Project description:Whole-transcriptome gene-expression analyses are commonly performed in species that have a sequenced genome and for which microarrays are commercially available. To do such analyses in species with no or limited genome data, i.e. non-model organisms, necessary transcriptomics resources, i.e. an annotated transcriptome and a validated gene-expression microarray, must first be developed. The aim of the present study was to establish an advanced approach for developing transcriptomics resources for non-model organisms by combining next-generation sequencing (NGS) and microarray technology. We applied our approach to the non-biting midge Chironomus riparius, an ecologically relevant species that is widely used in sediment ecotoxicity testing. We sampled extensively covering all C. riparius developmental stages as well as toxicant exposed larvae and obtained from a normalized cDNA library 1.5 M NGS reads totalling 501 Mbp. Using the NGS data we developed transcriptomics resources in several steps. First, we designed 844 k probes directly on the NGS reads, as well as 76 k probes targeting expressed sequence tags of related species. These probes were tested for their affinity to C. riparius DNA and mRNA, by performing two biological experiments with a 1 M probe-selection microarray that contained the entire probe-library. Subsequently, the 1.5 M NGS reads were assembled into 23,709 isotigs and 135,082 singletons, which were associated to ~55 k, respectively, ~61 k gene ontology terms and which corresponded together to 22,593 unique protein accessions. An algorithm was developed that took the assembly and the probe affinities to DNA and mRNA into account, what resulted in 59 k highly-reliable probes that targeted uniquely 95% of the isotigs and 18% of the singletons. Concluding, our approach allowed the development of high-quality transcriptomics resources for C. riparius, and is applicable to any non-model organism. It is expected, that these resources will advance ecotoxicity testing with C. riparius as whole-transcriptome gene-expression analysis are now possible with this species. 1x 1M CGH array with Cy3 labeled C. riparius gDNA and Cy5 labeled A. gambiae gDNA. The microarray was designed against C. riparius mRNA sequencing reads, and has been used to identify trustworthy sequencing reads to design an expression array. This 1M array is therefore not functionally annotated.

Project description:Cellular stress responses are frequently presumed to be more sensitive than traditional ecotoxicological life cycle endpoints such as survival and growth. Yet, the focus to reduce test duration and to generate more sensitive endpoints has caused transcriptomics studies to be performed at low doses during short exposures, separately and independently from traditional ecotoxicity tests, making comparisons with life cycle endpoints indirect. Therefore we aimed to directly compare the effects on growth, survival and gene expression of the non-biting midge Chironomus riparius. To this purpose, we analyzed simultaneously life cycle and transcriptomics responses of chironomid larvae exposed to four model toxicants. We observed that already at the lowest test concentrations many transcripts were significantly differentially expressed, while the life cycle endpoints of C. riparius were hardly affected. Analysis of the differentially expressed transcripts showed that at the lowest test concentrations substantial and biologically relevant cellular stress was induced and that many transcripts responded already maximally at these lowest test concentrations. The direct comparison between molecular en life cycle responses after fourteen days of exposure revealed that gene expression is more sensitive to toxicant exposure than life cycle endpoints, underlining the potential of transcriptomics for ecotoxicity testing and environmental risk assessment. Cellular stress responses are frequently presumed to be more sensitive than traditional ecotoxicological life cycle endpoints such as survival and growth. Yet, the focus to reduce test duration and to generate more sensitive endpoints has caused transcriptomics studies to be performed at low doses during short exposures, separately and independently from traditional ecotoxicity tests, making comparisons with life cycle endpoints indirect. Therefore we aimed to directly compare the effects on growth, survival and gene expression of the non-biting midge Chironomus riparius. To this purpose, we analyzed simultaneously life cycle and transcriptomics responses of chironomid larvae exposed to four model toxicants. We observed that already at the lowest test concentrations many transcripts were significantly differentially expressed, while the life cycle endpoints of C. riparius were hardly affected. Analysis of the differentially expressed transcripts showed that at the lowest test concentrations substantial and biologically relevant cellular stress was induced and that many transcripts responded already maximally at these lowest test concentrations. The direct comparison between molecular en life cycle responses after fourteen days of exposure revealed that gene expression is more sensitive to toxicant exposure than life cycle endpoints, underlining the potential of transcriptomics for ecotoxicity testing and environmental risk assessment. Four 14-day Chironomus riparius sediment toxicity tests were conducted, one with each toxicant. The surviving larvae were individually flash frozen in liquid nitrogen. For each toxicant we analyzed the gene expression of larvae exposed to low, intermediate and high concentrations. We also included a control and a solvent control. For each treatment we analyzed 10 replicates (individual larvae).

Project description:Cellular stress responses are frequently presumed to be more sensitive than traditional ecotoxicological life cycle endpoints such as survival and growth. Yet, the focus to reduce test duration and to generate more sensitive endpoints has caused transcriptomics studies to be performed at low doses during short exposures, separately and independently from traditional ecotoxicity tests, making comparisons with life cycle endpoints indirect. Therefore we aimed to directly compare the effects on growth, survival and gene expression of the non-biting midge Chironomus riparius. To this purpose, we analyzed simultaneously life cycle and transcriptomics responses of chironomid larvae exposed to four model toxicants. We observed that already at the lowest test concentrations many transcripts were significantly differentially expressed, while the life cycle endpoints of C. riparius were hardly affected. Analysis of the differentially expressed transcripts showed that at the lowest test concentrations substantial and biologically relevant cellular stress was induced and that many transcripts responded already maximally at these lowest test concentrations. The direct comparison between molecular en life cycle responses after fourteen days of exposure revealed that gene expression is more sensitive to toxicant exposure than life cycle endpoints, underlining the potential of transcriptomics for ecotoxicity testing and environmental risk assessment. Cellular stress responses are frequently presumed to be more sensitive than traditional ecotoxicological life cycle endpoints such as survival and growth. Yet, the focus to reduce test duration and to generate more sensitive endpoints has caused transcriptomics studies to be performed at low doses during short exposures, separately and independently from traditional ecotoxicity tests, making comparisons with life cycle endpoints indirect. Therefore we aimed to directly compare the effects on growth, survival and gene expression of the non-biting midge Chironomus riparius. To this purpose, we analyzed simultaneously life cycle and transcriptomics responses of chironomid larvae exposed to four model toxicants. We observed that already at the lowest test concentrations many transcripts were significantly differentially expressed, while the life cycle endpoints of C. riparius were hardly affected. Analysis of the differentially expressed transcripts showed that at the lowest test concentrations substantial and biologically relevant cellular stress was induced and that many transcripts responded already maximally at these lowest test concentrations. The direct comparison between molecular en life cycle responses after fourteen days of exposure revealed that gene expression is more sensitive to toxicant exposure than life cycle endpoints, underlining the potential of transcriptomics for ecotoxicity testing and environmental risk assessment.

Project description:The soil worm Enchytraeus crypticus (oligochaete) is an ecotoxicology model species although without genome or transcriptome sequence information. The present research aimed at studying, via high-throughput pyrosequencing, the transcriptome of Enchytraeus crypticus, sampled from multiple test conditions, and the construction of a high-density microarray for functional genomic studies. A pyrosequencing run retrieved approximately 1.5 million reads representing 645 million bases. After assembly, 27,296 contigs and 87,686 singletons were obtained. from which 44% and 25% were annotated as protein-coding genes. We show that the high amount of orphan genes is not due to poor sequence or assemble quality: 84% of the contig sequences contains an open reading frame with a start codon and E. crypticus homologs were identified for 92% of the core eukaryotic genes. Moreover, 65 and 77% of the unknown singletons and contigs, respectively, showed transcriptional activity. An Agilent 180K microarray platform was designed and validated by hybridizing cDNA from 3 day zinc- exposed E. crypticus to the concentration corresponding to 50% reduction in reproduction (EC50). Overall, 70% of all probes exerted a hybridization signal above background level. More specifically, the probes derived from contigs showed a wider range of average intensities when compared to probes derived from singletons. In total, 522 significantly regulated transcripts were identifying upon zinc exposure. Several significantly regulated genes exerted predicted functions (e.g. zinc efflux, zinc transport) associated with zinc stress. Unexpectedly, the microarray data suggest that zinc exposure alters retrotransposon activity in the E. crypticus genome. In conclusion, characterization of the presented E. crypticus transcriptome and associated microarray platform is a valuable and high quality resource that permits further functional genomics experiments examining gene expression patterns underlying distinct environmental stress conditions. We show that unknown sequences are not the result of technical errors but mostly represent functional genes that are actively transcribed.

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Combining Next-Generation Sequencing and Microarray Technology into a Transcriptomics Approach for the Non-Model Organism Chironomus riparius

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