Transcriptomic and phylogenetic analysis of Culex pipiens quinquefasciatus for three detoxification gene families
© Yan et al.; licensee BioMed Central Ltd. 2012
Received: 29 June 2012
Accepted: 8 November 2012
Published: 10 November 2012
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.
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.
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.
KeywordsCarboxyl/cholinesterases Cytochrome P450 monooxygenases Glutathione S-transferases Insecticide resistance Gene expansion Gene expression
Mosquitoes are the most important vectors of human diseases. The Culex pipiens complex has a broad geographic distribution and is the vector of the West Nile virus and the Wuchereria bancrofti nematode, which causes filariasis. Over the last several decades, chemical insecticides have been intensively applied to control disease transmission. However, such control is undermined seriously by the increased insecticide resistance of vector mosquitoes. Three gene families are implicated in insecticide metabolism in mosquitoes: cytochrome P450 monooxygenases (P450s) are responsible for pyrethroid resistance, glutathione S-transferases (GSTs) are responsible for DDT resistance, and carboxyl/cholinesterases (CCEs) are responsible for organophosphate and carbamate resistance. Many insect species show rapid expansion and diversification of detoxification genes, as disclosed by their sequenced genomes. The expansion or restriction of detoxification genes likely helps insects adapt to their particular ecological niches and enable them to survive natural and man-made insecticide selection.
The genomes of three major taxonomic mosquitoes, including Anopheles gambiae, Aedes aegypti, and Culex pipiens quinquefasciatus, have been analyzed and released to the public[4–6]. Of the three, Ae. aegypti has the largest genome size (1376 Mb), while C. p. quinquefasciatus has the largest number of predicted protein-coding genes (18883), which is 22% larger than that of Ae. aegypti and 52% larger than that of An. gambiae. The extra number of protein-coding genes partially results from the expansion of its three detoxification gene families. However, unlike An. gambiae and Ae. aegypti, which have large amounts of gene expression data, such as various expressed sequence tag libraries and transcriptomes, C. p. quinquefasciatus has limited gene expression resources, with only several salivary gland transcriptomes currently reported[7, 8]. Knowledge of complete gene expression information is very important for the exploration of the functions of genes involved in specific biological processes and for the discovery of new candidate genes.
In the present study, the three detoxification gene families of C. p. quinquefasciatus were subjected to phylogenetic analysis and compared with those of three other dipteran insects. The CCE and P450 families were found to undergo large gene expansion. Digital gene expression tag profiling (DGE) technology was used to perform a deep transcriptome analysis of C. p. quinquefasciatus during development and in response to organophosphate insecticide selection. The gene expression profiles obtained provide an invaluable resource for the identification of genes involved in the development and insecticide resistance of C. p. quinquefasciatus.
Results and discussion
C. p. quinquefasciatus detoxification gene families
Classification of detoxification gene families in Drosophila melanogaster , Anopheles gambiae , Aedes aegypti , and Culex pipiens quinquefasciatus
C. p. quinquefasciatus
B class (α-esterases)
D class (integument esterases)
E class (β-esterases)
F class (dipteran JH esterases)
G class (lepidopteran JH esterases)
H class (glutactins)
I class (unknown)
J class (acetylcholinesterases)
K class (gliotactins)
L class (neuroligins)
M class (neurotactins)
CYP3 (include CYP6 and CYP9)
The number of CCEs in the neuro/developmental class was relatively conserved among the four dipteran insects. Similar conservation occurs in hymenopteran (Nasonia vitripennis, A. mellifera) and coleopteran (Tribolium castaneum) genomes, which reflects the relatively ancient origins of this class, where all members are catalytically inactive except for the acetylcholinesterases.
The secreted β-esterases (clade E) are comparatively conserved among the three mosquito species: from 2 to 4 β-esterase genes were found in mosquito genomes, largely different from the expansion (11 β-esterase genes) found in N. vitripennis genome. Some members of the β-esterases have well-described functions in other insects, such as E4 and FE4 esterases, which confer OP insecticide resistance in Myzus persicae, and the antennal Apo1PDE esterase of the silkworm Antheraea polyphemus, which degrades sex pheromones. The functions of the β-esterases in Culex need to be further investigated.
Why does C. p. quinquefasciatus have such an abundance of detoxification genes compared to other insect species? Several biological characteristics of mosquitoes may provide clues. The aquatic breeding sites of larvae and pupae contain numerous microorganisms, phenolic products of plant degradation, and pesticides. Adults feed on plant nectars and mammalian blood, which contain some harmful substances, such as heme and plant toxins. As viral pathogen vectors, mosquitoes have to deal with the generation of toxic endogenous compounds and reactive oxygen species during the immune response. But these cannot account for the gene expansion in C. p. quinquefasciatus compared to Anopheles and Aedes species. Perhaps its more polluted larval habitat and more diverse geographic range have exerted a greater selective pressure on C. p. quinquefasciatus so as to produce a larger repertoire of detoxification enzymes.
DGE library sequencing and mapping to genome
DGE sequencing statistics
Total raw tag
Total clean tag
Distinct clean tag
Distinct tag mapping to gene
Distinct unambiguous tag mapping to gene
Unambiguous tag-mapped genes
Distinct tag mapping to genome
Total unknown tag
Distinct unknown tag
GO and KEGG pathway classification of the genes expressed in C. p. quinquefasciatus
Life-stage specific detected genes
Developmental-stage specifically expressed genes of carboxylcholinesterase (CCE), glutathione-S-transferase (GST), and cytochrome P450 monooxygenase (P450)
For gene expansion clusters of detoxification genes, the expression profiles were different among the members. For example, among the six members of one expanded α-esterase cluster (Figure 2), CpipJ_CPIJ007825 was detected for expression in pupae and adults while the other five members were not detected in any stage. For the seven members of another expanded α-esterase cluster (Figure 2), CpipJ_CPIJ016025, CpipJ_CPIJ005694, and CpipJ_CPIJ008749 were not detected for expression; CpipJ_CPIJ000049 and CpipJ_CPIJ000051 were expressed in pupae and adults, CpipJ_CPIJ000050 in larvae and pupae, and CpipJ_CPIJ016026 only in eggs. Similar phenomenon was observed in the three expansion clusters of juvenile hormone esterases (Figure 3). For the five members of cluster, only CpipJ_CPIJ013027 was detected for expression and in eggs. No expression was found in the three members of cluster. For the four members of cluster, three were detected for expression: CpipJ_CPIJ016681 and CpipJ_CPIJ016682 in adults, and CpipJ_CPIJ017763 in larvae and adults. The different expression patterns of these duplicated detoxification genes are probably indicative of their subfunctionalization or retrogression as pseudogenes.
Differentially expressed genes between parathion resistant and susceptible larvae
When the third instar DGE library of the parathion-resistant strain SG was compared with the same stage in the susceptible strain S-lab, a total of 1511 genes showed different expression levels, among which 619 genes had up-regulated expression levels in the SG strain (Additional file8). The most prominent GO functions of these up-regulated genes were endopeptidase or serine-type peptidase activity, such as genes encoding trypsin, chymotrypsin, mast cell protease 2, urokinase-type plasminogen activator, and elastase. However, not all of the differentially expressed genes are responsible for parathion resistance because comparison strains were not selected from the same panmictic population such that genetic background differences could be ruled out.
Detoxification genes up-regulated in parathion resistant larvae of the SG strain
Egg, larva, adult
Larva, pupa, adult
C. p. quinquefasciatus is an important vector that transmits human diseases different from those by An. gambiae and Ae. aegypti. The lack of transcriptomic data available for this species has hampered characterization of the molecular mechanisms underlying the different biological characters of the three major mosquito vectors. The five DGE libraries described in the present study represent a dramatic expansion of the existing transcriptomic sequence available for C. p. quinquefasciatus. This expansion will facilitate the investigation of the fundamental biology of C. p. quinquefasciatus and its pathogenic interactions. In addition, the results of the present study provide new insights into the functions and evolution of the three detoxification gene families of mosquitoes. A larger number of detoxification genes were identified on the genome of C. p. quinquefasciatus compared with three other dipteran insect genomes, representing the widest gene expansion sequenced thus far. Comparative genomic analysis suggested that gene expansion mainly occurs in α-esterases, juvenile hormone esterases, and P450 CYP325. Some detoxification genes were expressed in all developmental stages, while some were developmentally regulated. The expression profiles were different among the members of gene expansion clusters, probably indicative of their subfunctionalization or retrogression as pseudogenes. Fifteen detoxification genes showed the potential to take part in the parathion resistance of Culex, including unexpected P450 genes.
Mosquito strains of C. p. quinquefasciatus used included S-lab, which was OP-susceptible and reared at the laboratory without any contact with insecticides for many years and Shengui (SG), a field population collected in Foshan, Guangdong Province, in 2007 and constantly treated with parathion at the laboratory. The parathion-resistance of SG was 115-fold that of S-lab before use in the DGE analysis. The mosquitoes were maintained at 26°C ± 1°C and a long-day photoperiod (14 h light/10 h darkness cycle). Fifty egg rafts, forty third instar larvae, forty pupae, and forty adults (twenty females and twenty males) of SG and forty third instar larvae of S-lab were collected and frozen at −80°C for further analysis.
Identification and phylogenetic classification of detoxification genes
Sequences encoding GSTs, P450s, and CCEs were identified from the protein set of the C. p. quinquefasciatus whole genome sequencing database at the Broad Institute (http://www.broadinstitute.org/annotation/genome/culex_pipiens) using the HMMER program (http://hmmer.janelia.org/) with the protein domains for CCEs (PF00135), GSTs (PF00043 and PF02798), and P450s (PF00067) as described in the Pfam database. A significance value of 1e-10 was used in the searches for CCEs and P450 and 2e-2 for GSTs. Community annotations and VectorBase were referred to verify the searches. Those candidate genes not supported by the community annotations as CCEs, P450 or GST were not accounted. P450s were named by the P450 nomenclature committee (http://drnelson.uthsc.edu/CytochromeP450.html). Known detoxification genes from An. gambiae, D. melanogaster, and Ae. aegypti were used as references for the phylogenetic classification of the detoxification genes from C. p. quinquefasciatus. Protein sequences were aligned with ClustalW2 at EMBL-EBI (http://www.ebi.ac.uk/Tools/msa/clustalw2). Unrooted distance neighbor-joining trees showing the phylogeny of detoxification gene families were constructed using the pairwise deletion and p-distance functions of Mega 4.0 software. Bootstrap analysis (1000 replicates) was applied to evaluate the internal support of the tree topology.
Pipeline of DGE
Six micrograms of total RNA from each of the above five mosquito samples were isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. Tag library preparation was performed with an Illumina Gene Expression Sample Prep Kit. The raw data (tag sequences and counts) were deposited in the NCBI Sequence Read Archive (SRA) database under submission number SRA049959.
Pipeline of bioinformatics analysis on DGE
Sequencing-received raw image data were transformed by base calling into raw sequence data. Clean tags were obtained after raw sequences were filtered to remove adaptor sequences, empty tags, low quality tags, tags that were too long or too short, and tags with a copy number of 1. The distribution of clean tags was used to evaluate the normality of the whole data. Saturation analysis was performed to determine whether or not the number of detected genes continues to increase when the sequencing amount increases. Pearson correlation analysis of two parallel libraries was performed to evaluate the reliability and operational stability of the experimental results. All clean tags were mapped to C. p. quinquefasciatus whole genome reference sequences and allowed no more than 1 nucleotide mismatch. The number of unambiguous clean tags for each gene was calculated and then normalized to TPM (number of transcripts per million clean tags). When the expression of a gene was not detected, TPM was set to 0.01.
A rigorous custom written algorithm using the method described by Audic et al. was developed to identify differentially expressed genes between two samples. The p value corresponded to the differential gene expression test. False discovery rate (FDR) was used to determine the p value threshold in multiple tests and analyses. FDR ≤ 0.001 and the absolute value of log2Ratio ≥ 1 were used as thresholds to judge the significance of the gene expression difference.
Unigenes matched by clean tags were assigned to Gene Ontology (GO) terms using Blast2GO and canonical pathways in KEGG (Kyoto Encyclopedia of Genes and Genomes). GO or pathway enrichment analysis of the differentially expressed genes was performed based on the algorithm presented by GOstat. The difference between the differentially expressed gene group and the whole gene expression background was represented by a p value, which was approximated by a chi-square test. The Fisher exact test was used when any expected count value was below 5, which will result in inaccurate chi-square test results. Benjamini multiple-testing correction of the p value was done by FDR.
This study was financially supported by the Major State Basic Research Development Program of China (973 Program, Grant No. 2012CB114102), the Important National Science & Technology Specific Project (Grant No. 2012ZX10004219), and the Knowledge Innovation Program of Chinese Academy of Sciences (Grant No. KSCX3-IOZ-1006).
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