The Feeding Response of Epilachna indica (Coleoptera: Coccinellidae: Epilachninae) Towards Extracts of Azadirachta indica

MATERIALS AND METHODS

Life Cycle Study of E. Indica in Outdoor Cage
Twenty pairs of ladybirds were collected from Malaysian Agriculture Research Development Institute (MARDI) and reared in a cage of 3x1x3 m containing S. melongena plant of 2 months old. Time taken for every pair of beetles to lay eggs was recorded. The eggs were then transferred to new cages containing S. melongena of 2 months old. Each cage was checked daily in order to record the time taken for eggs to hatch. Twenty newly hatched larvae were collected and reared in new cage. The time taken for larvae to pupate was also recorded. Newly developed pupae were identified from the S. melongena plant and their location was marked. Duration of time for pupae to become adult ladybird beetles was recorded (Table 1).

Beetles
Wild adult beetles were collected from a farm of Malaysian Agriculture Research Development Institute (MARDI) at Jalan Kebun in order to start a colony of E. indica for future experiments. The beetles were maintained on 2 months old S. melongena plants placed in a rearing cage of size 3x1x3 m. Adult beetles of the same age were used in all experiments. Beetles to be used in feeding bioassay were only fed with water for three days before used.

Plant Material
The leaves of the neem tree Azadirachta indica (6 years old of 10 meter height) were collected from Section 12, Petaling Jaya, Selangor, Malaysia. One hundred gram (dried weight) of leaves were soaked in 1000 mL methanol for 2 h. The mixture was then filtered and the filtrate was concentrated into 20 mL using a rotary evaporator at 40°C. This concentrate was used as stock extract.

Table 1:	Period of the ladybird beetles’ life cycle at Somme Estate, Serdang, Kedah

Fig. 2:	Feeding bioassay in agar

Dual Choice Feeding Bioassay
Five starved adult beetles were placed in a petri dish (Fig. 2) containing methanolic crude extract in 2% agar in one half of compartment. The other half compartment contained 2% agar as control. The petri dishes were covered with a nylon mesh of aperture 0.5 mm. After every 24 h for three consecutive days, the numbers of triangular bite marks made by the beetles were examined under binocular microscope. Ten petri dish were given the treatment above. Ten fractions were collected from 2 mL methanolic extract isolated by column chromatography with silica gel 60 (230-400 mesh). The agar were also treated with fractions from column chromatography then subjected to the same behaviour bioassay as above.

Thin Layer Chromatography Analysis (TLC)
Five microliter of the fraction which gave positive anti feeding response in the bioassay was analysed with Thin Layer Chromatography (TLC) plate silica gel F254 using diethyl ether and methanol at 70:30 solvent mixtures. The separation that occurred on the TLC plate was observed under UVGL-58 UV light of short wave 254 nm long^-1 wave 366 nm. Each compounds of different Rf was subjected to the same bioassay as in crude and fractions.

High Performance Liquid Chromatography Analysis (HPLC)
The fractions that gave positive result from bioassay were analyzed using HPLC to identify the active components in them. Analyses were performed on a Shimadzu HPLC model that build in combination of LC-10AT pump, fitted with ODS Hypersil C₁₈ column (250x4.6 mm I.D.). The injection system (Rheodyne) used was 20 μL sample loop. Detection was done by a SPD-MIOA Variable Wavelength Detector at wavelength of 220 nm. KT- 25S degassing device (Shodex degasser, Tokyo, Japan) was used to degas the solvents. Mobile phase consisted of an isocratic mixture of acetonitrile-water (43:57) at a flow rate of 1.5 mL min^-1. The peaks were confirmed by comparing with standards of pure Azadirachtin. Five Azadirachtin solutions at concentrations ranging from 0.01, 0.05, 0.1, 0.125 and 0.25 mg mL^-1 were used for analysis. Each concentration of standard Azadirachtin were injected 3 times into HPLC and peak area responses were obtained. The calibration curve for Azadirachtin was prepared by plotting concentration of Azadirachtin versus peak area (average of three runs). Fraction 3 from TLC which had evoked positive antifeedant response from the beetles, with known amounts of standard Azadirachtin was analyzed.

Feeding Bioassay Using Synthetic Compound (SC) of Azadirachtin
Synthetic neem compound, Azadirachtin supplied by Sigma-Aldrich was prepared in concentration of 50, 100 and 200 ppm with methanol. One half of the agar compartment was treated with 50 ppm and the other half of the agar compartment was treated with methanol as control. Each different concentration was subjected to the same bioassay method as in crude and fractions.

RESULTS

Life Cycle Study of Epilachna indica in Outdoor Cage
In this study, it was observed that the life cycle of Epilachna indica has four distinct life stages; egg, larva, pupa and adult. A male beetle finds a female beetle by using antennae to locate its mate. A single female beetle laid 10 to 15 eggs on the underside of a leaf after 6.95±0.67 days of mating. The eggs were tiny, elongated and yellow jellybeans like which hatched in about 4.05±0.80 days and larvae begin searching on plants. The larvae are white when they hatch, but soon turn black. Larvae were spindle-shaped, wrinkled and have short antennae. As the larva grows it sheds its skin four times before it is fully grown after feeding for 12.85±0.73 days. The larva attached its rear end to the back of a leaf with a sticky liquid and for the final time sheded its skin to reveal the soft orange colored skin. During the immobile pupa stage, the wings and other adult body parts developed. Pupation lasted 5.50±0.50 days until the adult beetles emerged from the pupa case. The time for development from an egg to an adult in this study was about 29.35±0.79 days.

Figure 3 shows the life cycle of the ladybird beetle at Somme Estate in October 2003. After the mating of 20 pairs, 211 eggs (100%) were laid. Every Epilachna indica laid 10 to 15 eggs. From the 211 eggs, 105 eggs (49.8%) were hatched to become larvae, 58 eggs (27.5%) did not hatch and the rest of 48 eggs (22.7%) were missed. Out of the living larvae, 83 larvae (39.4%) lived to become pupae and 22 larvae (10.4%) died in the process of becoming pupae. Among the surviving pupae, 62 pupae (29.4%) lived and become adult beetles and 21 pupae (10%) died before they became adult beetles. According to this study, 62 new born adult beetles (29.4%) were produced from the mating of 20 pairs Epilachna indica.

Figure 4 shows the life cycle of ladybird beetles, Epilachna indica at Somme estate during October 2003.

Feeding Bioassay Using Fraction from Column Chromatography
Table 2 shows the number of bites by ladybird beetles in fraction 1, 2 and 3. Table 3 shows the number of bites by ladybird beetles in fraction 4, 5 and 6. Table 4 shows the number of bites by ladybird beetles in fraction 7 and 8. Table 5 shows the number of bites by ladybird beetles in fraction 9 and 10.

Fig. 3:	Life cycle sequence of ladybird beetles, Epilahna indica at Somme estate during October 2003

Fig. 4:	Life cycle of ladybird beetles, Epilahna indica at Somme estate during October 2003

Table 2:	The No. of bites by ladybird beetles in fractions 1, 2 and 3

Total number of bites was analysed using ANOVA. Power of the test was more than 80% showing that 10 replicates are sufficient to show the difference between treated and untreated (control). Effect Size (ES) of the test was more than 0.14, showing that there was a clear difference between the treated and untreated (control). Probability value (p-value) was less than 0.05, meaning that there is a significant difference between control and treatment. Regardless of time there is a significant difference between the treated agar and untreated agar. Based on observation from Table 2-5 fraction 3 was considered as giving the positive results in antifeeding bioassay as it gave maximum differences between treated agar and untreated agar (control). The other fractions (fraction 1, 2, 4, 5, 6, 7, 8, 9 and 10) show negative results in antifeeding bioassay. Fraction 3 was then analysed with Thin Layer Chromatography.

Table 3:	The number of bites by ladybird beetles in fractions 4, 5 and 6

Thin Layer Chromatography Analysis (TLC)
Fraction 3 was analysed using Thin Layer Chromatography plate. Three spots were observed for fraction 3. The retention time (Rf) of the three spots from fraction 3 was 0.44, 0.77 and 0.94. On the basis of number of bites by ladybird beetle after every 24 h for 3 days in 10 replicates (Table 6) fraction spot 2 was considered most promising and was therefore further analysed using High Pressure Liquid Chromatography Analysis (HPLC).

Table 4:	The number of bites by ladybird beetles in fractions 7 and 8

Table 5:	The number of bites by ladybird beetles in fractions 9 and 10

Table 6:	The number of bites by ladybird beetle for every 24 h in 10 replicates

Table 7:	The number of bites by ladybird beetle for every 24 h towards Synthetic Compound (SC)

Feeding Bioassay Using Synthetic Compound Azadirachtin
Table 7 represents the number of bites by ladybird beetle for every 24 h towards Synthetic Compound (SC) of Azadirachtin. Total numbers of bites from Table 7 were analysed using ANOVA. Feeding Bioassay using 50 ppm concentration of synthetic Compound revealed negative antifeedant activity while 100 and 200 ppm showed positive antifeedant response. Further, 100 ppm concentration of synthetic compound was considered as minimum concentration that could cause optimal antifeedant effect towards Epilachna indica.

High Pressure Liquid Chromatography Analysis (HPLC)
Major organic compound that was found in fraction 3 by HPLC was Azadirachtin as shown in result. Table 8 represents area for 5 concentrations to plot standard curve. Azadirachtin was resolved as single peak in all samples analyzed with no interference from other compounds. The identity of the Azadirachtin peak was confirmed by determination of retention time and by spiking with standard Azadirachtin. A calibration curve was derived from three injections of six concentrations of Azadirachtin. Linearity was found in the range and it has a good reproducibility and accuracy (Fig. 5). The following regression equation was obtained y = 1571.9; x-78.784, where y is the peak area and x is the concentration of Azadirachtin. The correlation coefficient of the calibration graph was ≥0.9821.

Table 8:	Area for 5 concentrations to plot standard curve

Fig. 5:	The linear relationship between the area and concentration of Azadirachtin

DISCUSSION

In this study, female ladybird beetle Epilachna indica laid eggs 6.95±0.67 days after mating at environmental at temperature of 30 to 32°C. The eggs hatched in about 4.05±0.80 days to become larvae. Larvae fed for 12.85±0.73 days before changing to immobile pupa stage. Pupation lasted 5.50±0.50 days until the adult beetles emerged from the pupa case. The time for development from an egg to an adult is about 29.35±0.79 days whereas Tung (1983) reported that the life cycle of E. indica is about a month and Epilachna sparsa has a life cycle of 22 to 27 days (Khoo et al., 1991).

Since only 62 newborn adult E. indica were produced from laboratory culture it was not enough for feeding experiment thus adults were used to establish laboratory colony. All experiments in this study used adult captured from wild from the S. melongena farm at Jalan Kebun. Methanolic extraction method was used in experiment was similar to Schlüter and Seifert (1988) who also used methanolic extraction method in studying the Mexican bean beetle, Epilachna varivesties.

About 2500 plants species had one or more active feeding insects but only neem was found to be highly effective, non-toxic and environmentally friendly agent for controlling insects by acting as feeding inhibitor and growth regulator (Warthen, 1979) and neem was projected as the insecticide of the future for protection against field pests (Jotwani and Srivastra, 1981).

Various studies on neem extracts are known to affect various insects in certain ways. The neem extracts disrupt or inhibit the development of eggs, larvae, or pupae. This ensures that the pests do not develop in numbers. The neem extracts also block the molting of larvae or nymphs (Schmutterer and Asher, 1986). Similarly, Kubo and Klocke (1982) isolated and identified azadirachtin as an antifeedant while looking for limonoids as insect controlling agents. It was also observed that these limonoids prevented the completion of larval moulting by inhibiting the exuviae after the formation of new cuticle. These compounds did not kill the insects directly but lowered their growth rate and made them more vulnerable to other mortality factors. Jaipal et al. (1983) also noted juvenile hormone-like activities in the bark of neem and observed that the metamorphosis of the insect was inhibited to varying degrees by these.

This study successfully identified Azadirachtin as the antifeedant from Azadirachta indica which elicited antifeeding behaviour in Epilachna indica (Coleoptera: Coccinellidae). Eventhough Azadirachtin is not a new active compound since has already been found by Kubo and Klocke (1982), Jaipal et al. (1983), Swaminathan (1983), Freeman and Andow (1983), Jacobson (1986), Schmutterer and Asher (1986), Saxena (1987), Kareem et al. (1987), Singh and Singh (1988). However this is the first time Azadirachtin has been tested against Epilachna indica. The result of this study is significant for the field of pest management in Malaysia since Epilachna indica feed on several Solanaceae plants. The use of purified extract of neem was suggested for pest control. Swaminathan (1983) brought forward the potential of neem in pest control. Freeman and Andow (1983) described the role of neem as tree for protection of other plants as an insect feeding deterrent. Jacobson (1986) gave the details of its insecticidal activity. Schmutterer and Asher (1986) edited the proceedings of a conference which had research papers on the pesticidal activity of neem. Saxena (1987) brought forward the use of neem as an antifeedant in pest management in the tropics and recommended quality control and standardization of its biological properties for introduction on a commercial scale.

Qi et al. (2001) in their studies found that 50 and 200 ppm azadirachtin treatments had effects on Schneider, Mallada signatus (Neuroptera: Chrysophidae) pupal survival, with the 200 ppm treatment killing all individuals and about 50% being affected by the 50 ppm treatment. In contrast this study found that in Epilachna indica, 50 ppm concentration Synthetic Compound of Azadirachtin did not give positive antifeedant response unlike Qi et al. (2001). However at 100 and 200 ppm positive antifeedant response were seen in this study on Epilachna indica similar to the study on Mallada signatus (Qi et al., 2001). Both studies show that 200 ppm give maximum response whereas 100 ppm concentration is considered as minimum concentration that can cause optimal antifeedant effect on Epilachna indica this study.

Neem inhibited oviposition, larval development and feeding and greatly increased mortality of cabbage pest, Mamestra brassicae L. (Seljasen and Meadow, 2006). Neem limonoids azadirachtin, salannin, deacetylgedunin, gedunin, 17-hydroxyazadiradione and deacetylnimbin may be used in IPM programs for Cnaphalocrocis medinalis, rice leaffolder and should be evaluated for efficacy under field conditions (Nathan et al., 2005). The potential of neem to control two important pests, the coffee leaf miner (Leucoptera coffeella) and the coffee red mite (Oligonychus ilicis) occurring in coffee plantations was demonstrated by Venzon et al. (2005). In that study, neem was not lethal to an important predator commonly found in coffee agro ecosystems in Brazil. Neem extracts may have a role to play in protecting seedling trees from attack by pine weevil, Hylobius abietis L. during their first year of growth in the field (Thacker et al., 2003). Neem kernel water extract may be recommended for plant protection against third instar nymphs of Jacobiasca lybica in vegetables in the Sudan (El Shafie and Basedow, 2003).

The results of this study confirms that Azadirachtin has the potential for furthering the development of broader scale integrated pest management programs in controlling the ladybird beetle, Epilachna indica in small-scale plantations of Solanum melongena which is an important vegetable in Malaysia. This study is significant to agriculture in Malaysia especially to control pest of eggplant, Epilachna indica in view of using non-toxic natural product as biopesticide, which is safe for the environment and for human health. No other researchers have done studies on antifeedant properties of Azadirachta indica against pest of eggplant, Epilachna indica (Coleoptera: Coccinellidae). For future studies, the compound should be tested in the field on S. melongena plant to compliment results obtained in this study.

ACKNOWLEDGMENTS

Funding for this project was provided by Vot F grant No. F0110/2003A and No. F0190/2004B from University of Malaya, Malaysia.