Microb Ecol (1984) 10:69-77
MICROBIAL ECOLOGY 9 1984 Springer-Verlag
Contribution by Symbiotically Luminous Fishes to the Occurrence and Bioluminescence of Luminous Bacteria in Seawater K e n n e t h H. Nealson, ~ Margo G. Haygood, ~ Bradley M. Tebo, ~ M a r k R o m a n , ~ Edward Miller, 2 and J o h n E. McCosker 2 ~Marine Biology Research Division, A-002, Scripps Institution of Oceanography, La Jolla, California 92093 USA; and 2Steinhart Aquarium, California Academy of Sciences, San Francisco, California 94118, USA
Abstract. Seawater samples from a variety o f locations contained viable luminous bacteria, but luminescence was not detectable although the system used to measure light was sensitive enough to measure light from a single, fully induced luminous bacterial cell. W h e n the symbiotically lum i n o u s fish Cleidopus gloriamaris was placed in a sterile aquarium, plate counts o f water samples showed an increase in luminous colony-forming units. Luminescence also increased, decreasing when the fish was removed. Light m e a s u r e m e n t s o f water samples f r o m a sterile aquarium containing Photoblepharon palpebratus, a n o t h e r symbiotically luminous fish, whose bacterial symbionts have not been cultured, showed a similar pattern o f increasing light which rapidly decreased upon removal o f the fish. These experiments suggest that symbiotically luminous fishes release brightly lum i n o u s bacteria from light organs into their e n v i r o n m e n t and may be a source o f planktonic luminous bacteria. Although planktonic luminous bacteria are generally not bright when found in seawater, water samples f r o m e n v i r o n m e n t s with populations o f symbiotically luminous fish m a y show detectable levels o f light.
Introduction Planktonic luminous bacteria are ubiquitous in seawater. T h e y can usually be isolated on agar media in n u m b e r s ranging from 102-104 per liter [7-9, 12-14, 16]. In a given e n v i r o n m e n t , m a j o r fluctuations in species composition m a y be seen on a seasonal scale [9, 10, 12, 13, 16]. T h e factors responsible for these variations are not yet completely understood, but they have been postulated to be due to the physical and chemical properties o f the e n v i r o n m e n t s studied. L u m i n o u s bacteria emit light in laboratory culture and in symbiotic associations. However, the question o f whether or not planktonic luminous bacteria emit light in situ has rarely been e x a m i n e d [ 1]. In this paper we present evidence that although planktonic luminous bacteria were ubiquitous in the environments we examined, they were not luminescent.
70
K.H. Nealson et al.
T h e s a m e species o f bacteria t h a t are f o u n d as p l a n k t o n i c f o r m s can also be isolated f r o m o n e or m o r e o f a v a r i e t y o f s y m b i o t i c a s s o c i a t i o n s (saprophytic, gut s y m b i o t i c , light o r g a n s y m b i o t i c , o r parasitic) [3, 7, 8, 10, 1 1]. I n these a s s o c i a t i o n s t h e y can a t t a i n v e r y high n u m b e r s (109 per m l o r m o r e ) a n d be visibly l u m i n o u s . W e r e p o r t here the r e l a t i o n s h i p b e t w e e n s y m b i o t i c a n d p l a n k t o n i c niches in 3 s y m b i o t i c a l l y l u m i n o u s fishes: Photoblepharon palpebratus (family A n o m alopidae), Cleidopus gloriamaris a n d Monocentrisjaponicus ( b o t h in the f a m i l y M o n o c e n t r i d a e ) [ 2 ] . T h e s e fishes o c c u p y s h a l l o w w a t e r m a r i n e habitats; the a n o m a l o p i d is a tropical species, w h e r e a s the m o n o c e n t r i d s are s u b t r o p i c a l a n d t e m p e r a t e species [5]. T h e specific s y m b i o n t o f the 2 m o n o c e n t r i d s is Vibrio fischeri [ 1 1]; t h a t o f P . palpebratus has n o t b e e n c u l t u r e d or identified. Structural studies o f the s u b o r b i t a l light o r g a n s o f P. palpebratus a n d the light organs l o c a t e d at the a n t e r i o r e n d o f the l o w e r j a w o f M . japonicus s h o w t h a t the light organs c o m m u n i c a t e w i t h the s u r r o u n d i n g s e a w a t e r via p o r e s o n the surface [4, 151. T h e r e l a t i o n s h i p b e t w e e n these a s s o c i a t e d p o p u l a t i o n s a n d the p l a n k t o n i c f o r m s is n o t k n o w n , a l t h o u g h it has b e e n suggested b o t h t h a t gut s y m b i o n t s m a y c o n t r i b u t e significantly to p o p u l a t i o n s o f l u m i n o u s bacteria in s e a w a t e r [10] a n d t h a t light o r g a n s o f certain m a r i n e fishes m a y serve as a specific source o f s o m e l u m i n o u s bacterial species [3, 14]. Fish gut tracts c o n t a i n l u m i n o u s b a c t e r i a b u t cell densities a n d relative species a b u n d a n c e m a y be quite variable. I n s o m e species o f fishes, species o f l u m i n o u s gut s y m b i o n t s h a v e b e e n s h o w n to v a r y seasonally [10]. O n the o t h e r h a n d , light o r g a n s c o n t a i n a fixed p o p u l a t i o n o f a pure culture o f l u m i n o u s b a c t e r i a w h i c h m a y p r o v i d e a c o n t i n u o u s i n p u t o f a given species to the e n v i r o n m e n t . W e h a v e f o c u s e d o u r research o n release o f b a c t e r i a f r o m light organs, a n d o u r results suggest t h a t this i n d e e d occurs. I n a d d i t i o n , o u r results suggest t h a t released s y m b i o n t s h a v e the p o tential to p r o d u c e m e a s u r e a b l e levels o f light in h a b i t a t s h e a v i l y p o p u l a t e d b y s y m b i o t i c a l l y l u m i n o u s fishes.
Materials and M e t h o d s
Collection of Samples, and Measurements ofLight and Growth of Bacteria Seawater samples were collected in a variety of ways (Niskin bottles, sterile syringes, open bottles, continuous pumping, and sterile tubes), from a number of sites and depths (Table 1), and throughout the day and night. The water was passed through a l0 micron mesh net to exclude dinoflagellates and placed in sterile glass scintillation vials that had been previously kept in the dark to minimize fluorescence. These samples, ranging from 1-20 ml, were then viewed and counted by an E.M.I. 9635 photomultiplier tube in the chamber of a modified "JRB-ATP photometer (S.A.I. Industries, Sorrento Valley, CA). In cases in which luminescence was seen, a second sample was obtained and counted and the pH then adjusted to approximately 4 by the addition of 1 M HC1, and recounted. This allowed distinction between bacterial light emission, which is characteristically sensitive to pH of less than 6 [2], and delayed fluorescence or phosphorescence of the seawater, which was common in the near-shore samples taken during the day. All samples except lhose treated with HC1 were then used for determination of bacterial populations. Two methods of enumeration and isolation of luminous bacteria were used, depending on the volume of the sample to be examined: either 0.1 ml of seawater was spread directly onto
71
Appearance of Symbiotic L u m i n o u s Bacteria in Seawater Table 1.
Measurements of bioluminescence and viable luminous bacteria pH sensitive luminescence C P M / m l a.b
Lum bact/ml
CPM/Lum bact
Field samples: Mission B a y : San Diego Scripps Pier: La Jolla Open ocean d ( > 5 mi from coast) G u l f of Calife
< < <
MICROBIAL ECOLOGY 9 1984 Springer-Verlag
Contribution by Symbiotically Luminous Fishes to the Occurrence and Bioluminescence of Luminous Bacteria in Seawater K e n n e t h H. Nealson, ~ Margo G. Haygood, ~ Bradley M. Tebo, ~ M a r k R o m a n , ~ Edward Miller, 2 and J o h n E. McCosker 2 ~Marine Biology Research Division, A-002, Scripps Institution of Oceanography, La Jolla, California 92093 USA; and 2Steinhart Aquarium, California Academy of Sciences, San Francisco, California 94118, USA
Abstract. Seawater samples from a variety o f locations contained viable luminous bacteria, but luminescence was not detectable although the system used to measure light was sensitive enough to measure light from a single, fully induced luminous bacterial cell. W h e n the symbiotically lum i n o u s fish Cleidopus gloriamaris was placed in a sterile aquarium, plate counts o f water samples showed an increase in luminous colony-forming units. Luminescence also increased, decreasing when the fish was removed. Light m e a s u r e m e n t s o f water samples f r o m a sterile aquarium containing Photoblepharon palpebratus, a n o t h e r symbiotically luminous fish, whose bacterial symbionts have not been cultured, showed a similar pattern o f increasing light which rapidly decreased upon removal o f the fish. These experiments suggest that symbiotically luminous fishes release brightly lum i n o u s bacteria from light organs into their e n v i r o n m e n t and may be a source o f planktonic luminous bacteria. Although planktonic luminous bacteria are generally not bright when found in seawater, water samples f r o m e n v i r o n m e n t s with populations o f symbiotically luminous fish m a y show detectable levels o f light.
Introduction Planktonic luminous bacteria are ubiquitous in seawater. T h e y can usually be isolated on agar media in n u m b e r s ranging from 102-104 per liter [7-9, 12-14, 16]. In a given e n v i r o n m e n t , m a j o r fluctuations in species composition m a y be seen on a seasonal scale [9, 10, 12, 13, 16]. T h e factors responsible for these variations are not yet completely understood, but they have been postulated to be due to the physical and chemical properties o f the e n v i r o n m e n t s studied. L u m i n o u s bacteria emit light in laboratory culture and in symbiotic associations. However, the question o f whether or not planktonic luminous bacteria emit light in situ has rarely been e x a m i n e d [ 1]. In this paper we present evidence that although planktonic luminous bacteria were ubiquitous in the environments we examined, they were not luminescent.
70
K.H. Nealson et al.
T h e s a m e species o f bacteria t h a t are f o u n d as p l a n k t o n i c f o r m s can also be isolated f r o m o n e or m o r e o f a v a r i e t y o f s y m b i o t i c a s s o c i a t i o n s (saprophytic, gut s y m b i o t i c , light o r g a n s y m b i o t i c , o r parasitic) [3, 7, 8, 10, 1 1]. I n these a s s o c i a t i o n s t h e y can a t t a i n v e r y high n u m b e r s (109 per m l o r m o r e ) a n d be visibly l u m i n o u s . W e r e p o r t here the r e l a t i o n s h i p b e t w e e n s y m b i o t i c a n d p l a n k t o n i c niches in 3 s y m b i o t i c a l l y l u m i n o u s fishes: Photoblepharon palpebratus (family A n o m alopidae), Cleidopus gloriamaris a n d Monocentrisjaponicus ( b o t h in the f a m i l y M o n o c e n t r i d a e ) [ 2 ] . T h e s e fishes o c c u p y s h a l l o w w a t e r m a r i n e habitats; the a n o m a l o p i d is a tropical species, w h e r e a s the m o n o c e n t r i d s are s u b t r o p i c a l a n d t e m p e r a t e species [5]. T h e specific s y m b i o n t o f the 2 m o n o c e n t r i d s is Vibrio fischeri [ 1 1]; t h a t o f P . palpebratus has n o t b e e n c u l t u r e d or identified. Structural studies o f the s u b o r b i t a l light o r g a n s o f P. palpebratus a n d the light organs l o c a t e d at the a n t e r i o r e n d o f the l o w e r j a w o f M . japonicus s h o w t h a t the light organs c o m m u n i c a t e w i t h the s u r r o u n d i n g s e a w a t e r via p o r e s o n the surface [4, 151. T h e r e l a t i o n s h i p b e t w e e n these a s s o c i a t e d p o p u l a t i o n s a n d the p l a n k t o n i c f o r m s is n o t k n o w n , a l t h o u g h it has b e e n suggested b o t h t h a t gut s y m b i o n t s m a y c o n t r i b u t e significantly to p o p u l a t i o n s o f l u m i n o u s bacteria in s e a w a t e r [10] a n d t h a t light o r g a n s o f certain m a r i n e fishes m a y serve as a specific source o f s o m e l u m i n o u s bacterial species [3, 14]. Fish gut tracts c o n t a i n l u m i n o u s b a c t e r i a b u t cell densities a n d relative species a b u n d a n c e m a y be quite variable. I n s o m e species o f fishes, species o f l u m i n o u s gut s y m b i o n t s h a v e b e e n s h o w n to v a r y seasonally [10]. O n the o t h e r h a n d , light o r g a n s c o n t a i n a fixed p o p u l a t i o n o f a pure culture o f l u m i n o u s b a c t e r i a w h i c h m a y p r o v i d e a c o n t i n u o u s i n p u t o f a given species to the e n v i r o n m e n t . W e h a v e f o c u s e d o u r research o n release o f b a c t e r i a f r o m light organs, a n d o u r results suggest t h a t this i n d e e d occurs. I n a d d i t i o n , o u r results suggest t h a t released s y m b i o n t s h a v e the p o tential to p r o d u c e m e a s u r e a b l e levels o f light in h a b i t a t s h e a v i l y p o p u l a t e d b y s y m b i o t i c a l l y l u m i n o u s fishes.
Materials and M e t h o d s
Collection of Samples, and Measurements ofLight and Growth of Bacteria Seawater samples were collected in a variety of ways (Niskin bottles, sterile syringes, open bottles, continuous pumping, and sterile tubes), from a number of sites and depths (Table 1), and throughout the day and night. The water was passed through a l0 micron mesh net to exclude dinoflagellates and placed in sterile glass scintillation vials that had been previously kept in the dark to minimize fluorescence. These samples, ranging from 1-20 ml, were then viewed and counted by an E.M.I. 9635 photomultiplier tube in the chamber of a modified "JRB-ATP photometer (S.A.I. Industries, Sorrento Valley, CA). In cases in which luminescence was seen, a second sample was obtained and counted and the pH then adjusted to approximately 4 by the addition of 1 M HC1, and recounted. This allowed distinction between bacterial light emission, which is characteristically sensitive to pH of less than 6 [2], and delayed fluorescence or phosphorescence of the seawater, which was common in the near-shore samples taken during the day. All samples except lhose treated with HC1 were then used for determination of bacterial populations. Two methods of enumeration and isolation of luminous bacteria were used, depending on the volume of the sample to be examined: either 0.1 ml of seawater was spread directly onto
71
Appearance of Symbiotic L u m i n o u s Bacteria in Seawater Table 1.
Measurements of bioluminescence and viable luminous bacteria pH sensitive luminescence C P M / m l a.b
Lum bact/ml
CPM/Lum bact
Field samples: Mission B a y : San Diego Scripps Pier: La Jolla Open ocean d ( > 5 mi from coast) G u l f of Calife
< < <
