Coral Coloration – Part 7: Coral Reflectance, Chromoproteins and Environmental Factors Affecting Non-fluorescent Pigmentation

by | Oct 15, 2007 | 0 comments

Some of the information within this article was presented at the Marine Aquarium Conference of North America (MACNA) in Pittsburgh. Since it is difficult at best to present even the highlights of a year’s research in a one-hour presentation, I appreciate the opportunity offered by the Advanced Aquarist staff for allowing me to ‘flesh out’ that presentation with many ‘new’ details in a written format.

Fig1.jpg

Figure 1. The purple portion of this Acropora specimen is merely reflecting light falling upon it and is not fluorescent.

A Review

All the information presented in the previous six installments of this series has been only a precursor to this article, where we identified coral pigments as described in scientific literature. In this and following articles, we will begin to apply what we’ve learned and make some sense of it.

 

Major Groups of Colorants

For our purposes, we can divide coral pigments into several different categories:

  1. Fluorescent proteins. These proteins absorb light energy at a certain wavelength and emit (fluoresce) it at another lower energy wavelength. These pigments ‘glow’ when viewed with actinic and ‘backlight’ lamps. There are about 85 identified fluorescent proteins, but there are likely thousands.
  2. Photosynthetic pigments. Chlorophyll, phycoerythrin, and marienne. Zooxanthellae are the most common photo-symbionts found within corals, and these dinoflagellates of course contain photopigments including chlorophyll a (which fluoresces a deep red color but is seen only under rather special circumstances). Phycoerythrin is found in cyanobacteria that can also infect coral tissues. Phycoerythrin fluoresces a bright orange color. This orange pigment is visible under conditions less demanding than chlorophyll. ‘Marienne’ is a non-fluorescent but highly reflective pigment found in algae infesting tissues of clams and corals.
  3. “Kindling” Chromoproteins. “Kindling” is a term that refers to various proteins’ abilities to change from a non-fluorescent chromoprotein to one that fluoresces.
  4. Non-fluorescent ‘reflective’ proteins – Chromoproteins. These pigments are produced by the coral animal and can appear red, purple, blue, mauve or other colors. There are only a dozen or so described chromoproteins but there are likely many more. These proteins appear rather dull (usually a drab bluish color) when illuminated by high kelvin lamps, but may appear reddish when the light source has a ‘warm’ (lower) kelvin rating. There are only two dozen or so chromoproteins found in corals.

In this article, we will examine how chromoproteins are identified and discuss other pertinent information.

The previous six articles in this series have examined fluorescent pigments (including photopigments) as well as non-fluorescent chromoproteins. In general, fluorescent pigments are produced by the coral host (but see exceptions below). There are at least 85 described fluorescent pigments, each with different peak emission wavelength. If we consider the molecular structure of these pigments, there are probably hundreds, if not thousands. Non-fluorescent chromoproteins have received less attention from researchers – there are only a couple of dozen described absorption peaks. Again, further investigations at finer scales will likely reveal many more non-fluorescent pigments.

As we have seen, pigments are often found in combination with other colorants (Figure 2 shows an excitation and emission spectra of 5 fluorescent pigments within a single coral colony). Some corals will contain more, perhaps a dozen or more fluorescent pigments.

Some fluorescent or reflective pigments are associated with photosynthetic symbionts. Chlorophyll (most often found in zooxanthellae, for our purposes anyway) can, under certain conditions, lend a deep red fluorescent coloration. Another photopigment, phycoerythrin, can, in some cases, make the animal host appear fluoresce orange. Marienne is a reflective pigment found in cyanobacteria and can make clams or corals appear blue-green (Note that marienne is not known to occur in Tridacna clams. Tridacna colors are known to be due to the presence of refractive tissue layers). Any of these pigments have the potential, either through fluorescence or reflectance, to modify the apparent coloration of the host animal.

Fig2.JPG

Figure 2. Some corals contain up to 12 fluorescent proteins – this Favia only has five! After Salih et al., 2006.

As reef hobbyists, most of us are probably interested in what it takes to make corals colorful, and once this coloration is expressed, how to maintain or, in certain cases, how to manipulate coloration for our viewing pleasure.

There is now little doubt that light, especially violet and blue wavelengths (and to a lesser degree, ultraviolet ‘light’), are important in rearranging molecular structures of chromoproteins and fluorescent proteins, thus producing the vivid colorations that are so desirable in display aquaria. For many years, there was anecdotal evidence within the hobby that spectra of high-kelvin lamps (10,000-20,000K) were responsible for apparent coral shifts in many captive corals (I personally believe this is partially true. Many of the fluorescent colors are visually striking when the viewing lamp emits a high proportion of excitation wavelengths -violet and blue – while also generating relatively little light in the corals’ fluorescent emission spectra – such as green, yellow, orange or red wavelengths. In addition, there seems little doubt that ‘energetic’ wavelengths such as violet and blue can cause proteins to rearrange their molecular structures and thus shift from drab to colorful. But, as we shall see, there are other factors involved.)

Little distinction has been drawn by hobbyists (and many researchers for that matter) in the differences of required illumination (in captive conditions) for fluorescent and non-fluorescent proteins. Chromoproteins also seem to be expressed by the coral host in conditions of relatively high light energy skewed towards the higher kelvin wavelengths (violet, blue). Since chromoproteins are reflective by nature, the spectral characteristic of the lamp(s) illuminating the aquarium again takes on importance. As we have seen, most chromoproteins found within corals absorb light energy at ~570-580 nanometers (since they do not preferentially absorb blue and red wavelengths, they reflect it), often giving corals an apparent blue-purple coloration. High kelvin metal halide lamps are generally poor in producing a large amount of red light, hence corals containing non-fluorescent reflective proteins are best viewed using lamps with a spectral output balanced between blue and red wavelengths, such as 6,500 – 10,000K lamps.

In order to understand coral coloration, we must identify individual pigments. A fiber optic spectrometer can ‘see’ how light energy is absorbed, fluoresced or reflected. This article will examine reflected light and reflectance.

 

What is Reflectance?

Reflectance is the ratio of the total amount of radiation (such ultraviolet, visible and infrared wavelengths) reflected by a surface to the total amount of radiation incident to the surface. Reflectance is important when determining how an object absorbs and reflects light. In this manner, pigments can sometimes be identified.

 

Procedure

Use of a fiber optic spectrometer determines reflectance and fluorescence of different corals in a non-invasive manner. Two spectrometers were used to make these observations – a USB-2000 for reflectance and a USB-2000FL (especially configured for fluorescence measurements), both manufactured by Ocean Optics of Dunedin, Florida. A cosine-corrected collection lens and 600-micron fiber optic patch cord collected and delivered light to the spectrometer. Calculation of reflectance involves several steps: First, light is reflected from a calibrated standard (Spectralon, >99% diffuse reflection) using a standardized light source – in this case, a 100-watt clear glass tungsten filament incandescent lamp operating on a line voltage of 110v (under these conditions, this lamp is also known by the exotic name of ‘CIE Standard Illuminant A’, where the relative spectral power distribution is the approximately the same as a Planckian blackbody radiator at a temperature of about 2856 K – Hunt, 1998). Reflectance ratios were calculated by using Ocean Optic’s OOBase32 software. Figures 3, 4 and 5 illustrate how reflectance is determined.

 

Determination of Incident Light

Fig3.JPG

Figure 3. The spectral signature of a Standard Illuminant ‘A’ light source -a fancy name for a tungsten incandescent lamp.

 

 

 

 

Determination of Reflected Light

Fig4.JPG

Figure 4. The light reflected from the coral’s surface.

 

 

 

 

Reflectance

Fig5.JPG

Figure 5. Reflectance is the ratio of reflected energy to incident energy it is an aid in understanding how corals/zooxanthellae utilize light energy. This is the reflectance of a brown Pocillopora meandrina. Note the spectral peaks at ~575nm, 600nm and 650nm (typical of many brown, green, yellow, orange or red corals).

 

 

 

 

 

What Reflectance Tells Us

In the grand scheme of things, reflectance is an important tool in coral reef remote monitoring via aircraft or satellite. In particular, seafloor reflectance allows large areas to be monitored with relative ease. Remote sensing is the subject of much research as it can identify coral bleaching and perhaps coral health, as well as discriminate between coral, algal and other benthic surfaces.

Fig6.JPG

Figure 6. “Brown corals” possess a triple-peaked reflectance with peaks and shoulders at ~575, 600 and 650nm. This spectral signature is due to the presence of photosynthetic pigments within the symbiotic zooxanthellae (see Figure 7 below).

Some of this information is important to us as hobbyists. It should be noted that corals, as a group, reflect little light (as little as 0.5% at 400nm (violet), 2.5% at 400-500nm (blue through green), 8% between 550 and 650nm (green-red), and as much as ~100% at 700nm (red); Hochberg et al., 2004). However, these ratios are variable and seemingly small variations in the overall spectral signature of a coral can make a profound difference in how it appears to the human eye.

We should note that reflectance is not independent of fluorescence. Since fluorescent pigments often occur in corals, and will absorb light energy and emit it at less energetic wavelengths, their presence will impact the spectral reflectance. Careful examination of reflectance is required to distinguish between reflectance and fluorescence (something we’ll discuss in more detail later in this series).

Stony corals generally fall into two categories (as defined by Hochberg et al., 2000) – ‘brown’ corals and ‘blue’ corals. At face value, this seems odd – corals are known to exhibit a rainbow of colors. Let’s examine what these two terms mean.

Fig7.JPG

Figure 7. Zooxanthellae photosynthetic pigments absorb various light wavelengths.

‘Brown’ Corals

Generally, coral reflectance most often falls into this category. Reflectance of ‘brown’ corals generally exhibits a distinctive pattern, with peaks at 575, 600 and 600 nm (likely due to the wavelengths not absorbed -and hence reflected – by zooxanthellae). This ‘triple peak’ or ‘crown’ reflectance is also seen in corals exhibiting (in addition to brown) red, orange, yellow or green coloration (see Figures 6, 7 and 8).

An ‘action spectrum’ shows the efficiency of light (by wavelength) in producing a photochemical reaction. If this image was inverted, it would resemble the reflectance shown in Figure 6 – light not absorbed is reflected! Absorption is due to the presence of photopigments including chlorophyll a, chlorophyll c2, peridinin and a few other minor pigments.

Fig8.JPG

Figure 8. Reflectance of an orange coral – a variation of ‘brown coral’ reflectance – note the presence of the triple-peaked reflectance.

‘Blue’ Corals

‘Blue’ coral reflectance is the name given to a group of corals exhibiting purple, blue, pink or grey coloration, which is indicative of the possible presence of non-fluorescent proteins. ‘Blue’ corals lack the peak at ~575nm (as seen in ‘brown’ corals, and in close proximity to the maximum absorbance of many non-fluorescent proteins).

Transitional Coloration – Colors Are Not Always There

Although many researchers have categorized corals’ reflectance as either ‘brown’ or ‘blue’ there must surely be transitional states. Although much research needs to be done, we have some data showing the reflectance of genetically-identical coral fragments (See Figure 10). In this particular experiment, Acropora (austera? – commonly called the ‘Purple Monster’) fragments were placed in similar conditions with the exception of light intensity. The fragment in ‘low’ light (138 µmol·m²·sec) lost its mauve coloration over the period of a few weeks. The ‘high’ light fragment (maintained at 875µmol·m²·sec) retained its brilliant (but non-fluorescent) ‘purple’ coloration.

Fig9.JPG

Figure 9. A spectral signature typical of ‘blue’ corals. This category includes purple, blue, pink or grey corals. Spectral reflectance from a ‘purple’ Cyphastrea specimen.

 

 

 

 

 

 

Fig10.JPG

Figure 10. Colors are not ‘always there’ as demonstrated by the reflectance of genetically-identical coral fragments grown under ‘low’ and ‘high’ high intensities.

 

 

 

 

 

 

Pigment 580

At the time of this writing, Pigment 580 is the only chromoprotein I have been able to identify, and this was seen in an Acropora (austera? – see Figure 11).

Fig11.jpg

Figure 11. The ‘Purple Monster’Acropora. It contains a non-fluorescent pigment with maximum absorption at 580nm; hence it preferentially reflects blue and red wavelengths thus making it appear ‘purple’.

Interestingly enough, Pigment 580 is likely a photo-activated pigment and has been described only in Acropora species, including A. aculeus and A. hyacinthus (Matz et al., 2005). However, note that a chromoprotein with maximum of 580nm has been described from Goniopora tenuidens (Martynov et al., 2003). This pigment can mature in darkness and its expression is probably not linked to any given light intensity. Although its color is similar to those pigments found in Acropora species, the Goniopora pigment is probably of an entirely different class of chromoproteins from those found in some Acropora specimens.

 

Where Are Corals Containing Chromoproteins Found in Nature?

Salih (2003) investigated how chromoproteins are distributed across depth on the Great Barrier Reef (see Figure 12). Interestingly, there appears to be a fairly strong correlation between shallow depth and chromoprotein abundance with corals containing these reflective pigments composing about 22% of all corals on the reef flat and forereef. (Also of interest is the fact that shallow tidepools did not contain many corals containing chromoproteins. Salih did not address this issue, but we can speculate that tidepool environments were not conducive to those coral species known to contain reflective pigments. These corals include Acropora, Pocillopora, Seriatopora, Montipora and a few other genera).

Fig12.JPG

Figure 12. Water depth and its relationship to reflective coral pigments. The chart illustrates the percentage of corals containing non-fluorescent proteins across a transect line at various depths. After Salih, 2003.

The Apparent Effect of Light on Generating Coral Coloration

It seems apparent that light can be responsible for generating at least some colors within coral tissues. Riddle (2003) examined the apparent effects of spectral quality on coloration of the stony coral Pocillopora meandrina. Blue light (generated by monochromatic light-emitting diodes) apparently caused a color shift within the coral’s tissues from a brown color to non-fluorescent pink. We can speculate that the blue light induced expression of the pink pigment.

See http://www.advancedaquarist.com/issues/nov2003/feature.htm

‘Coloration Threshold’

The term ‘coloration threshold’ describes the point at which colorful coral pigments become apparent. Although many environmental factors must be proper (such as temperature, pH, etc.) in order for a color to ‘color up’, ‘coloration threshold’ will be used to describe the point at which light intensity is sufficient to induce expression of these pigments.

Fig13.JPG

Figure 13. Coloration of Acropora austera specimens at different light intensities and spectra. The mauve coloration is not seen in this Acropora species until a light intensity of ~400 µmol·m²·sec is achieved. Interestingly, green coloration was noted at 310 µmol·m²·sec when the metal halide lamp was unshielded for ultraviolet radiation. Apparently, the strong UV encouraged the expression, or dominance, of the green fluorescent protein.

It seems certain that different light intensities are required for expression of various coral pigments. Some pigments, such as pink ‘pocilloporans’ are seen at relatively low light intensity. On the other hand, some pigments seem to require a great deal of light (at least in terms of aquaria illumination) for observation of colorful pigments, and such seems the case with the color of ‘Purple Monster’ Acropora specimens.

“Alkali Effect” on Chromoproteins

Battad et al., 2007 report that pH values above 9.0 can ‘kindle’ (that is, cause a chromoprotein to become a fluorescent protein) many coral pigments and cause dramatic (20-100 fold) increases in fluorescence efficiencies. Of course, we’re talking about corals’ tissue pH and not that of the ambient water.

Photosynthesis (such as that by corals’ symbiotic algae) is known to have an effect on pH. Removal of carbon dioxide (CO2) by algae will raise the pH during times of ‘sufficient’ illumination.

Kawaguti (1944) demonstrated that coral pigments changed colors in response to pH. This seminal researcher found Green Fluorescent Pigments (GFPs) reached a maximum intensity at pH 8 – 9. At higher and lower pH, the pigment was less intense but coloration changes were reversible when using certain chemicals (the same held true for pink and purple pigments, but not yellow. As a footnote, he found the GFP did not “bleach” when exposed to hydrogen peroxide – an oxygen radical believed by some today to destroy coral pigments). Gentien (1981) reported disappearance of blue fluorescence at pH 2 and, at pH 10, a shift in maximum fluorescence from 432 nm to 450. Interestingly, the maximum absorption by photoprotective pigments found in zooxanthellae (diadinoxanthin, diatoxanthin and beta-carotene) all have a maximum absorption in 445-451nm range. Are pH-induced shifts in coloration a small part of the arsenal against photo-damage and photo-destruction of zooxanthellae and coral animals?

This could very well be the case since the pH of photosynthetic invertebrates’ tissues and fluids are altered by rates of photosynthesis (that is, the removal or addition of carbon dioxide). Fitt et al. (1995) found diurnal pH values of 7.2 – 8.1 in Tridacna derasa and T. gigas tissues. Coral tissue pH demonstrate diurnal swings as well; Acropora and Favia tissue pH may be as high as 8.5 in light and fall rapidly to as low as 7.3 in darkness. Natural pH swings likely help regulate (or alter) the expression of coloration within coral tissues. In experiments with water-soluble and variously colored pigments extracted from corals, pH was lowered in the samples with CO² instead of caustic or acidic reagents used by Kawaguti (1944). Transmittance (as measured with a spectrophotometer) rose as the pH fell (personal observations). In other words, it seems that pH-modulated light transmission (along with shifts in fluorescence) could possibly help prevent photo-damage to zooxanthellae and hence the coral animal.

 

Temperature

Kawaguti (1944) also reported heat (boiling) could destroy some pigments, while other pigments (such as pocilloporins) may be stable at relatively high temperatures (Dove et al., 2001). The potential for loss of ‘photo-protective’ coloration due to high temperatures could have devastating effects on coral colonies and zooxanthellae.

That high temperature could denature (destroy), or at least alter the absorbance capacity of, pigment proteins with possible photoprotective abilities is a quite interesting thought, especially when considering temperatures apparent role in coral “bleaching.” Denaturing of proteins is often an irreversible process, such as the changes seen in an egg yolk when its proteins are heated and denatured. Colorful corals collected and frozen for later lab work do not lose their coloration (personal observation).

In general, reef aquarists are advised to keep water temperatures in the mid-to-high 70ºF range. Under certain conditions, it is possible for coral skeletons to act as heat-sinks and actually become slightly warmer than the ambient water temperature. This rather disturbing news (especially for those corals maintained in aquaria teetering on the edge of acceptable upper-scale temperatures) is discussed by Riddle, 2006 (see www.advanceaquarist.com/2006/2/aafeature2 ).

 

In Closing

Spectrometry and determination of reflectance allows us to investigate coral coloration at a level previously unknown within the hobby. Once we have identified the pigment(s) within a particular coral, we can then begin to understand the environmental conditions that are favorable to the expression of the pigment(s).

We must be careful to avoid the pitfall of thinking a single environmental parameter alone is responsible for coloration – many factors are synergistically linked. Although light certainly seems important, photosynthesis depends upon a source of inorganic carbon (such as bicarbonates – ‘alkalinity’ – or CO2) which in turn could be potentially limited by insufficient water flow. By the same token, CO2, due to coral and zooxanthellae respiration, could possibly accumulate within the stagnant boundary layer surrounding the coral and thus drive pH downwards. Temperature is probably less important to the coral animal than its resident zooxanthellae. However, loss of zooxanthellae could be catastrophic to the coral so, naturally, avoid temperatures approaching 80ºF or so. Carefully monitor any corals directly under metal halide lamps for loss of pigmentation due to ‘spot warming’ resulting in bleaching.

The only chromoprotein identified in an aquarium coral, so far, is Pigment 580. Currently, it is known to occur in only Acropora specimens. This chromoprotein seems to be expressed at relatively high light levels (~400 µmol·m²·sec) when other factors (such as those mentioned above) are correct.

Though only briefly discussed, hobbyists should abandon the thought that ultraviolet radiation is responsible and therefore necessary for inducing the expression of chromoproteins. It is apparent that violet and blue wavelengths can also cause coloration shifts. Excessive ultraviolet radiation usually isn’t a problem with fluorescent lamps and it is definitely not a problem with the newer LED luminaires. However, hobbyists should shield their metal halide lamps for UV radiation by using acrylic panes. For those doubters, I’ll present some information on the effects of UV on some fluorescent proteins in the next installment of this series.

As mentioned, some of this information was presented at MACNA XIX in Pittsburgh. For those who have never attended a national conference such as MACNA, there are many reasons you should do so. You’ll benefit from the presentations and be one of the first to see new products aimed at the reef aquaria market, but the real treat is the fellowship with 1,000 other hobbyists. Atlanta is hosting MACNA XX in September 2008. I hope to see you there! See www.masna.org for conference details and registration.

I wish to thank Steve Ruddy and Coral Reef Ecosystems (www.coralreefecosystems.com) for his help during the preparation of this and future articles. I simply could not have done it without his help.

 

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  • Dana Riddle

    I have been an aquarist since 1964 and a reef hobbyist since the mid-1980’s. I am the owner of a small laboratory (Riddle Aquatic Laboratories) that specializes in investigation of interactions between light and water motion & photosynthetic organisms (especially corals). The results of this research, resulting in almost 250 articles, have been published in Advanced Aquarist Online, Aquarium Frontiers, Koralle, Freshwater and Marine Aquarium, The Breeders’ Registry, Aquarium Fish, Marine Fish Monthly and others. My first article was published in a 1984 SeaScope and relayed my experiences with a refugium – an idea that would catch fire about a decade later. I have had the honor of making over 60 presentations to various groups, including national conferences such as the Marine Aquarium Conference of North America (MACNA) International Marine Aquarium Conference (IMAC), PetsFestival (Italy), regional conferences, and local clubs. I received the Marine Aquarium Society of North America (MASNA) Aquarist of the Year Award in 2011 at the MACNA conference in Des Moines.

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