The Biochemical Composition of Planulae from the Scleractinian Coral Porites astreoides

 Fitsum Yohannes

University of Miami, Department of Biology

P.O. Box 249118, Coral Gables, Florida 33124, U.S.A.

Introduction

World wide, coral reefs are being threatened by human activities. Agricultural runoff, sewage, and land clearing lead to nutrient enrichment, one of the most important hazards facing coral reefs world wide (Roberts, 1993). Although this phenomenon is known to cause algal overgrowth, drops in fertilization and embryo development, and reduced coral recruit settlement, the direct effect on scleractinian reef building corals species is not well known (Richmond, 1993; Roberts, 1993). The following experiment was designed to examine the effects of ammonium enrichment on the biochemical composition of the planulae of the Atlantic coral Porites astreoides.

P. astreoides is known to have hermaphroditic polyps (Szmant, 1986), as well as individuals that are female and male (Chornesky & Peters, 1987; Soong, 1991). This species is thought to brood its larvae for approximately two weeks before releasing them (McGuire, pers. comm.). The planulae released by P. astreoides, pear-shaped or elongate along the oral-aboral axis, are similar to those of other scleractinian planulae (Fadlallah, 1983; McGuire, unpublished data). Their dry weights range from 85-150 µg, and they are approximately 1 mm in length (McGuire, unpublished data).

In Florida, the planulae are released during the summer months over a range of six days before to six days after the new moon (McGuire, unpublished data). Szmant (1986) noticed a correlation between brooding species of corals in the Caribbean, multiple planulating cycles per year, and unstable habitats that result in high rates of adult mortality. It is thought that these behaviors are an adaptation of coral species that lead to higher levels of recruitment to replace newly killed adults (Szmant, 1986). Reproduction of many scleractinian corals are synchronized with lunar cycles (Harrison, 1990). Scleractinians reported to have lunar periodicity of reproduction include the spawning species Diploria stringosa, Acropora cervicornis (Szmant, 1986), and the brooding species Favia fragum, (Szmant-Froelich, 1985), Porites astreoides (Chornesky & Peters, 1987), Agaricia agaricites, and Agaricia humilis (Delvoye, 1982). Previous research has shown that the adult colonies of P. astreoides have shown physiological responses to nutrient enrichment after 2-4 week incubations (McGuire, unpublished data). This experiment was designed to test the effect of ammonium enrichment on the biochemical composition of planulae released from the aforementioned colonies.

Muscatine (1989) proposed that zooxanthellae are nitrogen-limited. Several studies have shown that ammonium enrichment results in increased zooxanthellae densities (Snidvongs, 1987; Hoegh-Guldberg & Smith, 1989; Muscatine, Fallowski, Dubinsky, Cook, & McCloskey, 1989; Dubinsky et al., 1990; Stambler, Popper, Dubinsky, Stimson, 1991; Muller-Parker, McCloskey, Hoegh-Guldberg, & McAuley, 1994; McGuire, unpublished data). Therefore planulae produced under ammonium treatment are expected to have an increased number of zooxanthellae per larva compared to controls. Muscatine et al, 1986, found increased proteins and decreased lipids per square centimeter when the coral Stylophora pistillata was exposed to nitrogen enrichment. A similar trend was expected in the "enriched" P. astreoides planulae. With the exception of Pocillopora damicornis, very little research has been done on the bio-chemical composition of planulae (Richmond, 1987, 1988).

Methods

Adult colonies of Porites astreoides were collected in the northern Florida Keys and kept in outdoor flow through chambers. Colonies were cut in half; one half of each colony was placed in an aquarium containing low-nutrient seawater (<0.5µM NH4 control), the other half was placed in a similar aquarium which was spiked with ammonium chloride to give a final NH4 concentration of 5µM (enriched). Larvae were collected from each individual colony between April -May 1995, and frozen separately in sets of fifty, twenty five, or ten in microcentrifuge tubes. Tubes with 50 larvae were frozen in 0.5 ml of sea water. Tubes with less than fifty larvae were frozen in 0.1 ml of sea water. Planulae were analyzed for protein, lipids, and zooxanthellae (symbiotic dinoflagellates) densities to detect any difference between enriched and unenriched colonies (see Figure 1). All samples were homogenized with a glass tissue homogenizer, and diluted with distilled water for a final volume of 1ml.

Figure 1. Flow diagram showing procedures (Click on image to view full size)

Zooxanthellae densities

10µl of Lugol’s fixant (20 g KI, 10 g I2, 19 ml Glacial Acetic Acid, 200 ml distilled water) was added to a 100µl sub-sample of each homogenate. Each sample was put onto a hemacytometer, and examined under a microscope. A minimum of 400 cells were counted and converted to the number of zooxanthellae per larva using the following formulae:

a. 80000/ number of squares = calibration factor

b. calibration factor x number of zooxanthellae = zooxanthellae/ml

c. zooxanthellae/ml x 1.1/# of larvae = zooxanthellae per larva

Protein assay

The amount of protein per larva was determined using Bicinchoninic Acid (BCA) reagents (Pierce) as described in the protocol by Smith et al., 1985. Standard concentrations of Bovine Serum Albumin (BSA) were prepared to determine the relationship between absorbance units and concentration (µg/ml).

For samples containing 50 planulae, a 50µl sub-sample of homogenate was diluted with 100µl of distilled water. For samples containing less than 50 larvae, a 100 µl sub-sample was diluted with 50 µl of water. Three ml of BCA reagent (Pierce) was added to each sample in a 1 cm disposable cuvette for a final volume of 3.15 ml. Samples were left at room temperature for a minimum of two hours before being read in a Biochrom ultraspec 4050 spectrophotometer at 562 nm.

The linear relationship between absorbance units and concentration (µg/ml) found using the standard BSA samples was determined. This relationship was used to find concentration of the planulae samples based on their absorbance. The following formula was used to convert µg/ml to µg per larva:

(µg/ml protein)/ dilution factora/ # of larvae= µg/larva

a. dilution factor= volume of planulae sample/total sample volume

for example:

50 larvae=0.05/0.15=0.33

<50 larvae=0.10/0.15=0.67

Lipid assay

The amount of lipids per larva was determined using a chloroform, methanol, and water extraction (Bligh & Dyer, 1959). This lipid assay is a conservative estimate because some of the lipids are lost as the homogenate/filtrate is transferred between containers. In a glass homogenizer, 6 ml of 2:1 chloroform: methanol was added to the remaining homogenate (800 µl for samples with 50, and 700 µl for samples with less than 50 larvae), and mixture was homogenized for about thirty seconds. 2 ml of chloroform was added and the mixture was rehomogenized. 0.6 ml of distilled water was used to rinse out the microcentrifuge tube and was then added to the mixture. The sample was homogenized once more. Another 0.6 ml of distilled water was added, the mixture was homogenized one last time and then filtered through Watman #1 filter paper. The homogenizer and the filter were rinsed with 2 ml of chloroform. The filter was rinsed once more with 1 ml of chloroform, and the filtrate was poured into a 30 ml separatory funnel. The funnel was shaken well, and allowed to stand until the chloroform and methanol separated into two distinct layers (less than 5 minutes). The bottom layer containing the chloroform and the lipids was drained into preweighed aluminum dishes. The chloroform was allowed to evaporate at room temperature leaving the lipids in the dish. The dish was then re-weighed and the µg of lipid per larva was determined as follows.

weight of lipid(µg) /(volume of sample x original # of larvae) = µg lipid/larva

Data Analysis

Amounts of protein and lipid, protein: lipid ratio and zooxanthellae densities were compared using a 1-way ANOVA. When necessary, a Kruskal-Wallis One Way Analysis of Variance on Ranks was used instead. Differences between groups were determined using a posteriori tests (student-Newman-Keuls or Dunn’s method).  

Results

120 samples containing 10, 25 or 50 Porites astreoides planulae released in April and May of 1995 from seven different colonies were analyzed for protein, lipid and zooxanthellae content. There were no significant differences in the biochemical composition between the planulae of enriched and control colonies. However there were differences seen in larvae produced by different colonies.

P. astreoides larvae contained about twice as much lipid as protein. Protein and lipid content fluctuated between larvae from different colonies. Protein content ranged from 19 + 2 µg/larva to 36 + 7 µg/larva (for n’s see figure 2). There were statistically significant differences in the protein content of larvae from different colonies (p<0.0001; 1-way ANOVA). Similar groups are indicated by letters on Figure 2.

Figure 2. Plot showing protein content of larvae released by each of the seven colonies. Bars are means, error bars are 1 standard deviation. The n for each bar, the number of sets of larvae analyzed (each set contained either 10, 25, of 50 larvae), is shown a t the bottom of the bar. Simmilar groups are denoted by simmilar letters.

Figure 3. As for Figure 2 but for lipid content.

Figure 4. As for Figure 2 but for protein: lipid ratio.

Figure 5. As for Figure 2 but for zooxanthellae density.

Figure 6. Plot showing zoaxanthellae densities in larva and adult colonies as well as larvae released per cm². Bars are means of the quantities measured.

Lipid content ranged from 41+ 12 µg/larva to 91+ 33 µg/larva. As with the protein levels, larvae from different colonies were statistically different from each other (Kruskal-Wallis One Way Analysis of Variance, p<0.0001). Similar groups are indicated by letters on Figure 3. As shown in Figure 4, despite the differences in protein and lipid levels from colony to colony, the protein: lipid ratio was consistent between colonies (ANOVA, p=0.8442).

The zooxanthellae densities in the larvae varied from 10683 + 3778 to 18756 + 8594. Larvae from different colonies were significantly different from each other (ANOVA, p<0.0001) Similar groups are indicated by letters on Figure 5. There was no correlation between the zooxanthellae density in the parent colonies and the planulae. There was also no correlation between the number of larvae released per colony with the zooxanthellae densities of the planulae (see Figure 6).

Discussion

The lack of difference between larvae released from enriched and control colonies is probably due to too short of an incubation period in the treatment. Sixteen days was the longest that a colony was incubated before releasing its planulae. Previous experiments have shown that differences in adult colonies were not found until after 2-4 weeks of enrichment (McGuire, unpublished data). Also, most of the time, only one half of the colony (either control or enriched) released larvae. Therefore, it was not possible to make direct daily comparisons.

Variation in lipids and proteins between larvae from different colonies is thought to be related to the size of the larvae. For example, larvae from colony six, which had the highest levels of proteins lipids and zooxanthellae, were very large. It is thought that the variety in size of the larvae is linked to the competency. The larger larvae which have more lipids will probably have a longer duration of time before they settle.

Overall, the planulae had about a 1:2 protein/lipid ratio. These findings are consistent with the ratio of protein: lipids for P. damicornis planulae ( Richmond, 1987). Protein: lipid ratios for several coral species are listed in Table 1. The average planula of P. damicornis had a dry weight of 150 µg, and was composed of 17% protein, 70% lipids, and 13% carbohydrates by dry weight. The high percent of lipids is thought to be reserve of energy for the planulae (Richmond, 1987). Because they have no feeding apparatus, the larvae probably rely on translocated metabolities from zooxanthellae and the aforementioned lipid reserves for energy. Tissue of adult P. astreoides typically has higher protein content than lipids (McGuire, unpublished data). It is not known why zooxanthellae densities in the larvae do not reflect those of the parent colonies. Further studies comparing mitotic indices of the zooxanthellae in planulae and parent colonies, or zoaxanthellae densities verses different sizes of larvae might be useful in shedding light on this subject. Overall, Porites astreoides colonies have been found to release planulae of different sizes with varying biochemical compositions. Although the larvae from colonies can differ greatly in absolute protein, lipid and zooxanthellae density, the protein/lipid ratio is consistent.

Table 1: Protein: lipid ratio for several coral species

Acknowledgements

This research was made possible by Dr. Micheal Gaines, the Howard Hughes Medical Institute and the National Institute of General Medical Sciences program who provided the funding, Maia P. McGuire who designed the experimental plan and did the statistical analysis, Dr. Alina Szmant who gave me the opportunity to work in her lab, and Robb Wright and Mark Chiappone for their technical expertise in putting together this poster.

References

Achituv, Y., Ben Zion M., & Mizrahi L. (1994). Carbohydrate, lipid and protein composition of zooxanthellae and animal fractions of the coral Pocillopora damicornis exposed to ammonium enrichment. Pacific Science, 48 , 224-233.

Bligh, E.G., & Dyer, W.J. (1959). A rapid method of total lipid extraction and purification. Canadian Journal of Biochemistry and Physiology, 37, 911-917.

Chornesky, E.A., & Peters, E.C. (1987). Sexual reproduction and colony growth in the scleractinian coral Porites astreoides. Biological Bulletin, 172, 161-177.

Delvoye, L. (1982). Aspects of sexual reproduction in some caribbean scleractinia. Thesis, Carribean Marine Biological Institute Curacao, N.A., 93pp.

Dubinsky , Z., Stambler N., Ben-Zion M., McCloskey L.R., Muscatine, L., & . Falkowski,P.G. (1990). The effect of external nutrient resources on the optical properties and photosynthetic efficiency of Stylophora pistillata. Proceedings of the Royal Society of London, B, 239-246.

Fadlallah, Y. H. (1983). Sexual reproduction, development and larval biology in scleractinian corals. Coral Reefs, 2, 129-150.

Falkowski, P.G., Dubinsky, Z., Muscatine, L., & McCloskey, L. 1993. Population control in symbiotic corals. BioScience, 43, 606-611.

Fang, L.S., Chen, Y.-W.J., & Chen, C.-S. (1989). Why does the white tip of stony coral grow so fast without zooxanthellae? Marine Biology, 103, 359-363.

Harland, A.D., Davies, P.S. & Fixter, L. M. (1992). Lipid content of some Caribbean corals in relation to depth and light. Marine Biology, 113, 357-361.

Harrison, P.L., & Wallace, C.C. (1990). Reproduction, dispersal and recruitment of scleractinian corals. In Z. Dubinsky (Ed.), Ecosystems of the World (pp.133-207). Netherlands: Elsevier Science Publishers.

Hoegh-Goldberg, O., & Smith G.J. (1989). Influence of the population density of zooxanthellae and supply of ammonium on the biomass and metabolic characteristics of the reef corals Seriatopora hystrix and Stylophora pistillata. Marine Ecology Progressive Series, 57, 173-186.

Muller-Parker, G., McCloskey, L.R. , Hoegh-Guldberg O., & McAuley, P.J. (1994). Effect of ammonium enrichment on animal and algal biomass of the coral Pocillopora damicornis. Pacific Science,48, 273-283.

Muscatine, Fallowski, P.G., Dubinsky, Z., Cook, P.A., & McCloskey. (1989). The effect of external nutrient resources on the population dynamics of zooxanthellae in a reef coral. Proceedings of the Royal Society of London, 311-324.

Richmond, R.H. (1987). Energetics, competency, and long-distance dispersal of planula larvae of the coral Pocillopora damicornis. Marine Biology, 93, 527-533.

Richmond, R. H. (1988). Competency and dispersal potential of planula larvae of a spawning versus a brooding coral. Proceedings of the 6th annual Coral Reef Symposium, 2, 827-831.

Richmond, R.H. (1993). Coral Reefs: Present problems and future concerns resulting from anthropogenic disturbance. American Zoologist, 33, 524-536.

Roberts, C.M. (1993). Coral reefs: health, hazards, and history. Trends in Ecology and Evolution, 8, 425- 427.

Soong, K. (1991). Sexual reproductive patterns of shallow-water reef corals in Panama Bulletin of Marine Science, 49 (3), 832-846.

Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E. K.,Goeke, N. M., Olson, B.J., and Klenk, D. C. (1985). Measurement of Protein Using Bicinchoninic Acid. Analytical BIochemistry, 150, 76-85.

Snidvongs, A. (1987). Effects of nitrogen and phosphorus enrichment on the reef coral Pocillopora damicornis and its zooxanthellae. MS Thesis, University of Hawaii, 76 pp.

Stambler, N., Popper, N., Dubinsky, Z., & Stimson, J. (1991). Effects of nutrient enrichment and water motion on the coral Pocillopora damicornis. Pacific Science, 45, 299-307.

Szmant, A.M. (1986). Reproductive ecology of Caribbean reef corals. Coral Reefs, 5, 43-53.

Szmant-Froelich, A., Reutter, M., & Riggs, L. (1985). Sexual reproduction of Favia fragum: Lunar Patterns of Gametogenesis, Embryogenesis and Planulation in Puerto Rico. Bulletin of Marine Science, 37 (3), 880-892.

Szmant-Froelich, A., & Pilson, M. E. Q. (1980). The effects of feeding frequency and symbgiosis with zooxanthellae on the biochemical composition of Astrangia danae Milne Edwards & Haime 1849. Journal of Experimental Marine Biology and Ecology, 48, 85-97.