A Lipidomic Approach to Understanding Free Fatty Acid Lipogenesis Derived from Dissolved Inorganic Carbon within Cnidarian-Dinoflagellate Symbiosis

The cnidarian-dinoflagellate symbiosis is arguably one of the most important within the marine environment in that it is integral to the formation of coral reefs. However, the regulatory processes that perpetuate this symbiosis remain unresolved. It is essential to understand these processes, if we are to elucidate the mechanisms that support growth and resource accumulation by coral host, and conversely, recently observed reduction and/or mortality of corals in response to rapid environmental change. This study specifically focused on one area of metabolic activity within the symbiosis, that of free fatty acid synthesis within both the dinoflagellate symbionts and cnidarian host. The main model system used was Aiptasia pulchella and Symbiodinium sp. in combination with aposymbiotic A. pulchella, the symbiotic coral Acropora millepora system and dinoflagellate culture. Fatty acids (FAs) were selected because of their multiple essential roles inclusive of energy storage (resource accumulation), membrane structure fluidity and cell signaling. The study addressed free FA lipogenesis by using a new method of enriched stable isotopic (¹³C) incorporation from dissolved inorganic carbon (DI¹³C) combined with HPLC-MS. FAs derived from DI¹³C aligned with a mixture of known lipogenesis pathways with the addition of some unusual FAs. After 120 hr, ¹³C-enriched FA synthesis rates were attributed to only a complex integration of both n–3 and n–6 lipogenesis pathways within the dinoflagellate symbionts. Furthermore, there was no detectible evidence of symbiont derived enriched isotope fatty acids, catabolized ¹³C derivatives or DI¹³C being directly utilized, in host late n–6 pathway long-chain FA lipogenesis. These findings do not align with a popular mutualistic translocation model with respect to the use of translocated symbiont photoassimilates in host long-chain FA lipogenesis, which has important connotations for linking nutrient sources with metabolite production and the dynamic regulation of this symbiosis.     

Comparative Proteomics of Symbiotic and Aposymbiotic Juvenile Soft Corals

The symbiotic association between corals and photosynthetic unicellular algae is of great importance in coral reef ecosystems. The study of symbiotic relationships is multidisciplinary and involves research in phylogeny, physiology, biochemistry, and ecology. An intriguing phase in each symbiotic relationship is its initiation, in which the partners interact for the first time. The examination of this phase in coral–algae symbiosis from a molecular point of view is still at an early stage. In the present study we used 2-dimensional polyacrylamide gel electrophoresis to compare patterns of proteins synthesized in symbiotic and aposymbiotic primary polyps of the Red Sea soft coral Heteroxenia fuscescens. This is the first work to search for symbiosis-specific proteins during the natural onset of symbiosis in early host ontogeny. The protein profiles reveal changes in the host soft coral proteome through development, but surprisingly virtually no changes in the host proteome as a function of symbiotic state.     

Phosphatidylcholine synthesis in Crithidia deanei: the influence of the endosymbiont

In this study, the role of phospholipid biosynthetic pathways was investigated in the establishment of the mutualistic relationship between the trypanosomatid protozoan Crithidia deanei and its symbiotic bacterium. Although the endosymbiont displays two unit membranes, it lacks a typical Gram-negative cell wall. As in other intracellular bacteria, phosphatidylcholine is a major component of the symbiont envelope. Here, it was shown that symbiont-bearing C. deanei incorporates more than two-fold (32)Pi into phospholipids as compared with the aposymbiotic strain. The major phospholipid synthesized by both strains was phosphatidylcholine, followed by phosphatidylethanolamine and phosphatidylinositol. Cellular fractioning indicated that (32)Pi-phosphatidylcholine is the major phospholipid component of the isolated symbionts, as well as of mitochondria. Although the data indicated that isolated symbionts synthesized phospholipids independently of the trypanosomatid host, a key finding was that the isolated bacteria synthesized mostly phosphatidylethanolamine, rather than phosphatidylcholine. These results indicate that phosphatidylcholine production by the symbiont depends on metabolic exchanges with the host protozoan. Insight about the mechanisms underlying lipid biosynthesis in symbiont-bearing C. deanei might help to understand how the prokaryote/trypanosomatid relation has evolved in the establishment of symbiosis.     

Ecological significance of endosymbionts in a mixotrophic ciliate--an experimental test of a simple model of growth coordination between host and symbiont

Ciliates with endosymbiotic algae (green ciliates) have oftenbeen found to be more viable than aposymbiotic (without endosymbionts)counterparts during periods of starvation. However, the possiblebenefit of algal endosymbionts to the growth of ciliate hostshas rarely been quantified. Growth coordination between hostand symbionts is essential to maintain the symbiosis, but themechanism behind this is also uncertain. Our hypothesis is thatthe growth rate of the symbionts is always close to its maximum,irrespective of the nutritional status of the host. To testthis hypothesis we built a model based on a constant symbiontgrowth rate, and performed an experiment where we observed thegrowth rate of aposymbiotic and green Coleps cells under differentlight conditions and food concentrations. The results were ingood agreement with the model, and showed that at low food concentrationthe growth rate of green Coleps was clearly higher than thatof aposymbiotic Coleps, while there were no significant differenceswhen food was abundant. Our results indicate that algal grossgrowth rate is always close to maximum, and that growth coordinationbetween host and symbiont is obtained by a variable degree ofleakage of photosynthetic products from the symbionts to theciliate host.     

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.     

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.     

On the functional significance of symbiotic microorganisms in the Homoptera: a comparative study of Acyrthosiphon pisum and Nilaparvata lugens

All phloem‐feeding Homoptera possess symbiotic microorganisms. Although the phylogenetic position and anatomical location of the micro‐ organisms differ, the underlying theme of the symbiosis is the same; the microorganisms improve the nutritional quality of the diet through the provision of essential amino acids. The symbiosis has been well documented in aphids, but little information is available from other homopteran groups. The impact of the loss of bacterial symbionts in the pea aphid Acyrthosiphon pisum Harris and eukaryotic yeast‐like symbionts in the Asian rice brown planthopper Nilaparvata lugens Stål was examined in parallel. The weight and relative growth rate of aphids and planthoppers was significantly reduced by symbiont loss, and characteristic features of aposymbiotic pea aphids, so‐called ‘metabolic signatures’, were, for the first time, observed in aposymbiotic N. lugens. For example, the amount of protein per unit fresh weight was reduced by 26 and 10%, and the free amino acid levels increased 1.8‐ and 1.4‐fold, in aposymbiotic A. pisum and N. lugens, respectively. In addition, the concentration of the amino acid glutamine was elevated in the tissues of aposymbiotic insects. The data are discussed in the context of our current understanding of the nutritional role of the symbiosis and the mechanisms of nitrogen metabolism in the two insect species. It is concluded that the metabolic adjustments of the insects to symbiont loss are broadly equivalent.     

Thermal Stress Promotes Host Mitochondrial Degradation in Symbiotic Cnidarians: Are the Batteries of the Reef Going to Run Out?

The symbiotic relationship between cnidarians and their dinoflagellate symbionts, Symbiodinium spp, which underpins the formation of tropical coral reefs, can be destabilized by rapid changes to environmental conditions. Although some studies have concluded that a breakdown in the symbiosis begins with increased reactive oxygen species (ROS) generation within the symbiont due to a decoupling of photosynthesis, others have reported the release of viable symbionts via a variety of host cell derived mechanisms. We explored an alternative model focused upon changes in host cnidarian mitochondrial integrity in response to thermal stress. Mitochondria are often likened to being batteries of the cell, providing energy in the form of ATP, and controlling cellular pathway activation and ROS generation. The overall morphology of host mitochondria was compared to that of associated symbionts under an experimental thermal stress using confocal and electron microscopy. The results demonstrate that hyperthermic stress induces the degradation of cnidarian host mitochondria that is independent of symbiont cellular deterioration. The potential sites of host mitochondrial disruption were also assessed by measuring changes in the expression of genes associated with electron transport and ATP synthesis using quantitative RT-PCR. The primary site of degradation appeared to be downstream of complex III of the electron transport chain with a significant reduction in host cytochrome c and ATP synthase expression. The consequences of reduced expression could limit the capacity of the host to mitigate ROS generation and maintain both organelle integrity and cellular energy supplies. The disruption of host mitochondria, cellular homeostasis, and subsequent cell death irrespective of symbiont integrity highlights the importance of the host response to thermal stress and in symbiosis dysfunction that has substantial implications for understanding how coral reefs will survive in the face of climate change.     

Trans-generational specificity within a cnidarian–algal symbiosis

Ocean warming and other anthropogenic stresses threaten the symbiosis between tropical reef cnidarians and their dinoflagellate endosymbionts (Symbiodinium). Offspring of many cnidarians acquire their algal symbionts from the environment, and such flexibility could allow corals to respond to environmental changes between generations. To investigate the effect of both habitat and host genotype on symbiont acquisition, we transplanted aposymbiotic offspring of the common Caribbean octocoral Briareum asbestinum to (1) an environmentally different habitat that lacked B. asbestinum and (2) an environmentally similar habitat where local adults harbored Symbiodinium phylotypes that differed from parental colonies. Symbiont acquisition and establishment of symbioses over time was followed using a within-clade DNA marker (23S chloroplast rDNA) and a within-phylotype marker (unique alleles at a single microsatellite locus). Early in the symbiosis, B. asbestinum juveniles harbored multiple symbiont phylotypes, regardless of source (parent or site). However, with time (~4 yr), offspring established symbioses with the symbiont phylotype dominant in the parental colonies, regardless of transplant location. Within-phylotype analyses of the symbionts revealed a similar pattern, with offspring acquiring the allelic variant common in symbionts in the parental population regardless of the environment in which the offspring was reared. These data suggest that in this host species, host–symbiont specificity is a genetically determined trait. If this level of specificity is widespread among other symbiotic cnidarians, many cnidarian–algal symbioses may not be able to respond to rapid, climate change-associated environmental changes by means of between-generation switching of symbionts.     

Flexibility of nutritional strategies within a mutualism: food availability affects algal symbiont productivity in two congeneric sea anemone species

Mutualistic symbioses are common, especially in nutrient-poor environments where an association between hosts and symbionts can allow the symbiotic partners to persist and collectively out-compete non-symbiotic species. Usually these mutualisms are built on an intimate transfer of energy and nutrients (e.g. carbon and nitrogen) between host and symbiont. However, resource availability is not consistent, and the benefit of the symbiotic association can depend on the availability of resources to mutualists. We manipulated the diets of two temperate sea anemone species in the genus Anthopleura in the field and recorded the responses of sea anemones and algal symbionts in the family Symbiodiniaceae to our treatments. Algal symbiont density, symbiont volume and photosynthetic efficiency of symbionts responded to changes in sea anemone diet, but the responses depended on the species of sea anemone. We suggest that temperate sea anemones and their symbionts can respond to changes in anemone diet, modifying the balance between heterotrophy and autotrophy in the symbiosis. Our data support the hypothesis that symbionts are upregulated or downregulated based on food availability, allowing for a flexible nutritional strategy based on external resources.     

An ecological cost associated with protective symbionts of aphids

Beneficial symbioses are widespread and diverse in the functions they provide to the host ranging from nutrition to protection. However, these partnerships with symbionts can be costly for the host. Such costs, so called “direct costs”, arise from a trade‐off between allocating resources to symbiosis and other functions such as reproduction or growth. Ecological costs may also exist when symbiosis negatively affects the interactions between the host and other organisms in the environment. Although ecological costs can deeply impact the evolution of symbiosis, they have received little attention. The pea aphid Acyrthosiphon pisum benefits a strong protection against its main parasitoids from protective bacterial symbionts. The ecological cost of symbiont‐mediated resistance to parasitism in aphids was here investigated by analyzing aphid behavior in the presence of predatory ladybirds. We showed that aphids harboring protective symbionts expressed less defensive behaviors, thus suffering a higher predation than symbiont‐free aphids. Consequently, our study indicates that this underlined ecological cost may affect both the coevolutionary processes between symbiotic partners and the prevalence of such beneficial bacterial symbionts in host natural populations.     

Acclimation effect on fatty acids of the coral Montipora digitata and its symbiotic algae

Lipids play a key role in thermal and photo-acclimation processes, yet they are often neglected in stress studies. We investigated the influence of different light intensities and an increase of temperature on the fatty acid composition of the coral Montipora digitata and its symbiotic algae (i.e., zooxanthellae). Coral branches were subjected to 3 different light intensities (7, 30 and 95% sea surface photosynthetic active radiation) in filtered seawater for 35 days. Fatty acids as methyl esters were determined using gas chromatography (GC) and verified by GC-mass spectrometry. Different light intensities, but only in combination with increased temperature, significantly affected the fatty acid composition of the coral host and zooxanthellae. Temperature and light intensity increases caused reductions in the proportion of polyunsaturated fatty acids in both the host and symbionts. Most changes occurred in the host coral, which suggests that the host is more susceptible to environmental change than the symbiont, or that the host shields the symbionts from environmental change.     

Oxidative stress causes coral bleaching during exposure to elevated temperatures

 Elevated temperatures and solar ultraviolet (UV) radiation have been implicated as recent causes for the loss of symbiotic algae (i.e., bleaching) in corals and other invertebrates with photoautotrophic symbionts. One hypothesized mechanism of coral bleaching involves the production of reduced oxygen intermediates, or toxic oxygen, in the dinoflagellate symbionts and host tissues that subsequently causes cellular damage and expulsion of symbionts. Measurements of photosynthesis in the Caribbean coral Agaricia tenuifolia, taken during temperature-induced stress and exposure to full solar radiation, showed a decrease in photosynthetic performance followed by bleaching. Exposure of corals to exogenous antioxidants that scavenge reactive oxygen species during temperature-induced stress improves maximum photosynthetic capacity to rates indistinguishable from corals measured at the ambient temperature of their site of collection. Additionally, these antioxidants prevent the coral from “ bleaching ” and affect the mechanism of symbiont loss from the coral host. These observations confirm a role for oxidative stress, whether caused by elevated temperatures or exposure to UV radiation, in the bleaching phenomenon. Accepted: 18 October 1996     

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.     

Genetic variation within <Emphasis Type="Italic">Symbiodinium</Emphasis> clade B from the coral genus <Emphasis Type="Italic">Madracis</Emphasis> in the Caribbean (Netherlands Antilles)

The internal transcribed spacer (ITS) region was sequenced in symbiotic dinoflagellates (zooxanthellae) from five morphospecies in the genus Madracis. The phylogeny of the symbionts is congruent with a companion phylogeny of the coral host. Comparison with known clade B symbiont ITS types reveals that M. mirabilis contains the B13 symbiont and that the other morphospecies contain the B7 symbiont. Madracis formosa also contains a previously undescribed type. The B7 and B13 symbionts appear to be highly specific to morphospecies in the genus Madracis. The host specificity between the B13 symbionts and its coral host may be the result of co-evolution of the coral-symbiont association and suggests that the brooding species, M. mirabilis, is reproductively isolated. Microhabitat differentiation associated with light utilization independent of depth is discussed.     

Water availability alters the tri-trophic consequences of a plant-fungal symbiosis

Plant–microbe protection symbioses occur when a symbiont defends its host against enemies (e.g., insect herbivores); these interactions can have important influences on arthropod abundance and composition. Understanding factors that generate context-dependency in protection symbioses will improve predictions on when and where symbionts are most likely to affect the ecology and evolution of host species and their associated communities. Of particular relevance are changes in abiotic contexts that are projected to accompany global warming. For example, increased drought stress can enhance the benefits of fungal symbiosis in plants, which may have multi-trophic consequences for plant-associated arthropods. Here, we tracked colonization of fungal endophyte-symbiotic and aposymbiotic Poa autumnalis (autumn bluegrass) by Rhopalosiphum padi (bird-cherry-oat aphids) and their parasitoids (Aphelinus sp.) following manipulations of soil water levels. Endophyte symbiosis significantly reduced plant colonization by aphids. Under low water, symbiotic plants also supported a significantly higher proportion of aphids that were parasitized by Aphelinus and had higher above-ground biomass than aposymbiotic plants, but these endophyte-mediated effects disappeared under high water. Thus, the multi-trophic consequences of plant-endophyte symbiosis were contingent on the abiotic context, suggesting the potential for complex responses in the arthropod community under future climate shifts.     

An experimental test of the symbiosis specificity between the ciliate Paramecium bursaria and strains of the unicellular green alga Chlorella

The ciliate Paramecium bursaria living in mutualistic relationship with the unicellular green alga Chlorella is known to be easily infected by various potential symbionts/parasites such as bacteria, yeasts and other algae. Permanent symbiosis, however, seems to be restricted to Chlorella taxa. To test the specificity of this association, we designed infection experiments with two aposymbiotic P. bursaria strains and Chlorella symbionts isolated from four Paramecium strains, seven other ciliate hosts and two Hydra strains, as well as three free-living Chlorella species. Paramecium bursaria established stable symbioses with all tested Chlorella symbionts of ciliates, but never with symbiotic Chlorella of Hydra viridissima or with free-living Chlorella. Furthermore, we tested the infection specificity of P. bursaria with a 1:1:1 mixture of three compatible Chlorella strains, including the native symbiont, and then identified the strain of the newly established symbiosis by sequencing the internal transcribed spacer region 1 of the 18S rRNA gene. The results indicated that P. bursaria established symbiosis with its native symbiont. We conclude that despite clear preferences for their native Chlorella, the host-symbiont relationship in P. bursaria is flexible.     

Multiple Symbiont Acquisition Strategies as an Adaptive Mechanism in the Coral Stylophora pistillata

In obligate symbioses, the host’s survival relies on the successful acquisition and maintenance of symbionts. Symbionts can either be transferred from parent to offspring via direct inheritance (vertical transmission) or acquired anew each generation from the environment (horizontal transmission). With vertical symbiont transmission, progeny benefit by not having to search for their obligate symbionts, and, with symbiont inheritance, a mechanism exists for perpetuating advantageous symbionts. But, if the progeny encounter an environment that differs from that of their parent, they may be disadvantaged if the inherited symbionts prove suboptimal. Conversely, while in horizontal symbiont acquisition host survival hinges on an unpredictable symbiont source, an individual host may acquire genetically diverse symbionts well suited to any given environment. In horizontal acquisition, however, a potentially advantageous symbiont will not be transmitted to subsequent generations. Adaptation in obligate symbioses may require mechanisms for both novel symbiont acquisition and symbiont inheritance. Using denaturing-gradient gel electrophoresis and real-time PCR, we identified the dinoflagellate symbionts (genus Symbiodinium) hosted by the Red Sea coral Stylophora pistillata throughout its ontogenesis and over depth. We present evidence that S. pistillata juvenile colonies may utilize both vertical and horizontal symbiont acquisition strategies. By releasing progeny with maternally derived symbionts, that are also capable of subsequent horizontal symbiont acquisition, coral colonies may acquire physiologically advantageous novel symbionts that are then perpetuated via vertical transmission to subsequent generations. With symbiont inheritance, natural selection can act upon the symbiotic variability, providing a mechanism for coral adaptation.