APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 1990, p. 2436-2440 Vol. 56, No. 8 0099-2240/90/082436-05$02.00/0 Copyright C) 1990, American Society for Microbiology

Description of a Novel Symbiotic Bacterium from the Brittle Star, Amphipholis squamata MICHAEL P. LESSERt* AND RICHARD P. BLAKEMORE Department of Microbiology, University of New Hampshire, Durham, New Hampshire 03824 Received 8 November 1989/Accepted 3 June 1990

A gram-negative, marine, facultatively anaerobic bacterial isolate designated strain AS-1 was isolated from the subcuticular space of the brittle star, Amphipholis squamata. Its sensitivity to 0/129 and novobiocin, overall morphology, and biochemical characteristics and the moles percent guanine-plus-cytosine composition of its DNA (42.9 to 44.4) suggest that this isolate should be placed in the genus Vibrio. Strain AS-1 was not isolated from ambient seawater and is distinct from described Vibrio species. This symbiotic bacterium may assist its host as one of several mechanisms of nutrient acquisition during the brooding of developing embryos.

The biology of bacterium-invertebrate symbiotic associa- isopropyl alcohol for 30 s and two rinses in sterile ASW. tions has elicited considerable interest, particularly since the Logarithmic dilutions were plated on Zobell modified 2216E discoveries during the past decade of chemoautotrophic medium (ASW, 1 g of peptone liter-1, 1 g of yeast extract symbiotic bacteria associated with several invertebrate spe- liter-' [pH 7.8 to 8.4]) (29), as were samples of ambient cies in sulfide-rich habitats (4, 5). Bacterial-invertebrate seawater from the site of collection and ASW controls. All symbioses (mutualistic) have been reported from many materials and equipment were sterilized, and all procedures invertebrate taxa, examples of which include cellulolytic were performed by aseptic techniques. Plates incubated nitrogen-fixing bacteria from the wood-boring bivalves of the aerobically at 20°C were examined daily, and numbers of family Teridinidae (28), methanogenic bacteria of protozoa CFU were determined by standard methods. 2216E medium (25, 26), methanotrophic bacteria of bivalves (6, 7), and was used throughout the study unless otherwise indicated. bacterial symbionts of echinoderms (11, 15). Experimental Electron microscopy. For transmission electron micros- evidence suggests many of these bacterial endosymbionts copy, whole animals were fixed (with glutaraldehyde and provide organic carbon to their hosts (3, 4, 12), as has been osmium tetroxide) by the method of Walker (27) and stained recently shown for the gutless bivalves Solemya velum and with uranyl acetate and lead citrate. Thin sections were Solemya reidi (8, 13). examined with a JEOL 200S transmission electron micro- Amphipholis squamata (Echinodermata: Ophiuroidea) is a scope operated at 80 kV. Bacterial isolates from A. squa- small (3- to 5-mm adult disk diameter), viviparous, and mata were fixed, negatively stained with uranyl acetate, and hermaphroditic brittle star that has symbiotic bacteria asso- examined as described above. ciated with its protective cuticle and the epithelial cells DNA extraction and base composition. The moles percent which secrete the cuticle (Fig. 1) (27a). Holland and Nealson guanine-plus-cytosine (G+C) content of DNA, extracted as (17) have shown by electron microscopy the presence of described by Marmur (22), was determined by the method of bacteria associated with cuticle-secreting epithelial cells in Marmur all five classes of echinoderms, but no isolation or charac- and Doty (23). Standards included DNA extracted terization of these bacteria has been done. We present here from Pseudomonas aeruginosa (ATCC 10145), Vibrio fis- a preliminary description of a gram-negative, facultatively cheri (ATCC 7744) (Photobacteriumfischeri) (19), and Esch- anaerobic, symbiotic bacterium isolated from the subcutic- erichia coli (ATCC 12435) which have moles percent G+C ular space ofA. squamata. This bacterium, designated strain values of 67, 41, and 51, respectively. AS-1, appears to be an undescribed Vibrio species. On the Temperature range of growth. The range of temperatures basis of the strong specific binding and localization of both supporting growth was determined in 250-ml Erlenmeyer the isolated bacterium and bacteria in situ with polyclonal flasks, each containing 125 ml of 2216E broth prepared with antibodies in an immunoperoxidase procedure (27a), it is 75% ASW and incubated at 4, 20, 30, 37, or 40°C for 14 days. likely that only strain AS-1 is present in the subcuticular Media were inoculated with 3 ml of a culture growing space. A description of the cellular interactions of the host exponentially at 20°C. Results were scored as positive or and negative on the basis of the presence of turbidity, which symbiont is presented elsewhere (27a). indicated growth in a particular medium. MATERIALS AND METHODS Salinity range of growth. The range of salinities supporting growth and the requirement for Na+ were determined in a Isolation and culture conditions. Freshly collected brittle manner similar to that of the temperature range. 2216E broth stars were maintained at 1 to 20°C, depending on the season was prepared with sterile distilled water (as opposed to of collection. Within 24 h of collection, whole, intact animals ASW) and adjusted with NaCl or KCI to final concentrations were homogenized in sterile 75% artificial seawater (ASW) of 0.0, 0.5, 1.0, 2.0, 2.5, 3.0, 3.5, 4.0, 6.0, 8.0, and 10.0% (18) with a glass tissue grinder after immersion in 70% (wt/vol). Antibiotic and inhibitor sensitivities. A saturated solution * Corresponding author. (10 ,ul) of 2,4-diamino-6,7-isopropylpteridine phosphate t Present address: Bigelow Laboratory for Ocean Sciences, (vibriostatic agent 0/129) in water was prepared on a sterile McKown Point, West Boothbay Harbor, ME 04575. paper disk and then placed in the center of an agar plate after 2436 VOL. 56, 1990 ECHINODERM-SYMBIOTIC BACTERIUM 2437

FIG. 1. Thin section of the bursal area ofA. squamata showing the relationship of numerous endosymbiotic bacteria (B) to the bursa (Bu), cuticle (C), and epithelial cells (E). Transmission electron microscopy was used for this figure. Bar = 3.0 ,.m. the plate was covered with cells of strain AS-1 from an RESULTS exponentially growing culture. Sterile antibiotic sensitivity disks (Difco Laboratories, Detroit, Mich.) were used to Morphology and ultrastructure. Translucent to opaque 1- determine novobiocin sensitivity (5 ,ug per disk). to 2-mm colonies of a single morphological type appeared The sensitivity to 1% (wt/vol) oxgall bile and 0.008% after 10 to 14 days on plates streaked with serial dilutions of (wt/vol) KCN was determined on 2216E agar. Results were homogenized whole animals. These colonies were mucoid, scored as positive or negative growth after 14 days (24). smooth, and convex with entire borders. Gram stains of the Biochemical tests. Preliminary biochemical tests were con- cells from these colonies revealed rod-shaped and comma- ducted with the API 20E system (Analytab Products, Plain- shaped gram-negative organisms averaging 0.7 by 1.6 ,um. view, N.Y.) inoculated as recommended by MacDonell et al. Spheroplasts formed in late-stationary-phase cultures as (20) for marine isolates. These tests were repeated on all reported for other species of the genus Vibrio (1, 2). isolates by standard techniques modified for marine bacteria The number of CFU per entire adult brittle star ranged (21, 24). Hugh-Liefson medium was made with 75% ASW from 5 x 106 to 9 x 106. All ASW controls produced no and supplemented with 1% glucose or mannitol, with bro- colonies, and colonies of similar characteristics, although mothymol blue as an indicator of fermentative metabolism. repeatedly sought, were never isolated from ambient seawa- The presence of cytochrome oxidase, catalase, coagulase, ter. Isolates of the same colony type were obtained in 10 urease, lipase, phosphatase, and sulfatase were determined separate isolation attempts, and all isolates were positive according to the methods of Smibert and Krieg (24) and with each other and the bacteria in situ by an immunoper- MacFaddin (21). oxidase assay (27a). Bacteria isolated from the brittle star Luminescence was examined by inoculating photobacte- homogenate were used as an antigen to produce polyclonal rium broth (Difco) under aerobic conditions at 20°C. Growth antibodies. These antibodies were then used as the primary and luminescence were determined daily for 14 days with V. antibody in the immunoperoxidase testing. Walker and fischeri (ATCC 7744) as a control. Lesser (27a) used tissue sections of the brittle star and the Hydrolysis of sodium hippurate, 0.1% (wt/vol) esculin, cultured bacteria in their assays. Appropriate controls and soluble starch, 12% gelatin, and cellulose was determined preabsorption of the antisera with E. coli to remove antibod- after 14 days of growth. Hydrogen sulfide production from ies against common antigens of the members of the family 0.03% sodium thiosulfate, indole production from 0.1% Enterobacteriaceae were used before any testing. These tryptophan, and acetoin production from glucose were de- isolates were maintained for more than a year on 2216E agar, termined after 14 days of growth (24). with the only detectable change being an increase in the Motility was determined by dark-field microscopy (hang- growth rate (M. P. Lesser, M.S. thesis, University of New ing drops) and motility test medium (Difco) made with 75% Hampshire, Durham, 1985). Occasional isolates from the A. ASW. squamata homogenate, morphologically resembling cells of Nitrate reduction was determined aerobically and anaer- the genus Flavobacterium, composed less than 1% of the obically with nitrate broth and nitrate agar (Difco) made with total colony counts in any isolation of the symbiotic bacteria 75% ASW. in which they appeared. 2438 LESSER AND BLAKEMORE APPL. ENVIRON. MICROBIOL.

FIG. 2. (a) Thin section ofA. squamata showing a subcuticular symbiotic bacterium within a pocket formed by the epithelial cell. (b) Thin (cross) section of a symbiotic bacterium showing the features of the gram-negative envelope. Abbreviations: C, cuticle; CM, cytoplasmic membrane; OM, outer membrane; PR, periplasmic region; PHB, poly-p-hydroxybutyrate storage body; G, glycocalyx. Transmission electron microscopy was used for this figure. Bars = 0.5 ,um.

The symbiotic bacteria possessed a typical gram-negative mm or more in diameter. Strain AS-1 was sensitive to oxgall cell envelope (9), except for the presence of additional bile and to KCN, as evidenced by its failure to grow after 14 electron-dense material within the periplasmic region (Fig. days with either substance in the medium. 2b). Cytoplasmic inclusions bounded by a single electron- Biochemical tests. Of the carbon sources tested, only dense membrane are believed to be poly-p-hydroxybutyrate glucose and mannitol supported aerobic and anaerobic storage bodies (Fig. 2a). These inclusions also stained posi- tively with Sudan black B (Lesser, M.S. thesis). An exten- sive extracellular glycocalyx (Fig. 2b) was present and is presumably used to anchor the symbiotic bacteria (10) in pockets formed by the brittle star epithelial cells (Fig. 1). Figure 1 shows the lining of a bursal chamber, in which the area labeled Bu is an open space created by the invagination of the cuticle-covered epidermis. The space has access to *i seawater which is constantly replaced by ciliary action at the opening of the chamber and has the same orientation as the brittle star mouth (towards the substratum). The entire 1,~ t epidermal layer of the brittle star, which is continuous with all 10 of the bursal chambers, has the same morphology, including the presence of endosymbiotic bacteria. The glycocalyx stained positively with ruthenium red, indicating that it is composed of acidic mucopolysaccharides (Lesser, M.S. thesis). Negatively stained symbiotic bacteria grown in culture (broth and agar) were shown by electron microscopy to possess a single polar flagellum (Fig. 3) with many cells exhibiting tubular appendages, as reported for other marine Vibrio species (Fig. 3) (1, 2). DNA base composition. The moles percent G+C content of strain AS-1 DNA (Tm method) ranged from 42.9 to 44.4. TA Temperature and salinity range. Strain AS-1 grew at 4 and .~~~~ 20°C. No growth occurred at 37 or 40°C. Growth at 30°C was suboptimal, and this temperature may be the upper limit for this strain. Strain AS-1 grew in 0.5, 1.0, 2.0, 2.5, 3.0, and 3.5% NaCl, while suboptimal growth occurred in 2.0, 2.5, FIG. 3. Negatively stained symbiotic bacterium from an expo- 3.0, and 3.5% KCl. nential-phase culture. Abbreviations: F, flagellum; TA, tubular Sensitivities. Strain AS-1 was sensitive to the vibriostatic appendage. Transmission electron microscopy was used for this agent 0/129 and to novobiocin, with zones of inhibition 20 figure. Bar = 0.5 Lrm. VOL. 56, 1990 ECHINODERM-SYMBIOTIC BACTERIUM 2439

TABLE 1. Biochemical and physiological characteristics of strain AS-1 Test Resulta Test Result'

Oxidation or fermentation...... F ONPGb . Gas from glucose ...... Rhamnose. Catalase ...... + Sorbitol. Oxidase ...... + Sucrose. Indole ...... Arginine (ADH, API). Methyl red...... Lysine (LDC, API) . Voges-Proskauer...... Ornithine (ODC, API). Growth in: Sensitivity to: 0% (NaCl or KCl) ...... -,- 0/129.+ 0.5% (NaCl or KCl)...... +, - Oxgall bile.+ 1.0% (NaCl or KCl)...... +, - KCN .+ 2.0% (NaCl or KCl)...... + , Novobiocin.+ 2.5% (NaCl or KCl)...... +, ± 3.0% (NaCl or KCl)...... + , ± Hydrolysis of: 3.5% (NaCl or KCl)...... +,.+ Esculin. 4.0% (NaCl or KCl)...... , - Gelatin. 6.0% (NaCl or KCl)...... , - Starch. 8.0% (NaCl or KCl)...... , - Hippurate. 10.0% (NaCl or KCl) ...... , - Cellulose . Growth at: 4-200C ...... + Phosphatase. 300C ...... + Urease. 370C ...... -DNase. Lipase.

Growth on citrate ...... -Sulfatase . H2S production. Acid production from: Luminescence . Amygdalin ...... Motility..+ Glucose ...... + Tryptophan deaminase. Inositol . Mannitol...... + Nitrate reductase Melibiose ...... -rAerobic. Arabinose ...... Anaerobic..+

Guanine plus cytosine (mol%) ...... 42.9-44.4

a F, Fermentative; +, positive; -, negative; +, suboptimal. b ONPG, o-Nitrophenyl-,-D-galactopyranoside. growth. The isolate requires sodium for growth, which is developing embryos, as observed by electron microscopy. shown by the absence of growth without sodium and poor Cells of bacterial strain AS-1 appear identical to these growth when only potassium was supplied. Cultures reduced endosymbionts. Previous work using antibodies produced nitrate anaerobically but not aerobically. The remaining against the isolates showed a strong positive immunoper- results of biochemical and physiological tests are listed in oxidase reaction (27a) with both strain AS-1 cells and Table 1. bacteria in situ, supporting the observations described above. DISCUSSION Available evidence does suggest that the endosymbiotic in the of Strain AS-1 is a gram-negative, marine, facultatively an- bacteria may be important developmental biology aerobic, motile, curved, and rod-shaped bacterium. It is the brooding brittle star. Embryos developing within the oxidase positive and susceptible to the vibriostatic agent bursal chambers, which are essentially chambers formed by 0/129. The G+C content is 42.9 to 44.4 mol%. Growth is the invagination of the cuticle and epithelial cells and are oxidative or fermentative, and the organism denitrifies continuous with the entire animal, do not have sufficient yolk anaerobically. These characteristics, together with the de- material in the oocyte to grow and differentiate through scribed morphology and biochemical reactions, tentatively complete metamorphosis without additional nutrients (27a). place this bacterium in the genus Vibrio. Phenotypically, Some of these essential nutrients could be obtained by AS-1 resembles a Vibrio species more closely than it does a transdermal uptake of dissolved organic matter by the em- Photobacterium species (2). Additional tests are needed bryonic cells of A. squamata, as suggested by previous work before a specific epithet is assigned. (14). Additionally, at least twice during their residence in the Results of direct examination by electron microscopy bursa, bacteria from the subcuticular space are transferred suggest that a dividing population of bacteria normally from the adult to the embryo (27a). This vertical transmis- resides in the subcuticular space of healthy specimens of A. sion of bacteria has been shown in at least one other squamata, and it appears that some of these bacteria may be invertebrate-bacterium symbiosis (16). The bacterial endo- directly utilized by the epithelial cells (phagocytized) of symbionts of A. squamata could represent another mecha- 2440 LESSER AND BLAKEMORE APPL. ENVIRON. 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