Toxic evaluation of Proclaim Fit® on adult and larval worker honey bees.

Impacts to honey bees due to exposure to agricultural pesticides is one of the most serious threats to the beekeeping industry. Our research evaluated toxicity of the formulated insecticides Lufenuron+Emamectin benzoate (Proclaim Fit®) on the European honey bee Apis mellifera L. at field-realistic concentration (worst-case scenario). Newly emerged (≤24-h old) and forager (unknown age) worker bees were treated with the field recommended concentration of Proclaim Fit® using three routes of exposure including residual contact, oral, and spray within the laboratory. We also assessed the effects of Proclaim Fit® on the specific activity of some well-known detoxifying enzymes including α-esterase, ß-esterase, and Glutathione S-transferase (GST) in the honey bees. In addition, toxicity of the formulation was tested on 4th instar larvae within the hive. Based on estimated median survival times (MSTs), Proclaim Fit® was highly toxic to the bees, especially when applied as spray. According to our estimated relative median potency (RMP) values, newly emerged bees were 1.72× more susceptible than foragers to Proclaim Fit® applied orally. Enzyme assays revealed the considerable involvement of the enzymes, especially GST and α-esterase, in detoxification of the Proclaim Fit®, but their activities were significantly influenced by route of exposure and age of bee. Notably, Proclaim Fit® was highly toxic to 4th instar honey bee larvae. Our results generally indicate a potent toxicity of Proclaim Fit® toward honey bees. Therefore, its application requires serious consideration and adherence to strict guidelines, especially during the flowering time of crops.

Monophosphoramide derivatives: synthesis and crystal structure, theoretical and experimental studies of their biological effects.

Synthesizing new chemical compounds and studying their biological applications have been important issues in scientific research. In this investigation, we synthesized and characterized ten new N-acetyl phosphoramidate compounds and explored the crystal structure of three others. Furthermore, not only were some kinetic inhibition parameters measured, like IC50, Ki, kp, KD for 7 compounds on human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), but also their hydrophobic parameter was determined by shake-flask technique. All compounds (number 1-10) were investigated for anti-bacterial activity against three Gram-positive and three Gram-negative bacteria, while chloramphenicol was used as a standard antibiotic. In order to find new insecticide, toxicities of 13 acephate (Ace)-derived compounds (number 20-32) were bioassayed on third larval instar of elm leaf beetle and Xanthogaleruca luteola. Additionally, screening in vivo tests revealed that two compounds had had the greatest insecticidal potential in comparison with others. It means these ones inhibited AChE (with mixed mechanisms) and general esterase more than the rest. According to ChE-QSAR models, the inhibitory potency for enzyme and bacteria is directly influenced by the electronic parameters versus structural descriptors. AChE-QSPR model of fluorescence assay indicated that the inhibitory power of AChE is primarily influenced by a set of electronic factors with the priority of: EHB > PL > δ(31P) versus structural descriptor (SA and Mv). Synthesizing new chemical compounds and studying their biological applications have been important issues in scientific research. Toxicities of 13 acephate (Ace)-derived compounds (number 20-32) were bioassayed on third larval instar of elm leaf beetle and Xanthogaleruca luteola. Insect-QSAR equations of these compounds, based on MLR and PCA, showed that non-descriptor net charge nitrogen atom (which was affected by the polarization of N-H group) had the greatest effect on insecticidal potential.

Cellular Energy Allocation of Tuta absoluta (Lepidoptera: Gelechiidae) treated with Flubendiamide and Thiocyclam Hydrogen Oxalate on Different Tomato Cultivars.

The tomato leaf miner, Tuta absoluta (Meyrick), is the most important pest for tomato production in Iran. The effect of flubendiamide and thiocyclam hydrogen oxalate insecticides was assessed on cellular energy allocation (CEA) of the third instar larvae of T. absoluta ingesting six different tomato cultivars. Plant leaves were treated with LC50 concentration of both insecticides and their energy available (Ea), energy consumption (Ec), and CEA were calculated. The results showed that total energy reserves (protein, carbohydrate, and lipid budgets) were significantly reduced after exposure to insecticides and tomato secondary metabolites. The larvae fed on Riogrande and Super Chief cultivars showed the least amount of energy available after treatment with both insecticides. The highest rate of oxygen consumption was observed in larvae fed on Calj, Super Luna, and Super strain B after treatment with flubendiamide. The amount of CEA decreased in treated larvae compared to untreated larvae. This reduction was statistically higher in treated larvae that fed on Riogrande and Super strain B cultivars. Reduction in CEA is probably due to the expenses of dealing with detoxification of insecticides and plant metabolites. CEA is a suitable and primary biomarker for the effects of cultivars and insecticides as integrate and summarizes insect energy allocation in variable situations.

Molecular and genetic analysis of resistance to METI-I acaricides in Iranian populations of the citrus red mite Panonychus citri.

The citrus red mite, Panonychus citri, is a major pest on citrus all around the world. Mitochondrial Electron Transport Inhibitors of complex I (METI-I) acaricides such as fenpyroximate have been used extensively to control P. citri populations, which resulted in multiple reports of METI-I resistant populations in the field. In this study, biochemical and molecular mechanisms of fenpyroximate resistance were investigated in P. citri. Seven populations were collected from Northern provinces of Iran. Resistance ratios were determined and reached up to 75-fold in comparison to a fenpyroximate susceptible population. Cross-resistance to two additional METI-I acaricides, pyridaben and tebufenpyrad, was detected. PBO synergism experiments, in vivo enzyme assays and gene expression analysis suggest a minor involvement of cytochrome P450 monooxygenases in fenpyroximate resistance, which is in contrast with many reported cases for the closely related Tetranychus urticae. Next, we determined the frequency of a well-known mutation in the target-site of METI-Is, the PSST subunit, associated with METI-I resistance. Indeed, the H92R substitution was detected in a highly fenpyroximate resistant P. citri population. Additionally, a new amino acid substitution at a conserved site in the PSST subunit was detected, A94V, with higher allele frequencies in a moderately resistant population. Marker-assisted back-crossing in a susceptible background confirmed the potential involvement of the newly discovered A94V mutation in fenpyroximate resistance. However, introduction of the A94V mutation in the PSST homologue of D. melanogaster using CRISPR-Cas9 did not result in fenpyroximate resistant flies. In addition, differences in binding curves between METI-Is and complex I measured directly, in isolated transgenic and wildtype mitochondria preparations, could not be found.

Nitrogen Fertilization Increases the Nutritional Quality of Aphis gossypii (Hemiptera: Aphididae) as Prey for Hippodamia variegata (Coleoptera: Coccinellidae) and Alters Predator Foraging Behavior.

Nitrogen (N) fertilization is a common agricultural practice, which, by increasing the quality of plants, also enhances their nutritional suitability for insect herbivores, creating the possibility of a cascade of N across trophic levels, from plant to herbivore to predator. We manipulated the quality of cucumber plants by fertilizing them with three different N rates (110, 160, and 210 ppm), which represented low, medium, and high N levels, respectively. Colonies of Aphis gossypii Glover (Hemiptera: Aphididae) were then reared on these plants and used as prey for adult Hippodamia variegata (Goeze) (Coleoptera: Coccinellidae) in experiments that characterized the predator's foraging behavior and functional response to different aphid densities. The nutritional content of plants and aphids was also measured. As N fertilization increased, so did the nutrient content (total energy) of aphids and this resulted in declining rates of aphid consumption by beetles at higher aphid densities. Females in the 110 N treatment, and males in all treatments, responded to aphids with a type II functional response (decelerating consumption at higher densities), but females in the 160 and 210 ppm N treatments exhibited a type III response (consuming a declining proportion of available aphids at high densities). Beetles fed aphids from the 110 N treatment consumed more prey in both assays than did those fed aphids from the 210 N treatment. Beetle searching time, handling time, and duration of digestive pauses all increased at high levels of N fertilization, especially for females. The results indicate that heavy N fertilization can increase prey nutritional quality to the point where it alters predator foraging and feeding behavior, resulting in slower rates of prey consumption and longer prey handling times.

Differential Change Patterns of Main Antimicrobial Peptide Genes During Infection of Entomopathogenic Nematodes and Their Symbiotic Bacteria.

The expression of antimicrobial peptides (AMPs) as the main humoral defense reactions of insects during infection by entomopathogenic nematodes (EPNs) and their symbiont is addressed herein. Three AMPs, attacin, cecropin, and spodoptericin, were evaluated in the fifth instar larvae of Spodoptera exigua Hübner (beet armyworm) when challenged with Steinernema carpocapsae or Heterorhabditis bacteriophora. The results indicated that attacin was expressed to a greater extent than either cecropin or spodoptericin. While spodoptericin was expressed to a much lesser extent, this AMP was induced against Gram-positive bacteria, and thus not expressed after penetration of Xenorhabdus nematophila and Photorhabdus luminescens. Attacin and cecropin in the larvae treated with S. carpocapsae at 8 hr post-injection (PI) attained the maximum expression levels and were 138.42-fold and 65.84-fold greater than those of larvae infected with H. bacteriophora, respectively. Generally, the ability of H. bacteriophora to suppress attacin, cecropin, and spodoptericin was greater than that of S. carpocapsae. According to the results, the expression of AMPs by Sp. exigua larvae against S. carpocapsae was determined in the 4 statuses of monoxenic nematode, axenic nematode, live symbiotic bacterium, and dead symbiotic bacterium. The expression of attacin in larvae treated with a monoxenic nematode and live bacterium at 8 and 2 hr PI, respectively, were increased to the maximum amount. Live X. nematophila was the strongest agent for the suppression of attacin. The expression of cecropin against monoxenic nematodes and live symbiotic bacteria at 8 and 4 hr PI, respectively, reached the maximum amount while the expression levels of attacin and cecropin for axenic nematodes were lesser and stable. The results highlighted that the ability of P. luminescens in AMPs suppression was much more than X. nematophila. The results also showed that the effect of symbiotic bacterium in suppressing attacin and cecropin expression was greater than that of a monoxenic nematode; this result provided deep insight into the expression pattern parallels and fluctuations of the main AMPs during nematode infection.

Characterization of acetylcholinesterase from elm left beetle, Xanthogaleruca luteola and QSAR of temephos derivatives against its activity.

Insect acetylcholinesterase (AChE) is the principal target for organophosphate (OP) and carbamate (CB) insecticides. In this research, an AChE from third instar larvae of elm left beetle, Xanthogaleruca luteola was purified by affinity chromatography. The enzyme was purified 75.29-fold with a total yield of 8.51%. As shown on denaturing SDS-PAGE, the molecular mass of purified AChE was 70kDa. The enzyme demonstrated maximum activity at pH7 and 35°C. Furthermore, a series of temephos (Tem) derivatives with the general structure of P(O)XP(O) (1-44) were prepared, synthesized and characterized by 31P, 13C, 1H NMR and FT-IR spectral techniques. The toxicity of 36 new Tem derivatives was screened on the third instar larvae and the compound compound 1,2 cyclohexane-N,N'-bis(N,N'-piperidine phosphoramidate) exhibited the highest insecticidal potential. The method of kinetic analysis is applied in order to obtain the maximum velocity (Vmax), the Michaelis constant (Km) and the parameters characterizing the inhibition type for inhibitors with >75% mortality in preliminary bioassay. The inhibition mechanism was mixed and inhibitory constant (Ki) was calculated as 4.70µM-1min-1 for this compound. Quantitative structure-activity relationship (QSAR) equations of these compounds indicated that the electron orbital energy has major effect on insecticidal properties.

Effects of 4-hexylresorcinol on the phenoloxidase from Hyphantria cunea (Lepidoptera: Arctiidae): In vivo and in vitro studies.

Insecticidal effects of 4-hexylresorcinol, a phenoloxidase (PO) inhibitor, were determined on Hyphantria cunea (Drury) under laboratory conditions. The LC50 for the 15-d-old larvae was estimated to be 2.95 g/L after 96 h exposure. The activities of glutathione S-transferase (GST) and PO showed a decrease in larvae treated with 4-hexylresorcinol, and the IC50 of GST and PO were estimated to be 0.8 and 0.43 g/L, respectively, 24 h after treatment. The PO from the hemolymph of fall webworm was purified by ammonium sulfate precipitation, gel-filtration, and ion-exchange chromatography, and then enzymatic characteristics and the mechanism of inhibition were determined using L-dihydroxyphenylalanine (L-DOPA) as the substrate. The purified PO showed a single band on SDS-PAGE with a molecular weight of about 70 kDa. The optimum pH for PO activity was observed at pH 7.0, optimum temperature was found to be 45 °C, and PO activity was strongly inhibited by Zn(2+) . IC50 values were estimated to be 8.2, 19.14, and 24.04 µmol/L for 4-hexylresorsinol, kojic acid, and quercetin, respectively. The inhibitory potencies (i.e., I50 of each compound/I50 of 4-hexylresorcinol) of kojic acid and quercetin on H. cunea PO were estimated to be 1.87 and 2.89, respectively. 4-hexylresorcinol was determined to be a competitive inhibitor, and kojic acid and quercetin were determined to be mixed inhibitors. PO is one of the most important enzymes in an insect's immune system, and the use of PO inhibitors seems to be a promising approach for pest control due to their potential safety for humans.

Toxicological and biochemical characterizations of AChE in phosalone-susceptible and resistant populations of the common pistachio psyllid, Agonoscena pistaciae.

The toxicological and biochemical characteristics of acetylcholinesterases (AChE) in nine populations of the common pistachio psyllid, Agonoscena pistaciae Burckhardt and Lauterer (Hemiptera: Psyllidae), were investigated in Kerman Province, Iran. Nine A. pistaciae populations were collected from pistachio orchards, Pistacia vera L. (Sapindales: Anacardiaceae), located in Rafsanjan, Anar, Bam, Kerman, Shahrbabak, Herat, Sirjan, Pariz, and Paghaleh regions of Kerman province. The previous bioassay results showed these populations were susceptible or resistant to phosalone, and the Rafsanjan population was most resistant, with a resistance ratio of 11.3. The specific activity of AChE in the Rafsanjan population was significantly higher than in the susceptible population (Bam). The affinity (K(M)) and hydrolyzing efficiency (Vmax) of AChE on acetylthiocholine iodide, butyrylthiocholine iodide, and propionylthiocholine odide as artificial substrates were clearly lower in the Bam population than that in the Rafsanjan population. These results indicated that the AChE of the Rafsanjan population had lower affinity to these substrates than that of the susceptible population. The higher Vmax value in the Rafsanjan population compared to the susceptible population suggests a possible over expression of AChE in the Rafsanjan population. The in vitro inhibitory effect of several organophosphates and carbamates on AChE of the Rafsanjan and Bam populations was determined. Based on I50, the results showed that the ratios of AChE insensitivity of the resistant to susceptible populations were 23 and 21.7-fold to monocrotophos and phosphamidon, respectively. Whereas, the insensitivity ratios for Rafsanjan population were 0.86, 0.8, 0.78, 0.46, and 0.43 for carbaryl, eserine, propoxur, m-tolyl methyl carbamate, and carbofuran, respectively, suggesting negatively correlated sensitivity to organophosphate-insensitive AChE. Therefore, AChE from the Rafsanjan population showed negatively correlated sensitivity, being insensitive to phosphamidon and monocrotophos and sensitive to N-methyl carbamates.

Linear-dendritic copolymers/indoxacarb supramolecular systems: biodegradable and efficient nano-pesticides.

Photodegradable and biocompatible nano-indoxacarb was prepared successfully by encapsulation of indoxacarb with poly(citric acid)-poly(ethylene glycol)-poly(citric acid) (PCA-PEG-PCA) ABA type linear-dendritic copolymers both with (nano-IND/TiO2) and without (nano-IND) TiO2 nanoparticles via supramolecular interactions. Preparation of nano-indoxacarb by both formulae was confirmed using spectroscopy and microscopy analyses. TEM images showed small particles with average sizes of 10 nm for nano-IND and 12 nm for nano-IND/TiO2. Interestingly, the rate of degradation of indoxacarb in both nano-IND/TiO2 and nano-IND exposed to UV and natural light was higher than that for free indoxacarb in the presence of TiO2 nanoparticles. Furthermore, results of bioassay experiments on the model pest showed that the essential dosage of pesticide for pest control significantly decreased when nano-IND and nano-IND/TiO2 were used. Because of the higher loading capacity and slower release rate for indoxacarb from nano-IND than from nano-IND/TiO2, and because TiO2 nanoparticles show toxicity in bioassay experiments, nano-IND is introduced as a promising and eco-friendly pesticide system.

Preparation, characterization and efficiency of nanoencapsulated imidacloprid under laboratory conditions.

In this work, nano-imidacloprid was prepared by direct encapsulation with ABA triblock linear dendritic copolymers composed of poly(citric acid) (PCA) as A block and poly(ethylene glycol) (PEG) as B block. Nanocapsules of imidacloprid were characterized using spectroscopy, microscopy and thermal analysis. The encapsulation process was performed by self-assembly of PCA-PEG-PCA in the presence of imidacloprid in different solvents. Comparison of the TEM images of nano-imidacloprid prepared in ethanol and water showed that, during the first day, self-assemblies appeared as small particles with an average size of 10-20nm. Depending upon the type of solvent, the time and concentration, morphology and size of the nano-imidacloprid varied from fiber-like to globular to tubular from 10nm to several mm in size. Higher loading capacity and slower release rate of imidacloprid from nano-imidacloprid at optimum pH of Glyphodes pyloalis׳s gut (pH=10) compared to neutral pH confirmed the selective and controllable action of nano-imidacloprid. Results of bioassays on the model insect showed that by using the nanoform of imidacloprid, essential dosage of pesticide and environmental risk decreased significantly and indicated good performance for this formulation.

Identification and partial characterization of midgut proteases in the lesser mulberry pyralid, Glyphodes pyloalis.

Proteolytic activities in digestive system extracts from the larval midgut of the lesser mulberry pyralid, Glyphodes pyloalis Walker (Lepidoptera: Pyralidae), were analyzed using different specific peptide substrates and proteinase inhibitors. High proteolytic activities were found at pH 10.0 and a temperature of 50° C using azocasein as substrate. The trypsin was active in the pH range of 9.5- 12.0, with its maximum activity at pH 11.5. Ethylene diamine tetraacetic acid had the most inhibitory effect, and 44% inhibition was detected in the presence of this inhibitor. Phenyl methane sulfonyl floride and N-tosyl-L-phe chloromethyl ketone also showed considerable inhibition of larval azocaseinolytic activity, with 40.2 and 35.1% inhibition respectively. These data suggest that the midgut of larvae contains mainly metalloproteases and serine proteases, mainly chymotrypsin. The effect of several metal ions on the activity of proteases showed that NaCl, CaCl2, CoCl2 (5 and 10 mM), and MnCl2 (5mM) reduced the protease activity. The kinetic parameters of trypsin-like proteases using N-benzoyl-L-arg-p-nitroanilide as substrate indicated that the Km and Vmax values of trypsin in the alimentary canal were 50.5 ± 2.0 µM and 116.06 ± 1.96 nmol min(-1) mg(-1) protein, respectively. Inhibition assays showed only small amounts of cysteine proteases were present in the G. pyloalis digestive system. The midgut digestive protease system of G. pyloalis is as diverse as that of any of the other polyphagous lepidopteran insect species, and the midgut of larvae contains mainly metalloproteases. Moreover, serine proteases and chymotrypsin also play main roles in protein digestion. Characterization of the proteolytic properties of the digestive enzymes of G. pyloalis offers an opportunity for developing appropriate and effective pest management strategies via metalloproteases and chymotrypsin inhibitors.

Effect of sweet wormwood Artemisia annua crude leaf extracts on some biological and physiological characteristics of the lesser mulberry pyralid, Glyphodes pyloalis.

The lesser mulberry pyralid, Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is a monophagous and dangerous pest of mulberry that has been recently observed in Guilan province, northern Iran. In this study, the crude methanol extract of sweet wormwood Artemisia annua L. (Asterales: Asteracaea) was investigated on toxicity, biological and physiological characteristics of this pest under controlled conditions (24 ± 1 °C, 75 ± 5% RH, and 16:8 L:D photoperiod). The effect of acute toxicity and sublethal doses on physiological characteristics was performed by topical application. The LC50 and LC20 values on fourth instar larvae were calculated as 0.33 and 0.22 gram leaf equivalent/ mL, respectively. The larval duration of fifth instar larvae in LC50 treatment was prolonged (5.8 ± 0.52 days) compared with the control group (4.26 ± 0.29 days). However larval duration was reduced in the LC20 treatment. The female adult longevity in the LC50 dose was the least (4.53 ± 0.3 days), while longevity among controls was the highest (9.2 ± 0.29 days). The mean fecundity of adults after larval treatment with LC50 was recorded as 105.6 ± 16.84 eggs/female, while the control was 392.74 ± 22.52 eggs/female. The percent hatchability was reduced in all treatments compared with the control. The effect of extract in 0.107, 0.053, 0.026 and 0.013 gle/mL on biochemical characteristics of this pest was also studied. The activity of α-amylase and protease 48 hours post-treatment was significantly reduced compared with the control. Similarly lipase, esterase, and glutathione S-transferase activity were significantly affected by A. annua extract.

Partial biochemical characterization of alpha- and beta-glucosidases of lesser mulberry pyralid, Glyphodes pyloalis Walker (Lep.: Pyralidae).

The Lesser Mulberry Pyralid, Glyphodes pyloalis, is an important pest of mulberry. This pest feeds on mulberry leaves, and causes some problems for the silk industries in the north of Iran. The study of digestive enzymes is highly imperative to identify and apply new pest management technologies. Glucosidases have an important role in the final stages of carbohydrate digestion. Some enzymatic properties of alpha- and beta-glucosidases from midgut and salivary glands of G. pyloalis larvae were determined. The activities of alpha- and beta-glucosidase in the midgut and salivary glands of 5th instar larvae were obtained as 0.195, 1.07, 0.194 and 0.072 micromol(-1) min(-1) mg protein(-1), respectively. Activity of alpha- and beta-glucosidase from whole body of larval stages was also determined. Data showed that the highest activity of alpha- and beta-glucosidase was observed in the 5th larval stage, 0.168 and 0.645 micromol(-1) min(-1) mg protein(-1), respectively and the lowest activity in the 2nd larval stage, 0.042 and 0.164 micromol(-1) min(-1) mg protein(-1), respectively. Results showed that the optimal pH for alpha- and beta-glucosidase activity in midgut and salivary glands were 7.5, 5.5, 8-9 and 8-9 respectively. Also, the optimal temperature for alpha- and beta-glucosidase activity in the midgut was obtained as 45 degrees C. The addition of CaCl(2) (40 mM) decreased midgut beta-glucosidase activity whereas alpha-glucosidase activity was significantly increased at this concentration. The alpha-glucosidase activity, in contrast to beta-glucosidase, was enhanced with increasing in concentration of EDTA. Urea (4 mM) and SDS (8 mM) significantly decreased digestive beta-glucosidase activity. Characterization studies of insect glucosidases are not only of interest for comparative investigations, but also understanding of their function is essential when developing methods of insect control such as the use of enzyme inhibitors and transgenic plants to control insect pest.

Enzymatic properties of alpha-amylase in the midgut and the salivary glands of mulberry moth, Glyphodes pyloalis Walker (Lepidoptera: Pyralidae).

The pyralid moth, Glyphode pyloalis Walker, is an important pest of the mulberry. Amylases are the hydrolytic enzymes that catalyze the hydrolysis of the alpha-D-(1,4)-glucan linkage in glycogen and other related carbohydrates. Laboratory-reared fifth stadium larvae were randomly selected; the midgut (MG) and the salivary glands (SG) were removed by dissection under a dissecting microscope and alpha-amylase activity was assayed using the dinitrosalicylic acid procedure. The activity of alpha-amylase in the MG and the SG were 0.011 and 0.0018 micromol/min, respectively. The optimal pH and temperature for alpha-amylase were 9 for MG at 37-40 degrees C and 10 for SG at 37 degrees C respectively. Various concentrations of compounds (NaCl, KCl, MgCl(2), Urea, EDTA, SDS and CaCl(2)) had differential effects on the enzyme activity. Plant amylase inhibitors may play an important role against insect pests. Hence, the characterization of digestive enzymes and the examination of their inhibitors may be a useful tool in future management of this important mulberry pest.

Diazinon resistance in different selected strains of Chilo suppressalis (Lepidoptera: Crambidae) in northern Iran.

Asiatic rice borer, Chilo suppressalis (Walker) (Lepidoptera: Crambidae), is a cosmopolitan and destructive pest in rice fields of the world. This pest was reported in 1973 in Iran, and it has since spread widely in rice, Oryza sativa L., fields throughout the country. In this study, we tried to evaluate comparative toxicity of diazinon in five colonies of C. suppressalis, collected from Babol (Ba), Amol (Am) of Mazandaran Province and Rasht (Ra), Sheikhmahale (Sh), and Gourabzarmikh (Go) of Guilan Province, northern Iran. The LD50 values were compared. We also evaluated the general esterases, alkaline phosphatase (ALP), glutathione transferase (GST), and acetylcholinesterase (AChE) activities from the five populations. The LD50 values of Ra, Ba, Am, and Sh (12.64, 11.4, 7.17, and 3.71 microg/mg larva(-1)) were 13.67-, 12.33-, 7.75-, and 4.02-fold higher than Go population (0.924 microg/mg larva(-1)). Using alpha-naphthyl acetate as substrate, the general esterase activities in Ra, Ba, Am, and Sh colonies were, respectively, 1.81-, 1.68-, 1.75-, and 1.35-fold more than those in Go population. When beta-naphthyl acetate was used as the substrate, activity ratio was measured 1.98-, 2.58-, 1.25-, and 1.24-fold compared with the Go population. Glutathione transferase activities in Ra, Ba, Am, and Sh populations were 1.27-, 1.68-, 0.98-, and 1.7-fold more than those in Go, when 1-chloro-2,4-dinitrobenzene was used as the substrate. When 1,2-dichloro-4-nitro-benzene was used as the substrate, activity ratio was measured 1.14-, 1.42-, 0.56-, and 0.95-fold compared with Go population. The ALP activity demonstrated a significant difference among these populations and in Ra, Ba, Am, and Sh larvae were 3.54-, 4.62-, 3.84-, and 2.18-fold more than Go. The AChE inhibition or I50 value was 0.19, 0.22, 0.31, 0.19, and 0.26 mM in Ra, Ba, Am, Sh and Go populations, respectively. However, the results showed no significant differences in studied colonies. These biochemical characterizations of general esterases ALP, GST, and AChE were consistent with diazinon bioassay in the five populations. It is inferred from increased esterase, alkaline phosphatase and glutathione transferase, activities that might play an important role in the increasing resistance in C. suppressalis to diazinon among these five populations.