Understanding Causes and Impacts of the Dinoflagellate,Cochlodinium polykrikoides, Blooms in the Chesapeake Bay

Margaret R. Mulholland & Ryan E. Morse & George E. Boneillo & Peter W. Bernhardt &Katherine C. Filippino & Leo A. Procise & Jose L. Blanco-Garcia & Harold G. Marshall &Todd A. Egerton & William S. Hunley & Kenneth A. Moore & Dianna L. Berry &

Christopher J. Gobler

Received: 13 January 2009 /Revised: 10 April 2009 /Accepted: 16 April 2009# Coastal and Estuarine Research Federation 2009

Abstract During August and September 2007, the lowerChesapeake Bay and its tributaries experienced a massivebloom of Cochlodinium polykrikoides Margalef (>104 cellsper milliliter) that persisted for over a month, wasextremely patchy, and at times penetrated into the AtlanticOcean. The onset of the bloom coincided with a period ofintense rainfall and stormwater runoff after a protractedsummer drought period. Genetic evidence showed thisspecies to be distinct from many Asian strains but similar toother North American strains. Populations dominated by C.polykrikoides took up a variety of nitrogenous compounds

to support their growth and were able to mobilize additionalsources of organic nutrients through peptide hydrolysis.Bioassays determined that C. polykrikoides exerted a lethalaffect on juvenile fish and shellfish, causing 100%mortality of juvenile fish (Cyprinodon variegates) in lessthan 24 h and 20% mortality in juvenile American oysters(~21 mm; Crassostrea virginica) within 72 h.

Keywords Cochlodinium polykrikoides . Nitrogen uptake .

Chesapeake Bay . Fish mortality

Introduction

Cochlodinium polykrikoides Margalef is a dinoflagellatethat forms blooms in coastal waters around the world whereit is well known for causing fish kills (Onoue et al. 1985;Yuki and Yoshimatsu 1989; Guzmán et al. 1990; Qi et al.1993; Gárrate-Lizárraga et al. 2004; Whyte et al. 2001;Gobler et al. 2008; Azanza et al. 2008). The ichthyotoxicproperties of C. polykrikoides blooms have been welldescribed (Onoue et al. 1985; Yuki and Yoshimatsu 1989;Guzmán et al. 1990; Qi et al. 1993; Kim 1998; Kim et al.1999; Gárrate-Lizárraga et al. 2004; Whyte et al. 2001;Gobler et al. 2008). In addition, studies have shown thatmetamorphosis of Pacific oyster, Crassostrea gigas, larvaeare slowed during Cochlodinium blooms (Matsuyama et al.2001) and that mortality of American oyster, Crassostreavirginica, larvae increases during exposures to Cochlodi-nium (Ho and Zubkoff 1979).

C. polykrikoides is a mixotrophic alga (Larsen andSournia 1991; Jeong et al. 2004) capable of cyst productionand, like other mixotrophs, may employ flexible nutrientacquisition strategies during blooms. Moreover, since thisphytoplankter is noxious to some planktonic grazers (Ho

Estuaries and CoastsDOI 10.1007/s12237-009-9169-5

M. R. Mulholland (*) : R. E. Morse :G. E. Boneillo :P. W. Bernhardt :K. C. Filippino : L. A. Procise :J. L. Blanco-GarciaDepartment of Ocean, Earth, and Atmospheric Sciences,Old Dominion University,4600 Elkhorn Avenue,Norfolk, VA 23529-0276, USAe-mail: mmulholl@odu.edu

H. G. Marshall : T. A. EgertonDepartment of Biological Sciences, Old Dominion University,Norfolk, VA 23529-0266, USA

W. S. HunleyHampton Roads Sanitation District,Virginia Beach, VA, USA

K. A. MooreThe Virginia Institute of Marine Science,School of Marine Science, College of William and Mary,Gloucester Point, VA 23061, USA

D. L. Berry : C. J. GoblerSchool of Marine and Atmospheric Sciences,Stony Brook University,Stony Brook, NY 11794-5000, USA

and Zubkoff 1979; Shin et al. 2003), it may escape top-down control by zooplankton (e.g., Gobler et al. 2002). Todate, there has been little research on Cochlodinium bloomsin North America and studies of factors that cause andimpact blooms on the eastern US coast have never beenconducted. Further, it now appears that the North AmericanCochlodinium strains are genetically distinct from theirAsian counterparts (Gobler et al. 2008). Consequently, thefactors promoting blooms and the ecosystem impacts ofthese blooms in North American estuaries are likely to bedistinct as well.

C. polykrikoides blooms in the York River, a tributaryof Chesapeake Bay, have been recorded for over40 years (Mackiernan 1968) and blooms in this tributaryhave continued to occur in recent years, concomitant witha general increase in their occurrence in this and otherlower Chesapeake Bay tributaries over the past twodecades (Marshall et al. 2005). The geographical spreadof this organism appears to have been enhanced by anextensive bloom of C. polykrikoides that developed fromAugust through September 1992, spreading from the YorkRiver into the lower Chesapeake Bay and tributaries alongits western shoreline, including the lower James Riverestuarine complex (Marshall 1995a). During this bloom,concentrations of C. polykrikoides in Chesapeake Bayranged from 102 to 103 cells per milliliter and coveredapproximately 215.7 km2 of the lower Bay, with watertemperatures of 24.6–27.0°C and salinity of >20. Thebloom spread into Atlantic coastal waters and along theNorth Carolina shoreline. Regional sites where blooms ofC. polykrikoides have occurred annually since 1992 haveincluded tributaries of the lower James River, includingthe Lafayette and Elizabeth Rivers, in addition to sitesnorth of the York River (Marshall, personal records).Sediment records have also established the presence ofcysts produced by Cochlodinium in lower ChesapeakeBay tributaries and a regional source or “seed bed” ofviable cells for initiating bloom events. In 1996, JamesRiver surface sediment (from 13 stations) had an averageCochlodinium dinocyst abundance of 96 cysts per gramsediment (Seaborn and Marshall 2008).

To ascertain possible hydrological and nutrient trig-gers as well as ecological impacts of the C. polykri-koides bloom in the Chesapeake during 2007, we usedboth field monitoring and laboratory bioassays toevaluate the spatial extent of the blooms and the effectsof river discharge, rainfall, nutrient concentrations, andnutrient uptake rates on bloom dynamics, as well as thedirect effects of C. polykrikoides on juvenile fish andoysters. We also genetically characterized the bloom toestablish the composition of this C. polykrikoides popula-tion and to compare it to others found in the USA andglobally.

Methods

During summer 2007, a massive bloom of C. polykrikoidesoccurred throughout August and into September in thelower James River and its tributaries, extending into theChesapeake Bay and Atlantic Ocean at times (Fig. 1). Tovisualize the extent of this bloom, we used DATAFLOWmeasurements, an underway, surface multiparameter mon-itoring system (including chlorophyll a (Chl a) fluores-cence). The Hampton Roads Sanitation District collectsinformation from DATAFLOW at least once per week aspart of the Chlorophyll Monitoring and AssessmentProgram for the lower James River region while theVirginia Institute of Marine Science (VIMS) collectsmonthly DATAFLOW data for the upper James Riverregion as well as all other major western Virginiatributaries. DATAFLOW data and visualizations, as wellas collection and interpretation methods, are described atthe Virginia Estuarine and Coastal Observing System(VECOS) website (http://www2.vims.edu/vecos). Data for

Fig. 1 Map of the lower Chesapeake Bay and its tributaries. Areasmarked with stars are the stations in the Lafayette River (LAF),Elizabeth River (ER and GSB), James River (JRM, PP, and JRB),Little Creek (LC1 and LC2), and the Chesapeake Bay (CH, BM0) thatwere sampled during the 2007 C. polykrikoides bloom (see text forstation identification). The dashed line indicates the location of theBay Mouth Transect (BMT) and the southernmost station, BM0, whichwas highly impacted by the bloom

Estuaries and Coasts

James River discharge were obtained from the USGeological Survey (USGS) station 02037500, in the JamesRiver near Richmond, VA, USA (http://va.water.usgs.gov/).Meteorological data were obtained from Old DominionUniversity’s meteorological station at Hoffler Creek, a tidalcreek entering the James River near Craney Island (http://www.lions.odu.edu/~cmpowell/hoffler/). Continuous(15 min) measurements of in vivo Chl a fluorescence fortwo fixed stations along the lower James River wereobtained from the VIMS, VECOS fixed station monitoringprogram (http://www2.vims.edu/vecos). These fixed stationChl a fluorescence data were measured using monitoringinstruments (YSI 6600 EDS datasondes, YSI, Inc.) identicalto those used for DATAFLOW monitoring.

Water column sampling was conducted within the C.polykrikoides bloom from docks, small boats, and aboardthe Research Vessel Fay Slover. Water samples werecollected at station in the Lafayette River (LAF and GSB),the Elizabeth River (ER), the James River (JRM, JRB, andPP), Little Creek (LC1 and LC2), and the Chesapeake Bay(BM0 and CH). Temperature, salinity, and dissolved oxygenwere measured either with a handheld probe (dock and smallboat sampling) or with a CTD. Water samples were collectedusing a clean bucket, a peristaltic pump, or Niskin bottles.Samples were gently transferred to 20-L carboys and eithertransported back to the laboratory or used for experimentsinitiated in the field during cruises on the Research Vessel FaySlover. C. polykrikoides cell densities were quantifiedmicroscopically from Lugol’s preserved samples. The iden-tity of C. polykrikoides was confirmed using light microsco-py and molecular techniques described below. Chlorophyll aand particulate carbon and nitrogen (PC, PN) providedadditional biomass estimates. Samples for Chl a, PC, and PNanalysis were collected onto precombusted (2 h at 450°C)GF/F glass fiber filters and frozen until analysis. Chl a wasanalyzed after acetone extraction using standard fluorometricmethods (Welschmeyer 1994) within 1 to 3 days of samplecollection. PC and PN were analyzed on a Europa 20/20isotope ratio mass spectrometer equipped with an automatednitrogen and carbon analyzer.

For dissolved nutrient analyses, filtered water wascollected in the field using a low-pressure peristalticpumping system equipped with an acid-cleaned Pallpolysulfone filter capsule (0.2 µm) and acid-washedTeflon tubing. Samples were stored on ice and frozenwithin 1 h. Dissolved urea and inorganic nutrients(nitrate, nitrite, phosphate, silicate) were analyzed usingstandard techniques with an Astoria Pacific nutrientautoanalyzer using standard colorimetric methods (sim-ilar to those in Parsons et al. 1984) and as recommendedby the manufacturer. Ammonium concentrations weredetermined colorimetrically using the manual phenolhypochlorite method (Solorzano 1969). Total dissolved

nitrogen (TDN) concentrations were determined in dupli-cate after persulfate oxidation (Valderrama 1981). Dis-solved organic nitrogen (DON) was calculated as thedifference between TDN and dissolved inorganic nitrogen(DIN).

For genetic analysis, one liter of water was collected on20-µm polycarbonate filters at sites occupied during the R/V Fay Slover sampling cruise. Filters were immediatelyresuspended in 600-µL cetyltrimethylammonium bromide(CTAB) solution (100 mM Tris–HCl [pH 8], 1.4 M NaCl,20 mM ethylenediaminetetraacetic acid, 2% [w/v] CTAB,0.4% [v/v] β-mercaptoethanol, 1% [w/v] polyvinylpyrroli-done; Dempster et al. 1999) and cells were disrupted byvortexing and pipetting. The resuspended cells were storedfrozen at −80°C. Nucleic acids were extracted as in Coyneet al. (2001). The hypervariable D1–D3 region of theribosomal DNA (rDNA) was amplified by polymerasechain reaction (PCR) using forward primer “D1RF” (5′-ACCCGCTGAATTTAAGCATA-3′) and reverse primer“D3Car” (5′-ACGAACGATTTGCACGTCAG-3′; Mikulskiet al. 2005). The D4–D6 region was amplified with forwardprimer 6F (5′-TAGTAGCTGGTTCCCTCCGA-3′) and re-verse primer 11R (5′-TTGCCGACTTCCCTTACCTA -3′;Iwataki et al. 2007). A 50-μL PCR reaction was performedwith the following components: 80–100-ng nucleic acids,0.5 μM of each primer, and 25-μL GoTaq Green 2X PCRMaster Mix (Promega). We used the following cyclingparameters: 94°C for 5 min, followed by 31 cycles of 94°Cfor 45 s, 45°C for 60 s, and 72°C for 45 s, followed by 72°Cfinal extension for 7 min (Mikulski et al. 2005). Sequencingwas done directly on the unmodified PCR product using50 ng of PCR product and 3.2-pmol primer on a ABI3730Genetic Analyzer using BigDye Terminator ® Cycle se-quencing kit (Applied Biosystems) at the Stony BrookUniversity DNA Sequencing Facility. We have used thisapproach to successfully sequence several isolated clones ofC. polykrikoides from New York estuaries (Gobler et al.2008).

We determined the dominant forms of nitrogen andcarbon assimilated by the plankton communities dominatedby C. polykrikoides by measuring the uptake of a suite of15N-labeled compounds (NO3

−, NO2−, NH4

+), 15N and 13Cdually labeled compounds (urea and amino acids), and 13C-labeled bicarbonate (Mulholland et al. 2006). Nitrogenadditions ranged from 2% to 70% of the ambient concen-trations but were normally between 2% and 10%. Previousresults suggest that enrichments as low as 1% producereliable results (Mulholland et al. 2009). Enrichments >10%might artificially stimulate uptake and result in over-estimates of ambient uptake rates. Mobilization of organiccompounds through peptide hydrolysis was also estimatedusing Lucifer-Yellow anhydride tetra-alanine (Mulhollandet al. 2002, 2003).

Estuaries and Coasts

Bioassay toxicity experiments with fish and shellfishCochlodinium sp. blooms in multiple ecosystems and haspreviously been found to be lethal to fish and shellfish.Experiments were conducted to determine the potentialimpact of C. polykrikoides bloom water on fish andshellfish using recently established protocols. One set ofexperiments was performed with juvenile (2 week old)sheepshead minnows (Cyprinodon variegates) spawnedfrom a line which has been laboratory-reared for more thana decade (E.M. Cosper, personal communication). For theseexperiments, individual minnows were transferred using amodified 200-µL pipette to 3 mL of treatment water held in24-well sterile polystyrene plates (n=24 per treatment)containing bloom water or filtered bloom water (0.2 µm)since prior research has shown filtered bloom water isnontoxic to fish (Gobler et al. 2008). Fish were monitoredinitially every 30 min for the first 2 h, hourly through 6 H,and then every 6 h.

Experiments were also conducted using juvenile Amer-ican oysters (~21 mm; C. virginica) obtained from CornellCooperative Extension, Southold, NY, USA. Experimentswere initiated by measuring the lengths of ten markedindividuals and placing them in triplicate 1-L plasticbuckets (ten individuals per bucket) containing 1 L of: (1)whole bloom water, (2) filtered (0.2 µm) water from thebloom site, (3) whole water from a control site, or (4)filtered (0.2 µm) water from a control site outside theinfluence of the bloom. All buckets were kept in atemperature-controlled room maintained at 24°C with lightfrom a bank of fluorescent bulbs which provided100 µE m−2 s−1 of light on a 14:10 h light–dark cycle.All buckets were bubbled with air, maintaining oxygenlevels above 5 mg L−1, and covered with white nylonscreening with 1-mm mesh openings. Experimental waterwas exchanged with newly obtained water from the fieldwith comparable cell numbers on the second day duringthe experiment. Survival was examined daily.

Results

Here, we present a comprehensive investigation of theextensive C. polykrikoides bloom that occurred in Ches-apeake Bay and its tributaries during the summer of 2007.The 2007 C. polykrikoides bloom appeared to initiate in theElizabeth River at the beginning of August 2007 (Fig. 2),after a protracted summer drought (Fig. 3) followed byperiods of intense storm activity (Fig. 4) that resulted inpulses of overland flow into the region’s waterways. Thisweather pattern is not unusual at this time of year; however,in 2007, while spring flow was close to normal, the regionexperienced extreme drought conditions throughout the late

spring and summer (Fig. 3). The James River is dammed,and, because upriver reservoirs were so low due to thedrought, water flow from the dams was restricted to allowthe reservoirs to be replenished; consequently, impacts fromthe summer rainfall events were local rather than a result ofincreased flow and nutrient loading from the entire JamesRiver watershed.

Following its initiation, the C. polykrikoides bloomextended up into the Lafayette River and through themouth of the Elizabeth River into the James River (Fig. 2,personal observations). During late August, after anotherintense period of storm activity, the bloom entered theChesapeake Bay, the Lynnhaven River, and extended outthrough the Bay mouth into the Atlantic Ocean resulting inbeach closures (personal observations). While blooms ofCochlodinium now occur annually, such an extensivebloom and its penetration into the Atlantic Ocean areunusual and have not been seen since 1992 (Marshall1995a).

Sampling during this bloom event was initiated on 11August 2007 and continued through 6 September 2007.During this time, observed salinities ranged from 18.9 to27.9 and observed temperatures were 27.0 to 29.5 (Table 1),salinities lower than and temperatures comparable to thosereported by Kudela et al. (2008). Samples were collectedonly during daylight hours and were not restricted toparticular stages in the tidal cycle and so the temperatureand salinity ranges should be considered conservative.

Analysis of the phytoplankton assemblage During the1-month sampling period, C. polykrikoides ranged from45.6% to 90.4% of the phytoplankton assemblage enumer-ated microscopically (Fig. 5). C. polykrikoides abundanceswere highest in the Elizabeth River on 11 August 2007,present at 11,137 cells per milliliter, and representing85.2% of the total phytoplankton abundance. Among thelower Bay phytoplankton samples (taken 29 August 2007),C. polykrikoides abundance ranged from 57.9% to 90.4% ofthe total phytoplankton community enumerated, but con-centrations were an order of magnitude lower than thoseobserved in the Lafayette River. Each of these samplescontained other dinoflagellates, diatoms, and cryptomonadsas subdominant taxa, and all the taxa present were floracommon to the Elizabeth River and the Chesapeake Baythis time of the year (Marshall et al. 2005). Thezooplankton composition in the Lafayette River was morediverse and included nauplii, polychaete larvae, and theciliate Strombidium sp. The lower Chesapeake Bay zoo-plankton contained several ciliates (Strombidium sp.,tintinnids), but none of these were in high abundance.Grazers were observed in preserved samples but theirquantitative abundance could not be ascertained given themethods employed.

Estuaries and Coasts

Characterization of the bloom organism Genetic analysissuggests that the Virginia/Chesapeake Bay bloom organism isidentical to the New York and other North American strainsbut is genetically distinct from most Asian Cochlodiniumisolates. We sequenced the D1–D3 and D4–D6 regions ofthe large subunit (LSU) rDNA from field material collectedat multiple sites during the 2007 Virginia blooms. Identicalsequences were found between samples at all sites. In theconserved D4–D6 region of the LSU rDNA, the Virginia

strain (accession number pending) showed 100% identitywith North American strains CPCB10 isolated from CotuitBay, MA, USA (accession EF506626), CPPV-1 isolatedfrom Bahia de La Paz, Mexico (accession EF506628),PR107 isolated from Phosphorescence Bay, Puerto Rico(accession AB295050), CpNY-06-1 isolated from FlandersBay, NY, USA (accession EF110557), and Malaysian strainscp1 and cp2 (accessions AB295048 and AB295049). Thisregion is 97% identical with strains isolated from Japan

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Sampling sites for this study

VECOS fixed shallow water monitoring

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JamesRiver

James River

08/20/07 08/21/07

Fig. 2 DATAFLOW maps ofsurface Chl a (μg L−1) insegments of the James River inAugust and September, 2007,during the C. polykrikoidesbloom. Red stars in the twoupper panels were used to indi-cate the location for twoshallow-water fixed monitoringstations, James River CountryClub (left) and Wythe Point(right). Yellow stars in the lowerpanels indicate sites sampledduring the bloom

Estuaries and Coasts

(accession EF506624) and South Korea (EF506621,EF506619, EF506617, and EF506615).

The hypervariable D1–D3 region sequences of oursamples (accession number pending) displayed 100%identity with four North American C. polykrikoides strains:CPCB10 (accession EF506625), CPPV-1 (accessionEF506627 and accession AB295050), and CpFB-06-1(accession EF110556). Additionally, the D1–D3 alsoshowed 100% identity with two clones isolated fromMalaysia (cp1 and cp2, accession numbers AB295048 andAB295049). In contrast, the D1–D3 region sequenceshowed only 91–92% similarity to GenBank sequencesfrom additional Asian isolates of C. polykrikoides isolatedfrom Omura Bay, Japan (accession AB295047); ManilaBay, Philippines (accession AB295046); South Korea(accession EF506618); and another strain from Japan(accession EF506623; Fig. 6). These results indicate thatthe Virginia strain is identical to the North American clonesof C. polykrikoides but only 91–92% similar to most Asianstrains (Fig. 6).

DATAFLOW mapping suggests that the C. polykrikoidesbloom was patchy in its intensity and distribution overspace and time for the duration of the bloom (Fig. 2).Further, Chl a data from fixed shallow-water monitoringstations within the impacted area confirmed the temporallyvariable nature of the bloom (Fig. 7). Discolored water wasfirst seen in the Lower James River and its tributaries at thebeginning of August. At this time, the James Riverdischarge was uniformly low and the region was experi-encing a drought (Fig. 3). The intense storm activity thatoccurred in August was localized and associated withintense but sporadic rainfall onto a drought-hardenedlandscape that resulted in overland runoff (Fig. 4). Detailedwater column hydrography during a monthly bay mouthmonitoring transect conducted during the bloom on 29August 2007 (Fig. 8) indicated that the bloom penetratedthe Atlantic through the southern side of the Bay mouthwhich was disproportionately influenced by the JamesRiver at this time (Filippino et al. 2009); the James River

in turn was highly influenced by overland flow into theElizabeth and Lafayette Rivers. During the time in whichthe bloom was pronounced and as is usually the case, watertemperatures were slightly higher and salinity lower on thesouth side of the Chesapeake Bay mouth relative to thenorth side, resulting in a lower sigma t (Fig. 8). Further,high Chl a due to Cochlodinium was also concentrated onthe south side of the Bay mouth (Fig. 8), suggesting thatpopulations intruding into the Atlantic Ocean near VirginiaBeach originated from Chesapeake Bay and the JamesRiver.

Throughout the bloom period (August–September),surface water temperatures ranged from 27.0°C to 29.5°Cand salinity at bloom sites ranged from 18.9 to 27.9(Table 1). Chl a concentrations were highly variable andranged from 13.1 to 350.1 μg Chl a L−1, and Cochlodiniumpolykrikoides cell density ranged from 680 to 11,137 cellsper milliliter at the sites sampled as part of this study(Table 1; although higher densities were found elsewhere;Marshall, unpublished data). N and C biomass was alsohighly variable over the study period; PN was 27.9 to226.5 μmol N per liter and PC was 175.8 to 1,724.8 μmolC per liter (Table 1). Compared to background levels of PN,PC, and Chl a, values were at least an order of magnitudehigher during the bloom.

Except for two stations near the Chesapeake Bay mouthand one in the Elizabeth River, nitrate concentrations weregenerally submicromolar (Table 2). Nitrite and ammoniumconcentrations were similarly low throughout the bloom.Phosphate concentrations on the other hand were fairly highranging from 0.32 to 3.83 μmol L−1 (Table 2) and the ratios

USGS Station 02037500 near Richmond, VA

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Fig. 4 Local rainfall as measured at an Old Dominion Universitymeteorological station located on a tidal creek exchanging with theJames River near Craney Island

Estuaries and Coasts

of DIN to dissolved inorganic phosphorus (DIP) werealways well below Redfield (0.63 to 6.97). DON concen-trations ranged from 5.5 to 24.4 μmol N per liter, and ureaconcentrations were a small fraction of this pool (0.7% to7.4%) and always less than 0.5 μmol L−1. Dissolved freeamino acid (DFAA) concentrations were another smallfraction of the DON pool (1.4% to 5.8%) and always lessthan 0.7 μmol L−1. Silicate was always present at highconcentrations (28.2 to 99.0 μmol L−1) and so was unlikelyto limit diatom production.

Results from uptake studies suggest that C. polykrikoidestook up a variety of N compounds to support their growth(Fig. 9a, c). Absolute versus N-specific uptake rates suggestthat, while uptake rates are biomass sensitive, N uptake isstrictly a function of biomass. In fact, total N-specificuptake rates were independent of PN or Chl a concen-trations (Fig. 10a, c). Combined urea and DFAA uptakeranged from 24.8% to 68.8% of the total measured Nuptake and DIN uptake ranged from 31.2% to 75.2%.Based on total measured N uptake and assuming uptakeover a 24-h day, N doubling times were 0.48 to1.30 days.

At most locations, the majority of the C assimilation wasphotosynthetic bicarbonate uptake, although the photosyn-thetic C uptake was often low compared with the totalmeasured N uptake (Fig. 9b, d); bicarbonate to total Nuptake ranged from 0.5 to 13.0 but was generally <6.0(Fig. 9). Based on photosynthetic bicarbonate C uptake, Cdoubling times ranged between 1.1 and 217 days, muchlonger than N-based doubling times. Further, the highestbicarbonate uptake rates were observed when biomass (PCand Chl a) were lower (Fig. 10b, d). Urea and DFAAcontributed to C uptake during some of the samplingperiods particularly during mid-August in the Elizabeth andLafayette Rivers.

Based on turnover of our labeled peptide, mobilizationof peptides through extracellular hydrolysis was substantialat bloom sites (1 to 2 day−1) compared to nonbloom sites(<0.5 day−1) and may also have contributed to regenerationof nutrients to support the growth of bloom organisms(Fig. 11).

Bioassay toxicity experiments with fish and shellfish Dur-ing this event, we were able to ascertain that blooms of C.polykrikoides could have had lethal impacts on larval fishpopulations; juvenile sheepshead minnows (C. variegates)experienced 100% mortality within 3.5 h of exposure tobloom water with 104 C. polykrikoides cells per milliliter(Fig. 12). Juvenile oysters were also severely impacted with20% mortality during 72 h of exposure to bloom watercontaining ~103 C. polykrikoides cells per milliliter (datanot shown). There were no mortalities observed in controlpopulations that were not exposed to C. polykrikoides foreither type of experiments. The fish-killing ability ofCochlodinium is consistent with earlier experiments usingbloom water from NY estuaries (Gobler et al. 2008).

Discussion

Blooms of C. polykrikoides typically occur along the lowerChesapeake Bay western shoreline between July and October.The distribution and magnitude of C. polykrikoides bloomsappear to have increased over time (years–decades) in theChesapeake Bay and its tributaries (Marshall et al. 2005).For example, in 1995, the highest cell densities in theElizabeth River were 15 cells per milliliter (Marshall,unpublished data). These concentrations subsequently in-creased to 810 cells per milliliter in 2005, 3,500 cells per

Table 1 Biomass and physical parameters during the 2007 C. polykrikoides bloom sampling period

Date Location Salinity Temp Chl a(μg L−1)

C. polykrikoides celldensity (cells mL−1)

Particulate N(μmol N L−1)

Particulate C(μmol C L−1)

C to Nmolar ratio

8/11/07 LAF 21.0 29.5 279.0 (35.0) 11,137 131.5 (22.1) 1,015.5 (190.8) 7.7

8/17/07 ER 22.1 28.4 350.1 (13.3) 9,139 226.5 (18.1) 1,724.8 (216.3) 7.6

8/29/07 CH 27.9 27.0 35.6 (1.0) 1,184 27.9 (1.5) 205.8 (19.8) 7.4

8/29/07 JRM 24.0 27.0 121.6 (1.8) 2,294 65.3 (5.8) 474.5 (41.8) 7.3

8/29/07 BM0 24.0 27.0 39.6 (4.8) 1,739 41.5 (2.6) 311.3 (63.8) 7.5

9/5/07 PP 21.2 28.3 56.5 (0.6) 2,660 80.2 (4.8) 1,090.1 (110.3) 13.6

9/5/07 GSB 22.2 28.4 44.8 (0.8) 2,440 78.4 (8.1) 733.2 (98.1) 9.4

9/6/07 LC1 23.9 27.0 13.1 (0.5) 888 29.4 (3.6) 175.8 (30.0) 6.0

9/6/07 LC2 24.2 27.2 28.2 (1.5) 1,813 50.3 (5.2) 425.5 (59.0) 8.5

9/6/07 JRB 18.9 28.0 81.5 (2.0) 680 36.3 (3.6) 330.1 (41.5) 9.1

Station locations are shown in Fig. 1. Standard deviations are in parentheses

Estuaries and Coasts

milliliter in 2006, and during the 2007 bloom reached 28,120and 31,000 cells per milliliter in the Elizabeth and Jamesrivers, respectively, with high concentrations extending allthe way to the Virginia ocean front. Further, C. polykrikoidesconsistently represented almost half to 90% of the micro-phytoplankton enumerated during bloom events (Fig. 5).

During the past 4 years, C. polykrikoides blooms havealso become annual events in the Peconic Estuary andShinnecock Estuary, NY, USA. Blooms have occurredduring late summer (2004–2007) when temperatures andsalinities ranged from 20°C to 25°C and 22°C to 30°C,respectively (Gobler et al. 2008), temperatures cooler thanbut salinities comparable to those observed during the 2007lower Chesapeake Bay bloom (Table 1). Bloom patches inNY have reached cell densities exceeding 104 mL−1 andChl a levels in excess of 100 µg L−1, comparable to thoseobserved in the Chesapeake Bay tributaries during 2007.

Cysts may be key to bloom formation and transport of thisorganism. Tyler et al. (1982) described how cysts from thedinoflagellate Gyrodinium uncatenum were deposited in the

Potomac River sediment, a Chesapeake Bay tributary, andsubsequently produced future generations of cells in the watercolumn there. A similar mechanism may be invoked forbloom-producing cells of C. polykrikoides. There is evidencethat cysts of this organism are deposited in the surfacesediments of several of the lower Chesapeake Bay tributar-ies, thereby providing a local source or “seed bed” for newgenerations of cells (Seaborn and Marshall 2008). C.polykrikoides cells originating from different sources withinthe Chesapeake Bay ecosystem may spread and causeblooms elsewhere as a result of current and tidal transport.

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6000

8000

10000

12000

14000

LAF ER CH JRM BM0 PP GSB LC1 LC2 JRB

Cel

l num

ber

(cel

ls m

l -1)

Cryptomonas

Diatoms

Other dinoflagellates

C. polykrikoides

*

* *

Fig. 5 Abundance of Cochlodinium sp. relative to co-occurringphytoplankton in samples collected during the bloom period. Theasterisks indicate stations where only Cochlodinium sp. wereenumerated

Fig. 6 Phylogenetic cladogramshowing the similarity of thehypervariable D1–D3 region ofthe large subunit of the ribo-somal DNA for Virginia C.polykrikoides bloom samples, ascompared to other globally iso-lated strains of C. polykrikoidesand C. fulvescens. Accessionnumbers for each strain andscores representing percent sim-ilarity to the Virginia isolate areshown

0

50100

150

200250

300

350400

450

8/1 8/11 8/21 8/31 9/10 9/20 9/30

Chl

orop

hyll

a (

µg

l -1) A

0

50100

150

200250

300

350400

450

8/1 8/11 8/21 8/31 9/10 9/20 9/30

Chl

orop

hyll

a (

µg

l -1) B

Fig. 7 Chl a concentrations at two fixed shallow-water stations,James River at Wythe Point (a) and James River at James RiverCountry Club (b) monitored by VECOS, and in areas impacted by the2007 C. polykrikoides bloom. Locations for these two stations areindicated with a red star in the upper left and right panels in Fig. 2

Estuaries and Coasts

The production of cysts by C. polykrikoides may play akey role in understanding the ecology and dispersal of thisspecies in both NY and VA (Anderson et al. 2003; Seabornand Marshall 2008). Ovoid cysts covered with fin-likeprojections were first observed after a bloom of C. cf.polykrikoides in Puerto Quetzale, Guatemala (Rosales-Loessener et al. 1996). Subsequently, cysts from other fieldsamples have also been identified as being C. cf. poly-krikoides (Matsuoka and Fukuyo 2000). Cysts were

successfully regenerated from motile cells produced in thelaboratory from hyaline cysts of C. polykrikoides cellscollected from Korean coastal water, after a 6-monthstorage at 4°C (Kim et al. 2002). More recently, Kim etal. (2007) observed resting cysts of C. polykrikoides strainsfrom Korean coastal waters in both laboratory cultures andfield samples. Although it has been demonstrated that theNorth American and Korean strains of C. polykrikoides aredifferent “ribotypes” (Iwataki et al. 2007; Fig. 6), compa-

Fig. 8 Hydrography during aChesapeake Bay mouth transect(BMT in Fig. 1) conductedduring the 2007 C. polykrikoidesbloom. The South to Northtransect appears from left toright with station numbers in-creasing in a northerly directionbeginning with 0 at the south(station BM0 in Fig. 1) andnumbered consecutively 0 to 18.The intense Chl a patch can beseen at the southern reach of thistransect

Table 2 Dissolved inorganic and organic nutrient constituents during the 2007 C. polykrikoides bloom

Date Location NO3−

(μmol L−1)NO2

−

(μmol L−1)NH4

+

(μmol L−1)PO4

3−

(μmol L−1)Urea(μmol L−1)

DON(μmol L−1)

SiO2

(μmol L−1)DFAA(μmol L−1)

8/11/07 LAF 0.58 (0.02) 0.27 (0.02) 0.73 (0.01) 0.87 (0.02) 0.23 (0.00) 13.3 35.3 (7.3) 0.38 (0.01)

8/17/07 ER 0.58 (0.00) 0.28 (0.00) 0.76 (0.02) 1.36 (0.01) 0.23 (0.01) 24.4 42.9 (0.6) 0.69 (0.01)

8/29/07 CH 2.00 (0.09) 0.14 (0.00) 0.09 (0.02) 0.32 (0.05) 0.37 (0.04) 9.1 29.7 (14.8) 0.21 (0.00)

8/29/07 JRM 2.17 (0.08) 0.09 (0.00) 0.10 (0.01) 0.48 (0.00) 0.41 (0.01) 5.5 28.2 (1.4) 0.32 (0.02)

8/29/07 BM0 0.48 (0.01) 0.25 (0.01) 0.10 (0.01) 0.48 (0.07) 0.09 (0.00) 13.3 28.6 (0.1) 0.25 (0.01)

9/5/07 PP 0.43 (0.01) 0.31 (0.01) 0.28 (0.01) 1.21 (0.01) 0.25 (0.02) 18.4 58.6 (7.9) 0.31 (0.01)

9/5/07 GSB 1.74 (0.00) 0.22 (0.00) 0.44 (0.01) 3.83 (0.02) 0.38 (0.02) 23.0 99.0 (4.9) 0.33 (0.00)

9/6/07 LC1 0.37 (0.00) 0.36 (0.00) 0.23 (0.05) 0.56 (0.01) 0.16 (0.03) 17.8 37.4 (1.9) 0.33 (0.03)

9/6/07 LC2 0.39 (0.01) 0.47 (0.01) 0.16 (0.02) 0.35 (0.00) 0.38 (0.01) 14.8 35.7 (1.0) 0.53 (0.02)

9/6/07 JRB 0.45 (0.00) 0.66 (0.00) 0.15 (0.02) 0.61 (0.00) 0.49 (0.01) 22.2 39.4 (1.6) 0.49 (0.02)

Station locations are shown in Fig. 1. Standard deviations are in parentheses

Estuaries and Coasts

rable studies of the presence of resting cysts in the NorthAmerican “ribotype” of C. polykrikoides are crucial forunderstanding blooms of this organism. Sediment recordshave already established the abundance of Cochlodiniumdinocysts in the lower Chesapeake Bay tributaries (Seabornand Marshall 2008). We know little about the presence ofresting cysts and their distribution in field sediments, whichis critical in understanding the population dynamics andbloom initiation for this cyst-forming species.

Although C. polykrikoides has been studied extensivelyin Asia, sequencing of a hypervariable region of the large

subunit rDNA suggests that North American C. polykri-koides strains are similar to each other but have lowsimilarity to most strains from Southeast Asia (88–92%;Gobler et al. 2008). The exceptions were the strains fromMalaysia, which are 100% similar to the North Americanstrains in the D1–D2 region of the large subunit of theribosome. This positive species identification is important,as recent research has demonstrated that multiple Cochlo-dinium blooms on the west coast of North America that hadbeen thought to be caused by C. polykrikoides are in factcaused by a new species of Cochlodinium, Cochlodinium

0

2

4

6

8

10

LAF ER CH JRM BM0 PP GSB LC1 LC2 JRB

N U

ptak

e (

µmo

l L

-1

h

-1

)

DFAA

Urea

Nitrite

Nitrate

Ammonium

A

0

5

10

15

20

25

LAF ER CH JRM BM0 PP GSB LC1 LC2 JRB

C U

ptak

e (

µmo

l L

-1

h

-1

)

DFAA

Urea

Bicarbonate

B

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

LAF ER CH JRM BM0 PP GSB LC1 LC2 JRB

N U

ptak

e (h

-1

)

DFAA

Urea

Nitrite

Nitrate

Ammonium

C

0

0.01

0.02

0.03

0.04

0.05

0.06

LAF ER CH JRM BM0 PP GSB LC1 LC2 JRB

C U

ptak

e (h

-1

)

DFAA

Urea

Bicarbonate

D

Fig. 9 Measured volumetric nitrogen (a) and carbon (c) uptake within Cochlodinium polykrikoides bloom patches on multiple dates. Panels onthe right are N-specific uptake rates (b) and C-specific bicarbonate uptake rates (d)

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0 50 100 150 200 250

PN (µmol L-1

)

Tot

al N

upt

ake

(h-1

)

A

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0 500 1000 1500 2000

PC (µmol L-1

)

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arbo

nate

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ake

(h-1

)

B

0

0.01

0.02

0.03

0.04

0.05

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0.07

0 100 200 300 400

Chl a (µg L-1

)

Tot

al N

upt

ake

(h-1

)

C

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0 100 200 300 400

Chl a (µmol L-1

)

Bic

arbo

nate

upt

ake

(h-1

)

D

Fig. 10 The relationship be-tween total specific N uptakeand PN (a) and Chl a (c). Therelationships between C-specificbicarbonate uptake and PC (b)and Chl a (d)

Estuaries and Coasts

fulvescens (Iwataki et al. 2007, 2008). The Virginia andother North American C. polykrikoides isolates are only77–78% genetically similar to C. fulvescens strains isolatedfrom North America. The genetic differences amongspecies and strains are likely to translate into importantecological differences among bloom populations.

Little is known about the nutrition of Cochlodiniumspecies. The low DIN to DIP ratio during these events (~2;Table 2) suggests that N was the limiting nutrient duringthese blooms. Our study is the first to measure N and Cuptake by populations dominated by these species anddemonstrates that, at least during blooms, plankton pop-ulations dominated by C. polykrikoides take advantage of avariety of inorganic and organic nitrogen sources (Fig. 9),similar to other bloom-forming dinoflagellates (Glibert etal. 2001). The extremely high biomass achieved duringblooms clearly suggests that there is a large nutrientdemand during these events; however, no one N sourceappeared to be responsible for the observed bloom in theLower Chesapeake Bay region. This finding is consistentwith a recent study of C. fulvescens blooms in Californiacoastal waters, during which both inorganic (nitrate,ammonium) and organic (urea) nitrogen sources wereassimilated (Kudela et al. 2008). During the 2007 bloomin the Chesapeake Bay, organic N represented 25% to 69%of the total measured N uptake. Combined with theobserved turnover times for peptide hydrolysis (Fig. 11),organic nitrogen is suspected to contribute substantially tothe maintenance of blooms. This hypothesis is consistentwith the availability of nutrients, as inorganic forms weregenerally present at submicromolar levels, while DONconcentrations averaged more than 16 µM (Table 2).

Similarly, photosynthetic C fixation appeared to beinsufficient for meeting cellular C demand at times(assuming Redfield uptake) and uptake of C from organiccompounds could alleviate some of the observed imbalancebetween N and C uptake (Fig. 9). While N-specificdoubling times fell within a relatively narrow range,

between 0.48 and 1.30 days, C-specific doubling timesestimated from rates of photosynthetic uptake of bicarbon-ate were much longer and more variable, ranging from 1.1to 217 days. Because bicarbonate uptake decreased withincreasing biomass (Fig. 10), C-specific doubling timeswere far higher (range 15.9 to 217 days) at the samplingtimes and locations where the highest biomass wasmeasured (stations LAF, ER, PP, and GSB). C. polykri-koides abundance was highest at stations ER and LAF(Fig. 5, Table 1). While C biomass at PP and GSB was alsohigh (Table 1), the C to N ratio was high suggesting thatdetrital material may have been abundant. At these twostations, even though Chl a concentrations were elevated,C. polykrikoides abundance was lower and so nonphoto-synthetic phytoplankton and other microbes not enumeratedduring this study may have contributed to the high N-specific doubling times. At high biomass levels, such asthose achieved at the four highest-biomass sample loca-tions, light can also limit photosynthesis.

Most of the bloom-forming photosynthetic dinoflagel-lates in the Chesapeake Bay can ingest other cells andhence are mixotrophic (Stoecker 1999). It has recently beenshown that Asian strains of Cochlodinium are capable ofphagotrophy, including grazing on small flagellates, cyano-bacteria, and cryptophytes (Jeong et al. 2004, 2005; Seonget al. 2006). Each of these prey categories are commoncomponents within Chesapeake Bay and its tributaries;concentrations of Cryptomonas present were quite highduring the start of this bloom event (Fig. 5). Highconcentrations of autotrophic picoplankton are also com-mon to these waters (Marshall 1995b; Marshall et al. 2006)but were not enumerated as part of this study. We knowlittle about the contribution of phagotrophy to the total Nand C nutrition of this, or most, organisms, nor howmixotrophy might contribute to the success of Cochlodi-

0

0.5

1

1.5

2

2.5

JRB LC1 LC2 Non-Bloom

Pep

tide

hyd

roly

sis

(d -1

)

Fig. 11 Turnover times for peptide hydrolysis during C. polykrikoidesbloom sampling conducted aboard the R/V Fay Slover on 6 Sept 2007

0

25

50

75

100

0 5 10 15Time (h)

Per

cent

mor

tali

ty

James River Bridge

Little Creek

Pig Point

Flitered Pig Point water

Fig. 12 Larval fish mortality after exposure to natural water samplescollected from the lower Chesapeake Bay system containing bloomconcentrations of C. polykrikoides on 6 Sept 2007

Estuaries and Coasts

nium. However, if C. polykrikoides cells were obtainingcellular C via phagotrophy during this bloom event, itmight account for the observed imbalance in C and N. Inaddition to phagotrophy, osmotrophic uptake of dissolvedorganic C compounds may also be an important source ofC. Our limited results suggest that C. polykrikoides can alsoassimilate C from urea and amino acids as well (Fig. 9).Consequently, any assessment of the C or N budget for thisorganism should include phagotrophy and osmotrophy.

The impacts of C. polykrikoides blooms in theChesapeake Bay and its tributaries may be severe. Weobserved rapid and complete mortality of juvenile fishand 20% mortality of juvenile oysters during short-termincubations within bloom water (Fig. 12). Similarly,bioassay experiments during a Peconic Estuary (NY, USA)bloom demonstrated that 24-h exposures to bloom water(>104 cells per milliliter) killed 100% of multiple fishspecies (1-week-old sheepshead minnow, C. variegates;adult striped mummichog, Fundulus majalis; adult silver-side, Menidia menidia; and adult common mummichog,Fundulus heteroclitus) and 9-day exposures to bloomwater yielded 20% mortality in C. virginica, the nativeoyster in the Chesapeake Bay (Gobler et al. 2008). Thesurvival of fish and oysters exposed to 0.2-µm-filteredwater (Fig. 12) or freeze-killed cells (Gobler et al. 2008)suggests that the toxic agent is associated with live C.polykrikoides cells. These results are consistent with thoseof Ho and Zubkoff (1979) who also found that removingcells from bloom water mitigated cellular toxicity. Givenour current results, the emergence and proliferation ofintense C. polykrikoides blooms in Chesapeake Bay maybe highly detrimental to the substantial efforts being madeto restore the native oyster fishery there. In fact, theblooms in 2007 coincided with major oyster plantingprojects organized by the Chesapeake Bay Foundation.

Uptake results suggest that no one N source wasresponsible for the observed Cochlodinium sp. growth.Both organic and inorganic N were taken up duringblooms. In addition, organic C from these compounds wasalso taken up indicating that uptake of dissolved organiccarbon may contribute to cellular C demands. The generalnutrient characteristics of this C. polykrikoides bloomcoupled with the prevailing nutrient and hydrologicaldynamics of the lower Chesapeake Bay during 2007 mayhave created a niche that favored dominance by thisspecies. While river flow rates were high during the springand early summer of 2007, the drought period whichfollowed (Fig. 3) likely led to a dramatic reduction innutrient supply to this region (as was reflected in the lowlevels of nitrogenous nutrients; Table 2) and consequentlyN-limiting conditions (as evidenced by the low DIN:DIPratios; Table 2). Under such conditions, a species capable ofutilizing organic nitrogen compounds (Figs. 9 and 10) and

perhaps exploiting phytoplankton prey phagotrophically(Jeong et al. 2004, 2005; Seong et al. 2006) would have aclear advantage over competing autotrophic phytoplanktonor even nonphagotrophic mixotrophs (i.e., osmotrophicphytoplankton). Given the ability of this species to rapidlykill grazers such as planktivorous fish (Fig. 12), shellfishlarvae (Ho and Zubkoff 1979), and probably zooplankton,this species may also gain dominance among the planktonby avoiding predation losses. Together, these physical,chemical, and biological factors all likely contributed to thelarge C. polykrikoides bloom in the lower Chesapeake Bayduring 2007.

No research program aimed at understanding ecosystemimpacts and algal bloom dynamics in general can hope toprovide forecasts for the future without considering climatechange (Pyke et al. 2008). Phytoplankton production andspecies composition generally follow predictable seasonalpatterns dictated primarily by annual river flow, light, andtemperature patterns (Malone et al. 1996; Marshall andNesius 1996) but these may change in unexpected waysunder future CO2 and temperature scenarios (Pyke et al.2008). In the Chesapeake Bay, blooms of Cochlodiniumappear to occur predominantly during the summer and earlyautumn when water temperatures are warm (Table 1;Marshall 1995a) but representatives of this genus may alsobloom in cooler waters (Kudela et al. 2008). Monitoringrecords (beginning in 1985) of phytoplankton in the lowerChesapeake Bay and its tidal rivers along its southwesternshoreline indicate that C. polykrikoides and blooms of thisorganism typically occur in this region from mid-Augustinto early October when water temperatures are the highest(Marshall, unpublished data). If global warming causeschanges in the timing of blooms as a result of warmer watertemperatures earlier in the year, blooms may impinge uponlarval recruitment of various fauna within this estuarinesystem and set back the attainment of restoration goals forthe Chesapeake Bay. Further, we also observed that thetiming of heavy rainfall events after the protracted 2007drought coincided with blooms (Figs. 3 and 4). Blooms ofCochlodinium may be more common under future climatescenarios due to projected changes in both the timing andintensity of precipitation (e.g., more rainfall during shorterintense events), temperature, and CO2 and are likely toexert greater impacts on ecosystems if blooms occur earlierand during larval recruitment seasons.

Clearly, US east coast estuaries (e.g., Peconic estuary,NY, USA and Chesapeake Bay and its tributaries, VA,USA) are experiencing intense C. polykrikoides blooms thatare likely to be impacting local fisheries both now and intothe future. Multiple research and managerial questions havearisen regarding these blooms as little research has beendone on blooms of this species in North America to date.Nutrients, grazing on and/or by C. polykrikoides, and

Estuaries and Coasts

physical dispersal may be key controls of Cochlodiniumblooms and have yet to be thoroughly examined.

Acknowledgements Funding was provided by the National Oceanicand Atmospheric Administration, Center for Sponsored Coastal OceanResearch Event Response Program. We thank the Hampton RoadsSanitation District and Virginia Department of Environmental Qualityfor supporting DATAFLOW mapping and shallow-water continuousmonitoring efforts. This work was also funded through grants from theVirginia Environmental Endowment to MRM.

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