Field Evidence for Bioluminescent Signaling in the Pony Fish, Leiognathus elongatus

Luminescence of the pony fish, Leiognathus elongatus, was observed in the natural environment during nighttime diving. The light was emitted from the lateroventral portion of the body, as bright rectangular-shaped luminescence patches turned on and off periodically. Luminescent fish had a distinct clear patch on the flank through which light was emitted, whereas non-luminous fish did not have such a clear patch. Both luminous and non-luminous fish were found within a shoal, where non-luminous individuals were chased by luminous ones. From previous morphological studies, the luminous and non-luminous individuals are likely to be male and female, respectively. Our observations provide field evidence that the luminescence functions as intraspecific communications in L. elongatus.     

Luminous bacteria and light emitting fish: ultrastructure of the symbiosis

The luminescent fish Monocentris japonicus uses symbiotic luminous bacteria as a source of light. These bacteria live in light organs, complex tissue compartments, consisting of richly vascularized tubules or canals (in which the bacteria are cultured) lined with mitochondria-rich epithelial cells. The structure is consistent with a proposed model of symbiosis in which nutrients and oxygen are supplied by the vertebrate blood (vascular system). The nutrients, oxidized by the bacteria for growth and light production, are returned in part to the fish as pyruvate, which by reacting with mitochondrial oxygen regulates the light organ oxygen tensions. The luminous bacteria provide steady light that is modulated by passage through the melanocyte-containing dermis of the fish. Both the fish and the bacteria are highly adapted for their symbiotic coexistence.     

Morphology of the Luminous Organ of the Squid Loligo duvaucelid'Orbigny, 1839

The present paper deals with the morphology of the luminous organ of the squid Loligo duvauceli, caught in the sea off Indonesia and Thailand. Two luminous organs are situated on the ventral surface of the ink sac, near the anus. Each organ consists of a luminous sac divided into numerous discrete chambers containing bacteria. On the ventral side the organ is bordered by a lens-like structure, consisting of muscle cells. On the remaining sides the bacterial chambers are limited by a cup-shaped reflector layer with numerous parallel lamellae. The reflector separates the bacterial chamber from the ink sac. A ciliated channel connects the interior of the bacterial chamber with the mantle cavity.     

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     

Carbohydrate specificity of lectins from luminous bacteria

The presence of lectins on a cell surface was demonstrated for 70 cultures of luminous bacteria using hemagglutination reactions. It was shown that hemagglutination of luminous bacteria is inhibited by glucose, maltose, fructose, mannose, and N-acetyl-D-glucosamine. The differences in the inhibition of hemagglutination of luminescent and nonluminescent (spontaneous mutants) symbiotic cultures by N-acetyl-D-galactosamine were revealed. The fact that N-acetyl-D-galactosamine inhibits hemagglutination of the luminescent symbiotic bacteria but does not inhibit hemagglutination of the symbiotic cultures lacking luminescence suggests that lectins with N-acetyl-D-galactosamine specificity are possibly involved in the formation and functioning of the symbiosis of luminous bacteria with marine animals possessing luminous organs.     

The specificity of symbiosis: Pony fish and luminescent bacteria

Luminescent bacteria isolated from light organs of seven different species (3 genera) of fishes of the family Leiognathidae were subjected to taxonomic analysis. Of the 733 isolated all but seven were identified as Photobacterium leiognathi; the others are considered to be either chance contaminants of the sampling procedure or transients within the organ. In most fish, the luminous organ appeared to contain a single predominating strain of P. leiognathi with small numbers of one to three other strains of the same species, differing by only one or two characters.     

Developmental and Microbiological Analysis of the Inception of Bioluminescent Symbiosis in the Marine Fish Nuchequula nuchalis (Perciformes: Leiognathidae)

Many marine fish harbor luminous bacteria as bioluminescent symbionts. Despite the diversity, abundance, and ecological importance of these fish and their apparent dependence on luminous bacteria for survival and reproduction, little is known about developmental and microbiological events surrounding the inception of their symbioses. To gain insight on these issues, we examined wild-caught larvae of the leiognathid fish Nuchequula nuchalis, a species that harbors Photobacterium leiognathi as its symbiont, for the presence, developmental state, and microbiological status of the fish's internal, supraesophageal light organ. Nascent light organs were evident in the smallest specimens obtained, flexion larvae of 6.0 to 6.5 mm in notochord length (NL), a developmental stage at which the stomach had not yet differentiated and the nascent gasbladder had not established an interface with the light organ. Light organs of certain of the specimens in this size range apparently lacked bacteria, whereas light organs of other specimens of 6.5 mm in NL and of all larger specimens harbored large populations of bacteria, representatives of which were identified as P. leiognathi. Bacteria identified as Vibrio harveyi were also present in the light organ of one larval specimen. Light organ populations were composed typically of two or three genetically distinct strain types of P. leiognathi, similar to the situation in adult fish, and the same strain type was only rarely found in light organs of different larval, juvenile, or adult specimens. Light organs of larvae carried a smaller proportion of strains merodiploid for the lux-rib operon, 79 of 249 strains, than those of adults (75 of 91 strains). These results indicate that light organs of N. nuchalis flexion and postflexion larvae of 6.0 to 6.7 mm in NL are at an early stage of development and that inception of the symbiosis apparently occurs in flexion larvae of 6.0 to 6.5 mm in NL. Ontogeny of the light organ therefore apparently precedes acquisition of the symbiotic bacteria. Furthermore, bacterial populations in larval light organs near inception of the symbiosis are genetically diverse, like those of adult fish.     

Dual bioluminescent systems in the anglerfish genus Linophryne (Pisces: Ceratioidea)

Females of the anglerfish genus Linophryne bear barbels containing luminous organs, in addition to an escal light organ. Luminescence has been observed from the barbels of four species of Linophryne , and the morphology of the luminous organs investigated. The barbel light organs do not contain bacteria but complex paracrystalline photogenic granules. The esca contains luminous bacteria. The esca is ectodermal in origin whereas the barbel organs may be derived from the mesoderm.
The possible significance of this unique dual system of luminous organs is discussed.     

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.     

Studies on the relation between density of Longidorus elongatus and growth of sugar beet, with supplementary observations on Trichodorus spp

Longidorus elongatus attacks sugar beet on light sandy soils in the West Midlands. Severely damaged plants may die or recover, producing fanged roots. Up to 335 L. elongatus/200 g of soil were found around attacked seedling plants and were often visible to the unaided eye on the roots of freshly lifted plants. Five experiments were made and regressions computed of yield of beet, total numbers of plants and numbers of normal and fanged beet on L. elongatus numbers. Between 24% and 50% of roots per 100 L. elongatus/ 200 g were killed or became fanged. Corresponding figures for loss of plant were between 7.5% and 33%. Estimated loss of yield varied between 0.8 and 7.3 tons (2.0–18.3 t/ha)/acre/100 L. elongatus/200 g, the former where the potential yield was high and the latter where poor growing conditions hindered recovery. Large numbers of Trichodorus occurred in two trial sites and there is some evidence of competition between the two genera. Significant negative regressions for Trichodorus spp. were obtained in one trial suggesting a loss of 12% total and 17% normal roots per 100 Trichodorus spp./200 g.     

Contribution by symbiotically luminous fishes to the occurrence and bioluminescence of luminous bacteria in seawater

Seawater samples from a variety of locations contained viable luminous bacteria, but luminescence was not detectable although the system used to measure light was sensitive enough to measure light from a single, fully induced luminous bacterial cell. When the symbiotically luminous fishCleidopus gloriamaris was placed in a sterile aquarium, plate counts of water samples showed an increase in luminous colony-forming units. Luminescence also increased, decreasing when the fish was removed. Light measurements of water samples from a sterile aquarium containingPhotoblepharon palpebratus, another symbiotically luminous fish, whose bacterial symbionts have not been cultured, showed a similar pattern of increasing light which rapidly decreased upon removal of the fish. These experiments suggest that symbiotically luminous fishes release brightly luminous bacteria from light organs into their environment and may be a source of planktonic luminous bacteria. Although planktonic luminous bacteria are generally not bright when found in seawater, water samples from environments with populations of symbiotically luminous fish may show detectable levels of light.     

Relationship of the luminous bacterial symbiont of the Caribbean flashlight fish,Kryptophanaron alfredi (family Anomalopidae) to other luminous bacteria based on bacterial luciferase (luxA) genes

Flashlight fishes (family Anomalopidae) have light organs that contain luminous bacterial symbionts. Although the symbionts have not yet been successfully cultured, the luciferase genes have been cloned directly from the light organ of the Caribbean species, Kryptophanaron alfredi. The goal of this project was to evaluate the relationship of the symbiont to free-living luminous bacteria by comparison of genes coding for bacterial luciferase (lux genes). Hybridization of a luxAB probe from the Kryptophanaron alfredi symbiont to DNAs from 9 strains (8 species) of luminous bacteria showed that none of the strains tested had lux genes highly similar to the symbiont. The most similar were a group consisting of Vibrio harveyi, Vibrio splendidus and Vibrio orientalis. The nucleotide sequence of the luciferase subunit gene luxA of the Kryptophanaron alfredi symbiont was determined in order to do a more detailed comparison with published luxA sequences from Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi. The hybridization results, sequence comparisons and the mol% G+C of the Kryptophanaron alfredi symbiont luxA gene suggest that the symbiont may be considered as a new species of luminous Vibrio related to Vibrio harveyi.The nucleotide sequence reported in this article has been deposited in Genbank under accession number M36597     

The stalked eyes of Bathothauma (Mollusca, Cephalopoda)

In the larvae of some cranchiid squids the eyes are carried sideways on long stalks and also possess a presumed luminous organ. The structure of this organ is described but the only sign of light producing tissues is some possible luminescent bacteria. The stalked eye may be connected with plankton feeding at great depths, perhaps improving distance judgement. The older animals of these species carry a different form of luminous organ.     

A New Niche for Vibrio logei, the Predominant Light Organ Symbiont of Squids in the Genus Sepiola

Two genera of sepiolid squids—Euprymna, found primarily in shallow, coastal waters of Hawaii and the Western Pacific, and Sepiola, the deeper-, colder-water-dwelling Mediterranean and Atlantic squids—are known to recruit luminous bacteria into light organ symbioses. The light organ symbiont of Euprymna spp. is Vibrio fischeri, but until now, the light organ symbionts of Sepiola spp. have remained inadequately identified. We used a combination of molecular and physiological characteristics to reveal that the light organs of Sepiola affinis and Sepiola robusta contain a mixed population of Vibrio logei and V. fischeri, with V. logei comprising between 63 and 100% of the bacteria in the light organs that we analyzed. V. logei had not previously been known to exist in such symbioses. In addition, this is the first report of two different species of luminous bacteria co-occurring within a single light organ. The luminescence of these symbiotic V. logei strains, as well as that of other isolates of V. logei tested, is reduced when they are grown at temperatures above 20°C, partly due to a limitation in the synthesis of aliphatic aldehyde, a substrate of the luminescence reaction. In contrast, the luminescence of the V. fischeri symbionts is optimal above 24°C and is not enhanced by aldehyde addition. Also, V. fischeri strains were markedly more successful than V. logei at colonizing the light organs of juvenile Euprymna scolopes, especially at 26°C. These findings have important implications for our understanding of the ecological dynamics and evolution of cooperative, and perhaps pathogenic, associations of Vibrio spp. with their animal hosts.     

Ultrastructural and immunocytochemical observations of the nervous systems of three macrodasyidan gastrotrichs

The nervous systems of three macrodasyidan gastrotrichs, Dactylopodola baltica, Macrodasys caudatus and Dolichodasys elongatus, were investigated using immunocytochemistry and electron microscopy. Labelling of neural structures against serotonin revealed the presence of two pairs of cerebral cells, a dorsal cerebral connective, and paired ventral nerve cords in D. baltica. In M. caudatus and D. elongatus serotonin immunoreactivity was present in a single pair of dorsal cerebral cells and the ventral nerve cords; the dorsal connective of D. elongatus was also immunoreactive to serotonin and acetylated α‐tubulin. The presence of paired, serotonin‐like immunoreactive cells in D. baltica and other species may represent the plesiomorphic condition in Macrodasyida. The fine structure of the photoreceptors in D. baltica was also investigated to explore the potential ground pattern for eyes in the Macrodasyida. The pigmented photoreceptors of D. baltica contain a unicellular pigment cup, sheath cell and sensory receptor. The pigment cup contains numerous osmiophilic granules that presumably function to shield the eyes from downwelling light in the red part of the spectrum. Projecting into the pigment cup and sheath cell are numerous microvilli from a bipolar sensory cell. A single sensory cell may represent the plesiomorphic condition in Macrodasyida, with multiplication of sensory cells representative of more derived taxa.     

Inception of bioluminescent symbiosis in early developmental stages of the deep-sea fish,Coelorinchus kishinouyei (Gadiformes: Macrouridae)

Larvae and juveniles of the macrourid fish Coelorinchus kishinouyei, captured from the near-bottom habitat (ca. 1–10 m above the seafloor) at 186 to 500 m depth in Suruga Bay, Honshu, Japan, were examined for the presence, developmental state, and bacterial colonization of the fish’s internal ventral light organ. The specimens ranged from 3.6 mm to 8.5 mm head length, and all exhibited an external cluster of melanophores expanding anteriorly from around the anus that is thought to indicate the presence of an internal light organ. Histological analysis revealed the presence of a light organ in all examined specimens. In smaller specimens, the light organ was seen as a small nub of tissue associated with the intestine near the anus; the light organ gradually elongated anteriorly in larger specimens to form a bean-shaped structure composed of hollow, finger-like chambers. Bacteria were present within the light organ chambers of some, but not all larvae and all juveniles. In light organs not yet colonized by bacteria, the chambers exhibited a generally uniform appearance over their entire length. In colonized light organs, the bacteria were consistently present at the anterior-most tips of the chambers; furthermore, cells comprising chambers colonized by bacteria were swollen, and upon bacterial colonization the orientation of the chambers began to change from anterior–posterior to dorsal–ventral. The colonizing bacteria were identified as Photobacterium kishitanii based on sequence analysis of the luxA gene. These results suggest that formation of the light organ in C. kishinouyei begins during the fish’s pelagic phase, but that bacterial colonization of the light organ occurs after the larvae have reached the near-bottom habitat. Furthermore, colonization of the nascent light organ by P. kishitanii induces morphogenetic changes in the light organ.     

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.     

Populations of Longidorus elongatus (Nematoda: Longidoridae) in two New Zealand soils under grazed pasture

Abstract

Information on the “needle nematode” Longidorus elongatus in New Zealand pastures has hitherto been limited. Monthly sampling of Pukepuke black sand and Manawatu fine sandy loam yielded L. elongatus populations up to 87 500 m^?2 (February) and 21 600 m^?2 (August). First stage juveniles appeared when soil temperature exceeded 15°C; females dominated populations every month and males were rare. Differences in abundance between soils may reflect the pore space available to these relatively large nematodes. Specimens survived 24 weeks storage at 5 and 15°C. While abundance tended to decline with depth, at 30–40 cm depth in Pukepuke sand, numbers increased, perhaps through the impact of groundwater levels on rooting patterns. In Pukepuke sand, plant species were associated with significantly different populations of L. elongatus, with Trifolium repens and T. subter‐raneum supporting more than Lolium perenne. More L. elongatus were found in grazed pasture with lower plant available P.     

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

SYNOPSIS. The association of the sepiolid squid Euprymna scolopeswith its marine luminous bacterial symbiont Vibrio fischeriis an emerging model system to study the initiation and developmentof bacterial symbioses in higher animals, in particular theinfluence of bacteria on the ontogenic development of symbiotic-specifichost tissues. Experiments comparing the development of juvenilesquid infected with symbiotic V. fischeri with that of uninfectedjuveniles suggest postembryonic development of the light organrequires cell-cell interactions with the bacterial symbionts.The presence of symbiotic bacteria induces specific morphologicalchanges by affecting such fundamental processes as cell deathand cell differentiation. The surface of the juvenile organis largely composed of ciliated cells that appear to facilitateinfection of the light organ. These cells begin to undergo celldeath within hours of infection with symbiotic V. fischeri.Within three days the epithelial cells that form the bacteriacontainingcrypts of the light organ increase in size; these cells do notappear mitotically active, and may represent a terminally differentiatedstate. The light organs of uninfected juvenile E. scolopes,however, do not exhibit any of these early postembryonic developmentalevents but remain in a state of arrested morphogenesis.     

Metabolic engineering of Synechococcus elongatus for photoautotrophic production of mannitol

With multiple applications in food, pharmaceutical, and chemical industries as antioxidant or nonmetabolizable sweetener; the bioproduction of d -mannitol is gaining global attention, especially with photosynthetic organisms as hosts. Considering the sustainability prospects, the current work encompasses metabolic engineering of a widely used cyanobacterial strain, Synechococcus elongatus PCC 7942, and two newly isolated fast-growing cyanobacterial strains; S. elongatus PCC 11801 and S. elongatus PCC 11802, for mannitol production. We engineered these strains with a two-step pathway by cloning genes for mannitol-1-phosphate dehydrogenase (mtlD) and mannitol-1-phosphatase (mlp), where the mtlD expression was under the control of different promoters from PCC 7942, namely, P_rbc225, P_cpcB300, P_cpcBm1, P_rbcLm17, and P_rbcLm15. The strains were tested under the “switch conditions,” where the growth conditions were switched after the first 3 days, thereby resulting in differential promoter activity. Among the engineered strains of PCC 11801 and PCC 11802, the strains possessing P_rbc225-mtlD module produced relatively high mannitol titers of 401 ± 18 mg/L and 537 ± 18 mg/L, respectively. The highest mannitol titer of 701 ± 15 mg/L (productivity 60 mg/L.d, yield 895 µM/OD₇₃₀) was exhibited by the engineered strain of PCC 7942 expressing P_cpcB300-mtlD module. It is by far the highest obtained mannitol yield from the engineered cyanobacteria.