Abstract: The saw-toothed grain beetle, Oryzaephilus surinamensis is a cosmopolitan insect pest that attacks stored grains and dried fruits, causing damage to stored dates in the United Arab Emirates (UAE). Stored dates may contain insecticide residues as a result of insecticide field applications and it is of interest to know the effect of insecticide residues on O. surinamensis. The aim of this study was to evaluate mortality among O. surinamensis adults exposed to residual levels of the insecticides carbosulfan, chlorpyrifos, cypermethrin, imidacloprid, malathion and spinosad which are commonly used on date palm trees in the UAE. Also to evaluate the Glutathione S-Transferase (GST) enzyme activity in the O. surinamensis adults exposed to insecticides. Adults of O. surinamensis were subjected to petri dish insecticide bioassays. Low insecticide concentrations, simulating field residues, were tested and the range of 0.713-3.31 μg mL-1 caused 50% mortality. Chlorpyrifos was the most toxic insecticide while cypermethrin was the least toxic. In addition to effect on mortality, insecticide exposure caused induction in GST enzyme activity in O. surinamensis adults. Because elevated GST enzyme activity can play a role in the detoxification of insecticides and in the development of insecticide metabolic resistance in insects it is likely to have a similar role in O. surinamensis population.
INTRODUCTION
The saw-toothed grain beetle, Oryzaephilus surinamensis (L.) (Coleoptera: Silvanidae) is the most widely distributed stored grain pest in the world. This insect attacks stored dates in the United Arab Emirates (UAE) (Zaid et al., 2002) and significantly lowers their market value. Several insect pests such as the red palm weevil, lesser date moth and date root borer attack date palm trees in the UAE and farmers use a number of insecticides to control them. Residues of some of these insecticides have been detected in ripe dates at concentrations reaching up to 5 ppm (μg mL-1) (Al-Dhaheri, 2011). Consequently, O. surinamensis and other stored date insects are exposed to these residues while feeding on stored dates. The exposure of insects to insecticides causes mortality and also can act as a selection pressure for resistance development in the treated insect population (Georghiou et al., 1987; Ahmad et al., 2007). Resistance to an insecticide can be defined as a genetic change in response to selection by toxicants that may impair control in the field (Yu, 2008). Several detoxifying enzymes confer insecticide metabolic resistance in insects.
Glutathione S-transferases (GSTs) are one the major detoxifying enzymes associated with insecticide metabolic resistance (Bull, 1981; Oppenoorth, 1985). In general, GSTs are involved in the detoxification of various xenobiotic chemicals (Motoyama and Dauterman, 1980) and act as catalysts for the conjugation of various electrophilic compounds with tripeptide glutathione (Chasseaud, 1979; Wilce and Parker, 1994). Resistant insects to all major classes of insecticides often exhibit elevated GST activity (Huang et al., 1998; Vontas et al., 2001). The objectives of this study were (1) to evaluate mortality among O. surinamensis adults exposed to residual levels of six insecticides commonly used on date palm trees in the UAE and (2) to evaluate the GST enzyme activity in the O. surinamensis adults exposed to insecticides.
MATERIALS AND METHODS
The study was conducted in 2010 at the Entomology laboratory of the UAE University and lasted for four months.
Insects: The O. surinamensis adults were collected from infested dates and were placed inside an incubator at 25°C in screw cap plastic jars (2500 mL) containing insecticide-free dates as food.
Chemicals: Insecticide reference standards (chlorpyrifos, imidacloprid, spinosad, carbosulfan, malathion and cypermethrin) (Fig. 1) all from Dr. Ehrenstorfer GmbH, (Augsburg, Germany) were purchased from a local supplier. Acetone was used for dissolving the insecticides.
| Fig. 1: | Structures of the insecticides used in the petri dish residue bioassay on O. surinamensis adults, Spinosad is a mixture of spinosin A and D |
Insecticide bioassay
LC50 tests: Mortality of O. surinamensis adults
exposed to each one of the six insecticides was evaluated using a petri dish
residue bioassay. For each bioassay, three replicates with at least seven concentrations
of each insecticide were prepared. Each glass petri dish (Pyrex®,
100x15 mm) was treated with 1 mL of an insecticide dissolved in acetone. Treated
dishes were left to air dry in a fume hood. Petri dishes treated only with acetone
served as a control. After the dishes dried, a group of 15 unsexed O. surinamensis
adults were transferred into each petri dish with a fine paintbrush and the
dish was covered with its lid. Mortality was assessed after a 24 h waiting interval
at 25°C with 12:12 (L:D) photoperiod. Adults of O. surinamensis were
considered dead if they failed to walk after light tapping on the petri dish
or being probed by the paintbrush. Mortality was corrected according to mortality
of the controls using Abbott’s formula (Abbott, 1925).
The values of the LC50, their 95% Confidence Intervals (CIs) and
slopes of regression lines were estimated using PROBIT analysis (SPSS program;
SPSS Inc., Chicago, IL). LC50 values of the tested insecticides were
considered not significantly different if the 95% CIs overlapped.
Glutathione S-transferase enzyme activity bioassay
Exposure to low insecticide concentrations: A GST bioassay was conducted
after determining the LC50 values for all tested insecticides. A
concentration (0.5 μg mL-1) which was lower than the least LC50
value among all of the tested insecticides was selected and used in this
bioassay which was done before conducing the GST enzyme activity test. This
concentration was chosen because it left an adequate number of live O. surinamensis
adults for preparing an enzyme homogenate and also simulated exposing O.
surinamensis adults to low post-harvest insecticide residue levels that
might be encountered when feeding on stored date palm fruits. Adults of O.
surinamensis were exposed to the selected concentration in a petri dish
bioassay as described above.
Enzyme preparation: After exposure to insecticides the surviving insects were collected and homogenized in a potassium phosphate buffer (pH 6.5) using a ceramic mortar placed on ice. The homogenate was centrifuged at 13,200 rpm at 4°C for 10 min and the supernatant was decanted and used in the GST enzyme activity assays.
Glutathione S-transferase assay: The specific activity of GST of O. surinamensis adults was determined by the spectrophotometric method of Habig et al. (1974) using CDNB (1-chloro-2,4- dinitrobenzene) as a substrate. Each cuvette contained an insect homogenate (250 μL) mixed with potassium phosphate buffer (500 μL). Cuvettes were incubated at 25°C for 5 min. Ethanol-dissolved CDNB (10 μL; 0.2 M) and reduced glutathione (GSH) (150 μL; 10 mM) then were added. After 1 min, absorbance was measured at 340 nm using a spectrophotometer (Lambda 25, Perkin Elmer). A cuvette containing all reactants except substrate was used as a blank. The assay was done in triplicate. GST specific activity was calculated as ΔOD/min/mg protein (Tomarev et al., 1991). Protein concentration of the insect homogenate was determined by the method of Bradford (1976) using Bovine Serum Albumin (BSA) as a standard. Data were analyzed using analysis of variance with insecticide treatments as the classification variable (SAS, 2001).
RESULTS
Insecticide bioassay: Table 1 shows the LC50 values of the tested insecticides. Chlorpyrifos was significantly the most toxic insecticide to O. surinamensis with an LC50 value of 0.713 μg mL-1, followed by malathion and carbosulfan at 1.197 and 1.637 μg mL-1, respectively.
| Table 1: | Mortality of O. surinamensis adults determined by petri dish bioassay |
| The LC50 values are expressed in micrograms of active ingredient of insecticide per milliliter of acetone and their 95% confidence intervals (95% CI). bA value of p>χ2 larger than or equal to 0.05 indicates a significant fit between the observed and expected regression lines. LC50 values of the tested insecticides were considered not significantly different if the 95% CIs overlapped each other | |
| Table 2: | Mean (±SE) of GST specific activity (ΔOD/min/mg protein) in O. surinamensis adults treated with different insecticides at concentration of 0.5 μg mL-1 |
| Means with different letters are significantly different at p<0.05 | |
Spinosad was less toxic with an LC50 value of 2.562 μg mL-1, whereas imidacloprid and cypermethrin were the least toxic with LC50 values of 3.122 and 3.310 μg mL-1, respectively.
Glutathione S-transferase enzyme assay: Insecticide exposure caused significant differences in GST specific activity of O. surinamensis adults (F = 4.85, df = 9, 20, p = 0.0016) (Table 2). GST activity of cypermethrin-treated insects was not significantly higher than GST activity of malathion-treated insects (t = -0.9004, p = 0.3786). However, GST activity of cypermethrin-treated insects was significantly higher than in carbosulfan-treated insects (t = -2.9416, p = 0.0081).
The GST activity of O. surinamensis adults treated with spinosad, imidacloprid and chlorpyrifos did not significantly differ from the control [(t = 0.2766, p = 0.7841), (t = 0.2742, p = 0.7867), (t = 0.3397, p = 0.7376), respectively]. Overall, the highest GST induction was caused by exposure to cypermethrin (activity was 3.4 fold of the control) followed by malathion (2.9 fold of the control). GST induction by malathion and cypermethrin was significantly higher than in the control insects [(t = 3.3032, p = 0.0035) and (t = 4.2037, p = 0.0004), respectively].
DISCUSSION
Insecticide bioassay: All the values of p>χ2 were larger than 0.05 indicating a significant fit between the observed and expected regression lines of all the tested insecticides. The O. surinamensis population was homogenous in terms of its response to insecticide exposure for all the tested insecticides, indicated by slopes >2. A flat line or a low slope (<1), is indicative of a heterogeneous population, showing a large variance in response (Yu, 2008).
Pesticide residues can be found in fresh fruits as well as in their products (Cabras and Conte, 2001). The tested insecticides in the current study are commonly applied on date palm trees in the UAE. The examined range of concentrations (0.05-5 μg mL-1) simulated the residue levels that can be found on dates collected from insecticide treated date palm trees. Exposure to such concentration levels can cause mortality among O. surinamensis, when feeding on dates in storage. This was manifested in the current study by recording 50% mortality caused by a concentration range of 0.713 μg mL-1 for chlorpyrifos and 3.310 μg mL-1 for cypermethrin. Chlorpyrifos was significantly the most toxic insecticide based on its LC50 value (0.713 μg mL-1) which indicates that the presence of this residue level in stored dates can cause a similar mortality in the O. surinamensis population. In accordance, chlorpyrifos-methyl and malathion (Lee and Lees, 2001) and spinosad (Fang et al., 2002) were reported causing mortality to O. surinamensis. Based on UAE weather conditions (temperature and humidity) and agricultural practices, harvested dates could contain insecticide residues reaching 5 μg mL-1 for some insecticides (Al-Dhaheri, 2011). Thus the presence of these insecticide residues can provide partial control against the insects including O. surinamensis that feed on stored dates. Although some insecticides such as chlorpyrifos were toxic to O. surinamensis at low concentrations and as mentioned can provide partial control, it is not recommended to apply insecticides to stored dates as a control measure because of human health risks. It was demonstrated that they show a potential dose-related chronic and acute toxicity against humans (Schilter and Huggett, 1998). If needed, such chemicals may be used to treat the walls and floor of the storeroom. From a pest management standpoint, the presence of insecticide residues at low concentrations, coupled with the extended exposure to these chemicals during the feeding on the dates in storage, may lead to development of insecticide resistance in O. surinamensis populations. Although the presence of insecticide residues can help in reducing the infestation of O. surinamensis in stored dates, it is better for date growers to use the recommended dose in the field to avoid human health risks and to delay the development of insecticide resistance.
Glutathione S-transferase (GST) enzyme assay: Glutathione S-transferase enzymes are useful biomarkers for metals and organic pollutants yielding oxidative stress (Yang et al., 2001) and have also been useful as an indicator of pesticide exposures (Taysse et al., 1998). They also have a significant role in insect resistance to Organophosphate (OP) insecticides (Motoyama and Dauterman, 1980). The results of the current study demonstrated that exposing O. surinamensis adults to insecticides at concentrations of 0.5 μg mL-1 caused a GST enzyme response in the form of activity induction. The maximum induction was caused by cypermethrin which is a pyrethroid insecticide. This insecticide was the least toxic to O. surinamensis based on its LC50. The high GST activity in O. surinamensis adults exposed to cypermethrin likely explains its low toxicity. This is because GST enzymes play a role in xenobiotic metabolic detoxification. In addition, the high GST activity indicates that this enzyme group participates in the detoxification of cypermethrin and malathion to a greater extent than it does in the other tested insecticides. The induction of GST enzyme activity in this study was in agreement with the findings of previous studies. Yadwad and Kallapur (1988) evaluated the effect of fenitrothion on the castor semilooper Achaea janata (Lepidoptera: Noctuidae) and measured the GST activity. Both sublethal and lethal doses of fenitrothion produced significant induction of enzyme activity. Sivori et al. (1997) reported a significant increase of GST activity in Triatoma infestans (Heteroptera: Reduviidae) adults which were toxicated with sublethal dose of tetramethrin. Not all of the tested insecticides significantly induced GST enzyme activity in O. surinamensis and this is in part because they belong to different insecticide chemical groups (pyrethroids, organophosphates and nicotinoids). Moreover, other detoxifying enzymes such as cytochrome P450 and esterases can also be involved in the detoxification process of insecticides. It is also likely that the 0.5 μg mL-1 concentration was not sufficient for some of the tested insecticides to cause induction in the GST enzyme activity.
CONCLUSION
In conclusion, exposure to date palm fruits containing insecticide residues at low concentrations can cause up to 50% mortality in O. surinamensis adults. Chlorpyrifos was the most toxic insecticide and cypermethrin was the least toxic. In addition to causing mortality, insecticide exposure caused GST enzyme induction in O. surinamensis adults, an effect that could play a role in the development of insecticide metabolic resistance. More work is recommended to determine parameters of development of insecticide resistance in O. surinamensis populations.
ACKNOWLEDGMENT
This study constitutes a part of a research project performed in partial fulfilment of the Master’s degree of Hamda A. Al-Dhaheri, supervised by Mohammad A. Al-Deeb at UAE University, UAE.