Annotation and analysis of low-complexity protein families of Anopheles gambiae that are associated with cuticle

We have characterized four new families of homologous genes of the mosquito, Anopheles gambiae , all of which include members shown by previous work to be cuticular in nature. The CPLCG, CPLCW, CPLCP, and CPLCA families (where CPLC is 'cuticular protein of low complexity') encode proteins with a high proportion of low-complexity sequence. We have also annotated the An. gambiae Tweedle genes, a family of cuticular protein genes first described in Drosophila , and additional ungrouped An. gambiae cuticular proteins identified by proteomics. Our annotations reveal multiple gene-family expansions that are specific to Diptera or Culicidae. The CPLCG and CPLCW families occur within a large and dynamic tandem array on chromosome 3R that includes sets of concertedly evolving genes. Most gene families exhibit two or more different expression profiles during development.     

Collaborative contribution of six cytochrome P450 monooxygenase genes to fenpropathrin resistance in Tetranychus cinnabarinus (Boisduval)

Cytochrome P450 monooxygenases (P450s), as an important family of detoxification enzymes, participate in the metabolism of agrochemicals in almost all agricultural pests and play important roles in the development of insecticide resistance. Two P450 genes (CYP389B1 and CYP392A26) were identified and their expression patterns were investigated in our previous study. In this study, four more P450 gene sequences (CYP391A1, CYP384A1, CYP392D11 and CYP392A28) from the Clan 2, Clan 3 and Clan 4 families were identified and characterized. Quantitative PCR analysis showed that these four P450 genes were highly expressed in a fenpropathrin‐resistant (FeR) strain of Tetranychus cinnabarinus. In addition, their expressions were much more sensitive to fenpropathrin induction in the FeR strain than the susceptible strain. Gene‐silencing experiments via double‐stranded RNA feeding were carried out. The results showed that mRNA levels of these six P450 genes were reduced in the FeR strain and the activities of P450s were decreased. Consequently mite susceptibilities to fenpropathrin were increased. Interestingly, silencing all six P450 genes simultaneously had an even greater effect on resistance than silencing them individually. This study increases our understanding of the molecular mechanisms of insecticide detoxification, suggesting that the overexpression of these six P450 genes might play important roles in fenpropathrin resistance in T. cinnabarinus collaboratively.     

The Aedes aegypti genome: a comparative perspective

The sequencing of the second mosquito genome, Aedes aegypti , in addition to Anopheles gambiae , is a major milestone that will drive molecular-level and genome-wide high-throughput studies of not only these but also other mosquito vectors of human pathogens. Here we overview the ancestry of the mosquito genes, list the major expansions of gene families that may relate to species adaptation processes, as exemplified by CYP9 cytochrome P450 genes, and discuss the conservation of chromosomal gene arrangements among the two mosquitoes and fruit fly. Many more invertebrate genomes are expected to be sequenced in the near future, including additional vectors of human pathogens (see http://www.vectorbase.org ), and further comparative analyses will become increasingly refined and informative, hopefully improving our understanding of the genetic basis of phenotypical differences among these species, their vectorial capacity, and ultimately leading to the development of novel disease control strategies.     

Identification of a distinct family of genes encoding atypical odorant-binding proteins in the malaria vector mosquito, Anopheles gambiae

We performed a genome-wide analysis for candidate odorant-binding protein (OBP) genes in the malaria vector Anopheles gambiae (Ag). We identified fifty-seven putative genes including sixteen genes predicted to encode distinct, higher molecular weight proteins that lack orthologues in Drosophila. Expression analysis indicates that several of these atypical AgOBPs are transcribed in chemosensory organs in adult and immature stages. Phylogenetic analysis of the Anopheles and Drosophila OBP families reveals these proteins fall into several clusters based on sequence similarity and suggests the atypical AgOBP genes arose in the mosquito lineage after the divergence of mosquitoes and flies. The identification of these AgOBP genes is the first step towards determining their biological roles in this economically and medically important insect.     

Anopheles gambiae P450 reductase is highly expressed in oenocytes and in vivo knockdown increases permethrin susceptibility

We describe an in vivo model for investigation of detoxification mechanisms of the mosquito Anopheles gambiae, important for the development of malaria control programmes. Cytochrome P450s are involved in metabolic insecticide resistance and require NADPH cytochrome P450 reductase (CPR) to function. Here we demonstrate that the major sites of adult mosquito CPR expression are oenocytes, mid-gut epithelia and head appendages. High CPR expression was also evident in Drosophila oenocytes indicating a general functional role in these insect cells. RNAi mediated knockdown drastically reduced CPR expression in oenocytes, and to a lesser extent in mid-gut epithelia; the head was unaffected. These flies showed enhanced sensitivity to permethrin, demonstrating a key role for abdominal/mid-gut P450s in pyrethroid metabolism, aiding the development of insecticides.     

Molecular characterization,expression pattern and metabolic activity of flavin‐dependent monooxygenases in Spodoptera exigua

Enhanced detoxification is one of the important mechanisms for insecticide resistance. Most research in this field to date has focused on the role of cytochrome P450s. Our previous work revealed that flavin‐dependent monooxygenases (FMOs) were involved in metabolic resistance of Spodoptera exigua. In the present study we investigated the molecular characteristics, expression patterns and oxidative activities of SeFMO on insecticides. Three FMO genes, which encode proteins with the typical FMO motifs, were cloned from S. exigua. The oxidative activities of eukaryotically expressed SeFMO enzymes were verified with the model substrate of FMO. Importantly, the SeFMOs had significantly higher oxidative activities on metaflumizone and lambda‐cyhalothrin than on model substrates and other insecticides tested. The three SeFMOs were mainly expressed in the midgut, fat body and Malpighian tubules. The tissues responsible for xenobiotic metabolism and their expression characteristics were similar to those of P450s acting as detoxification genes. The study also revealed that the expression of SeFMOs could be induced by insecticide exposure, and that SeFMOs were over‐expressed in a metaflumizone‐resistant strain of S. exigua. These results suggest that SeFMOs are important insecticide detoxifying enzymes, and that over‐expression of FMO genes may be one of the mechanisms for metabolic resistance in S. exigua.     

Developmental expression and gene/enzyme identifications in the alpha esterase gene cluster of Drosophila melanogaster

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.     

Effects of CFTR, interleukin-10, and Pseudomonas aeruginosa on gene expression profiles in a CF bronchial epithelial cell Line.

Mutations in CFTR lead to a complex phenotype that includes increased susceptibility to Pseudomonas infections, a functional deficiency of IL-10, and an exaggerated proinflammatory cytokine response. We examined the effects of CFTR gene correction on the gene expression profile of a CF bronchial epithelial cell line (IB3-1) and determined which CF-related gene expression changes could be reversed by IL-10 expression. We performed microarray experiments to monitor the gene expression profile of three cell lines over a time course of exposure to Pseudomonas. At baseline, we identified 843 genes with statistically different levels of expression in CFTR-corrected (S9) cells compared to the IB3-1 line or the IL-10-expressing line. K-means clustering and functional group analysis revealed a primary up-regulation of ubiquitination enzymes and TNF pathway components and a primary down-regulation of protease inhibitors and protein glycosylation enzymes in CF. Key gene expression changes were confirmed by real-time RT-PCR. Massive reprogramming of gene expression occurred 3 h after Pseudomonas exposure. Changes specific to CF included exaggerated activation of cytokines, blunted activation of anti-proteases, and repression of protein glycosylation enzymes. In conclusion, the CFTR genotype changes the expression of multiple genes at baseline and in response to bacterial challenge, and only a subset of these changes is secondary to IL-10 deficiency.     

Gene expression in insecticide resistant and susceptible Anopheles gambiae strains constitutively or after insecticide exposure

A microarray containing approximately 20 000 expressed sequence tags (ESTs; 11 760 unique EST clusters) from the malaria vector, Anopheles gambiae, was used to monitor differences in global gene expression in two insecticide resistant and one susceptible strains. Statistical analysis identified 77 ESTs that were differentially transcribed among the three strains. These include the cytochrome P450 CYP314A1, over-transcribed in the DDT resistant ZAN/U strain, and many genes that belong to families not usually associated with insecticide resistance, such as peptidases, sodium/calcium exchangers and genes implicated in lipid and carbohydrate metabolism. Short-term (6 and 10 h) effects of exposure of the pyrethroid resistant RSP strain to permethrin were also detected. Several genes belonging to enzyme families already implicated in insecticide or xenobiotic detoxification were induced, including the carboxylesterase COEAE2F gene and members of the UDP-glucuronosyl transferase and nitrilase families.     

Knockout of cytochrome P450 3A yields new mouse models for understanding xenobiotic metabolism

Cytochrome P450 3A (CYP3A) enzymes constitute an important detoxification system that contributes to primary metabolism of more than half of all prescribed medications. To investigate the physiological and pharmacological roles of CYP3A, we generated Cyp3a-knockout (Cyp3a-/-) mice lacking all functional Cyp3a genes. Cyp3a-/- mice were viable, fertile, and without marked physiological abnormalities. However, these mice exhibited severely impaired detoxification capacity when exposed to the chemotherapeutic agent docetaxel, displaying higher exposure levels in response to both oral and intravenous administration. These mice also demonstrated increased sensitivity to docetaxel toxicity, suggesting a primary role for Cyp3a in xenobiotic detoxification. To determine the relative importance of intestinal versus hepatic Cyp3a in first-pass metabolism, we generated transgenic Cyp3a-/- mice expressing human CYP3A4 in either the intestine or the liver. Expression of CYP3A4 in the intestine dramatically decreased absorption of docetaxel into the bloodstream, while hepatic expression aided systemic docetaxel clearance. These results suggest that CYP3A expression determines impairment of drug absorption and efficient systemic clearance in a tissue-specific manner. The genetic models used in this study provide powerful tools to further study CYP3A-mediated xenobiotic metabolism, as well as interactions between CYP3A and other detoxification systems.     

Functional validation of the carbon dioxide receptor genes in Aedes aegypti mosquitoes using RNA interference

Carbon dioxide (CO(2)) is an important long-range chemosensory cue used by blood-feeding female mosquitoes to find their hosts. The CO(2) receptor in Drosophila melanogaster was previously determined to be a heterodimer comprised of two gustatory receptor (Gr) proteins, DmGr21a and DmGr63a. In the mosquito Aedes aegypti, two putative orthologous genes, AaGr1 and AaGr3, were identified in the genome database, along with an apparent paralogue of AaGr1, AaGr2. In this study, RNA interference (RNAi)-mediated gene knockdown of either AaGr1 or AaGr3 resulted in a loss of CO(2) sensitivity in both male and female mosquitoes, suggesting that these two proteins, like the Drosophila orthologues, function as a heterodimer. RNAi-mediated knockdown of AaGr2 expression had no impact on CO(2) reception. All three Gr genes were expressed in the maxillary palps of both Ae. aegypti and the West Nile virus vector mosquito, Culex pipiens quinquefasciatus. Interestingly, expression of the two CO(2) receptor genes was not equivalent in the two sexes and the implications of differential sex expression of the CO(2) receptor in different species are discussed. The functional identification of the CO(2) receptor in a mosquito could prove invaluable in the strategic design of compounds that disrupt the mosquito's ability to find hosts.     

Cytochrome P450 CYP4DE1 and CYP6CW3v2 contribute to ethiprole resistance in Laodelphax striatellus (Fallén)

Laodelphax striatellus Fallén (Hemiptera: Delphacidae), a destructive pest of rice, has developed high resistance to multiple insecticides, threatening the success of pest management programmes. The present study investigated ethiprole resistance mechanisms in a field population that is highly resistant to ethiprole. That population was used to establish a laboratory population that was subjected to further selection to produce a resistant strain. Target genes were cloned and compared between the resistant and the susceptible strains, the role of detoxification enzymes was examined, and the relative expression levels of 71 detoxification enzyme genes were tested using quantitative real time (RT)‐PCR. The laboratory selection enhanced the resistance from 107‐fold to 180‐fold. The Rdl‐type target site mutation seldom occurred in the resistant strain and is unlikely to represent the major mechanism underlying the observed resistance. Of the three important detoxification enzymes, only P450 monooxygenase was found to be associated with ethiprole resistance. Moreover, two genes, CYP4DE1 and CYP6CW3v2, were found to be overexpressed in the resistant strain. Furthermore, gene‐silencing via a double‐stranded RNA feeding test was carried out, and the results showed that the mRNA levels of CYP4DE1 and CYP6CW3v2 were reduced in the resistant strain, whereas ethiprole susceptibility was increased. These results suggest that CYP4DE1 and CYP6CW3v2 play an important role in ethiprole resistance in L. striatellus.     

cpbAg1 encodes an active carboxypeptidase B expressed in the midgut of Anopheles gambiae

We previously used differential display to identify several Anopheles gambiae genes, whose expression in the mosquito midgut was regulated upon ingestion of Plasmodium falciparum. Here, we report the characterization of one of these genes, cpbAg1, which codes for the first zinc-carboxypeptidase B identified in An. gambiae and in any insect. Expression of cpbAg1 in baculovirus gave rise to an active enzyme, and determination of the N-terminal amino acids confirmed that CPBAg1 contains a signal peptide and a pro-peptide, typical features of digestive zinc carboxypeptidases. cpbAg1 mRNA was mainly produced in the mosquito midgut, where it accumulated in unfed females and was rapidly down-regulated upon blood feeding. Annotation of the An. gambiae genome predicts twenty-three sequences coding for zinc-carboxypeptidases of which only two (cpbAg1 and cpbAg2) are expressed at a significant level in the mosquito midgut.