Rethinking Prehospital Response to Mass Casualty Events: Move, Treat, and Transport.

Herein, we present a simplified approach to prehospital mass casualty event (MASCAL) management called "Move, Treat, and Transport." Prior publications demonstrate a disconnect between MASCAL response training and actions taken during real-world incidents. Overly complex algorithms, infrequent training on their use, and chaotic events all contribute to the low utilization of formal triage systems in the real world. A review of published studies on prehospital MASCAL management and a recent series of military prehospital MASCAL responses highlight the need for an intuitive MASCAL management system that accounts for expected resource limitations and tactical constraints. "Move, Treat, and Transport" is a simple and pragmatic approach that emphasizes speed and efficiency of response; considers time, tactics, and scale of the event; and focuses on interventions and evacuation to definitive care if needed.

Limitations of Triage in Military Mass Casualty Response: A Case Series.

INTRODUCTION: Mass casualty events (MASCALs) in the combat environment, which involve large numbers of casualties that overwhelm immediately available resources, are fundamentally chaotic and dynamic and inherently dangerous. Formal triage systems use diagnostic algorithms, colored markers, and four or more named categories. We hypothesized that formal triage systems are inadequately trained and practiced and too complex to successfully implement in true MASCAL events. This retrospective analysis evaluates the real-world application of triage systems in prehospital military MASCALs and other aspects of MASCAL management. METHODS: We surveyed Special Operations Forces (SOF) medics known to us who have participated in military prehospital MASCALs and analyzed them. Aggregated data describing the scope of the incidents, the use of formal triage algorithms and colored markers, the number of categories, and the interventions on scene were analyzed using descriptive statistics, and lessons learned were consolidated. RESULTS: From 1996 to 2022 we identified 29 MASCALs that were managed by military medics in the prehospital setting. There was a median of three providers (range 1-85) and 15 casualties (range 6-519) per event. Four or more formal triage categories were used in only one event. Colored markers and formal algorithms were not used. Life-saving interventions were performed in 27 of 29 (93%) missions and blood transfusions were performed in four (17%) MASCALs. The top lessons learned were: 1) security and accountability are cornerstones of MASCAL management; 2) casualty movement is a priority; 3) intuitive triage categories are the default; 4) life-saving interventions are performed as time and tactics permit. CONCLUSION: Formal triage systems requiring the use ofdiagnostic algorithms, colored tags, and four or five categories are seldom implemented in real-world military prehospital MASCAL management. The training of field triage should be simplified and pragmatic, as exemplified by these instances.

Humanitarian Assistance Lessons Learned From Providing Acute Medical Care for Afghan Nationals on a U.S. Military Base.

Operation Allies Welcome provided a unique opportunity for military medical personnel to engage in humanitarian assistance operations on military bases in the USA. With thousands of Afghan nationals evacuated from Kabul in August 2021 to various military installations across the USA, the Military Health System was tasked with health screening, emergency care, and disease prevention and surveillance in resource-limited settings. Marine Corps Base Quantico served as a "safe haven" site from August to December 2021, providing refuge to nearly 5000 travelers awaiting resettlement. During this time, active duty medical personnel provided 10,122 primary and acute patient encounters to patients aged < 1 to 90 years. Pediatrics represented 44% of the total encounters and children aged less than 5 years represented nearly 62% of the pediatric visits. In caring for this population, the authors were able to learn important lessons about humanitarian assistance capabilities, the difficulties of establishing acute care centers in a resource-limited environment, and the importance of cultural competency. Recommendations include staffing with medical providers that can see a large volume of pediatric, obstetrics, and urgent care visits, with less emphasis on trauma and surgical capabilities, which are the more traditional focus of military medicine. To this end, the authors advocate for the creation of specific humanitarian assistance supply blocks that would focus more on urgent and primary care treatments as well as an ample supply of pediatric, neonatal, and prenatal medicines. Further, early engagement with telecommunications companies when practicing in a remote location can be crucial to mission success. Finally, the medical care team should maintain continued mindfulness of the cultural norms of the population to which aid is given, particularly the gender norms and expectations of Afghan nationals. The authors hope that these lessons can prove informative and may provide increased readiness for future humanitarian assistance missions.

Effectiveness of and Adherence to Triage Algorithms During Prehospital Response to Mass Casualty Incidents.

Mass casualty incidents (MCIs) can rapidly exhaust available resources and demand the prioritization of medical response efforts and materials. Principles of triage (i.e., sorting) from the 18th century have evolved into a number of modern-day triage algorithms designed to systematically train responders managing these chaotic events. We reviewed reports and studies of MCIs to determine the use and efficacy of triage algorithms. Despite efforts to standardize MCI responses and improve the triage process, studies and recent experience demonstrate that these methods have limited accuracy and are infrequently used.

"Failure To Launch": Development of a Reproductive Organ Linked to Symbiotic Bacteria.

Developmental processes in animals are influenced by colonization and/or signaling from microbial symbionts. Here, we show that bacteria from the environment are linked to development of a symbiotic organ that houses a bacterial consortium in female Hawaiian bobtail squid, Euprymna scolopes. In addition to the well-characterized light organ association with the bioluminescent bacterium Vibrio fischeri, female E. scolopes house a simple bacterial community in a reproductive organ, the accessory nidamental gland (ANG). In order to understand the influences of bacteria on ANG development, squid were raised in the laboratory under conditions where exposure to environmental microorganisms was experimentally manipulated. Under conditions where hosts were exposed to depleted environmental bacteria, ANGs were completely absent or stunted, a result independent of the presence of the light organ symbiont V. fischeri. When squid were raised in the laboratory with substrate from the host's natural environment containing the native microbiota, normal ANG development was observed, and the bacterial communities were similar to wild-caught animals. Analysis of the bacterial communities from ANGs and substrates of wild-caught and laboratory-raised animals suggests that certain bacterial groups, namely, the Verrucomicrobia, are linked to ANG development. The ANG community composition was also experimentally manipulated. Squid raised with natural substrate supplemented with a specific ANG bacterial strain, Leisingera sp. JC1, had high proportions of this strain in the ANG, suggesting that once ANG development is initiated, specific strains can be introduced and subsequently colonize the organ. Overall, these data suggest that environmental bacteria are required for development of the ANG in E. scolopes. IMPORTANCE Microbiota have profound effects on animal and plant development. Hosts raised axenically or without symbionts often suffer negative outcomes resulting in developmental defects or reduced organ function. Using defined experimental conditions, we demonstrate that environmental bacteria are required for the formation of a female-specific symbiotic organ in the Hawaiian bobtail squid, Euprymna scolopes. Although nascent tissues from this organ that are involved with bacterial recruitment formed initially, the mature organ failed to develop and was absent or severely reduced in sexually mature animals that were not exposed to microbiota from the host's natural environment. This is the first example of complete organ development relying on exposure to symbiotic bacteria in an animal host. This study broadens the use of E. scolopes as a model organism for studying the influence of beneficial bacteria on animal development.

Evidence of Genomic Diversification in a Natural Symbiotic Population Within Its Host.

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.

Feasibility of Obtaining Intraosseous and Intravenous Access Using Night Vision Goggle Focusing Adaptors.

BACKGROUND: The optimal tactical lighting for performing medical procedures under low-light conditions is unclear. METHODS: United States Navy medical personnel (N = 23) performed intravenous (IV) and intraosseous (IO) procedures on mannequins using a tactical headlamp, night vision goggles (NVGs), and night vision goggles with focusing adaptors (NVG+A) utilizing a randomized within-subjects design. Procedure success, time to completion, and user preferences were analyzed using analysis of variance (ANOVA) and nonparametric statistics at p < .05. RESULTS: IV success rates were significantly greater for the headlamp (74%) than for NVG (35%; p < .03) and somewhat greater than for NVG+A (52%; p = .18). IO success rates were high under each lighting condition (96% to 100%). Time to completion was significantly faster using headlamp (IV, 106 ± 28 s; IO, 47 ± 11 s) than NVG (IV, 168 ± 80 s; IO, 56 ± 17 s) or NVG-A (IV, 157 ± 52 s; IO, 59 ± 27 s; each p < .01). Posttesting confidence on a 1-to-5 scale was somewhat higher for NVG+A (IV, 2.9 ± 0.2; IO, 4.2 ± 0.2) than for NVG (IV, 2.6 ± 0.2; IO, 4.0 ± 0.2). Participants cited concerns with NVG+A depth perception and with adjusting the adaptors, and that the adaptors were not integrated into the NVG. CONCLUSION: While this mannequin study was limited by laboratory conditions and by the lack of practice opportunities, we found some small advantages of focusing adaptors over NVG alone but not over headlamp for IV and IO access in low-light conditions.

Variation of anti-A and anti-B titers in group O potential blood donors: A pilot study.

BACKGROUND: Resuscitation with fresh whole blood is vital to preserving life on the battlefield. Transfusing low titer O whole blood (LTOWB), defined as anti-A and anti-B titer levels of <1:256, is safe because LTOWB alleviates the risk for hemolytic transfusion reactions. Because of possible variations in titer levels over time, a study was needed using US Navy and Marine Corps personnel to assess how these titers change across two assessments. METHODS: Retrospective data from group O marines and sailors (M = 25 years of age; range, 19-35 years) stationed in the San Diego region were acquired from the Armed Services Blood Program and the Composite Health Care System. Of 972 group O donors between January 2016 and November 2019, 55 donors with 2 samples were identified (N = 55). Analysis included contrasting rates of high (≥1:256) and low (<1:256) anti-A and anti-B titers on the initial and second blood tests, along with the time between testings. RESULTS: The average time between testing was 332 days (range, 35-1,121 days), which far exceeded the recommended 90-day interval (p < 0.00001). Only 45% met the 90-day recommendation. Titer status changed frequently, from low to high (anti-A, 18%; anti-B, 13%; LTOWB to not LTOWB, 21%) or from high to low (anti-A, 62%; anti-B, 78%; not LTOWB to LTOWB, 62%). CONCLUSIONS: Anti-A and anti-B titers change frequently enough to warrant testing immediately before deployment and even during deployment. The observed time elapsed between testing is unacceptably long. The present pilot study provides a foundation for a larger formal study to more fully characterize titer changes over repeated testing. LEVEL OF EVIDENCE: Diagnostic test, level IV.

The cytokine MIF controls daily rhythms of symbiont nutrition in an animal-bacterial association.

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.

Mode and Intensity of Physical Activity During the Postacute Phase of Sport-Related Concussion: A Systematic Review.

INTRODUCTION: Sports-related concussions (SRCs) have received attention due to their prevalence in youth. An SRC results from a strong force causing neurological impairment. Recent research has recommended rehabilitation within the first week post-SRC after 24 to 48 hours of rest. The postacute phase is defined as 48 hours to 7 days post-SRC. It is imperative to evaluate the most effective mode and intensity of physical activity to reduce symptoms and improve outcomes. METHODS: CINAHL, PubMed, SPORTDiscus, and Web of Science databases were used to search the terms "brain concussion" AND "exercise" and variations of these terms. The evidence level for each study was evaluated using the 2011 Oxford Center for Evidence-Based Medicine Guide. The methodological rigor of each study was evaluated using a scale adapted from Medlicott and Harris. RESULTS: Two thousand sixty-eight records were identified. Six studies were included in this systematic review. Three studies were classified as moderately strong. The remaining 3 studies were considered weak. Five of the studies used either a cycle ergometer or a treadmill. The sixth study used walking, cycling, and swimming, as well as sports drills. All of these modes of exercise were determined to be safe. All studies utilized low- and moderate-intensity interventions, which were found to be nondetrimental and showed improved recovery time and symptom resolution. Five of the studies also incorporated components of high-intensity exercise that was also found to be nondetrimental, and they showed a positive influence on recovery time and symptom resolution. However, all activity in each of the reviewed studies started at a low level and progressed up to a higher level only as each individual client's symptoms permitted. DISCUSSION: Overall, this review found that various modes of activity at light-, moderate-, and high-intensity levels are efficacious and can be safely used during the postacute phase of SRC. CONCLUSION: Though the volume of literature at this time is limited, therapists should consider prescribing closely monitored individualized exercise programs utilizing progressive intensities when treating patients during the postacute phase of SRC.

The impact of persistent colonization by Vibrio fischeri on the metabolome of the host squid Euprymna scolopes.

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.

Critical symbiont signals drive both local and systemic changes in diel and developmental host gene expression.

The colonization of an animal's tissues by its microbial partners creates networks of communication across the host's body. We used the natural binary light-organ symbiosis between the squid Euprymna scolopes and its luminous bacterial partner, Vibrio fischeri, to define the impact of colonization on transcriptomic networks in the host. A night-active predator, E. scolopes coordinates the bioluminescence of its symbiont with visual cues from the environment to camouflage against moon and starlight. Like mammals, this symbiosis has a complex developmental program and a strong day/night rhythm. We determined how symbiont colonization impacted gene expression in the light organ itself, as well as in two anatomically remote organs: the eye and gill. While the overall transcriptional signature of light organ and gill were more alike, the impact of symbiosis was most pronounced and similar in light organ and eye, both in juvenile and adult animals. Furthermore, the presence of a symbiosis drove daily rhythms of transcription within all three organs. Finally, a single mutation in V. fischeri-specifically, deletion of the lux operon, which abrogates symbiont luminescence-reduced the symbiosis-dependent transcriptome of the light organ by two-thirds. In addition, while the gills responded similarly to light-organ colonization by either the wild-type or mutant, luminescence was required for all of the colonization-associated transcriptional responses in the juvenile eye. This study defines not only the impact of symbiont colonization on the coordination of animal transcriptomes, but also provides insight into how such changes might impact the behavior and ecology of the host.

Symbiotic organs shaped by distinct modes of genome evolution in cephalopods.

Microbes have been critical drivers of evolutionary innovation in animals. To understand the processes that influence the origin of specialized symbiotic organs, we report the sequencing and analysis of the genome of Euprymna scolopes, a model cephalopod with richly characterized host-microbe interactions. We identified large-scale genomic reorganization shared between E. scolopes and Octopus bimaculoides and posit that this reorganization has contributed to the evolution of cephalopod complexity. To reveal genomic signatures of host-symbiont interactions, we focused on two specialized organs of E. scolopes: the light organ, which harbors a monoculture of Vibrio fischeri, and the accessory nidamental gland (ANG), a reproductive organ containing a bacterial consortium. Our findings suggest that the two symbiotic organs within E. scolopes originated by different evolutionary mechanisms. Transcripts expressed in these microbe-associated tissues displayed their own unique signatures in both coding sequences and the surrounding regulatory regions. Compared with other tissues, the light organ showed an abundance of genes associated with immunity and mediating light, whereas the ANG was enriched in orphan genes known only from E. scolopes Together, these analyses provide evidence for different patterns of genomic evolution of symbiotic organs within a single host.

Persistent Interactions with Bacterial Symbionts Direct Mature-Host Cell Morphology and Gene Expression in the Squid-Vibrio Symbiosis.

In horizontally transmitted symbioses, structural, biochemical, and molecular features both facilitate host colonization by specific symbionts and mediate their persistent carriage. In the association between the squid Euprymna scolopes and its luminous bacterial partner Vibrio fischeri, the symbionts interact with two epithelial fields; they interact (i) transiently with the superficial ciliated field that potentiates colonization and regresses within days of colonization and (ii) persistently with the cells that line the internal crypts, whose ultrastructure changes in response to the symbionts. Development of the association creates conditions that promote the symbiotic partner over the lifetime of the host. To determine whether light organ maturation requires continuous interactions with V. fischeri or only the signaling that occurs during its initiation, we compared 4-week-old squid that were uncolonized with those colonized either persistently by wild-type V. fischeri or transiently by a V. fischeri mutant that triggers early events in morphogenesis but does not persist. Microscopic analysis of the light organs showed that, while morphogenesis of the superficial ciliated field is greatly accelerated by V. fischeri colonization, its eventual outcome is largely independent of colonization state. In contrast, the symbiont-induced changes in crypt cell shape require persistent host-symbiont interaction, reflected in the similarity between uncolonized and transiently colonized animals. Transcriptomic analyses reflected the microscopy results; host gene expression at 4 weeks was due primarily to the persistent interactions of host and symbiont cells. Further, the transcriptomic signature of specific pathways reflected the daily rhythm of symbiont release and regrowth and required the presence of the symbionts. IMPORTANCE A long-term relationship between symbiotic partners is often characterized by development and maturation of host structures that harbor the symbiont cells over the host's lifetime. To understand the mechanisms involved in symbiosis maintenance more fully, we studied the mature bobtail squid, whose light-emitting organ, under experimental conditions, can be transiently or persistently colonized by Vibrio fischeri or remain uncolonized. Superficial anatomical changes in the organ were largely independent of symbiosis. However, both the microanatomy of cells with which symbionts interact and the patterns of gene expression in the mature animal were due principally to the persistent interactions of host and symbiont cells rather than to a response to early colonization events. Further, the characteristic pronounced daily rhythm on the host transcriptome required persistent V. fischeri colonization of the organ. This experimental study provides a window into how persistent symbiotic colonization influences the form and function of host animal tissues.

Model systems for the study of how symbiotic associations between animals and extracellular bacterial partners are established and maintained.

This contribution describes the current state of experimental model development and use as a strategy for gaining insight into the form and function of certain types of host-microbe associations. Development of quality models for the study of symbiotic systems will be critical not only to facilitate an understanding of mechanisms underlying symbiosis, but also for providing insights into how drug development can promote healthy animal-microbe interactions as well as the treatment of pathogenic infections. Because of the growing awareness over the last decade of the importance of symbiosis in biology, a number of model systems has emerged to examine how these partnerships are maintained within and across generations of the host. The focus here will be upon host-bacterial symbiotic systems that, as in humans, (i) are acquired from the environment each generation, or horizontally transmitted, and (ii) are defined by interactions at the interface of their cellular boundaries, i.e., extracellular symbiotic associations. As with the use of models in other fields of biology where complexity is daunting (e.g., developmental biology or brain circuitry), each model has its strengths and weaknesses, i.e., no one model system will provide easy access to all the questions defining what is conserved in cell-cell interactions in symbiosis and what creates diversity within such partnerships. Rather, as discussed here, the more models explored, the richer our understanding of these associations is likely to be.

Motile cilia create fluid-mechanical microhabitats for the active recruitment of the host microbiome.

We show that mucociliary membranes of animal epithelia can create fluid-mechanical microenvironments for the active recruitment of the specific microbiome of the host. In terrestrial vertebrates, these tissues are typically colonized by complex consortia and are inaccessible to observation. Such tissues can be directly examined in aquatic animals, providing valuable opportunities for the analysis of mucociliary activity in relation to bacteria recruitment. Using the squid-vibrio model system, we provide a characterization of the initial engagement of microbial symbionts along ciliated tissues. Specifically, we developed an empirical and theoretical framework to conduct a census of ciliated cell types, create structural maps, and resolve the spatiotemporal flow dynamics. Our multiscale analyses revealed two distinct, highly organized populations of cilia on the host tissues. An array of long cilia ([Formula: see text]25 [Formula: see text]m) with metachronal beat creates a flow that focuses bacteria-sized particles, at the exclusion of larger particles, into sheltered zones; there, a field of randomly beating short cilia ([Formula: see text]10 [Formula: see text]m) mixes the local fluid environment, which contains host biochemical signals known to prime symbionts for colonization. This cilia-mediated process represents a previously unrecognized mechanism for symbiont recruitment. Each mucociliary surface that recruits a microbiome such as the case described here is likely to have system-specific features. However, all mucociliary surfaces are subject to the same physical and biological constraints that are imposed by the fluid environment and the evolutionary conserved structure of cilia. As such, our study promises to provide insight into universal mechanisms that drive the recruitment of symbiotic partners.

Rotation of Vibrio fischeri Flagella Produces Outer Membrane Vesicles That Induce Host Development.

UNLABELLED: Using the squid-vibrio association, we aimed to characterize the mechanism through which Vibrio fischeri cells signal morphogenesis of the symbiotic light-emitting organ. The symbiont releases two cell envelope molecules, peptidoglycan (PG) and lipopolysaccharide (LPS) that, within 12 h of light organ colonization, act in synergy to trigger normal tissue development. Recent work has shown that outer membrane vesicles (OMVs) produced by V. fischeri are sufficient to induce PG-dependent morphogenesis; however, the mechanism(s) of OMV release by these bacteria has not been described. Like several genera of both beneficial and pathogenic bacteria, V. fischeri cells elaborate polar flagella that are enclosed by an extension of the outer membrane, whose function remains unclear. Here, we present evidence that along with the well-recognized phenomenon of blebbing from the cell's surface, rotation of this sheathed flagellum also results in the release of OMVs. In addition, we demonstrate that most of the development-inducing LPS is associated with these OMVs and that the presence of the outer membrane protein OmpU but not the LPS O antigen on these OMVs is important in triggering normal host development. These results also present insights into a possible new mechanism of LPS release by pathogens with sheathed flagella. IMPORTANCE: Determining the function(s) of sheathed flagella in bacteria has been challenging, because no known mutation results only in the loss of this outer membrane-derived casing. Nevertheless, the presence of a sheathed flagellum in such host-associated genera as Vibrio, Helicobacter, and Brucella has led to several proposed functions, including physical protection of the flagella and masking of their immunogenic flagellins. Using the squid-vibrio light organ symbiosis, we demonstrate another role, that of V. fischeri cells require rotating flagella to induce apoptotic cell death within surface epithelium, which is a normal step in the organ's development. Further, we present evidence that this rotation releases apoptosis-triggering lipopolysaccharide in the form of outer membrane vesicles. Such release may also occur by pathogens but with different outcomes for the host.

A genomic comparison of 13 symbiotic Vibrio fischeri isolates from the perspective of their host source and colonization behavior.

Newly hatched Euprymna scolopes squid obtain their specific light-organ symbionts from an array of Vibrio (Allivibrio) fischeri strains present in their environment. Two genetically distinct populations of this squid species have been identified, one in Kaneohe Bay (KB), and another in Maunaloa Bay (MB), Oahu. We asked whether symbionts isolated from squid in each of these populations outcompete isolates from the other population in mixed-infection experiments. No relationship was found between a strain's host source (KB or MB) and its ability to competitively colonize KB or MB juveniles in a mixed inoculum. Instead, two colonization behaviors were identified among the 11 KB and MB strains tested: a 'dominant' outcome, in which one strain outcompetes the other for colonization, and a 'sharing' outcome, in which two strains co-colonize the squid. A genome-level comparison of these and other V. fischeri strains suggested that the core genomic structure of this species is both syntenous and highly conserved over time and geographical distance. We also identified ~250 Kb of sequence, encoding 194 dispersed orfs, that was specific to those strains that expressed the dominant colonization behavior. Taken together, the results indicate a link between the genome content of V. fischeri strains and their colonization behavior when initiating a light-organ symbiosis.

The chemistry of negotiation: rhythmic, glycan-driven acidification in a symbiotic conversation.

Glycans have emerged as critical determinants of immune maturation, microbial nutrition, and host health in diverse symbioses. In this study, we asked how cyclic delivery of a single host-derived glycan contributes to the dynamic stability of the mutualism between the squid Euprymna scolopes and its specific, bioluminescent symbiont, Vibrio fischeri. V. fischeri colonizes the crypts of a host organ that is used for behavioral light production. E. scolopes synthesizes the polymeric glycan chitin in macrophage-like immune cells called hemocytes. We show here that, just before dusk, hemocytes migrate from the vasculature into the symbiotic crypts, where they lyse and release particulate chitin, a behavior that is established only in the mature symbiosis. Diel transcriptional rhythms in both partners further indicate that the chitin is provided and metabolized only at night. A V. fischeri mutant defective in chitin catabolism was able to maintain a normal symbiont population level, but only until the symbiotic organ reached maturity (∼ 4 wk after colonization); this result provided a direct link between chitin utilization and symbiont persistence. Finally, catabolism of chitin by the symbionts was also specifically required for a periodic acidification of the adult crypts each night. This acidification, which increases the level of oxygen available to the symbionts, enhances their capacity to produce bioluminescence at night. We propose that other animal hosts may similarly regulate the activities of epithelium-associated microbial communities through the strategic provision of specific nutrients, whose catabolism modulates conditions like pH or anoxia in their symbionts' habitat.

Features governing symbiont persistence in the squid-vibrio association.

Experimental studies of the interaction between host and symbiont in a maturing symbiotic organ have presented a challenge for most animal-bacterial associations. Advances in the rearing of the host squid Euprymna scolopes have enabled us to explore the relationship between a defect in symbiont light production and late-stage development (e.g. symbiont persistence and tissue morphogenesis) by experimental colonization with specific strains of the symbiont Vibrio fischeri. During the first 4 weeks postinoculation of juvenile squid, the population of wild-type V. fischeri increased 100-fold; in contrast, a strain defective in light production (Δlux) colonized normally the first day, but exhibited an exponential decline to undetectable levels over subsequent weeks. Co-colonization of organs by both strains affected neither the trajectory of colonization by wild type nor the decline of Δlux levels. Uninfected animals retained the ability to be colonized for at least 2 weeks posthatch. However, once colonized by the wild-type strain for 5 days, a subsequent experimentally induced loss of the symbionts could not be followed by a successful recolonization, indicating the host's entry into a refractory state. However, animals colonized by the Δlux before the loss of their symbionts were receptive to recolonization. Analyses of animals colonized with either a wild-type or a Δlux strain revealed slight, if any, differences in the developmental regression of the ciliated light-organ tissues that facilitate the colonization process. Thus, some other feature(s) of the Δlux strain's defect also may be responsible for its inability to persist, and its failure to induce a refractory state in the host.