Toxicity of Chlorantraniliprole and Lufenuron Against the Cotton Leafworm Larvae in Relation to Their Effects on AST, ALT and ALP Enzymes Activity

INTRODUCTION

The cotton leafworm (CLW), Spodoptera littoralis (Boisd.) is one of the most destructive agriculture lepidopterous insect-pests. In Egypt, it can attack numerous economically important crops throughout the year (Hatem et al., 2009). The chemical control of S. littoralis has been extensively reported especially in relation to cotton in Egypt (Abo-El-Ghar et al., 1986). Extensive use of insecticides, multiple generations of CLW per annum and the availability of host crops, all over the year have contributed to the development of resistance in this insect-pest to many insecticide groups (Abo Elghar et al., 2005; Abou-Taleb, 2010). Therefore, searching for an effective alternatives and/or pest control strategies is needed to avoid the increase of the selection pressure of the insect population to insecticides and provide adequate crop protection for sustainable food, feed and crops of fiber production. This need is met in part by the insecticide chlorantraniliprole, which has a novel mechanism of action and can be applied as an alternative insecticide for managing CLW (Bentley et al., 2010).

Chlorantraniliprole is discovered by DuPont; it is also known as rynaxypyr (Bentley et al., 2010). Studies have shown that chlorantraniliprole has exceptional insecticidal activity on a range of lepidopteran pests and many other orders, such as Coleoptera, Diptera, Isoptera and Hemiptera (Sattelle et al., 2008; Lahm et al., 2009). Chlorantraniliprole activates the unregulated release of internal calcium stores leading to Ca^2+‏ depletion, feeding cessation, lethargy, muscle paralysis and finally insect death (Lahm et al., 2005). It blocks the feeding of lepidopteran larvae rapidly; the feeding cessation time being equivalent to that of nerve agents. Accordingly, it was ranked as the fastest-acting insecticide for lepidopteran control (Hannig et al., 2009). In addition, the low ecotoxicology to non-target organisms such as birds, fish, mammals, earthworms and many other arthropods (Lahm et al., 2007; Larson et al., 2012) and no cross-resistance with other older classes of chemistry (Cao et al., 2010; Sial et al., 2010; Wang et al., 2010) make it an excellent pest management tool.

It is critically important to establish the susceptibility of insects to newly developed insecticides before their widespread use. Therefore, the aim of this work was to assess the insecticidal activity of chlorantraniliprole compared to lufenuron against the 2^nd and 4^th larval instars (laboratory strain) of the cotton leafworm. The in vivo effects of chlorantraniliprole and lufenuron on certain enzymes activities [aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatases (ALP)] of the 4^th instar larvae of the cotton leafworm (laboratory strain) were also investigated.

MATERIALS AND METHODS

Experimental insect:

A laboratory strain of the cotton leafworm, Spodoptera littoralis larvae used for testing the evaluated insecticides was reared under the laboratory conditions of 25±2 ºC and 65±5 % RH according to Eldefrawi et al. (1964).

Tested insecticides:

Chlorantraniliprole (Coragen^® 20% SC) was provided by DuPont Agricultural Chemicals Ltd.). Lufenuron (Match^®5% EC) was supplied by Syngenta.

Bioassay studies:

 Toxicity of the formulated chlorantraniliprole (Coragen^® 20% SC) and lufenuron (Match^®5% EC) against 2^nd and 4^th instar larvae of S. littoralis laboratory strains was evaluated. Homogenous pieces of castor oil leaves were dipped in a series of chlorantraniliprole or lufenuron concentrations for 10 sec., held vertically to allow excess solution to drip off and dried at room temperature. Treated pieces of castor oil leaf were transferred to a plastic cups, and an appropriate and fixed number (10 larvae per cup) of starved larvae were added. Each concentration was replicated four times. Mortality percentages were recorded after 24, 48, 72, 96 hrs of treatment. Mortality counts were recorded and corrected according to Abbott equation (Abbott, 1925) and subjected to probit analysis (Finney, 1971).

Biochemical studies

In vivo effect of chlorantraniliprole and lufenuron on AST and ALT activities of 4^th instar larvaeof S. littoralis (Lab. strain)

Tissue preparation: Starved 4^th instar larvae of S. littoralis laboratory strain were fed on castor oil leaves dipped in chlorantraniliprole and lufenuron solution at ¹/₁₀ LC₅₀, ½ LC₅₀ and LC₅₀. Larvae fed on untreated castor oil leaves were used as control. Suitable numbers of total larvae of each treatment were collected after 96 hrs post-treatment. These larvae were homogenized (1:10 w/v) in glass distilled water (pH=7) using glass homogenizer. The homogenate was centrifuged at 5000 rpm for 30 min at 4ºC using IEC-CRU 5000 cooling centrifuge. The supernatant was used for the estimation of AST (Aspartate aminotransferase), and ALT (Alanine aminotransferase) activities.

Enzyme assay: Activity of both enzymes AST and ALT was measured according to the method of Reitman and Frankel (1957), using Diamond Diagnostic kit (Diamond Co. Egypt). In this method, 100 μl of enzyme source was added to 500 μl of 100 mM phosphate buffer of pH=7.2 containing 80 mM L-aspartate as a substrate for AST or 80 mM D-L-alanine as a substrate for ALT, and 4 mM ά-ketoglutarate. This mixture was incubated for 30 min at 37˚C. After that, 500 μl of developing color reagent (4 mM 2, 4-dinitrophenylhydrazine) was added and the solution was incubated for 20 min at room temperature. Lastly, 5 ml of 0.4 N NaOH was added then mixed and left at room temperature for five min. An assay mixture without enzyme source was used as the blank and the absorption was measured at the wave length of 546 nm using spectrophotometer (Milton Roy Spectronic 601). AST and ALT specific activities were determined as IU/mg protein/hr and calculated as a percentage of control.

In vivo effect of S. littoralis (Lab. strain) 4^th instar larvae ALP activity by chlorantraniliprole and lufenuron

Tissue preparation: Starved laboratory strain 4^th instar larvae of S. littoralis were fed on castor oil leaves dipped in chlorantraniliprole and lufenuron solution at ¹/₁₀ LC₅₀, ½ LC₅₀ and LC₅₀ plus the untreated larvae which have been used as control. Midguts were collected, after 96 hrs post-treatment, excised, repeatedly washed with ice-cold saline solution (0.9% NaCl) to remove foodstuff. These midguts were homogenized (1:10 w/v) in 100 mM phosphate buffer (pH= 9.8), using glass homogenizer. The homogenate was centrifuged at 5000 rpm for 30 min at 4ºC using IEC-CRU 5000 cooling centrifuge. The supernatant was used for ALP (alkaline phosphatases) activity estimation.

Enzyme assay: Activity of ALP was determined according to the method of Dgkc (1972), using Diamond Diagnostic kit (Diamond Co. Egypt). In this method, 20 μl of the enzyme source was added to 1000 μl of 0.9 M diethanolamine buffer (pH 9.8) containing 0.6 mM magnesium ions and 1 mM p-nitrophenyl phosphate, then mixed in the cuvette, incubated for 30 seconds in the spectrophotometer (Milton Roy Spectronic 601), using a stopwatch simultaneously and the reading was done again after exactly 1, 2 and 3 minutes at 405 nm. ALP specific activity was determined as IU/mg protein/hr and calculated as a percent age of control.

Statistical analysis: Data was subjected to analysis of variance (ANOVA) (CoStat Statistical Software, 1990). The standard deviation (SD) of four replications was calculated. Means were compared with each other using Student-Newman Keuls (SNK) test (LSD at P < 0.05).

Insecticides relative potency was calculated as the following equation:

RESULTS:

Toxicity of chlorantraniliprole and lufenuron against the 2^nd and 4^th larval instars of S. littoralis

Toxicity of chlorantraniliprole and lufenuron against the 2^nd and 4^th larval instars of S. littoralis by leaves dipping technique (mixing the insecticide with food) after different exposure times is shown in Tables 1 and 2. Regarding the 2^nd instar larvae, chlorantraniliprole (LC₅₀ = 0.009, 0.005 and 0.003 mg/l) was approximately 234.33, 206.20 and 226.33 fold more toxic than lufenuron (LC₅₀ = 2.019, 1.031 and 0.679 mg/l) after 48, 72 and 96 hrs post-treatment, respectively (Table 1).

Table (1): Toxicity of chlorantraniliprole and lufenuron against 2^nd instar larvae of S. littoralis after different exposure times

Table (2): Toxicity of chlorantraniliprole and lufenuron against 4^th instar larvae of S. littoralis after different exposure times

Concerning the treated 4^th instar larvae, chlorantraniliprole (LC₅₀ = 6.645, 0.028 and 0.006 mg/l) was approximately 2.01, 105.36 and 311.67 times more toxic than lufenuron (LC₅₀ = 13.37, 2.95 and 1.87 mg/l) after 48, 72 and 96 hrs post-treatment, respectively (Table 2).

In vivo effect of chlorantraniliprole and lufenuron on AST activity of S. littoralis 4^th instar larvae

The in vivo effects of chlorantraniliprole and lufenuron on S. littoralis AST activity after different exposure times are presented in Tables 3 and 4. It is clear that, the activity of AST is increased with the increase of chlorantraniliprole and lufenuron concentrations and as will as the increase of the exposure time. The AST activity reached to its highest levels after 96 hrs of exposure, where it was 190.1% as compared to control at the concentration of 0.006 mg/l (Lc₅₀) of chlorantraniliprole (Table 3). In the case of lufenuron, AST reached to the highest activity (316.2% as compared to control) after 96 hrs at 1.870 mg/l (Lc₅₀) (Table 4).

In vivo effect of chlorantraniliprole and lufenuron on ALT activity of S. littoralis 4^th instar larvae

In vivo effects of chlorantraniliprole and lufenuron on ALT activity of S. littoralis 4^th instar larvae after different exposure times are presented in Tables 5 and 6. It is clear that the exposure of CLW 4^th instar larvae to different tested concentrations of chlorantraniliprole or lufenuron after different exposure times resulted in the increase of the ALT activity. When the 4^th instar S. littoralis larvae were exposed to chlorantraniliprole at 0.006 mg/l for an exposure period of 96hrs, ALT activity was 173.4% as a percentage of control (Table 5).  The activity of ALT was 221.1% as that of control (100%) when CLW 4^th instar larvae were exposed to lufenuron at concentrations of 1.870 mg/l (Table 6).

In vivo inhibition of alkaline phosphatase (ALP) activity of S. littoralis 4^th instar larvae by chlorantraniliprole and lufenuron at different exposure times

In vivo inhibition of ALP activity within treated S. littoralis 4^th instar larvae by chlorantraniliprole after different exposure times was investigated (Table 7). When S. littoralis larvae were treated with chlorantraniliprole at concentrations of 0.0006, 0.003 and 0.006 mg/l, ALP activity was 92.7, 81.8, and 68.2% as a percentage of control (100%), respectively after 48 hrs of exposure. ALP activity after 72 hrs of exposure was 78.1, 61.6, and 50.5% of that of control at the same concentrations, respectively. After 96 hrs of exposure, the ALP activity was 68.2, 59.5 and 47.3% of that of control at the same concentrations, respectively. The in vivo inhibition of ALP activity of S. littoralis 4^th instar larvae treated with lufenuron was also investigated after different exposure times (Table 8).

Table (3): In vivo effect of chlorantraniliprole on the AST activity of S. littoralis 4^th instar larvae after different exposure times

*S.A = Specific activity (IU / mg protein / hr). Numbers within the same column followed by the same letter(s) are not significantly different according to Student-Newman Keuls (SNK) test (LSD_0.05).

Table (4): In vivo effect of lufenuron on the AST activity of S. littoralis 4^th instar larvae after different exposure times

*S.A = Specific activity (IU / mg protein / hr). Numbers within the same column followed by the same letter are not significantly different according to Student-Newman Keuls (SNK) test (LSD_0.05).

Table (5): In vivo effect of chlorantraniliprole on the ALT activity of S. littoralis 4^th instar larvae after different exposure times

*S.A = Specific activity (IU / mg protein / hr). Numbers within the same column followed by the same letter are not significantly different according to Student-Newman Keuls (SNK) test (LSD_0.05).

Table (6): In vivo effect of lufenuron on the ALT activity of S. littoralis 4^th instar larvae after different exposure times

*S.A = Specific activity (IU / mg protein / hr). Numbers within the same column followed by the same letter are not significantly different according to Student-Newman Keuls (SNK) test (LSD_0.05).

Table (7): In vivo effect of chlorantraniliprole on the ALP activity of S. littoralis 4^th instar larvae after different exposure times

*S.A = Specific activity (IU / mg protein / hr). Numbers within the same column followed by the same letter (s) are not significantly different according to Student-Newman Keuls (SNK) test (LSD_0.05).

Table (8): In vivo effect of lufenuron on the ALP activity of S. littoralis 4^th instar larvae after different exposure times.

*S.A = Specific activity (IU / mg protein / hr). Numbers within the same column followed by the same letter are not significantly different according to Student-Newman Keuls (SNK) test (LSD_0.05).

When S. littoralis larvae were treated with lufenuron at concentrations of 0.187, 0.935, and 1.870 mg/l, the ALP activity was 77.03, 69.43 and 54.76% of that of control, respectively, after 48 hrs of exposure. ALP activity after 72 hrs of exposure was 65.0, 36.4, and 10.1% (as percentages of control), at the same concentrations, respectively. After 96 hrs of exposure, ALP activity was 59.3, 35.8, and 15.2 % of that of control at the same concentrations, respectively. From these data it could be concluded that the inhibition of ALP by chlorantraniliprole and lufenuron is concentration and time dependent.

DISCUSSION

Many insect pests present an ongoing battle between the grower’s ability to control the pest and the pest’s ability to resist the available control methods. The discovery of new, novel insect control agents for use against insect pests has served as a focal point for insecticide research for more than five decades, since the insects were actually conquered for the first time about 55 years ago. Because there are several problems resulted from the intensive use of synthetic insecticides, trends in pest management now are emphasis on methods of controlling insect pests apart from it. Chlorantraniliprole is an example for searching about new compounds with high activity in insect control and low ecotoxicity.

In the present study, experiments were conducted to evaluate the susceptibility of S. littoralis to chlorantraniliprole and lufenuron. The carried out bioassay enhances the understanding of differential toxicity of these compounds against the insect. From the data obtained in this study, it is obvious that chlorantraniliprole exert high toxicity against the 2^nd and 4^th larval instars of S. littoralis. The toxicity is increased as the concentration rates and exposure time increased while decreased as the insect instars increased. Temple et al. (2009) stated that Rynaxypyr^® (chlorantraniliprole) demonstrated very good activity at relatively low rates against all three tested major caterpillar pests of cotton in their study, including tobacco budworm, bollworm, and fall armyworm. Because of the high insecticidal activity of Rynaxypyr^® (chlorantraniliprole), Lahm et al. (2007) mentioned that Rynaxypyr^® (chlorantraniliprole) could be an excellent option for resistance management strategies as an additional class of chemistry and mode of action for management of lepidopteran pests in cotton. Also, they declared that the high degree of mammalian safety, relatively low use rates compared to standard insecticides (pyrethroids, organophosphates, and carbamates), long residual properties, and broad spectrum of activity against lepidopteran pests will make chlorantraniliprole an excellent control option in an overall integrated pest management system.

Results obtained with both AST and ALT enzymes revealed that the exposure of S. littoralis 4^th instar larvae to different concentrations of chlorantraniliprole and lufenuron at different exposure times resulted in an increase in the activity levels of these enzymes. The determined changes in the AST and ALT activity levels in the 4^th instar larvae of S. littoralis following different periods of sub-lethal and lethal chlorantraniliprole and lufenuron exposure suggested that S. littoralis exhibited adaptive elevation in the activity levels of both the aminotransferase enzymes, thereby probably aiding gluconeogenesis through transamination of glucogenic amino acids to meet the energy demand under chlorantraniliprole and lufenuron toxicity. These data is in accordance with the arrived at results by other authors. Ramaswamy et al. (1999) reported that the activity levels of AST and ALT enzymes were elevated when the fish Sarotherodon mossambicus had been exposed to sub-lethal (3 mg/l) and lethal (25 mg/l) concentrations of the carbamate insecticide carbaryl. Radwan et al. (1992) reported that the possible mechanism involved in the elevation of AST and ALT levels may be due to the tissue damage, as a result of the increased synthesis and/or the decreased metabolism of both enzymes.

Alkaline phosphatases (LPs) are classically described as homodimeric nonspecific metalloenzymes which catalyze phosphomonesterase reactions (Trowsdale et al., 1990). Phosphatases have been included in the list of detoxifying enzymes of insecticides; mostly of organophosphorus (Oppenoorth, 1985), however, fenvalerate and cypermethrin resistant larvae of Helicoverpa armigera showed higher activities of esterases, phosphatases and methyl paraoxon hydrolase compared with susceptible larvae (Srinivas et al., 2003). In the present study, chlorantraniliprole and lufenuron inhibited ALP activity. Based on the toxicity of chlorantraniliprole and lufenuron against the laboratory strain and the in vivo inhibition of ALP activity, chlorantraniliprole and lufenuron could be usefully incorporated and used for developing a more effective management program for S. littoralis.