Microbes colonize the apical surfaces of polarized epithelia in nearly all animal taxa. In one example, the luminous bacterium Vibrio fischeri enters, grows to a dense population within, and persists for months inside, the light-emitting organ of the squid Euprymna scolopes. Crucial to the symbiont's success after entry is the ability to trigger the constriction of a host tissue region (the "bottleneck") at the entrance to the colonization site. Bottleneck constriction begins at about the same time as bioluminescence, which is induced in V. fischeri through an autoinduction process called quorum sensing. Here, we asked the following questions: (i) Are the quorum signals that induce symbiont bioluminescence also involved in triggering the constriction? (ii) Does improper signaling of constriction affect the normal maintenance of the symbiont population? We manipulated the presence of three factors, the two V. fischeri quorum signal synthases, AinS and LuxI, the transcriptional regulator LuxR, and light emission itself, and found that the major factor triggering and maintaining bottleneck constriction is an as yet unknown effector(s) regulated by LuxIR. Treating the animal with chemical inhibitors of actin polymerization reopened the bottlenecks, recapitulating the host's response to quorum-sensing defective symbionts, as well as suggesting that actin polymerization is the primary mechanism underlying constriction. Finally, we found that these host responses to the presence of symbionts changed as a function of tissue maturation. Taken together, this work broadens our concept of how quorum sensing can regulate host development, thereby allowing bacteria to maintain long-term tissue associations.

The development of powerful model systems has been a critical strategy for understanding the mechanisms underlying the progression of an animal through its ontogeny. Here we provide two examples that allow deep and mechanistic insight into the development of specific animal systems. Species of the cnidarian genus Hydra have provided excellent models for studying host-microbe interactions and how metaorganisms function in vivo. Studies of the Hawaiian bobtail squid Euprymna scolopes and its luminous bacterial partner Vibrio fischeri have been used for over 30 years to understand the impact of a broad array of levels, from ecology to genomics, on the development and persistence of symbiosis. These examples provide an integrated perspective of how developmental processes work and evolve within the context of a microbial world, a new view that opens vast horizons for developmental biology research. The Hydra and the squid systems also lend an example of how profound insights can be discovered by taking advantage of the "experiments" that evolution had done in shaping conserved developmental processes.

Animals have evolved within the framework of the microbes and are constantly exposed to diverse microbiota. This dominance of the microbial world is forcing all fields of biology to question some of their most basic premises, with developmental biology being no exception. While animals under laboratory conditions can develop and live without microbes, they are far from normal, and would not survive under natural conditions, where their fitness would be strongly compromised. Since much of the undescribed biodiversity on Earth is microbial, any consideration of animal development in the absence of the recognition of microbes will be incomplete. Here, we show that animal development may never have been autonomous, rather it requires transient or persistent interactions with the microbial world. We propose that to formulate a comprehensive understanding of embryogenesis and post-embryonic development, we must recognize that symbiotic microbes provide important developmental signals and contribute in significant ways to phenotype production. This offers limitless opportunities for the field of developmental biology to expand.

Planktonic cells of the luminous marine bacterium Vibrio fischeri establish themselves in the light-emitting organ of each generation of newly hatched Euprymna scolopes bobtail squid. A symbiont population is maintained within the 6 separated crypts of the organ for the similar to 9-month life of the host. In the wild, the initial colonization step is typically accomplished by a handful of planktonic V. fischeri cells, leading to a species-specific, but often multi-strain, symbiont population. Within a few hours, the inoculating cells proliferate within the organ's individual crypts, after which there is evidently no supernumerary colonization. Nevertheless, every day at dawn, the majority of the symbionts is expelled, and the regrowth of the remaining similar to 5% of cells provides a daily opportunity for the population to evolve and diverge, thereby increasing its genomic diversity. To begin to understand the extent of this diversification, we characterized the light-organ population of an adult animal. First, we used 16S sequencing to determine that species in the V. fischeri clade were essentially the only ones detectable within a field-caught E. scolopes. Efforts to colonize the host with a minor species that appeared to be identified, V. litoralis, revealed that, although some cells could be imaged within the organ, they were V. fischeri population, and did not persist. Next, we determined the genome sequences of seventy-two isolates from one side of the organ. While all these isolates were associated with one of three clusters of V. fischeri strains, there was considerable genomic diversity within this natural symbiotic population. Comparative analyses revealed a significant difference in both the number and the presence/absence of genes within each cluster; in contrast, there was little accumulation of single-nucleotide polymorphisms. These data suggest that, in nature, the light organ is colonized by a small number of V. fischeri strains that can undergo significant genetic diversification, including by horizontal-gene transfer, over the course of similar to 1500 generations of growth in the organ. When the resulting population of symbionts is expelled into seawater, its genomic mix provides the genetic basis for selection during the subsequent environmental dispersal, and transmission to the next host.

Microbes are found in nearly every habitat and organism on the planet, where they are critical to host health, fitness, and metabolism. In most organisms, few microbes are inherited at birth; instead, acquiring microbiomes generally involves complicated interactions between the environment, hosts, and symbionts. Despite the criticality of microbiome acquisition, we know little about where hosts' microbes reside when not in or on hosts of interest. Because microbes span a continuum ranging from generalists associating with multiple hosts and habitats to specialists with narrower host ranges, identifying potential sources of microbial diversity that can contribute to the microbiomes of unrelated hosts is a gap in our understanding of microbiome assembly. Microbial dispersal attenuates with distance, so identifying sources and sinks requires data from microbiomes that are contemporary and near enough for potential microbial transmission. Here, we characterize microbiomes across adjacent terrestrial and aquatic hosts and habitats throughout an entire watershed, showing that the most species-poor microbiomes are partial subsets of the most species-rich and that microbiomes of plants and animals are nested within those of their environments. Furthermore, we show that the host and habitat range of a microbe within a single ecosystem predicts its global distribution, a relationship with implications for global microbial assembly processes. Thus, the tendency for microbes to occupy multiple habitats and unrelated hosts enables persistent microbiomes, even when host populations are disjunct. Our whole-watershed census demonstrates how a nested distribution of microbes, following the trophic hierarchies of hosts, can shape microbial acquisition.

Symbiotic bacteria often navigate complex environments before colonizing privileged sites in their host organism. Chemical gradients are known to facilitate directional taxis of these bacteria, guiding them toward their eventual destination. How-ever, less is known about the role of physical features in shaping the path the bacteria take and defining how they traverse a given space. The flagellated marine bacterium Vibrio fischeri, which forms a binary symbiosis with the Hawaiian bobtail squid, Euprymna scolopes, must navigate tight physical confinement during colonization, squeezing through a tissue bottleneck constricting to-2 mm in width on the way to its eventual home. Using microfluidic in vitro experiments, we discovered that V. fischeri cells alter their behavior upon entry into confined space, straightening their swimming paths and promoting escape from confinement. Using a computational model, we attributed this escape response to two factors: reduced directional fluctuation and a refractory period be-tween reversals. Additional experiments in asymmetric capillary tubes confirmed that V. fischeri quickly escape from confined ends, even when drawn into the ends by chemoattraction. This avoidance was apparent down to a limit of confinement approaching the diameter of the cell itself, resulting in a balance between chemoattraction and evasion of physical confinement. Our findings demon-strate that nontrivial distributions of swimming bacteria can emerge from simple physical gradients in the level of confinement. Tight spaces may serve as an additional, crucial cue for bacteria while they navigate complex environments to enter specific habitats.

The Hawaiian bobtail squid, Euprymna scolopes, harvests its luminous symbiont, Vibrio fischeri, from the surrounding seawater within hours of hatching. During embryogenesis, the host animal develops a nascent light organ with ciliated fields on each lateral surface. We hypothesized that these fields function to increase the efficiency of symbiont colonization of host tissues. Within minutes of hatching from the egg, the host's ciliated fields shed copious amounts of mucus in a non-specific response to bacterial surface molecules, specifically peptidoglycan (PGN), from the bacterioplankton in the surrounding seawater. Experimental manipulation of the system provided evidence that nitric oxide in the mucus drives an increase in ciliary beat frequency (CBF), and exposure to even small numbers of V. fischeri cells for short periods resulted in an additional increase in CBF. These results indicate that the light-organ ciliated fields respond specifically, sensitively, and rapidly, to the presence of nonspecific PGN as well as symbiont cells in the ambient seawater. Notably, the study provides the first evidence that this induction of an increase in CBF occurs as part of a thus far undiscovered initial phase in colonization of the squid host by its symbiont, i.e., host recognition of V. fischeri cues in the environment within minutes. Using a biophysics-based mathematical analysis, we showed that this rapid induction of increased CBF, while accelerating bacterial advection, is unlikely to be signaled by V. fischeri cells interacting directly with the organ surface. These overall changes in CBF were shown to significantly impact the efficiency of V. fischeri colonization of the host organ. Further, once V. fischeri has fully colonized the host tissues, i.e., about 12-24h after initial host-symbiont interactions, the symbionts drove an attenuation of mucus shedding from the ciliated fields, concomitant with an attenuation of the CBF. Taken together, these findings offer a window into the very first interactions of ciliated surfaces with their coevolved microbial partners.