Light intensity as a factor in the regulation of the density of symbiotic zooxanthellae in Aiptasia tagetes (Coelenterata, Anthozoa)

Experiments designed to investigate the effect of different levels of illumination on the density of symbiotic zooxanthellae in the anemone Aiptasia tagetes (Duch. & Mich.) are described.
The anemones are found to regulate the densities of their zooxanthella populations to fixed levels which are dependent upon ambient light intensity.
Regulation is continuous and results from the interaction of increase in algal numbers, growth of host tissues, population of new host tissues by symbionts and extrusion of zooxanthellae by the host.
The nature of the process by which zooxanthellae are selected for extrusion is discussed and a scheme is outlined indicating the pathways through which the level of the algal population in Aiptasia , and other coelenterates, is believed to be controlled.     

The effect of different zooxanthellae on the growth of experimentally reinfected hosts.

1. A method is given enabling the differential effects of different strains of zooxanthellae on host growth to be assessed. This technique uses the increase in the number of tentacles as the measure of growth. 2. Aposymbiotic polyps of the anemone Aiptasia pulchella reinfected with strains of Symbiodinium microadriaticum isolated from the anemone Aiptasia pulchella and the scyphozoan Cassiopea xamachana grow as well as normal Aiptasia polyps. 3. Aposymbiotic Aiptasia polyps reinfected with zooxanthellae from the gastropod Melibe pilosa and the clam Tridacna maxima grew no better than polyps lacking zooxanthellae. 4. These results lead to the conclusion that strains of zooxanthellae differ in their ability to enhance growth of Aiptasia polyps under the experimental conditions and that these differences may have important ecological consequences.     

The role of symbiotic dinoflagellates in the temperature-induced bleaching response of the subtropical sea anemone Aiptasia pallida

Coral bleaching involves the loss of symbiotic dinoflagellates (zooxanthellae) from reef corals and other cnidarians and may be a stress response of the host, algae or both. To determine the role of zooxanthellae in the bleaching process, aposymbiotic sea anemones from Bermuda (Aiptasia pallida) were infected with symbionts from other sea anemones (Aiptasia pallida from Florida, Bartholomea annulata and Condylactis gigantea). The expulsion of algae was measured during 24-h incubations at 25, 32 and 34 degrees C. Photosynthetic rates of freshly isolated zooxanthellae were also measured at these temperatures. The C. gigantea (Cg) symbionts were expelled in higher numbers than the other algae at 32 degrees C. Photosynthesis by the Cg algae was completely inhibited at this temperature, in contrast to the other symbionts. At 34 degrees all of the symbionts had increased expulsion rates, and at this temperature only the symbionts from Florida A. pallida exhibited any photosynthesis. These results provide the first evidence that the differential release of symbionts from the same host species is related to decreased photosynthesis at elevated temperatures, and support other findings suggesting that zooxanthellae are directly affected by elevated temperatures during bleaching events.     

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.     

Regulation and control of intracellular algae (= zooxanthellae) in hard corals

To examine algal (= zooxanthellae) regulation and control, and the factors determining algal densities in hard corals, the zooxanthellae mitotic index and release rates were regularly determined in branch tips from a colony of a staghorn coral, Acropora formosa, recovering from a coral ''bleaching'' event (the stress-related dissociation of the coral–algal symbiosis). Mathematical models based upon density-dependent decreases in the algal division frequency and increases in algal release rates during the post-bleaching recovery period accurately predict the observed recovery period (ca. 20 weeks). The models suggest that (i) the colony recovered its algal population from the division of the remaining zooxanthellae, and (ii) the continual loss of zooxanthellae significantly slowed the recovery of the coral. Possible reasons for the ''paradoxical'' loss of healthy zooxanthellae from the bleached coral are discussed in terms of endodermal processes occurring in the recovering coral and the redistribution of newly formed zooxanthellae to aposymbiotic host cells. At a steady-state algal density of 2.1 x 10⁶ zooxanthellae cm^-2 at the end of the recovery period, the zooxanthellae would have to form a double layer of cells in the coral tissues, consistent with microscopic observations. Neighbouring colonies of A. formosa with inherently higher algal densities possess proportionately smaller zooxanthellae. Results suggest that space availability and the size of the algal symbionts determines the algal densities in the coral colonies. The large increases in the algal densities reported in corals exposed to elevated nutrient concentrations (i.e between a two- and five-fold increase in the algal standing stock) are not consistent with this theory. We suggest that increases of this magnitude are a product of the experimental conditions: reasons for this statement are discussed. We propose that the stability of the coral–algal symbiosis under non-stress conditions, and the constancy of zooxanthellae densities in corals reported across growth form, depth and geographic range, are related to space availability limiting algal densities. However, at these densities, zooxanthellae have attributes consistent with nutrient limitation.     

Heat stress induces different forms of cell death in sea anemones and their endosymbiotic algae depending on temperature and duration

Bleaching of reef building corals and other symbiotic cnidarians due to the loss of their dinoflagellate algal symbionts (=zooxanthellae), and/or their photosynthetic pigments, is a common sign of environmental stress. Mass bleaching events are becoming an increasingly important cause of mortality and reef degradation on a global scale, linked by many to global climate change. However, the cellular mechanisms of stress-induced bleaching remain largely unresolved. In this study, the frequency of apoptosis-like and necrosis-like cell death was determined in the symbiotic sea anemone Aiptasia sp. using criteria that had previously been validated for this symbiosis as indicators of programmed cell death (PCD) and necrosis. Results indicate that PCD and necrosis occur simultaneously in both host tissues and zooxanthellae subject to environmentally relevant doses of heat stress. Frequency of PCD in the anemone endoderm increased within minutes of treatment. Peak rates of apoptosis-like cell death in the host were coincident with the timing of loss of zooxanthellae during bleaching. The proportion of apoptosis-like host cells subsequently declined while cell necrosis increased. In the zooxanthellae, both apoptosis-like and necrosis-like activity increased throughout the duration of the experiment (6 days), dependent on temperature dose. A stress-mediated PCD pathway is an important part of the thermal stress response in the sea anemone symbiosis and this study suggests that PCD may play different roles in different components of the symbiosis during bleaching.     

Nutritional role of two algal symbionts in the temperate sea anemone Anthopleura elegantissima brandt

The intertidal sea anemone Anthopleura elegantissima in the Pacific Northwest may host a single type of algal symbiont or two different algal symbionts simultaneously: zooxanthellae (Symbiodinium muscatinei) and zoochlorellae (green algae; Trebouxiophyceae, Chlorophyta). A seasonal comparison of zooxanthellate and zoochlorellate anemones showed stable symbiont population densities in summer and winter, with densities of zoochlorellae about 4 times those of zooxanthellae. Photosynthesis-irradiance curves of freshly isolated symbionts show that the productivity (P(max) cell) of freshly isolated zooxanthellae was about 2.5 times that of zoochlorellae during July; comparable rates were obtained in other months. Models of algal carbon flux show that zoochlorellae may supply the host with more photosynthetic carbon per unit anemone biomass than zooxanthellae supply. Zooxanthellate anemone tissue was 2 per thousand ((13)C) and 5 per thousand ((15)N) enriched and zoochlorellate anemone tissue was 6 per thousand ((13)C) and 8 per thousand ((15)N) enriched over their respective symbionts, suggesting that zoochlorellate anemones receive less nutrition from their symbionts than do zooxanthellate individuals. The disparity between predicted contributions from the algal carbon budgets and the stable isotopic composition suggests that short-term measures of algal contributions may not reflect actual nutritional inputs to the host. Isotopic data support the hypothesis of substantial reliance on external food sources. This additional nutrition may allow both algae to persist in this temperate intertidal anemone in spite of differences in seasonal photosynthetic carbon contributions.     

Specificity of a model cnidarian-dinoflagellate symbiosis

To understand the flexibility of symbiotic associations in coral reefs, we investigated the specificity of the Aiptasia (cf. insignis)-Symbiodinium association in the laboratory by rendering the anemones aposymbiotic and inoculating them with different isolates of SYMBIODINIUM: Infective algal symbionts were monitored over 3 months by re-isolation and identification using denaturing-gradient gel electrophoresis and sequence comparison of their amplified 18S rRNA hypervariable V1 + V2 gene region. Despite similarity in their external morphology, the algal isolates differed in their infectivity towards the host. Within days of single-isolate inoculation, aposymbiotic anemones formed associations with fresh or cultured isolates (clade B) from the anemones Aiptasia sp. or A. tagetes, respectively. They associated to a limited extent with cultured isolates (clade A) from the tridacnids Tridacna crocea or Hippopus hippopus, and not at all with a cultured isolate (clade C) from the stony coral Montipora verrucosa, nor with a free-living isolate (clade A) from subtidal sands. Aposymbiotic anemones inoculated with a mixture of all isolates had only the anemone taxon as their detectable symbionts. Re-inoculation of induced symbioses with a mixture of all isolates and incubation with wild anemones showed that the initial induced symbioses with the anemone taxon were stable. Anemones originally infected with tridacnid isolates either additionally acquired the anemone taxon or had the former outgrown by the latter. These results demonstrate the presence of a host-symbiont recognition mechanism, and possibly competition among potential algal symbionts in the Aiptasia-Symbiodinium association. Here we present a method that may be useful in monitoring the algal population dynamics in symbiotic corals in the field, along with an efficient method of rendering Aiptasia aposymbiotic for further laboratory investigation of Aiptasia-Symbiodinium symbioses.     

Allozyme electrophoresis as a tool for distinguishing different zooxanthellae symbiotic with giant clams

The taxonomy of zooxanthellae in marine invertebrate symbioses is not well understood owing mainly to their lack of reliable morphological differences. Nevertheless, previous work using protein and DNA electrophoreses has set the stage for advancing our taxonomic understanding of cnidarian zooxanthellae. Here we present the use of allozymes as genetic markers for distinguishing algal isolates from tridacnid hosts. Zooxanthellae from seven Tridacna and Hippopus species were isolated and maintained in axenic clonal cultures over many generations. Of 16 enzyme systems, α- and β-esterase (EST), esterase-F (EST-F), glucose phosphate isomerase (GPI), and malate dehydrogenase (MDH) were found suitable polymorphic markers of genetic differences among clonal cultures. Of 39 clonal isolates, 97% were found to be genetically distinguishable. This high extent of genetic variation in zooxanthellae within and between clam species was unexpected, and is difficult to explain based solely on the general notion of asexual reproduction in symbiotic zooxanthellae. Our results are also consistent with the occurrence of sexual reproduction in clam zooxanthellae. The close genetic similarity of the symbionts of Tridacna gigas, the largest and fastest-growing clam species, and the difficulty of initiating their clonal cultures in the given nutrient medium, compared with the symbionts of other clam species, are further indicative of possibly distinct algal symbionts in T. gigas. These findings are discussed in light of current taxonomic understanding of these organisms.     

Molecular cloning of Rab5 (ApRab5) in Aiptasia pulchella and its retention in phagosomes harboring live zooxanthellae

The intracellular association of symbiotic dinoflagellates (zooxanthellae) with marine cnidarians is the very foundation of the highly productive and diversified coral reef ecosystems. To reveal its underlying molecular mechanisms, we previously cloned ApRab7, a Rab7 homologue of the sea anemone Aiptasia pulchella, and demonstrated its selective exclusion from phagosomes containing live zooxanthellae, but not from those containing either dead or photosynthesis-impaired algae. In this study, Rab5 was characterized, due to its key role in endocytosis and phagocytosis acting upstream of Rab7. The Aiptasia Rab5 homologue (ApRab5) is 79.5% identical to human Rab5C and contains all Rab-specific signature motifs. Subcellular fractionation study showed that ApRab5 is mainly cytosolic. EGFP reporter and phagocytosis studies indicated that membrane-associated ApRab5 is present in early endocytic and phagocytic compartments, and is able to promote their fusion. Significantly, immunofluorescence study showed that the majority of phagosomes containing either resident or newly internalized live zooxanthellae were labeled with ApRab5, while those containing either heat-killed or photosynthesis-impaired algae were mostly negative for ApRab5 staining whereas the opposite was observed for ApRab7. We propose that active phagosomal retention of ApRab5 is part of the mechanisms employed by live zooxanthellae to: (1) persist inside their host cells and (2) exclude ApRab7 from their phagosomes, thereby, establishing and/or maintaining an endosymbiotic relationship with their cnidarian hosts.     

On the origin of terpenes in symbiotic associations between marine invertebrates and algae (zooxanthellae). Culture studies and an application of 13C/12C isotope ratio mass spectrometry

13C/12C ratios of sets of compounds, algal sterols and terpenes, isolated from dinoflagellate symbiont (zooxanthellae)-bearing soft corals and gorgonians were determined. In most cases, a significant difference was found between the delta 13C values of the terpenes and of the algal sterols from the same set, the algal sterols containing less 13C than the terpenes. These results can only be explained if terpenes are synthesized by the host. Cultured zooxanthellae, isolated from symbiotic associations, do not make terpenes. Algal sterols of the various sets do not all have the same delta 13C value: average values range from -18.2 to -24.3%. A consistent difference of about 7% in the delta 13C values of sterols of cultured symbionts isolated from two of the gorgonians was found. This has potential applications for the taxonomy of zooxanthellae, most of which are believed by some specialists to be one discrete species.     

The influence of "host release factor" on carbon release by zooxanthellae isolated from fed and starved Aiptasia pallida (Verrill).

Symbiotic dinoflagellates (zooxanthellae) typically respond to extracts of host tissue with enhanced release of short-term photosynthetic products. We examined this "host release factor" (HRF) response using freshly isolated zooxanthellae of differing nutritional status. The nutritional status was manipulated by either feeding or starving the sea anemone Aiptasia pallida (Verrill). The release of fixed carbon from isolated zooxanthellae was measured using 14C in 30 min experiments. Zooxanthellae in filtered seawater alone released approximately 5% of photosynthate irrespective of host feeding history. When we used a 10-kDa ultrafiltrate of A. pallida host tissue as a source of HRF, approximately 14% of photosynthate was released to the medium. This increased to over 25% for zooxanthellae from anemones starved for 29 days or more. The cell-specific photosynthetic rate declined with starvation in these filtrate experiments, but the decline was offset by the increased percentage release. Indeed, the total amount of released photosynthate remained unchanged, or even increased, as zooxanthellae became more nutrient deficient. Similar trends were also observed when zooxanthellae from A. pallida were incubated in a 3-kDa ultrafiltrate of the coral Montastraea annularis, suggesting that HRF in the different filtrates operated in a similar manner. Our results support the suggestion that HRF diverts surplus carbon away from storage compounds to translocated compounds such as glycerol.     

Cellular growth of host and symbiont in a cnidarian-zooxanthellar symbiosis

The hydroid Myrionema ambionense, a fast-growing cnidarian (doubling time = 8 days) found in shallow water on tropical back-reefs, lives in symbiosis with symbiotic dinoflagellates of the genus Symbiodinium (hereafter also referred to as zooxanthellae). The symbionts live in vacuoles near the base of host digestive cells, whereas unhealthy looking zooxanthellae are generally located closer to the apical end of the host cell. Cytokinesis of zooxanthellae occurred at night, with a peak in number of symbionts with division furrows (mitotic index, MI = 12%-20%) observed at dawn. The MI of zooxanthellae decreased to near zero by the middle of the afternoon and remained there until the middle of the next night. Densities of live zooxanthellae living inside of host digestive cells peaked following cytokinesis, whereas densities of unhealthy looking symbionts were highest just before the division peak. Mitosis of host digestive cells was highest in the evening, also preceding the peak in zooxanthellar MI. This is the first study relating phased host cell division to diel zooxanthellar division in marine cnidarians. Food vacuoles were prevalent inside of digestive cells of field-collected hydroids within a few hours after sunset and throughout the night, coinciding with digestion of captured demersal plankton. Laboratory experiments showed that food vacuoles appeared in digestive cell cytoplasm within 2 h of feeding with nauplii of Artemia. The number and size of food vacuoles per digestive cell and the percentage of digestive cells with food vacuoles all decreased 5-7 h following feeding in laboratory experiments, and by mid-day in field-collected hydroids. Light and external food supply were important in maintaining phased division of the symbionts, with a lag in response time to both parameters of 11-36 h. Altering light and feeding during the night did not influence the level of the peak MI the next morning, though in one experiment the absence of light slowed final separation of daughter cells at the end of cytokinesis. In another experiment, hydroids starved for 3-7 d and "pulse-fed" Artemia nauplii for 1 h at the beginning of the dark period showed continued low symbiont division (< 5%) after 11 h, whether maintained in constant light or darkness, implying that most algal division is set more than 24 h prior to actual cytokinesis. Transferred to a 14:10 h light:dark cycle for another 24 h (36 h after feeding), the same hydroids exhibited a "normal" peak MI (ca. 15%) at dawn, but zooxanthellae from hydroids kept in constant darkness still showed a low MI. These results show that mitosis of symbiotic dinoflagellates requires three factors: external food; a minimum period of time following feeding (11-36 h), presumably for digestion; and a period of light following feeding, presumably to provide carbon skeletons necessary for completing cytokinesis.     

The adaptive bleaching hypothesis: experimental tests of critical assumptions

Coral bleaching, the loss of color due to loss of symbiotic zooxanthellae or their pigment, appears to be increasing in intensity and geographic extent, perhaps related to increasing sea surface temperatures. The adaptive bleaching hypothesis (ABH) posits that when environmental circumstances change, the loss of one or more kinds of zooxanthellae is rapidly, sometimes unnoticeably, followed by formation of a new symbiotic consortium with different zooxanthellae that are more suited to the new conditions in the host's habitat. Fundamental assumptions of the ABH include (1) different types of zooxanthellae respond differently to environmental conditions, specifically temperature, and (2) bleached adults can secondarily acquire zooxanthellae from the environment. We present simple tests of these assumptions and show that (1) genetically different strains of zooxanthellae exhibit different responses to elevated temperature, (2) bleached adult hosts can acquire algal symbionts with an apparently dose-dependent relationship between the concentration of zooxanthellae and the rate of establishment of the symbiosis, (3) and finally, bleached adult hosts can acquire symbionts from the water column.     

Entry of algal symbionts into oocytes of the coral Litophyton arboreum

On the Red Sea coral reefs Litophyton arboreum is a common octocoral whose endodermal cells are associated with endosymbiotic dinoflagellates (zooxanthellae). Colonies of this species are gonochoric and brood planulae which, upon release, are already associated with the algal symbionts. Algal cells within membrane-bound vacuoles are observed within the gastrovascular cavity of the polyps, adjacent to the oocytes and are gradually phagocytized by the follicular cells which surround the oocytes. During oogenesis, temporary gaps open in the mesoglea underlying the follicular cells. Symbionts within vacuoles, along with cytoplasm and various organelles derived from the follicular cells, are translocated through these gaps. Subsequently, groups of zooxanthellae accumulate at the perioocytic zone, flanked between the mesoglea and oocytic microvilli. At a later stage, prior to the commencement of the breeding season, symbionts pass through the oolemma and rest inside the periphery of the oocytes. It is proposed that early uptake of zooxanthellae by sexual progeny at the oocyte stage, indicates a highly specialized mode of interaction between this algal symbiont and its host.     

Uric acid deposits in symbiotic marine algae

The symbiosis between cnidarians and dinoflagellate algae is not understood at the cell or molecular level, yet this relationship is responsible for the formation of thousands of square kilometres of coral reefs. We have investigated the nature of crystalline material prominent within marine algal symbionts of Aiptasia sp. anemones. This material, which has historically been considered to be calcium oxalate, is shown to be uric acid. We demonstrate that these abundant uric acid stores can be mobilized rapidly, thereby allowing the algal symbionts to flourish in an otherwise N-poor environment. This is the first report of uric acid accumulation by symbiotic marine algae. These data provide new insight and considerations for understanding the physiological basis of algal symbioses, and represent a new and previously unconsidered aspect of N metabolism in cnidarian, and a variety of other, marine symbioses.     

Response of sponges with autotrophic endosymbionts during the coral-bleaching episode in Puerto Rico

An updated list of sponges with algal endosymbionts including new records for Puerto Rico and the Caribbean, indicates that thirty-five species of common Caribbean sponges possess photosynthetic endosymbionts. Of these, 23 (67.6%) species in seven orders, were found with unicellular chroococcoid cyanobacteria (Aphanocapsa-like) and 5 (14.7%) hadromerid species were found with zooxanthellae. Sponges with other algae as symbionts occur less frequently (6%). Thirty-one common sponge species were inspected for bleaching during coral-bleaching months (July-September 1987; January 1988) in Puerto Rico. Anthosigmella varians, Xestospongia muta and Petrosia pellasarca bleached partially, but only few individuals within any given population became bleached and the bleaching of sponges was very localized. Adaptations between cyanobacterial symbionts and sponges, acquired during the long evolutionary history of these two taxa may explain the paucity of bleached sponges when compared to the high incidence of bleached corals reported.     

Ability of Monaco shrimp Lysmata seticaudata (Decapoda: Hippolytidae) to control the pest glass anemone Aiptasia pallida (Actiniaria: Aiptasidae)

The present work highlights the ability of wild and cultured Monaco shrimp Lysmata seticaudata to control the glass anemone Aiptasia pallida. Starved shrimp ingested the highest percentages of glass anemones [85.7% and 89.3% for wild (W) and cultured (C) shrimp, respectively]. The absence of symbiotic zooxanthellae in glass anemones did not influence the shrimps feeding rate, with shrimp offered aposymbiotic anemones displaying the same feeding percentages (67.3% and 70.7% for W and C shrimp, respectively) as those offered sea anemones with symbiotic zooxanthellae (70.0% and 74.4% for W and C shrimp, respectively). Shrimp offered larger sea anemones had the lowest feeding percentages (33.0% and 36.3% for W and C shrimp, respectively), along with shrimp offered an alternative food (27.3% and 36.0% for W and C shrimp, respectively). There were no significant differences in the percentage of glass anemones ingested by cultured and wild Monaco shrimp in the same feeding trial.