Phylogenetic analysis of cell-cycle regulatory proteins within the Symbiodiniaceae.

In oligotrophic waters, cnidarian hosts rely on symbiosis with their photosynthetic dinoflagellate partners (family Symbiodiniaceae) to obtain the nutrients they need to grow, reproduce and survive. For this symbiosis to persist, the host must regulate the growth and proliferation of its symbionts. One of the proposed regulatory mechanisms is arrest of the symbiont cell cycle in the G1 phase, though the cellular mechanisms involved remain unknown. Cell-cycle progression in eukaryotes is controlled by the conserved family of cyclin-dependent kinases (CDKs) and their partner cyclins. We identified CDKs and cyclins in different Symbiodiniaceae species and examined their relationship to homologs in other eukaryotes. Cyclin proteins related to eumetazoan cell-cycle-related cyclins A, B, D, G/I and Y, and transcriptional cyclin L, were identified in the Symbiodiniaceae, alongside several alveolate-specific cyclin A/B proteins, and proteins related to protist P/U-type cyclins and apicomplexan cyclins. The largest expansion of Symbiodiniaceae cyclins was in the P/U-type cyclin groups. Proteins related to eumetazoan cell-cycle-related CDKs (CDK1) were identified as well as transcription-related CDKs. The largest expansion of CDK groups was, however, in alveolate-specific groups which comprised 11 distinct CDK groups (CDKA-J) with CDKB being the most widely distributed CDK protein. As a result of its phylogenetic position, conservation across Symbiodiniaceae species, and the presence of the canonical CDK motif, CDKB emerged as a likely candidate for a Saccharomyces cerevisiae Cdc28/Pho85-like homolog in Symbiodiniaceae. Similar to cyclins, two CDK-groups found in Symbiodiniaceae species were solely associated with apicomplexan taxa. A comparison of Breviolum minutum CDK and cyclin gene expression between free-living and symbiotic states showed that several alveolate-specific CDKs and two P/U-type cyclins exhibited altered expression in hospite, suggesting that symbiosis influences the cell cycle of symbionts on a molecular level. These results highlight the divergence of Symbiodiniaceae cell-cycle proteins across species. These results have important implications for host control of the symbiont cell cycle in novel cnidarian-dinoflagellate symbioses.

Myxozoans: Ancient metazoan parasites find a home in phylum Cnidaria.

Myxozoans are endoparasites with complex life cycles that alternate between invertebrate and vertebrate hosts. Though considered protozoans for over 150 years, they are now recognized as metazoans, given their multicellularity and ultrastructural features. In recognition of synapomorphies and cnidarian-specific genes, myxozoans were placed recently within the phylum Cnidaria. Although they have lost genetic and structural complexity on the path to parasitism, myxozoans have retained characteristic cnidarian cnidocysts, but use them for initiating host infection. Myxozoans represent at least 20% of phylum Cnidaria, but as a result of rapid evolution, extensive diversification and host specialization, they are probably at least as diverse as their free-living relatives. The ability of myxozoans to infect freshwater, marine and terrestrial hosts implies that Cnidaria are no longer constrained to the aquatic environment.

A de novo approach to disentangle partner identity and function in holobiont systems.

BACKGROUND: Study of meta-transcriptomic datasets involving non-model organisms represents bioinformatic challenges. The production of chimeric sequences and our inability to distinguish the taxonomic origins of the sequences produced are inherent and recurrent difficulties in de novo assembly analyses. As the study of holobiont meta-transcriptomes is affected by challenges invoked above, we propose an innovative bioinformatic approach to tackle such difficulties and tested it on marine models as a proof of concept. RESULTS: We considered three holobiont models, of which two transcriptomes were previously published and a yet unpublished transcriptome, to analyze and sort their raw reads using Short Read Connector, a k-mer based similarity method. Before assembly, we thus defined four distinct categories for each holobiont meta-transcriptome: host reads, symbiont reads, shared reads, and unassigned reads. Afterwards, we observed that independent de novo assemblies for each category led to a diminution of the number of chimeras compared to classical assembly methods. Moreover, the separation of each partner's transcriptome offered the independent and comparative exploration of their functional diversity in the holobiont. Finally, our strategy allowed to propose new functional annotations for two well-studied holobionts (a Cnidaria-Dinophyta, a Porifera-Bacteria) and a first meta-transcriptome from a planktonic Radiolaria-Dinophyta system forming widespread symbiotic association for which our knowledge is considerably limited. CONCLUSIONS: In contrast to classical assembly approaches, our bioinformatic strategy generates less de novo assembled chimera and allows biologists to study separately host and symbiont data from a holobiont mixture. The pre-assembly separation of reads using an efficient tool as Short Read Connector is an effective way to tackle meta-transcriptomic challenges and offers bright perpectives to study holobiont systems composed of either well-studied or poorly characterized symbiotic lineages and ultimately expand our knowledge about these associations.

Symbiont Identity Influences Patterns of Symbiosis Establishment, Host Growth, and Asexual Reproduction in a Model Cnidarian-Dinoflagellate Symbiosis.

The genus Symbiodinium is physiologically diverse and so may differentially influence symbiosis establishment and function. To explore this, we inoculated aposymbiotic individuals of the sea anemone Exaiptasia pallida (commonly referred to as "Aiptasia"), a model for coral symbiosis, with one of five Symbiodinium species or types (S. microadriaticum, S. minutum, phylotype C3, S. trenchii, or S. voratum). The spatial pattern of colonization was monitored over time via confocal microscopy, and various physiological parameters were measured to assess symbiosis functionality. Anemones rapidly formed a symbiosis with the homologous symbiont, S. minutum, but struggled or failed to form a long-lasting symbiosis with Symbiodinium C3 or S. voratum, respectively. Symbiodinium microadriaticum and S. trenchii were successful but reached their peak density two weeks after S. minutum. The spatial pattern of colonization was identical for all Symbiodinium taxa that were ultimately successful, starting in the oral disk and progressing to the tentacles, before invading the column and, finally, the pedal disk. In all cases, proliferation through the anemone's tentacles was patchy, suggesting that symbionts were being expelled into the gastrovascular cavity and re-phagocytosed by the host. However, the timing of these various spatial events differed between the different Symbiodinium taxa. Furthermore, S. microadriaticum and S. trenchii were less beneficial to the host, as indicated by lower rates of photosynthesis, anemone growth, and pedal laceration. This study enhances our understanding of the link between symbiont identity and the performance of the overall symbiosis, which is important for understanding the potential establishment and persistence of novel host-symbiont pairings. Importantly, we also provide a baseline for further studies on this topic with the globally adopted "Aiptasia" model system.

The joint evolution of the Myxozoa and their alternate hosts: A cnidarian recipe for success and vast biodiversity.

The relationships between parasites and their hosts are intimate, dynamic and complex; the evolution of one is inevitably linked to the other. Despite multiple origins of parasitism in the Cnidaria, only parasites belonging to the Myxozoa are characterized by a complex life cycle, alternating between fish and invertebrate hosts, as well as by high species diversity. This inspired us to examine the history of adaptive radiations in myxozoans and their hosts by determining the degree of congruence between their phylogenies and by timing the emergence of myxozoan lineages in relation to their hosts. Recent genomic analyses suggested a common origin of Polypodium hydriforme, a cnidarian parasite of acipenseriform fishes, and the Myxozoa, and proposed fish as original hosts for both sister lineages. We demonstrate that the Myxozoa emerged long before fish populated Earth and that phylogenetic congruence with their invertebrate hosts is evident down to the most basal branches of the tree, indicating bryozoans and annelids as original hosts and challenging previous evolutionary hypotheses. We provide evidence that, following invertebrate invasion, fish hosts were acquired multiple times, leading to parallel cospeciation patterns in all major phylogenetic lineages. We identify the acquisition of vertebrate hosts that facilitate alternative transmission and dispersion strategies as reason for the distinct success of the Myxozoa, and identify massive host specification-linked parasite diversification events. The results of this study transform our understanding of the origins and evolution of parasitism in the most basal metazoan parasites known.

A diverse host thrombospondin-type-1 repeat protein repertoire promotes symbiont colonization during establishment of cnidarian-dinoflagellate symbiosis.

The mutualistic endosymbiosis between cnidarians and dinoflagellates is mediated by complex inter-partner signaling events, where the host cnidarian innate immune system plays a crucial role in recognition and regulation of symbionts. To date, little is known about the diversity of thrombospondin-type-1 repeat (TSR) domain proteins in basal metazoans or their potential role in regulation of cnidarian-dinoflagellate mutualisms. We reveal a large and diverse repertoire of TSR proteins in seven anthozoan species, and show that in the model sea anemone Aiptasia pallida the TSR domain promotes colonization of the host by the symbiotic dinoflagellate Symbiodinium minutum. Blocking TSR domains led to decreased colonization success, while adding exogenous TSRs resulted in a 'super colonization'. Furthermore, gene expression of TSR proteins was highest at early time-points during symbiosis establishment. Our work characterizes the diversity of cnidarian TSR proteins and provides evidence that these proteins play an important role in the establishment of cnidarian-dinoflagellate symbiosis.

Symbiodinium isolation by NaOH treatment.

The presence of photosynthetic zooxanthellae (dinoflagellates) in the tissue of many cnidarians is the main reason for their ecological success (i.e. coral reefs). It could also be the main cause of their demise, as the worldwide bleaching of reef-building coral is nothing less than the breakdown of this symbiotic association. The stability of this relationship is the principal marker for the biomonitoring of cnidarian health. We have therefore developed a new, simple method to isolate zooxanthellae in a few steps using NaOH solution. The protocol was validated in three symbiotic cnidarian species: a sea anemone, a gorgonian and a coral. Our method allows the isolation of intact and viable zooxanthellae with better yields than classic methods, especially for species with a calcareous skeleton. Moreover, the isolated zooxanthellae were free of host nucleic contaminants, facilitating subsequent specific molecular analyses.

Neglected biological features in cnidarians self-nonself recognition.

Cnidarian taxa, currently of the most morphologically simplest extant metazoans, exhibit many salient properties of innate immunity that are shared by most Animalia. One hallmark constituent of immunity exhibit by most cnidarians is histocompatibility, marked by wide spectrum of allogeneic and xenogeneic effector arms, progressing into tissue fusions or inflammatory rejections. Scientific propensity on cnidarians immunity, while discussing historecognition as the ground for immunity in these organisms, concentrates on host-parasitic and disease oriented studies, or focuses on genome approaches that search for gene homologies with the vertebrates. Above tendency for mixing up between historecognition and host-parasitic/disease, highlights a serious obstacle for the progress in our understanding of cnidarian immunobiology. Here I critically overview four 'forgotten' cnidarian immune features, namely, specificity, immunological memory, allogeneic maturation and natural chimerism, presenting insights into perspectives that are prerequisite for any discussion on cnidarian evolution. It is evident that cnidarian historecognition embraces elements that the traditional field of vertebrate immunology has never encountered (i.e., variety of cytotoxic outcomes, different types of effector mechanisms, chimerism, etc.). Also, cnidarian immune features dictating that different individuals within the same species seem to respond differently to the same immunological challenge, is far from that recorded in the vertebrates' adaptive immunity. While above features may be connected to host-parasitic and disease phenomena and effector arms, they clearly attest to their unique critical roles in shaping cnidarians historecognition, calling for improved distinction between historecognition and host-response/ disease disciplines. The research on cnidarians immunity still suffers from the lack of accepted synthesis of what historecognition is or does. Mounting of an immune response against conspecifics or xenogeneic organisms should therefore be clearly demarcated from other paths of immunity, till cnidarian innate immunity as a whole is expounded.

Defense of benthic invertebrates against surface colonization by larvae: a chemical arms race.

Sessile invertebrates evolved in a competitive milieu where space is a limiting resource, setting off an arms race between adults that must maintain clean surfaces and larvae that must locate and attach to a suitable substratum. I review the evidence that invertebrates chemically deter or kill the propagules of fouling animals and protists under natural conditions, and that chemosensory mechanisms may allow larvae to detect and avoid settling on chemically protected organisms. The fouling process is an ecologically complex web of interactions between basibionts, surface-colonizing microbes, and fouling larvae, all mediated by chemical signaling. Host-specific bacterial communities are maintained by many invertebrates, and may inhibit fouling by chemical deterrence of larvae, or by preventing biofilm formation by inductive strains. Larval settlement naturally occurs in a turbulent environment, yet the effects of waterborne versus surface-adsorbed chemical defenses have not been compared in flow, limiting our understanding of how larvae respond to toxic surfaces in the field. The importance of evaluating alternative hypotheses such as mechanical and physical defense is discussed, as is the need for ecologically relevant bioassays that quantify effects on larval behavior and identify compounds likely to play a defensive role in situ.

Molecular diversity of dinoflagellate symbionts of Cnidaria: the psbA minicircle of Symbiodinium.

Dinoflagellate algae of the genus Symbiodinium are important symbionts within corals and other benthic marine animals. The molecular diversity of Symbiodinium has been described mainly by use of ribosomal DNA sequence data. We tested whether minicircle sequences, which appear to form the chloroplast genome in many dinoflagellates, could be used as a marker for molecular diversity among symbionts found in corals and sea anemones. Partial and full-length sequences for psbA were obtained from environmental samples of coral and sea anemones of wide-ranging geographical distribution. Phylogenetic trees constructed with partial psbA sequences were consistent with the known phylotypes of the isolates. Further sequencing suggested that the psbA gene is present on a minicircle in all Symbiodinium phylotypes. The length and DNA sequence of the non-coding portion of the minicircles varied considerably among Symbiodinium phylotypes. In two Symbiodinium isolates from different phylotypes an elaborate pattern of repeat sequences of unknown function was found in the non-coding region. Phylogenetic analysis of the non-coding region of the psbA minicircle indicates that minicircle sequences could be a useful chloroplast-derived marker for differentiating both closely related and distantly related Symbiodinium isolates.

Species boundaries, populations and colour morphs in the coral reef three-spot damselfish (Dascyllus trimaculatus) species complex.

Coloration patterns of tropical reef fishes is commonly used for taxonomic purposes, yet few studies have focused on the relationship between species boundaries and coloration types. The three-spot damselfish (Dascyllus trimaculatus) species complex comprises four species that vary both in geographical ranges and colour patterns making them an ideal model to study these relationships. We analysed the mitochondrial control region of 122 individuals from all four species collected from 13 localities. Individuals from two species (Dascyllus albisella and D. strasburgi) grouped into monophyletic clades, while the two other species (D. trimaculatus and D. auripinnis) were found to be paraphyletic. Coloration patterns were therefore not found to be good predictors of genetic isolation. In contrast, geographical origin was always consistent with the observed genetic pattern.

Zooxanthellae of the Montastraea annularis species complex: patterns of distribution of four taxa of Symbiodinium on different reefs and across depths.

Corals of the Montastraea annularis complex host several different dinoflagellates in the genus Symbiodinium. Here we address two questions arising from our previous studies of these associations on an offshore reef. First, do the same taxa and patterns of association (Symbiodinium A and B found in higher irradiance habitats than Symbiodinium C) occur on an inshore reef? Second, does M. franksi at the limits of its depth range host only Symbiodinium C, as it does at intermediate depths? In both surveys, a new Symbiodinium taxon and different patterns of distribution (assayed by analyses of small ribosomal subunit RNA genes [srDNA]) were observed. Inshore, a taxon we name Symbiodinium E predominated in higher irradiance habitats in M. franksi and its two sibling species; the only other zooxanthella observed was Symbiodinium C. Offshore, M. franksi mainly hosted Symbiodinium C, but hosted Symbiodinium A, B, C, and E in shallow water and Symbiodinium E and C in very deep water. Symbiodinium E may be stress-tolerant. Observed srDNA heterogeneity within samples of Symbiodinium B, C, and E is interpreted as variation across copies within this multigene family. Experimental bleaching of Symbiodinium C supported this interpretation. Thus sequences from natural samples should be interpreted cautiously.

Repopulation of Zooxanthellae in the Caribbean corals Montastraea annularis and M. faveolata following experimental and disease-associated bleaching.

Caribbean corals of the Montastraea annularis species complex associate with four taxa of symbiotic dinoflagellates (zooxanthellae; genus Symbiodinium) in ecologically predictable patterns. To investigate the resilience of these host-zooxanthella associations, we conducted field experiments in which we experimentally reduced the numbers of zooxanthellae (by transplanting to shallow water or by shading) and then allowed treated corals to recover. When depletion was not extreme, recovering corals generally contained the same types of zooxanthellae as they did prior to treatment. After severe depletion, however, recovering corals were always repopulated by zooxanthellae atypical for their habitat (and in some cases atypical for the coral species). These unusual zooxanthellar associations were often (but not always) established in experimentally bleached tissues even when adjacent tissues were untreated. Atypical zooxanthellae were also observed in bleached tissues of unmanipulated Montastraea with yellow-blotch disease. In colonies where unusual associations were established, the original taxa of zooxanthellae were not detected even 9 months after the end of treatment. These observations suggest that zooxanthellae in Montastraea range from fugitive opportunists and stress-tolerant generalists (Symbiodinium A and E) to narrowly adapted specialists (Symbiodinium B and C), and may undergo succession.

Patterns of coral-dinoflagellate associations in Acropora: significance of local availability and physiology of Symbiodinium strains and host-symbiont selectivity.

Like other reef-building corals, members of the genus Acropora form obligate endosymbioses with dinoflagellates (zooxanthellae) belonging to the genus Symbiodinium. Both Symbiodinium and its hosts are diverse assemblages, and the relationships between host and algal genotypes are unclear. In this study, we determined phylogenetic relationships between Symbiodinium isolates from a wide range of Acropora species and plotted the algal genotypes onto a molecular phylogeny of 28 Acropora species, using the same samples for the host and symbiont genotyping. In addition, we performed a preliminary survey of zooxanthella distribution in Acropora species from the central Great Barrier Reef. Three of the four known major zooxanthellae clades were represented in the 168 samples examined, and within the major clade C, three distinct subclades were identified. No evidence was found for coevolution, but several clear patterns of specificity were identified. Moreover, composition of the zooxanthella pool varied among locales and in one host species we found light-related patterns of zooxanthella distribution.

Development of host- and symbiont-specific monoclonal antibodies and confirmation of the origin of the symbiosome membrane in a cnidarian-dinoflagellate symbiosis.

The "symbiosome membrane" as defined by Roth et al. (1988) is a single, host-derived membrane that surrounds an endosymbiotic organism, separating it from the cytoplasm of the host cell. However, in the case of cnidarian-dinoflagellate endosymbioses, clear identification of the symbiosome membrane is complicated by the fact that each algal symbiont is surrounded by multiple layers of apparent membrane. The origin and molecular nature of these membranes has been the subject of considerable debate in the literature. Here we report the development of host-specific (G12) and symbiont-specific (PC3) monoclonal antibodies that allow separation of the host and symbiont components of these multiple membranes. Using immunocytochemistry at both the light and the electron microscopic level, we present data supporting the conclusion that the definitive symbiosome membrane is a single, host-derived membrane, whereas the remainder of the underlying apparent membranes surrounding the algal cell are symbiont-derived. The potential for macromolecules associated with these membranes to act as cellular signals critical to recruiting symbionts and maintaining established symbioses is discussed.

Host-associated speciation in a coral-inhabiting barnacle.

Host specificity of symbionts is considered an important factor associated with sympatric speciation. Here, we examine host specificity and the degree of host-associated speciation in the barnacle Savignium milleporum, an obligate symbiont of the hydrocoral Millepora (the "fire coral"). Little morphological variability was revealed between barnacles collected from two morphs of the hydrocoral Millepora dichotoma (encrusting or branching) or from its congener Millepora platyphylla, but a molecular analysis revealed an unexpected pattern of DNA sequence divergence. The sequences of the 12S mitochondrial rDNA were nearly identical within each of the three barnacle populations (average sequence divergence <1%), and the sequences obtained for barnacles collected from the two different morphs of M. dichotoma differed considerably (ca. 9% average sequence divergence). However, S. milleporum collected from M. platyphylla were nearly identical to the barnacles from the branching M. dichotoma (<0.5% average sequence divergence). The pattern of speciation demonstrated by Savignium barnacles indicates the gradual colonization of similar hosts (i.e., sequential evolution), rather then "casual" colonization, as indicated for other systems. If this is indeed so, then symbiont phylogeny should roughly correlate with host phylogeny. Additionally, the data support the "rendezvous host" hypothesis, which invokes the opportunity of both sexes to meet as a major component for which selection favors the costly habit of host specificity.

Early development of zooxanthella-containing eggs of the corals Pocillopora verrucosa and P. eydouxi with special reference to the distribution of zooxanthellae.

Some hermatypic corals spawn eggs that contain zooxanthellae. We followed development of zooxanthella-containing eggs of two such species, Pocillopora verrucosa and P. eydouxi. We also documented changes in the distribution pattern of zooxanthellae during development. Oocytes of both species took up zooxanthellae 3 to 4 days before spawning. At first, zooxanthellae were evenly distributed in oocytes, but they later moved to the hemisphere that contained the germinal vesicle. After fertilization, early cleavage events were holoblastic, progressing by furrow formation. The first cleavage furrow started at the hemisphere that contained zooxanthellae, dividing the zooxanthellate complement of the zygote about equally into the two blastomeres. The second division divided each blastomere into one zooxanthellae-rich cell and one with few zooxanthellae. With continued cell division, blastomeres containing zooxanthellae moved into the blastocoel. The blastocoel disappeared at about 5 h after the first cleavage, and the central region of the embryo was filled with cells containing either zooxanthellae or lipid droplets, forming a stereogastrula. Our results suggest that only blastomeres that had been determined to develop into gastrodermal cells receive zooxanthellae during cleavage. This determination appears to take place, at the latest, by the second cell division at the four-cell stage.

Revised description of the fine structure of in situ "zooxanthellae" genus Symbiodinium.

The fine structure of the symbiotic dinoflagellate genus Symbiodinium has been well described. All of the published descriptions are based on tissue that was fixed in standard aldehyde and osmium fixatives and dehydrated in an ethanol series before embedding. When the technique of freeze-substitution was used to fix tissue from Cassiopeia xamachana, Aiptasia pallida, and Phyllactis flosculifera and prepare it for embedding, thecal vesicles were revealed within the in situ symbionts of all three species. Although these structures have been identified in cultured symbionts, they have never been described in the in situ symbionts. A review of the literature has revealed several instances where thecal vesicles were either overlooked or identified incorrectly. Thus the formal description of the genus Symbiodinium, which describes the in situ symbionts, contains information that is based on artifact and should be revised. A revision of the genus is suggested, and the true nature of these structures and their significance in the symbiotic association are discussed.

Speciation versus phenotypic plasticity in coral inhabiting barnacles: Darwin's observations in an ecological context.

Speciation and phenotypic plasticity are two extreme strategic modes enabling a given taxon to populate a broad ecological niche. One of the organismal models which stimulated Darwin's ideas on speciation was the Cirripedia (barnacles), to which he dedicated a large monograph. In several cases, including the coral-inhabiting barnacle genera Savignium and Cantellius (formerly Pyrgoma and Creusia, respectively), Darwin assigned barnacle specimens to morphological "varieties" (as opposed to species) within a genus. Despite having been the subject of taxonomic investigations and revisions ever since, the significance of these varieties has never been examined with respect to host-associated speciation processes. Here we provide evidence from molecular (12S mt rDNA sequences) and micromorphological (SEM) studies, suggesting that these closely related barnacle genera utilize opposite strategies for populating a suite of live-coral substrates. Cantellius demonstrates a relatively low genetic variability, despite inhabiting a wide range of corals. The species C. pallidus alone was found on three coral families, belonging to distinct higher-order classification units. In contrast, Savignium barnacles exhibit large between- and within-species variations with respect to both micromorphology and DNA sequences, with S. dentatum "varieties" clustering phylogenetically according to their coral host species (all of which are members of a single family). Thus, whereas Savignium seems to have undergone intense host-associated speciation over a relatively narrow taxonomic range of hosts, Cantellius shows phenotypic plasticity over a much larger range. This dichotomy correlates with differences in life-history parameters between these barnacle taxa, including host-infestation characteristics, reproductive strategies, and larval trophic type.

A digenean metacercaria from the reef coral, Porites compressa, experimentally identified as Podocotyloides stenometra.

Feeding Porites compressa infected with a digenean metacercaria to the coral-feeding butterflyfish, Chaetodon multicinctus, established that the metacercaria was Podocotyloides stenometra. Those and field examinations finding a prevalence of 100% and an average intensity of infection of 6.5 worms/fish in 28 C. multicinctus off Kaneohe Bay, Oahu, Hawaii, established this fish as a definitive host. Plagioporus sp. of Cheng and Wong, 1974 is a synonym of P. stenometra.