ABSTRACT. Tumor-like growths associated with necrosis in the Caribbean octocorallian, Gorgonia ventalina. contain an infesting filamentous green alga which ...
BULLETIN
OF MARINE SCIENCE. 31(2): 399-409. 1981
CORAL REEF PAPER
ALGAL TUMORS IN THE CARIBBEAN OCTOCORALLIAN, GORGONIA VENTALINA: II. BIOCHEMICAL CHARACTERIZATION OF THE ALGAE, AND FIRST EPIDEMIOLOGICAL OBSERVATIONS
Daniel E. Morse, Aileen Morse, Helen Duncan, and Robert K. Trench ABSTRACT Tumor-like growths associated with necrosis in the Caribbean octocorallian, Gorgonia ventalina. contain an infesting filamentous green alga which is not detectable in normal, nontumorous colonies. The infesting alga is identified here by the chromatographic resolution and spectrophometric characterization of pigments unique to the algal Order Siphonales. Similar algae commonly are found as endolithic, non-pathogenic and non-tumorigenic associates of many scleractinian species. The tumorigenic response in G. ventalina includes hyperplasia of the coral mesenchyme; this tissue secretes abnormal elements of endoskeletal collagen-like gorgonin, elaborated as a dense network of fine tubules which encapsulate but do not kill the infesting algal filaments. The infesting alga and host response described for G. ventalina differ from those we have observed in other Caribbean gorgonian species. The incidence of tumorigenic algal infestation in G. ventalina, in contrast to that seen in other species, is non-uniform. The incidence of these tumors may prove useful as an indicator of environmental stress, damage by predation, or other as yet unidentified ecological factors controlling growth, parasitic infestation and mortality in reef communities.
We previously reported the occurrence of tumor-like growths associated with algal infestation, peripheral necrosis and erosion of the Caribbean sea-fan, Gorgonia ventalina [A1cyonaria (=Octocorallia): Gorgonacea] (Morse et aI., 1977). These growths (Fig. 1) occur principally as spherical nodules (2-28 mm diameter) and as irregular, flat plaques (2-300 mm), often growing confluently on the affected colonies. Histological and electron-micrographic analyses of these tumorlike growths revealed the presence of filamentous algal networks extensively proliferated within a hyperplastic coral mesenchyme. Each infesting algal filament, or group of filaments, was found to be enclosed within a surrounding sheath of endoskeletal collagen-like gorgonin, present as an abnormal proliferation of fine tubular elements produced by the hyperplastic mesenchymal cells (Figs. 2, 3). Tumor-free colonies of the gorgonian from the same locale appear free of these algae, and contain neither hyperplastic mesenchyme nor ramifications of the axial endoskeleton. Very few pathological conditions responsible for the erosion and death of corals have been well characterized. These notably include two kinds of bacterial infection of hard corals [Zoantharia (=Hexacorallia): Scleractinia] described by Garrett and Ducklow (1975), and a blue-green algal infestation of scleractinia reported by Antonius (1977). Tumor-like growths previously had been observed in scleractinia from the Pacific, although the etiology of these growths has remained largely obscure (Squires, 1965a; 1965b; White, 1965; Soule, 1965; Cheney, 1975). Non-tumorigenic endolithic growth of siphonous green algae within the calcium carbonate skeletons of living scleractinian corals is widespread, and, apparently, non-pathogenic (Halldal, 1968; Jeffrey, 1968; Shibata and Haxo, 1969; Lukas, 1973; 1974; van den Hoek et aI., 1975). The algal tumor-like growths in G. ventalina represent the first such infestation 399
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Figure 1. Algal tumors in G, ventalina, showing associated peripheral necrosis and erosion of the coral (Specimen from study-site E; cf. Fig. 7)
reported to occur in soft corals (Morse et al., 1977). We therefore have characterized these infesting algae biochemically, and have made preliminary epidemiological observations of the incidence and distribution of these tumors in populations of the susceptible gorgonian. A preliminary characterization of similar algal infestations in other Caribbean soft corals also is presented. These algal infestations are of interest as potential sources of mortality in corals, as readily identifiable potential (secondary) indicators of environmental stress or of damage by predation to reef communities, and as newly described parasitic and apparently pathological associates of a relatively simple animal group. METHODS
Observation, Collection, and Preservation of Specimens Observations and collections of G. ventalina were performed with SCUBA in depths of 1-20 m surrounding the island of Bonaire, Netherlands Antilles, (12°, 6'N; 68°, 25'W) in July, 1976. Samples of tissue (10-40 g) were removed from living colonies bearing tumors and from colonies which were tumor-free. Samples taken for microscopic examination were preserved, immediately after collection, in seawater containing formaldehyde (4% w/v) and glycerol (0.5% v/v); these subsequently were decalcified, fixed, and stained for microscopy as described previously (Morse et aI., 1977). Samples taken for the extraction, purification and characterization of algal pigments were frozen quickly without other treatment; these were maintained at temperatures below -20°C until thawed for the extraction of pigments.
Extraction and Analysis of Pigments Pigments were extracted from the coral tissues and analyzed by methods described previously (Muscatine, 1971; Trench, 1975); samples were protected from light during all stages of extraction,
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Figure 2. Cross-section through nodule from Figure I, showing profusion of abnormal tubules of gorgonin (with irregular, dense walls) throughout the coral mesenchyme. Filamentous algal cells, cut in transverse section, fill the gorgonin tubules; the empty-appearing central axis of the colony also is visible. (Methylene blue, 165x).
Figure 3. Algal cells within gorgonin tubule from Figure 2. Note thick algal walls and prominent starch-like inclusion bodies. (Methylene blue, 450x).
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purification, and anlysis. Frozen tissue and associated endoskeleton (10-40 g) were crushed in a ball mill (Baldor Electric Co., Ft. Smith, Ark.) at 2_5°C; this material then was extracted by continuous stirring (I h, at 2°C) with 1.2 volumes of absolute methanol saturated with MgC03• Following clarification of the aqueous methanol extracts by centrifugation, pigments were extracted into 2 volumes of ethyl ether; phase separation was facilitated by addition of small volumes of 15% (w/v) aqueous NaCI. The separated ether extracts subsequently were washed twice with 15% NaCI, and then washed repeatedly with water to remove all traces of this salt. The washed ether extracts were concentrated by evaporation under nitrogen and chromatographed on thin layers (250 /-Lm) of silica gel G on glass plates (Brinkman, Sit G-25) developed with ascending 15% n-hexane in ethyl ether. Purified pigments were eluted from the plates with methanol, concentrated by evaporation under nitrogen, and analyzed by absorption spectroscopy using a Varian Techtron series 634 recording spectrophotometer. Samples from three colonies of each type gave identical results. RESULTS
Identification of Tumor-specific Algal Pigments Microscopic examination ofthe tumor-like nodules and plaques on G. venta/ina clearly reveals infestation by densely packed algal filaments encapsulated within fine tubules of gorgonin (Figs. 2, 3; Morse et al., 1977). To further identify these filamentous algae seen within the tumor-like growths, we therefore undertook the chemical extraction and chromatographic and spectrophotometric analysis of those pigments which might prove to be unique to the tumor-specific algae. Pigments were extracted from the tumor-like nodules and from comparable portions of tissue from unaffected colonies of G. venta/ina as described in Methods. The extracted pigments were resolved by chromatography on silica gel G (Fig. 4). Organic solvent extracts of both tissues are seen to contain those pigments characteristic of the normal endosymbiotic dinoflagellate Symbiodinium (=Gymnodinium) microadriaticum (Schoenberg and Trench, 1980a, 1980b, 1980c), principally including chlorophylls a and c, peridinin, t3-carotene, and an unidentified yellow carotenoid (probably dinoxanthin; Muscatine, 1971). In addition, extracts of the infested tissue reveal the presence of appreciable quantities of chlorophyll b (yellow-green), and tumor-specific siphonoxanthin-like orange carotenoids. Identification of the chlorophyll b in these extracts was confirmed by spectrophotometric analysis of the pigment after preparative resolution and purification by thin-layer chromatography (Fig. 5); the principal absorption maximum at 452 nm, secondary peak at 642 nm and shoulder at 428 nm make this identification unequivocal (Strain, 1951). This identification of chlorophyll b in extracts only of the tumor material confirms the previous histological identification (Morse et al., 1977) of a eukaryotic green alga specifically associated with the tumor-like nodules. Further characterization of the carotenoids unique to the tumor extracts (Fig. 4) also was performed by absorption spectroscopy following chromatographic purification on silica gel G (Fig. 6). The spectra of carotenoid # 1 (Fig. 6A) and carotenoid #2 (Fig. 6B) identify these compounds as derivatives or structural isomers of siphonoxanthin and siphonein, respectively (Strain, 1951; Kleinig and Egger, 1967; Walton et aI., 1970; Isler, 1971). These spectral determinations confirm the preliminary identifications of these siphonoxanthins based upon their chromatographic mobilities (Fig. 4; Muscatine, 1971; Trench, 1975; Strain and Svec, 1969). Oxidation prevented further characterization of the material available. On the basis of the pigment content thus established, the tumor-specific alga tentatively is identified as a member of the Order Siphonales, apparently representing either a member or close relative of the Genus Ostreobium (Strain, 1965; Kleinig and Egger, 1967; Kleinig, 1969; Strain and Svec, 1969; Jeffrey 1968; Halldal, 1968; Shibata and Haxo, 1969).
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Chromatographic Resolution of Pigments from Algal Tumors
-
f3 -
-.-
Carotene
Chlorophyll A Chlorophyll B
-•• =__ -
Peridinin
Normal
•• Carotenoid '*1 -- Carotenoid #2 .• .• Chlorophyll C Tumor
Figure 4. Resolution of pigments from equivalent samples of normal and infested specimens of G. venlalina. Exlraclion and chromatography were performed as described in Methods.
Incidence and Distribution of Algal Tumors in G. ventalina The incidence of algal tumors within populations of G. ventalina at Bonaire clearly is non-uniform. Sites A-P, surveyed thoroughly by SCUBA-reconnaissance to depths of 20 m, were identified as sites with ;::.20 individual mature colonies of G. ventalina (Fig. 7). Of these, only site E, comprising ca. 1 km of coastline centered at the abandoned aloe plantation at Karpata, was found to contain fans with algal tumors. The affected population at site E consisted of ;::.22 tumor-bearing colonies; these were growing at depths of 2-8 m, and clustered closely within a radius of 25 m. Within this cluster, the incidence of visible infestation exceeded 90% of all colonies present. Tumor-bearing individuals were encountered more rarely, among unaffected colonies, at distances up to 300 m from the apparent primary center of infestation. Most of the fans of G. ventalina with visible tumors were large specimens (;::'1 m colony height), containing 2-ca. 50 nodules or plaques per fan. These growths were either spherical (2-28 mm diam.; see Fig. 1) or flat and irregular (2-4 mm height x 30 cm longest dimension), frequently forming large confluent growths extending up to 60 cm along the major branches of the colony (Morse et al.,
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