**Seasonal Changes of Bioluminescence in Photosynthetic and Heterotrophic Dinoflagellates at San Clemente Island**

David Lapota *Space and Naval Warfare Systems Center, Pacific USA* 

## **1. Introduction**

26 Bioluminescence – Recent Advances in Oceanic Measurements and Laboratory Applications

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and chlorophyll during the late summer in the North Atlantic: Maps and a

A significant portion of bioluminescence in all oceans is produced by dinoflagellates. Numerous studies have documented the ubiquitous distribution of bioluminescent dinoflagellates in near surface waters (Seliger et al., 1961; Yentsch and Laird 1968; Tett 1971; Tett and Kelly 1973). The number of bioluminescent species and their relative abundance change temporally, with depth, and geographically. Dinoflagellates are most abundant in coastal waters and inland seas and are less abundant in the open ocean (Colebrook and Robinson, 1965; Dodge and Hart-Jones, 1977). Studies have been conducted to determine the species contributing to bioluminescence. In several studies, this involved making plankton collections, isolation and measurement of cells with a laboratory photometer to quantify the light output of several species of bioluminescent dinoflagellates (Lapota and Losee 1984; Batchelder and Swift 1989; Lapota et al., 1992a,b; Swift et al., 1995). These studies were limited to short sampling periods (days-weeks) and to specific locations. There is also evidence that dinoflagellates undergo changes in light output which may be attributable to environmental conditions. For example, cells of *Protoperidinium* spp. produce more bioluminescence when nutritional requirements were optimized in the laboratory (Buskey 1992; Latz 1993). Others have observed that the bioluminescence potential of a dinoflagellate is related to its surface area or cell volume for several species, which might be related to light and nutrient history (Seliger et al. 1969; Seliger and Biggley 1982; Swift et al. 1995; Sullivan and Swift 1995). Bioluminescence may also be a function of light, temperature, and nutrient history (Sweeney, 1981). Other data have suggested that cells of the same species in the same study display differences in bioluminescence. These observations may indicate that cells are exposed to a wide range of environmental conditions affecting light output on a short time scale such as light history, nutrient history, grazing pressure by herbivores and consequent loss of potential bioluminescent capacity (Swift et al., 1981; Sullivan and Swift, 1995).

Despite strong interest in short term process effects on dinoflagellates there have been few investigations on the seasonality of marine bioluminescence (Tett 1971; Bityukov et al. 1967; Lapota et al. 1997). Long term aspects of the development of bioluminescence are unknown for most oceans. The present study was designed to cast light on this question. A station for

Seasonal Changes of Bioluminescence in

Photosynthetic and Heterotrophic Dinoflagellates at San Clemente Island 29

Fig. 1. Bioluminescence study area within the Gulf of Santa Catalina. Boxed area identifies

Single cells in cuvettes were measured in a laboratory photometer system (Lapota et al. 1994b). This system is similar to another photometer system used in previous studies (Lapota and Losee 1984; Lapota et al. 1988, 1989, 1992) and consists of a horizontally mounted 2-inch diameter end window photomultiplier tube (PMT - RCA 8575 with an S-20 response) attached to a darkened chamber with removable opening to receive the cuvettes. Cells were stimulated to luminescence by stirring with an adjustable speed motor driving a plastic stirrer for 30 sec after which the total bioluminescence (PMT counts) was displayed. PMT counts were either logged on a notebook computer operating under Windows™ or hand recorded on data sheets. PMT dark counts were subtracted from all light output values prior to conversion to photons cell-1. The system was calibrated with aliquots of the luminescent bacterium *Vibrio harveyii* measured by the Quantalum 2000 silicon-photodiode detector (Matheson et al., 1984). The detector calibration is traceable to a luminol light

Following testing, the cells were individually placed in borosilicate vials and preserved in a 5% formalin solution for later microscopic identification to species level. Mean light output

NOTS pier where all plankton collections were made.

**2.1.1 Laboratory measurements** 

standard.

measuring bioluminescence was established in August 1993 at San Clemente Island (SCI), 100 km offshore of Southern California. Bioluminescence was measured with a moored bathyphotometer (MOORDEX) hourly through February 1996. Other environmental parameters such as nutrients, chlorophyll, and associated plankton species were measured and collected on a monthly and quarterly basis( Lapota et al. 1997). In the present study, plankton samples were collected and tested for bioluminescence on a quarterly basis to: 1) determine which dinoflagellate species were bioluminescent and 2) observe differences in light output on a seasonal basis. The latter is an important consideration because seasonal changes in bioluminescence from dinoflagellates might possibly indicate a response to regional seasonal environmental changes. These factors include the available nutrients and light for the photosynthetic species (*Ceratium, Gonyaulax1, Pyrocystis*) and the availability of diatoms and smaller algal cells consumed by the heterotrophic *Protoperidinium* dinoflagellates. Seasonal changes in light output will affect the bioluminescence light budget of all species. Published light budgets are limited and specific for limited oceanic areas (Swift et al. 1983, 1985a,b; 1995; Batchelder and Swift 1989; Lapota et al. 1988, 1989, 1992a,b; Buskey 1991) , the number of species tested, or modeled to predict bioluminescence output based on the calculated cell surface area (Seliger and Biggley 1982). This study will complement earlier laboratory work and enlarge these observations by identifying distinct seasonal differences in bioluminescence of open ocean dinoflagellates over a two year period.

#### **2. Methods and materials**

#### **2.1 Plankton collections**

Plankton samples were collected from the Naval Ordinance Test Station (NOTS) pier at SCI on a quarterly basis from the summer of 1994 through spring 1996. The pier is on the leeward side of SCI and is in a water depth of approximately 15 meters. The island shelf begins to deepen to greater than 100 meters within 150 meters of the pier (Figure 1). A MOORDEX bathyphotometer was suspended under the pier by a 1cm diameter stainless steel cable. Depending on the height of the tide, MOORDEX was usually at a depth of 2-3 meters below the sea surface (Lapota et al. 1994a). A similar MOORDEX bathyphotometer was also deployed in San Diego Bay (SDB) from 1992-1996 for comparative coastal measurements of bioluminescence (Lapota et al., 1997). Plankton samples were always collected in the late afternoon. A 20-m mesh plankton net and attached collection cup was lowered off the pier to a depth of approximately 10 meters and vertically retrieved. Samples were diluted with fresh filtered (0.45 m) seawater and transported to a field trailer and kept in a temperature-controlled incubator (Coolatron) at ambient seawater temperature. Individual cells were viewed microscopically and isolated by pipet and placed in 4-ml spectrophotometric disposable cuvettes with 3-ml of (0.45 µm) filtered seawater. All isolations were completed no later than 1 hour prior to sunset to prevent premature stimulation of bioluminescence. Bioluminescence measurements were always conducted midway into scotophase, about 8-9 hours after collection. Two consecutive nights of isolation and testing were conducted each season (i.e., winter: December 21- March 21; spring: March 22-June 21; summer: June 22-September 21; fall: September 22-December 21).

<sup>1</sup> *Gonyaulax polyedra* has recently been renamed *Lingulodinium polyedrum* Stein (Dodge 1989), but for simplicity we will use the former name because of its wider use in earlier literature.

measuring bioluminescence was established in August 1993 at San Clemente Island (SCI), 100 km offshore of Southern California. Bioluminescence was measured with a moored bathyphotometer (MOORDEX) hourly through February 1996. Other environmental parameters such as nutrients, chlorophyll, and associated plankton species were measured and collected on a monthly and quarterly basis( Lapota et al. 1997). In the present study, plankton samples were collected and tested for bioluminescence on a quarterly basis to: 1) determine which dinoflagellate species were bioluminescent and 2) observe differences in light output on a seasonal basis. The latter is an important consideration because seasonal changes in bioluminescence from dinoflagellates might possibly indicate a response to regional seasonal environmental changes. These factors include the available nutrients and light for the photosynthetic species (*Ceratium, Gonyaulax1, Pyrocystis*) and the availability of diatoms and smaller algal cells consumed by the heterotrophic *Protoperidinium* dinoflagellates. Seasonal changes in light output will affect the bioluminescence light budget of all species. Published light budgets are limited and specific for limited oceanic areas (Swift et al. 1983, 1985a,b; 1995; Batchelder and Swift 1989; Lapota et al. 1988, 1989, 1992a,b; Buskey 1991) , the number of species tested, or modeled to predict bioluminescence output based on the calculated cell surface area (Seliger and Biggley 1982). This study will complement earlier laboratory work and enlarge these observations by identifying distinct seasonal differences in bioluminescence

Plankton samples were collected from the Naval Ordinance Test Station (NOTS) pier at SCI on a quarterly basis from the summer of 1994 through spring 1996. The pier is on the leeward side of SCI and is in a water depth of approximately 15 meters. The island shelf begins to deepen to greater than 100 meters within 150 meters of the pier (Figure 1). A MOORDEX bathyphotometer was suspended under the pier by a 1cm diameter stainless steel cable. Depending on the height of the tide, MOORDEX was usually at a depth of 2-3 meters below the sea surface (Lapota et al. 1994a). A similar MOORDEX bathyphotometer was also deployed in San Diego Bay (SDB) from 1992-1996 for comparative coastal measurements of bioluminescence (Lapota et al., 1997). Plankton samples were always collected in the late afternoon. A 20-m mesh plankton net and attached collection cup was lowered off the pier to a depth of approximately 10 meters and vertically retrieved. Samples were diluted with fresh filtered (0.45 m) seawater and transported to a field trailer and kept in a temperature-controlled incubator (Coolatron) at ambient seawater temperature. Individual cells were viewed microscopically and isolated by pipet and placed in 4-ml spectrophotometric disposable cuvettes with 3-ml of (0.45 µm) filtered seawater. All isolations were completed no later than 1 hour prior to sunset to prevent premature stimulation of bioluminescence. Bioluminescence measurements were always conducted midway into scotophase, about 8-9 hours after collection. Two consecutive nights of isolation and testing were conducted each season (i.e., winter: December 21- March 21; spring: March 22-June 21; summer: June 22-September 21; fall: September 22-December 21).

<sup>1</sup> *Gonyaulax polyedra* has recently been renamed *Lingulodinium polyedrum* Stein (Dodge 1989), but for

simplicity we will use the former name because of its wider use in earlier literature.

of open ocean dinoflagellates over a two year period.

**2. Methods and materials** 

**2.1 Plankton collections** 

Fig. 1. Bioluminescence study area within the Gulf of Santa Catalina. Boxed area identifies NOTS pier where all plankton collections were made.

#### **2.1.1 Laboratory measurements**

Single cells in cuvettes were measured in a laboratory photometer system (Lapota et al. 1994b). This system is similar to another photometer system used in previous studies (Lapota and Losee 1984; Lapota et al. 1988, 1989, 1992) and consists of a horizontally mounted 2-inch diameter end window photomultiplier tube (PMT - RCA 8575 with an S-20 response) attached to a darkened chamber with removable opening to receive the cuvettes. Cells were stimulated to luminescence by stirring with an adjustable speed motor driving a plastic stirrer for 30 sec after which the total bioluminescence (PMT counts) was displayed. PMT counts were either logged on a notebook computer operating under Windows™ or hand recorded on data sheets. PMT dark counts were subtracted from all light output values prior to conversion to photons cell-1. The system was calibrated with aliquots of the luminescent bacterium *Vibrio harveyii* measured by the Quantalum 2000 silicon-photodiode detector (Matheson et al., 1984). The detector calibration is traceable to a luminol light standard.

Following testing, the cells were individually placed in borosilicate vials and preserved in a 5% formalin solution for later microscopic identification to species level. Mean light output

Seasonal Changes of Bioluminescence in

from 1992-1996.

Island from 1993-1996.

Photosynthetic and Heterotrophic Dinoflagellates at San Clemente Island 31

Fig. 2. Mean monthly bioluminescence trends at San Diego Bay and San Clemente Island

Fig. 3. The abundance of *Protoperidinium* and *Gonyaulax* dinoflagellates at San Clemente

values for each species tested for all seasons were calculated. The Student's t test for comparison of two means was used to calculate significant differences between means of cells within species for all seasons. Critical values of the Student's t distribution were calculated using n-2 degrees of freedom.

## **2.2 Nutrient and chlorophyll data**

Nitrate and Chl *a* levels were obtained from archived CalCOFI data bases from 1994 - 1996 for the Southern California Bight and were averaged along CalCOFI lines 90 and 93 which extend offshore west of San Diego to the north and south of SCI (Hayward et al. 1996). Nitrates (m L-1) and Chl *a* (g L-1) along each of the CalCOFI transit lines 93.26 to 93.45 and 90.28 to 90.53 were averaged from the surface to a depth of 50 m, seasonally (fall, winter, spring, summer), from spring 1994 through spring 1996. These data were used to calculate correlations with seasonal means of bioluminescence cell-1 of the photosynthetic dinoflagellate *Pyrocystis noctiluca* and the heterotrophic dinoflagellate *Protoperidnium pellucidum*. Nutrient data was lagged by one season to calculate correlation coefficients with mean bioluminescence cell-1. The Student t-test was used to determine significant differences among seasonal bioluminescence means for *Gonyaulax, Ceratium, Pyrocystis*, and *Protoperidinium* species.
