Vol. 460: 277–287, 2012 MARINE ECOLOGY PROGRESS SERIES Published July 24 doi: 10.3354/meps09776 Mar Ecol Prog Ser REVIEW Zooxanthellae that open calcium channels: implications for reef corals Ted A. McConnaughey* 2906 Norman Dr., Boise, Idaho 83704, USA ABSTRACT: Toxins that open cell membrane calcium channels have been found in the dinoflagel- late genus Symbiodinium, and likely occur in most zooxanthellae. I used published observations to examine some potentially far-reaching consequences to reef corals. Algal toxins may stimulate coral calcification by opening Ca2+ channels on the calcifying ectoderm. The coral discharges the 2+ − → + resulting protons (Ca + HCO3 CaCO3 + H ) into its coelenteron cavity, where they improve algal bicarbonate and nutrient assimilation. Coupling calcification with autotrophic physiologies contributes to the success of highly calcareous zooxanthellar symbioses, and to their associations with nutrient-poor tropical waters. Nutrient shortages freeze zooxanthellae in the G1 phase of the cell cycle. Dinoflagellates are often most toxic at such times, perhaps because toxins modulate their nuclear mix of cations, to control DNA conformation and activity. Increased Ca2+ influx into host cells disrupts cell adhesion and induces apoptosis. Zooxanthellae assimilate host nutrients, complete G1, divide, and disperse to new hosts. Nutrient shortages associate with high sea surface temperatures (SST), producing correlations between SST, calcification, and algal exit. Zooxan- thellae proliferate when nutrients are abundant, but when nutrients later disappear, usually as SST warms, toxins and the departure of over-abundant zooxanthellae potentially overwhelm the coral and cause coral bleaching. KEY WORDS: Toxin · Coral · Polyketide · Symbiodinium · Calcification · Photosynthesis · Bleaching Resale or republication not permitted without written consent of the publisher INTRODUCTION the jellyfish Cassiopeia (Fukatsu et al. 2007). Al - though the first of these toxins were discovered Several ‘bioactive compounds’ or ‘toxins’ that open nearly 20 yr ago, little is known about how zooxan- membrane Ca2+ channels have been discovered in thellae use them. This article provides a roadmap for the dinoflagellate genus Symbiodinium. A given experiments. strain of algae may produce several structurally re - Most of these alcohol-soluble polyketides consist of lated toxins. Zooxanthellatoxins A and B (A: C140H232 long continuous looped carbon chains, about 9 nm O57NS) and symbiodinolide (C137H232O57NS) were long, decorated with alcohols and ketones, plus bis- found in zooxanthellae strain Y-6, likely clade A2, epoxides in the zooxanthellatoxins and symbiodino- from the acoel flatworm Amphiscolops (Nakamura et lide. Zooxanthellatoxins A and B contract mam- al. 1993, Kita et al. 2007). Clade A1 algae from a malian muscle tissues at 0.7 µM (Nakamura et al. Hawaiian tidepool make zooxanthellamides C1-5 1993), and symbiodinolide opens mammalian N-type 2+ (C128H220O53N2S2; Onodera et al. 2005). Zooxanthel- Ca channels at 7 nM and immediately ruptures host lamide-D (C54H83O19N) occurs in clade B algae from cells at 2.5 µM (Kita et al. 2007). Symbiodinium toxins *Email: [email protected] © Inter-Research 2012 · www.int-res.com 278 Mar Ecol Prog Ser 460: 277–287, 2012 are, however, much weaker than the Ca2+ influx ago- cluding motility, secretion, exocytosis, transcription, nist maitotoxin, which may be the most potent dino- cell adhesion, apoptosis, dehydrogenation reactions, flagellate toxin. and immunological responses including the genera- Coral symbionts have not yet been examined for tion of reactive oxygen species (ROS) and nitric oxide toxins, but all analyzed strains of Symbiodinium (NO; Clapham 2007). Intracellular parasites and sym- apparently make Ca2+ channel openers, so coral sym- bionts often hijack the regulation of host cell Ca2+, bionts likely do also. Symbiodinium furthermore and Symbiodinium may do likewise (Fang et al. 1998, retains the specialized machinery for making elabo- Sawyer & Muscatine 2001, DeSalvo et al. 2008, Kita rate polyketides despite being a small dinoflagellate et al. 2010, Yuyama et al. 2011). Ca2+ channel open- with the smallest known dinoflagellate genome ers provide a mechanism. Algal colonization presum- (LaJeunesse et al. 2005). ably succeeds best in hosts with compatible physio- Algal toxins appear abundant enough to affect the logies, especially if toxins suppress harmful host host coral. Cultured zooxanthellae strain Y-6 con- physiologies or stimulate beneficial ones. tains 75 and 40 µM of zooxanthellatoxins A and B, Many of Symbiodinium’s prominent hosts are plus 76 µM symbiodinolide (Nakamura et al. 1993, highly calcareous, including foraminifera, sponges, Kita et al. 2007). These values may not be maxima, giant clams, and corals. Symbiotic corals generally due to incomplete yield on toxin extractions, and calcify faster than non-symbiotic corals, especially variable toxin levels in the algae. Symbiodinium can during the daytime (e.g. Gattuso et al. 1999). Open- occupy most of the volume of infected cells and ing Ca2+ channels on the coral’s calcifying ectoderm reaches densities of millions of cells cm−2 in the coral would stimulate calcification more directly than feed- endoderm. ing the coral and raising pH and O2 levels. Calcifica- 2+ 2+ − → + Ca channel openers can be potent physiological tion generates protons (Ca + HCO3 CaCO3 + H ) modulators. Eukaryotic cells maintain sub-micromo- which the coral discharges into its semi-enclosed lar levels of cytosolic free Ca2+. Small increases in coelenteron cavity. That potentially improves algal 2+ − intracellular Ca affect many cellular functions, in- HCO3 and nutrient uptake (Fig. 1). The following a – 2 HCO3 b 10 Oral Surface 9 pH 11 (mM) 2 CO 8 10 2+ 2 pH Ca2+ Ca 7 Zooxanthella 10 Coelenteron 9 11 + Ca2+ 2H 8 10 NO – CO nH+ 3 2 7 ) ( 3– PO4 10 Skeleton 2– + + 9 11 Seawater CO3 2H 8 10 CaCO3 LLD 7 Fig. 1. Coral physiological model. (a) Coral calcification generates protons, which the coral discharges into its coelenteron cavity. Zooxanthellae inhabit adjacent cells of the oral endoderm, and potentially benefit from improved CO2 and nutrient uptake result- ing from this proton discharge. (b) pH and Ca2+ measured above the coral, in the coelenteron, and at the calcification site, in light (L) and dark (D), based on Al-Horani et al. (2003a,b). Ries (2011) reported pH values exceeding 10 at the calcification site McConnaughey: Zooxanthellae opening calcium channels 279 sections explore these ideas. Later sections explore et al. 2002; Fig. 2a). High coral photosynthetic rates how algal toxins may contribute to algal dispersal, and mild coelenteron pH values (Al-Horani et al. coral bleaching, and algal cell cycles. 2003a,b; Fig. 1b, middle panel) suggest that the pro- tons from calcification largely counteract photosyn- thetic alkalinization and CO2 depletion. CALCIFICATION AND PHOTOSYNTHESIS Fig. 2b estimates how calcification and photosyn- thesis affect seawater CO2 and pH. Fig. 2c estimates Corals bring seawater into the calcification site carboxylation efficiency (%Vmax), based on kinetics (Bentov et al. 2009, Tambutté et al. 2012), and appar- in Fig. 2a. The shaded diagonal arrows depict a 1.3 ently raise its [Ca2+] and pH through Ca2+/2H+ ex - ratio of calcification to net photosynthesis (C:P). This change pumping by Ca2+ ATPase (Niggli et al. 1982, was the average for several corals examined by Gat- Dixon & Haynes 1989). [Ca2+] increases <10%, while tuso et al. (1999), and for corals not supplemented pH rises 1 to 2 pH units (Al-Horani et al. 2003a,b, with nutrients by Tanaka et al. (2007; see Fig. 4c). Ries 2011; Fig. 1b bottom panel). CO2 diffuses into Photosynthetic removal of 30% of the dissolved inor- = this alkaline fluid and reacts to produce CO3 ganic carbon (DIC) without calcification raises pH 2+ (McConnaughey 1989, 2003). This raises the [Ca ] from 8.0 to 8.8, reduces CO2 from 10 to 1 µM, and 2– [CO3 ] ion product and speeds calcification (Cohen reduces carboxylation efficiency to 8%Vmax. Yet the & McConnaughey 2003). same amount of photosynthesis at C:P = 1.3 reduces 2+ Corals have abundant Ca channels (Zoccola et CO2 only to 5 µM, and carboxylation efficiency to al. 1999), which probably concentrate on the coral’s 30% Vmax. In this extreme example, calcification calcifying ectoderm, along with Ca2+ ATPase (Zoc- triples photosynthetic efficiency. cola et al. 2004). This localization of Ca2+ channels Coral calcification is generally considered a weak may allow zooxanthellae to selectively stimulate photosynthetic stimulus (Tanaka et al. 2007). Most coral calcification. experiments minimize CO2 stress, however, and that The coral discharges the protons from calcification minimizes the stimulus. Gattuso et al. (2000) also into its coelenteron cavity. There they react with showed that reduced-Ca2+ seawater prevents net cal- − HCO3 to produce CO2, which is captured by zooxan- cification without inhibiting photosynthesis. These thellae in the surrounding endoderm (Fig. 1a). CO2 experiments probably did not stop the proton trans- levels below ambient (~10 µM) strongly inhibit pho- port into the coelenteron. Finally, branching and tosynthesis in freshly isolated zooxanthellae (Leggat foliose corals calcify fastest in their apical polyps, and Fig. 2. Photosynthesis, calcification, and CO2. (a) Photosynthetic kinetics of freshly isolated zooxanthellae (Leggat et al. 2002). Photosynthesis is half saturated at ~10 µM CO2, which