Reference genes for QRT-PCR tested under various stress conditions in Folsomia candida and Orchesella cincta (Insecta, Collembola)

<p>Abstract</p> <p>Background</p> <p>Genomic studies measuring transcriptional responses to changing environments and stress currently make their way into the field of evolutionary ecology and ecotoxicology. To investigate a small to medium number of genes or to confirm large scale microarray studies, Quantitative Reverse Transcriptase PCR (QRT-PCR) can achieve high accuracy of quantification when key standards, such as normalization, are carefully set. In this study, we validated potential reference genes for their use as endogenous controls under different chemical and physical stresses in two species of soil-living Collembola, <it>Folsomia candida </it>and <it>Orchesella cincta</it>. Treatments for <it>F. candida </it>were cadmium exposure, phenanthrene exposure, desiccation, heat shock and pH stress, and for <it>O. cincta </it>cadmium, desiccation, heat shock and starvation.</p> <p>Results</p> <p>Eight potential reference genes for <it>F. candida </it>and seven for <it>O. cincta </it>were ranked by their stability per stress factor using the programs geNorm and Normfinder. For <it>F. candida </it>the succinate dehydrogenase (<it>SDHA</it>) and eukaryotic transcription initiation factor 1A (<it>ETIF</it>) genes were found the most stable over the different treatments, while for <it>O. cincta</it>, the beta actin (<it>ACTb</it>) and tyrosine 3-monooxygenase (<it>YWHAZ</it>) genes were the most stable.</p> <p>Conclusion</p> <p>We present a panel of reference genes for two emerging ecological genomic model species tested under a variety of treatments. Within each species, different treatments resulted in differences in the top stable reference genes. Moreover, the two species differed in suitable reference genes even when exposed to similar stresses. This might be attributed to dissimilarity of physiology. It is vital to rigorously test a panel of reference genes for each species and treatment, in advance of relative quantification of QRT-PCR gene expression measurements.</p

Collembase: a repository for springtail genomics and soil quality assessment

<p>Abstract</p> <p>Background</p> <p>Environmental quality assessment is traditionally based on responses of reproduction and survival of indicator organisms. For soil assessment the springtail <it>Folsomia candida </it>(Collembola) is an accepted standard test organism. We argue that environmental quality assessment using gene expression profiles of indicator organisms exposed to test substrates is more sensitive, more toxicant specific and significantly faster than current risk assessment methods. To apply this species as a genomic model for soil quality testing we conducted an EST sequencing project and developed an online database.</p> <p>Description</p> <p>Collembase is a web-accessible database comprising springtail (<it>F. candida</it>) genomic data. Presently, the database contains information on 8686 ESTs that are assembled into 5952 unique gene objects. Of those gene objects ~40% showed homology to other protein sequences available in GenBank (blastx analysis; non-redundant (nr) database; expect-value < 10^-5). Software was applied to infer protein sequences. The putative peptides, which had an average length of 115 amino-acids (ranging between 23 and 440) were annotated with Gene Ontology (GO) terms. In total 1025 peptides (~17% of the gene objects) were assigned at least one GO term (expect-value < 10^-25). Within Collembase searches can be conducted based on BLAST and GO annotation, cluster name or using a BLAST server. The system furthermore enables easy sequence retrieval for functional genomic and Quantitative-PCR experiments. Sequences are submitted to GenBank (Accession numbers: <ext-link ext-link-type="gen" ext-link-id="EV473060">EV473060</ext-link> – <ext-link ext-link-type="gen" ext-link-id="EV481745">EV481745</ext-link>).</p> <p>Conclusion</p> <p>Collembase <url>http://www.collembase.org</url> is a resource of sequence data on the springtail <it>F. candida</it>. The information within the database will be linked to a custom made microarray, based on the Agilent platform, which can be applied for soil quality testing. In addition, Collembase supplies information that is valuable for related scientific disciplines such as molecular ecology, ecogenomics, molecular evolution and phylogenetics.</p

Surface coating and particle size are main factors explaining the transcriptome-wide responses of the earthworm Lumbricus rubellus to silver nanoparticles

Due to the unique properties of differently sized and coated silver nanoparticles (AgNPs), they are used in important industrial and biomedical applications. However, their environmental fate in soil ecosystems and potential mechanisms of toxicity remain elusive, especially at the level of transcriptional regulation. We investigated the transcriptome-wide responses of the earthworm Lumbricus rubellus exposed to nine AgNPs differing in surface coating/charge (bovine serum albumin/negative AgNP_BSA, chitosan/positive AgNP_Chit, and polyvinylpyrrolidone/neutral AgNP_PVP) and sizes (20, 35 and 50 nm) at concentrations close to the EC50 value related to reproduction. AgNO3 was used in two concentrations to benchmark the AgNP effects against those of the Ag salt. A correlation was observed between the number of differentially expressed genes (DEGs) and Ag internal body concentration. Only metallothionein was regulated by all treatments. Medium sized AgNPs caused the most pronounced transcriptional responses, while AgNO3 affected the transcriptome less. Medium sized AgNP_BSA exposure caused the most extensive transcriptional responses with 684 DEGs. Gene ontology enrichment analysis of medium sized AgNP_BSA affected DEGs revealed that mitochondrial electron transport, autophagy and phagocytosis, mesoderm and heart development and microtubule organisation were affected. This was also confirmed by gene set enrichment for KEGG pathway analysis, indicating that phagocytosis, autophagy and signalling pathways related to mesoderm formation were significantly up regulated. All AgNP_BSA and AgNP_PVP exposures caused severe down regulation of ribosomal translation, suggesting that the high energy-demanding protein synthesis process is inhibited. Our data confirm the mechanisms previously identified among other animal models and human cell lines. To conclude, coating formulation and particle size severely impact transcriptional responses at a particular nanoparticle size, suggesting diverse mechanistic responses depending on the coating type

Molecular mechanisms of zinc toxicity in the potworm Enchytraeus crypticus, analysed by high-throughput gene expression profiling

Zinc (Zn) is known to be relatively toxic to some soil-living invertebrates including the ecologically important enchytraeid worms. To reveal the molecular mechanisms of zinc toxicity we assessed the gene expression profile of Enchytraeus crypticus (Enchytraeidae), exposed to the reproduction effect concentrations EC10 and EC50, over 4 consecutive days, using a high-throughput microarray (species customized). Three main mechanisms of toxicity to Zn were observed: 1) Zn trafficking (upregulation of zinc transporters, a defence response to regulate the cellular zinc level), 2) oxidative stress (variety of defence mechanisms, triggered by Reactive Oxygen Species (ROS)), and 3) effects on the nervous system (possibly the primary lesion explaining the avoidance behaviour and also why enchytraeids are relatively susceptible to Zn). The adverse outcome at the organism level (reproduction EC50) could be predicted based on gene expression (male gonad development, oocyte maturation), with Zn at the EC50 affecting processes related to higher stress levels. The gene expression response was time-dependent and reflected the cascade of events taking place over-time. The 1 to 4 days of exposure design was a good strategy as it captured the time for sequence of events towards zinc adverse outcomes in E. crypticus

Transcriptome assembly and microarray construction for Enchytraeus crypticus, a model oligochaete to assess stress response mechanisms derived from soil conditions

The soil worm Enchytraeus crypticus (Oligochaeta) is an ecotoxicology model species that, until now, was without genome or transcriptome sequence information. The present research aims at studying the transcriptome of Enchytraeus crypticus, sampled from multiple test conditions, and the construction of a high-density microarray for functional genomic studies. Over 1.5 million cDNA sequence reads were obtained representing 645 million nucleotides. After assembly, 27,296 contigs and 87,686 singletons were obtained, from which 44% and 25% are annotated as protein-coding genes, respectively, sharing homology with other animal proteomes. Concerning assembly quality, 84% of the contig sequences contain an open reading frame with a start codon while E. crypticus homologs were identified for 92% of the core eukaryotic genes. Moreover, 65% and 77% of the singletons and contigs without known homologs, respectively, were shown to be transcribed in an independent microarray experiment. An Agilent 180 K microarray platform was designed and validated by hybridizing cDNA from 4 day zinc- exposed E. crypticus to the concentration corresponding to 50% reduction in reproduction after three weeks (EC50). Overall, 70% of all probes signaled expression above background levels (mean signal + 1x standard deviation). 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 differentially regulated transcripts were identified 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 retro transposon activity in the E. crypticus genome. An initial investigation of the E. crypticus transcriptome including an associated microarray platform for future studies proves to be a valuable resource to investigate functional genomics mechanisms of toxicity in soil environments and to annotate a potentially large number of lineage specific genes that are responsive to environmental stress conditions

Population-specific transcriptional differences associated with freeze tolerance in a terrestrial worm

Enchytraeus albidus is a terrestrial earthworm widespread along the coasts of northern Europe and the Arctic. This species tolerates freezing of body fluids and survives winters in a frozen state. Their acclimatory physiological mechanisms behind freeze tolerance involve increased fluidity of membrane lipids during cold exposure and accumulation of cryoprotectants (glucose) during the freezing process. Gene regulatory processes of these physiological responses have not been studied, partly because no gene expression tools were developed. The main aim of this study was to understand whether the freeze tolerance mechanisms have a transcriptomic basis in E. albidus. For that purpose, first the transcriptome of E. albidus was assembled with RNAseq data. Second, two strains from contrasting thermal environments (Germany and Greenland) were compared by mapping barcoded RNAseq data onto the assembled transcriptome. Both of these strains are freeze tolerant, but Greenland is extremely freeze tolerant. Results showed more plastic responses in the Greenland strain as well as higher constitutive expression of particular stress response genes. These altered transcriptional networks are associated with an adapted homeostasis coping with prolonged freezing conditions in Greenland animals. Previously identified physiological alterations in freeze-tolerant strains of E. albidus are underpinned at the transcriptome level. These processes involve anion transport in the hemolymph, fatty acid metabolism, metabolism, and transport of cryoprotective sugars as well as protection against oxidative stress. Pathway analysis supported most of these processes, and identified additional differentially expressed pathways such as peroxisome and Toll-like receptor signaling. We propose that the freeze-tolerant phenotype is the consequence of genetic adaptation to cold stress and may have driven evolutionary divergence of the two strains

Elucidating fungal Rigidoporus species FMD21 lignin-modifying enzyme genes and 2,3,7,8-tetrachlorodibenzo-p-dioxin degradation by laccase isozymes

White-rot fungus Rigidoporus sp. FMD21 is a lignin-modifying enzyme producing fungus that can degrade dioxin. Extracellular enzymes from FMD21 include laccase and manganese peroxidase which are promising enzymes for myco-remediation because of their wide substrate specificity and mild catalysis conditions. The FMD21 genome was sequenced using Ion Torrent technology and consists of 38.98 Mbps with a GC content of 47.4 %. Gene prediction using Augustus with Basidiomycota reference setting resulted in 8245 genes. Functional gene annotations were carried out by using several programs and databases. We focused on laccase and ligninolytic peroxidase genes, which are most likely involved in the degradation of aromatic pollutants. The genome of FMD21 contains 12 predicted laccase genes (10 out of 12 predicted as full length) and 13 putative ligninolytic peroxidases which were annotated as MnP or versatile peroxidases. Four predicted laccases showed a higher than 65 % binding chance to 2,3,7,8-TCDD with the highest at 72 % in in silico docking analysis. Heterologous expressed laccases showed activity towards three tested substrates included ABTS, guaiacol and 2,6-DMP. ABTS displayed two-stage oxidation which differed from natural FMD21 laccases. 2,3,7,8-TCDD was degraded by 50 % after two weeks of enzymatic treatment by three out of five laccase isozymes which were natural laccases secreted by FMD21. In this study, we provide direct evidence for the 2,3,7,8-TCDD biodegradation capability of fungal laccases

Characterization of 2,3,7,8-tetrachlorodibenzo-p-dioxin biodegradation by extracellular lignin-modifying enzymes from ligninolytic fungus

Ligninolytic fungi secrete extracellular lignin-modifying enzymes (LME) that degrade plant polymers for fungal nutrition but that are, because of their broad substrate specificity, also applicable for the degradation of many hazardous pollutants. Laccase is one of the most well characterized LME and is involved in the removal and degradation of recalcitrant aromatic compounds with or without the assistance of laccase-mediators. The Ligninolytic fungus Rigidoporus sp. FMD21 can degrade 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) with a half-life of 6.2 days. Using Rigidoporus sp. FMD21 crude extracellular enzyme extract (ExE) that mainly consisted of laccase, 77.4% of 2,3,7,8-TCDD was degraded within 36 days. The degradation rate did not depend on the 2,3,7,8-TCDD concentration in the tested range between 0.005 and 0.5 pgTEQ/μL. 2,3,7,8-TCDD was analysed by DR-CALUX® bioassay and the degradation was confirmed by GC-HRMS. In this study, we found evidence for cleavage of the diaryl ether bond in the 2,3,7,8-TCDD molecule and here we propose a new degradation mechanism in which 3,4-dichlorophenol is the main metabolite of 2,3,7,8-TCDD degradation by FMD21’s ExE. Six laccase-mediators were tested. Three of them 1-hydroxybenzotriazole (HBT), syringaldehyde (Syr) and violuric acid (Vio) showed an equipotent added effect on 2,3,7,8-TCDD degradation by ExE, however only in case of Vio a level of significance was reached. The others showed no effect or negatively impacted degradation. In conclusion, we have shown that Rigidoporus sp. FMD21 produces extracellular enzymes, mainly laccases that apparently are able to degrade the highly recalcitrant and most toxic 2,3,7,8-congener of TCDD via diaryl bond cleavage into 3,4-dichlorophenol

Screening white-rot fungi for bioremediation potential of 2,3,7,8-tetrachlorodibenzo-p-dioxin

Ligninolytic fungi contain a number of representative strains consisting of mainly white-rot fungi (WRF) that produce lignin-modifying enzymes (LME) such as laccases and manganese peroxidases. Lignin-modifying enzymes are multipurpose enzymes which have potential for application in various fields such as, for example, bioremediation and biomass conversion. Because of the non-specific nature of these enzymes, they are also capable of biodegradation and removal of xenobiotic pollutants. In this study we used a tiered screening process where we screened over 70 Vietnamese WRF fungal isolates for LME activity and subsequently for the ability to breakdown the dioxin TCDD. After the initial screening we selected four fungal strains, which belong to the order of Polyporales, which excreted high laccase enzyme levels. The most active fungus being isolate FMD21, a species of Rigidoporus, which was isolated from a forest in the South of Vietnam and which produced both laccase and manganese peroxidase. In the optimized PDSRb medium, FMD21 laccase levels reaced activities of 238800 U/L after 10 days while MnP activity showed the highest activity at day 4 of aproximately 40 U/L. 2,3,7,8-TCDD, which is the most toxic dioxin congener, is a persistent organic pollutant of which few organisms are known that break it down. After the final screening, FMD21 was the only fungus capable of degrading TCDD and was able to reach a breakdown percentage of 73% after 28 days culture with a start concentration of 0.5 pg TEQ/μL TCDD. Co-cultivation experiments of up to three fungi were performed to test for a synergistic breakdown effect of TCDD but such an effect was not observed. FMD21 is a fungus that shows a potential to be used as a bioremediation agent to clean up dioxin contamination in the environment