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OBJECTIVES To assess the effectiveness of nanopermethrin as a potential new formulation for pest and vector control. METHODS Permethrin nanoparticles were prepared by the ionic gelation method and its structure and the formulations were designed using Box-Behnken statistical technique. The effect of independent variables (Chitosan/Permethrin ratio, tripolyphosphate quantity, sonication time) on the properties of nanoparticles was investigated to determine the optimal formulation. RESULTS The size of the nanoparticles ranged from 135.27 ± 5.88 to 539.5 ± 24.01 nm and the insecticide entrapment efficiency per cent (EE%) ranged from 7.72 ± 1.36 to 63.59 ± 3.17%. Anopheles stephensi larvae were then bioassayed with the nanopermethrin and compared with the results of the bioassay with the mother molecule of permethrin using a standard WHO-recommended mosquito larval bioassay kit. LC50 with permethrin and nanopermethrin on larvae of An. stephensi were 0.125 and 0.026 ppm showing a 4.8 times difference. The LC50 for permethrin and nanopermethrin on Culex pipiens were 0.003 and 0.00032 ppm, respectively, showing a 9.4-fold difference. CONCLUSION Nanopermethrin is much more potent than its mother molecule against larvae of An. stephensi and Cx. pipiens.

Resistance to acaricides in ticks is becoming increasingly widespread throughout the world; therefore, tick control requires resistance monitoring for each tick species. The aims of this study were to monitor the susceptibility status of the cattle tick, Rhipicephalus (Boophilus) annulatus (Acari: Ixodidae), against pyrethroid acaricides from Mazandaran Province, northern Iran, and where resistance was evident, and establish the possible underlying mechanisms. Fully engorged adult R. (B.) annulatus females collected on cattle from Mazandaran Province. Twenty-nine tick populations produced 10-18days old larvae and bioassayed with cypermethrin and λ-cyhalothrin by larval packet test and the levels of detoxification enzymes were measured. Population AM-29 had a maximum resistance ratio (RR99) of 20.21 to cypermethrin and 53.57% of the tick populations were resistant at LC99 level. With λ-cyhalothrin, 17.86% of the tick populations were resistant and AM-29 was the most resistant population with RR99=4.54. AM-29 also showed significant elevation of cytochrome P450 monooxygenase (P450) and glutathione S-transferase (GST) (2.76- and 2.39-fold, respectively) (P

Residual effects of abamectin (0.02%), propargite (0.1%), dichlorvos (DDVP) (0.15%) and pymetrozine (0.15%) were determined on Orius albidipennis at laboratory conditions. Planted cucumbers were sprayed with the highest recommended concentrations of all insecticides and experimental treatments were monitored during 20 days based on the period of pesticides residues. On the first day after spraying, the greatest of mortality belonged to dichlorvos and pymetrozine (97 ± 1.22, 57 ± 3.1, respectively, p

Rhipicephalus (Boophilus) annulatus is one of the most important hard ticks parasitizing cattle in northern Iran. The aim of this study was to evaluate pyrethroid resistance levels of this species from Nur County, northern Iran. The hard ticks were collected through a multistage cluster randomized sampling method from the study area and fully engorged female R. (B.) annulatus were reared in a controlled insectary until they produced larvae for bioassay. Seventeen populations of the hard ticks were bioassayed with cypermethrin and 12 populations with lambda-cyhalothrin using a modified larval packet test (LPT). Biochemical assays to measure the contents/activity of different enzyme groups including mixed function oxidases (MFOs), glutathione S-transferases (GSTs) and general esterases were performed. Population 75 showed a resistance ratio of 4.05 with cypermethrin when compared with the most susceptible field population 66 at the LC50 level. With lambda-cyhalothrin the resistance ratio based on LC50 was 3.67 when compared with the susceptible population. The results of biochemical assays demonstrated significantly elevated levels of GSTs and esterases in populations tested compared with the heterozygous susceptible filed population and a correlation coefficient of these enzymes was found in association to lambda-cyhalothrin resistance. Based on the results, pyrethroid acaricides may operationally fail to control R. (B.) annulatus in North of Iran. This study is the first document of pyrethroid resistance in R. (B.) annulatus populations from Iran.

Efficient control of Chilo suppressalis Walker is always controversial due to highly economic damage, resistance to insecticides and environmental pollutions. So, combination of safe pest controls e.g. biocontrol agents and insect growth regulators seems to be promising via integrated pest management program. Bioassay of hexaflumuron on 4th larval instars revealed concentrations of 44.34, 179.74 and 474.94µg/ml as LC10-50 values. Numbers of total hemocytes, plasmatocytes and granulocytes as well as phenoloxidase activity increased in the different time intervals following treatment by hexaflumuron. Combined effects of hexaflumuron and Beauveria bassiana Vuillemin also increased hemocyte numbers and phenoloxidase activity at different time intervals using all concentrations. Activities of general esterases assayed by α- and β-naphtyl acetate and glutathione S-transferase using CDNB and DCNB increased 1-12h post-treatment. Activities of aspartate aminotransferase, alanine aminotransferase, γ-glutamyl transferase and aldolase increased in the larvae treated by hexaflumuron. However enhanced activity of lactate dehydrogenase was only obtained by treating 180 and 470µg/ml concentrations of hexaflumuron. Activities of ACP and ALP were found to be higher than control for all time intervals even 1-12h post-treatment. The amounts of HDL and LDL increased in the highest concentrations of hexaflumuron after 12-24h of post-treatment. Amount of triglyceride was higher than that of control after 1 and 3h but it was lower in other time intervals. Amounts of glycogen and protein were lower than those of control for all time intervals except for 6 and 12h of post-treatment in case of protein. Results of the current study revealed negative effects of hexaflumuron on intermediary metabolism of Chilo suppressalis but it increased the number of hemocytes and activity of phenoloxidase which are responsible for spore removal from hemolymph. It can be concluded that hexaflumuron is able to decrease survival and biological performance of C. suppressalis via intervening in intermediary metabolism but the given results showed incompatibility of the IGR with possible microbial control.

In the current study, two phenoloxidases (POs) from the larvae of Chilo suppressalis Walker were extracted and purified by column chromatography using Sepharyl G-100 and DEAE-Cellulose fast flow column. Two proteins possessing PO activity, named as POI and POII, were extracted by purification, 5.08- and 5.62-fold, respectively, with 8.94% and 7.31% recoveries, respectively. Also, the specific activities of POI and POII were 0.478 and 0.529 U/mg protein, respectively. Finally, the molecular weights of POI and POII were calculated as 94.6 and 95.7 kDa, respectively. Kinetic parameters of the purified phenoloxidases by Lineweaver-Burk analysis were Vmax of 2.27 and 1.11 U/mg protein and Km of 15.51 and 17.31 mM for POI and POII, respectively. Mg2+ and Cu2+ significantly increased the PO activities. Ca2+ decreased the activity of POI and showed no statistical effects on POII activity. EDTA and DTC significantly inhibited the activities of the purified enzymes, while triethylenetetramine hexaacetic acid (TTHA) and RGTA showed no significant effects on enzymatic activities.

Background and purpose: Paederus associated dermatitis has always been considered as a health problem in the northern of Iran. Since until now, the traditional method and some corticosteroids have been used in the treatment of Paederus dermatitis (PD) and no comprehensive studies have been carried out on the new method of treatment. Therefore, this study was aimed to compare the anti-inflammatory effects of triamcinolone with placebo in treatment of PD. Materials and Methods: This randomized double-blind clinical trial was performed during 6 months period in the clinics of Sari and Neka, Iran. Experimental group received triamcinolone and control group received placebo. The therapeutic effects of topically applied triamcinolone in 15 dermatitis patients and 15 control subjects were compared. The study subjects were visited in three separate times in the 1st, 7th and 14th day of the treatment. The data were collected in the questionnaire and compared in both groups by introducing the data into SPSS 11 software and analyzed by means of χ 2 test. Results: A total of 15 patients, 10 and 5 cases were undertaken for treatment in the Sari and Neka Townships, respectively. 40% and 50% of the patients from Sari and Neka Townships had lesion size 6-10 cm2, respectively. In this study, 90% and 100% of the patients from Sari and Neka had complete recovery 7 days after treatment, respectively. Statistical analysis indicated a significant difference between the case and control groups (P < 0.05). Conclusion: Insignificant difference was observed for treatment between the patients from the two Townships under study.

Roles of salivary proteases in the extra-oral digestion of the predatory bug, Andrallus spinidens Fabricius (Hemiptera: Pentatomidae) were studied by using 2% azocasein as a general substrate and specific protease substrates, as well as synthetic and endogenous inhibitors. It was found that salivary glands of A. spinidens have two anterior, two lateral, and two posterior lobes. Azocasein was used to measure the activity of general proteases in the salivary glands using different buffer solutions. The enzyme had the highest activity at pH 8. General protease activity was highest at 40 °C and was stable for 6-16 hours. The use of specific substrates showed that trypsin-like, chymotrypsin-like, aminopeptidase, and carboxypeptidase are the active proteases present in salivary glands, by the maximum activity of trypsin-like protease in addition to their optimal pH between 8-9. Ca(2+) and Mg(2+) increased proteolytic activity about 216%, while other ions decreased it. Specific inhibitors including SBTI, PMSF, TLCK, and TPCK significantly decreased enzyme activity, as well as the specific inhibitors of methalloproteases including phenanthroline, EGTA, and TTHA. Extracted endogenous trypsin inhibitors extracted from potential prey, Chilo suppressalis, Naranga aenescens, Pieris brassicae, Hyphantria cunea, and Ephestia kuhniella, had different effects on trypsin-like protease activity of A. spinidens salivary glands. With the exception of C. suppressalis, the endogenous inhibitors significantly decreased enzyme activity in A. spinidens.

Lipases are some of the crucial enzymes during the fungal penetrating process of the insect integument. Due to the importance and lack of information on the microbial lipases of Beauveria bassiana, investigations were carried out to purify and biochemically characterize these enzymes. The results obtained on growth medium demonstrated the highest activity of lipase 6 days after inoculation while the pH of the medium was 7.1. After three purification steps, the purified enzyme was 9.91-fold with specific activity of 20816 U/mg protein, recovery of 25% and molecular weight of 25 kDa. The purified lipase had the optimal pH and temperature at 7 and 35°C and was stable for 36–72 h under those conditions. Ca2 + significantly increased the enzyme activity and NaCl decreased it at all the tested concentrations. In addition, Mn2 + had no effect on enzyme activity but Mg2 + and Zn2 + increased it only at the highest concentration used. Three out of the four inhibitors used, significantly decreased the purified lipa...

Characteristics of mosquito larval habitats are important in determining whether they can survive and successfully complete their developmental stages. Therefore, data on the ecological factors affecting mosquito density and abundance especially the physicochemical properties of water of their breeding sites, can possibly be helpful in implementing larval management programs. Mosquito larvae were collected using a standard 350 ml dipper from fixed habitats including: artificial pool, river edge, creek and etc, in 30 villages of 16 counties from May-December 2014. Water samples were collected during larval collection and temperature (°C), acidity (pH), turbidity (NTU), electrical conductivity (μS/cm), alkalinity (mg/l CaCO3), total hardness (mg/l), nitrate (mg/l), chloride (mg/l), phosphate (mg/l) and sulphate (mg/l) were measured using standard methods. Spearman correlation coefficient, Kruskal-Wallis test of nonparametric analysis, Chi-square (χ2) analysis, regression analysis and C8 interspecific correlation coefficient were used for data analysis. A total of 7,566 mosquito larvae belonging to 15 species representing three genera were collected from fixed larval breeding places. Culex pipiens was the dominant species except in four villages where An. maculipennis s.l. and Cx. torrentium were predominant. There was a significant positive correlation between the density of Cx. pipiens and electrical conductivity, alkalinity, total hardness and chloride, whereas no significant negative correlation was observed between physicochemical factors and larval density. The highest interspecific association of up to 0.596 was observed between An. maculipennis s.l/An. pseudopictus followed by up to 0.435 between An. maculipennis s.l/An. hyrcanus and An. hyrcanus/An. pseudopictus. The correlations observed between physicochemical factors and larval density, can possibly confirm the effect of these parameters on the breeding activities of mosquitoes, and may be indicative of the presence of certain mosquito fauna in a given region., Author summary Determination of association between mosquito larval abundance and physicochemical factors in aquatic habitats in Mazandaran Province was the purpose of this study. In 30 villages of 16 counties in the province, Culex pipiens, Cx. tritaeniorhynchus and An. maculipennis s.l. were the most abundant species discovered in larval habitats. The pair of species An. maculipennis s.l/An. pseudopictus has the highest interspecific association in the area. Culex pipiens showed the greatest compatibility compared with other species with a range of densities from the lowest to the highest in different habitats containing varying levels of physicochemical factors. Measuring physicochemical factors of water in larval habitats indicated that values of chloride, electrical conductivity, alkalinity and total hardness were significantly associated with the larval abundance in different larval habitats. The electrical conductivity, alkalinity, total hardness and chloride had significant positive correlation with the density of Cx. pipiens, whereas no negative correlation was obtained between larval density and physicochemical factors. These differences in preferences and association with varying levels of physicochemical factors can have vector-borne diseases control implications, because habitat changes through the manipulation of these factors to the detriment of vector species can ultimately lead to reduced abundance of vector species. The data may also be useful to predict changes in the densities of mosquito larvae or their transition should natural or artificial environmental modifications lead to alteration in the physicochemical factors.

Digestive proteolytic activity in the alimentary canal of Andrallus spinidens, a potential biocontrol agent of lepidopteran larvae, was studied by considering enzyme compartmentalization and diversity. The alimentary canal of adults consists of a foregut, a four- sectioned midgut, namely V1 to V4 (ventriculus), and a hindgut. The optimal pH for general proteolytic activity was found to be at pH 8 with a small peak at pH 6. Results revealed that there are several specific proteases in the midgut of A. spinidens, including trypsin-like, chymotrypsin-like, and elastase as serine proteases, and cathepsins B, L and D as cysteine proteases, in addition to two exopeptidases of carboxy- and aminopetidases. Compartmentalization of digestive proteolytic activity showed that V3 is the main area of proteolytic secretion for both general and specific proteases and that V4 has the lowest enzymatic role, so that four out of the eight specific proteases found showed no activity in this section. The lowest and the highest proteolytic activity was found to be in the 1st and 4th nymphal instars, respectively. Using the specific inhibitors phenylmethylsulfonyl fluoride, Na-p-tosyl-L-lysine chloromethyl ketone, Ntosyl- L-phenylalanine chloromethyl ketone, L-trans-epoxysuccinyl-leucylamido-( 4-guanidino)-butane, cystatin, phenanthroline and ethylendiamidetetraacetic acid, we verified the presence of all specific proteases noted using both biochemical assays and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Our findings demonstrated that A. spinidens could utilize several caterpillars because of the presence of various of proteases in its midgut.

The rice green semi looper, Naranga aenescens Moore (Lepidoptera: Noctuidae) causes severe damage to rice fields in Eastern Asia and Middle East. We demonstrate that two types of serine proteases are active in the midgut of the third instar larvae of N. aenescens, but trypsin-like proteases are considerably more active than chymotrypsin-like proteases. To develop better control strategies, purification and biochemical characterization of a major trypsin-like digestive protease from the midgut of the third instar larvae of N. aenescens was achieved by gel filtration and anion exchange chromatography. After the final purification step, the enzyme was purified 9.62-fold with a recovery of 16.1% and a specific activity of 4.12 U/mg protein and a molecular mass of approximately 88.5 kDa. Biochemical characterization indicated that the purified protease had highest activity at pH 10 and 30°C and was stable for up to 6 h under those conditions. Divalent cations, especially Ca2+, Mg2+, and Cu2+, increased the enzyme activity and synthetic inhibitors that target trypsin-like activity caused a significant reduction in caseinolytic activity. These data may be used to develop inhibitors that decrease the damage of N. aenescens to rice cultivars in the field.

Background: Culex pipiens play an important role in transmission of infectious diseases. Vector control by chemical pesticides, leads inevitably to resistance development. Understanding the underlying resistance mechanisms can help improve the control programmes and insecticide resistance management. Methods: The total contents of cytochrome p450s and the activities of glutathione S-transferases, alpha- and beta-esterases and inhibition rates of acetylcholine esterase (by propoxur) were measured in the field population of Cx. pipiens collected from Sari County, North of Iran, in 2016 and the results were compared with those of the laboratory susceptible strain according to the biochemical assay methods of WHO for adult mosquitoes. Independent sample t-test was used to compare the mean values of enzyme activities/contents between filed and laboratory susceptible popula-tions. Results: The enzyme ratio of cytochrome p450s, alpha- and beta-esterases in the field population was 2.07, 3.72 and 1.36 respectively when compared with the results of the laboratory population. Although not statistically significant, the mean GSTs activities in the field population was marginally less than the laboratory population (ER=0.92). Ace-tylcholinesterase was insensitive to propoxur in 62.82% of the individuals of the tested field population. There was a significant difference (P< 0.05) between all values of the activities/contents of the enzyme in the field population except for GSTs compared with the laboratory susceptible strain. The highest enzyme activity was related to alpha esterase. Conclusion: The present study showed a range of metabolic mechanisms, comprising p450s and esterases combined with target site insensitivity of AChE, contributing to organophosphate, carbamate and pyrethroid resistance in the field population of Cx. pipiens.

Background\ud \ud Malaria is endemic in most parts of Afghanistan and insecticide-based vector control measures are central in controlling the disease. Insecticide resistance in the main malaria vector Anopheles stephensi from Afghanistan is increasing and attempts should be made to determine the underlying resistance mechanisms for its adequate management.\ud \ud Methods\ud \ud The contents of cytochrome P450s, esterases, glutathione S-transferases (GSTs) and acetylcholine esterase (AChE) activities were measured in the Kunar and Nangarhar populations of An. stephensi from Afghanistan and the results were compared with those of the susceptible Beech strain using the World Health Organization approved biochemical assay methods for adult mosquitoes.\ud \ud Results\ud \ud The cytochrome P450s enzyme ratios were 2.23- and 2.54-fold in the Kunar and Nangarhar populations compared with the susceptible Beech strain. The enzyme ratios for esterases with alpha-naphthyl acetate were 1.45 and 2.11 and with beta-naphthyl acetate were 1.62 and 1.85 in the Kunar and Nangarhar populations respectively compared with the susceptible Beech strain. Esterase ratios with para-nitrophenyl acetate (pNPA) were 1.61 and 1.75 in the Kunar and Nangarhar populations compared with the susceptible Beech strain. The GSTs enzyme ratios were 1.33 and 1.8 in the Kunar and Nangarhar populations compared with the susceptible Beech strain. The inhibition of AChE was 70.9 in the susceptible Beech strain, and 56.7 and 51.5 in the Kunar and Nangarhar populations. The differences between all values of the enzymes activities/contents and AChE inhibition rates in the Kunar and Nangarhar populations were statistically significant when compared with those of the susceptible Beech strain.\ud \ud Conclusions\ud \ud Based on the results, the reported resistance to pyrethroid and organophosphate insecticides, and tolerance to bendiocarb in the Kunar and Nangarhar populations of An. stephensi from Afghanistan are likely to be caused by a range of metabolic mechanisms, including esterases, P450s and GSTs combined with target site insensitivity in AChE.