Nonspecific Esterase Activity Research Articles

Monitoring Insecticide Susceptibility in Aedes Aegypti Populations from the Two Biggest Cities, Ouagadougou and Bobo-Dioulasso, in Burkina Faso: Implication of Metabolic Resistance.

In West Africa, Aedes aegypti remains the major vector of dengue virus. Since 2013, dengue fever has been reemerging in Burkina Faso with annual outbreaks, thus becoming a major public health problem. Its control relies on vector control, which is unfortunately facing the problem of insecticide resistance. At the time of this study, although data on phenotypic resistance were available, information related to the metabolic resistance in Aedes populations from Burkina Faso remained very scarce. Here, we assessed the phenotypic and the metabolic resistance of Ae. aegypti populations sampled from the two main urban areas (Ouagadougou and Bobo-Dioulasso) of Burkina Faso. Insecticide susceptibility bioassays to chlorpyriphos-methyl 0.4%, bendiocarb 0.1% and deltamethrin 0.05% were performed on natural populations of Ae. aegypti using the WHO protocol. The activity of enzymes involved in the rapid detoxification of insecticides, especially non-specific esterases, oxidases (cytochrome P450) and glutathione-S-transferases, was measured on individual mosquitos. The mortality rates for deltamethrin 0.05% were low and ranged from 20.72% to 89.62% in the Bobo-Dioulasso and Ouagadougou sites, respectively. When bendiocarb 0.1% was tested, the mortality rates ranged from 7.73% to 71.23%. Interestingly, in the two urban areas, mosquitoes were found to be fully susceptible to chlorpyriphos-methyl 0.4%. Elevated activity of non-specific esterases and glutathione-S-transferases was reported, suggesting multiple resistance mechanisms involved in Ae. aegypti populations from Bobo-Dioulasso and Ouagadougou (including cytochrome P450). This update to the insecticide resistance status within Ae. aegypti populations in the two biggest cities is important to better plan dengue vectors control in the country and provides valuable information for improving vector control strategies in Burkina Faso, West Africa.

Sublethal Doses of Essential Oils Impact Growth and Esterase Levels in German Cockroach (Blattodea: Ectobiidae) with and without Symbiotic Bacteria

The inhibition by three essential oils of the growth and esterase production of German cockroaches, Blattella germanica (L.) (Blattodea: Ectobiidae), with and without symbiotic bacteria, was determined in laboratory assays. Essential oils extracted from Lilium brownii var. viridulum Baker, Croton tiglium L., and Lonicera japonica Thunberg were mixed with a powdered rat food at a 10% concentration and fed to cockroach nymphs. Body mass and body length were measured in adults in all three treatments. Acetylcholinesterase and nonspecific esterase activities were compared between the Lilium and Croton essential oil treatments. The nonlethal doses of these essential oils reduced cockroach growth, while the growth of the females lacking symbiotic bacteria was significantly reduced when fed the diet containing Lilium essential oil. Lilium and Croton essential oils significantly inhibited esterase activity. Our results indicate that symbiotic gut bacteria might be involved in mediating detoxification in the German cockroach.

Analysis of the Bioaugmentation Potential of Pseudomonas putida OR45a and Pseudomonas putida KB3 in the Sequencing Batch Reactors Fed with the Phenolic Landfill Leachate

The treatment of landfill leachate could be challenging for the biological wastewater treatment systems due to its high toxicity and the presence of poorly biodegradable contaminants. In this study, the bioaugmentation technology was successfully applied in sequencing batch reactors (SBRs) fed with the phenolic landfill leachate by inoculation of the activated sludge (AS) with two phenol-degrading Pseudomonas putida OR45a and Pseudomonas putida KB3 strains. According to the results, the SBRs bioaugmented with Pseudomonas strains withstood the increasing concentrations of the leachate. This resulted in the higher removal efficiency of the chemical oxygen demand (COD) of 79–86%, ammonia nitrogen of 87–88% and phenolic compounds of 85–96% as compared to 45%, 64%, and 50% for the noninoculated SBR. Simultaneously, the bioaugmentation of the AS allowed to maintain the high enzymatic activity of dehydrogenases, nonspecific esterases, and catalase in this ecosystem, which contributed to the higher functional capacity of indigenous microorganisms than in the noninoculated AS. Herein, the stress level experienced by the microorganisms in the SBRs fed with the leachate computed based on the cellular ATP measurements showed that the abundance of exogenous Pseudomonas strains in the bioreactors contributed to the reduction in effluent toxicity, which was reflected by a decrease in the stress biomass index to 32–45% as compared to the nonbioaugmented AS (76%).

Metallothionein level, non-specific esterases, fitness-related traits and integrated biomarker response (IBR) in larvae of Lymantria dispar L. (Lepidoptera) originating from unpolluted and polluted locations after chronic cadmium treatment

Long-term exposure of invertebrate populations to pollution might result in adaptations that must be taken into account when evaluating physiological parameters as biomarkers of contamination. In the present study we investigated whether previous exposure of gypsy moth, Lymantria dispar L. (Lepidoptera), population to pollution affects the sensitivity of larvae to cadmium treatment under laboratory conditions. Our goals in the current study were to compare metallothionein (MT) level and non-specific esterase activity including isoform expression in the midgut and fitness-related traits between caterpillars originating from polluted and unpolluted localities after chronic oral intake (50 and 100 µg Cd/g dry food). Besides being individually assessed as biomarkers of cadmium contamination, the responses of above parameters related to the midgut (MT concentration, specific activity of esterases and midgut mass) were summarized into the integrated biomarker response (IBR). Upon cadmium exposure, MT concentration increased significantly only in larvae originating from the polluted site, whereas non-specific esterase activity decreased in caterpillars from the unpolluted forest. We noticed similar patterns of esterase isoforms in both populations, including isoform number three that appeared only after exposure to the lower cadmium concentration. Midgut mass was reduced only in the group from the unpolluted locality exposed to the higher cadmium concentration. IBR index increased in a concentration-dependent manner in both populations, reflecting higher sensitivity of larvae from the unpolluted locality to the metal. Neither cadmium exposure nor population origin influenced larval survival. While MT concentration could be applied as a biomarker of cadmium exposure in populations that have been previously exposed to pollution for a longer time, non-specific esterase activity could indicate metal presence at unpolluted locations. IBR index and expression of esterase isoform number three could be used universally, regardless of the exposure history, except the latter might be limited to lower cadmium concentrations.

Aedes aegypti vector resistance status on malation and activity of non specific esteration enzymes in Tembalang district, Semarang city

Background: Controlling of dengue vectors around dengue haemorrhagic fever cases is often conducted by using insecticides, including a malathion insecticide in the city of Semarang. A research on susceptibility status of Ae. aegypti to insecticides, used for fogging and based on a high number of hemorrhagic fever cases in Tembalang district, Semarang city, needs to be observed. Objective: This study aimed to determine resistance status of Ae. aegypti mosquitoes based on bioassay and biochemical tests of esterase non-specific enzyme activity in the Tembalang District. Methods: This study used a cross sectional design, it was conducted in June-September 2018 in a population of Aedes sp. from Tembalang District, Semarang City. Samples of F2 generations of female Ae. aegypti were obtained by ovitraps in the Tembalang District consisting of 12 urban villages, and their resistance status were tested by impregnated paper bioassay with malathion at a diagnostic dose of 0.8%. Esterase enzyme activity in the Ae. aegypti mosquito body of this population was tested biochemically to prove an increase in a non-specific esterase enzyme on the a-naphthyl acetate substrate. Results: The bioassay results showed that mortality rates of Ae. aegypti at the 12 villages in Tembalang district after 24 hourtest by impregnated paper indicated a range of 0-62%. Mechanism of resistance indicating an increase of non-specific enzyme esterase activity on a-naphthyl acetate at Rowosari, Kedungmundu, Sambiroto, and Meteseh could not be seen, It began to be seen in a low percentage (15%) at Sendang Mulyo, Tandang, Sendangguwo and Bubusan, and in a moderate percentage (45-75%) in Tembalang, Jangli and Mangunharjo. The mechanism of high esterase enzyme activity could be seen in the Ae. aegypti population at Kramas with AV=0,700-0,900 by a percentage of 20%, and AV=0,900 by a percentage of 80%. Conclusion: This study provided information about some Ae. aegypti mosquitoes from Tembalang District that showed resistance to the malathion insecticide with an elevation of non-specific esterase enzyme activity ona-naphthyl acetate substrate in several villages except Kelurahan Rowosari, Kedungmundu, Sambiroto, and Meteseh.

Gill epithelium of an angler catfish, Chaca chaca (Siluriformes, Chacidae): Enzyme and glycoprotein histochemistry.

A series of histochemical techniques have been employed to localize alkaline phosphatase, acid phosphatase, non-specific esterase, catalase and peroxidase; and to visualize and characterize glycoprotein (GPs) moieties in the epithelium of gill arch, gill filaments and secondary lamellae of an angler catfish Chaca chaca. The epithelium of gill arch and gill filament shows strong alkaline phosphatase activity in the deeper layer epithelial cells; strong non-specific esterase activities in the outer layer epithelial cells; and weak acid phosphatase activity throughout the epithelium. The activity of these enzymes in the secondary lamellae is weak. The catalase and peroxidase show strong activities in the blood cells of the secondary lamellae. Various classes of GPs have been identified and characterized in the mucous secretions of the gill epithelium of C.chaca. These include-GPs with oxidizable vicinal diols, GPs with sialic acid residues without O-acyl substitution and GPs with O-sulphate esters. The functional significance of different enzymes in gill epithelium and the GPs in the mucus secreted on the surface has been discussed with the physiology of the gills in relation to the characteristic habit and habitat of the fish.

Histochemical Distribution of Three Types of Enzymes and Mucous Cells in the Gill, Mantle and Hepatopancreas of the Ark Shell Scapharca subcrenata

The present study was aimed at evaluating the histochemical distribution of alkaline phosphatase (ALP), nonspecific esterase (NSE), peroxidase (POD) and various types of mucous cells in the gill, mantle and hepatopancreas of the ark shell Scapharca subcrenata. The ALP activity was found in the epithelial cells (EP) of the gill filament and the mantle as well as in the basal lamina of the glandular duct in the hepatopancreas. The staining intensity of ALP in the mantle and hepatopancreas was stronger than that in the gill. The NSE activity was detected in the EP in the gill filament, mantle and hepatopancreas, with the hepatopancreas showing a stronger intensity than the gill. The POD activity was located in the EP of the mantle and glandular duct of the hepatopancreas as well as in the connective tissue of the gill. The mantle showed stronger staining intensity of POD than the hepatopancreas. The Alcian blue (pH 2.5) periodic acid Schiff (AB-PAS) histochemical results revealed four types of mucous cells in the gill, mantle and hepatopancreas. Type III cells (nPAS+AB+) and type IV cells (aPAS+AB+) were observed in the gill, mantle and hepatopancreas. Type I cells (PAS+AB–) were not detected in the hepatopancreas but were distributed ubiquitously throughout the gill filament and mantle. Type II cells (PAS–AB+) were not detected in the gill but were distributed ubiquitously in the digestive ducts of the hepatopancreas.

Histochemical distribution of four types of enzymes and mucous cells in the intestine of koi carp (Cyprinus carpio var. koi).

The main purpose of this study was to investigate the distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non-specific esterase (NSE), peroxidase (POD), and mucous cells in the intestine of the koi carp Cyprinus carpio var. koi. ACP activity was located in the striated border, enterocytes, and lamina propria of the anterior and middle intestines. The ACP activity in the anterior intestine was higher than that in the middle and posterior intestines. ALP existed in the striated border of enterocytes and lamina propria, serosa, muscular layer, and the junction between muscular layer and submucosa layer of the intestine. The ALP activity in the anterior intestine was higher than that in the middle and posterior intestines. NSE activity was localized in the cytoplasm of enterocytes in the whole intestine, and the middle intestine showed the lower NSE activity than the anterior and posterior intestines. POD activity was localized in the blood cells of the lamina propria and cytoplasm of enterocytes in all intestinal segments. The POD activity among the anterior, middle, and posterior intestines was non-significantly different. Alcian blue periodic acid-Schiff histochemical results revealed three types of mucous cells in the intestine. The total number of mucous cells and percentage of type I cells among the anterior, middle, and posterior intestines were non-significantly different. The percentage of the type II cells was the highest in the posterior intestine, while the lowest in the anterior intestine. The percentage of the type III cells was the highest in the anterior intestine, while the lowest in the posterior intestine.

Dual application of entomopathogenic nematodes and fungi on immune and antioxidant enzymes of the greater wax moth, Galleria mellonella L.

Pathogenicity and immunity effects of both the entomopathogenic nematode, (Heterorhabditis zealandica) and the entomopathogenic fungus (Beauveria bassiana) on the last larval instar of the greater wax moth, Galleria mellonella L. (Lepidoptera: Pyralidae), were investigated. Both pathogens were either applied separately or in a combination. The fungus was inoculated first, followed by the nematode on time manner 0, 2, 4, and 6 days. LC50 values for both pathogens were calculated and then used for determination of the changes in immune response-mediated phenoloxidase (PO) and detoxifying enzymes glutathione S-transferase (GST) and non-specific esterase (EST) activities. The results indicated that a positive correlation was found between pathogen concentration and host mortality percentage. LC50 values were 6.49 IJs/larva for H. zealandica and 3.1 × 102 conidia/ml for B. bassiana. Synergistic interactions were found in all combined applications. The degree of synergism increased (reaching 100% mortality), when the nematode was applied 2 days post-fungal infection. PO activity increased significantly (p < 0.05) in a time-dependent manner post-B. bassiana infection. In contrary, the combination of H. zealandica + B. bassiana or H. zealandica alone produced a significant suppressive effect on PO activity over time. GST activity increased significantly (p < 0.05) in 36 h, then decreased at 48 h post-combined application, while the fungal infection enhanced significantly GST activity in time-dependent manner than the control and other treatments. EST activity increased significantly (p < 0.05) in both combined application and the single nematode infection than the single fungal infection, which increased during the initial period only. The increased mortality rates and suppression of phenoloxidase and glutathione S-transferase enzymes, following the combined application suggests a strong synergistic effect between both pathogens. It could be concluded that the tested combined pathogens are compatible element for integrated pest management.

Spheroid-3D and Monolayer-2D Intestinal Electrochemical Biosensor for Toxicity/Viability Testing: Applications in Drug Screening, Food Safety, and Environmental Pollutant Analysis

The rise of three-dimensional cell culture systems that provide in vivo-like environments for pharmaco-toxicological models has prompted the need for simple and robust viability assays suitable for complex cell architectural structures. This study addresses that challenge with the development of an in vitro enzyme based electrochemical sensor for viability/cytotoxicity assessment of two-dimensional (2D) monolayer and three-dimensional (3D) spheroid culture formats. The biosensor measures the cell viability/toxicity via electrochemical monitoring of the enzymatic activity of nonspecific esterases of viable cells, through the hydrolysis of 1-naphthyl acetate to 1-naphthol. The proposed sensor demonstrated strong correlation ( r = 0.979) with viable cell numbers. Furthermore, the model intestinal toxicants diclofenac (DFC, pharmaceutical), okadaic acid (OA, food-safety), and mancozeb (MZB, environmental) were used for the functional evaluation of the proposed sensor using 2D and 3D culture formats. Sensor performance showed high consistency with conventional cell viability/cytotoxicity assays (MTT/CFDA-AM) for all toxicants, with the sensor IC50 values matching the relevant viability LC50 values at the 95% confidence interval range for 2D (DCF: 1.19-1.26 mM, MZB: 10.28-14.18 μM, OA: 40.91-77.13 nM) and 3D culture formats (DCF: 1.02-4.78 mM, MZB: 11.26-15.16 μM, OA: 162.09-179.67 nM). The presented results demonstrate the feasibility of the proposed sensor as a robust endpoint screening tool for both 2D and 3D cytotoxicity assessment.

Isoniazid induces a monocytic-like phenotype in HL-60 cells

Isoniazid (INH) is one of the oldest drugs for the treatment of tuberculosis (TB) and is of continual clinical and research interest. The aim of the current study is to investigate the ability of INH to induce monocyte differentiation and the underlying signaling pathway involved in this phenomenon using HL-60 cells. In this study, HL-60 cells were treated with different non-cytotoxic concentrations of INH or vitamin D (a well-known inducer of monocytic differentiation) to determine key functional changes in the phenotype of these cells using several biochemical and cytobiological experiments. HL-60 cells are derived from human promyelocytic leukemia and bear some resemblance to promyelocytes, which differentiate into various cell types. INH-induced differentiation was confirmed to occur in a concentration-dependent manner through several functional markers such as nonspecific esterase activity, NADPH oxidase activity and expression of surface markers CD14 and CD16 (characteristic of monocytes). INH-induced monocytic-like differentiation in HL-60 cells and demonstrated that at least 25% of cells were differentiated within the range of the pharmacological concentrations of INH. To determine the effects of INH on HL-60 cells, we applied quantitative proteomics that revealed 32 proteins were altered significantly in pathways that could involve differentiation signals. Lastly, INH activated the ERK-1/MAPK signaling pathway based on detection of phosphorylated ERK-1. These in vitro findings in HL-60 cells warrant further study using promyelocytes or hematopoietic stem cells to evaluate the physiological capability of INH to induce monocytic differentiation that may aid in host defense against TB.

The Synergistic Antibacterial Properties of Glycinin Basic Peptide against Bacteria via Membrane Damage and Inactivation of Enzymes

This study investigated the antibacterial properties of glycinin basic peptide (GBP), a natural antibacterial component from soybean protein, against Staphylococcus aureus (S. aureus). The minimum inhibitory and bactericidal concentrations of GBP against S. aureus were 0.2 mg/mL and 0.8 mg/mL, respectively. Flow cytometry analysis manifested that GBP decreased the number of intact and normal cells. Higher concentrations of GBP induced more severe damage of the bacterial membrane; the maximal percentage of injured and dead cells was 93.8% with 0.8 mg/mL GBP. Electron microscopy imaging visually showed the morphological damage of S. aureus by GBP. Intracellular K+ leakage and the membrane depolarization of S. aureus further verified that GBP could destroy the bacterial membrane. Moreover, GBP decreased the activity of nonspecific esterase and ATPase of S. aureus in a concentration-dependent manner. These results demonstrated that GBP exhibited antibacterial properties against S. aureus via synergistic actions of damage to the cell membrane and inactivation of metabolic enzymes.

Chronic effects of copper and zinc on the fish, Etroplus suratensis (Bloch, 1790) by continuous flow through (CFT) bioassay

Copper (Cu) and zinc (Zn) play a vital role in the growth and development, however increased uptake causes deleterious effects in normal functioning of organisms. We have demonstrated in this contribution the tolerance limit of Cu and Zn on Etroplus suratensis (pearl spot) by Continuous Flow Through (CFT) bioassay and the biomarker responses. The accumulation for Cu, Zn and selected trace metals (Cr, Cd, Ni and Pb) from field conditions, as well Geo-accumulation index (I geo) and Contamination factor (C.F) suggested moderate to heavy pollution in the Cochin estuarine system. The 96 h LC50 values for Cu was 1.74 ± 0.04 mg L−1and that for Zn was 24.36 ± 0.58 mg L−1 at 95% confidence interval. No observed effect concentration (NOEC) and low observed effect concentration (LOEC) for Cu and Zn were derived based on the survival rates. Chronic toxicity values for Cu and Zn were 0.23 mg L−1 and 2.005 mg L−1 respectively for 30 days period. The histological, biochemical, hematological and behavioral parameters showed significant variations at sublethal concentrations. Lamellar hyperplasia in gills, vacuolation combined with necrosis in liver, increased occurrence of melanomacrophage centres in spleen were noticed at chronic levels for both Cu and Zn. Tissue specific bioconcentration was observed for zinc and copper in gill and liver respectively, with least rate of bioconcentration observed in muscle tissues. Malaonate Dehydrogenase (MDH), Super oxide dismutase (SOD), Nonspecific esterase (EST) activity significantly varied compared to control at NOEC and LOEC values in both the metals. The hematological and genotoxic alterations as decrease in erythrocyte count, lymphocytes, hemoglobin concentration and hematocrit percentage were significantly reduced (p < 0.05) and increased thrombocytes and neutrophils, increased frequency of micronuclei, lobed, blebbed and notched nuclei and binucleate cells were characteristic for the metals at the sublethal concentrations. The frequency of behavioral changes remained significantly higher at chronic level than the control group. Thus such CFT based studies are important for precisely mapping the toxicity changes in organisms and also to develop suitable water quality guidelines.

The Role of Acetylcholine Esterase in Resistance Mechanism of Plutella xylostella to Emamektin Benzoate

One of the resistance mechanism of P. xylostellato emamektin benzoate is target insensitivity which is acetylcholine esterase that responsible for resistance occurrence. The objective of this study was to determine the role of acetylcholinesterase in the resistance mechanism of P. xylostella population to emamektin benzoate. For enzyme activity analysis, larvae homogenate of the third instar of P. xylostella was prepared. The number of insects required for each scour is 1 for each field population. The protein content in P. xylostella homogenate was measured by the Folin-Ciocalteu test. Non-specific esterase activity with an absorption rate was read using ELISA reader tool with λ = 450 nm. The inhibition level of acetylcholinesterase activity by emamectin benzoate in the tested population was 36.84%. The highest inhibition occurs in Puasan (Ngablak) population. The result shows that a α-naphthyl acetate substrate was used so that it was recorded as non-specific esterase activity and did not exhibit esterase activity which specifically describes emamectin benzoate. Non-specific esterase enzyme activity of either α or β-naphthyl acetate substances to benzoic emamectin in the tested population most of the population was still susceptible. On α-naphthyl acetate substrate, the highest absorbance value found in susceptible population to benzoate emamectin (0.773), while the lowest found in Babrik (Ngablak) population (0.083).

The current insecticide resistance status of Anopheles gambiae (s.l.) (Culicidae) in rural and urban areas of Bouak\xe9, C\xf4te d\u2019Ivoire

BackgroundSeveral studies were carried out in experimental hut station in areas surrounding the city of Bouaké, after the crisis in Côte d’Ivoire. They reported increasing resistance levels to insecticide for malaria transmiting mosquitoes. The present work aims to evaluate the current resistance level of An. gambiae(s.l.) in rural and urban areas in the city of Bouaké.MethodsLarvae of Anopheles gambiae (s.l.) were collected from five different study sites and reared to adult stages. The resistance status was assessed using the WHO bioassay test kits for adult mosquitoes, with eight insecticides belonging to pyrethroids, organochlorines, carbamates and organophosphates classes. Molecular assays were performed to identify the molecular forms of An. gambiae (s.l.), the L1014F kdr and the ace-1R alleles in individual mosquitoes. The synergist PBO was used to investigate the role of enzymes in resistance. Biochemical assays were performed to detect potential increased activities in mixed function oxidase (MFO) levels, non-specific esterases (NSE) and glutathione S-transferases (GST).ResultsHigh resistance levels to pyrethroids, organochlorines, and carbamates were observed in Anopheles gambiae (s.l.) from Bouaké. Mortalities ranged between 0 and 73% for the eight tested insecticides. The pre-exposure to PBO restored full or partial susceptibility to pyrethroids in the different sites. The same trend was observed with the carbamates in five sites, but to a lesser extent. With DDT, pre-exposure to PBO did not increase the mortality rate of An. gambiae (s.l.) from the same sites. Tolerance to organophosphates was observed. An increased activity of NSE and higher level of MFO were found compared to the Kisumu susceptible reference strain. Two molecular forms, S form [(An. gambiae (s.s)] and M form (An. coluzzi) were identified. The kdr allele frequencies vary from 85.9 to 99.8% for An. gambiae (s.s.) and from 81.7 to 99.6% for An. coluzzii. The ace-1R frequencies vary between 25.6 and 38.8% for An. gambiae (s.s.) and from 28.6 to 36.7% for An. coluzzii.ConclusionResistance to insecticides is widespread within both An. gambiae (s.s.) and An. coluzzii. Two mechanisms of resistance, i.e. metabolic and target-site mutation seemed to largely explain the high resistance level of mosquitoes in Bouaké. Pyrethroid resistance was found exclusively due to the metabolic mechanism.

Insecticide susceptibility status of invasiveAedes albopictusacross dengue endemic districts of Odisha, India

Aedes albopictus is currently the most invasive mosquito species in the world. Keeping in view the wide emergence of insecticide resistance, it is imperative to focus on the current susceptibility status for various insecticides in Ae. albopictus. This study focused on understanding the insecticide resistance mechanism of Ae. albopictus collected from dengue-endemic districts of Odisha. Insecticide resistance was evaluated by using standardized bioassay kits (WHO) and biochemical analysis. Larval bioassays revealed the highest level of resistance from Jaipur (JP) population with a RR50 of 15.3 and LC50 of 1.177 ppm compared with an LC50 of 0.077 for the susceptible strain LabS. Results indicated the presence of dichlorodiphenyltrichloroethane resistance in the majority of adult populations. Elevated activity of nonspecific esterases and cytochrome P450s MFO indicated probable resistance to organophosphates and pyrethroids. Molecular screening for common insecticide target-site mutations confirmed the absence of the 'knockdown resistance' response for pyrethroid insecticide in Ae. albopictus population, suggesting its continual effectiveness as the major insecticide of significant importance in future vector-control programmes. This is the first report of a kdr mutation in Ae. albopictus in India and highlights the need for intensive research on other unexplored target-site mutations that might also contribute to pyrethroid resistance. Effective management and sustainable use of insecticides can be implemented by understanding resistance mechanisms and development of appropriate diagnostic tools. © 2017 Society of Chemical Industry.

Flow cytometic analysis of Penaeus monodon haemocyte responses to poly I:C

This study was aimed at investigating the cellular responses of Penaeus monodon haemocytes to poly I:C stimulation using flow cytometric assay. Total haemocyte count (THC), percentages of different haemocyte subpopulations [hyaline cells (HC), semigranular cells (SGC) and granular cells (GC)], non-specific esterase activity (EA), total reactive oxygen species/reactive nitrogen species (ROS/RNS) production, nitric oxide (NO) production, apoptotic haemocyte ratio and plasmic phenoloxidase (PO) activity were determined in poly I:C-injected shrimp. Results showed that poly I:C at a low dose (5 μg shrimp−1) caused obvious increases in THC, GC proportion, ROS/RNS production and NO production, but had no significant effect on EA, apoptosis and PO activity. In the early stage of poly I:C injection at a high dose (20 μg shrimp−1), THC and GC proportion improvements could also be observed, suggesting that GC might be induced to release from hemocytopoietic or other tissues to participate in immune response, and this subpopulation might be the main cell type involved in the cellular defence against virus. In the later period, proportions of both GC and SGC reduced paralleled by THC reduction, indicating that depletion of GC and SGC was mainly contributed to the reduced count of circulating haemocyte. Obvious increases in ROS/RNS production and NO production were induced in haemocyte of shrimp under a high dose of poly I:C stimulation, but only slight rise of EA and suppression of PO activity could be observed in poly I:C-stimulated shrimp, suggesting that ROS/RNS-dependent system was vital in the immune defence of shrimp against virus. On the other hand, increase of apoptotic haemocyte ratio and THC reduction were presented after the drastic increases of ROS/RNS and NO productions, implying that the stimulated ROS/RNS might be excess and harmful, and was the major factor for the haemocyte apoptosis and depletion. THC recovered after 48 h injection, while haemocyte apoptosis also returned to the control level, suggesting that apoptosis might be contributed to eliminate damaged, weak or infected haemocytes to renew the circulating haemocytes, and it could be considered as an important defending strategy against virus.

Histochemical distribution of four types of enzymes and mucous cells in the gastrointestinal tract of reared half-smooth tongue sole Cynoglossus semilaevis.

The histochemical distribution of acid phosphatase (ACP), alkaline phosphatase (ALP), non-specific esterase (NSE), peroxidase (POD) and mucous-cell types was evaluated in the gastrointestinal tract of the half-smooth tongue sole Cynoglossus semilaevis. The enzymes were detected in the entire stretch of the gastrointestinal tract. ACP activity was found in the supranuclear region of enterocytes and the lamina propria of the intestine, as well as the cytoplasm of epithelial cells of the stomach. The staining intensity of ACP in the anterior and posterior intestines was stronger than in the stomach. ALP activity was detected in the striated border of enterocytes and muscularis of the whole intestine, lamina propria and supranuclear cytoplasm of the enterocytes in the anterior intestine, as well as in the blood vessels of the stomach. The staining intensity for ALP in the anterior intestine was stronger than in the posterior segment and the latter was stronger than in the stomach. NSE activity was detected in the cytoplasm of the epithelial cells in the entire gastrointestinal tract, with the anterior intestine showing stronger intensity than the stomach. POD activity was located in the blood cells of the lamina propria of the gastrointestinal tract and the levels in the stomach were similar to the anterior and posterior intestines. Alcian blue (pH2·5) periodic acid Schiff (AB-PAS) histochemical results revealed three types of mucous cells in the gastrointestinal tract. Type I cells (PAS+AB-) were observed among the gastric mucosa columnar cells in the stomach and enterocytes in the basal region of the villi and in the middle and top regions of the intestinal villi. Type II cells (PAS-AB+) and type III cells (PAS+AB+) were not detected in the stomach but were distributed ubiquitously among enterocytes in the middle and top regions of the intestinal villi.

Osteoblast-derived Laminin-332 is a novel negative regulator of osteoclastogenesis in bone microenvironments

Laminin-332 (Lm-332), a major basement membrane protein, has been shown to provide a niche for some stem cells. Here, we found that Lm-332 was expressed in osteoblasts, and is implicated in the regulation of osteoclast differentiation. Immunofluorescence analysis of laminin-β3, a unique component of Lm-332, indicated specific expression of laminin-β3 in osteoblast-like cells localized on bone surface. RT-PCR analysis confirmed that α3, β3, and γ2 chains of Lm-332 were all expressed in primary osteoblasts prepared from mouse calvaria. Lm-332 markedly inhibited osteoclastogenesis induced by receptor activator of nuclear factor kappa B (NF-κB) ligand (RANKL) when bone marrow-derived macrophages (BMMs) were cultured on Lm-332-coated plates. Lm-332 also blocked RANKL-induced activation of mitogen-activated protein kinases (MAPKs) (ERK, JNK, and p38) and expression of NFATc1, c-Fos, and c-Jun. Lm-332 suppressed osteoclast differentiation while retaining macrophage phenotypes, including nonspecific esterase activity and gene expression of lysozyme and EGF-like module-containing mucin-like hormone receptor-like 1 (Emr1). Furthermore, the treatment of primary osteoblasts with osteoclastogenic factors dramatically suppressed expression of Lm-332. These findings suggest that Lm-332 produced by osteoblasts in bone tissues has a pivotal role in controlling normal bone remodeling through suppressing osteoclastogenesis.

Various cellular responses of different shrimp haemocyte subpopulations to lipopolysaccharide stimulation

Different haemocyte types have been reported to play diverse roles in immune defense of shrimp. To investigate the roles of the three haemocyte types [hyaline cells (HC), semigranular cells (SGC) and granular cells (GC)] of shrimp in immune responses against lipopolysaccharide (LPS), percentage, non-specific esterase activity (EA), reactive oxygen species (ROS) production and nitric oxide (NO) production of the three haemocyte subpopulations were analyzed in LPS-injected Penaeus monodon using flow cytometry. Results showed that percentage of HC increased after 3 h injection, and returned to the original level after 48 h. Proportion of SGC and GC reduced after 6–36 h and 3–12 h respectively, and recovered to the initial level after 48 and 24 h respectively. Loss of SGC and GC might be related to degranulation to release proPO system, and degranulation of GC seemed more sensitive to LPS stimulation. EA of both HC and SGC improved after 3–6 h injection, while EA of GC was induced after 3–24 h. No significant effect of LPS injection could be found in ROS production and NO production of HC. Enhanced ROS levels was observed in SGC and GC after 3–24 h and 3–36 h respectively, and NO production of SGC and GC improved after 3–48 h injection. These results demonstrated that SGC and GC possessed strong capabilities for LPS-induced EA, ROS production and NO production, while HC only displayed EA response to LPS, suggesting that GC and SGC play the main role in immune defense of shrimp against Gram-negative bacteria.