Efficiency of some new insecticides on physiological , histological and molecular level of cotton leafworm

Efficiency of indoxacarb, spinetoram and methoxyfenozide on cotton leaf worm (Spodoptera littoralis) was determined through exposure of second and fourth instar larvae to dipped castor bean leaves. LC50 estimates of second instar larvae ranged from 0.004 to 0.006 ppm of indoxacarb, 0.022 to 0.033ppm of spinetoram and 0.006 to 0.011ppm of methoxyfenozide. LC50 estimates of fourth instar larvae ranged from 0.36 to 0.54 ppm of indoxacarb, 1.78 to 2.64ppm of spinetoram and 1.36 to 1.69 ppm of methoxyfenozide. Also, the ovicidal activity of these compounds was studied. Three day old eggs are more affected than that of one or two days old in case of indoxacarb and spinetoram while the reverse was in the case of methoxyfenozide. In nutritional assays, all tested compounds reduce food intake and affected growth rate (GR) where indoxacarb caused the highest rate of efficiency and followed by methoxyfenozide and spinetoram against 4 larval instar fed for two days on castor leaves treated with LC50s of the previous coumpounds. The histological examinations of 6 larval instar cuticle (after treatment of fourth larval instar by LC50 of methoxyfenozide) showed destruction in the cuticle layers, fissures in the endocuticle and irregular distribution of the hypodermal cells. While indoxacarb and spinetoram treatments showed slight effect in the cuticle layers as compared to methoxyfenozide. In addition, the deteriorative potentialities of aldehyde oxidase and α glycerophosphate dehydrogenase pattern of S. littoralis were screened.


INTRODUCTION
The cotton leaf worm, Spodoptera littoralis (Boisd) is a well polyphagous herbivore and is regarded as one of the most important agricultural pest in the Middle East.It is a very harmful potential pest of many field crops and vegetables, causing important economic losses in both greenhouses and open field on a broad range of ornamental, industrial and vegetable crops.To overcome the losses and to increase the yield, chemical insecticide application is utmost important (Aslam et al., 2004).Currently new groups of chemical compounds are being tested against lepidopteran pests such as, indoxacarb (Steward 15% SC), spinetoram (Radiant 12% SC) and methoxyfenozide ( Runner 24% SC).
Indoxacarb represents a new class of insecticides (the oxidiazines).The primary route of entry into target insects is through ingestion (Wise et al., 2006), although it also is absorbed through the cuticle.It has a novel mode of action that blocks the movement of sodium ions into certain nerve cell ion channels, resulting in paralysis and death of the pest species.
Spinetoram is a new generation of spinosyn group.It causes excitation of the insect nervous system by altering the function of nicotine and GABA-gated ion channels.It dose not interact with the known binding sites of other classes of insecticides such as of neonicitinoids, fiproles or avermectins (Crouse and Sparks, 1998).
Methoxyfenocide is classified as a diacylhydrazine insecticide.It acts as ecdysone agonists with enormous potential for development as insect specific control agents with little or no effect on non target species (Dhadialla et al., 1998;Toya et al., 2002;Sawada et al., 2003).Also, it provides effective control of a wide range of lepidopteran insects.The chemical upon absorption into the haemolymph of the insect, binds to the ecdysone receptor which initiates the moulting process.As the normal process disrupted, the insects prevented from shedding its old cuticle.The larvae die of dehydration and starvation within 2-5 days (Kumar and Santharam, 2008).
Present studies were designed to evaluate some toxicological, physiological, histopathological and molecular effects of these compounds against S. littoralis.

MATERIALS AND METHODS
The cultural of S. littoralis used in this study originated from eggs obtained from a susceptible strain established in the cotton leaf worm Department, Plant Protection Research Institute, Dokki, Giza.This strain was reared in the laboratory under constant laboratory conditions of 27±2 o C and 65±5 % RH (EL-Defrawy et al., 1964).

Experimental Insecticides
Three new chemical insecticides, indoxacarb (Steward 15% SC, DuPont), spinetoram (Radiant 12% SC, Dow Agro sciences) and methoxyfenozide (Runner 24% SC, Dow Agro sciences) were obtained from their respective manufacturers and used in the present studies.

Susceptibility Test
To assess the insecticidal activity of the tested compounds, different concentrations were prepared ranged from 8 to 3x10 -3 ppm.The larvae used in the experiments were freshly moulted 2 nd and 4 th instar larvae within 6 hrs after ecdysis.The leaf dipping technique was adopted, where freshly castor oil beans leaves were dipped for 30 second in one of the prepared concentrations.The treated leaves were left to dry for approximately 15 minutes at room temperature before being offer to S. littoralis larvae.Three replicates contained 20 larvae / jar were used for each treatment and also for the control experiments.The mortality percentages of treated larvae were corrected against those of the control by using Abbotts formula, (Abbott, 1925).Probit analysis was determined to calculate LC 50 and slope values of the tested compounds (Finney, 1971), through software computer program.Statistical analysis of results according to (SAS, 1996).

Treatment of Egg Masses
Different ages of S. littoralis egg masses ( 0-24; 24-48 and 48-72 hrs old) which deposited on Nerium oleander leaves were dipped in different concentrations of the tested compounds for 15 seconds, left to dry, placed in petri-dishes and incubated at 27± 2 о C and 70± 5 RH.The control one was dipped in water and left to dry.Hatchability was recorded after 2 to 4 days.

Food Consumption and Utilization
Three replicates contained 20 second and/or fourth instar larvae / jar were left to feed on treated leaves with LC 50 of the tested compounds for 48 hrs, then the survived larvae were transferred to untreated leaves in clean jars and left to feed until death or pupation.The fresh weight of larvae, faeces and castor bean leaves in each rearing jar were recorded daily.Fresh leaves were kept in a similar rearing jar under the same condition to estimate the actual loss of moisture, which was used for calculating the corrected weight of consumed fresh leaves.Food consumption and utilization were calculated according to the equations given by (Waldbauer, 1968) and (Slansky and Scriber, 1982) as follows: Food ingested ( I ) = [1-a/2] [w-(L+bL)] Where : a = the ratio of loss of water to the initial weight of leaf.b = the ratio of loss of water to the final weight of leaf.w = weight of food introduced.L = weight of uneaten food.Consumption index (CI) = ___I___ TA Where: A = mean body weight of larvae during the feeding period (mg).
Relative growth rate (RGR) = W TA Where: W = weight gain of larvae (mg).Approximate weight of digested food (mg) (D) = I -F Where: F = weight of fasces (mg).Approximate digestibility (AD) = Approximate weight of digested food X100 Amount of food ingested Efficiency of conversion of ingested food into body matter (ECI) = Weight gain of larva (mg) X 100 Amount of food ingested Efficiency of conversion of digested food into body matter (ECD) = Weight gain of larva (mg) X 100 Amount of food digested

Histopathological Studies
The tested compound were selected for a comparative study to clarify their effects on the histopathology of the cuticle of 6 th larval instar surviving after treatment of the 4 th larval instar with the LC 50 values of the tested compounds.These larvae were dissected in 0.85% Saline solution, and the cuticle was taken and fixed in carnoy's fixative for half an hour, then two changes (for about 15 minute) in absolute ethyl alcohol (100%) for dehydration.The dehydrated organs were cleared in methyl benzoate for about 24 hrs., washed in toluene for three-five minutes, then transferred to liquid Paraffin (melting point 58 o C ) for two-three hrs.changes.The different organs were embedded in liquid paraffin, sectioned at 5 µm, and stained with haematoxylin and eosin for microscopic examination.Control sections of non-treated larvae were also studied.

Molecular Studies
Simplified procedure of electrophoretic separation of protein is the best method for the separation of isoenzymes.Protein molecules migration in an electric field depends on its net electric charge and size and pH of buffers.In alkaline solution proteins are negatively changed and travel towards anode.To visualize the location of enzyme bands on the gels, we follow Aldehyde oxidase (AO) After electrophoresis, the gel was rinsed in 100 ml tris/HCl, pH 8.5, which mixed with 20 mg NBT, 10 mg EDTA, 25 mg NAD, 100 mg KCl and 1ml Heba A. Hassan 200 Benzaldehyde for 30min, then 5mg PMS were put.The gel was stained in dark place at (35-37 o C) from (3 -3.30 h.)After incubation, the gel was destained in 7% acetic acid ( Ayala et al., 1972 andPasteur et al., 1988).

Susceptibility of Spodoptera littoralis toward tested compounds
Susceptibility test on a laboratory strain of the 2 nd and 4 th larval instars of cotton leaf worm, Spodoptera littoralis was carried out at different concentrations of tested compounds.The mortalities were recorded after 120 hrs.post treatment .Data in Table 1 indicated that, 2 nd and 4 th larval instars of S. littoralis were susceptible to all tested coumpound, however, in different levels according to insecticide classes.The earlier instar was found to be more susceptible with lesser LC 50 values and as the instars advances the values of LC 50 was found to be increasing.Indoxacarb was the most effective followed by methoxyfenozide and finally spinetoram.The LC 50 values of indoxacarb, methoxyfenozide and spinetoram were 0.005, 0.008 and 0.027 ppm, for 2 nd larval instars, while LC 50 values were 0.45, 1.52 and 2.17 ppm, for 4 th larval instars respectively.These results are in conformity with that obtained by (Ahmed et al., 2004;Abdou-Gehan and Abdalla, 2006;Ademczyk et al., 1999;Moulton et al., 2002).

Effect of Tested Compounds on Spodoptera littoralis Eggs.
Table (2) show that the three day old eggs are more affected than that of one or two days old in case of indoxacarb and spinetoram while the reverse was in the case of methoxyfenozide i.e. the highest ovicidal activity was showed at the early eggs.Laboratory data indicate that the probable mechanism of ovicidal activity of indoxacarb and spinetoram are due to adsorption of them into the chorion and subsequent oral uptake as the neonate chews through the chorion to hatch.At this point the larva ingests a dose of them that is sufficient to affect the larva to die within the egg.Sometimes, eggs can be seen with the head of the neonate larvae protruding (Plate 1-B,C and D).These findings are in full agreement with (Elbarky Nehad et al., 2008).In the case of methoxyfenozide, failure of the eggs hatching could be explained by the known mode of action of this compound, which act as ecdysone agonists.Lepidopterans eggs have high concentrations of ecdysteroids that are made available to developing embryos and prehatching larvae (Hoffman et al. 1980) thus, methoxyfenozide may accelerate embryo development lead to premature lethal molt (Plate 1-E and F).

Food Consumption and Utilization
Results in Table 3 reveal that food consumption of the 4 th instar of S. littoralis larva was significantly reduced by LC 50 treatments.The inhibitory effect of larval feeding on treated leaves revealed the superior action of indoxacarb.This reduction varied in case of other treatment.The data were always significant and showed that the activity of indoxacarb> methoxyfenozide> spinetoram in descending order Plate 2. This indicated that the tested compound, especially indoxacarb act as antifeedant.These results are in agreement with the findings of (Hussien, 2000;Abdel-Al and Abdel-Khalek, 2006) who revealed that, IGR s act as antifeedant.Data indicated that the treatments had significant reduced the digestibility this may be due to the high percent of excretion or food consumption by larvae as compared to the control.All tested compounds decrease the relative growth rate.The efficiency of conversion of ingested food to body tissue (ECI) increased in larvae fed on tested compounds.Also results showed a significant increase in the efficiency of conversion of digested food to body tissue (ECD).

Histopathological Studies:
Normal cuticle of S. littoralis larva composed of an outermost distinct layer, the epicuticle and the inner layer called procuticle.Procuticle, parts of which are hardened to form the exocuticle, while other parts remain flexible and colourless to form endocuticle .Finally there is commonly a distinct layer called hypodermal layer which composed of columnar or cuboidal cells (Plate 3-A).
Histopathological examination of the 6 th larval instar of S. littoralis resulting from the 4 th larval instar treated with LC 50 of methoxyfenozide revealed a separation of the hypodermal cells from the endocuticle and the hypodermal cells showed several Heba A. Hassan 202 mitotic divisions and some fissure.Distortion in the endocuticle was also quite visible.This distortion revealed blockage of its formation (Plate 3-B).Hegazy (1990) reported that benzoylphenyle ureas induced a great disturbance in cuticle deposition of S. littoralis larvae.
Indoxacarb caused abnormalities in the shape of the exocuticle and the hypodermal cells separated from the endocuticle (Plate 3-C).While spinetoram has slight effect on the cuticle as compared with other compounds.As shown in (Plate 3-D) the thickness of the cuticle decreased, some hypodermal cells became separated from the endocuticle.Elbarky et al. (2008) showed that, the amount of total carbohydrates, total proteins, carbohydrate hydrolyzing enzymes were significantly decreased when S. littoralis treated with spinetoram.These components are necessary for building cuticle and lake of them may lead to abnormal endocuticular deposition and abortive molting.These results accordance with the demonstrated by El-Sheikh et al. (2005).

Molecular Studies
The enzymes, which act on a substrate and tend to remove hydrogen from it are known as dehydrogenases while those involved in the oxidative chain are termed oxidases.These enzymes are rather specific.The alcohols are produced inside the living organism through the hydrogenation of aldehyde and ketones or through metabolism of fats (production of glycerol).The high concentration of alcohols inside the living organism causes high disturbance in different metabolic pathways, so the living organism must regulate these alcohols through alcohol metabolizing enzymes such as alcoholic dehyrogenase or aldehyde oxidase (AO) (Parkash and Shamina, 1994).
The present study refers to the pattern of aldehyde oxidase (Plate 4-A), which is recorded at higher diversity in each of control and treated samples Table 4.This may be due to the higher metabolic rate in these samples.Aldehyde oxidase isozyme patterns were studied in several insects; (Garcin el al., 1983) who demonstrated the capacity of (AO) in D. melanogaster to detoxify acetaldehyde and use it for energy production.(Barnes, 1983) who stated that the inversion-allozme polymorphism of (AO) is directly involved in the adaptation to a specific environmental component.Also, (AO) isozyme in adult Hypera postica can be influenced by developmental age and environmental conditions (Romero et al., 1986).Dehydrogenases are important tools for the investigation of insect metabolic activities during the course of development.The relative activities of insect dehydrogenases may be related to the energy yielding (Dickinson and Sullivan, 1975).

Efficiency of some new insecticides on physiological, histological and molecular level 203
The α-GPDH, is a soluble, very active enzyme which is heavily concentrated in the muscles to fulfill the energy utilization from high carbohydrate metabolic rate in such organs (Sacktor, 1974).It generates NAD + via reduction of dihydroxy acetone phosphate during glycolysis.Therefor, any rise in α-GPDH activity normally corresponds to the drastic fall in LDH activity (Sacktor, 1976).
The assayed α-GPDH activity in the present study Table 5 and (Plate 4-B) recorded high effect due to the treatment with methoxyfenozide .These results are agreement with many authors, (Mostafa, 1994) who stated that IGR s lead to a high activity of α-GPDH in larvae of M. domestica.(Abdel-Moty et al., 1996) declared that IGR s effect on the pattern of enzymes activity, quite fluctuation in the ontogenic spectrum of α-GPDH differing from the normal behavior.Finally, any unusual change of α-GPDH and AO patterns in treated and evoluted stages might be, on molecular levels and referred to depression or mutations of the regulating genes responsible for biosynthesis of polypeptide chain building these enzymes.

Table 1 :
Susceptibility of 2 nd and 4 th larval instars of Spodoptera littoralis toward tested compounds.

Table 2 :
Ovicidal activity of tested compounds on egg masses of Spodoptera littoralis.

Table 3 :
Some nutritional values of 4 th larval instar of Spodoptera littoralis treated with LC 50 of tested

Table 4 :
Relative fragmentation R f and amount percentage of haemolymph aldehyde oxidase pattern of S. littoralis after treated the 4 th instar larvae with Tested compounds.

Table 5 :
Relative fragmentation R f and amount percentage of haemolymph α-GPDH pattern of S. littoralis after treated the 4 th instar larvae with Tested compounds.