Sepiolid Squid Euprymna Scolopes Research Articles

Characterization of a Vibrio fischeri Aminopeptidase and Evidence for Its Influence on an Early Stage of Squid Colonization

Vibrio fischeri cells are the sole colonists of a specialized light organ in the mantle cavity of the sepiolid squid Euprymna scolopes. The process begins when the bacteria aggregate in mucus secretions outside the light organ. The cells eventually leave the aggregate, enter the light organ, and encounter a rich supply of peptides. The need to dissociate from mucus and presumably utilize peptides led us to hypothesize that protease activity is integral to the colonization process. Protease activity associated with whole cells of Vibrio fischeri strain ES114 was identified as the product of a putative cell membrane-associated aminopeptidase (PepN). To characterize this activity, the aminopeptidase was cloned, overexpressed, and purified. Initial steady-state kinetic studies revealed that the aminopeptidase has broad activity, with a preference for basic and hydrophobic side chains and k(cat) and K(m) values that are lower and smaller, respectively, than those of Escherichia coli PepN. A V. fischeri mutant unable to produce PepN is significantly delayed in its ability to colonize squid within the first 12 h, but eventually it establishes a wild-type colonization level. Likewise, in competition with the wild type for colonization, the mutant is outcompeted at 12 h postinoculation but then competes evenly by 24 h. Also, the PepN-deficient strain fails to achieve wild-type levels of cells in aggregates, suggesting an explanation for the initial colonization delay. This study provides a foundation for more studies on PepN expression, localization, and role in the early stages of squid colonization.

Abstracts of the 17th International Symposium on Bioluminescence and Chemiluminescence ‐ (ISBC 2012)

WARNING : The light-emitting molecular structures responsible for the chemiluminescence and fluorescence phenomena are not necessarily the same!

Developmental expression ofapterous/Lhx2/9in the sepiolid squidEuprymna scolopessupports an ancestral role in neural development

The transcription factors Apterous/Lhx2/9 play many pivotal roles in the development of protostomes and deuterostomes, most notably limb patterning, eye morphogenesis, and brain development. Full-length apterous/lhx2/9 homologs have been isolated from several invertebrate species, but hitherto not from a lophotrochozoan. Here, we report the isolation, characterization, and spatio-temporal expression of apterous in the sepiolid squid Euprymna scolopes. The isolated composite cDNA encodes a hypothetical protein of 448 amino acid residues with a typical LIM-homeodomain (LIM-HD) structure and the greatest overall sequence similarity to vertebrate Lhx2/9 proteins. The Euprymna scolopes apterous (Es-ap) expression patterns provided no indication of a role in the early dorso/ventral patterning or growth of the arm crown that showed expression only in two ventral cords running in parallel inside the arms and tentacles and at the base of the suckers, a region rich in nerve endings and chemosensory neurons. The Es-ap hybridization signal was also conspicuous in the eyes, olfactory organs, optic lobes, and in several lobes of the supraesophageal mass, among these the olfactory and vertical lobes, and paravertical bodies. The observed expression patterns suggest gene involvement in eye morphogenesis and neural wiring of sensory structures, including those for olfaction and vision.

Wnt gene expression during appendage development of the sepiolid squid Euprymna scolopes

Wnt gene expression during appendage development of the sepiolid squid Euprymna scolopes

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.

The Squid-Vibrio Symbioses: From Demes to Genes

The monospecific light organ association between the Hawaiian sepiolid squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri has been used as a model for the study of the most common type of coevolved animal-bacterial interaction; i.e., the association of Gram-negative bacteria with the extracellular apical surfaces of polarized epithelia. Analysis of the squid-vibrio symbiosis has ranged from characterizations of the harvesting mechanisms by which the host ensures colonization by the appropriate symbiont to identification of bacteria-induced changes in host gene expression that accompany the establishment and maintenance of the relationship. Studies of this model have been enhanced by extensive collaboration with microbiologists, who are able to manipulate the genetics of the bacterial symbiont. The results of our studies have indicated that initiation and persistence of the association requires a complex, reciprocal molecular dialogue between these two phylogenetically distant partners.

Pax6 in the sepiolid squid Euprymna scolopes: evidence for a role in eye, sensory organ and brain development

The cloning of a Pax6 orthologue from the sepiolid squid Euprymna scolopes and its developmental expression pattern are described. The data are consistent with the presence of a single gene encoding a protein with highly conserved DNA-binding paired and homeodomains. A detailed expression analysis by in situ hybridization and immunodetection revealed Pax6 mRNA and protein with predominantly nuclear localization in the developing eye, olfactory organ, brain lobes (optic lobe, olfactory lobe, peduncle lobe, superior frontal lobe and dorsal basal lobe), arms and mantle, suggestive of a role in eye, brain, and sensory organ development.

LitR, a new transcriptional activator in Vibrio fischeri, regulates luminescence and symbiotic light organ colonization.

Vibrio fischeri is the bacterial symbiont within the light-emitting organ of the sepiolid squid Euprymna scolopes. Upon colonizing juvenile squids, bacterial symbionts grow on host-supplied nutrients, while providing a bioluminescence that the host uses during its nocturnal activities. Mutant bacterial strains that are unable to emit light have been shown to be defective in normal colonization. A 606 bp open reading frame was cloned from V. fischeri that encoded a protein, which we named LitR, that had about 60% identity to four related regulator proteins: Vibrio cholerae HapR, Vibrio harveyi LuxR, Vibrio parahaemolyticus OpaR and Vibrio vulnificus SmcR. When grown in culture, cells of V. fischeri strain PMF8, in which litR was insertionally inactivated, were delayed in the onset of luminescence induction and emitted only about 20% as much light per cell as its parent. Protein-binding studies suggested that LitR enhances quorum sensing by regulating the transcription of the luxR gene. Interestingly, when competed against its parent in mixed inocula, PMF8 became the predominant symbiont present in 83% of light organs. Thus, the litR mutation appears to represent a novel class of mutations in which the loss of a regulatory gene function enhances the bacterium's competence in initiating a benign infection.

HOX genes in the sepiolid squid Euprymna scolopes: implications for the evolution of complex body plans.

Molluscs display a rich diversity of body plans ranging from the wormlike appearance of aplacophorans to the complex body plan of the cephalopods with highly developed sensory organs, a complex central nervous system, and cognitive abilities unrivaled among the invertebrates. The aim of the current study is to define molecular parameters relevant to the developmental evolution of cephalopods by using the sepiolid squid Euprymna scolopes as a model system. Using PCR-based approaches, we identified one anterior, one paralog group 3, five central, and two posterior group Hox genes. The deduced homeodomain sequences of the E. scolopes Hox cluster genes are most similar to known annelid, brachiopod, and nemertean Hox gene homeodomain sequences. Our results are consistent with the presence of a single Hox gene cluster in cephalopods. Our data also corroborate the proposed existence of a differentiated Hox gene cluster in the last common ancestor of Bilaterians. Furthermore, our phylogenetic analysis and in particular the identification of Post-1 and Post-2 homologs support the Lophotrochozoan clade.

A description of the nuchal organ, a possible photoreceptor, in Euprymna scolopes and other cephalopods

A type of apparent photoreceptor, the nuchal organ, is described in the sepiolid squid Euprymna scolopes. These vesicles are identified in a variety of other cephalopod species. The nuchal organs have specific structural similarities to the already well-described photosensitive vesicles. Both organs have a distinct vesiculate structure and possess a rind consisting of several types of cells. Certain rind cells from both organs send processes with microvilli into the lumen of the vesicle forming a core area. Within the cores of both organs numerous microvilli occupy much of the core volume. These histological similarities provide strong evidence that the nuchal organs, like the photosensitive vesicles, are photoreceptors. Three distinct differences also exist between the nuchal organs and photosensitive vesicles: (1) the location of the nuchal organs differs from that of all known photosensitive vesicles in cephalopods; (2) the location of the nuchal organs is highly conservative in decapod cephalopods compared to the varying locations of the photosensitive vesicles; (3) the degree of structural order generally found within photosensitive vesicles is not apparent in the nuchal organs. The function of the nuchal organ is probably different from previously described photoreceptors in cephalopods.

Aposymbiotic culture of the sepiolid squid Euprymna scolopes: Role of the symbiotic bacterium Vibrio fischeri in host animal growth, development, and light organ morphogenesis

The sepiolid squid Euprymna scolopes forms a bioluminescent mutualism with the luminous bacterium Vibrio fischeri, harboring V. fischeri cells in a complex ventral light organ and using the bacterial light in predator avoidance. To characterize the contribution of V. fischeri to the growth and development of E. scolopes and to define the long-term effects of bacterial colonization on light organ morphogenesis, we developed a mariculture system for the culture of E. scolopes from hatching to adulthood, employing artificial seawater, lighting that mimicked that of the natural environment, and provision of prey sized to match the developmental stage of E. scolopes. Animals colonized by V. fischeri and animals cultured in the absence of V. fischeri (aposymbiotic) grew and survived equally well, developed similarly, and reached sexual maturity at a similar age. Development of the light organ accessory tissues (lens, reflectors, and ink sac) was similar in colonized and aposymbiotic animals with no obvious morphometric or histological differences. Colonization by V. fischeri influenced regression of the ciliated epithelial appendages (CEAs), the long-term growth of the light organ epithelial tubules, and the appearance of the cells composing the ciliated ducts, which exhibit characteristics of secretory tissue. In certain cases, aposymbiotic animals retained the CEAs in a partially regressed state and remained competent to initiate symbiosis with V. fischeri into adulthood. In other cases, the CEAs regressed fully in aposymbiotic animals, and these animals were not colonizable. The results demonstrate that V. fischeri is not required for normal growth and development of the animal or for development of the accessory light organ tissues and that morphogenesis of only those tissues coming in contact with the bacteria (CEAs, ciliated ducts, and light organ epithelium) is altered by bacterial colonization of the light organ. Therefore, V. fischeri apparently makes no major metabolic contribution to E. scolopes beyond light production, and post-embryonic development of the light organ is essentially symbiont independent. J. Exp. Zool. 286:280–296, 2000. © 2000 Wiley-Liss, Inc.

Aposymbiotic culture of the sepiolid squidEuprymna scolopes: Role of the symbiotic bacteriumVibrio fischeri in host animal growth, development, and light organ morphogenesis

The sepiolid squid Euprymna scolopes forms a bioluminescent mutualism with the luminous bacterium Vibrio fischeri, harboring V. fischeri cells in a complex ventral light organ and using the bacterial light in predator avoidance. To characterize the contribution of V. fischeri to the growth and development of E. scolopes and to define the long-term effects of bacterial colonization on light organ morphogenesis, we developed a mariculture system for the culture of E. scolopes from hatching to adulthood, employing artificial seawater, lighting that mimicked that of the natural environment, and provision of prey sized to match the developmental stage of E. scolopes. Animals colonized by V. fischeri and animals cultured in the absence of V. fischeri (aposymbiotic) grew and survived equally well, developed similarly, and reached sexual maturity at a similar age. Development of the light organ accessory tissues (lens, reflectors, and ink sac) was similar in colonized and aposymbiotic animals with no obvious morphometric or histological differences. Colonization by V. fischeri influenced regression of the ciliated epithelial appendages (CEAs), the long-term growth of the light organ epithelial tubules, and the appearance of the cells composing the ciliated ducts, which exhibit characteristics of secretory tissue. In certain cases, aposymbiotic animals retained the CEAs in a partially regressed state and remained competent to initiate symbiosis with V. fischeri into adulthood. In other cases, the CEAs regressed fully in aposymbiotic animals, and these animals were not colonizable. The results demonstrate that V. fischeri is not required for normal growth and development of the animal or for development of the accessory light organ tissues and that morphogenesis of only those tissues coming in contact with the bacteria (CEAs, ciliated ducts, and light organ epithelium) is altered by bacterial colonization of the light organ. Therefore, V. fischeri apparently makes no major metabolic contribution to E. scolopes beyond light production, and post-embryonic development of the light organ is essentially symbiont independent. J. Exp. Zool. 286:280-296, 2000.

Consequences of Evolving With Bacterial Symbionts: Insights from the Squid-Vibrio Associations

▪ Abstract The squid-vibrio light-organ symbioses, which have been under investigation for just over 10 years, offer the opportunity to decipher aspects of the dynamics of stable associations between animals and bacteria. The two best-studied partners, the Hawaiian sepiolid squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri, engage in the most common type of animal-bacterial association, i.e., between extracellular, gram-negative bacteria and animal epithelia. Similar to most such symbioses, the squid-vibrio relationship begins anew each generation when thehost animal acquires the symbiont from the surrounding environment. To establish a specific association, mechanisms have evolved to ensure recognition between the host and symbiont and the exclusion of other potential partnerships. Once the association has been established, the bacteria induce significant morphological changes in the host that result in a transition of the light organ from a form associated with initiation of the symbiosis to one characteristic of the mature, functional relationship.

Late postembryonic development of the symbiotic light organ of Euprymna scolopes (Cephalopoda: Sepiolidae).

The symbiotic light organ of the sepiolid squid Euprymna scolopes undergoes significant anatomical, morphological, and biochemical changes during development. Previously we described the embryonic organogenesis and early postembryonic development of the light organ. During embryogenesis, tissues are developed that will promote the onset of an association with Vibrio fischeri, the light organ symbiont. Upon inoculation, and in response to the first interactions with the bacterial symbionts, the light organ undergoes a dramatic morphogenesis during the first 4-5 days of postembryonic development. Here we describe the final developmental stage of the light organ system, a period of late postembryonic development in which particular tissues of the light organ mature that eventually mediate the functional symbiosis. The maturation of the light organ occurs within 1 to 2 weeks posthatch and entails two principal processes: (1) changes in the shape of the organ and elaboration of the accessory tissues that modify the bacterially produced light; and (2) branching of the epithelial crypts, where the bacterial symbionts reside, and restriction of epithelial cell proliferation to the deepest branches of the crypts. The gross morphological changes of the organ occur in the absence of V. fischeri, although rudiments of the ciliated field of the hatchling remain in animals not exposed to the microbial symbiont.

Induction of apoptosis by cooperative bacteria in the morphogenesis of host epithelial tissues.

Associations with pathogenic bacteria have recently been shown to initiate apoptotic programs in the cells of their animal hosts, where host cell death is hypothesized to be a response of the immune system, either initiated as a mechanism of host defense or bacterial offense. In this study, we present evidence that bacterial initiation of apoptosis is neither restricted to pathogenesis nor to the initation of an immune response. In the cooperative association between the sepiolid squid Euprymna scolopes and the luminous bacterium Vibrio fischeri, the bacteria induce a dramatic morphogenesis of the host tissues during the first few days of interaction between these partners. The most striking change is the bacteria-triggered loss of an extensive superficial epithelium that potentiates the infection process. Our analyses of these tissues revealed that the bacteria induce apoptosis in the cells that comprise this epithelium within hours of the interaction with bacteria. Ultrastructural analysis revealed that after 24 h the integrity of the epithelium had been lost, i.e., the basement membrane had degenerated and the majority of the cells exhibited signs of apoptosis, most notably chromatin condensation. Analysis of these tissues with probes that reveal intracellular acidification showed that the cells first undergo an initial acidification beginning about 6-8 h after exposure to V. fischeri. As determined by end-labeling of DNA fragments, extensive endonuclease activity was detected at approximately 16-20 h post-infection. These data provide evidence that cooperative bacteria can participate in the remodeling of host tissues through the induction of host apoptotic programs.

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.

A Transient Exposure to Symbiosis-Competent Bacteria Induces Light Organ Morphogenesis in the Host Squid.

Recent studies of the symbiotic association between the Hawaiian sepiolid squid Euprymna scolopes and the luminous bacterium Vibrio fischeri have shown that colonization of juvenile squid with symbiosis-competent bacteria induces morphogenetic changes of the light organ. These changes occur over a 4-day period and include cell death and tissue regression of the external ciliated epithelium. In the absence of bacterial colonization, morphogenesis does not occur. To determine whether the bacteria must be present throughout the morphogenetic process, we used the antibiotic chloramphenicol to clear the light organ of bacteria at various times during the initial colonization. We provide evidence in this study that a transient, 12-hour exposure to symbiosis-competent bacteria is necessary and sufficient to induce tissue regression in the light organ over the next several days. Further, we show that successful entrance into the light organ is necessary to induce morphogenesis, suggesting that induction results from bacterial interaction with internal crypt cells and not with the external ciliated epithelium. Finally, no difference in development was observed when the light organ was colonized by a mutant strain of V. fischeri that did not produce autoinducer, a potential light organ morphogen.

Isolation and characterization of a visibly luminous variant of Vibrio fischeri strain ES114 from the sepiolid squid Euprymna scolopes

Isolation and characterization of a visibly luminous variant of Vibrio fischeri strain ES114 from the sepiolid squid Euprymna scolopes

Isolation and characterization of a visibly luminous variant ofVibrio fischeri strain ES114 from the sepiolid squidEuprymna scolopes

Vibrio fischeri strains isolated from light organs of the sepiolid squidEuprymna scolpes are non-visibly luminous and fast growing in laboratory culture, whereas in the symbiosis they are visibly luminous and slow growing. A spontaneous, visibly luminous, slowgrowing variant was isolated from a laboratory culture of the squid-symbioticV. fischeri strain ES114. Taxonomic and DNA-homology analyses demonstrated that the variant wasV. fischeri and was very similar to the original form. However, the variant grew at one-fourth the rate of the original form, produced 30,000-fold more luminescence, induced luminescence at a lower cell density, and produced a higher level ofV. fischeri luminescence autoinducer. Regulation of luminescence, nonetheless, was similar in the two forms and typical ofV. fischeri with respect to responses to autoinducer, glucose, the iron chelator ethylenediamine-di(o-hydroxyphenyl acetic acid), and 3′:5′-cyclic AMP. Compared to the original form, cells of the variant were smaller, exhibited from zero to two polar, sheathed flagella instead of a tuft of three to eight flagella, produced a deeper yellow-orange pigment, did not acidify media containing glycerol, and produced a more distinct pellicle. The two forms also differed in the levels of several outer membrane and soluble proteins. These results establish a distinctive physiological, morphological, and biochemical dimorphism inV. fischeri ES114 in which the variant exhibits several traits similar toV. fischeri cells in the symbiotic state. The variant and its conversion from the original form in laboratory culture may provide insight into the properties ofV. fischeri cells in the symbiosis and may serve as a model for elucidating the mechanism for their pleiotropic conversion upon colonization of the squid.

The Inductive Role of Bacterial Symbionts in the Morphogenesis of a Squid Light Organ

SYNOPSIS. The association of the sepiolid squid Euprymna scolopes with its marine luminous bacterial symbiont Vibrio fischeri is an emerging model system to study the initiation and development of bacterial symbioses in higher animals, in particular the influence of bacteria on the ontogenic development of symbiotic-specific host tissues. Experiments comparing the development of juvenile squid infected with symbiotic V. fischeri with that of uninfected juveniles suggest postembryonic development of the light organ requires cell-cell interactions with the bacterial symbionts. The presence of symbiotic bacteria induces specific morphological changes by affecting such fundamental processes as cell death and cell differentiation. The surface of the juvenile organ is largely composed of ciliated cells that appear to facilitate infection of the light organ. These cells begin to undergo cell death within hours of infection with symbiotic V. fischeri . Within three days the epithelial cells that form the bacteriacontaining crypts of the light organ increase in size; these cells do not appear mitotically active, and may represent a terminally differentiated state. The light organs of uninfected juvenile E. scolopes , however, do not exhibit any of these early postembryonic developmental events but remain in a state of arrested morphogenesis.