Imidaclopride, a Potent Inhibitor of Array of Digestive Enzymes of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae)

The Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) a phytophagous insect which feeds on more than hundred plant species, brought crop loss more than half the yield and annual loss of more than US $500 million [1]. The devastating pest have developed resistance to almost all class of insecticides [2,3], have become serious constraint to crop production in Asia, Africa, Australia and Mediterranean Europe for the last four to five decades [4]. Enhanced metabolism rate in insects also plays an important role on fitness of the insect [5]. Digestive proteolytic activity in the insect gut might be the key in the adaptation of resistance against plant defensive compounds. Defensive role of lucine aminopeptidases (LAPs) have extensively studied in lepidopteran gut. The decreased utilization of food, delayed growth, reduction in survival, weight or size and reproduction of new generation adults could be due to the interference of the toxicants with amino acid metabolism, inhibited by most of the plant metabolites [6]. The H. armigera has a large multigene family for serine proteinases, which includes two main types, trypsin and chymotrypsin [7]. It has been shown [7] that the differential regulation and expression of protease enzymes in response to chronic exposure to protease inhibitors [8]. Studies were carried out with respect to the effect of imidacloprid on reproduction, fecundity, and survivorship of female in H. armigera [9]. The litrature survay shown a minimul/ lack of information regarding metabolic changes in H. armigera under imidacloprid stress conditions which can be correlated with the other neonicotinoids and lepidopteran insect pest. Here we report the effect of imidacloprid on serine proteases, aminopeptidase and detoxification enzymes on the fitness of the H. armigera larvae.


Introduction
The Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) a phytophagous insect which feeds on more than hundred plant species, brought crop loss more than half the yield and annual loss of more than US $500 million [1]. The devastating pest have developed resistance to almost all class of insecticides [2,3], have become serious constraint to crop production in Asia, Africa, Australia and Mediterranean Europe for the last four to five decades [4]. Enhanced metabolism rate in insects also plays an important role on fitness of the insect [5]. Digestive proteolytic activity in the insect gut might be the key in the adaptation of resistance against plant defensive compounds. Defensive role of lucine aminopeptidases (LAPs) have extensively studied in lepidopteran gut. The decreased utilization of food, delayed growth, reduction in survival, weight or size and reproduction of new generation adults could be due to the interference of the toxicants with amino acid metabolism, inhibited by most of the plant metabolites [6]. The H. armigera has a large multigene family for serine proteinases, which includes two main types, trypsin and chymotrypsin [7]. It has been shown [7] that the differential regulation and expression of protease enzymes in response to chronic exposure to protease inhibitors [8]. Studies were carried out with respect to the effect of imidacloprid on reproduction, fecundity, and survivorship of female in H. armigera [9]. The litrature survay shown a minimul/ lack of information regarding metabolic changes in H. armigera under imidacloprid stress conditions which can be correlated with the other neonicotinoids and lepidopteran insect pest. Here we report the effect of imidacloprid on serine proteases, aminopeptidase and detoxification enzymes on the fitness of the H. armigera larvae.

Insects
The present study was carried out at International Crops Research Instituite for Semi-Arid Tropics (ICRISAT), Patancheru, Telangana State, India. H. armigera larvae were reared on a chickpea based semi-synthetic diet under laboratory conditions at 27±1 °C, 65±5% RH, and 12 h photoperiod, obtained from the insect rearing laboratory.

Bioassay
Initial weights of third instar larvae were recorded before releasing on the diet containing different concentrations of imidacloprid (0-600µM). Three replicates for each treatment containing 10 larvae in each replication. The larvae fed with insecticide for 7 days and the final weights and mortality were receded [10].

Enzyme preparations
Imidacloprid treated and untreated larval midguts were homogenised with two volumes of 0.1 M glycine-NaOH buffer (pH 10.0), centrifuged at 8000 ×g for 20 min at 4 °C. The supernatants were used as enzyme source for protease assay [11].
Brush border membrane vesicles (BBMVs) were prepared by the method adopted from [12], where guts were removed from imidacloprid treated and untreated larvae were homogenised with 20mM Tris-HCl buffer (pH 7.5) containing 5mM EGTA, 1mM PMSF and 300mM mannitol, at cold condition. The homogenates were added with equal volumes of 20mM MgCl 2 and allowed to stand on ice for 20min followed by centrifugation at 2500 ×g for 15min at 4 °C. The supernatants were stand on ice where as the pelletes were resuspended with the extraction buffer and the process repeated for twice. The resulting supernatants were pooled and centrifuged at 30000×g for 30min at 4 °C. The pellets containing BBMVs obtained were suspended in minimum volumes of extraction buffer for aminopeptidase assay.

Effect of imidacloprid on gut proteases
Aminopeptidase activity was measured by incubating brush border membrane vesicles (BBMVs) from imidacloprid fed and unfed larvae using leucine p-nitroanilide (LpNA) as a substrate. Reaction mixture consisted of 50µL of enzyme, 20µL of 2mM substrate and 630µL of Glycine-NaOH buffer (pH 10.0) incubated for 20 min at 37 °C. The reaction mixtures were centrifuged at 5000 ×g for 15min after adding 300µL of 30% acetic acid. The absorbance was measured at 410 nm [11].
Elastase activity was measured similarly as aminopeptidase, except using N-succinyl-alanine-alanine-alanine p-nitroanilide (SAAApNA), as a substrate. One unit of enzyme activity was defined as the micromoles of p-nitroaniline released per minute per milligram of gut protein [11].
Total protease activity was measured by using 1% azocasein as a substrate in 0.1M glycine-NaOH buffer (pH 10.0). 100µL of gut extract was incubated with 500µL of substrate and incubated for 30min at 37 °C. Then add 200µL of 5% TCA followed by centrifugation at 5000 ×g for 15min. Equal volumes of 1N NaOH were added to the supernatants to read the absorbance at 450 nm [11].
Chymotrypsin and trypsin activities were determined by using 30µL of 1mM N-glutaryl-L-phenylalanine p-nitroanilide and 10µL of 1.2mM N-α-benzoyl-DL-arginine-p-nitroanilide as substrates, respectively. Enzyme (50µL) mixed with respective substrates and 0.1M glycine-NaOH buffer (pH 10.0) (to make 700µL of final volume), was incubated for 20 min at 37 °C and then adds 300µL of 30% acetic acid and stand for 10min. The absorbance was measured at 410nm after centrifugation of the samples at 8000 ×g for 10min. Amount of enzyme catalyzing the hydrolysis of 1µM substrate per minute at 37 °C, was defined as one unit of enzyme activity [13].

Alkaline phosphatase assay
Alkaline phosphatase activity was measured using p-nitrophenyl phosphate (p-NP) as substrate [13]. In brief, assay mixture consisted 50µL of 0.1mM p-NP, 900µL of 0.1 M Tris-HCl buffer (pH 8.6) and 50µL of enzyme, change in the absorbance was measured at 405 nm for 3min. Protein concentrations for all the enzymes assays were measured using bovine serum albumin (BSA) as a standard [14].

Statistical analysis
Data were subjected to One-way Analysis of Variance (ANOVA) using Genstat (14th edition, Version 14.1.0.5943, VSN International Ltd, United Kingdom) software to judge the significance of differences between the treatments by F-test, while the treatment means were compared by least significant difference (LSD) at p < 0.05. Ducan's Multiple Range Test (DMRT) was used to know the differences between treatments. LD50 values were determined by using EPA PROBIT analysis program (Version 1.5).

Bioassay
Imidacloprid inhibited the growth of third instar H. armigera larvae in a dose-dependent manner. There was ~67.5% larval growth inhibition and 73.33% reduction in survival rate observed. LD 50 value for the insecticide was found to be 531.24µM (95% confidence limit) (data not shown).

Effect of imidacloprid on gut proteases
Dose dependent response was observed in gut protease profile of larvae fed with insecticide compared with the control larvae for 24h. A significant decrease in of chymotrypsin (57.14%), trypsin (57.07%) and total proteases (14.81%) were observed at 600µM of imidacloprid. The activities in the control larvae were found to be 0.133±0.008U/mg, 2.05±0.190U/mg and 0.027±0.001 UA, respectively, and the activities of imidaclopride (600µM) treated larvae were found to be 0.057±0.005U/mg, 0.88±0.008U/mg and 0.023±0.005 UA, respectively (Table 1). Elastas (0.37±0.008 to 0.18±0.049U/mg) and aminopeptidase (0.13±0.002-0.11±0.003U/mg) activities were also inhibited significantly by 51.35%, and 15.38%, respectively, compared with the control larvae, in a dose dependent manner, of the larvae fed with increasing concentrations of imidacloprid (0-600µM) ( Table 1). Column values followed by same letters are not significantly different from each other at p<0.05%.

Alkaline phosphatase activity (ALP)
Significant dose-dependent reduction of alkaline phosphatase activity in the larvae fed on diet containing increasing concentration of imidacloprid (0-600µM). The decline was measured to be 0.33±0.0004 to 0.13±0.001µM/min/ mg of protein ( Figure 1).

Discussion
Proteases play a vital role in the proper development of insects as they hydrolyze the peptide bonds to liberate free amino acids by the action of endopeptidases and exopeptidases, as they are needed for the growth, survival and reproduction [15]. The decreased activities of chymotrypsin, trypsin and total protease confers to the antifeedant nature of imidacloprid where the larvae unable to metabolise the diet provided for its survival, growth, and reproduction. Insects when exposed to protease inhibitors alter their midgut composition by monitoring the proteases secretion [16]. Studies have shown that the food, temperature, and acidity are key factors responsible for production of energy needed for larval development. The food containing imidacloprid directly affected the feeding behaviour of the insect thereby leading to the reduced body weight and delayed growth in a dose dependent pattern (data not shown). Aminopeptidases, catalyses the amino acids hydrolysis from N-terminus of the peptides and proteins was moderately inhibited at higher concentration of insecticide. The physiological condition of insects are related to levels of alkaline phosphatase which can also reflects digestion, absorption, and positive transport of nutrients in the midgut [17]. Tyrosine O-phosphate was shown to be the natural substrate for Alkaline phosphatase [18] in Drosophila. Decreased levels of ALP activities provides the lower levels of tyrosine, precursor of dopamine and octopamine, play a vital role in regulation of juvenile hormone (JH), and 20-hydroxyecdysone (20E) [18]. The decreased level of alkaline phosphatase activity in response to increased concentrations of imidacloprid indicates insecticide's harmful effect on insect's digestive system and development.

Conclusion
Our results demonstrate that imidacloprid acts as potent protease inhibitor by inhibiting elastase, aminopeptidase, chymotrypsine, trypsine, total proteases and ALP.

Advances in Biotechnology & Microbiology
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