Confocal Microscopy of the Light Organ Crypts in Juvenile Euprymna scolopes Reveals Their Morphological Complexity and Dynamic Function in Symbiosis Laura K. Sycuro, Edward G. Ruby, and Margaret McFall-Ngai* Department of Medical Microbiology and Immunology, University of Wisconsin, Madison, Wisconsin 53706 ABSTRACT In the hours to days following hatching, the Hawaiian bobtail squid, Euprymna scolopes, obtains its light-emitting symbiont, Vibrio fischeri, from the sur- rounding environment and propagates the bacteria in the epithelial crypts of a specialized light organ. Three- dimensional analyses using confocal microscopy revealed that each of the three crypts on either side of the juvenile light organ is composed of four morphological regions. Progressing from the lateral pore to the medial blind end of each crypt, the regions consist of 1) a duct, 2) an ante- chamber, 3) a bottleneck, and 4) a deep region. Only the deep region houses a persistent bacterial population, whereas the duct, antechamber, and bottleneck serve as conduits through which the bacteria enter during initial colonization and exit during diel venting, a behavior in which 90% of the symbionts are expelled each dawn. Our data suggest that, like the duct, the antechamber and bottleneck may function to promote and maintain the specificity of the symbiosis. Pronounced structural and functional differences among the deep regions of the three crypts, along with previously reported characterizations of embryogenesis, suggest a continued developmental pro- gression in the first few days after hatching. Taken to- gether, the results of this study reveal a high degree of complexity in the morphology of the crypts, as well as in the extent to which the three crypts and their constituent regions differ in function during the early stages of the symbiosis. J. Morphol. 267:555–568, 2006. © 2006 Wiley-Liss, Inc. KEY WORDS: Euprymna scolopes; Vibrio fischeri; symbi- osis; light organ,; confocal microscopy A juvenile Euprymna scolopes squid (Cephalopo- da:Sepiolidae) emerges from its egg poised to recruit cells of its luminous bacterial symbiont, Vibrio fis- cheri, from the surrounding seawater into nascent light organ tissues (for review, see Nyholm and McFall-Ngai, 2004). Although V. fischeri constitutes less than 0.1% of the microbial population in ambi- ent seawater, the hatchling squid acquire a mono- specific culture of this species of luminous bacteria in as little as 12 h (McFall-Ngai and Ruby, 1991; Ruby and Lee, 1998). Even in the absence of the symbiont, other environmental bacteria do not colo- nize the light organ (McFall-Ngai and Ruby, 1991). Once initiated, the squid will maintain the symbio- sis with a high degree of specificity throughout its life, apparently using the bacterial luminescence for counterillumination defense against predation (Moynihan, 1983). Each morning at dawn the squid vents 90% of its symbionts, allowing the remain- ing cells to grow up into a healthy, luminescent culture by evening (Boettcher et al., 1996). The biological mechanisms by which the squid establishes and sustains symbiosis are remarkably complex and highly localized (Nyholm and McFall- Ngai, 2004). The single light organ in the center of the hatchling squid’s body cavity (Fig. 1A) provides the stage for the diverse and highly specialized func- tions of symbiont collection, colonization, and prop- agation, as well as the determination of specificity and the control of bacterial luminescence. The tis- sues that mediate these complex processes develop both embryonically and postembryonically (Mont- gomery and McFall-Ngai, 1998). Embryogenesis creates a nascent light organ in the hatchling that functions to ensure colonization by the proper symbiont species (Montgomery and McFall-Ngai, 1993; Nyholm et al., 2000; Nyholm et al., 2002; Nyholm and McFall-Ngai, 2003). The bi- lateral organ is roughly heart-shaped (Fig. 1B) and its dimensions average 500 – 600 m laterally and 400 –500 m along the anterior–posterior axis Contract grant sponsor: National Science Foundation (NSF); Con- tract grant number: IOB-0517007; Contract grant sponsor: National Institutes of Health (NIH); Contract grant numbers: RR-12292, AI- 50661; Contract grant sponsor: WM Keck Foundation. Present address for L.K. Sycuro: Department of Microbiology, Uni- versity of Washington, and Department of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA 98109. *Correspondence to: Margaret McFall-Ngai, Department of Medical Microbiology and Immunology, University of Wisconsin, 1300 Univer- sity Ave., Madison, WI 53706. E-mail: mjmcfallngai@wisc.edu Published online 20 January 2006 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/jmor.10422 JOURNAL OF MORPHOLOGY 267:555–568 (2006) © 2006 WILEY-LISS, INC.