Phylogenetic placement of "zoochlorellae" (Chlorophyta), algal symbiont of the temperate sea anemone Anthopleura elegantissima

At northern latitudes the sea anemones Anthopleura elegantissima and its congener A. xanthogrammica contain unidentified green chlorophytes (zoochlorellae) in addition to dinophytes belonging to the genus Symbiodinium. This dual algal symbiosis, involving members of distinct algal phyla in one host, has been extensively studied from the perspective of the ecological and energetic consequences of hosting one symbiotic type over the other. However, the identity of the green algal symbiont has remained elusive. We determined the phylogenetic position of the marine zoochlorellae inhabiting A. elegantissima by comparing sequence data from two cellular compartments, the nuclear 18S ribosomal RNA gene region and the plastid-encoded rbcL gene. The results support the inclusion of these zoochlorellae in a clade of green algae that form symbioses with animal (Anthopleura elegantissima), fungal (the lichen genus Nephroma), and seed plant (Ginkgo) partners. This clade is distinct from the Chlorella symbionts of Hydra. The phylogenetic diversity of algal hosts observed in this clade indicates a predisposition for this group of algae to participate in symbioses. An integrative approach to the study of these algae, both within the host and in culture, should yield important clues about how algae become symbionts in other organisms.     

Body size and symbiotic status influence gonad development in <Emphasis Type="Italic">Aiptasia pallida</Emphasis> anemones

Pale anemones (Aiptasia pallida) coexist with dinoflagellates (primarily Symbiodinium minutum) in a mutualistic relationship. The purpose of this study was to investigate the role of these symbionts in gonad development of anemone hosts. Symbiotic and aposymbiotic anemones were subjected to light cycles that induced gametogenesis. These anemones were then sampled weekly for nine weeks, and gonad development was analyzed histologically. Anemone size was measured as mean body column diameter, and oocytes or sperm follicles were counted for each anemone. Generalized linear models were used to evaluate the influence of body size and symbiotic status on whether gonads were present and on the number of oocytes or sperm follicles produced. Body size predicted whether gonads were present, with larger anemones being more likely than smaller anemones to develop gonads. Both body size and symbiotic status predicted gonad size, such that larger and symbiotic anemones produced more oocytes and sperm follicles than smaller and aposymbiotic anemones. Overall, only 22 % of aposymbiotic females produced oocytes, whereas 63 % of symbiotic females produced oocytes. Similarly, 6 % of aposymbiotic males produced sperm follicles, whereas 60 % of symbiotic males produced sperm follicles. Thus, while gonads were present in 62 % of symbiotic anemones, they were present in only 11 % of aposymbiotic anemones. These results indicate that dinoflagellate symbionts influence gonad development and thus sexual maturation in both female and male Aiptasia pallida anemones. This finding substantiates and expands our current understanding of the importance of symbionts in the development and physiology of cnidarian hosts.     

The Hydra viridis/Chlorella symbiosis. Growth and sexual differentiation in polyps without symbionts

To investigate interactions between the basal metazoan Hydra viridis and its symbiotic Chlorella algae, we generated aposymbiotic hydra lacking algae and compared them to symbiotic ones with regard to growth and sexual differentiation. Under standard feeding conditions aposymbiotic polyps proliferated similarly to symbiotic polyps. Under moderate and low feeding conditions asexual growth was reduced in polyps lacking algae, indicating that the symbionts supply nutrients to their hosts. In addition, the Chlorella symbionts had a strong influence on the sexual reproduction of Hydra viridis: in most cases female gonads were produced only when symbiotic algae were present. Spermatogenesis proceeded similarly in symbiotic and aposymbiotic polyps. Since during oogenesis symbionts are actively transferred from endodermal epithelial cells to the ectodermal oocytes, this oogenesis promoting role could indicate that the symbionts are critically involved in the control of sexual differentiation in green hydra.     

Biogeography and microhabitat variation in temperate algal-invertebrate symbioses: zooxanthellae and zoochlorellae in two Pacific intertidal sea anemones, Anthopleura elegantissima and A. xanthogrammica

Abstract. Temperate sea anemones in the genus Anthopleura are unique among cnidarians in harboring two phylogenetically distinct symbiotic algae, zooxanthellae (golden-brown dinophytes, Symbiodinium ) and zoochlorellae (green chlorophytes). To determine whether their physiological differences generate patterns in anemone habitat and biogeographic distribution, we sampled symbiotic algae in the small clonal A. elegantissima and the large solitary A. xanthogrammica at 8 field sites (and the other large solitary Anthopleura species at one site) spanning 18° of latitude along 2500 km of the Pacific coast of North America. We found that zoochlorellae predominate in low intertidal habitats and northerly latitudes and in A. xanthogrammica , while zooxanthellae constitute the majority of symbionts in high intertidal habitats and more southerly latitudes and in A. elegantissima. These data are consistent with published predictions based on photosynthetic efficiency of the two algae under varied temperature and light regimes in the laboratory. This anemone-algal system provides a potential biological signal of benthic intertidal communities' responses to El Niño events and long-term climate changes in the Pacific.     

Ratiometric imaging of gastrodermal lipid bodies in coral–dinoflagellate endosymbiosis

Cnidaria–dinoflagellate endosymbiosis is the phenomenon of autotrophic symbionts living inside the gastrodermal cells of their animal hosts. The molecular mechanism that regulates this association remains unclear. Using quantitative microscopy, we now provide evidence that the dynamic lipid changes in gastrodermal “lipid bodies” (LBs) reflect the symbiotic status of the host cell and its symbiont in the hermatypic coral Euphyllia glabrescens. By dual-emission ratiometric imaging with a solvatochromic fluorescent probe, Nile red (9-diethylamino-5H-benzo[α]phenoxazine-5-one), we showed that the in situ distribution of polar versus neutral lipids in LBs in living gastrodermal cells and symbionts can be analyzed. The ratio of Nile red fluorescence at red (R) versus green (G) wavelength region (i.e., R/G ratio) correlated with the relative molar ratio of polar (P) versus neutral (NP) lipids (i.e., P/NP ratio). The R/G ratio in host LBs increased after bleaching, indicating a decrease in neutral lipid accumulation in gastrodermal cells. On the other hand, neutral lipid accumulation inside the symbiont LBs resulted in gradual decreases of the R/G ratio as a result of bleaching. In comparison with the bleaching event, there was no relative lipid concentration change in host LBs under continual light or dark treatments as shown by insignificant R/G ratio shift. Patterns of R/G ratio shift in symbiont LBs were also different between corals undergoing bleaching and continual light/dark treatment. In the latter, there was little lipid accumulation in symbionts, with no resulting R/G ratio decrease. These results, demonstrating that the symbiotic status positively correlated with morphological and compositional changes of lipid bodies, not only highlight the pivotal role of LBs, but also implicate an involvement of lipid trafficking in regulating the endosymbiosis.     

Comparative gene expression in the symbiotic and aposymbiotic Aiptasia pulchella by expressed sequence tag analysis

Intracellular symbiotic relationships are prevalent between cnidarians, such as corals and sea anemones, and the photosynthetic dinoflagellate symbionts. However, there is little understanding about how the genes express when the symbiotic relationship is set up. To characterize genes involved in this association, the endosymbiosis between sea anemone, Aiptasia pulchella, and dinoflagellate zooxanthellae, Symbiodinium spp., was employed as a model. Two complementary DNA (cDNA) libraries were constructed from RNA isolated from symbiotic and aposymbiotic A. pulchella. Using single-pass sequencing of cDNA clones, a total of 870 expressed sequence tags (ESTs) clones were generated from the two libraries: 474 from symbiotic animal and 396 from aposymbiotic animal. The initial ESTs consisted of 143 clusters and 231 singletons. A BLASTX search revealed that 147 unique genes had similarities with protein sequences available from databases; 120 of these clones were categorized according to their putative function. However, many ESTs could not assign functionally. The putative roles of some of the identified genes relative to endosymbiosis were discussed. This is the first report of the use of EST analysis to examine the gene expression in symbiotic and aposymbiotic states of the cnidarians. The systematic analysis of EST from this study provides a useful database for future investigations of the molecular mechanisms involved in algal-cnidarian symbiosis.     

IPD3 controls the formation of nitrogen-fixing symbiosomes in pea and Medicago Spp

A successful nitrogen-fixing symbiosis requires the accommodation of rhizobial bacteria as new organelle-like structures, called symbiosomes, inside the cells of their legume hosts. Two legume mutants that are most strongly impaired in their ability to form symbiosomes are sym1/TE7 in Medicago truncatula and sym33 in Pisum sativum. We have cloned both MtSYM1 and PsSYM33 and show that both encode the recently identified interacting protein of DMI3 (IPD3), an ortholog of Lotus japonicus (Lotus) CYCLOPS. IPD3 and CYCLOPS were shown to interact with DMI3/CCaMK, which encodes a calcium- and calmodulin-dependent kinase that is an essential component of the common symbiotic signaling pathway for both rhizobial and mycorrhizal symbioses. Our data reveal a novel, key role for IPD3 in symbiosome formation and development. We show that MtIPD3 participates in but is not essential for infection thread formation and that MtIPD3 also affects DMI3-induced spontaneous nodule formation upstream of cytokinin signaling. Further, MtIPD3 appears to be required for the expression of a nodule-specific remorin, which controls proper infection thread growth and is essential for symbiosome formation.     

The magnesium inhibition and arrested phagosome hypotheses: new perspectives on the evolution and ecology of Symbiodinium symbioses

Zooxanthella symbioses are arguably the most important ecological interaction on coral reefs because they energetically subsidize the entire community, and enhance the calcification process that provides structure for all other organisms. While we have developed a detailed understanding of the diversity among and within the Symbiodinium clades, we currently lack a mechanistic explanation for which factors favoured zooxanthella invasion of the intracellular habitat in heterotrophic hosts, and for what molecular mechanisms permit residence within the cell. We propose two hypotheses that explain important evolutionary and ecological features of zooxanthella symbioses. The magnesium inhibition hypothesis (MIH) states that increases in the Mg/Ca ratio in sea water that occurred over the last 100 million years created a situation where Mg(2+) inhibited Ca(2+) transport to zooxanthellae. The MIH predicts, among other things, that the intracellular niche was invaded as a response to this abiotic stressor. The arrested phagosome hypothesis (APH) states that Symbiodinium spp. mimic host cell endosomal digestive machinery via the symbiosome to appear like digesting prey through perpetual release of zooxanthella-derived compounds. The APH represents a subtle but important distinction from previous hypotheses regarding interactions between symbiont and host at the cellular level. The APH predicts that symbionts tune rates of material release to match expectations of host cellular machinery. An outcome of the APH is that intra-host residence time becomes a vital parameter to consider. Both hypotheses shift control of the symbiosis away from the host, and instead focus attention on the niche requirements of Symbiodinium spp.     

Molecular identification of Rab7 (ApRab7) in Aiptasia pulchella and its exclusion from phagosomes harboring zooxanthellae

The establishment and maintenance of the intracellular association between marine cnidarians and their symbiotic microalgae is essential to the well being of coral reef ecosystems; however, little is known concerning its underlying molecular mechanisms. In light of the critical roles of the small GTPase, Rab7, as a key regulator of vesicular trafficking, we cloned and characterized the Rab7 protein in the endosymbiosis system between the sea anemone, Aiptasia pulchella and its algal symbiont, Symbiodinium spp. The Aiptasia homologue of Rab7 proteins, ApRab7 is 88% identical to human Rab7 protein and contains all Rab-specific signature motifs. Results of EGFP reporter analysis, protein fractionation, and immunocytochemistry support that ApRab7 is located in late endocytic and phagocytic compartments and is able to promote their fusion. Significantly, the majority of phagosomes containing live symbionts that either have taken long residency in, or were newly internalized by Aiptasia digestive cells did not contain detectable levels of ApRab7, while most phagosomes containing either heat-killed or photosynthesis-impaired symbionts were positive for ApRab7 staining. Overall, our data suggest that live algal symbionts persist inside their host cells by actively excluding ApRab7 from their phagosomes, and thereby, establish and/or maintain an endosymbiotic relationship with their cnidarian hosts.     

Nutrient availability for zooxanthellae derived from physiological activities of Condylactus spp

Differences in phosphate metabolism of symbiotic and aposymbiotic Condylactus suggest that the host animal makes available quantities of phosphate to support growth of zooxanthellae. Nitrite may serve as a nitrogen source for symbionts as indicated by host removal of nitrite from sea water.The presence of zooxanthellae is responsible for removal of phosphate from sea water in the dark whereas there is excretion during light periods. There is a greater uptake of nitrite from sea water in the light compared with the dark in symbiotic animals.Since nitrate is removed from sea water by aposymbiotic animals, the presence of nitrate reducing bacteria is proposed.     

Bacterial symbiosis maintenance in the asexually reproducing and regenerating flatworm Paracatenula galateia

Bacteriocytes set the stage for some of the most intimate interactions between animal and bacterial cells. In all bacteriocyte possessing systems studied so far, de novo formation of bacteriocytes occurs only once in the host development, at the time of symbiosis establishment. Here, we present the free-living symbiotic flatworm Paracatenula galateia and its intracellular, sulfur-oxidizing bacteria as a system with previously undescribed strategies of bacteriocyte formation and bacterial symbiont transmission. Using thymidine analogue S-phase labeling and immunohistochemistry, we show that all somatic cells in adult worms - including bacteriocytes - originate exclusively from aposymbiotic stem cells (neoblasts). The continued bacteriocyte formation from aposymbiotic stem cells in adult animals represents a previously undescribed strategy of symbiosis maintenance and makes P. galateia a unique system to study bacteriocyte differentiation and development. We also provide morphological and immunohistochemical evidence that P. galateia reproduces by asexual fragmentation and regeneration (paratomy) and, thereby, vertically transmits numerous symbiont-containing bacteriocytes to its asexual progeny. Our data support the earlier reported hypothesis that the symbiont population is subjected to reduced bottleneck effects. This would justify both the codiversification between Paracatenula hosts and their Candidatus Riegeria symbionts, and the slow evolutionary rates observed for several symbiont genes.     

Molecular evidence for host-symbiont specificity in soritid foraminifera

Symbiosis between the dinoflagellate genus Symbiodinium and various invertebrates and protists is an ubiquitous phenomenon in shallow tropical and subtropical waters. Molecular studies undertaken on cnidarian symbionts revealed the presence of several distinctive lineages or subgeneric clades of Symbiodinium whose taxonomic level provides limited information about the specificity between invertebrate hosts and their symbionts. This contrasts with the finding of several Symbiodinium clades being present almost exclusively in foraminifera and belonging to the subfamily Soritinae. To test whether such specificity also exists at a lower taxonomic level within Soritinae, we obtained the SSU rDNA sequences from 159 soritid individuals collected in nine localities worldwide and representing all known morphospecies of this subfamily. For each individual, the symbionts were determined either by sequencing or by RFLP analysis. We distinguished 22 phylotypes of Soritinae in relation with a number of symbiont "groups" corresponding to 3 clades and 5 subclades of Symbiodinium. Among the 22 soritid phylotypes, 14 show strict symbiont specificity and only one was found to be a host for more than two "groups" of Symbiodinium. It is suggested that the strong host-symbiont specificity observed in Soritinae is a combined effect of a selective recognition mechanism, vertical transmission of symbionts, and biogeographical isolation.