Obtaining Hemocytes from the Hawaiian Bobtail Squid Euprymna scolopes and Observing their Adherence to Symbiotic and Non-Symbiotic Bacteria
Summary
This video will demonstrate how to obtain hemocytes (blood cells) from the Hawaiian bobtail squid, Euprymna scolopes for use in cell biological and bacterial adhesion assays. Hemocytes will be stained with a fluorescent dye and exposed to GFP-labeled bacteria.
Abstract
Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made significant contributions to our understanding of the co-evolution of eukaryotes with their microbiota. The symbiotic association between the Hawaiian bobtail squid, Euprymna scolopes and the bioluminescent bacterium Vibrio fischeri has been utilized as a model system for understanding the effects of beneficial bacteria on animal development. Recent studies have shown that macrophage-like hemocytes, the sole cellular component of the squid host’s innate immune system, likely play an important role in mediating the establishment and maintenance of this association. This protocol will demonstrate how to obtain hemocytes from E. scolopes and then use these cells in bacterial binding assays. Adult squid are first anesthetized before hemolymph is collected by syringe from the main cephalic blood vessel. The host hemocytes, contained in the extracted hemolymph, are adhered to chambered glass coverslips and then exposed to green fluorescent protein-labeled symbiotic Vibrio fischeri and non-symbiotic Vibrio harveyi. The hemocytes are counterstained with a fluorescent dye (Cell Tracker Orange, Invitrogen) and then visualized using fluorescent microscopy.
Protocol
Prepare 500 mLs of 0.22 μm filter-sterilized artificial seawater (FSW; salinity 35 ppt). Filter artificial or natural seawater through a 0.22 μm micron filter to remove particles and bacteria. Anesthetize one adult Hawaiian bobtail squid (Euprymna scolopes) by placing in a 2% solution of ethanol in FSW. Place the animal in anesthetic for approximately 10 minutes. The squid will cease swimming and will not actively respond to touch. Continued respiration, indicated by movement of the mantle, and chromat…
Discussion
Studies concerning the role of the immune system in mediating molecular signaling between beneficial bacteria and their hosts have, in recent years, made significant contributions to our understanding of the co-evolution of eukaryotes with their microbiota. The squid/vibrio system has proven itself as a tractable model system to answer fundamental questions in this field2,3,5,6,8. The light-organ of the squid Euprymna scolopes permits colonization exclusively by the luminous bacterium Vibrio fisc…
Acknowledgements
Funding sources: University of Connecticut Research Foundation and the Department of Molecular and Cell Biology to SVN, Sigma Xi Grant-in-Aid-of Research and Antonio H. and Majorie R.
Romano Graduate Education Fellowship to AJC
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Lab-Tek chambered #1.0 borosilicate coverglass system (8-chambers) | Thermo-Fisher Scientific | 155411 | ||
| 26.5 G 1ml latex-free insulin syringe | Becton, Dickinson and Company | C34551 | ||
| Cell Tracker Orange | Invitrogen | C34551 | ||
| SteREO Discovery V20 Microscope | Zeiss | |||
| SP2 Confocal Microscope | Leica |
References
- 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).
- McFall-Ngai, M. J. Unseen forces: the influence of bacteria on animal development. Dev. Biol. 242, 1-14 (2002).
- 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).
- 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).
- Nyholm, S. V., McFall-Ngai, M. J. The winnowing: establishing the squid-Vibrio symbiosis. Nat. Rev. Microbiol. 2, 632-642 (2004).
- Nyholm, S. V., Stabb, E. V., Ruby, E. G., McFall-Ngai, M. J. Establishment of an animal-bacterial association: recruiting symbiotic Vibrios from the environment. Proc. Natl. Acad. Sci. USA. 97, 10231-10235 (2000).
- Nyholm, S. V., McFall-Ngai, M. J. Sampling the light-organ microenvironment of Euprymna scolopes: description of a population of host cells in association with the bacterial symbiont Vibrio fischeri. Biol Bull. 195, 89-97 (1998).
- Visick, K. L., Ruby, E. G. Vibrio fischeri and its host: it takes two to tango. Curr. Opin. Microbiol. 9, 632-638 (2006).
Tags
HemocytesHawaiian Bobtail SquidEuprymna ScolopesAdherenceSymbiotic BacteriaNon-symbiotic BacteriaImmune SystemMolecular SignalingBeneficial BacteriaCo-evolutionEukaryotesMicrobiotaModel SystemAnimal DevelopmentMacrophage-like HemocytesInnate Immune SystemEstablishmentMaintenanceProtocolAnesthetizedHemolymph CollectionCephalic Blood VesselChambered Glass CoverslipsGreen Fluorescent Protein-labeled Symbiotic Vibrio FischeriNon-symbiotic Vibrio HarveyiFluorescent DyeCell Tracker OrangeVisualizedFluorescent Microscopy
Cite This Article
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. (36), e1714, doi:10.3791/1714 (2010).