Influence of water quality and climate variables on growth of the harmful alga, Prymnesium parvum

Golden alga Prymnesium parvum Carter is a euryhaline, ichthyotoxic haptophyte (Chromista) that typically inhabits marine and estuarine habitats, but it has recently invaded brackish inland waters where it is capable of forming fish-killing blooms and cause major ecological damages. In recent years, some field studies reported new and sometimes unexpected information concerning environmental variables that associate with golden alga presence and abundance in inland waters of the USA. Important examples include (1) a biphasic association between golden alga abundance and salinity, where the association is hypothesized to be positive at low salinity and negative at high salinity; (2) a positive association between abundance and sulfate concentration; and (3) a positive association between abundance and organic nitrogen concentrations. These working hypotheses, however, are based on descriptive observations in the field and cannot be used as conclusive evidence of causal associations. There is a need, therefore, to test these field-generated hypotheses under the controlled environment of a laboratory, where only the independent variable or variables of interest are allowed to vary. A fourth question being addressed in this dissertation is whether changes in air CO2 concentration can influence growth of golden alga by providing additional carbon for fixation. Results with other algae have been inconsistent and this question has never been addressed for golden alga. To address the first working hypothesis, in Chapter 2 salinity (5–30 psu) effects on golden alga growth were determined at a standard laboratory temperature (22 °C) and one associated with natural blooms (13 °C). Studies reported a minimum initial cell density (inoculum size) requires for golden algal survival in field conditions, however, information regarding the inoculum-size effects in laboratory conditions is not available for this algal species. Inoculum-size effects were determined over a wide size range (100–100,000 cells ml-1), as it may influence golden algal growth. A strain widely distributed in the USA, UTEX-2797 was the primary study subject but another of limited distribution, UTEX-995 was used to evaluate growth responses in relation to genetic background. Variables examined were exponential growth rate (r), maximum cell density and, when inoculum size was held constant (100 cells ml-1), density at onset of exponential growth (early cell density). In UTEX-2797, maximum cell density increased as salinity increased from 5 to ~10–15 psu and declined thereafter regardless of temperature but r remained generally stable and only declined at salinity of 25–30 psu. In addition, maximum cell density correlated positively with r and early cell density, the latter also being numerically highest at salinity of 15 psu. In UTEX-995, maximum cell density and r responded similarly to changes in salinity – they remained stable at salinity of 5–10 psu and 5–15 psu, respectively, and declined at higher salinity. Also, maximum cell density correlated with r but not early cell density. Inoculum size positively and negatively influenced maximum cell density and r, respectively, in both strains and these effects were significant even when the absolute size difference was small (100 versus 1000 cells ml-1). When cultured under similar conditions, UTEX-2797 grew faster and to far higher density than UTEX-995. In conclusion, (1) UTEX-2797’s superior growth performance may explain its relatively wide distribution in the USA, (2) the biphasic growth response of UTEX-2797 to salinity variation, with peak abundance at salinity of 10–15 psu, generally mirrors golden alga abundance-salinity associations in US inland waters, and (3) early cell density – whether artificially manipulated or naturally attained – can influence UTEX-2797 bloom potential. Because of its presumed coastal/marine origin where SO42- levels are high, the relatively high SO42- concentration of its brackish inland habitats, and the sensitivity of marine chromists to sulfur deficiency, Chapter 3 examined whether golden alga growth is sensitive to SO42- concentration. Fluoride is a ubiquitous ion that has been reported at higher levels in golden alga habitat; thus, the influence of F- on growth also was examined. In low-salinity (5 psu) artificial seawater medium, overall growth was SO42--dependent up to 1000 mg l-1 using MgSO4 or Na2SO4 as source; the influence on growth rate, however, was more evident with MgSO4. Transfer from 5 to 30 psu inhibited growth when salinity was raised with NaCl but in the presence of seawater levels of SO42-, these effects were fully reversed with MgSO4 as source and only partially reversed with Na2SO4. Growth inhibition was not observed after acute transfer to 30 psu in a commercial sea salt mixture. In 5-psu medium, F- inhibited growth at all concentrations tested. These observations support the hypothesis that spatial differences in SO42- – but not F- – concentration help drive the inland distribution and growth of golden alga and also provide physiological relevance to reports of relatively high Mg2+ concentrations in golden alga habitat. At high salinity, however, the ability of sulfate to maintain growth under osmotic stress was weak and overshadowed by the importance of Mg2+. A mechanistic understanding of growth responses of golden alga to SO42-, Mg2+ and other ions at environmentally relevant levels and under different salinity scenarios will be necessary to clarify their ecophysiological and evolutionary relevance. As a mixotroph, P. parvum can utilize organic and inorganic nitrogen (N) for growth but the relative importance of each fraction when present in combination is uncertain. Some field studies have suggested there is a positive association between organic N and golden alga distribution or abundance, but experimental evidence supporting this observation is unavailable. The objective of this study is to determine if different molar ratios of inorganic to organic N affect growth of golden alga in a standard culture medium (5 psu). Sodium nitrate was used as source of inorganic N and urea and glycine as sources of organic N. Concentrations of total N (880 μM) and phosphorus (36 μM) were kept constant at F/2 levels. The inorganic:organic N ratios tested were 1:0, 0.75:0.25, 0.5:0.5, 0.75:0.25, and 0:1. Cultures were conducted under standard conditions (initial density, 100 cells ml-1; 22°C, ~6500 lux), and endpoints measured were early cell density (day 3, cells ml-1), exponential growth rate (r, day-1) and maximum cell density (cells ml-1). Growth rate was unaffected by changes in inorganic:organic N ratio. Early and maximum cell density, however, increased gradually as the fraction of organic N increased from 0 to 0.75 followed by a decrease at 1, and this pattern was stronger when using glycine as source. In conclusion, while golden alga can grow in cultures supplemented exclusively with organic or inorganic N, optimal growth occurs when both are present and the organic fraction is predominant. These findings are consistent with field observations and provide context for a better understanding of the association between nutrient stoichiometry and golden alga growth. Carbon dioxide is the primary source of carbon for photosynthetic fixation by plants and algae. Because it is highly soluble in water, changes in air CO2 concentration can lead to corresponding changes in dissolved CO2 concentration of surface waters. This has led to concerns over the potential effects of the rising air CO2 concentration on growth of harmful algae. Current knowledge suggests that these effects may be species-specific and no information is available for Prymnesium parvum, a euryhaline haptophyte that forms toxic blooms. This study determined and compared the effects on P. parvum growth by air CO2 at concentrations of 280 (pre-industrial era), 400 (current), and 670 ppm (a projected scenario by 2100). Batch cultures were conducted in two different media, Artificial Seawater Medium at salinity of 5 and Instant Ocean® at salinity of 30. Treatments were done in triplicate and experiments were conducted twice. Early (pre-exponential) growth was not affected by air CO2. Exponential growth rates were positively stimulated by air CO2 concentration in the higher but not the lower salinity medium. In both media, maximum population density was strongly and positively associated with CO2 concentration. Medium pH increased during incubation but to a similar level in both media regardless of CO2 concentration. This increase in pH is likely due to the onset of carbon limitation as cell populations neared their maximum density which could not be overcome at even the highest air CO2 concentration tested. Our results suggest that relative to pre-industrial times, current concentrations of atmospheric CO2 may already be enhancing growth of P. parvum in the field and as CO2 levels continue to rise, so may the magnitude of this effect. In conclusion, this current study results are consistent with field observations and confirmed all working hypotheses (field-generated) tested in this study, except the effects of fluoride on golden algal growth. In this study, results showed F- negatively associated with golden alga abundance, however, field study hypothesized a positive association. The results of this study are expected to provide basic and comparative information to a better understanding of the effects of environmental factors on golden alga growth.