The authors acknowledge funding provided by the Edwin. Pauley Foundation .... Mizuno, H., T. K. Mal, K. I. Tong, R. Ando, T. Furuta, M. Ikura, and. A. Miyawaki.
Reference: Biol. Bull. 216: 68 –74. (February 2009) © 2009 Marine Biological Laboratory
Red Fluorescent Protein Responsible for Pigmentation in Trematode-Infected Porites compressa Tissues CAROLINE V. PALMER*,1,2, MELISSA S. ROTH3, AND RUTH D. GATES Hawai’i Institute of Marine Biology, University of Hawai’i at Manoa P.O. Box 1346, Kaneohe, Hawaii 96744
Abstract. Reports of coral disease have increased dramatically over the last decade; however, the biological mechanisms that corals utilize to limit infection and resist disease remain poorly understood. Compromised coral tissues often display non-normal pigmentation that potentially represents an inflammation-like response, although these pigments remain uncharacterized. Using spectral emission analysis and cryo-histological and electrophoretic techniques, we investigated the pink pigmentation associated with trematodiasis, infection with Podocotyloides stenometre larval trematode, in Porites compressa. Spectral emission analysis reveals that macroscopic areas of pink pigmentation fluoresce under blue light excitation (450 nm) and produce a broad emission peak at 590 nm (⫾6) with a 60-nm full width at half maximum. Electrophoretic protein separation of pigmented tissue extract confirms the red fluorescence to be a protein rather than a low-molecular-weight compound. Histological sections demonstrate green fluorescence in healthy coral tissue and red fluorescence in the trematodiasis-compromised tissue. The red fluorescent protein (FP) is limited to the epidermis, is not associated with cells or granules, and appears unstructured. These data collectively suggest that the red FP is produced and localized in tissue infected by larval trematodes and plays a role in the immune response in corals.
INTRODUCTION A more comprehensive understanding of resistance mechanisms in corals is a critical component of the knowledge base necessary to design strategies aimed at mitigating the increasing incidence of coral disease (Peters, 1997; Harvell et al., 1999, 2007; Hoegh-Guldberg, 1999; Sutherland et al., 2004; Aeby, 2006) associated with reduced water quality (Bruno et al., 2003) and ocean warming (Harvell et al., 2007). Disease resistance mechanisms in invertebrates are primarily limited to the innate immune system, which provides immediate, effective, and nonspecific internal defense against invading organisms via a series of cellular pathways (Rinkevich, 1999; Cooper, 2002; Cerenius and So¨derha¨ll, 2004). Of the innate immune pathways, inflammation is a biphasic response (Rowley, 1996) involving rapid infiltration of phagocytic cells (Olano and Bigger, 2000; Cooper, 2002) into compromised tissue, frequently followed by encapsulation. The associated inflammatory responses therefore destroy infected, damaged or dead host cells or wall them off from healthy tissue (Sparks, 1972), and they frequently involve the up-regulation of the melanin pathway. For anthozoans, inflammation-like responses primarily involving cell infiltration have been described for several species (Olano and Bigger, 2000; Domart-Coulon et al., 2006; Ellner et al., 2007; Mydlarz et al., 2008; Palmer et al., 2008). Additional support for the presence of inflammatory-like responses in anthozoans is the presence and up-regulation of the melanin pathway in diseased gorgonians (Mydlarz and Harvell, 2007; Mydlarz et al., 2008) and compromised scleractinian corals (Palmer et al., 2008). Compromised tissues of anthozoans are frequently characterized by non-normal pigmentation (referred to as pigmentation henceforth), which appears pink in Porites spp. (Aeby, 2003; Ravindran and Raghukumar, 2006a, b), blue in Acropora spp. (Bongiorni and Rinkevich, 2005), and
Received 12 March 2008; accepted 20 October 2008. * To whom correspondence should be addressed. E-mail: c.v.palmer@ newcastle.ac.uk 1 School of Biology, Newcastle University, Newcastle upon Tyne, NE1 7RU. UK. 2 ARC Centre of Excellence for Coral Reef Studies and School of Marine and Tropical Biology, James Cook University, Townsville, QLD 4811, Australia. 3 Scripps Institution of Oceanography, University of California San Diego, 9500 Gilman Dive, La Jolla, CA 92093-0208. 68
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purple in Gorgonia ventalina (Petes et al., 2003). Although this pigmentation is inducible by numerous agents (Willis et al., 2004; Palmer et al., 2008), descriptions of several coral and other anthozoan infections rely on the distinct macroscopic characteristics of this host response. These include the purple lesions of Gorgonia ventalina (the Caribbean gorgonian sea fan) that reflect fungal infection by Aspergillus sydowii (Petes et al., 2003) and the pink swollen nodules of Porites spp. infected by larval trematodes of the species Podocotyloides stenometre, a condition called trematodiasis (Aeby, 2003). The presence of the melanin pathway directly associated with these pigmented areas of compromised tissue (Mydlarz and Harvell, 2007; Palmer et al., 2008) combined with the frequency and diversity of causes suggest that pigmentation in corals represents a general immune response. Fluorescent proteins (FPs) are largely responsible for the brightly colored tissues found in many coral genera (Matz et al., 1999; Labas et al., 2002; Mazel et al., 2003). Their capacity to emit light at a wavelength different from the excitation wavelength has driven independent hypotheses regarding their role as photo-protective molecules (Salih et al., 1998) and as promoters of photosynthesis (Salih et al., 2000; Dove et al., 2001). FPs have also been demonstrated to quench oxygen radicals (Bou-Abdallah et al., 2006), a property that could serve an important function in corals. The potential involvement of FPs in the immune response of corals was examined in the pink tissue associated with the swollen nodules of Porites compressa trematodiasis infections (Aeby, 2003). This interaction presents a unique opportunity to investigate the characteristics of scleractinian coral tissue compromised by a known foreign organism, Podocotyloides stenometre (Aeby, 2003). MATERIALS AND METHODS Sample collection Six trematode-infected branches, nubbins, of Porites compressa (Dana, 1846) (Fig. 1) were collected from three colonies within Kaneohe Bay, during June 2007. Nubbins were collected from similarly oriented areas of the colonies, and all were moderately infected with larval trematodes. Distinct infections were indicated by pink swellings surrounded by brown healthy tissue on individual polyps. Pigmented and healthy tissue from each nubbin was compared. Spectral emission An airgun was used to blast areas of pigmented and healthy tissue from live P. compressa nubbins (n ⫽ 3) into an extraction buffer containing 30 mmol l⫺1 phosphate buffer and 5 mmol l⫺1 2-mercaptoethanol (Sigma-Aldrich M7522). The tissue slurry was homogenized and centrifuged for 7 min at 8050 ⫻ g. Samples were maintained at 4
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Figure 1. Pink pigmentation in response to larval trematode infections of Porites compressa from Kane’ohe Bay. (Photo credited to C. S. McKeon; used with permission.)
°C during processing. Three 200-l aliquots of the supernatant from each sample were placed in wells of a black (with transparent bottom) 96-well microtiter plate. Parallel 200-l aliquots of extraction buffer were used to control for extract-independent effects. Each well was excited at 450 nm, using a spectrophotometer (Spectramax M2, Molecular Devices), and the emission spectrum was measured in 6-nm increments. The data were normalized to the highest peak and the full width at half maximum (FWHM) of the emission peak was calculated. Cryo-histology Pigmented and healthy samples (n ⫽ 3) of P. compressa were fixed in 4% paraformaldehyde phosphate buffer and decalcified in 20% disodium EDTA, pH 7.0, that was changed twice daily for 4 days. Samples were rinsed with deionized water and stored in 15% sucrose solution overnight at 4 °C, then transferred to optimum cutting temperature (OCT) compound. Samples were frozen onto histological chucks using OCT and isopropanol cooled with liquid nitrogen. Sections were cut at either 10 m or 5 m, using a cryo-stat at ⫺30 °C. Sections were air dried on slides overnight and covered using aqueous coverslip solution. Slides were observed and photographed using a Zeiss 510 laser scanning confocal microscope with excitations of 633 nm at 60%, 488 nm at 58%, and 543 nm at 100%. Filters used were LP 650, LP 505-550, BP 560-615. Gel electrophoresis The supernatants of pigmented and healthy tissue extracts (obtained as described above) were analyzed using polyacrylamide gel electrophoresis (10% resolving, 4% stacking gel). Auto-fluorescent protein bands were visualized and
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RESULTS Spectral emission The emission spectrum of pigmented Porites compressa tissue extract is distinct from that of the healthy tissue (Fig. 2). The pigmented tissue extract has a broad emission peak at 590 ⫾ 6 nm (60 nm FWHM) with a shoulder extending through 650 nm; this peak is absent in healthy tissue. Both healthy and pigmented tissue samples have a chlorophyll emission peak in the far-red (674 ⫾ 6 nm).
Cryo-histology
Figure 2. Representative emission spectra for Porites compressa healthy (solid) and pigmented (dashed) tissue extracts with an excitation of 450 nm.
photographed on a Typhoon 8600 variable mode imaging system, at an excitation of 532 nm. An auto-fluorescent protein band was excised from the gel and excited at 450 nm in the spectrophotometer (Spectramax M2, Molecular Devices) to determine whether the band had the same spectral properties as the original sample extract.
Confocal microscopy of pigmented and healthy tissue cryo-sections of P. compressa show green fluorescence within the coral tissue. The pigmented tissue (Fig. 3a) has a high density of granular cells in a relatively enlarged gastrodermal layer, which is devoid of zooxanthellae. Melanincontaining granular cells are also present in high densities within the epidermis of the pigmented tissue, although they are partially obscured by an unstructured red fluorescent substance. Both epithelial layers of healthy tissue (Fig. 3b) also have melanin-containing granular cells, although these are in lower abundance in healthy versus pigmented tissues (data not shown). The gastrodermis of healthy tissues also contains many zooxanthellae, and there is no red fluorescence in the healthy tissue sections.
Figure 3. Autofluorescence of longitudinal sections of Porites compressa tissue. (a) Pigmented tissue section (5 m). (b) Healthy tissue (10 m). E, epithelium, G, gastrodermis, Z, zooxanthellae, Me, melanin. Scale bars represent 50 m.
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Gel electrophoresis Electrophoretic separation of proteins resolved two bands in the pigmented tissue extracts and one band in the healthy tissue (Fig. 4). The emission spectrum of the additional band from the pigmented tissue was equivalent to that of the raw pink tissue extract, displaying the peak at 590 nm when excited with 450 nm (data not shown). DISCUSSION Numerous fluorescent proteins (FPs) have been characterized from a variety of anthozoans (Matz et al., 1999; Labas et al., 2002; Ando et al., 2002; Sun et al., 2004; Leutenegger et al., 2007; Alieva et al., 2008). FPs identified from anthozoans generally are cyan, green, or the more complex red fluorescence (Sacchetti et al., 2002; Ulgalde et al., 2004; Alieva et al., 2008); in addition, nonfluorescent FPs have been identified (Dove et al., 1995; Alieva et al., 2008). The pigmented tissue extracts of trematode-infected
Figure 4. Auto-fluorescent photograph at 532-nm excitation of acrylamide gel with lanes of healthy (H) and pigmented (P) tissue extract.
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Porites compressa (Aeby, 2003) fluoresce at a wavelength in the red spectrum (590 nm ⫾ 6) when excited with blue light (450 nm), similar to that of cyanobacteria that have been found in coral; however, the location and structure of their red fluorescence is very distinct (Lesser et al., 2004) and bears no resemblance to the pigmentation patterns in P. compressa. In addition, the native gel confirms that a fluorescent protein is responsible for the pink tissue in P. compressa. Red FPs have evolved as several independent evolutionary lineages (Shagin et al., 2004; Alieva et al., 2008), and each type possesses unique structural and functional characteristics. Red FPs have previously been reported for the scleractinian corals Montastraea cavernosa (Labas et al., 2002; Leutenegger et al., 2007), Lobophyllia hemprichii (Leutenegger et al., 2007), and Trachyphyllia geoffroyi (Ando et al., 2002), and more recently, for several species within the family groups Acroporiidae, Faviidae, Mussidae, and Poritidae (Alieva et al., 2008). These red FPs can be categorized into either Kaede (Mizuno et al., 2003) or DsRed-types (Gross et al., 2000) and represent alternative methods of extending the green chromophore complex to red (Alieva et al., 2008). The distinct broad emission spectra of DsRed-type chromophores with a shoulder at 630 nm implies that the FP located in infected P. compressa tissue is equivalent to that identified in Porites porites with its emission maximum at 595 nm (Alieva et al., 2008). Despite the identification of FPs, the specific role associated with them remains elusive; however, their prevalence among the anthozoans suggests multiple or common functionality (Leutenegger et al., 2007). The highly repeated and specific patterns of color in corals and other anthozoans have led to the generation of hypotheses regarding location-specific functional roles for FPs (Labas et al., 2002). This is logical when one considers the purple-blue chromoprotein in the tentacle tips (Labas et al., 2002) and in colony extremities (Dove et al., 1995; Shagin et al., 2004) of corals and anemones, for which there is currently limited physiological explanation (Kawaguti, 1944; Takabayashi and Hoegh-Guldberg, 1995). The areas of pink pigmentation in Porites spp. are also highly localized, being found exclusively in compromised tissue such as at the site of trematodiasis. One explanation for this pattern is that the pink pigment attracts the fish necessary to continue the trematode life cycle (Aeby, 1992, 2002). However, pigmentation is found in corals that are not infected with larval trematodes but are alternatively compromised (Willis et al., 2004; Bongiorni and Rinkevich, 2005; Ravindran and Raghukumar, 2006a, b; Palmer et al., 2008), reinforcing its potential importance during an immune response. Support for the hypothesis of color-related differential functions of FPs is found here in the histological sections of P. compressa. Healthy tissues show heavy localization of green fluorescence in healthy gastrodermis and a complete
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lack of red fluorescence, confirmed by the absence of an emission peak within the red spectrum. In contrast, the compromised tissue has highly reduced green fluorescence in the gastrodermis and high red fluorescence in the epidermis, again confirmed in the emission spectrum. The first of two functions that have been proposed for FPs in anthozoans is light optimization (Salih et al., 1998, 2000; Dove et al., 2001). The histological evidence presented here for compromised tissue areas of P. compressa localizes the red FP exclusively in the epidermis and documents the low zooxanthellae density in the gastrodermis. These results corroborate the findings of Mazel et al. (2003) for Caribbean corals. However, the highly reduced number of symbionts in the tissues directly underlying the red FP suggests redundancy in the production of the protein or its ineffectiveness as a photo-protector. The requirement for photoprotection suggests an alteration to the local tissue environment leading to an increased light level, which is undetermined for compromised tissue. In addition, the location of the red FP in the epidermis and an emission wavelength that is not effective for photosynthesis (Levy et al., 2003) do not support a photo-enhancing, screen-scattering role (Salih et al., 2000) for the P. compressa red FP. The second major function proposed for FPs is as oxygen-radical quenchers (Tsein, 1998; Mazel et al., 2003; Bou-Abdallah et al., 2006). During periods of thermal or UV stress, the symbiotic dinoflagellates and coral host increase production of superoxide dismutase, a reactive oxygen species (ROS) scavenger (Lesser, 1996, 1997). Mazel et al. (2003) initially proposed a role for FPs as non-enzymatic scavengers of superoxide radicals, and the potential of a green FP for oxygen-radical quenching was demonstrated by Bou-Abdallah et al. (2006), although the mechanisms involved are unknown. Despite the focus on stress and the endosymbiosis, the potential involvement of FP in ROS scavenging opens new doors in the context of immunology. The cytotoxic, melanin-producing pathway (Nappi and Christensen, 2005) is a characteristic part of invertebrate innate immune responses (Rowley, 1996; So¨derha¨ll and Cerenius, 1998; Cerenius and So¨derha¨ll, 2004; Butt and Raftos, 2008). In anthozoans, the melanin synthesis pathway is associated with compromised pigmented tissue (Petes et al., 2003; Mydlarz and Harvell, 2007; Palmer et al., 2008), coincident with inflammation-like responses (Palmer et al., 2008). The phenoloxidase-activating melanin pathway produces ROS intermediates that provide cytotoxic defense against invading organisms (Nappi and Christensen, 2005; Mydlarz and Jacobs, 2006). However, in excess, ROS has the potential to damage the host, which implies that the host has the capacity to regulate and control local cytotoxicity. The histology reveals that the red FP in P. compressa is coincident with an increase in granular cells. These cells are very similar to the melanin-containing granular cells proposed as putative amoebocytes in two
other Porites spp. (Domart-Coulon et al., 2006; Palmer et al., 2008) and to melanin deposits reported in gorgonian sea fans (Mydlarz et al., 2008). The presence of melanin-containing granular cells (Palmer et al., 2008) in both epithelia of compromised tissue further negates the role of the red FP as a photo-protector, because melanin has light-absorbing properties (Meredith et al., 2006). This does, however, suggest a direct relationship between an inflammation-like response and the presence of red FP, and perhaps lends additional support for a role in ROS quenching and cytotoxic defense. Further investigation is required to elucidate the structure and the potential role or roles of the red FP in areas of localized damage or invasion in Porites spp. Determining the protein structure will indicate whether there is potential homology of the red FP to the well-characterized specific structure of the GFP from Aequorea victoria (Tsien, 1998) and the GFP-like proteins of other anthozoans (Miyawaki et al., 2003). The investigation of similar appearances in response to foreign organisms in other coral species would help to determine the generality of the presence of FPs in compromised tissue, with the goal of better defining immune responses in scleractinian corals. In summary, this study demonstrates the presence of a red fluorescent protein in compromised tissue of a scleractinian coral, lends support for a role for this protein in cytotoxic defense, and provides new insights into the biological mechanisms involved in immune resistance in the anthozoans. Acknowledgments The authors acknowledge funding provided by the Edwin Pauley Foundation, the Coral Reef Targeted Research Project, and the US National Science Foundation. We thank T. Ainsworth for assistance with histological protocols, and Kewalo Marine Laboratory for access to the confocal microscope. This is Hawaii Institute of Marine Biology Contribution Number 1003 and 2007 Pauley Program Contribution Number 2. LITERATURE CITED Aeby, G. S. 1992. The potential effect the ability of a coral intermediate host to regenerate has had on the evolution of its association with a marine parasite. Pp. 809 – 815 in Proceedings of the 7th International Coral Reef Symposium, Vol. 2, R. H. Richmond, ed. University of Guam Press, UOG Station, Guam. Aeby, G. S. 2002. Trade-offs for the butterflyfish, Chaetodon multicinctus, when feeding on coral prey infected with trematode metacercariae. Behav. Ecol. Sociobiol. 52: 158 –163. Aeby, G. S. 2003. Corals in the genus Porites are susceptible to infection by a larval trematode. Coral Reefs 22: 216. Aeby, G. S. 2006. Baseline levels of coral disease in the Northwestern Hawaiian Islands. Atoll Res. Bull. 543: 471– 488. Alieva, N. O., K. A. Konzen, S. F. Field, E. A. Meleshkevitch, M. E. Hunt, V. Beltran-Ramirez, D. J. Miller, J. Wiedenmann, A. Salih,
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