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Effects of azadirachtin on post-embryonic development, energy reserves and α-amylase activity of Plodia interpunctella Hübner (Lepidoptera: Pyralidae)
ARTICLE IN PRESS Journal of Stored Products Research 44 (2008) 290– 294
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Effects of azadirachtin on post-embryonic development, energy reserves and a-amylase activity of Plodia interpunctella Hu¨bner (Lepidoptera: Pyralidae) Kacem Rharrabe, Haitam Amri, Noureddin Bouayad, Fouad Sayah ´diterrane´ennes, Laboratoire de Biologie Applique´e & Sciences de l’Environnement, PER-Centre des Etudes Environnementales Me ˆdi, BP:416, Tanger, Morocco ´ des Sciences et Techniques, Universite´ Abdelmalek Essaa Faculte
a r t i c l e in f o
a b s t r a c t
Article history: Accepted 18 March 2008
The effects of azadirachtin on the fourth instar larvae of Plodia interpunctella (Lepidoptera) were investigated. When incorporated into the diet at 2 and 4 ppm, azadirachtin provoked larval weight loss, developmental delay and high larval and pupal mortality. Spectrophotometric assays showed that azadirachtin caused a severe reduction in protein, glycogen and lipid contents 7 days after the beginning of the treatment. In addition, a-amylase activity was reduced in larvaefed azadirachtin. & 2008 Elsevier Ltd. All rights reserved.
Keywords: Plodia interpunctella Azadirachtin Development Energy reserves a-Amylase
1. Introduction Azadirachtin is a triterpenoid compound (Butterworth and Morgan, 1968) isolated from the seed kernels of the Indian neem tree Azadirachta indica A. Jussieu (Meliaceae) and the fruits of the bark of Chinaball tree Melia azedarach (L.) (Morgan and Thornton, 1973). Much research has focused upon the insecticidal effects induced by azadirachtin (Mordue and Blackwell, 1993; Mordue et al., 2005). Azadirachtin, apart from its unique mode of action against insects, can also affect other organisms including nematodes, fungi, viruses and protozoa (Mordue and Blackwell, 1993). It is a potent antifeedant disrupting growth, development and reproduction of several insect species (Mordue and Blackwell, 1993; Mordue et al., 1998; Walter, 1999; Mordue et al., 2005). Azadirachtin, as a biopesticide, has minimal effect on non-target organisms such as natural enemies and pollinators (Lowery and Isman, 1995; Naumann and Isman, 1996) and is non-toxic to vertebrates (Scott et al., 1999; Salehzadeh et al., 2002). However, azadirachtin is not effective against all pest insects. Its effects depend on the concentrations used, on the method of application (e.g. ingestion, injection and topical application) and the species (Mordue and Blackwell, 1993). Though there is a large amount of data on the effects of this compound on insects, there have been no studies on the Indian meal moth, Plodia interpunctella (Hu¨bner) (Lepidoptera: Pyralidae). This insect has a worldwide distribution. Larvae have been recorded feeding upon
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E-mail address: [email protected] (F. Sayah). 0022-474X/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jspr.2008.03.003
and contaminating many commodities (Simmons and Nelson, 1975). Living on a starch-rich diet, many insects depend on the effectiveness of their amylases for survival. The a-amylases (a-1,4-glucan-4-glucanohydrolases; EC 3.2.1.1) are hydrolytic enzymes that are found in microorganisms, plants and animals. These enzymes catalyse the hydrolysis of a-D-(1,4)-glucan linkages in starch and related carbohydrates (Strobl et al., 1998). They are synthesized and secreted by exocytosis from midgut epithelial cells (Terra and Ferreira, 2005). Azadirachtin action on the physiology of digestion has been related to the efficiency of digestion and inhibition of digestive enzymes (Mordue et al., 2005). In the present work, the effects of azadirachtin on P. interpunctella development, on larval energy reserves (protein, glycogen and lipid contents), and on the activity of the enzyme a-amylase were studied.
2. Materials and methods 2.1. Insect rearing The stock of P. interpunctella used in this research was initially collected as larvae from Errachidia province in the south-east region of Morocco. The stock was reared in the laboratory from 1999 under standard conditions of 2872 1C with a relative humidity (r.h.) of 7075% and a photoperiod of 16:8 (L:D). Larvae were placed in 0.25-l glass containers half full of dates as a diet.
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2.2. Azadirachtin treatment Azadirachtin (Sigma Chemical) was dissolved in 10% ethanol in distilled water. A volume of 2 ml was incorporated into 4 g of dates at 2 and 4 ppm. For control larvae, 2 ml of 10% ethanol in distilled water alone was added to the dates. In each case the ethanol was then evaporated from the diet. 2.3. Post-embryonic development Fourth instar larvae were starved for 24 h prior to use to induce a higher feeding rate, and then groups of 10 larvae were placed in the Petri dishes in the presence of 4 g of treated or control diet. Observations including weight change, larval and pupal mortality were recorded every 2 days for 20 days. Larval mortality was determined by a brown colouration with no observable movement. Pupal mortality was determined by the appearance of black colour with no emergence. Insects were maintained at 2872 1C and 7075% r.h. during the whole experiment. Five replicates were set up for treated and for control larvae. 2.4. Protein determination For protein and other biochemical analyses, fourth instar larvae were reared individually and taken 7 days after the beginning of the treatment. Each sample was composed of cold-anesthetized larvae. Six to ten replicates were run for the analyses and for each dose. Each larva was homogenized in 1 ml of Tris 50 mM buffer at pH 7. Protein content was quantified as described by Bradford (1976) using BSA as a standard. 2.5. Glycogen determination Glycogen content was quantified by the method of Roe (1955) using anthrone reagent. Each larva was homogenized in 1 ml of ethanol saturated with sodium sulphate. It was then centrifuged at 1000 rpm for 10 min and the supernatant discarded. The pellet was resuspended in 0.5 ml of 70% ethanol then centrifuged as before and the supernatant was discarded. The final pellet was heated to drive off residual ethanol and then dissolved in 0.5 ml of 30% potassium hydroxide and heated at 100 1C for 15 min. The digest was cooled, and 1 ml of absolute ethanol was added. This was spun down by centrifugation at 1000 rpm for 10 min. The supernatant was carefully removed and the pellet was dissolved in 0.5 ml of distilled water. Two millilitres of anthrone reagent (0.05% in sulphuric acid) was then added. After mixing, the mixtures were heated at 90 1C for 15 min and then cooled. The absorbance was read at 620 nm, and glycogen level was calculated by reference to a standard curve prepared using glycogen. 2.6. Lipid determination Lipid was extracted according to the method of Van Handel (1965). Each larva was homogenized in 1 ml of chloroform/ methanol (1:1 v/v). The homogenate was centrifuged at 1000 rpm for 10 min at 4 1C. The supernatant was mixed with 1 ml of chloroform and 0.5 ml of distilled water and centrifuged at 1500 rpm for 1 h at 4 1C. The aqueous phase was discarded (this operation was repeated twice). The lipid extract was evaporated to dryness. Lipid content was quantified by the method of Zo¨llner and Krich (1962). The sample was digested with 1 ml of sulphuric acid at 100 1C for 10 min. The tube was cooled and 5 ml of
sulphosphovanillin reagent (orthophosphoric acid/0.6% aqueous vanillin solution 4:1) was added to the mixture. After 40 min, the absorbance was measured at 530 nm and lipid level was calculated by reference to a standard curve prepared using cholesterol palmitate. 2.7. a-Amylase activity Polyacrylamide gel electrophoresis was carried out in a vertical slab gel apparatus as described by Laemmli (1970) at 4 1C. Samples were homogenized in 50 ml of buffer (0.5-M Tris, 10% glycerol, 0.1% bromophenol blue) and run on a 12% polyacrylamide gel in Tris-glycine buffer at pH 8.5, with constant current set at 100 mA. A zymogram of amylase activity was carried out as described by Campos et al. (1989). After separation, the gel was placed in a solution of 0.1-M Tris-borate, 0.8% amidon solution and 20-mM CaCl2 at pH 8.5, and incubated for 90 min at 30 1C. a-Amylase activity appeared on the polyacrylamide gel as clear bands on a purple-coloured background after staining with I2–KI solution. 2.8. Statistical analysis Developmental results were analysed by one-way ANOVA using Statistica Software (Statistica, 1997). Tukey honest significant difference (HSD) test was carried out for biochemical analyses. A significance level of 0.05 was used for all statistical tests.
3. Results 3.1. Effects of azadirachtin on development Exposure of larvae to diet containing azadirachtin caused a reduction of their weight (Fig. 1). Eight days after the treatment, the weight loss was 36% and 37% for the doses of 2 and 4 ppm, respectively. In contrast, control larvae gained 39% in weight. The statistical analysis showed that the treatment at both doses had a highly significant effect (F(1,62) ¼ 70.53 and F(1,77) ¼ 61.66 respectively, Po0.001 in each case). Azadirachtin caused both larval and pupal mortality. The larval mortality (Fig. 2) began 4 days after treatment with 7% in larvae treated at 2 ppm and 10% at 4 ppm. After 16 days of treatment, the mortality of larvae was 34% at 2 ppm and 25% at 4 ppm. There was no mortality in untreated larvae. As in larvae, azadirachtin caused pupal mortality (Fig. 3). It began 8 days after treatment. It reached 33% and 20% respectively
20 Control 2 ppm 4 ppm
16 Weight (mg)
Emerging adults were removed and allowed to mate in new 0.25-l glass containers.
291
12 8 4 0 0
2
4 Time (Days)
6
8
Fig. 1. Effect of azadirachtin on Plodia interpunctella larval weight. Each point represents the mean7standard error of five replicates.
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% of cumulated larval mortality
50
Table 1 Effect of azadirachtin on protein, glycogen and lipid content of Plodia interpunctella larvae 7 days after exposure
Control 2 ppm 4 ppm
40 30 20
Azadirachtin (ppm)
Protein (mg/larva)
Glycogen (mg/larva)
Lipid (mg/larva)
0 2 4
586768a 297725b 431752b
153738a 772b 1377b
866732a 779795a 5607126b
Means in the same column followed by different letters indicate that the difference between control and treated larvae is highly significant (Po0.001) as determined by the Tukey HSD test. Each point represents the mean7standard error of 6–10 replicates.
10 0 0
2
4
6
10 8 Time (Days)
12
14
16 4 ppm
2 ppm
Control
Fig. 2. Effect of azadirachtin on Plodia interpunctella larval mortality. Each point represents the mean7standard error of five replicates.
% of cumulated pupal mortality
50
Control 2 ppm 4 ppm
40
1
30 20 10 2
0 0
2
4
6
10 12 8 Time (Days)
14
16
18
20
Fig. 3. Effect of azadirachtin on Plodia interpunctella pupal mortality. Each point represents the mean7standard error of five replicates.
for the doses of 2 and 4 ppm after 20 days. Mortality of control pupae did not exceed 8% during the observation period. The statistical analysis showed that the treatment at both doses had a highly significant effect on larval and pupal mortality (F(1,64) ¼ 315.92 and F(1,64) ¼ 110.21, respectively, Po0.001 for both). The difference between the doses was also significant for the both larval and pupal mortality (F(1,64) ¼ 36.55, Po0.05). 3.2. Effect of azadirachtin on biochemical parameters Biochemical results showed clearly that azadirachtin induced a decrease in protein, glycogen and lipid contents compared with control larvae (Table 1). The a-amylase activity on polyacrylamide gel showed the presence of two isoenzymes: a major one (1) with a highmolecular weight and a minor one (2) with a low-molecular weight. According to the zymogram, we noted very weak enzymatic activity in larvae fed azadirachtin indicating a severe reduction in a-amylase activity (Fig. 4).
4. Discussion Azadirachtin significantly reduced larval growth and was toxic to P. interpunctella. This toxicity was displayed by a decrease of larvae weight, inhibition of development, together with significant larval and pupal mortality. Such effects on development and
Fig. 4. Effect of azadirachtin on a-amylase activity, in gel expression, of Plodia interpunctella larvae 7 days after exposure: (1) major isoenzyme and (2) minor isoenzyme (not evident).
survival have often been described in other insects treated with azadirachtin (Mordue and Blackwell, 1993). Feeding larvae of Spodoptera litura (F.), with azadirachtin caused a reduction of feeding and death of larvae and pupae (Huang et al., 2004). The effect of ingested azadirachtin in P. interpunctella could be due to antifeedant action as well as a toxic effect. An antifeedant and repellent action of azadirachtin has been noted in insects as diverse as Rhyzopertha dominica (F.) (Malik and Mujtaba Naqvi, 1984), Ostrinia nubilalis (Hu¨bner) (Arnason et al., 1985), Cryptolestes ferrugineus (Stephens), Sitophilus oryzae (L.) and Tribolium castaneum (Herbst) (Xie et al., 1995). A reduction in larval feeding was also seen in four keratinophagous insect species after neem seed extract treatment (Gerard and Ruf, 1995). Even if an antifeedant effect is often observed after azadirachtin treatment (Mordue and Blackwell, 1993), it is not a generalized phenomenon among insect species. For example, in Epilachna varivestis (Mulsant) (Schmutterer, 1990) and Rhodnius prolixus (Stal) (Garcia and Rembold, 1984), azadirachtin does not inhibit feeding, and, consequently, growth and development are not affected by lack of nutrition. In Locusta migratoria L., last instar
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larvae feeding inhibition is not the primary cause for growth disruption (Sieber and Rembold, 1983). In Labidura riparia (Pallas), inhibition of vitellogenesis does not result from antifeeding effects, since starved females exhibited vitellogenic ovaries (Sayah et al., 1996). Pavela et al. (2004) in experiments with the aphid, Brevicoryne brassicae (L.), showed that the systemic application of low concentrations of azadirachtin on the diet caused a significant increase in mortality during the nymphal stage. Similar effects were seen with Spodoptera littoralis (Boisduval) exposed to azadirachtin (Adel and Sehnal, 2000). Neem seed extract ingestion also provoked significant mortality in Tinea dubiella (Stainton) and Tineola bisselliella (Hummel) (Gerard and Ruf, 1995). These adverse developmental effects may be due to the action of azadirachtin on the endocrine and neuroendocrine system regulating the developmental processes of insects (Mordue et al., 2005). For instance, in Tenebrio molitor (L.) (Marco et al., 1990), Oncopeltus fasciatus (Dallas) (Dorn et al., 1986), Manduca sexta (L.) (Schlu¨ter et al., 1985) and Galleria mellonella (L.) (Malczewska et al., 1988), azadirachtin caused a depletion of the moulting hormone ecdysteroid. In parallel with ecdysteroid deficiencies, developmental anomalies were also related to the lack of the juvenile hormone (Schlu¨ter et al., 1985; Koul et al., 1987; Malczewska et al., 1988). Moreover, the developmental effects of azadirachtin were attributed to a disruption of ecdysteroid and juvenile hormone titres through a blockage of morphogenetic peptide hormone release from the neuroendocrine system (Koolman et al., 1988; Subrahmanyam and Rembold, 1989; Sayah et al., 1998). As a result of exposure to azadirachtin, a significant decrease in protein, glycogen and lipid contents was observed in P. interpunctella. The depletion of such biochemical constituents can be due to major mobilization of these substances in response to the lack of nutrients caused by the toxic effect of azadirachtin on the midgut as well as a reduction of their synthesis. Azadirachtin may exert its effect on insects by modifying the protein synthesis capacity of the fat body as was seen in L. migratoria (Rembold et al., 1987), R. prolixus (Feder et al., 1988), L. riparia (Sayah et al., 1996) and S. litura (Huang et al., 2004). Our results showed that a-amylase activity was reduced after azadirachtin ingestion. This reduction can be due to an inhibition of enzyme activity or to the cytotoxic effect of azadirachtin on midgut epithelial cells which synthesize this enzyme. In R. prolixus (Nogueira et al., 1997) and L. migratoria (Nasiruddin and Mordue, 1993) azadirachtin caused severe cytotoxicity to midgut epithelial cells. Azadirachtin affects many enzymatic activities in different tissues where cells are producing enzymes. For example, azadirachtin inhibits secretion of trypsin in the midgut of M. sexta (Timmins and Reynolds, 1992). Ingestion of azadirachtin by S. litura and Cnaphalocrocis medinalis (Guene´e) (Senthil Nathan et al., 2005a, b) decreases gut enzyme activities. In summary, this work shows clearly that azadirachtin provoked potent toxic and growth inhibiting effects in P. interpunctella. It also induced physiological depression of energy reserves and a-amylase activity.
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Effects of azadirachtin on post-embryonic development, energy reserves and a-amylase activity of Plodia interpunctella Hu¨bner (Lepidoptera: Pyralidae) Kacem Rharrabe, Haitam Amri, Noureddin Bouayad, Fouad Sayah ´diterrane´ennes, Laboratoire de Biologie Applique´e & Sciences de l’Environnement, PER-Centre des Etudes Environnementales Me ˆdi, BP:416, Tanger, Morocco ´ des Sciences et Techniques, Universite´ Abdelmalek Essaa Faculte
a r t i c l e in f o
a b s t r a c t
Article history: Accepted 18 March 2008
The effects of azadirachtin on the fourth instar larvae of Plodia interpunctella (Lepidoptera) were investigated. When incorporated into the diet at 2 and 4 ppm, azadirachtin provoked larval weight loss, developmental delay and high larval and pupal mortality. Spectrophotometric assays showed that azadirachtin caused a severe reduction in protein, glycogen and lipid contents 7 days after the beginning of the treatment. In addition, a-amylase activity was reduced in larvaefed azadirachtin. & 2008 Elsevier Ltd. All rights reserved.
Keywords: Plodia interpunctella Azadirachtin Development Energy reserves a-Amylase
1. Introduction Azadirachtin is a triterpenoid compound (Butterworth and Morgan, 1968) isolated from the seed kernels of the Indian neem tree Azadirachta indica A. Jussieu (Meliaceae) and the fruits of the bark of Chinaball tree Melia azedarach (L.) (Morgan and Thornton, 1973). Much research has focused upon the insecticidal effects induced by azadirachtin (Mordue and Blackwell, 1993; Mordue et al., 2005). Azadirachtin, apart from its unique mode of action against insects, can also affect other organisms including nematodes, fungi, viruses and protozoa (Mordue and Blackwell, 1993). It is a potent antifeedant disrupting growth, development and reproduction of several insect species (Mordue and Blackwell, 1993; Mordue et al., 1998; Walter, 1999; Mordue et al., 2005). Azadirachtin, as a biopesticide, has minimal effect on non-target organisms such as natural enemies and pollinators (Lowery and Isman, 1995; Naumann and Isman, 1996) and is non-toxic to vertebrates (Scott et al., 1999; Salehzadeh et al., 2002). However, azadirachtin is not effective against all pest insects. Its effects depend on the concentrations used, on the method of application (e.g. ingestion, injection and topical application) and the species (Mordue and Blackwell, 1993). Though there is a large amount of data on the effects of this compound on insects, there have been no studies on the Indian meal moth, Plodia interpunctella (Hu¨bner) (Lepidoptera: Pyralidae). This insect has a worldwide distribution. Larvae have been recorded feeding upon
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E-mail address: [email protected] (F. Sayah). 0022-474X/$ - see front matter & 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.jspr.2008.03.003
and contaminating many commodities (Simmons and Nelson, 1975). Living on a starch-rich diet, many insects depend on the effectiveness of their amylases for survival. The a-amylases (a-1,4-glucan-4-glucanohydrolases; EC 3.2.1.1) are hydrolytic enzymes that are found in microorganisms, plants and animals. These enzymes catalyse the hydrolysis of a-D-(1,4)-glucan linkages in starch and related carbohydrates (Strobl et al., 1998). They are synthesized and secreted by exocytosis from midgut epithelial cells (Terra and Ferreira, 2005). Azadirachtin action on the physiology of digestion has been related to the efficiency of digestion and inhibition of digestive enzymes (Mordue et al., 2005). In the present work, the effects of azadirachtin on P. interpunctella development, on larval energy reserves (protein, glycogen and lipid contents), and on the activity of the enzyme a-amylase were studied.
2. Materials and methods 2.1. Insect rearing The stock of P. interpunctella used in this research was initially collected as larvae from Errachidia province in the south-east region of Morocco. The stock was reared in the laboratory from 1999 under standard conditions of 2872 1C with a relative humidity (r.h.) of 7075% and a photoperiod of 16:8 (L:D). Larvae were placed in 0.25-l glass containers half full of dates as a diet.
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2.2. Azadirachtin treatment Azadirachtin (Sigma Chemical) was dissolved in 10% ethanol in distilled water. A volume of 2 ml was incorporated into 4 g of dates at 2 and 4 ppm. For control larvae, 2 ml of 10% ethanol in distilled water alone was added to the dates. In each case the ethanol was then evaporated from the diet. 2.3. Post-embryonic development Fourth instar larvae were starved for 24 h prior to use to induce a higher feeding rate, and then groups of 10 larvae were placed in the Petri dishes in the presence of 4 g of treated or control diet. Observations including weight change, larval and pupal mortality were recorded every 2 days for 20 days. Larval mortality was determined by a brown colouration with no observable movement. Pupal mortality was determined by the appearance of black colour with no emergence. Insects were maintained at 2872 1C and 7075% r.h. during the whole experiment. Five replicates were set up for treated and for control larvae. 2.4. Protein determination For protein and other biochemical analyses, fourth instar larvae were reared individually and taken 7 days after the beginning of the treatment. Each sample was composed of cold-anesthetized larvae. Six to ten replicates were run for the analyses and for each dose. Each larva was homogenized in 1 ml of Tris 50 mM buffer at pH 7. Protein content was quantified as described by Bradford (1976) using BSA as a standard. 2.5. Glycogen determination Glycogen content was quantified by the method of Roe (1955) using anthrone reagent. Each larva was homogenized in 1 ml of ethanol saturated with sodium sulphate. It was then centrifuged at 1000 rpm for 10 min and the supernatant discarded. The pellet was resuspended in 0.5 ml of 70% ethanol then centrifuged as before and the supernatant was discarded. The final pellet was heated to drive off residual ethanol and then dissolved in 0.5 ml of 30% potassium hydroxide and heated at 100 1C for 15 min. The digest was cooled, and 1 ml of absolute ethanol was added. This was spun down by centrifugation at 1000 rpm for 10 min. The supernatant was carefully removed and the pellet was dissolved in 0.5 ml of distilled water. Two millilitres of anthrone reagent (0.05% in sulphuric acid) was then added. After mixing, the mixtures were heated at 90 1C for 15 min and then cooled. The absorbance was read at 620 nm, and glycogen level was calculated by reference to a standard curve prepared using glycogen. 2.6. Lipid determination Lipid was extracted according to the method of Van Handel (1965). Each larva was homogenized in 1 ml of chloroform/ methanol (1:1 v/v). The homogenate was centrifuged at 1000 rpm for 10 min at 4 1C. The supernatant was mixed with 1 ml of chloroform and 0.5 ml of distilled water and centrifuged at 1500 rpm for 1 h at 4 1C. The aqueous phase was discarded (this operation was repeated twice). The lipid extract was evaporated to dryness. Lipid content was quantified by the method of Zo¨llner and Krich (1962). The sample was digested with 1 ml of sulphuric acid at 100 1C for 10 min. The tube was cooled and 5 ml of
sulphosphovanillin reagent (orthophosphoric acid/0.6% aqueous vanillin solution 4:1) was added to the mixture. After 40 min, the absorbance was measured at 530 nm and lipid level was calculated by reference to a standard curve prepared using cholesterol palmitate. 2.7. a-Amylase activity Polyacrylamide gel electrophoresis was carried out in a vertical slab gel apparatus as described by Laemmli (1970) at 4 1C. Samples were homogenized in 50 ml of buffer (0.5-M Tris, 10% glycerol, 0.1% bromophenol blue) and run on a 12% polyacrylamide gel in Tris-glycine buffer at pH 8.5, with constant current set at 100 mA. A zymogram of amylase activity was carried out as described by Campos et al. (1989). After separation, the gel was placed in a solution of 0.1-M Tris-borate, 0.8% amidon solution and 20-mM CaCl2 at pH 8.5, and incubated for 90 min at 30 1C. a-Amylase activity appeared on the polyacrylamide gel as clear bands on a purple-coloured background after staining with I2–KI solution. 2.8. Statistical analysis Developmental results were analysed by one-way ANOVA using Statistica Software (Statistica, 1997). Tukey honest significant difference (HSD) test was carried out for biochemical analyses. A significance level of 0.05 was used for all statistical tests.
3. Results 3.1. Effects of azadirachtin on development Exposure of larvae to diet containing azadirachtin caused a reduction of their weight (Fig. 1). Eight days after the treatment, the weight loss was 36% and 37% for the doses of 2 and 4 ppm, respectively. In contrast, control larvae gained 39% in weight. The statistical analysis showed that the treatment at both doses had a highly significant effect (F(1,62) ¼ 70.53 and F(1,77) ¼ 61.66 respectively, Po0.001 in each case). Azadirachtin caused both larval and pupal mortality. The larval mortality (Fig. 2) began 4 days after treatment with 7% in larvae treated at 2 ppm and 10% at 4 ppm. After 16 days of treatment, the mortality of larvae was 34% at 2 ppm and 25% at 4 ppm. There was no mortality in untreated larvae. As in larvae, azadirachtin caused pupal mortality (Fig. 3). It began 8 days after treatment. It reached 33% and 20% respectively
20 Control 2 ppm 4 ppm
16 Weight (mg)
Emerging adults were removed and allowed to mate in new 0.25-l glass containers.
291
12 8 4 0 0
2
4 Time (Days)
6
8
Fig. 1. Effect of azadirachtin on Plodia interpunctella larval weight. Each point represents the mean7standard error of five replicates.
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% of cumulated larval mortality
50
Table 1 Effect of azadirachtin on protein, glycogen and lipid content of Plodia interpunctella larvae 7 days after exposure
Control 2 ppm 4 ppm
40 30 20
Azadirachtin (ppm)
Protein (mg/larva)
Glycogen (mg/larva)
Lipid (mg/larva)
0 2 4
586768a 297725b 431752b
153738a 772b 1377b
866732a 779795a 5607126b
Means in the same column followed by different letters indicate that the difference between control and treated larvae is highly significant (Po0.001) as determined by the Tukey HSD test. Each point represents the mean7standard error of 6–10 replicates.
10 0 0
2
4
6
10 8 Time (Days)
12
14
16 4 ppm
2 ppm
Control
Fig. 2. Effect of azadirachtin on Plodia interpunctella larval mortality. Each point represents the mean7standard error of five replicates.
% of cumulated pupal mortality
50
Control 2 ppm 4 ppm
40
1
30 20 10 2
0 0
2
4
6
10 12 8 Time (Days)
14
16
18
20
Fig. 3. Effect of azadirachtin on Plodia interpunctella pupal mortality. Each point represents the mean7standard error of five replicates.
for the doses of 2 and 4 ppm after 20 days. Mortality of control pupae did not exceed 8% during the observation period. The statistical analysis showed that the treatment at both doses had a highly significant effect on larval and pupal mortality (F(1,64) ¼ 315.92 and F(1,64) ¼ 110.21, respectively, Po0.001 for both). The difference between the doses was also significant for the both larval and pupal mortality (F(1,64) ¼ 36.55, Po0.05). 3.2. Effect of azadirachtin on biochemical parameters Biochemical results showed clearly that azadirachtin induced a decrease in protein, glycogen and lipid contents compared with control larvae (Table 1). The a-amylase activity on polyacrylamide gel showed the presence of two isoenzymes: a major one (1) with a highmolecular weight and a minor one (2) with a low-molecular weight. According to the zymogram, we noted very weak enzymatic activity in larvae fed azadirachtin indicating a severe reduction in a-amylase activity (Fig. 4).
4. Discussion Azadirachtin significantly reduced larval growth and was toxic to P. interpunctella. This toxicity was displayed by a decrease of larvae weight, inhibition of development, together with significant larval and pupal mortality. Such effects on development and
Fig. 4. Effect of azadirachtin on a-amylase activity, in gel expression, of Plodia interpunctella larvae 7 days after exposure: (1) major isoenzyme and (2) minor isoenzyme (not evident).
survival have often been described in other insects treated with azadirachtin (Mordue and Blackwell, 1993). Feeding larvae of Spodoptera litura (F.), with azadirachtin caused a reduction of feeding and death of larvae and pupae (Huang et al., 2004). The effect of ingested azadirachtin in P. interpunctella could be due to antifeedant action as well as a toxic effect. An antifeedant and repellent action of azadirachtin has been noted in insects as diverse as Rhyzopertha dominica (F.) (Malik and Mujtaba Naqvi, 1984), Ostrinia nubilalis (Hu¨bner) (Arnason et al., 1985), Cryptolestes ferrugineus (Stephens), Sitophilus oryzae (L.) and Tribolium castaneum (Herbst) (Xie et al., 1995). A reduction in larval feeding was also seen in four keratinophagous insect species after neem seed extract treatment (Gerard and Ruf, 1995). Even if an antifeedant effect is often observed after azadirachtin treatment (Mordue and Blackwell, 1993), it is not a generalized phenomenon among insect species. For example, in Epilachna varivestis (Mulsant) (Schmutterer, 1990) and Rhodnius prolixus (Stal) (Garcia and Rembold, 1984), azadirachtin does not inhibit feeding, and, consequently, growth and development are not affected by lack of nutrition. In Locusta migratoria L., last instar
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larvae feeding inhibition is not the primary cause for growth disruption (Sieber and Rembold, 1983). In Labidura riparia (Pallas), inhibition of vitellogenesis does not result from antifeeding effects, since starved females exhibited vitellogenic ovaries (Sayah et al., 1996). Pavela et al. (2004) in experiments with the aphid, Brevicoryne brassicae (L.), showed that the systemic application of low concentrations of azadirachtin on the diet caused a significant increase in mortality during the nymphal stage. Similar effects were seen with Spodoptera littoralis (Boisduval) exposed to azadirachtin (Adel and Sehnal, 2000). Neem seed extract ingestion also provoked significant mortality in Tinea dubiella (Stainton) and Tineola bisselliella (Hummel) (Gerard and Ruf, 1995). These adverse developmental effects may be due to the action of azadirachtin on the endocrine and neuroendocrine system regulating the developmental processes of insects (Mordue et al., 2005). For instance, in Tenebrio molitor (L.) (Marco et al., 1990), Oncopeltus fasciatus (Dallas) (Dorn et al., 1986), Manduca sexta (L.) (Schlu¨ter et al., 1985) and Galleria mellonella (L.) (Malczewska et al., 1988), azadirachtin caused a depletion of the moulting hormone ecdysteroid. In parallel with ecdysteroid deficiencies, developmental anomalies were also related to the lack of the juvenile hormone (Schlu¨ter et al., 1985; Koul et al., 1987; Malczewska et al., 1988). Moreover, the developmental effects of azadirachtin were attributed to a disruption of ecdysteroid and juvenile hormone titres through a blockage of morphogenetic peptide hormone release from the neuroendocrine system (Koolman et al., 1988; Subrahmanyam and Rembold, 1989; Sayah et al., 1998). As a result of exposure to azadirachtin, a significant decrease in protein, glycogen and lipid contents was observed in P. interpunctella. The depletion of such biochemical constituents can be due to major mobilization of these substances in response to the lack of nutrients caused by the toxic effect of azadirachtin on the midgut as well as a reduction of their synthesis. Azadirachtin may exert its effect on insects by modifying the protein synthesis capacity of the fat body as was seen in L. migratoria (Rembold et al., 1987), R. prolixus (Feder et al., 1988), L. riparia (Sayah et al., 1996) and S. litura (Huang et al., 2004). Our results showed that a-amylase activity was reduced after azadirachtin ingestion. This reduction can be due to an inhibition of enzyme activity or to the cytotoxic effect of azadirachtin on midgut epithelial cells which synthesize this enzyme. In R. prolixus (Nogueira et al., 1997) and L. migratoria (Nasiruddin and Mordue, 1993) azadirachtin caused severe cytotoxicity to midgut epithelial cells. Azadirachtin affects many enzymatic activities in different tissues where cells are producing enzymes. For example, azadirachtin inhibits secretion of trypsin in the midgut of M. sexta (Timmins and Reynolds, 1992). Ingestion of azadirachtin by S. litura and Cnaphalocrocis medinalis (Guene´e) (Senthil Nathan et al., 2005a, b) decreases gut enzyme activities. In summary, this work shows clearly that azadirachtin provoked potent toxic and growth inhibiting effects in P. interpunctella. It also induced physiological depression of energy reserves and a-amylase activity.
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