Cloning, Expression, Characterization and Immobilization of a Recombinant Carboxylesterase from the Halophilic Archaeon, Halobacterium salinarum NCR-1.

Only a few halophilic archaea producing carboxylesterases have been reported. The limited research on biocatalytic characteristics of archaeal esterases is primarily due to their very low production in native organisms. A gene encoding carboxylesterase from Halobacterium salinarum NRC-1 was cloned and successfully expressed in Haloferax volcanii. The recombinant carboxylesterase (rHsEst) was purified by affinity chromatography with a yield of 81%, and its molecular weight was estimated by SDS-PAGE (33 kDa). The best kinetic parameters of rHsEst were achieved using p-nitrophenyl valerate as substrate (KM = 78 µM, kcat = 0.67 s-1). rHsEst exhibited great stability to most metal ions tested and some solvents (diethyl ether, n-hexane, n-heptane). Purified rHsEst was effectively immobilized using Celite 545. Esterase activities of rHsEst were confirmed by substrate specificity studies. The presence of a serine residue in rHsEst active site was revealed through inhibition with PMSF. The pH for optimal activity of free rHsEst was 8, while for immobilized rHsEst, maximal activity was at a pH range between 8 to 10. Immobilization of rHsEst increased its thermostability, halophilicity and protection against inhibitors such as EDTA, BME and PMSF. Remarkably, immobilized rHsEst was stable and active in NaCl concentrations as high as 5M. These biochemical characteristics of immobilized rHsEst reveal its potential as a biocatalyst for industrial applications.

A Single Nucleotide Polymorphism Translates into a Radical Amino Acid Substitution at the Ligand-Binding Site in Fasciola hepatica Carboxylesterase B.

Fasciola hepatica anthelmintic resistance may be associated with the catalytic activity of xenobiotic metabolizing enzymes. The gene expression of one of these enzymes, identified as carboxylesterase B (CestB), was previously described as inducible in adult parasites under anthelmintic treatment and exhibited a single nucleotide polymorphism at position 643 that translates into a radical amino acid substitution at position 215 from Glutamic acid to Lysine. Alphafold 3D models of both allelic sequences exhibited a significant affinity pocket rearrangement and different ligand-docking modeling results. Further bioinformatics analysis confirmed that the radical amino acid substitution is located at the ligand affinity site of the enzyme, affecting its affinity to serine hydrolase inhibitors and preferences for ester ligands. A field genotyping survey from parasite samples obtained from two developmental stages isolated from different host species from Argentina and Mexico exhibited a 37% allele distribution for 215E and a 29% allele distribution for 215K as well as a 34% E/K heterozygous distribution. No linkage to host species or geographic origin was found in any of the allele variants.

Gut transcriptome of two bark beetle species stimulated with the same kairomones reveals molecular differences in detoxification pathways.

Dendroctonus bark beetles are the most destructive agents in coniferous forests. These beetles come into contact with the toxic compounds of their host's chemical defenses throughout their life cycle, some of which are also used by the insects as kairomones to select their host trees during the colonization process. However, little is known about the molecular mechanisms by which the insects counteract the toxicity of these compounds. Here, two sibling species of bark beetles, D. valens and D. rhizophagus, were stimulated with vapors of a blend of their main kairomones (α-pinene, ß-pinene and 3-carene), in order to compare the transcriptional response of their gut. A total of 48 180 unigenes were identified in D. valens and 43 704 in D. rhizophagus, in response to kairomones blend. The analysis of differential gene expression showed a transcriptional response in D. valens (739 unigenes, 0.58-10.36 Log2FC) related to digestive process and in D. rhizophagus (322 unigenes 0.87-13.08 Log2FC) related to xenobiotics metabolism. The expression profiles of detoxification genes mainly evidenced the up-regulation of COEs and GSTs in D. valens, and the up-regulation of P450s in D. rhizophagus. Results suggest that terpenes metabolism comes accompanied by an integral hormetic response, result of compensatory mechanisms, including the activation of other metabolic pathways, to ensure the supply of energy and the survival of organisms which is specific for each species, according to its life history and ecological strategy.

Transcriptome-Based Identification of a Functional Fasciola hepatica Carboxylesterase B.

Bioinformatics analysis of the complete transcriptome of Fasciola hepatica, identified a total of ten putative carboxylesterase transcripts, including a 3146 bp mRNA transcript coding a 2205 bp open reading frame that translates into a protein of 735 amino acids, resulting in a predicted protein mass of 83.5 kDa and a putative carboxylesterase B enzyme. The gene coding for this enzyme was found in two reported F. hepatica complete genomes stretching 23,230 bp, containing two exons of 1282 and 1864 bp, respectively, as well as a 20,084 bp intron between the exons. The enzymatic activity was experimentally assayed on F. hepatica protein extracts by SDS-PAGE zymograms using synthetic chromogenic substrates, confirming both the theoretical molecular weight and carboxylesterase enzymatic activity. Further bioinformatics predicted that this enzyme is an integral component of the cellular membrane that should be active as a 167 kDa homodimer complex and polyacrylamide gel electrophoresis (PAGE) zymograms experiments confirmed the analysis. Additional bioinformatics analysis showed that DNA sequences that code for this particular enzyme are highly conserved in other parasitic trematodes, although they are labeled hypothetical proteins.

Nuclear-lipid-droplet proteome: carboxylesterase as a nuclear lipase involved in lipid-droplet homeostasis.

Nuclear-lipid droplets (nLD)-a dynamic cellular organelle that stores neutral lipids, within the nucleus of eukaryotic cells-consists of a hydrophobic triacylglycerol -cholesterol-ester core enriched in oleic acid (OA) surrounded by a monolayer of polar lipids, cholesterol, and proteins. nLD are probably involved in nuclear-lipid homeostasis serving as an endonuclear buffer that provides or incorporates lipids and proteins participating in signaling pathways, as transcription factors and enzymes of lipid metabolism and nuclear processes. In the present work, we analyzed the nLD proteome and hypothesized that nLD-monolayer proteins could be involved in processes similar as the ones occurring in the cLD including lipid metabolism and other cellular functions. We evaluated the rat-liver-nLD proteome under physiological and nonpathological conditions by GeLC-MS2. Since isolated nLD are highly diluted, a protein-concentrating isolation protocol was designed. Thirty-five proteins were identified within the functional categories: cytoskeleton and structural, transcription and translation, histones, protein-folding and posttranslational modification, cellular proliferation and/or cancer, lipid metabolism, and transport. Purified nLD contained an enzyme from the lipid-metabolism pathway, carboxylesterase 1d (Ces1d/Ces3). Nuclear Carboxylesterase localization was confirmed by Western blotting. By in-silico analyses rat Ces1d/Ces3 secondary and tertiary structure predicted would be equivalent to human CES1. These results-the first nLD proteome-demonstrate that a tandem-GeLC-MS2-analysis protocol facilitates studies like these on rat-liver nuclei. A diversity of cellular-protein function was identified indicating the direct or indirect nLD participation and involving Ces1d/Ces3 in the LD-population homeostasis.

Biological responses of mangrove oysters (Crassostrea rhizophorae) and mercury contamination in an urban tropical estuary.

This study aimed to assess the biological responses of oysters from an urban estuary in Northeast Brazil, through the evaluation of biochemical and physiological biomarkers, and integrate these responses with the investigation of mercury seasonal contamination. Oysters and sediment were collected from three sites in the estuary of the Ceará River during dry and rainy seasons. Biomarkers (AchE, CaE, GST, CAT, and Condition Index) were analyzed in different tissues. Hg bioaccumulation was higher in animals sampled in the rainy season, with increases varying from 5% to 136%, compared to the dry season. The changes in biomarkers highlight already elevated stresses for the organisms at the inner portion of the estuary, near the confluence with the Maranguapinho River, mainly during the rainy season, corroborating other studies that showed ecotoxicological effects with water and sediment samples. Finally, no correlation between Hg in sediment/oyster and biomarker results was observed.

Acute toxicity and esterase response to carbaryl exposure in two different populations of amphipods Hyalella curvispina.

During the last years, a carbaryl insecticide was extensively applied in the valley of Río Negro and Neuquén, North Patagonia Argentina, to manage codling moths (Cydia pomonella), the main pest of pear and apple trees. In this study carbaryl susceptibility and B-esterase activity from both insecticide-exposed and non-exposed field populations of amphipods Hyalella curvispina were studied. Two subpopulations, one susceptible to carbaryl (LC50=213±7.5µg/L carbaryl) and one resistant to it (LC50=14,663±2379µg/L carbaryl), were found in the agricultural area selected in this study. Both populations were, in turn, more resistant to carbaryl than the population from a pristine area (LC50=11.31±2.27µg/L carbaryl). The in vivo 48h-IC50 values for cholinesterase (ChE) were close to the corresponding 48h-LC50 values as determined for the non-exposed population (IC50=7.16±0.86µg/L carbaryl) and for the susceptible subpopulation from the insecticide-exposed site (IC50=193±99µg/L carbaryl). Carbaryl exposure of the amphipods from the agricultural area mentioned above produced a significant decrease of carboxylesterase (CabE) activity, at a sublethal concentration (10µg/L) that was not able to significantly inhibit ChE, thereby showing a protective role of CabE and its usefulness as early biomarker. However, at lethal concentrations the inhibition of ChE activity was higher than that of CabE. On the other hand, CabE of amphipods from the pristine site was less sensitive to carbaryl than ChE, suggesting a different participation of CabE in ChE protection in the susceptible population of H. curvispina. Pulse exposure to carbaryl for 2h caused a significant inhibition of ChE in amphipods from both populations, with a fast recovery as expected for a carbamate insecticide. In conclusion, we proved that amphipods from the said agricultural area have developed resistance to carbaryl and showed the presence of two subpopulations with a different response to the insecticide. Moreover, these results reinforce the use of ChE together with CabE inhibition as indicators of carbamate exposure in H. curvispina.

Biochemical response of the Africanized honeybee exposed to fipronil.

Bees are recognized worldwide for their social, economic, and environmental value. In recent decades they have been seriously threatened by diseases and high levels of pesticide use. The susceptibility of bees to insecticides makes them an important terrestrial model for assessing environmental quality, and various biomarkers have been developed for such assessments. The present study aimed to evaluate the activity of the enzymes acetylcholinesterase (AChE), carboxylesterase (CaE), and glutathione-S-transferase (GST) in Africanized honeybees exposed to fipronil. The results showed that fipronil at a sublethal dose (0.01 ng/bee) modulates the activity of CaE in all isoforms analyzed (CaE-1, CaE-2, and CaE-3) in both newly emerged and aged bees, and does not affect the activity of AChE or GST. The recovery of the bees after fipronil exposure was also investigated, and these results demonstrated that even the cessation of fipronil ingestion might not lead to complete recovery of individual bees. Even at low doses, fipronil was shown to cause changes in the activity of key enzymes in bees. The possible consequences of these changes are discussed. Environ Toxicol Chem 2017;36:1652-1660. © 2016 SETAC.

Effect of diet on carboxylesterase activity of tadpoles (Rhinella arenarum) exposed to chlorpyrifos.

An outdoor microcosm was performed with tadpoles (Rhinella arenarum) exposed to 125µgL-1 chlorpyrifos and fed two types of food, i.e., lettuce (Lactuca sativa) and a formulated commercial pellet. Acetylcholinesterase (AChE) and carboxylesterase (CbE) activities were measured in liver and intestine after 10 days of pesticide exposure. Non-exposed tadpoles fed lettuce had an intestinal AChE activity almost two-fold higher than that of pellet-fed tadpoles. No significant differences were observed, however, in liver AChE activity between diets. Likewise, intestinal CbE activity - measured using two substrates, i.e. 1-naphthyl acetate (1-NA) and 4-nitrophenyl valerate (4-NPV) - was higher in tadpoles fed lettuce than in those fed pellets. However, the diet-dependent response of liver CbE activity was opposite to that in the intestine. Chlorpyrifos caused a significant inhibition of both esterase activities, which was tissue- and diet-specific. The highest inhibition degree was found in the intestinal AChE and CbE activities of lettuce-fed tadpoles (42-78% of controls) compared with pellet-fed tadpoles (<60%). Although chlorpyrifos significantly inhibited liver CbE activity of the group fed lettuce, this effect was not observed in the group fed pellets. In general, intestinal CbE activity was more sensitive to chlorpyrifos inhibition than AChE activity. This finding, together with the high levels of basal CbE activity found in the intestine, may be understood as a detoxification system able to reduce intestinal OP uptake. Moreover, the results of this study suggest that diet is a determinant factor in toxicity testing with tadpoles to assess OP toxicity, because it modulates levels of this potential detoxifying enzyme activity.

Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus), from India and its possible role in indoxacarb degradation

Abstract Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n = 13) and adults (n = 12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32 µmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus – KC985225 and Pantoea agglomerans – KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26 µmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM.

Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus), from India and its possible role in indoxacarb degradation

Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n = 13) and adults (n = 12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32 µmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus KC985225 and Pantoea agglomerans KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26 µmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM.(AU)

Detection of carboxylesterase and esterase activity in culturable gut bacterial flora isolated from diamondback moth, Plutella xylostella (Linnaeus), from India and its possible role in indoxacarb degradation.

Diamondback moth (DBM), Plutella xylostella (Linnaeus), is a notorious pest of brassica crops worldwide and is resistant to all groups of insecticides. The insect system harbors diverse groups of microbiota, which in turn helps in enzymatic degradation of xenobiotic-like insecticides. The present study aimed to determine the diversity of gut microflora in DBM, quantify esterase activity and elucidate their possible role in degradation of indoxacarb. We screened 11 geographic populations of DBM in India and analyzed them for bacterial diversity. The culturable gut bacterial flora underwent molecular characterization with 16S rRNA. We obtained 25 bacterial isolates from larvae (n=13) and adults (n=12) of DBM. In larval gut isolates, gammaproteobacteria was the most abundant (76%), followed by bacilli (15.4%). Molecular characterization placed adult gut bacterial strains into three major classes based on abundance: gammaproteobacteria (66%), bacilli (16.7%) and flavobacteria (16.7%). Esterase activity from 19 gut bacterial isolates ranged from 0.072 to 2.32µmol/min/mg protein. Esterase bands were observed in 15 bacterial strains and the banding pattern differed in Bacillus cereus - KC985225 and Pantoea agglomerans - KC985229. The bands were characterized as carboxylesterase with profenofos used as an inhibitor. Minimal media study showed that B. cereus degraded indoxacarb up to 20%, so it could use indoxacarb for metabolism and growth. Furthermore, esterase activity was greater with minimal media than control media: 1.87 versus 0.26µmol/min/mg protein. Apart from the insect esterases, bacterial carboxylesterase may aid in the degradation of insecticides in DBM.

B-esterase determination and organophosphate insecticide inhibitory effects in JEG-3 trophoblasts.

The placenta and trophoblasts express several B-esterases. This family includes acethylcholinesterase (AChE), carboxylesterase (CES) and butyrylcholinesterase (BChE), which are important targets of organophosphate insecticide (OP) toxicity. To better understand OP effects on trophoblasts, B-esterase basal activity and kinetic behavior were studied in JEG-3 choriocarcinoma cell cultures. Effects of the OP azinphos-methyl (Am) and chlorpyrifos (Cp) on cellular enzyme activity were also evaluated. JEG-3 cells showed measurable activity levels of AChE and CES, while BChE was undetected. Recorded Km for AChE and CES were 0.33 and 0.26 mM respectively. Native gel electrophoresis and RT-PCR analysis demonstrated CES1 and CES2 isoform expression. Cells exposed for 4 and 24 h to the OP Am or Cp, showed a differential CES and AChE inhibition profiles. Am inhibited CES and AChE at 4 h treatment while Cp showed the highest inhibition profile at 24 h. Interestingly, both insecticides differentially affected CES1 and CES2 activities. Results demonstrated that JEG-3 trophoblasts express AChE, CES1 and CES2. B-esterase enzymes were inhibited by in vitro OP exposure, indicating that JEG-3 cells metabolization capabilities include phase I enzymes, able to bioactivate OP. In addition, since CES enzymes are important for medicinal drug activation/deactivation, OP exposure may interfere with trophoblast CES metabolization, probably being relevant in a co-exposure scenario during pregnancy.

Isolation of an aryloxyphenoxy propanoate (AOPP) herbicide-degrading strain Rhodococcus ruber JPL-2 and the cloning of a novel carboxylesterase gene (feh).

The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L(-1) FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.

Isolation of an aryloxyphenoxy propanoate (AOPP) herbicide-degrading strain Rhodococcus ruber JPL-2 and the cloning of a novel carboxylesterase gene (feh)

The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L−1 FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.(AU)

Isolation of an aryloxyphenoxy propanoate (AOPP) herbicide-degrading strain Rhodococcus ruber JPL-2 and the cloning of a novel carboxylesterase gene (feh)

The strain JPL-2, capable of degrading fenoxaprop-P-ethyl (FE), was isolated from the soil of a wheat field and identified as Rhodococcus ruber. This strain could utilize FE as its sole carbon source and degrade 94.6% of 100 mg L−1 FE in 54 h. Strain JPL-2 could also degrade other aryloxyphenoxy propanoate (AOPP) herbicides. The initial step of the degradation pathway is to hydrolyze the carboxylic acid ester bond. A novel esterase gene feh, encoding the FE-hydrolyzing carboxylesterase (FeH) responsible for this initial step, was cloned from strain JPL-2. Its molecular mass was approximately 39 kDa, and the catalytic efficiency of FeH followed the order of FE > quizalofop-P-ethyl > clodinafop-propargyl > cyhalofop-butyl > fluazifop-P-butyl > haloxyfop-P-methyl > diclofop-methy, which indicated that the chain length of the alcohol moiety strongly affected the hydrolysis activity of the FeH toward AOPP herbicides.

Comparative intestinal esterases amongst passerine species: Assessing vulnerability to toxic chemicals in a phylogenetically explicit context.

Inhibition of blood esterase activities by organophosphate (OP) pesticides has been used as a sensitive biomarker in birds. Furthermore, compared to mammalian vertebrates, less is known about the role of these enzyme activities in the digestive tracts of non-mammalian vertebrates, as well as the environmental and biological stressors that contribute to their natural variation. To fill this gap, we examined butyrylcholinesterase (BChE) and carboxylesterases (CbE) in the digestive tracts of sixteen passerine species from central Chile. Whole intestine enzyme activities were positively and significantly correlated with body mass. After correcting for body mass and phylogenetic effect, we found only a marginal effect of dietary category on BChE activity, but a positive and significant association between the percentage of dietary nitrogen and the mass-corrected lipase activity. Our results suggest that observed differences may be due to the dietary composition in the case of lipases and BChE, and also we predict that all model species belonging to the same order will probably respond differently to pesticide exposure, in light of differences in the activity levels of esterase activities.

Identificación de un polimorfismo del gen Est9 relacionado con resistencia a piretroides en Rhipicephalus (Boophilus) microplus / Identification of a gene polymorphism Est9 associated with resistance to pyrethroids in Rhipicephalus (Boophilus) microplus

Objetivo. Mediante procedimientos de PCR-RFLP, detectar un polimorfismo en el gen Est9 de garrapatas Rhipicephalus (Boophilus) microplus resistentes a piretroides en Ibagué, Colombia, determinando el grado de asociación entre los fenotipos y los genotipos resultantes. Materiales y métodos. El ADN de 30 teleoginas R. (Boophilus) microplus fenotípicamente susceptibles, resistentes o medianamente resistentes a piretroides en una prueba de Drummond modificada, fue amplificado por PCR con cebadores específicos para obtener un fragmento de 372 pb del gen Est9, que fue sometido a digestión con la enzima EcoRI para estudiar los RFLPs generados y poder diferenciar los respectivos genotipos. El grado de asociación entre los fenotipos y los genotipos resultantes se determinó mediante la prueba exacta de Fisher. Resultados. Luego de digerir el fragmento con la endonucleasa, se generaron dos segmentos en teleoginas con algún nivel de resistencia, mientras en las teleoginas susceptibles no hubo división del fragmento de 372 pb, demostrándose así la presencia de una mutación puntual y los genotipos homocigoto natural, homocigoto mutante y heterocigoto. Las diferencias altamente significativas (p<0.01) entre teleoginas susceptibles y aquellas con algún nivel de resistencia, mostraron una relación directa entre el genotipo y el fenotipo con un nivel de confianza de p=0.0009852. Conclusiones. Se comprobó, por primera vez en Colombia, la presencia de una mutación puntual en el gen Est9 de garrapatas R. (Boophilus) microplus resistentes a piretroides, sugiriendo la necesidad de realizar estudios para detectar alteraciones moleculares en otros genes relacionados con quimioresistencia.

Objective. Using PCR-RFLP procedures, to detect a polymorphism of the Est9 gene in Rhipicephalus (Boophilus) microplus ticks resistant to pyrethroids in Ibague, Colombia, determining the degree of association between phenotypes and resulting genotypes. Materials and methods. The DNA of 30 engorged R. (Boohilus) microplus phenotypically susceptible females, resistant or moderately resistant to pyrethroids in a modified Drummond test was amplified by PCR with specific primers. We obtained a 372 bp fragment of the Est9 gene which was digested with the EcoRI enzyme to study RFLPs generated in order to differentiate the respective genotypes. The degree of association between phenotypes and resulting genotypes was determined by Fisher’s exact test. Results. After digesting the fragment with the endonuclease, two segments were generated in engorged females with some level of resistance, while, in those susceptible, there was no fragment division. The presence of a mutation of 372 pb was demonstrated, as well as the natural homozygous genotypes, mutant homozygous and heterozygous. The highly significant differences (p<0.01) between engorged susceptible females and those with some level of resistance, revealed a direct relationship between genotype and phenotype with a confidence level of p = 0.0009852. Conclusions. For the first time in Colombia, the presence of a mutation in the Est9 gene of R. (B.) microplus tick resistant to pyrethroids was found, suggesting the need for studies to detect molecular alterations in other genes associated with chemical resistance.

Cholinesterases and neurotoxicity as highly sensitive biomarkers for an organophosphate insecticide in a freshwater gastropod (Chilina gibbosa) with low sensitivity carboxylesterases.

In the Upper Valley of Río Negro and Río Neuquén in Argentina, agriculture represents the second most important economic activity. Azinphos-methyl has been found in water from this region throughout the year at a maximum concentration of 22.48 µg L(-1) during the application period. Toxicological studies on local non-target species have been performed mostly on vertebrates, while mollusks, which could be more sensitive, have not been studied so far. This work aims to characterize cholinesterase (ChE) and carboxilesterase (CE) activities of Chilina gibbosa, a freshwater gastropod native to southern Argentina and Chile. These enzymes, together with neurotoxicity signals, are evaluated herein after as sensitive biomarkers of exposure to azinphos-methyl at environmentally relevant concentrations. Effects of azinphos-methyl on antioxidant defenses: glutathione (GSH), catalase (CAT), superoxide dismutase (SOD) and glutathione S-transferase (GST) are also studied in order to complete a set of biomarkers with different sensitivity and specificity, to propose C. gibbosa as a sentinel species. The highest specific activity was obtained with acetylthiocholine as substrate, followed by propionylthiocholine (83% in comparison to acetylthiocholine) and butyrylthiocholine (19%).The lowest Km and the highest efficiency for ChE were obtained with acetylthiocholine. Regarding CEs activities, a higher efficiency was obtained with p-nitrophenyl butyrate than with p-nitrophenyl acetate. Eserine produced significant inhibition of ChE activity (81% with 0.001 mM and 98% with 1mM) while iso-OMPA did not produce any significant effect on ChE. Our results show that C. gibbosa ChE is very sensitive to azinphos-methyl (CI50 0.02 µg L(-1)) while CEs are inhibited at higher concentrations (CI50 1,000 µg L(-1)). CEs have been reported to be more sensitive to OPs than ChEs in most of the aquatic invertebrates protecting the organisms from neurotoxic effects. In contrast, C. gibbosa, has ChE which are much more sensitive to azinphos-methyl than CEs and shows marked signs of neurotoxicity. Regarding antioxidant defenses, GSH levels were significantly increased by 0.02 and 20 µg L(-1) azinphos-methyl (80 and 103%, respectively), CAT activity was increased 85% only at 0.02 µg L(-1) and SOD and GST did not show any significant response. Since ChE activity, neurotoxicity signs, GSH and CAT are sensitive biomarkers of acute exposure to azinphos-methyl at environmental concentrations C. gibbosa could be included as sentinel species in monitoring programs of pesticide hazard in regions of Argentina and Chile.

In vitro and in vivo inhibition of acetylcholinesterase and carboxylesterase by metals in zebrafish (Danio rerio).

Metals are natural components in ecosystems; however, if these elements are in excess, they can have adverse effects on living organisms. This study analyzes the interference of copper, lead, iron and cadmium in acetylcholinesterase (AChE) and carboxylesterase (CbE) activities in zebrafish. AChE was significantly inhibited in vitro by copper, iron, lead and cadmium at higher concentrations (10 and 20 mmol/L), whereas CbE was inhibited only at a concentration of 20 mmol/L. In vivo, only lead and cadmium were able to cause AChE inhibition at higher concentrations, while iron didn't cause any changes, and copper promoted an increase in AChE activity at a concentration of 0.06 mg/L. CbE activity did not change at any of the times (two and seven days) and concentrations tested, except in the case of copper exposure, which resulted in a decrease in CbE activity. Indeed, iodoacetamide treatment didn't changed AChE neither CbE activities, results which indicate that the metal inhibiting effect is probably not due to its biding to thiol groups close the active site of the enzyme. This outcome reveals that metals are important esterase inhibitors in zebrafish, and should be considered in environmental monitoring studies that use esterase inhibition as exposure biomarkers of organophosphate and carbamate pesticides.