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Photoreception and signal transduction in corals: proteomic and behavioral evidence for cytoplasmic calcium as a mediator of light responsivity.

Introduction

How do corals sense light and respond to the reception of light? The cellular pathways that respond to the detection of photons by photoreceptors have been studied in considerable detail, mostly in complex invertebrates such as the arthropod Drosophila or in vertebrates (Yarfitz and Hurley, 1994). These pathways fall into two classes. The first is the cyclic nucleotide pathway utilized by mammals. In this example a photon striking a rhodopsin or opsin class photoreceptor activates a trimeric G-protein that in turn regulates cyclic nucleotide levels in the cytoplasm, and the levels of this second messenger relay the effect of the photon strike to the cellular metabolism (Yarfitz and Hurley, 1994). The second class of pathway is exemplified by arthropods, in which light striking a rhodopsin photoreceptor also changes the activity of a G-protein, although in this example the G-protein regulates the activity of a membrane-associated enzyme called phospholipase C (PLC). PLC drives the production of inositol triphosphate (1P3), a soluble messenger that crosses from the membrane to the endoplasmic reticulum where it binds to 1P3 gated calcium channels, opening them and allowing the calcium sequestered in the endoplasmic reticulum to flood into the cytoplasm and propagate the effects of the photon strike (Hardie, 2001). Mammals use a similar [IP.sub.3]/calcium pathway to mediate the effects of the nonvisual photoreceptor melanopsin (Sexton et al., 2012), which is responsible for entraining light-driven circadian rhythms (Kumbalasiri et al., 2007).

Light sensing in corals has been poorly studied, and is better understood in the related cnidarian, the jellyfish. Some jellyfish, in particular cubozoan box jelly medusae, have light-sensing organs called ocelli. Within ocelli a cyclic nucleotide transduction pathway similar to that described above for mammalian eyes is utilized (Koyanagi et al., 2008; Fain et at., 2010); in this case the cyclic nucleotide is cAMP, and levels increase in response to light. Light sensing is relayed to muscles and allows the jellyfish to swim in the appropriate direction relative to light. In jellyfish planulae, the early planktonic larval phase of development in cnidaria, single dispersed cells are photoreceptive (Nordstrom et al., 2003). These light-responsive microvillar cells have a motor cilium, so once again the planula can orient its movement relative to a fight source, though in this case the effect is directly on the ciliary beating pattern--no eyes, nerves, or muscles are involved. In this later example the signal transduction pathway remains uncharacterized, but microvillar-type photoreceptive cells typically signal via calcium as a second messenger (Koyanagi et al., 2008).

The issue of how corals sense and respond to light is relevant to many aspects of coral biology. Large-scale biochemical shifts occur between day and night, exemplified by cycles in RNA transcripts, cell division rates, energetics, and behavior (Sweeney, 1976; Brady et at., 2011; Levy et al., 2011). We are particularly interested in how coral broadcast-spawning is temporally regulated, and especially how sunset and the onset of darkness controls the time at which spawning occurs. In this report we demonstrate that two-dimensional protein electrophoresis is a powerful tool for examining the biochemical status of coral tissue, and we use it to show that light-mediated changes in the proteome and in coral behavior can both be achieved in darkness by altering cytoplasmic calcium levels.

Materials and Methods

Calcium-modulating reagents

Ionomycin (Sigma 10634) was used at a final concentration of 4 [mu]mol [l.sup.-1]. Stock was dissolved in 100% ethanol to a concentration of 4 mmol [l.sup.-1] and diluted in seawater 1:1000 for a final working solution. Thapsigargin (Sigma T9033) was used at a final concentration of 2 [mu]mol [l.sup.-1]. A 1000X stock was made by dissolving thapsigargin in 100% ethanol to a concentration of 2 mmol [l.sup.-1], which was diluted in fresh seawater for final use. Ruthenium red (Sigma R2751) was dissolved in seawater to a final concentration of 1 mmol [l.sup.-1] for use.

Coral sampling and treatment

Montastraea cavernosa (Linnaeus, 1767) samples were collected from the Flower Garden Banks National Marine Sanctuary in the Gulf of Mexico (27[degrees]59'N 93[degrees]35'W) under permit (FGBNMS-2008-008). Samples were collected from the vicinity of East Bank buoy 4 at 0900h on 23 August 2008 from a depth of about 23 m. All culture and treatments were performed on the deck of the ship while at sea. After collection the single fragment was split into three pieces, each with a surface area of about 10 [cm.sup.2] of coral tissue, and kept in a flow-through tank at ambient light for the remainder of the day. One sample was collected at 1620h by scraping tissue into a cryotube containing three tissue volumes of phosphosafe (EMD cat#71296), a protein-preserving reagent that contains protein phosphatase inhibitors, and freezing it on dry ice. The remaining two samples were kept alive in large buckets of seawater. After sunset, one sample was left in the dark and screened from stray light using black plastic. The third sample was in an identical bucket, but illuminated by a 10-W incandescent light (~ 100 lux, standard Osram household bulb). These two samples were collected by tissue scraping into phosphosafe at 0420 on the following day and frozen on dry ice.

Specimens of Montastraea franksi (Gregory, 1895) were collected from the same site, but on 4 September 2007 (permit numbers FGBNMS-2007-003 and -006). Six samples were collected mid-morning and kept in flow-through tanks on the ship's deck. Each fragment was split into four clonal fragments, each with about 5 cm x 5 cm of coral tissue. Shortly before sunset (1932 EST) one fragment of each individual was placed into a beaker containing seawater plus one of four options--control: seawater; iono-mycin: seawater plus 4 [micro]mol [l.sup.-1] ionomycin; ruthenium red: seawater plus 1 mmol [l.sup.-1] ruthenium red; thapsigargin: seawater plus 2 [micro]mol thapsigargin. Corals were kept in the dark after sunset and periodically monitored, using a dim red flashlight (Pelican 353244), for spawning behavior. Shortly after sunset and also post-spawning, the set-up was documented using flash photography.

Five-day post-fertilization Acropora millepora (Ehrenberg, 1834) planulae were kindly made available by Francois Seneca and Bette Willis at Orpheus Island Research Station, Great Barrier Reef (18[degrees]35'S 146[degrees]29'E)(CITES permit 2009-AU-563189). At this stage planula are mostly elongated and highly motile. Planulae were kept in petri dishes in filtered seawater at ambient temperature. Day samples of 100-200 planulae were collected, seawater was removed with a pipette, and the sample was solubilized in 2% (w/v) sodium dodecyl sulfate (SDS) and frozen. At night, to ensure darkness, petri dishes were covered with a low tent of aluminum foil. Three hours after the onset of darkness, planulae were treated with ionomycin or thapsi-gargin for a further 3.5 h, then harvested as described above, frozen in 2% SDS, and returned to the laboratory for analysis.

Two-dimensional protein electrophoresis

The Montastraea cavernosa, M. franksi, and Acropora millepora samples were all subjected to two-dimensional protein electrophoresis and silver staining. 2D gels were run by Kendrick Laboratories (Wisconsin, USA) using carrier ampholine isoelectric focusing (IEF) for the first dimension and SDS/PAGE for molecular weight in the second dimension of separation, according to the method of O'Farrell (1975). About 50 tkg of protein was loaded on each gel. For IEF of adult whole tissue, pH 3.5 to 10 ampholines were used, and for larval samples, pH 4 to 8 ampholines. Polyacrylamide (10%, w/v) was used to separate in the second dimension. One microgram of an isoelectric focusing control, tropomyosin, which migrates with an isoelectric point of 5.2 and has a molecular weight of 33,000, was added to each sample loaded. A small arrowhead indicates this marker on whole gels. After the two dimensions of separation, gels were silver-stained and photographed.

Results

Proteomic shifts between day and night in Montastraea cavernosa holobiont

Light-based proteomic shifts assayed by 2D protein electrophoresis were first studied in Montastraea cavernosa. This species was chosen because its large polyps were expected to provide good protein yields. A 20 X 10-cm sample was collected from a large individual colony that was then split into three smaller pieces. All three were exposed to shaded sunlight in a flow-through tank during the normal daytime window. One sample was harvested and frozen at 1620, 2 h and 27 min before sunset. The two other fragments were transferred to large buckets with aeration, one of which was covered with light-proof plastic at sunset, and the other that was exposed to constant illumination from a 10-W incandescent bulb. These two samples were both harvested and frozen at 0420 the following morning.

The proteins in all three samples were analyzed by 2D electrophoresis. The first dimension of separation is based on isoelectrical point (pI) and performed by isoelectrical focusing with a pH4-10 gradient; the second dimension was molecular weight. Detection was via silver staining. The results of the 2D protein electrophoresis on Montastraea cavernosa are presented in Figure 1. Hundreds of spots, each representing a unique pl and molecular weight, were detected in each sample. Detailed comparison of the patterns indicates that many differences in the intensity of individual spots (or species) occur between the three samples, and this is illustrated by enlarging the same area (see box in panel 1A) for all three gels in Figure 1, panels D, E, and F. A change in intensity indicates a change in abundance of a particular species of protein. The amount of the protein present may have increased or decreased, or the polypeptide may have been post-translationally modified so that it now migrates at a different position--either way, the relative level of a specific protein species is altered if a spot changes. As Figure 1 shows, some spots are present only in the daylight sample and are absent from the night sample, while others are present only in the night sample and absent in the day sample. These results clearly show that there are distinct patterns of protein species present in the same individual coral in the daytime and at night.

The third sample from the same individual, which was collected at night but kept under constant illumination, was included to show that differences in proteins were due to light exposure and not to the extended incubation time or to entrained circadian oscillations in the proteome. The protein species marked with black arrows in Figure 1A (day) that are absent in the night sample are also present in Figure IC (night + light). These data show that shifts in the proteome occur as a direct response to light exposure in mature adult tissue.

Proteomic shifts between day and night in Acropora millepora planulae lacking zooxanthellae

The adult Montastraea cavernosa tissue used in the above experiment contains both coral and endosymbiotic zooxanthellae, and perhaps other holobiont organisms as well; thus the spots that change in intensity could belong to any of the constituents. To determine if similar changes occur in coral tissue in the absence of endosymbionts, this experiment was repeated using larvae of Acropora mille-pora, a species that has azooxanthellar early development (Van Oppen, 2001). Five-day-old motile planula of A. mille-pora collected under light and dark conditions were subjected to 2D protein electrophoresis. As the isoelectric gradient optimized for these samples was slightly different (pH 4-8), this is a different coral species, and these planula lack zooxanthellae, the patterns are expected to be different from those shown in Figure 1. Once again, clear differences are observed between day and night samples (Fig. 2), indicating that proteome shifts also occur in this species and in the absence of zooxanthellae. Once again some protein spots are present in the day sample but missing from the night sample (black and white arrows, Fig. 2C and D), while others are present in the night sample but absent in the day (Fig. 2A and B). On the entire gel at least six protein spots are present in day samples that are missing in night samples, and at least five spots in night samples are missing from day samples (Fig. 2A, B).

Modulation of cytoplasmic calcium levels causes shifts in the proteome similar to those from light exposure

To test whether transduction of light signals uses cellular pathways similar to those of other invertebrates, Acropora planulae were treated with two pharmacological compounds that alter calcium levels in different ways. The first compound is the ionophore, ionomycin. Ionomycin acts by releasing the cell's sequestered calcium, normally stored in the endoplasmic reticulum, into the cytoplasm where it acts as a second messenger (Morgan and Jacob, 1994). The second compound tested is thapsigargin, which acts by blocking reuptake of calcium from the cytoplasm into the endoplasmic reticulum, thereby raising cytoplasmic calcium levels (Thastrup et al., 1990).

Coral planulae kept under dark conditions were then exposed to ionomycin or thapsigargin for 3.5 hours, along with controls, then frozen. Plan ula proteins were then processed by 2D electrophoresis and silver staining. Enlargements of the key regions from light, dark, and dark+drug-treated planula are shown in Figure 2C--F. Protein species that are visible in this small area that are present only in light and absent in untreated dark controls not only are present in dark planula exposed to either compound, but the spots are even stronger than they are in light-exposed sampies. This finding is akin to the result in adult tissue where light from a 10-W incandescent bulb elicited a stronger response than did sunlight. This is best observed by comparing the intensity of the indicated protein spots to the reference spot marked with an asterisk. This result shows that two different compounds that increase cytoplasmic calcium levels in different ways both cause changes in the proteome that match those induced by light. These results were replicated on duplicate sets of 2D gels.

Timing of coral broadcast-spawning is sensitive to shifts in cytoplasmic calcium levels

Broadcast-spawning by both Montastraea and Acropora occurs at night, as it does for almost all broadcast-spawners (Willis et at., 1985; Vize et al., 2005). Darkening corals early on the night of spawning triggers correspondingly early spawn release, while illuminating corals delays spawn release (Willis et at., 1985; Vize et at., 2005). The data presented above indicates that the transduction of light signals in corals may be mediated by elevated cytoplasmic calcium levels. If this is the case, elevating calcium levels should impact spawning behavior by mimicking the response to light and delaying spawn release. This was tested by collecting fragments of an extremely predictable broadcast spawning species, the hermaphrodite Montastraea franksi. Samples from six individuals were collected on the predicted spawning day and kept in flow-through tanks exposed to ambient sunlight. We have previously shown that corals treated in this manner spawn gamete packets containing both eggs and sperm at the correct time, and that spawning time can be manipulated in about 50% of collected samples (Brady et al., 2009). Each individual sample was split into four fragments, generating a series of clonal replicates, and one clonal sample from each individual was exposed to four different treatments. These were control, ionomycin, ruthenium red and thapsigargin. Ruthenium red was included as this compound has multiple cellular targets that impact cytoplasmic calcium levels in other organisms, including calcium ATPases (Tapia and Velasco, 1997; Marshall and Clode, 2003).

All samples were added to beakers containing seawater plus the test compound shortly before sunset (7:47 p.m. EST), some 2 hours prior to predicted spawning times (Vize et at., 2005). Corals were monitored for spawning activity by brief visual inspections every few minutes using a dim red flashlight, and also photographed periodically (Figure 3). Of the six individuals tested, four spawned under control conditions between 2139 and 2149h, within the normal range for this species which was from 2123 to 2155 in the 2007 season (Vize et al., 2005). Treatment with ruthenium red had no impact on spawning, and fragments of each coral that spawned under control conditions also spawned in ruthenium red at around the same time (Table 1). The ruthenium red was supplied in ethanol, so this result also demonstrates that ethanol does not inhibit spawning in this assay. However, both ionomycin and thapsigargin had impacts. Only one sample treated with ionomycin spawned, indicating that this compound effectively blocked spawning behavior in three out of four individuals. Treatment with thapsigargin did not completely block spawning except in sample 3, but it did result in a shift in spawn timing in sample 2, where the treated sample spawned a small number of packets an hour later than did its control treatment (Figure 3, Table ), indicating that it blocked or delayed spawning in two out of four samples. While preliminary due to sample sizes, these results show that increasing the levels of cytoplasmic calcium with ionomycin or thapsigargin has an impact on spawning behavior similar to that of light exposure. Unfortunately these sample sizes are too low to apply meaningful statistics, and larger sample sizes will be difficult to obtain due to permit restrictions and the practical limitations with working in the field.

Table 1 Pharmacological modulation of cytoplasmic calcium
levels delays spawn timing in Monlastraeu franksi

                                    Spawn time (USA EST)
Treatment     Individ#l  Individ#2             Individ#3  Individ#4

None               2139       2144                  2139       none

Ionomycin          2139       none                  none       none

Ruthenium          2134       2149                  2134       none
red

Thapsigargin       2140       2249                  none       none

Treatment     Individ#5  Individ#6

None               none       2149

Ionomycin          none       none

Ruthenium          none       2154
red

Thapsigargin       none       2154


Discussion

The use of two-dimensional protein electrophoresis is a powerful new approach for exploring coral responses to environmental factors. It has previously been used to study changes in the proteome of soft corals as they develop and are populated by zooxanthellae (Barneah et al., 2006). In this report we show that it is a robust method for characterizing differences that exist under different conditions of illumination. A large number of protein species vary between light and dark conditions (Figs. 1, 2). In each case, the change in intensity relative to other spots indicates that a specific protein has changed in abundance or has been post-translationally modified (acetylation, phosphorylation, etc.) so that it migrates with a different isoelectric point or molecular weight and therefore at a different position on the gel. Species that vary between environmental conditions can be used as molecular markers for those conditions, just as protein expressed in a specific tissue--for example, muscle myosin--can be used as a molecular marker for the presence of muscle in a sample.

2D electrophoresis of adult Montastraea tissue followed by silver staining conclusively demonstrated that the proteome of an individual varies between day and night. The data also show that this is a direct response to the presence or absence of light and is not under the control of an entrained biological clock, as corals at night under a lamp have a day-like proteome. Similar shifts in the proteome occur in Acropora larvae that lack zooxanthellae, so these shifts can occur in the absence of photosynthesis by endo-symbionts. We also explored whether the phosphoproteome could be used in a similar way, using 2D electrophoresis, western blotting, and development with anti-phospho-serine and anti-phospho-threonine antibodies (results not shown). This approach also detected differences between day and night samples, but it was no more informative, and much more complex. than was silver staining, so was not pursued further.

Shifts in the proteome were then used as indicators of coral light response to explore whether calcium signaling mediates light responses in scleractinians. Two compounds that act via different mechanisms (Thastrup et al., 1990; Morgan and Jacob, 1994) to raise levels of cytoplasmic calcium both converted a night proteome pattern to a day pattern after planulae were treated in the dark for only 3.5 h. In the examples illustrated in Figure 2, the day-specific protein spots are stained more strongly in night samples treated with ionomycin or thapsigargin than in day samples, indicating that these compounds elicit an even stronger response than does exposure to light--presumably by elevating calcium levels higher than did ambient light. These two independent routes both producing the same proteomic shift conclusively demonstrates that increasing cytoplasmic calcium generates the same response as does exposure to light. Given that light acts via calcium signals in other systems (Yarfitz and Hurley, 1994; Hardie, 2001; Kum-balasiti et al., 2007; Fain et al., 2010; Sexton et al., 2012), the conclusion that it also does so in corals is strongly supported by these data. Direct measurement of cytoplasmic calcium levels via calcium-sensitive dye ratiometric fluoroscopy will be required to unequivocally measure calcium flux in response to light.

It has previously been proposed that corals use crypto-chromes (Cry), transcription factors that shuttle between the cytoplasm and the nucleus, as photoreceptors (Levy et al., 2007). While Cry does act as a photoreceptor in plants (Salome and McClung, 2005), in animals it serves as a transcription factor regulating entrained circadian clocks (Griffin et at., 1999; Fan et at., 2007). Although animal Cry is photosensitive in some invertebrates, there is no evidence that is acts as a photoreceptor (an initiator of light signaling cascades)--an important distinction--in corals. The data presented in this report show that calcium acting as a second messenger mimics the response to light and is the likely mechanism of light signal propagation. Since corals express rhodopsins and melanopsins (Anctil et al., 2007; Vize, 2009), which are classic visual and nonvisual photoreceptors that act via calcium second messengers in many marine invertebrates (Yarfitz and Hurley, 1994; Vize, 2009), this is more likely to be the mechanism responsible for sensing and responding to light in coral. While the photosensitivity of cryptochromes may be important in some way in corals, this remains to be demonstrated (e.g., see Fukushiro et at., 2011).

Behavior is a top-level test of a biological process, and broadcast-spawning behavior is one of the largest scale biological processes on the planet. Vast numbers of corals release their yearly reproductive output, often on the scale of hundreds of millions of gametes, within narrowly defined time windows of less than an hour that are set by lunar and solar light cycles (Babcock et al., 1986). Spawning occurs at a specific time after sunset, and moving sunset forward shifts spawn time by an equal amount; however, if corals are illuminated at night with bright lights, such as video lights, it delays spawning (Brady et al., 2009). If, as we hypothesize, light raises cytosolic calcium levels, these levels would decline with decreasing light as calcium is pumped back into the endoplasmic reticulum, and spawning behavior is triggered when a minimum threshold concentration is reached. Exposing corals to light would increase calcium levels and delay the reduction to this critical level. Chemically maintaining high cytosolic calcium levels in darkened corals with ionomycin or thapsigargin would also be expected to delay or block spawn release if the hypothesis is correct, and this is what was observed (Table 1). Two compounds that act in different ways to increase cytosolic calcium levels either blocked or delayed spawning in the majority of samples. One tested compound, ruthenium red, had no effect on the time of spawn. This compound has also been reported to fail to block calcium uptake at the surface of coral ectoderm (Marshall and Clode, 2003), so for unexplained reasons, it apparently does not work well on cnidarians. All samples treated with ruthenium red spawned within 5 min of control clonal fragments, emphasizing the major difference brought about by exposure to ionomycin or thapsigargin, which in most cases blocked spawning completely or, in one example, delayed it by an hour. Further experiments with larger sample sizes and a wider range of pharmacological compounds will enable the biochemistry of light transduction in corals to be dissected at the molecular level.

The data presented in this report have implications for understanding diverse processes in coral reproduction, including the impact of weather on spawn timing and the mechanism of reproductive isolation during speciation. If light exposure increases cytoplasmic calcium levels, more light may result in higher levels, dependent on limitations such as photoreceptor sensitivity, calcium stockpiles, receptor numbers, and reuptake rates. This implies that on days with stronger illumination, calcium levels may be higher and take longer to be sequestered in the endoplasmic reticulum, resulting in later spawning times. Cloudy weather would have the opposite effect. This prediction is testable and perhaps can be validated by integrating spawning data already collected with detailed weather records. In fact, spawn timing has been shown to be slightly earlier in deeper corals where daytime light levels are lower (Levitan et at., 2004), consistent with this hypothesis.

Another prediction is that if spawn time is regulated by reaching a low threshold value of calcium and achieved by endoplasmic reticulum calcium pumps acting in darkness, a shift in spawn time would result from either changing the rate at which calcium is sequestered or changing the sensitivity of a cytoplasmic process to calcium levels. Changing the number of pumps present in the endoplasmic reticulum membrane or mutations altering pumping rates of calcium transporters could generate differences in spawn timing. As this class of calcium pumps require ATP to drive translocation against concentration gradients (Ikemoto, 1982), changes in ATP levels could also impact pump activity, as could changes in any of the proteins required to generate the inositol triphosphate that opens the channels (e.g., rhodopsins, trimeric G proteins, phospholipase C). It will be useful to measure and contrast cytoplasmic calcium levels, pumping rates, and protein sequences between closely related species that display differences in spawn timing--for example. Montastraea franksi and M. annularis (Levitan et at., 2004)--to ascertain if changes in calcium levels or sensitivity to calcium levels drive reproductive isolation and speciation.

A more general role for light-responsive changes in cytoplasmic calcium levels in corals may involve entrainment of biological clocks and circadian rhythms. Over a daily light/darkness cycle in an organism that uses calcium as a second messenger in light transduction, cytoplasmic calcium levels will oscillate accordingly and can be used to synchronize a circadian rhythm to the phase of the current light cycle. Oscillating cycles of second messengers, including calcium and cyclic nucleotides, entrain biological clocks in systems as diverse as plants (Johnson et at., 1995), Drosophila (Harrisingh et at., 2007), and the mammalian brain (Lowrey and Takahashi, 2000): there is a high probability that calcium functions in the same manner in corals. Further exploration of the role of calcium second messengers and light transduction in corals will allow a variety of biological processes to be better understood.

Acknowledgments

Field work was supported by Sarah Davies, Mike Nickell, Kurt Carson, and the crew of the M.V. Fling. In the 2008 season, Galatee productions and the crew of "Oceans" (Disney, 2010), especially Antoine Colette, played a major role by helping us run field experiments in the midst of their busy documentary production schedule. At Orpheus Island field station the station managers, Kylie and Rob Eddie, provided extraordinary support, and Francois Seneca and Bette Willis supplied both academic and practical advice. The NOAA FGB staff provided ship time in 2007 and administrative support. AKB was supported by an NSERC PGS and an Alberta Innovates graduate award. PDV is supported by NIH P41 HD064556.

Appendix

Complete gels for two-dimensional protein electrophoresis of Acropora larvae.

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J. DANIEL HILTON, AISLING K. BRADY, SKENDER A. SPAHO, AND PETER D. VIZE *

Department of Biological Sciences, University of Calgary, 2500 University Drive NW, Calgary, AB T2N1N4, Canada

Received 10 May 2012; accepted 22 October 2012.

* To whom correspondence should be addressed. E-mail: pvize@ucalgary.ca
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Author:Hilton, J. Daniel; Brady, Aisling K.; Spaho, Skender A.; Vize, Peter D.
Publication:The Biological Bulletin
Article Type:Report
Geographic Code:1CANA
Date:Dec 1, 2012
Words:5540
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