Aerial exposure and body temperature of the intertidal sea anemone Anthopleura elegantissima

The sea anemone Anthopleura elegantissima is a common member of intertidal communities along the west coast of North America, and can experience extended periods of increased temperature during summertime low tides. Internal body temperatures of emersed individuals of A. elegantissima were monitored in a laboratory wind tunnel and in the field, and factors influencing the anemones’ thermal experience were examined. Larger body size and aggregation with conspecifics slowed body temperature increases in controlled wind tunnel conditions. In the field, anemones in the interior of an aggregation stayed cooler than those on the edges, and microhabitat features related to light exposure and surface orientation overshadowed any direct effects of body size. In the warmest month only (July), aggregations of A. elegantissima were significantly larger at the upper limit of their distribution than they were at the mid and lower limits, suggesting aggregation in high intertidal zones may be a behavioral response to desiccation and temperature stress. As this sea anemone can host multiple species of symbiotic algae with different thermal tolerances, the ability to slow body heating may affect the type of algae hosted and thus the potential contribution of this abundant anemone to primary production in the intertidal zone.     

Physiological energetics of the intertidal sea anemone Anthopleura elegantissima

Summary Lipid is the major energy storage molecule, and protein is highly conserved when food availability is low in high- and low-intertidal individuals of Anthopleura elegantissima. Under laboratory conditions zooxanthellae were lost from anemones, and tissue carbohydrate was mobilized to support metabolic demands. Routine catabolic requirements for carbohydrate are probably met by translocated photosynthate. Substrate-specific absorption efficiencies were greater than 95% for protein and lipid, but considerably lower (10 to 60%) for carbohydrate. This pattern of absorption is directly related to very low levels of dietary carbohydrate compared to the other substrates. High-intertidal anemones absorbed more lipid than low-intertidal anemones. Integrated budgets for organic nitrogen, the first reported for any cnidarian, indicate that low-intertidal anemones require larger daily rations than high-intertidal anemones to meet their higher metabolic demands for protein. These results are considered in relation to potential importance of zooxanthellae in nutrient cycling.     

Ammonium metabolism in the symbiotic sea anemone Anemonia viridis

The temperate sea anemone Anemonia viridis (Forskål) forms an endosymbiotic association with dinoflagellate algae commonly referred to as zooxanthellae. It is now well established that under appropriate environmental conditions, these associations can be autotrophic for carbon. Under such conditions, many of these symbioses, including A. viridis, not only retain excretory ammonium, but can take up ammonium added to the surrounding seawater. The flux from inorganic to organic nitrogen will be via the free amino acid pools and in A. viridis these were found to be markedly different between zooxanthellae and host with glycine and taurine dominant in the latter. When anemones were maintained with 20 M ammonium, the concentration of free amino groups increased in the zooxanthellae but appeared not to change in the host. There was no evidence that the ratio of glutamine – glutamate in zooxanthellae changed when anemones were maintained with 20 M ammonium for 47 days. These ratios imply that zooxanthellae from this temperate symbiosis may not be nitrogen-limited. GDH was detected in both zooxanthellae and host where it was most active with the coenzyme NADPH. In addition, GDH showed activity when glutamine replaced ammonium as the substrate, indicating that the host may have alternative means to assimilate ammonium. Zooxanthellae were shown to possess GOGAT activity in the presence of a ferredoxin analogue. This suggests that in vivo zooxanthellae could assimilate ammonium via the activity of GS linked with ferredoxin-dependent GOGAT. Given evidence from other studies of rapid ammonium assimilation and essential amino acid synthesis in symbiotic host tissue, it appears that the capacity of cnidarians to metabolise nitrogen may at present be underestimated.     

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.     

Characterization of a novel EF-hand homologue, CnidEF, in the sea anemone Anthopleura elegantissima

The superfamily of EF-hand proteins is comprised of a large and diverse group of proteins that contain one or more characteristic EF-hand calcium-binding domains. This study describes and characterizes a novel EF-hand cDNA, CnidEF, from the sea anemone Anthopleura elegantissima (Phylum Cnidaria, Class Anthozoa). CnidEF was found to contain two EF-hand motifs near the C-terminus of the deduced amino acid sequence and two regions near the N-terminus that could represent degenerate EF-hand motifs. CnidEF homologues were also identified from two other sea anemone species. A combination of bioinformatic and molecular phylogenetic analyses was used to compare CnidEF to EF-hand proteins in other organisms. The closest homologues identified from these analyses were a luciferin binding protein (LBP) involved in the bioluminescence of the anthozoan Renilla reniformis, and a sarcoplasmic calcium-binding protein (SARC) involved in fluorescence of the annelid worm Nereis diversicolor. Predicted structure and folding analysis revealed a close association with bioluminescent aequorin (AEQ) proteins from the hydrozoan cnidarian Aequorea aequorea. Neighbor-joining analyses grouped CnidEF within the SARC lineage along with AEQ and other cnidarian bioluminescent proteins rather than in the lineage containing calmodulin (CAM) and troponin-C (TNC).     

The energetics of asexual reproduction: Pedal laceration in the symbiotic sea anemone Aiptasia pulchella (Carlgren, 1943)

Asexual reproduction of the symbiotic sea anemone Aiptasia pulchella (Carlgren, 1943) was studied in the context of energy budgets determined under a variety of light and feeding conditions. Continuous darkness significantly increased rates of pedal laceration (the method of asexual reproduction), while different feeding rates had no significant effect. Digestive assimilation efficiency averaged 63% for whole brine shrimp, and conversion efficiencies (assimilated energy into biomass) averaged 31%. There were no significant differences between the conversion efficiencies of energy obtained from light or food. The index of reproductive effort (RE) for pedal laceration was very low, ranging from 0.004 to 0.044. This low RE associated with a substantial rate of asexual reproduction may, in part, explain the widespread success of certain sea anemones.     

Annual fluctuations of lipid levels in the sea anemone Anthopleura elegantissima (Brandt, 1835)

The lipid content of sea anemones from central California was measured over a three-year period by ether extraction. These levels were significantly above those which would be considered as solely structural, and they cycled in a manner roughly paralleling the reproductive cycle. The lipid content was shown to be independent of sex, and directly proportional to size of anemone. Animals from the thermal outfall canal of a power plant had lower levels of lipid than control animals, although these levels rose to equal control values during periods of decreased power plant activity. There was no effect of a one-month starvation on the lipid content in anemones from either site.     

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.     

Fine structural studies of the nervous system and the apical organ in the planula larva of the sea anemone Anthopleura elegantissima

The nervous system of the planula larva of Anthopleura elegantissima consists of an apical organ, one type of endodermal receptor cell, two types of ectodermal receptor cells, central neurons and nerve plexus. Both interneural and neuromuscular synapses are found in the nerve plexus. The apical organ is a collection of about 100 long, columnar cells each bearing a long cilium and a collar of about 10 microvilli. The cilia of the apical organ are twisted together to form an apical tuft. The ciliary rootlets of the apical organ cells are extremely long, reaching to the basal processes of the cells adjacent to the mesoglea. All three types of sensory cells are tall and slender in profile and are identified by the presence of one or more of the following features: microtubules, small vesicles, membrane-bound granules and synapses. The interneurons are bipolar cells with somas restricted to the aboral end, adjacent to the apical organ. All synapses observed are polarized or asymmetrical. A diagram including all the elements of the nervous system is presented and the possible functions of the nervous system are discussed in relation to larval behavior.     

Biogeography of two species of Symbiodinium (Freudenthal) inhabiting the intertidal sea anemone Anthopleura elegantissima (Brandt)

We have analyzed the genetic profiles of dinoflagellate populations obtained from the Pacific coast sea anemone Anthopleura elegantissima (Brandt) at collection sites from Washington to California. Genetic differences within the symbiont populations of California anemones have been uncovered by restriction length polymorphism (RFLP) analysis of the small subunit (SSU) and large subunit (LSU) ribosomal RNA genes, and by denaturing gradient gel electrophoresis (DGGE) of the internal transcribed spacer region 2 (ITS 2). The existence of two Symbiodinium species is substantiated by sequence analysis of the variable regions V1, V2, and V3 of the SSUrDNA, which also establishes their phylogenetic relatedness to other members of the genus Symbiodinium. Anemones from Washington and Oregon harbor a single dinoflagellate species, for which we propose the name S. muscatinei sp. nov. At these northern locations, S. muscatinei either exists alone or co-occurs with the Chlorella-like green algal symbiont. Our results indicate that S. muscatinei co-occurs with a second dinoflagellate, S. californium, in mixed populations in central and southern California. We suggest that the geographic distribution of these dinoflagellates is related to the temperature cline created by latitude.     

Solution structure of APETx1 from the sea anemone Anthopleura elegantissima: a new fold for an HERG toxin

APETx1 is a 42-amino acid toxin purified from the venom of the sea anemone Anthopleura elegantissima. This cysteine-rich peptide possesses three disulfide bridges (C4-C37, C6-C30, and C20-C38). Its pharmacological target is the Ether-a-gogo potassium channel. We herein determine the solution structure of APETx1 by use of conventional two-dimensional 1H-NMR techniques followed by torsion angle dynamics and refinement protocols. The calculated structure of APETx1 belongs to the disulfide-rich all-beta structural family, in which a three-stranded anti-parallel beta-sheet is the only secondary structure. APETx1 is the first Ether-a-gogo effector discovered to fold in this way. We therefore compare the structure of APETx1 to those of the two other known effectors of the Ether-a-gogo potassium channel, CnErg1 and BeKm-1, and analyze the topological disposition of key functional residues proposed by analysis of the electrostatic anisotropy. The interacting surface is made of a patch of aromatic residues (Y5, Y32, and F33) together with two basic residues (K8 and K18) at the periphery of the surface. We pinpoint the absence of the central lysine present in the functional surface of the two other Ether-a-gogo effectors.     

Early development and acquisition of Zooxanthellae in the temperate symbiotic sea anemone Anthopleura ballii (Cocks)

The ova of Anthopleura ballii become infected with zooxanthellae (endosymbiotic dinoflagellates) of maternal origin just prior to spawning. After fertilization, the zygotes undergo radial, holoblastic cleavage, and then gastrulate by invagination to form ciliated planulae. Because the zooxanthellae are localized on one side of the ovum-and later, within the blastomeres at one end of the embryo-invagination leads to the zooxanthellae being restricted to the planular endoderm and hence to the gastrodermal cells of the adult anemone. We propose that maternal inheritance of zooxanthellae plays an important part in the success of these temperate sea anemones, which live in regions where potential sources of zooxanthellae are scarce.     

The impact of coral bleaching on the pigment profile of the symbiotic alga, Symbiodinium

Bleaching of corals by loss of symbiotic dinoflagellate algae and/or photosynthetic pigments is commonly triggered by elevated temperatures coupled with high irradiance, and is a first-order threat to coral reef communities. In this study, a high-resolution high-performance liquid chromatography method integrated with mass spectrometry was applied to obtain the first definitive identification of chlorophyll and carotenoid pigments of three clades of symbiotic dinoflagellate algae (Symbiodinium) in corals, and their response to experimentally elevated temperature and irradiance. The carotenoids peridinin, dinoxanthin, diadinoxanthin (Dn), diatoxanthin (Dt) and beta-carotene were detected, together with chlorophylls a and c2, and phaeophytin a, in all three algal clades in unstressed corals. On exposure to elevated temperature and irradiance, three coral species (Montastrea franksi and Favia fragum with clade B algae, and Montastrea cavernosa with clade C) bleached by loss of 50-80% of their algal cells, with no significant impact to chlorophyll a or c2, or peridinin in retained algal cells. One species (Agaricia sp. with clade C) showed no significant reduction in algal cells at elevated temperature and irradiance, but lost substantial amounts of chlorophyll a and carotenoid pigments, presumably through photo-oxidative processes. Two coral species (Porites astreoides and Porites porites both bearing clade A algae) did not bleach. The impact of elevated temperature and irradiance on the levels of the photoprotective xanthophylls (Dn + Dt) and beta-carotene varied among the corals, both in pool size and xanthophyll cycling, and was not correlated to coral bleaching resistance.     

Individual and population growth in the asexually reproducing anemone Aiptasia pallida Verrill

The effects of feeding regime and zooxanthellae on individual and population growth of Aiptasia pallida (Verrill) were studied in a series of laboratory experiments. Individual and population growth were measured as biomass increase. Both individual and population growth were significantly affected by feeding regime while zooxanthellae enhanced growth only at the most infrequent feeding regime. Feeding regime had no significant effect on the number of individual anemones in the population produced asexually by pedal laceration after 8 wk, although there were temporal differences in pedal laceration between feeding regimes during this 8-wk period. Anemones fed three times per wk underwent little pedal laceration prior to week 4 with most individuals produced after week 4. In contrast, anemones fed once per 4 wk produced almost all anemones prior to week 4 with little subsequent pedal laceration. Zooxanthellae significantly increased the number of anemones produced by pedal laceration only among individuals fed at 4-wk intervals. Zooxanthellae had no effect on pedal laceration among individuals fed three times per week.

Population growth, measured as weight change, after 4 wk was greater than individual growth for all treatments except aposymbiotic anemones fed at 4-wk intervals for which there was no significant difference between individual and population percent weight change. These results suggest that zooxanthellae enhance growth only during periods of prey scarcity and that asexual reproduction by symbiotic individuals increases biomass at a greater rate than does individual anemone growth.     

Gonad development of the sea anemone Anthopleura asiatica in clonal populations

Seasonal fluctuations in relative gonad volume and oocyte size of the sea anemone Anthopleura asiatica were examined in 3 unisexual (male) populations and one bisexual population in the Seto Inland Sea of Japan from December 1982 to December 1985. A distinct annual cycle of gonadal maturation with a peak in the summer was found in all of the populations, although they appeared to be sustained only by asexual reproduction. Spawning occured synchronously between the 2 sexes early in the fall in the bisexual population while it was one to one and a half months later in the unisexual populations.     

Gene flow in the anemone Anthopleura elegantissima limits signatures of local adaptation across an extensive geographic range

Species inhabiting marine environments face a wide range of environmental conditions that vary spatially across several orders of magnitude. The selective pressures that these conditions impose on marine organisms, in combination with potentially high rates of gene flow between distant populations, make it difficult to predict the extent to which these populations can locally adapt. Here, I identify how selection and gene flow influence the population genetic structure of the anemone Anthopleura elegantissima along the Pacific coast of North America. Isolation by distance is the dominant pattern across the range of this species, with a genetic break near Pt. Conception, CA. Furthermore, demographic modelling suggests that this species was historically confined to southerly latitudes before expanding northward. Outlier analyses identify 24 loci under selection (out of ~1,100), but the same analysis on simulated genetic data generated using the most likely demographic model erroneously identified the same number of loci under selection, if not more. Taken together, these results suggest that demographic processes are the dominant force shaping population genetic patterns in A. elegantissima along the Pacific coast of North America. I discuss these patterns in terms of the evolutionary history of A. elegantissima, the potential for local adaptation, and their consequences with respect to interactions with the endosymbiont Breviolum muscatinei across their geographic range.     

Functional expression and characterization of four novel neurotoxins from sea anemone Anthopleura sp

The genes of four novel neurotoxins, named Hk2a, Hk7a, Hk8a, and Hk16a, were obtained from sea anemone Anthopleura sp. All four neurotoxins were composed of 47 amino acid residues and the variable residues among them were found in positions 14, 22, 25, and 37. To study their activities, the four toxins fused to the Escherichia coli thioredoxin were overexpressed by BL21 (DE3), cleaved off from the fusion partner, purified, and characterized with MALDI-TOF and CD assays. Contractile force studies of isolated SD atria indicated that rHk2a had the strongest and rHk7a the longest heart stimulation effect. Consequently, the Arg14, a highly conserved residue in various sea anemone neurotoxins, can be inferred to contribute to the duration but not the intensity of contraction-stimulating activity. Our work renders useful information to studies of sea anemone neurotoxins, especially to the clarification of the function of the disputative Arg14.