Large scale changes in the transcriptome of Eisenia fetida during regeneration.
ABSTRACT: Earthworms show a wide spectrum of regenerative potential with certain species like Eisenia fetida capable of regenerating more than two-thirds of their body while other closely related species, such as Paranais litoralis seem to have lost this ability. Earthworms belong to the phylum Annelida, in which the genomes of the marine oligochaete Capitella telata and the freshwater leech Helobdella robusta have been sequenced and studied. Herein, we report the transcriptomic changes in Eisenia fetida (Indian isolate) during regeneration. Following injury, E. fetida regenerates the posterior segments in a time spanning several weeks. We analyzed gene expression changes both in the newly regenerating cells and in the adjacent tissue, at early (15days post amputation), intermediate (20days post amputation) and late (30 days post amputation) by RNAseq based de novo assembly and comparison of transcriptomes. We also generated a draft genome sequence of this terrestrial red worm using short reads and mate-pair reads. An in-depth analysis of the miRNome of the worm showed that many miRNA gene families have undergone extensive duplications. Sox4, a master regulator of TGF-beta mediated epithelial-mesenchymal transition was induced in the newly regenerated tissue. Genes for several proteins such as sialidases and neurotrophins were identified amongst the differentially expressed transcripts. The regeneration of the ventral nerve cord was also accompanied by the induction of nerve growth factor and neurofilament genes. We identified 315 novel differentially expressed transcripts in the transcriptome, that have no homolog in any other species. Surprisingly, 82% of these novel differentially expressed transcripts showed poor potential for coding proteins, suggesting that novel ncRNAs may play a critical role in regeneration of earthworm.
Project description:Earthworms show a wide spectrum of regenerative potential with certain species like Eisenia fetida, a terrestrial redworm, capable of regenerating more than two-thirds of their body while other closely related species, such as Paranais litoralis seem to have lost this ability. Earthworms belong to the phylum annelida, in which the genomes of the marine oligochaete Capitella telata, and the freshwater leech Helobdella robusta have been sequenced and studied. Herein, we report the de novo assembled transcriptome of Eisenia fetida (Indian isolate), along with an analysis of the transcriptomic changes during regeneration. We also used de novo assembled RNAseq data to identify genes that are differentially expressed during regeneration, both in the newly regenerating cells and in the adjacent tissue. Overall design: Gene Expression profiles of E.fetida, before and 15,20 or 30 days after cutting were generated by deep sequencing, in triplicate, using Illumina.
Project description:Earthworms are sensitive to toxic chemicals present in the soil and so are useful indicator organisms for soil health. Eisenia fetida are commonly used in ecotoxicological studies; therefore the assembly of a baseline transcriptome is important for subsequent analyses exploring the impact of toxin exposure on genome wide gene expression.This paper reports on the de novo transcriptome assembly of E. fetida using Trinity, a freely available software tool. Trinotate was used to carry out functional annotation of the Trinity generated transcriptome file and the transdecoder generated peptide sequence file along with BLASTX, BLASTP and HMMER searches and were loaded into a Sqlite3 database. To identify differentially expressed transcripts; each of the original sequence files were aligned to the de novo assembled transcriptome using Bowtie and then RSEM was used to estimate expression values based on the alignment. EdgeR was used to calculate differential expression between the two conditions, with an FDR corrected P value cut off of 0.001, this returned six significantly differentially expressed genes. Initial BLASTX hits of these putative genes included hits with annelid ferritin and lysozyme proteins, as well as fungal NADH cytochrome b5 reductase and senescence associated proteins. At a cut off of P = 0.01 there were a further 26 differentially expressed genes.These data have been made publicly available, and to our knowledge represent the most comprehensive available transcriptome for E. fetida assembled from RNA sequencing data. This provides important groundwork for subsequent ecotoxicogenomic studies exploring the impact of the environment on global gene expression in E. fetida and other earthworm species.
Project description:BACKGROUND: Explosive compounds such as TNT and RDX are recalcitrant contaminants often found co-existing in the environment. In order to understand the joint effects of TNT and RDX on earthworms, an important ecological and bioindicator species at the molecular level, we sampled worms (Eisenia fetida) exposed singly or jointly to TNT (50 mg/kg soil) and RDX (30 mg/kg soil) for 28 days and profiled gene expression in an interwoven loop designed microarray experiment using a 4k-cDNA array. Lethality, growth and reproductive endpoints were measured. RESULTS: Sublethal doses of TNT and RDX had no significant effects on the survival and growth of earthworms, but significantly reduced cocoon and juvenile counts. The mixture exhibited more pronounced reproductive toxicity than each single compound, suggesting an additive interaction between the two compounds. In comparison with the controls, we identified 321 differentially expressed transcripts in TNT treated worms, 32 in RDX treated worms, and only 6 in mixture treated worms. Of the 329 unique differentially expressed transcripts, 294 were affected only by TNT, 24 were common to both TNT and RDX treatments, and 3 were common to all treatments. The reduced effects on gene expression in the mixture exposure suggest that RDX might interact in an antagonistic manner with TNT at the gene expression level. The disagreement between gene expression and reproduction results may be attributed to sampling time, absence of known reproduction-related genes, and lack of functional information for many differentially expressed transcripts. A gene potentially related to reproduction (echinonectin) was significantly depressed in TNT or RDX exposed worms and may be linked to reduced fecundity. CONCLUSIONS: Sublethal doses of TNT and RDX affected many biological pathways from innate immune response to oogenesis, leading to reduced reproduction without affecting survival and growth. A complex interaction between mixtures of RDX and TNT was observed at the gene expression level that requires further study of the dynamics of gene expression and reproductive activities in E. fetida. These efforts will be essential to gain an understanding of the additive reproductive toxicity between RDX and TNT.
Project description:The earthworm Eisenia fetida is one of the most used species in standardized soil ecotoxicity tests. End points such as survival, growth, and reproduction are eco-toxicologically relevant but provide little mechanistic insight into toxicity pathways, especially at the molecular level. Here we apply a toxicogenomic approach to investigate the mode of action underlying the reversible neurotoxicity of hexanitrohexaazaisowurtzitane (CL-20), a cyclic nitroamine explosives compound. We developed an E. fetida-specific shotgun microarray targeting 15119 unique E. fetida transcripts. Using this array we profiled gene expression in E. fetida in response to exposure to CL-20. Eighteen earthworms were exposed for 6 days to 0.2 ?g/cm(2) of CL-20 on filter paper, half of which were allowed to recover in a clean environment for 7 days. Nine vehicle control earthworms were sacrificed at days 6 and 13, separately. Electrophysiological measurements indicated that the conduction velocity of earthworm medial giant nerve fiber decreased significantly after 6-day exposure to CL-20, but was restored after 7 days of recovery. Total RNA was isolated from the four treatment groups including 6-day control, 6-day exposed, 13-day control, and 13-day exposed (i.e., 6-day exposure followed by 7-day recovery), and was hybridized to the 15K shotgun oligo array. Statistical and bioinformatic analyses suggest that CL-20 initiated neurotoxicity by noncompetitively blocking the ligand-gated GABA(A) receptor ion channel, leading to altered expression of genes involved in GABAergic, cholinergic, and Agrin-MuSK pathways. In the recovery phase, expression of affected genes returned to normality, possibly as a result of autophagy and CL-20 dissociation/metabolism. This study provides significant insights into potential mechanisms of CL-20-induced neurotoxicity and the recovery of earthworms from transient neurotoxicity stress.
Project description:The bacterial symbiont Verminephrobacter eiseniae colonizes nephridia, the excretory organs, of the lumbricid earthworm Eisenia fetida. E. fetida transfers V. eisenia into the egg capsule albumin during capsule formation and V. eiseniae cells migrate into the earthworm nephridia during embryogenesis, where they bind and persist. In order to characterize the mechanistic basis of selective tissue colonization, methods for site-directed mutagenesis and colonization competence were developed and used to evaluate the consequences of individual gene disruptions. Using these newly developed tools, two distinct modes of bacterial motility were shown to be required for V. eiseniae colonization of nascent earthworm nephridia. Flagella and type IV pili mutants lacked motility in culture and were not able to colonize embryonic earthworms, indicating that both twitching and flagellar motility are required for entrance into the nephridia.
Project description:In annelid worms, the nerve cord serves as a crucial organ to control the sensory and behavioral physiology. The inadequate genome resource of earthworms has prioritized the comprehensive analysis of their transcriptome dataset to monitor the genes express in the nerve cord and predict their role in the neurotransmission and sensory perception of the species. The present study focuses on identifying the potential transcripts and predicting their functional features by annotating the transcriptome dataset of nerve cord tissues prepared by Gong et al., 2010 from the earthworm Eisenia fetida. Totally 9762 transcripts were successfully annotated against the NCBI nr database using the BLASTX algorithm and among them 7680 transcripts were assigned to a total of 44,354 GO terms. The conserve domain analysis indicated the over representation of P-loop NTPase domain and calcium binding EF-hand domain. The COG functional annotation classified 5860 transcript sequences into 25 functional categories. Further, 4502 contig sequences were found to map with 124 KEGG pathways. The annotated contig dataset exhibited 22 crucial neuropeptides having considerable matches to the marine annelid Platynereis dumerilii, suggesting their possible role in neurotransmission and neuromodulation. In addition, 108 human stem cell marker homologs were identified including the crucial epigenetic regulators, transcriptional repressors and cell cycle regulators, which may contribute to the neuronal and segmental regeneration. The complete functional annotation of this nerve cord transcriptome can be further utilized to interpret genetic and molecular mechanisms associated with neuronal development, nervous system regeneration and nerve cord function.
Project description:Two new megadrile earthworms from the steppes, the first species wholly from Outer Mongolia, are ascribed to the partially parthenogenetic Eisenia nordenskioldi (Eisen, 1879) species-complex. Taxonomic justification of sympatric Eisenia nordenskioldi mongol and Eisenia nordenskioldi onon ssp. n. are supported by mtDNA COI barcodes. The unreliability of molecular differentiation based on voucher names compared to definitive types is again demonstrated, as pertains to the ultimate Eisenia andrei Bouché, 1972 synonym of the Eisenia fetida (Savigny, 1826) sibling species-complex composed of more than a dozen prior names. Similar species described from Northeast China [formerly Manchuria] and North Korea are briefly considered, albeit they are intermittently held in synonymy of cosmopolitan Aporrectodea rosea (Savigny, 1826) along with many other taxa including some exotic lumbricids initially found in India. Japanese and North American lumbricids are also mentioned. Distributions are discussed and an annotated checklist of all nine Siberian/sub-arctic Eisenia nordenskioldi ssp. is appended.
Project description:The data described are related to the article "Lysenin family proteins in earthworm coelomocytes - comparative approach" (B. Swiderska, S. Kedracka-Krok, T. Panz, A.J. Morgan, A. Falniowski, P.Grzmil, B. Plytycz, 2016) . Lysenin family proteins were identified based on unique peptides sequenced by tandem mass spectrometry coupled with liquid chromatography (LC-MS/MS) in lumbricid earthworms Eisenia andrei and E. fetida, the latter with or without the MUG-like fluorophore. Lysenin and lysenin-related protein 2 (LRP-2, fetidin) were identified in all 9 investigated specimens of Eisenia sp. LRP-1 was identified in 5 of 6 specimens of E. fetida, while LRP-3 was present in 2 of 3 investigated specimens of E. andrei. Here, the detailed characteristics of identified peptides unique to the particular members of lysenin family present in each particular earthworm specimen was provided. The information concerning mass to charge ratio, retention time, modifications and score of unique peptides was given.
Project description:Survival of earthworms in the environment depends on their ability to recognize and eliminate potential pathogens. This work is aimed to compare the innate defense mechanisms of two closely related earthworm species, Eisenia andrei and Eisenia fetida, that inhabit substantially different ecological niches. While E. andrei lives in a compost and manure, E. fetida can be found in the litter layer in forests. Therefore, the influence of environment-specific microbiota on the immune response of both species was followed. Firstly, a reliable method to discern between E. andrei and E. fetida based on species-specific primers for cytochrome c oxidase I (COI) and stringent PCR conditions was developed. Secondly, to analyze the immunological profile in both earthworm species, the activity and expression of lysozyme, pattern recognition protein CCF, and antimicrobial proteins with hemolytic function, fetidin and lysenins, have been assessed. Whereas, CCF and lysozyme showed only slight differences in the expression and activity, fetidin/lysenins expression as well as the hemolytic activity was considerably higher in E. andrei as compared to E. fetida. The expression of fetidin/lysenins in E. fetida was not affected upon the challenge with compost microbiota, suggesting more substantial changes in the regulation of the gene expression. Genomic DNA analyses revealed significantly higher level of fetidin/lysenins (determined using universal primer pairs) in E. andrei compared to E. fetida. It can be hypothesized that E. andrei colonizing compost as a new habitat acquired an evolutionary selection advantage resulting in a higher expression of antimicrobial proteins.
Project description:Animals start interactions with the bacteria that will constitute their microbiomes at embryonic stage. After mating, earthworms produce cocoons externally which will be colonized with bacteria from their parents and the environment. Due to the key role bacterial symbionts play on earthworm fitness, it is important to study bacterial colonization during cocoon formation. Here we describe the cocoon microbiome of the earthworms Eisenia andrei and E. fetida, which included 275 and 176 bacterial species, respectively. They were dominated by three vertically-transmitted symbionts, Microbacteriaceae, Verminephrobacter and Ca. Nephrothrix, which accounted for 88% and 66% of the sequences respectively. Verminephrobacter and Ca. Nephrothrix showed a high rate of sequence variation, suggesting that they could be biparentally acquired during mating. The other bacterial species inhabiting the cocoons came from the bedding, where they accounted for a small fraction of the diversity (27% and 7% of bacterial species for E. andrei and E. fetida bedding). Hence, earthworm cocoon microbiome includes a large fraction of the vertically-transmitted symbionts and a minor fraction, but more diverse, horizontally and non-randomly acquired from the environment. These data suggest that horizontally-transmitted bacteria to cocoons may play an important role in the adaptation of earthworms to new environments or diets.