Toxicity and Biological effect of Capparis leaves extracts to the black cutworm, Agrotis ipsilon (Hufn.)

Three different concentrations (20, 10 and 5%) of ethanol, ethyl acetate, diethyl ether and chloroform extracts of Capparis aegyptia plant leaves were tested against 2 instar larvae of Agrotis ipsilon to study the toxicity and their effects on some biological parameters under laboratory conditions. Maximum mortality percentage was 40% at 5% of ethyl acetate extract, while treatment with ethanol extract recorded the lowest mortality percentages especially at 5%. Calculated LC50 was ranged between 5.752 and 8.027 at treatments with ethanol extract and chloroform extract, respectively. Low concentrations were almost more toxic than high. Malformation percentages of emerged adults were observed with ethanol and diethyl ether extracts. Fecundity reduction percentages at treatments in comparison with control reached the maximum (100%) at treatment with ethyl acetate extract while the minimum was about 50% at chloroform extract treatment. INTRODUCTION Several crops naturally infested with many noctuid insect pests in the field resulting unlimited yield loss. The black cutworm, Agrotis ipsilon (Hufn.) is considered the most notorious and destructive phytophagous insect pest in Egypt, not only to cotton but also to many field crops and vegetables (Salama et al., 1970 and Kandil et al., 2003). The excessive use of insecticides, particularly those with long residual effect, resulted in several harm to the natural balance between pests and their enemies by killing many non-target organisms, long storage and some times very slow degradation of the insecticides and in many cases high toxicity to mammals (Schmidt, 1986). In an endeavour to overcome this defect, entomologists are concerned to use alternative approaches to control insect pests. One of these approaches is the use of natural pesticides. These materials have a relatively low mammalian toxicity as compared to many chemical pesticides (Ahmed et al., 1984) Plants of the genus Capparis (Family: Capparaceae) have been a subject of interest to some investigators from the phytochemical point of view and in particular its glucosinolate content (Kjaer, 1961 and Brown, 1964). Investigation of the medicinal plant Capparis aegyptia L., revealed the presence of alkaloids, glucosinolates, sterols and flavonoids (Hammouda et al., 1975) as well as cumarins, rutic acid, saponins and pectic acid (Ahmed et al., 1972). The objective of the present work is to study the efficacy of ethanol, ethyl acetate, diethyl ether and chloroform extracts of Capparis aegyptia plant leaves on its toxicity and some biological parameters of black cutworm, Agrotis ipsilon. MATERIAL AND METHODS 1Rearing of Experimental Insect Pupae of the black cutworm, A. ipsilon were supplied from a laboratory, Department of Cutworm and Mole Crickets, Plant Protection, Research Institute, Agriculture Research Center. Emerged adults were kept in a glass jar Mahasen M. A. El-Shershaby 46 at ratio (1 ♂: 1♀), jars were supplied with onion–skin paper and covered with black gauze for laying eggs. For adults feeding a piece of cotton soaked with 20% honey solution was used. Hatched larvae were reared in plastic cages (20 x 30 x 50cm) supplied with tissue paper for moisture and castor oil leaves for feeding. After the third moulting, larvae reared individually to avoid cannibalistic behaviour (El-Shershaby, 2010). 2Preparation of Extracts a)Plant Materials Leaves of the wild plant, C. aegyptia (C. spinosa var. aegyptia Boiss.) (Family: Capparaceae), were collected from different areas of South Sinai and left to dry at room temperature. b) Extract Preparation Crude extract of dry powder of plant leaves was prepared using 4 different solvents, Ethanol, Ethyl acetate, Chloroform and Diethyl ether according to Su and Horvat (1981). c) -Tested Concentrations Three different concentrations (20, 10 and 5%) of each extract were prepared using distilled water and tween-20 (3-5 drops) as an emulsifier and a magnetic stirrer was used for mixing. 3Experiments Newly moulted 2 instar larvae of A.ipsilon were collected from the stock culture to use in the experiments. Plastic cups (5 cm in diameter and 4 cm height) were used; each cup contains 5 larvae of 2 instar, confined with treated piece of castor oil leaf (using dipping technique) for feeding and a tissue paper to reduce the moisture. Cups were covered with gauze material and incubated at controlled constant conditions of 25 ±2C and 50-60% RH and day light. Five cups were used as replicates/concentration/extract. Untreated food was used in check cups. Two days post treatment; larvae were transferred individually into clean cups, which supplied with untreated food and a small piece of tissue paper until pupation. The whole tests were repeated 5 times. Percentages of larval, prepupal and pupal mortality, deformed and nondeformed emerged adults in addition to fecundity and fertility of treatments and control were recorded. To determine the effect of tested extracts on fecundity and fertility of emerged adult females from treated larvae, moths were kept in a glass jar and fed on 20% honey solution. Black paper sheets were hanged up inside the jars for oviposition. Five jars were used as replicates per concentration and the same number were used as control. Total number of laid eggs per replicate was counted and the percentage of hatchability was calculated for treatments and control. Insect rearing and all experiments were carried out at controlled conditions of 25 ±20C, 5060% RH and day light. 1. Statistical analysis Statistical analysis was carried out using Analysis of Variance (one way ANOVA) test through "SPSS-Computer Program". Means were compared using Duncann’s Multiple Range test. Percentage of mortality was corrected according to Abbott ُ ◌ s formula (Abbott, 1925), the Probit Analysis was applying for calculating LC50 according to Finney (1952). RESULTS Data in table (1) reflect the different mortality responses of the treated Agrotis ipsilon 2 larval instar. The highest percent mortality (40%) was recorded for ethyl acetate extract at 5% concentration, where the lowest percentage mortality (5%) was recorded for ethanol extract at the same concentration. The other extracts with the tested concentrations reflects different mortality percentage ranged between 10 and 30%; being statistically different either between extract or between tested concentrations (Table 1). Toxicity and Biological effect of Capparis leaves extracts to the black cutworm 47 Table (1): Percentage of mortality of A. ipsilon larvae treated at 2 instar with C. aegyptia extracted leaves using different solvents Concentration (%) Mortality percentage (%) of treated larvae Ethanol Ethyl acetate Chloroform Diethyl ether Control F1-value 20 15.0+1.3 bB 10.0+0.7 cC 10.0+0.9 cC 30.0+1.3 aA 8.0+0.7 79.873** 10 20.0+0.9 aB 15.0+0.9 bC 30.0+0.6 aA 10.0+0.8 bD 8.0+0.7 113.016** 5 5.0+0.3 dD 40.2+1.1 aA 25.0+0.6 bB 10.0+0.7 bC 8.0+0.7 424.860** Control 8.0+0.7 c 8.0+0.7 8.0+0.7 8.0+0.7 F2-value 57.880** 292.069** 247.228** 125.522** **= Highly Significant Means in columns followed with the same SMALL letters(s) are not significantly different at 5% level of probability (F1-value). Means in rows followed with the same CAPITAL letters(s) are not significantly different at 5% level of probability (F2-value). The corrected percentage mortality for larval and pupal stages and the LC-values reflect the reverse effect of all extracts except of diethyl ether, which induced normal effect shape (Table2). The highest concentration (20%) of ethanol, ethyl acetate and chloroform extracts, recorded the lowest percentage mortality, followed by 10% concentration then the lowest concentration (5%) which recorded the highest percent mortality. Table (2): Mortality percentage of larval and pupal stages of treated 2 instar larvae Concentration (%) Corrected mortality (%) Ethanol Ethyl acetate Chloroform Diethyl ether


INTRODUCTION
Several crops naturally infested with many noctuid insect pests in the field resulting unlimited yield loss.The black cutworm, Agrotis ipsilon (Hufn.) is considered the most notorious and destructive phytophagous insect pest in Egypt, not only to cotton but also to many field crops and vegetables (Salama et al., 1970 andKandil et al., 2003).
The excessive use of insecticides, particularly those with long residual effect, resulted in several harm to the natural balance between pests and their enemies by killing many non-target organisms, long storage and some times very slow degradation of the insecticides and in many cases high toxicity to mammals (Schmidt, 1986).In an endeavour to overcome this defect, entomologists are concerned to use alternative approaches to control insect pests.One of these approaches is the use of natural pesticides.These materials have a relatively low mammalian toxicity as compared to many chemical pesticides (Ahmed et al., 1984) Plants of the genus Capparis (Family: Capparaceae) have been a subject of interest to some investigators from the phytochemical point of view and in particular its glucosinolate content (Kjaer, 1961 andBrown, 1964).Investigation of the medicinal plant Capparis aegyptia L., revealed the presence of alkaloids, glucosinolates, sterols and flavonoids (Hammouda et al., 1975) as well as cumarins, rutic acid, saponins and pectic acid (Ahmed et al., 1972).
The objective of the present work is to study the efficacy of ethanol, ethyl acetate, diethyl ether and chloroform extracts of Capparis aegyptia plant leaves on its toxicity and some biological parameters of black cutworm, Agrotis ipsilon.

MATERIAL AND METHODS 1-Rearing of Experimental Insect
Pupae of the black cutworm, A. ipsilon were supplied from a laboratory, Department of Cutworm and Mole Crickets, Plant Protection, Research Institute, Agriculture Research Center.Emerged adults were kept in a glass jar at ratio (1 ♂: 1♀), jars were supplied with onion-skin paper and covered with black gauze for laying eggs.For adults feeding a piece of cotton soaked with 20% honey solution was used.Hatched larvae were reared in plastic cages (20 x 30 x 50cm) supplied with tissue paper for moisture and castor oil leaves for feeding.After the third moulting, larvae reared individually to avoid cannibalistic behaviour (El-Shershaby, 2010).

2-Preparation of Extracts a)-Plant Materials
Leaves of the wild plant, C. aegyptia (C.spinosa var.aegyptia Boiss.)(Family: Capparaceae), were collected from different areas of South Sinai and left to dry at room temperature.

b) -Extract Preparation
Crude extract of dry powder of plant leaves was prepared using 4 different solvents, Ethanol, Ethyl acetate, Chloroform and Diethyl ether according to Su and Horvat (1981).

c) -Tested Concentrations
Three different concentrations (20, 10 and 5%) of each extract were prepared using distilled water and tween-20 (3-5 drops) as an emulsifier and a magnetic stirrer was used for mixing.

3-Experiments
Newly moulted 2 nd instar larvae of A.ipsilon were collected from the stock culture to use in the experiments.Plastic cups (5 cm in diameter and 4 cm height) were used; each cup contains 5 larvae of 2 nd instar, confined with treated piece of castor oil leaf (using dipping technique) for feeding and a tissue paper to reduce the moisture.Cups were covered with gauze material and incubated at controlled constant conditions of 25 ±2 0 C and 50-60% RH and day light.
Five cups were used as replicates/concentration/extract.Untreated food was used in check cups.
Two days post treatment; larvae were transferred individually into clean cups, which supplied with untreated food and a small piece of tissue paper until pupation.The whole tests were repeated 5 times.
Percentages of larval, prepupal and pupal mortality, deformed and nondeformed emerged adults in addition to fecundity and fertility of treatments and control were recorded.
To determine the effect of tested extracts on fecundity and fertility of emerged adult females from treated larvae, moths were kept in a glass jar and fed on 20% honey solution.Black paper sheets were hanged up inside the jars for oviposition.Five jars were used as replicates per concentration and the same number were used as control.Total number of laid eggs per replicate was counted and the percentage of hatchability was calculated for treatments and control.
Insect rearing and all experiments were carried out at controlled conditions of 25 ±2⁰C, 50-60% RH and day light.

Statistical analysis
Statistical analysis was carried out using Analysis of Variance (one way ANOVA) test through "SPSS-Computer Program".Means were compared using Duncann's Multiple Range test.Percentage of mortality was corrected according to Abbott ُ◌ s formula (Abbott, 1925), the Probit Analysis was applying for calculating LC 50 according to Finney (1952).

RESULTS
Data in table (1) reflect the different mortality responses of the treated Agrotis ipsilon 2 nd larval instar.The highest percent mortality (40%) was recorded for ethyl acetate extract at 5% concentration, where the lowest percentage mortality (5%) was recorded for ethanol extract at the same concentration.The other extracts with the tested concentrations reflects different mortality percentage ranged between 10 and 30%; being statistically different either between extract or between tested concentrations (Table 1).The vies versa, diethyl ether at the highest concentration induced the highest percent mortality, while the lowest concentration (5%) recorded the lowest percentage mortality (Table 2) the same trends were observed in table (3) when the needed concentration for killing 25% of the pupation represented by the highest value, and the needed concentration for killing 90% of the population was the lowest in case of ethanol, ethyl acetate and chloroform extract; but in case of diethyl ether, the lowest LC-value (LC 25 ) represented the lowest concentration (1.866%) and the highest (LC 90 ) represented the highest concentration (88.890%).The survived larvae of A.ipsilon 2 nd instar, post treatment, pupate normally as illustrated in figures (1, 2& 3).It was observed that the latent effects of different extracts could be arranged according to its strength as follows: chloroform>ethanol > ethyl acetate > diethyl ether (at 20% concentration); being significantly different either among each other or when compared with the check group.The corresponding figures for 10 and 5% concentrations of the extracts were more or less gave the same trends (Figs.2&3).It was clear that the 5% concentration of extracts was more drastically affected all given parameters when compared with 10 or 20% of extracts concentration (Table 4).

DISCUSSION
The black cutworm Agrotis ipsilon (Hufn.) is a major pest attack seedlings of many economic plants.It's actually one of the most important insect pests of vegetables and several field crops in Egypt (Salem et al., 1982).The wide range of natural host of the target insect could be increase the resistance to the natural and /or alternative toxic chemicals.
It's interesting that on strictly biochemical criteria polyphagy may enhance the potential of a species to develop resistance, Krieger et al. (1971).Slight toxicity of tested Capparis extracts that obtained in our results may be due to developed resistance of target insect.
Significant reduction in the total number of eggs laid by Tetranychus urticae

Fig. 2 :
Fig. 2: Latent effects of the treatment of the newly moulted 2 nd instar larvae with 10% concentration of different Capparis extracts.

Table ( 1
): Percentage of mortality of A. ipsilon larvae treated at 2 nd instar with C. aegyptia extracted leaves using different solvents

Table ( 3
): Calculated lethal concentrations of extracted C. aegyptia leaves using different solvents Latent effects of the treatment of the newly moulted 2 nd instar larvae with 20% concentration of different Capparis extracts.