Degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) by fungi originating from Vietnam.

Three different fungi were tested for their ability to degrade 2,4-dichlorophenoxyacetic acid and 2,4,5-trichlorophenoxyacetic acid and for the role of laccases and cytochromes P450-type in this process. We studied a white-rot fungus Rigidoporus sp. FMD21, which has a high laccase activity, for its efficiency to degrade these herbicides. A positive correlation was found between its laccase activity and the corresponding herbicide degradation rate. Even more, the doubling of the enzyme activity in this phase corresponded with a doubling of the herbicide degradation rate. It is, therefore, tempting to speculate that laccase is the most dominant enzyme in the degradation of 2,4-D and 2,4,5-T under these conditions. In addition, it was shown that Rigidoporus sp. FMD21 partly relies on cytochromes P450-type for the breakdown of the herbicides as well. Two filamentous fungi were isolated from soil contaminated with herbicides and dioxins located at Bien Hoa airbase. They belong to genera Fusarium and Verticillium of the phylum Ascomycota as judged by their 18S rRNA gene sequences. Both isolated fungi were able to degrade the herbicides but with different rates. Their laccase activity, however, was very low and did not correlate with the rate of breakdown of the herbicides. These data indicate that the white-rot fungus most likely synthesizes laccase and cytochromes P450-type for the breakdown of the herbicides, while the types of enzyme used for the breakdown of the herbicides by the two Ascomycota remain unclear.

Combined Transcriptomics Analysis for Classification of Adverse Effects As a Potential End Point in Effect Based Screening.

Environmental risk assessment relies on the use of bioassays to assess the environmental impact of chemicals. Gene expression is gaining acceptance as a valuable mechanistic end point in bioassays and effect-based screening. Data analysis and its results, however, are complex and often not directly applicable in risk assessment. Classifier analysis is a promising method to turn complex gene expression analysis results into answers suitable for risk assessment. We have assembled a large gene expression data set assembled from multiple studies and experiments in the springtail Folsomia candida, with the aim of selecting a set of genes that can be trained to classify general toxic stress. By performing differential expression analysis prior to classifier training, we were able to select a set of 135 genes which was enriched in stress related processes. Classifier models from this set were used to classify two test sets comprised of chemical spiked, polluted, and clean soils and compared to another, more traditional classifier feature selection. The gene set presented here outperformed the more traditionally selected gene set. This gene set has the potential to be used as a biomarker to test for adverse effects caused by chemicals in springtails to provide end points in environmental risk assessment.

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

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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.

A functional isopenicillin N synthase in an animal genome.

Horizontal transfer of genes is widespread among prokaryotes, but is less common between microorganisms and animals. Here, we present evidence for the presence of a gene encoding functional isopenicillin N synthase, an enzyme in the ß-lactam antibiotics biosynthesis pathway, in the genome of the soil-living collembolan species, Folsomia candida (FcIPNS). At present, this gene is only known from bacteria and fungi, as is the capacity to produce ß-lactam antibiotics. The FcIPNS gene was located on two genomic contigs, was physically linked to a predicted insect ATP-binding cassette transporter gene, and contained three introns each flanked by eukaryotic splicing recognition sites (GT/AG). Homology searches revealed no similarity between these introns and the FcIPNS regions of bacteria or fungi. All amino acids conserved across bacteria and fungi were also conserved in F. candida. Recombinant FcIPNS was able to convert its substrate amino δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine into isopenicillin N, providing strong evidence that FcIPNS is functional. Phylogenetic analysis clustered FcIPNS outside the bacterial IPNS clade, and also outside the fungal IPNS clade, suggesting an ancient gene transfer followed by divergence in the F. candida genome. In conclusion, the data suggest that the soil-living collembolan F. candida has assimilated the capacity for antibacterial activity by horizontal gene transfer, which may be an important adaptive trait in the microbe-dominated soil ecosystem.

The influence of long-term copper contaminated agricultural soil at different pH levels on microbial communities and springtail transcriptional regulation.

Copper has long been applied for agricultural practises. Like other metals, copper is highly persistent in the environment and biologically active long after its use has ceased. Here we present a unique study on the long-term effects (27 years) of copper and pH on soil microbial communities and on the springtail Folsomia candida an important representative of the soil macrofauna, in an experiment with a full factorial, random block design. Bacterial communities were mostly affected by pH. These effects were prominent in Acidobacteria, while Actinobacteria and Gammaroteobacteria communities were affected by original and bioavailable copper. Reproduction and survival of the collembolan F. candida was not affected by the studied copper concentrations. However, the transcriptomic responses to copper reflected a mechanism of copper transport and detoxification, while pH exerted effects on nucleotide and protein metabolism and (acute) inflammatory response. We conclude that microbial community structure reflected the history of copper contamination, while gene expression analysis of F. candida is associated with the current level of bioavailable copper. The study is a first step in the development of a molecular strategy aiming at a more comprehensive assessment of various aspects of soil quality and ecotoxicology.

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.

Transcriptional plasticity of a soil arthropod across different ecological conditions.

Ecological functional genomics, dealing with the responses of organisms to their natural environment is confronted with a complex pattern of variation and a large number of confounding environmental factors. For gene expression studies to provide meaningful information on conditions deviating from normal, a baseline or normal operating range (NOR) response needs to be established which indicates how an organism's transcriptome reacts to naturally varying ecological factors. Here we determine the transcriptional plasticity of a soil arthropod, Folsomia candida, exposed to various natural environments, as part of a first attempt in establishing such a NOR. Animals were exposed to 26 different field soils after which gene expression levels were measured. The main factor found to regulate gene expression was soil-type (sand or clay). Cell homeostasis and DNA replication were affected in collembolans exposed to sandy soil, indicating general stress. Multivariate analysis identified soil fertility as the main factor influencing gene expression. Regarding land-use, only forest soils showed an expression pattern deviating from the others. No significant effect of land-use, agricultural practice or soil type on fitness was observed, but arsenic concentration was negatively correlated with reproductive output. In conclusion, transcriptional responses remained within a limited range across the different land-uses but were significantly affected by soil-type. This may be caused by the contrasting soil physicochemical properties to which F. candida strongly responds. The broad range of conditions over which this soil-living detritivore is able to survive and reproduce, indicates a strategy of high plasticity, which comes with extensive gene expression regulation.

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.

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

BACKGROUND: 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, Folsomia candida and Orchesella cincta. Treatments for F. candida were cadmium exposure, phenanthrene exposure, desiccation, heat shock and pH stress, and for O. cincta cadmium, desiccation, heat shock and starvation. RESULTS: Eight potential reference genes for F. candida and seven for O. cincta were ranked by their stability per stress factor using the programs geNorm and Normfinder. For F. candida the succinate dehydrogenase (SDHA) and eukaryotic transcription initiation factor 1A (ETIF) genes were found the most stable over the different treatments, while for O. cincta, the beta actin (ACTb) and tyrosine 3-monooxygenase (YWHAZ) genes were the most stable. CONCLUSION: 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.

Species-specific transcriptomic responses in Daphnia magna exposed to a bio-plastic production intermediate.

Hydroxymethylfurfural (HMF) is a plant-based chemical building block that could potentially substitute petroleum-based equivalents, yet ecotoxicological data of this compound is currently limited. In this study, the effects of HMF on the reproduction and survival of Daphnia magna were assessed through validated ecotoxicological tests. The mechanism of toxicity was determined by analysis of transcriptomic responses induced by exposure to different concentrations of HMF using RNA sequencing. HMF exerted toxicity to D. magna with an EC50 for effects on reproduction of 17.2 mg/l. HMF exposure affected molecular pathways including sugar and polysaccharide metabolism, lipid metabolism, general stress metabolism and red blood cell metabolism, although most molecular pathways affected by HMF exposure were dose specific. Hemoglobin genes, however, responded in a sensitive and dose-related manner. No induction of genes involved in the xenobiotic metabolism or oxidative stress metabolism pathway could be observed, which contrasted earlier observations on transcriptional responses of the terrestrial model Folsomia candida exposed to the same compound in a similar dose. We found 4189 orthologue genes between D. magna and F. candida, yet only twenty-one genes of those orthologues were co-regulated in both species. The contrasting transcriptional responses to the same compound exposed at a similar dose between D. magna and F. candida indicates limited overlap in stress responses among soil and aquatic invertebrates. The dose-related expression of hemoglobin provides further support for using hemoglobin expression as a biomarker for general stress responses in daphnids.

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.

Collembase: a repository for springtail genomics and soil quality assessment.

BACKGROUND: Environmental quality assessment is traditionally based on responses of reproduction and survival of indicator organisms. For soil assessment the springtail Folsomia candida (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. DESCRIPTION: Collembase is a web-accessible database comprising springtail (F. candida) genomic data. Presently, the database contains information on 8686 ESTs that are assembled into 5952 unique gene objects. Of those gene objects approximately 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 (approximately 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: EV473060 - EV481745). CONCLUSION: Collembase http://www.collembase.org is a resource of sequence data on the springtail F. candida. 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.

Draft Genome Sequence of Bacillus toyonensis VU-DES13, Isolated from Folsomia candida (Collembola: Entomobryidae).

We present here the draft genome of Bacillus toyonensis VU-DES13, which was isolated from the midgut of the soil-living springtail Folsomia candida Previous research revealed the presence of gene clusters for the biosynthesis of various secondary metabolites, including ß-lactam antibiotics, in the host's genome. The genome data are discussed in the light of the antimicrobial properties against fungi and oomycetes and a high level of ß-lactam resistance of the isolate.

Mechanisms of phenanthrene toxicity in the soil invertebrate, Enchytraeus crypticus.

Polycyclic aromatic hydrocarbons (PAHs) continue to cause environmental challenges as a result of their release into the environment by a great variety of anthropogenic activities and their accumulation in soils. Studies were conducted on the toxicological effect of the model PAH phenanthrene using the soil invertebrate model Enchytraeus crypticus at the individual, tissue, and molecular levels. Animals were exposed for 2 d and 21 d to phenanthrene concentrations corresponding to the (previously estimated) 3-wk effective concentration, 10% (EC10) and EC50 for effects on reproduction. Gene expression profiling did not reveal a typical phenanthrene-induced biotransformation signature, as it usually does in arthropods and vertebrates. Instead, after 2 d of exposure, only general metabolic processes were affected, such as translation and adenosine triphosphate synthesis-coupled electron transport. Histological sections of tissues of 2-d exposed animals showed no deviations from control. In contrast, after prolonged exposure of up to 21 d, histopathological effects were found: chloragogenous cells were highly vacuolated and hypertrophic. This was corroborated by differential expression of genes related to immune response and oxidative stress at the transcriptomic level. The data exemplify the complexity and species-specific features of PAH toxicity among soil invertebrate communities, which restricts read-across and extrapolation in the context of soil ecological risk assessment. Environ Toxicol Chem 2016;35:2713-2720. © 2016 SETAC.

Integrating transcriptomics into triad-based soil-quality assessment.

The present study examined how transcriptomics tools can be included in a triad-based soil-quality assessment to assess the toxicity of soils from riverbanks polluted by metals. To that end, the authors measured chemical soil properties and used the International Organization for Standardization guideline for ecotoxicological tests and a newly developed microarray for gene expression in the indicator soil arthropod Folsomia candida. Microarray analysis revealed that the oxidative stress response pathway was significantly affected in all soils except one. The data indicate that changes in cell redox homeostasis are a significant signature of metal stress. Finally, 32 genes showed significant dose-dependent expression with metal concentrations. They are promising genetic markers providing an early indication of the need for higher-tier testing of soil quality. During the bioassay, the toxicity of the least polluted soils could be removed by sterilization. The gene expression profile for this soil did not show a metal-related signature, confirming that a factor other than metals (most likely of biological origin) caused the toxicity. The present study demonstrates the feasibility and advantages of integrating transcriptomics into triad-based soil-quality assessment. Combining molecular and organismal life-history trait stress responses helps to identify causes of adverse effects in bioassays. Further validation is needed for verifying the set of genes with dose-dependent expression patterns linked with toxic stress.

The effect of soil pH and temperature on Folsomia candida transcriptional regulation.

Differences in abiotic factors like temperature and soil pH can have a significant physiological impact on soil dwelling invertebrates and may confound results in ecotoxicological testing. In this study we exposed Folsomia candida to a range of two abiotic stress treatments (pH and temperature) for 3 days and measured gene expression of a panel of nine stress response genes with real-time Q-PCR. The exposure to different pH values had a minimal effect on the expression of the nine selected genes: only V-ATPase expression was significantly increased due to decreasing pH. ATPase expression was up-regulated, possibly due to increased proton trafficking across the cell membrane, at a lower pH. HSP70 was up-regulated in collembolans exposed to 30 degrees C, and along with HSP40 at 0 degrees C. We speculate that the minor pH effect on gene expression, compared to the temperature treatment, can be explained by the spatial restricted exposure to the external pH in the gut. Our data showed that only 1 or 2 stress response genes were transcriptionally affected by pH and temperature thus exerting minimal effects. The physiological effects of these treatments on F. candida might indicate interesting novel molecular mechanisms.

Gene expression analysis of collembola in cadmium containing soil.

Increasing concern about pollution of our environment calls for advanced and rapid methods to estimate ecological toxicity. The use of gene expression microarrays in environmental studies can potentially meet this challenge. We present a novel method to examine soil toxicity. We exposed the collembolan Folsomia candida to soil containing an ecologically relevant cadmium concentration, and found a cumulative total of 1586 differentially expressed transcripts across three exposure durations, including transcripts involved in stress response, detoxification, and hypoxia. Additional enrichment analysis of gene ontology (GO) terms revealed that antibiotic biosynthesis is important at all time points examined. Interestingly, genes involved in the "penicillin and cephalosporin biosynthesis pathway" have never been identified in animals before, but are expressed in F. candida's tissue. The synthesis of antibiotics can possibly be a response to increased cadmium-induced susceptibility to invading pathogens, which might be caused by repression of genes involved in the immune-system (C-type lectins and Toll receptor). This study presents a first global view on the environmental stress response of an arthropod species exposed to contaminated soil, and provides a mechanistic basis for the development of a gene expression soil quality test.