hc.) PHENOTYPIC BIOLUMINESCENCE AS AN INDICATOR OF COMPETITIVE DOMINANCE IN THE EUPRYMNA-WBKIO SYMBIOSIS nof tbr M.l. McFall-Ngai rr'ied M.K. Nishiguchi, E.G. Ruby, and rom Department of Biologicnl Sciences,Llniaersity of Southern California, rhe Los Angeles,CA 90089-0371,USA snd ent. Pacific BiomedicalResearch Center, Uniaersity of Hawai'i rom 41 Ahui St., Honolulu, HI 9681-3,USA mid -o8 Introduction ned The study of coevolutionary relationships among symbiotic associations has been an important avenue for understanding the establishment and development of two independent, but closely integrated organisms (1-3)' Most uu studies investigating the evolutionary relatedness amongst host-symbiont pairs I1re have alluded to the parallelisms that occur with the onset of a particular host laI] leads to the isolation of the symbiont his speciation event that eventually population (4). Hence, symbionts from an "ancestral" host species may have similar biochemical, physiological, or molecular characteristics that group them within the same "species". Yet, the delineation between strains or biovars within a species is much harder to define; strain characteristics that would normally define a particular bacterial population may be consistent throughout the entire group of symbionts. Although sequencing hypervariable regions of +. specific loci is a technique that can genetically differentiate between the strains (5), there are few ways that one can phenotypically distinguish similar strains e from one other. )n The sepiolid squid-bioluminescent bacterial association offers several l. advantages as a model system to study the coordinated influence of luminous bacteria on the coevolution and speciation between partners (6). Both the allowing the specific fs bacteria and the host squid can be cultured separately, d comparison between different symbiotic strains during colonization of a particular host squid light organ. Initiation, colonization, and persistence of each strain can then be monitored individually, or, in a competition experiment, where two strains compete for dominance in a particular species of host squid (7). Previous research has clearly indicated that symbiotic bacterial strains can be genetically differentiated from each other, either through restriction fragment length polymorphisms from specific DNA fragments (Lee and Ruby, unpubl. data), or through direct sequencing of loci that are variable between strains (8). Atthough molecular probes designed from these types of experiments can be used to distinguish and identify various strains in each individual light organ, this method is laborious and is limited by the number of colony blots one utilizes per infection assay (most infection/competition experiments utilize approximately 100 juveniles) (9). It has been recently discovered that variation in luminescence intensity of individual strains of symbiotic bacteria can be utilized to phenotypically distinguish strains from each other in an infected juvenile sepiolid light organ (8, Figure 1). This phenotypic r24 Bioluminescenceand Chemiluminescence Symbiosesof LuminousBacteria with Hi;

had colonized a light organ 48 hours variation allows the direct observing the percentage of brightl physiological comparison between arising from platings of light organ different strains of symbiotic bacteria agar medium (Table 1). The relatir-' in a particular host squid, and can be distinguished by viewing them in a d compared to other types of data for determining the evolutionary Results and Discussion relatednessof different host species When presented individuallr-. and the symbiotic bacteria thit have isolated from Euprymna ot Sepilla. \\' coevolved with them symbiotic population in E' scoloTtes: did not infect E. scolopesjuveniles rr-; Figure1. Light organ symbionts(left to right: EM17,ES1,I4,and ET101)from three speciesof which belongs to the sPecies F sepiolidsqrrid (Euprymna morsei, E. scolopes,and E. tasmanica,respectively) have similar growth concentrations of bacteria homogeruz characteristicson agarplates. However,when observedin the dark, it canbe seenthat the light the symbiotic strains, luminescence per output bacterialcell differs significantlybetween the strains. between visibly luminous and non-r-l indicates that a symbiont associated ' Methods and Matdrials in the luminescence it produces out: For colonization assays, luminescent symbiotic bacteria isolated were occurred luminescence in the sq.i from frozen or freshly collected light organs of several species of squid: symbiosis is established it can be ded Euprymna scolopes(Hawaii), E. morsei (]apan), E. (Australia), tasmanica Sepiola on the production of light and lumr (France), and Loliolus nlctiluca (Australia) (10, Table ffinis 1). of which host it has evolved. Prer-i, shown direct correlation with the a: Table1. Sepiolidand loliginid squidsand their respectivelight organs)..rnbionts organ (11), irrespective of the elolut: Representative luminous symbiotic bacteria. Species lightorgan Concentration in Luminescence on seawater The evidence of bacterial sPec symbiont shain E. scolopaslight organ nutrient agar medium symbiotically competent strains that sequence identity) provides nen' ins Euprymnascolopes ES114* 2.6x16 non-visible events that have occurred after the speciation within symbiotic popul; Euprymna morsei EMLT* 5x1d bright molecular clock of either host or svr that alludes to the predisposition ol Euprymna tasmanica ET101* 3xld dim the symbioses (12). Present dar', r biological mechanisms that contrc Sepiola affinis SAl* 4x16 moderately bright distinct methods for defining cha: Loliolus noctiluca LN101+ 0 bright evolutionary tracking is obscure phenotypic character in the sepiolid * = Vibriosp. a unique and informative quallt I = Photobacterium leiognathi competency of individual strains. a diversification have occurred after th All strains isolated from the sepiolid squids were identified as Vibrio species;the bacterium isolated from the loliginid squid was identified as Photobacterium Acknowledgements Ieiognathi. For experiments, cell suspensions consisttng of approximately equal We would like to thank S.v. Boletzl concentrations [about 103 colony forming units (CFU's)] were used as the for help collecting and obtaimnE s: bacterial inocula. Using isolates of symbiotically competent bacteria from NSF #OCE-9327645to MKN, O\R = Euprymna, Sepiola, and Loliolus species, infection experiments were completed 9220492to MMN and ECR. to determine whether non-native strains could infect juvniles from a closely related sepiolid host, E. scolopes. 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9ZI srustue8rg :eq8r11q1r.,ra, eualJeg snoulrun'I Jo sesorqru,{5 eJUJJSJUttunltueqJpu€ JJuJJseu.*-. - 126 Bioluminescenceand Chemiiuminescence References 1. Doyle |J. Phylogeny of the Legume Family: An approach to understanding the origins of nodulation. Annu Rev Ecol Syst 1994;25: FREE -LIVING AND ASSOCA. 325-49. GA Vydryakov 2. Haygood MG, Distel DL. Polymerase chain reaction and 16s rRNA Siberian Bro' gene sequencesfrom the luminous bacterial symbionts of two deep sea Iflstitute of Biophysics, anglerfishes. Nature 1993;363 154-56. Kra.woycsN t 3. Hinkle G, Wetterer ]K, Schultz TR, Sogin ML. Phylogeny of the Attine ant fungi based on analysis of small subunit rRNA gene sequences. Science 1994;266: 1695-97. Intro&rction The investigation of the World Oc€a! 4. Baumann P, Lai C-Y, Baumann L, Rougbakhsh D, Moran NA, Clarke bacteria to be among the most numerol MA. Mutualistic associationsof aphids and prokaryotes: Biology of the and gastroenteric tract sf psrins anim genus Buchnera. Appl Environ Microbiol 1,995;61:1,-6. marinc fauna are represcnted by slml 5. Cary SC, Warren W, Anderson E, Giovannoni S]. Identification and protocooperadvs lurninous' baetena i localization of bacterial endosymbionts in hydrothermal vent taxa with Amoug the luminous bacteria isolated t symbiont-specific polymerase chain reaction amplification and in situ frequcntly found arc the foller hybridization techniques. Mar Mol Biol Biotech 1993;2: 51-62. Phot obact eiwn leiognathi, Vibrio Jischc 6. McFall-Ngai M], Ruby EG. Symbiont recognition and subsequent luminous bacteria are afirong symbioo' morphogenesis as early events in an animal-bacterial mutualism. from light organs of marine animals. Th Science 1991;254: 1.491.-94. obtain and maintaia the propcr s1.ml 7. Lee K-H, Ruby EG. Competition between Vibrio fischerl strains during However. direct evidenceof it still remd initiation and maintenance of a light organ symbiosis. ] Bacteriol 1994;176:1985-91. Materials and mefhods 8. Nishiguchi MK, Ruby EG, McFall-Ngai MJ. Competitive dominance $enrples of sea water aod during colonization is an indicator of coevolution in an animal- mariae animals were taken bacterial symbioses. Submitted. from 64 stations during the 9. Lee K-H, Ruby EG. Detection of the light organ symbtont, Vibrio l7-rh trip of Scientific fischeri in Hawaiian seawater by using lux gene probes. Appl Environ hvestigation Ship "Vityaz" in Microbiol 1992;58: 942-47. the westcrn part of the Indian 10. Boettcher K], Ruby EG. Depressed light emission by symbioti c Vibrio Occan in winter period (Figure 1), For isolation of fischeri of the sepiolid squid Euprymna scolopes.] Bacteriol 1990;\72: 3701.-05. luminous bactEria from 1,1,. Nealson KH. Autoinduction of bacterial luciferase. Arch Microbiol samples,Photobacterium agar 7977;772: 73-9. ("Difco") was used, Cultures were grown at 20-24o C. 12. Moran NA, Baumann P, Ishikawa H. A molecular clock in t Physiological and biochemical endosymbiotic bacteria is calibrated using the insect host. Proc R Soc Lond i propertie"s of luminous I B. 1993;253: 167-71. baeteria were used for their identification.

Fisrrre l. Map of same nric;,, l,irlogical stations

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