SUMMARY
The aim of this study was to evaluate the bio-pesticidal potential of (Peceutia viridans, Family Oxyopidae, Order Araneae) against armyworm (Spodoptera exigua: order Lepidoptera: Noctuidae). Spiders were collected from the trees of Acacia in the vicinity of Sahiwal (Sargodha) Punjab, Pakistan. They were placed in different container in order to avoid cannibalism. The spiders were immobilized by placing them at low temperature of -4ºC for 5-10 minutes in the freezer. The immobilized spiders were dissected under stereomicroscope (Labomed-7GA9) and their chelicerae along with the venom gland were separated with the help of a set of needles and forceps (Guerrero et al., 2010). The venom glands were immediately placed in eppendorffs of 1.5 ml having 0.5 ml of 0.05 M tris HCl buffer (pH 8.2) (Frontali et al., 1976).
The biopesticidal potential of venom (different dilutions) of Peceutia viridians against armyworm was evaluated. Different dilutions of insecticide Chlorantraniliprole were also prepared and percent mortality was observed in each dilutions of insecticide (Chlorantraniliprole). The comparison was also done to see the percent mortality in different dilutions of venom and insecticide.
The outcomes of the study showed that venom of Peceutia viridians has biopesticidal potential against armyworm. The results also showed the efficacy of spider venom as biopesticide is comparable to insecticide (Chlorantraniliprole). The results of the present study will be useful for further study on spider venom and will give information about applications of spider’s venom in agriculture. The results will also beneficial for industry of pesticides.
INTRODUCTION
Pest organisms cause destruction of approximately 14% of the world’s crops and 20% of the reserved food grains (Oerke & Dehne, 2004; Peshin et al., 2007) that results in an approximate damage of 100 billion USD every year (Carlini & Grossi-de-Sá, 2002). Including other insect pests, Armyworm (Spodoptera exigua) is one of the major pest of cotton in Pakistan. It causes destructive damage to different fields and ornamental crops. Agrochemicals have been used to effectively control insect pests. However, these agrochemicals have adverse impacts on human health and environment.
Recurring use of insecticides have developed resistance in almost all insect vector species (WHO 1992; Georghiou, 2008). Natural enemies play a vital role in the pest management of agricultural crops (Lang et al., 1999; Marc et al., 1999; Tahir & Butt, 2009). Insecticides impart negative impacts on the non target animals including natural enemies (Devine et al., 2007; Paul & Thygarajan, 1992). Due to these problems researchers are being focused to discover novel insecticides with target specific and eco-friendly nature.
The venom of spiders consists of different biologically active compounds that have different chemical nature. Their venom adapts the activity of neuronal ion channels and receptors to paralyse the prey. The venom of spiders consists of 10 millions bioactive peptides that have different applications in agriculture (King et al., 2008; Tahir et al., 2016). Spider’s venom comprises of a diverse range of compounds including peptides, proteins salts, and small organic molecules (Escoubas et al., 2000; Escoubas, & Tarantulas, 2004; Estrada et al., 2007; Rash & Hodgson, 2002; Tedford et al., 2004; Vassilevski, 2009). The peptides are the major fraction of spider venom and some species have more than 1000 distinctive peptides of 2–8 kDa mass (Escoubas et al., 2006). These peptides have high affinity and specificity for varied range of sites including membranes, receptors, enzymes and ion channels (Saez et al., 2010).
Using molecular mass the compounds found in spider venom are generally classified which are:-
- Those having low molecular weight (˂1 kDa). In this category the most common are amines, nucleosides, polyamines, organic acids, nucleotides, amino acids 2. Peptides having size 1-10 kDa.
- Peptides having high molecular weight (Adams, 2004; Escoubas et al., 2000; Vassilevski et al., 2009).
The composition of venom varies widely from species to species and its composition depends on various factors that may include sex, diet, natural habitat and climate etc (Adams, 2004; Bettini et al., 1978; Escoubas et al., 2008; Herzig et al., 2008; Kuhn-Nentwig et al., 2004; Liang, 2008; Mebs, 2002; Vapenik & Nentwig, 2000; Wullschleger et al., 2005). The amount of venom put in the prey depends upon the size of the prey and their vulnerability. If the amount of venom exceeds than needed then spiders wisely retreat it (Kuhn-Nentwig et al., 2004; Wigger et al., 2002; Malli et al., 1999; Boeve et al., 1995).
Bioinsecticides are more efficient and safer alternatives to the agrochemicals. It is investigated that the peptides obtained from the spider venom are potential source of biopesticides. Spiders are able to efficiently kill or paralyse their prey or predators rapidly. They use their venom to paralyse or kill their prey. Their venom consists of various types of peptides and insect selective toxins. These neurotoxins affect the nervous system of prey and thus cause lethality to their prey. These insect selective toxins in the venom provide the base for the improvement of biopesticides (Windley et al., 2012).
Agriculture plays a vital role in the economy of Pakistan. It is very important to introduce insect selective bio-pesticide to increase the crop yield while protecting non target species and environment. Previous studies have shown that spider venom has bio-pesticide potential but in Pakistan, very little work has been conducted in this regard. There is a dire need to conduct studies on spider venom to cope with agricultural challenges. There is no published data on characterization and bio-pesticidal potential of the venom of Peceutia viridians (Araneae: Oxyopidae). The present study was conducted on the spider’s venom of Peceutia viridians and bio-pesticidal potential was evaluated against armyworm (Spodoptera exigua) (2nd instar) as pest organism. The results of the present research work will be valuable to give information about applications of spider’s venom in agriculture. It will also beneficial for industry of pesticides. It is the need of time to control the armyworm (Spodoptera exigua) in order to save our crops. The present study was aimed to evaluate the activity of venom (Peceutia viridian) venom against the larvae of armyworm (Spodoptera exigua).
Study objectives
The followings are main objectives of the present studies:
- To extract venom of Peceutia viridans
- To evaluate biopesticidal potential of venom against 2nd instar of armyworm in the laboratory
- To compare biopesticidal potential of Peceutia viridans venom with a commonly used insecticide.
REVIEW OF LITERATURE
Spider’s venom is very important and varied widely because of their neurotoxic (Corzo et al., 2000; Gwee et al., 2002), cytotoxic (Corzo et al., 2003), necrotic (Richardson, 2007; Goddard, 2007) properties and have diverse applications in agriculture. Their venom and haemolymph consist of both neurotoxic and cytotoxic ingredients, which are chemically related to glucose, nucleic acids, free acids, inorganic ions for example Calcium, Magnesium, Sodium, Potassium, Chloride ions and neurotransmitters. These ions cause prey immobilization and improve venomous properties (Escoubas et al., 2000; Wullschleger et al., 2005; Pukala et al., 2007).
Several studies have been done to see the impact of spider venom in the ion channels of victims (Vieira et al., 2007). The Brazilian wondering spider Phoneutria nigriventer contains a variety of neurotoxins in their venom, that are active in the presence of Na+, K+ and Ca2+ ion channels (Carneiro et al., 2003; Matavel et al., 2002).
Vetter et al. (2011) evaluated that the most distinguishing and the most well-known targets of spider-venom are lipid bilayer, voltage gated channels of Na+, K+ and Ca2+.
Wei et al. (2005) noted that Voltage-activated channels of potassium cause the neurons excitation, regulation of heart rate, cell volume, contraction in smooth muscles, release of neurotransmitter and cell signaling.
Windley et al. (2012) also revealed that the peptides present in the spider venom are potent source of bioinsecticides. They demonstrated that the spider venom has plenty of hyperstable insecticidal mini-proteins that inflict the lethality in insects by modulating ion channels, receptors or enzymes. They also evaluated that about 800 bioactive peptides have characterized from spider-venom and out of these, 136 peptides exhibit insecticidal activity. It is also found that the effective insect-selectivity is shown by 38 peptides among these 136 peptides and 34 are revealed to be non-selective and 64 have indefinite selectivity.
Sollod et al. (2005) found that spider’s venom has developed a diverse peptide library of enzymes, cytolytic peptides and neurotoxins. Spiders have possessed these peptides in their venom glands to expand their toxin pool.
Gao et al. (2005) determined that in spite of rich in neurotoxic peptides, spider venom also pocesses a variety of peptidic toxins having cytolytic, hemolytic, and enzyme inhibitory activity. The peptides present in the spider venom have selective insecticidal activity.
The widow spider’s (Theridiidae) venom has five peptides (latroinsectotoxins) specific for insect. These peptides have selective insecticidal action (Grishin, 1998; Graudins et al., 2012).
Nicholson, (2007) noted that for various types of insecticides and neurotoxins, the voltage-gated channel of sodium is an important target. These insecticides and neurotoxins attach to minimum seven known neurotoxin binding sites and cause either modulation in voltage-gated sodium (Nav) channel or block nerve conductance. Various types of peptide neurotoxins are isolated and characterized from the venoms of araneomorph and mygalomorph and it is also evaluated that these peptides interact with a number of these sites. Out of these toxins, numerous spider toxins prove to be phyla-specific and are believed to be as leading chemicals for the enhancement of biopesticides. It is found that Hainantoxin-I from Ornithoctonus hainana interact with target site-1 and block the conductivity of sodium voltage channels. Magi 2 and Tx4 (6-1) interact with site-3 and cause slow Nav channel inactivation.
It is revealed that the selective peptide, lycotoxin-1 from the wolf spider’s venom has insect neuroactive properties. It has amphipathic nature and various physiological actions. It forms pores in the membrane and thus increases the membrane permeability, disperse voltage gradient and leads to cells’ lysis in insects. These properties proposed the lycotoxin-1 as a potential use of bioinsecticides (Yan et al., 1998).
Catterall et al. (2007) determined that toxins are produced by spiders (araneomorph and mygalomorph family) and these toxins affect the channels of NaV. They also found that these toxins change the neuronal excitability and thus cause paralysis and eventually lead towards the death of insects.
ω-Hexatoxin-Hv1a is seperated from the Australian funnel-web spiders’ venom. This toxin is highly target specific for the insect’s CaV channels. That’s why it can block Ca voltage gated channels of insects (Chong et al., 2007; Fletcher et al., 1997; Wang et al., 1999). In addition, it has no influence on the calcium currents present in the nervous system of rats (Atkinson et al., 1996). For this reason, it is non toxic for vertebrates still when used in 10,000 times higher concentrations (Khan et al., 2006).
Gunning et al. (2008) revealed that κ-Hexatoxin-1, is obtained from Australian funnel-web spider’s venom that has potential to selectively target the potassium channels of insects. Rash and Hodgson (2002) found that venoms of arachnids consist of proteins which are biologically active toxins when these are inserted into latent prey. Almost all are small proteins consisting of amino acid residues (known as peptides or proteins). These cause paralysis of prey mainly by targetting the neuronal ion channels and to some extent neuronal receptors and presynaptic membrane proteins.
Vassilevski et al. (2009) determined that some toxins of spiders’ venom cause a high toxicity for insects but have no effects on members of other taxons due to evolutionary selection. Whetstone and Hammock (2007) suggested that spider’s neurotoxins can be used in pest management techniques due to effectiveness, target specific mode of action and environment friendly.
Catterall et al. (2005) evaluated that in excitable cells, NaV channels are very important in increasing action potential. In contrast to vertebrates, insects explicit only one type of Sodium Voltage channel (King et al., 2008a). These are very responsive toward NaV modulators, as it is a verity that numerous classes of insecticides (e.g., DDT, indoxacarb, N-alkylamides, dihydropyrazoles and pyrethroids) are modulators of NaV channel (Bloomquist et al., 1996; Soderlund & Knipple, 1995; Zlotkin, 1999). The presence of toxins of NaV channel in the venom of spider proposed that these peptides have the capacity to be used as functional insecticide to manage the arthropod pests (Maggio et al., 2010; Windley et al., 2012).
Ekberg et al. (2008) determined that there are two main mechanisms by which the venom peptides alter channel activity. They either stop the channel pore or they bring conformational changes in the channel by binding to allosteric site that modify the balance among the open, closed and inactivated stages.
Vassilevski et al. (2013) found that the venom extracted from lynx spider Oxyopes takobius has two-domain modular toxins namely spiderines (OtTx1a, 1b, 2a, 2b) other than the conservative neurotoxins and cytotoxins. They also determined that these toxins have effective insecticidal activity.
Spiders are extensively recognized to make strong and selective toxins (Nentwig et al., 2011; Vassilevski et al., 2009). The black widows (genus Latrodectus, some are hazardous to humans) produce a range of latrotoxins (a-latrotoxin, a-latrocrustatoxin, latroinsectotoxins), that causes definite fatal influences in mammals, crustaceans and insects, respectively (Grishin, 1998).
Adams (2004) noted that venom of spiders consists of very different compounds having definite biological potential. For example, Agelenopsis aperta generates a range of α-agatoxins (that block post-synaptic glutamate receptors), l-agatoxins (Na channel peptide activators) and x-agatoxins (various Ca channel peptide blockers). In most examined species it is found that the peptidic components are mainly abundant and functionally important and there are two types of peptides either neurotoxins or cytotoxins. Neurotoxins typically consist of disulfide bridges that are similar to the ICK/knottin fold (Escoubas & Rash, 2004). Cytotoxins are characteristically linear peptides and these peptides take on helical conformation when they interact with lipid membranes (Nentwig, 2003).
Ushkaryov et al. (2004) determined that Latrotoxins are larger toxins obtained by the black widow spider and associated species provoke neurotransmitter discharge and thus played a central role in the blockage of exocytosis of the synaptic vesicle. The majority of peptides extracted from spider venoms target the voltage-gated potassium (KV) (Swartz & MacKinnon, 1995), calcium (CaV) (Adams, 2004; King, 2007) or sodium (NaV) channels (Adams, 2004; King et al., 2008).
Grishin in 1999 noted that venoms of spider have various types of toxic units. The polypeptide toxins have been categorized into two types, low and high molecular weight. Small polypeptide toxins act together with cationic channels and show spatial structure homology. These polypeptides toxins can influence the activity of Ca+2, K+, or Na+1 channels. A group of toxic proteins having high molecular weight was found in the venom of the spider genus Latrodectus. These neurotoxins, latrotoxins, cause an immense release of neurotransmitters by a variety of nerve endings. The latrotoxins have nature of proteins and consist of about one thousand residues of amino acids. These are highly identical in structure.
In the last few years, some transgenic plants have been produced that express spider toxins. The ώ-ACTX-Hv1a toxin gene from the Australian funnel web spider (codes for a 37-amino-acid and antagonistic to calcium channel) was artificially formed for plants with the most effective use of codon. This toxin gene has power to express in all tissues of tobacco (Khan et al., 2006). Hundred percent mortality occurred in two types of caterpillars, (i. cotton bollworm Helicoverpa armigera, ii. cotton leafworm Spodoptera littoralis) by these transgenic plants within 2 days.
Similar toxin was isolated from species Hadronyche versuta, which was very efficient against cotton bollworm caterpillars when it has shown expression only in the phloem of transgenic tobacco (Shah et al., 2011). It is forecasted that toxin obtained by a spider Macrothele gigas, have insecticidal potential, but their mode of activity is unknown (Campuzano et al., 2009). It is reported that in the transgenic plants there is no morphological abnormalities caused by the expression of the spider toxin (Campuzano et al., 2009; Khan et al., 2006), thus demonstrating that these toxins have no targets for plants. The studies revealed that expression of spider toxin peptides in transgenic plant is viable for the control of insects.
In the venom of Chinese black earth tiger tarantula, the major constituent is μ-Theraphotoxin-Hhn2b (μ-TRTX-Hhn2b) (Liang et al., 1999). It has high affinity to block the channels of insects (Li et al., 2003). Insecticidal toxins have been extracted from Japanese funnel web spider Macrothele gigas (Corzo et al., 2003). It is revealed that μ-hexatoxin-Mg1a (μ-HXTX-Mg1a) particularly have much affinity and specificity for NaV channel of insect that leads towards the paralysis in insects larvae.
MATERIALS AND METHODS
3.1. Collection of spiders:
Green lynx spiders (Peceutia viridans, Family Oxyopidae, Order Araneae, and Genus Oxyopes) were collected from the trees of acacia present in Sahiwal (Sargodha) Punjab, Pakistan. A total of 250 spiders were collected by beating the branches of acacia trees. Spiders were kept in separate containers (7cm height × 8.5cm diameter and 250cm3 volume) to avoid cannibalism. To provide proper ventilation, the containers were covered with a mesh cloth protected with a rubber band.
3.2. Extraction of venom:
Spiders were kept without food for 2 days before venom extraction to retrieve the maximum amount of venom (Nagaraju et al., 2007). In order to immobilize the spiders, they were placed at low temperature of -4ºC for 5-10 minutes in the freezer. The immobilized spiders were dissected under stereomicroscope (Labomed-7GA9) and their chelicerae along with the venom gland were separated with the help of a set of needles and forceps (Guerrero et al., 2010). The venom glands were immediately placed in eppendorffs of 1.5 ml having 0.5 ml of tris HCl buffer (0.05 M, pH 8.2) (Frontali et al., 1976). On average 70 to 80 venom glands were placed in each eppendrof and venom glands were homogenized by using small plastic rod. The homogenate were centrifuged in refrigerated centrifuge at 20,000 G for 20 minutes at 4ºC (MPW-352R). The supernatant were separated by using micropipette and kept it at −20°C in Ultra Freezer (Arkteco ULTF 220) until use (Frontali et al., 1976).
3.3. Collection of Armyworms (pest):
Armyworm (Spodoptera exigua: order Lepidoptera: Noctuidae) were collected from the agriculture college of Sargodha in august 2017. The experiment was conducted on the 2nd instars larvae of armyworm. The second instars larvae of armyworm were identified by having green body without patches and head of yellowish orange color.
3.4. Evaluation of bio-pesticidal potential of venom through laboratory bioassay:
A laboratory Bioassay of spider venom was conducted on armyworms. In order to evaluate the biopesticidal potential of venom, 5µl of different venom concentrations were applied topically. 10 larvae were placed in separate plastic cups. These are divided into four groups, control (C) and experimental (Ex) group i.e. PV1 (160 µL of venom), PV2 (80µL of venom) and PV3 (53.3 µL of venom).
3.5. Venom’s dilutions of Peucetia viridans:
Each venom gland of Peucetia viridans contains approximately 0.5 µL venom. Three dilutions of venom were prepared. Control group contains Tris-HCl buffer of 8.8 pH and 0.05 Molarity. In order to make Tris-HCl buffer (0.05M, 8.8 pH), 121.1 g of tris base was mixed in 800ml of water and pH was maintained at 8.8. Water was added to make a final volume up to 1000ml.
1st dilution was prepared by mixing 160 µL of venom of Peucetia viridans in 0.5ml of Tris-HCl buffer (PV1). 2nd dilution was prepared by mixing 80 µL of venom of Peucetia viridans in 1ml of Tris-HCl buffer (PV2). 3rd dilution was prepared by mixing 53.3 µL of venom of Peucetia viridans in 1.5ml of Tris-HCl buffer (PV3).
3.6. Laboratory Bioassay:
The experimental groups were treated topically with 5µl of different concentrations (PV1, PV2, and PV3) of Peucetia viridans venom by using micropipette. The control group was treated with 5µl of Tris-HCl buffer. The mortality was evaluated in the control and experimental groups. In order to get consistent results each experiment was repeated thrice.
3.7. Dilutions of Insecticide (Chlorantraniliprole, 40SC):
Different dilutions of insecticide Chlorantraniliprole i.e. field dose (C1), half field dose (C2), quarter field dose (C3) and 1/10th (C4) field dose were also prepared to check comparison of mortality of armyworm (Spodoptera exigua) in different dilutions of venom and insecticide (Chlorantraniliprole).
0.8ml of Chlorantraniliprole was mixed in 1000ml of distilled water to form field dose or we can say 0.8µL/ml. Half field dose was prepared by taking 0.4ml solution of field dose and mixed it with 1000ml of Distilled water or it can be 0.4µL/ml solution of Chlorantraniliprole (40SC). In order to make quarter field dose 0.2ml of Chlorantraniliprole was mixed in 1000ml of distilled water or we can say 0.2 µL of Chlorantraniliprole in 1mL of distilled water. 0.08ml of Chlorantraniliprole was mixed in 1000mL of distilled water to form one-tenth of field dose or it can be 0.08 µL of Chlorantraniliprole (40SC)in 1mL of distilled water.
Different concentrations of Chlorantraniliprole (40SC) were applied topically on armyworm and mortality was checked for each concentrations of insecticide Chlorantraniliprole. Mortality of armyworms was evaluated after one hour till 24 hours of exposure.
RESULTS
4.1. Bioassay for Spodoptera exigua:
Laboratory bioassay was performed to check the bio-pesticidal potential of crude venom of Peucetia viridans at different dilutions i.e. PV1 (160 µL/ml), PV2 (80µL/ml) and PV3 (53.3 µL/ml) against armyworm (Spodoptera exigua). Percent mortality of armyworm was recorded for 24 hours. In PV1 treated group, higher mortality (100%) of armyworm was observed at 18 hours of exposure as compared to PV2 and PV3 (80%), whereas 100% mortality could be achieved only after 24 hours in the last two dilutions. In the control group, Tris-HCl buffer, the percent mortality was very low (10%) than the experimental groups after 24 hours (Table 1).
Table.1. Percent mortality in army worm (Spodoptera exigua) exposed to the venom (different dilutions) of Peucetia viridans.
| Treatments
(Concentrations µl/ml) |
% Mortality | |||
| Time in hours | ||||
| 6h | 12h | 18h | 24h | |
| Control(Tris-HCl buffer) | 0 | 0 | 0 | 10 |
| PV1 (160 µL/ml) | 40 | 80 | 100 | 100 |
| PV2 (80µL/ml) | 20 | 50 | 80 | 100 |
| PV3 (53.3 µL/ml) | 20 | 40 | 80 | 100 |
Armyworm (S. exigua) were also exposed to different concentrations of Chlorantraniliprole i.e. field dose (C1), half field dose (C2), quarter field dose (C3) and 1/10th of the field dose (C4) and percent mortality was observed in each treated group. After 18 hours of exposure, the highest mortality (100%) was examined in C1 and C2 followed by C3 (90%) and C4 (80%) in which 100 % mortality was found after 24 hours (Table 2).
Table.2. Percent mortality in army worms (Spodoptera exigua) treated with different concentrations of Chlorantraniliprole.
| Treatments
(Concentrations µl/ml) |
% Mortality | |||
| Time in hours | ||||
| 6h | 12h | 18h | 24h | |
| Control(H2oµL/ml) | 0 | 0 | 0 | 0 |
| C1 (0.8µL/ml) | 60 | 90 | 100 | 100 |
| C2 (0.4µL/ml) | 40 | 70 | 100 | 100 |
| C3 (0.2µL/ml) | 30 | 60 | 90 | 100 |
| C4 (0.08µL/ml) | 20 | 50 | 80 | 100 |
Lethal Time for armyworm (S. exigua) was also calculated from probit analysis. The LT50 and LT95 was 8.04± 0.72 and 15.26± 1.35, respectively when treated with PV1 (160 µL/ml) at 12 hours (Figure.3).
LT50 8.04± 0.72
LT95 15.26± 1.35
In PV2 (80 µL/ml) treated group the LT50 was 11.95± 0.84 and LT95 was 21.53± 1.64 after 12 hours exposure (Figure.4).
LT50 11.95± 0.84
LT95 21.53± 1.64
LT50 and LT95 for armyworm (S. exigua) treated with PV3 (53.3 µL/ml) was 12.90± 0.91 and 23.99± 1.97, respectively at 12 hours post treatment (Figure.5).
LT50 12.90± 0.91
LT95 23.99± 1.97
Armyworm (S. exigua) was treated with field dose (C1) (0.8µL/ml) of insecticide Chlorantraniliprole. The LT50 and LT95 of armyworm (S. exigua) at 12 hours exposure of, insecticide, Chlorantraniliprole was also calculated through probit analysis. The LT50 and LT95 was 4.73± 0.99 and 12.94± 1.54, respectively (Figure.6).
LT50 4.73± 0.99
LT95 12.94± 1.54
The LT50 and LT95 for armyworm (S. exigua) was 7.015± 1.01 and 17.35± 1.74, respectively when exposed to Half Field Dose (C2) (0.4µL/ml) of insecticide Chlorantraniliprole at 12 hours (Figure.7).
LT50 7.015± 1.01
LT95 17.35± 1.74
When armyworm (S. exigua) treated with Quarter Field Dose (C3) (0.2µL/ml) of insecticide (Chlorantraniliprole) the LT50 was 8.06± 0.88 and LT95 was 17.42± 1.60 at 12 hours of exposure (Figure.8).
LT50 8.06± 0.88
LT95 17.42± 1.60
At 1/10th Field Dose (C4) (0.08µL/ml) of insecticide Chlorantraniliprole, the LT50 for armyworm (S. exigua) was 12.17± 0.87 and LT95 was 22.59± 1.78 at 12 hours post treatment Figure.9.
LT50 12.17± 0.87
LT95 22.59± 1.78
Statistical Analysis
Biopesticidal potential of different dilutions of venom, Peucetia viridian, was compared with half field dose (C2:0.4µL/ml) of insecticide, Chlorantraniliprole, by applying one way ANOVA (SPSS-13). Percent mortality was observed against armyworm (S. exigua) at different time intervals of exposure i.e. 6, 12, 18 and 24 hours. After 12 hours exposure of C2 (0.4µL Chlorantraniliprole/ml) the percent mortality (73.33±3.33) was observed in armyworm (S. exigua). In PV1 (160 µL/ml) treated group higher mortality (76.66±3.33) was observed at 12 hours post treatment. Percent mortality of armyworm (S. exigua) in PV2 and PV3 was lower as compared to PV1 and C2 at 12 hours (Table.3). At 18 hours post treatment, PV1 (160 µL/ml) and C2 (0.4µL/ml) showed same mortality (100±0.00). After 24 hours of exposure, similar mortality (100±0.00) was observed in PV1, PV2, PV3 and C2 treated groups. Little mortality (10±0.00) was observed in control group at 24 hours post treatment (Table.3).
Table.3. Comparison of activity of venom (different dilutions) of Peucetia viridans and half field dose of Chlorantraniliprole C2 (0.4µL/ml) on armyworms.
| Treatments (Concentrations µl/ml) | % Mortality | |||
| Time in hours | ||||
| 6h | 12h | 18h | 24h | |
| Control(Tris-HCl buffer) | 0.00± 0.00 | 0.00±0.00 | 0.00±0.00 | 10.00±0.00 |
| PV1 (160 µL/ml) | 36.66±3.33 | 76.66±3.33 | 100±0.00 | 100±0.00 |
| PV2 (80µL/ml) | 23.33±3.33 | 46.66±3.33 | 80.00±0.00 | 100±0.00 |
| PV3 (53.3 µL/ml) | 20.00±0.00 | 40.00±0.00 | 76.66±3.33 | 100±0.00 |
| C2 (0.4µL/ml) | 43.33±3.33 | 73.33±3.33 | 100±0.00 | 100±0.00 |
| P value | 0.00 | 0.00 | 0.00 | . |
| df | 4,10 | 4,10 | 4,10 | 4,10 |
| F | 42.167 | 143.667 | 769.00 | . |
Percent mortality in armyworms (S. exigua) was examined against venom of Peucetia viridians at different time intervals. At the end of experiment, 24 hours post treatment, control group showed (10.00±0.00) mortality. Maximum mortality (100±0.00) was observed in PV1 and C2 treated group after 18 hours of exposure. PV2 and PV3 treated groups showed (80.00±0.00) and (76.66±3.33) mortality, respectively in which (100±0.00) mortality could be achieved at 24 hours post treatment (table.4).
Table.4. Comparison of activity of different concentrations of Peucetia viridans venom and half field dose C2 (0.4µL/ml) at different time intervals.
| Time in hours
|
% Mortality | ||||
| Treatments (Concentrations µl/ml) | |||||
| Control(Tris-HCl buffer) | PV1 (160µL/ml) | PV2 (80µL/ml) | PV3 (53.3µL/ml) | C2 (0.4µL/ml) | |
| 6 Hrs | 0.00±0.00 | 36.66±3.33 | 23.33±3.33 | 20.00±0.00 | 43.33±3.33 |
| 12 Hrs | 0.00±0.00 | 76.66±3.33 | 46.66±3.33 | 40.00±0.00 | 73.33±3.33 |
| 18 Hrs | 0.00±0.00 | 100±0.00 | 80.00±0.00 | 76.66±3.33 | 100±0.00 |
| 24 Hrs | 10.00±0.00 | 100±0.00 | 100±0.00 | 100±0.00 | 100±0.00 |
| P | . | 0.00 | 0.00 | 0.00 | 0.00 |
| df | 3,8 | 3,8 | 3,8 | 3,8 | 3,8 |
| F | . | 160.667 | 209.83 | 465.00 | 131.167 |
DISCUSSION
In the present study, bio-pesticidal potential of spider venom of Peceutia viridans (Araneae: Oxyopidae) has been assessed against armyworm (Spodoptera exigua). Different dilutions of spider’s venom were applied topically on dorsal side of armyworm (Spodoptera exigua) and percent mortality was observed for 24 hours.
The outcomes of present study demonstrated that the percent mortality of armyworm was higher in PV1 (160 µL/ml) as compared to other dilutions (PV2 and PV3) of venom (Peceutia viridians). Concentrated venom (PV1) of Peceutia viridans showed the highest mortality (100%) at 18 hours exposure and other dilutions of venom PV2 and PV3 showed (80%) and (76.66%) mortality, respectively.
PV2 (80µl in 1 ml of buffer) and PV3 (53.33µl in 1.5 ml of buffer) showed lesser mortality as compared to PV1 (160µl in 0.5ml of buffer).
In control group (Tris-HCl buffer), very small mortality i.e. (10.00±0.00) was observed in armyworm. The probable reason of mortality in control group is that, it may be due to the stress or it might be due to the changed environmental conditions in laboratory and field.
The percent mortality of armyworm was also observed with different concentrations of insecticide, Chlorantraniliprole, at different time intervals. At 18 hours post treatment, the highest mortality was observed in C1 and C2 followed by C3 (90%) and C4 (80%) in which 100% mortality was achieved at 24 hours of exposure. It shows that as concentration of insecticide is diluted, the percent mortality of armyworms decreased. The control group (water) showed no mortality after 24 hours of exposure.
The comparison of different concentration of venom (Peceutia viridans) and half field dose of Chlorantraniliprole (insecticide) was also made. The results of crude venom (PV1) and half field dose (C2) of Chlorantraniliprole (insecticide) were comparable. Both showed same mortality (100%) at 18 hours post treatment. This shows that spider venom is quite effective as insecticide (Chlorantraniliprole) against armyworm.
Various studies have been conducted on spider’s venom of different species. Many researchers have accounted that spider venom has bio-pesticidal potential (Windley et al., 2012). Spider venom contains various inorganic and organic compounds of low molecular weight like biogenic amines, salts, amino acids, carbohydrates and AP etc (Bettini et al., 1978; Mebs et al., 2002; Rash & Hodgson, 2002; Schroeder et al., 2008).
It is reported by (Herzig et al. 2011) that the peptide toxins present in spider venom have selective insecticidal effect. This biological action of spider venom is a consequence of its main components: amino acids, polypeptides, proteins, enzymes, neurotoxic and biogenic amines, neurotransmitters, inorganic salts and nucleic acids (Jackson & Parks, 1989; Meinwald & Eisner 1995; Horni et al., 2000; Mellor & Usherwood, 2004). In order to control insect pests specifically insect selective toxins are very effective to use them as bioinsecticides.
Fletcher et al. (1997) successfully extracted ω-Hexatoxins (previously known as ω-atracotoxins) from the fatal Blue Mountains funnel-web spider venom. They also determined that ω-Hexatoxins are peptides consist of ~37 residues.
The main constituents in the Australian funnel-web spider’s venom are also the ω-hexatoxins (Fletcher et al., 1997; Chong et al., 2007; Wang et al., 1999). They have the capacity to particularly block the voltage gated calcium (CaV) channels of insects, but no effect on vertebrate, and so play a vital role in prey immobilization (Chong et al., 2007; Fletcher et al., 1997; Tedford et al., 2004; Tedford et al., 2001; Tedford et al., 2007). These peptides can be used as bioinsecticides due to their effective insecticidal activity (King et al., 2013; Khan et al., 2006; Tedford et al., 2004).
The cation concentrations in the venom of wandering spider C. salei are ~10 mM Na+, ~200 mM K+, and ~1 mM Ca2+ (Kuhn-Nentwig et al., 2004; Kuhn-Nentwig et al., 1994). Due to high concentration of potassium ions, the venom draws in extraordinary consideration. At this concentration they cause lethality in prey due to depolarization induced by potassium.
Armyworm (Lepidoptera: Noctuidae) is the main insect pests that cause destructive damage to cotton crops (Holloway, 1989). In India, its larvae induce 60% damage to various crops and vegetables (Grand et al., 1984). The study was conducted by Gary et al. (1992) to see the effect of spider venom against army worm’s mortality. The venom was extracted from different species of spiders and was applied topically on armyworm. The mortality was recorded in armyworm (5th instars), spotted cucumber beetle and cucumber beetle (3rd instar). The LD50 of 0.64(0.58-0.55) was noted. After 24 hours exposure these species of pests undergoes paralysis and died due to effective action of spider venom.
Basit et al. (2013) reported that in Pakistan, farmers are totally dependent on insecticides in order to control lepidopterous insect pests. Repeated application of insecticides caused resistance in S. litura species against various insecticides (Ahmad et al., 2007; Saleem et al., 2007; Sayyed et al., 2006). Different insecticides also cause environmental problems and hazardous to human health.
Due to these problems it is better to use spider venom against different insect’s pest as compared to insecticides. But further studies on spider venom are required to see its potential against different insect pest species in the fields. The present study was conducted in the laboratory. The venom of Peceutia viridians showed biopesticidal potential in the laboratory but it is necessary to see that either it is thermostable or not in the fields.
CONCLUSION
It can be concluded from the above discussion that venom of Peceutia viridians has biopesticidal potential against armyworm (Spodoptera exigua). The possible mechanism of the venom (Peceutia viridians) is either alters the activity of channels (Na+1, K+, Ca+2) or blocks them. Our present study showed that mortality caused by concentrated venom (PV1: 160 µL of venom), was higher as compared to other dilutions, PV2 (80µL of venom) and PV3 (53.3 µL of venom).
Our present study also revealed that mortality caused by venom of Peceutia viridians in armyworm was comparable to insecticide (Chlorantraniliprole). Both PV1 (160 µL of venom) and C2 (0.4µL/ml) showed same mortality (100%) after 18 hours post treatment.
Thus these outcomes indicate the biopesticidal potential of spider’s venom and its uses in further research. Although the biopesticidal potential of spiders venom has generated a hope for effective management of insects pest but further studies is needed to see the effect of spiders venom on other biological fauna such as honey bees and other pollinators.