Characterization of the Bacterial Diversity in Indo-West Pacific Loliginid and Sepiolid Squid Light Organs

Loliginid and sepiolid squid light organs are known to host a variety of bacterial species from the family Vibrionaceae, yet little is known about the species diversity and characteristics among different host squids. Here we present a broad-ranging molecular and physiological analysis of the bacteria colonizing light organs in loliginid and sepiolid squids from various field locations of the Indo-West Pacific (Australia and Thailand). Our PCR-RFLP analysis, physiological characterization, carbon utilization profiling, and electron microscopy data indicate that loliginid squid in the Indo-West Pacific carry a consortium of bacterial species from the families Vibrionaceae and Photobacteriaceae. This research also confirms our previous report of the presence of Vibrio harveyi as a member of the bacterial population colonizing light organs in loliginid squid. pyrH sequence data were used to confirm isolate identity, and indicates that Vibrio and Photobacterium comprise most of the light organ colonizers of squids from Australia, confirming previous reports for Australian loliginid and sepiolid squids. In addition, combined phylogenetic analysis of PCR-RFLP and 16S rDNA data from Australian and Thai isolates associated both Photobacterium and Vibrio clades with both loliginid and sepiolid strains, providing support that geographical origin does not correlate with their relatedness. These results indicate that both loliginid and sepiolid squids demonstrate symbiont specificity (Vibrionaceae), but their distribution is more likely due to environmental factors that are present during the infection process. This study adds significantly to the growing evidence for complex and dynamic associations in nature and highlights the importance of exploring symbiotic relationships in which non-virulent strains of pathogenic Vibrio species could establish associations with marine invertebrates.     

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.     

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.     

Phylogeographical patterns among Mediterranean sepiolid squids and their Vibrio symbionts: environment drives specificity among sympatric species

Bobtail squid from the genera Sepiola and Rondeletiola (Cephalopoda: Sepiolidae) form mutualistic associations with luminous Gram-negative bacteria (Gammaproteobacteria: Vibrionaceae) from the genera Vibrio and Photobacterium. Symbiotic bacteria proliferate inside a bilobed light organ until they are actively expelled by the host into the surrounding environment on a diel basis. This event results in a dynamic symbiont population with the potential to establish the symbiosis with newly hatched sterile (axenic) juvenile sepiolids. In this study, we examined the genetic diversity found in populations of sympatric sepiolid squid species and their symbionts by the use of nested clade analysis with multiple gene analyses. Variation found in the distribution of different species of symbiotic bacteria suggests a strong influence of abiotic factors in the local environment, affecting bacterial distribution among sympatric populations of hosts. These abiotic factors include temperature differences incurred by a shallow thermocline, as well as a lack of strong coastal water movement accompanied by seasonal temperature changes in overlapping niches. Host populations are stable and do not appear to have a significant role in the formation of symbiont populations relative to their distribution across the Mediterranean Sea. Additionally, all squid species examined (Sepiola affinis, S. robusta, S. ligulata, S. intermedia, and Rondeletiola minor) are genetically distinct from one another regardless of location and demonstrate very little intraspecific variation within species. These findings suggest that physical boundaries and distance in relation to population size, and not host specificity, are important factors in limiting or defining gene flow within sympatric marine squids and their associated bacterial symbionts in the Mediterranean Sea.     

Enlightenment of old ideas from new investigations: more questions regarding the evolution of bacteriogenic light organs in squids

Bioluminescence is widespread among many different types of marine organisms. Metazoans contain two types of luminescence production, bacteriogenic (symbiotic with bacteria) or autogenic, via the production of a luminous secretion or the intrinsic properties of luminous cells. Several species in two families of squids, the Loliginidae and the Sepiolidae (Mollusca: Cephalopoda) harbor bacteriogenic light organs that are found central in the mantle cavity. These light organs are exceptional in function, that is, the morphology and the complexity suggests that the organ has evolved to enhance and direct light emission from bacteria that are harbored inside. Although light organs are widespread among taxa within the Sepiolidae, the origin and development of this important feature is not well studied. We compared light organ morphology from several closely related taxa within the Sepiolidae and combined molecular phylogenetic data using four loci (nuclear ribosomal 28S rRNA and the mitochondrial cytochrome c oxidase subunit I and 12S and 16S rRNA) to determine whether this character was an ancestral trait repeatedly lost among both families or whether it evolved independently as an adaptation to the pelagic and benthic lifestyles. By comparing other closely related extant taxa that do not contain symbiotic light organs, we hypothesized that the ancestral state of sepiolid light organs most likely evolved from part of a separate accessory gland open to the environment that allowed colonization of bacteria to occur and further specialize in the eventual development of the modern light organ.     

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.     

Lights out: the evolution of bacterial bioluminescence in Loliginidae

Representatives of several metazoan clades engage in symbiotic interactions with bioluminescent bacteria, but the evolution and maintenance of these interactions remain poorly understood. Uroteuthis is a genus of loliginid squid (Cephalopoda: Loliginidae) characterized by paired ventral photophores (light organs) housing bioluminescent bacteria. While previous phylogenetic studies have suggested that Uroteuthis is closely related to Loliolus, a genus of non-bioluminescent species, this relationship remains unresolved. To illuminate Uroteuthis and Loliolus phylogeny and its implications for the evolution of bioluminescence in Loliginidae, we generated sequences from two mitochondrial genes from Uroteuthis specimens sampled from several sites in the Indian and western Pacific Oceans. We combined these data with data from GenBank, analyzed the concatenated data set using maximum likelihood and Bayesian methods, and reconstructed the evolution of bacterial bioluminescence on the resulting phylogenies. Our analyses support the hypothesis that Uroteuthis is paraphyletic with respect to Loliolus. Furthermore, our reconstructions suggest that the symbiosis between loliginid squid and bioluminescent bacteria evolved once in the ancestor of Loliolini (the clade comprising Uroteuthis and Loliolus), but was subsequently lost in the ancestor of Loliolus. These findings could have profound implications for our understanding of the evolution of symbiotic bioluminescence in squid.     

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.     

Occurrence of plasmid DNA in the sepiolid squid symbiontVibrio fischeri

Because of the importance of plasmids in many bacterial associations with plants and animals, we determined the occurrence and distribution of plasmid DNA in symbioticVibrio fischeri from the light organ of the sepiolid squidEuprymna scolopes. Analyses of 225 isolates of symbioticV. fischeri from 25 individual squids revealed an overall plasmid-carriage rate of 56%. A large plasmid (39 kb) was detected in 96% of those isolates carrying plasmids, and multiple small plasmids were found to co-occur with one of the large plasmids in 81% of plasmid-carrying strains. In addition, these plasmids appear to be restricted toV. fischeri strains isolated fromE. scolopes and from seawater at sites of squid populations. We were unable to assign a role or function to these plasmids, but they do not carry genes required for the establishment of the light organ symbiosis. We conclude that the essential bacterial symbiotic determinants must be encoded on the chromosome and that the plasmids may carry genes that are important for the survival of theseV. fischeri strains outside of the symbiotic association.     

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.     

Gene-Swapping Mediates Host Specificity among Symbiotic Bacteria in a Beneficial Symbiosis

Environmentally acquired beneficial associations are comprised of a wide variety of symbiotic species that vary both genetically and phenotypically, and therefore have differential colonization abilities, even when symbionts are of the same species. Strain variation is common among conspecific hosts, where subtle differences can lead to competitive exclusion between closely related strains. One example where symbiont specificity is observed is in the sepiolid squid-Vibrio mutualism, where competitive dominance exists among V. fischeri isolates due to subtle genetic differences between strains. Although key symbiotic loci are responsible for the establishment of this association, the genetic mechanisms that dictate strain specificity are not fully understood. We examined several symbiotic loci (lux-bioluminescence, pil = pili, and msh-mannose sensitive hemagglutinin) from mutualistic V. fischeri strains isolated from two geographically distinct squid host species (Euprymna tasmanica-Australia and E. scolopes-Hawaii) to determine whether slight genetic differences regulated host specificity. Through colonization studies performed in naïve squid hatchlings from both hosts, we found that all loci examined are important for specificity and host recognition. Complementation of null mutations in non-native V. fischeri with loci from the native V. fischeri caused a gain in fitness, resulting in competitive dominance in the non-native host. The competitive ability of these symbiotic loci depended upon the locus tested and the specific squid species in which colonization was measured. Our results demonstrate that multiple bacterial genetic elements can determine V. fischeri strain specificity between two closely related squid hosts, indicating how important genetic variation is for regulating conspecific beneficial interactions that are acquired from the environment.     

Bioluminescence in the symbiotic squid Euprymna scolopes is controlled by a daily biological rhythm

In most symbioses between animals and luminous bacteria it has been assumed that the bacterial symbionts luminesce continuously, and that the control of luminescent output by the animal is mediated through elaborate accessory structures, such as chromatophores and muscular shutters that surround the host light organ. However, we have found that while in the light organ of the sepiolid squid Euprymna scolopes, symbiotic cells of Vibrio fischeri do not produce a continuously uniform level of luminescence, but instead exhibit predictable cyclic fluctuations in the amount of light emitted per cell. This daily biological rhythm exhibits many features of a circadian pattern, and produces an elevated intensity of symbiont luminescence in juvenile animals during the hours preceding the onset of ambient darkness. Comparisons of the specific luminescence of bacteria in the intact light organ with that of newly released bacteria support the existence of a direct host regulation of the specific activity of symbiont luminescence that does not require the intervention of accessory tissues. A model encompassing the currently available evidence is proposed for the control of growth and luminescence activity in the E. scolopes/V. fischeri light organ symbiosis.Abbreviations CFU colony-forming-unit - LD light-dark     

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 degrees C. In contrast, strains of V. logei grew faster at 18 degrees 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 degrees C, strains of V. fischeri were present in sepiolid light organs in greater concentrations at 26 degrees C, whereas strains of V. logei were present in greater concentrations at 18 degrees 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.     

Phylogenetic analysis of host–symbiont specificity and codivergence in bioluminescent symbioses

Several groups of marine fishes and squids form mutualistic bioluminescent symbioses with luminous bacteria. The dependence of the animal on its symbiont for light production, the animal's specialized anatomical adaptations for harboring bacteria and controlling light emission, and the host family bacterial species specificity characteristic of these associations suggest that bioluminescent symbioses are tightly coupled associations that might involve coevolutionary interactions. Consistent with this possibility, evidence of parallel cladogenesis has been reported for squid–bacterial associations. However, genetic adaptations in the bacteria necessary for and specific to symbiosis have not been identified, and unlike obligate endosymbiotic associations in which the bacteria are transferred vertically, bacterially bioluminescent hosts acquire their light‐organ symbionts from the environment with each new host generation. These contrasting observations led us to test the hypotheses of species specificity and codivergence in bioluminescent symbioses, using an extensive sampling of naturally formed associations. Thirty‐five species of fish in seven teleost families (Chlorophthalmidae, Macrouridae, Moridae, Trachichthyidae, Monocentridae, Acropomatidae, Leiognathidae) and their light‐organ bacteria were examined. Phylogenetic analysis of a taxonomically broad sampling of associations was based on mitochondrial 16S rRNA and cytochrome oxidase I gene sequences for the fish and on recA, gyrB and luxA sequences for bacteria isolated from the light organs of these specimens. In a fine‐scale test focused on Leiognathidae, phylogenetic analysis was based also on histone H3 subunit and 28S rRNA gene sequences for the fish and on gyrB, luxA, luxB, luxF and luxE sequences for the bacteria. Deep divergences were revealed among the fishes, and clear resolution was obtained between clades of the bacteria. In several associations, bacterial species identities contradicted strict host family bacterial species specificity. Furthermore, the fish and bacterial phylogenies exhibited no meaningful topological congruence; evolutionary divergence of host fishes was not matched by a similar pattern of diversification in the symbiotic bacteria. Re‐analysis of data reported for squids and their luminous bacteria also revealed no convincing evidence of codivergence. These results refute the hypothesis of strict host family bacterial species specificity and the hypothesis of codivergence in bioluminescent symbioses. © The Willi Hennig Society 2007.     

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.     

Competitive Dominance among Strains of Luminous Bacteria Provides an Unusual Form of Evidence for Parallel Evolution in Sepiolid Squid-Vibrio Symbioses

One of the principal assumptions in symbiosis research is that associated partners have evolved in parallel. We report here experimental evidence for parallel speciation patterns among several partners of the sepiolid squid-luminous bacterial symbioses. Molecular phylogenies for 14 species of host squids were derived from sequences of both the nuclear internal transcribed spacer region and the mitochondrial cytochrome oxidase subunit I; the glyceraldehyde phosphate dehydrogenase locus was sequenced for phylogenetic determinations of 7 strains of bacterial symbionts. Comparisons of trees constructed for each of the three loci revealed a parallel phylogeny between the sepiolids and their respective symbionts. Because both the squids and their bacterial partners can be easily cultured independently in the laboratory, we were able to couple these phylogenetic analyses with experiments to examine the ability of the different symbiont strains to compete with each other during the colonization of one of the host species. Our results not only indicate a pronounced dominance of native symbiont strains over nonnative strains, but also reveal a hierarchy of symbiont competency that reflects the phylogenetic relationships of the partners. For the first time, molecular systematics has been coupled with experimental colonization assays to provide evidence for the existence of parallel speciation among a set of animal-bacterial associations.     

Effect of transposon-induced motility mutations on colonization of the host light organ by Vibrio fischeri.

Vibrio fischeri is found both as a free-living bacterium in seawater and as the specific, mutualistic light organ symbiont of several fish and squid species. To identify those characteristics of symbiosis-competent strains that are required for successful colonization of the nascent light organ of juvenile Euprymna scolopes squids, we generated a mutant pool by using the transposon Mu dI 1681 and screened this pool for strains that were no longer motile. Eighteen independently isolated nonmotile mutants that were either flagellated or nonflagellated were obtained. In contrast to the parent strain, none of these nonmotile mutants was able to colonize the juvenile squid light organ. The flagellated nonmotile mutant strain NM200 possessed a bundle of sheathed polar flagella indistinguishable from that of the wild-type strain, indicating that the presence of flagella alone is not sufficient for colonization and that it is motility itself that is required for successful light organ colonization. This study identifies motility as the first required symbiotic phenotype of V. fischeri.