Project description:The aquatic midge, Chironomus tentans, is a keystone species in aquatic ecosystem and used as a model organism to assess chemicals toxicity in aquatic environment. To characterize midge’s cellular and molecular responses to pesticides, we established cDNA library with 10,000 cDNA elements representing 2,456 C. tentans unique genes. Blast2go identified 49 genes potentially involved in xenobiotics metabolism, including 24 cytochrome p450s (CYPs), 14 esterases (ESTs) and 11 glutathione-s-transferases (GSTs). Based on 2,456 unique genes, a cDNA microarray was developed to monitor gene expression profiles in 4th instar larvae under chlopyrifos (0.1 µg/L and 0.5 µg/L) and 1000 µg/L atrazine 48-hr exposure. We identified 149, 435 and 244 genes were significantly differentially expressed (p-value ≤0.05 with expression ratios ≥2.0) after 0.1 µg/L, 0.5 µg/L chlopyrifos, and 1000 µg/L atrazine application, respectively. Sixteen insect detoxification genes (11 CYPs, 3 GSTs and 2 esterases) were validated by qPCR and their expressions were significantly either up- or down-regulated under chlorpyrifos and atrazine exposure, especially the expression of 10 CYPs were significantly induced after chlopyrifos and atrazine exposure. The up-regulated CYPs might be involved in xenobiotic activation and/or degradation. Furthermore, we also found 5 differentially expressed hemoglobin genes. The expression changes of hemoglobins might be an adaption mechanism of C. tentans to hypoxic condition caused by xenobiotic exposure. This study provides a platform for further functional studies of pesticide-insect interactions in C. tentans.
Project description:Analysis of expressed sequence tags by pyrosequencing: Identification and characterization of glutathione S-transferase (GST) genes in the aquatic midge Chironomus riparius (Diptera: Chironomidae) fourth instar larvae
Project description:Analysis of expressed sequence tags by pyrosequencing: Identification and characterization of delta, sigma, omega, epsilon, theta and zeta class glutathione S-transferase (GST) genes in the aquatic midge Chironomus riparius (Diptera: Chironomidae) fourth
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.
Project description:MicroRNAs (miRNAs) are small noncoding RNAs that play critical roles in regulating post transcriptional gene expression. Gall midges encompass a large group of insects that are of economic importance and also possess fascinating biological traits. The gall midge Mayetiola destructor, commonly known as the Hessian fly, is a model organism for studying gall midge biology and insect – host plant interactions. In this study, we systematically analyzed miRNAs from the Hessian fly. Deep-sequencing a Hessian fly larval transcriptome led to the identification of 89 miRNA species that are either identical or very similar to known miRNAs from other insects, and 184 novel miRNAs that have not been reported from other species. Microarray analyses revealed the expression of miRNA genes was strictly regulated during Hessian fly larval development and abundance of many miRNA genes were affected by host genotypes. The identification of a large number of miRNAs for the first time from a gall midge provides a foundation for further studies of miRNA functions in gall midge biology and behavior.
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:During an incompatible or compatible interaction between rice (Oryza sativa) and the Asian rice gall midge (Orseolia oryzae), a lot of genetic reprogamming occurs in the plant host We used microarray to know the changes occuring in the resistant host (indica rice variety RP2068-18-3-5) when challenged by avirulent biotype of gall midge (GMB 1). During this incompatible interaction the resistance in the host is manifested by a hypersenstive response. Using microarray data, we identified distinct classes of up- and down-regulated genes during this process. Tissues from the stem region (feeding site of insect larvae) of the plants that were exposed to gall midge, were taken for RNA extraction and hybridization on Affymetric microarrays. For control, tissues were taken from the corresponding region of plants that were not exposed to gall midge.