Abstract
All plants and animals form symbiotic associations with microbes, yet many of the underlying mechanisms associated with these interactions remain uncharacterized. There are inherent challenges to studying the cellular and metabolic interactions between eukaryotes and their microbial symbionts, thus new methodologies that enable the discovery of symbiotic processes are continually needed. Here, we explored the use of magnetic nanoparticles (MNPs) as a tool to track aspects of the host innate immune response to symbionts under both ex vivo and in vivo conditions. The symbiotic association between the Hawaiian bobtail squid Euprymna scolopes and its bioluminescent partner Vibrio fischeri was used as a model to explore the potential of MNPs as non-toxic, manipulable agents to investigate aquatic symbiotic associations. Results suggest that host cells can be effectively labeled with MNPs under ex vivo conditions and that the particles can be visualized and tracked within the host animal in vivo using magnetic particle imaging. Proteomic and metabolomic analyses also revealed minimal changes to the host innate immune cells after uptake of MNPs in the presence and absence of V. fischeri. Together, these results suggest that MNPs have minimal biochemical impact on the host cells and can serve as an effective tool to explore aquatic symbiotic interactions.
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Data availability
All data generated as part of this study is available within this manuscript, including figures, and supplemental materials. The mass spectrometry proteomics data have been deposited in the EMBL-EBI PRoteomics IDEntifications Database (PRIDE) under the dataset identifiers PXD074352 and 10.6019/PXD074352. Spectra are available in the MassIVE data repository MSV000098647.
References
Munzi, S., Cruz, C. & Correa, A. When the exception becomes the rule: An integrative approach to symbiosis. Sci. Total Environ. 672, 855–861 (2019).
McFall-Ngai, M. Symbiosis takes a front and center role in biology. PLoS Biol. 22, e3002571 (2024).
Margulis, L. & Fester, R. Symbiosis as a source of evolutionary innovation: speciation and morphogenesis (MIT Press, 1991).
Maher, R. L. et al. Coral microbiomes demonstrate flexibility and resiliance through a reduction in community diversity following a thermal stress event. Front. Ecol. Evol. 14, 555698 (2020).
McFall-Ngai, M. Divining the essence of symbiosis: insights from the squid-vibrio model. PLoS Biol. 12, e1001783 (2014).
Perreau, J. & Moran, N. A. Genetic innovations in animal-microbe symbioses. Nat. Rev. Genet. 23, 23–39 (2022).
Engelberts, J. P., Robbins, S. J., D’amjanovic, K. & Webster, N. S. Integrating novel tools to elucidate the metabolic basis of microbial symbiosis in reef holobionts. Mar. Biol. 168, 175 (2021).
Jain, D., Jones, L. & Roy, S. Gene editing to improve legume-rhizobia symbiosis in a changing climate. Curr. Opin. Plant. Biol. 71, 102324 (2023).
Krishnan, K. M. Biomedical nanomagnetics: a spin through possibilities in imaging, diagnostics, and therapy. IEEE Trans. Magn. 46, 2523–2558 (2010).
Kianfar, E. Magnetic nanoparticles in targeted drug delivery: a review. J. Supercond Nov Magn. 34, 1709–1735 (2021).
Tay, Z. W. et al. Magnetic particle imaging-guided heating in vivo using gradient fields for arbitrary localization of magnetic hyperthermia therapy. ACS nano. 12, 3699–3713 (2018).
Fuller, E. G. et al. Theranostic nanocarriers combining high drug loading and magnetic particle imaging. Int. J. Pharm. 572, 118796 (2019).
Stueber, D. D., Villanova, J., Aponte, I., Xiao, Z. & Colvin, V. L. Magnetic nanoparticles in biology and medicine: past, present, and future trends. Pharmaceutics 13 (2021).
Liu, S. et al. Long circulating tracer tailored for magnetic particle imaging. Nanotheranostics 5, 348–361 (2021).
Mittal, A., Roy, I. & Gandhi, S. Magnetic nanoparticles: an overview for biomedical applications. Magnetochemistry 8, 107 (2022).
Stiufiuc, G. F. & Stiufiuc, R. I. Magnetic nanoparticles: synthesis, characterization, and their use in the biomedical field. Appl. Sci. 14, 1623 (2024).
Baki, A., Wiekhorst, F. & Bleul, R. Advances in magnetic nanoparticles engineering for biomedical applications: A review. Bioengineering (Basel) 8 (2021).
Nyholm, S. V. & McFall-Ngai, M. J. A lasting symbiosis: how the Hawaiian bobtail squid finds and keeps its bioluminescent bacterial partner. Nat. Rev. Microbiol. 19, 666–679 (2021).
Visick, K. L., Stabb, E. V. & Ruby, E. G. A lasting symbiosis: how Vibrio fischeri finds a squid partner and persists within its natural host. Nat. Rev. Microbiol. 19, 654–665 (2021).
McFall-Ngai, M. J. & Ruby, E. G. Symbiont recognition and subsequent morphogenesis as early events in an animal-bacterial mutualism. Science 254, 1491–1494 (1991).
Imes, A. M. et al. Euprymna berryi as a comparative model host for Vibrio fischeri light organ symbiosis. Appl. Environ. Microbiol., e0000125 (2025).
McAnulty, S. J. & Nyholm, S. V. The role of hemocytes in the Hawaiian bobtail squid, Euprymna scolopes: a model organism for studying beneficial host-microbe interactions. Front. Microbiol. 7, 2013 (2016).
Koropatnick, T. A., Kimbell, J. R. & McFall-Ngai, M. J. Responses of host hemocytes during the initiation of the squid-vibrio symbiosis. Biol. Bull. 212, 29–39 (2007).
Rader, B., McAnulty, S., J, S. & Nyholm, V. Persistent symbiont colonization leads to a maturation of hemocyte response in the Euprymna scolopes/Vibrio fischeri symbiosis. MicrobiologyOpen 8, e858 (2019).
Schwartzman, J. A. et al. The chemistry of negotiation: rhythmic, glycan-driven acidification in a symbiotic conversation. Proc. Natl. Acad. Sci. USA 112, 566–571 (2015).
Tay, R. E. et al. High-efficiency magnetophoretic labelling of adoptively-transferred T cells for longitudinal in vivo magnetic particle imaging. Theranostics 14, 6138–6160 (2024).
Searle, B. C. Scaffold: a bioinformatic tool for validating MS/MS-based proteomic studies. Proteomics 10, 1265–1269 (2010).
Li, J. & Hu, Z. Research progress on damage-associated molecular patterns in acute kidney injury. Front. Immunol. 16, 1590822 (2025).
Richards, C. M., McRae, S. A., Ranger, A. L. & Klegeris, A. Extracellular histones as damage-associated molecular patterns in neuroinflammatory responses. Rev. Neurosci. 34, 533–558 (2023).
Schleicher, T. R., VerBerkmoes, N. C., Shah, M. & Nyholm, S. V. Colonization state influences the hemocyte proteome in a beneficial squid-Vibrio symbiosis. Mol. Cell. Proteom. 13, 2673–2686 (2014).
Longo, N., Frigeni, M. & Pasquali, M. Carnitine transport and fatty acid oxidation. Biochim. Biophys. Acta. 1863, 2422–2435 (2016).
Chen, X. et al. A non-invasive nanoparticles for multimodal imaging of ischemic myocardium in rats. J. Nanobiotechnol. 19, 82 (2021).
Rubio, J. M. et al. Group V secreted phospholipase A2 is upregulated by IL-4 in human macrophages and mediates phagocytosis via hydrolysis of ethanolamine phospholipids. J. Immunol. 194, 3327–3339 (2015).
Nyholm, S. V., Stewart, J. J. & Ruby, E. G. McFall-Ngai, M. J. Recognition between symbiotic Vibrio fischeri and the haemocytes of Euprymna scolopes. Environ. Microbiol. 11, 483–493 (2009).
Collins, A. J., Schleicher, T. R., Rader, B. A. & Nyholm, S. V. Understanding the role of host hemocytes in a squid/vibrio symbiosis using transcriptomics and proteomics. Front. Immunol. 3, 91 (2012).
Murphy, F. & Quinn, B. The effects of microplastic on freshwater Hydra attenuata feeding, morphology & reproduction. Environ. Pollut. 234, 487–494 (2018).
Jacobovitz, M. R. et al. Dinoflagellate symbionts escape vomocytosis by host cell immune suppression. Nat. Microbiol. 6, 769–782 (2021).
Marulanda-Gomez, A. M., Bayer, K., Pita, L. & Hentschel, U. A novel in-vivo phagocytosis assay to gain cellular insights on sponge-microbe interactions. Front. Mar. Sci. (2023).
Velazquez-Albino, A. C. & Imhoff, E. D. Rinaldi-Ramos, C. M. Advances in engineering nanoparticles for magnetic particle imaging (MPI). Sci. Adv. 11, eado7356 (2025).
Estelrich, J., Escribano, E., Queralt, J. & Busquets, M. A. Iron oxide nanoparticles for magnetically-guided and magnetically-responsive drug delivery. Int. J. Mol. Sci. 16, 8070–8101 (2015).
Zhao, Z., Torres-Díaz, I., Vélez, C., Arnold, D. P. & Rinaldi, C. Brownian dynamics simulations of magnetic nanoparticles captured in strong magnetic field gradients. J. Phys. Chem. C. 121, 801–810 (2016).
Bushra, R. et al. Recent advances in magnetic nanoparticles: key applications, environmental insights and future strategies. SM&T 40, e00985 (2024).
Pan, Y., Du, X., Zhao, F. & Xu, B. Magnetic nanoparticles for the manipulation of proteins and cells. Chem. Soc. Rev. 41, 2912–2942 (2012).
Zwi-Dantsis, L. et al. Remote Magnetic Nanoparticle Manipulation Enables the Dynamic Patterning of Cardiac Tissues. Adv. Mater. 32, e1904598 (2020).
Al-Obaidy, R., Haider, A. J., Al-Musawi, S. & Arsad, N. Targeted delivery of paclitaxel drug using polymer-coated magnetic nanoparticles for fibrosarcoma therapy: in vitro and in vivo studies. Sci. Rep. 13, 3180 (2023).
He, N. et al. Nano pom-poms prepared exosomes enable highly specific cancer biomarker detection. Commun. Biol. 5, 660 (2022).
Chen, Y. & Hou, S. Recent progress in the effect of magnetic iron oxide nanoparticles on cells and extracellular vesicles. Cell. Death Discov. 9, 195 (2023).
Unni, M. et al. Thermal decomposition synthesis of iron oxide nanoparticles with diminished magnetic dead layer by controlled addition of oxygen. ACS nano. 11, 2284–2303 (2017).
Butler-Struben, H. M., Brophy, S. M., Johnson, N. A. & Crook, R. J. vivo recording of neural and behavioral correlates of anesthesia induction, reversal, and euthanasia in cephalopod molluscs. Front. Physiol. 9, 109 (2018).
Fiorito, G. et al. Guidelines for the care and welfare of cephalopods in research -a consensus based on an initiative by CephRes, FELASA and the Boyd Group. Lab. Anim. 49, 1–90 (2015).
Collins, A. J. & Nyholm, S. V. Obtaining hemocytes from the Hawaiian bobtail squid Euprymna scolopes and observing their adherence to symbiotic and non-symbiotic bacteria. J. Vis. Exp. (2010).
Koch, E. et al. (ed, J.) The cytokine MIF controls daily rhythms of symbiont nutrition in an animal-bacterial association. Proc. Natl. Acad. Sci. USA 117 27578–27586 (2020).
Boettcher, K. J. & Ruby, E. G. Depressed light emission by symbiotic Vibrio fischeri of the sepiolid squid Euprymna scolopes. J. Bacteriol. 172, 3701–3706 (1990).
Koch, E. J. & Foster, J. S. Labeling of host immune cells with magnetic nanoparticles (protocols.io, 2024).
Cox, J. & Mann, M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008).
Belcaid, M. et al. Symbiotic organs shaped by distinct modes of genome evolution in cephalopods. Proc. Natl. Acad. Sci. USA. 116, 3030–3035 (2019).
Tsugawa, H. et al. MS-DIAL: data-independent MS/MS deconvolution for comprehensive metabolome analysis. Nat. Methods. 12, 523–526 (2015).
NIST23. NIST23: updates of the NIST tandem and electron ionization spectral libraries (2023). https://www.nist.gov/programs-projects/nist23-updates-nist-tandem-and-electron-ionization-spectral-libraries
Dixon, P. VEGAN, a package of R functions for community ecology. J. Veg. Sci. 14, 927–930 (2003).
Team, R. C. R: A language and environment for statistical computing (2013). http://www.R-project.org/
Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multple testing. J. R. Stat. Soc. B 289–300 (1995).
Ward, J. H. Jr & Hook, M. E. Application of an hiercrchical grouping procedure to a problem of grouping profiles. Educ. Psychol. Meas. 23, 69–81 (1963).
Chen, H. & Boutros, P. C. VennDiagram: a package for the generation of highly-customizable Venn and Euler diagrams in R. BMC Bioinform. 12, 35 (2011).
Conway, J. R., Lex, A. & Gehlenborg, N. UpSetR: an R package for the visualization of intersecting sets and their properties. Bioinformatics 33, 2938–2940 (2017).
Good, H. J. et al. On the partial volume effect in magnetic particle imaging. Phys. Med. Biol. 70 (2025).
Kerwin, A. H., McAnulty, S. J. & Nyholm, S. V. Development of the accessory nidamental gland and associated bacterial community in the Hawaiian bobtail squid, Euprymna scolopes. Biol. Bull. 240, 205–218 (2021).
Acknowledgements
The authors thank by Dr. Jen Liddle and Dr. Jeremy Balsbaugh, of the Proteomics & Metabolomics Facility, part of the Center for Open Research Resources and Equipment at the University of Connecticut for quantitative proteomics analysis. NIH S10 High-End Instrumentation Award 1S10-OD028445-01A1 supported this work by providing funds to acquire the Orbitrap Eclipse Tribrid mass spectrometer housed in the UConn Proteomics & Metabolomics Facility. The authors would also like to thank Y. Zhang and J. Richards of the Microgravity Simulation Support Facility at the Kennedy Space Center for use of the confocal microscope.
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The work was supported by the Gordon and Betty Moore Foundation award number 9349.
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H.G. completed the magnetic particle characterization. E.K. completed the hemocyte labeling optimization and imaging. R.S. completed the initial hemocyte extraction and acquisition of mass spectrometry data. N.V. and S.N. completed the proteomics analyses. D.G.M. and M.B. completed the metabolomics analyses. H.G., C.R., S.N. and J.F. completed the magnetic particle imaging. D.A., C.R., S.N., M.B. and J.F. conceived of the experimental plan and design. All authors contributed to the writing and editing of the manuscript.
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All animal experiments were performed in accordance with ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments). For example, all cephalopod procedures were approved by both the University of Florida (Protocol 201910899) and the University of Connecticut (Protocol A25-004) Institutional Animal Care and Use Committees and were performed in accordance with the approved protocols and guidelines.
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Guillen Matus, D.G., Koch, E.J., Vijayan, N. et al. Using magnetic nanoparticles to explore symbiotic interactions. Sci Rep (2026). https://doi.org/10.1038/s41598-026-46489-8
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DOI: https://doi.org/10.1038/s41598-026-46489-8
