A genetically tractable jellyfish model for systems and evolutionary neuroscience.

Jellyfish are radially symmetric organisms without a brain that arose more than 500 million years ago. They achieve organismal behaviors through coordinated interactions between autonomously functioning body parts. Jellyfish neurons have been studied electrophysiologically, but not at the systems level. We introduce Clytia hemisphaerica as a transparent, genetically tractable jellyfish model for systems and evolutionary neuroscience. We generate stable F1 transgenic lines for cell-type-specific conditional ablation and whole-organism GCaMP imaging. Using these tools and computational analyses, we find that an apparently diffuse network of RFamide-expressing umbrellar neurons is functionally subdivided into a series of spatially localized subassemblies whose synchronous activation controls directional food transfer from the tentacles to the mouth. These data reveal an unanticipated degree of structured neural organization in this species. Clytia affords a platform for systems-level studies of neural function, behavior, and evolution within a clade of marine organisms with growing ecological and economic importance.

The evolution of siphonophore tentilla for specialized prey capture in the open ocean.

Predator specialization has often been considered an evolutionary "dead end" due to the constraints associated with the evolution of morphological and functional optimizations throughout the organism. However, in some predators, these changes are localized in separate structures dedicated to prey capture. One of the most extreme cases of this modularity can be observed in siphonophores, a clade of pelagic colonial cnidarians that use tentilla (tentacle side branches armed with nematocysts) exclusively for prey capture. Here we study how siphonophore specialists and generalists evolve, and what morphological changes are associated with these transitions. To answer these questions, we: a) Measured 29 morphological characters of tentacles from 45 siphonophore species, b) mapped these data to a phylogenetic tree, and c) analyzed the evolutionary associations between morphological characters and prey-type data from the literature. Instead of a dead end, we found that siphonophore specialists can evolve into generalists, and that specialists on one prey type have directly evolved into specialists on other prey types. Our results show that siphonophore tentillum morphology has strong evolutionary associations with prey type, and suggest that shifts between prey types are linked to shifts in the morphology, mode of evolution, and evolutionary correlations of tentilla and their nematocysts. The evolutionary history of siphonophore specialization helps build a broader perspective on predatory niche diversification via morphological innovation and evolution. These findings contribute to understanding how specialization and morphological evolution have shaped present-day food webs.

A G protein-coupled receptor mediates neuropeptide-induced oocyte maturation in the jellyfish Clytia.

The reproductive hormones that trigger oocyte meiotic maturation and release from the ovary vary greatly between animal species. Identification of receptors for these maturation-inducing hormones (MIHs) and understanding how they initiate the largely conserved maturation process remain important challenges. In hydrozoan cnidarians including the jellyfish Clytia hemisphaerica, MIH comprises neuropeptides released from somatic cells of the gonad. We identified the receptor (MIHR) for these MIH neuropeptides in Clytia using cell culture-based "deorphanization" of candidate oocyte-expressed G protein-coupled receptors (GPCRs). MIHR mutant jellyfish generated using CRISPR-Cas9 editing had severe defects in gamete development or in spawning both in males and females. Female gonads, or oocytes isolated from MIHR mutants, failed to respond to synthetic MIH. Treatment with the cAMP analogue Br-cAMP to mimic cAMP rise at maturation onset rescued meiotic maturation and spawning. Injection of inhibitory antibodies to the alpha subunit of the Gs heterodimeric protein (GαS) into wild-type oocytes phenocopied the MIHR mutants. These results provide the molecular links between MIH stimulation and meiotic maturation initiation in hydrozoan oocytes. Molecular phylogeny grouped Clytia MIHR with a subset of bilaterian neuropeptide receptors, including neuropeptide Y, gonadotropin inhibitory hormone (GnIH), pyroglutamylated RFamide, and luqin, all upstream regulators of sexual reproduction. This identification and functional characterization of a cnidarian peptide GPCR advances our understanding of oocyte maturation initiation and sheds light on the evolution of neuropeptide-hormone systems.

Identification of jellyfish neuropeptides that act directly as oocyte maturation-inducing hormones.

Oocyte meiotic maturation is crucial for sexually reproducing animals, and its core cytoplasmic regulators are highly conserved between species. By contrast, the few known maturation-inducing hormones (MIHs) that act on oocytes to initiate this process are highly variable in their molecular nature. Using the hydrozoan jellyfish species Clytia and Cladonema, which undergo oocyte maturation in response to dark-light and light-dark transitions, respectively, we deduced amidated tetrapeptide sequences from gonad transcriptome data and found that synthetic peptides could induce maturation of isolated oocytes at nanomolar concentrations. Antibody preabsorption experiments conclusively demonstrated that these W/RPRPamide-related neuropeptides account for endogenous MIH activity produced by isolated gonads. We show that the MIH peptides are synthesised by neural-type cells in the gonad, are released following dark-light/light-dark transitions, and probably act on the oocyte surface. They are produced by male as well as female jellyfish and can trigger both sperm and egg release, suggesting a role in spawning coordination. We propose an evolutionary link between hydrozoan MIHs and the neuropeptide hormones that regulate reproduction upstream of MIHs in bilaterian species.

Frizzled3 expression and colony development in hydractiniid hydrozoans.

Hydractiniid hydrozoan colonies are comprised of individual polyps connected by tube-like stolons or a sheet-like mat. Mat and stolons function to integrate the colony through continuous epithelia and shared gastrovascular cavity. Although mechanisms of hydrozoan polyp development have been well studied, little is known about the signaling processes governing the patterning of colonies. Here we investigate the Wnt receptor family Frizzled. Phylogenetic analysis reveals that hydrozoans possess four Frizzled orthologs. We find that one of these genes, Frizzled3, shows a spatially restricted expression pattern in colony-specific tissue in two hydractiniid hydrozoans, Hydractinia symbiolongicarpus and Podocoryna carnea, in a manner that corresponds to their distinct colony forms (stolonal mat in Hydractinia and free stolons in Podocoryna). Interestingly, Frizzled3 was lost in the genome of Hydra, which is a solitary polyp and thus lacks colony-specific tissue. Current evidence suggests that the Wnt signaling pathway plays a key role in the evolution of colony diversity and colony loss in Hydrozoa.

Isolation and Characterization of Feeding-Deficient Strains in Inbred Lines of the Hydrozoan Jellyfish Cladonema pacificum.

Feeding behavior in cnidarians has been studied as a model experimental system in physiology and neurobiology. Although the feeding response in cnidarians, such as Hydra, is triggered by chemical signals, the underlying molecular mechanisms that ensure their precise execution are not well understood. It could be largely due to the lack of genetic analysis in cnidarian experimental systems. Cladonema pacificum is a hydrozoan jellyfish that is easy to maintain and cross for genetic analysis in the laboratory. To establish C. pacificum as a model experimental animal in cnidarians, we have been inbreeding strains of jellyfish. Here, we document our progress in developing C. pacificum inbred lines and feeding-defective strains that we isolated in the course of inbreeding. In the inbred lines, an increasing number of feeding-defective strains appeared as descending generations and finally all the F5 progeny showed a feeding-deficient phenotype presumably owing to inbreeding depression. Feeding behaviors of these strains were analyzed by video microscopy and we found that the feeding-defective strains captured prey, but could not kill them. After trapping prey, wild-type medusae contracted their tentacles tightly and then bent the tentacles to bring the prey to the mouth; however, feeding-defective medusae rarely contracted their tentacles and did not bend. These feeding-defective phenotypes are caused by lack of stinging nematocytes in their tentacle batteries. These findings furnish a clue to the regulatory aspects of feeding behavior, but also reveal the mechanisms of stinging nematocyte transport in tentacles.

Phenotypic Plasticity in Animals Exposed to Osmotic Stress - Is it Always Adaptive?

Hyperplasia and hypertrophy are elements of phenotypic plasticity adjusting organ size and function. Because they are costly, we assume that they are beneficial. In this review, the authors discuss examples of tissue and organ systems that respond with plastic changes to osmotic stress to raise awareness that we do not always have sufficient experimental evidence to conclude that such processes provide fitness advantages. Changes in hydranth architecture in the hydroid Cordylophora caspia or variations in size in the anal papillae of insect larvae upon changes in medium salinity may be adaptive or not. The restructuring of salt glands in ducklings upon salt-loading is an example of phenotypic plasticity which indeed seems beneficial. As the genomes of model species are recently sequenced and the animals are easy to rear, these species are suitable study objects to investigate the biological significance of phenotypic plasticity and to study potential epigenetic and other mechanisms underlying phenotypic changes.

Propulsive design principles in a multi-jet siphonophore.

Coordination of multiple propulsors can provide performance benefits in swimming organisms. Siphonophores are marine colonial organisms that orchestrate the motion of multiple swimming zooids for effective swimming. However, the kinematics at the level of individual swimming zooids (nectophores) have not been examined in detail. We used high-speed, high-resolution microvideography and particle image velocimetry of the physonect siphonophore Nanomia bijuga to study the motion of the nectophores and the associated fluid motion during jetting and refilling. The integration of nectophore and velum kinematics allow for a high-speed (maximum ∼1 m s-1), narrow (1-2 mm) jet and rapid refill, as well as a 1:1 ratio of jetting to refill time. Scaled to the 3 mm nectophore length, jet speeds reach >300 lengths s-1 Overall swimming performance is enhanced by velocity gradients produced in the nectophore during refill, which lead to a high-pressure region that produces forward thrust. Generating thrust during both the jet and refill phases augments the distance traveled by 17% over theoretical animals, which generate thrust only during the jet phase. The details of velum kinematics and associated fluid mechanics elucidate how siphonophores effectively navigate three-dimensional space, and could be applied to exit flow parameters in multijet underwater vehicles.

Widespread utilization of passive energy recapture in swimming medusae.

Recently, it has been shown that some medusae are capable of swimming very efficiently, i.e. with a low cost of transport, and that this is in part due to passive energy recapture (PER) which occurs during bell relaxation. We compared the swimming kinematics among a diverse array of medusae, varying in taxonomy, morphology and propulsive and foraging modes, in order to evaluate the prevalence of PER in medusae. We found that while PER was common among taxa, the magnitude of the contribution to overall swimming varied greatly. The ability of medusae to utilize PER was not related to morphology and swimming performance but was controlled by their swimming kinematics. Utilizing PER required the medusae to pause after bell expansion and individuals could modulate their PER by changing their pause duration. PER can greatly enhance swimming efficiency but there appear to be trade-offs associated with utilizing PER.

Corals hosting symbiotic hydrozoans are less susceptible to predation and disease.

In spite of growing evidence that climate change may dramatically affect networks of interacting species, whether-and to what extent-ecological interactions can mediate species' responses to disturbances is an open question. Here we show how a largely overseen association such as that between hydrozoans and scleractinian corals could be possibly associated with a reduction in coral susceptibility to ever-increasing predator and disease outbreaks. We examined 2455 scleractinian colonies (from both Maldivian and the Saudi Arabian coral reefs) searching for non-random patterns in the occurrence of hydrozoans on corals showing signs of different health conditions (i.e. bleaching, algal overgrowth, corallivory and different coral diseases). We show that, after accounting for geographical, ecological and co-evolutionary factors, signs of disease and corallivory are significantly lower in coral colonies hosting hydrozoans than in hydrozoan-free ones. This finding has important implications for our understanding of the ecology of coral reefs, and for their conservation in the current scenario of global change, because it suggests that symbiotic hydrozoans may play an active role in protecting their scleractinian hosts from stresses induced by warming water temperatures.

Kleptopredation: a mechanism to facilitate planktivory in a benthic mollusc.

Predation occurs when an organism completely or partially consumes its prey. Partial consumption is typical of herbivores but is also common in some marine microbenthic carnivores that feed on colonial organisms. Associations between nudibranch molluscs and colonial hydroids have long been assumed to be simple predator-prey relationships. Here we show that while the aeolid nudibranch Cratena peregrina does prey directly on the hydranths of Eudendrium racemosum, it is stimulated to feed when hydranths have captured and are handling prey, thus ingesting recently captured plankton along with the hydroid polyp such that plankton form at least half of the nudibranch diet. The nudibranch is thus largely planktivorous, facilitated by use of the hydroid for prey capture. At the scale of the colony this combines predation with kleptoparasitism, a type of competition that involves the theft of already-procured items to form a feeding mode that does not fit into existing classifications, which we term kleptopredation. This strategy of subsidized predation helps explain how obligate-feeding nudibranchs obtain sufficient energy for reproduction from an ephemeral food source.

Benthic assemblages, biodiversity and invasiveness in marinas and commercial harbours: an investigation using a bioindicator group.

Fouling communities on artificial marine structures are generally different from benthic communities in natural rocky habitats. However, they may also differ among different types of artificial structures. Two artificial structures in direct contact with arriving vessels were compared: floating pontoons within recreational marinas, and sea-walls within commercial harbours. Natural rocky habitats were used as a reference, and the genus Eudendrium (Cnidaria, Hydrozoa) was chosen as a bioindicator. The assemblages were different among the three types of habitat studied, with different species characterising each habitat. The probability of finding an invasive Eudendrium species was significantly higher on pontoons. Diversity was the lowest on pontoons, but it was not significantly different between sea-walls and natural rocks. In general, a barrier to the spread of exotic species exists between harbours and natural rocky habitats. Floating pontoons seem to be a less suitable habitat for native fauna and a key element in marine biological invasions.

Polyspermy block in jellyfish eggs: collaborative controls by Ca(2+) and MAPK.

Jellyfish eggs neither undergo apparent cortical reaction nor show any significant change in the membrane potential at fertilization, but nevertheless show monospermy. Utilizing the perfectly transparent eggs of the hydrozoan jellyfish Cytaeis uchidae, here we show that the polyspermy block is accomplished via a novel mechanism: a collaboration between Ca(2+) and mitogen-activated protein kinase (MAPK). In Cytaeis, adhesion of a sperm to the animal pole surface of an egg was immediately followed by sperm-egg fusion and initiation of an intracellular Ca(2+) rise from this site. The elevated Ca(2+) levels lasted for several minutes following the sperm-egg fusion. The Ca(2+) rise proved to be necessary and sufficient for a polyspermy block, as inhibiting a Ca(2+) rise with EGTA promoted polyspermy, and conversely, triggering a Ca(2+) rise by inositol 1,4,5-trisphosphate (IP3) or excess K(+) immediately abolished the egg's capacity for sperm-egg fusion. A Ca(2+) rise at fertilization or by artificial stimulations evoked dephosphorylation of MAPK in eggs. The eggs in which phosphorylated MAPK was maintained by injection of mRNA for MAPK kinase kinase (Mos), like intact eggs, exhibited a Ca(2+) rise at fertilization or by IP3 injection, and shut down the subsequent sperm-egg fusion. However, the Mos-expressing eggs became capable of accepting sperm following the arrest of Ca(2+) rise. In contrast, addition of inhibitors of MAPK kinase (MEK) to unfertilized eggs caused MAPK dephosphorylation without elevating Ca(2+) levels, and prevented sperm-egg fusion. Rephosphorylation of MAPK by injecting Mos mRNA after fertilization recovered sperm attraction, which is known to be another MAPK-dependent event, but did not permit subsequent sperm-egg fusion. Thus, it is possible that MAPK dephosphorylation irreversibly blocks sperm-egg fusion and reversibly suppresses sperm attraction. Collectively, our data suggest that both the fast and late mechanisms dependent on Ca(2+) and MAPK, respectively, ensure a polyspermy block in jellyfish eggs.

Ontogenetic propulsive transitions by Sarsia tubulosa medusae.

While swimming in their natural environment, marine organisms must successfully forage, escape from predation, and search for mates to reproduce. In the process, planktonic organisms interact with their fluid environment, generating fluid signatures around their body and in their downstream wake through ontogeny. In the early stages of their life cycle, marine organisms operate in environments where viscous effects dominate and govern physical processes. Ontogenetic propulsive transitions in swimming organisms often involve dramatic changes in morphology and swimming behavior. However, for organisms that do not undergo significant changes in morphology, swimming behavior or propulsive mode, how is their swimming performance affected? We investigated the ontogenetic propulsive transitions of the hydromedusa Sarsia tubulosa, which utilizes jet propulsion and possesses a similar bell morphology throughout its life cycle. We used digital particle image velocimetry and high-speed imaging to measure the body kinematics, velocity fields and wake structures induced by swimming S. tubulosa with bell exit diameters from 1 to 10 mm. Our experimental observations revealed three distinct classes of hydrodynamic wakes: elongated vortex rings for 1030 (larger than 2 mm bell exit diameter) and elliptical vortex rings (or leading vortex rings) followed by trailing jets for most instances where Re>100 (larger than 4 or 5 mm bell exit diameter). The relative travel distance and propulsive efficiency remained unchanged throughout ontogeny, and the swimming proficiency and hydrodynamic cost of transport decreased non-linearly.

Differential gene expression between functionally specialized polyps of the colonial hydrozoan Hydractinia symbiolongicarpus (Phylum Cnidaria).

BACKGROUND: A colony of the hydrozoan Hydractinia symbiolongicarpus comprises genetically identical yet morphologically distinct and functionally specialized polyp types. The main labor divisions are between feeding, reproduction and defense. In H. symbiolongicarpus, the feeding polyp (called a gastrozooid) has elongated tentacles and a mouth, which are absent in the reproductive polyp (gonozooid) and defensive polyp (dactylozooid). Instead, the dactylozooid has an extended body column with an abundance of stinging cells (nematocysts) and the gonozooid bears gonophores on its body column. Morphological differences between polyp types can be attributed to simple changes in their axial patterning during development, and it has long been hypothesized that these specialized polyps arose through evolutionary alterations in oral-aboral patterning of the ancestral gastrozooid. RESULTS: An assembly of 66,508 transcripts (>200 bp) were generated using short-read Illumina RNA-Seq libraries constructed from feeding, reproductive, and defensive polyps of H. symbiolongicarpus. Using several different annotation methods, approximately 54% of the transcripts were annotated. Differential expression analyses were conducted between these three polyp types to isolate genes that may be involved in functional, histological, and pattering differences between polyp types. Nearly 7 K transcripts were differentially expressed in a polyp-specific manner, including members of the homeodomain, myosin, toxin and BMP gene families. We report the spatial expression of a subset of these polyp-specific transcripts to validate our differential expression analyses. CONCLUSIONS: While potentially originating through simple changes in patterning, polymorphic polyps in Hydractinia are the result of differentially expressed functional, structural, and patterning genes.The differentially expressed genes identified in our study provide a starting point for future investigations of the developmental patterning and functional differences that are displayed in the different polyp types that confer a division of labor within a colony of H. symbiolongicarpus.

Microsatellite abundance across the Anthozoa and Hydrozoa in the phylum Cnidaria.

BACKGROUND: Microsatellite loci have high mutation rates and thus are indicative of mutational processes within the genome. By concentrating on the symbiotic and aposymbiotic cnidarians, we investigated if microsatellite abundances follow a phylogenetic or ecological pattern. Individuals from eight species were shotgun sequenced using 454 GS-FLX Titanium technology. Sequences from the three available cnidarian genomes (Nematostella vectensis, Hydra magnipapillata and Acropora digitifera) were added to the analysis for a total of eleven species representing two classes, three subclasses and eight orders within the phylum Cnidaria. RESULTS: Trinucleotide and tetranucleotide repeats were the most abundant motifs, followed by hexa- and dinucleotides. Pentanucleotides were the least abundant motif in the data set. Hierarchical clustering and log likelihood ratio tests revealed a weak relationship between phylogeny and microsatellite content. Further, comparisons between cnidaria harboring intracellular dinoflagellates and those that do not, show microsatellite coverage is higher in the latter group. CONCLUSIONS: Our results support previous studies that found tri- and tetranucleotides to be the most abundant motifs in invertebrates. Differences in microsatellite coverage and composition between symbiotic and non-symbiotic cnidaria suggest the presence/absence of dinoflagellates might place restrictions on the host genome.

The effect of external flow on 3D orientation of a microscopic sessile suspension feeder, Vorticella convallaria.

Vorticella convallaria are microscopic sessile suspension feeders that live attached to substrates in aquatic environments. They feed using a self-generated current and help maintain the health of aquatic ecosystems and wastewater treatment facilities by consuming bacteria and detritus. Their environmental impact is mediated by their feeding rate. In ambient flow, feeding rates are highly dependent on an individual's orientation relative to the substrate and the flow. Here, we investigate how this orientation is impacted by flow speed. Furthermore, we examined whether individuals actively avoid orientations unfavorable for feeding. We exposed individuals to unidirectional laminar flow at shear rates of 0, 0.5, 1.0, and 1.5 s-1, and recorded their 3D orientation using a custom biplanar microscope. We determined that V. convallaria orientation became progressively tilted downstream as the shear rate increased, but individuals were still able to actively reorient. Additionally, at higher shear rates, individuals spent a larger fraction of their time in orientations with reduced feeding rates. Our shear rates correspond to freestream flows on the scale of mm s-1 to cm s-1 in natural environments.

New tricks with old genes: the genetic bases of novel cnidarian traits.

Recent thought on genome evolution has focused on the creation of new genes and changes in regulatory mechanisms while ignoring the role of selective gene loss in shaping genomes. Using data from two cnidarians, the jellyfish Clytia and the coral Acropora, we examined the relative significance of new 'taxonomically restricted' genes and selectively retained ancestral genes in enabling the evolution of novel traits. Consistent with its more complex life-cycle, the proportion of novel genes identified in Clytia was higher than that in the 'polyp only' cnidarians Nematostella and Hydra, but each of these cnidarians has retained a proportion of ancestral genes not present in the other two. The ubiquity and near-stochastic nature of gene loss can explain the discord between patterns of gene distribution and taxonomy.

Wnt signaling promotes oral but suppresses aboral structures in Hydractinia metamorphosis and regeneration.

We studied the role of Wnt signaling in axis formation during metamorphosis and regeneration in the cnidarian Hydractinia. Activation of Wnt downstream events during metamorphosis resulted in a complete oralization of the animals and repression of aboral structures (i.e. stolons). The expression of Wnt3, Tcf and Brachyury was upregulated and became ubiquitous. Rescue experiments using Tcf RNAi resulted in normal metamorphosis and quantitatively normal Wnt3 and Brachyury expression. Isolated, decapitated polyps regenerated only heads but no stolons. Activation of Wnt downstream targets in regenerating animals resulted in oralization of the polyps. Knocking down Tcf or Wnt3 by RNAi inhibited head regeneration and resulted in complex phenotypes that included ectopic aboral structures. Multiple heads then grew when the RNAi effect had dissipated. Our results provide functional evidence that Wnt promotes head formation but represses the formation of stolons, whereas downregulation of Wnt promotes stolons and represses head formation.

The fouling hydroid Ectopleura larynx: a lack of effect of next generation antifouling technologies.

The hydroid Ectopleura larynx is one of the main fouling organisms on salmon aquaculture cages in Norway; this study investigated novel surface materials and microtopographies to deter its settlement. The settlement preferences of hydroid larvae for 12 materials with wettabilities ranging from hydrophobic (54°) to hydrophilic (112°) were tested in a no-choice bioassay. Although settlement differed between materials, with the highest average settlement on polytetrafluoroethylene (95%) and the lowest on untreated polyurethane (53%), no trend regarding the tested wettabilities could be found and none of the tested materials was able to reduce average settlement below 50%. Furthermore, nine high-density polyethylene (HDPE, 100-600 µm microtopographies) and seven polydimethylsiloxane (PDMS; 40-400 µm microtopographies) microtextured surfaces were tested. There was no systematic effect of microtopography on the settlement of E. larynx larvae. However, there was a preference for settlement in channels on PDMS microtopographies between 80 and 300 µm. Similarly, there were no preferences for any of the examined microtopographies in a 12-day field test using PDMS surfaces at a commercial fish farm. The study indicated that neither surface wettability (hydrophilicity-phobicity) nor microtopographies were effective at deterring the settlement of the hydroid E. larynx. The high plasticity of the aboral pole and the hydrorhiza of the hydroids may explain settlement even under unfavourable conditions, highlighting the successful colonisation traits of this dominant biofouling species.