Toxicity and Biochemical Effects of Four Plant Essential Oils Against Cotton Leafworm, Spodoptera littoralis (Boisd)

The journal of Toxicology and pest control is one of the series issued twice by the Egyptian Academic Journal of Biological Sciences, and is devoted to publication of original papers related to the interaction between insects and their environment. The goal of the journal is to advance the scientific understanding of mechanisms of toxicity. Emphasis will be placed on toxic effects observed at relevant exposures, which have direct impact on safety evaluation and risk assessment. The journal therefore welcomes papers on biology ranging from molecular and cell biology, biochemistry and physiology to ecology and environment, also systematics, microbiology, toxicology, hydrobiology, radiobiology and biotechnology. www.eajbs.eg.net Provided for non-commercial research and education use. Not for reproduction, distribution or commercial use.


INTRODUCTION
Chemical control methods using insecticides had been favored so far because of their speedy action and easy application.However, the public concern over harmful effect of chemical insecticides on the environment and human health has enhanced the search for safer and environmentally friendly control alternatives where plant oils seem to be relevant and had great promise as an alternative to the conventional pesticides.
Terpenes and terpenoids are the most representative molecules constituting 90% of the essential oils and allow a great variety of structures with diverse functions (Bakkali et al., 2008).Insecticidal properties of numerous essential oils and some monoterpenes have been extensively studied against various insects (Lee et al., 2003, Abdel Aziz, et al., 2007, Bashir et al., 2013. and Hany, 2013).Eugenol, citronellal and thymol are reported as toxic to Spodoptera litura and Musca domestica (Lee et al., 1997: Hummel runner & Isman, 2001).
Estragole is an example of a toxic fumigant compound in the essential oils from coriander (Coriandrum sativum), caraway (Carum carvi) and basil (Ocimum basilicum) that is active against insect pests (Lopez et al., 2008).
Compounds extracted from plants, or the derivatives of such compounds may affect insect physiology in various ways (Shekari et al., 2008).
AChE is a key enzyme that terminates nerve impulses by catalyzing the hydrolysis of neurotransmitter, acetylcholine, in the nervous system of various organisms (Wang et al. 2004).
The possible sites of action of essential oil toxicity are acetylcholinesterase and the octopamingeric system in insects (Kostyukovsky et al. 2002;Evans 1981).
Transamination has been demonstrated in a number of insect tissues, particularly that concerning glutamate, aspartate and alanine (Gilmour, 1961).The glutamic oxaloacetate transaminase (GOT) and glutamic pyruvic transaminase (GPT) are key enzymes in the formation of nonessential amino acids, in metabolism of nitrogen waste, gluconeogenesis and correlated with protein anabolism and catabolism (Mordue and Golworthy, 1973).
The present study was conducted to investigate the toxicity level and biochemical effects of four plant essential oils against 4 th larval instar of Spodoptera littoralis.

Test insects:
Fourth instar larvae of S. littoralis were obtained from a continuous stock susceptible colony maintained in the Central Agricultural Pesticides Laboratory (CAPL), Dokki, Giza, Egypt.Larvae were reared throughout the experiments as described by (El-Defrawi et al., 1964) under laboratory conditions (25±2°C and 65±5% R/H.).

Experimental plant essential oils:
The four plant essential oils used in this study (Trigonella foenum graecum Sesamum indica, Eucalyptus camaldulensis and Nigella sativa) were purchased from El-captain Company (CAP.PHARM., Cairo), Egypt.

Method of application:
Thin film technique was used as a method of application in this study (Asher and Mirion, 1981), where the tested concentrations were applied through acetone to the surface of 9 cm in petri-dish.One ml of each concentration of the tested oils was spread on the inner surface of a petri-dish, by moving the dish gently in circles.Petri-dish used as control was treated with 1 ml of acetone only.The solvent was evaporated under room conditions leaving a thin film of oil on the surface of petri-dish.Ten newly moulted 4 th larval instar of S. littoralis were exposed to the tested oils for 6 hrs in each petri-dish, then transferred to clean glass containers and fed on fresh castor bean leaves.Five replicates of each concentration and the control were made.The mortality percentages were recorded after 24 hr from treatment and corrected as compared to control larvae according to Abbott formula (Abbott, 1925).The LC 25 , LC 50 and LC 90 values for each oil were calculated according to Finney (1971).
To evaluate the toxicity index (TI) of the tested oils, the following equation (Sun, 1950) was applied: To investigate the biochemical effects of the tested oils, newly moulted 4 th larval instars were treated with LC 50 of tested essential oils as described before to determine their potential effects on the activities of GOT, GPT, AChE and total protein content as well.

Sample preparation for biochemical assays:
The larvae were collected after treatment and starved for about 4 h before being homogenized in (1/5 w/v) homogenization buffer (pH 7.8) which prepared by dissolved 15 ml of glycerol into 75 ml distilled water and added 606 mg Tris, 292 mg EDTA and 5 mg phenyl thiourea.The pH was adjusted to 7.8, then final volume was completed to 100 ml.Homogenates were centrifuged at 10000 rpm for 15 min at 4°C and resulting supernatants were held on ice and used to determine the activity of enzymes and total protein content (Mohamady, 2005).

Biochemical assays:
GOT and GPT activities were determined in homogenate of larvae according to Harold (1975).AChE activity was determined according to Ellman et al. (1961).The total protein content in the samples was determined according to Lowry et al. (1951).

Statistical Analysis:
The data obtained from the present study was analysed using one-way ANOVA at ( < 0.05).The data was expressed as mean ± SE.Probitanalysis was performed for calculating LC 25 , LC 50 , and LC 90 according to Finney (1971).Based on LC 50 values and toxicity index in Table (1) data revealed that, the T. foenum gaecum was the most effective oil against 4 th larval instar of S. littoalis, where (TI =100%) followed by S. indeca (TI =84.86 %) and E. camaldulensis (TI = 73.71%),while N. sativa showed the lowest toxic effect (TI = 35.53%).(2) declared enhancing effect in the activity of GOT after treatment of larvae with tested plant essential oils.One exception was exhibited.This exceptional was the remarkable reduction in enzyme activity in the homogenate of larvae (Change %: -5.76) after treatment with N. sativa oil.The highest inducing effect was exhibited in the activity of GOT enzyme after treatment of larvae with E. camaldulensis oil (Change %:55.23) in comparison with control.On the other hand, the least inducing effect was detected in homogenate of larvae treated with T. foenum graecum (Change %:6.85) as compared to control.

Data in
Significant elevation in GPT activity was recorded in homogenate of larvae treated with LC 50 of all tested plant essential oils as shown in Table (2).The strongest inducing activity was detected in homogenate of larvae treated with S. indica (Change %:300.99).In respect to the other three plant essential oils, treatment with E. camaldulensis significantly increased GPT activity to193.43%followed by T. foenum graecum (Change %:81.06) and finally N. sativa (Change %:56.67) as compared to control.
No significant changes in the total protein content in larvae homogenate after treatment with E. camaldulensis and S. indica (Change %:-7.55 and -10.98, respectively).However treatment with N. sativa induced significant decrease in total protein content with Change % -21.61 as compared to control.On the other hand the protein content was increased significantly (Change %:17.84)from control when larvae treated with T. foenum graecum oil.

DISCUSSION
Many researchers have reported on the effectiveness of plant essential oils against insects, especially storedproducts insects.In our study, we are concentrated on S. littoralis.
It is clear from the obtained results that, T. foenum graecu was the most toxic compound and has low LC 50 value followed by S. indica, E. camaldulensis and finally N.sativa which has high value of LC 50 .These results are in agreement with the findings of Pavela (2004) and Krishnappa et al. (2010)  that some medicinal plants essential oils are larvicidal to the larval instar of S. littoralis.Hazrat and Soaib (2012) showed that those essential oils from C. sativum are effective against Mosquito larvae.Rana and Rana (2012) found that EO from F. vulgare kills 100% of Culex quinquefasciatus (Linnaeus) larvae at 250 ppm after 40 min.Similar toxic effects also were observed for essential and edible oils including sesame oil in the control of the pulse beetle C. chinensis (Ali et al., 1983, Khalequzzaman et al., 2007and Kumar et al., 2008 ), larvae of S. littoralis (Mesbah et al., 2006) andC. maculatus (Abd El-Razik andZayed, 2014).Likewise Kanat and Alma (2003) and Sampson et al., (2005) found insecticidal effects of essential oils from Eucalyptus camaldulensis against the larvae of pine processionary moth, T. pityocampa and adult turnip aphids, Lipaphis pseudobrassicae.Also Chaubey (2008) evaluated the toxic effects of seven different essential oils against Callosobruchus chinensis and found that Nigella sativa was the most effective at all stages.
On the other hand Omara et al. (2014) reported that clove and sesame oils can be used as repellent botanical insecticides, against P. Americana.Ebadollahi (2011) studied the antifeedant activity of essential oils from Eucalyptus globulus Labill and Lavandula stoechas L. on Tribolium castaneum Herbst Marei et al. (2009) showed that sesame oil has a latent effect on larvae up to certain limit while pupal mortality was affected with jojoba and sesame oil extracts at 3% concentration, being 50% and 80% respectively.
Essential oils or some of their substances, respectively, may exhibit mutual synergistic effects.For example the susceptibility of S. littoralis larvae to cyhalothrin increased when treated after treatment with LC 50 of essential oils, (Ismail and Shaker 2014).Visetson et al. (2003) found that sesame oil showed good synergism with cypermethrin.
Also the obtained results agree with Souguir et al. (2013) who recorded that the essential oils of S. officinalis leaves, C. sativum seeds, D. carota flowers, and F. vulgare seeds, may be serving as a lepidopteran agricultural pest control of S. littoralis.
In the present study change in the level of some biochemical paramatersin larvae homogenate of S. littoralis may be due to physiological alterations which are induced by compounds found in tested plant essential oils.
Regarding the mode of action of essential oils against insect pests, little information is available but treatment with various essential oils or their constituents cause symptoms that suggest a neurotoxic mode of action (Kostyukovsky et al., 2002;Priestley et al., 2003;Lu and Wu, 2010).In the present study, activity of AChE decreased in all treatments in comparison to control.These findings are coincide with that reported by Chaubey (2011) who found that, fumigation of S. oryzae adults with sublethal concentrations of C. cyminum and P. nigrum essential oils inhibited AChE activity.Previous researchers have reported the competitive inhibition of AChE activity by monoterpenes and monoterpenoids.Most of the essential oil components like cuminaldehyde, limonene, αpinene and βphellendrene inhibiting AChE activity (Lee et al., 2001, Abdelgaleil et al., 2009and Zapata & Snagghe, 2010).Several essential oils from aromatic plants, monoterpenes, and natural products act as AChE inhibitors (Shaaya and Rafaeli 2007;Ló opez et al., 2010).
On the other hand the obtained results indicated that, treatment with all plant essential oils induced an elevation of GOT and GPT activities except N. sativa induced a reduction in GOT activity.
Early studies were revealed that, the GPT activity was disrupted in S. gregaria by Neemazal (a neem preparation) and N. sativa extracts (Hamadah, 2009) as well by F. bruguieri extracts (Tanani et al., 2009).A considerable inducing effect on GOT and GPT activities in haemolymph of nymphs and adults of S. gregaria after treatment with P. granatum peel extracts, was recorded by Ghoneim et al. ( 2014) .
It is of interest to mention that GOT and GPT serve as a strategic link between the carbohydrate and protein metabolism and are known to be altered during various physiological and pathological conditions (Etebari et al., 2005).Accordingly, the disturbance in GOT and GPT levels will be closely related to metabolism of proteins and amino acids.Thus it will disrupt many physiological functions and ultimately lead to death, in other way control the pest (Ezz and Fahmy, 2009).
Khatter and Abuldahb (2010) revealed that, the botanicals and mineral oil inhibited the anabolism of the treated insects.The metabolic activity is mostly of catabolic pattern.Also total protein content decreased after treatment with tested plant essential oils except T. foenum graecu increased the level of protein content.Medhini et al. (2012) studied that the highest reduction in protein content of the larvae of Spodoptera litura when treated with Calendula olficinalis extracts.The level of protein content in the body of larva is dependent upon the rate of synthesis, the breakdown of proteins and even water movement between tissues.Moreover, Krishnaveni et al. (2013) revealed that, treatment of the larvae of Spodoptera litura with Pongam oil and neem oil decreased the total protein content and this reduction may be due to increased breakdown of proteins to detoxify the active principles present in the pongam oil.

CONCLUSION
The results indicated that tested plant essential oils (T.foenum graecu, S. indica, E. camaldulensis and N. sativa) gave toxicity and biochemical effects against 4 th larval instar of S. littoralis.Based on these results we recommended the use of plant essential oils in control of S. littoralis as alternative to chemicals insecticides.Because of these oils come from natural resources, safe, cheap and efficient and they will help to decrease the negative effects of synthetic chemicals such as residues in products, insect resistance and environmental pollution.

Table 1 :
Toxicity values of tested plant essential oils against 4 th larval instar of S. littoralis.

Table 2 :
Activity levels of some biochemical parameters in larvae homogenate of S. littoralis after treatment by LC 50 of tested plant essential oils.
Results were expressed as mean ±SE.*significance difference versus control at P<0.05; ns nonsignificance difference versus control at P<0.05.