Kewalo Research Featured on Journal Covers

(Portrait of He'e, the Hawaiian Day Octopus)

Eric Roettinger

This image of the Hawaiian Day Octopus, also known as he'e, was submitted to the EMBO Journal Cover Contest 2011 by Eric Roettinger, a post-doctoral research fellow in the Martindale Lab. His image was selected as the cover image for the June 1, 2011 issue (Volume 30, Number 11) of the EMBO Journal. It was also selected as one of the "Favorites of the Jury" for the 2011 Cover Contest.

Ventralization of an indirect developing hemichordate by NiCl(2) suggests a conserved mechanism of dorso-ventral (D/V) patterning in Ambulacraria (hemichordates and echinoderms)

Eric Roettinger and Mark Q. Martindale

One of the earliest steps in embryonic development is the establishment of the future body axes. Morphological and molecular data place the Ambulacraria (echinoderms and hemichordates) within the Deuterostomia and as the sister taxon to chordates. Extensive work over the last decades in echinoid (sea urchins) echinoderms has led to the characterization of gene regulatory networks underlying germ layer specification and axis formation during embryogenesis. However, with the exception of recent studies from a direct developing hemichordate (Saccoglossus kowalevskii), very little is known about the molecular mechanism underlying early hemichordate development. Unlike echinoids, indirect developing hemichordates retain the larval body axes and major larval tissues after metamorphosis into the adult worm. In order to gain insight into dorso-ventral (D/V) patterning, we used nickel chloride (NiCl(2)), a potent ventralizing agent on echinoderm embryos, on the indirect developing enteropneust hemichordate, Ptychodera flava. Our present study shows that NiCl(2) disrupts the D/V axis and induces formation of a circumferential mouth when treated before the onset of gastrulation. Molecular analysis, using newly isolated tissue-specific markers, shows that the ventral ectoderm is expanded at expense of dorsal ectoderm in treated embryos, but has little effect on germ layer or anterior-posterior markers. The resulting ventralized phenotype, the effective dose, and the NiCl(2) sensitive response period of Ptychodera flava, is very similar to the effects of nickel on embryonic development described in larval echinoderms. These strong similarities allow one to speculate that a NiCl(2) sensitive pathway involved in dorso-ventral patterning may be shared between echinoderms, hemichordates and a putative ambulacrarian ancestor. Furthermore, nickel treatments ventralize the direct developing hemichordate, S. kowalevskii indicating that a common pathway patterns both larval and adult body plans of the ambulacrarian ancestor and provides insight in to the origin of the chordate body plan.

Cover photos by Aldine Amiel, Eric Roettinger, & Mattias Ormestad (all of www.kahikai.org); CSLM by Aldine Amiel of the Seaver lab.

A Twist in Time—The Evolution of Spiral Cleavage in the Light of Animal Phylogeny

Andreas Hejnol

β-catenin and Early Development in the Gastropod, Crepidula fornicata

Jonathan Q. Henry, Kimberly J. Perry, and Mark Q. Martindale

Cell Lineage and Fate Map of the Primary Somatoblast of the Polychaete Annelid Capitella teleta

Néva P. Meyer and Elaine C. Seaver

Invited cover image by Eric Roettinger.

Close-up of the mouth region of the cushion star, Culcita novaeguineae, an echinoderm found in Hawaiian waters. The photographer, Eric Roettinger, works as a postdoc in Mark Martindales lab at the Kewalo Marine Laboratory (Hawai'i). Visit www.kahikai.com or www.livingonabeach.com for more of Eric's fascinating photographic portraits of the ocean and the creatures in it.

Centralization of the Deuterostome Nervous System Predates Chordates

Marc Nomaksteinsky, Eric Roettinger, Heloise D. Dufour, Zoubida Chettouh, Chris J. Lowe, Mark Q. Martindale and Jean-Francois Brunet

The origin of the chordate central nervous system (CNS) is unknown. One theory is that a CNS was present in the first bilaterian and that it gave rise to both the ventral cord of protostomes and the dorsal cord of deuterostomes. Another theory proposes that the chordate CNS arose by a dramatic process of dorsalization and internalization from a diffuse nerve net coextensive with the skin of the animal, such as enteropneust worms (Hemichordata, Ambulacraria) are supposed to have. We show here that juvenile and adult enteropneust worms in fact have a bona fide CNS, i.e., dense agglomerations of neurons associated with a neuropil, forming two cords, ventral and dorsal. The latter is internalized in the collar as a chordate-like neural tube. Contrary to previous assumptions, the greater part of the adult enteropneust skin is nonneural, although elements of the peripheral nervous system (PNS) are found there. We use molecular markers to show that several neuronal types are anatomically segregated in the CNS and PNS. These neuroanatomical features, whatever their homologies with the chordate CNS, imply that nervous system centralization predates the evolutionary separation of chordate and hemichordate lineages.

The Hawaiian Bobtail Squid (Euprymna scolopes): A Model to Study the Molecular Basis of Eukaryote-Prokaryote Mutualism and the Development and Evolution of Morphological Novelties in Cephalopods

Patricia N. Lee, Margaret J. McFall-Ngai, Patrick Callaerts, and H. Gert de Couet

The Hawaiian bobtail squid, Euprymna scolopes, is a cephalopod whose small size, short lifespan, rapid growth, and year-round availability make it suitable as a model organism. E. scolopes is studied in three principal contexts: (1) as a model of cephalopod development; (2) as a model of animal-bacterial symbioses; and (3) as a system for studying adaptations of tissues that interact with light. E. scolopes embryos can be obtained continually and can be reared in the laboratory over an entire generation. The embryos and protective chorions are optically clear, facilitating in situ developmental observations, and can be manipulated experimentally. Many molecular protocols have been developed for studying E. scolopes development. This species is best known, however, for its symbiosis with the luminous marine bacterium Vibrio fischeri and has been used to study determinants of symbiont specificity, the influence of symbiosis on development of the squid light organ, and the mechanisms by which a stable association is achieved. Both partners can be grown independently under laboratory conditions, a feature that offers the unusual opportunity to manipulate the symbiosis experimentally. Molecular and genetic tools have been developed for V. fischeri, and a large expressed sequence tag (EST) database is available for the host symbiotic tissues. Additionally, comparisons between light organ form and function to those of the eye can be made. Both types of tissue interact with light, but have divergent embryonic development. As such, they offer an opportunity to study the molecular basis for the evolution of morphological novelties

Comb Jellies (Ctenophora): A Model for Basal Metazoan Evolution and Development

Kevin Pang and Mark Q. Martindale

Ctenophores, or comb jellies, are a group of marine organisms whose unique biological features and phylogenetic placement make them a key taxon for understanding animal evolution. These gelatinous creatures are clearly distinct from cnidarian medusae (i.e., jellyfish). Key features present in the ctenophore body plan include biradial symmetry, an oral-aboral axis delimited by a mouth and an apical sensory organ, two tentacles, eight comb rows composed of interconnected cilia, and thick mesoglea. Other morphological features include definitive muscle cells, a nerve net, basal lamina, a sperm acrosome, and light-producing photocytes. Aspects of their development made them attractive to experimental embryologists as early as the 19th century. Recently, because of their role as an invasive species, studies on their role in ecology and fisheries-related fields have increased. Although the phylogenetic placement of ctenophores with respect to other animals has proven difficult, it is clear that, along with poriferans, placozoans, and cnidarians, ctenophores are one of the earliest diverging extant animal groups. It is important to determine if some of the complex features of ctenophores are examples of convergence or if they were lost in other animal branches. Because ctenophores are amenable to modern technical approaches, they could prove to be a highly useful emerging model.

Developmental expression of homeobox genes in the ctenophore Mnemiopsis leidyi

Kevin Pang and Mark Q. Martindale

Homeobox genes are a large family of genes that encode helix-turn-helix transcription factors that play fundamental roles in such developmental processes including body axis formation and cell specification. They have been found in a wide variety of organisms, from fungi to plants and animals, with some classes being specific to the Metazoa. While it was once thought that organismal complexity was tied to gene complexity, sequencing of genomes from a cnidarian, poriferan, and placozoan have shown no clear correlation. However, little attention has been paid to ctenophores, another early branching taxon. Ctenophores are mostly pelagic marine animals, with complex morphological features, so understanding the gene content and expression of this nonbilaterian phylum is of key interest to evolutionary biology. Expression information from developmental genes in ctenophores is sparse. In this study, we isolated seven homeobox genes from the ctenophore Mnemiopsis leidyi and examined their expression through development. Phylogenetic analyses of these genes placed four in the ANTP class and three in the PRD class. These are the first reported full-length PRD class genes, although our analyses could not place them into specific families. We have found that most of these homeobox genes begin expression at gastrulation, and their expression patterns suggest a possible role in patterning of the tentacle apparati and pharynx.

Invited cover image by Eric Roettinger.

A close encounter with Dendrodoris nigra, a nudibranch from Hawaiian shore lines. The photographer, Eric Roettinger, is a postdoctoral fellow in Mark Martindale's group at the University of Hawaii Kewalo Marine Laboratory. Besides his work on evolutionary and developmental biology, Eric has a keen interest in portraying the fascinating, colourful and highly endangered marine world, and is an active contributor to the Kahi Kai ("one ocean") online community Kahi Kai. For more amazing photographs of sea creatures, be sure to also visit the new website of Eric and his colleagues.

Broad phylogenomic sampling improves resolution of the animal tree of life

Dunn CW, Hejnol A, Matus DQ, Pang K, Browne WE, Smith SA, Seaver E, Rouse GW, Obst M, Edgecombe GD, Sørensen MV, Haddock SH, Schmidt-Rhaesa A, Okusu A, Kristensen RM, Wheeler WC, Martindale MQ, Giribet G

Long-held ideas regarding the evolutionary relationships among animals have recently been upended by sometimes controversial hypotheses based largely on insights from molecular data. These new hypotheses include a clade of moulting animals (Ecdysozoa) and the close relationship of the lophophorates to molluscs and annelids (Lophotrochozoa). Many relationships remain disputed, including those that are required to polarize key features of character evolution, and support for deep nodes is often low. Phylogenomic approaches, which use data from many genes, have shown promise for resolving deep animal relationships, but are hindered by a lack of data from many important groups. Here we report a total of 39.9 Mb of expressed sequence tags from 29 animals belonging to 21 phyla, including 11 phyla previously lacking genomic or expressed-sequence-tag data. Analysed in combination with existing sequences, our data reinforce several previously identified clades that split deeply in the animal tree (including Protostomia, Ecdysozoa and Lophotrochozoa), unambiguously resolve multiple long-standing issues for which there was strong conflicting support in earlier studies with less data (such as velvet worms rather than tardigrades as the sister group of arthropods), and provide molecular support for the monophyly of molluscs, a group long recognized by morphologists. In addition, we find strong support for several new hypotheses. These include a clade that unites annelids (including sipunculans and echiurans) with nemerteans, phoronids and brachiopods, molluscs as sister to that assemblage, and the placement of ctenophores as the earliest diverging extant multicellular animals. A single origin of spiral cleavage (with subsequent losses) is inferred from well-supported nodes. Many relationships between a stable subset of taxa find strong support, and a diminishing number of lineages remain recalcitrant to placement on the tree.

Phylogenetic analysis of lineage relationships among hyperiid amphipods as revealed by examination of the mitochondrial gene, cytochrome oxidase I (COI)

William E. Browne, Steven H. D. Haddock, and Mark Q. Martindale

The Hox gene complement of a pelagic chaetognath, Flaccisagitta enflata

David Q. Matus, Kenneth M. Halanych, and Mark Q. Martindale

Homology of ciliary bands in Spiralian Trochophores

Jonathan Q. Henry, Andreas Hejnol, Kimberly J. Perry, and Mark Q. Martindale

High-resolution fate map of the snail Crepidula fornicata: The origins of ciliary bands, nervous system, and muscular elements

Andreas Hejnol, Mark Q. Martindale, and Jonathan Q. Henry

The littorinimorph gastropod Crepidula fornicata shows a spiralian cleavage pattern and has been the subject of studies in experimental embryology, cell lineage, and the organization of the larval nervous system. To investigate the contribution of early blastomeres to the veliger larva, we used intracellular cell lineage tracers in combination with high-resolution confocal imaging. This study corroborates many features derived from other spiralian fate maps (such as the origins of the hindgut and mesoderm from the 4d mesentoblast), but also yields new findings, particularly with respect to the origins of internal structures, such as the nervous system and musculature that have never been described in detail. The ectomesoderm in C. fornicata is mainly formed by micromeres of the 3rd quartet (principally 3a and 3b), which presumably represents a plesiomorphic condition for molluscs. The larval central nervous system is mainly formed by the micromeres of the 1st and 2nd quartet, of which 1a, 1c, and 1d form the anterior apical ganglion and nerve tracks to the foot and velum, and 2b and 2d form the visceral loop and the mantle cell. Our study shows that both first and second velar ciliary bands are generated by the same cells that form the prototroch in other spiralians and apparently bear no homology to the metatroch found in annelids.

Expression of otd orthologs in the amphipod crustacean, Parhyale hawaiensis

Browne WE, Schmid BG, Wimmer EA, Martindale MQ

The arthropod head is a complex metameric structure. In insects, orthodenticle (otd) functions as a 'head gap gene' and plays a significant role in patterning and development of the anterior head ectoderm, the protocerebrum, and the ventral midline. In this study, we characterize the structure and developmental deployment of two otd paralogs in the amphipod crustacean, Parhyale hawaiensis. Photd1 is initially expressed at gastrulation through germband stages in a bilaterally symmetric, restricted region of the anterior head ectoderm and also in a single column of cells along the ventral midline. Late in embryogenesis, Photd1 is expressed within the developing anterior brain and the expression along the embryonic midline has become restricted to a stereotypic group of segmentally reiterated cells. The second ortholog Photd2, however, has a unique temporal-spatial expression pattern and is not detected until after the head lobes have been organized in the developing ectoderm of the germband during late germband stages. Anteriorly, Photd2 is coincident with the Photd1 head expression domain; however, Photd2 is not detected along the ventral midline during formation of the germband and only appears in the ventral midline late in embryonic development in a restricted group of cells distinct from those expressing Photd1. The early expression of Photd1 in the anterior head ectoderm is consistent with a role as a head gap gene. The more posterior expression of Photd1 is suggestive of a role in patterning the embryonic ventral midline. Photd2 expression appears too late to play a role in early head patterning but may contribute to latter patterning in restricted regions of both the head and the ventral midline. The comparative analysis of otd reveals the divergence of gene expression and gene function associated with duplication of this important developmental gene.

Stages of embryonic development in the amphipod crustacean, Parhyale hawaiensis

Browne WE, Price AL, Gerberding M, Patel NH

Studying the relationship between development and evolution and its role in the generation of biological diversity has been reinvigorated by new techniques in genetics and molecular biology. However, exploiting these techniques to examine the evolution of development requires that a great deal of detail be known regarding the embryonic development of multiple species studied in a phylogenetic context. Crustaceans are an enormously successful group of arthropods and extant species demonstrate a wide diversity of morphologies and life histories. One of the most speciose orders within the Crustacea is the Amphipoda. The embryonic development of a new crustacean model system, the amphipod Parhyale hawaiensis, is described in a series of discrete stages easily identified by examination of living animals and the use of commonly available molecular markers on fixed specimens. Complete embryogenesis occurs in 250 h at 26 degrees C and has been divided into 30 stages. This staging data will facilitate comparative analyses of embryonic development among crustaceans in particular, as well as between different arthropod groups. In addition, several aspects of Parhyale embryonic development make this species particularly suitable for a broad range of experimental manipulations.

Unexpected complexity of Wnt gene family in a sea anemone

Kusserow, A., Pang, K., Sturm, C., Hrouda, M., Lentfer, J., Technau, U., Hobmayer, B., Martindale, M.Q., and Holstein, T.W.

The Wnt gene family encodes secreted signalling molecules that control cell fate in animal development and human diseases. Despite its significance, the evolution of this metazoan-specific protein family is unclear. In vertebrates, twelve Wnt subfamilies were defined, of which only six have counterparts in Ecdysozoa (for example, Drosophila and Caenorhabditis). Here, we report the isolation of twelve Wnt genes from the sea anemone Nematostella vectensis, a species representing the basal group within cnidarians. Cnidarians are diploblastic animals and the sister-group to bilaterian metazoans. Phylogenetic analyses of N. vectensis Wnt genes reveal a thus far unpredicted ancestral diversity within the Wnt family. Cnidarians and bilaterians have at least eleven of the twelve known Wnt gene subfamilies in common; five subfamilies appear to be lost in the protostome lineage. Expression patterns of Wnt genes during N. vectensis embryogenesis indicate distinct roles of Wnts in gastrulation, resulting in serial overlapping expression domains along the primary axis of the planula larva. This unexpectedly complex inventory of Wnt family signalling factors evolved in early multi-cellular animals about 650 million years (Myr) ago, predating the Cambrian explosion by at least 100 Myr (refs 5, 8). It emphasizes the crucial function of Wnt genes in the diversification of eumetazoan body plans.

Rapid behavioral responses of an invertebrate larva to dissolved settlement cue

Michael G. Hadfield and M. A. R. Koehl

Larvae of the nudibranch Phestilla sibogae were used to study whether a natural dissolved settlement cue (from their prey, Porites compressa, an abundant coral on Hawaiian reefs) induces behavioral responses that can affect larval transport to suitable settlement sites. As cue and larvae are mixed in the turbulent flow over a reef, cue is distributed in fine-scale filaments that the larva experiences as rapid (seconds) on/off encounters. To examine larval responses in this setting, individual larvae were tethered in a small flume with flow simulating water velocity relative to a freely swimming larva, and their responses to realistic temporal patterns of cue encounter were videotaped. Competent larvae quickly ceased swimming in cue filaments and resumed swimming after exiting filaments. The threshold cue concentration eliciting a response was 3%–17% of concentrations within heads of P. compressa in nature. When moving freely in filtered seawater, competent larvae swam along straight paths in all directions at ~0.2 cm s–1, whereas in water conditioned by P. compressa, most ceased swimming and sank at ~0.1 cm s–1. The ability of larvae to rapidly respond (by sinking) to brief encounters with dissolved settlement cues can enhance their rapid transport to the substratum, even in wave-driven turbulent flow.

Investigating the origins of triploblasty: "Mesodermal" gene expression in a diploblastic animal, the sea anemone, Nematostella vectensis (phylum, Cnidaria; Class Anthozoa)

Mark Q. Martindale, Kevin Pang, and John R. Finnerty

Mesoderm played a crucial role in the radiation of the triploblastic Bilateria, permitting the evolution of larger and more complex body plans than in the diploblastic, non-bilaterian animals. The sea anemone Nematostella is a non-bilaterian animal, a member of the phylum Cnidaria. The phylum Cnidaria (sea anemones, corals, hydras and jellyfish) is the likely sister group of the triploblastic Bilateria. Cnidarians are generally regarded as diploblastic animals, possessing endoderm and ectoderm, but lacking mesoderm. To investigate the origin of triploblasty, we studied the developmental expression of seven genes from Nematostella whose bilaterian homologs are implicated in mesodermal specification and the differentiation of mesodermal cell types (twist, snailA, snailB, forkhead, mef2, a GATA transcription factor and a LIM transcription factor). Except for mef2, the expression of these genes is largely restricted to the endodermal layer, the gastrodermis. mef2 is restricted to the ectoderm. The temporal and spatial expression of these 'mesoderm' genes suggests that they may play a role in germ layer specification. Furthermore, the predominantly endodermal expression of these genes reinforces the hypothesis that the mesoderm and endoderm of triploblastic animals could be derived from the endoderm of a diploblastic ancestor. Alternatively, we consider the possibility that the diploblastic condition of cnidarians is a secondary simplification, derived from an ancestral condition of triploblasty.

Invited cover image by Elaine Seaver.

Cover Image: Ventral view of a late stage larva of the polychaete Capitella capitata. The anterior end is marked by two orange larval eye spots. Annelids are one of the three major segmented animal groups and represent an important study group for understanding the evolution of body plan segmentation in the Metazoa. At this stage the first 12 segments are already formed and additional segments are added after metamorphosis during juvenile and adult stages. Image provided by Elaine Seaver (Kewalo Marine Lab, University of Hawai?i

Characterization of gill-specific genes of the acorn worm Ptychodera flava

Noko Okai, Kunifumi Tagawa, Tom Humphreys, Nori Satoh, Michio Ogasawara

Acorn worms are hemichordate deuterostomes that have remarkable gills thought to be homologous to pharyngeal gills in urochordates and cephalochordates, and pharyngeal pouches in vertebrates. In search of molecular keys to analyzing the origin and evolution of the anterior gut and neck region of the chordate body, the present study isolated cDNA clones for six gill-specific genes, designated PfG1 to PfG6, from Ptychodera flava using differential screening of a cDNA library of RNA from gills. Northern blotting confirmed that these genes were all expressed only in the gills. In situ hybridization showed that the expression of these genes is limited to the endodermally derived columnar epithelium of the pharynx. PfG1 encodes a 42-kDa polypeptide containing sequence similar to D-domains, protein domains characteristic of extracellular proteins. Expression of PfG1 is localized in a delimited pattern along the columnar epithelium of the inner gill apparatus. Expression in the epibranchial ridge appears as two stripes running longitudinally in the epithelium just lateral of the midline. A stripe of expression also appears in a slightly posterior portion on the curve of each band of columnar epithelium on the pharyngeal surface of the secondary gill bars. The five other gill-specific genes, PfG2 to PfG6, encode a family of C-type lectin polypeptides that appear to be secreted proteins. PfG2 to PfG6 are also expressed in the columnar epithelium of the epibranchial ridge as two parallel stripes, but at the lateral margin of the ridge. One of the genes, PfG6, is additionally expressed in the innermost curve of the epithelium on the pharyngeal surface of each secondary gill bar. The localization of expression of PfPax1/9, a gill-specific transcription factor gene, was examined and shown to also be primarily in the endodermal columnar epithelium on the pharyngeal faces of the gill bars. On the secondary gill bars, where PfG1 and PfG6 are also expressed in the columnar epithelium, PfPax1/9 is expressed in the anterior and posterior portions but signal is not evident in the epithelium on the central, innermost curve of the gill bar. The anterior domain of PfPax1/9 expression is more extensive but overlaps the anterior domain of PfG1 expression, whereas its posterior domain of expression is more posterior and complementary to that of PfG6.

Oxygen-utilizing reactions and symbiotic colonization of the squid light organ by Vibrio fischeri

Edward G Ruby, Margaret J McFall-Ngai

A major goal in microbiology is to understand the processes by which bacteria successfully colonize host tissue. Although a wealth of studies focusing on pathogenic microorganisms has revealed much about the rare interactions that result in disease, far less is known about the regulation of the ubiquitous, long-term, cooperative associations of bacteria with their animal hosts.

Cellular and subcellular structure of anterior sensory pathways in Phestilla sibogae (Gastropoda, Nudibranchia)

Boudko DY, Switzer-Dunlap M, Hadfield MG

Two sensory-cell types, subepithelial sensory cells (SSCs) and intraepithelial sensory cells (ISCs), were identified in the anterior sensory organs (ASO: pairs of rhinophores and oral tentacles, and the anterior field formed by the oral plate and cephalic shield) of the nudibranch Phestilla sibogae after filling through anterior nerves with the neuronal tracers biocytin and Lucifer Yellow. A third type of sensory cells, with subepithelial somata and tufts of stiff-cilia (TSCs, presumably rheoreceptors), was identified after uptake of the mitochondrial dye DASPEI. Each sensory-cell type has a specific spatial distribution in the ASO. The highest density of ISCs is in the oral tentacles (approximately 1,200/mm2), SSCs in the middle parts of the rhinophores (>4,000/mm2), and TSCs in the tips of cephalic tentacles (100/mm2). These morphologic data, together with electrophysiologic evidence for greater chemical sensitivity of the rhinophores than the oral tentacles (Murphy and Hadfield [1997] Comp. Biochem. Physiol. 118A:727-735; Boudko et al. [1997] Soc. Neurosci. Abstr. 23:1787), led us to conclude that the two pairs of chemosensory tentacles serve different chemosensory functions in P. sibogae; i.e., ISCs and the oral tentacles serve contact- or short-distance chemoreception, and SSCs and the rhinophores function for long-distance chemoreception or olfaction. If this is true, then the ISC subsystem probably represents an earlier stage in the evolution and adaptations of gastropod chemosensory biology, whereas among the opisthobranchs, the SSC subsystem evolved with the rhinophores from ancestral cephalaspidean opisthobranchs.

Sampling the light-organ microenvironment of Euprymna scolopes: Description of a population of host cells in association with the bacterial symbiont Vibrio fischeri

S. V. Nyholm and M. J. McFall-Ngai

The symbiosis between the squid Euprymna scolopes and the luminous bacterium Vibrio fischeri has a pronounced diel rhythm, one component of which is the venting of the contents of the light organ into the surrounding seawater each day at dawn. In this study, we explored the use of this behavior to sample the microenvironment of the light-organ crypts. Intact crypt contents, which emerge from the lateral pores of the organ as a thick paste-like exudate, were collected from anesthetized host animals that had been exposed to a light cue. Microscopy revealed that the expelled material is composed of a conspicuous population of host cells in association with the bacterial symbionts, all of which are embedded in a dense acellular matrix that strongly resembles the bacteria-based biofilms described in other systems. Assays of the viability of expelled crypt cells revealed no dead bacterial symbionts and a mixture of live and dead host cells. Analyses of the ultrastructure, biochemistry, and phagocytic activity of a subset of the host cell population suggested that some of these cells are macrophage-like molluscan hemocytes.

Bobtail squid and their luminous bacteria: when first they meet

McFall-Ngai, M.J., and E.G. Ruby

Stimulation of metamorphosis in the polychaete Hydroides elegans Haswell (Serpulidae)

E. Carpizo-Ituarte and M. G. Hadfield

The serpulid polychaete Hydroides elegans is a common, cosmopolitan warm-water biofouling organism. Competent larvae of H. elegans metamorphose rapidly after induction by marine biofilms. Only 15 min after coming in contact with the metamorphic cue, larvae have completed secretion of the primary tube; secretion of the secondary, calcareous tube begins 1.5 h after the primary tube has been deposited. Metamorphosis is characterized by disappearance of the prototroch and differentiation of the tentacular crown in the head region, the collar and thoracic membrane in the thoracic region, and the pygidium at the tip of the abdomen. These morphogenetic events were used to gauge the responses of larvae to biofilms, as well as to the artificial inducers Cs+ and K+. A maximal metamorphic response to the two ions requires exposure to different concentrations and durations, i.e., a 3-h pulse of 10 mM CsCl, or a 24-h continuous exposure to 50 mM excess KCl. The metamorphic response to Cs+ or K+ is much slower than the response to biofilms, demonstrating that the tissues respond differently to artificial inducers. The differences in the kinetics of the responses to the natural and cationic inducers suggest that the induction mechanisms are not the same. When these artificial inducers were used, some, but not all, of the metamorphosed juveniles never attached to the substratum or secreted a primary tube, probably as a result of secondary effects of the ions on processes of tube formation. The exact mechanisms by which Cs+ and excess K+ induce metamorphosis are still unclear, although we assume, as do others, that these agents act by depolarizing the membranes of excitable sensory cells and not by interacting with specific receptors.

Symbiont recognition and subsequent morphogenesis as early events in an animal-bacterial mutualism

McFall-Ngai MJ, Ruby EG

Bacterial colonization of the developing light organ of the squid Euprymna scolopes is shown to be highly specific, with the establishment of a successful association resulting only when the juvenile host is exposed to seawater containing one of a subset of Vibrio fischeri strains. Before a symbiotic infection the organ has elaborate epithelial structures covered with cilia and microvilli that are involved in the transfer of bacteria to the incipient symbiotic tissue. These structures regressed within days following infection; however, they were retained in uninfected animals, suggesting that the initiation of symbiosis influences, and is perhaps a prerequisite for, the normal developmental program of the juvenile host.