Trophic coupling of a testate amoeba and <Emphasis Type="Italic">Microcystis</Emphasis> species in a hypertrophic pond

Seasonal changes in abundance of the testate amoeba Penardochlamys sp. and its food vacuole contents were investigated in relation to blooms of the cyanobacteria Microcystis spp. in a hypertrophic pond from April 1999 to March 2000. The behavior of the amoeba feeding on M. aeruginosa and M. wesenbergii was also observed in the laboratory. The amoeba was detectable from late May to November 1999 during the blooms of Microcystis spp. Cell densities of the amoeba fluctuated between 1.4 and 350 cells ml^–1 with some sporadic peaks, which did not coincide with rapid decreases in the abundance of Microcystis spp. Food vacuoles contained only Microcystis cells; other prey items were not found, suggesting that this amoeba utilized only the cyanobacteria as food. The amoeba was frequently found attached to Microcystis colonies, but was not associated with other suspended particles. Observation of the amoeba feeding revealed the feeding mechanism and that the amoeba was able to graze on both species of Microcystis. These results suggest that the trophic coupling of these organisms is substantial, although grazing by the amoeba is not sufficient to regulate the dynamics of Microcystis populations in this hypertrophic pond.     

Ultrastructure of the Giant Amoeba Pelomyxa palustris*

SYNOPSIS. The ultrastructure of the herbivorous amoeba Pelomyxapalustris was studied. Nuclear division is not understood in this amoeba, and evidence for the method of nuclear division was sought. This species typically has many spheroidal nuclei which are similar within a given cell. However, some amoebae from our collections differed from this common type in both the number and structure of their nuclei. This suggested stages associated with nuclear division. One current hypothesis of nuclear division in this organism is that of nuclear budding. Our evidence is more in accord with this method than with mitosis. The cytoplasm contained no mitochondria, Golgi bodies, contractile vacuoles or crystals. Most amoebae had 2 types of bacteria (bacteroids or endosymbionts) in their cytoplasm; a separate vesicle enclosed each of these. Characteristically, only 1 type of bacterium (B_n) surrounded the nucleus. Another type (B) was found elsewhere in the cytoplasm. Also in the cytoplasm were the following: food vacuoles enclosing various algae, relatively clear vacuoles and vesicles, glycogen, various electron-opaque particles, and occasional microtubules. The plasmalemma was smooth, lacking the external fringe which characterizes other large fresh-water amoebae.     

A GLYCOPROTEIN NONCOVALENTLY ASSOCIATED WITH CELL‐WALL POLYSACCHARIDE OF THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA)1

The cells of the red microalga Porphyridium sp. (UTEX 637) are encapsulated in a cell wall of a negatively charged mucilaginous polysaccharide complex composed of 10 different sugars, sulfate, and proteins. In this work, we studied the proteins associated with the cell‐wall polysaccharide. A number of noncovalently associated proteins were resolved by SDS‐PAGE, but no covalently bound proteins were detected. The most prominent protein detected was a 66‐kDa glycoprotein consisting of a polypeptide of approximately 58 kDa and a glycan moiety of approximately 8 kDa containing N‐linked terminal mannose. In size‐exclusion chromatography, the 66‐kDa protein was coeluted with the polysaccharide and could be separated from the polysaccharide only after denaturation of the protein, indicating that the 66‐kDa protein was tightly bound to the polysaccharide. Western blot analysis revealed that the 66‐kDa protein was specific to Porphyridium sp. and P. cruentum, because it was not detected in the other species of red microalgae examined. Indirect immunofluorescence assay confirmed the location of the protein in the algal cell wall. The sequence of cDNA clone encoding the 66‐kDa glycoprotein, detected in our in‐house expressed sequence tag database of Porphyridium sp., revealed that this is a novel protein with no similarity to any protein in the public domain databases and our in‐house expressed sequence tag database of the red microalga Rhodella reticulata. The 66‐kDa protein bound polysaccharides from red algae but not from those of other origins tested. Possible roles of the 66‐kDa protein in the biosynthesis of the polysaccharide are discussed.     

Chemotaxonomic notes on Porphyridium Näg. (Rhodophyta,Porphyridiales)

Abstract

The taxonomic attribution of Porphyridium Näg. to Rhodophyta is a question from a long time debated since Porphyridium lacks both the morphological and the reproductive features of the red algae.

In the present paper we report the presence of floridosides and γ-linolenic acid in P. aerugineum and P. cruentum. Both these compounds are believed to be useful in the elucidation of the rhodophycean phylogeny; thus we support the attribution of Porphyridium to Rhodophyta.

Phylogenetic relationships between Porphyridium and other red algae are discussed.     

Immunocytochemical Localization of Phosphoribulokinase in Microalgae

Employing immunogold electron microscopy, the subcellular location of the Calvin cycle enzyme phosphoribulokinase (PRK) was determined for two diverse species of microalgae. In both the red alga Porphyridium cruentum and the green alga Chlamydomonas reinhardtii, PRK was distributed throughout the thylakoid-containing chloroplast stroma. In contrast, the next enzyme in the pathway, ribulose 1,5-bisphosphate carboxylase/oxygenase, was predominantly pyrenoid-localized in both species. In Porphyridium, the chloroplast stroma abuts the pyrenoid but in Chlamydomonas and other green algae, the pyrenoid appears encased in a starch sheath. Unique inclusions found in the pyrenoid of Chlamydomonas were immunolabelled by anti-PRK and thus identified as regions of chloroplast stroma. It is postulated that such PRK-containing stromal inclusions in the pyrenoids of Chlamydomonas and perhaps other green algae provide a means for exchange of Calvin cycle metabolites between pyrenoid and stroma.     

CHANGES IN THE ULTRASTRUCTURE OF SYMBIOTIC ZOOXANTHELLAE (SYMBIODINIUM SP., DINOPHYCEAE) IN FED AND STARVED SEA ANEMONES MAINTAINED UNDER HIGH AND LOW LIGHT1

The ultrastructure of symbiotic dinoflagellates (Symbiodinium sp., zooxanthellae) in the sea anemone Aiptasia pallida Verrill was examined in well-fed or starved (up to 120 days) anemones maintained under two light levels (5 and 50 μmol · m^?2· s^?1). Cell size of zooxanthellae was not affected by feeding history; however, both light and feeding history affected the relative cell volume of chloroplasts, lipids, and vacuoles. Stereological analysis of transmission electron micrographs showed that algae in low-light starved anemones had 10 times as much lipid (17.4% of cell volume) as those in well-fed anemones under the same light conditions (1.8%). The lipid content of algae from anemones in high light increased from 15.4% in well-fed anemones to 30.1% in starved anemones. The starch content of zooxanthellae in low-light anemones was law (4.1%) and not affected by feeding history, while the starch content of zooxanthellae in high-light anemones was greater (10.7%), with some differences among groups. Algal photoacclimation to low light included an increase in chloroplast relative volume from 17% (in well-fed high-light anemones) to 33% in well-fed low-light anemones. Starvation of the host resulted in a significant decrease in chloroplast volume in zooxanthellae in anemones at both light levels. Morphometry provides quantitative confirmation of biochemical and physiological data on zooxanthellae, because the changes in zooxanthellae with starvation of the host are consistent with other indicators of nutrient limitation of zooxanthellae of A. pallida held without food for long periods of time.     

Inhibition of lysosomal fusion with symbiont-containing vacuoles in Paramecium bursaria

Paramecium bursaria harbors several hundred intracellular Chlorella symbionts which remain undigested at the same time that the host cell phagocytizes and digests other organisms. Using electron microscopy and thorotrast labelling, we have shown that secondary lysosomes fuse with food vacuoles, but do not fuse with vacuoles containing symbiotic algae. From these and other data we suggest that the symbiotic algae alter the membrane of the vacuole which surrounds them, thus inhibiting fusion with secondary lysosomes.     

Relationship between the Unicellular Red Alga Porphyridium sp. and Its Predator, the Dinoflagellate Gymnodinium sp

Contamination of algae cultivated outdoors by various microorganisms, such as bacteria, fungi, algae, and protozoa, can affect growth and product quality, sometimes causing fast collapse of the cultures. The main contaminant of Porphyridium cultures grown outdoors in Israel is a Gymnodinium sp., a dinoflagellate that feeds on the alga. Comparison of the effects of various environmental conditions, i.e., pH, salinity, and temperature, on Gymnodinium and Porphyridium species revealed that the Gymnodinium sp. has sharp optimum curves, whereas the Porphyridium sp. has a wider range of optimum conditions and is also more resistant to extreme environmental variables. The mode of preying on the alga was observed, and the specificity of the Gymnodinium sp. for the Porphyridium sp. was shown. In addition, Gymnodinium extract was shown to contain enzymatic degrading activity specific to the Porphyridium sp. cell wall polysaccharide.     

FLOW‐CYTOMETRIC CHARACTERIZATION OF THE CELL‐SURFACE GLYCANS OF SYMBIOTIC DINOFLAGELLATES (SYMBIODINIUM SPP.)1

Symbiodinium spp. dinoflagellates are common symbionts of marine invertebrates. The cell‐surface glycan profile may determine whether a particular Symbiodinium is able to establish and maintain a stable symbiotic relationship. To characterize this profile, eight Symbiodinium cultures were examined using eight glycan‐specific fluorescent lectin probes. Confocal imaging and flow‐cytometric analysis were used to determine significant levels of binding of each probe to the cell surface. No significant variation in glycan profile was seen within each Symbiodinium culture, either over time or over growth phase. No cladal trends in glycan profile were found, but of note, two different Symbiodinium cultures (from clades A and B) isolated from one host species had very similar profiles, and two other cultures (from clades B and F) from different host species had identical profiles. Two lectin probes were particularly interesting: concanavalin A (ConA) and Griffonia simplicifolia‐II (GS‐II). The ConA probe showed significant binding to all Symbiodinium cultures, suggesting the widespread presence of cell‐surface mannose residues, while the GS‐II probe, which is specific for glycans possessing N‐acetyl groups, showed significant binding to six of eight Symbiodinium cultures. Other probes showed significant binding to the following percentage of Symbiodinium cultures examined: wheat germ agglutinin (WGA), 37.5%; peanut agglutinin (PNA), 50%; Helix pomatia agglutinin (HPA), 50%; phytohemagglutinin‐L (PHA‐L), 62.5%; soybean agglutinin (SBA), 50%; and Griffonia simplicifolia‐IB₄ (GS‐IB₄), 12.5%. This study highlights the complexity of cell‐surface glycan assemblages and their potential role in the discrimination of different dinoflagellate symbionts by cnidarian hosts.     

The effect of temperature stress on coral–<Emphasis Type="Italic">Symbiodinium</Emphasis> associations containing distinct symbiont types

Several studies have demonstrated that the temperature tolerance of scleractinian reef-building corals is controlled, in part, by hosting physiologically distinct symbiotic algae. We investigated the thermal tolerance of coral–algal associations within seven common species of reef-building corals hosting distinct Symbiodinium sub-clades collected from Heron Island during experimentally induced bleaching conditions. During experimental heating, photosynthetic fitness was assessed by the dark-adapted yield of PSII (F _v/F _m), and excitation pressure across PSII (Q _m) of each coral–algal association using pulse amplitude modulation fluorometry. The onset of bleaching was determined by the measurement of Symbiodinium cell density. Using the ribosomal internal transcribed spacer 2 (ITS-2) region, we showed that Symbiodinium type–coral host associations were temporally and spatially conserved in a high proportion of the colonies sampled within each species. Generally, the species Acropora millepora, Platygyra daedalea, Acropora aspera and Acropora formosa contained Symbiodinium ITS-2 type C3, whereas the species Montipora digitata, Porites cylindrica and Porites lutea contained Symbiodinium type C15. Bleaching susceptibility showed some association with Symbiodinium type, but further research is required to confirm this. Corals hosting C3 Symbiodinium displayed higher reductions in F _v/F _m during heating compared to their C15 counterparts, irrespective of host species. However, a corresponding reduction in Symbiodinium density was not observed. Nonetheless, A. aspera and A. formosa showed significant reductions in Symbiodinium density relative to controls. This correlated with large increases in Q _m and decreases in F _v/F _m in heated explants. Our results suggest a range of bleaching susceptibilities for the coral species investigated, with A. aspera and A. formosa showing the greatest susceptibility to bleaching and M. digitata showing the lowest bleaching susceptibility. The data provide strong evidence for distinct differences in temperature tolerance between C3 and C15 Symbiodinium types when in-hospite; however, future studies addressing the confounding effect of host species would help to confirm this.     

The diversity of bacteria,eukaryotic cells and viruses in an oligotrophic lake

An in situ transmission electron microscopic study of biomass samples concentrated from oligotrophic lake water revealed a variety of virus-infected microbial cells and many free viruses and virus-like particles. The most abundant group of microorganisms in screened and filtered water-column samples were 2 μm or less in diameter, and included representatives of several oligotrophic genera, Prosthecomicrobium, Ancyclobacter, Caulobacter and Hyphomicrobium. Among the prokaryotic host cells, which included both heterotrophs and autotrophs, on the basis of electron microscope observations, approximately 17% were infected with bacteriophage or bore adherent phage particles on their surfaces. Several bacterial morphotypes were observed among the prokaryotic hosts. Water samples passed through a 20-μm Nitex screen allowed us to concentrate and examine the larger host cells as well, including several species of single-celled algae and two amoeba species. The infected algal cells included those Chlorella-like in appearance, photosynthetic flagellates and others that could not be positively identified. About one-third of the eukaryotic cells were infected by viruses that were larger (150–200 nm) and structurally more complex than bacteriophages (50–60 nm). None of the viruses have been isolated, but when 0.2 μm filtrate from a biomass sample was spotted onto lawns of four representative heterotrophs and a Chlorella, the clearing observed was taken as evidence of lysis. Cyanobacterial lawns showed no plaques. Thin sections of two amoeba showed food vacuoles containing what appeared to be virus particles of a type seen in certain prokaryotic and eukaryotic cells in the biomass. Received: 26 January 1996 / Received revision: 10 July 1996 / Accepted: 5 August 1996     

Symbiotic Association Between Symbiodinium and the Gastropod Strombus gigas: Larval Acquisition of Symbionts

The importance of the dinoflagellate Symbiodinium sp. was studied in the early life stages of the gastropod Strombus gigas. This dinoflagellate was not found in the eggs or the gelatinous mass surrounding the eggs of the mollusk; therefore, Symbiodinium is not inherited directly. To determine whether the planktonic veligers can acquire these algae from the environment, they were exposed to freshly isolated Symbiodinium from adult S. gigas (homologous). The optimal stage for Symbiodinium inoculation was found at 48 h post-hatching. Survival and growth rates of veligers and juveniles were higher when inoculated with freshly isolated Symbiodinium in conjunction with daily feeding of Isochrysis spp. Veligers inoculated with Symbiodinium freshly isolated from three host species elicited distinct responses: (1) veligers did not take up Symbiodinium isolated from the hydrozoan Millepora alcicornis suggesting that there is discrimination on contact prior to ingestion, (2) veligers did take up Symbiodinium isolated from the anemone Bartholomea annulata, but the algae did not persist in the host tissue suggesting that selection against this type took place after ingestion or that the algae did not divide in the host, and (3) veligers did take up Symbiodinium isolated from Pterogorgia anceps where it persisted and was associated with metamorphosis of the larvae. In contrast, the Symbiodinium freshly isolated from S. gigas were not associated with metamorphosis and required an inducer such as the red alga Laurencia poitei. These data present a significant advancement for the establishment of a new approach in the aquaculture of this important but declining Caribbean species.     

PHYLOGENETIC POSITION OF SYMBIODINIUM (DINOPHYCEAE) ISOLATES FROM TRIDACNIDS (BIVALVIA), CARDIIDS (BIVALVIA), A SPONGE (PORIFERA), A SOFT CORAL (ANTHOZOA), AND A FREE-LIVING STRAIN

The genus Symbiodinium is the commonly observed symbiotic dinoflagellate (zooxanthellae) that forms mutual associations with various marine invertebrates. Numerous studies have revealed that the genus is comprised of a group of diverse taxa, and information on the phylogenetic relationships among the genus’ members is increasing. In this study, small subunit (SSU) ribosomal RNA (ssrRNA) gene sequences were determined for 15 more Symbiodinium strains from 12 relatively unstudied host taxa (Indo-Pacific tridacnids, cardiids, sponge, and soft coral), 1 hitherto unreported free-living Symbiodinium strain, and 4 other Symbiodinium strains from four other host taxa (Indo-Pacific zoanthid, foraminifer, jellyfish, and mid-Pacific hard coral). Their respective phylogenetic positions were inferred, and strains that are either closely related to or distinct from previously reported Symbiodinium taxa were revealed. The cultured Symbiodinium strains isolated from individuals of six species of tridacnids and three species of cardiids all had identical ssrRNA gene sequences, are closely related to S. microadriaticum Freudenthal, and are indistinguishable from the RFLP Type A strain previously reported. However, the ssrRNA gene sequences of clam symbionts that were obtained via gene cloning were different from those of the cultured isolates and represent strains that are close to the RFLP Type C strains. The Symbiodinium-like dinoflagellate from the Indo-Pacific sponge Haliclona koremella De Laubenfels is distinct from any of the Symbiodinium taxa studied and may be similar to the symbiont previously isolated from the stony coral Montipora patula Quelch. The isolates from the soft coral Sarcophyton glaucum Quoy et Gaimard and from the zoanthid Zoanthus sp. are both very closely related to S. pilosum Trench et Blank. The free-living Symbiodinium isolate is very closely related to the symbiont isolated from the Indo-Pacific foraminifer Amphisorus hemprichii Ehrenberg, which in turn is distinct from the Red Sea strain isolated from a similar host. Theisolate from Cassiopeia sp. is different from S. microadriaticum F., the type species harbored by Cassiopeia xamachana Bigelow, and is instead very closely related to S. pulchrorum Trench isolated from a sea anemone. The symbiont from the stony coral M. verrucosa Lamarck is a sister taxon to the symbionts isolated from the foraminifera Marginopora kudakajimensis Gudmundsson and Sorites orbiculus Forskål. These data suggest that polymorphic symbioses extend from cnidarians to some bivalve, foraminifer, and jellyfish host species.     

Feeding Characteristics of an Amoeba (Lobosea: Naegleria) Grazing Upon Cyanobacteria: Food Selection, Ingestion and Digestion Progress

Bacterivory by heterotrophic nanoflagellates and ciliates has been widely studied in aquatic environments, but data on the grazing of amoebae, are still scarce. From the water samples of Dianchi Lake (Kunming, Yunnan Province, China), we isolated an amoeba, designated as Naegleria sp. strain W2, which had potent grazing effects on some kind of cyanobacteria. The food selection mechanism and the digestion process of the amoeba were investigated in batch experiments. Predation experiments showed that filamentous cyanobacteria (e.g., Anabaena, Cylindrospermum, Gloeotrichia, and Phormidium) were readily consumed, with clearance rates ranging from 0.332 to 0.513 nL amoeba⁻¹ h⁻¹. The tight threads (Oscilltoria) and aggregates (Aphanizomenon) could not be ingested; however, their sonicated fragments were observed inside food vacuoles, suggesting that their morphologies prevent them from being ingested. Live video microscopy noted that unicellular Chroococcaceae (e.g., Synechococcus, Aphanocapsa, and Microcystis) were excreted after ingestion, indicating that food selection takes place inside food vacuoles. To determine whether the tastes or the toxins prevented them from being digested, heat-killed cells were retested for predation. Digestion rates and ingestion rates of the amoebae for filamentous cyanobacteria were estimated from food vacuole content volume. Through a “cold-chase” method, we found that the food vacuole contents declined exponentially in diluted amoebae cells, and digestion rates were relatively constant, averaging about 1.5% food vacuole content min⁻¹ at 28°C. Ingestion strongly depended on the satiation status of the amoebae, starved amoebae fed at higher rates compared with satiated amoebae. Our results suggest that the food selection and food processing mechanisms of the amoeba are similar to those of interception feeding flagellates; however, filamentous cyanobacteria cannot obtain a refuge under the grazing pressure of phagotrophic amoebae, which may widen our knowledge on the grazing of protists.     

A FREEZE-ETCHING STUDY OF THE RED ALGA PORPHYRIDIUM

Freeze-etched cells of Porphyridium cruentum and P. aerugineum closely resemble those fixed with glutaraldehyde and post-fixed with osmium tetroxide. Freeze-etching reveals diversity in the non-membranous and membranous parts of the cell. All the membranes are asymmetrical. The application of a double-replica technique illustrates how the two sides of several cell membranes fit together. Interpretation of fracture patterns through the thylakoids and stroma leads to the suggestion that the thylakoids are composed of repeating structural units. A model of the photosynthetic apparatus is proposed. It is suggested that the thylakoids of Porphyridium and other red algae are not always “free” but can be stacked much like those of other plants.     

Symbiotic dinoflagellates in marine Cnidaria: diversity and function

Dinoflagellates of the genus Symbiodinium are the most common symbiotic algae in benthic marine Cnidaria. This review addresses our current understanding of the molecular diversity of Symbiodinium and the function of these algae in symbiosis. Ribosomal DNA sequence data indicate that Symbiodinium is a diverse but probably monophyletic group. They also provide a phylogenetic framework for the analysis of the functional diversity of Symbiodinium (i.e. the variation in phenotype among various Symbiodinium genotypes), especially in relation to their nutritional role in the symbiosis. Symbiodinium provides the animal host with photosynthetic carbon and may also recycle animal nitrogenous waste. These interactions are advantageous to animals in shallow, oligotrophic waters. Recent developments in understanding of both photosynthate release and nitrogen relations in the symbiosis are reviewed. They provide the basis to explore the variation in nutritional interactions among different Symbiodinium genotypes. This review highlights areas of current uncertainty and controversy and addressess possible fulture directions of research.     

Bacteriolytic activities of the free-living soil amoebae,Acanthamoeba castellanii,Acanthamoeba polyphaga andHartmannella vermiformis

Bacteriolytic activities of axenically grown free-living soil amoebaeAcanthamoeba castellanii, Acanthamoeba polyphaga andHartmannella vermiformis towards various Gram-positive and Gram-negative bacteria were determined. A spectrophotometric assay revealed that the specific bacteriolytic activities of bothAcanthamoeba species were higher as those of the threeHartmannella strains.Bacillus megaterium, Bacillus subtilis, Chromatium vinosum, Micrococcus luteus andPseudomonas fluorescens were more easily lysed than the other bacteria tested.Agrobacterium tumefaciens, Klebsiella aerogenes andSerratia marcescens were hardly affected at all by the amoebal bacteriolytic activities. Among the Gram-negative bacteria we observed differences in lysis sensitivity while the Gram-positive bacteria tested were sensitive to lysis. Isoelectric focusing (IEF) gel-electrophoresis in the pH range 3–10 was performed to separate the bacteriolytic isoenzymes of amoebae. Bacteriolytic patterns were shown by using an activity assay in which lysis bands were formed in the agar/bacteria gel-overlay. The activity assay revealed remarkable differences in typical banding patterns for bacteriolytic activities among amoebae. Distinct differences between typical pI points of bacteriolytic activities inAcanthamoeba andHartmannella were shown. Bacteriolytic activities ofHartmannella were more pronounced and observed in the isoelectric points (pI) range of 4.0–9.3 while forAcanthamoeba the range was pI 4.5–8.9.Abbreviations IEF isoelectric focusing - PAA-IEF polyacrylamide-isoelectric focusing - CCAP culture collection of algae and protozoa - AS amoeba saline medium - pI isoelectric points     

Evolution and function of eukaryotic‐like proteins from sponge symbionts

Sponges (Porifera) are ancient metazoans that harbour diverse microorganisms, whose symbiotic interactions are essential for the host's health and function. Although symbiosis between bacteria and sponges are ubiquitous, the molecular mechanisms that control these associations are largely unknown. Recent (meta‐) genomic analyses discovered an abundance of genes encoding for eukaryotic‐like proteins (ELPs) in bacterial symbionts from different sponge species. ELPs belonging to the ankyrin repeat (AR) class from a bacterial symbiont of the sponge Cymbastela concentrica were subsequently found to modulate amoebal phagocytosis. This might be a molecular mechanism, by which symbionts can control their interaction with the sponge. In this study, we investigated the evolution and function of ELPs from other classes and from symbionts found in other sponges to better understand the importance of ELPs for bacteria–eukaryote interactions. Phylogenetic analyses showed that all of the nine ELPs investigated were most closely related to proteins found either in eukaryotes or in bacteria that can live in association with eukaryotes. ELPs were then recombinantly expressed in Escherichia coli and exposed to the amoeba Acanthamoeba castellanii, which is functionally analogous to phagocytic cells in sponges. Phagocytosis assays with E. coli containing three ELP classes (AR, TPR‐SEL1 and NHL) showed a significantly higher percentage of amoeba containing bacteria and average number of intracellular bacteria per amoeba when compared to negative controls. The result that various classes of ELPs found in symbionts of different sponges can modulate phagocytosis indicates that they have a broader function in mediating bacteria–sponge interactions.     

HOST‐SPECIALIST LINEAGES DOMINATE THE ADAPTIVE RADIATION OF REEF CORAL ENDOSYMBIONTS

Bursts in species diversification are well documented among animals and plants, yet few studies have assessed recent adaptive radiations of eukaryotic microbes. Consequently, we examined the radiation of the most ecologically dominant group of endosymbiotic dinoflagellates found in reef‐building corals, Symbiodinium Clade C, using nuclear ribosomal (ITS2), chloroplast (psbA^ncr), and multilocus microsatellite genotyping. Through a hierarchical analysis of high‐resolution genetic data, we assessed whether ecologically distinct Symbiodinium, differentiated by seemingly equivocal rDNA sequence differences, are independent species lineages. We also considered the role of host specificity in Symbiodinium speciation and the correspondence between endosymbiont diversification and Caribbean paleo‐history. According to phylogenetic, biological, and ecological species concepts, Symbiodinium Clade C comprises many distinct species. Although regional factors contributed to population‐genetic structuring of these lineages, Symbiodinium diversification was mainly driven by host specialization. By combining patterns of the endosymbiont's host specificity, water depth distribution, and phylogeography with paleo‐historical signals of climate change, we inferred that present‐day species diversity on Atlantic coral reefs stemmed mostly from a post‐Miocene adaptive radiation. Host‐generalist progenitors spread, specialized, and diversified during the ensuing epochs of prolonged global cooling and change in reef‐faunal assemblages. Our evolutionary reconstruction thus suggests that Symbiodinium undergoes “boom and bust” phases in diversification and extinction during major climate shifts.     

Physiology and cryosensitivity of coral endosymbiotic algae (Symbiodinium)

Coral throughout the world are under threat. To save coral via cryopreservation methods, the Symbiodinium algae that live within many coral cells must also be considered. Coral juvenile must often take up these important cells from their surrounding water and when adult coral bleach, they lose their endosymbiotic algae and will die if they are not regained. The focus of this paper was to understand some of the cryo-physiology of the endosymbiotic algae, Symbiodinium, living within three species of Hawaiian coral, Fungia scutaria, Porites compressa and Pocillopora damicornis in Kaneohe Bay, Hawaii. Although cryopreservation of algae is common, the successful cryopreservation of these important coral endosymbionts is not common, and these species are often maintained in live serial cultures within stock centers worldwide. Freshly-extracted Symbiodinium were exposed to cryobiologically appropriate physiological stresses and their viability assessed with a Pulse Amplitude Fluorometer. Stresses included sensitivity to chilling temperatures, osmotic stress, and toxic effects of various concentrations and types of cryoprotectants (i.e., dimethyl sulfoxide, propylene glycol, glycerol and methanol). To determine the water and cryoprotectant permeabilities of Symbiodinium, uptake of radio-labeled glycerol and heavy water (D₂O) were measured. The three different Symbiodinium subtypes studied demonstrated remarkable similarities in their morphology, sensitivity to cryoprotectants and permeability characteristics; however, they differed greatly in their sensitivity to hypo- and hyposmotic challenges and sensitivity to chilling, suggesting that standard slow freezing cryopreservation may not work well for all Symbiodinium. An appendix describes our H₂O:D₂O water exchange experiments and compares the diffusionally determined permeability with the two parameter model osmotic permeability.