**4. Discussion**

The data agree that dinoflagellate bioluminescence has a marked seasonality in the open ocean which is affected by regional environmental events such as upwelling and rainstorms resulting in enhanced terrestrial runoff. Other laboratory observations support the view that nutritional requirements are important in determining bioluminescent capacity (Sweeney 1971). For example, *Protoperidinium* dinoflagellates underwent increases in bioluminescence potential when fed to excess with diatoms (Buskey et al., 1992; Latz 1993) In an earlier study, when the heterotroph *Noctiluca miliaris* was fed with the flagellate *Dunaliella* more light was emitted than from unfed *Noctiluca* (Sweeney 1981). It was also reported that a strain of this dinoflagellate carrying a photosynthetic algal symbiont produced bioluminescence which was proportional to the light intensity at which the symbiont was grown, suggesting a nutritive contribution by the algal symbiont (Sweeney 1981; Sullivan and Swift 1995). Certainly other observations support the view that nutritional requirements are important in determining bioluminescent capacity (Sweeney 1971). Laboratory investigations have also shown that increased irradiance elevates photosynthesis with consequent increased bioluminescence (Sweeney et al., 1959; Sweeney 1981; Swift et al., 1981; Sullivan and Swift 1995).

When cultures of *Gonyaulax polyedra* were maintained in artificial seawater media for periods of up to 37 days, mean bioluminescence decreased by almost a factor of 10 when compared to cells after 5-13 days in culture (Sweeney 1981). Because cell numbers increased throughout the study (from 6,300 cells ml-1 to 21,830 cells ml-1), auto-toxicity is not a likely explanation of this significant decrease in bioluminescence, leaving nutrient limitation a possibility. This was tested by Sweeney in the same report with the finding that nitrates and phosphates appeared to enhance cell growth, but not bioluminescence capacity, while only trace levels of iron sequestrine supported maximum bioluminescence and cell growth. At higher concentrations, iron sequestrine appeared to reduce the bioluminescent capacity and cell division in *G. polyedra* (Sweeney 1981). The bioavailability of nutrients and trace metals are often not addressed with respect to impact on different physiological mechanisms (cell division *vs* bioluminescent capacity) within the same cell. Particularly in coastal waters environmental contaminants might complicate interpretation of nutrient effects. Thus bioluminescence enhancement has been observed in toxicity studies using *G. polyedra* incubated for up to 4 days with bay sediment pore waters. Ammonia is commonly found in sediment pore waters and levels of 200-400 µg L-1 have been observed to increase light output 3-4 times above controls. The data suggest that the organism may be responding to a readily available increased source of nitrogen, possibly an example of hormesis (Unpublished data, Lapota and Liu 1997).

In the present study, we have observed seasonal trends in nitrates, Chl *a*, and bioluminescence in numerous species of bioluminescent dinoflagellates. Maximum bioluminescence in *Protoperidinium pellucidum* was observed in winter '95 and in fall '96

Seasonal and yearly differences of bioluminescence in *Gonyaulax polyedra* and *Ceratium fusus* were also observed (Table 8, 10). While both data sets are incomplete with respect to a continuous record, the data do show a maximum bioluminescence in fall '94 for *C. fusus* and

The data agree that dinoflagellate bioluminescence has a marked seasonality in the open ocean which is affected by regional environmental events such as upwelling and rainstorms resulting in enhanced terrestrial runoff. Other laboratory observations support the view that nutritional requirements are important in determining bioluminescent capacity (Sweeney 1971). For example, *Protoperidinium* dinoflagellates underwent increases in bioluminescence potential when fed to excess with diatoms (Buskey et al., 1992; Latz 1993) In an earlier study, when the heterotroph *Noctiluca miliaris* was fed with the flagellate *Dunaliella* more light was emitted than from unfed *Noctiluca* (Sweeney 1981). It was also reported that a strain of this dinoflagellate carrying a photosynthetic algal symbiont produced bioluminescence which was proportional to the light intensity at which the symbiont was grown, suggesting a nutritive contribution by the algal symbiont (Sweeney 1981; Sullivan and Swift 1995). Certainly other observations support the view that nutritional requirements are important in determining bioluminescent capacity (Sweeney 1971). Laboratory investigations have also shown that increased irradiance elevates photosynthesis with consequent increased bioluminescence (Sweeney et al., 1959; Sweeney 1981; Swift et al.,

When cultures of *Gonyaulax polyedra* were maintained in artificial seawater media for periods of up to 37 days, mean bioluminescence decreased by almost a factor of 10 when compared to cells after 5-13 days in culture (Sweeney 1981). Because cell numbers increased throughout the study (from 6,300 cells ml-1 to 21,830 cells ml-1), auto-toxicity is not a likely explanation of this significant decrease in bioluminescence, leaving nutrient limitation a possibility. This was tested by Sweeney in the same report with the finding that nitrates and phosphates appeared to enhance cell growth, but not bioluminescence capacity, while only trace levels of iron sequestrine supported maximum bioluminescence and cell growth. At higher concentrations, iron sequestrine appeared to reduce the bioluminescent capacity and cell division in *G. polyedra* (Sweeney 1981). The bioavailability of nutrients and trace metals are often not addressed with respect to impact on different physiological mechanisms (cell division *vs* bioluminescent capacity) within the same cell. Particularly in coastal waters environmental contaminants might complicate interpretation of nutrient effects. Thus bioluminescence enhancement has been observed in toxicity studies using *G. polyedra* incubated for up to 4 days with bay sediment pore waters. Ammonia is commonly found in sediment pore waters and levels of 200-400 µg L-1 have been observed to increase light output 3-4 times above controls. The data suggest that the organism may be responding to a readily available increased source of nitrogen, possibly an example of hormesis

In the present study, we have observed seasonal trends in nitrates, Chl *a*, and bioluminescence in numerous species of bioluminescent dinoflagellates. Maximum bioluminescence in *Protoperidinium pellucidum* was observed in winter '95 and in fall '96

differences in bioluminescence in *G. polyedra* between winter '95 and winter '96.

**4. Discussion** 

1981; Sullivan and Swift 1995).

(Unpublished data, Lapota and Liu 1997).

which might be explained by the availability in the diet of diatoms and *Gonyaulax polyedra* (Figure 7a).

 Increased levels of Chl *a* were measured in the winter and spring '95 and were strongly correlated with increased *P. pellucidum* bioluminescence. Species of *Protoperidinium* are known to graze on *G. polyedra* in laboratory studies (Buskey et al., 1992; Latz and Jeong 1993; Jeong and Latz 1994). Latz (1993) demonstrated the maintenance of *Protoperidinium divergens* growth, survival, and bioluminescence capacity when grazing on a variety of dinoflagellates, but found maximum bioluminescence when the diet was solely *G. polyedra*. The winter '95 period within the Southern California Bight was characterized by an extensive red tide and extended from Santa Barbara south to San Diego and west to San Clemente Island*. G. polyedra* was the principal dinoflagellate present, reaching concentrations of approximately 16,000 cells l-1 in January 1995, although increases in *Protoperidinium* spp. were also observed (Lapota et al., 1997). Heavy rainfall was recorded during this winter period (17-18 inches, as compared with the norm of 5-10 inches) and consequently extensive runoff was observed along the entire southern California coast. Total bioluminescence (photons ml-1 year-1) was positively correlated with rainfall (inches year-1) for a 4 year period in San Diego Bay (1992-1996) (Lapota et al., 1997). Nitrates and trace metals are carried off from land with the runoff into surface waters (Dugdale and Goering 1967). Thus, the *G. polyedra* red tide was probably triggered by extensive runoff including nutrients such as nitrates from this "wet" year which in turn stimulated growth of phytoplankton grazed by *Protoperidinium pellucidum* and other *Protoperidinium* species. Increases in bioluminescence were observed in more than 60% of all rain events from 1992- 1996 in San Diego Bay (Lapota et al., 1997). Others have observed these sudden blooms and they often occur in spring or summer following heavy rains that produce nutrient-rich land runoff (Eppley, 1986). The reason for a fall '95 peak in *P. pellucidum* bioluminescence is unknown but may be due to grazing by *P. pellucidum* on lower numbers of *G. polyedra* and other algal cells. Upwelling and nitrate levels (Figure 8b) were greatest during the summer months and could result in a later increase in photosynthetic biomass in the fall. However, Chl *a* levels were actually low during the period when bioluminescence was high and may indicate previous grazing by *P. pellucidum.* Seasonal mean Chl *a* and mean bioluminescence cell-1 were strongly correlated (r = 0.962; p < 0.02) for 1994-1995 which may suggest that as Chl *a* levels increased, so did bioluminescence cell-1 (Figure 7c). These field measurements support previous laboratory studies (Buskey et al., 1992; Latz and Jeong 1993).

 Both nitrates and mean bioluminescence cell-1 in *Pyrocystis noctiluca* show similar trends temporally and in magnitude (Figures 7b, 7c). Peak levels of nitrates were found in the summer months followed by increases in bioluminescence during the fall months. Nitrate levels were greater in summer '95 than in summer '94. Peak bioluminescence was also greater in fall '95 than in fall '94. Lagging data comparisons + 1 season for nitrates produced a strong correlation with bioluminescence for the entire 2 year period (r = 0.859; p < 0.02). That is, lower nitrate levels measured in 1994 correlated with lower bioluminescence in 1994 while greater nitrate levels correlated with an increased bioluminescence (Figure 7c). Peak bioluminescence was also observed to occur in the fall for both years (Figure 7d). The effect of nitrate on bioluminescent capacity within photosynthetic dinoflagellates is unclear, but perhaps may be related to the overall health of the cell and how *Pyrocystis, Gonyaulax* spp. and *Ceratium* partition their metabolic resources when nitrate satiated. Others have

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observed the photosynthesis-irradiance relation to bioluminescence capacity. That is, cells grown at higher irradiance levels produce more photosynthetic products which may be diverted to the bioluminescence system (Sweeney et al., 1959; Sweeney 1981; Swift et al., 1981; Sullivan and Swift 1995). It is very possible that increased levels of nutrients from upwelling and storm runoff events may override diminished irradiance levels found during the fall and winter months to explain maximum bioluminescence observed in these species.
