Cnidarian Larvae Research Articles

Live imaging of Aiptasia larvae, a model system for coral and anemone bleaching, using a simple microfluidic device

Coral reefs, and their associated diverse ecosystems, are of enormous ecological importance. In recent years, coral health has been severely impacted by environmental stressors brought on by human activity and climate change, threatening the extinction of several major reef ecosystems. Reef damage is mediated by a process called ‘coral bleaching’ where corals, sea anemones, and other cnidarians lose their photosynthetic algal symbionts (family Symbiodiniaceae) upon stress induction, resulting in drastically decreased host energy harvest and, ultimately, coral death. The mechanism by which this critical cnidarian-algal symbiosis is lost remains poorly understood. The larvae of the sea anemone, Exaiptasia pallida (commonly referred to as ‘Aiptasia’) are an attractive model organism to study this process, but they are large (∼100 mm in length, ∼75 mm in diameter), deformable, and highly motile, complicating long-term imaging and limiting study of this critical endosymbiotic relationship in live organisms. Here, we report ‘Traptasia’, a simple microfluidic device with multiple traps designed to isolate and image individual, live larvae of Aiptasia and their algal symbionts over extended time courses. Using a trap design parameterized via fluid flow simulations and polymer bead loading tests, we trapped Aiptasia larvae containing algal symbionts and demonstrated stable imaging for >10 hours. We visualized algae within Aiptasia larvae and observed algal expulsion under an environmental stressor. To our knowledge, this device is the first to enable time-lapsed, high-throughput live imaging of cnidarian larvae and their algal symbionts and, in further implementation, could provide important insights into the cellular mechanisms of cnidarian bleaching under different environmental stressors. The ‘Traptasia’ device is simple to use, requires minimal external equipment and no specialized training to operate, and can easily be adapted using the trap optimization data presented here to study a variety of large, motile organisms.

Development and Symbiosis Establishment in the Cnidarian Endosymbiosis Model Aiptasia sp.

Symbiosis between photosynthetic algae and heterotrophic organisms is widespread. One prominent example of high ecological relevance is the endosymbiosis between dinoflagellate algae of the genus Symbiodinium and reef-building corals, which typically acquire symbionts anew each generation during larval stages. The tropical sea anemone Aiptasia sp. is a laboratory model system for this endosymbiosis and, similar to corals, produces non-symbiotic larvae that establish symbiosis by phagocytosing Symbiodinium from the environment into the endoderm. Here we generate the first overview of Aiptasia embryogenesis and larval development and establish in situ hybridization to analyze expression patterns of key early developmental regulators. Next, we quantify morphological changes in developing larvae and find a substantial enlargement of the gastric cavity over time. Symbiont acquisition starts soon after mouth formation and symbionts occupy a major portion of the host cell in which they reside. During the first 14 days of development, infection efficiency remains constant while in contrast, localization of phagocytosed symbionts changes, indicating that the occurrence of functional phagocytosing cells may be developmentally regulated. Taken together, here we provide the essential framework to further develop Aiptasia as a model system for the analysis of symbiosis establishment in cnidarian larvae at the molecular level.

Trehalose is a chemical attractant in the establishment of coral symbiosis.

Coral reefs have evolved with a crucial symbiosis between photosynthetic dinoflagellates (genus Symbiodinium) and their cnidarian hosts (Scleractinians). Most coral larvae take up Symbiodinium from their environment; however, the earliest steps in this process have been elusive. Here we demonstrate that the disaccharide trehalose may be an important signal from the symbiont to potential larval hosts. Symbiodinium freshly isolated from Fungia scutaria corals constantly released trehalose (but not sucrose, maltose or glucose) into seawater, and released glycerol only in the presence of coral tissue. Spawning Fungia adults increased symbiont number in their immediate area by excreting pellets of Symbiodinium, and when these naturally discharged Symbiodinium were cultured, they also released trehalose. In Y-maze experiments, coral larvae demonstrated chemoattractant and feeding behaviors only towards a chamber with trehalose or glycerol. Concomitantly, coral larvae and adult tissue, but not symbionts, had significant trehalase enzymatic activities, suggesting the capacity to utilize trehalose. Trehalase activity was developmentally regulated in F. scutaria larvae, rising as the time for symbiont uptake occurs. Consistent with the enzymatic assays, gene finding demonstrated the presence of a trehalase enzyme in the genome of a related coral, Acropora digitifera, and a likely trehalase in the transcriptome of F. scutaria. Taken together, these data suggest that adult F. scutaria seed the reef with Symbiodinium during spawning and the exuded Symbiodinium release trehalose into the environment, which acts as a chemoattractant for F. scutaria larvae and as an initiator of feeding behavior- the first stages toward establishing the coral-Symbiodinium relationship. Because trehalose is a fixed carbon compound, this cue would accurately demonstrate to the cnidarian larvae the photosynthetic ability of the potential symbiont in the ambient environment. To our knowledge, this is the first report of a chemical cue attracting the motile coral larvae to the symbiont.

The genetics of colony form and function in Caribbean Acropora corals.

BackgroundColonial reef-building corals have evolved a broad spectrum of colony morphologies based on coordinated asexual reproduction of polyps on a secreted calcium carbonate skeleton. Though cnidarians have been shown to possess and use similar developmental genes to bilaterians during larval development and polyp formation, little is known about genetic regulation of colony morphology in hard corals. We used RNA-seq to evaluate transcriptomic differences between functionally distinct regions of the coral (apical branch tips and branch bases) in two species of Caribbean Acropora, the staghorn coral, A. cervicornis, and the elkhorn coral, A. palmata.ResultsTranscriptome-wide gene profiles differed significantly between different parts of the coral colony as well as between species. Genes showing differential expression between branch tips and bases were involved in developmental signaling pathways, such as Wnt, Notch, and BMP, as well as pH regulation, ion transport, extracellular matrix production and other processes. Differences both within colonies and between species identify a relatively small number of genes that may contribute to the distinct “staghorn” versus “elkhorn” morphologies of these two sister species.ConclusionsThe large number of differentially expressed genes supports a strong division of labor between coral branch tips and branch bases. Genes involved in growth of mature Acropora colonies include the classical signaling pathways associated with development of cnidarian larvae and polyps as well as morphological determination in higher metazoans.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1133) contains supplementary material, which is available to authorized users.

Pattern of serotonin‐like immunoreactive cells in scyphozoan and hydrozoan planulae and their relation to settlement

AbstractThe planulae of almost all investigated cnidarian species possess neuron‐like cells. The distribution of these cells is usually uneven throughout the long axis of the planula. The majority of these cells are located in the anterior half of the planula body. Scyphozoan planulae, as well as anthozoan planulae, have a sensory structure at the anterior pole called an apical organ, which is believed to take part in metamorphosis induction. Hydrozoan planulae also possess sensory cells. It has been previously shown in several cnidarian larvae that their neuronal cells contain the neurotransmitter, serotonin. The present study describes the peculiarities of serotonin‐like immunoreactive cells in Aurelia aurita (Scyphozoa) and Gonothyraea loveni (Hydrozoa) planulae. We show that several cells in the presumptive apical organ of A. aurita are immunoreactive to antibodies against serotonin, while G. loveni planulae have an accumulation of serotonin‐positive cells near the anterior pole. Additional serotonin‐like immunoreactive cells are found in the lateral ectoderm of both planulae. Treatment of A. aurita and G. loveni planulae with serotonin or its blockers show that serotonin is likely involved in the initiation of planula settlement.

Are G-protein-Coupled Receptors Involved in Mediating Larval Settlement and Metamorphosis of Coral Planulae?

Larvae of the scleractinian coral Pocillopora damicornis are induced to settle and metamorphose by the presence of marine bacterial biofilms, and the larvae of Montipora capitata respond to a combination of filamentous and crustose coralline algae. The primary goal of this study was to better understand metamorphosis of cnidarian larvae by determining what types of receptors and signal-transduction pathways are involved during stimulation of metamorphosis of P. damicornis and M. capitata. Evidence from studies on larvae of hydrozoans suggests that G-protein-coupled receptors (GPCRs) are good candidates. Settlement experiments were conducted in which competent larvae were exposed to neuropharmacological agents that affect GPCRs and their associated signal-transduction pathways, AC/cAMP and PI/DAG/PKC. On the basis of the results of these experiments, we conclude that GPCRs and these pathways do not mediate settlement and metamorphosis in either coral species. Two compounds that had an effect on both species, forskolin and phorbol-12-myristate-13-acetate (TPA), may be acting on other cellular processes not related to GPCRs. This study strengthens our understanding of the underlying physiological mechanisms that regulate metamorphosis in coral larvae.

Biochemical Responses of Cnidarian Larvae to Mercury and Benzo(a)pyrene Exposure

The biochemical responses of planulae from the coral Porites astreoides exposed to 10 microg/L of benzo(a)pyrene (B(a)P) and to 10 microg/L of mercury (Hg) was evaluated. The survivorship of larvae only dropped significantly after 48 h of B(a)P exposure, whereas it remained at 98% for Hg exposure and up to 96 h. Exposure to B(a)P significantly increased free thiols, and the activity of glutathione-S-transferase and catalase were unaltered under exposure of any of the contaminants. This study is the first contribution of the biochemical effects in cnidarian larvae exposed to contaminants.

Induction of settlement and metamorphosis of Cnidarian larvae: Signals and signal transduction

Summary Settlement and metamorphosis are induced by environmental cues in many marine invertebrates. These signals emanate from biotic or abiotic material indicating the presence of a suitable habitat. In Cnidarians, pelagic larvae undergo metamorphosis to a sessile polyp. Environmental signals reactivate the morphogenetically inactive larvae and internal mechanisms are turned onto coordinate the development of the polyp. In recent years developmental biologists, ecophysiologists and ecologists have shown a considerable interest in the metamorphosis of Cnidarians. The purpose of this review is to give a survey on what is known about the natural induction of metamorphosis and to provide information on the biochemical and cellular mechanisms underlying the metamorphic events in Cnidarians. The emphasis is on the external and internal signals and their transduction mechanisms used to control metamorphosis.

Ultrastructural Localization of RFamide-like Peptides in Neuronal Dense-Cored Vesicles of a Cnidarian Planula Larva

The location of Arg-Phe-amide (RFamide)-like peptides in dense-cored vesicles in neurons of the planula larva of the marine hydrozoan Pennaria tiarella was shown using immunogold electron microscopy. Expression of the peptide was detected as early as 16 h after fertilization. Gold-labeled dense-cored vesicles ranging from 80 nm to 140 nm in diameter were found in both cell bodies and neurites of ganglion cells (non-ciliated bipolar and multipolar neurons found at the base of the ectoderm just above the mesoglea) located in the aboral (anterior) and middle regions of the planulae. Within the neurites, the labeled dense-cored vesicles were found in distinct clusters all along their lengths in close association with the cell membranes, a distribution which suggests that release of the peptide in planulae probably does not occur at specialized synaptic sites. Labeled cells also contained unlabeled dense-cored vesicles, unlabeled clear vesicles (60 nm to 100 nm in diameter), and unlabeled electron-dense droplets (100 nm to 240 nm in diameter). Localization of RFamide peptides to dense-cored vesicles of specific neurons in early planulae is significant because of growing evidence on the role of these substances in morphogenesis. Additional key words: neuropeptide, neural development, hydrozoans, Pennaria tiarella Peptides structurally related to the neuropeptide Phe-Met-Arg-Phe-amide (FMRFamide) are abundant in the nervous systems of cnidarians (Grimmelikhuijzen 1984; Grimmelikhuijzen & Graff 1985; Grimmelikhuijzen et al. 1992; Martin 1992; Westfall & Grimmelikhuijzen 1993). When antiserum to FMRFamide or its carboxyterminal fragment RFamide is applied to cnidarians, subsets of nerve cells are identified (Grimmelikhuijzen 1984). The exact subcellular location of these immunoreactive substances within neurons has been shown only in adults of a few species of cnidarians. In the freshwater hydrozoan Hydra littoralis, RFamide was found in dense-cored vesicles in both neurites and nerve terminals (Koizumi et al. 1989); in the sea anemone Anthopleura elegantissima, RFamide activity was seen in four different types of neuronal vesicles (opaque vesicles, heterogranular vesicles, dense-cored vesicles, and light-cored vesicles) in the tentacle plexus (Westfall & Grimmelikhuijzen 1993); and in the hydromedusa Aglantha digitale, FMRFamide-like immunoreactivity was found in large dense-cored vesicles (Singla & Mackie 1991). That at least some cnidarian larvae contain RFama To whom correspondence should be addressed. ide-like material was first demonstrated in planulae of the hydroid Pennaria tiarella (= Halocordyle disticha) (Martin 1992). When antiserum to RFamide was applied to 48and 72-h planulae and visualized with FITC (fluorescein isothiocyanate), some of the ganglion cells in the aboral (anterior) and middle regions and a few ganglion cells in the oral (posterior) regions exhibited immunoactivity. Ganglion cells (also referred to as ganglionic cells in previous publications) are non-ciliated bipolar and multipolar neurons found at the base of the ectoderm just above the mesoglea. A very small number of sensory neurons in the aboral end of planulae also expressed RFamide; however, the great majority of the stained neurons were ganglion cells. Not determined, however, was the subcellular location of the neuropeptide in the planula and how early in development expression of RFamide began. This study uses the superior sensitivity of immunogold to demonstrate that RFamide-like material is located in non-synaptic dense-cored vesicles of cell bodies and processes of ganglion cells of the planula of Pennaria tiarella. The immunogold method reveals subcellular localization of neuropeptide and also detects lower levels of neuropeptide than does the previously used FITC method. These results are the first This content downloaded from 157.55.39.189 on Sun, 12 Mar 2017 19:06:04 UTC All use subject to http://about.jstor.org/terms

Seabather's eruption: Clinical, histologic, and immunologic features

Seabather's eruption (SE) is a highly pruritic eruption under swimwear that occurs after bathing in the ocean. Its cause has been unknown. Few data have been collected since the classic description by Sams in 1949. Our purpose was to describe the clinical and histopathologic findings in SE and to confirm the cause. Patients with a pruritic eruption that developed after swimming were seen within 1 week of onset. Skin biopsy specimens and sera were obtained in selected cases. Water samples taken from areas of active SE outbreaks were examined for a causative organism. Sera were tested by enzyme-linked immunosorbent assay for reactivity to this organism. In southeast Florida, during a 4-month period, 70 patients with SE were seen. Inflammatory papules and pruritus were noted within hours of exposure. Eruptions were maximal in areas covered by a bathing suit. Children were more likely than adults to have systemic symptoms. The average duration of the eruption and pruritus was 12.5 days, with recurrences in 4.3% of patients. Histopathologic examination revealed a superficial and deep perivascular and interstitial infiltrate consisting of lymphocytes, neutrophils, and eosinophils. Water samples contained many cnidarian larvae, later grown to maturity and identified as Linuche unguiculata (thimble jellyfish). Enzyme-linked immunosorbent assay demonstrated in patients' sera high IgG levels specific for L. unguiculata. SE is a severely pruritic marine dermatosis that resolves spontaneously within 2 weeks. Therapy is symptomatic but often ineffective. Sera from affected persons showed specific reactivity to L. unguiculata.

Acquisition de cnidocystes et différenciation de pseudocolloblastes chez les larves et les adultes de deux cténophores du genre Haeckelia Carus, 1863

The development of two species of cydippid Ctenophora, Haeckelia rubra and Haeckelia bimaculata, was studied to determine the origin and acquisition of exogenous cnidocysts in these two species, which lack colloblasts. A new type of cell, the pseudocolloblasts, is described. In H. rubra, the outer layer of the egg contains cnidocysts which are ingested by the larva after gastrulation; these cnidocysts make up the initial stock of exogenous cnidocysts that will allow the ctenophore to catch its first Cnidaria. In H. bimaculata, the outer layer of the egg is devoid of cnidocysts. The hatching larva has no cnidocysts either, but its tentacles are lined by an hitherto unknown type of glandular cell that we have named the pseudocolloblast. Pseudocolloblasts are composed of a secretory head bearing regularly interspaced, striated structures and an anchoring stalk. They eject their content progressively during successive phases of exocytosis. Pseudocolloblasts differ morphologically from real colloblasts, but they probably have the same function and they allow larvae of H. bimaculata to catch cnidarian larvae and thus acquire the exogenous cnidocysts present in postlarval and adult stages. Cnidocysts are identical in both species of Haeckelia in the Mediterranean Sea, consisting of macroisorhizas and microisorhizas in which the intracapsular filament has five circumferential pleats, a character typical of Narcomedusae. They differ in size from the cnidocysts found in H. rubra in the Pacific and in the Sea of Japan.[Journal translation]

Development of Nerve Cells in Hydrozoan Planulae: II. Examination of Sensory Cell Differentiation Using Electron Microscopy and Immunocytochemistry

The development of sensory cells in hydrozoan planulae of Halocordyle disticha was examined using transmission electron microscopy and light immunocytochemistry. Sensory cells arise in the anterior end of the planula in the ectoderm at 24 hours postfertilization. These cells extend from the free surface of the planula to a ganglionic plexus located just above the mesoglea. The cytomorphosis of sensory cells is characterized by the appearance of a single apical cilium, microtubules, mitochondria, one to several Golgi complexes, electron-dense droplets, dense-cored vesicles, and neurites. The basal end of the sensory cell forms one to several processes (neurites) which contribute to the ganglionic plexus. Apical specialization of the sensory cell precedes basal differentiation. Sensory cells increase in number as planulae develop and many become organized into clusters of 3-6 cells distributed along the entire length of the planula. Within some of these clusters, two morphological types of sensory cells are discernible: light sensory cells and dark sensory cells. Light sensory cells outnumber the dark sensory cells and are the first sensory cells to appear at 24 hours postfertilization. Use of immunocytochemical techniques on wholemounts and paraffin-embedded sections of planulae demonstrates the presence of FMRFamide-like immunoactivity associated with some of the sensory cells. Such FMRFamide-like expression is first detected at 24 hours postfertilization in the anterior ectoderm of the planula. By 96 hours postfertilization, the spatial distribution of FMRFamide-like positive sensory cells is such that many are found in clusters along the entire anterior-posterior axis of the planula. There is, however, an abundance of FMRFamide-like positive cells in the anterior region of the planula just prior to metamorphosis. The apices and cell bodies of the sensory cells exhibit intense immunostaining, whereas the basal processes stain faintly. This study identifies neuropeptide-like substances in nerve cells of cnidarian larvae and demonstrates a developmental correlation between the time of appearance of the synthetic machinery of sensory cells with the pattern of expression of the FMRFamide-like peptide.