Confocal microscopy of the light organ crypts in juvenile Euprymna scolopes reveals their morphological complexity and dynamic function in symbiosis

In the hours to days following hatching, the Hawaiian bobtail squid, Euprymna scolopes, obtains its light-emitting symbiont, Vibrio fischeri, from the surrounding environment and propagates the bacteria in the epithelial crypts of a specialized light organ. Three-dimensional analyses using confocal microscopy revealed that each of the three crypts on either side of the juvenile light organ is composed of four morphological regions. Progressing from the lateral pore to the medial blind end of each crypt, the regions consist of 1) a duct, 2) an antechamber, 3) a bottleneck, and 4) a deep region. Only the deep region houses a persistent bacterial population, whereas the duct, antechamber, and bottleneck serve as conduits through which the bacteria enter during initial colonization and exit during diel venting, a behavior in which approximately 90% of the symbionts are expelled each dawn. Our data suggest that, like the duct, the antechamber and bottleneck may function to promote and maintain the specificity of the symbiosis. Pronounced structural and functional differences among the deep regions of the three crypts, along with previously reported characterizations of embryogenesis, suggest a continued developmental progression in the first few days after hatching. Taken together, the results of this study reveal a high degree of complexity in the morphology of the crypts, as well as in the extent to which the three crypts and their constituent regions differ in function during the early stages of the symbiosis.     

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     

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 squid Euprymna 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, slow-growing variant was isolated from a laboratory culture of the squid-symbiotic V. fischeri strain ES114. Taxonomic and DNA-homology analyses demonstrated that the variant was V. 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 of V. fischeri luminescence autoinducer. Regulation of luminescence, nonetheless, was similar in the two forms and typical of V. 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 in V. fischeri ES114 in which the variant exhibits several traits similar to V. fischeri cells in the symbiotic state. The variant and its conversion from the original form in laboratory culture may provide insight into the properties of V. fischeri cells in the symbiosis and may serve as a model for elucidating the mechanism for their pleiotropic conversion upon colonization of the squid. Received: 10 January 1995 / Accepted: 24 May 1995     

A possible ciliary photoreceptor in a juvenile polyopisthocotylean monogenean, Sphyranura sp.

A pair of multiciliate, saccular structures containing multilayered lamellar bodies is reported in the head region of freshly hatched juveniles of the polyopisthocotylean monogenean Sphyranura sp. from Necturus maculosus. Similar structures have been recorded in only one other monogenean, in the oncomiracidium of Entobdella soleae, and the looser packing of the lamellar bodies in Sphyranura has permitted a more detailed ultrastructural study. A comparison is made with similar structures found in digenean miracidia.     

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.     

Vascular architecture in the bacteriogenic light organ of Euprymna tasmanica (Cephalopoda: Sepiolidae)

Symbiosis between southern dumpling squid, Euprymna tasmanica (Cephalopoda: Sepiolidae), and its luminescent symbiont, the bacterium Vibrio fischeri, provides an experimentally tractable system to examine interactions between the eukaryotic host and its bacterial partner. Luminescence emitted by the symbiotic bacteria provides light for the squid in a behavior termed “counter‐illumination,” which allows the squid to mask its shadow amidst downwelling moonlight. Although this association is beneficial, light generated from the bacteria requires large quantities of oxygen to maintain this energy‐consuming reaction. Therefore, we examined the vascular network within the light organ of juveniles of E. tasmanica with and without V. fischeri. Vessel type, diameter, and location of vessels were measured. Although differences between symbiotic and aposymbiotic squid demonstrated that the presence of V. fischeri does not significantly influence the extent of vascular branching at early stages of symbiotic development, these finding do provide an atlas of blood vessel distribution in the organ. Thus, these results provide a framework to understand how beneficial bacteria influence the development of a eukaryotic closed vascular network and provide insight to the evolutionary developmental dynamics that form during mutualistic interactions.     

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.     

Vibrio fischeri lipopolysaccharide induces developmental apoptosis, but not complete morphogenesis, of the Euprymna scolopes symbiotic light organ

During initiation of the association between the squid host Euprymna scolopes and its bacterial partner Vibrio fischeri, the bacteria induce dramatic morphogenesis of the host symbiotic organ, a portion of which involves the signaling of widespread apoptosis of the cells in a superficial ciliated epithelium on the colonized organ. In this study, we investigated the role in this process of lipopolysaccharide (LPS), a bacterial cell-surface molecule implicated in the induction of animal cell apoptosis in other systems. Purified V. fischeri LPS, as well as the LPS of V. cholerae, Haemophilus influenzae, Escherichia coli, and Shigella flexneri, added in the concentration range of pg/ml to ng/ml, induced apoptosis in epithelial cells 10- to 100-fold above background levels. The absence of species specificity suggested that the conserved lipid A portion of the LPS was the responsible component of the LPS molecule. Lipid A from V. fischeri, E. coli, or S. flexneri induced apoptosis. In addition, strains of H. influenzae carrying a mutation in the htrB gene, which is involved in the synthesis of virulent lipid A, showed a diminished ability to induce apoptosis of host cells. Confocal microscopy using fluorescently labeled LPS indicated that the LPS behaves similar to intact bacterial symbionts, interacting with host cells in the internal crypt spaces and not directly with the superficial epithelium. Although LPS was able to induce apoptosis, it did not induce the full morphogenesis of the ciliated surface, suggesting that multiple signals are necessary to mediate the development of this animal-bacterial mutualism.     

Roles of Vibrio fischeri and nonsymbiotic bacteria in the dynamics of mucus secretion during symbiont colonization of the Euprymna scolopes light organ

During light organ colonization of the squid Euprymna scolopes by Vibrio fischeri, host-derived mucus provides a surface upon which environmental V. fischeri forms a biofilm and aggregates prior to colonization. In this study we defined the temporal and spatial characteristics of this process. Although permanent colonization is specific to certain strains of V. fischeri, confocal microscopy analyses revealed that light organ crypt spaces took up nonspecific bacteria and particles that were less than 2 micro m in diameter during the first hour after hatching. However, within 2 h after inoculation, these cells or particles were not detectable, and further entry by nonspecific bacteria or particles appeared to be blocked. Exposure to environmental gram-negative or -positive bacteria or bacterial peptidoglycan caused the cells of the organ's superficial ciliated epithelium to release dense mucin stores at 1 to 2 h after hatching that were used to form the substrate upon which V. fischeri formed a biofilm and aggregated. Whereas the uncolonized organ surface continued to shed mucus, within 48 h of symbiont colonization mucus shedding ceased and the formation of bacterial aggregations was no longer observed. Eliminating the symbiont from the crypts with antibiotics restored the ability of the ciliated fields to secrete mucus and aggregate bacteria. While colonization by V. fischeri inhibited mucus secretion by the surface epithelium, secretion of host-derived mucus was induced in the crypt spaces. Together, these data indicate that although initiation of mucus secretion from the superficial epithelium is nonspecific, the inhibition of mucus secretion in these cells and the concomitant induction of secretion in the crypt cells are specific to natural colonization by V. fischeri.     

Dominance of Vibrio fischeri in secreted mucus outside the light organ of Euprymna scolopes: the first site of symbiont specificity

Previous studies of the Euprymna scolopes-Vibrio fischeri symbiosis have demonstrated that, during colonization, the hatchling host secretes mucus in which gram-negative environmental bacteria amass in dense aggregations outside the sites of infection. In this study, experiments with green fluorescent protein-labeled symbiotic and nonsymbiotic species of gram-negative bacteria were used to characterize the behavior of cells in the aggregates. When hatchling animals were exposed to 10(3) to 10(6) V. fischeri cells/ml added to natural seawater, which contains a mix of approximately 10(6) nonspecific bacterial cells/ml, V. fischeri cells were the principal bacterial cells present in the aggregations. Furthermore, when animals were exposed to equal cell numbers of V. fischeri (either a motile or a nonmotile strain) and either Vibrio parahaemolyticus or Photobacterium leiognathi, phylogenetically related gram-negative bacteria that also occur in the host's habitat, the symbiont cells were dominant in the aggregations. The presence of V. fischeri did not compromise the viability of these other species in the aggregations, and no significant growth of V. fischeri cells was detected. These findings suggested that dominance results from the ability of V. fischeri either to accumulate or to be retained more effectively within the mucus. Viability of the V. fischeri cells was required for both the formation of tight aggregates and their dominance in the mucus. Neither of the V. fischeri quorum-sensing compounds accumulated in the aggregations, which suggested that the effects of these small signal molecules are not critical to V. fischeri dominance. Taken together, these data provide evidence that the specificity of the squid-vibrio symbiosis begins early in the interaction, in the mucus where the symbionts aggregate outside of the light organ.     

O-antigen and core carbohydrate of Vibrio fischeri lipopolysaccharide: composition and analysis of their role in Euprymna scolopes light organ colonization

Vibrio fischeri exists in a symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes, where the squid provides a home for the bacteria, and the bacteria in turn provide camouflage that helps protect the squid from night-time predators. Like other gram-negative organisms, V. fischeri expresses lipopolysaccharide (LPS) on its cell surface. The structure of the O-antigen and the core components of the LPS and their possible role in colonization of the squid have not previously been determined. In these studies, an O-antigen ligase mutant, waaL, was utilized to determine the structures of these LPS components and their roles in colonization of the squid. WaaL ligates the O-antigen to the core of the LPS; thus, LPS from waaL mutants lacks O-antigen. Our results show that the V. fischeri waaL mutant has a motility defect, is significantly delayed in colonization, and is unable to compete with the wild-type strain in co-colonization assays. Comparative analyses of the LPS from the wild-type and waaL strains showed that the V. fischeri LPS has a single O-antigen repeat composed of yersiniose, 8-epi-legionaminic acid, and N-acetylfucosamine. In addition, the LPS from the waaL strain showed that the core structure consists of L-glycero-D-manno-heptose, D-glycero-D-manno-heptose, glucose, 3-deoxy-D-manno-octulosonic acid, N-acetylgalactosamine, 8-epi-legionaminic acid, phosphate, and phosphoethanolamine. These studies indicate that the unusual V. fischeri O-antigen sugars play a role in the early phases of bacterial colonization of the squid.     

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.     

Examination of Phycomyces blakesleeanus for nitrate reductase as a possible blue light photoreceptor

Phycomyces blakesleeanus is unable to grow on media which contain nitrate as the sole nitrogen source. Further, according to a number of assay procedures, there is no significant nitrate reductase activity in Phycomyces. Thus, although nitrate reductase has been proposed to be a blue-light receptor in Neurospora, no active nitrate reductase is available to serve this function in Phycomyces.     

Draft genome of Phaeobacter gallaeciensis ANG1, a dominant member of the accessory nidamental gland of Euprymna scolopes

Phaeobacter gallaeciensis strain ANG1 represents the dominant member of the bacterial consortium within the reproductive accessory nidamental gland (ANG) of the squid Euprymna scolopes. We present a 4.59-Mb assembly of its genome, which may provide clues as to how it benefits its host.     

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.     

Membrane particles and gap junctions in the retinas of two species of cephalopods,Octopus ocellatus and Sepiella japonica

To study correlation between membrane structure and photoreceptor function, we compared the size and density of intramembrane particles (IMPs) in various membrane compartments of freeze-fractured retinas in a cuttle-fish, Sepiella japonica, and an octopus, Octopus ocellatus. Distribution of gap junctions in the retinas was also examined. Similar results were obtained in the two species. P-faces of both rhabdomeric microvillar membrane and non-rhabdomeric plasma membrane of the apical process were characterized by a random distribution of dense IMPs (ca. 5500-6500/microns2), which showed a unimodal size distribution with a mean diameter of ca. 10 nm. Unlike other invertebrate ocelli, the plasma membrane of the cell body in both the outer and inner segments had significantly denser P-face particles (ca. 7500-8000/microns2) than the rhabdomeric microvillar membrane. The size distribution of IMPs in each part of the membrane was also unimodal, but with a mean diameter of ca. 8 nm. In tangential fractures, each lamella of the myeloid body showed a patchwork of P-faces with irregularly arranged, dense particles and E-faces with orderly patterened granulation. Density and size distribution of the P-face particles in the myeloid membrane resembled those in the rhabdomeric microvillar membrane. The plasma membranes of the supporting cell and the gial cell had relatively sparse P-face particles (ca. 1500-3000/microns2). In addition to the previously reported gap junctions, which connected visual cell inner segments with each other, directly or via collaterals, small gap junctions were found between the visual cell axons and presumed efferent nerve fibres in the plexiform layer. Large-sized gap junctions provided mutual connections for both supporting cells and glial cells. In conclusion, IMPs of 10 nm in mean diameter in the microvillar and non-microvillar parts of the apical process plasma membrane and in the myeloid membrane represent the molecules or their clusters of two photopigments in the cephalopod visual cell, rhodopsin and retinochrome, respectively, and electrical transmission plays a role in visual cell-efferent nerve interactions.     

Localization of the photoreceptor for photoperiodism in the stink bug, Plautia crossota stali

The brown-winged green bug Plautia crossota stali Scott (Heteroptera: Pentatomidae) shows a long-day photoperiodic response with respect to the control of adult diapause. The location of the photoreceptor for this response was examined by surgical removal of putative photoreceptor organs. Even after both ocelli were removed, the insects responded normally to the photoperiod. After bilateral removal of the compound eyes, the insects developed reproductive organs and the volume of the corpus allatum increased regardless of photoperiod. Therefore, the compound eyes play a major role in the reception of photic information for photoperiodism in P. c. stali. However, because removal of the bilateral compound eyes did not completely prevent the response to photoperiod, photoreceptors other than the compound eyes can also receive photic information for photoperiodism.     

Characterizing the host and symbiont proteomes in the association between the Bobtail squid, Euprymna scolopes, and the bacterium, Vibrio fischeri

The beneficial symbiosis between the Hawaiian bobtail squid, Euprymna scolopes, and the bioluminescent bacterium, Vibrio fischeri, provides a unique opportunity to study host/microbe interactions within a natural microenvironment. Colonization of the squid light organ by V. fischeri begins a lifelong association with a regulated daily rhythm. Each morning the host expels an exudate from the light organ consisting of 95% of the symbiont population in addition to host hemocytes and shed epithelial cells. We analyzed the host and symbiont proteomes of adult squid exudate and surrounding light organ epithelial tissue using 1D- and 2D-polyacrylamide gel electrophoresis and multidimensional protein identification technology (MudPIT) in an effort to understand the contribution of both partners to the maintenance of this association. These proteomic analyses putatively identified 1581 unique proteins, 870 proteins originating from the symbiont and 711 from the host. Identified host proteins indicate a role of the innate immune system and reactive oxygen species (ROS) in regulating the symbiosis. Symbiont proteins detected enhance our understanding of the role of quorum sensing, two-component signaling, motility, and detoxification of ROS and reactive nitrogen species (RNS) inside the light organ. This study offers the first proteomic analysis of the symbiotic microenvironment of the adult light organ and provides the identification of proteins important to the regulation of this beneficial association.