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J Hemingway

Publications and source records attributed to J Hemingway.

At least 55 records · Page 3Linked to original sources
PubMed2000

Biochemical characterization of insecticide resistance in the German cockroach, Blattella germanica, from Malaysia.

The possible insecticide resistance mechanisms of four Malaysian field-collected strains of the German cockroach, Blattella germanica (Linnaeus) (Dictyoptera: Blattellidae), were characterized with biochemical assays and native polyacrylamide gel electrophoresis (PAGE). Elevated esterase activity (at low to moderate frequency) and altered acetylcholinesterase (low frequency) were detected in all field strains, while elevated glutathione S-transferase levels were present in only two strains. Seven esterase bands were separated by native PAGE; a greater intensity occurred in three bands in the resistant strains compared to the susceptible strain. Inhibition studies using specific inhibitors on polyacrylamide gels suggested that the slowest of these three esterases is a cholinesterase, while the other two are carboxylesterases with a preference for beta- over alpha-naphthyl acetate.

Acetylcholinesterase↗
PubMed2000

Insecticide resistance spectra and resistance mechanisms in populations of Japanese encephalitis vector mosquitoes, Culex tritaeniorhynchus and Cx. gelidus, in Sri Lanka.

Culex tritaeniorhynchus Giles and Cx. gelidus Theobald (Diptera: Culicidae), both vectors of Japanese encephalitis, were collected in 1984 and 1998 from two disease endemic localities in Sri Lanka: Anaradhapura and Kandy. Using wild-caught adult mosquitoes from light traps, log dosage-probit mortality curves for insecticide bioassays were obtained for three insecticides: malathion (organophosphate), propoxur (carbamate) and permethrin (pyrethroid). LD50 values showed that, in 1998, Cx. tritaeniorhynchus was -100-fold more resistant to malathion and 10-fold more resistant to propoxur than was Cx. gelidus. This difference was attributed to Cx. tritaeniorhynchus breeding mostly in irrigated rice paddy fields, where it would have been exposed to pesticide selection pressure, whereas Cx. gelidus breeds in other types of aquatic habitats less prone to pesticide applications. Resistance in Cx. tritaeniorhynchus increased between 1984 and 1998, whereas Cx. gelidus remained predominantly susceptible. Propoxur inhibition of acetylcholinesterase (AChE) activity (the target site of organophosphates and carbamates) indicated that in 1998, frequencies of insensitive AChE-based resistance were 9% in Cx. gelidus and 2-23% in Cx. tritaeniorhynchus, whereas in 1984 this resistance mechanism was detected only in 2% of the latter species from Anaradhapura. The AChE inhibition coefficient (ki) with propoxur was 1.86+/-0.24 x 10(5) M(-)1 min(-1) for Cx. tritaeniorhynchus from Anaradhapura in 1998. Both species were tested for activity levels of detoxifying glutathione S-transferases (GSTs) and malathion-specific as well as general carboxylesterases. High activities of GSTs and carboxylesterases were detected in Cx. tritaeniorhynchus but not Cx. gelidus. Malathion-specific carboxylesterase was absent from both species. Native polyacrylamide gel electrophoresis resolved two elevated general carboxylesterases, CtrEstbeta1 and CtrEstalpha1, from Cx. tritaeniorhynchus and none from Cx. gelidus. CtrEstbeta1 was the most intensely staining band. Gel inhibition experiments showed that both elevated esterases were inhibited by organophosphates and carbamates but not by pyrethroids. The major elevated esterase CtrEstbeta1 was partially purified (15-fold) by sequential Q-Sepharose and phenyl Sepharose column chromatography. The bimolecular rate constant (ka) and the deacylation rate constant (k3) for the malaoxon/enzyme interaction were 9.9+/-1.1 x 10(3) M(-1) min(-1) and 3.5+/-0.05 x 10(-4) M(-1) min(-1), respectively, demonstrating that the role of this enzyme in organophosphorus insecticide resistance is sequestration.

Animals↗
PubMed2000

Insecticide resistance in insect vectors of human disease.

Insecticide resistance is an increasing problem in many insect vectors of disease. Our knowledge of the basic mechanisms underlying resistance to commonly used insecticides is well established. Molecular techniques have recently allowed us to start and dissect most of these mechanisms at the DNA level. The next major challenge will be to use this molecular understanding of resistance to develop novel strategies with which we can truly manage resistance. State-of-the-art information on resistance in insect vectors of disease is reviewed in this context.

Animals↗
PubMed1999

Pyrethroid resistance mechanisms in the head louse Pediculus capitis from Israel: implications for control.

In Israel, the head louse, Pediculus capitis, developed resistance to DDT through the extensive use of this insecticide until the 1980s. In 1991, permethrin was introduced for control of DDT resistant P. capitis in Israel, leading to control failure of this pyrethroid insecticide by 1994. Pyrethroid resistance of P. capitis in Israel extends to phenothrin, which has not been used for louse control. We identified a glutathione S-transferase(GST)-based mechanism of DDT resistance in the Israeli head lice. This GST mechanism occurred before 1989, while permethrin resistance in P. capitis developed after 1994, suggesting that the main GST resistance mechanism selected by DDT use does not confer any pyrethroid cross-resistance. Esterase activity levels were equivalent in pyrethroid resistant and susceptible P. capitis field-collected in Israel, and in a susceptible strain of P. humanus, the body louse, indicating no involvement of any esterase-based mechanism in resistance. A weak monooxygenase-based permethrin metabolism resistance mechanism was the only factor identified which could account for any of the observed pyrethroid resistance in P. capitis. However, the lack of synergism of phenothrin resistance by piperonyl butoxide suggests that a non-oxidative mechanism is also present in the resistant lice. Therefore it seems probable that pyrethroid resistance in Israeli P. capitis is due to a combination of nerve insensitivity (knockdown resistance or 'kdr') and monooxygenase resistance mechanisms.

Animals↗
PubMed1999

Molecular and kinetic evidence for allelic variants of esterase Estbeta1 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.

Alleles↗
PubMed1999

Can multiple-copy sequences of prey DNA be detected amongst the gut contents of invertebrate predators?

The first experiments to clearly demonstrate that DNA techniques might be used to detect predator-prey interactions between arthropods are reported. The accurate modelling of such interactions has depended until now upon a mixture of laboratory experiments, population monitoring and biochemical tests. The latter involve gut-content analyses, and have most recently depended upon the development of prey-specific monoclonal antibodies. Although these are excellent for detecting predation on a target prey, they are impractical for analysing the prey range of a particular predator. Molecular detection depends upon the ability of DNA to resist digestion in the predator gut and of the polymerase chain reaction (PCR) to amplify prey-specific DNA from semidigested material. As a first step, experiments using carabid beetles, Pterostichus cupreus L., as predators and mosquitoes as prey are reported. The target sequences were fully characterized multiple-copy esterase genes from two laboratory strains of Culex quinquefasciatus Say. Although DNA was extracted from homogenates of whole beetles (minus appendages), a 146 bp product could be amplified from both mosquito strains digested in the beetle gut for 28 h. The larger, 263 bp product was detectable for 28 h in one mosquito strain, but could not be amplified after 5 h from the other. Whether the beetles had eaten one mosquito or six, digested for zero or 28 h, the prey were equally detectable. Having demonstrated that shorter, multiple-copy sequences survive digestion for a considerable period in the gut of a predator, the opportunity exists to develop new detection systems for studying predation in the field.

Animals↗
PubMed1999

Insecticide resistance in malaria vectors: a new approach to an old subject.

The application of biochemical and molecular biological techniques to the study of insecticide resistance has revolutionized our understanding of the underlying genetic basis of resistance. Using the examples of glutathione S-transferase and esterase-based metabolic insecticide resistance, three different routes via which increased insecticide detoxication can be achieved are elucidated. An understanding of these molecular pathways opens up new avenues for manipulating mosquito populations to restore insecticide susceptibility to the vectors.

Animals↗
PubMed1998

The role of alternative mRNA splicing in generating heterogeneity within the Anopheles gambiae class I glutathione S-transferase family.

The class I glutathione S-transferases (GSTs) of Anopheles gambiae are encoded by a complex gene family. We describe the genomic organization of three members of this family, which are sequentially arranged on the chromosome in divergent orientations. One of these genes, aggst1-2, is intronless and has been described. In contrast, the two A. gambiae GST genes (aggst1alpha and aggst1beta) reported within are interrupted by introns. The gene aggst1alpha contains five coding exons that are alternatively spliced to produce four mature GST transcripts, each of which contains a common 5' exon encoding the N termini of the GST protein spliced to one of four distinct 3' exons encoding the carboxyl termini. All four of the alternative transcripts of aggst1alpha are expressed in A. gambiae larvae, pupae, and adults. We report on the involvement of alternative RNA splicing in generating multiple functional GST transcripts. A cDNA from the aggst1beta gene was detected in adult mosquitoes, demonstrating that this GST gene is actively transcribed. The percentage similarity of the six cDNAs transcribed from the three GST genes range from 49.5% to 83.1% at the nucleotide level.

Alternative Splicing↗
PubMed1998

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.

Alternative Splicing↗
PubMed1998

Amplification of a serine esterase gene is involved in insecticide resistance in Sri Lankan Culex tritaeniorhynchus.

Culex tritaeniorhynchus, the major vector of Japanese encephalitis in Sri Lanka, is resistant to organophosphorus insecticides, with a 10-fold resistance ratio at the LC50 for chlorpyrifos, and a high heterogenelty factor in the insect field population. The major mechanism of resistance in this species, as in the mosquito C. quinquefasciatus, is elevation of esterase activity. Basic biochemical, immunological and molecular analysis suggests that the C. tritaeniorhynchus CtrEstbeta1 gene is orthologous to the C. quinquefasciatus amplified Estbetas. The Estbeta2(1) antiserum cross-reacts strongly with CtrEstbeta1(1). Its corresponding cDNA, over the 545 base pairs sequenced, has approximately 84% identity with the various C. quinquefasciatus Estbetas. The gene is amplified in C. tritaeniorhynchus. Amplification of the same esterase in two independent species, along with multiple amplification events involving this esterase gene in C. quinquefasciatus suggests that the location of this gene within the genome predisposes it to amplification.

Animals↗
PubMed1998

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.

Animals↗
PubMed1998

Characterization of amplified esterase Estbeta1(2) associated with organophosphate resistance in a multi-resistant population of the mosquito Culex quinquefasciatus from Cuba.

Esterase amplification is the major organophosphorus (OP) insecticide resistance mechanism in Culex mosquitoes. The amplified Estalpha2(1)¿Estbeta2(1) esterases are found in > 90% of resistant populations worldwide, whereas amplified DNAs (amplicons) containing Estbeta1s are much rarer. Individuals with the Estbeta1 amplicons appear to be at a selective disadvantage in competition with those carrying the Estalpha2(1)¿Estbeta2(1) amplicons. To test the hypothesis that this is because Estbeta1 is less able to bind insecticide than the common amplified esterases, Estbeta1(2) was purified from the multi-resistant Habana strain of Culex quinquefasciatus, from Cuba. In its native form Estbeta1 is a monomeric enzyme of 66 kDa, with a pI of 4.8. The bimolecular rate constants for interaction of Estbeta1(2) with several OP insecticides were similar to those for the commonly elevated esterases Estalpha2(1) and Estbeta2(1), and much higher than for the electrophoretically identical non-elevated Estbeta1(3) and Estalpha3. Hence the apparent selective advantage of the Estalpha2(1)¿Estbeta2(1) amplicon is not due to its greater efficiency of insecticide binding, as OP insecticides are significantly better inhibitors of all the amplified esterases than of their non-amplified counterparts and therefore should be equally effective at conferring resistance.

Animals↗
PubMed1998

Resistance management strategies in malaria vector mosquito control. Baseline data for a large-scale field trial against Anopheles albimanus in Mexico.

A high level of DDT resistance and low levels of resistance to organophosphorus, carbamate and pyrethroid insecticides were detected by discriminating dose assays in field populations of Anopheles albimanus in Chiapas, southern Mexico, prior to a large-scale resistance management project described by Hemingway et al. (1997). Biochemical assays showed that the DDT resistance was caused by elevated levels of glutathione S-transferase (GST) activity leading to increased rates of metabolism of DDT to DDE. The numbers of individuals with elevated GST and DDT resistance were well correlated, suggesting that this is the only major DDT resistance mechanism in this population. The carbamate resistance in this population is conferred by an altered acetylcholinesterase (AChE)-based resistance mechanism. The level of resistance observed in the bioassays correlates with the frequency of individuals homozygous for the altered AChE allele. This suggests that the level of resistance conferred by this mechanism in its heterozygous state is below the level of detection by the WHO carbamate discriminating dosage bioassay. The low levels of organophosphate (OP) and pyrethroid resistance could be conferred by either the elevated esterase or monooxygenase enzymes. The esterases were elevated only with the substrate pNPA, and are unlikely to be causing broad spectrum OP resistance. The altered AChE mechanism may also be contributing to the OP but not the pyrethroid resistance. Significant differences in resistance gene frequencies were obtained from the F1 mosquitoes resulting from adults obtained by different collection methods. This may be caused by different insecticide selection pressures on the insects immediately prior to collection, or may be an indication that the indoor- and outdoor-resting A. albimanus collections are not from a randomly mating single population. The underlying genetic variability of the populations is currently being investigated by molecular methods.

Acetylcholinesterase↗
PubMed1997

Co-amplification explains linkage disequilibrium of two mosquito esterase genes in insecticide-resistant Culex quinquefasciatus.

The mosquito Culex quinquefasciatus (Say) is a vector of human disease and a world-wide biting nuisance. Organophosphorus insecticides (OPs) have been widely used to control C. quinquefasciatus populations and this has led to the emergence of OP-resistance. Predominantly, resistance is caused by increased production of two non-specific carboxylesterases, Estalpha2(1) and Estbeta2(1). Increased abundance of these esterases is associated with the amplification of their respective genes. The estalpha21 and estbeta21 genes were cloned and sequenced from OP-resistant Sri Lankan C. quinquefasciatus; the two adjacent genes are in a head to head configuration, within a single amplification unit (amplicon). The homology between the two genes suggests that they arose from an ancient duplication event. The two genes have different numbers of exons (estalpha21 has seven and estbeta21 has four); however, the intron/exon boundaries in estbeta21 are all conserved in estalpha21. The two genes are co-amplified in three other mosquito strains with the elevated Estalpha2(1)/Estbeta2(1) phenotype. Their complete linkage disequilibrium is explained by the location of the two genes involved in resistance within a single amplicon. In insecticide-susceptible C. quinquefasciatus, the non-amplified estalpha and estbeta gene loci are also found in a similar head to head configuration, but the size of the intergenic non-coding region is approx. 1 kb less than in the amplicon. The smaller intergenic spacer is also found in a strain with amplified estbeta11, which suggests that extensive laboratory selection for this amplified esterase has not eliminated the non-amplified genes. The intergenic spacer regions have been subcloned and sequenced. They contain numerous possible TATA boxes, promoters and a number of possible regulatory elements with high homology to the consensus sequence of the Barbie box. These latter putative regulatory elements are more numerous in the larger intergenic spacer, which differs from the non-amplified spacer by two large (>>420 bp) and one small (5 bp) insertions.

Amino Acid Sequence↗
PubMed1997

Cloning and characterization of two glutathione S-transferases from a DDT-resistant strain of Anopheles gambiae.

Two cDNA species, aggst1-5 and aggst1-6, comprising the entire coding region of two distinct glutathione S-transferases (GSTs) have been isolated from a 1,1,1-trichloro-2,2-bis-(p-chlorophenyl)ethane (DDT) resistant strain (ZANDS) of Anopheles gambiae. The nucleotide sequences of these cDNA species share 80.2% identity and their derived amino acid sequences are 82.3% similar. They have been classified as insect class I GSTs on the basis of their high sequence similarity to class I GSTs from Drosophila melanogaster and Musca domestica and they are localized to a region of an An. gambiae chromosome known to contain further class I GSTs. The genes aggst1-5 and aggst1-6 were expressed at high levels in Escherichia coli and the recombinant GSTs were purified by affinity chromatography and characterized. Both agGST1-5 and agGST1-6 showed high activity with the substrates 1-chloro-2,4-dinitrobenzene and 1, 2-dichloro-4-nitrobenzene but negligible activity with the mammalian theta class substrates, 1,2-epoxy-3-(4-nitrophenoxy)propane and p-nitrophenyl bromide. Despite their high level of sequence identity, agGST1-5 and agGST1-6 displayed different kinetic properties. Both enzymes were able to metabolize DDT and were localized to a subset of GSTs that, from earlier biochemical studies, are known to be involved in insecticide resistance in An. gambiae. This subset of enzymes is one of three in which the DDT metabolism levels are elevated in resistant insects.

Amino Acid Sequence↗
PubMed1997

Cloning and localization of a glutathione S-transferase class I gene from Anopheles gambiae.

1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane (DDT) resistance in both adults and larvae of Anopheles gambiae is mediated by stage-specific glutathione S-transferases (GSTs). On the basis of their biochemical characteristics the larval resistance-associated GSTs are likely to be insect class I GSTs. Aggst1-2, a class I GST gene, which is expressed in larvae, has been cloned from the malaria vector A. gambiae. The gene was inserted into a bacterial expression system, and the detection of 1-chloro-2,4-dinitrobenzene (CDNB) conjugating activity in Eschericia coli expressing the recombinant enzyme confirmed that aggst1-2 encodes a catalytically active GST. The gene encodes a 209 amino acid protein with 46% sequence similarity to a Drosophila melanogaster class I GST (GST-D1), 44% similarity with a Musca domestica class I GST (MdGST-1), but only low levels of homology with class II insect GSTs, including the adult specific AgGST2-1 from A. gambiae. Southern analysis of genomic DNA indicated that A. gambiae has multiple class I GSTs. In situ hybridization of class I genomic and cDNA clones to polytene chromosomes identified a single region of complementarity on chromosome 2R division 18B, suggesting that these class I GSTs in A. gambiae are arranged sequentially in the genome. Three positive overlapping recombinant clones were identified from an A. gambiae genomic library. Mapping and partial sequencing of these clones suggests that there are several GSTs and truncated GST pseudogenes within the 30kb of DNA that these clones span.

Amino Acid Sequence↗
PubMed1997

Resistance to insecticides in insect vectors of disease: est alpha 3, a novel amplified esterase associated with amplified est beta 1 from insecticide resistant strains of the mosquito Culex quinquesfasciatus.

Vector control programmes in many countries face the dual problems of parasite drug resistance and insecticide resistance in the insect vectors of the disease. Here we report for the first time a new esterase-based insecticide resistance mechanism in the filariasis vector Culex quinquefasciatus. The field collected COL strain of C. quinquefasciatus from Columbia was heterogeneous for organophosphorus insecticide resistance. On native polyacrylamide gels it had an elevated beta-naphthyl acetate specific esterase with the same Rf as that for the Est beta 1s involved in insecticide resistance in other strains of this mosquito species. After five generations of temephos insecticide selection, both the esterase specific activity with p-nitrophenyl acetate and the temephos LC50 values were increased, suggesting that elevation of esterase activity was the underlying mechanism of resistance. Western blots with antisera raised to Est alpha 2(1) and Est beta 2(1) from C. quinquefasciatus indicated that the COL strain had an elevated Est alpha 3 enzyme which co-migrated on native gels with Est beta 1. Southern blots indicated that an est alpha 3 gene was amplified in the COL strain and a Cuban mosquito strain (MRes), although the restriction digest patterns of the est beta 1 genes in these two strains are different. In contrast, the Californian TEMR strain, with the amplified est beta 1(1) gene, had no associated elevated Est alpha. Restriction digest patterns for COL and TEMR DNA suggest that they contain an identical est beta 1(1) gene, but our data suggest that the est alpha 3 gene occurs on the same amplicon as an est beta 1 gene although the genes are probably > 10 kb apart. Hence, either the COL strain has two est beta 1 genes or the est beta 1(1) amplicon in TEMR has been disrupted at some stage during the long colonisation of this strain and the amplified est alpha has been lost.

Animals↗