Esterase activity in whiteflies (Bemisia tabaci) in Israel in relation to insecticide resistance

A cotton-field population of the whitefly Bemisia tabaci (Genn.) was 3–5 times more resistant to an organophosphorous insecticide than two control populations. Esterase activity levels in individuals from the resistant population were considerable lower than in control individuals. The distribution of activity values of large numbers of adults from the cotton field was strongly skewed to the right, while the control populations had symmetrical distributions. This may reflect selection against individuals with high EST activity. Control populations are almost fixed for a fast esterase allozyme, which preferentially cleaves -Naphthyl Acetate. In field populations the alternative slow allozyme, which prefers the -form of the substrate, is dominant. Low EST activity may be used as a marker for resistant individuals in Israeli B. tabaci.

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Résumé La population de l'aleurode, B. tabaci Genn., d'un champ de coton s'est montrée 3 à 5 fois plus résistante aux organophosphorés que 2 populations témoins. Le niveau d'activité des estérases était considérablement plus faible chez les individus de la population résistante que chez les individus des populations témoins. La distribution des activités estérasiques de nombreux adultes du champ de conton étaient biaisée vers la droite, alors qu'elle était symétrique pour les populations témoins, ce qui révèle une sélection à l'encontre des individus ayant une forte activité estérasique. Cette hypothèse est étayée par l'affinité pour le substrat et des preuves électrophorétiques. Une faible activité estérasique peut être utilisée comme marqueur des individus résistants de B. tabaci en Israël.

     

Further Study on the Esterase Patterns of Sibling Species in the Drosophila saltans Subgroup (saltans Group): Intraspecific and Interspecific Variations in the Development

Twenty of the 32 esterase bands previously detected in the adults of D. prosaltans, D. saltans and D.␣austrosaltans were found in larvae and pupae studied in this work. The results showed that, in addition to expressing the highest number of esterase bands, the adult stage of the three species exhibited the highest degree of expression (amount of synthesis) for most of the bands. Differences between larval and pupal stages were detected in the degree of expression (amount of synthesis) of the bands and in the frequency of samples expressing them. The frequencies of expression of the bands corresponding to genes in loci 1–3 were greater in pupae than in larvae while the frequencies of expression of the bands corresponding to genes in loci 4–9 were predominantly expressed in larvae or were equal in both developmental stages. Like the adults, larvae, pupae and empty pupal cases (which were also studied in this work) showed specific esterases. Taken together, the observations showed that, in the species studied, every developmental stage is characterized by specific bands and by specific frequency and degree of expression of the bands shared with other stages.     

发酵性丝孢酵母酯酶的稳定性研究

以发酵性丝孢酵母胞壁酯酶为研究对象,研究了其在不同理化条件下的稳定性。结果表明：0 ℃保存24 h,酯酶仍保持81.5%原酶活;在40 ℃完全失活;5 mmol/L K⁺、Mn²⁺和Fe²⁺提高酶活力6.9%以下,5 mmol/L Ca²⁺、Cu²⁺分别降低酶活力90.7%和73.3%;1%(质量分数,下同) Tween-40和TritonX-100保持77%以上原酶活,1% Tween-80提高酶活力23.3%,1%十二烷基磺酸钠（Sodium dodecyl sulfate,SDS）和十六烷基三甲基溴化铵 （Cetyl trimethyl ammonium bromide,CTAB）完全抑制酶活;5%甘油、阿拉伯胶和糊精分别提高酶活力57%、29.6% 和2%,5%可溶性淀粉和牛血清蛋白（Albumin from bovine serum,BSA）分别降低酶活力20.4%和71.5%。发酵性丝孢酵母酯酶热稳定性差,宜10 ℃以下贮存,甘油对其保护作用良好,BSA强烈抑制该酶活性。该酯酶对处理含油脂废水和增强洗涤剂洗涤效果有潜在的应用价值。     

Esterase Patterns and Phylogenetic Relationships of Drosophila Species in the Saltans Subgroup (Saltans Group)

The esterase patterns of sixteen strains from four species in the saltans subgroup were analyzed using polyacrylamide gel electrophoresis. Thirty-four esterase bands were detected. By using and naphthyl acetates as substrates, they were classified in 18 -esterases (they hydrolyse the -naphtyl substrate), 15 -esterases (they hydrolyse the -naphtyl substrate) and 1 /-esterase (it hydrolyses the and -naphtyl substrates). Among the -esterases, three were detected exclusively in males. Malathion, Eserine and pCMB were used as inhibitors in order to characterize biochemically the esterases. The results indicated the presence of cholinesterases, carboxylesterases and acetylesterases. The degree of mobility of the bands in the gels, their specificity to and naphthyl acetates and the results of the inhibition tests allowed us to recognize tentatively nine genetic loci. Phylogenetic relationships among species inferred on the basis of the esterase patterns by PAUP 4.0b8, with neighbor-joining search and a bootstrap analysis showed that, although the four species are closely related, D. septentriosaltans, D. saltans and D. austrosaltans are closer to each other than to D. prosaltans. These results showed to be consistent with phylogenetic relationships previously inferred from inversion polymorphism.     

On-line monitoring of lipolytic activity by sequential injection analysis

An automated sequential injection analysis using stop-flow technique for the on-line determination of lipolytic activity has been developed. It is based on a colorimetric method using a chromogenic substrate, 1,2-O-dilauryl-rac-glycero-3-glutaric acid-(6-methylresorufin)-ester. The system permits a linear range analysis between 5–100 lipolytic activity units ml^–1, without external dilution of the sample, a sampling frequency of 5 samples per hour and a relative standard deviation (RSD) of 5%. The analyser has been used for the on-line monitoring of Candida rugosa fed-batch fermentation with excellent performance, regarding its reliability and reproducibility.     

Biodegradation of p-nitrophenol by methyl parathion-degrading Ochrobactrum sp. B2

Ochrobactrum sp. B2, a methyl parathion-degrading bacterium, was proved to be capable of using p-nitrophenol (PNP) as carbon and energy source. The effect of factors, such as temperature, pH value, and nutrition, on the growth of Ochrobactrum sp. B2 and its ability to degrade p-nitrophenol (PNP) at a higher concentration (100 mg l⁻¹) was investigated in this study.The greatest growth of B2 was observed at a temperature of 30 °C and alkaline pH (pH 9–10). pH condition was proved to be a crucial factor affecting PNP degradation. Enhanced growth of B2 or PNP degradation was consistent with the increase of pH in the minimal medium, and acidic pH (6.0) did not support PNP degradation. Addition of glucose (0.05%, 0.1%) decreased the rate of PNP degradation even if increased cell growth occurred. Addition of supplemental inorganic nitrogen (ammonium chloride or ammonium sulphate) inhibited PNP degradation, whereas organic nitrogen (peptone, yeast extract, urea) accelerated degradation.     

On-line monitoring of enzymes in downstream processing by flow injection analysis (FIA)

Flow injection analysis (FIA) has been employed to automate enzyme assays for formate dehydrogenase (FDH) and l-leucine dehydrogenase (l-LeuDH). Coupled to a special sampling device the FIA assays were used to monitor on-line downstream processes, e.g. disintegration of microbial cells and cross-flow filtration of cell homogenates.     

Rapid on-line protein detection in biotechnological processes by flow injection analysis (FIA)

Two rapid methods for on-line protein determination useful for control purposes in the automation of biotechnological processes such as fermentation and downstream processing are described. Both methods are derived from colorimetric laboratory biuret and Bradford protein assays adapted to a flow injection analyser.     

Esterase electrophoretic analysis to distinguish isolates between Bipolaris spp. and Drechslera tritici-repentis from wheat

Diseases caused in wheat by Bipolaris sorokiniana and Drechslera tritici-repentis have led to considerable yield and production losses. In wheat seeds another isolate has recently been identified, resembling Bipolaris bicolor. The objective of the present trial was to differentiate and identify isolates of these fungi based on electrophoretic analyses and morphology. Esterase electrophoresis enabled the differentiation between Drechslera sp. and Bipolaris sp. isolates. In relation to morphology, conidia from D. tritici-repentis isolates were significantly longer than the isolates of B. sorokiniana. Bipolaris bicolor isolates, on the other hand, presented wider conidia than those of D. tritici-repentis and B. sorokiniana.     

Rapid and simple assay for feruloyl and p-coumaroyl esterases

A rapid, simple and sensitive method for detection of ferulic and p-coumaric acids using HPLC has been developed which can be used to determine the respective phenolic acid esterase activities of microorganisms. Prior concentration, purification or derivatization of the samples are not required. As little as 0.5 mg ferulic or p-coumaric acid/I could be detected and estimated in < 1 h. The method is specific for the two phenolic acids sice no interference by other components was observed.The authors are with the Department of Microbiology and Biochemistry, University of the Orange Free State (UOFS), PO Box 339, Bloemfontein 93000, South Africa     

Anthelmintic efficacy of flemingia vestita (fabaceae): Genistein-induced alterations in the esterase activity in the cestode,raillietina echinobothrida

To ascertain the anthelmintic efficacy ofFlemingia vestita (an indigenous leguminous plant of Meghalaya, having putative anthelmintic usage), its crude root-tuber peel extract and active chemical principle, genistein, were testedin vitro with reference to esterase activity in the fowl tapeworm,Raillietina echinobothrida. With the localization of non-specific esterases (NSE) and cholinesterase (ChE), the organization of the cholinergic components of the nervous system in toto could be visualized in the cestodeo The specific ChE in the parasite is acetylcholinesterase (AChE). Both NSE and ChE were found in close association with the central and peripheral nervous components, besides being present in the tegument and muscular parts of the terminal male genitalia. The whole tissue homogenate of the parasite also showed a high AChE activity. After exposure to the crude peel extract (50 mg/ml of the incubation medium) and to genistein (0.5 mg/ml), a pronounced decline in the visible stain intensity in the cholinergic components of the nervous system and in the tegument was noticeable, indicating extremely reduced activity of NSE and ChE in these sites. The total AChE activity was also reduced to 4907% and 56–77%, following treatment with the peel extract and genistein, respectively. The reference drug, praziquantel (0.01 mg/ml) also caused reduction in the enzyme activity, somewhat at par with the genistein treatment. Genistein appears to have a transtegumental mode of action. Alteration in the AChE activity points towards acetylcholine, an inhibitory neurotransmitter in cestodes, as the potential target of action.     

High-performance liquid chromatography of the renal blood flow marker p-aminohippuric acid (PAH) and its metabolite N-acetyl PAH improves PAH clearance measurements

PAH (N-(4-aminobenzoyl)glycin) clearance measurements have been used for 50 years in clinical research for the determination of renal plasma flow. The quantitation of PAH in plasma or urine is generally performed by colorimetric method after diazotation reaction but the measurements must be corrected for the unspecific residual response observed in blank plasma. We have developed a HPLC method to specifically determine PAH and its metabolite NAc-PAH using a gradient elution ion-pair reversed-phase chromatography with UV detection at 273 and 265 nm, respectively. The separations were performed at room temperature on a ChromCart^® (125 mm×4 mm I.D.) Nucleosil 100-5 μm C₁₈ AB cartridge column, using a gradient elution of MeOH–buffer pH 3.9 1:99→15:85 over 15 min. The pH 3.9 buffered aqueous solution consisted in a mixture of 375 ml sodium citrate–citric acid solution (21.01 g citric acid and 8.0 g NaOH per liter), added up with 2.7 ml H₃PO₄ 85%, 1.0 g of sodium heptanesulfonate and completed ad 1000 ml with ultrapure water. The N-acetyltransferase activity does not seem to notably affect PAH clearances, although NAc-PAH represents 10.2±2.7% of PAH excreted unchanged in 12 healthy subjects. The performance of the HPLC and the colorimetric method have been compared using urine and plasma samples collected from healthy volunteers. Good correlations (r=0.94 and 0.97, for plasma and urine, respectively) are found between the results obtained with both techniques. However, the colorimetric method gives higher concentrations of PAH in urine and lower concentrations in plasma than those determined by HPLC. Hence, both renal (Cl_R) and systemic (Cl_S) clearances are systematically higher (35.1 and 17.8%, respectively) with the colorimetric method. The fraction of PAH excreted by the kidney Cl_R/Cl_S calculated from HPLC data (n=143) is, as expected, always <1 (mean=0.73±0.11), whereas the colorimetric method gives a mean extraction ratio of 0.87±0.13 implying some unphysiological values (>1). In conclusion, HPLC not only enables the simultaneous quantitation of PAH and NAc-PAH, but may also provide more accurate and precise PAH clearance measurements.     

Effects of organic compounds on the degradation ofp-nitrophenol in lake and industrial wastewater by inoculated bacteria

Many microorganisms fail to degrade pollutants when introduced in different natural environments. This is a problem in selecting inocula for bioremediation of polluted sites. Thus, a study was conducted to determine the success of four inoculants to degradep-nitrophenol (PNP) in lake and industrial wastewater and the effects of organic compounds on the degradation of high and low concentrations of PNP in these environments.Corynebacterium strain Z4 when inoculated into the lake and wastewater samples containing 20 µg/ml of PNP degraded 90% of PNP in one day. Addition of 100 µg/ml of glucose as a second substrate did not enhance the degradation of PNP and the bacterium utilized the two substrates simultaneously. Glucose used at the same concentration (100 µg/ml), inhibited degradation of 20 µg of PNP in wastewater byPseudomonas strain MS. However, glucose increased the extent of degradation of PNP byPseudomonas strain GR. Phenol also enhanced the degradation of PNP in wastewater byPseudomonas strain GR, but had no effect on the degradation of PNP byCorynebacterium strain Z4.Addition of 100 µg/ml of glucose as a second substrate into the lake water samples containing low concentration of PNP (26 ng/ml) enhanced the degradation of PNP and the growth ofCorynebacterium strain Z4. In the presence of glucose, it grew from 2×10⁴ to 4×10⁴ cells/ml in 3 days and degraded 70% of PNP as compared to samples without glucose in which the bacterium declined in cell number from 2×10⁴ to 8×10³ cells/ml and degraded only 30% PNP. The results suggest that in inoculation to enhance biodegradation, depending on the inoculant, second organic substrate many play an important role in controlling the rate and extent of biodegradation of organic compounds.Abbreviations PNP p-nitrophenol     

Methanol determination in Pichia pastoris cultures by flow injection analysis

An automated reverse flow injection analysis (r-FIA) system using stop-flow technique for quantifying methanol based on the enzymatic reactions of alcohol oxidase and peroxidase was developed. The system permitted methanol analysis in a linear range of 0.006-0.1 g methanol l^–1 without external dilution, and with a sampling frequency of 12 analyses per hour, with a relative standard deviation of 1.16%. The analyser was validated analysing samples from a Pichia pastoris fermentation producing a heterologous protein.     

Esterase isozyme variation in theEragrostis curvula complex (Poaceae)

The biosystematic relationships of the apomictic complexEragrostis curvula s. lato, is investigated by disc electrophoresis of seed extracts to obtain esterase patterns of 23 accessions representing the morphological variants of this complex: curvula, conferta, robusta, chloromelas and lehmanniana. The zymograms thus obtained were classified into four groups on the basis of the presence of certain bands taken as characteristic and constant markers. Within each group variations were found in strict accordance with the morphological and cytogenetic data available on the complex. Cluster analysis showed similarity levels between the strains studied, representing different genomic groups. The esterase pattern proved useful as an additional criterion for identifying the individual taxa making up the complex and for evaluating their reciprocal relationships.     

Increasing glucose determination range by flow injection analysis (FIA) using glucose oxidase immobilised on polyaniline

A Flow Injection Analysis (FIA) for glucose using glucose oxidase (E.C.1.1.3.4) was developed. The enzyme was immobilised on polyaniline chemically synthetized on internal surface of silicon tube (2.0 mm of diameter and length from 1 to 5 meters), using glutaraldehyde as a bifunctional agent. The system was able to measure glucose at levels from 5 to 500mM with a response time of 2min using 250l of sample. The system has been operated satisfactorily for one month with more than 300 assays with loss of 60% of activity.     

Genetic Variation Along Time in a Brazilian Population of <Emphasis Type="Italic">Aedes Aegypti</Emphasis> (Diptera: Culicidae), Detected by Changes in the Esterase Patterns

Aedes aegypti from the Brazilian cities of São José do Rio Preto (SJ) and Goiânia (GO) were analyzed as to their esterase patterns and the results were compared with data obtained about 5 years before for SJ population. Esterase bands not detected in the previous study were now observed in mosquitoes from both SJ and GO populations, being the last considered a population resistant to insecticides. Other similarities between SJ and GO populations in this study, and some differences in comparison with the previous data on SJ were observed, involving, in addition to changes in band type, changes in frequency of mosquitoes expressing them and differential gene activation during development. As it is generally true for genetic features, changes in the esterase patterns are expected to be the result of factors such as selection by environmental conditions and genetic drift. In the present case, continuous use of insecticides aiming mosquito population size control in SJ by sanitary authorities could be involved in the observed changes. Changed esterases were classified as carboxylesterases and cholinesterases, which are enzymes already shown to take part in the development of resistance in several organisms. In addition, data obtained in the elapsed time by authorities responsible for the mosquito control has shown increasing insecticide resistance of SJ population mosquitoes parallel to increase in the total amount of esterases, reinforcing the mentioned possibility.     

Insight into the metabolism of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by biphenyl dioxygenases

In this work we have investigated the ability of the biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE_LB400) and of Pandoraea pnomenusa B356 (BphAE_B356) to metabolize DDT. Data show BphAE_LB400 is unable to metabolize this substrate but BphAE_B356 metabolizes DDT to produce two stereoisomers. Structural analysis of DDT-docked BphAE_LB400 and BphAE_B356 identified residue Phe336 of BphAE_LB400 as critical to prevent productive binding of DDT to BphAE_LB400. Furthermore, the fact that residue Gly319 of BphAE_B356 is less constrained than Gly321 of BphAE_LB400 most likely contributes to the ability of BphAE_B356 to bind DDT productively. This was confirmed by examining the ability of BphAE chimeras obtained by shuffling bphA genes from strain B356 and LB400. Chimeras where residues Thr335 (which modulates the constraints on Gly321) and Phe336 (which contacts the substrate) of BphAE_LB400 were replaced by Gly and Ile respectively were able to metabolize DDT. However their stereospecificities varied depending on the presence of other segments or residues from BphAE_B356. Structural analysis suggests that either one or both of residue 267 and a segments comprised of residue 247–260 are likely involved in stereospecificity.     

On-line determination of intracellular beta-galactosidase activity in recombinant Escherichia coli using flow injection analysis (FIA).

A flow injection analysis (FIA) system was developed for the determination of cytoplasmic beta-galactosidase activity in recombinant Escherichia coli. The FIA system and its application for on-line monitoring of beta-galactosidase production during cultivation of recombinant E. coli in a 60-l airlift tower loop reactor is described. The results demonstrate that an FIA assay in conjunction with a cell disintegration step can be applied successfully for on-line monitoring of intracellular protein formation.     

Esterase variation and insecticide resistance in Japanese Aphis gossypii

Nine clones of Aphis gossypii Glover (Homoptera: Aphididae) collected from five localities in Japan were tested for esterase activity and resistance to two insecticides. These clones were classified into six types according to esterase pattern and activity detected by electrophoresis. Three clones with very high esterase activity (Type-1) were moderately resistant to malathion and very highly so to pirimicarb. Of six clones with moderate esterase activity one clone (T-4a) was susceptible to malathion but highly resistant to pirimicarb, whereas five clones (T-2, 3a, 3b and 4b) were susceptible to both insecticides. Thus malathion resistance is positively correlated with high esterase activity, whereas pirimicarb resistance is not necessarily so. It is suggested that high esterase activity plays an important role in resistance to these insecticides but that another mechanism is also responsible for resistance to pirimicarb.

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Variations de l'activité estérase et résistance aux insecticides chez des Aphis gossypii japonais

Résumé L'activité estérase et la résistance aux insecticides ont été examinées chez 9 clones de A. gossypii, récoltés dans 5 sites du Japon. Ces clones ont été groupés en 6 types suivant le comportement et l'activité de l'estérase mis en évidence par électrophorèse. 3 clones ont présenté une activité estérase très élevée (type 1) et des résistances, moyenne pour le malathion et très élevée pour le pirimicarbe. Parmi les 6 clones ayant une activité estérase modérée, un clone (T-4a) était sensible au malathion mais très résistant au pirimicarbe, tandis que les 5 autres clones (T-2, 3a, 3b et 4b) étaient très sensibles aux deux insecticides. Ainsi, la résistance au malathion présente une forte corrélation avec une activité estérase élevée, sans qu'il n'en soit nécessairement de même pour la résistance au pirimicarbe. Une hypothèse est émise suivant laquelle l'activité estérase élevée joue un rôle important dans la résistance à ces deux insecticides, mais qu'un autre mécanisme intervient aussi dans la résistance au pirimicarbe.