De novo assembly, gene annotation, and marker discovery in stored-product pest Liposcelis entomophila (Enderlein) using transcriptome sequences.

BACKGROUND: As a major stored-product pest insect, Liposcelis entomophila has developed high levels of resistance to various insecticides in grain storage systems. However, the molecular mechanisms underlying resistance and environmental stress have not been characterized. To date, there is a lack of genomic information for this species. Therefore, studies aimed at profiling the L. entomophila transcriptome would provide a better understanding of the biological functions at the molecular levels. METHODOLOGY/PRINCIPAL FINDINGS: We applied Illumina sequencing technology to sequence the transcriptome of L. entomophila. A total of 54,406,328 clean reads were obtained and that de novo assembled into 54,220 unigenes, with an average length of 571 bp. Through a similarity search, 33,404 (61.61%) unigenes were matched to known proteins in the NCBI non-redundant (Nr) protein database. These unigenes were further functionally annotated with gene ontology (GO), cluster of orthologous groups of proteins (COG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. A large number of genes potentially involved in insecticide resistance were manually curated, including 68 putative cytochrome P450 genes, 37 putative glutathione S-transferase (GST) genes, 19 putative carboxyl/cholinesterase (CCE) genes, and other 126 transcripts to contain target site sequences or encoding detoxification genes representing eight types of resistance enzymes. Furthermore, to gain insight into the molecular basis of the L. entomophila toward thermal stresses, 25 heat shock protein (Hsp) genes were identified. In addition, 1,100 SSRs and 57,757 SNPs were detected and 231 pairs of SSR primes were designed for investigating the genetic diversity in future. CONCLUSIONS/SIGNIFICANCE: We developed a comprehensive transcriptomic database for L. entomophila. These sequences and putative molecular markers would further promote our understanding of the molecular mechanisms underlying insecticide resistance or environmental stress, and will facilitate studies on population genetics for psocids, as well as providing useful information for functional genomic research in the future.

Transcriptomic and phylogenetic analysis of Culex pipiens quinquefasciatus for three detoxification gene families.

BACKGROUND: The genomes of three major mosquito vectors of human diseases, Anopheles gambiae, Aedes aegypti, and Culex pipiens quinquefasciatus, have been previously sequenced. C. p. quinquefasciatus has the largest number of predicted protein-coding genes, which partially results from the expansion of three detoxification gene families: cytochrome P450 monooxygenases (P450), glutathione S-transferases (GST), and carboxyl/cholinesterases (CCE). However, unlike An. gambiae and Ae. aegypti, which have large amounts of gene expression data, C. p. quinquefasciatus has limited transcriptomic resources. Knowledge of complete gene expression information is very important for the exploration of the functions of genes involved in specific biological processes. In the present study, the three detoxification gene families of C. p. quinquefasciatus were analyzed for phylogenetic classification and compared with those of three other dipteran insects. Gene expression during various developmental stages and the differential expression responsible for parathion resistance were profiled using the digital gene expression (DGE) technique. RESULTS: A total of 302 detoxification genes were found in C. p. quinquefasciatus, including 71 CCE, 196 P450, and 35 cytosolic GST genes. Compared with three other dipteran species, gene expansion in Culex mainly occurred in the CCE and P450 families, where the genes of α-esterases, juvenile hormone esterases, and CYP325 of the CYP4 subfamily showed the most pronounced expansion on the genome. For the five DGE libraries, 3.5-3.8 million raw tags were generated and mapped to 13314 reference genes. Among 302 detoxification genes, 225 (75%) were detected for expression in at least one DGE library. One fourth of the CCE and P450 genes were detected uniquely in one stage, indicating potential developmentally regulated expression. A total of 1511 genes showed different expression levels between a parathion-resistant and a susceptible strain. Fifteen detoxification genes, including 2 CCEs, 6 GSTs, and 7 P450s, were expressed at higher levels in the resistant strain. CONCLUSIONS: The results of the present study provide new insights into the functions and evolution of three detoxification gene families in mosquitoes and comprehensive transcriptomic resources for C. p. quinquefasciatus, which will facilitate the elucidation of molecular mechanisms underlying the different biological characteristics of the three major mosquito vectors.

Cholinesterase activity in Gammarus pulex (Crustacea Amphipoda): characterization and effects of chlorpyrifos.

The aim of this study was to characterize cholinesterase (ChE) activity in Gammarus pulex, an abundant and ecologically relevant species of the European stream environment. Biochemical and pharmacological properties were tested using different substrates (acetylthiocholine iodide, propionylthiocholine iodide and butyrylthiocholine iodide) and selective inhibitors (eserine sulfate, BW284c51 and iso-OMPA). In a second part, the in vitro and in vivo effects of a widely used organophosphorous pesticide, chlorpyrifos, on ChE activity were investigated. The results suggest that G. pulex possess only one ChE which displays the typical properties of an acetylcholinesterase, since: (1) it hydrolyses to the substrate acetylthiocholine at a higher rate than all other tested substrates and (2) it is highly sensitive to eserine sulphate and BW284c51, but not to iso-OMPA. In vitro and in vivo inhibitions were observed for highly different contamination levels, which suggests that bioaccumulation and biotransformation mechanisms are involved. In vivo AChE inhibition was observed at realistic environmental concentrations, with lethal effects appearing at inhibitions higher than 50%. The results of this study show the value of G. pulex as a sentinel organism for environmental assessment.

New friendly tools for users of ESTHER, the database of the alpha/beta-hydrolase fold superfamily of proteins.

The structural alpha/beta-hydrolase fold is characterized by a beta-sheet core of five to eight strands connected by alpha-helices to form a alpha/beta/alpha sandwich. The superfamily members, exemplified by the cholinesterases, diverged from a common ancestor into a number of hydrolytic enzymes displaying a wide range of substrate specificities, along with proteins with no recognized hydrolytic activity. In the enzymes, the catalytic triad residues are presented on loops of which one, the nucleophile elbow, is the most conserved feature of the fold. Of the other proteins, which all lack from one to all of the catalytic residues, some may simply be 'inactive' enzymes while others have been shown to be involved in heterologous surface recognition functions. The ESTHER (for esterases, alpha/beta-hydrolase enzymes and relatives) database (http://bioweb.ensam.inra.fr.esther) gathers and annotates all the published pieces of information (gene and protein sequences; biochemical, pharmacological, and structural data) related to the superfamily, and connects them together to provide the bases for studying structure-function relationships within the superfamily. The most recent developments of the database are presented.

The origin of the molecular diversity and functional anchoring of cholinesterases.

Vertebrates possess two cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) which both hydrolyze acetylcholine, but differ in their specificity towards other substrates, and in their sensitivity to inhibitors. In mammals, the AChE gene produces three types of coding regions through the choice of 3' splice acceptor sites, generating proteins which possess the same catalytic domain, associated with distinct C-terminal peptides. AChE subunits of type R ('readthrough') produce soluble monomers; they are expressed during development and induced by stress in the mouse brain. AChE subunits of type H ('hydrophobic') produce GPI-anchored dimers, but also secreted molecules; they are mostly expressed in blood cells. Subunits of type T ('tailed') exist for both AChE and BChE. They represent the enzyme forms expressed in brain and muscle. These subunits generate a variety of quaternary structures, including homomeric oligomers (monomers, dimers, tetramers), as well as hetero-oligomeric assemblies with anchoring proteins, ColQ and PRiMA. Mutations in the four-helix bundle (FHB) zone of the catalytic domain indicate that subunits of type H and T use the same interaction for dimerization. On the other hand, the C-terminal T peptide is necessary for tetramerization. Four T peptides, organized as amphiphilic alpha helices, can assemble around proline-rich motifs of ColQ or PRiMA. The association of AChE(T) or BChE subunits with ColQ produces collagen-tailed molecules, which are inserted in the extracellular matrix, e.g. in the basal lamina of neuromuscular junctions. Their association with PRiMA produces membrane-bound tetramers which constitute the predominant form of cholinesterases in the mammalian brain; in muscles, the level of PRiMA-anchored tetramers is regulated by exercise, but their functional significance remains unknown. In brain and muscles, the hydrolysis of acetylcholine by cholinesterases, in different contexts, and their possible noncatalytic functions clearly depend on their localization by ColQ or PRiMA.

Distribution profiles of paraoxonase and cholinesterase phenotypes in a Spanish population.

The paraoxonase/arylesterase phenotype was measured in a Spanish population as previous studies have reported that the polymorphic variation in serum paraoxonase activity may affect the metabolism of organophosphates in individuals at risk of chronic intoxication. The prevalence of congenital deficiency in serum cholinesterase was also established in order to ascertain whether individuals with a congenital defect would be at a higher risk against a potential organophosphate exposure. We consider it useful to incorporate these two biomarkers into the health programme of agricultural workers with the purpose of monitoring workers who spray organophosphate pesticides, as they provide reliable indications of early-stage effects related to biochemical alterations that might precede overt clinical pictures.

[Cholinesterase].

Since the chromatographic separation of cholinesterase (ChE) by Malström in 1956 many investigator studied ChE isozyme, Harris divided five spots by two dimensional paper electrophoresis and starchgel electrophoresis, and referred as C1 C2 C3 C4. Clinically, Juul separated ChE 12 bands by polyacrylamidegel electrophoresis. We separated ChE as five bands using polyacrylamidegel electrophoresis, revealing fusion and deformity of the band. Takahashi et al reported separation of band using acetyl and butyrylthiocholine as substrate. They found abnormal band in liver cirrhosis, however they have thought it acetyl cholinesterase. Hada et al revealed a defect of band II in liver cirrhosis. They investigated ChE isozyme using affinity electrophoresis with Concanavalin A (Con A) and wheat germ agglutinin (WGA). They found disappearance of band 2, Con A and WGA containing agarose gel electrophoresis seem to be useful method in differentiating liver cirrhosis from chronic hepatitis. The number of isozyme fraction exhibited a species related variations in laboratory animals. Rats, hamsters guinea pigs, rabbits, dogs, monkeys, pigs, horses and quails have 4, 3, 4, 3-5, 3, 3, 4 and 3 isozyme bands, respectively.

Nature of cholinesterase in the rat retina.

The occurrence of cholinesterases has been demonstrated in retinas of several mammalian species. Histochemical staining techniques indicate that the acetylcholinesterases (AChE) are present in amacrine cells and their neighboring bipolar cells. However, the nature of retinal cholinesterases and their interactions with specific cholinesterase inhibitors are not known. Therefore, we have studied the inhibition of the rat retinal cholinesterase activity by BW284C51, a selective inhibitor of AChE, and iso-OMPA, a selective inhibitor of butyrylcholinesterase (BChE). Retinas from Zivic-Miller rats were solubilized by sonication in phosphate buffer (0.134 M, pH 7.2) at 4 degrees C for 20 min. The cholinesterase activity in the sonicate was determined by a radiometric method using 14C-acetylcholine (ACh) as substrate (10(-2) M). Excess 14C-ACh was adsorbed by Amberlite CG-120 cation exchange resin. 14C-acetate formed and retained in the aqueous medium was determined by liquid scintillation counting. This study gave the following results: (a) Rat retinal sonicate gave total cholinesterase activity of 3.76 mumol of ACh hydrolyzed/mg protein/15 min; (b) This activity was inhibited by BW284C51 (IC50, 0.115 microM). Iso-OMPA (IC50, 500 microM) did not cause significant inhibition at 0.115 microM. These observations suggest that the rat retinal cholinesterase is predominantly AChE.

Interactions of bambuterol with human serum cholinesterase of the genotypes EuEu (normal), EaEa (atypical) and EuEa.

Bambuterol, a carbamate ester prodrug of the bronchodilator terbutaline, was tested as inhibitor and substrate of human serum cholinesterases of the genotypes EuEu (the normal enzyme), EaEa (the atypical enzyme) and EuEa. The IC50 for the normal enzyme was 11 +/- 2.2 nM (mean, SD, N = 10) and for the atypical enzyme 140 +/- 6 nM (N = 13), indicating a much higher affinity of bambuterol to the normal enzyme. The heterozygotes showed a mixed behaviour; the major activity was inhibited like the normal enzyme (IC50 = 9.3 +/- 1.9 nM, N = 9), while a residual activity (10-15%) was inhibited by bambuterol like the atypical enzyme. At a bambuterol concentration of 100 nM each of the three cholinesterase genotypes responded uniquely to bambuterol; the normal enzyme was inhibited to 2.2 +/- 0.9%, the atypical enzyme to 58 +/- 4.6%, and the heterozygote to 10 +/- 1.2% of the basal activity. Bambuterol may therefore be added to the list of inhibitors useful in the genotyping of cholinesterases. Bambuterol was much less efficiently hydrolysed in serum containing the atypical cholinesterase than in serum containing the normal enzyme. The results of the hydrolysis experiments once again illustrate the difference in affinity of bambuterol to the genetic forms of cholinesterase, and also strengthen the evidence that cholinesterase is the major serum enzyme catalysing the hydrolysis of bambuterol.

Interspecific variations of cerebral endothelial cholinesterases in rodents and carnivores.

The cholinesterase equipment of cerebral microvessels was studied in some rodents and carnivores using the Koelle-Friedenwald histochemical method with 3 artificial substrates and specific inhibitors for butyrylcholinesterase or acetylcholinesterase. Our observations reveal a great heterogeneity in cholinesterase types and their distribution in each of the different species studied. Only in the rat, butyrylcholinesterase appears to be a marker for the microvessels provided with a blood-brain barrier.