Box Extension 2.2

**Squid and Bioluminescent Bacteria, A Study in Cross-Phylum Coordination: The Euprymna scolopes-Vibrio fischeri Symbiosis**

Margaret McFall-Ngai

The Hawaiian bobtail squid (Euprymna scolopes) forms a lifelong symbiotic relationship with the Gram-negative bioluminescent bacterium Vibrio fischeri. The animal houses populations of the bacterium in a bi-lobed light organ in the center of its mantle (body) (Figure A). This squid is nocturnally active and uses the light produced by the bacterial symbiont as an antipredatory mechanism. Specifically, bacterial light is emitted from the ventral surface of the squid at an intensity that matches the intensity of moonlight and starlight shining down through the water (a phenomenon termed counterillumination), so that the animal does not cast a shadow that can be perceived by a predator looking up from below. Each squid acquires its own bacteria from its environment early in life: A juvenile squid recruits V. fischeri cells from the seawater in which it develops within hours of hatching from its egg. Careful studies have revealed that this recruitment—the formation of the symbiosis—entails an intimate interaction between the squid and the bacteria (Figure B). A young squid presents specialized epithelia to its seawater environment to acquire the specific bacterial symbionts, which populate deep crypts within the squid’s light organ. Once acquired, the symbionts initiate the lifelong loss of those very tissues, making further acquisition impossible! We examine this fascinating story in Box Extension 2.2.

Figure A The ecological function of the symbiosis for the squid (Photo courtesy of Margaret McFall-Ngai.)

Figure B Acquisition of bacterial symbionts Both images were obtained by use of immunocytochemistry and confocal microscopy. (Images courtesy of J. Foster [upper] and E. Ziegelhoffer [lower].)

During embryogenesis, a young squid develops an early form of its light organ that is poised to interact with V. fischeri immediately upon the squid’s hatching. Two complex ciliated epithelial fields are present on the lateral faces of this nascent light organ. These fields play a critical role in populating the light organ with bacteria.

More than 25 years of research into the steps that follow hatching—the actual formation of the symbiosis—have revealed some interesting features about this system as well as symbioses in general. The evidence to date suggests that to obtain V. fischeri to the exclusion of other possible bacterial partners, a young squid creates a biochemical environment around its light organ that encourages a specific interaction with V. fischeri. The cells of the ciliated epithelial fields, on either side of the light organ, secrete mucus with antimicrobial molecules, such as nitric oxide. As the squid pumps water through its mantle cavity for breathing, V. fischeri cells are swept over the epithelial fields. The bacteria attach to the surfaces of the fields and reside there, adapting to the chemical environment presented by the animal. The attached V. fischeri cells soon form an aggregate, a process that requires production of a capsular polysaccharide by the bacteria. About 3 h after this process begins, the aggregated bacteria, in response to some unknown signal, begin to migrate into the squid’s tissues. The juvenile light organ has six pores on its surface—three in each ciliated field. The colonizing bacteria stream to the pores and head inside. Each pore leads to an independent ciliated duct, which, after a length of about 50 μm, widens into an antechamber, which then leads into a deep crypt. At the medial side of the antechamber is a bottleneck that is only a few micrometers in diameter. Experiments in which inoculation size has been varied have revealed that, while an aggregate can contain hundreds of bacterial cells, generally only a single cell makes its way past the bottleneck into the deep crypt. Single cells enter each of the six crypts and multiply to fill the crypt spaces.

Thus far we have emphasized the effects of the squid on the bacteria during the formation of the symbiosis. The interaction is two-way, however, because the bacteria affect the maturation of the host’s light-organ tissues. Most dramatically, the bacteria induce the loss of the ciliated epithelial surfaces that were critical for their recruitment. This morphogenetic effect is induced, soon after bacteria are acquired, by derivatives of bacterial surface molecules, specifically a lipopolysaccharide and a peptidoglycan. The finding that these molecules act as morphogens in this system is surprising, because before this discovery, these molecules had only been described as mediators of tissue destruction by pathogenic bacteria. The molecules induce apoptosis and other processes that lead to loss of the epithelial surfaces within about 4 days after bacteria are acquired.

Once the symbiosis between squid and bacteria has become established, it is maintained by a complex daily rhythm. Each day at dawn, as a squid buries itself in the sand where it will rest during the day, the squid releases most of its current population of bacterial symbionts into the surrounding water. Thus the symbiont population is knocked down at the beginning of each day, then increases again as the remaining bacteria multiply. This behavior is tightly controlled and allows the squid to manage its number of symbionts. In addition, the behavior increases the population of V. fischeri in the surrounding seawater, which certainly increases the chances that a newly hatched squid will obtain a symbiotic partner.

Although the bacterial symbionts are likely to be important for the ecological survival of the squid host under field conditions, the symbionts do not seem to be critical for host physiology. This is probably because the symbionts provide only light, and do not provide their host with nutrients or vitamins (as is the case in most animal–bacterial symbioses). Evidence for this conclusion is that the squids do quite well in the laboratory without their symbionts.

The formation of the squid–vibrio symbiosis is an example of perhaps the most prevalent type of such a process between animal hosts and bacterial partners, namely the extracellular colonization of the apical surfaces of polarized epithelia. The characteristics of the squid–vibrio symbiosis have made it an ideal model for the study of the establishment and maintenance of animal–bacterial symbioses in general. Using the squid–vibrio system, researchers have asked: How is the correct symbiont acquired with fidelity each generation—that is, how do the partners recognize one another? How do the partners affect one another’s development? How does the relationship mature so that a healthy association is maintained throughout the life of the host? And finally, what are the cellular and molecular differences between this beneficial symbiosis and a symbiosis in which the bacterial partner is a pathogen?