Activity and heavy metal resistance of non-specific esterases in leaf beetle Chrysomela lapponica from polluted and unpolluted habitats

We compared the general activity and heavy metal resistance of non-specific esterases in two populations of the leaf beetle Chrysomela lapponica from habitats severely contaminated by heavy metals (mostly Ni and Cu) and two populations from unpolluted habitats. Concentrations of Ni and Cu in adult beetles from the most polluted site were 7.7 and 3.6 times higher that in beetles from unpolluted habitats. Larval esterases showed higher activity and lower susceptibility to heavy metals than esterases of adults. Larval esterase activity did not differ between populations from polluted and unpolluted sites, but adult beetles from polluted localities had lower esterase activity than beetles from unpolluted habitats. Both Cu and Ni sulfates in millimolar concentrations in vitro suppressed esterase activity of larvae from unpolluted habitats, but caused no negative effect on esterases of larvae from polluted sites. Similarly, inhibition of adult esterase activity by Ni was stronger in beetles from unpolluted localities than in beetles from polluted localities. This indicates that resistance of non-specific esterases to heavy metals is higher in leaf beetle populations from contaminated environment.     

Bodily heat resistance and polymorphism of liver esterases of grass frogs]

The determination of organismal heat resistance and qualitative composition of polymorphous liver esterases during heat acclimation (25 degrees) has been made on frogs Rana temporaria. During hybernation the most heat resistant frogs possess the homozygous allele of A2 esterase. Heat acclimation and the summer rise in temperature in nature lead to an increase in heat resistance of frogs and to the disappearance of selective advantage of animals possessing the isoenzyme of the A2A2 esterase. The functional homeostasis of populations can maintain biochemical polymorphism regardless of the selective advantage of individuals possessing one of the homozygous alleles of the isoenzyme.     

Alterations in esterases are associated with malathion resistance in Habrobracon hebetor (Hymenoptera: Braconidae)

Biochemical mechanisms of malathion resistance were investigated in a malathion-resistant strain of the parasitoid Habrobracon hebetor Say collected from a farm storage in Kansas. General esterase activities were significantly lower in the resistant strain compared with those in a susceptible strain. However, no significant differences were found in activities of malathion specific carboxylesterase (MCE), glutathione S-transferase and cytochrome P450 dependent O-demethylase activities, cytochrome P450 contents, and sensitivity of acetylcholinesterase to inhibition by malaoxon between the 2 strains. Because MCE was not elevated in the resistant strain, the weak malathion resistance in H. hebetor may result from a different mechanism compared with that hypothesized for some insect species in which reduced general esterase activity is accompanied by an elevated MCE. Decreased esterase activity in the resistant strain suggested that null alleles of some esterases were associated with the resistance. Indeed, E1 and E2, major esterases in the susceptible strain, were not present in the resistant strain on polyacrylamide gels that were stained for esterase activity using the model substrate 1-naphthyl acetate. In contrast, the activity of esterase E3 on the gels was much higher in the resistant strain as compared with that of the susceptible strain. These findings indicate that malathion resistance in H. hebetor is associated with both an increased activity of the esterase E3 and null alleles of the esterases E1 and E2.     

Identification of factors responsible for insecticide resistance in Helicoverpa armigera

Moth larvae (Helicoverpa armigera Hübner) collected from field crops were tested for resistance to cypermethrin, fenvalerate, endosulfan, monocrotophos and quinolphos. Larvae were treated with a dose of the pesticide that would kill 99% of the susceptible insects. The percent survival of the resistant strains was determined. Highest seasonal average percentage survival was recorded by fenvalerate (65.0%) followed by cypermethrin (62.4%). Acetylcholinesterase of resistant larvae was less sensitive to monocrotophos and methyl paraoxon. Resistant larvae showed higher activities of esterases, phosphatases and methyl paraoxon hydrolase compared with susceptible larvae. The presence of high activity of esterases was attributed to appearance of extra bands of esterases in native PAGE. The presence of P-glycoprotein expression was detected in resistant larvae using P-gp antibodies; this was not detected in the susceptible larvae. Our results indicate that the high level of resistance detected in the field pests could be because of a combined effect of decreased sensitivity to AChE, higher levels of esterases, phosphatases and the expression of P-gp.     

Immunological assessment of an insecticide resistance-associated esterase in the Western corn rootworm

In previous investigations, we have determined that organophosphate resistance in the western corn rootworm, Diabrotica virgifera virgifera, is at least partially attributed to a group of non-specific carboxylesterases referred to as group II. Antiserum raised against a purified 66-kDa group II esterase is specific for the denatured enzyme. This antiserum reacts similarly with both beetle homogenates from resistant and susceptible populations, although there is much higher signal intensity in immunoblots of resistant relative to susceptible beetles. These results suggest that overproduction of group II esterases is the underlying basis of esterase-mediated resistance in D. v. virgifera by demonstrating that (1) group II esterases are immunologically indistinguishable between the resistant and susceptible populations, and (2) the intensity differences are due to increased group II esterase proteins in the resistant population. The diagnostic potential of immunological-based assays was tested with a traditional diagnostic concentration bioassay and a biochemical-based native PAGE assay. Significant correlations were observed among all three diagnostic assays (regression coefficients ranging from 0.95 to 0.96). These results demonstrate the importance of the 66-kDa protein as a resistance-associated biochemical marker, thus emphasizing the potential for 66-kDa protein-targeted immunoassays in resistance monitoring programs.     

家蝇对二氯苯醚菊酯的抗性及增效磷的增效作用Ⅰ:水解代谢

水解代谢在家蝇对二氯苯醚菊酯抗性中起重要作用。正常家蝇和抗性家蝇酯酶在对SV₁、SV₂等抑制剂的敏感性和电泳性质上存在着差异。抗性家蝇酯酶水解二氯苯醚菊酯的活性较高,水解乙酸-α-萘酯的活性相对比正常家蝇要低。SV₁及其在体内的代谢产物SV₂在离体和活体情况下对家蝇酯酶都有明显的抑制作用。SV₁和SV₂抑制相同的酯酶电泳条带,但SV₁的抑制作用相对小一些。SV_t对酯酶的抑制是它在家蝇体内对二氯苯醚菊酯增效的机理之一。     

Molecular and kinetic evidence for allelic variants of esterase Estβ1 in the mosquito Culex quinquefasciatus

Elevated esterase Estbeta1 was purified from larvae of newly isolated strains of the mosquito Culex quinquefasciatus from Colombia (COL) and Trinidad (TRI) with resistance to organophosphate (OP) insecticides. Insecticide interactions were compared with those of elevated Estbeta1(2) from the OP-resistant Habana strain and the non-elevated Estbeta1(3) from the susceptible PelSS strain. On the basis of insecticide binding efficiency, all elevated Estbeta1 esterases were readily distinguishable. Differences between the EcoRI restriction fragment patterns of the amplified estbeta1 gene in COL and TRI strains compared with each other, and between amplified estbeta1(1), estbeta1(2) and the non-amplified estbeta1(3), suggest differences in their nucleotide sequence. Considering their variable insecticide binding efficiencies, these genetic differences would imply that, in contrast to estalpha2 and estbeta2, amplification of estbeta1 has occurred several times independently. Generally, the elevated Estbeta1s were more reactive with insecticides than the non-elevated Estbeta1(3). This supports the hypothesis that the elevated esterase-based mechanism confers resistance through amplification of alleles coding for esterases which have a greater specificity for the insecticides they sequester than the esterases coded by their non-amplified counterparts.     

Mosquito carboxylesterases: a review of the molecular biology and biochemistry of a major insecticide resistance mechanism

The major mechanism of organophosphorus insecticide resistance in Culex mosquitoes involves the elevation of one or more esterases. The general mechanism underlying this resistance is the amplification of the structural genes. This review covers the classification of the mosquito esterases in the context of classical esterase nomenclature. The function of the amplified esterases and the structure of the amplified DNA on which they occur are also described. Implications of information on the esterase amplicons are discussed in relation to the evolution and migration of insecticide resistance in Culex.     

The role of gene splicing, gene amplification and regulation in mosquito insecticide resistance

The primary routes of insecticide resistance in all insects are alterations in the insecticide target sites or changes in the rate at which the insecticide is detoxified. Three enzyme systems, glutathione S-transferases, esterases and monooxygenases, are involved in the detoxification of the four major insecticide classes. These enzymes act by rapidly metabolizing the insecticide to non-toxic products, or by rapidly binding and very slowly turning over the insecticide (sequestration). In Culex mosquitoes, the most common organophosphate insecticide resistance mechanism is caused by co-amplification of two esterases. The amplified esterases are differentially regulated, with three times more Est beta 2(1) being produced than Est alpha 2(1). Cis-acting regulatory sequences associated with these esterases are under investigation. All the amplified esterases in different Culex species act through sequestration. The rates at which they bind with insecticides are more rapid than those for their non-amplified counterparts in the insecticide-susceptible insects. In contrast, esterase-based organophosphate resistance in Anopheles is invariably based on changes in substrate specificities and increased turnover rates of a small subset of insecticides. The up-regulation of both glutathione S-transferases and monooxygenases in resistant mosquitoes is due to the effects of a single major gene in each case. The products of these major genes up-regulate a broad range of enzymes. The diversity of glutathione S-transferases produced by Anopheles mosquitoes is increased by the splicing of different 5' ends of genes, with a single 3' end, within one class of this enzyme family. The trans-acting regulatory factors responsible for the up-regulation of both the monooxygenase and glutathione S-transferases still need to be identified, but the recent development of molecular tools for positional cloning in Anopheles gambiae now makes this possible.     

Purification and properties of three esterases from Brevibacterium sp. R312

C. LAMBRECHTS, J. ESCUDERO AND P. GALZY. 1995. The esterases of Brevibacterium sp. R312 were found to have an intracellular location. Electrophoresis of lysed cell supernatant fluids revealed seven bands of esterase activity in the presence of α-naphthyl acetate. Eight esterases were separated by anion exchange chromatography. The three main esterases (esterase 4b, 2 and 4a) of Brevibacterium sp. R312 were purified. The molar masses, the pH optima, the temperature optima and heat stabilities were determined. Esterase 2 differed from the two others in sensitivity to inhibitors. Esterase 4b differed from esterases 2 and 4a in its substrate specificity. This enzyme hydrolyses aliphatic and nitrophenyl esters. The spectrum of activity of the two other esterases is narrower. They hydrolysed only naphthyl esters and, in the case of esterase 2, tributyrate and ethyl butyrate.     

Immunological comparison of cholesterol esterases.

Rat pancreas cholesterol esterase has been immunologically compared with rat intestinal cholesterol esterase. Monospecific precipitating antisera against purified rat pancreas cholesterol esterase were produced in rabbits. Immune IgG, isolated from the antisera, crossreacted with the cholesterol esterase of intestine in the immunodiffusion assay with a pattern of complete identity. Titration of the pancreatic and intestinal enzyme with immune IgG revealed a maximum precipitation (99 and 98%) and maximum inhibition of enzyme activity (66 and 65%) when the ratio of enzyme activity (units) to immune IgG (mg) was 4.1 and 4.0, respectively. The immunological identity demonstrated in these studies lend support to the concept that intestinal cholesterol esterase is derived from the pancreatic enzyme. In additional studies, the immune IgG was employed in the immunodiffusion assay to test for cross-reaction with cholesterol esterases prepared from rat aorta, adrenal, and liver and with cholesterol esterases prepared from the pancreas of rabbit, dog, cow, and guinea pig. There was no evidence of cross-reaction in any case. Further, cholesterol esterase prepared from the pancreas of rabbit, dog, and cow retained full enzymatic activity when titrated with immune IgG.     

Organophosphate and Pyrethroid Hydrolase Activities of Mutant Esterases from the Cotton Bollworm Helicoverpa armigera

Two mutations have been found in five closely related insect esterases (from four higher Diptera and a hymenopteran) which each confer organophosphate (OP) hydrolase activity on the enzyme and OP resistance on the insect. One mutation converts a Glycine to an Aspartate, and the other converts a Tryptophan to a Leucine in the enzymes’ active site. One of the dipteran enzymes with the Leucine mutation also shows enhanced activity against pyrethroids. Introduction of the two mutations in vitro into eight esterases from six other widely separated insect groups has also been reported to increase substantially the OP hydrolase activity of most of them. These data suggest that the two mutations could contribute to OP, and possibly pyrethroid, resistance in a variety of insects. We therefore introduced them in vitro into eight Helicoverpa armigera esterases from a clade that has already been implicated in OP and pyrethroid resistance. We found that they do not generally enhance either OP or pyrethroid hydrolysis in these esterases but the Aspartate mutation did increase OP hydrolysis in one enzyme by about 14 fold and the Leucine mutation caused a 4–6 fold increase in activity (more in one case) of another three against some of the most insecticidal isomers of fenvalerate and cypermethrin. The Aspartate enzyme and one of the Leucine enzymes occur in regions of the H. armigera esterase isozyme profile that have been previously implicated in OP and pyrethroid resistance, respectively.     

Variation in general esterase activity within a population of Haematobia irritans (Diptera: Muscidae).

Control of the horn fly, Hematobia irritans (L.), is generally dependent on chemical insecticides. However, the biology and behavior of the horn fly favors rapid development of insecticide resistance. To prolong the effectiveness of the insecticide option, information is required regarding the mechanisms of insecticide resistance. Metabolic hydrolysis of insecticides by esterases is a detoxification mechanism in many insect species. Measurement of general esterase activity within populations of horn flies may provide a diagnostic tool for resistance management. In this study we evaluated the amount of variation in general esterase activity within female and male horn fly samples from a population that had not been exposed to insecticides for 8 yr. We found considerable variation in general esterase activity within samples of each sex, with females demonstrating the greater variation. The observed variation is thought to be the result of age-structure dynamics within the population. The amount of inherent variation makes it difficult to detect small mean differences between populations, thus limiting the utility of general esterase assays. Thus, effective diagnosis of esterase-mediated resistance mechanisms can only be achieved by the identification of specific detoxification esterases and the design of assays, either biochemical or molecular, for their detection and measurement.     

Biosynthesis of rat-liver pI-5.0 esterases in cell-free systems and in cultured hepatocytes

Rat liver esterases focusing at pH 5.0 (referred to below as pI-5.0 esterases) are structurally related glycoproteins which differ slightly in their mobility in sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). They reside in the lumen of the endoplasmic reticulum. We have studied their biosynthesis in cell-free systems programmed by total liver RNA, using sheep and rabbit antibodies to isolate the translation products related to these enzymes. Our results show that they are assembled as a precursor polypeptide chain (62 kDa) larger than the mature proteins. The pI-5.0 esterase mRNA could be extracted from bound but not free polysomes. Reticulocyte lysates supplemented with dog pancreas microsomes produced four esterase-related components in segregated form (61, 60, 58 and 56 kDa). The largest three correspond in electrophoretic mobility to the mature enzymes. They are glycoproteins that bind to concanavalin A, and can be reduced to the size of the shortest component by endo-beta-N-acetylglucosaminidase H (endo-H). Immunoprecipitation after biosynthetic labeling of the proteins in cultured hepatocytes also gave three glycosylated components that had the same mobility in SDS-PAGE as the mature enzymes. When tunicamycin was present in the culture medium, a single immunoprecipitable form was observed. Its apparent Mr was similar to that of the unglycosylated pI 5.0 esterase form synthesized in vitro in the presence of dog pancreas microsomes. Thus the biosynthesis of these esterases has characteristics in common with that of numerous secretory proteins, except for the rather large difference in size (approximately equal to 6 kDa) resulting from the proteolytic processing of their in-vitro-synthesized precursor.     

Evaluation of manometric temperature measurement (MTM), a process analytical technology tool in freeze drying, part III: Heat and mass transfer measurement

This article evaluates the procedures for determining the vial heat transfer coefficient and the extent of primary drying through manometric temperature measurement (MTM). The vial heat transfer coefficients (K_v) were calculated from the MTM-determined temperature and resistance and compared with K_v values determined by a gravimetric method. The differences between the MTM vial heat transfer coefficients and the gravimetric values are large at low shelf temperature but smaller when higher shelf temperatures were used. The differences also became smaller at higher chamber pressure and smaller when higher resistance materials were being freeze-dried. In all cases, using thermal shields greatly improved the accuracy of the MTM K_v measurement. With use of thermal shields, the thickness of the frozen layer calculated from MTM is in good agreement with values obtained gravimetrically. The heat transfer coefficient “error” is largely a direct result of the error in the dry layer resistance (ie, MTM-determined resistance is too low). This problem can be minimized if thermal shields are used for freeze-drying. With suitable use of thermal shields, accurate K_v values are obtained by MTM; thus allowing accurate calculations of heat and mass flow rates. The extent of primary drying can be monitored by real-time calculation of the amount of remaining ice using MTM data, thus providing a process analytical tool that greatly improves the freeze-drying process design and control.     

INSECTICIDE RESISTANCE IN THE GROUND SPIDER,Pardosa sumatrana (THORELL, 1890; ARANEAE: LYCOSIDAE)

Elevated levels of insecticides detoxifying enzymes, such as esterases, glutathione S‐transferases (GSTs), and cytochrome P‐450 monooxygenases, act in the resistance mechanisms in insects. In the present study, levels of these enzymes in the insecticide‐resistant ground spider Pardosa sumatrana (Thorell, 1890) were compared with a susceptible population (control) of the same species. Standard protocols were used for biochemical estimation of enzymes. The results showed significantly higher levels of nonspecific esterases and monooxygenases in resistant spiders compared to controls. The activity of GSTs was lower in the resistant spiders. Elevated levels of nonspecific esterases and monooxygenases suggest their role in metabolic resistance in P. sumatrana. The reduced levels of total protein contents revealed its possible consumption to meet energy demands.     

Genetics of esterases in Drosophila. V. Characteristics of the “pupal” esterase in D. virilis

From analysis of the properties of the pupal esterase (p-esterase) in Drosophila virilis, it is concluded that it is heat stable, its electrophoretic detection depends on culture density, its expression is stage specific, and it is not a variant of esterase 2. It was also demonstrated that p-esterase, like esterase 6, is activated by injections of the juvenile hormone into larvae. Heat treatment of heat-resistant D. virilis stocks led to decreased activities of the juvenile hormone dependent esterases but did not affect those of the heat-sensitive stocks. It is suggested that heat resistance in D. virilis is related to some functional features of the system of modifier genes controlling the phenotypic expression of esterases.     

High yields in advanced lines of Pima cotton are associated with higher stomatal conductance, reduced leaf area and lower leaf temperature

Advanced lines of Pima cotton ( Gossypium barbadense L.) bred for higher yield potential and heat resistance have higher stomata conductance and smaller leaf areas than those of obsolete lines. In controlled experiments, five commercial lines of Pima cotton having increasing lint yield and heat resistance showed a gradient of increasing stomatal conductance and decreasing leaf size. In field experiments, heat-sensitive, low yield Pima lines showed a lower stomatal conductance than high yielding, advanced lines. This indicates that selection for high yield potential and heat resistance has imposed a selection pressure for higher stomatal conductance and smaller leaf areas. The higher stomatal conductance and smaller leaf area in the advanced lines resulted in a lower leaf temperature in both controlled environments and in the field. The largest leaf temperature differences between obsolete and advanced lines were observed in the afternoon. These differences coincided with the largest differences in stomatal conductance and the highest air temperatures. Measurements of stomatal conductance and leaf temperature in field-grown progeny from a cross between the advanced line, Pima S-6. and the obsolete line, Pima 32, showed that genetically determined differences in stomatal conductance resulted in corresponding differences in leaf temperature. None of the altered physiological traits were selected for in the breeding program, indicating that selection for the desired agronomic traits imposed selection pressures on the altered physiological traits. The increases in stomatal conductance and decreases in leaf area could represent an integrated response to selection pressures on enhanced evaporative cooling, ensuing from selection for heat resistance.     

Heat resistance studies of yeasts; vegetative cells versus ascospores: erythromycin inhibition of sporulation in Kluyveromyces and Saccharomyces species

A comparative study of the heat resistance ( D ₆₀ values) of four Saccharomyces spp. and two Kluyveromyces spp. (21 strains) showed a 30–350-fold higher heat resistance of ascospores than of vegetative cells. It was also observed that small numbers of ascospores exhibiting a considerably higher heat resistance can easily be formed, even in a complete vegetative growth medium. This phenomenon may have led most other authors to report none or only slight differences between the heat resistance of yeast ascospores and their vegetative cells. Until more information has been collected about the ascospore load of acid (fruit) products and their heat resistance, accurate calculations of the minimum F values for heat preservation of these products may not be possible.