Noise and light pollution elicit endocrine responses in urban but not forest frogs.

Urban areas are characterised by the presence of sensory pollutants, such as anthropogenic noise and artificial light at night (ALAN). Animals can quickly adapt to novel environmental conditions by adjusting their behaviour, which is proximately regulated by endocrine systems. While endocrine responses to sensory pollution have been widely reported, this has not often been linked to changes in behaviour, hampering the understanding of adaptiveness of endocrine responses. Our aim was, therefore, to investigate the effects of urbanisation, specifically urban noise and light pollution, on hormone levels in male urban and forest túngara frogs (Engystomops pustulosus), a species with reported population divergence in behaviour in response to urbanisation. We quantified testosterone and corticosterone release rates in the field and in the lab before and after exposure to urban noise and/or light. We show that urban and forest frogs differ in their endocrine phenotypes under field as well as lab conditions. Moreover, in urban frogs exposure to urban noise and light led, respectively, to an increase in testosterone and decrease in corticosterone, whereas in forest frogs sensory pollutants did not elicit any endocrine response. Our results show that urbanisation, specifically noise and light pollution, can modulate hormone levels in urban and forest populations differentially. The observed endocrine responses are consistent with the observed behavioural changes in urban frogs, providing a proximate explanation for the presumably adaptive behavioural changes in response to urbanisation.

Validation of biomarkers for neonicotinoid exposure in Folsomia candida under mutual exposure to diethyl maleate.

Neonicotinoid insecticides are harmful to non-target soil invertebrates, which are crucial for sustainable agriculture. Gene expression biomarkers could provide economic and high-throughput metrics of neonicotinoid exposure and toxicity to non-target invertebrates. Thereby, biomarkers can help guide remediation efforts or policy enforcement. Gene expression of Glutathione S-Transferase 3 (GST3) has previously been proposed as a biomarker for the neonicotinoid imidacloprid in the soil ecotoxicological model species Folsomia candida (Collembola). However, it remains unclear how reliably gene expression of neonicotinoid biomarkers, such as GST3, can indicate the exposure to the broader neonicotinoid family under putative GST enzymatic inhibition. In this work, we exposed springtails to two neonicotinoids, thiacloprid and imidacloprid, alongside diethyl maleate (DEM), a known GST metabolic inhibitor that imposes oxidative stress. First, we determined the influence of DEM on neonicotinoid toxicity to springtail fecundity. Second, we surveyed the gene expression of four biomarkers, including GST3, under mutual exposure to neonicotinoids and DEM. We observed no effect of DEM on springtail fecundity. Moreover, the expression of GST3 was only influenced by DEM under mutual exposure with thiacloprid but not with imidacloprid. The results indicate that GST3 is not a robust indicator of neonicotinoid exposure and that probable GST enzymatic inhibition mediates the toxicity of imidacloprid and thiacloprid differentially. Future research should investigate biomarker reliability under shifting metabolic conditions such as provided by DEM exposure.

Combining time-resolved transcriptomics and proteomics data for Adverse Outcome Pathway refinement in ecotoxicology.

Conventional Environmental Risk Assessment (ERA) of pesticide pollution is based on soil concentrations and apical endpoints, such as the reproduction of test organisms, but has traditionally disregarded information along the organismal response cascade leading to an adverse outcome. The Adverse Outcome Pathway (AOP) framework includes response information at any level of biological organization, providing opportunities to use intermediate responses as a predictive read-out for adverse outcomes instead. Transcriptomic and proteomic data can provide thousands of data points on the response to toxic exposure. Combining multiple omics data types is necessary for a comprehensive overview of the response cascade and, therefore, AOP development. However, it is unclear if transcript and protein responses are synchronized in time or time lagged. To understand if analysis of multi-omics data obtained at the same timepoint reveal one synchronized response cascade, we studied time-resolved shifts in gene transcript and protein abundance in the springtail Folsomia candida, a soil ecotoxicological model, after exposure to the neonicotinoid insecticide imidacloprid. We analyzed transcriptome and proteome data every 12 h up to 72 h after onset of exposure. The most pronounced shift in both transcript and protein abundances was observed after 48 h exposure. Moreover, cross-correlation analyses indicate that most genes displayed the highest correlation between transcript and protein abundances without a time-lag. This demonstrates that a combined analysis of transcriptomic and proteomic data from the same time-point can be used for AOP improvement. This data will promote the development of biomarkers for the presence of neonicotinoid insecticides or chemicals with a similar mechanism of action in soils.

Biomarker development for neonicotinoid exposure in soil under interaction with the synergist piperonyl butoxide in Folsomia candida.

Pesticide toxicity is typically assessed by exposing model organisms to individual compounds and measuring effects on survival and reproduction. These tests are time-consuming, labor-intensive, and do not accurately capture the effect of pesticide mixtures. Moreover, it is unfeasible to screen the nearly infinite combinations of mixtures for synergistic effects on model organisms. Therefore, reliable molecular indicators of pesticide exposure have to be identified, i.e., biomarkers. These biomarkers can form the basis of rapid and economical screening procedures to assess the toxicity of pesticides even under synergistic interaction with other pollutants. In this study, we screened the expression patterns of eight genes for suitability as a biomarker for neonicotinoid exposure in the soil ecotoxicological model Folsomia candida (springtails). Springtails were exposed to the neonicotinoids imidacloprid and thiacloprid either alone or with various levels of piperonyl butoxide (PBO), which inhibits cytochrome P450 enzymes (CYPs): a common point of synergistic interaction between neonicotinoid and other pesticides. First, we confirmed PBO as a potency enhancer for neonicotinoid toxicity to springtail fecundity, and then used it as a tool to confirm biomarker robustness. We identified two genes that are reliably indicative for neonicotinoid exposure even under metabolic inhibition of CYPs by PBO, nicotinic acetylcholine receptor-subunit alpha 1 (nAchR) and sodium-coupled monocarboxylate transporter (SMCT). These results can form the basis for developing high-throughput screening procedures for neonicotinoid exposure in varying mixture compositions.

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.

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.

Glutathione serves an extracellular defence function to decrease arsenite accumulation and toxicity in yeast.

Arsenic is an environmental toxin and a worldwide health hazard. Since this metalloid is ubiquitous in nature, virtually all living organisms require systems for detoxification and tolerance acquisition. Here, we show that during chronic exposure to arsenite [As(III)], Saccharomyces cerevisiae (budding yeast) exports and accumulates the low-molecular-weight thiol molecule glutathione (GSH) outside of cells. Extracellular accumulation of the arsenite triglutathione complex As(GS)3 was also detected and direct transport assays demonstrate that As(GS)3 does not readily enter cells. Yeast cells with increased extracellular GSH levels accumulate less arsenic and display improved growth when challenged with As(III). Conversely, cells defective in export and extracellular accumulation of GSH are As(III) sensitive. Taken together, our data are consistent with a novel detoxification mechanism in which GSH is exported to protect yeast cells from arsenite toxicity by preventing its uptake.

Expression of mtc in Folsomia candida indicative of metal pollution in soil.

The soil-living springtail Folsomia candida is frequently used in reproduction bioassays to assess soil contamination. Alternatively, the response of genes to contamination is assessed. In this study the expression of F. candida's gene encoding the deduced metallothionein-like motif containing protein (MTC) was assessed, using quantitative PCR, in response to six different metals, each at two concentrations in soil. The expression of mtc was induced after exposure to all metals, except for one chromium concentration. Exposure to soil originating from metal-contaminated field sites also induced mtc, while the expression did not change in response to a polycyclic aromatic hydrocarbon. Since this transcript is induced by most of the tested metals, it may potentially be a good indicator of metal contamination. The presented gene expression assay might become a useful tool to screen potentially polluted sites, in order to identify the ones that need further ecotoxicological investigation.

Chelation by histidine inhibits the vacuolar sequestration of nickel in roots of the hyperaccumulator Thlaspi caerulescens.

* The mechanisms of enhanced root to shoot metal transport in heavy metal hyperaccumulators are incompletely understood. Here, we compared the distribution of nickel (Ni) over root segments and tissues in the hyperaccumulator Thlaspi caerulescens and the nonhyperaccumulator Thlaspi arvense, and investigated the role of free histidine in Ni xylem loading and Ni transport across the tonoplast. * Nickel accumulation in mature cortical root cells was apparent in T. arvense and in a high-Ni-accumulating T. caerulescens accession, but not in a low-accumulating T. caerulescens accession. * Compared with T. arvense, the concentration of free histidine in T. caerulescens was 10-fold enhanced in roots, but was only slightly higher in leaves, regardless of Ni exposure. Nickel uptake in MgATP-energized root- and shoot-derived tonoplast vesicles was almost completely blocked in T. caerulescens when Ni was supplied as a 1 : 1 Ni-histidine complex, but was uninhibited in T. arvense. Exogenous histidine supply enhanced Ni xylem loading in T. caerulescens but not in T. arvense. * The high rate of root to shoot translocation of Ni in T. caerulescens compared with T. arvense seems to depend on the combination of two distinct characters, that is, a greatly enhanced root histidine concentration and a strongly decreased ability to accumulate histidine-bound Ni in root cell vacuoles.

High-level Zn and Cd tolerance in Silene paradoxa L. from a moderately Cd- and Zn-contaminated copper mine tailing.

Cadmium and zinc tolerance were examined in populations of Silene paradoxa, one from uncontaminated calcareous soil (CVD) and one from a mine tailing (FC) (Cd<1-15 ppm, Zn 400-1300 ppm, pH 2-6). The mine population exhibited extremely high Zn and Cd tolerance levels, although the degrees of Cd and Zn enrichment relatively low at the population site. Cd and Zn hypertolerance in FC were associated with reduced rates of accumulation of these metals, both in roots and shoots (Cd), or exclusively in shoots (Zn). However, exclusion potentially explained only a minor part of the superior tolerance in FC. Cd hypertolerance in FC was associated with decreased, rather than enhanced phytochelatin accumulation. The remarkably high levels of Cd and Zn hypertolerance in FC might relate to the low soil pH, due to oxidation of sulphide minerals, and the absence of soil organic matter at the FC site.

Arsenate tolerance in Silene paradoxa does not rely on phytochelatin-dependent sequestration.

Arsenate tolerance, As accumulation and As-induced phytochelatin accumulation were compared in populations of Silene paradoxa, one from a mine site enriched in As, Cu and Zn, the other from an uncontaminated site. The mine population was significantly more arsenate-tolerant. Arsenate uptake and root-to-shoot transport were slightly but significantly higher in the non-mine plants. The difference in uptake was quantitatively insufficient to explain the difference in tolerance between the populations. As accumulation in the roots was similar in both populations, but the mine plants accumulated much less phytochelatins than the non-mine plants. The mean phytochelatin chain length, however, was higher in the mine population, possibly due to a constitutively lower cellular glutathione level. It is argued that the mine plants must possess an arsenic detoxification mechanism other than arsenate reduction and subsequent phytochelatin-based sequestration. This alternative mechanism might explain at least some part of the superior tolerance in the mine plants.

The role of thiol species in the hypertolerance of Aspergillus sp. P37 to arsenic.

Aspergillus sp. P37 is an arsenate-hypertolerant fungus isolated from a river in Spain with a long history of contamination with metals. This strain is able to grow in the presence of 0.2 M arsenate, i.e. 20-fold higher than the reference strain, Aspergillus nidulans TS1. Although Aspergillus sp. P37 reduces As(V) to As(III), which is slowly pumped out of the cell, the measured efflux of oxyanions is insufficient to explain the high tolerance levels of this strain. To gain an insight into this paradox, the accumulation of acid-soluble thiol species in Aspergillus sp. P37 when exposed to arsenic was compared with that of the arsenic-sensitive A. nidulans TS1 strain. Increasing levels of arsenic in the medium did not diminish the intracellular pool of reduced glutathione in Aspergillus sp. P37, in sharp contrast with the decline of glutathione in A. nidulans under the same conditions. Furthermore, concentrations of arsenic that were inhibitory for the sensitive A. nidulans strain (e.g. 50 mM and above) provoked a massive formation of vacuoles filled with thiol species. Because the major fraction of the cellular arsenic was present as the glutathione conjugate As(GS)3, it is plausible that the arsenic-hypertolerant phenotype of Aspergillus sp. P37 is in part due to an enhanced capacity to maintain a large intracellular glutathione pool under conditions of arsenic exposure and to sequester As(GS)3 in vacuoles. High pressure liquid chromatography analysis of cell extracts revealed that the contact of Aspergillus sp. P37 (but not A. nidulans) with high arsenic concentrations (> or =150 mM) induced the production of small quantities of a distinct thiol species indistinguishable from plant phytochelatin-2. Yet, we argue that phytochelatins do not explain arsenic resistance in Aspergillus, and we advocate the role of As(GS)3 complexes in arsenic detoxification.

Differential metal-specific tolerance and accumulation patterns among Thlaspi caerulescens populations originating from different soil types.

• Here, Thlaspi caerulescens populations from contrasting soil types (serpentine, calamine and nonmetalliferous) were characterized with regard to tolerance, uptake and translocation of zinc (Zn), cadmium (Cd) and nickel (Ni) in hydroponic culture. • Results showed that high-level tolerances were apparently metal-specific and confined to the metals that were enriched at toxic levels in the soil at the population site. • With regard to metal accumulation, results suggested that, unlike Zn hyperaccumulation, Cd and Ni hyperaccumulation were not constitutive at the species level in T. caerulescens. • In general, the populations under study exhibited a pronounced uncorrelated and metal-specific variation in uptake, root to shoot translocation, and tolerance of Zn, Cd and Ni. The distinct intraspecific variation of these characters provides excellent opportunities for further genetic and physiological dissection of the hyperaccumulation trait.

A cosegregation analysis of zinc (Zn) accumulation and Zn tolerance in the Zn hyperaccumulator Thlaspi caerulescens.

• To analyse the relation between zinc (Zn) accumulation and Zn tolerance in the Zn hyperaccumulator, Thlaspi caerulescens, a cross was made between a plant from a nonmetallicolous population (LE: high accumulation, low tolerance) and one from a calamine population (LC: low accumulation, high tolerance). • More or less homogeneous F3 lines with contrasting extreme accumulation phenotypes were selected and phenotyped for tolerance, using the threshold exposure level for chlorosis as a tolerance measure. Zn accumulation and tolerance segregated largely independently, although there was a significant degree of association between low accumulation and high tolerance. • Plants from an F2 family were phenotyped for Zn tolerance and their Zn accumulation rates were compared. The plants with low Zn tolerance exhibited significantly higher Zn accumulation than did the more tolerant plants. • The results suggest that the superior Zn tolerance in LC plants compared with LE plants results from a superior plant-internal Zn sequestration capacity and, although to a lower degree, a reduced rate of Zn accumulation. It is argued that the relatively low Zn accumulation capacity levels found in LC and several other calamine T. caerulescens populations might represent an adaptive response to Zn-toxic soil.

Mechanisms of arsenate tolerance in Cytisus striatus.

• Arsenate tolerance, uptake and arsenate-induced phytochelatin (PC) accumulation were compared at different phosphorus supply rates in two populations of the broom, Cytisus striatus , one from an arsenic-enriched gold mine and one from a nonmetalliferous site. • After 7 d of exposure, arsenate tolerance was higher in the mine population. Arsenate uptake was phosphate-suppressible, and much lower in the mine plants. When compared at equal levels of stress, the mine plants and the nonmetallicolous plants exhibited similar arsenic accumulation, suggesting that reduced arsenate uptake is mainly responsible for superior tolerance. • Arsenate-induced PC accumulation occurred in both plant types. The γ-glutamylcysteine synthetase inhibitor, L-buthioninesulfoximine, caused arsenate hypersensitivity in both plant types, suggesting that PC-based arsenic sequestration is essential for both normal and enhanced arsenate tolerance. Mine plants produced longer PCs than the nonmetallicolous plants, possibly due to a differential temporal pattern of arsenate accumulation. • Our results are consistent with a similar mechanism underlying arsenate hypertolerance in C. striatus and grasses, that is reduced arsenate uptake through suppression of phosphate transporter activity.

The role of phytochelatins in constitutive and adaptive heavy metal tolerances in hyperaccumulator and non-hyperaccumulator metallophytes.

Using the gamma-glutamylcysteine synthetase inhibitor, L-buthionine-[S,R]-sulphoximine (BSO), the role for phytochelatins (PCs) was evaluated in Cu, Cd, Zn, As, Ni, and Co tolerance in non-metallicolous and metallicolous, hypertolerant populations of Silene vulgaris (Moench) Garcke, Thlaspi caerulescens J.&C. Presl., Holcus lanatus L., and Agrostis castellana Boiss. et Reuter. Based on plant-internal PC-thiol to metal molar ratios, the metals' tendency to induce PC accumulation decreased in the order As/Cd/Cu > Zn > Ni/Co, and was consistently higher in non-metallicolous plants than in hypertolerant ones, except for the case of As. The sensitivities to Cu, Zn, Ni, and Co were consistently unaffected by BSO treatment, both in non-metallicolous and hypertolerant plants, suggesting that PC-based sequestration is not essential for constitutive tolerance or hypertolerance to these metals. Cd sensitivity was considerably increased by BSO, though exclusively in plants lacking Cd hypertolerance, suggesting that adaptive cadmium hypertolerance is not dependent on PC-mediated sequestration. BSO dramatically increased As sensitivity, both in non-adapted and As-hypertolerant plants, showing that PC-based sequestration is essential for both normal constitutive tolerance and adaptive hypertolerance to this metalloid. The primary function of PC synthase in plants and algae remains elusive.

In vitro alleviation of heavy metal-induced enzyme inhibition by proline.

Accumulation of proline in response to toxic heavy metal exposure seems to be wide-spread among plants. To elucidate the role for proline in plant responses to heavy metal stress, we studied the effect of proline on Cd-induced and Zn-induced inhibition of glucose-6-phosphate dehydrogenase (G-6-PDH; EC 1.1.1.49) and nitrate reductase (NR; EC 1.6.6.2) in vitro. Proline appeared to protect both enzymes against Zn and, though less effectively, against Cd. Measurements with a Cd(2+)-specific electrode strongly suggested that this protection was based on a reduction of the free metal ion activity in the assay buffer, due to the formation of metal-proline complexes. There were no indications of any significant role for proline-water or proline-protein interactions. The significance of these findings with regard to heavy metal-induced proline accumulation in vivo is discussed.

Multiple tolerance and co-tolerance to heavy metals in Silene vulgaris: a co-segregation analysis.

Plants from metalliferous soils often exhibit combined tolerance to different heavy metals. Such tolerance could rely either on the possession of a combination of different metal-specific tolerance mechanisms ('multiple tolerance'), or on less specific mechanisms that pleiotropically confer tolerance to different metals ('co-tolerance'). In this study, we examined the co-segregation of tolerances to Cu, Zn, Ni, Co and Cd in crosses between distinctly tolerant ecotypes of Silene vulgaris (Moench) Garcke. The results demonstrate non-pleiotropic genetic control of tolerance to Cu, Zn and Cd. Tolerance to Ni and Co, on the other hand, seems to represent the pleiotropic byproduct of the tolerance allele of one particular locus for zinc tolerance.

IDENTICAL MAJOR GENE LOCI FOR HEAVY METAL TOLERANCES THAT HAVE INDEPENDENTLY EVOLVED IN DIFFERENT LOCAL POPULATIONS AND SUBSPECIES OF SILENE VULGARIS.

Heavy metal tolerant Silene vulgaris plants, originating from different metalliferous sites in Germany and one in Ireland, were crossed to each other and to nontolerant plants from a nonmetalliferous site in The Netherlands. Analysis of the crosses suggested that there were two distinct major gene loci for zinc tolerance among a total of five tolerant populations. The tolerance loci for zinc, copper, and cadmium in the Irish plants were shown to be identical with those in the German populations. It is argued that the occurrence of common major genes for tolerance among different geographically isolated populations must have resulted from independent parallel evolution in local nontolerant ancestral populations. Each of the tolerances studied seems to be controlled by only a few specific major genes.