Nanomaterials (NMs) can interact with the innate immunity of organisms. It remains, however, unclear whether these interactions can compromise the immune functioning of the host when faced with a disease threat. Co-exposure with pathogens is thus a powerful approach to assess the immuno-safety of NMs. In this paper, we studied the impacts of in vivo exposure to a biocidal NM on the gut microbiome, host immune responses, and susceptibility of the host to a bacterial challenge in an earthworm. Eisenia fetida were exposed to CuO-nanoparticles in soil for 28 days, after which the earthworms were challenged with the soil bacterium Bacillus subtilis. Immune responses were monitored by measuring mRNA levels of known earthworm immune genes. Effects of treatments on the gut microbiome were also assessed to link microbiome changes to immune responses. Treatments caused a shift in the earthworm gut microbiome. Despite these effects, no impacts of treatment on the expression of earthworm immune markers were recorded. The methodological approach applied in this paper provides a useful framework for improved assessment of immuno-safety of NMs. In addition, we highlight the need to investigate time as a factor in earthworm immune responses to NM exposure.

• Klíčová slova
• Eisenia fetida, copper, earthworms, infection, innate immunity, microbiome, nanomaterials, nanoparticles, survival,
• Publikační typ
• časopisecké články MeSH

Earthworms are not endowed with adaptive immunity and they are rely on the tools of innate immunity. Cells of the innate immune system utilize pattern recognition receptors, such as Toll-like receptors, to detect the pathogen-associated molecular patterns (PAMPs). The first earthworm TLR was isolated from Eisenia andrei earthworms (EaTLR), which belongs to the single cysteine cluster TLR (sccTLR). Here, we identified a new multiple cysteine cluster TLR (mccTLR) in E. andrei earthworms. Phylogenetic DNA analysis revealed that it has no variability within one earthworm as well as in the population. By screening of the tissue expression profile, the TLR was expressed primarily in earthworm seminal vesicles and receptacles suggesting a connection to sperm cells. Seminal vesicles are often heavily infected by gregarine parasites. As a sign of immune response, a strong melanization reaction is visible around parasites. Stimulation experiments with profilin from related parasite Toxoplasma gondii, led to the upregulation of mccEaTLR in the earthworm seminal vesicles. Also, profilin activated prophenoloxidase cascade, the efficient mechanism of innate immunity. However, its involvement in the NF-κB signaling was not proven. Further, we provide evidence that the antibiotics metronidazole and griseofulvin destroyed the developing spermatocytes. The observed decrease in the mccEaTLR mRNA levels after the antibiotic treatment of parasites is caused by the decline of sperm cells numbers rather than by diminution of the parasites. Since earthworms with extensively reduced parasite load had a similar amount of mccEaTLR mRNA, presumably, earthworm sperm cells have a certain level of mccEaTLR expressed as a standard, which can be augmented by particular antigenic stimulation. Also, mccEaTLR was expressed mainly in the early stages of earthworm development and presumably is primarily involved in early embryonic development. Expression of mccEaTLR in seminal vesicles correlates with the expression of endothelial monocyte-activation polypeptide II. High-throughput sequencing of gregarine DNA from seminal vesicles of individual earthworms resulted in great diversity of the observed genotypes. Phylogenetically, all observed OTUs belong to the clade of earthworm gregarines suggesting host specificity. Overall, mccEaTLR is supposed to play a function role in early embryonic development and potentially it participates in immune response against parasites.

• Klíčová slova
• PRR, TLR, development, earthworm, gregarine, innate immunity, invertebrate, parasite,
• MeSH
• cystein MeSH
• cytokiny imunologie   MeSH
• embryonální vývoj imunologie   MeSH
• fylogeneze MeSH
• messenger RNA imunologie   MeSH
• nádorové proteiny imunologie   MeSH
• NF-kappa B imunologie   MeSH
• Oligochaeta imunologie   MeSH
• přirozená imunita imunologie   MeSH
• proteiny vázající RNA imunologie   MeSH
• receptory rozpoznávající vzory imunologie   MeSH
• signální transdukce imunologie   MeSH
• toll-like receptory imunologie   MeSH
• Toxoplasma imunologie   MeSH
• upregulace imunologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• cystein MeSH
• cytokiny MeSH
• messenger RNA MeSH
• nádorové proteiny MeSH
• NF-kappa B MeSH
• proteiny vázající RNA MeSH
• receptory rozpoznávající vzory MeSH
• small inducible cytokine subfamily E, member 1 MeSH Prohlížeč
• toll-like receptory MeSH

Vermicomposting is a process of degradation of biowaste which involves complex interactions between earthworms and microorganisms. This process lacks a thermophilic stage and thus, the possible presence of pathogens poses a potential health hazard. To assess the contribution of earthworms during the selective reduction of various pathogens, apple pomace substrate was artificially inoculated with Escherichia coli, Salmonella spp., thermotolerant coliform bacteria, and Enterococci. The artificial bacterial load did not influence the weight, reproduction, or intestinal enzymatic activity of the earthworms, but it caused reversible histological changes to the epithelial layer and chloragogen tissue of their intestines. The reduction of pathogenic Enterococci and E. coli from the substrate was accelerated by earthworms (63-fold, 77-fold, and 840-fold for Enterococci and 6-fold, 36-fold, and 7-fold for E. coli inoculated substrates after 2, 4, and 6 weeks, respectively). Moreover, the rapid elimination of Salmonella spp. was supported by the upregulated expression of two pattern recognition receptors which bind lipopolysaccharide, coelomic cytolytic factor, and lipopolysaccharide-binding protein. Further, the microbiomes of the intestine and the composting substrate differed significantly. Graphical abstract.

• Klíčová slova
• Biowaste, Earthworm, Eisenia, Immunity, Microbiome, Pathogen, Pattern recognition receptor, Vermicompost,
• MeSH
• Escherichia coli MeSH
• kompostování metody   MeSH
• Oligochaeta mikrobiologie   fyziologie   MeSH
• půdní mikrobiologie * MeSH
• střevní mikroflóra * MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH

Survival of earthworms in the environment depends on their ability to recognize and eliminate potential pathogens. This work is aimed to compare the innate defense mechanisms of two closely related earthworm species, Eisenia andrei and Eisenia fetida, that inhabit substantially different ecological niches. While E. andrei lives in a compost and manure, E. fetida can be found in the litter layer in forests. Therefore, the influence of environment-specific microbiota on the immune response of both species was followed. Firstly, a reliable method to discern between E. andrei and E. fetida based on species-specific primers for cytochrome c oxidase I (COI) and stringent PCR conditions was developed. Secondly, to analyze the immunological profile in both earthworm species, the activity and expression of lysozyme, pattern recognition protein CCF, and antimicrobial proteins with hemolytic function, fetidin and lysenins, have been assessed. Whereas, CCF and lysozyme showed only slight differences in the expression and activity, fetidin/lysenins expression as well as the hemolytic activity was considerably higher in E. andrei as compared to E. fetida. The expression of fetidin/lysenins in E. fetida was not affected upon the challenge with compost microbiota, suggesting more substantial changes in the regulation of the gene expression. Genomic DNA analyses revealed significantly higher level of fetidin/lysenins (determined using universal primer pairs) in E. andrei compared to E. fetida. It can be hypothesized that E. andrei colonizing compost as a new habitat acquired an evolutionary selection advantage resulting in a higher expression of antimicrobial proteins.

• MeSH
• Bacteria klasifikace   genetika   imunologie   MeSH
• biologické toxiny genetika   imunologie   MeSH
• cytotoxicita imunologická genetika   imunologie   MeSH
• druhová specificita MeSH
• ekosystém * MeSH
• exprese genu MeSH
• hemolýza genetika   imunologie   MeSH
• hnůj mikrobiologie   parazitologie   MeSH
• molekulární sekvence - údaje MeSH
• muramidasa genetika   imunologie   MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• Oligochaeta klasifikace   genetika   imunologie   MeSH
• polymerázová řetězová reakce s reverzní transkripcí MeSH
• přirozená imunita genetika   imunologie   MeSH
• proteiny genetika   imunologie   MeSH
• půdní mikrobiologie MeSH
• respirační komplex IV genetika   MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sekvenční homologie nukleových kyselin MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• biologické toxiny MeSH
• fetidin MeSH Prohlížeč
• hnůj MeSH
• lysenin MeSH Prohlížeč
• muramidasa MeSH
• proteiny MeSH
• respirační komplex IV MeSH