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Research Article | DOI: https://doi.org/http
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Copyright: © 2018. Mohammad Saadati.This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Received: 30 November -0001 | Accepted: 01 January 1970 | Published: 01 January 1970
Keywords: Gut, Sunn Pest, Nutrition, Carbohydrate, Lipase.
Insecticide- enzyme interaction is the new field of insecticide physiology researches. Digestive enzyme like Lipase and amylase are responsible of lipid and carbohydrate metabolism. In this study, the effects of two insecticides fenitrothion and fenvalerate, on the lipase and amylase activity were studied. The used concentrations were 2000, 1500, 1000, 500, 100 and 0 ppm. Results showed that enzyme activities were reduced after incubation with different concentrations of insecticides, significantly. Nutrition processes were disrupted after reducing or suppressing of digestive enzyme activities. Energy production and metabolism can be considered as secondary targets for some of insecticides. Results suggest that fenitrothion and fenvalerate can be reported as ideal candidates to disrupt carbohydrates and lipase metabolism.
Digestive system is the main place to secrete many digestive enzymes as intermediate proteins in the animal world. Similarity between structure and function of the digestive systems are very different in the vertebrate and invertebrate. Various alimentary canals were observed in the insects as more frequent animals that take place different biochemical process in it’s as the first line. Sunn pest, Eurygaster integriceps, is the most important of crop pest in the Iran. It is an oligophagus insect that causes serious damage to wheat and barley. Many approaches to management of this pest were used for several decades but non them could not recommended as effective action in the applied entomology. Nowadays, using of safer methods for non-target organism and environment converted it to the first choice in the pest management plane in the case of sunn pest (Critchley, 1998; Javaheri et al, 2009). Biological and ecological control as safe tactic not expanded in the economical scales. Nowadays, new ways are focusing on the disruption of normal biochemical process such as using of enzyme inhibitors and BT toxins (Jouanian, 1998). Having a minimal risk to the non-target organisms is the most beneficial effect to using of insecticidal proteins in the current studies.
Extra oral digestion as a unique method in feeding has evolved in the hemipterous insects like sunn pest (Boyd, 2003; Habibi et al, 2008). In this way, salivary enzymes are injected to the plant tissues and after liquefying, foods pumped to the gut for final digestion (Liu et al., 2009; Barbehenn, 2002; Saadati et al, 2012a, b, c). Food digestion in the organisms catalyzed with various set of enzymes like a protease, lipase and carbohydrates which were reported from gut and salivary glands of the sunn pest (Saadati et al, 2007, 2008, 2012b, c). Any disruption in the digestive enzymes activity can be lead to stop and/ or reduce of the feeding and it will be effective to reduce the other life process that are necessary to reproduction in the next generations. Pesticides, plant metabolites, intrinsic regulators, environmental factors and unknown agent are main disruptors in the enzyme activities in the different organisms.
Carbohydrates are main food of the phytophagus insects that digested with carbohydrases like α and β-galactosidae, α and β-glucosidae and α-amylase which were reported in the gut and salivary glands of the sunn pest (Saadati et al., 2008, 2012c). Degradation of starch need to α-amylase (EC 3.2.1.1) activity that breakdowns α-(1, 4) glycosidic bond in the starch and its derivations (Kunieda et al., 2006).This enzyme has addressed for inhibiting with protein and nonproteins compounds (Saadati et al., 2007).Inhibiting of gut amylase in the some pest like Callosobruchus maculates is reached to practical step in the pest management (Jouanian et al., 1998). Lipids are lower valuable in comparison to the carbohydrates, but amount of released energy from them are very more than to carbohydrates in the similar volume .Lipase (EC 3.1.1.3) is water-soluble enzyme that causes to hydrolysis of ester bonds in the lipid compounds into free fatty acids and glycerol (Horne et al., 2009). The many isozymes from lipase and α-amylase have reported in the insect world particularly in the midgut of sunn pest (Saadati et al., 2012c). Hence they can be used as potential candidates in the field of enzyme inhibiting.
Using of insecticides is commonplace application in the sunn pest management in Iran (Javaheri et al., 2009). Different insecticides which belonged to various chemical groups are recommended to E.integricepsmanagement. Occurrence environmental pollution especially adverse effects on the non-target organisms created serious concerns for now and future. New researches are needed to investigate about effective ways to reduce ecological risks of the chemicals. Insect- insecticide interaction, as dynamic system, increases our knowledge to improve chemical application. In the previous work, we studied some of these systems like cypermethrin, chlorpyrifos, diazinon and deltamethrin with α-amylase and lipase (Saadati and Mirzaei, 2016). Although modes of action in the most insecticides are obvious (direct action) but the other targets which may be affected after treatment is not determined (indirect action). To elucidate the effects of insecticides on the nutrition process, digestive enzyme- insecticide interaction experiments should be used as in vivo and in vitro system.
In this study, for the first time, interaction of lipase and α-amylase from the gut of adult sunn pest with fenitrothion and fenvalerate were considered as in vitro experiments .We undertook this study to gain a better understanding of digestive enzyme behaviors after exposure to different concentrations of chemicals. Also, we hope that result of these research lead to new opportunities for finding of new enzyme inhibitors and increase our knowledge about side effects of some insecticides on the digestive enzymes activities.
Overwintering adults, E. integriceps (Hemiptera, Scutelleridae) were collected from Torbat-e Jam, Iran, in February 2017 and reared in an insectary room on wheat, Triticium aestivum L. Poales: Poaceae) variety Alvand at 27◦C with a 16:8 L: D photoperiod.
The midgut of adult insects were dissected under a stereomicroscope in ice cold phosphate buffer (4 ºC, pH=6.9). The midguts were separated from the insect bodies, rinsed in ice-cold phosphate buffer and three number placed in a micro tube containing one ml of cold phosphate buffer. The midguts were homogenized by using a homogenizer immediately after dissection. The homogenates were centrifuged at 12000 rpm for 10 minutes at 4 ºC. The supernatants were stored at -20 ºC for later analyses.
Two insecticides contain Fenitrothion (50EC, Partonar Co., Iran) and Fenvalerate (20 EC Samiran Co., Iran) were ordered for this experiments. After preliminary test 2500, 2000, 1500, 1000, 500, 100 and 0 ppm (active ingredient) were selected for interaction with enzymes. Fifty micro liter enzyme solution with 450 μl toxic solutions incubated 30 min in the room temperature before enzyme assay.
α-Amylase activity assay
Amylase activity in the midgut was determined using a diagnostic kit (Amylase kit®, Pars Azmoon Co., Iran). The substrate was ethylidene-pnitrophenyl malto heptaoside (EPS-G7). Absorbance, which is directly related to α-amylase activity, was measured at 405 nm and 37 ºC using an auto analyzer (Alcyon 300® Plus, Molecular Devices Corporation, Sunnyvale, CA). Before application, the auto analyzer calibrated with the control sera N and P (TrueLab N®and TrueLab P®, respectively; Pars Azmoon Co., Iran) and a calibrator solution (TrueCal U®, Pars Azmoon Co., Iran). After calibration, the auto analyzer mixed4 μl of enzyme sample with 300 μl of substrate solution, automatically, and calculates the enzyme activity (IU/L) after a reaction delay of 1 minute and 36 seconds. Optimized pH was eight for all treatments. The assays were replicated three times. Finally, the specific α-amylase activity calculated as U/mg protein that known Specific activity.
Lipase activity assay
Lipase activity in the midgut was determined using a diagnostic kit (Lipase kit®, Biorexfars Co., Iran). The substrate was 1, 2-o-dilauryl-rac-glycero-3-glutaric acid-(6'-methylresorufin) (DGGR). Absorbance, which is directly related toproduction of methyl resorufin and lipase activity, was measured at 580 nm and 37 ºC using an auto analyzer. Optimized pH was eight for all treatments. Recommended calibrators and the other considerations were similar to amylase assay procedure.
Total protein were assayed according to Biuret test using diagnostic kit (Total protein kit®, Pars Azmoon Co., Iran). In this procedure, copper sulfate ions react with the peptide bonds to produce purple (or pink) color. The final amount of proteins in the samples is estimated by comparing their intensity color with standard solution that contains defined bovine serum albumin (BSA) concentration.
Data were compared by one-way analysis of variance (ANOVA) and factorial design that followed by Duncan’s studentized test at p< 0.05(Mstat-C and Spss ver. 15). Differentially activities were shown (as different letters) in figures.
Fenitrothion- α-amylase interaction
Mixture of enzyme solution and different concentrations of fenitrothion were incubated for 30 min before calculating of enzyme activities. Our results showed that there was a significant difference in the amylase activity after exposure with different concentrations of this chemical (Fig 1).
Figure 1. α-amylase activity after incubation with different concentration of fenitrothion in the Adult sunn pest. Values are the average specific activity± SE from three independent experiments. Different letters showed significantly difference among concentrations (P< 0.05).
The highest and lowest activities were observed in 100 and 2500 ppm, respectively (Fig.1). The highest level of enzyme inhibitory was occurred in the 2500 ppm (94.2%). On the other hand, 100 ppm of fenitrothion was caused the minimum of enzyme inhibitory (46.7%).
Table 1: showed the amount of inhibitory and activity of α-amylase in the response of increasing doses of fenitrothion. Nearly, all doses of fenitrothion reduced enzyme activity more than fifty percent (exceptly 100 ppm).
Table1. Effects of six concentrations of fenitrothion on the α-amylase activity in the midgut of sunn pest 30 minutes after incubation. Values are average of Inhibitory effects and calculated based to the control treatment (% inhibitory).
Fenitrothion- lipase interaction
Mixture of lipase solution and different concentrations of fenitrothion were incubated for 30 min before calculating of enzyme activities. Result showed that maximum and minimum of enzyme activity were occurred in the 100 and 2500 ppm in comparison to the control treatment (Fig 2).
Table 2: showed the details of lipase inhibitory and activity percent in response to different concentrations. Our result proposed that 2500 and 2000 ppm reduced enzyme activity more than 99%.
Table 2. Effects of six concentrations of fenitrothion on the Lipase activity in the midgut of sunn pest 30 minutes after incubation. Values are average of Inhibitory effects and calculated based to the control treatment (% inhibitory).
Fenvalerate- α-amylase activity
Mixture of enzyme solution and different concentrations of insecticide were incubated for 30 min in temperature room before measuring of enzyme inhibitory. Significant difference between doses of chemical was observed as the rate of enzyme activity after incubating treatments were reduced hardly with increasing of concentration to 2500 ppm (Fig. 3).
Table 3 showed the enzyme behavior in the response to the different concentrations of fenvalerate. Interesting result was that all doses of fenvalerate inhibited amylase activity more than 75%.
Table 3. Effects of six concentrations of fenvalearte on the amylase activity in the midgut of sunn pest 30 minutes after incubation. Values are average of Inhibitory effects and calculated based to the control treatment (% inhibitory).
Fenvalerate- lipase activity
Mixture of enzyme solution and different concentrations of fenvalerate were incubated for 30 min in temperature room before measuring of enzyme inhibitory. There were significant differences among various concentrations (Fig.4).
Table 4. Effects of six concentrations of fenvalerate on the Lipase activity in the midgut of sunn pest 30 minutes after incubation. Values are average of Inhibitory effects and calculated based to the control treatment (% inhibitory).
Lipase was classified as serine hydrolyses those breakdown lipids as energy sources. These enzymes have key role in the physiological process like lipid metabolism and transport, regulation of plasma membrane lipid and cell signal transduction (Yao et al., 2009). Lipase activity after exposing to the fenitrothion, drop sharply. The doses 2500, 2000 and 1500 ppm were caused 99.5, 99.2 and 97.3 percent lipase inhibitory. Saadati and Mirzaei (2014) reported that 63 percent lipase inhibitory was occurred in 2000 ppm of chlorpyrifos. Also, they showed that lipase activity completely removed in 1500 and 2000 ppm of cypermethrin.
Fenvalerate- enzyme interaction showed that lipase and amylase were more sensitive to this chemical. Amylase activity was reduced 25 to 99 percent in 100 and 2500 ppm, respectively. This result suggests that amylase inhibiting can be considered as secondary target to fenvalerate. More than 90 percent inhibitory was occurred in the 1000, 1500, 2000 and 2500 ppm. Saadati and Mirzaei (2016) reported that diazinon and deltamethrin reduced amylase activity 64 and 0 percent in the maximum concentration of insecticide (2000 ppm).
Lipase activity after incubating with fenvalerate was suppressed from 62 to 100 percent in the 100 and 2500 ppm, respectively. Lipase inhibitory was occurred more that 90 percent in the 1000, 1500, 2000 and 2500 ppm. Saadati and Mirezaei (2016) showed that lipase activity hardly suppressed after treatment with diazinon and deltamethrin. They explained that deltamethrin completely removed lipase activity in the concentrations upper 500 ppm.
In the fenvalerate- amylase interaction, there are no significant differences among high concentration (Fig. 3) but in the fenitrothion- amylase interaction, significant difference occurred among all concentrations (exceptionally between 1000 and 1500 ppm) (Fig. 1). Occurrence of different sensitivity of amylase to the insecticides was related to various factors like intrinsic difference of protein sequencing and structure, nature of insecticides, different doses, age and health of organism and environmental factors (Temperature, humidity, pH) (Saadati and Mirzaei, 2016).
There are no significant differences among 1000, 1500, 2000 and 2500 in the fenvalerate-lipase test (Fig. 4). But in the fenitrothion treatment after incubating with lipase, significant differences were occurred in the all used concentrations (Fig. 2). Saadati and Mirzaei (2016) reported that pyrethroid insecticides have more inhibitory effects on the digestive enzyme in comparison to the organophosphate compounds. Gunes and Yerli (2011) reported that deltamethrin inhibited lipase activity in the Poecilia reticulate. Pyrethroids type II like cypermethrin and deltamethrin have Cyanide factor in their structures. Cyanides are strong inhibitors for different enzymes in the live cells. They can bind to enzymes and after changing enzyme conformations, convert them to inactive form (Veerapan et al., 2012). According our results, there was no difference between organophosphate and carbamate insecticides. Deltamethrin and cyper metrin as pyrtheroid insecticide affected enzyme activity in the in vitro test (Saadati and Mirzaei, 2016). Their result showed that deltamethrin completely inhibited lipase activity in all concentration except 100 ppm. Also, in the 1500, 2000 and 2500 ppm of cypermethrin was not recorded any lipase activity. On the other hand, in the all concentration of chlorpyrifos and diazinon lipase activity was recorded (Saadati and Mirzaei, 2016).
Reducing of amylase and lipase activity after interfering with insecticides led to decrease of insect ability in the starch digestion. A number of different proteinous and nonproteinous inhibitors were reported against α-amylas in the salivary glands of sunn pest and the other insects (Saadati et al., 2007). Eraslan et al (2007) reported that inserting of deltamethrin to food of mice was not reduced amylase activity in the serum. Also their results showed that deltamethrin reduced acetylcholinesterase in the serum of mics. Amylase activity in the serum of rat showed no appreciable changes after treatment with cypermethrin (Veerapan et al., 2012). Also, amylase activity in the serum of Gallus domesticus had not showed significantly change after cypermethrin treatment (Anwar, 2004).
Although, complete inhibition of target enzymes were considered as successful results but even partial inhibition recommended as substantial strategy for insect control (Ishimoto & Kitamura, 1989). Data from this study indicated that ganophosphate and carbamate insecticides can inhibit digestive enzyme activities. Also, enzymatic assays with energy-metabolism enzymes, including Lipase and amylase, after mixture with insecticide showed that effects of insecticides is not limit to the one target and set of different proteins may be affected after treatment (Saadati and Mirzaei, 2016).
One of the major challenges is to link the results from in vitro studies to in vivo condition. The in vivo studies are very complicate and need to consider various factors to gain desirable results (Saadati and Mirzaei, 2016). We will focus on the enzyme-insecticide interaction to elucidate correlation between in vivo and in vitro condition for insect systems. Although, there are a few studies that investigate the effects of insecticides on digestive enzymes of sunn pest, new researches should be focus to clear mechanism of inhibitory effects and their interaction with assimilation process.
Many advantages have mentioned from the use of pesticides in agriculture, medicine and veterinary. On the other hand, several environmental concerns reported because of overusing of pesticides. Our knowledge about direct and indirect effects of insecticides is not complete and need to increase continuously (Saadati and Mirzaei, 2016). Nowadays, acetylcholinesterase were addressed as final targets of organophosphate and carbamate insecticides, but new researches suggests that set of various proteins in the target organism may be affected by pesticides. Saadati et al (2012 c) showed that proteome analysis of digestive system in the sunn pest can be used as appropriate technique for studying of changed proteins after exposure to the specific treatment. Some of effective proteins in the carbohydrates metabolism in the brown plant hoppers were down regulated after exposure to carbamate insecticides (Sharma et al, 2004). Because of environmental disadvantages of insecticides, finding of new aspects of insecticides to be safe is necessary. Using of insecticidal proteins or different inhibitors against digestive enzymes is a new strategy to pest control (Pauchet et al., 2008). Occurrence of the side effects of insecticides on the nutrition process of insects can be used as secondary factor to help us for choosing of suitable insecticide and their doses (Saadati and Mirzaei, 2016).
Many important enzymes, α-amylase, protease and lipase were identified in the gut of sunn pest using proteomics technique (Saadati et al, 2012 b, c). These enzymes play key roles in the carbohydrates, protein and lipid metabolism. In the present study, two enzyme, α-amylase and lipase, were selected to incubate with different doses of fenitrothion and fenvalerate, and then to be studied enzyme- insecticide interactions.
Our results showed that enzyme activities were decreased after treatment action, sharply (Fig.1 and Fig. 2). Fenitrothion- enzyme interaction showed that amylase and lipase activity were inhibited strongly at higher concentrations in comparison with lower concentrations (Tab. 1, Tab 2). Amylase activity was decreased more than 50 percent in the all treatment exceptionally 100 ppm concentration. On the other hand, 94 percent inhibitory was occurred in the 2500 ppm (Tab 1). Saadati and Mirzaei (2016) reported that chlorpyrifos, organophosphate insecticide, reduced amylase activity about 20 percent in comparison to control treatment. Also, they explained that cypermethrin, pyrtheroid insecticide, inhibited amylase activity more than 80 percent. It is not possible to describe a reason/s for this behavior but it is guessable that fenithrothion was an effective inhibitor in comparison to the chlorpyrifos. Organophosphate compounds such pesticides were reported as potential lipase inhibitors in the vertebrate animals (Quistad et al., 2006). Our results proved that fenitrothion is strong inhibitors for insect lipase, too.
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