The diversity of symbioses
Dinoflagellates form symbioses with a variety of cnidarians including corals, sea anemones and jellyfish. In all cases, the symbionts are found within a host-derived vacuole within the gastrodermal cell layer. This cellular arrangement presumably allows the cnidarian host some control over metabolite transfer to the alga. Dinoflagellate cell numbers can range from one symbiont per host cell (Muscatine et al. 1998) to over 60 in some hydroids (Fitt 2000). Although these symbiotic dinoflagellates are generally referred to as either Symbiodinium sp., or the common name of zooxanthellae, they actually represent a large species complex. To date, sexual reproduction has not been observed in this group, although it has been inferred (LaJeunesse 2001; Santos et al. 2003), and taxonomy is generally based upon sequence variation of taxonomic markers (e.g. ITS1, ITS2, 28s) (LaJeunesse 2001; Baker 2003; Coffroth & Santos 2005), with latest estimates suggesting that at least 160 distinct Symbiodinium types can be identified (LaJeunesse, personal communication), each of which may represent distinct species. Despite this diversity of both cnidarian hosts and symbionts, there is high host–symbiont fidelity (LaJeunesse et al. 2003, 2004; Santos et al. 2004; Thornhill et al. 2006). However, we know very little about how this specificity is maintained. Symbionts may be passed ‘maternally’ from one generation to the next through the eggs, termed maternal or vertical transmission, or each generation may acquire symbionts from their surrounding environment, so-called open or horizontal transmission. Despite the fact that those cnidarians with an open system of symbiont transmission should be susceptible to infection by all the available symbionts, there is a great degree of specificity (e.g. LaJeunesse 2002). In open transmission, symbionts are indiscriminately ingested in a manner similar to any other food particle. However, those vacuoles containing the correct symbionts avoid lysosome fusion (Colley & Trench 1983), possibly because of Rab protein interactions between the host and symbiont (Chen et al. 2003, 2004), and begin to proliferate. A variety of different symbionts can be taken up via this process and are observed in the early juvenile stages, which can lead to different host growth rates (Little, van Oppen & Willis 2004). However, generally, the adult is dominated by one symbiont type (del Gómez-Cabrera et al. 2007) with other symbionts maintaining a much smaller, residual population.
The evolution of symbiosis requires a significant alteration of the metabolism of both partners to coordinate and facilitate the symbiotic life history strategy. A key question then is: how large are these changes, and are there novel symbiotic genes? Intraspecific comparisons of symbiotic versus non-symbiotic anemones demonstrated significantly different protein profiles (Weis & Levine 1996); however, subsequently microarray analysis has failed to identify any ‘symbiosis-specific’ genes. This suggests that existing protein pathways have been seconded for a role in symbiosis and that differences in expression may be extremely subtle (Rodriguez-Lanetty, Phillips & Weis 2006; deBoer, Krupp & Weis 2007). Recently, there have been a number of studies that have indicated a role for lectin/glycan interaction in symbiont recognition and persistence (Jimbo et al. 2000; Lin, Wang & Fang 2000; Koike et al. 2004; Wood-Charlson et al. 2006) following the initial identification of strain-specific difference of symbiont surface antigens (Kinzie & Chee 1982). This is analogous to the roles for lectin–glycan interactions found in other well-characterized symbioses (Nyholm et al. 2000; Rodriguez-Navarro, Dardanelli & Ruiz-Sainz 2007). A number of α-mannose/α-glucose and α-galactose glycans on the dinoflagellate cell wall have been identified as potential targets for cnidarian lectins, with modification of these glycans leading to reduced dinoflagellate infection in larvae (Wood-Charlson et al. 2006) and adults (Lin et al. 2000). The potential for a sophisticated interaction between cnidarians and their symbionts can be illustrated by the surprising fact that cnidarians have a gene complement which contains many of those pathways involved in immunity and cell recognition, which were previously thought to be restricted to higher animals (Kortschak et al. 2003; Kuo et al. 2004; Miller, Ball & Technau 2005; Technau et al. 2005; Miller et al. 2007). In addition, the Symbiodinium genome is at least as large as that of humans (LaJeunesse et al. 2005) with a wide variety of unique genes, including a number which appear to be most closely related to that of bacteria (Leggat et al. 2007).
Inorganic carbon uptake and fixation
The success of the cnidarian–dinoflagellate symbioses is demonstrated by the dominance of coral reefs in the shallow waters of the tropics, with estimates of over 700 coral species found in symbiosis with dinoflagellates. A large amount of effort has gone into understanding the metabolic exchange between these two partners, in particular the transfer of carbon (both organic and inorganic), nitrogen and phosphate. The presence of photosynthetic symbionts within the gastrodermal layer in cnidarians necessitates the host playing a key role in the transport of Ci from the surrounding seawater to the photosynthetic dinoflagellate. This transport of Ci from an external environment to a sink within the tissue (the alga) contrasts with other animals where Ci is excreted from the body to the external media. This necessitates a number of physiological changes including localization of a variety of enzymes. The currently accepted model for Ci uptake by the host involves an H+-ATPase acidifying the boundary layer where bicarbonate is converted to CO2 by an external CA isoform. This CO2 subsequently diffuses into the host where it is trapped by conversion to bicarbonate and is then transported to the symbiosome by an unknown pathway (for reviews, see Furla, Allemand & Orsenigo 2000a; Furla et al. 2000b, 2005; Leggat et al. 2002).
Once in the symbiosome, the Ci is taken up by the Symbiodinium CCM (Goiran et al. 1996; Leggat et al. 1999) and fixed by a form II Rubisco (Whitney, Shaw & Yellowlees 1995; Whitney & Yellowlees 1995; Rowan et al. 1996). This Rubisco isoform has a CO2/O2 specificity which, although much higher than other form II Rubiscos, is significantly less than the form I Rubisco found in all other eukaryotic photoautotrophs (Whitney & Andrews 1998). Despite this limitation, photosynthetic CO2 fixation is sufficient to meet not only the dinoflagellates' requirements for growth and respiration, but can also meet up to 95% of the host's energy requirements (Falkowski et al. 1993). These high photosynthetic rates generate significant diel variation in oxygen tensions within the coral tissue. At midday, during periods of peak photosynthesis, oxygen concentrations can be up to 250% air saturation and decrease to less than 5% during darkness (Kühl et al. 1995). As with other photosynthetic organisms, CO2 fixation in cnidarians follows a diurnal hysteresis. However, unlike the majority of other organisms, higher photosynthetic rates occur in the afternoon rather than the morning at the same PAR levels, although this may vary significantly between species (Levy et al. 2004).
Understanding Ci transport and use in scleractinian corals is further complicated by the calcification process which produces the calcium carbonate skeletons of coral reefs. This also utilizes the coral intracellular Ci pool, and the relationship between Ci utilized for photosynthesis and calcification is still unknown. Recent analysis of the calcium carbonate skeleton of corals demonstrates that the coral skeleton is one of the most efficient collectors of solar radiation in nature and, by increasing the length of the internal light path, is able to increase the internal light field up to fivefold (Enriquez, Mendez & Iglesias-Prieto 2005). As with photosynthesis, Ci for calcification can be derived from host/algal respiration (Al-Horani, Al-Moghrabi & de Beer 2003; Al-Horani et al. 2005) or the surrounding seawater Ci pool. However, the exact pathway by which Ci is transported to the aboral ectoderm, where skeletogenesis occurs, is unknown (Gattuso, Allemand & Frankignoulle 1999). In addition to Ci, calcium must also be transported to the aboral ectoderm, and a putative plasma membrane calcium pump has been characterized and localized to the site of calcification (Zoccola et al. 2004). Elucidation of the exact mechanisms of Ci movement for calcification has taken on a greater urgency given the increasing acidification seen in the world's oceans as anthropogenically generated CO2 enters the oceans, reducing pH and subsequently leading to reduced calcification rates (Hoegh-Guldberg 2005). Moreover, we know little about the effect of increased CO2 levels (hypercapnia) on the physiology of these associations.
In cnidarian symbioses, photosynthetically fixed carbon appears to be transported to the host in the form of glycerol, glucose, amino acids or lipids (Muscatine 1967; Trench 1971a; Kellogg & Patton 1983; Patton & Burris 1983; Whitehead & Douglas 2003). Although no transporters have yet been characterized in the dinoflagellate or host, it has been suggested that the process is controlled by cnidarian host factors or host release factors (HRFs), which can stimulate the release of over 40% of the photosynthetically fixed carbon from the dinoflagellate (Trench 1971b; Muscatine, Pool & Cernichiari 1972; Sutton & Hoegh-Guldberg 1990). The exact nature of these HRFs is unclear, although some evidence points to them being low-molecular weight compounds, possibly amino acids (Gates et al. 1995; Cook & Davy 2001). In addition, there is evidence for a host-synthesized photosynthetic inhibitory factor which decreases the photosynthetic rates of the alga (Grant et al. 2001). If confirmed, this ability of the host to modulate photosynthate release may be one way in which it controls the intracellular algal population, as intracellular algal growth rates in culture, where doubling times are approximately 3 d (Fitt & Trench 1983), are significantly higher than that found when in symbiosis, where doubling times can range from 6 to 74 d (Muscatine et al. 1984; Wilkerson, Kobayashi & Muscatine 1988). Recently, it was reported that the export of photosynthate from different Symbiodinium strains varies significantly when in symbiosis with the same host (Loram, Trapido-Rosenthal & Douglas 2007), indicating that different algal strains may display significantly different behaviour under similar conditions.
Another way in which cnidarian hosts may control their algal populations is through nutrient limitation. It is well known that addition of nitrogen and/or phosphate sources leads to increased dinoflagellate growth rates, population densities and photosynthetic rates within the symbiosis (Taylor 1978; Marubini & Davies 1996; Koop et al. 2001). Indeed, nitrogen limitation has been shown to prolong the progression from G1 to S phase in dinoflagellates including Symbiodinium (Olson & Chisholm 1986; Smith & Muscatine 1999). Two possible conclusions from this are that the host may be restricting the availability of nutrients, in particular nitrogen, to limit algal growth rates while maintaining high photosynthetic rates (Gunnerson, Yellowlees & Miller 1988; Rees 1991; Falkowski et al. 1993), or alternatively available seawater nitrogen concentrations may be limiting. Under N-limited conditions, photosynthetically fixed carbon that exceeds the alga's reduced carbon requirement, because of the high C : N ratio, must then be exported from the cell, where it is utilized by the host.
In cnidarian symbioses, both the host and the alga are capable of ammonium assimilation, with both possessing the enzymes glutamine synthetase (GS) and glutamate dehydrogenase (Anderson & Burris 1987; Catmull, Yellowlees & Miller 1987; Rahav et al. 1989; Yellowlees, Rees & Fitt 1994). The algal symbiont also possesses glutamine:2-oxoglutarate aminotransferase activity (Rahav et al. 1989; Roberts, Fixter & Davies 2001) which is required for the regeneration of glutamate for ammonium assimilation by GS to proceed. This ability to assimilate ammonium from the surrounding seawater, and that generated by host and algal metabolism, is thought to be a prime reason for the success of the symbiosis in the nutrient-poor tropical oceans. A variety of ammonium transporters have recently been found in Symbiodinium with similarity to bacterial transporters (Leggat et al. 2007).
Given that both host and alga are capable of ammonium assimilation, one of the main questions is: where is the primary site of ammonium assimilation in the symbiosis, the host or alga? D'Elia, Domotor & Webb (1983) proposed the depletion–diffusion hypothesis where the alga was responsible for the majority of ammonium assimilation, thereby creating a concentration gradient of ammonium from the seawater into the animal host and subsequently to the alga. In contrast, a number of studies have suggested that the host is limiting ammonium availability to the alga as a way of population control. If so, it would be expected that the majority of ammonium is assimilated in the animal fraction by GS which has a high affinity for ammonia. Early work found ammonium assimilation rates in the intact symbioses were enhanced in the light (Muscatine & D'Elia 1978) and that aposymbiotic cnidarians excreted ammonium supported the depletion–diffusion hypothesis. However, Wang & Douglas (1998), in a set of elegant experiments, found that dark ammonium assimilation, and protein and free amino acid pool sizes were the same as in the light following the addition of organic carbon (e.g. α-ketoglutarate) to the symbiosis. This supports the proposition that host ammonium assimilation is high and requires a constant supply of carbon skeletons, presumably from photosynthesis, for ammonium assimilation, which in the dark can be replaced by exogenous organic carbon. This evidence suggests that host control of ammonium concentrations is possible. Despite this, there is evidence that some nitrogen is assimilated by Symbiodinium and transported back to the host, in particular the essential amino acids histidine, isoleucine, leucine, lysine, phenylalanine, tyrosine and valine (Wang & Douglas 1999). This interplay between host and algal ammonium assimilation is yet to be clearly elucidated. Moreover, it is likely to vary significantly under different nitrogen regimes and between different host–algal combinations.
Symbiodinium is also capable of utilizing nitrate as a nitrogen source with long-term field studies showing that zooxanthellae densities are positively correlated to seawater nitrate concentrations (Fagoonee et al. 1999). Transporters for both nitrate and nitrite have been found among expressed sequence tags from Symbiodinium (Leggat et al. 2007), and the intact cnidarian symbiosis has been shown to remove nitrate from the water column (Crossland & Barnes 1977; Bythell 1990) which can lead to increased algal cell densities within the symbiosis (Marubini & Davies 1996, although see Ferrier-Pages et al. 2001) and mitotic indices (Fitt & Cook 2001). Nitrate is converted to nitrite through the action of the enzyme nitrate reductase whose cDNA sequence has been found (Leggat et al. 2007). Nitrite reductase subsequently reduces nitrite to ammonium. As would be expected, given that only the alga is capable of nitrate utilization, the majority of 15N in stable isotope studies is found in the algal fraction (Grover et al. 2003; Tanaka et al. 2006), but this nitrogen can be quickly translocated to the host (Tanaka et al. 2006) presumably as amino acids. Given that both nitrate and ammonium can serve as nitrogen sources, and are present at varying levels in seawater, a number of studies have examined the interaction between these two sources. Generally, nitrate levels in the oligotrophic waters that surround coral reefs, which are normally in the low micromolar range, are higher than ammonium, which is quickly assimilated by a variety of organisms and is found in sub-micromolar concentrations. Therefore, it is not surprising that cnidarian symbioses are capable of reasonably high rates of nitrate uptake. However, once ammonium, the preferred nitrogen source, is available, nitrate uptake rates decrease significantly (Grover et al. 2003). Elevated levels of either nitrate or ammonium through eutrophication of near-shore waters have the potential to severely disrupt the metabolism of both host and algal symbionts (Fabricius 2005).
In addition, ammonium and nitrate cnidarian symbioses are also capable of N2 fixation. The ability of some corals to fix nitrogen has only recently been discovered and is attributed to intracellular cyanobacterial symbionts and not the cnidarian host or alga (Lesser et al. 2004, 2007). However, these bacterial symbionts are not always present. Nitrogen fixation occurs in a diurnal fashion with peak fixation in the early morning and late evening, thereby avoiding the high O2 tensions in the coral tissue. The majority of the fixed nitrogen is utilized by Symbiodinium (Lesser et al. 2007). A variety of other genes associated with nitrogen fixation and metabolism have also recently been characterized from a metagenome study of the microbial associates of corals (Wegley et al. 2007), suggesting that a variety of coral-associated bacteria may be capable of nitrogen fixation.
Perhaps the least well-understood nutrient in cnidarian symbioses is phosphate. It has been suggested by a number of studies that cnidarian–Symbiodinium symbioses are phosphate limited (Jackson et al. 1989; Jackson and Yellowlees 1990) with increased coral growth and calcification rates seen in the presence of elevated phosphate (although see Renegar & Riegl 2005) in addition to higher algal densities (Koop et al. 2001). However, there are few studies that have extensively examined phosphate effects. As with nitrogen, phosphate uptake does not occur in aposymbiotic cnidarians, while uptake rates by the intact symbiosis are greater in the light than in the dark (D'Elia 1977). Phosphate uptake rates reduce after exposure to phosphate after as little as 1 h (Todd, Thornhill & Fitt 2006), suggesting that phosphate uptake becomes saturated or phosphate transporters are down-regulated. However, the exact mechanism is still unknown.
Despite decades of research, we still know very little about the more complex aspects of metabolic interplay between cnidarian–algal partners. The uptake and assimilation rates of various nutrients have been characterized; however, their ultimate metabolic destinations are often still unclear and will probably vary depending upon the relative nutrient concentrations. For example, up to half of the photosynthetically fixed carbon exported to the host is released from the host as mucus (Wild et al. 2004). This may change when nitrogen is not limiting. We still know little about the flux of metabolites between symbiotic partners and how they alter over a variety of time-scales (hours to days to months). Perhaps the most clear gap in our current knowledge is about the communication that exists between the symbiotic partners under various conditions. An example of this is the osmoregulation of both the host and alga (Mayfield & Gates 2007). Given their evolutionary success, there must be a complex conversation which coordinates the metabolism of host and symbiont. What form that takes and the molecular mechanism involved are still to be resolved. This may be of particular importance given that the greatest threat to the survival of coral reefs is the disassociation of the symbiosis, so-called coral bleaching, because of anthropogenic factors such as global warming (Hughes et al. 2003).