[Coral-List] Summary of Methods for Zoox Density with References

[Sara Edge]

For those interested in what I found regarding counting zooxanthellae numbers
and correlating with coral surface area.

Here's a summary of what I plan to do:
1. freeze coral as soon after collecting as possible and keep in dark
2. water-pick tissue (I might try the air-brush method also)
3. centrifuge to pellet zoox.
4. fix zoox. in 5 – 10% formalin:filtered seawater
5. hemocytometer count
6. surface area – wax (branching morph) or aluminum foil (mounding morph)

Below is a very brief outline of the different methods I found. I've included
references for each (numbers in parentheses). It will be a few months before I
know exactly what works for my system. (I will be in the field July/August).

Preserve Samples
o  Wrap in foil to keep out light and freeze (3, 5)

Zooxanthellae Count
- Tissue removal:
   o  Water pick (1, 2, 3, 8, 9, 10, 11, 12, 13, 17)
       * creates large volume and foaming from mucus
   o  Airbrush (5, 11)
       * less volume, not as quantitative
   o  Dissolve tissue or skeleton (4, 14)
   o  Scrape tissue and homogenize (15)
- Fix/preserve:
   o  4% formalin:96% FSW (4)
   o  5% buffered formalin (9)
   o  10% formalin:90% FSW (5, 15)
- Count:
   o  Hemacytometer (all)

Surface Area
   o  Dry-to-wet weight ratio (4)
   o  Wax coating (5, 6, 7) – good for branching morphologies (11)
   o  Foil (9, 14, 16, 17) – good for mounding morphologies (11)
   o  Varathane/dye (9, 18)
   o  Photometric etc.
       * Leaf-area analyzer (8)
       * Weigh, photograph, cut out & weigh image, calculate s.a. (12)

References:

1.  Johannes, R.E. and W.J. Wiebe, Method for Determination of Coral
Tissue Biomass and Composition. Limnology and Oceanography, 1970. 15(5): p.
822 - 824.

2.  Tytler, E.M. and P.S. Davies, A method of isolating clean and viable
zooxanthellae by density gradient centrifugation. Limnology and Oceanography,
1983. 28(6): p. 1266 - 1268.

3.  Broadbent, A.D., G.B. Jones, and R.J. Jones, DMSP in corals and
benthic algae from the Great Barrier Reef. Estuarine, Coastal and Shelf
Science, 2002. 55: p. 547 - 555.

4.  Van Alstyne, K.L., P. Schupp, and M. Slattery, The distribution of
dimethylsulfoniopropionate in tropical Pacific coral reef invertebrates. Coral
Reefs, 2006.

5.  Pillay, R.M., B. Willis, and H. Terashima, Trends in the density of
zooxanthellae in Acropora millepora (Ehrenberg, 1834)at the Palm Island Group,
Great Barrier Reef, Australia. Symbiosis, 2005. 38(209 - 226).

6.  Vytopil, E. and B.L. Willis, Epifaunal community structure in Acropora
spp. (Scleractinia) on the Great Barrier Reef: implications of coral
morphology and habitat complexity. Coral Reefs, 2001. 20(281 - 288).

7.  Stimson, J. and R.A. Kinzie III, The temporal pattern and rate of
release of zooxanthellae from the reef coral Pocillopora damicornis (Linnaeus)
under nitrogen-enrichment and control conditions. Journal of Experimental
Marine Biology and Ecology, 1991. 153: p. 63 - 74.

8.  Muscatine, L., et al., The effect of external nutrient resources on
the population dynamics of zooxanthellae in a reef coral. Proceedings of the
Royal Society of London B, 1989. 236: p. 311 - 324.

9.  Hoegh-Guldberg, O. and J. Smith, The effect of sudden changes in
temperature, light and salinity on the population density and export of
zooxanthellae from the reef corals Stylophora pistillata Esper and Seriatopora
hystrix Dana. Journal of Experimental Marine Biology and Ecology, 1989. 129:
p. 279 - 303.

10.  Dustan, P., Distribution of zooxanthellae and photosynthetic
chloroplast pigments of the reef-building coral Montastrea annularis Ellis and
Solander in relation to depth on a West Indian coral reef. Bulletin of Marine
Science, 1979. 29(1): p. 79 - 95.

11.  Shokri, N., Protocol: The effect of reduced irradiance on coral growth
and zooxanthellae density. (lab protocol provided via email)

12.  Falkowski, P.G. and Z. Dubinsky, Light-shade adaptation of Stylophora
pistillata, a hermatypic coral from the Gulf of Eilat. Nature, 1981. 289
(January 15): p. 172 - 174.

13.  Cervino, J.M., et al., Zooxanthellae regulation in yellow blotch/band
and other coral diseases contrasted with temperature related bleaching: in
situ destruction vs expulsion
Symbiosis 2004. 37(63 - 85).

14.  Fagoonee, I., et al., The dynamics of zooxanthellae populations: A
long-term study in the field. Science, 1999. 283(5403): p. 843-845.

15.  Wilkerson, F.P., G.M. Parker, and L. Muscatine, Temporal patterns of
cell division in natural populations of endosymbiotic algae. Limnology and
Oceanography, 1983. 28(5): p. 1009 - 1014.

16.  Marsh Jr., J.A., Primary productivity of reef-building calcareous red
algae. Ecology, 1969. 51(2): p. 255 - 263.

17.  Edmunds, P.J. and R.D. Gates, Normalizing physiological data for
scleractinian corals. Coral Reefs, 2002. 21: p. 193–197.

18.  Hoegh-Guldberg, O., A method for determining the surface area of corals.
Coral Reefs, 1988. 7: p. 113-116.

I hope this helps.

Sara Edge
Georgia Institute of Technology
School of Biology
310 Ferst Drive
Atlanta, Georgia 30332-0230
gte405r at prism.gatech.edu


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