In Part 1 of this series, we began our review of fluorescent pigments and also discussed the concepts of fluorescence excitation and emission, Stokes shift, photoconversion and so on. A review of that article is recommended if you are unfamiliar with any of these terms. This time, our discussion continues with green, yellow-green and yellow fluorescent pigments and a few hypotheses concerning their function. We’ll also see a couple of cases where light energy of specific bandwidths has induced coloration changes.

Green fluorescence is perhaps the most common of any seen in aquaria. Well over half of the 70 or so known fluorescent pigments spanning 9 color categories can be described as ‘greenish.’ Fluorescence of green fluorescent proteins (GFPs) can also combine with other ‘glowing’ pigments to form any number of colors.

Table 4 lists the fluorescent proteins by emission maxima as described in various works:

Table 4: Fluorescent Proteins by Emission Maxima

Pigment	Emission	2	3	Excitation	2	Found in:	Reference
P-510	510	479	*	*	*	Montastraea annularis	Mazel, 1995
P-510	510	*	*	*	*	Ricordea florida	Mazel, 1995
P-510	510	*	*	498	*	Renilla muelleri (sea pansy)	Labas et al., 2002
P-510	510	*	*	490	420	Heteractis magnifica	Tu et al., 2003
P-510	510	*	*	440	*	Montastraea cavernosa	Mazel et al., 2003
P-510-520	510-520	*	*	440	*	Montastraea faveolata	Lesser et al., 2000
P-510-520	510-520	*	*	440	*	Montastraea cavernosa	Lesser et al., 2000
P-510-623	510-623	*	*	*	*	Montastraea cavernosa @ 40m	Vermeij et al., 2002
P-511	511	*	*	*	*	Plesiastrea verispora (green morph)	Salih et al., 2004
P-512	512	*	*	503	*	Discosoma sp. 3	Labas et al., 2002
P-512	512	*	*	*	*	Plesiastrea verispora (blue morph)	Salih et al., 2004
P-512	512	*	*	*	*	Plesiastrea verispora (green morph)	Salih et al., 2004
P-513	513	545	490	*	*	Agaricia sp.	Mazel, 1995
P-513	513	*	*	*	*	Ricordea florida	Mazel, 1995
P-514	514	490	*	501	480	Acropora aspera	Papina et al., 2002
P-514	514	*	*	*	*	Montastraea cavernosa	Kelmanson & Matz, 2003
P-515	515	*	*	*	*	Mycetophyllia lamarckiana	Mazel, 1997
P-515	515	*	*	~494	*	Mycetophyllia sp.	Fux and Mazel, unpublished
P-515	515	*	*	505	*	Montastraea annularis	Mazel, 1997; Manica & Carter, 2000
P-515	515±3	*	*	505±3	*	Montastraea cavernosa (mc2/3/4)	Kelmanson & Matz, 2003
P-515	515	*	*	*	*	Lobophyllia hemprichii (red)	Salih et al., 2004
P-515	515	*	*	*	*	Colpophyllia natans	Fux and Mazel, unpublished
P-515	515	*	*	*	*	Scolymia sp.	Mazel, 1997
P-515	515	*	*	*	*	Plesiastrea verispora (green morph)	Gilmore et al., 2003
P-515	515	*	*	*	*	Plesiastrea verispora (green morph)	Salih et al., 2004
P-515	515	*	*	*	*	Agaricia sp.	Mazel, 1995
P-515	515	*	*	*	*	Ricordea sp.	Mazel, 1995
P-516	516	*	*	486	*	Pocillopora damicornis	Salih et al., 2000
P-516	516	*	*	506	~480	Montastraea cavernosa (=mc2/3/4 – see P-515)	Labas et al., 2002
P-516	516	*	*	*	*	Lobophyllia hemprichii	Nienhaus et al., 2005
P-517	517	555	485	505	465	Acropora tenuis	Papina et al., 2002
P-517	517	574	*	506	566	Ricordea florida	Labas et al., 2002
P-517	517	*	*	506	*	Ricordea florida	Mazel, 1995
P-517	517	*	*	507	*	Favia favus	Tsutsui et al., 2005
P-517	517	551	483	*	*	Montastraea annularis	Mazel, 1995
P-518	518	*	*	508	475	Ricordea florida	Labas et al., 2002
P-518	518	*	*	*	*	Acropora cytheria @ Waikiki Aquarium	Hochberg et al., 2004
P-518	518	*	*	*	*	Acropora digitifera	Hochberg et al., 2004
P-518	518	*	*	*	*	Plesiastrea verispora (green morph)	Salih et al., 2004
P-518	518	*	*	*	*	Montastraea cavernosa	Kelmanson & Matz, 2003
P-518	518	*	*	503	*	Family Pectiniidae	Ando et al., 2004
P-518	518	*	*	*	*	Ricordea florida	Mazel, 1995
P-519	519	*	*	~505	*	Montastraea cavernosa	Kelmanson & Matz, 2003
P-519	519	*	*	*	*	Lobophyllia hemprichii (red)	Salih et al., 2004
P-519-557	519-557	590	*	*	*	Madracis carmabi	Vermeij et al., 2002
P-519-559	519-559	590	*	*	*	Madracis senaria	Vermeij et al., 2002
P-519-570	519-570	590-615	*	*	*	Madracis pharensis (green tissue)	Vermeij et al., 2002
P-520	520	*	*	*	*	Ricordea florida	Mazel, 1995
P-520	520	*	*	488	*	Goniopora tenuidens	Salih et al., 1999
P-520-555	520-555	590	*	*	*	Madracis carmabi	Vermeij et al., 2002
P-520-558	520-558	590	*	*	*	Madracis senaria	Vermeij et al., 2002
520-570	520-570	590	*	*	*	Madracis pharensis (green tissue @ 10m)	Vermeij et al., 2002
520-570	520-570	590	*	*	*	Madracis pharensis (green tissue @ 20m)	Vermeij et al., 2002
520-570	520-570	590-620	*	*	*	Madracis pharensis (green tissue @ 40m)	Vermeij et al., 2002
520-570	520-570	590	*	*	*	Madracis pharensis (green tissue @ 60m)	Vermeij et al., 2002
P-522	522	*	*	499	**	Anemonia sculata var. rufescens	Wiedenmann et al., 2000
P-522	522	*	*	*	*	Montastraea cavernosa	Kelmanson & Matz, 2003
P-522-623	522-623	*	*	*	*	Madracis formosa @ 40m	Vermeij et al., 2002
P-530	530	*	*	450	**	Porites astreoides	Mazel, 2003
P-532	532	590	*	*	*	Madracis pharensis (brown tissue @ 10m)	Vermeij et al., 2002
P-532	532	*	*	*	*	Madracis pharensis (red tissue @ 10m)	Vermeij et al., 2002
P-533	533	590	*	*	*	Madracis pharensis (brown tissue @ 20m)	Vermeij et al., 2002
P-534	534	575	*	*	*	Madracis formosa @ 60m	Vermeij et al., 2002
P-534	534	593	*	*	*	Montastraea cavernosa @ 60m	Vermeij et al., 2002
P-535	535	587	*	*	*	Madracis pharensis (brown tissue @ 60m)	Vermeij et al., 2002
P-537	537	590	*	*	*	Madracis pharensis (grey and blue tissue @ 10m)	Vermeij et al., 2002
P-538	538	~580	*	528	494	Zoanthus 2	Matz et al., 1999
P-538	538	*	*	525	494	Zoanthus sp.	Yanushevich et al., 2002
P-540	540	*	*	*	*	Plesiastrea verispora (blue morph)	Salih et al., 2004
P-540	540	*	*	*	*	Plesiastrea verispora (green morph)	Salih et al., 2004
P-542	542	*	*	*	*	Agaricia undata @ 40m	Vermeij et al., 2002
P-550	550	*	*	530	*	Porites murrayensis	Dove et al., 2001
P-557	557	600	545	*	*	Agaricia sp.	Mazel, 1995
P-559	559	590	*	*	*	Madracis senaria @ 60m	Vermeij et al., 2002
P-560	560	590	*	*	*	Madracis pharensis @ 10m (grey tentacles)	Vermeij et al., 2002
P-561	561	*	*	548	*	Fungia concinna	Karasawa et al., 2004
P-561	561	587-616	*	*	*	Madracis senaria @ 40m	Vermeij et al., 2002
CP-562	*	*	*	562	*	Anemonia sulcata, immature P-595	Wiedenmann et al., 2002
P-565	565	*	*	490	*	Agaricia humilis	Mazel et al., 2003
P-565	565	*	*	548	*	Cerianthus sp.	Ip et al., 2004
P-573	573	510	*	*	*	Ricordea florida	Mazel, 1995
P-574	574	517	*	506	566	Ricordea florida	Labas et al., 2002
P-574	574	550	*	*	*	Plesiastrea verispora	Dove et al., 2001
P-575	575	~630	*	~525	~570	Montastraea cavernosa	Mazel, 1997
P-575	575	*	*	506	555	Montipora (digitata/angulata)	Mazel, unpublished
P-575	575	*	*	557	*	Dendronephthya	Pakhomov et al., 2004
P-575	575	630	*	520	*	Scolymia sp.	Mazel et al., 2003
P-575	575	*	*	~569	~540	Phycoerythrin within symbiotic cyanobacteria in coral	Mazel et al., 2004

Comments about Green Fluorescent Proteins

It seems quite clear that GFPs emissions are quite varied and abundant in coral reef animals. At least some GFPs require the presence of oxygen for maturation – the transition from a colorless state to one of fluorescence with the emission peaking in the green portion of the spectrum, yet once mature, oxygen apparently has no further effect (Tsien, 1998). However, modification of environment can cause further color-shifting, resulting in various apparent colorations (Labas, 2002).

Fluorescent Coloration is Not Produced by Zooxanthellae! This is one of the persistent myths within the hobby yet there is no data to support this view. To the contrary, the evidence does support fluorescent pigment production by the host. Kawaguti (1966) shows several electron photomicrographs of pigment granules apparently being produced by a coral cell’s endoplasmic reticulum. With that said, zooxanthellae pigmentation (photopigments including chlorophyll a, c², peridinin and perhaps others) can greatly influence the non-fluorescent coloration ranging from brown to yellow-green.

 

Possible Functions of GFP-like Compounds

Opinions on possible function (or non-function) of fluorescent proteins are almost as numerous as the number of papers addressing the subject. Here are a few:

Photoprotection? Perhaps the most popular concept is one where pigments act as sunscreens – photoprotectants – against ultraviolet or visible radiation. Fairly early on in the course of ‘serious’ coral reef studies, Kawaguti (1944) was of the opinion that fluorescent pigments in corals act to shield symbiotic zooxanthellae from ‘strong sunlight.’ His idea became mantra for decades and sporadic re-visitations seemed to confirm his thoughts (see Salih et al., 1998; Salih et al., 2000 and others).

Fluorescent pigments can cause a reflection of light, which can be 40-80% higher than that of adjacent non-fluorescent cells (Salih et al., 1998), leading to the conclusion that polyp retraction can produce a highly reflective layer of varying optical density thereby protecting gonads and high-growth areas (polyp tips, ‘anchoring’ basal tissues).

However, Mazel et al. (2003) found no photoprotective quality associated with green fluorescent proteins (with emission spectra peaking at 500nm to 518nm bandwidth).

A Photosynthetic Aid? Schlichter et al. (1985) were perhaps the first to describe fluorescence as a potential aid to photosynthesis. These researchers documented behavior of fluorescent granules and dispersed pigmentation in the deep-water coral Leptoseris fragilis (found at depths of 100-145m, or ~325-475’ at Eilat, Israel on the Red Sea). Normally, this coral appears dark brown since its chromatophores (pigment-containing granules) are beneath a layer of symbiotic zooxanthellae. However, the color changes from to green in less than 1 minute after coral is transferred to daylight conditions. It is thought that host tissue contractions could force zooxanthellae downwards, or move chromatophores upwards through displacement, possibly via a vascular microtubule system within the coral’s tissue. Schlichter states that violet light is absorbed by the host pigment and is fluoresced as bluish-green light that is usable in photosynthesis. These deep-water corals receive only up to 10 µmol·m²·s PAR at noon and would need to efficiently collect light – much radiation making it through a densely packed zooxanthellae layer above the fluorescent pigment could be absorbed and fluoresced in an altered wavelength. Though the paper does not state as much, it could be that fluorescence shifts absorbed wavelengths to a bandwidth normally rarified at this depth thus avoiding photosaturation at those wavelengths usually transmitted through the water column. See Figure 36.

In a later paper, Schlichter et al. (1986) further discuss that pigment granules (chromatophores) fluoresce reddish while the intense turquoise autofluorescence seems dispersed throughout the cytoplasm, and is not in any specialized structure. Interestingly, we could speculate that the red fluorescence is also of benefit to photosynthesis.

Phototaxis: A Beacon for Zooxanthellae? This rather interesting concept involves phototaxis (a movement of an animal or plant towards light) and was proposed by Hollinsworth et al. in 2005. Cultured motile (non-symbiotic) zooxanthellae were exposed to white light which was split into a spectrum and projected on the culture flask. The idea was to determine if these dinoflagellates had a preference for any particular ‘color.’ Apparently they did – they swarmed in the area illuminated by green light. Blue light attracted significantly fewer zooxanthellae. Interestingly, previous observations suggested the motile zooxanthellae would move out of the area irradiated with ultraviolet wavelengths. These observations prompted a hypothesis – green fluorescence may act as a beacon, attracting free swimming zooxanthellae to coral recruits or planulae. Of course, other factors are undoubtedly in involved with corals attracting symbionts – corals are known to release chemical cues to the environment and it has been proven that zooxanthellae can swim against weak currents to reach a host (Pasternak et al., 2006). If we were to mesh these observations, we might arrive at the hypothesis that chemical cues released by the coral act as a navigational beacon to potential symbionts while green fluorescence is akin to landing strip lights.

GFP: A Shading Function for Photoreceptors? This thought was advanced in a paper by Shagin et al. (2004) and briefly discussed the possibility that GFP might shield the photoreceptors of the jellyfish Aequorea victoria. In addition, Burr et al. (2000) advanced the possibility of a protein (hemoglobin) in a nematode as possibly possessing an optical function. (Hemoglobin concentrates densely around Mermis nigrescens’ photoreceptor and is thought to play a role in phototaxis.) Some coral species have photoreceptors as well (Gorbunov and Falkowski, 2002), but to my knowledge no one has advanced a hypothesis linking fluorescence and protection of coral photoreceptors via optical shading. However a shielding function by GFP has been suggested for the non-photosynthetic soft coral Carijoa riisei (Khang and Salih, 2005).

This ends our discussion of potential functions of GFPs, and we will now turn our attention to specific green fluorescent proteins beginning with P-512.

 

 

Green-Blue Pigments

P-512

Host: Discosoma, Pigment 3

• Excitation: 503nm
• Emission: 512nm
• Stokes shift: 9nm
• Reference: Labas et al., 2002
• Comments: P-512 is one of several identified in the Discosoma genus. See Figure 37.
  

  Figure 38. Note that the chart’s line represents the emission spectrum. The absorption curve probably resembles that in Figure 37. After Mazel, 1995.

P-513

Host: Agaricia sp.

• Excitation: Not listed
• Emission: 513nm
• Stokes shift: N/A
• Reference: Mazel, 1995
• Comments: With shoulders at 545 nm and 490 nm. See Figure 38.

Host: Ricordea florida

• Excitation: Not listed
• Emission: 513nm
• Stokes shift: N/A
• Reference: Mazel, 1995
• Comments: In addition to the peak fluorescence at 513nm, Ricordea florida reportedly has a single shoulder at ~550-565nm. (R. florida specimens are variously colored green, blue, fluorescent orange, pink, copper, yellow and multiple combinations – Sprung and Delbeek, 1997).

P-514

Host: Acropora aspera

• Excitation: 501nm, shoulder at 480nm
• Emission: 514nm, shoulder at 490nm
• Stokes shift: 13nm
• Reference: Papina et al., 2002
• Comments: Collected off the coast of Okinawa, Japan in 1.5m of water, in August of 2001. This Acropora specimen appeared yellow-green in natural light and fluoresced bluish-purple when excited with UV-A radiation peaking at 365nm. Shifts in pH (5.0-8.0) do not significantly affect excitation and emission wavelengths or fluorescent quantum yield. See Figure 39.
  

  Figure 39. More than a few hobbyists have seen this particular fluorescent pigment. After Papina et al., 2002.

Host: Montastraea cavernosa

• Excitation: ~505nm
• Emission: 514nm
• Stokes shift: ~9nm
• Reference: Kelmanson and Matz, 2003
• Comments: Found in a red M. cavernosa specimen collected in the Florida Keys National Marine Sanctuary.

Host: Entacmaea quadricolor (Actinaria)

• Excitation: N/A
• Emission: 514nm
• Stokes shift: N/A
• Reference: Wiedenmann et al., 2002
• Comments: Precursor to P-611. The fluorescence is stable over a pH range of 4-10.

P-515

P-515’s fluorescence can contribute significantly to daylight appearance and is often found in conjunction with other fluorescent pigments (Fux and Mazel, 1999).

Host: Mycetophyllia lamarckiana

• Excitation: ~500-505nm
• Emission: 515nm
• Stokes shift: 10-15nm
• Reference: Mazel, 1997
• Comments: See Figure 40.

Host: Montastraea annularis

• Excitation: Not listed
• Emission: 515nm
• Stokes shift: N/A
• Reference: Mazel, 1997

Host: Montastraea sp.

• Excitation: Not listed
• Emission: 515nm
• Stokes shift: N/A
• Reference: Fux and Mazel, unpublished
  

  Figure 41. The spectral signature of this pigment is very similar to a number of pigments found with stony corals Montastraea. After Kelmanson and Matz, 2003.

Host: Montastraea cavernosa

• Excitation: 505±3
• Emission: 515±3nm
• Stokes shift: ~10nm
• Reference: Kelmanson and Matz, 2003.
• Comments: M. cavernosa can contain a number of pigments. There are several with similar emissions, and are referred to as P-515±3 by Kelmanson and Matz. See Figure 41.

Host: Scolymia sp.

• Excitation: Not listed
• Emission: 515nm
• Stokes shift: N/A
• Reference: Fux and Mazel, unpublished

Host: Mycetophyllia sp.

• Excitation: Not listed
• Emission: 515nm
• Stokes shift: N/A
• Reference: Fux and Mazel, unpublished
  

  Figure 42. Green fluorescence comes in many variations, as this photograph from LA hobbyist Dave Botwin’s reef aquarium demonstrates.

Host: Colpophyllia sp.

• Excitation: Not listed
• Emission: 515nm
• Stokes shift: N/A
• Reference: Fux and Mazel, unpublished

Host: Plesiastrea verispora

• Excitation: Not listed
• Emission: 515nm
• Stokes shift: N/A
• Reference: Gilmore et al., 2003.
• Comments: These Plesiastrea specimens (appearing blue in natural light due to a non-fluorescent chromoprotein) were collected in 5-9m of water at Port Jackson, Australia.

P-516: Precursor of Pigment 581

Commercially available as EosFP.

Host: Montastrea cavernosa

• Excitation: 506nm
• Emission: 516nm
• Stokes shift: 10nm
• Reference: Labas et al., 2002
• Comments: Relative brightness of this green form is 1.28 (when compared to a modified form of A. victoria GFP).

Host: Pocillopora damicornis

• Excitation: 486nm
• Emission: 516nm
• Stokes shift: 30nm
• Reference: Salih et al., 2000.
  

  Figure 43. A photoconvertible pigment found in Lobophyllia stony corals. After Nienhaus et al., 2005.

Host: Lobophyllia hemprichii

• Excitation: ~510nm
• Emission: 516nm
• Stokes shift: 6nm
• Reference: Nienhaus et al., 2005
• Comments: These researchers observed this green form (emission peak at 516nm) maturing to red fluorescence (peak at 581nm) when irradiated with ‘blue’ light (actually violet) at ~400nm. This maturation is driven by light energy and not chemical oxidation. See Figure 43 for excitation/emission spectra.

P-517

Host: Ricordea florida

• Excitation: Not listed
• Emission: 517nm
• Stokes shift: N/A
• Reference: Mazel, 1995

Host: Montastraea annularis

• Excitation: Not listed
• Emission: 517nm
• Stokes shift: N/A
• Reference: Mazel, 1995
• Comments: Emission has shoulders at 551nm and 483nm.
  

  Figure 44. Acropora tenuis specimens are not rare in coral aquaria. After Papina et al., 2002.

Host: Acropora tenuis

• Excitation: 505nm, shoulder at 465nm.
• Emission: 517nm, 555nm and 485nm.
• Stokes shift: 12nm
• Reference: Papina et al., 2002
• Comments: This A. tenuis specimen was collected in August 2001 at a depth of 1.5m in Okinawa, Japan. It appeared to the observer as brown in natural light, and fluoresced green under a black light. Spectral chart is that of an intact coral. Papina et al. noted no significant shifts of either fluorescent quantum yield or spectral characteristics over a pH range of 5.0 to 8.0. See Figure 44.

P-517 variant: Precursor of Pigment 593

Commercially available as Kikume or KikGR.

Host: Favia favus

• Excitation: 507nm
• Emission: 517nm
• Stokes shift: 10nm
• Reference: Tsutsui et al., 2005
• Comments: Pigment 517 from Favia favus is photoconvertible to P-593 (green to red). Relative brightness (compared to an engineered GFP from Aequorea victoria) is 1.12. See Figure 45.

Pigment 517 variant: Precursor to Pigment 574

Host: Ricordea florida

• Excitation: 506nm
  

  Figure 46. If we are to believe the thoughts of some researchers, this ‘double-peaked’ spectrum is indicative of a pigment in transition from green to red. This pigment was isolated from Ricordea florida. After Labas et al., 2002.

• Emission: 517nm
• Stokes shift: 11nm
• Reference: Labas et al., 2002
• Comments: See Figure 46.

P-518

Host: Ricordea florida

• Excitation: Not listed
• Emission: 518nm
• Stokes shift: N/A
• Reference: Mazel, 1995

Host: Acropora cytheria

• Excitation: Not listed
• Emission: 518nm
• Stokes shift: Not listed
• Reference: Hochberg et al., 2004
• Comments: This particular Acropora cytheria specimen was grown at the Waikiki Aquarium under a combination of artificial light (metal halide lamps) supplemented with afternoon sunlight (J.C. Delbeek, personal communication).

Host: Acropora digitifera

• Excitation: Not listed
• Emission: 518nm
• Stokes shift: Not listed
• Reference: Hochberg et al., 2004
• Comments: Found in an Acropora digitifera specimen on a shallow reef flat (depth of 0.1m; ~4”), Mayoette, Comorros.

Host: Family Pectiniidae

• Excitation: 503nm
• Emission: 518nm
• Stokes shift: 15nm
• Reference: Ando et al., 2004
• Comments: This is an interesting protein – its fluorescence is ‘switchable.’ The green pigment is bleached upon exposure to intense light at ~490nm. The green fluorescence returns upon exposure to violet light at about 400nm. This photoconvertible pigment (from green fluorescence to non-fluorescent) is commercially available as “Dronpa” (“Dron” being a ninja term for ‘vanishing’ and ‘pa’ for photoactivatible) and was isolated from a coral in the family ‘Pectiniidae’ (which includes genera Echinophyllia, Oxypora, Mycedium and Pectinia – Veron, 1986). Relative brightness of this green form is 2.40 (when compared to a modified form of A. victoria GFP).
  

  Figure 47. Spectral signatures of P-518 isolated from Ricordea florida by Labas et al., 2002. The excitation and emission spectra are identical to that of P-518 described in Montastraea cavernosa by Kelmanson and Matz (2003).

Host: Ricordea florida

• Excitation: 508nm
• Emission: 518nm
• Stokes shift: 10nm
• Reference: Labas et al., 2002

Host: Montastraea cavernosa

• Excitation: ~505nm
• Emission: 518nm
• Stokes shift: ~13nm
• Reference: Kelmanson and Matz, 2003
• Comments: This pigment was found in a green fluorescent M. cavernosa collected in the Florida Keys.

P-518 could also be an immature green form of the red fluorescent protein Kaede (P-582), although it is difficult to believe the pigment would not mature in shallow water as described by Hochberg et al., 2004. This could simply be a non-photoconvertible form of P-518.

P-519

Host: Montastraea cavernosa

• Excitation: Not listed
• Emission: 519nm
• Stokes shift: N/A
• Reference: Kelmanson and Matz, 2003
• Comments: Pigment 519 was found in a red fluorescent M. cavernosa collected in the Florida Keys. No excitation wavelength is listed; however, its emission spectrum is almost identical to P-518.

P-520: Precursor of Pigment
580.

• Host: Ricordea florida
• Excitation: Not listed
• Emission: 520nm
• Stokes shift: N/A
• Reference: Mazel, 1995.
• Comments: This pigment might be the immature form of the ‘red’ fluorescent P-580. See below.

Host: Montastraea cavernosa

• Excitation: 508nm, with a shoulder at 572nm
• Emission: 520nm, with shoulder at 580nm
• Stokes shift: 12nm and 8nm
• Reference: Labas et al., 2002.
• Comments: Possibly the same pigment found in Ricordea florida by Mazel (1995)?

P-520: Precursor of Pigment
611.

• Host: Entacmaea quadricolor
• Excitation: ~500nm
• Emission: ~520nm
• Stokes shift: 20nm
• Reference: Nienhaus et al., 2003.

P-522

Host: Montastraea cavernosa

• Excitation: Not listed
• Emission: 522nm
• Stokes shift: N/A
• Reference: Kelmanson and Matz, 2003.
• Comments: This pigment was found in a red Montastraea cavernosa collected in the Florida Keys National Marine Sanctuary.
  

  Figure 48. The excitation and emission suggest photoconversion is possible in this pigment. After Wiedenmann et al., 2000.

Host: Anemonia sculata

• Excitation: 511nm
• Emission: 522nm
• Stokes shift: 11nm
• Reference: Wiedenmann et al., 2000
• Comments: Note the double-peaked emission in Figure 48.

P-530

Host: Porites astreoides

• Excitation: ~500nm
  

  Figure 49. Spectral qualities of a pigment found in the Caribbean coral Porites astreoides. After Mazel et al., 2003.

• Emission: 530nm
• Stokes shift: ~30nm
• Reference: Mazel et al., 2003
• Comments: See Figure 49 for excitation and emission spectra.

Yellow-Green Pigments

P-538

Host: Zoanthus 2.

• Excitation: 528nm
  

  Figure 50. After Yanushevich et al., 2002. Excitation and emission spectra are practically identical to those presented by Matz et al., 1999.

• Emission: 494nm and 538nm
• Stokes shift: 10nm
• Reference: Matz et al., 1999; Labas et al., 2002
• Comments: P-538 was isolated by Matz et al. (1999) from a Zoanthus species and found in combination with another fluorescent pigment (P-506). Fluorescent Quantum Yield and Relative Brightness are fairly low – 0.42 and 0.38, respectively. See data of Yanushevich et al. (2002) in Figure 50.

P-550

Host: Porites murrayensis

• Excitation: 530nm
  

  Figure 51. Compare the spectral qualities of this pigment to that in Figure 50 – they are almost identical. After Matz et al., 1999.

• Emission: 550nm
• Stokes shift: 20nm
• Reference: Dove et al., 2001
• Comments: This coral was purple when viewed in natural light due to the presence of a non-fluorescent protein. Maximum host tissue/zooxanthellae absorbance is at 576nm (data not shown).

P-557

Host: Agaricia sp.

• Excitation: Not listed
• Emission: 557nm
• Stokes shift: N/A
  

  Figure 52. A rather unique yellowish fluorescent protein found it in the Caribbean stony oral Agaricia. After Mazel, 1995.

• Reference: Mazel, 1995
• Comments: Emission also has shoulders at 600nm and 545nm. Reports of this particular pigment are practically non-existent, with Mazel’s reports being the only ones of which I am aware. See Figure 52. (Mazel reports yellow Agaricia are common in Bonaire – personal communication).

P-561 (Kusabira-Orange)

Host: Fungia concinna

• Excitation: 548nm
• Emission: 565nm
• Stokes shift: 17nm
• Reference: Karasawa et al., 2004.
• Comments: Commercially available from MBL International under the trade name “Kusabira-Orange”. Wild type P-561 has a fluorescent quantum yield of 0.45, and is not pH sensitive (pKa <5.0). The Japanese name for Fungia is “Kusabira-ishi.” See Figure 53.
  

  Figure 53. A true orange fluorescent protein found in the popular ‘mushroom’ stony coral (Fungia). After Karasawa et al., 2004.

P-562 (or asCP-562)

Host: Anemonia sculata

• Excitation: 559nm
• Emission: 562nm (immature form)
• Stokes shift: 52nm
• Reference: Wiedenmann et al., 2002
• Comments: This yellow green pigment is found in the anemone (Anemonia sculata) and is technically fluorescent although laboratory instruments are needed to detect it. Under certain conditions, this pigment can transform from yellow-green to red with a very weak emission maximum at 611nm.

P-565

Host: Agaricia humilis

• Excitation: 490nm
  

  Figure 54. A yellowish pigment from the stony coral Agaricia. After Mazel et al., 2003.

• Emission: 565nm
• Stokes shift: 75nm
• Reference: Mazel et al., 2003.
• Comments: See Figure 54.

Host: Cerianthus sp.

• Excitation: 548nm
• Emission: 565nm
• Stokes shift: 17nm
• Reference: Ip et al., 2004.
• Comments: This pigment is apparently spectrally different from the P-565 found in Agaricia stony corals.

An Early GFP Description

In homage to Siro Kawaguti, one of the first researchers of coral coloration, Table 3 describes some of the properties of fluorescent pigments found in Pacific corals. Though much of Kawaguti’s early works were completed and published in the late 1930’s and 40’s, his ideas, for the most part, have withstood the test of time. His equipment was not as sophisticated as that of today and did not offer great precision, yet his descriptions are remarkable. His works offer perspectives on GFPs and the varieties of coral hosts and complement later research. Note that his work deals with mostly emission wavelengths (as seen in the ‘1^st Band’ and ‘2^nd Band’ columns) while absorbance (‘Max. Abs. 1’ and ‘2’ columns is rarely described. See Table 5.

Table 5. Kawaguti’s early observations of Pacific corals’ fluorescence.

Green Fluorescence
Coral	1st Abs. Band	2nd Abs. Band	Maxima Abs.1	Maxima Abs. 2
Fungia actiniformis	498-521	475-484	512	480
Montipora verrucosa	505-514	*	*	*
Porites (arboreal)	515-525	495	*	*
Fungia repunda	504-517	476-483	*	*
Montipora ramosa	498-511	486	504-507	*
Tridacophyllia lacinata	488-511	467-471	*	*
Pachyseris rugosa	498-509	*	*	*
Caulastrea tumida	498-510	*	*	*
Hydnophora microconos	498-511	*	*	*
Acropora hyacynthus	498-512	474	*	*
Acropora haimei	498-513	471	*	*
Pocillopora acuta	498-514	*	*	*
Favia sp.	500-507	*	*	*
Mycedium elephantotus	491-505	*	*	*
Herpetolitha limax	493-508	*	*	*
Lobophyllia hemprichii	494-514	*	*	*
Lobophyllia corymbosa	501-514	473	*	*
Psammocora contigua	482-498	463	*	*
Euphyllia glabrescens	489-500	*	*	*
Galaxea fascicularis	488-501	*	*	*
Pocillopora eydouxi	473-489	*	*	*
Plerogyra sinuosa	488-507	*	*	*
Echinopora lamellosa	491-503	*	*	*
Goniopora sp.	485-501	458-466	*	*
Acropora arcuata	494-503	*	*	*
Cyphastrea mocrophthalma	482-499	462-466	*	*
Tridacophyllia lacinata	493-504	*	*	*

Next time, we’ll look at more fluorescent pigments and discuss how light can cause coloration shifts in at least some pigments. Interested parties can correspond with the author at [email protected].

 

References

Please see Part 1 (Volume V, Issue IX) for a full listing of references used in this series.