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Neonicotinoids are currently licensed for use in 120 countries, making accurate nontarget species sensitivity predictions critical. Unfortunately, such predictions are fraught with uncertainty, as sensitivity is extrapolated from only a few test species and neonicotinoid sensitivities can differ greatly between closely related taxa. Combining classical toxicology with de novotoxicogenomics could greatly improve sensitivity predictions and identify unexpectedly susceptible species. We show that there is a >30-fold differential species sensitivity (DSS) for the neonicotinoid imidacloprid between five earthworm species, a critical nontarget taxon. This variation could not be explained by differential toxicokinetics. Furthermore, comparing key motif expression in subunit genes of the classical nicotinic acetylcholine receptor (nAChR) target predicts only minor differences in the ligand binding domains (LBDs). In contrast, predicted dissimilarities in LBDs do occur in the highly expressed but nonclassical targets, acetylcholine binding proteins (AChBPs). Critically, the predicted AChBP divergence is capable of explaining DSS. We propose that high expression levels of putative nonsynaptic AChBPs with high imidacloprid affinities reduce imidacloprid binding to critical nAChRs involved in vital synaptic neurotransmission. This study provides a clear example of how pragmatic interrogation of key motif expression in complex multisubunit receptors can predict observed DSS, thereby informing sensitivity predictions for essential nontarget species.

Understanding how engineered nanomaterials affect immune responses of living organisms requires a strong collaborative effort between immunologists, toxicologists, ecologists, physiologists, inorganic chemists, nanomaterial scientists and experts in law and risk management. This perspective aims to provide a new viewpoint on the interaction between engineered nanomaterials and the immune defensive systems across living species, gained within the EU Horizon 2020 project PANDORA. We consider the effects of nanoparticle exposure on immune functions in plants, marine and terrestrial invertebrates and their relation to the current state of knowledge for vertebrates (in particular humans). These studies can shed light on the broader perspective of defensive and homeostatic mechanisms (immunity, inflammation, stress responses, microbiota, stem cell differentiation) suggesting ways to: i) perform a comparative analysis of the nanoparticle impact on immunity across model organisms; ii) inspire best practices in experimental methodologies for nanosafety/nanotoxicity studies; iii) regroup and harmonise fragmented research activities; iv) improve knowledge transfer strategies and nano-security; v) propose innovative tools and realistic solutions, thereby helping in identifying future research needs and tackling their challenges.

A critical question for read across of the hazards of nanomaterials is the degree to which their mechanisms of action differ from those of their bulk chemical constituents. It has been established that metal and metal oxide nanoparticles (NPs) can be accumulated by invertebrate species. Moreover, it has been hypothesised that the observed toxicity resulting on exposure to these nanomaterials is most likely to be associated with the releases of ions from external or internal dissolution leading ultimately to toxicity. However, mechanistic confirmation of the similar modes of action for metal oxide nanomaterials and metal ions in studies invertebrates are largely lacking. Therefore, here we present a toxicogenomic study using exposed individuals of the earthworm Eisenia fetidafrom a single genetic lineage. We compared gene expression and pathway responses through RNA-seq analysis at equitoxic concentrations (EC50for reproduction) of ZnO NPs and ionic Zn. We found similar transcriptomic effects for both Zn forms with genetic signatures of tight control of cytosolic Zn concentrations through expression changes of genes encoding several Zn transporters. Activation and regulation of JUN, MAP and JNK kinases indicated a cellular response to the increased Zn concentrations of both forms with compound binding terms also enriched. Our results illustrate the need to consider both toxicokinetic and toxicodynamic mechanisms in the development of adverse outcome pathways for nanomaterials.

Supplemental digital content is available in the text.The role of earthworms as one of the most important groups of ecosystem engineers in human-modified and natural environments has been increasingly recognized only during the last 30 years, yet earthworms and humans have been acting together in building landscapes for millennia. This relationship is well represented in the pre-Columbian raised fields, in flood-prone savannas around the rim of Amazonia, but also by the potentially significant role of earthworms in the formation and resilience of Amazonian Dark Earths. Through the bioturbating action of earthworms, soil is biologically, chemically, and physically altered; nutrients are translocated; organic matter is decomposed and transformed; and the surrounding biota interacts as a large orchestra where the soil musicians play together on the various instruments but where earthworms take a leading role, enhancing microbial activity and generally stimulating plant growth. In this article, we assess the remarkable role of earthworms at the center of soil pedogenetic processes within anthropogenic landscapes, dissecting their functions with a special focus on Amazonian Dark Earths.

The ‘Atribacteria’ is a candidate phylum in the Bacteria recently proposed to include members of the OP9 and JS1 lineages. OP9 and JS1 are globally distributed, and in some cases abundant, in anaerobic marine sediments, geothermal environments, anaerobic digesters and reactors and petroleum reservoirs. However, the monophyly of OP9 and JS1 has been questioned and their physiology and ecology remain largely enigmatic due to a lack of cultivated representatives. Here cultivation-independent genomic approaches were used to provide a first comprehensive view of the phylogeny, conserved genomic features and metabolic potential of members of this ubiquitous candidate phylum. Previously available and heretofore unpublished OP9 and JS1 single-cell genomic data sets were used as recruitment platforms for the reconstruction of atribacterial metagenome bins from a terephthalate-degrading reactor biofilm and from the monimolimnion of meromictic Sakinaw Lake. The single-cell genomes and metagenome bins together comprise six species- to genus-level groups that represent most major lineages within OP9 and JS1. Phylogenomic analyses of these combined data sets confirmed the monophyly of the ‘Atribacteria’ inclusive of OP9 and JS1. Additional conserved features within the ‘Atribacteria’ were identified, including a gene cluster encoding putative bacterial microcompartments that may be involved in aldehyde and sugar metabolism, energy conservation and carbon storage. Comparative analysis of the metabolic potential inferred from these data sets revealed that members of the ‘Atribacteria’ are likely to be heterotrophic anaerobes that lack respiratory capacity, with some lineages predicted to specialize in either primary fermentation of carbohydrates or secondary fermentation of organic acids, such as propionate.

Changes to host behaviour induced by some trematode species, as a means of increased trophic transmission, represents one of the seminal examples of host manipulation by a parasite. The amphipod Echinogammarus marinus (Leach, 1815) is infected with a previously undescribed parasite, with infected individuals displaying positive phototaxic and negative geotaxic behaviour. This study reveals that the unknown parasite encysts in the brain, nerve cord and the body cavity of E. marinus, and belongs to the Microphallidae family. An 18 month population study revealed that host abundance significantly and negatively correlated with parasite prevalence. Investigation of the trematode's influence at the transcriptomic level revealed genes with putative neurological functions, such as serotonin receptor 1A, an inebriated-like neurotransmitter, tryptophan hydroxylase and amino acid decarboxylase, present consistent altered expression in infected animals. Therefore, this study provides one of the first transcriptomic insights into the neuronal gene pathways altered in amphipods infected with a trematode parasite associated with changes to its host's behaviour and population structure. [ABSTRACT FROM PUBLISHER]

C16H15NO5, monoclinic, P21/n(no. 14), a= 6.7689(5) Å, b= 45.219(3) Å, c= 10.1102(6) Å, β= 101.360(7)°, V= 3033.9(4) Å3, T= 298(2) K.

Abstract: One of the promises of environmental metabolomics, together with other ecotoxicogenomic approaches, is that it can give information on toxic compound mechanism of action (MOA), by providing a specific response profile or fingerprint. This could then be used either for screening in the context of chemical risk assessment, or potentially in contaminated site assessment for determining what compound classes were causing a toxic effect. However for either of these two ends to be achievable, it is first necessary to know if different compounds do indeed elicit specific and distinct metabolic profile responses. Such a comparative study has not yet been carried out for the earthworm Lumbricus rubellus. We exposed L. rubellus to sub-lethal concentrations of three very different toxicants (CdCl2, atrazine, and fluoranthene, representing three compound classes with different expected MOA), by semi-chronic exposures in a laboratory test, and used NMR spectroscopy to obtain metabolic profiles. We were able to use simple multivariate pattern-recognition analyses to distinguish different compounds to some degree. In addition, following the ranking of individual spectral bins according to their mutual information with compound concentrations, it was possible to identify both general and specific metabolite responses to different toxic compounds, and to relate these to concentration levels causing reproductive effects in the worms.

The ubiquitous protein metallothionein (MT) has proven to be a major player not only in the homeostasis of CuIand ZnII, but also binds all the Group 11 and 12 metals. Metallothioneins are characterised by the presence of numerous cys‐x‐cys and cys–cys motifs in the sequence and are found naturally with either one domain or two, linked, metal‐binding domains. The use of chains of these metal–thiolate domains offers the possibility of creating chemically tuneable and, therefore, chemically dependent electrochemical or photochemical surface modifiers or as nanomachinery with nanomechanical properties. In this work, the metal‐binding properties of the Cd4‐containing domain of α‐rhMT1a assembled into chains of two and three concatenated domains, that is, “necklaces”, have been studied by spectrometric techniques, and the interactions within the structures modelled and interpreted by using molecular dynamics. These chains are metallated with 4, 8 or 12 CdIIions to the 11, 22, and 33 cysteinyl sulfur atoms in the α‐rhMT1a, αα‐rhMT1a, and ααα‐rhMT1a proteins, respectively. The effect of pH on the folding of each protein was studied by ESI‐MS and optical spectroscopy. MM3/MD simulations were carried out over a period of up to 500 ps by using force‐field parameters based on the reported structural data. These calculations provide novel information about the motion of the clustered metallated, partially demetallated, and metal‐free peptide chains, with special interest in the region of the metal‐binding site. The MD energy/time trajectory conformations show for the first time the flexibility of the metal–sulfur clusters and the bound amino acid chains. We report unexpected and very different sizes for the metallated and demetallated proteins from the combination of experimental data, with molecular dynamics simulations.

We report the use of a PTFE‐based microfluidic device for the encapsulation of living, therapeutically‐active cells within monodisperse alginate microspheres. We present a novel microfluidic platform and a flexible experimental method for the production of alginate microspheres. Cell lines HEK293, U‐2 OS and PC12 were separately encapsulated using this method, with minimal loss of cell viability.

Researchers working on environmentally relevant organisms, populations, and communities are increasingly turning to the application of OMICS technologies to answer fundamental questions about the natural world, how it changes over time, and how it is influenced by anthropogenic factors. In doing so, the need to capture meta-data that accurately describes the biological "source" material used in such experiments is growing in importance. Here, we provide an overview of the formation of the "Env" community of environmental OMICS researchers and its efforts at considering the meta-data capture needs of those working in environmental OMICS. Specifically, we discuss the development to date of the Env specification, an informal specification including descriptors related to geographic location, environment, organism relationship, and phenotype. We then describe its application to the description of environmental transcriptomic experiments and how we have used it to extend the Minimum Information About a Microarray Experiment (MIAME) data standard to create a domain-specific extension that we have termed MIAME/Env. Finally, we make an open call to the community for participation in the Env Community and its future activities. This paper is part of the special issue of OMICS on data standards.

The seaweed Fucus vesiculosus is unusual when compared with other algal species, in that it can survive in toxic-metal-contaminated aquatic environments. The metallothionein gene has been identified in F. vesiculosus by Kille and co-workers (Morris, C. A., Nicolaus, B., Sampson, V., Harwood, J. L., and Kille, P. (1999) Biochem. J.338, 553), which suggests a possible protective mechanism against toxic metals for this species. We report the first detailed study of the metal binding properties of F. vesiculosus metallothionein using UV absorption, circular dichroism (CD), and electrospray mass spectral techniques. The overall metal-to-sulfur ratios of this novel algal protein when bound to divalent cadmium and zinc were determined to be Cd6S16 and Zn6S16, respectively. Mixed Cd/Zn species were also formed when Cd2+ was added to the Zn-containing Fucus metallothionein. Only one conformation was identified at low pH for the native protein. Analysis of the UV absorption, CD, and ESI-MS spectral data recorded during stepwise, acid-induced demetalation supports a two-domain structure for the protein, with two 3-metal binding sites. The data suggest that one of the domains is significantly less stable than the other, and we tentatively propose from the arrangement of cysteines in the sequence that the two domains are M3S7 and M3S9 (where M = Cd2+ or Zn2+). While the M3S9 cluster is known in the β clusters of crab, lobster, and mammalian metallothioneins, the M3S7 is a hitherto unknown cluster structure. Metallothionein in F. vesiculosus is thought to act as a protective mechanism against incoming toxic metals. The metal binding studies reported are a putative model for metal binding in vivo.

Exposure to cadmium poses a considerable risk to human health and environmental safety. Earthworms reside in the most contaminated sites on earth, displaying a phenomenal tolerance to toxic heavy metals. They exhibit a distinct metabolic pathway that allows the bio-accumulation of cadmium to yield body burdens in excess of 1/1000th of total dry body weight, a most impressive figure by any standard. However, the precise molecular mechanism underlying this phenomenon remains to be unraveled. This study meets this challenge by fully characterizing the major metal-binding protein in earthworms, namely the two isoforms of metallothionein. Chemical analysis of recombinant protein showed that although both isoforms bind equimolar amounts of cadmium (6 mol), wMT-2 is more stable during proton competition. Furthermore, isoform-specific transcript analysis demonstrated that only wMT-2 is responsive to cadmium in a dose and temporal manner. The specific sequestration of cadmium to wMT-2 protein was confirmed in situusing polyclonal antisera. The latter also provided the means for mapping the cellular and intracellular distribution of metallothionein, thus yielding a holistic insight into its involvement in cadmium transit during absorption, storage, and excretion. The structure-function relationship of wMT-2 and its role in cadmium detoxification through sequestration and compartmentalization is discussed.

Metals accumulated within the tissues of earthworms are not homogeneously distributed. Rather, they are sequestered predominantly within the posterior alimentary fraction. The clear organotropism of non-essential metals is reinforced by the subcellular compartmentation of metals according to their known ligand-binding affinities: in earthworm chloragocytes and intestinal epithelial cells, in situ X-ray mapping in high-pressure frozen thin cryosections revealed that Pb, Zn, Ca and P were co-distributed in a discrete subcellular compartment, whilst Cd and S were co-distributed in another. Tissue homogenization and fractionation confirmed these differences. Cd is sequestered by cysteine-rich metallothionein isoforms; one isoform may be a true Cd-detoxification protein because it bears two putative glycosylation sites, and may thus deliver Cd to the lysosomal system for cytological isolation. Metal accumulation imposes metabolic costs that can reduce somatic growth and/or reproduction in earthworms: the sequestered bioinactive metal fraction “diverts” energy into enhanced metallothionein synthesis and associated processes; the bioavailable metal fraction(s) may cause direct toxicological damage by competitive inhibition of metalloenzymes and metal-dependent transcription factors, and indirect damage if repair and compensatory stress responses are invoked. Molecular responses to metal-exposure stress are, in principle, linked to whole organism life-cycle parameters which, in turn, are ecologically relevant.

A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique ‘linker ’ region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of ∼ 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper.

A metallothionein cDNA was generated from pea ( Pisum sativumL.) roots, amplified by PCR and inserted into a plasmid for expression in E. coli. Purification of the resultant product generated 3 pools of cadmium-containing material after DEAE-cellulose chromatography. The amino acid composition of each was in excellent agreement with that predicted for pea metallothionein. A cadmium content of ∼6 g.atoms per mole of protein was estimated. N-terminal sequence analysis revealed that the recombinant molecule had been proteolysed within the extended region linking the 2 cysteine-rich (putative) metal-binding regions. The significance of these findings in terms of the protein folding/targeting of the molecule are considered.

Reverse transcriptase-mediated PCR has been used to isolate two distinct metallothionein (MT) cDNA species from RNA extracted from icefish liver, namely MT-I and MT-II. Northern blot analysis with these cDNA species revealed that significant endogenous levels of MT mRNA were present in liver tissues of normal animals despite the fact that no MT protein could be found accumulating in the same tissue. However, multiple injections of CdCl2 induced high levels of both MT mRNA and MT protein. Sequence analysis of the cDNA species that were present after cadmium injection revealed the presence of both isoforms. Quantification of the MT-I and MT-II transcripts from normal and heavy-metal-treated fish showed an alteration in the ratio of the MT isoform transcripts. Endogenous transcripts consisted mostly of MT-II, whereas the MT-I transcript was preferentially accumulated only in response to the cadmium salt. The protein encoded by each cDNA isoform was isolated from the heavy-metal-treated fish and the availability of the specific MT mRNA for translation was demonstrated by translation in vitro. These results show that: (1) there is a discrepancy between the significant endogenous levels of MT mRNA and the absence of MT protein; (2) the accumulation of MT in icefish liver can be triggered by heavy metals; (3) genes encoding distinct MT isoforms are differentially regulated by heavy metals.

A full-length cDNA clone encoding beta-actin (β-actin) was isolated from a sea bream (Sparus aurata) liver cDNA library. Sequencing of this clone reveals an open reading frame encoding a 375 amino acid protein that shares a high degree of conservation to other known actins. The sea bream β-actin sequence showed 98% identity to carp and human β-actin and 95% and 94% identity to sea squirt and Dictyosteliumcytoplasmic actins, respectively.

Icefish (family Channichthyidae, suborder Nothothenioidei) are a group of Antarctic fish that have evolved unique phenotypes in order to adapt to the environment in which they live. Besides the lack of haemoglobin and the drastic reduction in the number of erythrocyte-like cells, another striking feature of the icefish is that their liver is devoid of metallothionein. These cysteine-rich heavy-metal-binding proteins are usually present in large amounts in a large variety of organisms, from bacteria to mammals. Despite the failure to detect appreciable levels of metallothionein in icefish liver, a cDNA encoding metallothionein was produced from total RNA by reverse transcriptase PCR. The icefish metallothionein showed high percentage identity with metallothionein from Trematomus bernacchii, a red-blooded Antarctic fish in which a normal content of hepatic metallothionein was found. Steady-state mRNA levels were assessed in fish liver by high-stringency hybridization of the metallothionein probe with total RNA. The results showed that icefish livers retain large amounts of untranslated metallothionein mRNA. The stability of the icefish transcript might be correlated with the lack of specific motifs in the untranslated 3ƀ ends of mRNA.

Total RNA from human reticulocytes was purified and reverse‐transcribed into cDNA using an oligo‐dT primer. This cDNA was used as a template for a polymerase chain reaction (PCR) with a primer specific for the N‐terminal sequence of mammalian metallothioneins (MT) and a universal primer. A single amplified fragment was thus generated which when cloned and sequenced revealed two distinct MT cDNAs of almost identical molecular weights. One sequence was identical to that previously reported for human MT II and the other encoded a novel MT I isoform (MT IR). The DNA sequence of MT 1R is distinct from those documented for other MT I isoforms.

Molecular genetic methods can distinguish divergent evolutionary lineages in what previously appeared to be single species, but it is not always clear what functional differences exist between such cryptic species. We used a metabolomic approach to profile biochemical phenotype (metabotype) differences between two putative cryptic species of the earthworm Lumbricus rubellus. There were no straightforward metabolite biomarkers of lineage, i.e. no metabolites that were always at higher concentration in one lineage. Multivariate methods, however, identified a small number of metabolites that together helped distinguish the lineages, including uncommon metabolites such as Nε-trimethyllysine, which is not usually found at high concentrations. This approach could be useful for characterizing functional trait differences, especially as it is applicable to essentially any species group, irrespective of its genome sequencing status.

Phosphorylation of the zinc channel ZIP7 releases zinc from the endoplasmic reticulum and promotes signals associated with proliferation and migration.