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Some of the resistance of Helicoverpa armigera to conventional insecticides such as organophosphates and synthetic pyrethroids appears to be due to metabolic detoxification by carboxylesterases. To investigate the H. armigera carboxyl/cholinesterases, we created a data set of 39 putative paralogous H. armigera carboxyl/cholinesterase sequences from cDNA libraries and other sources. Phylogenetic analysis revealed a close relationship between these sequences and 70 carboxyl/cholinesterases from the recently sequenced genome of the silkworm, Bombyx mori, including several conserved clades of non-catalytic proteins. A juvenile hormone esterase candidate from H. armigera was identified, and B. mori orthologues were proposed for 31% of the sequences examined, however low similarity was found between lepidopteran sequences and esterases previously associated with insecticide resistance from other insect orders. A proteomic analysis of larval esterases then enabled us to match seven of the H. armigera carboxyl/cholinesterase sequences to specific esterase isozymes. All identified sequences were predicted to encode catalytically active carboxylesterases, including six proteins with N-terminal signal peptides and N-glycans, with two also containing C-terminal signals for glycosylphosphatidylinositol anchor attachment. Five of these sequences were matched to zones of activity on native PAGE at relative mobility values previously associated with insecticide resistance in this species.

Background Neurexins and neuroligins, which have recently been associated with neurological disorders such as autism in humans, are highly conserved adhesive proteins found on synaptic membranes of neurons. These binding partners produce a trans-synaptic bridge that facilitates maturation and specification of synapses. It is believed that there exists an optimal spatio-temporal code of neurexin and neuroligin interactions that guide synapse formation in the postnatal developing brain. Therefore, we investigated whether neuroligins and neurexin are differentially regulated by sensory input using a behavioural model system with an advanced capacity for sensory processing, learning and memory, the honeybee. Methodology/Principal Findings Whole brain expression levels of neuroligin 1–5 (NLG1–5) and neurexin I (NrxI) were estimated by qRT-PCR analysis in three different behavioural paradigms: sensory deprivation, associative scent learning, and lateralised sensory input. Sensory deprived bees had a lower level of NLG1 expression, but a generally increased level of NLG2–5 and NrxI expression compared to hive bees. Bees that had undergone associative scent training had significantly increased levels of NrxI, NLG1 and NLG3 expression compared to untrained control bees. Bees that had lateralised sensory input after antennal amputation showed a specific increase in NLG1 expression compared to control bees, which only happened over time. Conclusions/Significance Our results suggest that (1) there is a lack of synaptic pruning during sensory deprivation; (2) NLG1 expression increases with sensory stimulation; (3) concomitant changes in gene expression suggests NrxI interacts with all neuroligins; (4) there is evidence for synaptic compensation after lateralised injury.

In silico substrate docking of both stereoisomers of the pesticide chlorfenvinphos (CVP) in the phosphotriesterase from Agrobacterium radiobacter identified two residues (F131 and W132) that prevent productive substrate binding and cause stereospecificity. A variant (W131H/F132A) was designed that exhibited ca. 480-fold and 8-fold increases in the rate of Z -CVP and E -CVP hydrolysis, respectively, eliminating stereospecificity.

The methyl carbamate-degrading hydrolase (MCD) of Achromobacter WM111 has considerable potential as a pesticide bioremediation agent. However this potential has been unrealisable until now because of an inability to express MCD in heterologous hosts such as Escherichia coli. Herein, we describe the first successful attempt to express appreciable quantities of MCD in active form in E. coli, and the subsequent characterisation of the heterologously expressed material. We find that the properties of this material closely match the previously reported properties of MCD produced from Achromobacter WM111. This includes the presence of two distinct forms of the enzyme that we show are most likely due to the presence of two functional translational start sites. The purified enzyme catalyses the hydrolysis of a carbamate (carbaryl), a carboxyl ester (alpha-naphthyl acetate) and a phophotriester (dimethyl umbelliferyl phosphate) and it is relatively resistant to thermal and solvent-mediated denaturation. The robust nature and catalytic promiscuity of MCD suggest that it could be exploited for various biotechnological applications.

The atrazine chlorohydrolase AtzA has evolved within the past 50 years to catalyze the hydrolytic dechlorination of the herbicide atrazine. It is of wide research interest for two reasons: first, catalytic improvement of the enzyme would facilitate its application in bioremediation, and second, because of its recent evolution, it presents a rare opportunity to examine the early stages in the acquisition of new catalytic activities. Using a structural model of the AtzA-atrazine complex, a region of the substrate-binding pocket was targeted for combinatorial randomization. Identification of improved variants through this process informed the construction of a variant AtzA enzyme with 20-fold improvement in its k cat / K m value compared with that of the wild-type enzyme. The reduction in K m observed in the AtzA variants has allowed the full kinetic profile for the AtzA-catalyzed dechlorination of atrazine to be determined for the first time, revealing the hitherto-unreported substrate cooperativity in AtzA. Since substrate cooperativity is common among deaminases, which are the closest structural homologs of AtzA, it is possible that this phenomenon is a remnant of the catalytic activity of the evolutionary progenitor of AtzA. A catalytic mechanism that suggests a plausible mechanistic route for the evolution of dechlorinase activity in AtzA from an ancestral deaminase is proposed.

Eight esterases identified using native polyacrylamide gel electrophoresis were characterized in a susceptible and an organophosphate-resistant strain of Lucilia cuprina. Developmental profiles and tissue distributions were determined for all eight esterases and substrate and inhibitor specificities were also determined for six of them. Esterases E1 and E2 were isozymes of acetylcholinesterase, with E1 present throughout development and E2 largely confined to adults. Esterases E8, which was mainly confined to the neuromuscular and digestive tissues of early larvae, and E7, which was only recovered from 2- to 3-day-old adults, were not abundant enough for biochemical analysis. E4 and E13, classified as carboxylesterases, were only moderately sensitive to inhibition by organophosphates and found mainly in hemolymph. E3 and E9 were also identified as carboxylesterases but were highly sensitive to inhibition by organophosphates. They were the only enzymes found to differ between the two strains, both being detected only in the organophosphate-susceptible strain. In this strain they were both abundant in pupae, and E3 was also abundant in larvae and sexually mature adults, localized mainly in digestive tissues and the adult reproductive tract. The inhibitor, developmental, and tissue specificities of E3 all support genetic data reported previously that E3 is encoded by the major organophosphate resistance locus, ROP-1. While genetic data are not available for E9, its concentration in pupae suggests that it is unlikely to confer major gene resistance in feeding stages of L. cuprina.

Orgamophosphorus (OP) pesticides poison more than 3,000,000 people everyyear in the developing world, mostly through intentional self-poisoning. Advances in medical therapy for OP poisoning have lagged, and current treatment is not highly effective with mortality of up to 40% in even the most advanced Western medical facilities. Administration of a broadly active bacterial OP hydrolase to patients in order to hydrolyze OPs in circulation might allow current therapies to be more effective. The objective of this work was to evaluate the efficacy of a new recombinant bacterial OP hydrolase (OpdA), cloned from Agrobacterium radiobacter, in rat models of two chemically distinct but highly toxic and rapidly acting OP pesticides: dichlorvos and parathion. Without OpdA treatment, median time to death in rats poisoned with 3 x LD50 of dichlorvos or parathion was 6 min and 25.5 min, respectively. Administration of a single dose of OpdA immediately after dichlorvos resulted in 100% survival at 24 h, with no additional antidotal therapy. After parathion poisoning, OpdA alone caused only a delay to death. However, an additional two doses of OpdA resulted in 62.5% survival at 24 h after parathion poisoning. In combination with pralidoxime therapy, a single dose of OpdA increased survival to 75% after parathion poisoning. Our results demonstrate that OpdA is able to improve survival after poisoning by two chemically distinct and highly toxic OP pesticides. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

Pyridoxine 5′-phosphate oxidases (PNPOxs) are known to catalyse the terminal step in pyridoxal 5′-phosphate biosynthesis in a flavin mononucleotide-dependent manner in humans and Escherichia coli. Recent reports of a putative PNPOx from Mycobacterium tuberculosis, Rv1155, suggest that the cofactor or catalytic mechanism may differ in Mycobacterium species. To investigate this, a putative PNPOx from M. smegmatis, Msmeg_3380, has been cloned. This enzyme has been recombinantly expressed in E. coli and purified to homo­geneity. Good-quality crystals of selenomethionine-substituted Msmeg_3380 were obtained by the hanging-drop vapour-diffusion technique and diffracted to 1.2 A using synchrotron radiation.

Enzymes are central to the biology of many pesticides, influencing their modes of action, environmental fates and mechanisms of target species resistance. Since the introduction of synthetic xenobiotic pesticides, enzymes responsible for pesticide turnover have evolved rapidly, in both the target organisms and incidentally exposed biota. Such enzymes are a source of significant biotechnological potential and form the basis of several bioremediation strategies intended to reduce the environmental impacts of pesticide residues. This review describes examples of enzymes possessing the major activities employed in the bioremediation of pesticide residues, and some of the strategies by which they are employed. In addition, several examples of specific achievements in enzyme engineering are considered, highlighting the growing trend in tailoring enzymatic activity to a specific biotechnologically relevant function.

Vertebrate studies show neuroligins and neurexins are binding partners in a trans-synaptic cell adhesion complex, implicated in human autism and mental retardation disorders. Here we report a genetic analysis of homologous proteins in the honey bee. As in humans, the honeybee has five large (31-246 kb, up to 12 exons each) neuroligin genes, three of which are tightly clustered. RNA analysis of the neuroligin-3 gene reveals five alternatively spliced transcripts, generated through alternative use of exons encoding the cholinesterase-like domain. Whereas vertebrates have three neurexins the bee has just one gene named neurexin I (400 kb, 28 exons). However alternative isoforms of bee neurexin I are generated by differential use of 12 splice sites, mostly located in regions encoding LNS subdomains. Some of the splice variants of bee neurexin I resemble the vertebrate alpha- and beta-neurexins, albeit in vertebrates these forms are generated by alternative promoters. Novel splicing variations in the 3' region generate transcripts encoding alternative trans-membrane and PDZ domains. Another 3' splicing variation predicts soluble neurexin I isoforms. Neurexin I and neuroligin expression was found in brain tissue, with expression present throughout development, and in most cases significantly up-regulated in adults. Transcripts of neurexin I and one neuroligin tested were abundant in mushroom bodies, a higher order processing centre in the bee brain. We show neuroligins and neurexins comprise a highly conserved molecular system with likely similar functional roles in insects as vertebrates, and with scope in the honeybee to generate substantial functional diversity through alternative splicing. Our study provides important prerequisite data for using the bee as a model for vertebrate synaptic development.

Polycyclic aromatic hydrocarbons (PAHs) are compounds of intense public concern due to their persistence in the environment and potentially deleterious effects on human, environmental and ecological health. The clean up of such contaminants using invasive technologies has proven to be expensive and more importantly often damaging to the natural resource properties of the soil, sediment or aquifer. Bioremediation, which exploits the metabolic potential of microbes for the clean-up of recalcitrant xenobiotic compounds, has come up as a promising alternative. Several approaches such as improvement in PAH solubilization and entry into the cell, pathway and enzyme engineering and control of enzyme expression etc. are in development but far from complete. Successful application of the microorganisms for the bioremediation of PAH-contaminated sites therefore requires a deeper understanding of the physiology, biochemistry and molecular genetics of potential catabolic pathways. In this review, we briefly summarize important strategies adopted for PAH bioremediation and discuss the potential for their improvement.

Diverse carbamate compounds are widely used as insecticides, herbicides and fungicides in agriculture around the world. Environmental and human health concerns about off-target and off-site residues of various pesticides have sparked interest in the development of technologies for their detoxification. One promising technology for this purpose is enzymatic bioremediation, which uses formulations of detoxifying enzymes to clean up residues in contaminated environments. Here we review the feasibility of developing carbamate detoxifying enzymes as bioremediation agents.

Lucilia cuprina lines, previously characterised by their resistance to diazinon and malathion, were tested for their resistance to another organophosphate, propetamphos. All 13 lines tested showed no difference in propetamphos tolerance, regardless of their resistance to diazinon, malathion or both. It is concluded that resistance to one structural type of organophosphate does not necessarily confer resistance to another.

Sulfur is essential for life on Earth, but its availability is limited in many environments. Here the sulfur-starvation response of the model soil bacterium Pseudomonas putida KT2440 is shown to be associated with an approximately fivefold reduction in the total soluble thiol content of the cell. A bioinformatic survey of the P. putida KT2440 genome identified 646 genes encoding proteins with a significantly lower than average sulfur content (low sulfur-content proteins, LSPs), the expression of which may have a role in the global reduction of cellular thiol content during sulfur starvation. Analysis of the genetic organization of the LSP-encoding genes showed that 31% were potentially transcriptionally associated with at least one other gene encoding a protein defined as an LSP. In particular, 55 LSP genes were located in three large clusters, termed low-sulfur islands (LSIs) here. The predicted identities of the proteins encoded by the LSIs strongly suggest that the LSIs have a role in acquiring sulfur from organic sulfur sources during sulfur starvation. This hypothesis was supported by transcription fusion studies on a limited number of LSP promoters under low-sulfur conditions. In a wider survey of bacterial species, LSIs were found to be more prevalent in free-living, Gram-negative bacteria than in Gram-positive or obligately intracellular bacteria.

The 458 amino acid sequence of a mature JHE protein from the cricket Gryllus assimilis was identifi ed after isolating the partial cDNA sequence encoding this protein from a fat body and midgut cDNA library. This hemimetabolan JHE sequence shows over 40% amino acid similarity to the known JHE sequences of several holometabolous insects. It also includes previously determined peptide sequences for G. assimilis JHE as well as two other motifs associated with JHE enzymes in holometabolous insects. The predicted molecular weight of the protein agrees with that of the JHE previously purifi ed from G. assimilis. Partial genomic sequence encoding the Jhe contains two large (1330 and 2918 bp) introns. No coding DNA sequence variation was observed over a 1293 bp region between selected lines differing six to eight-fold in hemolymph JHE activity. However, a 19 bp indel was found in one of the introns; the insertion was strongly associated with elevated hemolymph activity, both in the selected lines and in the F 2 progeny of crosses between them. Phylogenetic analyses localised the G. assimilis JHE to a clade containing dipteran and coleopteran JHEs, with lepidopteran JHEs occurring in a separate clade.

The honeybee genome has substantially fewer protein coding genes (approximate to 11 000 genes) than Drosophila melanogaster (approximate to 13 500) and Anopheles gambiae (approximate to 14 000). Some of the most marked differences occur in three superfamilies encoding xenobiotic detoxifying enzymes. Specifically there are only about half as many glutathione-S-transferases (GSTs), cytochrome P450 monooxygenases (P450s) and carboxyl/cholinesterases (CCEs) in the honeybee. This includes 10-fold or greater shortfalls in the numbers of Delta and Epsilon GSTs and CYP4 P450s, members of which clades have been recurrently associated with insecticide resistance in other species. These shortfalls may contribute to the sensitivity of the honeybee to insecticides. On the other hand there are some recent radiations in CYP6, CYP9 and certain CCE clades in A. mellifera that could be associated with the evolution of the hormonal and chemosensory processes underpinning its highly organized eusociality.

The phosphotriesterase OpdA from Agrobacterium sp. P230 has about 10-fold higher activity for dimethyl organophosphate (OP) insecticides, than its homologue from Flavobacterium sp. ATCC27551, organophosphate hydrolase (OPH). OpdA shows about 10% amino acid sequence divergence from OPH and also has a 20 residue C-terminal extension. Here we show that the difference in kinetics is largely explained by just two amino acid differences between the two proteins. A truncated form of OpdA demonstrated that the C-terminal extension has no effect on its preference for dimethyl organophosphate substrates. Chimeric proteins of OPH and OpdA were then analysed to show that replacement of a central region of OpdA sequence, which encodes the residues in the large subsite of the active site, with the homologous region in OPH decreased the activity of OpdA towards dimethyl OPs, to values close to those for OPH. Site-directed mutagenesis in this region identified two differences between the proteins, Y257H and F272L (with the OpdA residues first) as being responsible for this reduction. These two differences were also responsible for the increased activity of OpdA towards the diisopropyl organophosphate, diisopropyl fluorophosphate, relative to OPH. Molecular modelling of triethyl phosphate in the active site of OpdA confirmed a reduction in the size of the large subsite relative to OPH.

Ce travail résulte de la collaboration de très nombreux chercheurs. Seuls les auteurs de la rubrique Physical and Genetic Mapping sont cités explicitement.; Here we report the genome sequence of the honeybee Apis mellifera, a key model for social behaviour and essential to global ecology through pollination. Compared with other sequenced insect genomes, the A. mellifera genome has high A1T and CpG contents, lacks major transposon families, evolves more slowly, and is more similar to vertebrates for circadian rhythm, RNA interference and DNA methylation genes, among others. Furthermore, A.mellifera has fewer genes for innate immunity, detoxification enzymes, cuticle-forming proteins and gustatory receptors, more genes for odorant receptors, and novel genes for nectar and pollen utilization, consistent with its ecology and social organization. Compared to Drosophila, genes in early developmental pathways differ in Apis, whereas similarities exist for functions that differ markedly, such as sex determination, brain function and behaviour. Population genetics suggests a novel African origin for the species A.mellifera and insights into whether Africanized bees spread throughout the New World via hybridization or displacement

Mutant insect carboxyl/cholinesterases underlie over 60 cases of resistance to organophosphorus and/or carbarnate insecticides. Biochemical and molecular data on about 20 of these show recurrent use of a very small number of mutational options to generate either target site or metabolic resistance. Moreover, the mutant enzymes are often kinetically inefficient and associated with significant fitness costs, due to impaired performance of the enzymes' original function. By contrast many bacterial enzymes are now known which can effectively detoxify these pesticides. It appears that the constraints of the genetic code and eukaryote genetic systems have severely limited the evolutionary response of insects to the widespread use of the insecticides over the last 60 years. (c) 2005 Elsevier Ireland Ltd. All rights reserved.

Several studies have suggested that esterase-2 (EST-2) may be the target of natural selection in the cactophilic fly Drosophila buzzatii. In this work, we analyzed nucleotide variation in a fragment of α-esterase5 (αE5), the gene encoding EST-2, in original (Argentinian) and colonized (Australian) populations of D. buzzatii and in its sibling D. koepferae. Estimates of nucleotide heterozygosity in D. buzzatii were similar in Australia and Argentina, although we detected a loss of singletons in colonized populations, suggesting a moderate founder effect. Interspecific comparisons revealed that D. buzzatii was more polymorphic for nonsynonymous variation, whereas D. koepferae was more variable for synonymous and noncoding sites. The two major chromosomal arrangements (2st and 2j) in D. buzzatii displayed similar levels of nucleotide variation, whereas 2jz 3 was monomorphic. The sequenced region allowed the discrimination of a greater number of EST-2 protein variants in the Australian sample than in the Argentinean sample. In D. koepferae, nucleotide variation in αE5 does not depart from neutral expectations, although tests of population structure were significant for silent variation. In contrast, D. buzzatii has probably undergone a recent population expansion in its South American range. In addition, the McDonald and Kreitman test revealed an excess of nonsynonymous polymorphism in both original and colonized populations of this species.

Brevibacillus choshinensis has previously been shown to be a useful strain for the secretion of heterologous proteins via the Sec secretory pathway. This pathway involves the secretion of proteins prior to folding, whereas the alternative TAT (twin-arginine translocation) pathway enables pre-folded proteins to be secreted. We have modified the signal peptide of the Brevibacillus expression vector pNCMO2 to accommodate a Sec avoidance signal as well as the twin arginines required for secretion via the TAT system. Use of this modified signal peptide with the phosphotriesterase OpdA enabled B. choshinensis transformants to express and secrete the enzyme in an active and substantially pure form. The system was also used successfully to secrete two cytoplasmic proteins, the phosphotriesterase HocA from Pseudomonas monteilii and the phenylcarbamate-degrading enzyme, PCD, from Arthrobacter oxydans. The inhibitors carbonyl cyanide m-chlorophenyl hydrazine and sodium azide were used to confirm that secretion was occurring via the TAT secretion pathway. The modified B. choshinensis system we have developed may have general utility in secreting a wide range of heterologous proteins in active and conveniently processed form.

"The publication of the extensive seven-volume work Comprehensive Molecular Insect Science provided a complete reference encompassing important developments and achievements in modern insect science, including reviews on the ecdysone receptor, lipocalins, and bacterial toxins. Now two editors of the original series, Drs. Lawrence I. Gilbert and Sarjeet S. Gill, have selected contributions for a special volume of compelling and timeless articles on key aspects of insect pharmacology. Insect Pharmacology includes articles and new addenda covering sodium channels, spider toxins and their potential for insect control, insect transformation for use in control, amino acid and neurotransmitter transporters, and more." "This single-volume was created for the researcher, entomologist, or student with a specialized interest in insect pharmacology." [Book Jacket]

The cloned genes encoding carboxylesterase E3 in the blowfly Lucilia cuprina and its orthologue in Drosophila melanogaster were expressed in Sf9 cells transfected with recombinant baculovirus. Resistance of L. cuprina to organophosphorus insecticides is due to mutations in the E3 gene that enhance the enzyme's ability to hydrolyse insecticides. Previous in vitro mutagenesis and expression of these modifications (G137D, in the oxyanion hole and W251L, in the acyl pocket) have confirmed their functional significance. We have systematically substituted these and nearby amino acids by others expected to affect the hydrolysis of pyrethroid insecticides. Most mutations of G137 markedly decreased pyrethroid hydrolysis. W251L was the most effective of five substitutions at this position. It increased activity with trans permethrin 10-fold, and the more insecticidal cis permethrin >130-fold, thereby decreasing the trans : cis hydrolysis ratio to only 2, compared with >25 in the wild-type enzyme. Other mutations near the bottom of the catalytic cleft generally enhanced pyrethroid hydrolysis, the most effective being F309L, also in the presumptive acyl binding pocket, which enhanced trans permethrin hydrolysis even more than W251L. In these assays with racemic 1RS cis and 1RS trans permethrin, two phases were apparent, one being much faster suggesting preferential hydrolysis of one enantiomer in each pair as found previously with other esterases. Complementary assays with individual enantiomers of deltamethrin and the dibromo analogue of cis permethrin showed that the wild type and most mutants showed a marked preference for the least insecticidal 1S configuration, but this was reversed by the F309L substitution. The W251L/F309L double mutant was best overall in hydrolysing the most insecticidal 1R cis isomers. The results are discussed in relation to likely steric effects on enzyme–substrate interactions, cross-resistance between pyrethroids and malathion, and the potential for bioremediation of pyrethroid residues.

The continued availability of endosulfan is desirable for the production of cotton, and various other crops, because of its particular suitability for use in IPM and resistance management strategies. However, ongoing residue problems threaten the availability of the insecticide. Data described here suggest a beta-enriched insecticide is worthy of investigation as a useful alternative organochloride insecticide, having the advantages of reduced environmental and health concerns. The alpha-isomer of endosulfan is an order of magnitude more volatile than the beta-isomer, which is reflected in its initial rapid disappearance in the field. Approximately 70% of endosulfan is lost within 2 d of application due to volatilization. Based on volatilization kinetics, the volatilization of a beta-endosulfan formulation would be less than 5% that of commercial endosulfan. However, while it has been established that endosulfan vapor does lead to contamination of the riverine environment, this contamination is below residue levels recorded in rivers during the cotton growing season and, as such, reducing the volatility of the insecticide will only partially alleviate residue problems. Initial field trial results suggest that beta-endosulfan insecticide can achieve equivalent efficacy to commercial endosulfan at half the recommended label application rate; presumably this is a reflection of its containment on site in comparison to the more volatile commercial mix of isomers. An insecticide composed primarily of the beta-isomer would have reduced volatility and equivalent efficacy at lower application rates compared to the commercial mix of isomers, reducing offsite endosulfan residues. An important advantage of a beta-enriched insecticide would be its potential to minimize endosulfan residues in locally grown production animals. The predominant endosulfan residue in animal fat is endosulfate, accumulated after the animal ingests the metabolite while grazing on pastures contaminated by endosulfan spray drift. As the beta-isomer is oxidized on the surface of plants and by microbes at much lower rates than the alpha-isomer, endosulfate levels would be lower as a result of a contamination event with a beta-endosulfan-based insecticide compared to the commercial mix. Finally, acute toxicity against mammals of the alpha-isomer is more than three times that of the beta-isomer, and the neurotoxicity of the insecticide has been attributed to the alpha-isomer. Therefore, a beta-enriched insecticide will be less acutely and chronically toxic to agricultural workers than the commercially available insecticide. In conclusion, these properties suggest that the alpha-isomer contributes more significantly to the residue problems associated with the insecticide than the beta-isomer and that the use of a beta-isomer-based insecticide would reduce residue problems yet retain the advantages to IPM and resistant management strategies unique to the current endosulfan formulation.

Many insecticidal products are sold as isomeric mixtures even though the toxicological properties and contribution to environmental residues can differ significantly between isomers. Therefore, increasingly there is a trend toward the use of single-isomer products. Commercial endosulfan (6,7,8,9,10,10-hexachloro-1,5,5a, 6,9,9a-hexahydro-6,9-methano-2,4,3-benzodioxanthiepine 3-oxide) is a broadspectrum insecticide that is used to control a large range of insects and mites in agricultural and horticultural crops. Endosulfan has traditionally been described as a mixture of two diastereoisomers in a ratio of 70% a-endosulfan and 30% sendosulfan. Since cancellation in many countries of most cyclodiene insecticide registrations, the ongoing availability of endosulfan has particular importance for use in rotation in resistance management strategies. However, endosulfan is extremely toxic to fish and aquatic invertebrates and has been implicated increasingly in mammalian gonadal toxicity (Singh and Pandey 1990; Sinha et al. 1995, 1997; Turner et al. 1997), genotoxicity (Chaudhuri et al. 1999), and neurotoxicity (Paul and Balasubramaniam 1997). No suitable alternatives have been identified. In this review, we investigate whether an insecticide containing only one of the endosulfan isomers, preferably β-endosulfan, would be likely to have reduced residue problems yet retain the advantages of being able to use a cyclodiene insecticide in integrated pest management (IPM) and resistance management strategies.

Here we show how the 10 genes of the alpha esterase cluster of Drosophila melanogaster have diverged substantially in their expression profiles. Together with previously described sequence divergence this suggests substantial functional diversification. By peptide mass fingerprinting and in vitro gene expression we have also shown that two of the genes encode the isozymes EST9 (formerly ESTC) and EST23. EST9 is the major ‘alpha staining’ esterase in zymograms of gut tissues in feeding stages while orthologues of EST23 confer resistance to organophosphorus insecticides in other higher Diptera. The results for EST9 and EST23 concur with previous suggestions that the products of the alpha esterase cluster function in digestion and detoxification of xenobiotic esters. However, many of the other genes in the cluster show developmental or tissue-specific expression that seems inconsistent with such roles. Furthermore, there is generally poor correspondence between the mRNA expression patterns of the remaining eight genes and isozymes previously characterized by standard techniques of electrophoresis and staining, suggesting that the alpha cluster might only account for a small minority of the esterase isozyme profile.

We report a transposase gene (tnpA) upstream of the opdA phosphotriesterase gene of Agrobacterium radiobacter P230, as well as inverted repeats indicative of insertion sequences, flanking the two genes. Both the tnpA gene and the inverted repeats resemble the Tn610 transposon from Mycobacterium fortuitum. Two additional putative open reading frames separate opdA and tnpA with inferred translation products with similarity to two proteins encoded on the Geobacillus stearothermophilus IS5376 transposon. To test the proposition that these genes were contained on a transposon, an artificial composite transposon was constructed. This artificial transposon was then delivered into Escherichia coli DH10beta cells. Transposition was demonstrated by the presence of opdA on the E. coli chromosome and confirmation of insertion by inverse polymerase chain reaction. The data presented suggest a possible role of transposition in the distribution of the opd/opdA genes across a wide range of soil bacteria.

A group of transposons, named maT, with characteristics intermediate between mariner and Tc1 transposons, is described. Two defective genomic copies of MdmaT from the housefly Musca domestica, with 85% identity, were found flanking and imbedded in the MdalphaE7 esterase gene involved in organophosphate insecticide resistance. Two cDNA clones, with 99% identity to each other and 72%-89% identity to the genomic copies were also obtained, but both represented truncated versions of the putative open reading frame. A third incomplete genomic copy of MdmaT was also identified upstream of the putative M. domestica period gene. The MdmaT sequences showed high identity to the transposable element Bmmar1 from the silk-worm moth, Bombyx mori, and to previously unidentified sequences in the genome of Caenorhabditis elegans. A total of 16 copies of full-length maT sequences were identified in the C elegans genome, representing three variants of the transposon, with 34%-100% identity amongst them. Twelve of the copies, named CemaT1, were virtually identical, with eight of them encoding a putative full length, intact transposase. Secondary structure predictions and phylogenetic analyses confirm that maT elements belong to the mariner-Tc1 superfamily of transposons, but their intermediate sequence and predicted structural characteristics suggest that they belong to a unique clade, distinct from either mariner-like or Tc1-like elements.

The cloning of a gene encoding the novel phosphotriesterase from Pseudomonas monteilii C11, which enabled it to use the organophosphate (OP) coroxon as its sole phosphorus source, is described. The gene, called hocA (hydrolysis of coroxon) consists of 501 bp and encodes a protein of 19 kDa. This protein had no sequence similarity to any proteins in the SWISS-PROT/GenBank databases. When a spectinomycin-resistance cassette was placed in this gene, phosphotriesterase activity was abolished and P. monteilii C11 could no longer grow with coroxon as the sole phosphorus source. Overexpression and purification of HocA as a maltose-binding protein fusion produced a protein having a broad substrate specificity across oxon and thion OPs. Michaelis-Menten kinetics were observed with the oxon OPs, but not with the thion OPs. End-product inhibition was observed for coroxon-hydrolytic activity. Increased expression of hocA was observed from an integrative hocA-lacZ fusion when cultures were grown in the absence of phosphate, suggesting that it might be part of the Pho regulon, but the phosphate-regulated promoter was not cloned in this study. This is believed to be the first study in which a gene required for an organism to grow with OP pesticides as a phosphorus source has been isolated.

Aim: The aim of this study was to isolate and characterize a bacterium capable of metabolizing endosulfan. Methods and Results: A endosulfan-degrading bacterium (strain ESD) was isolated from soil inoculum after repeated culture with the insecticide as the sole source of sulfur. Analysis of its 16S rRNA gene sequence, and morphological and physiological characteristics revealed it to be a new fast-growing Mycobacterium, closely related to other Mycobacterium species with xenobiotic-degrading capabilities. Degradation of endosulfan by strain ESD involved both oxidative and sulfur-separation reactions. Strain ESD did not degrade endosulfan when sulfite, sulphate or methionine were present in the medium along with the insecticide. Partial degradation occurred when the culture was grown, with endosulfan, in the presence of MOPS (3-(N-morpholino)propane sulphonic acid), DMSO (dimethyl sulfoxide), cysteine or sulphonane and complete degradation occurred in the presence of gutathione. When both beta-endosulfan and low levels of sulphate were provided as the only sources of sulfur, biphasic exponential growth was observed with endosulfan metabolism being restricted to the latter phase of exponential growth. Conclusions: This study isolated a Mycobacterium strain (strain ESD) capable of metabolizing endosulfan by both oxidative and sulfur-separation reactions. The endosulfan-degrading reactions are a result of the sulfur-starvation response of this bacterium. Significance and Impact of the Study: This describes the isolation of a Mycobacterium strain capable of degrading the insecticide endosulfan. This bacterium is a valuable source of enzymes for use in enzymatic bioremediation of endosulfan residues.

Aims: To develop a simple, rapid and sensitive fluorimetric assay to detect, isolate and characterize a soil bacterium capable of degrading the organophosphorus pesticide, coumaphos. Methods and Results: A high throughput microtitre plate-based method was used to quantify coumaphos hydrolysis by the bacterium. The fluorescent hydrolysis product of coumaphos, chlorferon, was detected at levels as low as 10 nmol l−1. Incorporation of coumaphos into agar plates allowed the rapid detection of coumaphos-hydrolysing bacteria when exposed to an excitation wavelength of approximately 340 nm. The coumaphos-hydrolysing enzyme could be visualized when bacterial cell extracts were separated on SDS-PAGE, incubated with coumaphos and exposed to an excitation source as above. Conclusions: This method is 100-fold more sensitive than the currently used spectrophotometric method for coumaphos. Significance and Impact of the Study: This is a unique and versatile tool to screen for bacteria possessing phosphotriesterase activity.

Juvenile hormone esterase (JHE, EC 3.1.1.1) from whole Drosophila melanogaster prepupae has previously been purified by selective precipitations, isoelectric focussing and two column chromatography steps. JHE bands from dried silver-stained SDS-PAGE gels of that material were digested with trypsin. The masses of the tryptic digest peptides were determined by MALDI-TOF mass spectrometry. Only one predicted gene product (CG8425) from the D. Melanogaster genome matches the JHE tryptic fingerprint with high confidence. This predicted JHE sequence includes features that are conserved among all active members of the serine carboxylesterase multigene family as well as features peculiar to JHEs from other species. Also we show that this JHE can be purified by an alternative method using anion exchange chromotography followed by trifluoromethylketone affinity chromatography. A cDNA encoding this JHE was isolated using 3' and 5' RACE. This sequence is in agreement with the Drosophila genome project's prediction except that the sixth predicted intron is not removed, instead there is a stop codon followed by a polyadenylation signal and a polyA tail. (C) 2001 Elsevier Science Ltd. All rights reserved.

Malathion resistance has been shown to be the result of a single point mutation in the LcalphaE7 gene in four independently isolated chromosomes of Lucilia cuprina. The resultant amino acid substitution specifies high malathion carboxylesterase (MCE) activity. We have assayed MCE activities and resistance to malathion in three sets of field-derived samples, two sets of isogenic lines and five mass populations, and show that resistance to malathion in these samples is associated with high MCE activity in both sets of isogenic lines and four of the five mass populations. Additional mechanisms contributing to MCE activity or malathion resistance may be present in one of the mass populations. A second point mutation in LcalphaE7 is responsible for conferring diazinon resistance by encoding an increased organophosphate (OP) hydrolase activity. We also assayed diazinon resistances from the same three samples and show that diazinon and malathion resistances were in complete disequilibrium, with two exceptions. One exception involves the mass population with additional resistance mechanism(s) and the other involves three isogenic lines that are resistant to both insecticides. The molecular data for these lines suggest that they carry a duplication of the LcalphaE7 gene.

Organophosphate (OP) insecticide resistance in certain strains of Musca domestica is associated with reduction in the carboxylesterase activity of a particular esterase isozyme. This has been attributed to a 'mutant ali-esterase hypothesis', which invokes a structural mutation to an ali-esterase resulting in the loss of its carboxylesterase activity but acquisition of OP hydrolase activity. It has been shown that the mutation in Lucilia cuprina is a Gly137--Asp substitution in the active site of an esterase encoded by the Lc alpha E7 gene (Newcomb, R.D., Campbell, P.M., Ollis, D.L., Cheah, E., Russell, R.J., Oakeshott, J.G., 1997. A single amino acid substitution converts a carboxylesterase to an organophosphate hydrolase and confers insecticide resistance on a blowfly. Proc. Natl. Acad. Sci. USA 94, 7464-7468). We now report the cloning and characterisation of the orthologous M. domestica Md alpha E7 gene, including the sequencing of cDNAs from the OP resistant Rutgers and OP susceptible sbo and WHO strains. The Md alpha E7 gene has the same intron structure as Lc alpha E7 and encodes a protein with 76% amino acid identity to Lc alpha E7. Comparisons between susceptible and resistance alleles show resistance in M. domestica is associated with the same Gly137--Asp mutation as in L. cuprina. Bacterial expression of the Rutgers allele shows its product has OP hydrolase activity. The data indicate identical catalytic mechanisms have evolved in orthologous Md alpha E7 and Lc alpha E7 molecules to endow diazinon-type resistance on the two species of higher Diptera.

Resistance to organophosphorus (OP) insecticides is associated with decreased carboxylesterase activity in several insect species. It has been proposed that the resistance may be the result of a mutation in a carboxylesterase that simultaneously reduces its carboxylesterase activity and confers an OP hydrolase activity (the “mutant ali-esterase hypothesis”). In the sheep blowfly, Lucilia cuprina , the association is due to a change in a specific esterase isozyme, E3, which, in resistant flies, has a null phenotype on gels stained using standard carboxylesterase substrates. Here we show that an OP-resistant allele of the gene that encodes E3 differs at five amino acid replacement sites from a previously described OP-susceptible allele. Knowledge of the structure of a related enzyme (acetylcholinesterase) suggests that one of these substitutions (Gly 137 → Asp) lies within the active site of the enzyme. The occurrence of this substitution is completely correlated with resistance across 15 isogenic strains. In vitro expression of two natural and two synthetic chimeric alleles shows that the Asp 137 substitution alone is responsible for both the loss of E3’s carboxylesterase activity and the acquisition of a novel OP hydrolase activity. Modeling of Asp 137 in the homologous position in acetylcholinesterase suggests that Asp 137 may act as a base to orientate a water molecule in the appropriate position for hydrolysis of the phosphorylated enzyme intermediate.

PCR primers designed from the alpha-esterase gene cluster of Drosophila melanogaster have been used to isolate fragments from eight esterase genes in the Australian sheep blowfly, Lucilia cuprina. Phylogenetic analysis suggests that three are homologues of the alpha E7, alpha E8 and alpha E9 genes of the alpha-esterase cluster of D. melanogaster. A further three are also probably alpha-esterases, whereas the remaining two more closely resemble beta-esterases. Transcripts for five of the alpha-esterase genes were detected by PCR in adult midgut, consistent with a role for these enzymes in digestion and/or detoxification. Based on the tissue distribution of these transcripts, Lc alpha E7 may possibly encode the esterase, E3, which is involved in organophosphate resistance.

The primary sequence of the esterase 6 (EST6) enzyme of Drosophila melanogaster contains four potential N-linked glycosylation sites, at residues 21, 399, 435, and 485. Here we determine the extent to which EST6 is glycosylated and how the glycosylation affects the biochemistry and physiology of the enzyme. We have abolished each of the four potential glycosylation sites by replacing the required Asn residues with Gln by in vitro mutagenesis. Five mutant genes were made, four containing mutations of each site individually and the fifth site containing all four mutations. Germline transformation was used to introduce the mutant genes into a strain of D. melanogaster null for EST6. Electrophoretic and Western blot comparisons of the mutant strains and wild-type controls showed that each of the four potential N-linked glycosylation sites in the wild-type protein is glycosylated. However, the fourth site is not utilized on all EST6 molecules, resulting in two molecular forms of the enzyme. Digestion with specific endoglycosidases showed that the glycan attached at the second site is of the high-mannose type, while the other three sites carry more complex oligosaccharides. The thermostability of the enzyme is not affected by abolition of the first, third, or fourth glycosylation sites but is reduced by abolition of the second site. Anomalously, abolition of all four sites together does not reduce thermostability. Quantitative comparisons of EST6 activities showed that abolition of glycosylation does not affect the secretion of the enzyme into the male sperm ejaculatory duct, its transfer to the female vagina during mating, or its subsequent translocation into her hemolymph. However, the activity of the mutant enzymes does not persist in the female's hemolymph for as long as wild-type esterase 6. The latter effect may compromise the role of the transferred enzyme in stimulating egg-laying and delaying receptivity to remating.

A 974-bp region immediately 5' of the esterase 6 gene was sequenced in 17 field derived third chromosome isoallelic lines. Twenty-three polymorphisms were identified, only two in the first 400 bp 5' but 16 in a 325-bp region from -494 to -819 bp. This distribution differs from previously published patterns in Drosophila simulans and D. mauritiana, where the first 800 bp are highly conserved. Fourteen common polymorphisms in the 325-bp region above are all in strong linkage disequilibrium with each other. Moreover, most of the haplotypes defined by the total of 23 polymorphisms fall into two groups that differ as a block at all 14 of these latter sites. Sequence differences between the two groups include some restriction sites that were scored in an earlier study of RFLPs and EST6 enzyme phenotypes among 42 isoallelic lines from the same population. By collating the two studies, we show that one haplotype group yields approximately 15% lower EST6 enzyme activity in adult males than the other. The promoter haplotypes show only weak disequilibrium with the esterase 6 fast/slow allozyme polymorphism, so it seems unlikely that previously reported latitudinal clines in the allozyme frequencies are due to their hitchhiking along with selection on the promoter difference.

The recently reported F420H2-dependent reductases (FDRs) catalyse the reduction of aflatoxins and coumarin via hydrogenation of the α,β-unsaturated moiety. We report that three FDRs (MSMEG_2027, MSMEG_6848 and MSMEG_3356) from Mycobacterium smegmatis also exhibit a different catalytic function towards some aflatoxins through the use of a different cofactor. When F420 was replaced by FMN in these three enzymes, the aflatoxins AFG1 and AFG2 were oxidised via dehydrogenation, producing the reduced cofactor (FMNH2) and an unstable aflatoxin derivative that hydrolyses to an enol with three distinct structural isomers. Both the oxidation and reduction reactions are discussed in detail. This is the first example of an enzyme showing promiscuity for its cofactor leading to divergence of function against the same substrate.

Three glutathione S-transferases from Lucilia cuprina (Australian sheep blowfly) pupae were purified by affinity chromatography and anion-exchange chromatography. One isoenzyme was composed of M(r)-24,800 subunits, and two isoenzymes had subunits of M(r) 23,900. The M(r)-23,900 subunits showed immunological identity and were immunologically distinct from the M(r)-24,800 subunits. All three enzymes were active with the substrate 1-chloro-2,4-dinitrobenzene and had low activity with 1,2-dichloro-4-nitrobenzene. A cDNA clone encoding a M(r)-23,900 subunit (LuGST1) was isolated and sequenced. The sequence has close similarities (> 81%) to that of GSTs from the fruitfly Drosophila melanogaster and Musca domestica (housefly). The deduced amino acid sequence of the Lu GST1 subunit showed no significant similarity to that of the mammalian GSTs to the Alpha, Mu and Pi classes, but shows some similarity (33%) over the first 100 residues with the rat subunit 12 Theta-class GST. Southern blots of genomic DNA hybridized with the LuGST1 cDNA identified many hybridizing fragments. Taken together, these data indicated that the L. cuprina genome contains multiple glutathione S-transferase genes.

Strain SG-6C (DSM 23264, CCM 7827) is a chemolithoautotrophic bacterium of the family Bradyrhizobiaceae . It can also grow heterotrophically under appropriate environmental conditions. Here we report the annotated genome sequence of this strain in a single 4.3-Mb circular scaffold.

Microfluidic droplet technology has gained popularity due to the advantages over conventional emulsion techniques and capabilities for a wide range of applications. In this paper, the development of a simple microfluidic-based double-emulsion system is reported. Such a system could be potentially used for in vitro protein synthesis. The system involves a two-step process to make water-in-oil-in-water (W/O/W) emulsions. A PMMA microchip is used for the formation of water-in-oil (W/O) single-emulsion droplets. Then, the single-emulsion droplets are transported to a PDMS/glass microchip to make the W/O/W double-emulsion droplets. The system was first characterized by detecting fluorescein sodium salt as a model dye in the internal aqueous droplets using laser-induced fluorescence. The effect of the flow rates of the internal aqueous phase and outer continuous aqueous phase on the formation of the double-emulsion droplets is investigated to provide information for system optimization. On-chip storage of double-emulsion droplets is also investigated to allow for protein synthesis from a PCR-generated DNA template using either commercial in vitro transcription and translation kits or crude Escherichia coli S30 extracts. In vitro expression of the green fluorescent protein is successfully demonstrated in this system.

Esterase 6 in Drosophila melanogaster, Drosophila simulans and Drosophila mauritiana is produced in several life stages and diverse tissues, but the major pulse of expression is in the sperm ejaculatory duct of adult males. Comparison of EST6 activity levels among several lines of D. melanogaster, D. simulans and D. mauritiana reveals two major quantitative differences among the species. First, newly eclosed females of both D. simulans and D. mauritiana show significantly higher EST6 activity than those of D. melanogaster. Secondly, 5-day-old adult D. simulans have significantly higher activities than D. mauritiana in both sexes and significantly higher activity than D. melanogaster in males. The genetic bases of the differences between D. melanogaster and the other species are investigated through germ line transfer of the D. simulans and D. mauritiana Est-6 genes plus 1.2 kb of 5' and 0.2 kb of their 3' flanking sequences into D. melanogaster. The newly eclosed female activities of the transformants resemble those of the two donor species, suggesting that the interspecific differences in this aspect of expression are due to cis-inherited factors contained within the transferred DNA. In contrast, the 5-day adult activity of the D. simulans transgene resembles the recipient species, D. melanogaster, suggesting that the difference between D. simulans and D. melanogaster in this aspect of expression is due to trans-acting factors. We also find that third instar larval activities of the D. simulans transgene and 5-day male activities of the D. mauritiana transgene are lower than those of either parental species, suggesting that not all the promoter elements relevant to these aspects of expression are included in the transferred DNA.

Corrigendum to “Gene identification and proteomic analysis of the esterases of the cotton bollworm, Helicoverpa armigera” [Insect Biochem. Mol. Biol. 40 (2010) 1e16] Mark G. Teese , Peter M. Campbell , Colin Scott , Karl H.J. Gordon , Adam Southon , Daniel Hovan , Charles Robin , Robyn J. Russell , John G. Oakeshott a,* CSIRO Entomology, GPO Box 1700, Canberra ACT 2601, Australia Research School of Chemistry, Australian National University, Canberra ACT 0200, Australia Department of Genetics, The University of Melbourne, Vic. 3010, Australia

Increasingly over the past two decades, biotechnologists have been exploiting various molecular technologies for high-throughput screening of genes and their protein products to isolate novel functionalities with a wide range of industrial applications. One particular technology now widely used for these purposes involves directed evolution, an artificial form of evolution in which genes and proteins are evolved towards new or improved functions by imposing intense selection pressures on libraries of mutant genes generated by molecular biology techniques and expressed in heterologous systems such as Escherichia coli. Most recently, the rapid development of droplet-based microfluidics has created the potential to dramatically increase the power of directed evolution by increasing the size of the libraries and the throughput of the screening by several orders of magnitude. Here, we review the methods for generating and controlling droplets in microfluidic systems, and their applications in directed evolution. We focus on the methodologies for cell-based assays, in vitro protein expression and DNA amplification, and the prospects for using such platforms for directed evolution in next-generation biotechnologies.

Approximately 50 kb of genomic DNA was isolated from polytene chromosome bands 19F1 and 2 of Drosophila melanogaster. Bands 19F1 and 2 are in the immediate vicinity of the beta-heterochromatin at the base of the X chromosome and encompass the little fly-like and lethal(1)B214 complementation groups. The cloned DNA consists of an approximately 21 kb stretch of unique or low copy number sequence that is bounded by repetitive elements interspersed with further unique sequences. The presence of repeated sequences is characteristic of regions within and adjacent to beta-heterochromatin. At least part of a tRNA gene cluster is present within the 50 kb of cloned DNA. The cloned region also produces at least 18 discrete size classes of developmentally regulated poly(A)+ RNA species. A 2 kb EcoRI fragment (E10), which lies in the 21 kb stretch of unique sequence, generates seven of these transcripts (of sizes 3.5, 3.35, 2.1, 2.0, 1.5, 1.2 and 1.0 kb) in wild-type flies. However, a small deletion of approximately 75 bp in E10 in a lethal(1)B214 mutant allele is associated with alterations in the production or processing of all seven of these transcripts. These data identify E10 sequences as belonging to the lethal(1)B214 gene and suggest that the wild-type lethal(1)B214 gene encodes multiple transcripts. Furthermore, no transcripts of the same size and having the same developmental profile as those generated by the wild-type E10 fragment were identified by probes covering the remainder of the cloned region. This suggests that at least the larger transcripts hybridizing to E10 are partly transcribed from sequences located outside the cloned region, which indicates that the lethal(1)B214 gene extends for more than 20 kb and contains other transcriptionally active sequences within it.

Droplet based microfluidics are promising new tools for biological and chemical assays. In this paper, a high throughput and high sensitivity microfluidic droplet platform is described for in vitro protein expression using crude Escherichia coli S30 extract. A flow-focusing polymethylmethacrylate (PMMA) microchip was designed and integrated with different functions involving droplet generation, storage, separation and detection. The material used for the chip is superior to the previously tested polydimethylsiloxane (PDMS) due to its mechanical and chemical properties. Droplet formation characteristics such as size and generation rate are investigated systematically. The effect of surfactants Abil EM90 and Span80 in the oil phase on droplet formation and optical detection is also studied. The performance of the system is demonstrated by the high throughput and stable droplet generation and ultralow detection limit. The robustness of the system is also demonstrated by the successful synthesis of a green fluorescent protein (GFP) using E. coli S30 extract as a source of RNA translation reagents.

Twenty-two soluble esterases have been identified in D. melanogaster by combining the techniques of native polyacrylamide gel electrophoresis and isoelectric focusing. The sensitivity of each isozyme to three types of inhibitors (organophosphates, eserine sulfate, and sulfydryl reagents) identified 10 as carboxylesterases, 6 as cholinesterases, and 3 as acetylesterases. Three isozymes could not be classified and no arylesterases were identified. The carboxyl- and cholinesterases could each be further divided into two subclasses on the basis of inhibition by organophosphates and sulfhydryl reagents, respectively. Choline- and acetylesterases have characteristic substrate preferences but both subclasses of carboxylesterases are heterogeneous in substrate utilization. Subclass 2 carboxylesterases exhibit diverse temporal expression patterns, with subclass 1 carboxylesterases generally found in larvae and subclass 1 cholinesterases and acetylesterases more characteristic of pupae and adults. Tissues showing the greatest number of isozymes are larval body wall (eight) and digestive tract (six in larvae, six in adults). Carboxylesterases are distributed across a wide range of tissues, but subclass 1 cholinesterases are generally associated with neural or neurosecretory tissues and subclass 2 cholinesterases with digestive tissues.

Thirty-five nucleotide polymorphisms were found in a 21.5-kbp region including the Est6 locus among 42 isoallelic lines extracted from a single natural population of Drosophila melanogaster. The heterozygosity per nucleotide pair was estimated to be 0.010 overall, but was lower in sequences hybridizing to transcripts than in those not hybridizing to transcripts. Eleven of 36 pairwise comparisons among the nine most common polymorphisms showed significant gametic disequilibrium. Four of these polymorphisms were also significantly associated with the major EST6-F/EST6-S allozyme polymorphism. Significant disequilibrium was generally restricted to polymorphisms less than 1-2 kbp apart. Previously reported measures of EST6 activity in virgin adult females proved not to be significantly associated with any of the six most common nucleotide polymorphisms located in the Est6 coding region or the 1.5 kbp immediately 5'. However, the 11 haplotypes for five of these polymorphisms that lie in the 1.5-kbp 5' region could explain about half of the previously reported variation among the lines for both EST6 activity and the amount of EST6 protein in virgin adult males. One particular polymorphism, for a RsaI site 530 bp 5' of the initiation codon, could explain 21% of the male activity variation among lines. This site is embedded in a large palindrome and we suggest that sequences including or close to this site may be involved in the regulation of EST6 synthesis in the ejaculatory duct of the adult male.