Cephalopods play a central ecological role across all oceans and depths. However, under the current climate crisis, their physiology and behaviour are impacted, and we are beginning to comprehend the effects of environmental stressors at a molecular level. Here, we study the Hawaiian bobtail squid (Euprymna scolopes), known for its specific binary symbiosis with the bioluminescent bacterium Vibrio fischeri acquired post-hatching. We aim to understand the response (i.e. developmental and molecular) of E. scolopes after embryogenetic exposure to different conditions: (i) standard conditions (control), (ii) increased CO2 (∆pH 0.4 units), (iii) warming (+3°C), or (iv) a combination of the two treatments. We observed a decrease in hatching success across all treatments relative to the control, and elevated temperature shortened the developmental time. Using transcriptomics, we identified modulation in metabolic pathways and energy production, at the expense of development under increased CO2. In addition to finding differentially expressed genes related to RNA editing, we also identified several splicing events linked to phenotypic plasticity in response to increased CO2 and temperature. The data also suggest that the initiation of the symbiosis may be negatively affected by these environmental drivers of change in the biosphere, although the animal may counter these via coping mechanisms.

The symbiotic relationship between Vibrio (Aliivibrio) fischeri and the Hawaiian bobtail squid, Euprymna scolopes, serves as a key model for understanding host-microbe interactions. Traditional culture-based methods have primarily isolated V. fischeri from the light organs of wild-caught squid, yet culture-independent analyses of this symbiotic microbiome remain limited. This study aims to enhance species-level resolution of bacterial communities associated with E. scolopes using hsp60 amplicon sequencing. We validated our hsp60 sequencing approach using pure cultures and mixed bacterial populations, demonstrating its ability to distinguish V. fischeri from other closely-related vibrios and the possibility of using this approach for strain-level diversity with further optimization. This approach was applied to whole-animal juvenile squid exposed to either seawater or a clonal V. fischeri inoculum, as well as ventate samples and light organ cores from wild-caught adults. V. fischeri accounted for the majority of the identifiable taxa for whole-animal juvenile samples and comprised 94%-99% of amplicon sequence variants (ASVs) for adult light organ core samples, confirming that V. fischeri is the dominant, if not sole, symbiont typically associated with E. scolopes light organs. In one ventate sample, V. fischeri comprised 82% of reads, indicating the potential for non-invasive community assessments using this approach. Analysis of non-V. fischeri ASVs revealed that Bradyrhizobium spp. and other members of the Rhodobacterales order are conserved across juvenile and adult samples. These findings provide insight into the presence of additional microbial associations with the squid host tissue outside of the light organ that have not been previously detected through traditional culture methods.

D-glu is a key component of peptidoglycan (PG) and is essential for growth in most bacteria. To assess constraints on PG evolution and bacterial requirements for D-glu, we sought to artificially evolve PG biosynthesis, leading to either replacement of D-glu in the PG peptide or alternative pathways to D-glu incorporation. We previously found that suppression of D-glu auxotrophy in a murI racD mutant of Vibrio fischeri grown on lysogeny broth salts (LBS) medium was rare but could be accomplished by mutation of bsrF, with restoration of wild-type PG structure. Here, we selected nine additional prototrophic suppressors of the same murI racD mutant from 1010 colony-forming units (CFU) plated on LBS supplemented with ~2.7 mM D-gln. Each suppressor had a mutation in gltS, which encodes a putative sodium:glutamate symporter. Increased copy numbers of mutant gltS alleles enabled growth on unsupplemented LBS and resulted in PG containing D-glu. Examination of media components suggests that D-gln supplementation had inadvertently added ~14 μM D-glu, and that LBS itself contains ~1.4 μM D-glu. The mutations in gltS enabled growth with similarly low D-glu concentrations, but also increased sensitivity to homocysteic acid, suggesting more promiscuous transport. Surprisingly, we discovered that expression of mutant gltS in the auxotroph leads to incorporation of lysine into PG, in addition to canonical D-glu. When seawater is supplemented with D-glu, this V. fischeri mutant still colonized Euprymna scolopes and triggered PG-induced morphogenesis. Our results shed light on glutamate transport, highlight trade-offs in GltS structure and function, and reveal an unusual PG modification.IMPORTANCED-glu is an important building block in the peptidoglycan (PG) component of the bacterial cell wall, and its endogenous production is considered essential in most bacteria, even when grown in complex media. In Vibrio fischeri, in trans expression of mutant GltS symporters allows D-glu auxotrophic strains to grow on lysogeny broth salts (LBS) medium without exogenous D-glu, although there is a fitness trade-off of increased sensitivity to homocysteic acid. Our finding that LBS contains sufficient D-glu to support robust growth highlights the undervalued importance of D-amino acid transport and the ubiquity of D-amino acids. Moreover, the discovery of D-lysine in the PG peptide is an unusual PG modification that warrants further study.

At a rapid pace, biologists are learning the many ways in which resident microbes influence, and sometimes even control, their hosts to shape both health and disease. Understanding the biochemistry behind these interactions promises to reveal completely novel and targeted ways of counteracting disease processes. However, in our protocols and publications, we continue to describe these new results using a language that originated in a completely different context. This language developed when microbial interactions with hosts were perceived to be primarily pathogenic, as threats that had to be vanquished. Biomedicine had one dominating thought: winning this war against microorganisms. Today, we know that beyond their defensive roles, host tissues, especially epithelia, are vital to ensuring association with the normal microbiota, the communities of microbes that persistently live with the host. Thus, we need to adopt a language that better encompasses the newly appreciated importance of host-microbiota associations. We also need a language that frames the onset and progression of pathogenic conditions within the context of the normal microbiota. Such a reimagined lexicon should make it clear, from the very nature of its words, that microorganisms are primarily vital to our health, and only more rarely the cause of disease. This article is part of the theme issue 'Sculpting the microbiome: how host factors determine and respond to microbial colonization'.

The symbiotic relationship between the bioluminescent bacterium Vibrio fischeri and the bobtail squid Euprymna scolopes serves as a valuable system to investigate bacterial growth and peptidoglycan (PG) synthesis within animal tissues. To better understand the growth dynamics of V. fischeri in the crypts of the light -emitting organ of its juvenile host, we showed that, after the daily dawn -triggered expulsion of most of the population, the remaining symbionts rapidly proliferate for similar to 6 h. At that point the population enters a period of extremely slow growth that continues throughout the night until the next dawn. Further, we found that PG synthesis by the symbionts decreases as they enter the slow -growing stage. Surprisingly, in contrast to the most mature crypts (i.e., Crypt 1) of juvenile animals, most of the symbiont cells in the least mature crypts (i.e., Crypt 3) were not expelled and, instead, remained in the slow -growing state throughout the day, with almost no cell division. Consistent with this observation, the expression of the gene encoding the PG -remodeling enzyme, L,D-transpeptidase (LdtA), was greatest during the slowly growing stage of Crypt 1 but, in contrast, remained continuously high in Crypt 3. Finally, deletion of the ldtA gene resulted in a symbiont that grew and survived normally in culture, but was increasingly defective in competing against its parent strain in the crypts. This result suggests that remodeling of the PG to generate additional 3-3 linkages contributes to the bacterium's fitness in the symbiosis, possibly in response to stresses encountered during the very slow -growing stage.

Animals often acquire their microbial symbionts from the environment, but the mechanisms underlying how specificity of the association is achieved are poorly understood. We demonstrate that the conserved proton pump, V-type ATPase (VHA), plays a key role in the establishment of the model light-organ symbiosis between the squid Euprymna scolopes and its bacterial partner, Vibrio fischeri. Recruitment of V. fischeri from the surrounding seawater is mediated by juvenile-specific ciliated fields on the organ's surface. These epithelia produce acidic mucus containing antimicrobials with low-pH optima, creating a chemical environment fostering specific recruitment of V. fischeri. We provide evidence that this critical acidic landscape is created by activity of VHA. VHA inhibition abolished epithelial-cell acidity, resulting in increased mucus pH and inefficient symbiont colonization. Thus, VHA provides a mechanistic link between host modulation of microenvironmental acidity, immune function, and selection of microbial symbionts, a strategy for specificity that may govern other symbioses.

Background Many animals and plants acquire their coevolved symbiotic partners shortly post-embryonic development. Thus, during embryogenesis, cellular features must be developed that will promote both symbiont colonization of the appropriate tissues, as well as persistence at those sites. While variation in the degree of maturation occurs in newborn tissues, little is unknown about how this variation influences the establishment and persistence of host-microbe associations. Results The binary symbiosis model, the squid-vibrio ( Euprymna scolopes - Vibrio fischeri ) system, offers a way to study how an environmental gram-negative bacterium establishes a beneficial, persistent, extracellular colonization of an animal host. Here, we show that bacterial symbionts occupy six different colonization sites in the light-emitting organ of the host that have both distinct morphologies and responses to antibiotic treatment. Vibrio fischeri was most resilient to antibiotic disturbance when contained within the smallest and least mature colonization sites. We show that this variability in crypt development at the time of hatching allows the immature sites to act as a symbiont reservoir that has the potential to reseed the more mature sites in the host organ when they have been cleared by antibiotic treatment. This strategy may produce an ecologically significant resiliency to the association. Conclusions The data presented here provide evidence that the evolution of the squid-vibrio association has been selected for a nascent organ with a range of host tissue maturity at the onset of symbiosis. The resulting variation in physical and chemical environments results in a spectrum of host-symbiont interactions, notably, variation in susceptibility to environmental disturbance. This “insurance policy” provides resiliency to the symbiosis during the critical period of its early development. While differences in tissue maturity at birth have been documented in other animals, such as along the infant gut tract of mammals, the impact of this variation on host-microbiome interactions has not been studied. Because a wide variety of symbiosis characters are highly conserved over animal evolution, studies of the squid-vibrio association have the promise of providing insights into basic strategies that ensure successful bacterial passage between hosts in horizontally transmitted symbioses.

Bacteriophages (phages) are diverse and abundant constituents of microbial communities worldwide, capable of modulating bacterial populations in diverse ways. Here, we describe the phage HNL01, which infects the marine bacterium Vibrio fischeri. We use culture-based approaches to demonstrate that mutations in the exopolysaccharide locus of V. fischeri render this bacterium resistant to infection by HNL01, highlighting the extracellular matrix as a key determinant of HNL01 infection. Additionally, using the natural symbiosis between V. fischeri and the squid Euprymna scolopes, we show that, during colonization, V. fischeri is protected from phages present in the ambient seawater. Taken together, these findings shed light on independent yet synergistic host- and bacterium-based strategies for resisting symbiosis-disrupting phage predation, and we present important implications for understanding these strategies in the context of diverse host-associated microbial ecosystems.

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 towards their eventual destination. However, 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, squeezing through a bottleneck constricting to ~2 μm 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 between reversals. Additional experiments in asymmetric capillary tubes confirmed that V. fischeri quickly escape from tapered 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 demonstrate that non-trivial 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. Significance Statement Symbiotic bacteria that navigate to and through specific host tissues often face tight physical confinement. This work reveals that confinement-associated changes in swimming can dramatically alter taxis, shaping bacterial localization in conjuncture with other motility-directing cues. This work helps explain how bacteria can avoid getting stuck in confined areas while transiting to privileged spaces, adding confinement as an environmental cue that symbiotic bacteria use to shape their motility behavior.

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 ∼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 ∼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 <0.1% of the typical 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 ∼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 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. IMPORTANCE Interbacterial signaling within a host-associated population can have profound effects on the behavior of the bacteria, for instance, in their production of virulence/colonization factors; in addition, such signaling can dictate the nature of the outcome for the host, in both pathogenic and beneficial associations. Using the mono specific squid-vibrio model of symbiosis, we examined how quorum-sensing regulation by the Vibrio fischeri population induces a biogeographic tissue phenotype that promotes the retention of this extracellular symbiont within the light organ of its host, Euprymna scolopes. Understanding the influence of bacterial symbionts on key sites of tissue architecture has implications for all horizontally transmitted symbioses, especially those that colonize an epithelial surface within the host.

Animals often acquire their microbiome from the environment at each generation, making the initial interaction of the partners a critical event in the establishment and development of a stable, healthy symbiosis. However, the molecular nature of these earliest interactions is generally difficult to study and poorly understood.

Reduced gravity, or microgravity, can have a pronounced impact on the physiology of animals, but the effects on their associated microbiomes are not well understood. Here, the impact of modeled microgravity on the shedding of Gram-negative lipopolysaccharides (LPS) by the symbiotic bacterium Vibrio fischeri was examined using high-aspect ratio vessels. LPS from V. fischeri is known to induce developmental apoptosis within its symbiotic tissues, which is accelerated under modeled microgravity conditions. In this study, we provide evidence that exposure to modeled microgravity increases the amount of LPS released by the bacterial symbiont in vitro. The higher rates of shedding under modeled microgravity conditions are associated with increased production of outer-membrane vesicles (OMV), which has been previously correlated to flagellar motility. Mutants of V. fischeri defective in the production and rotation of their flagella show significant decreases in LPS shedding in all treatments, but levels of LPS are higher under modeled microgravity despite loss of motility. Modeled microgravity also appears to affect the outer-membrane integrity of V. fischeri, as cells incubated under modeled microgravity conditions are more susceptible to cell-membrane-disrupting agents. These results suggest that, like their animal hosts, the physiology of symbiotic microbes can be altered under microgravity-like conditions, which may have important implications for host health during spaceflight.

Symbiosis, by its basic nature, depends on partner interactions that are mediated by cues and signals. This kind of critical reciprocal communication shapes the trajectory of host-microbe associations from their onset through their maturation and is typically mediated by both biochemical and biomechanical influences. Symbiotic partnerships often involve communities composed of dozens to hundreds of microbial species, for which resolving the precise nature of these partner interactions is highly challenging. Naturally occurring binary associations, such as those between certain legumes, nematodes, fishes, and squids, and their specific bacterial partner species offer the opportunity to examine interactions with high resolution and at the scale at which the interactions occur. The goals of this review are to provide the conceptual framework for evolutionarily conserved drivers of host-symbiont communication in animal associations and to offer a window into some mechanisms of this phenomenon as discovered through the study of the squid-vibrio model. The discussion focuses upon the early events that lead to persistence of the symbiotic partnership. The biophysical and biochemical determinants of the initial hours of dialogue between partners and how the symbiosis is shaped by the environment that is created by their reciprocal interactions are key topics that have been difficult to approach in more complex systems. Through our research on the squid-vibrio system, we provide insight into the intricate temporal and spatial complexity that underlies the molecular and cellular events mediating successful microbial colonization of the host animal.

As our understanding of the human microbiome progresses, so does the need for natural experimental animal models that promote a mechanistic understanding of beneficial microorganism-host interactions. Years of research into the exclusive symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium Vibrio fischeri have permitted a detailed understanding of those bacterial genes underlying signal exchange and rhythmic activities that result in a persistent, beneficial association, as well as glimpses into the evolution of symbiotic competence. Migrating from the ambient seawater to regions deep inside the light-emitting organ of the squid, V. fischeri experiences, recognizes and adjusts to the changing environmental conditions. Here, we review key advances over the past 15 years that are deepening our understanding of these events.

Extracellular bacterial symbionts communicate biochemically with their hosts to establish niches that foster the partnership. Using quantitative ion microprobe isotopic imaging (nanoscale secondary ion mass spectrometry [NanoSIMS]), we surveyed localization of 15 N-labelled molecules produced by the bacterium Vibrio fischeri within the cells of the symbiotic organ of its host, the Hawaiian bobtail squid, and compared that with either labelled non-specific species or amino acids. In all cases, two areas of the organ's epithelia were significantly more 15 N enriched: (a) surface ciliated cells, where environmental symbionts are recruited, and (b) the organ's crypts, where the symbiont population resides in the host. Label enrichment in all cases was strongest inside host cell nuclei, preferentially in the euchromatin regions and the nucleoli. This permissiveness demonstrated that uptake of biomolecules is a general mechanism of the epithelia, but the specific responses to V. fischeri cells recruited to the organ's surface are due to some property exclusive to this species. Similarly, in the organ's deeper crypts, the host responds to common bacterial products that only the specific symbiont can present in that location. The application of NanoSIMS allows the discovery of such distinct modes of downstream signalling dependent on location within the host and provides a unique opportunity to study the microbiogeographical patterns of symbiotic dialogue.

The recent recognition that many symbioses exhibit daily rhythms has encouraged research into the partner dialogue that drives these biological oscillations. Here we characterized the pivotal role of the versatile cytokine macrophage migration inhibitory factor (MIF) in regulating a metabolic rhythm in the model light-organ symbiosis between Euprymna scolopes and Vibrio fischeri. As the juvenile host matures, it develops complex daily rhythms characterized by profound changes in the association, from gene expression to behavior. One such rhythm is a diurnal shift in symbiont metabolism triggered by the periodic provision of a specific nutrient by the mature host: each night the symbionts catabolize chitin released from hemocytes (phagocytic immune cells) that traffic into the light-organ crypts, where the population of V. fischeri cells resides. Nocturnal migration of these macrophage-like cells, together with identification of an E. scolopes MIF (EsMIF) in the light-organ transcriptome, led us to ask whether EsMIF might be the gatekeeper controlling the periodic movement of the hemocytes. Western blots, ELISAs, and confocal immunocytochemistry showed EsMIF was at highest abundance in the light organ. Its concentration there was lowest at night, when hemocytes entered the crypts. EsMIF inhibited migration of isolated hemocytes, whereas exported bacterial products, including peptidoglycan derivatives and secreted chitin catabolites, induced migration. These results provide evidence that the nocturnal decrease in EsMIF concentration permits the hemocytes to be drawn into the crypts, delivering chitin. This nutritional function for a cytokine offers the basis for the diurnal rhythms underlying a dynamic symbiotic conversation.

Associations between animals and microbes affect not only the immediate tissues where they occur, but also the entire host. Metabolomics, the study of small biomolecules generated during metabolic processes, provides a window into how mutualistic interactions shape host biochemistry. The Hawaiian bobtail squid, Euprymna scolopes, is amenable to metabolomic studies of symbiosis because the host can be reared with or without its species-specific symbiont, Vibrio fischeri. In addition, unlike many invertebrates, the host squid has a closed circulatory system. This feature allows a direct sampling of the refined collection of metabolites circulating through the body, a focused approach that has been highly successful with mammals. Here, we show that rearing E. scolopes without its natural symbiont significantly affected one-quarter of the more than 100 hemolymph metabolites defined by gas chromatography mass spectrometry analysis. Furthermore, as in mammals, which harbor complex consortia of bacterial symbionts, the metabolite signature oscillated on symbiont-driven daily rhythms and was dependent on the sex of the host. Thus, our results provide evidence that the population of even a single symbiont species can influence host hemolymph biochemistry as a function of symbiotic state, host sex and circadian rhythm.

A major source of interest in Bdellovibrio has been its usefulness as a model of bacterial adaptation to an intracellular environment. In particular, one of the most studied set of these adaptations involves specific, developmentally expressed modifications that are brought about in the structure and function of both the bdellovibrio's and the invaded cell's envelope. The common, unifying characteristic of all the bdellovibrios is the presence of a developmental life cycle in which growth and proliferation is confined to the period of residence within another bacterial cell. The free-living or attack-phase bdellovibrio cell is a small, highly motile bacterium with a single, polar, sheathed flagellum. A biologically significant and baffling characteristic of Bdellovibrio as an intracellular organism is the broad range of Gram-negative bacteria that it can attack, differentiate within, and grow upon. The possible importance of such unusual stability and hydrophobic properties on the ability of bdellovibrio to successfully attack and enter another cell has yet to be explored.

The regulatory noncoding small RNAs (sRNAs) of bacteria are key elements influencing gene expression; however, there has been little evidence that beneficial bacteria use these molecules to communicate with their animal hosts. We report here that the bacterial sRNA SsrA plays an essential role in the light-organ symbiosis between Vibrio fischeri and the squid Euprymna scolopes . The symbionts load SsrA into outer membrane vesicles, which are transported specifically into the epithelial cells surrounding the symbiont population in the light organ. Although an SsrA-deletion mutant (Δ ssrA ) colonized the host to a normal level after 24 h, it produced only 2/10 the luminescence per bacterium, and its persistence began to decline by 48 h. The host’s response to colonization by the Δ ssrA strain was also abnormal: the epithelial cells underwent premature swelling, and host robustness was reduced. Most notably, when colonized by the Δ ssrA strain, the light organ differentially up-regulated 10 genes, including several encoding heightened immune-function or antimicrobial activities. This study reveals the potential for a bacterial symbiont’s sRNAs not only to control its own activities but also to trigger critical responses promoting homeostasis in its host. In the absence of this communication, there are dramatic fitness consequences for both partners.