Can cryptophyte abundance trigger toxic Karlodinium veneficum blooms in eutrophic estuaries?

Harmful Algae 8 (2008) 119–128

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Can cryptophyte abundance trigger toxic Karlodinium veneficum blooms in eutrophic estuaries? Jason E. Adolf 1,*, Tsvetan Bachvaroff, Allen R. Place * UMBI Center of Marine Biotechnology, Columbus Center, Suite 236, 701 E. Pratt St., Baltimore, MD 21202, United States

A R T I C L E I N F O

A B S T R A C T

Article history: Received 31 July 2007 Received in revised form 5 April 2008 Accepted 1 August 2008

Karlodinium veneficum is a common member of the phytoplankton in coastal ecosystems, usually present at relatively low cell abundance (102 to 103 mL1), but capable of forming blooms of 104 to 105 cells mL1 under appropriate conditions. We present evidence consistent with the hypothesis that prey abundance, particularly the abundance of nano-planktonic cryptophytes, is a key factor driving the formation of toxic K. veneficum blooms in eutrophic environments. K. veneficum is known to increase growth rate 2- to 3-fold in culture through mixotrophic nutrition, but the role of feeding in bloom formation has not been directly examined. We find that toxic K. veneficum blooms are correlated with cryptophytes abundance changes. We find a wide range of mixotrophic feeding capabilities (0–4 prey per predator per day) among genetically distinct strains of K. veneficum when fed a common prey. Finally, we find that toxic K. veneficum is capable of feeding on a wide range of cryptophyte species varying in size (31–421 mm3 per cell) and phylogenetic affinity, although ingestion rates of different prey vary significantly. While abiotic conditions (e.g. nutrients and advection) are an important aspect of K. veneficum bloom formation in eutrophic environments, our results reinforce the need for a broader view of conditions leading to toxic K. veneficum blooms including biotic factors such as prey availability. ß 2008 Elsevier B.V. All rights reserved.

Keywords: Bloom initiation Cryptophyte Karlodinium Mixotrophy

1. Introduction Anthropogenic eutrophication is the process of organic enrichment of an aquatic system, accelerated by human-related inputs of nutrients (Nixon, 1995). In coastal aquatic ecosystems, eutrophication results in increases in phytoplankton biomass and primary production resulting in autochthonous organic enrichment (Cloern, 2001). Associated with the general increase in phytoplankton biomass in eutrophic systems has been an increase in the frequency, duration, and types of harmful algal blooms (HABs) (Anderson et al., 2002). The relationship between eutrophication and HABs is complex, however, and the specifics of why a particular species blooms at a given time and place remain elusive. Chesapeake Bay is a temperate, eutrophic estuary (reviewed by Kemp et al., 2005) with a well-described seasonal succession of phytoplankton and historically elevated phytoplankton biomass levels related to anthropogenic eutrophication (Harding, 1994; Harding and Perry, 1997). Chesapeake Bay also experiences

* Corresponding authors. Tel.: +1 410 234 8828; fax: +1 410 234 8896. E-mail addresses: [email protected] (J.E. Adolf), [email protected] (A.R. Place). 1 Present address: University of Hawaii at Hilo, 200 W. Kawili St., Hilo, HI 96720, United States. 1568-9883/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.hal.2008.08.003

recurring, aperiodic incidences of HABs (Glibert et al., 2001; Tango et al., 2004) including mixotrophic Karlodinium veneficum (Goshorn et al., 2004), and thus serves as a good model system in which to examine the relationship between eutrophication and harmful algae. K. veneficum has previously been called Gyrodinium estuariale, Gymnodinium galatheanum, Gymnodinium veneficum and K. micrum, all of which are now synonymous with K. veneficum (Bergholtz et al., 2005). Here, we postulate that cryptophyte abundance in eutrophic estuaries is a triggering factor for toxic K. veneficum blooms. Mixotrophic nutrition, involving phagotrophic ingestion of prey by a primarily phototrophic organism, is common among harmful algae and has recently been reviewed by Stoecker et al. (2006). Studies examining the benefits of mixotrophic nutrition to K. veneficum, particularly the 2- to 3-fold growth enhancement associated with ingestion of prey (Li et al., 1999, 2000a,b, 2002; Adolf et al., 2003, 2006b) and the role that karlotoxin plays in prey capture (Adolf et al., 2007), strongly suggest mixotrophy as an important strategy involved in bloom formation, but this has not been directly examined (Stoecker et al., 2006). Current forecast models of K. veneficum in Chesapeake Bay rely on salinity, temperature and month, providing a probabilistic forecast of bloom habitat conditions (http://155.206.18.162/cbay_hab/) but these models do not consider the availability of prey as a factor

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increasing the probability of a bloom. This is in part due to the need for more information regarding the K. veneficum–prey relationship such as the range of mixotrophic capacity in different strains of K. veneficum and the prey preference (if any) of K. veneficum. Deeds et al. (2004) described significant strain variability among K. veneficum toxicity, suggesting physiological variability in karlotoxin production that could impact feeding capacity (Adolf et al., 2006a), although most feeding and growth studies with K. veneficum have been conducted with one strain (CCMP 1974). Likewise, most experiments have used only one prey source (the cryptophyte S. major) but we know that in situ the biodiversity of cryptophytes (i.e. Marshall et al., 2005) includes species that differ dramatically both in phylogenetic and functional (i.e. size) aspects that may impact feeding. Li et al. (2000b) described seasonal distributions of K. veneficum in the mainstem Chesapeake Bay, documenting cell densities (<4000 cells mL1) well below bloom levels (>10,000 cells mL1) associated with fish kills (Goshorn et al., 2004). In situ feeding on cryptophytes was observed but the rates were very low (0–12% cell carbon per day; Li et al., 2002) and unlikely to have a significant impact on growth rate, in contrast to observations made in culture. Possible explanations for the low feeding rates observed in situ by Li et al. (2002) include that K. veneficum was feeding on alternate prey not detected by the methodology used in that study, or that non-feeding strains of K. veneficum were examined. Thus, while culture studies suggest a potentially large growth rate enhancement associated with feeding, observations of feeding in situ (Li et al., 2002) suggest only modest gains in growth rate. A better understanding of strain variability in K. veneficum feeding capacity, and strain variability in cryptophyte prey quality will aid interpretations of K. veneficum feeding patterns observed in the field. Cryptophytes thrive under conditions typical of eutrophic estuaries (Bergmann, 2004) including low light and high concentrations of organic matter. The abundance of cryptophytes in eutrophic estuaries has been recently established through pigment analysis, using the unique accessory pigment alloxanthin as a biomarker. Analysis of Chesapeake Bay phytoplankton floral composition found cryptophytes to be a significant taxonomic group in this system, with a long-term (1995–2000) summer average at 20% of total Chlorophyll a (Chl a) and equivalent to the long-term average summer contribution for dinoflagellates in the Chesapeake Bay (Adolf et al., 2006c). Similarly, photopigment analysis of Neuse River Estuary phytoplankton found cryptophyte abundance at 20% of Chl a in long-term datasets, again often equivalent or greater than abundance of more familiar groups such as dinoflagellates and cyanobacteria (Pinckney et al., 1998; Paerl et al., 2003, 2006; Valdes-Weaver et al., 2006). A time series of phytoplankton photopigment data from Galveston Bay, Texas (USA) show a sequence of a coastal ecosystem freshening due to tropical-storm related precipitation, leading to an initial cryptophyte bloom that was dense enough to discolor oysters that fed upon the bloom (Paerl et al., 2003). This cryptophyte bloom was followed by a dinoflagellate bloom over a two-week period of time. The dinoflagellate bloom was comprised of the mixotrophic species, Prorocentrum minimum, that can prey upon cryptophytes (Stoecker et al., 1997) although a mixotrophic connection between cryptophyte and dinoflagellate was not established (J. Pinckney, personal communication). These data support the hypothesis that cryptophytes may respond first to episodic nutrient events, thus repackaging nutrients in a form that is preferential for their predators and possibly leading to a bloom of mixotrophic HA. Alternatively, growth rate differences between the cryptophytes and dinoflagellates may have staggered their respective responses to the nutrient inputs. A detailed time series study is needed to test

the hypothesis that prey abundance triggers mixotrophic bloom formation. Given the advantage that karlotoxin provides toxic K. veneficum strains (Adolf et al., 2007), it is also possible that increases in cryptophyte abundance may drive the overall population toxicity of K. veneficum blooms in situ. In order to examine the potential role of mixotrophy in K. veneficum bloom formation, we investigated aspects of the K. veneficum–cryptophyte (predator–prey) relationship focusing on spatial and temporal patterns of K. veneficum bloom dynamics in the Inner Harbor of Baltimore, MD, a highly eutrophic environment (Sellner et al., 2001). We further examined the feeding capabilities of different strains of K. veneficum with different toxicities and the cryptophyte prey preference for a toxic strain of K. veneficum. 2. Materials and methods 2.1. In situ feeding assay The in situ feeding experiment was conducted on board the RV Cape Henlopen, June 1999. A natural plankton assemblage from the mainstem Chesapeake Bay (388580 latitude) that contained 4000 K. veneficum mL1 and 3000 cryptophytes mL1 was incubated for 5 h un-amended (‘Not FED’) or after addition of 6000 cells mL1 additional cryptophytes (S. major) from a culture. Water samples were incubated in acid-washed 2-L polycarbonate bottles under two layers of neutral density screening in a flow through water table on the deck of the ship. The percentage of K. veneficum cells with orange fluorescent inclusions (OFIs) was used as an index of grazing (Li et al., 2000b). Comparison of OFIs in K. veneficum with the total loss of cryptophytes from the experimental treatment (compared to control treatments containing cryptophytes in filtered sea water) was used to calculate the percentage of cryptophyte grazing attributable to K. veneficum. 2.2. K. veneficum strains feeding assays K. veneficum strains were maintained for two weeks at <100,000 cells mL1 (in 25 cm2 polystyrene tissue culture flasks) by batch culture dilution in ESAW H1 medium (Enriched Seawater Artificial Water as in Berges et al., 2001 but with 1 mM HEPES). A culture of the cryptophyte Storeatula major was kept in exponential growth between 150,000 and 300,000 cells mL1 during this period to be used as prey. Experiments were conducted at 100 mmol photons m2 s1 PAR (12:12 L:D), 20 8C, salinity 15. Growth irradiance was measured below cool white fluorescent lamps with a Li-Cor QUANTUM probe attached to a Li-Cor LI-250 light meter. On the day of the feeding, K. veneficum cultures were diluted (if necessary) with ESAW H1 to a target density of 50,000 cells mL1. Samples for KmTx analysis were taken from the stock cultures at this point according to the procedures of Bachvaroff et al. (2008). The S. major culture density was 250,000 cells mL1. The feeding assay was conducted in triplicate for each strain in a polystyrene 24-well plate. Each well received 1 mL of S. major culture followed by 1 mL of each K. veneficum strain in triplicate. Thus, the predator to prey ratio in each feeding assay was approximately 1:5. The plate was covered and wrapped with clear poly vinyl chloride film to reduce evaporation. The plate was incubated under the growth conditions described above for 6.5 h and then the cultures killed by adding gluteraldehyde directly to the wells (final 1% concentration). The plate was again wrapped in PVC film and the fixed cells allowed to settle overnight at 4 8C before being counted at the inverted microscope (200–400). Wells were examined for feeding by looking for cryptophytes inside K. veneficum, which were obvious as darker orange cells inside the light yellow K. veneficum. Three fields were counted for

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each well equaling 9 total fields per treatment. Wells were scored as average ingested cryptophytes per K. veneficum divided by the length (h) of the incubation to derive an hourly ingestion rate. Feeding assays with the type culture of K. veneficum (strain PLY 103) were performed separately at the Marine Biological Association of the U.K. Citadel Hill laboratory because this strain cannot be shipped or grown outside of the lab where it was originally isolated in 1950 by Mary Parke (Ballantine, 1956). PLY 103 feeding was tested on the strains of cryptophytes shown in Table 1 with the exception of S. major and Rhodomonas. 2.3. K. veneficum feeding on various cryptophyte strains K. veneficum strain CCMP 2064 was maintained in exponential phototrophic growth by serial dilution in F/2 medium (Guillard, 1975) (30 ppt, 20 8C, 14:10 L:D, 75 mmol photons m2 s1) before the start of the experiment. Potential prey cryptophyte cultures were maintained under similar conditions before the start of the experiment. K. veneficum and twelve different cryptophyte strains were combined and allowed to incubate under the growth conditions described above for 12 h. Each feeding trial was performed in triplicate in 24-well polystyrene plates with approximately 33,000 K. veneficum per mL. The prey:predator ratio (cell numbers) averaged 4 (2.0) among treatments. Samples were then killed by addition of 1% final (v/v) gluteraldehyde and examined at the epifluorescent microscope (Zeiss Axioplan 2 microscope at 500 with a Zeiss filter set No. 9 (ex. 450–490 nm; em. LP 550 nm)) to look for orange fluorescent inclusions inside K. veneficum that indicated feeding had occurred. Feeding was quantified by enumerating the number of orange fluorescent inclusions in the first 100 cells encountered in each experimental replicate, resulting in calculation of feeding based on the observation of at least 300 random cells per treatment. 2.4. Field observations Field sampling was performed at foot-accessible stations around the Inner Harbor, Baltimore, MD, USA. Samples were collected from the upper 1 m of water with a Niskin bottle or a bucket. Cell densities were measured with a Coulter Counter (2005, 2006 sampling), using the size range of 7–20 mm as a proxy for K. veneficum. Microscopic observations of live samples confirmed the dominance of K. veneficum at this size range. In 2007, cell densities were measured by direct epifluorescence microscope counts (Zeiss Axioplan 2 microscope at 250 with a Zeiss filter set No. 9 (ex. 450–490 nm; em. LP 550 nm)) of samples preserved in 1% (final, v/

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v) gluteraldehyde filtered onto 0.22 mm blackened polycarbonate membranes. Two types of cryptophytes were enumerated here, distinguished at the microscope based on size (<10 mm or >10 mm). Phycocyanin was visualized using a rhodamine filter set (Zeiss Filter #15 ex. BP 546 em. LP 590) with comparison to known phycocyanin-containing cryptophyte cultures. Additionally, preliminary 18S sequence data obtained from this sample showed the presence of a phycocyanin-containing cryptophyte, Hemiselmis virescens. Salinity and temperature were measured with a YSI-30 probe. Toxin analysis of field samples was performed by liquid chromatography–mass spectrometry (LC–MS) according to Bachvaroff et al. (2008). Based on morphological similarities to cryptophyte cultures for which we had direct measurements of biovolume, biovolumes of 40 and 400 mm3 per cell were assigned to the small and large categories of cryptophytes, respectively, and used to compute cryptophyte biovolume in situ. Size-class specific cryptophyte biovolume was regressed on total cryptophyte biovolume to determine if one size-class dominated biovolume. K. veneficum biovolume (600 mm3 per cell based on cultures) was plotted against total cryptophyte biovolume, incorporating a lag of 0–3 days between cryptophytes and K. veneficum. 3. Results Natural assemblages containing feeding K. veneficum respond rapidly to added cryptophyte prey (Fig. 1). The control treatment in this experiment (‘Not FED’) showed a frequency of feeding that was low (10–15%) and similar to observations made in situ by Li et al. (2002) in Chesapeake Bay. When an additional 6000 cryptophytes/ mL were added, the percentage of K. veneficum cells with OFI’s increased to nearly 60% over 4 h, indicating rapid uptake of S. major by the in situ population of K. veneficum (Fig. 1). Overall, K. veneficum accounted for an estimated 33% of the grazing pressure on the added cryptophytes in this experiment. Toxic blooms of K. veneficum were observed in the Inner Harbor of Baltimore (Patapsco R., MD) in 2005, 2006 and 2007. Figs. 2 and 3 show K. veneficum cells tended to accumulate to high density (104 cells mL1) between the piers, with associated increases in karlotoxin concentration. The temporal duration of K. veneficum blooms observed in the Inner Harbor was on the order of several days to two weeks. In 2006, peak K. veneficum cell density was associated with reduced salinity and temperature (Fig. 3C and D), indicative of a local run off event. Table 2 illustrates the

Table 1 Cryptophyte strains tested as prey for K. veneficum strain CCMP 2064 STRAIN

Genus species

ESD

S.D.

ESV

S.D.

PLY PLY PLY ‘g’ PLY PLY

Chroomonas salina Hemiselmis brunnescens Rhinomonas reticulata Storeatula major Rhinomonas reticulata Hemiselmis sp.

6.5 4.3 9.3 6.6 7.3 4.1

0.82 0.58 0.98 0.78 0.9 0.63

144 42 421 151 204 36

18.1 5.6 44.4 17.8 25.1 5.5

PLY 563

Hemiselmis rufescens Rhodomonas sp.

3.9 6.5

0.45 0.761

31 144

3.6 16.8

PLY 412 PLY 23 PLY 29 PLY175

Cryptomonas Cryptomonas Cryptomonas Cryptomonas

7.8 7.9 7.9 8.8

1.18 1.11 1.01 1.11

249 258 258 357

37.6 36.3 33.0 45.0

544 14 358 530 631

calceiformis appendiculata maculata maculata

ESD = equivalent spherical diameter (mm). ESV = equivalent spherical volume (mm3). PLY cultures are from the Plymouth Culture Collection, U.K. Strain ‘g’ was isolated from Chesapeake Bay in 1990 by Alan Lewitus (NOAA).

Fig. 1. Experiment showing that natural K. veneficum assemblages respond rapidly to added cryptophyte prey. The % of K. veneficum cells with orange fluorescent inclusions (OFIs) was used as an index of grazing. K. veneficum accounted for approx. 33% of the grazing pressure on the added cryptophytes in this experiment.

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Fig. 2. Sampling map (top) showing the areas along the Baltimore Inner Harbor that were sampled for Karlodinium veneficum abundance and karlotoxin concentration, September 2005. The results from three sampling dates are shown in the lower panel where height of the data plane corresponds to toxin concentration on the zaxis, and shading of the data plane corresponds to cell abundance (right legend).

interannual differences among blooms, particularly higher cellular toxicity associated with 2006 and 2007 (ANOVA F2,78 = 7.7; P = 0.0009), and differences between all three years in the ratio of KmTx 1–1 to KmTx 1–3 (ANOVA F2,88 = 22.1; P < 0.0001), the common congeners of karlotoxin seen in the Chesapeake Bay. A detailed time series collected during summer 2007 in the Baltimore Inner Harbor showed an association between cryptophytes and K. veneficum preceding a bloom (Fig. 4). Cell density changes happened rapidly at this site, which may be due to advection, growth or a combination of the two. Salinity ranged between 5 and 10 ppt with an average of 8 ppt. Temperature ranged between 25 and 30 8C with an average of 27 8C. Cryptophyte biomass in situ was dominated by the larger size class of cells (Fig. 5A). With no lag or a 1 day lag between cryptophytes and K. veneficum, the correlation coefficient, R, was negative or below 0.20, but with a 3 days lag the correlation coefficient was 0.46. Three distinct morphological types of cryptophytes were associated with the K. veneficum blooms; a small cryptophyte in the first (day 189–190) and second (day 198) cases, and a large cryptophyte in the third case (day 206–209). While not obvious on the graph due to scaling, the large cryptophyte increased from 1400 mL1 on day 203 to

5600 mL1 on day 206. Then, between day 206 and 208, K. veneficum increased from 21,000 mL to over 750,000 per mL. Grazing on cryptophytes by K. veneficum is indicated by the number of orange fluorescent inclusions (OFI) in K. veneficum (Fig. 5B). We observed 13% OFI associated with the K. veneficum bloom on day 190, 24% and 36% in the days following this bloom when K. veneficum densities were low. Few OFI were observed associated with the bloom on day 198 because the small cryptophytes on that day were dominated by a phycocyanincontaining species which would not produce OFI under the microscope conditions we used. We observed spikes in feeding to 50% and 138% OFI (>100% indicating more than one OFI per K. veneficum) on days 204 and 205, respectively, preceding the accumulation of K. veneficum between days 206 and 208. Feeding rates varied among strains of K. veneficum, between 0 and 4 prey pred1 d1, with no feeding observed among the nontoxic strains tested (Fig. 6). ANOVA (F16,115 = 53.4; P < 0.001) supported the distinction of three feeding groups (a–c in Fig. 6). No mixotrophic feeding of K. veneficum PLY 103 on cryptophytes was observed under replete, N- or P-limited conditions (10 strains of cryptophytes, not including S. major, tested). K. veneficum strain CCMP 2064 ingested all cryptophytes we tested (Fig. 7), ranging in size from 31 to 431 mm3 in cell volume (Fig. 8 and Table 1). ANOVA (F11,65 = 22.5; P < 0.0001) showed statistically significant differences in ingestion rate for different cryptophytes (Fig. 7). There was no statistically significant relationship between ingestion rate and cryptophyte cell volume (linear regression P = 0.28) or starting prey:predator ratio (number of cells) (linear regression P = 0.41), which averaged 4 (2.0) across all cryptophytes tested. Prey to predator ratio expressed as a biovolume, which averaged 1  1.3 across all cryptophytes tested, was weakly negatively correlated with ingestion rate (ingestion rate = 2.60–0.43 (ratio), r2 = 0.34; P = 0.05). 4. Discussion K. veneficum has a worldwide distribution in temperate coastal aquatic habitats (Deeds et al., 2004; Bachvaroff et al., in press; Copenhagen, 1953; Pieterse and Van Der Post, 1967; Deeds et al., 2002; Kempton et al., 2002; Goshorn et al., 2004; Fensin, 2004). High density, ichthyotoxic blooms tend to occur in eutrophic environments where cryptophytes are abundant. The majority of the previous work detailing the benefits of mixotrophic nutrition to K. veneficum has used one strain (CCMP 1974) and one type of cryptophyte prey (S. major), under-representing both predator and prey biodiversity that is likely to be found in situ. Here, we show that, while most strains of K. veneficum feed mixotrophically, a broad range of variability exists among genetically distinct strains. Further, we show that K. veneficum can ingest a broad range of cryptophyte prey and that in situ associations between cryptophytes and K. veneficum leading to a bloom can involve several cryptophyte species over a short period of time. The three years of observation we show for the Inner Harbor, Baltimore, MD underscore the association between highly eutrophic environments and K. veneficum blooms. Sellner et al.

Table 2 Statistics for cell abundance and KmTx1 for three years of blooms in the Inner Harbor, Baltimore, MD Year

Max. cells per mL

Max. ng KmTx per mL

Avg. pg KmTx per cell

Avg. ratio (w/w) (KmTx 1–1:1–3)

2005 (n = 21) 2006 (n = 52) 2007 (n = 6)

400,000 110,000 750,000

460 232 3586

0.4 (0.22)a 2.8 (3.73)b 5.4 (3.04)b

0.69 (0.214)a 0.58 (0.099)b 0.78 (0.130)c

Standard deviations for average toxin per cell and average ratio (1–1:1–3) are shown in parentheses. Results of ANOVA for average toxin per cell and average ratio (1–1:1–3) are shown as superscripts.

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Fig. 3. Contour plots showing (A) K. veneficum density, (B) karlotoxin concentration (KmTx1), (C) salinity, and (D) temperature in the Inner Harbor, Baltimore MD, USA, June 2006.

Fig. 4. Time series (2007) of K. veneficum, two size classes of phycoerythrincontaining cryptophytes, and Eutreptiella sp. at Sta. 4 in the Inner Harbor, Baltimore, MD, USA, July 2007.

(2001) described a combination of excessive nutrients (summer DIN and DIP >30 and 0.5 mM, respectively), low turbulence, and low grazing pressure in the mesohaline Patapsco R, adjacent to where the Inner Harbor is located, that allows dinoflagellates to dominate. Sellner’s et al. (2001) finding confirms the paradigm of Margalef (1978) in terms of environmental conditions that favor phytoplankton with motile life forms, but does not discriminate between dinoflagellates and cryptophytes as both of these life forms of phytoplankton should benefit from the conditions present in the Patapsco River. Other reports documenting K. veneficum blooms in highly eutrophic environments include a hybrid striped bass aquaculture facility (Glibert and Terlizzi, 1999; Deeds et al., 2002), a South Carolina retention pond (Kempton et al., 2002), recent fish kills in 2005/2006 in the highly eutrophic Chesapeake Bay tributary, the Corsica River (http://mddnr.chesapeakebay.net/ hab/news_100505.cfm), a large fish kill-associated bloom in the Neuse River Estuary (Hall et al., 2008), and mixed K. veneficum/ Gymnodinium pulchellum complex blooms in highly eutrophic shrimp aquaculture farms (Burford, 2005). The issue of whether or not nutrient quality favored K. veneficum was investigated in some studies but the results were equivocal, with different studies reporting different results (Burford, 2005; Burford and Pearson, 1998; Burford and Glibert, 1999; Glibert and Terlizzi, 1999). Although high density K. veneficum blooms are found in highly nutrient-enriched eutrophic conditions, and clearly benefit from appropriate physical conditions (Hall et al., 2008), it is not clear from available reports how important other factors, such as biotic interactions, are to bloom initiation.

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Fig. 5. Cryptophyte–K. veneficum biovolume relationships during the 2007 Inner Harbor bloom. (A) Total cryptophyte and size-class cryptophyte biovolume. Correlations of cryptophyte and K. veneficum biomass including lags of (B) 0 days, (C) 1 day, (D) 3 days.

The Baltimore Inner Harbor K. veneficum bloom of 2007 (Fig. 5) shows co-occurrence of cryptophytes and K. veneficum in a time

series leading up to a high density K. veneficum bloom, supporting our hypothesis that cryptophytes can trigger blooms of K. veneficum. The association of K. veneficum with at least three different functional types of cryptophytes (small phycoerythrin (PE) form, small phycocyanin form, and large PE form) underscores

Fig. 6. Feeding of various strains of K. veneficum on the cryptophyte, S. major. All assays were conducted under nutrient replete conditions with approximately 30,000 K. veneficum per mL and a prey:predator ratio of approximately 4.5:1.

Fig. 7. Feeding of K. veneficum strain CCMP 2064 on various cryptophyte strains. Left axis (bars) shows ingestion rate, with statistically different (ANOVA) groups noted by letters at the tops of the bars. The right axis is cell volume for the different cryptophytes tested.

4.1. Cryptophytes and mixotrophic HAB species co-occur in eutrophic environments

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Fig. 8. K. veneficum ingested cryptophytes of a range of cell sizes. Here, K. veneficum is shown with ingested 7.9  1.01 mm Cryptomonas maculata (PLY 29) and 4.1  0.63 mm Hemiselmis sp. (PLY 631).

the need to further understand prey preference in K. veneficum, as well as the need for a better understanding of the biodiversity and population dynamics of cryptophytes in marine and estuarine environments. Spatial correlations between cryptophytes and mixotrophic HABs have been observed in several environments. Burkholder and Glasgow (1997) reported a positive correlation between Pfiesteria piscicida non-toxic zoospores and prey abundance, including cryptophytes. Li et al. (2000b) found a strong correlation between cryptophytes and K. veneficum in the mainstem Chesapeake Bay in a dataset spanning two-years. Hall et al. (2008) found abundant cryptophytes co-occurring with K. veneficum leading up to a bloom in the Neuse River Estuary. Noble et al. (2003) found statistically significant positive correlations between peridinin (dinoflagellates) and alloxanthin (cryptophytes) in two Southeastern U.S. salt marsh estuaries. Thompson and Hosja (1996) describe frequent co-occurrence between cryptophytes and dinoflagellates in the upper Swan River Estuary. K. veneficum can respond quickly to ephemeral blooms of cryptophytes in situ, as illustrated by our in situ feeding experiment (Fig. 1). It is unclear whether the increased grazing observed upon addition of S. major to this natural K. veneficum assemblage was due to preferential selection of the added S. major relative to the in situ cryptophyte, or simply an increased availability of prey, since the species diversity of cryptophytes in the Chesapeake Bay is largely uncharacterized. According to Reynolds (1993) freshwater cryptophytes (exemplified by Rhodomonas) are one of the functional types of phytoplankton likely to first exploit new nutrients introduced by events with time scales as short as the diurnal cycle of mixing or local run off. Similar observations of cryptophyte dynamics have been made in marine and estuarine environments (Bergmann, 2004; Pinckney et al., 2001, 1998). Rapid blooms of cryptophytes in response to newly available nutrients may set the stage for a successional shift from cryptophytes to K. veneficum. Cryptophytes are likely to respond first due to their higher growth rate (average for strains in Table 2 = 0.42  0.09 d1, n = 12 strains) compared to K. veneficum (average phototrophic growth rate = 0.26  0.05 d1, n = 20 strains, T.R.B. unpublished data). The succession from cryptophyte bloom to K. veneficum bloom would be aided by allelochemicals produced by K. veneficum. This was experimentally supported by observations that a non-toxic strain of K. veneficum (MD5) was outgrown by the cryptophyte, S. major, in co-culture while a toxic strain of K. veneficum (CCMP 2064) was not (Adolf et al., in press). Allelochemicals, secondary metabolites that exert a negative influence on co-occurring

microalgae and other protists including potential predators, and/or potential prey, are now recognized as an important strategy used by a number of HAB species (Legrand et al., 2003; Tillmann, 2004; Graneli and Hansen, 2006). Karlotoxin, the linear polyketide produced by K. veneficum with hemolytic, cytotoxic and ichthyotoxic bioactivity (Deeds et al., 2002; Deeds and Place, 2006) shows allelochemical properties by rendering prey more easily captured (Adolf et al., 2006c) and by deterring grazing of the predator, Oxyrrhis marina, on K. veneficum (Adolf et al., 2007). Similarly, actively toxic strains of P. piscicida are grazed less by microzooplankton than non-toxic strains, although toxic strains of P. piscicida grew and ate less than non-toxic strains (Stoecker et al., 2002; Lewitus et al., 2006). A role of toxin production in prey capture by mixotrophic HA has been previously described, particularly for those mixotrophs without morphological adaptations suited to this purpose (Tillmann, 1998; Skovgaard and Hansen, 2003). Increased growth rate due to mixotrophic feeding, and decreased losses to grazers, likely act in concert to allow K. veneficum to bloom. 4.2. Toxin and feeding variability among K. veneficum strains Strain variability in physiological capabilities is an important aspect of understanding and predicting HAB dynamics (Burkholder et al., 2001; Glibert et al., 2005). Deeds et al. (2004) described differences in cellular KmTx quotas among geographically distinct isolates. Interannual comparisons of KmTx per cell and the ratio of 1–1:1–3 are consistent with the dominance of different strains of K. veneficum in different years. Our evidence suggests that, within a given geographical location, temporal variability in K. veneficum strains may also influence cellular toxicity. This is important for predicting the toxicity of blooms year to year and reinforces the need for monitoring toxicity in addition to cell abundance in managing K. veneficum blooms. Our results suggest that non-toxic K. veneficum strains do not feed as actively as toxic strains, although strain PLY 103 is an exception to this generalization (Abbot and Ballantine, 1957; Galimany et al., 2008). Our results with K. veneficum strain PLY 103 confirms the original observation that PLY 103 does not feed (Ballantine, 1956). This underscores the point that while karlotoxin may aid K. veneficum in feeding (Adolf et al., 2006c), similar to other toxic and mixotrophic harmful algae (Tillmann, 1998; Skovgaard and Hansen, 2003), other factors also affect the ability to feed on cryptophytes. Nonetheless, the variability in feeding rate among strains of K. veneficum is significant and our data suggests that

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mixotrophy is probably not an important factor directly contributing to blooms of non-toxic strains of K. veneficum. In contrast to Dinophysis sp. (Koike et al., 2007; Park et al., 2006; Nishitani et al., 2005) and Myrionecta rubra (Gustafson et al., 2000), our results suggest K. veneficum has a wide range of cryptophyte prey that it can use for enhanced mixotrophic growth. K. veneficum does ingest non-cryptophyte prey, but Li et al. (1996) reported >10-fold lower feeding rates of K. veneficum on a non-cryptophyte prey, Isochrysis galbana, suggesting cryptophytes are a preferred prey item. Our experiments showed differences in the ingestion rate of different strains of cryptophytes, suggesting that ingestion rates in situ may depend upon the species of cryptophyte present. K. veneficum feeds by phagocytotic engulfment of prey (Li et al., 2000a), similar to the heterotrophic dinoflagellates, Gymnodinium sp. (Jakobsen and Hansen, 1997) and Gyrodinium spirale (Hansen, 1992), which show a preference for prey of approximately the same size as (or smaller than) itself, similar to the prey size ranges we determined for K. veneficum. As a corollary, lower ingestion rates probably indicate lower growth rates but additional study on the prey quality of different cryptophytes is necessary. Our results point to the need to better understand and monitor cryptophyte abundance and biodiversity in the context of understanding K. veneficum bloom dynamics. Cryptophyte abundance and biodiversity is greatly undersampled in situ due to methodological hurdles with preservation and identification (Cerino and Zingone, 2006) although they are a preferred prey species for a number of mixotrophic plankton. Li et al. (1996) reported experimental feeding rates of a natural K. veneficum assemblage on added cryptophytes (S. major) that were 10-fold higher than feeding rates on I. galbana (Haptophyte). Jakobsen et al. (2000) showed that the plastid-retaining dinoflagellate, Gymnodinium gracilentum, prefers cryptophyte prey as do five other plastid-retaining dinoflagellates listed in their study. Gustafson et al. (2000) showed that a polar isolate of the phototrophic ciliate, M. rubra (syn. Mesodinium rubrum), ingests and retains functional chloroplasts from a polar cryptophyte, Teleaulax acuta (syn. Geminigera cryophila). Subsequently, Johnson et al. (2007) showed that M. rubra sequesters cryptophyte nuclei as well which are transcriptionally active and play a role in maintaining sequestered cryptophyte plastids within the ciliate, but the kleptoplastidic nature of M. rubra may be restricted to particular strains (Hansen and Fenchel, 2006). Park et al. (2006) recently demonstrated the dependence of D. acuminata on plastids of the cryptophyte Teleaulax acuta, which it acquires along with other cell contents from M. rubra as it cannot directly uptake plastids from the free cryptophyte. In situ associations between Dinophysis spp. and small (<5 mm) cryptophytes (Nishitani et al., 2005), or with T. acuta specifically (Koike et al., 2007), further demonstrate the relationship between cryptophytes and Dinophysis HABs in Japanese coastal waters. Interestingly, Dinophysis has recently been reported for the first time in the Virginia portion of Chesapeake Bay (Marshall et al., 2004), and has bloomed recently in the Maryland portion of the Bay where it had been observed at low levels previously (Tango et al., 2004) although the link to cryptophytes has not been directly examined. We suggest that the key elements leading to toxic K. veneficum blooms include (1) eutrophic environments, (2) co-occurrence of cryptophytes and K. veneficum, (3) a rapid response of cryptophytes to environmental opportunities (e.g. nutrient input) to bloom, (4) mixotrophic predation of K. veneficum on cryptophytes, aided by allelochemicals (karlotoxins) produced by K. veneficum that improve prey capture and reduce grazing mortality of toxic strains (Adolf et al., 2006c, 2007, in press). We expect this scenario to favor accumulations of toxic K. veneficum due to their generally higher feeding capacity compared to non-toxic cells (Fig. 9). Once

Fig. 9. Conceptual diagram showing four phases in K. veneficum HAB dynamics. The hypothesis presented in this paper addresses the transition from phase 1 to phase 2.

the mixotrophic HA population has achieved bloom density, mixotrophic feeding is probably not important because prey are likely to be significantly reduced (sensu Tittel et al., 2003), thus predator avoidance may be of greater importance to maintaining population levels once a bloom is established. Our conceptual model of K. veneficum bloom formation shares some features with that of Glasgow et al. (2001), which suggests availability of cryptophyte prey as a factor stimulating springtime abundance of Pfiesteria spp. Considering Glasgow’s suggestion and the conceptual model we have proposed it is not surprising that Pfiesteria and Karlodinium tend to co-occur in eutrophic estuaries (Place et al., 2008). Our model differs from other recent models (Stoecker and Gustafson, 2002; Irigoien et al., 2005; Sunda et al., 2006; Mitra and Flynn, 2006) in that it stresses predatory grazing by the HAB species rather than predation (or lack thereof) on the HAB species. In the model presented by Mitra and Flynn (2006), nutrient limitation is essential for the production of grazing deterrents by HAB species, although this is not the case for K. veneficum (Adolf et al., 2007). In brief, HAB species existing as part of a mixed assemblage are avoided by predators that selectively graze on abundant non-HAB species. Regenerated nutrients from this grazing activity are preferentially taken up by the un-grazed HAB species, promoting their increase while the non-HA is grazed away. By the time the predator switches to eating the HAB species it has become nutrient limited and has begun to produce grazing deterrents. Adapting this perspective to K. veneficum would have the cryptophytes playing the role of the rapidly growing non-toxic phytoplankton preceding the HAB-species, but we suggest that toxic K. veneficum then ingests the cryptophytes leading to their own rapid growth. The mixotrophic, predatory nature of K. veneficum and many other HAB-species clearly demonstrates the importance of considering the biotic environment along with the physical–chemical environment when trying to understand HAB dynamics. Acknowledgements This work was funded in part by grants from National Oceanic and Atmospheric Administration Coastal Ocean Program under ECOHAB award #NA04NOS4780276 to University of Maryland Biotechnology Institute, Grant # U50/CCU 323376, Centers for Disease Control and Prevention and the Maryland Department of Health and Mental Hygiene; and MD Sea Grant award R/EH-5 (2007–2008) to A.R.P. This is contribution # 07-166 the UMBI Center of Marine Biotechnology, and #221 from the Ecology and Oceanography of Harmful Algal Blooms (ECOHAB) program. J.E.A. acknowledges the assistance of Dr. Richard Pipe, Ms. Maria Jutson, and Ms. Magali Guenevere at the Marine Biological Association of the U.K., and partial support on a Mary Parke Fellowship from the

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