Article | Published:

Achieving a multi-strain symbiosis: strain behavior and infection dynamics

The ISME Journalvolume 13pages698706 (2019) | Download Citation


Strain diversity, while now recognized as a key driver underlying partner dynamics in symbioses, is usually difficult to experimentally manipulate and image in hosts with complex microbiota. To address this problem, we have used the luminous marine bacterium Vibrio fischeri, which establishes a symbiosis within the crypts of the nascent light organ of the squid Euprymna scolopes. Competition assays in newly hatched juvenile squid have shown that symbiotic V. fischeri are either niche-sharing “S strains”, which share the light organ when co-inoculated with other S strains, or niche-dominant “D strains”, which are typically found alone in the light organ after a co-colonization. To understand this D strain advantage, we determined the minimum time that different V. fischeri strains needed to initiate colonization and used confocal microscopy to localize the symbionts along their infection track. Further, we determined whether symbiont-induced host morphogenic events also occurred earlier during a D strain colonization. We conclude that D strains colonized more quickly than S strains. Nevertheless, light-organ populations in field-caught adult squid often contain both D and S strains. We determined experimentally that this symbiont population heterogeneity might be achieved in nature by a serial encounter of different strains in the environment.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Bongrand C, Koch EJ, Moriano-Gutierrez S, Cordero OX, McFall-Ngai M, Polz MF, et al. A genomic comparison of 13 symbiotic Vibrio fischeri isolates from the perspective of their host source and colonization behavior. ISME J. 2016;10:2907–017.

  2. 2.

    Lee KWK, Periasamy S, Mukherjee M, Xie C, Kjelleberg S, Rice SA. Biofilm development and enhanced stress resistance of a model, mixed-species community biofilm. ISME J. 2014;8:894–907.

  3. 3.

    Kerwin AH, Nyholm SV. Symbiotic bacteria associated with a bobtail squid reproductive system are detectable in the environment, and stable in the host and developing eggs. Environ Microbiol. 2017;19:1463–75.

  4. 4.

    Lozupone CA, Stombaugh JI, Gordon JI, Jansson JK, Knight R. Diversity, stability and resilience of the human gut microbiota. Nature. 2012;489:220–30.

  5. 5.

    Kwong WK, Moran NA. Gut microbial communities of social bees. Nat Rev Microbiol. 2016;14:374–84.

  6. 6.

    McLean AHC, Parker BJ, Hrček J, Kavanagh JC, Wellham PAD, Godfray HCJ. Consequences of symbiont co-infections for insect host phenotypes. J Anim Ecol. 2018;87:478–88.

  7. 7.

    Yawata Y, Cordero OX, Menolascina F, Hehemann J-H, Polz MF, Stocker R. Competition-dispersal tradeoff ecologically differentiates recently speciated marine bacterioplankton populations. Proc Natl Acad Sci USA. 2014;111:5622–7.

  8. 8.

    Duar RM, Frese SA, Lin XB, Fernando SC, Burkey TE, Tasseva G, et al. Experimental evaluation of host adaptation of Lactobacillus reuteri to different vertebrate species. Appl Environ Microbiol. 2017;83:e00132–17.

  9. 9.

    Jones BW, Nishiguchi MK. Counterillumination in the Hawaiian bobtail squid, Euprymna scolopes Berry (Mollusca: Cephalopoda). Mar Biol. 2004;144:1151–5.

  10. 10.

    McFall-Ngai M, Ruby E. Symbiont recognition and subsequent morphogenesis as early events in an animal-bacterial mutualism. Science. 1991;254:1491–4.

  11. 11.

    Nawroth JC, Guo H, Koch E, Heath-Heckman EAC, Hermanson JC, Ruby EG, et al. Motile cilia create fluid-mechanical microhabitats for the active recruitment of the host microbiome. Proc Natl Acad Sci USA. 2017;114:9510–6.

  12. 12.

    Nyholm SV, McFall-Ngai M. The winnowing: establishing the squid–vibrio symbiosis. Nat Rev Microbiol. 2004;2:632–42.

  13. 13.

    Koropatnick TA, Kimbell JR, McFall-Ngai MJ. Responses of host hemocytes during the initiation of the squid-vibrio symbiosis. Biol Bull. 2007;212:29–39.

  14. 14.

    McFall-Ngai M, Nyholm SV, Castillo MG. The role of the immune system in the initiation and persistence of the Euprymna scolopes-Vibrio fischeri symbiosis. Sem Immunol. 2010;22:48–53.

  15. 15.

    Graf J, Ruby EG. Host-derived amino acids support the proliferation of symbiotic bacteria. Proc Natl Acad Sci USA. 1998;95:1818–22.

  16. 16.

    McFall-Ngai MJ. The importance of microbes in animal development: lessons from the squid-vibrio symbiosis. Annu Rev Microbiol. 2014;68:177–94.

  17. 17.

    Boettcher KJ, Ruby EG. Depressed light emission by symbiotic Vibrio fischeri of the sepiolid squid Euprymna scolopes. J Bacteriol. 1990;172:3701–6.

  18. 18.

    Wollenberg MS, Ruby EG. Phylogeny and fitness of Vibrio fischeri from the light organs of Euprymna scolopes in two Oahu, Hawaii populations. ISME J. 2012;6:352–62.

  19. 19.

    Wollenberg MS, Ruby EG. Population structure of Vibrio fischeri within the light organs of Euprymna scolopes squid from Two Oahu (Hawaii) populations. Appl Environ Microbiol. 2009;75:193–202.

  20. 20.

    Speare L, Cecere AG, Guckes KR, Smith S, Wollenberg MS, Mandel MJ, et al. Bacterial symbionts use a type VI secretion system to eliminate competitors in their natural host. Proc Natl Acad Sci USA. 2018;115:8528–37.

  21. 21.

    Dunn AK, Millikan DS, Adin DM, Bose JL, Stabb EV. New rfp- and pES213-derived tools for analyzing symbiotic Vibrio fischeri reveal patterns of infection and lux expression in situ. Appl Environ Microbiol. 2006;72:802–10.

  22. 22.

    Graf J, Dunlap PV, Ruby EG. Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri. J Bacteriol. 1994;176:6986–91.

  23. 23.

    McCann J, Stabb EV, Millikan DS, Ruby EG. Population dynamics of Vibrio fischeri during Infection of Euprymna scolopes. Appl Environ Microbiol. 2003;69:5928–34.

  24. 24.

    Lee KH, Ruby EG. Symbiotic role of the viable but nonculturable state of Vibrio fischeri in Hawaiian coastal seawater. Appl Environ Microbiol. 1995;61:278–83.

  25. 25.

    Jones BW, Maruyama A, Ouverney CC, Nishiguchi MK. Spatial and temporal distribution of the Vibrionaceae in coastal waters of Hawaii, Australia, and France. Microb Ecol. 2007;54:314–23.

  26. 26.

    Heath-Heckman EAC, McFall-Ngai MJ. The occurrence of chitin in the hemocytes of invertebrates. Zoology. 2011;114:191–8.

  27. 27.

    Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: An open-source platform for biological-image analysis. Nat Meth. 2012;9:676–82.

  28. 28.

    Doino JA, McFall-Ngai MJ. A transient exposure to symbiosis-competent bacteria induces light organ morphogenesis in the host squid. Biol Bull. 1995;189:347–55.

  29. 29.

    Montgomery MK, McFall-Ngai MJ. Embryonic development of the light organ of the sepiolid squid Euprymna scolopes Berry. Biol Bull. 1993;184:296–308.

  30. 30.

    Sycuro LK, Ruby EG, McFall-Ngai M. Confocal microscopy of the light organ crypts in juvenile Euprymna scolopes reveals their morphological complexity and dynamic function in symbiosis. J Morphol. 2006;267:555–68.

  31. 31.

    Visick KL, Hodge-Hanson KM, Tischler AH, Bennett AK, Mastrodomenico V. Tools for rapid genetic engineering of Vibrio fischeri. Appl Environ Microbiol. 2018;84:e00850–18.

  32. 32.

    Kinosita Y, Kikuchi Y, Mikami N, Nakane D, Nishizaka T. Unforeseen swimming and gliding mode of an insect gut symbiont, Burkholderia sp. RPE64, with wrapping of the flagella around its cell body. ISME J. 2018;12:838–48.

  33. 33.

    Mandel MJ, Schaefer AL, Brennan CA, Heath-Heckman EAC, DeLoney-Marino CR, McFall-Ngai MJ, et al. Squid-derived chitin oligosaccharides are a chemotactic signal during colonization by Vibrio fischeri. Appl Environ Microbiol. 2012;78:4620–6.

  34. 34.

    Kremer N, Philipp EER, Carpentier MC, Brennan CA, Kraemer L, Altura MA, et al. Initial symbiont contact orchestrates host-organ-wide transcriptional changes that prime tissue colonization. Cell Host Microbe. 2013;14:183–94.

  35. 35.

    Schleicher TR, Nyholm SV. Characterizing the host and symbiont proteomes in the association between the bobtail squid, Euprymna scolopes, and the bacterium, Vibrio fischeri. PLoS ONE. 2011;6:e25649.

  36. 36.

    Chen F, Krasity BC, Peyer SM, Koehler S, Ruby EG, Zhang X, et al. Bactericidal permeability-increasing proteins shape host-microbe interactions. mBio. 2017;8:e00040–17.

  37. 37.

    Koch EJ, Miyashiro T, McFall-Ngai MJ, Ruby EG. Features governing symbiont persistence in the squid-vibrio association. Mol Ecol. 2014;23:1624–34.

  38. 38.

    Koehler S, Gaedeke R, Thompson C, Bongrand C, Visick K, Ruby E, et al. The model squid-vibrio symbiosis provides a window into the impact of strain- and species-level differences during the initial stages of symbiont engagement. Environ Microbiol. 2018.

  39. 39.

    Williams JH, Mazer SJ. Pollen—tiny and ephemeral but not forgotten: new ideas on their ecology and evolution. Am J Bot. 2016;103:365–74.

  40. 40.

    Devevey G, Dang T, Graves CJ, Murray S, Brisson D. First arrived takes all: Inhibitory priority effects dominate competition between co-infecting Borrelia burgdorferi strains. BMC Microbiol. 2015;15:61.

  41. 41.

    Wiles TJ, Jemielita M, Baker RP, Schlomann BH, Logan SL, Ganz J, et al. Host gut motility promotes competitive exclusion within a model intestinal microbiota. PLoS Biol. 2016;14:e1002517.

  42. 42.

    Visick KL, Foster J, Doino J, McFall-Ngai M, Ruby EG. Vibrio fischeri lux genes play an important role in colonization and development of the host light organ. J Bacteriol. 2000;182:4578–86.

  43. 43.

    Checcucci A, Azzarello E, Bazzicalupo M, Galardini M, Lagomarsino A, Mancuso S, et al. Mixed nodule infection in Sinorhizobium meliloti–Medicago sativa symbiosis suggest the presence of cheating behavior. Front Plant Sci. 2016;7:835.

  44. 44.

    Aanen DK, De Fine Licht HH, Debets AJM, Kerstes NAG, Hoekstra RF, Boomsma JJ. High symbiont relatedness stabilizes mutualistic cooperation in fungus growing termites. Science. 2009;326:1103–6.

  45. 45.

    Kuzdzal-Fick JJ, Fox SA, Strassmann JE, Queller DC. High relatedness is necessary and sufficient to maintain multicellularity in Dictyostelium. Science. 2011;334:1548–51.

  46. 46.

    Lee KH, Ruby EG. Detection of the light organ symbiont, Vibrio fischeri, in Hawaiian seawater by using lux gene probes. Appl Environ Microbiol. 1992;58:942–7.

  47. 47.

    Betts A, Gray C, Zelek M, MacLean RC, King KC. High parasite diversity accelerates host adaptation and diversification. Science. 2018;360:907–11.

  48. 48.

    Wein T, Dagan T, Fraune S, Bosch TCG, Reusch TBH, Hülter NF. Carrying capacity and colonization dynamics of Curvibacter in the hydra host habitat. Front Microbiol. 2018;9:443.

  49. 49.

    Lee SM, Donaldson GP, Mikulski Z, Boyajian S, Ley K, Mazmanian SK. Bacterial colonization factors control specificity and stability of the gut microbiota. Nature. 2013;501:426–9.

  50. 50.

    Lemire A, Goudenège D, Versigny T, Petton B, Calteau A, Labreuche Y, et al. Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters. ISME J. 2015;9:1523–31.

  51. 51.

    Le Roux F, Wegner KM, Polz MF. Oysters and vibrios as a model for disease dynamics in wild animals. Trends Microbiol. 2016;24:568–80.

Download references


We thank the Ruby and McFall-Ngai lab members, together with Émilie Koch, for their helpful discussions and support. We also thank Tina Carvalho for imaging training and use of the BEMF facility of the University of Hawaii at Manoa. Support was provided by NIH grants from NIGMS (GM099507), NIAID (AI050661) and ORIP (RR012294/OD011024).

Author information


  1. Kewalo Marine Laboratory, University of Hawaii-Manoa, Honolulu, HI, USA

    • Clotilde Bongrand
    •  & Edward G. Ruby


  1. Search for Clotilde Bongrand in:

  2. Search for Edward G. Ruby in:

Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding author

Correspondence to Edward G. Ruby.

Electronic supplementary material

About this article

Publication history





Issue Date