chironomus riparius ( chironomidae , diptera ) , is widely used in aquatic exotoxicological studies for assessing acute and sub - lethal toxicities of contaminated sediments and for water monitoring due to their widespread occurrence , short life - cycle , easy to be reared in the laboratory , physiological tolerance to various environmental conditions . to date , the endpoints used for monitoring such effects in c. riparius are based on a small number of specific biomarkers and measurements of organism level effects , such as survival and reproduction . genomic - based techniques based on expression analysis of genes are important tools for investigating molecular level effects caused by exposure to environmental pollutants , which will provide the ability to detect mechanisms of action and subsequent adverse cellular level effects and associated with different types of toxicity . as a pre - requisite for genomic based eco - toxicological studies knowledge of the c. riparius transcriptome is important but despite its eco - toxicological importance , no large scale transcriptome analysis of c. riparius has been done so far . in a previous report arvestad et al . reported the transcriptome analysis of c. tentans midgut and an epithelial cell line using cdna sequencing using conventional cdna synthesis and sequencing method . however , with the advent of new high throughput pyrosequencing technologies using several genome sequencers , such as gs - flx , gs - flx - titanium and solexa , extensive cdna sequence information can be obtained in a short period of time . the gs - flx pyrosequencer , from 454 life science / roche , is well - suited for de novo transcriptome sequencing for the rapid production of sequence data with reduced time , labor and cost , and generates the longest reads . moreover , well characterized reference genomes of insects [ 8 - 10 ] could provide platforms for comparative genome analyses of non model organisms like c. riparius . in this study , we present the first comprehensive characterization of the transcriptome of c. riparius 4 instar larvae using 454 pyrosequencing . based on data corresponding to one single run on the flx gene sequencer from 454 life science , almost 49,774,676 bases were assembled into transcripts and the majority of these have been annotated and functionally classified . in light of limited genomic and transcriptomic information , these data would significantly enrich the molecular aspects of c. riparius and its role in genomics based ecotoxicological studies . c. riparius strains were obtained from korea institute of chemical technology ( daejon , korea ) . the larvae were reared on an artificial diet of fish flake food ( tetramin , tetrawerke , melle , germany ) in glass chambers containing dechlorinated tap water and acid washed sand , with aeration under a 16 - 8 h light - dark photoperiod at room temperature ( 201 ) . the larvae were collected and total rna samples were isolated using trizol reagent ( invitrogen life technology , usa ) . the rna samples ( a260/a280>1.8 ) were collected and mrna was purified from the pooled total rna ( 500 g ) by binding to oligo ( dt ) cellulose twice ( poly ( a ) purist , ambion ) . for first strand synthesis , 10 l of the purified mrna ( 5 g ) , denatured at 65 for 10 minutes in a rnase free tube , rapidly chilled on ice , mixed with 5 l of 10x first - strand buffer , 5 lof 100 mm dtt , 5 lof dntps ( 2.5 mm each ) , 5 lof oligo dt20 ( 50 m ) , 2.5 lof strata script reverse transcriptase ( 200 u/l ) in a 50 lreaction volume . first strand cdna was synthesized at 42 for 60 minutes and cdna synthesis and heat inactivated at 70 for 15 minutes and the tubes were placed on ice . for second strand cdna synthesis , h2o ( 61 l ) , 100 m tris - hcl , 20 l of second - strand buffer , dntps ( 6 l , 10 mm each ) , dna polymerase i ( 4 l , 10 u/l ) , and rnase h ( 2 l , 1.5 u/l ) were mixed with the first strand synthesis reaction and incubated at 16 for 150 minutes . for end blunting , 23 l of blunting dntp mix , 2 l of cloned pfu dna polymerase ( 2.5 u/l ) was incubated with the second strand synthesis reaction at 16 for 5 minutes . the cdna was purified using qiaquick pcr purification kit ( qiagen , ca , usa ) in a final elution volume of 50 l . approximately , 1 g of the final pcr product dna was used to generate dna library for genome sequencer flx titanium ( roche , mannheim , ge ) . the fragments ends were polished ( blunted ) , and short two adapters were ligated onto both ends . the adapters provide priming sequences for both amplification and sequencing of the sample library fragments , as well as the " sequencing key " , a short sequence of 4 nucleotides used by the system software for base calling and , following repair of any nicks in the double - stranded library , release of the unbound strand of each fragment ( with 5,-adaptor a ) . finally the quality of the library of single - stranded template dna fragments ( ssdna library ) was assessed using 2100 bioanalyzer ( agilent , waldbronn , ge ) , and the library was quantitated , including a functional quantitation to determine the optimal amount of the library to use as input for emulsion - based clonal amplification . single " effective " copies of template species from the dna library to be sequenced were hybridized to dna capture beads . the immobilized library was then resuspended in the amplification solution , and the mixture is emulsified , followed by pcr amplification . after amplification , the dna - carrying beads were recovered from the emulsion and enriched . the second strands of the amplification products were melted away as part of the enrichment process , leaving the amplified single - stranded dna library bound to the beads . after amplification , the dna - carrying beads were set into the wells of a picotiterplate device ( ptp ) such that wells contain single dna beads . the loaded ptp was then inserted into the genome sequencer flx instrument , and sequencing reagents were sequentially flowed over the plate . information from all the wells of the ptp is captured simultaneously by the camera , and can be processed in real time by the onboard computer . after assembly , the resulting contigs and singlets were blastx searched   against the protein databases " nr " and " uniprot " ( the uniprot consortium , 2008 ) . the sequences were annotated using the gene ontology ( go ) terms where possible according to molecular function , biological process and cellular component using database ( http://www.geneontology.org/ ) . pathway assignments according to kyoto encyclopedia of genes and genomes ( kegg ) mapping was carried out using unique sequences that had blastx scores with a cut off value of e = 10 the sequences were mapped to different kegg biochemical pathways according to the ec distribution in the pathway database ( http://www.genome.ad.jp/kegg/ ) . c. riparius strains were obtained from korea institute of chemical technology ( daejon , korea ) . the larvae were reared on an artificial diet of fish flake food ( tetramin , tetrawerke , melle , germany ) in glass chambers containing dechlorinated tap water and acid washed sand , with aeration under a 16 - 8 h light - dark photoperiod at room temperature ( 201 ) . the larvae were collected and total rna samples were isolated using trizol reagent ( invitrogen life technology , usa ) . the rna samples ( a260/a280>1.8 ) were collected and mrna was purified from the pooled total rna ( 500 g ) by binding to oligo ( dt ) cellulose twice ( poly ( a ) purist , ambion ) . for first strand synthesis , 10 l of the purified mrna ( 5 g ) , denatured at 65 for 10 minutes in a rnase free tube , rapidly chilled on ice , mixed with 5 l of 10x first - strand buffer , 5 lof 100 mm dtt , 5 lof dntps ( 2.5 mm each ) , 5 lof oligo dt20 ( 50 m ) , 2.5 lof strata script reverse transcriptase ( 200 u/l ) in a 50 lreaction volume . first strand cdna was synthesized at 42 for 60 minutes and cdna synthesis and heat inactivated at 70 for 15 minutes and the tubes were placed on ice . for second strand cdna synthesis , h2o ( 61 l ) , 100 m tris - hcl , 20 l of second - strand buffer , dntps ( 6 l , 10 mm each ) , dna polymerase i ( 4 l , 10 u/l ) , and rnase h ( 2 l , 1.5 u/l ) were mixed with the first strand synthesis reaction and incubated at 16 for 150 minutes . for end blunting , 23 l of blunting dntp mix , 2 l of cloned pfu dna polymerase ( 2.5 u/l ) was incubated with the second strand synthesis reaction at 16 for 5 minutes . the cdna was purified using qiaquick pcr purification kit ( qiagen , ca , usa ) in a final elution volume of 50 l . approximately , 1 g of the final pcr product dna was used to generate dna library for genome sequencer flx titanium ( roche , mannheim , ge ) . the fragments ends were polished ( blunted ) , and short two adapters were ligated onto both ends . the adapters provide priming sequences for both amplification and sequencing of the sample library fragments , as well as the " sequencing key " , a short sequence of 4 nucleotides used by the system software for base calling and , following repair of any nicks in the double - stranded library , release of the unbound strand of each fragment ( with 5,-adaptor a ) . finally the quality of the library of single - stranded template dna fragments ( ssdna library ) was assessed using 2100 bioanalyzer ( agilent , waldbronn , ge ) , and the library was quantitated , including a functional quantitation to determine the optimal amount of the library to use as input for emulsion - based clonal amplification . single " effective " copies of template species from the dna library to be sequenced were hybridized to dna capture beads . the immobilized library was then resuspended in the amplification solution , and the mixture is emulsified , followed by pcr amplification . after amplification , the dna - carrying beads were recovered from the emulsion and enriched . the second strands of the amplification products were melted away as part of the enrichment process , leaving the amplified single - stranded dna library bound to the beads . the sequencing primer is then annealed to the immobilized amplified dna templates . after amplification , the dna - carrying beads were set into the wells of a picotiterplate device ( ptp ) such that wells contain single dna beads . the loaded ptp was then inserted into the genome sequencer flx instrument , and sequencing reagents were sequentially flowed over the plate . information from all the wells of the ptp is captured simultaneously by the camera , and can be processed in real time by the onboard computer . after assembly , the resulting contigs and singlets were blastx searched   against the protein databases " nr " and " uniprot " ( the uniprot consortium , 2008 ) . the sequences were annotated using the gene ontology ( go ) terms where possible according to molecular function , biological process and cellular component using database ( http://www.geneontology.org/ ) . pathway assignments according to kyoto encyclopedia of genes and genomes ( kegg ) mapping was carried out using unique sequences that had blastx scores with a cut off value of e = 10 the sequences were mapped to different kegg biochemical pathways according to the ec distribution in the pathway database ( http://www.genome.ad.jp/kegg/ ) . to get an overview of c. riparius transcriptome , total rna was isolated from fourth instar larvae , and mrna purification , cdna synthesis , and sequence determination was done . these were then pyrosequenced , and in total , 138,091 reads were obtained , constituting a total of 49,774,676 bases of cdna . following the assembly of the sequences , a total of 8,565 contigs and 11,455 singletons were obtained ( table 1 ) . among the 8,565 contigs , 3,131 and 5,434 had lengths more than 500 and 100 base pairs , respectively ( table 2 ) . after assembly , the contigs and singletons were blastx searched   against the protein databases " nr " and " uniprot " ( the uniprot consortium , 2008 ) . of the 8,565 c. riparius contigs , 5,102 matched proteins in " uniprot " and 5,114 matched proteins in " nr " , while the numbers for the 11,455 singlets where 4,697 and 4,710 , respectively ( table 3 ) . after removing all redundant sequences , 9,512 sequences were obtained , representing a significant part of the c. riparius transcriptome . gene ontology ( go ) has been widely used to characterize gene function , annotation and classification . as a whole , 24% of the sequences matched with known function genes to existing gene models in blastx searches ( e - value 1 ) , 24% showed no significant match and 52% of the pyrosequencing assemblies ( e - value  1 ) did not match with any known sequences in the existing genbank database and ; thus , are likely to represent novel transcripts identified in this study ( figure 1 ) . similarities based on the results of blastx searches showed the highest percentage of sequences match with aedes aegypti ( 26% ) , followed by culex quinquefasciatus ( 21% ) , drosophila spp . ( 18% ) , anopheles gambiae ( 15% ) , tribolium castaneum ( 4% ) , apis mellifera ( 2% ) and nasonia vitripennis ( 2% ) . a small number of sequences ( 1% ) showed similarities with previously characterized genes of different chironomus species and 6% of sequences matched with other insects and rest of the 5% matched with other ( human , chicken , mouse , zebrafish , c. elegans or other organisms ) species ( figure 2 ) . kegg pathway analyses are widely used as a reference for the systematic interpretation of sequence data by linking individual genes to components of the kegg biochemical pathways . the pyrosequenced transcriptome of c. riparius was searched for the number of annotated gene sequences involved in shared specific kegg pathways among animal phyla , using the unique sequences that had blastx scores with a cut off value of e = 10 . it was found that 2908 ( 34.73% ) genes were involved in proteins with a binding function or cofactor requirement , 1,165 ( 13.91% ) genes were involved in subcellular localizations , 915 ( 11% ) in metabolism , 917 ( 10.95% ) in regulation of metabolism and protein function and 700 ( 8.36% ) in cellular transport , transport facilities and transport routes representing the largest groups with putative function , indicating the important metabolic activities in c. riparius ( table 4 ) . these results showed the effectiveness of our transcriptome analysis of c. riparius , and indicated many of the candidate genes involved in many pathways and cellular processes . a closer examination of the annotations revealed several genes that are of particular interest for environmental monitoring and ecological research . several proteins catalyzing biotransformation of many xenobiotic and a number of important biomarkers for a large number of different compounds were present . among the sequenced c. riparius transcripts , 117 different cytochrome p450 variants from 13 different families could be identified . other interesting classes of proteins such as heat shock protein ( 24.1 , 27 , 67b2 , 70 , 90 ) , genes coding for oxidative stress such as catalase , glutathione peroxidase , glutathione s - transferase , superoxide dismutase , thioredoxin reductase and several other biomarker genes such as metallothionein , vitellogenin were also present ( table 5 ) . to get an overview of c. riparius transcriptome , total rna was isolated from fourth instar larvae , and mrna purification , cdna synthesis , and sequence determination was done . these were then pyrosequenced , and in total , 138,091 reads were obtained , constituting a total of 49,774,676 bases of cdna . following the assembly of the sequences , a total of 8,565 contigs and 11,455 singletons were obtained ( table 1 ) . among the 8,565 contigs , 3,131 and 5,434 had lengths more than 500 and 100 base pairs , respectively ( table 2 ) . after assembly , the contigs and singletons were blastx searched   against the protein databases " nr " and " uniprot " ( the uniprot consortium , 2008 ) . of the 8,565 c. riparius contigs , 5,102 matched proteins in " uniprot " and 5,114 matched proteins in " nr " , while the numbers for the 11,455 singlets where 4,697 and 4,710 , respectively ( table 3 ) . after removing all redundant sequences , 9,512 sequences were obtained , representing a significant part of the c. riparius transcriptome . gene ontology ( go ) has been widely used to characterize gene function , annotation and classification . as a whole , 24% of the sequences matched with known function genes to existing gene models in blastx searches ( e - value 1 ) , 24% showed no significant match and 52% of the pyrosequencing assemblies ( e - value  1 ) did not match with any known sequences in the existing genbank database and ; thus , are likely to represent novel transcripts identified in this study ( figure 1 ) . similarities based on the results of blastx searches showed the highest percentage of sequences match with aedes aegypti ( 26% ) , followed by culex quinquefasciatus ( 21% ) , drosophila spp . ( 18% ) , anopheles gambiae ( 15% ) , tribolium castaneum ( 4% ) , apis mellifera ( 2% ) and nasonia vitripennis ( 2% ) . a small number of sequences ( 1% ) showed similarities with previously characterized genes of different chironomus species and 6% of sequences matched with other insects and rest of the 5% matched with other ( human , chicken , mouse , zebrafish , c. elegans or other organisms ) species ( figure 2 ) . kegg pathway analyses are widely used as a reference for the systematic interpretation of sequence data by linking individual genes to components of the kegg biochemical pathways . the pyrosequenced transcriptome of c. riparius was searched for the number of annotated gene sequences involved in shared specific kegg pathways among animal phyla , using the unique sequences that had blastx scores with a cut off value of e = 10 . it was found that 2908 ( 34.73% ) genes were involved in proteins with a binding function or cofactor requirement , 1,165 ( 13.91% ) genes were involved in subcellular localizations , 915 ( 11% ) in metabolism , 917 ( 10.95% ) in regulation of metabolism and protein function and 700 ( 8.36% ) in cellular transport , transport facilities and transport routes representing the largest groups with putative function , indicating the important metabolic activities in c. riparius ( table 4 ) . these results showed the effectiveness of our transcriptome analysis of c. riparius , and indicated many of the candidate genes involved in many pathways and cellular processes . a closer examination of the annotations revealed several genes that are of particular interest for environmental monitoring and ecological research . several proteins catalyzing biotransformation of many xenobiotic and a number of important biomarkers for a large number of different compounds were present . among the sequenced c. riparius transcripts , 117 different cytochrome p450 variants from 13 different families could be identified . other interesting classes of proteins such as heat shock protein ( 24.1 , 27 , 67b2 , 70 , 90 ) , genes coding for oxidative stress such as catalase , glutathione peroxidase , glutathione s - transferase , superoxide dismutase , thioredoxin reductase and several other biomarker genes such as metallothionein , vitellogenin were also present ( table 5 ) . c. riparius has been studied extensively because of their importance as an ecologically important biomonitoring species . however , due to limited knowledge of genomic resources necessary for mechanistic study , the effect of toxicants at the genomic level was rarely studied . this work describes the first assessment of the use of pyrosequencing in c. riparius and we have obtained a significant portion of the c. riparius transcriptome using this approach . to facilitate identifying sets of genes involved in a broad range of processes we developed our ests set from a normalized whole - body library . as compared to sanger - based approaches , which require cdna cloning and bacterial transformation , transcriptome sequencing using massively parallel pyrosequencing exhibits high sensitivity and detection of low - abundant transcripts . transcripts that previously have been hard to sequence can therefore be detected as in the case of arabidopsis transcriptome profiling using pyrosequecing as reported by weber et al . . even though , the length of the sequence is shorter than as compared to sanger sequencing , the flx gene sequencer used in this study generated 3,131 contigs with an average of 924 bp length which is longer compared to previous studies . in our study , sequence names to the assembled sequences were given based on the best blast match for that sequence available in the public sequence data base and almost 50% of the genes were assigned gene names . however , another 50% of the sequences not matching to known genes in public sequence databases . in our studies we obtained 9,512 , non - redundant genes and thus , a major part of the transcriptome of c. riparius has been obtained . one of the limitations in non - model organisms lacking fully sequenced genome where the transcriptome pyrosequencing is based on the number of genes expressed and without a fully - sequenced genome no clear data is available . genome sequences for the insects d. melanogaster , a. gambiae   and a. aegypti   have been reported and many other species are nearing completion . in our studies 81% of the c. riparius transcriptome closely related to insects a. aegypti , d. melanogaster , c. quinquefasciatus , and a. gambiae and therefore will provide a rich source of information for further investigation using comparative genome analyses . the expression levels of unknown transcripts were also as high as those aligning in annotated regions and these transcripts are likely to represent novel transcripts , which offer possibility to study new genes which may be specific to c. riparius . in earlier reports , many unique genes are observed in transcriptome studies of m. sexta . in data analysis , many genes involved in different pathways , cellular processes and genes involved in metabolism of toxic substances or well - known biomarker genes ( table 4 ) are identified . since c. riparius is extensively used in ecotoxicological studies , gene expression analysis are needed for mechanistic studies to understand changes in aquatic environment and the large collection of annotated sequences produced in this study represents a reasonably complete description of the c. riparius larval transcriptome . by correlating morphological as well as physiological charecters with molecular - level responses , caused by exposure to various toxicants , the subtle effect of various toxicants pyrosequencing offers rapid characterization of a large portion of the transcriptome and therefore provides a comprehensive tool for gene discovery . pyrosequencing the ests of 4 instar c. riparius larvae resulted in the identification of many sequences of ecotoxicological relevance . analysis of the c. riparius transcriptome has revealed several gene candidates of ecotoxicological interest and further functional characterization will identify genes with relevance to ecotoxicology . the obtained transcriptome offers the additional option to design microarrays to study transcript regulation to understand the effect of environmental stressors . transcriptome comparison with well - studied organisms will facilitate further the understanding of how environmental stressors effect at higher organisms levels using c. riparius as a model system . the platform will allow correlation of molecular - level responses , caused by exposure to various toxicants , to the unique morphological and physiological characters of c. riparius .