Absence/presence calling in microarray-based CGH experiments with non-model organisms.

Structural variations in genomes are commonly studied by (micro)array-based comparative genomic hybridization. The data analysis methods to infer copy number variation in model organisms (human, mouse) are established. In principle, the procedures are based on signal ratios between test and reference samples and the order of the probe targets in the genome. These procedures are less applicable to experiments with non-model organisms, which frequently comprise non-sequenced genomes with an unknown order of probe targets. We therefore present an additional analysis approach, which does not depend on the structural information of a reference genome, and quantifies the presence or absence of a probe target in an unknown genome. The principle is that intensity values of target probes are compared with the intensities of negative-control probes and positive-control probes from a control hybridization, to determine if a probe target is absent or present. In a test, analyzing the genome content of a known bacterial strain: Staphylococcus aureus MRSA252, this approach proved to be successful, demonstrated by receiver operating characteristic area under the curve values larger than 0.9995. We show its usability in various applications, such as comparing genome content and validating next-generation sequencing reads from eukaryotic non-model organisms.

Response of the nonbiting midge Chironomus riparius to multigeneration toxicant exposure.

The ability of the nonbiting midge Chironomus riparius to withstand long-term toxicant exposure has been attributed to genetic adaptation. Recently, however, evidence has arisen that supports phenotypic plasticity. Therefore, the present study aimed to investigate if Chironomus riparius indeed copes with prolonged toxicant exposure through phenotypic plasticity. To this purpose, we performed a multigeneration experiment in which we exposed C. riparius laboratory cultures for nine consecutive generations to two exposure scenarios of, respectively, copper, cadmium, and tributyltin. Total emergence and mean emergence time were monitored each generation, while the sensitivity of the cultures was assessed at least every third generation using acute toxicity tests. We observed that the sublethally exposed cultures were hardly affected, while the cultures that were exposed to substantially higher toxicant concentrations after the sixth generation were severely affected in the eighth generation followed by signs of recovery. A marginal lowered sensitivity was only observed for the highly exposed cadmium culture, but this was lost again within one generation. We conclude that C. riparius can indeed withstand long-term sublethal toxicant exposure through phenotypic plasticity without genetic adaption.

Gene expression patterns and life cycle responses of toxicant-exposed chironomids.

Cellular stress responses are frequently presumed to be more sensitive than traditional ecotoxicological life cycle end points such as survival and growth. Yet, the focus to reduce test duration and to generate more sensitive end points 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 end points indirect. Therefore we aimed to directly compare the effects on growth, survival, and gene expression of the nonbiting midge Chironomus riparius. To this purpose, we simultaneously analyzed 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 end points 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 end life cycle responses after fourteen days of exposure revealed that gene expression is more sensitive to toxicant exposure than life cycle end points, underlining the potential of transcriptomics for ecotoxicity testing and environmental risk assessment.

Life cycle responses of the midge Chironomus riparius to compounds with different modes of action.

Compounds with different modes of action may affect life cycles of biota differently. The aim of the present study was therefore to investigate the impact of four chemicals with different modes of action, including the essential metal copper, the nonessential metal cadmium, the organometal tributyltin, and the polycyclic aromatic compound phenanthrene, on chronic lethal and sublethal life-cycle effect parameters of the nonbiting midge Chironomus riparius, applying a 28-day sediment toxicity test. Tributyltin and cadmium delayed emergence significantly over a wide range of sublethal concentrations, while this range was narrow for copper and almost absent for phenanthrene. The chronic LC50/LOECEmT50 ratio, expressing these differences, amounted to 1.5, 3.5, 12.0, and 18.2 for respectively phenanthrene, copper, cadmium, and tributyltin. Thus the more specific the compounds mode of action, the higher the chronic LC50/LOECEmT50 ratio, as previously observed for acute-to-chronic ratios (ACRs). Comparison of our results with literature derived LC50/LOEC ratios showed a comparable trend and a lower variability compared to ACRs. We therefore conclude that the presently proposed chronic ratio is indicative for the specificity of a chemicals mode of action and that it is less variable than the ACR.

Loss of imprinting of IGF2 and H19, loss of heterozygosity of IGF2R and CTCF, and Helicobacter pylori infection in laryngeal squamous cell carcinoma.

Imprinting analyses of IGF2 and H19, loss of heterozygosity (LOH) analyses of IGF2R and CTCF and Helicobacter pylori detection, were performed on 35 human laryngeal squamous cell carcinomas (LSCC). Forty-six percent of the tumors were heterozygous for IGF2, and 54% were informative for the H19. Biallelic expression of IGF2 was observed in 33% (5 out of 15) of the tumors and in 27% (4 out of 15) of adjacent non-tumorous laryngeal tissues. H19 loss of imprinting (LOI) was observed in 24% (4 out of 17) of the tumors. For IGF2R and CTCF, 71% (25 out of 35) and 50% (17/34), respectively, of the samples were heterozygous, and LOH was detected in 12% (3 out of 25) and 6% (1 out of 17), respectively, of the tumors. H. pylori was found in 26% (9/35) of these tumors. Among them, four were informative for the imprinting analysis. The presence of H. pylori had no effect on IGF2/H19 imprinting. Only the H. pylori detection was further broadened with an additional 47 laryngeal tumors, resulting in a total final positivity of close to 16% (13 out of 82). This study represents the largest comprehensive IGF2/H19 imprinting study done to date on well-defined samples of human laryngeal carcinomas and corresponding non-tumorous tissue. For the first time, the analyses of IGF2/H19 imprinting have been broadened with LOH analyses of IGF2R and CTCF, with both of these genes acting as modulators of IGF2 and H19 activity. Although there were indications that H. pylori may be present in LSCC, we are the first to show its presence in LSCC by two direct techniques: Giemsa staining and nested-PCR.

Combining next-generation sequencing and microarray technology into a transcriptomics approach for the non-model organism Chironomus riparius.

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.