maximum in T. guatamelensis (70µL/ml). Whereas T .javana shows minimum mortality (43.33±3.33) in subject pest. There is no significant mortality seen in control groups. The venom of spider of family Tetragnathidae has neither been characterized nor been tested as biopesticidal potential against any insect pest. The previous studies on other species of spider venom showed that their venom has strong biopesticidal potential.
. Many researchers have indicated that spider venom is effective against insect pests. To check the toxicity of spider and scorpion venom, Manzoli et al., 1996 conducted experiments, he extracted venom from arachnid specie Tityas serrulatus & injected its venom in Diatraea saccharalis (Lepidoptera), which was used as model pest. The mortality test was of 24 hours. The calculated LD50 was 1057.00 (1190.00-1922.00). Helicoverpa armigera in its 2nd instar stage was injected with the venom of spider (Hadronyche). The toxicity was checked at different intervals. The LD50 was calculated at 12 hour of post treatment. It was observed that toxin Hvt-thioredoxin from spider venom cause reduce feeding activites in Helicoverpa armigera. The larvae was died after 48h of post treatment. So it was concluded that spider venom is useful for agriculture protection against insect pest (Khan et al., 2006).
Quistad et al., 1992 extracted venom from different spider species, and topically applied on pest. The mortality was tested against armyworm (5th instar), cucumber beetle (3rd instar), and spotted cucumber beetle. The LD50 0.64(0.58-0.55) was calculated. It was observed after 24 hour of post treatment that these pest species paralysed and dead due to potent activity of venom.
Crude venom from Agelenopsis aperta was extracted. And injected into abdomen of housefly, after 1h of post treatment it was seen 55% of insects lost their right response.The houseflies show tremors followed by paralysis due to venom toxins. The LD50 was calculated. The mortality (0.075±0.005) was seen after 24h of post treatment by µ Aga-1,(1.38± 0.08) by µ Aga-11, (0.58± 0.06) by µ Aga-111, (0.03 ±0.003) by µ Aga-1V, (0.08 ± 0.02) by µ Aga-V & (0.15±0.005) µ Aga-V1. This shows that Agelenopsis has powerful action potentials on housefly neuromuscular junction (Adams et al., 1989). Exposure of pesticides to honey bees cause mental impairment and effect on the foraging behavior of honeybees (Decourtye et al., 2008 and williumson et al., 2013). It is hence sensible to expect that more testing of pesticide cause poisoning to the pollinating bees , V-hexatoxin-Hv1 obtained from Australian web specie of spider, when injected into insects and mammals, it was observed acute toxicity in insects, but mammals were unaffected by it. Hv1a never show any lethal affect in insects when it is given orally, except when Galanthus nivalis agglutinin (GNA) is binded by Hv1a, so by the fusion of carrier molecule, the Hv1a is orally active, and cause toxicity in moths, armyworm, leafhoppers , and flies but there was even no lethality seen in butterflies. Following these tests, intense oral and contact toxicity of Hv1a/GNA is minor (LD50. 100 mg bee) (Fitches et al., 2012). When honey bees were infused with Hv1a/GNA, their mortality was 17% in 48 h. In contrast, lepidopteran larvae treated with small amount of fusion protein shows 90– 100% mortality (Fitches et al., 2012). We expect this level of mortality in honey bees is low when compared with the record of USEPA (United States Environmental Protection Agency), as it considered acute toxicity LD50 of compound is high when 11 mg of bees are are not harmed (Wright et al., 2014). This proposes the poison reach to the site of action in the CNS of lepidopteran species more than in bees, the reason is that the ion channel sites to which toxin binds are different. The survival of honey bees infused with GNA was decreased (ca 60%).
In their research, GNA was just utilized as a control. Hv1a/GNA did not impact on the survival in adult bees even after intense oral exposure. The low toxicity of Hv1a/GNA as compared to deadly impacts of neonicotinoids utilized it as positive control. Acetaminophen was intensely harmful for honey bees (Iwasa et al., 2003), and Tamoxifen ingestion at a field dosage had deadly impacts in nectar and pollen (Stoner et al., 2012 & Pohorecka et al., 2013). Other arthropod venom were also found effective, VHv1.1 obtained from the venom of wasp Campoletis sonorensis and fed to Heliothis virescens (moth) and Spodoptera exigua (armyworm), it was found lethal and both pests were died (Fath-Goodin et al., 2006).
OAIP-1 is a toxic component obtained from the venom of spider Australian feather leg tarantula. It is potent chemical, can easily block calcium activated potassium channels in pest (Gunning et al., 2008) and is the best activator of voltage gated ion channels, it was used against isoptera (termites), Tenebrio molitor (mealworms) and bollworms, LD50 value for bollworm was 104gmol/g (within 24–48 h),, which is higher than for mealworm and termites which were 171nmol/g and 350 nmol/g respectively. This shows that spider venom is more toxic than chemical insecticides.
Krapcho et al., 1995 conducted experiment to enhance the activity of bio pesticide. He used recombinant DNA technology. In this process toxic genes (vAcDTX9.2 and vAcTalTX-1) from desert bush spider and hobo spider was isolated and inserted into alfalfa looper nuclear polyhedrosis virus to enhance its activity. The response was observed in the lepidopteran pest and its feeding activity was noted. It was seen the cessation of feeding activity by 33% and mortality by 42% in those lepidopteran pests which were treated with vAcDTX9.2 and vAcTalTX-1 recombinant virus.
Acute toxicity of spider venom was observed by Nicholson et al., 2007. V-ACTX-Ar1a was isolated from venom and injected into in the house crickets. It was observed an increased muscle contraction, spasms, and cessation in feeding activity followed by paralysis of crickets. The LD50 value of v-ACTX-Ar1a was 236.28 pmol/g, after 48 hours The toxicity reach to an extent that they could not recover themselves even after 72 hours of post treatment.
A spider Macrothele gigas contain a Magi 6 toxic peptide in its venom, Hernández-Campuzano et al., 2009 derived the transgene of Magi 6 and its bioassay was used against the larval stages of armyworm (Spodoptera frugipera) on tobacco. The mortality was found 100% in larval stages of pest and scientist consider Magi 6 as one of the most suitable commercial biopesticide.
Conclusion
In the light of above discussion it can be concluded that the topical bioassays of Tetragnatha guatamalensis and Tetragnatha javana venom on Lepidopteran pest (Spodoptera exigua) showed more insectidal potentials. Lepidopteran species are regular pests in agriculture and cause great losses in annual crop production. The results of laboratory bioassays confirmed that pests couldn’t protect itself from lethal impacts of spider venom.
Spiders are found in great diversity and abundance, their venom has shown lethal effects on the subject pest. Based on the power of its poisons, it can be prescribed as lead organism for bio-pesticides. To cope with current challenges of loss in crop production, there is need to conduct more research on spider venom bio pesticidal potentials, its insect selective potency can be used for the biological control of pests. However there is dire need for the safety concerns, accessibility and proper profiling of new bio-pesticides.