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.

Laboratory of Cellular and Molecular Immunology Institute of Microbiology of the Czech Academy of Sciences Videnska 1083 142 20 Prague 4 Czech Republic

School of Biosciences Cardiff University Sir Martin Evans Building Museum Avenue Cardiff CF10 3AX UK

UK Centre for Ecology and Hydrology Maclean Building Benson Lane Wallingford OX10 8BB UK

Zobrazit více v PubMed

Sun T.Y., Bornhöft N.A., Hungerbühler K., Nowack B. Dynamic Probabilistic Modeling of Environmental Emissions of Engineered Nanomaterials. Environ. Sci. Technol. 2016;50:4701–4711. doi: 10.1021/acs.est.5b05828. PubMed DOI

Keller A.A., McFerran S., Lazareva A., Suh S. Glob al life cycle releases of engineered nanomaterials. J. Nanopart. Res. 2013;15 doi: 10.1007/s11051-013-1692-4. DOI

Colvin V.L. The potential environmental impact of engineered nanomaterials. Nat. Biotechnol. 2003;21:1166–1170. doi: 10.1038/nbt875. PubMed DOI

Moore M.N. Do nanoparticles present ecotoxicological risks for the health of the aquatic environment? Environ. Int. 2006;32:967–976. doi: 10.1016/j.envint.2006.06.014. PubMed DOI

Handy R.D., Owen R., Valsami-Jones E. The ecotoxicology of nanoparticles and nanomaterials: Current status, knowledge gaps, challenges, and future needs. Ecotoxicology. 2008;17:315–325. doi: 10.1007/s10646-008-0206-0. PubMed DOI

Dobrovolskaia M.A., McNeil S.E. Immunological properties of engineered nanomaterials. Nat. Nanotechnol. 2007;2:469–478. doi: 10.1038/nnano.2007.223. PubMed DOI

Boraschi D., Oostingh G.J., Casals E., Italiani P., Nelissen I., Puntes V.F., Duschl A. Nano-immunosafety: Issues in assay validation. J. Phys. Conf. Ser. 2011;304:012077. doi: 10.1088/1742-6596/304/1/012077. DOI

Boraschi D., Alijagic A., Auguste M., Barbero F., Ferrari E., Hernadi S., Mayall C., Michelini S., Navarro Pacheco N.I., Prinelli A., et al. Addressing Nanomaterial Immunosafety by Evaluating Innate Immunity across Living Species. Small. 2020;16 doi: 10.1002/smll.202000598. PubMed DOI

Fadeel B. Hide and seek: Nanomaterial interactions with the immune system. Front. Immunol. 2019;10:133. doi: 10.3389/fimmu.2019.00133. PubMed DOI PMC

Boraschi D., Italiani P., Palomba R., Decuzzi P., Duschl A., Fadeel B., Moghimi S.M. Nanoparticles and innate immunity: New perspectives on host defence. Semin. Immunol. 2017;34:33–51. doi: 10.1016/j.smim.2017.08.013. PubMed DOI

Pallardy M.J., Turbica I., Biola-Vidamment A. Why the immune system should be concerned by nanomaterials? Front. Immunol. 2017;8:544. doi: 10.3389/fimmu.2017.00544. PubMed DOI PMC

Alsaleh N.B., Brown J.M. Immune responses to engineered nanomaterials: Current understanding and challenges. Curr. Opin. Toxicol. 2018;10:8–14. doi: 10.1016/j.cotox.2017.11.011. PubMed DOI PMC

Bhattacharya K., Kiliç G., Costa P.M., Fadeel B. Cytotoxicity screening and cytokine profiling of nineteen nanomaterials enables hazard ranking and grouping based on inflammogenic potential. Nanotoxicology. 2017;11:809–826. doi: 10.1080/17435390.2017.1363309. PubMed DOI

Simonin M., Richaume A. Impact of engineered nanoparticles on the activity, abundance, and diversity of soil microbial communities: A review. Environ. Sci. Pollut. Res. 2015;22:13710–13723. doi: 10.1007/s11356-015-4171-x. PubMed DOI

McKee M.S., Filser J. Impacts of metal-based engineered nanomaterials on soil communities. Environ. Sci. Nano. 2016;3:506. doi: 10.1039/C6EN00007J. DOI

Courtois P., Rorat A., Lemiere S., Guyoneaud R., Attard E., Levard C., Vandenbulcke F. Ecotoxicology of silver nanoparticles and their derivatives introduced in soil with or without sewage sludge: A review of effects on microorganisms, plants and animals. Environ. Pollut. 2019;253:578–598. doi: 10.1016/j.envpol.2019.07.053. PubMed DOI

Judy J.D., McNear D.H., Chen C., Lewis R.W., Tsyusko O.V., Bertsch P.M., Rao W., Stegemeier J., Lowry G.V., McGrath S.P., et al. Nanomaterials in Biosolids Inhibit Nodulation, Shift Microbial Community Composition, and Result in Increased Metal Uptake Relative to Bulk/Dissolved Metals. Environ. Sci. Technol. 2015;49:8751–8758. doi: 10.1021/acs.est.5b01208. PubMed DOI

Brestoff J.R., Artis D. Commensal bacteria at the interface of host metabolism and the immune system. Nat. Immunol. 2013;14:676–684. doi: 10.1038/ni.2640. PubMed DOI PMC

Buffie C.G., Pamer E.G. Microbiota-mediated colonization resistance against intestinal pathogens. Nat. Rev. Immunol. 2013;13:790–801. doi: 10.1038/nri3535. PubMed DOI PMC

Nyholm S.V., Graf J. Knowing your friends: Invertebrate innate immunity fosters beneficial bacterial symbioses. Nat. Rev. Microbiol. 2012;10:815–827. doi: 10.1038/nrmicro2894. PubMed DOI PMC

Koch H., Schmid-Hempel P. Socially transmitted gut microbiota protect bumble bees against an intestinal parasite. Proc. Natl. Acad. Sci. USA. 2011;108:19288–19292. doi: 10.1073/pnas.1110474108. PubMed DOI PMC

Dillon R.J., Vennard C.T., Buckling A., Charnley A.K. Diversity of locust gut bacteria protects against pathogen invasion. Ecol. Lett. 2005;8:1291–1298. doi: 10.1111/j.1461-0248.2005.00828.x. DOI

Cirimotich C.M., Dong Y., Clayton A.M., Sandiford S.L., Souza-Neto J., Mulenga M., Dimopoulos G. Natural microbe-mediated refractoriness to Plasmodium infection in Anopheles gambiae. Science. 2011;332:855–858. doi: 10.1126/science.1201618. PubMed DOI PMC

Kwong W.K., Mancenido A.L., Moran N.A. Immune system stimulation by the native gut microbiota of honey bees. R. Soc. Open Sci. 2017;4:1–9. doi: 10.1098/rsos.170003. PubMed DOI PMC

Weiss B.L., Maltz M., Aksoy S. Obligate symbionts activate immune system development in the tsetse fly. J. Immunol. 2012;188:3395–3403. doi: 10.4049/jimmunol.1103691. PubMed DOI PMC

Motta E.V.S., Raymann K., Moran N.A. Glyphosate perturbs the gut microbiota of honey bees. Proc. Natl. Acad. Sci. USA. 2018;115:10305–10310. doi: 10.1073/pnas.1803880115. PubMed DOI PMC

Kim J.K., Lee J.B., Huh Y.R., Jang H.A., Kim C.H., Yoo J.W., Lee B.L. Burkholderia gut symbionts enhance the innate immunity of host Riptortus pedestris. Dev. Comp. Immunol. 2015;53:265–269. doi: 10.1016/j.dci.2015.07.006. PubMed DOI

Chen H., Zhao R., Wang B., Cai C., Zheng L., Wang H., Wang M., Ouyang H., Zhou X., Chai Z., et al. The effects of orally administered Ag, TiO2 and SiO2 nanoparticles on gut microbiota composition and colitis induction in mice. NanoImpact. 2017;8:80–88. doi: 10.1016/j.impact.2017.07.005. DOI

Williams K., Milner J., Boudreau M.D., Gokulan K., Cerniglia C.E., Khare S. Effects of subchronic exposure of silver nanoparticles on intestinal microbiota and gut-associated immune responses in the ileum of Sprague-Dawley rats. Nanotoxicology. 2015;9:279–289. doi: 10.3109/17435390.2014.921346. PubMed DOI

Auguste M., Balbi T., Montagna M., Fabbri R., Sendra M., Blasco J., Canesi L. In vivo immunomodulatory and antioxidant properties of nanoceria (nCeO2) in the marine mussel Mytilus galloprovincialis. Comp. Biochem. Physiol. Part—C Toxicol. Pharmacol. 2019;219:95–102. doi: 10.1016/j.cbpc.2019.02.006. PubMed DOI

Auguste M., Lasa A., Pallavicini A., Gualdi S., Vezzulli L., Canesi L. Exposure to TiO2 nanoparticles induces shifts in the microbiota composition of Mytilus galloprovincialis hemolymph. Sci. Total Environ. 2019;670:129–137. doi: 10.1016/j.scitotenv.2019.03.133. PubMed DOI

Alijagic A., Pinsino A. Probing safety of nanoparticles by outlining sea urchin sensing and signaling cascades. Ecotoxicol. Environ. Saf. 2017;144:416–421. doi: 10.1016/j.ecoenv.2017.06.060. PubMed DOI

Hayashi Y., Heckmann L.H., Simonsen V., Scott-Fordsmand J.J. Time-course profiling of molecular stress responses to silver nanoparticles in the earthworm Eisenia fetida. Ecotoxicol. Environ. Saf. 2013;98:219–226. doi: 10.1016/j.ecoenv.2013.08.017. PubMed DOI

Edwards C.A., Bohlen P.J. Biology and Ecology of Earthworms. Chapman & Hall; London, UK: 1996. The role of earthworms in organic matter and nutrient cycles; pp. 155–180.

Beschin A., Bilej M., Hanssens F., Raymakers J., Van Dyck E., Revets H., Brys L., Gomez J., De Baetselier P., Timmermans M. Identification and Cloning of a Glucan- and Lipopolysaccharide-binding Protein from Eisenia foetida Earthworm Involved in the Activation of Prophenoloxidase Cascade. J. Biol. Chem. 1998;273:24948–24954. doi: 10.1074/jbc.273.38.24948. PubMed DOI

Bilej M., De Baetselier P., Van Dijck E., Stijlemans B., Colige A., Beschin A. Distinct Carbohydrate Recognition Domains of an Invertebrate Defense Molecule Recognize Gram-negative and Gram-positive Bacteria. J. Biol. Chem. 2001;276:45840–45847. doi: 10.1074/jbc.M107220200. PubMed DOI

Šilerová M., Procházková P., Josková R., Josens G., Beschin A., De Baetselier P., Bilej M. Comparative study of the CCF-like pattern recognition protein in different Lumbricid species. Dev. Comp. Immunol. 2006;30:765–771. doi: 10.1016/j.dci.2005.11.002. PubMed DOI

Josková R., Šilerová M., Procházková P., Bilej M. Identification and cloning of an invertebrate-type lysozyme from Eisenia andrei. Dev. Comp. Immunol. 2009;33:932–938. doi: 10.1016/j.dci.2009.03.002. PubMed DOI

Sekizawa Y., Hagiwara K., Nakajima T., Kobayashi H. A novel protein, lysenin, that causes contraction of the isolated rat aorta: Its purification from the coelomic fluid of the earthworm Eisenia foetida. Biomed. Res. 1996;17:197–203. doi: 10.2220/biomedres.17.197. DOI

Lassegues M., Milochau A., Doignon F., Du Pasquier L., Valembois P. Sequence and expression of an Eisenia-fetida-derived cDNA clone that encodes the 40-kDa fetidin antibacterial protein. Eur. J. Biochem. 1997;246:756–762. doi: 10.1111/j.1432-1033.1997.00756.x. PubMed DOI

Cooper E.L., Roch P. Earthworm immunity: A model of immune competence. Pedobiologia. 2003;47:676–688. doi: 10.1078/0031-4056-00245. DOI

Bruhn H., Winkelmann J., Andersen C., Andrä J., Leippe M. Dissection of the mechanisms of cytolytic and antibacterial activity of lysenin, a defence protein of the annelid Eisenia fetida. Dev. Comp. Immunol. 2006;30:597–606. doi: 10.1016/j.dci.2005.09.002. PubMed DOI

Dvořák J., Mančíková V., Pižl V., Elhottová D., Šilerová M., Roubalová R., Škanta F., Procházková P., Bilej M. Microbial environment affects innate immunity in two closely related earthworm species Eisenia andrei and Eisenia fetida. PLoS ONE. 2013;8:e0079257. doi: 10.1371/journal.pone.0079257. PubMed DOI PMC

Dvořák J., Roubalová R., Procházková P., Rossmann P., Škanta F., Bilej M. Sensing microorganisms in the gut triggers the immune response in Eisenia andrei earthworms. Dev. Comp. Immunol. 2016;57:67–74. doi: 10.1016/j.dci.2015.12.001. PubMed DOI

Hayashi Y., Engelmann P., Foldbjerg R., Szabó M., Somogyi I., Pollák E., Molnár L., Autrup H., Sutherland D.S., Scott-Fordsmand J., et al. Earthworms and humans in vitro: Characterizing evolutionarily conserved stress and immune responses to silver nanoparticles. Environ. Sci. Technol. 2012;46:4166–4173. doi: 10.1021/es3000905. PubMed DOI

Hayashi Y., Miclaus T., Engelmann P., Autrup H., Sutherland D.S., Scott-Fordsmand J.J. Nanosilver pathophysiology in earthworms: Transcriptional profiling of secretory proteins and the implication for the protein corona. Nanotoxicology. 2016;10:303–311. doi: 10.3109/17435390.2015.1054909. PubMed DOI

Zeibich L., Schmidt O., Drake H.L. Fermenters in the earthworm gut: Do transients matter? FEMS Microbiol. Ecol. 2019;95:1–12. doi: 10.1093/femsec/fiy221. PubMed DOI

Singleton D.R., Hendrix P.F., Coleman D.C., Whitman W.B. Identification of uncultured bacteria tightly associated with the intestine of the earthworm Lumbricus rubellus (Lumbricidae; Oligochaeta) Soil Biol. Biochem. 2003;35:1547–1555. doi: 10.1016/S0038-0717(03)00244-X. DOI

Thakuria D., Schmidt O., Finan D., Egan D., Doohan F.M. Gut wall bacteria of earthworms: A natural selection process. ISME J. 2010;4:357–366. doi: 10.1038/ismej.2009.124. PubMed DOI

Swart E., Newbold L., Kille P., Spurgeon D., Svendsen C. The midgut of the earthworm Eisenia fetida harbours a resident host specific bacterial community independent from soil. Environ. Microbiol. under review.

Lund M.B., Holmstrup M., Lomstein B.A., Damgaard C., Schramm A. Beneficial effect of verminephrobacter nephridial symbionts on the fitness of the earthworm aporrectodea tuberculata. Appl. Environ. Microbiol. 2010;76:4738–4743. doi: 10.1128/AEM.00108-10. PubMed DOI PMC

Viana F., Paz L.C., Methling K., Damgaard C.F., Lalk M., Schramm A., Lund M.B. Distinct effects of the nephridial symbionts Verminephrobacter and Candidatus Nephrothrix on reproduction and maturation of its earthworm host Eisenia andrei. FEMS Microbiol. Ecol. 2018;94:1–7. doi: 10.1093/femsec/fix178. PubMed DOI

Šrut M., Menke S., Höckner M., Sommer S. Earthworms and cadmium—Heavy metal resistant gut bacteria as indicators for heavy metal pollution in soils? Ecotoxicol. Environ. Saf. 2019;171:843–853. doi: 10.1016/j.ecoenv.2018.12.102. PubMed DOI

Yausheva E., Sizova E., Lebedev S., Skalny A., Miroshnikov S., Plotnikov A., Khlopko Y., Gogoleva N., Cherkasov S. Influence of zinc nanoparticles on survival of worms Eisenia fetida and taxonomic diversity of the gut microflora. Environ. Sci. Pollut. Res. 2016;23:13245–13254. doi: 10.1007/s11356-016-6474-y. PubMed DOI

Ma L., Xie Y., Han Z., Giesy J.P., Zhang X. Responses of earthworms and microbial communities in their guts to Triclosan. Chemosphere. 2017;168:1194–1202. doi: 10.1016/j.chemosphere.2016.10.079. PubMed DOI

Swart E., Goodall T., Kille P., Spurgeon D., Svendsen C. The earthworm microbiome is resilient to exposure to biocidal metal nanoparticles. Environ. Pollut. accepted. PubMed

Pass D.A., Morgan A.J., Read D.S., Field D., Weightman A.J., Kille P. The effect of anthropogenic arsenic contamination on the earthworm microbiome. Environ. Microbiol. 2015;17:1884–1896. doi: 10.1111/1462-2920.12712. PubMed DOI

Ma J., Chen Q.-L., O’Connor P., Sheng G.D. Does soil CuO nanoparticles pollution alter the gut microbiota and resistome of Enchytraeus crypticus? Environ. Pollut. 2019:113463. doi: 10.1016/j.envpol.2019.113463. PubMed DOI

Zhu D., Zheng F., Chen Q.-L., Yang X.-R., Christie P., Ke X., Zhu Y.-G. Exposure of a soil collembolan to Ag nanoparticles and AgNO3 disturbs its associated microbiota and lowers the incidence of antibiotic resistance genes in the gut. Environ. Sci. Technol. 2018;52:12748–12756. doi: 10.1021/acs.est.8b02825. PubMed DOI

Ma J., Sheng G.D., Chen Q.L., O’Connor P. Do combined nanoscale polystyrene and tetracycline impact on the incidence of resistance genes and microbial community disturbance in Enchytraeus crypticus? J. Hazard. Mater. 2020;387:122012. doi: 10.1016/j.jhazmat.2019.122012. PubMed DOI

Keller A.A., Adeleye A.S., Conway J.R., Garner K.L., Zhao L., Cherr G.N., Hong J., Gardea-Torresdey J.L., Godwin H.A., Hanna S., et al. Comparative environmental fate and toxicity of copper nanomaterials. NanoImpact. 2017;7:28–40. doi: 10.1016/j.impact.2017.05.003. DOI

Mincarelli L., Tiano L., Craft J., Marcheggiani F., Vischetti C. Evaluation of gene expression of different molecular biomarkers of stress response as an effect of copper exposure on the earthworm Eisenia Andrei. Ecotoxicology. 2019;28:938–948. doi: 10.1007/s10646-019-02093-3. PubMed DOI

Smit C.E., Van Gestel C.A.M. Effects of soil type, prepercolation, and ageing on bioaccumulation and toxicity of zinc for the springtail Folsomia candida. Environ. Toxicol. Chem. 1998;17:1132–1141. doi: 10.1002/etc.5620170621. DOI

Waalewijn-Kool P.L., Ortiz M.D., Van Gestel C.A.M. Effect of different spiking procedures on the distribution and toxicity of ZnO nanoparticles in soil. Ecotoxicology. 2012;21:1797–1804. doi: 10.1007/s10646-012-0914-3. PubMed DOI PMC

Kiernan J.A. Histological and Histochemical Methods: Theory and Practice. 4th ed. Scion Publishing Ltd.; Banbury, UK: 2008.

Gibson-Corley K.N., Olivier A.K., Meyerholz D.K. Principles for Valid Histopathologic Scoring in Research. Vet. Pathol. 2013;50:1007–1015. doi: 10.1177/0300985813485099. PubMed DOI PMC

Novo M., Almodóvar A., Fernández R., Trigo D., Díaz Cosín D.J. Cryptic speciation of hormogastrid earthworms revealed by mitochondrial and nuclear data. Mol. Phylogenet. Evol. 2010;56:507–512. doi: 10.1016/j.ympev.2010.04.010. PubMed DOI

King R.A., Tibble A.L., Symondson W.O.C. Opening a can of worms: Unprecedented sympatric cryptic diversity within British lumbricid earthworms. Mol. Ecol. 2008;17:4684–4698. doi: 10.1111/j.1365-294X.2008.03931.x. PubMed DOI

Anderson C., Cunha L., Sechi P., Kille P., Spurgeon D. Genetic variation in populations of the earthworm, Lumbricus rubellus, across contaminated mine sites. BMC Genet. 2017;18 doi: 10.1186/s12863-017-0557-8. PubMed DOI PMC

Pérez-Losada M., Eiroa J., Mato S., Domínguez J. Phylogenetic species delimitation of the earthworms Eisenia fetida (Savigny, 1826) and Eisenia andrei Bouché, 1972 (Oligochaeta, Lumbricidae) based on mitochondrial and nuclear DNA sequences. Pedobiologia. 2005;49:317–324. doi: 10.1016/j.pedobi.2005.02.004. DOI

Folmer O., Black M., Hoeh W., Lutz R., Vrijenhoek R. DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 1994;3:294–299. doi: 10.1071/ZO9660275. PubMed DOI

Sharma A., Sonah H., Deshmukh R.K., Gupta N.K., Singh N.K., Sharma T.R. Analysis of Genetic Diversity in Earthworms using DNA Markers. Zool. Sci. 2010;28:25. doi: 10.2108/zsj.28.25. PubMed DOI

Paradis E., Schliep K. ape 5.0: An environment for modern phylogenetics and evolutionary analyses in R. Bioinformatics. 2019;35:526–528. doi: 10.1093/bioinformatics/bty633. PubMed DOI

Kozich J.J., Westcott S.L., Baxter N.T., Highlander S.K., Schloss P.D. Development of a dual-index sequencing strategy and curation pipeline for analyzing amplicon sequence data on the MiSeq Illumina sequencing platform. Appl. Environ. Microbiol. 2013;79:5112–5120. doi: 10.1128/AEM.01043-13. PubMed DOI PMC

Callahan B.J., McMurdie P.J., Rosen M.J., Han A.W., Johnson A.J.A., Holmes S.P. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods. 2016;13:581–583. doi: 10.1038/nmeth.3869. PubMed DOI PMC

Callahan B. Silva taxonomic training data formatted for DADA2 (Silva version 132) [Data set] Zenodo. 2018

Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262. PubMed DOI

Oksanen J., Blanchet F.G., Friendly M., Kindt R., Legendre P., McGlinn D., Minchin P.R., O’Hara R.B., Simpson G.L., Solymos P., et al. [(accessed on 25 October 2018)];Vegan: Community Ecology Package. R Package Version 2.5-3. 2018 Available online: https://CRAN.R-project.org/package=vegan.

Procházková P., Hanč A., Dvořák J., Roubalová R., Drešlová M., Částková T., Šustr V., Škanta F., Pacheco N.I.N., Bilej M. Contribution of Eisenia andrei earthworms in pathogen reduction during vermicomposting. Environ. Sci. Pollut. Res. 2018;25:26267–26278. doi: 10.1007/s11356-018-2662-2. PubMed DOI

Hayashi Y., Miclaus T., Scavenius C., Kwiatkowska K., Sobota A., Engelmann P., Scott-Fordsmand J.J., Enghild J.J., Sutherland D.S. Species differences take shape at nanoparticles: Protein corona made of the native repertoire assists cellular interaction. Environ. Sci. Technol. 2013;47:14367–14375. doi: 10.1021/es404132w. PubMed DOI

Bigorgne E., Foucaud L., Caillet C., Giambérini L., Nahmani J., Thomas F., Rodius F. Cellular and molecular responses of E. fetida cœlomocytes exposed to TiO 2 nanoparticles. J. Nanoparticle Res. 2012;14 doi: 10.1007/s11051-012-0959-5. DOI

Patricia C.S., Nerea G.V., Erik U., Elena S.M., Eider B., Darío D.M.W., Manu S. Responses to silver nanoparticles and silver nitrate in a battery of biomarkers measured in coelomocytes and in target tissues of Eisenia fetida earthworms. Ecotoxicol. Environ. Saf. 2017;141:57–63. doi: 10.1016/j.ecoenv.2017.03.008. PubMed DOI

Garcia-Velasco N., Irizar A., Urionabarrenetxea E., Scott-Fordsmand J.J., Soto M. Selection of an optimal culture medium and the most responsive viability assay to assess AgNPs toxicity with primary cultures of Eisenia fetida coelomocytes. Ecotoxicol. Environ. Saf. 2019;183:109545. doi: 10.1016/j.ecoenv.2019.109545. PubMed DOI

Alijagic A., Gaglio D., Napodano E., Russo R., Costa C., Benada O., Kofroňová O., Pinsino A. Titanium dioxide nanoparticles temporarily influence the sea urchin immunological state suppressing inflammatory-relate gene transcription and boosting antioxidant metabolic activity. J. Hazard. Mater. 2020;384:121389. doi: 10.1016/j.jhazmat.2019.121389. PubMed DOI

Meier C., Voegelin A., Pradas Del Real A., Sarret G., Mueller C.R., Kaegi R. Transformation of Silver Nanoparticles in Sewage Sludge during Incineration. Environ. Sci. Technol. 2016;50:3503–3510. doi: 10.1021/acs.est.5b04804. PubMed DOI

Sekine R., Marzouk E.R., Khaksar M., Scheckel K.G., Stegemeier J.P., Lowry G.V., Donner E., Lombi E. Aging of dissolved copper and copper-based nanoparticles in five different soils: Short-term kinetics vs. long-term fate. J. Environ. Qual. 2017;46:1198–1205. doi: 10.2134/jeq2016.12.0485. PubMed DOI PMC

Levard C., Hotze E.M., Lowry G.V., Brown G.E. Environmental Transformations of Silver Nanoparticles: Impact on Stability and Toxicity. Environ. Sci. Technol. 2012;46:6900–6914. doi: 10.1021/es2037405. PubMed DOI

Baccaro M., Undas A.K., De Vriendt J., Van Den Berg J.H.J., Peters R.J.B., Van Den Brink N.W. Ageing, dissolution and biogenic formation of nanoparticles: How do these factors affect the uptake kinetics of silver nanoparticles in earthworms? Environ. Sci. Nano. 2018;5:1107–1116. doi: 10.1039/C7EN01212H. DOI

Peng C., Duan D., Xu C., Chen Y., Sun L., Zhang H., Yuan X., Zheng L., Yang Y., Yang J., et al. Translocation and biotransformation of CuO nanoparticles in rice (Oryza sativa L.) plants. Environ. Pollut. 2015;197:99–107. doi: 10.1016/j.envpol.2014.12.008. PubMed DOI

Xiang Q., Zhu D., Chen Q.L., O’Connor P., Yang X.R., Qiao M., Zhu Y.G. Adsorbed Sulfamethoxazole Exacerbates the Effects of Polystyrene (∼2 μm) on Gut Microbiota and the Antibiotic Resistome of a Soil Collembolan. Environ. Sci. Technol. 2019;53:12823–12834. doi: 10.1021/acs.est.9b04795. PubMed DOI

Nechitaylo T.Y., Timmis K.N., Golyshin P.N. “Candidatus Lumbricincola”, a novel lineage of uncultured Mollicutes from earthworms of family Lumbricidae. Environ. Microbiol. 2009;11:1016–1026. doi: 10.1111/j.1462-2920.2008.01837.x. PubMed DOI

Tak E.S., Cho S., Park S.C. Gene expression profiling of coelomic cells and discovery of immune-related genes in the earthworm, Eisenia andrei, using expressed sequence tags. Biosci. Biotechnol. Biochem. 2015;8451:1–7. doi: 10.1080/09168451.2014.988677. PubMed DOI

Opper B., Bognár A., Heidt D., Németh P., Engelmann P. Revising lysenin expression of earthworm coelomocytes. Dev. Comp. Immunol. 2013;39:214–218. doi: 10.1016/j.dci.2012.11.006. PubMed DOI

Adamowicz A. Morphology and ultrastructure of the earthworm Dendrobaena veneta (Lumbricidae) coelomocytes. Tissue Cell. 2005;37:125–133. doi: 10.1016/j.tice.2004.11.002. PubMed DOI

Engelmann P., Cooper E.L., Opper B., Németh P. Earthworm Innate Immune System. In: Karaca A., editor. Biology of Earthworms. Volume 24. Springer; Berlin/Heidelberg, Germany: 2011. pp. 229–245. Soil Biology.

Bodó K., Ernszt D., Németh P., Engelmann P. Distinct immune-and defense-related molecular fingerprints in sepatated coelomocyte subsets of Eisenia andrei earthworms. Invertebr. Surviv. J. 2018;15:338–345.

Irizar A., Rivas C., García-Velasco N., de Cerio F.G., Etxebarria J., Marigómez I., Soto M. Establishment of toxicity thresholds in subpopulations of coelomocytes (amoebocytes vs. eleocytes) of Eisenia fetida exposed in vitro to a variety of metals: Implications for biomarker measurements. Ecotoxicology. 2015;24:1004–1013. doi: 10.1007/s10646-015-1441-9. PubMed DOI

Homa J., Zorska A., Wesolowski D., Chadzinska M. Dermal exposure to immunostimulants induces changes in activity and proliferation of coelomocytes of Eisenia andrei. J. Comp. Physiol. B. 2013:313–322. doi: 10.1007/s00360-012-0710-7. PubMed DOI PMC

Olchawa E., Bzowska M., Stürzenbaum S.R., Morgan A.J., Plytycz B. Heavy metals affect the coelomocyte-bacteria balance in earthworms: Environmental interactions between abiotic and biotic stressors. Environ. Pollut. 2006;142:373–381. doi: 10.1016/j.envpol.2005.09.023. PubMed DOI

Procházková P., Silerová M., Felsberg J., Josková R., Beschin A., De Baetselier P., Bilej M. Relationship between hemolytic molecules in Eisenia fetida earthworms. Dev. Comp. Immunol. 2006;30:381–392. doi: 10.1016/j.dci.2005.06.014. PubMed DOI

Plytycz B., Bigaj J., Osikowski A., Hofman S., Falniowski A., Panz T., Grzmil P., Vandenbulcke F. The existence of fertile hybrids of closely related model earthworm species, Eisenia andrei and E. fetida. PLoS ONE. 2018;13:e0191711. doi: 10.1371/journal.pone.0191711. PubMed DOI PMC

Martinsson S., Erséus C. Hybridisation and species delimitation of Scandinavian Eisenia spp. (Clitellata: Lumbricidae) Eur. J. Soil Biol. 2018;88:41–47. doi: 10.1016/j.ejsobi.2018.06.003. DOI

Römbke J., Aira M., Backeljau T., Breugelmans K., Domínguez J., Funke E., Graf N., Hajibabaei M., Pérez-Losada M., Porto P.G., et al. DNA barcoding of earthworms (Eisenia fetida/andrei complex) from 28 ecotoxicological test laboratories. Appl. Soil Ecol. 2016;104:3–11. doi: 10.1016/j.apsoil.2015.02.010. DOI

Bouché M.B. Lombriciens de France. Ecol. Syst. 1972;72:671.

Domínguez J., Velando A., Ferreiro A. Are Eisenia fetida (Savigny, 1826) and Eisenia andrei Bouche (1972) (Oligochaeta, Lumbricidae) different biological species? Pedobiologia. 2005;49:81–87. doi: 10.1016/j.pedobi.2004.08.005. DOI

Plytycz B., Bigaj J., Panz T., Grzmil P. Asymmetrical hybridization and gene flow between Eisenia andrei and E. fetida lumbricid earthworms. PLoS ONE. 2018;13:e0204469. doi: 10.1371/journal.pone.0204469. PubMed DOI PMC

Goodrich J.K., Davenport E.R., Beaumont M., Bell J.T., Clark A.G., Ley R.E., Goodrich J.K., Davenport E.R., Beaumont M., Jackson M.A., et al. Genetic Determinants of the Gut Microbiome in UK Twins Resource Genetic Determinants of the Gut Microbiome in UK Twins. Cell Host Microbe. 2016:731–743. doi: 10.1016/j.chom.2016.04.017. PubMed DOI PMC

Kolde R., Franzosa E.A., Rahnavard G., Hall A.B., Vlamakis H., Stevens C., Daly M.J., Xavier R.J., Huttenhower C. Host genetic variation and its microbiome interactions within the Human Microbiome Project. Genome Med. 2018;10:1–13. doi: 10.1186/s13073-018-0515-8. PubMed DOI PMC

Buhnik-Rosenblau K., Danin-Poleg Y., Kashi Y. Predominant effect of host genetics on levels of Lactobacillus johnsonii bacteria in the mouse gut. Appl. Environ. Microbiol. 2011;77:6531–6538. doi: 10.1128/AEM.00324-11. PubMed DOI PMC

Macke E., Callens M., De Meester L., Decaestecker E. Host-genotype dependent gut microbiota drives zooplankton tolerance to toxic cyanobacteria. Nat. Commun. 2017;8 doi: 10.1038/s41467-017-01714-x. PubMed DOI PMC

Davidson S.K., Davidson S.K., Stahl D.A. Transmission of Nephridial Bacteria of the Earthworm Eisenia fetida. Appl. Environ. Microbiol. 2006;72:769–775. doi: 10.1128/AEM.72.1.769-775.2006. PubMed DOI PMC

Cross-Species Comparisons of Nanoparticle Interactions with Innate Immune Systems: A Methodological Review