The Journal of Published by the International Society of Eukaryotic Microbiology Protistologists
Journal of Eukaryotic Microbiology ISSN 1066-5234
ORIGINAL ARTICLE Inducible Mixotrophy in the Dinoflagellate Prorocentrum minimum
Matthew D. Johnson
Woods Hole Oceanographic Institution, 266 Woods Hole Road, Woods Hole, Massachusetts, 02543
Keywords ABSTRACT Alveolate; cryptophyte; grazing; nutrient starvation; phagotrophic phototroph; stress; Prorocentrum minimum is a neritic dinoflagellate that forms seasonal blooms Teleaulax amphioxeia. and red tides in estuarine ecosystems. While known to be mixotrophic, previ- ous attempts to document feeding on algal prey have yielded low grazing Correspondence rates. In this study, growth and ingestion rates of P. minimum were measured M.D. Johnson, Biology Department, Woods as a function of nitrogen (-N) and phosphorous (-P) starvation. A P. minimum Hole Oceanographic Institution, 266 Woods isolate from Chesapeake Bay was found to ingest cryptophyte prey when in Hole Road, Woods Hole, MA 02543, USA stationary phase and when starved of N or P. Prorocentrum minimum ingested Telephone number: +1(508) 289-2584; two strains of Teleaulax amphioxeia at higher rates than six other cryptophyte FAX number: +1(508) 289-457-2076; species. In all cases -P treatments resulted in the highest grazing. Ingestion e-mail: [email protected] rates of -P cells on T. amphioxeia saturated at ~5 prey per predator per day, while ingestion by -N cells saturated at 1 prey per predator per day. In the Received: 22 August 2014; revised 12 presence of prey, -P treated cells reached a maximum mixotrophic growth rate November 2014; accepted November 12, 1 1 (lmax) of 0.5 d , while -N cells had a lmax of 0.18 d . Calculations of 2014. ingested C, N, and P due to feeding on T. amphioxeia revealed that phagotro- phy can be an important source of all three elements. While P. minimum is a doi:10.1111/jeu.12198 proficient phototroph, inducible phagotrophy is an important nutritional source for this dinoflagellate.
MIXOTROPHY is a widely practiced nutritional mode for 1998; Stoecker et al. 2009). Phagotrophy is an ancestral marine protists that combines phagotrophic heterotrophy trait that persists in all eukaryotic supergroups, and con- and photoautotrophy (Jones 1994; Sanders 1991; Stoecker stitutive mixotrophy has been documented within the Stra- 1998). The important ecological role of mixotrophy in pro- menopiles (e.g. chrysophytes), Alveolates (dinoflagellates), viding access to limiting nutrients has long been recog- Haptophytes, Archaeplastids (green algae), and Hacrobians nized (Bird and Kalff 1986; Caron et al. 1990; Keller et al. (cryptophytes). The retention of this trait among plastid- 1994; Nygaard and Tobiesen 1993). In oligotrophic ocean containing protists in marine foodwebs is particularly con- regions, where micro and/or macronutrients are chronically spicuous within the dinoflagellates (Jeong et al. 2010). limiting, bacterivory by mixotrophic flagellates has been Among constitutive mixotrophs, phagotrophy can be shown to be ubiquitous, shaping both the diversity and broadly categorized as being an inducible or persistent productivity of eukaryotic phytoplankton (Hartmann et al. trait, and this phenomenology of feeding reflects its physi- 2012). In coastal ecosystems, mixotrophy can be impor- ological role as either a source for limiting nutrients or as tant for both maximizing growth potential of certain spe- a major nutritional source for maximizing growth. Photo- cies (Li et al. 1999), or as a nutrient source when synthetic and heterotrophic metabolism in mixotrophs is dissolved nutrients are limiting (Smalley et al. 2003). rarely balanced (Flynn and Mitra 2009; Stoecker 1998), Mixotrophy may be divided into two major classes: (1) and the relative importance of each process varies by spe- the phagotrophic phototrophs or constitutive mixotrophs, cies due to factors such as their photosynthetic growth which possess intrinsic plastids and feed to supplement efficiency (Adolf et al. 2006a; Skovgaard et al. 2000) and their nutrition, and (2) photosynthetic protozoa, acquired need for “growth factors” (Skovgaard 2000). phototrophs, or nonconstitutive mixotrophs, which lack The dinoflagellate Prorocentrum minimum has been docu- their own plastids but sequester them from algal prey or mented to form annual blooms in numerous coastal and host endosymbionts (Flynn and Mitra 2009; Stoecker estuarine ecosystems (Glibert et al. 2012; Heil et al. 2005).
© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 431–443 431 P. minimum Mixotrophy Johnson
In Chesapeake Bay P. minimum is transported annually from extracting overnight in 90% acetone at 20 °C, and reading the mouth of the Bay during winter, to the upper bay the sample on a TD-700 (Turner Designs, Sunnyvale, CA) (~240 km) during spring (Tyler and Seliger 1978). Blooms of fluorometer. Photophysiology was evaluated using a fluo- P. minimum in Chesapeake Bay are most frequent between rescence induction and relaxation (FIRe) fluorometer sys- April and May, between temperatures of 12 and 28 °Cand tem (Satlantic, Halifax, NS, Canada). Dark-adapted (15 min) salinities of 4.5–12.8 PSU, and can reach concentrations in samples were put into a cylindrical 1 cm path length cuv- excess of 105 cells/ml (Tango et al. 2005). Nuisance blooms ette and placed in the FIRe fluorometer, where a blue of P. minimum have become more persistent in many tem- (450 nm) LED source was used to analyze samples. This perate estuaries in response to coastal eutrophication (Gli- consisted of a single turnover flash excitation of 80 lsto bert et al. 2012; Heil et al. 2005; Heisler et al. 2008; saturate photosystem II (PSII) and to push fluorescence to a Hodgkiss and Ho 1997; Paerl 1997). The persistence of these maximum, followed by a relaxation sequence that consisted blooms, under high N:P ratios, relative to Redfield propor- of 40 pulses of weak modulated light separated by 60 ls tions, points to flexible nutrient acquisition pathways (Glibert intervals. All curves were fitted using the software FIREP- et al. 2012). In P. minimum these pathways include mixotro- RO (v.1.20, Satlantic, Inc). A summary of how this method- phy (Stoecker et al. 1997), expression of alkaline phospha- ology works and how various physiological parameters are tase during P-starvation (Dyhrman and Palenik 1999), and determined may be found elsewhere (Kolber et al. 1998). ectocellular aminopeptidases that cleave amino acids from Cellular carbon (C), nitrogen (N), and phosphorous (P) dissolved organic matter (Salerno and Stoecker 2009; Stoec- concentrations were determined by filtering 10–30 ml of ker and Gustafson 2003). While previous studies have docu- cells onto a pre-combusted Whatman GF/F filter, for CHN mented mixotrophy in P. minimum in field (Stoecker 1998) and particulate P (PP) analysis. Samples were analyzed and laboratory (Jeong et al. 2005; Salerno 2005) conditions, using a CE-440 Elemental Autoanalyzer (Exeter Analytical details of their grazing kinetics and how feeding relates to Inc., Chelmsford, MA) and a Technicon AutoAnalyzer II nutrient starvation is lacking. The aims of this study were to (SEAL Analytical, Mequon, WI) for CHN and PP analyses, determine: (1) the optimal cryptophyte prey for P. minimum, respectively, at the Analytical Services Department of Uni- (2) the role of inorganic N and P starvation in inducing mixo- versity of Maryland, Center for Environmental Science, trophic feeding, and (3) to determine grazing rates on crypto- Horn Point Laboratory. Cell dimensions for acid Lugol’s phyte prey and its impact on growth of P. minimum. preserved cryptophyte prey were determined using a Ze- iss Axioscope and correcting for the average cell volume shrinkage (Montagnes et al. 1994) MATERIALS AND METHODS
Algal cultures Experimental conditions Prorocentrum minimum (CBJR10) was isolated by MDJ Nutrient starvation was induced by transferring log-phase from the mouth of the James River, Chesapeake Bay, dur- P. minimum cells into f/2 (-Si) media without either phos- ing a cruise in October of 2011. Teleaulax amphioxeia phorous (-P treatment) or nitrate (-N treatment), and allow- strains GCEP01 and GCEP02 were isolated from Eel Pond, ing cells to starve under batch culture conditions. Falmouth, MA, in May 2008 by Dr. Mengmeng Tong, Assessment of P. minimum grazing on various crypto- Hanusia phi (CCMP 325), Hemiselis andersenii (CCMP phyte species was determined by adding nutrient replete 439), Guillardia theta (CCMP 2712), Proteomonas sulcata (NR), -P (starved 2 wk), or -N (starved 2 wk) P. minimum (CCMP 704), and Rhodomonas salina (CCMP 757) were to 24-well polystyrene plates in filtered seawater at obtained from the National Center for Marine Algae and 1 9 103 cells/ml, and adding each cryptophyte prey at Microbiota (formally CCMP), and Storeatula major (SM or 1 9 104 cells/ml. After 24 h, 1 ml was removed and fixed g) was isolated from Chesapeake Bay by Dr. Allen Lewitus with 1% gluteraldehyde (Sigma Chemicals, St. Louis, MO) (Table 1). All cultures were maintained in batch at 18 °C, for at least 1 h at 4 °C, filtered onto a black 2.0 lm poly- using f/2 medium (-Si) (Guillard and Ryther 1962) added to carbonate filter under low pressure, mounted on a micro- seawater (35 PSU), 0.2 lm-filtered and autoclaved (FSW), scope slide with fluorescence-grade immersion oil, and collected from Martha’s Vineyard Sound, MA. Irradiance enumerated using a Zeiss AxioScope A1 under blue (Ex: was measured using a QMSW-SS light meter (Spectrum BP 450-490, BS FT 510; Em: LP 515 nm) and green (Ex: Technologies, Inc. Aurora, IL), and maintained at 14:10 h BP 546/12, BS FT 560; Em: BP 575–640 nm) fluorescent light-dark cycle, at 50 lmol photons/m2/s. light. Frequency and number of phycoerythrin-containing food vacuoles in feeding cells, which appear as circular orange bodies, were enumerated. For all prey treatments, Cellular attributes and enumeration at least 300 P. minimum cells were enumerated. A Cell concentrations were obtained using an Accuri C6 (BD representative micrograph of a P. minimum cell with T. Biosciences, San Jose, CA) flow cytometer, equipped with amphioxeia-containing food vacuoles was obtained using a laser excitation at 488 and 640 nm and emission at 533/30, Zeiss laser-scanning microscope (LSM) 700 fitted with a 585/40, and > 670 nm, or a Zeiss Axioscope (Zeiss, C-apochromat 40x/1.20-objective (Fig. 1). For this image Thornwood, NJ). Chlorophyll a (chl a) was determined by cells were fixed (as above) and stained with 1 lM of the filtering 10–30 ml of culture into a Whatman GF/C filter, nucleic acid dye 40,6-diamidino-2-phenylindole (DAPI;
© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists 432 Journal of Eukaryotic Microbiology 2015, 62, 431–443 Johnson P. minimum Mixotrophy
Table 1. Cryptophyte cultures used in feeding preference experiments and their cell dimensions
Cryptophyte Clone n Length (lm) Width (lm) Volume (lm3)
Rhodomonas salina CCMP 757 27 12.10 (0.97) 6.94 (0.58) 385.29 (78.91) Storeatula major SM, g 34 11.37 (1.15) 6.64 (0.62) 332.79 (75.69) Teleaulax amphioxeia GCEP01 44 12.26 (2.26) 6.28 (0.45) 321.41 (80.81) Guillardia theta CCMP 2712 19 8.62 (2.48) 5.83 (1.30) 172.99 (60.57) Hanusia phi CCMP 325 26 8.24 (1.61) 5.44 (1.07) 172.17 (38.57) Proteomonas sulcata CCMP 704 15 9.63 (1.92) 5.38 (1.23) 157.17 (39.81) Teleaulax amphioxeia GCEP02 27 7.16 (0.57) 4.41 (0.28) 91.75 (16.04) Hemiselmis andersenii CCMP 439 18 6.70 (1.49) 4.44 (0.97) 70.83 (13.64)
Sigma Chemicals) for 10 min, and then filtered onto a and P3, respectively, prey were washed prior to adding to membrane for microscopy (as above). A layered image P. minimum. T. amphioxeia were centrifuged in 50-ml fal- micrograph was constructed with images using differential con tubes, at 1,500 rpm for 30 min, and the supernatant interference contrast and lasers at 405 nm (DAPI) emitting was gently removed and replaced with supernatant from at 445 nm, 488 nm (chlorophyll) emitting at 515 nm, and either the -N or -P, P. minimum culture. This treatment 543 nm (phycoerythrin) emitting at 600 nm (Fig. 1). was designed to determine if elimination of “carry-over” To determine grazing rates of -N and -P P. minimum, nutrients would alter grazing rates. Each well was sampled cultures were maintained (n = 2) in batch, and sampled in at time 0, 24, and 48 h after addition of prey. Prey-only independent experiments for cellular attributes and grazing controls were sampled for each prey concentration in spent rates, after 2, 3, and 4 wk of nutrient starvation. Using 12- media (GF/C filtered) of P. minimum from the respective well plates, P. minimum from batch cultures were starvation treatment, in order to monitor T. amphioxeia combined with late log phase T. amphioxeia prey at prey/ growth in the absence of P. minimum.AP. minimum-only predator ratios between 0.5 and 20, and T. amphioxeia control was used to measure phototrophic growth P. mini- concentrations between 0 and 110,000 cells/ml. In one mum for each treatment. All cell counts for grazing assays experiment for both -N and -P treatments, experiments N3 were conducted on live cells immediately after sampling, using a flow cytometer (above). Growth (l) and grazing rates, including the grazing coefficient (g), clearance rate (F), and Ingestion rate (IR), were calculated using equations described by Frost (1972) and Heinbokel (1978), and refined by Jeong and Latz (1994) and Kim et al. (2008)
Statistical analysis Differences in P. minimum cellular attributes across differ- ent treatments were determined using a one-way ANOVA, and comparisons between treatments were clarified using a Tukey HSD test. Differences in log-transformed growth rate of P. minimum and its grazing rates (g, F, I)onT. am- phioxeia between nutrient treatments for pooled prey/ predator ratios (2–10) were determined using a multivari- ate analysis of variance (MANOVA), and significant differ- ences for each variable were clarified using a univariate ANOVA with Tukey HSD post-hoc tests (Bonferroni adjusted a level of 0.025). One-way ANOVAs and MANOVA were done using R (stats packages: AOV, MANOVA, and TukeyHSD func- tions) (R Development Core Team 2012).
Figure 1 A confocal laser scanning micrograph image of Prorocen- RESULTS trum minimum with four food vacuoles (FV) containing Teleaulax am- phioxeia prey. Image is layered with four channels: a differential Effects of nutrient stress on P. minimum interference contrast (DIC) image showing the P. minimum cell in visi- ble light, a UV light (Ex: 405 nm) image exciting the blue DAPI-stained Both N and P starvation resulted in significant changes in nucleus (N) of P. minimum, a blue-light (Ex: 488 nm) image exciting photosynthetic parameters and intracellular nutrient pools. chlorophyll (red) within the plastids (P) of P. minimum as well as T. Photochemical efficiency (Fv/Fm) was significantly lower amphioxeia in the food vacuoles, and a green light (Ex: 543 nm) in all nutrient stress treatments, dropping to 0.179 in -P image exciting the T. amphioxeia phycoerythrin (yellow) in the plastids cells after about 4 wk of stress (Table 2). Chlorophyll within the food vacuoles. Scale bar = 5 lm. concentrations were also lower in all treatments relative
© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 431–443 433 P. minimum Mixotrophy Johnson
Table 2. Cellular attributes for Teleaulax amphioxeia (TA) prey and Prorocentrum minimum (PM) under nutrient replete (R), nitrogen (N), or phos- phorous (P) limiting conditions for 2, 3, and 4 wk
Sample Fv/Fm Chl per cell C per cell Chl:C (9103) N per cell P per cell C:N C:P N:P
TA-Ra 0.511b (0.001) 1.79 (0.07) 89.6 (5.3) 20.2 (2.3) 13.1 (1.26) 3.13 (0.15) 6.73 (0.23) 29.6 (3.1) 4.42 (0.62) PM-R 0.550 (0.002) 4.36 (0.09) 210 (34.1) 21.0 (3.0) 31.5 (5.78) 4.39 (0.94) 6.66 (0.14) 48.1 (2.6) 7.22 (0.24) PM-P2c 0.413* (0.005) 3.51* (0.26) 308 (23.7) 11.4* (1.72) 26.2 (0.56) 1.52* (0.11) 11.8 (0.65) 204 (30.3) 17.3 (1.62) PM-P3 0.342* (0.003) 2.99* (0.02) 337* (38.2) 8.92* (0.95) 24.5 (1.87) 1.04* (0.09) 13.7* (0.51) 327* (64.5) 23.8 (3.82) PM-P4 0.179* (0.014) 1.33* (0.08) 332* (31.8) 4.00* (0.13) 22.8 (2.45) 0.89* (0.26) 14.7* (2.97) 384* (75.0) 27.2 (10.6) PM-N2 0.412* (0.007) 0.55* (0.01) 249 (4.5) 1.47* (0.04) 20.2* (1.95) – 12.4* (0.98) –– PM-N3 0.346* (0.013) 0.89* (0.06) 276 (24.6) 3.21* (0.05) 15.7* (1.67) – 17.7* (0.31) –– PM-N4 0.084* (0.002) 0.26* (0.01) 271 (18.5) 0.96* (0.03) 10.4* (0.71) – 26.2* (0.01) –– a b c TA-R not included in ANOVA tests; all values are means (n = 2) with the standard deviation in parentheses; weeks of starvation; shaded cells:
*p < 0.05, indicates significant difference from PM-R parameter, ANOVA, Tukey’s HSD, performed for each column: Fv/Fm: F(6,7) = 713, p < 0.05;
Chl per cell: F(6,7) = 416, p < 0.05; C per cell: F(6,7) = 6.3, p < 0.05; Chl:C: F(6,7) = 55.8, p < 0.05; N per cell: F(6,7) = 13.8, < 0.05; P per cell:
F(3,4) = 20.3, p < 0.05; C:N: F(6,7) = 44.9, p < 0.05; C:P: F(3,4) = 16.6, p < 0.05; N:P: F(3,4) = 154, N.S. to the control, and in -P cells varied between 1.33 and oxeia, at least 30% of P. minimum had ingested prey (Fig. 2), 3.51 pg chl a per cell. In contrast, chlorophyll concentra- with an average of 3.9 food vacuoles per feeding cell (not tions of -N cells were all below 1 pg chl a per cell follow- shown). In a few cases, cells were packed with so many ing ~2 wk of stress; however Fv/Fm remained above 0.2 food vacuoles (> 10), that it was not possible to accurately until ~3–4 wk of N starvation (Table 2). C per cell determine their number (not shown). Two cryptophyte spe- increased in the -P treatment after 3 wk of stress, while cies, G. theta and P. sulcata, were not ingested by P. mini- chl:C was significantly lower in all treatments compared to mum. Results of a one-way ANOVA (F(7,8) = 310.4; p < 0.001) P. minimum under nutrient replete conditions (PM-R). As revealed that feeding by P. minimum on the two strains of T. expected, N per cell was lower in all -N treatments, while amphioxeia was significantly higher than on all other species P per cell was lower in -P treatments relative to PM-R; (Tukey HSD, p < 0.001), however feeding was also higher however, P per cell was not measured in -N cells on the larger (Table 1) T. amphioxeia strain GCEP01 relative (Table 2). Among the -P experiments, C:P varied between to GCEP02 (Tukey HSD, p < 0.001). No relationship was 204 and 384, while N:P varied from 17.34 to 27.18 found between cryptophyte prey size (Table 1) and fre- (Table 2). In contrast, PM-R cells had a C:P of 48 and a N: quency of feeding by P. minimum when all species were P of 7.22. N-starved P. minimum cells had C:N ratios of considered (not shown). Based on these results, all further 12.4–26.2, while PM-R cells were 6.7 (Table 2). experiments, focused on the grazing kinetics of P. minimum, used T. amphioxeia (GCEP01). Cryptophyte prey preference Grazing and growth response of P. minimum on T. Prorocentrum minimum did not ingest all cryptophyte spe- amphioxeia cies it was offered, and frequency of feeding was dramati- cally higher (5–89) when in the presence of T. amphioxeia Several experiments were conducted for -P and -N treat- (Fig. 1, 2). After 24 h exposure to two strains of T. amphi- ments in order to determine the role of nutrient starvation on the induction of phagotrophy and the impact of feeding on the growth of P. minimum. P starvation resulted in sig- nificantly higher feeding by P. minimum than all other treatments. In both -N and stationary phase (PM-S) cells (not shown), feeding was difficult to measure, and nutrient replete (PM-R) cells were never observed to feed. Mea- sureable grazing for N-starved cells was only found in one experiment (N3), where T. amphioxeia culture media was removed and replaced with supernatant from -N treated P. minimum, prior to introducing to P. minimum cells. The rationale for this treatment was to remove any dissolved inorganic N that may carry over when T. amphioxeia prey was added. PM-R cultures had a growth rate (l) of 0.538 0.08, which was the highest observed for any treatment. Nutri- Figure 2 Frequency of phycoerythrin-containing food vacuoles in ent-starved P. minimum had higher l when fed T. amphi- phosphorous (P) and nitrogen (N) starved Prorocentrum minimum oxeia compared to unfed cultures, however the effects of cells fed a variety of cryptophyte species and strains. feeding on the growth rate of P. minimum were
© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists 434 Journal of Eukaryotic Microbiology 2015, 62, 431–443 Johnson P. minimum Mixotrophy dependent upon the T. amphioxeia to P. minimum ratio (T/ In all cases when T/P exceeded 5, g dropped below 1 d 1 P) and the duration of nutrient stress prior to feeding. and approached zero when T/P exceeded 10 (Table 3). Maximum impact of feeding on growth was seen after Clearance rates (F) of -P treated cells also declined with 2–3 wk of P-starvation (experiments P2, P3), and resulted increasing prey density, and reached a maximum rate of in a dramatic increase of l after 48 h (Fig. 3). In experi- 162 nl per P. minimum per day (Fig. 4). Ingestion by -N P. ment P2, l increased from < 0.1 d 1 without prey to over minimum on washed T. amphioxeia cells was low, relative 0.5 d 1 above a T/P of 1. In contrast, P3 l was negative to P-starved cells for the same duration, reaching a maxi- without prey, and increased to ~0.3 d 1 above a T/P of mum of about 1 prey per P. minimum per day, and clear- 2.5 (Fig. 3). In experiment P4, which was the most P- ance rates of nearly 100 nl per P. minimum per day. All stressed (Table 2), average growth due to mixotrophy ingestion rates (IR) for P. minimum on T. amphioxeia fol- never exceeded 0.2 d 1 and declined when T/P exceeded lowed a hyperbolic function, and were highest when P 5 (Fig. 3). Prorocentrum minimum l in the -N experiment starved, reaching a maximum between 3.5 and 5 prey per was also strongly negative without prey and remained so P. minimum per day (Fig. 5). The differences between until T/P reached 5 (Fig. 3). nutrient treatments based on the combined dependent Grazing coefficients (g) declined exponentially with variables for growth and grazing were highly significant, F increasing T. amphioxeia concentration in all treatments, (3,12) = 87.6, p < 0.0001; Wilks’ Λ = 0.023. This MANOVA and maximum g varied between 1.3 and 2.5 d 1 (Table 3). was conducted by pooling all saturated parameter values, which included prey: predator ratios between 2 and 10. Post hoc univariate ANOVAs for each parameter revealed significant differences between all -P treatments and N3 for F and IR, while only one of the l comparisons were different (Table 4). Relatively few differences were found for these parameters among the P treatments, while no differences were found for treatment comparisons for g (Table 4). Both the maximum IR (IRmax) and the initial slope of the IR (aIR) increased with increasing duration of P-starvation. Prorocentrum minimum treated for 4 wk in - P conditions (P4) had the highest overall IRmax (5.18 prey per P. minimum per day), which reached saturation the fastest, with an aIR of 1.34 0.24. In contrast, N-starved P. minimum (N3) had an IRmax of 1.30 and a aIR of 0.32 0.11. A one-way ANOVA (F(3,4) = 12.76, p = 0.016) and post hoc Tukey comparisons between aIR parameters revealed that P4 was significantly different from P3, and N3 (p < 0.05), but not P2.
DISCUSSION Prorocentrum minimum was found to ingest a variety of cryptophyte algae, but had a strong preference for T. am- phioxeia that was not based on size alone (Table 1). Feed- ing on the cryptophyte T. amphioxeia was strongly induced by P-starvation, and moderately induced under N- starvation (Fig. 4, 5). Inducible mixotrophy by P. minimum appeared to be particularly sensitive to dissolved inorganic nitrogen (DIN) availability, as washed prey were readily ingested by –N P. minimum while unwashed prey were not. However, the role of DIN in regulating feeding responses needs to be studied further. Feeding was never observed in P. minimum when in nutrient replete condi- tions, and was observed at low rates for cells in stationary phase (not shown). These characteristics suggest that P. minimum is an inducible mixotroph, feeding in response Figure 3 Growth rate (l) of Prorocentrum minimum as a function of to limiting inorganic P or N, and fitting with Stoecker’s Teleaulax amphioxeia prey levels. Prorocentrum minimum was grown (1998) type II mixotroph designation. These findings sup- under identical conditions (n = 2), except either starved of phospho- port previous studies on field assemblages of P. minimum rous (P) for 2, 3, or 4 wk, or nitrogen (N) for 3 wk. Prorocentrum mini- in Chesapeake Bay, where the frequency of mixotrophy mum l is plotted vs. T. amphioxeia cell and carbon (C) concentrations was observed to increase when sample bottles were (A) or prey/predator ratios (B). Dashed line shows growth rate of zero. enriched with nitrate or phosphate, but not both (Stoecker
© 2014 The Author(s) Journal of Eukaryotic Microbiology © 2014 International Society of Protistologists Journal of Eukaryotic Microbiology 2015, 62, 431–443 435 P. minimum Mixotrophy Johnson
Table 3. Prorocentrum minimum grazing on Teleaulax amphioxeia. Prey (C) and predator (P) concentrations, ratio, growth rate of predator (l) and prey (k), grazing constant (g), and growth efficiency (GE) after 48 h based on ingested nitrogen (N) or phosphorous (P), for each experiment. Data are mean values with standard deviation in parentheses (n = 2)