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AMER. ZOOL., 34:554-561 (1994) Animal-Bacterial Interactions in the Early Life History of Marine Invertebrates: The Euprymna scolopesIVibrio fischeri Symbiosis1 MARGARET J. MCFALL-NGAI University of Southern California, Department of Biological Sciences, Los Angeles, California 90089-0371 SYNOPSIS. The symbiotic association between the Hawaiian sepiolid squid Euprymna scolopes and the marine luminous bacterium Vibriofischeriis being developed as a model system for the study of animal-bacterial interactions during development. Changes in light organ morphology during embryogenesis foster successful infection of the light organ with the proper bacterial partner. These embryonic events of light organ morphogenesis include the elaboration of an epithelial surface with a complex ciliated, microvillous field. The squid host hatches without the bacterial symbionts, but acquires them within hours from the free-living population of the bacteria in the water column. Upon exposure to the proper symbionts, the host organ undergoes a series of morphogenetic changes, including loss of the ciliated, microvillous field. The light organ then goes on to mature into a morphological configuration that serves to promote the maintenance of a stable association with the bacteria and that correlates with the use of the bacterial bioluminescence in behavior of the host. This symbiosis is discussed in light of other cyclically transmitted animal-bacterial associations. INTRODUCTION A growing database exists suggesting that many marine invertebrate larvae and juveniles incorporate bacteria into specific stages of their development (see e.g., Zimmer and Woollacott, 1983; Hofmann and Brand, 1987; Gil-Turnes et al, 1989; Walker and Lesser, 1989; McFall-Ngai and Ruby, 1991; Bosch, 1992). In this contribution, I explore some areas of animal-bacterial interactions that should be relevant to the future study of the role of such associations in animal development. In addition, I review aspects of recent research on the symbiosis between the sepiolid squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri, which is an emerging, experimental model system for the study of the establishment and maintenance of stable animalbacterial cooperative associations. SPATIAL AND TEMPORAL PATTERNS OF ANIMAL-MICROBIAL INTERACTIONS The intimacy of the relationship All aspects of the relationship between animals and microbes will be affected by the physical, spatial relationship that the microbe has with the cells of the host. Traditionally, biologists have categorized microbial symbionts as being either extracellular or intracellular (Smith and Douglas, 1987). Extracellular microbial partners can occur, depending on the symbiosis, over a spectrum of topographical locations in which they are anywhere from superficial to invested deeply in microvilli of specifically evolved symbiotic tissues. Intracellular microbes occur either inside vacuoles or free in the host cytoplasm. Recent research on the mobility of DNA, often referred to as horizontal gene transfer {e.g., transposition, plasmid translocation), has revealed mechanisms for the direct interaction between the DNA of two unre1 From the symposium Evolutionary Morphology of lated organisms (Doolittle et al., 1990; Marine Invertebrate Larvae and Juveniles at the Annual Meeting of the American Society of Zoologists, 27-30 Amabile-Cuevas and Chicure, 1992; Smith et al, 1992). Thus, we must now include in December 1993, at Los Angeles, California. 554 THE EUPRYMNA/VIBRIO an equation considering the spatial relationship of the cells of the partners not only how the entire host cell interacts with the entire microbial cell, but also how the genomes of the two organisms may physically communicate; i.e., this communication can be mediated by intervening cascades of biochemical messages mediated at the membranes where two cells interface (Fisher and Long, 1992), or through direct intercalation of the foreign DNA into the host or symbiont (Chilton, 1983). Further, any given symbiosis may include, in either its evolution or its ontogeny, extracellular and intracellular stages, as well as indirect and direct interaction with the foreign genome. These complex levels of spatial interaction will define how changes in gene expression in host and microbe are achieved in the symbiosis. Such processes are currently best understood in several well studied plantmicrobial symbioses. The early stages of the root-nodule symbiosis between nitrogenfixing rhizobia and leguminous plants are extracellular, while later stages are intracellular (Fisher and Long, 1992;Hirsch, 1992). In contrast, in the classic example of horizontal gene transfer, the extracellular symbiont Agrobacterium tumifaciens transfers a plasmid to the host genome of a dicotyledonous plant (Chilton, 1983; Zambryski et al., 1989). This plasmid contains genes that encode enzymes which function to convert host primary metabolites to opines, substances that cannot be metabolized by the host. The opines are translocated to the bacterium, providing the microbial symbiont with a well balanced complement of nitrogen- and carbon-rich biomolecules. Further, other genes carried on the plasmid contribute to the mechanism by which the bacterial parasite promotes the transcription and overproduction of host hormones, thereby causing hyperplasia in the plant and the eventual formation of the mature gall. In contrast to these plant-bacterial symbioses, the development of animal-bacterial symbioses has not been well studied. Thus, the possible roles that the spatial relationships and the degree of genomic exchange between the two partners play have not been explored extensively. Nevertheless, there is SYMBIOSIS 555 some evidence that genetic exchange can occur between bacterial symbionts and heterotrophic hosts. For example, gene sequence comparisons have provided strong evidence for the acquisition by the prokaryotic enteric symbiont Escherichia coli of a eukaryotic glyceraldehyde-3-phosphate dehydrogenase gene (Doolittle et al., 1990), and for the acquisition by the eukaryote Entamoeba histolytica of a prokaryotic iron superoxide dismutase gene (Smith et al., 1992). Thus, evidence for genetic exchange in development of any organism does formally exist. Transmissionfrom generation to generation The development of the above-described spatial relationships during the life history of the host are, to a large extent, determined by the mechanisms by which the symbiosis persists from generation to generation. Two different modes are recognized: transovarian and cyclic, with cyclic transmission being the more commonly occurring (Douglas, 1987; Dyer, 1989; Saffo, 1992). In transovarian transmission, the symbionts are passed from adult to progeny in, or on, the egg {e.g., insect "mycetocyte" symbioses; Douglas, 1987). Thus, the microbes are incorporated into the events of embryogenesis and are considered "self; i.e., they are present as a component of the animal during the developmental period when non-self recognition mechanisms are established. In contrast, cyclic transmission occurs when the animal acquires its microbial symbiont from the environment as a larva or juvenile; i.e., the microbe is not directly included in events of embryogenesis. In those associations where the microorganisms are rare in the environment, behaviors have evolved in the adult host which facilitate the transfer of symbionts to the juveniles via the enteric tract [e.g., ruminants (Coleman, 1975); termite (Wilson, 1971)]. Often, however, the symbiont is a common component of the environmental microbiota and is acquired by the juvenile without a direct interaction with the adult host [e.g., non-ruminant mammalian intestinal microbes (Smith and Crabb, 1961); zooxanthellae (Muscatine and Porter, 1977); luminous bacteria of squid and fish light organs (McFall-Ngai, 1991; 556 MARGARET J. MCFALL-NGAI McFall-Ngai and Ruby, 1991; Haygood, 1993); the vent tube worm Riftia pachyptila (Caryetal., 1993)]. in response to dietary changes during ontogeny (Smith and Crabbe, 1961). In the case of cyclic transmission, inter- THE EUPRYMNA SCOLOPES-VIBRIO FISCHERI SYMBIOSIS AS A SYSTEM FOR THE STUDY actions between the host and symbionts OF THE CYCLIC TRANSMISSION OF occur stepwise in two distinct temporal BACTERIAL SYMBIONTS phases: 1) preparation before the initiation of the relationship; and, subsequently, 2) While most, if not all, animals have speevents triggered by the first direct interac- cific associations with microbes, this area tions. In the first phase, the host expresses of biology has been little studied, largely due a developmental program that results in a to the absence of a technically feasible model population of the cells poised to interact system. Many animals are known to harbor with specific, appropriate microorganisms essential microorganisms in their gastroinin the environment; e.g., specific receptors testinal tracts, but usually they exist as a must be in place to ensure that the proper dynamic consortium of numerous interactrelationship is fostered. The second phase ing species (Smith and Crabbe, 1961). Thus, encompasses those responses on the part of in experimental studies, it is not possible to both the host and the symbiont that occur distinguish the influence of any one species as a result of their direct interaction. The on the community as a whole. In other cases, interactions during this phase mediate the such as the symbiosis between chemoaudevelopment of the "active" symbiotic state, totrophic bacteria and various marine in which genes or gene products are ulti- invertebrates (Childress and Fisher, 1992), mately translocated between partners. where the symbiosis is characteristically one Often these two phases correspond to the host and one microbial species, the host embryonic and postembryonic periods of organisms either are rare, or one (or both) development. However, in animals such as of the partners cannot be raised indepenmarine invertebrates with complex life his- dently under laboratory conditions. Furtories involving larval stages, the interac- ther, in many symbioses that have been tions can occur exclusively at distinct and studied thus far, pivotal in the relationship specific time periods in the life history after is the derivation of nutrients from the hatching. For example, in the alternation of microbial component. Separation of the generations of the scyphozoan Cassiopea sp., partners results in a state of compromised metamorphosis of the planula larva requires health for the host animal (Gordon and Pesti, an interaction with ambient seawater bac- 1971; Nardon and Grenier, 1989). teria (Hofmann and Brand, 1987), and the The symbiotic association between the maturation of the medusa also depends upon sepiolid squid Euprymna scolopes and the interactions with the zooxanthellae (Trench gram negative luminous bacterium Vibrio et ai, 1981). Recently it has been reported fischeri offers several advantages as a system that specific bacteria associate with the sur- for the study of cooperative interactions faces of the larvae of some shrimp (Gil- between bacteria and animals (McFall-Ngai Turnes et al, 1989) and lobster (Gil-Turnes and Ruby, 1991; Ruby and McFall-Ngai, and Fenical, 1992) species. These associa- 1992). The host is a relatively abundant tions are believed to be obligate in nature, inhabitant of the shallow sandflatsof Hawaii because the microbe produces an antifungal (Singley, 1983), and the bacteria occur not compound that serves to protect the larvae. only as symbionts with the squid, but also In addition, although the author is not aware as free-living components of the bacterioof any studies on the subject, the essential plankton (Lee and Ruby, 1992). The symgut microbiota of marine invertebrate lar- biosis that has evolved between these two vae may vary from stage to stage as diet organisms consists of one host and one bacchanges, in much the same way as the essen- terial species, both of which are culturable tial microbiota of the mammalian infant is outside of the symbiosis. The host provides considerably different than that of the adult a nutrient-rich environment for this hetero- THE EUPRYMNA/ VIBRIO SYMBIOSIS 537 FIG. 1. A significant portion of the Euprymna scolopes life cycle involves development and maturation of the light organ system. Mature females lay eggs that hatch after approximately 20 days (A). During this embryonic period, the light organ develops those tissues that will ensure inoculation of the organ with symbionts. At hatching, the light organ bears superficially a ciliated, microvillous epithelium with three pores (upper left figure, left half). The pores lead to three independent crypts deep in the organ (upper left figure, right half). Upon exposure to symbiosis-competent Vibrio fischeri, the light organ undergoes a morphogenetic process over an approximately 5 day period (B) that results in the loss of the superficial structures, coalescence of the three pores into one (upper middle figure, left half), and enlargement of the crypts to accommodate the growing culture of symbionts (upper middle figure, right half). Further maturation of the light organ over the next several weeks (C) results in a mature light organ that maintains a stable association with the symbiont. During this period, the tissues associated with the behavioral use of the bacterial bioluminescence are elaborated. Scale bars on the figures representing the juvenile light organs = 100 microns; scale bar on the figure of the mature light organ = 1 mm. trophic bacterium, and the host derives luminescence (rather than a nutritional benefit) from the culture of approximately 1 billion bacteria that live in the extracellular epithelial crypts of the light organ. While experimental data on the bioluminescent behavior of the squid are few (Moynihan, 1983), the morphology of the light organ suggests that the bacterial light is used in predatory and antipredatory behavior of the squid (McFall-Ngai and Montgomery, 1990). The mature light organ consists of a core of epithelial tissue that occurs as narrow crypts within which are housed the bacterial symbionts (Fig. 1; McFall-Ngai and Montgomery, 1990). The epithelial cells are strongly polarized, and their apical surfaces show elaborate, lobate microvilli that invest the extracellular bacterial cells. The crypts communicate with the mantle cavity through two pores, one on each lateral face of the mature light organ. Surrounding much of this epithelial-cell core is a thick reflector which is itself surrounded by the ink sac. The ink sac covers the entire dorsal surface of the light organ. The amount of light emitted by the light organ into the environment is controlled by the dynamic behavior of diverticula of the ink sac in much the same way that the ocular iris controls the amount of environmental light admitted into the eye. Finally, the entire ventral surface of the light organ is covered by a transparent, muscle-derived lens (Montgomery and McFall-Ngai, 1992; Weis et al, 1993). These components of the light organ system 558 MARGARET J. MCFALL-NGAI FIG. 2. Scanning electron micrographs of the light organ of the juvenile Euprymna scolopes. The top panel is the light organ of a newly hatched juvenile; the bottom panel is the light organ of a juvenile after a few days of symbiosis with Vibriofischeri.Scale bar = 100 microns. are thought to work in concert to effect counterillumination behavior; i.e., emission of a ventral glow that is of the same quality as downwelling light, and thus camouflages their silhouette from predators below (McFall-Ngai, 1990, 1991). The persistence of the Euprymna-Vibrio relationship between generations is mediated through a process of cyclic transmission. Female squid lay between 50 and 400 fertilized eggs in each of a series of clutches placed on hard substrates, such as the rocks and coral rubble associated with the backreef habitats where the squid are abundant. After an embryonic period of approximately 20 days, the juvenile squids hatch. Within hours of hatching, the incipient symbiotic organ of the juvenile becomes colonized by planktonic V. fischeri (Ruby and Asato, 1993). Distinct morphological and anatomical features characterize both the initiation phase and the subsequent maintenance phase of the interaction between squid and the bacteria (Fig. 1). During embryogenesis, the squid develops tissue that has been selected over evolutionary time to be competent to interact only with the V. fischeri cells present among the bacterioplankton in the ambient seawater. The "conversation" between the two partners during this initiation phase must be such that only V. fischeri colonizes the symbiotic organ. At approximately 100 cells/ml, V. fischeri occurs as only about 0.001% of the bacteria in the water column (K. H. Lee, personal communication). In the few days following successful colonization of the light organ, this structure "metamorphoses" into an organ whose primary function is the maintenance of the symbiotic state (Figs. 1 and 2; McFall-Ngai and Ruby, 1991; Montgomery and McFall-Ngai, 1994). The complex changes in morphology of both the host and the symbiont that occur during this period suggest an intimate dialogue between the partners. The exact biochemical and genetic nature of this early dialogue between host and symbiont has yet to be elucidated. However, early studies have revealed some interesting features of the light organ system that have, in turn, suggested some avenues for future investigation. In the absence of symbiosiscompetent strains of V. fischeri, the light organ remains uncolonized by any other bacterial species. This observation suggests that V.fischeridoes not simply outcompete other bacteria for a habitat that would be suitable for a wide variety of bacteria. Instead, either specific recognition events mediate infection, or the light organ creates an environment where only symbiosiscompetent V. fischeri can persist; alternatively, some combination of both of these possibilities may exist. Preliminary data suggest that recognition may be partially or wholly mediated by lectin-glycan interactions between the host and symbiont (Weis, Brennan, and McFall-Ngai, unpublished data). Further, characterizations of the light THE EUPRYMNA/VIBRIO organ indicate that the animal may create a stressful environment in which only the proper symbiont can persist (Weis et ai, 1992; Tomarev et al, 1993). SYMBIOSIS 559 divisions in the underlying mesenchyme layer. Over the last half of embryogenesis, two distinct areas of the embryonic light organ are elaborated—a set of superficial structures, and a set of pockets or crypts. The superficial structures consist of complex ciliary, microvillous fields (Fig. 2). Thesefieldsoccur on each of the lateral faces of the incipient light organ in two forms: (i) a pair of two ciliated, microvillous epithelial appendages (CEA), one anterior and one posterior, and (ii) an area that extends out from the base of these structures. At the base of the CEA, on each of the two sides, are three pores that lead to three epithelial-lined pockets or crypts; i.e., the light organ at hatching has a total of 6 pores, leading to 6 non-connected crypts. The crypts form sequentially during development, with the first beginning to form about % of the way through embryogenesis and the last of the three beginning to form only a couple of days before hatching. Embryonic development of the light organ: Selection for tissues that mediate colonization In the case of most invertebrate larvae, special structures for the handling of the establishment and maintenance of mature interactions with microbes have not evolved without precursors. Instead, already existing structures have been co-opted, that may be otherwise available throughout the life history in relatives, but are lost in the mature symbiosis. For example, Riftia pachyptila, the vent tube worm, has a transient mouth, through which the symbionts are presumably introduced during the inoculation process at each generation (Jones and Gardiner, 1989). In these cases, it may be difficult to discern those processes that are involved in initiation of the symbiosis from those that are vestigial. The Euprymna-Vibrio sym- The functional morphology of the biosis, however, is the only recognized juvenile light organ example in which structures are present In the hatchling, the light organ is located whose sole function may be to insure the in the posterior portion of the funnel. Thus, inoculation of the appropriate symbiont. much of the water that is brought into the However, it should be noted that it is quite mantle cavity during ventilation passes likely that other such structures will be found across the surface of the juvenile light organ in related sepiolid-bacterial symbioses (Montgomery and McFall-Ngai, 1993). (Pierantoni, 1918). In E. scolopes these When the animal is anesthetized, the chrostructures respond dramatically to interac- matophores become punctate rendering the tions with the symbiont (see below). Thus, animal nearly transparent. Under these conwhile the presence of such structures is unusual to symbioses, it offers the unique ditions, the CEA on the juvenile light organ opportunity to study the developing inter- can be seen to form a ring, with the tips of actions between a host tissue and its sym- the anterior and posterior appendages combionts, without the confounding influences ing in close apposition (Fig. 1; McFall-Ngai, of any non-symbiotic functions of that tis- personal observation). Dissection of the light organ from the anesthetized juvenile does sue. not appear to alter the configuration of the components of the organ. Preliminary highEmbryonic development of the Euprymna speed cinematographic studies of the juvescolopes light organ nile organ have revealed that movements The development of the E. scolopes light of the cilia entrain water particles around organ system, and the structures associated the vicinity of the light organ (Emlet and with the inoculation process, begins approx- McFall-Ngai, unpublished data). imately halfway through embryogenesis in Upon colonization with V. fischeri, the the area of the nascent hindgut-ink sac com- light organ undergoes a dramatic metamorplex (Montgomery and McFall-Ngai, 1993). phosis (Fig. 1). Over a period of four to five First apparent is a lateral thickening of this days, the ciliated, microvillous field is lost region that appears to be the result of cell (Fig. 2), at least partially through the process 560 MARGARET J. MCFALL-NGAI of cell death (Montgomery and McFall-Ngai, 1994). In addition, the three pores on each side of the light organ coalesce into one single pore. Further, the volumes of the light organ crypts enlarge, primarily by an increase in the volumes of the epithelial cells lining the crypts. These changes in the light organ do not occur if the symbiont is withheld. In the absence of experimental genetics in these squids, such as that available in Drosophila, to select for a juvenile without CEA, it is difficult to prove definitively that the CEA function in the colonization of the light organ with V. fischeri. However, their disappearance upon infection is strong correlative evidence that they are involved in the successful colonization of the juvenile light organ. CONCLUSIONS In the squid-luminous bacteria symbiosis the exact biochemical and molecular mechanisms underlying the establishment of the association remain to be elucidated. In the broader view, however, there exists a wide variety of interesting questions that pertain to the role of bacteria in invertebrate larval development. These include: (i) which of the marine invertebrate larvae associate with, or require for metamorphosis, specific microbiota?; (ii) in those that have such a requirement, what is the nature of the interaction?; and, (iii) similarly, how does the microbial complement change with each successive larval stage? ACKNOWLEDGMENTS I thank R. Emlet and E. Ruppert for the invitation to participate in this symposium. E. Ruby made invaluable suggestions on the manuscript. I am grateful to the faculty and staff of the Hawaiian Institute of Marine Biology, University of Hawaii, who have provided field support in the development of these studies. The research on the squidbacterial mutualism discussed in this paper is funded by NSF grant #IBN 9220482 and ONR grant #N00014-91-J-1357 to the author. HIMB Contribution number 938. 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