Iron Availability Modulates the Response of Endosymbiotic Dinoflagellates to Heat Stress.

Warming and nutrient limitation are stressors known to weaken the health of microalgae. In situations of stress, access to energy reserves can minimize physiological damage. Because of its widespread requirements in biochemical processes, iron is an important trace metal, especially for photosynthetic organisms. Lowered iron availability in oceans experiencing rising temperatures may contribute to the thermal sensitivity of reef-building corals, which rely on mutualisms with dinoflagellates to survive. To test the influence of iron concentration on thermal sensitivity, the physiological responses of cultured symbiotic dinoflagellates (genus Breviolum; family Symbiodiniaceae) were evaluated when exposed to increasing temperatures (26 to 30°C) and iron concentrations ranging from replete (500 pM Fe') to limiting (50 pM Fe') under a diurnal light cycle with saturating radiance. Declines in photosynthetic efficiency at elevated temperatures indicated sensitivity to heat stress. Furthermore, five times the amount of iron was needed to reach exponential growth during heat stress (50 pM Fe' at 26-28°C vs. 250 pM Fe' at 30°C). In treatments where exponential growth was reached, Breviolum psygmophilum grew faster than B.minutum, possibly due to greater cellular contents of iron and other trace metals. The metal composition of B.psygmophilum shifted only at the highest temperature (30°C), whereas changes in B.minutum were observed at lower temperatures (28°C). The influence of iron availability in modulating each alga's response to thermal stress suggests the importance of trace metals to the health of coral-algal mutualisms. Ultimately, a greater ability to acquire scarce metals may improve the tolerance of corals to physiological stressors and contribute to the differences in performance associated with hosting one symbiont species over another.

RNA-seq profiling of Fugacium kawagutii reveals strong responses in metabolic processes and symbiosis potential to deficiencies of iron and other trace metals.

A healthy symbiotic relationship between corals and Symbiodiniaceae relies on suitable temperature and adequate nutrients including trace metals. Besides global warming, trace metal deficiency has been shown to cause coral bleaching, a phenomenon responsible for extensive coral reef degradation around the world. How trace metal deficiency impacts Symbiodiniaceae and coral symbiosis is poorly understood, however. In this study, we applied RNA-seq to investigate how Fugacium kawagutii responds to the deficiency of five trace metals (Fe2+, Zn2+, Cu2+, Mn2+, Ni2+). We identified 685 to 2805 differentially expressed genes (DEGs) from these trace metal deficiency conditions, among which 372 were commonly regulated by all the five trace metals and were significantly enriched in energy metabolism (e.g. fatty acid synthesis). Furthermore, genes associated with extracellular matrix (ECM), cell surface structure and cell adhesion were impacted, suggesting that the ability of recognition and adhesion of F. kawagutii may be altered by trace metal deficiencies. In addition, among the five metals, Fe2+ deficiency exhibited the strongest influence, with Fe-rich redox elements and many antioxidant synthesis genes being markedly down-regulated, indicative of adaptive reduction of Fe demand but a compromised ability to combat oxidative stress. Overall, deficiency of trace metals (especially Fe) seems to repress growth and ability of ROS scavenging, elevate energy metabolism and innate immunity, and alter cell adhesion capability, with implications in symbiosis disruption and coral bleaching.

Trace Metal Requirements and Interactions in Symbiodinium kawagutii.

Photosynthetic organisms need trace metals for various biological processes and different groups of microalgae have distinctive obligate necessities due to their respective biochemical requirements and ecological niches. We have previously shown that the dinoflagellate Symbiodinium kawagutii requires high concentrations of bioavailable Fe to achieve optimum growth. Here, we further explored the trace metal requirements of S. kawagutii with intensive focus on the effect of individual metal and its interaction with other divalent metals. We found that low Zn availability significantly decreases growth rates and results in elevated intracellular Mn, Co, Ni, and Fe quotas in the dinoflagellate. The results highlight the complex interaction among trace metals in S. kawagutii and suggest either metal replacement strategy to counter low Zn availability or enhanced uptake of other metals by non-specific divalent metal transporters. In this work, we also examined the Fe requirement of S. kawagutii using continuous cultures. We validated that 500 pM of Fe' was sufficient to support maximum cell density during steady state growth period either at 26 or 28°C. This study shows that growth of S. kawagutii was limited by metal availability in the following order, Fe > Zn > Mn > Cu > Ni > Co. The fundamental information obtained for the free-living Symbiodinium shall provide insights into how trace metal availability, either from ambient seawater or hosts, affects growth and proliferation of symbiotic dinoflagellates and the interaction between symbiont and their hosts.

Interactive effects of spectral quality and trace metal availability on the growth of Trichodesmium and Symbiodinium.

Light and trace metals are critical growth factors for algae but how the interdependence of light quality and metal availability affects algal growth remains largely unknown. Our previous studies have demonstrated the importance of Ni and Fe on the growth of Trichodesmium and Symbiodinium, respectively, two important marine primary producers inhabiting environments with high light intensities. Here, we investigated the effects of light quality and intensity with availability of either Ni or Fe on their growth. For Trichodesmium, we found that specific growth rates for high Ni treatments were all significantly higher than in corresponding low Ni treatments with varying light quality and intensity. The inhibitory effect of low intensity red light was also countered by sufficient Ni supply. For Symbiodinium, we found that growth rates and biomass were reduced by 75% under low intensity red light and the stress can only be partially relieved by sufficient Fe supply. The results show that trace metal availability plays an important role in relieving the stress induced by low red light condition for both Trichodesmium and Symbiodinium although the cyanobacterium performs better in this growth condition. The difference may be attributed to the presence of phycocyanin, a unique pigment attuned to absorption of red light, in Trichodesmium. Our study shows that the concerted effects of light intensity and quality compounded with trace metal availability may influence the growth of photosynthetic organisms in the ocean.

Effects of Trace Metal Concentrations on the Growth of the Coral Endosymbiont Symbiodinium kawagutii.

Symbiodinium is an indispensable endosymbiont in corals and the most important primary producer in coral reef ecosystems. During the past decades, coral bleaching attributed to the disruption of the symbiosis has frequently occurred resulting in reduction of coral reef coverage globally. Growth and proliferation of corals require some specific trace metals that are essential components of pertinent biochemical processes, such as in photosynthetic systems and electron transport chains. In addition, trace metals are vital in the survival of corals against oxidative stress because these metals serve as enzymatic cofactors in antioxidative defense mechanisms. The basic knowledge about trace metal requirements of Symbiodinium is lacking. Here we show that the requirement of Symbiodinium kawagutii for antioxidant-associated trace metals exhibits the following order: Fe >> Cu/Zn/Mn >> Ni. In growth media with Cu, Zn, Mn, and varying Fe concentrations, we observed that Cu, Zn, and Mn cellular quotas were inversely related to Fe concentrations. In the absence of Cu, Zn, and Mn, growth rates increased with increasing inorganic Fe concentrations up to 1250 pM, indicating the relatively high Fe requirement for Symbiodinium growth and potential functional complementarity of these metals. These results demonstrate the relative importance of trace metals to sustain Symbiodinium growth and a potential metal inter replacement strategy in Symbiodinium to ensure survival of coral reefs in an oligotrophic and stressful environment.

Influence of Co and B 12 on the growth and nitrogen fixation of Trichodesmium.

We investigated the influence of varying cobalt (Co) and B12 concentrations to growth and nitrogen fixation of Trichodesmium, a major diazotroph in the tropical and subtropical oligotrophic ocean. Here we show that sufficient inorganic Co, 20 pmol L(-1), sustains the growth of Trichodesmium either with or without an additional B12 supply. We also found that in these culture conditions, nitrogen levels fixed by Trichodesmium were higher in treatments with insufficient B12 than in treatments with higher B12 availability. Under limited inorganic Co availability, ranging from 0.2 to 2 pmol L(-1), Trichodesmium growth was significantly compromised in cultures without B12. In these low Co concentrations, addition of 400 pmol L(-1) of B12 supported phytoplankton growth indicating that B12 supply augmented for the low Co concentrations. Our study demonstrates that Trichodesmium has an absolute Co requirement, which is not replaceable with Zn, and that B12 supply alleviates stress in cases where Co is limiting. These results show that the interlocking availabilities of Co and B12 may influence the growth and nitrogen fixation of Trichodesmium in the ocean.

Diel nitrogen fixation pattern of Trichodesmium: the interactive control of light and Ni.

Trichodesmium, a nonheterocystous cyanobacterium widely abundant in the surface water of the tropical and subtropical ocean, fixes dinitrogen under high light conditions while concurrently undergoing photosynthesis. The new production considerably influences the cycling of nitrogen and carbon in the ocean. Here, we investigated how light intensity and nickel (Ni) availability interplay to control daily rates and diel patterns of N2 fixation in Trichodesmium. We found that increasing Ni concentration increased N2 fixation rates by up to 30-fold in the high light treatment. Cultures subjected to high Ni and light levels fixed nitrogen throughout most of the 24 H light:dark regime with the highest rate coinciding with the end of the 12 H light period. Our study demonstrates the importance of Ni on nitrogen fixation rates for Trichodesmium under high light conditions.