Population Dynamics of Vibrio fischeri during Infection of Euprymna scolopes

The luminous bacterium Vibrio fischeri colonizes a specialized light-emitting organ within its squid host, Euprymna scolopes. Newly hatched juvenile squid must acquire their symbiont from ambient seawater, where the bacteria are present at low concentrations. To understand the population dynamics of V. fischeri during colonization more fully, we used mini-Tn7 transposons to mark bacteria with antibiotic resistance so that the growth of their progeny could be monitored. When grown in culture, there was no detectable metabolic burden on V. fischeri cells carrying the transposon, which inserts in single copy in a specific intergenic region of the V. fischeri genome. Strains marked with mini-Tn7 also appeared to be equivalent to the wild type in their ability to infect and multiply within the host during coinoculation experiments. Studies of the early stages of colonization suggested that only a few bacteria became associated with symbiotic tissue when animals were exposed for a discrete period (3 h) to an inoculum of V. fischeri cells equivalent to natural population levels; nevertheless, all these hosts became infected. When three differentially marked strains of V. fischeri were coincubated with juvenile squid, the number of strains recovered from an individual symbiotic organ was directly dependent on the size of the inoculum. Further, these results indicated that, when exposed to low numbers of V. fischeri, the host may become colonized by only one or a few bacterial cells, suggesting that symbiotic infection is highly efficient.     

Salinity and Temperature Effects on Physiological Responses of <Emphasis Type="Italic">Vibrio fischeri</Emphasis> from Diverse Ecological Niches

Vibrio fischeri is a bioluminescent bacterial symbiont of sepiolid squids (Cephalopoda: Sepiolidae) and monocentrid fishes (Actinopterygii: Monocentridae). V. fischeri exhibit competitive dominance within the allopatrically distributed squid genus Euprymna, which have led to the evolution of V. fischeri host specialists. In contrast, the host genus Sepiola contains sympatric species that is thought to have given rise to V. fischeri that have evolved as host generalists. Given that these ecological lifestyles may have a direct effect upon the growth spectrum and survival limits in contrasting environments, optimal growth ranges were obtained for numerous V. fischeri isolates from both free-living and host environments. Upper and lower limits of growth were observed in sodium chloride concentrations ranging from 0.0% to 9.0%. Sepiola symbiotic isolates possessed the least variation in growth throughout the entire salinity gradient, whereas isolates from Euprymna were the least uniform at <2.0% NaCl. V. fischeri fish symbionts (CG101 and MJ101) and all free-living strains were the most dissimilar at >5.0% NaCl. Growth kinetics of symbiotic V. fischeri strains were also measured under a range of salinity and temperature combinations. Symbiotic V. fischeri ES114 and ET101 exhibited a synergistic effect for salinity and temperature, where significant differences in growth rates due to salinity existed only at low temperatures. Thus, abiotic factors such as temperature and salinity have differential effects between free-living and symbiotic strains of V. fischeri, which may alter colonization efficiency prior to infection.     

TfoX-Based Genetic Mapping Identifies Vibrio fischeri Strain-Level Differences and Reveals a Common Lineage of Laboratory Strains

Bacterial strain variation exists in natural populations of bacteria and can be generated experimentally through directed or random mutation. The advent of rapid and cost-efficient whole-genome sequencing has facilitated strain-level genotyping. Even with modern tools, however, it often remains a challenge to map specific traits to individual genetic loci, especially for traits that cannot be selected under culture conditions (e.g., colonization level or pathogenicity). Using a combination of classical and modern approaches, we analyzed strain-level variation in Vibrio fischeri and identified the basis by which some strains lack the ability to utilize glycerol as a carbon source. We proceeded to reconstruct the lineage of the commonly used V. fischeri laboratory strains. Compared to the wild-type ES114 strain, we identify in ES114-L a 9.9-kb deletion with endpoints in tadB2 and glpF; restoration of the missing portion of glpF restores the wild-type phenotype. The widely used strains ESR1, JRM100, and JRM200 contain the same deletion, and ES114-L is likely a previously unrecognized intermediate strain in the construction of many ES114 derivatives. ES114-L does not exhibit a defect in competitive squid colonization but ESR1 does, demonstrating that glycerol utilization is not required for early squid colonization. Our genetic mapping approach capitalizes on the recently discovered chitin-based transformation pathway, which is conserved in the Vibrionaceae; therefore, the specific approach used is likely to be useful for mapping genetic traits in other Vibrio species.     

Alterations in the Proteome of the Euprymna scolopes Light Organ in Response to Symbiotic Vibrio fischeri

During the onset of the cooperative association between the Hawaiian sepiolid squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri, the anatomy and morphology of the host's symbiotic organ undergo dramatic changes that require interaction with the bacteria. This morphogenetic process involves an array of tissues, including those in direct contact with, as well as those remote from, the symbiotic bacteria. The bacteria induce the developmental program soon after colonization of the organ, although complete morphogenesis requires 96 h. In this study, to determine critical time points, we examined the biochemistry underlying bacterium-induced host development using two-dimensional polyacrylamide gel electrophoresis. Specifically, V. fischeri-induced changes in the soluble proteome of the symbiotic organ during the first 96 h of symbiosis were identified by comparing the protein profiles of symbiont-colonized and uncolonized organs. Both symbiosis-related changes and age-related changes were analyzed to determine what proportion of the differences in the proteomes was the result of specific responses to interaction with bacteria. Although no differences were detected over the first 24 h, numerous symbiosis-related changes became apparent at 48 and 96 h and were more abundant than age-related changes. In addition, many age-related protein changes occurred 48 h sooner in symbiotic animals, suggesting that the interaction of squid tissue with V. fischeri cells accelerates certain developmental processes of the symbiotic organ. These data suggest that V. fischeri-induced modifications in host tissues that occur in the first 24 h of the symbiosis are independent of marked alterations in the patterns of abundant proteins but that the full 4-day morphogenetic program requires significant alteration of the host soluble proteome.     

Ecological Diversification of Vibrio fischeri Serially Passaged for 500 Generations in Novel Squid Host Euprymna tasmanica

Vibrio fischeri isolated from Euprymna scolopes (Cephalopoda: Sepiolidae) was used to create 24 lines that were serially passaged through the non-native host Euprymna tasmanica for 500 generations. These derived lines were characterized for biofilm formation, swarming motility, carbon source utilization, and in vitro bioluminescence. Phenotypic assays were compared between “ES” (E. scolopes) and “ET” (E. tasmanica) V. fischeri wild isolates to determine if convergent evolution was apparent between E. tasmanica evolved lines and ET V. fischeri. Ecological diversification was observed in utilization of most carbon sources examined. Convergent evolution was evident in motility, biofilm formation, and select carbon sources displaying hyperpolymorphic usage in V. fischeri. Convergence in bioluminescence (a 2.5-fold increase in brightness) was collectively evident in the derived lines relative to the ancestor. However, dramatic changes in other properties—time points and cell densities of first light emission and maximal light output and emergence of a lag phase in growth curves of derived lines—suggest that increased light intensity per se was not the only important factor. Convergent evolution implies that gnotobiotic squid light organs subject colonizing V. fischeri to similar selection pressures. Adaptation to novel hosts appears to involve flexible microbial metabolism, establishment of biofilm and swarmer V. fischeri ecotypes, and complex changes in bioluminescence. Our data demonstrate that numerous alternate fitness optima or peaks are available to V. fischeri in host adaptive landscapes, where novel host squids serve as habitat islands. Thus, V. fischeri founder flushes occur during the initiation of light organ colonization that ultimately trigger founder effect diversification.     

Chemoattraction of Vibrio fischeri to Serine, Nucleosides, and N-Acetylneuraminic Acid, a Component of Squid Light-Organ Mucus

Newlyhatched juveniles of the Hawaiian squid Euprymna scolopes rapidly become colonized by the bioluminescent marine bacterium Vibrio fischeri. Motility is required to establish the symbiotic colonization, but the role of chemotaxis is unknown. In this study we analyzed chemotaxis of V. fischeri to a number of potential attractants. The bacterium migrated toward serine and most sugars tested. V. fischeri also exhibited the unusual ability to migrate to nucleosides and nucleotides as well as to N-acetylneuraminic acid, a component of squid mucus.     

Phylogeny and fitness of Vibrio fischeri from the light organs of Euprymna scolopes in two Oahu,Hawaii populations

The evolutionary relationship among Vibrio fischeri isolates obtained from the light organs of Euprymna scolopes collected around Oahu, Hawaii, were examined in this study. Phylogenetic reconstructions based on a concatenation of fragments of four housekeeping loci (recA, mdh, katA, pyrC) identified one monophyletic group (‘Group-A'') of V. fischeri from Oahu. Group-A V. fischeri strains could also be identified by a single DNA fingerprint type. V. fischeri strains with this fingerprint type had been observed to be at a significantly higher abundance than other strains in the light organs of adult squid collected from Maunalua Bay, Oahu, in 2005. We hypothesized that these previous observations might be related to a growth/survival advantage of the Group-A strains in the Maunalua Bay environments. Competition experiments between Group-A strains and non-Group-A strains demonstrated an advantage of the former in colonizing juvenile Maunalua Bay hosts. Growth and survival assays in Maunalua Bay seawater microcosms revealed a reduced fitness of Group-A strains relative to non-Group-A strains. From these results, we hypothesize that there may exist trade-offs between growth in the light organ and in seawater environments for local V. fischeri strains from Oahu. Alternatively, Group-A V. fischeri may represent an example of rapid, evolutionarily significant, specialization of a horizontally transmitted symbiont to a local host population.     

Host-symbiont recognition in the environmentally transmitted sepiolid squid-Vibrio mutualism

Associations between environmentally transmitted symbionts and their hosts provide a unique opportunity to study the evolution of specificity and subsequent radiation of tightly coupled host-symbiont assemblages [3, 8, 24]. The evidence provided here from the environmentally transmitted bacterial symbiont Vibrio fischeri and its sepiolid squid host (Sepiolidae: Euprymna) demonstrates how host-symbiont specificity can still evolve without vertical transmission of the symbiont [1]. Infection by intraspecific V. fischeri symbionts exhibited preferential colonization over interspecific V. fischeri symbionts, indicating a high degree of specificity for the native symbiotic strains. Inoculation with symbiotic bacteria from other taxa (monocentrid fish and loliginid squids) produced little or no colonization in two species of Euprymna, despite their presence in the same or similar habitats as these squids. These findings of host specificity between native Vibrios and sepiolid squids provides evidence that the presence of multiple strains of symbionts does not dictate the composition of bacterial symbionts in the host.     

Effect of the Squid Host on the Abundance and Distribution of Symbiotic Vibrio fischeri in Nature

Euprymna scolopes, a Hawaiian species of bioluminescent squid, harbors Vibrio fischeri as its specific light organ symbiont. The population of symbionts grew inside the adult light organ with an average doubling time of about 5 h, which produced an excess of cells that were expelled into the surrounding seawater on a diurnal basis at the beginning of each period of daylight. These symbionts, when expelled into the ambient seawater, maintain or slightly increase their numbers for at least 24 h. Hence, locations inhabited by their hosts periodically receive a daily input of symbiotic V. fischeri cells and, as a result, become significantly enriched with these bacteria. As estimated by hybridization with a species-specific luxA gene probe, the typical number of V. fischeri CFU, both in the water column and in the sediments of E. scolopes habitats, was as much as 24 to 30 times that in similar locations where squids were not observed. In addition, the number of symbiotic V. fischeri CFU in seawater samples that were collected along a transect through Kaneohe Bay, Hawaii, decreased as a function of the distance from a location inhabited by E. scolopes. These findings constitute evidence for the first recognized instance of the abundance and distribution of a marine bacterium being driven primarily by its symbiotic association with an animal host.     

Population Structure of Vibrio fischeri within the Light Organs of Euprymna scolopes Squid from Two Oahu (Hawaii) Populations

We resolved the intraspecific diversity of Vibrio fischeri, the bioluminescent symbiont of the Hawaiian sepiolid squid Euprymna scolopes, at two previously unexplored morphological and geographical scales. These scales ranged from submillimeter regions within the host light organ to the several kilometers encompassing two host populations around Oahu. To facilitate this effort, we employed both novel and standard genetic and phenotypic assays of light-organ symbiont populations. A V. fischeri-specific fingerprinting method and five phenotypic assays were used to gauge the genetic richness of V. fischeri populations; these methods confirmed that the symbiont population present in each adult host's light organ is polyclonal. Upon statistical analysis of these genetic and phenotypic population data, we concluded that the characteristics of symbiotic populations were more similar within individual host populations than between the two distinct Oahu populations of E. scolopes, providing evidence that local geographic symbiont population structure exists. Finally, to better understand the genesis of symbiont diversity within host light organs, the process of symbiosis initiation in newly hatched juvenile squid was examined both experimentally and by mathematical modeling. We concluded that, after the juvenile hatches, only one or two cells of V. fischeri enter each of six internal epithelium-lined crypts present in the developing light organ. We hypothesize that the expansion of different, crypt-segregated, clonal populations creates the polyclonal adult light-organ population structure observed in this study. The stability of the luminous-bacterium-sepiolid squid mutualism in the presence of a polyclonal symbiont population structure is discussed in the context of contemporary evolutionary theory.     

Breaking the language barrier: experimental evolution of non-native <Emphasis Type="Italic">Vibrio fischeri</Emphasis> in squid tailors luminescence to the host

Although most Vibrio fischeri isolates are capable of symbiosis, the coevolution of certain strains with the Hawaiian bobtail squid, Euprymna scolopes, has led to specific adaptation to this partnership. For instance, strains from different hosts or from a planktonic environment are ineffective squid colonists. Even though bioluminescence is a symbiotic requirement, curiously, symbionts of E. scolopes are dim in culture relative to fish symbionts and free-living isolates. It is unclear whether this dim phenotype is related to the symbiosis or simply coincidental. To further explore the basis of symbiont specificity, we developed an experimental evolution model that utilizes the daily light organ venting behavior of the squid and horizontal acquisition of symbionts for serial passage of cultures. We passaged six populations each derived from the squid-naïve strains of V. fischeri MJ11 (a fish symbiont) and WH1 (a free-living isolate) through a series of juvenile squid light organs. After 15 serially colonized squid for each population, or an estimated 290–360 bacterial generations, we isolated representatives of the light organ populations and characterized their bioluminescence. Multiple evolved lines of both strains produced significantly less bioluminescence both in vitro and in vivo. This reduction in bioluminescence did not correlate with reduced quorum sensing for most isolates tested. The remarkable phenotypic convergence with squid symbionts further emphasizes the importance of bioluminescence in this symbiosis, and suggests that reduced light production is a specific adaptation to the squid.     

Evolutionary perspectives in a mutualism of sepiolid squid and bioluminescent bacteria: combined usage of microbial experimental evolution and temporal population genetics

The symbiosis between marine bioluminescent Vibrio bacteria and the sepiolid squid Euprymna is a model for studying animal-bacterial Interactions. Vibrio symbionts native to particular Euprymna species are competitively dominant, capable of outcompeting foreign Vibrio strains from other Euprymna host species. Despite competitive dominance, secondary colonization events by invading nonnative Vibrio fischeri have occurred. Competitive dominance can be offset through superior nonnative numbers and advantage of early start host colonization by nonnatives, granting nonnative vibrios an opportunity to establish beachheads in foreign Euprymna hosts. Here, we show that nonnative V. fischeri are capable of rapid adaptation to novel sepiolid squid hosts by serially passaging V. fischeri JRM200 (native to Hawaiian Euprymna scolopes) lines through the novel Australian squid host E. tasmanica for 500 generations. These experiments were complemented by a temporal population genetics survey of V. fischeri, collected from E. tasmanica over a decade, which provided a perspective from the natural history of V. fischeri evolution over 15,000-20,000 generations in E. tasmanica. No symbiont anagenic evolution within squids was observed, as competitive dominance does not purge V. fischeri genetic diversity through time. Instead, abiotic factors affecting abundance of V. fischeri variants in the planktonic phase sustain temporal symbiont diversity, a property itself of ecological constraints imposed by V. fischeri host adaptation.     

Contribution of pilA to Competitive Colonization of the Squid Euprymna scolopes by Vibrio fischeri

Vibrio fischeri colonizes the squid Euprymna scolopes in a mutualistic symbiosis. Hatchling squid lack these bacterial symbionts, and V. fischeri strains must compete to occupy this privileged niche. We cloned a V. fischeri gene, designated pilA, that contributes to colonization competitiveness and encodes a protein similar to type IV-A pilins. Unlike its closest known relatives, Vibrio cholerae mshA and vcfA, pilA is monocistronic and not clustered with genes associated with pilin export or assembly. Using wild-type strain ES114 as the parent, we generated an in-frame pilA deletion mutant, as well as pilA mutants marked with a kanamycin resistance gene. In mixed inocula, marked mutants were repeatedly outcompeted by ES114 (P < 0.05) but not by an unmarked pilA mutant, for squid colonization. In contrast, the ratio of mutant to ES114 CFUs did not change during 70 generations of coculturing. The competitive defect of pilA mutants ranged from 1.7- to 10-fold and was more pronounced when inocula were within the range estimated for V. fischeri populations in Hawaiian seawater (200 to 2,000 cells/ml) than when higher densities were used. ES114 also outcompeted a pilA mutant by an average of twofold at lower inoculum densities, when only a fraction of the squid became infected, most by only one strain. V. fischeri strain ET101, which was isolated from Euprymna tasmanica and is outcompeted by ES114, lacks pilA; however, 11 other diverse V. fischeri isolates apparently possess pilA. The competitive defect of pilA mutants suggests that cell surface molecules may play important roles in the initiation of beneficial symbioses in which animals must acquire symbionts from a mixed community of environmental bacteria.     

Detection of the Light Organ Symbiont, Vibrio fischeri, in Hawaiian Seawater by Using lux Gene Probes

Symbiotic bacteria that inhabit the light-emitting organ of the Hawaiian squid Euprymna scolopes are distinctive from typical Vibrio fischeri organisms in that they are not visibly luminous when grown in laboratory culture. Therefore, the abundance of these bacteria in seawater samples cannot be estimated simply by identifying them among luminous colonies that arise on nutrient agar plates. Instead, we have used luxR and polymerase chain reaction generated luxA gene probes to identify both luminous and non-visibly luminous V. fischeri colonies by DNA-DNA hybridization. The probes were specific, hybridizing at least 50 to 100 times more strongly to immobilized DNAs from V. fischeri strains than to those of pure cultures of other related species. Thus, even non-visibly luminous V. fischeri colonies could be identified among colonies obtained from natural seawater samples by their probe-positive reaction. Bacteria in seawater samples, obtained either within or distant from squid habitats, were collected on membrane filters and incubated until colonies appeared. The filters were then observed for visibly luminous V. fischeri colonies and hybridized with the lux gene probes to determine the number of total V. fischeri colonies (both luminous and non-visibly luminous). We detected no significant differences in the abundance of luminous V. fischeri CFU in any of the water samples observed (≤1 to 3 CFU/100 ml). However, probe-positive colonies of V. fischeri (up to 900 CFU/100 ml) were found only in seawater collected from within the natural habitats of the squids. A number of criteria were used to confirm that these probe-positive strains were indistinguishable from symbiotic V. fischeri. Therefore, the luxA and luxR gene probes were species specific and gave a reliable estimate of the number of culturable V. fischeri colonies in natural water samples.     

Characterization of two host-specific genes, mannose-sensitive hemagglutinin (mshA) and uridyl phosphate dehydrogenase (UDPDH) that are involved in the Vibrio fischeri–Euprymna tasmanica mutualism

While much has been known about the mutualistic associations between the sepiolid squid Euprymna tasmanica and the luminescent bacterium, Vibrio fischeri , less is known about the connectivity between the microscopic and molecular basis of initial attachment and persistence in the light organ. Here, we examine the possible effects of two symbiotic genes on specificity and biofilm formation of V. fischeri in squid light organs. Uridine diphosphate glucose-6-dehydrogenase (UDPDH) and mannose-sensitive hemagglutinin ( mshA ) mutants were generated in V. fischeri to determine whether each gene has an effect on host colonization, specificity, and biofilm formation. Both squid light organ colonization assays and transmission electron microscopy confirmed differences in host colonization between wild-type and mutant strains, and also demonstrated the importance of both UDPDH and mshA gene expression for successful light organ colonization. This furthers our understanding of the genetic factors playing important roles in this environmentally transmitted symbiosis.     

Temperature Affects Species Distribution in Symbiotic Populations of Vibrio spp.

The genus Sepiola (Cephalopoda: Sepiolidae) contains 10 known species that occur in the Mediterranean Sea today. All Sepiola species have a light organ that contains at least one of two species of luminous bacteria, Vibrio fischeri and Vibrio logei. The two Vibrio species coexist in at least four Sepiola species (S. affinis, S. intermedia, S. ligulata, and S. robusta), and their concentrations in the light organ depend on changes in certain abiotic factors, including temperature. Strains of V. fischeri grew faster in vitro and in Sepiola juveniles when they were incubated at 26°C. In contrast, strains of V. logei grew faster at 18°C in culture and in Sepiola juveniles. When aposymbiotic S. affinis or S. ligulata juveniles were inoculated with one Vibrio species, all strains of V. fischeri and V. logei were capable of infecting both squid species at the optimum growth temperatures, regardless of the squid host from which the bacteria were initially isolated. However, when two different strains of V. fischeri and V. logei were placed in direct competition with each other at either 18 or 26°C, strains of V. fischeri were present in sepiolid light organs in greater concentrations at 26°C, whereas strains of V. logei were present in greater concentrations at 18°C. In addition to the competition experiments, the ratios of the two bacterial species in adult Sepiola specimens caught throughout the season at various depths differed, and these differences were correlated with the temperature in the surrounding environment. My findings contribute additional data concerning the ecological and environmental factors that affect host-symbiont recognition and may provide insight into the evolution of animal-bacterium specificity.     

Vibrio fischeri‐derived outer membrane vesicles trigger host development

Outer membrane vesicles (OMV) are critical elements in many host‐cell/microbe interactions. Previous studies of the symbiotic association between Euprymna scolopes and Vibrio fischeri had shown that within 12 h of colonizing crypts deep within the squid's light organ, the symbionts trigger an irreversible programme of tissue development in the host. Here, we report that OMV produced by V. fischeri are powerful contributors to this process. The first detectable host response to the OMV is an increased trafficking of macrophage‐like cells called haemocytes into surface epithelial tissues. We showed that exposing the squid to other Vibrio species fails to induce this trafficking; however, addition of a high concentration of their OMV, which can diffuse into the crypts, does. We also provide evidence that tracheal cytotoxin released by the symbionts, which can induce haemocyte trafficking, is not part of the OMV cargo, suggesting two distinct mechanisms to induce the same morphogenesis event. By manipulating the timing and localization of OMV signal delivery, we showed that haemocyte trafficking is fully induced only when V. fischeri, the sole species able to reach and grow in the crypts, succeeds in establishing a sustained colonization. Further, our data suggest that the host's detection of OMV serves as a symbiotic checkpoint prior to inducing irreversible morphogenesis.