Dataset Information

Several Grassland Soil Nematode Species Are Insensitive to RNA-Mediated Interference.

ABSTRACT: Phenotypic analysis of defects caused by RNA mediated interference (RNAi) in Caenorhabditis elegans has proven to be a powerful tool for determining gene function. In this study we investigated the effectiveness of RNAi in four non-model grassland soil nematodes, Oscheius sp FVV-2., Rhabditis sp, Mesorhabditis sp., and Acrobeloides sp. In contrast to reference experiments performed using C. elegans and Caenorhabditis briggsae, feeding bacteria expressing dsRNA and injecting dsRNA into the gonad did not produce the expected RNAi knockdown phenotypes in any of the grassland nematodes. Quantitative reverse-transcribed PCR (qRT-PCR) assays did not detect a statistically significant reduction in the mRNA levels of endogenous genes targeted by RNAi in Oscheius sp., and Mesorhabditis sp. From these studies we conclude that due to low effectiveness and inconsistent reproducibility, RNAi knockdown phenotypes in non-Caenorhabditis nematodes should be interpreted cautiously.

SUBMITTER: Wheeler D

PROVIDER: S-EPMC3593264 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Several Grassland Soil Nematode Species Are Insensitive to RNA-Mediated Interference.

Wheeler David D Darby Brian J BJ Todd Timothy C TC Herman Michael A MA

Journal of nematology 20120301 1

Phenotypic analysis of defects caused by RNA mediated interference (RNAi) in Caenorhabditis elegans has proven to be a powerful tool for determining gene function. In this study we investigated the effectiveness of RNAi in four non-model grassland soil nematodes, Oscheius sp FVV-2., Rhabditis sp, Mesorhabditis sp., and Acrobeloides sp. In contrast to reference experiments performed using C. elegans and Caenorhabditis briggsae, feeding bacteria expressing dsRNA and injecting dsRNA into the gonad ...[more]

PMID: 23483038

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Project description:BackgroundChanges in plant diversity may induce distinct changes in soil food web structure and accompanying soil feedbacks to plants. However, knowledge of the long-term consequences of plant community simplification for soil animal food webs and functioning is scarce. Nematodes, the most abundant and diverse soil Metazoa, represent the complexity of soil food webs as they comprise all major trophic groups and allow calculation of a number of functional indices.Methodology/principal findingsWe studied the functional composition of nematode communities three and five years after establishment of a grassland plant diversity experiment (Jena Experiment). In response to plant community simplification common nematode species disappeared and pronounced functional shifts in community structure occurred. The relevance of the fungal energy channel was higher in spring 2007 than in autumn 2005, particularly in species-rich plant assemblages. This resulted in a significant positive relationship between plant species richness and the ratio of fungal-to-bacterial feeders. Moreover, the density of predators increased significantly with plant diversity after five years, pointing to increased soil food web complexity in species-rich plant assemblages. Remarkably, in complex plant communities the nematode community shifted in favour of microbivores and predators, thereby reducing the relative abundance of plant feeders after five years.Conclusions/significanceThe results suggest that species-poor plant assemblages may suffer from nematode communities detrimental to plants, whereas species-rich plant assemblages support a higher proportion of microbivorous nematodes stimulating nutrient cycling and hence plant performance; i.e. effects of nematodes on plants may switch from negative to positive. Overall, food web complexity is likely to decrease in response to plant community simplification and results of this study suggest that this results mainly from the loss of common species which likely alter plant-nematode interactions.

Project description:Some organisms can survive extreme desiccation by entering a state of suspended animation known as anhydrobiosis. The free-living mycophagous nematode Aphelenchus avenae can be induced to enter anhydrobiosis by pre-exposure to moderate reductions in relative humidity (RH) prior to extreme desiccation. This preconditioning phase is thought to allow modification of the transcriptome by activation of genes required for desiccation tolerance.To identify such genes, a panel of expressed sequence tags (ESTs) enriched for sequences upregulated in A. avenae during preconditioning was created. A subset of 30 genes with significant matches in databases, together with a number of apparently novel sequences, were chosen for further study. Several of the recognisable genes are associated with water stress, encoding, for example, two new hydrophilic proteins related to the late embryogenesis abundant (LEA) protein family. Expression studies confirmed EST panel members to be upregulated by evaporative water loss, and the majority of genes was also induced by osmotic stress and cold, but rather fewer by heat. We attempted to use RNA interference (RNAi) to demonstrate the importance of this gene set for anhydrobiosis, but found A. avenae to be recalcitrant with the techniques used. Instead, therefore, we developed a cross-species RNAi procedure using A. avenae sequences in another anhydrobiotic nematode, Panagrolaimus superbus, which is amenable to gene silencing. Of 20 A. avenae ESTs screened, a significant reduction in survival of desiccation in treated P. superbus populations was observed with two sequences, one of which was novel, while the other encoded a glutathione peroxidase. To confirm a role for glutathione peroxidases in anhydrobiosis, RNAi with cognate sequences from P. superbus was performed and was also shown to reduce desiccation tolerance in this species.This study has identified and characterised the expression profiles of members of the anhydrobiotic gene set in A. avenae. It also demonstrates the potential of RNAi for the analysis of anhydrobiosis and provides the first genetic data to underline the importance of effective antioxidant systems in metazoan desiccation tolerance.

Project description:BACKGROUND:Ecotoxicological studies on the insensitive munitions formulation IMX-101 and its components 2,4-dinitroanisole (DNAN), nitroguanidine (NQ) and nitrotriazolone (NTO) in various organisms showed that DNAN was the main contributor to the overall toxicity of IMX-101 and suggested that the three compounds acted independently. These results motivated this toxicogenomics study to discern toxicological mechanisms for these compounds at the molecular level. METHODS:Here we used the soil nematode Caenorhabditis elegans, a well-characterized genomics model, as the test organism and a species-specific, transcriptome-wide 44 K-oligo probe microarray for gene expression analysis. In addition to the control treatment, C. elegans were exposed for 24 h to 6 concentrations of DNAN (1.95-62.5 ppm) or NQ (83-2667 ppm) or 5 concentrations of NTO (187-3000 ppm) with ten replicates per treatment. The nematodes were transferred to a clean environment after exposure. Reproduction endpoints (egg and larvae counts) were measured at three time points (i.e., 24-, 48- and 72-h). Gene expression profiling was performed immediately after 24-h exposure to each chemical at the lowest, medium and highest concentrations plus the control with four replicates per treatment. RESULTS:Statistical analyses indicated that chemical treatment did not significantly affect nematode reproduction but did induce 2175, 378, and 118 differentially expressed genes (DEGs) in NQ-, DNAN-, and NTO-treated nematodes, respectively. Bioinformatic analysis indicated that the three compounds shared both DEGs and DEG-mapped Reactome pathways. Gene set enrichment analysis further demonstrated that DNAN and NTO significantly altered 12 and 6 KEGG pathways, separately, with three pathways in common. NTO mainly affected carbohydrate, amino acid and xenobiotics metabolism while DNAN disrupted protein processing, ABC transporters and several signal transduction pathways. NQ-induced DEGs were mapped to a wide variety of metabolism, cell cycle, immune system and extracellular matrix organization pathways. CONCLUSION:Despite the absence of significant effects on apical reproduction endpoints, DNAN, NTO and NQ caused significant alterations in gene expression and pathways at 1.95 ppm, 187 ppm and 83 ppm, respectively. This study provided supporting evidence that the three chemicals may exert independent toxicity by acting on distinct molecular targets and pathways.

Dataset Information

Several Grassland Soil Nematode Species Are Insensitive to RNA-Mediated Interference.

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Several Grassland Soil Nematode Species Are Insensitive to RNA-Mediated Interference.

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