@article{burkholder_kinder_allen_2023, title={Watershed Development and Eutrophying Potable Source-Water Reservoirs in a Warming Temperate/Subtropical Region}, volume={15}, ISSN={["2073-4441"]}, DOI={10.3390/w15224007}, abstractNote={Reservoirs are increasingly valuable worldwide as potable source waters, yet in many geographic regions, their limnology and trophic status are poorly known. We characterized 14 drinking water reservoirs and their watersheds across the warming temperate/subtropical southeastern USA. Selected reservoirs had at least three years of accessible summer water quality data during 2010–2020, including Secchi depth, nutrients, and algal biomass as chlorophyll a, and depth profiles for temperature and dissolved oxygen. Most watersheds, including lands within a 10-km radius of the reservoirs, had sustained substantial urbanization and/or intensive industrialized animal production, in some cases including the discharge of partially treated human sewage or livestock slaughterhouse wastes near or into the reservoirs. Five reservoirs were assessed as mesotrophic; the others were eutrophic. Most were stratified, but ephemeral near-surface thermoclines were common, and many were too shallow (median depth 5.0 m) to maintain uniform temperatures in the relatively warm hypolimnia. Bottom-water hypoxia/anoxia occurred throughout the summers but, surprisingly, in 8 of 14 reservoirs hypoxia commonly extended to surface waters. In the Southeast as in many regions, drinking water reservoirs are poorly protected and degrading as livestock production and/or urban development increasingly characterize their watersheds. The eutrophication trajectory of these valuable resources should be used as an indicator of ecosystem health and water quality in developing more protective management and policy actions.}, number={22}, journal={WATER}, author={Burkholder, JoAnn M. and Kinder, Carol A. and Allen, Elle H.}, year={2023}, month={Nov} }
 @article{burkholder_kinder_dickey_reed_arellano_james_mackenzie_allen_lindor_mathis_et al._2022, title={Classic indicators and diel dissolved oxygen versus trend analysis in assessing eutrophication of potable-water reservoirs}, volume={3}, ISSN={["1939-5582"]}, DOI={10.1002/eap.2541}, abstractNote={Abstract}, journal={ECOLOGICAL APPLICATIONS}, author={Burkholder, JoAnn M. and Kinder, Carol A. and Dickey, David A. and Reed, Robert E. and Arellano, Consuelo and James, Jennifer L. and Mackenzie, Linda M. and Allen, Elle H. and Lindor, Nicole L. and Mathis, Joshua G. and et al.}, year={2022}, month={Mar} }
 @article{huang_pietrasiak_gobler_johansen_burkholder_d'antonio_zimba_2021, title={Diversity of bioactive compound content across 71 genera of marine, freshwater, and terrestrial cyanobacteria}, volume={109}, ISSN={["1878-1470"]}, DOI={10.1016/j.hal.2021.102116}, abstractNote={Cyanobacterial blooms have increased in frequency, distribution, and intensity due to climate change and anthropogenic nutrient input. The release of bioactive compounds accumulated in these blooms can affect the health of humans and the environment. The co-occurrence of bioactive metabolites is well-documented in bloom samples from marine and freshwater ecosystems, with fewer reports from unialgal isolates. Cyanobacteria also are important terrestrial ecosystem components, especially in drylands, but reports on bioactive molecules from terrestrial cyanobacteria are sparse. This study determined bioactive metabolite profiles for 71 genera of cyanobacteria from seven orders isolated from freshwater (12 genera), marine (15 genera), and terrestrial (44 genera) habitats originally. Cultures were harvested for bioactive metabolites when entering the late-exponential phase for all 157 strains, and 33 were sampled at both early and late exponential phases. Bioactive metabolites were analyzed using an ultra high performance/pressure liquid chromatography in-line with a time-of-flight mass spectrometer. Overall, 12 bioactive classes of the 28 identified were ubiquitous in all samples. On average, each freshwater genus produced ∼12 bioactive classes, whereas each marine genus contained > 4 bioactive classes, and each terrestrial genus contained ∼6 bioactive classes. While 10 of 12 freshwater genera produced at least 10 bioactive classes, only a single genus each from marine and terrestrial habitats had the same number of bioactive classed accumulated. Aeruginosin was found in 58 of 71 total genera, carmabin in 51 of 71 genera, and anabaenopeptin in 48 of 71 genera. Chemotaxonomic use of these secondary metabolites may help resolve higher-level genetic classification(s). An additional growth curve experiment showed that bioactive metabolites were produced at both early and late exponential growth phases. The bioactive metabolite accumulation pattern between early and late exponential phases differed by bioactive classes, genera, and habitats. This survey of 55 bioactive classes in cyanobacteria isolated from freshwater, marine, and terrestrial habitats (71 genera) provides as one of the first systematic bioactive metabolite profiles for cyanobacteria, which should be useful in environmental and drinking water management. Further, it offers novel insights about the toxin potential of selected terrestrial cyanobacteria.}, journal={HARMFUL ALGAE}, author={Huang, I-Shuo and Pietrasiak, Nicole and Gobler, Christopher J. and Johansen, Jeffrey R. and Burkholder, JoAnn M. and D'Antonio, Sue and Zimba, Paul V}, year={2021}, month={Nov} }
 @article{mallin_burkholder_cahoon_grogan_sanger_smith_2019, title={An Environmental Assessment of the North and South Carolina Coasts}, ISBN={["978-0-12-805068-2"]}, DOI={10.1016/B978-0-12-805068-2.00044-9}, abstractNote={Coastal waters of the Carolinas include some magnificent resources that are increasingly degraded from upstream pollution and poorly controlled local development. Escalating water demands have rapidly depressed coastal water tables. Upper watershed industries, cities, and croplands contaminate coastal riverine potable source waters with a wide array of unregulated/poorly regulated toxic substances, and with nutrients that fuel nuisance algal blooms. Industrialized livestock production adds fecal microbes and many other contaminants to surface and groundwaters. Toxic cyanobacteria blooms have spread into newly affected freshwaters including potable source waters. Physical reengineering of coastal landscapes with numerous detention ponds has created ideal incubation sites for noxious estuarine/marine dinoflagellate and raphidiophycean blooms. Stormwater runoff from sprawling coastal development alters the sediment composition and benthic fauna of receiving waters, while also adding toxic chemicals and fecal contaminants that threaten human health and seafood safety. These impacts are expected to be exacerbated by warming trends in climate change.}, journal={WORLD SEAS: AN ENVIRONMENTAL EVALUATION, VOL I: EUROPE, THE AMERICAS AND WEST AFRICA, 2ND EDITION}, author={Mallin, Michael and Burkholder, Joann and Cahoon, Lawrence and Grogan, Amy and Sanger, Denise and Smith, Erik}, year={2019}, pages={405–426} }
 @article{shalygin_huang_allen_burkholder_zimba_2019, title={Odorella benthonica gen. & sp. nov. (Pleurocapsales, Cyanobacteria): an odor and prolific toxin producer isolated from a California aqueduct}, volume={55}, ISSN={["1529-8817"]}, DOI={10.1111/jpy.12834}, abstractNote={Pleurocapsales are one of the least understood groups of cyanobacteria in terms of molecular systematics and biochemistry. Considering the high number of cryptic taxa within the Synechococcales and Oscillatoriales, it is likely that such taxa also occur in the Pleurocapsales. The new genus described in our research is the first known pleurocapsalean cryptic taxon. It produces off‐flavor and a large number of bioactive metabolites (n = 38) some of which can be toxic including four known microcystins. Using a polyphasic approach, we propose the establishment of the genus Odorella with the new species O. benthonica from material originally isolated from the California Aqueduct near Los Angeles.}, number={3}, journal={JOURNAL OF PHYCOLOGY}, author={Shalygin, Sergei and Huang, I-Shuo and Allen, Elle H. and Burkholder, JoAnn M. and Zimba, Paul V.}, year={2019}, month={Jun}, pages={509–520} }
 @article{leles_mitra_flynn_tillmann_stoecker_jeong_burkholder_hansen_caron_glibert_et al._2019, title={Sampling bias misrepresents the biogeographical significance of constitutive mixotrophs across global oceans}, volume={28}, ISSN={["1466-8238"]}, DOI={10.1111/geb.12853}, abstractNote={Abstract}, number={4}, journal={GLOBAL ECOLOGY AND BIOGEOGRAPHY}, author={Leles, Suzana Goncalves and Mitra, Aditee and Flynn, Kevin John and Tillmann, Urban and Stoecker, Diane and Jeong, Hae Jin and Burkholder, JoAnn and Hansen, Per Juel and Caron, David A. and Glibert, Patricia M. and et al.}, year={2019}, month={Apr}, pages={418–428} }
 @article{flood_burkholder_cope_2018, title={Assessment of atrazine toxicity to the estuarine phytoplankter, Dunaliella tertiolecta (Chlorophyta), under varying nutrient conditions}, volume={25}, ISSN={["1614-7499"]}, DOI={10.1007/s11356-018-1310-1}, abstractNote={Anthropogenic inputs of chemical environmental contaminants are frequently associated with developing harmful algal blooms, but little is known about how estuarine phytoplankton assemblages respond to multiple, co-occurring chemical stressors in chronically disturbed habitats. The goals of this research were to establish a robust protocol for testing the effects of atrazine on estuarine phytoplankton, and then to use that protocol to compare the effects of atrazine exposure with and without nutrient enrichment on a cosmopolitan estuarine/marine alga, Dunaliella tertiolecta (Chlorophyta). Atrazine sensitivity in nutrient-replete media (96-h growth inhibition [Formula: see text]) was 159.16 μg l -1 , but sensitivity was influenced by exposure duration, and inhibitory effects of herbicide on algal growth decreased under imbalanced nutrient regimes and low nitrogen and phosphorus supplies. These findings advance knowledge about how nutrient regimes and herbicides interact to control estuarine phytoplankton population dynamics.}, number={12}, journal={ENVIRONMENTAL SCIENCE AND POLLUTION RESEARCH}, author={Flood, Stacie and Burkholder, JoAnn and Cope, Greg}, year={2018}, month={Apr}, pages={11409–11423} }
 @article{flood_burkholder_2018, title={Chattonella subsalsa (Raphidophyceae) growth and hemolytic activity in response to agriculturally-derived estuarine contaminants}, volume={76}, ISSN={["1878-1470"]}, DOI={10.1016/j.hal.2018.05.006}, abstractNote={The potential for toxic contaminants and nutrient pollution to alter natural cycles of estuarine phytoplankton blooms is well known, yet few studies have examined how these combined stressors affect harmful algal species. Here, a robust testing protocol was developed to enable an ecotoxicological assessment of responses to commonly co-occurring estuarine contaminants by harmful algal bloom species. The population growth and toxicity (as cell density and hemolytic activity, respectively) of a cultured strain of the toxigenic raphidiophycean, Chattonella subsalsa, were assessed in two experiments (duration 10 days and 28 days) across a gradient of atrazine concentrations and N:P ratios simulating nutrient-rich versus nutrient-depleted regimes. The response of this large-celled, slowly growing alga to atrazine × nutrients depended on growth phase; atrazine was most inhibitory during early exponential population growth (day 10), whereas nutrient regime was a more important influence during later phases of growth (day 28). Without atrazine, toxicity toward fish was highest in low-P cultures. At atrazine levels >25 μg L-1, hemolytic activity was highest in low-N cultures, and increased with increasing atrazine concentration in all nutrient-limited cultures. Hemolytic activity varied inversely with atrazine concentration in N,P-replete conditions. Overall, atrazine inhibitory effects on population growth of this C. subsalsa strain depended on the growth phase and the nutrient regime; hemolytic activity was higher and further enhanced by atrazine in low N-P regimes; and atrazine inhibited hemolytic activity in nutrient-replete conditions. The data suggest that, depending on the growth phase and nutrient regime, atrazine can help promote toxic C. subsalsa blooms.}, journal={HARMFUL ALGAE}, author={Flood, Stacie L. and Burkholder, JoAnn M.}, year={2018}, month={Jun}, pages={66–79} }
 @book{shumway_burkholder_morton_2018, place={Hoboken, NJ}, title={Harmful Algal Blooms: a compendium desk reference}, ISBN={9781118994672}, url={http://dx.doi.org/10.1002/9781118994672}, DOI={10.1002/9781118994672}, abstractNote={It is recommended that a long term Lake Management Plans be developed to effectively manage lake issues. All stakeholders should participate in the development of the plan and include homeowners, recreational users, lake management associations, park districts, townships or any other entity involved in managing Lake Louise. Lake Management plans should educate the public about specific lake issues, provide a concise assessment of the problem, outline methods and techniques that will be employed to control the problems and clearly define the goals of the program. Mechanisms for monitoring and evaluation should be developed as well and information gathered during these efforts should be used to implement management efforts ( Biology and Control of Aquatic Plants, Gettys et al., 2009).}, publisher={Wiley}, year={2018}, month={Jun} }
 @article{flood_burkholder_2018, title={Imbalanced nutrient regimes increase Prymnesium parvum resilience to herbicide exposure}, volume={75}, ISSN={1568-9883}, url={http://dx.doi.org/10.1016/J.HAL.2018.04.006}, DOI={10.1016/J.HAL.2018.04.006}, abstractNote={The toxigenic haptophyte Prymnesium parvum is a mixotrophic phytoplankter with an extensive historic record of forming nearly monospecific, high-biomass, ecosystem-disrupting blooms, and it has been responsible for major fish kills in brackish waters and aquaculture facilities in many regions of the world. Little is known about how this species responds to commonly occurring environmental contaminants, or how nutrient (nitrogen, phosphorus) pollution may interact with environmentally relevant pesticide exposures to affect this harmful algal species. Here, standard algal toxicity bioassays from pesticide hazard assessments were used along with modified erythrocyte lysis assays to evaluate how atrazine exposures, imbalanced nutrient supplies, and salinity interact to influence the growth and toxicity in P. parvum isolates from three different regions. In nutrient-replete media, P. parvum 96 h IC50s ranged from 73.0 to 88.3 μg atrazine L−1 at salinity 10 and from 118 to >200 μg atrazine μg L−1 at salinity 20, and the response depended on the strain and the test duration. Relative hemolytic activity, used as an indication of toxicity, was a function of herbicide exposure, nutrient availability, salinity, geographic origin, and interactions among these factors. Highest levels of hemolytic activity were measured from a South Carolina strain in low-nitrogen media with high atrazine concentrations. Herbicide concentration was related to relative hemolytic activity, although a consistent relationship between growth phase and toxicity was not observed. Overall, these findings suggest that increasing chemical contamination is helping to promote ecosystem-disruptive, strongly mixotrophic algal blooms.}, journal={Harmful Algae}, publisher={Elsevier BV}, author={Flood, Stacie L. and Burkholder, JoAnn M.}, year={2018}, month={May}, pages={57–74} }
 @article{flynn_mitra_glibert_burkholder_2018, title={Mixotrophy in Harmful Algal Blooms: By Whom, on Whom, When, Why, and What Next}, volume={232}, ISBN={["978-3-319-70068-7"]}, ISSN={["0070-8356"]}, DOI={10.1007/978-3-319-70069-4_7}, abstractNote={The traditional view of the planktonic food web is simplistic: nutrients are consumed by phytoplankton that, in turn, support zooplankton, which ultimately support fish. This structure is the foundation of most models used to explore fisheries production, biogeochemical cycling, and climate change. In recent years, however, the importance of mixotrophs increasingly has been recognized. Mixotrophy, the combination of phototrophy and heterotrophy (the latter, including phago- and/or osmotrophy), enables planktonic protists traditionally labeled as “phytoplankton” or “microzooplankton” to function at multiple trophic levels. Mixotrophy enables primary producers to acquire nutrients directly from ingestion of prey such as bacteria and algal competitors and even from their own potential predators. Mixotrophy is not simply additive or substitutional; rather, it is synergistic. While most harmful algal species (except diatoms and cyanobacteria) are mixotrophic via phagotrophy, little is known about how these organisms modulate their phototrophic and phagotrophic activities or how the flow of energy and material through mixotrophic predator-prey interactions is altered under varying nutrient, temperature, light, pH, or pCO2 conditions. All of these factors are also rapidly changing in coastal and oceanic environments with accelerating eutrophication and climate change that, in turn, alters the potential for harmful algal blooms. Accurate parameterization, including consideration of mixotrophy in water quality or fisheries models that are used as aids to regional and/or international policy development, should be a high priority.}, journal={GLOBAL ECOLOGY AND OCEANOGRAPHY OF HARMFUL ALGAL BLOOMS}, author={Flynn, Kevin J. and Mitra, Aditee and Glibert, Patricia M. and Burkholder, JoAnn M.}, year={2018}, pages={113–132} }
 @article{byrd_burkholder_zimba_2017, title={Environmental stressors and lipid production by Dunaliella spp. I. Salinity}, volume={487}, ISSN={["1879-1697"]}, DOI={10.1016/j.jembe.2016.11.004}, abstractNote={Fourteen strains within four species of the marine chlorophyte genus, Dunaliella were assessed for their potential utility in sustainable biofuel production by tracking lipid production under salinity stress. A modified technique with Nile Red stain was used to screen cultures rapidly for the presence of neutral lipid content. Promising strains with visually high lipid content and high growth as cell production were selected to enhance lipid production using high salinity (hyperosmotic) stress in short-term (s to h) and long-term (≥ 24 h) bench-scale experiments (culture volume 0.1 to 3.5 L). These strains were also grown at mass culture scale (culture volume ~ 150 to 175 L). The difference in experimental scale was imposed because of the container effects shown for various algae, and in recognition of the importance of scale-up feasibility in harnessing algae for biofuel production. Saponifiable lipids were converted to fatty acid methyl esters, here referred to as total fatty acids (FAs), by direct transesterification. High salinity stress generally resulted in maximal total fatty acid (FA) content (up to 65% by dry weight) in comparison to controls (~ 10–25% total FAs by dry weight). Glycerol production, a known mechanism of osmoregulation in Dunaliella, was measured in a short-term salinity stress experiment on a promising strain and found to increase significantly 30 min to 24 h after exposure to high salinity. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to evaluate the relative expression of glyceryl-3-phosphate dehydrogenase (GPDH), one of the primary glycerol biosynthesis genes for glycerol production, during a short-term experiment with high salinity stress. GPDH was significantly expressed (≥ 2-fold when compared to the endogenous gene ACTIN) 30 min after exposure and continued to be expressed for 2 h. In general, when cellular glycerol content was low, total FAs increased as an immediate or short-term response (30 s to 30 min) to hyperosmotic stress. Responses were strain-specific and indicated both inter- and intraspecific variation. Overall, a simple high salinity adjustment significantly increased lipid production in selected strains of Dunaliella spp. The data suggest that these Dunaliella strains may incorporate a portion of the available glycerol as triacylglycerols (TAGs) or neutral lipids under short-term high salinity stress.}, journal={JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY}, author={Byrd, Stephanie Mixson and Burkholder, JoAnn M. and Zimba, Paul V.}, year={2017}, month={Feb}, pages={18–32} }
 @article{byrd_burkholder_2017, title={Environmental stressors and lipid production in Dunaliella spp. II. Nutrients, pH, and light under optimal or low salinity}, volume={487}, ISSN={["1879-1697"]}, DOI={10.1016/j.jembe.2016.11.006}, abstractNote={Fourteen strains within four species of the halophytic microalgal genus, Dunaliella, were rapidly screened for cellular lipid content during senescence using an adapted Nile Red technique. Four promising strains that exhibited both rapid population growth and high lipid content, including three strains of Dunaliella tertiolecta and one strain of D. viridis, were selected to attempt to enhance lipid production as total fatty acids (FAs) in bench-scale cultures (0.5–3.5 L) under environmental stressors including nutrient limitation or deprivation of inorganic nitrogen (nitrate, N) and/or phosphorus (P), pH stress (control—early senescent cultures, versus pH 10 or pH 7), a 12-h: 12-h light:dark (L:D) photoperiod versus continuous light, and enrichment versus no enrichment with carbon dioxide (± 4-h pulse daily of bubbling with CO2). These strains were also tested in mass culture (~ 150–175 L) to assess feasibility of scale-up. All but one strain had minimal lipid production during active growth, and increased lipid production under senescence. The unique strain Dunaliella viridis UTEX ZZ1150 produced high amounts of total FAs during active growth as well as senescence, whether in a 12:12 L:D photoperiod or under continuous light. High pH × low salinity, low pH (7), and continuous light + pulsed CO2 yielded the maximum total FA content of 56%, 43%, and 42% (dry weight basis), respectively, in comparison to controls (~ 10–25% total FAs). Under continuous light ± pulsed CO2, cells likely incorporated the excess carbon as FAs and other products. Low N and/or P under a 12:12 L:D photoperiod or continuous light, and a 12:12 L:D with pulsed CO2, did not significantly enhance total FA production in these strains. High intraspecific and interspecific variation in growth and lipid production were found in Dunaliella in response to each environmental stressor, suggesting fundamental metabolic differences.}, journal={JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY}, author={Byrd, Stephanie Mixson and Burkholder, JoAnn M.}, year={2017}, month={Feb}, pages={33–44} }
 @article{mitra_flynn_tillmann_raven_caron_stoecker_not_hansen_hallegraeff_sanders_et al._2016, title={Defining Planktonic Protist Functional Groups on Mechanisms for Energy and Nutrient Acquisition: Incorporation of Diverse Mixotrophic Strategies}, volume={167}, ISSN={1434-4610}, url={http://dx.doi.org/10.1016/J.PROTIS.2016.01.003}, DOI={10.1016/J.PROTIS.2016.01.003}, abstractNote={Arranging organisms into functional groups aids ecological research by grouping organisms (irrespective of phylogenetic origin) that interact with environmental factors in similar ways. Planktonic protists traditionally have been split between photoautotrophic “phytoplankton” and phagotrophic “microzooplankton”. However, there is a growing recognition of the importance of mixotrophy in euphotic aquatic systems, where many protists often combine photoautotrophic and phagotrophic modes of nutrition. Such organisms do not align with the traditional dichotomy of phytoplankton and microzooplankton. To reflect this understanding, we propose a new functional grouping of planktonic protists in an eco-physiological context: (i) phagoheterotrophs lacking phototrophic capacity, (ii) photoautotrophs lacking phagotrophic capacity, (iii) constitutive mixotrophs (CMs) as phagotrophs with an inherent capacity for phototrophy, and (iv) non-constitutive mixotrophs (NCMs) that acquire their phototrophic capacity by ingesting specific (SNCM) or general non-specific (GNCM) prey. For the first time, we incorporate these functional groups within a foodweb structure and show, using model outputs, that there is scope for significant changes in trophic dynamics depending on the protist functional type description. Accordingly, to better reflect the role of mixotrophy, we recommend that as important tools for explanatory and predictive research, aquatic food-web and biogeochemical models need to redefine the protist groups within their frameworks.}, number={2}, journal={Protist}, publisher={Elsevier BV}, author={Mitra, Aditee and Flynn, Kevin J. and Tillmann, Urban and Raven, John A. and Caron, David and Stoecker, Diane K. and Not, Fabrice and Hansen, Per J. and Hallegraeff, Gustaaf and Sanders, Robert and et al.}, year={2016}, month={Apr}, pages={106–120} }
 @misc{burkholder_2016, title={Marine Benthic Dinoflagellates—Unveiling Their Worldwide Biodiversity. Kleine Senckenberg-Reihe, Volume 54. By Mona Hoppenrath, Shauna A. Murray, Nicolas Chomérat, and Takeo Horiguchi. Stuttgart (Germany): E. Schweizerbart’sche Verlagsbuchhandlung. 19.90€ (paper). 276 p.; ill.; taxonomic index. ISBN: 978-3-510-61402-8. 2014.}, volume={91}, ISSN={0033-5770 1539-7718}, url={http://dx.doi.org/10.1086/689525}, DOI={10.1086/689525}, abstractNote={Previous articleNext article No AccessMicrobiologyMarine Benthic Dinoflagellates—Unveiling Their Worldwide Biodiversity. Kleine Senckenberg-Reihe, Volume 54. By Mona Hoppenrath, Shauna A. Murray, Nicolas Chomérat, and Takeo Horiguchi. Stuttgart (Germany): E. Schweizerbart’sche Verlagsbuchhandlung. 19.90€ (paper). 276 p.; ill.; taxonomic index. ISBN: 978-3-510-61402-8. 2014.JoAnn BurkholderJoAnn BurkholderApplied Ecology, North Carolina State University, Raleigh, North Carolina Search for more articles by this author Applied Ecology, North Carolina State University, Raleigh, North CarolinaPDFPDF PLUSFull Text Add to favoritesDownload CitationTrack CitationsPermissionsReprints Share onFacebookTwitterLinkedInRedditEmail SectionsMoreDetailsFiguresReferencesCited by The Quarterly Review of Biology Volume 91, Number 4December 2016 Published in association with Stony Brook University Article DOIhttps://doi.org/10.1086/689525 Views: 17Total views on this site For permission to reuse, please contact [email protected]PDF download Crossref reports no articles citing this article.}, number={4}, journal={The Quarterly Review of Biology}, publisher={University of Chicago Press}, author={Burkholder, JoAnn}, year={2016}, month={Dec}, pages={518–518} }
 @article{glibert_wilkerson_dugdale_raven_dupont_leavitt_parker_burkholder_kana_2016, title={Pluses and minuses of ammonium and nitrate uptake and assimilation by phytoplankton and implications for productivity and community composition, with emphasis on nitrogen-enriched conditions}, volume={61}, ISSN={["1939-5590"]}, DOI={10.1002/lno.10203}, abstractNote={Anthropogenic activities are altering total nutrient loads to many estuaries and freshwaters, resulting in high loads not only of total nitrogen (N), but in some cases, of chemically reduced forms, notably NH4+ . Long thought to be the preferred form of N for phytoplankton uptake, NH4+ may actually suppress overall growth when concentrations are sufficiently high. NH4+ has been well known to be inhibitory or repressive for NO3‐ uptake and assimilation, but the concentrations of NH4+ that promote vs. repress NO3‐ uptake, assimilation, and growth in different phytoplankton groups and under different growth conditions are not well understood. Here, we review N metabolism first in a “generic” eukaryotic cell, and the contrasting metabolic pathways and regulation of NH4+ and NO3− when these substrates are provided individually under equivalent growth conditions. Then the metabolic interactions of these substrates are described when both are provided together, emphasizing the cellular challenge of balancing nutrient acquisition with photosynthetic energy balance in dynamic environments. Conditions under which dissipatory pathways such as dissimilatory NO3− / NO2− reduction to NH4+ and photorespiration that may lead to growth suppression are highlighted. While more is known about diatoms, taxon‐specific differences in NH4+ and NO3− metabolism that may contribute to changes in phytoplankton community composition when the composition of the N pool changes are presented. These relationships have important implications for harmful algal blooms, development of nutrient criteria for management, and modeling of nutrient uptake by phytoplankton, particularly in conditions where eutrophication is increasing and the redox state of N loads is changing.}, number={1}, journal={LIMNOLOGY AND OCEANOGRAPHY}, author={Glibert, Patricia M. and Wilkerson, Frances P. and Dugdale, Richard C. and Raven, John A. and Dupont, Christopher L. and Leavitt, Peter R. and Parker, Alexander E. and Burkholder, JoAnn M. and Kana, Todd M.}, year={2016}, month={Jan}, pages={165–197} }
 @misc{gobler_burkholder_davis_harke_johengen_stow_waal_2016, title={The dual role of nitrogen supply in controlling the growth and toxicity of cyanobacterial blooms}, volume={54}, ISSN={["1878-1470"]}, DOI={10.1016/j.hal.2016.01.010}, abstractNote={Historically, phosphorus (P) has been considered the primary limiting nutrient for phytoplankton assemblages in freshwater ecosystems. This review, supported by new findings from Lake Erie, highlights recent molecular, laboratory, and field evidence that the growth and toxicity of some non-diazotrophic blooms of cyanobacteria can be controlled by nitrogen (N). Cyanobacteria such as Microcystis possess physiological adaptations that allow them to dominate low-P surface waters, and in temperate lakes, cyanobacterial densities can be controlled by N availability. Beyond total cyanobacterial biomass, N loading has been shown to selectively promote the abundance of Microcystis and Planktothrix strains capable of synthesizing microcystins over strains that do not possess this ability. Among strains of cyanobacteria capable of synthesizing the N-rich microcystins, cellular toxin quotas have been found to depend upon exogenous N supplies. Herein, multi-year observations from western Lake Erie are presented demonstrating that microcystin concentrations peak in parallel with inorganic N, but not orthophosphate, concentrations and are significantly lower (p<0.01) during years of reduced inorganic nitrogen loading and concentrations. Collectively, this information underscores the importance of N as well as P in controlling toxic cyanobacteria blooms. Furthermore, it supports the premise that management actions to reduce P in the absence of concurrent restrictions on N loading may not effectively control the growth and/or toxicity of non-diazotrophic toxic cyanobacteria such as the cosmopolitan, toxin-producing genus, Microcystis.}, journal={HARMFUL ALGAE}, author={Gobler, Christopher J. and Burkholder, JoAnn M. and Davis, Timothy W. and Harke, Matthew J. and Johengen, Tom and Stow, Craig A. and Waal, Dedmer B.}, year={2016}, month={Apr}, pages={87–97} }
 @article{mitra_flynn_burkholder_berge_calbet_raven_graneli_glibert_hansen_stoecker_et al._2014, title={The role of mixotrophic protists in the biological carbon pump}, volume={11}, ISSN={["1726-4189"]}, DOI={10.5194/bg-11-995-2014}, abstractNote={Abstract. The traditional view of the planktonic food web describes consumption of inorganic nutrients by photoautotrophic phytoplankton, which in turn supports zooplankton and ultimately higher trophic levels. Pathways centred on bacteria provide mechanisms for nutrient recycling. This structure lies at the foundation of most models used to explore biogeochemical cycling, functioning of the biological pump, and the impact of climate change on these processes. We suggest an alternative new paradigm, which sees the bulk of the base of this food web supported by protist plankton communities that are mixotrophic – combining phototrophy and phagotrophy within a single cell. The photoautotrophic eukaryotic plankton and their heterotrophic microzooplankton grazers dominate only during the developmental phases of ecosystems (e.g. spring bloom in temperate systems). With their flexible nutrition, mixotrophic protists dominate in more-mature systems (e.g. temperate summer, established eutrophic systems and oligotrophic systems); the more-stable water columns suggested under climate change may also be expected to favour these mixotrophs. We explore how such a predominantly mixotrophic structure affects microbial trophic dynamics and the biological pump. The mixotroph-dominated structure differs fundamentally in its flow of energy and nutrients, with a shortened and potentially more efficient chain from nutrient regeneration to primary production. Furthermore, mixotrophy enables a direct conduit for the support of primary production from bacterial production. We show how the exclusion of an explicit mixotrophic component in studies of the pelagic microbial communities leads to a failure to capture the true dynamics of the carbon flow. In order to prevent a misinterpretation of the full implications of climate change upon biogeochemical cycling and the functioning of the biological pump, we recommend inclusion of multi-nutrient mixotroph models within ecosystem studies.}, number={4}, journal={BIOGEOSCIENCES}, author={Mitra, A. and Flynn, K. J. and Burkholder, J. M. and Berge, T. and Calbet, A. and Raven, J. A. and Graneli, E. and Glibert, P. M. and Hansen, P. J. and Stoecker, D. K. and et al.}, year={2014}, pages={995–1005} }
 @article{mixson_stikeleather_simmons_wilson_burkholder_2014, title={pH-induced flocculation, indirect electrocoagulation, and hollow fiber filtration techniques for harvesting the saltwater microalga Dunaliella}, volume={26}, ISSN={0921-8971 1573-5176}, url={http://dx.doi.org/10.1007/s10811-013-0232-z}, DOI={10.1007/s10811-013-0232-z}, number={4}, journal={Journal of Applied Phycology}, publisher={Springer Science and Business Media LLC}, author={Mixson, Stephanie M. and Stikeleather, Larry F. and Simmons, Otto D., III and Wilson, Cameron W. and Burkholder, JoAnn M.}, year={2014}, month={Jan}, pages={1701–1709} }
 @article{wang_allen_campos_cade_dean_dvora_immer_mixson_srirangan_sauer_et al._2013, title={ASI:Dunaliellamarine microalgae to drop-in replacement liquid transportation fuel}, volume={32}, ISSN={1944-7442}, url={http://dx.doi.org/10.1002/EP.11855}, DOI={10.1002/EP.11855}, abstractNote={Microalgae are a promising biofuels feedstock, theoretically yielding concentrations of triacylglycerides (TAGs) per unit area that are far higher than traditional feedstocks due to their rapid growth. Dunaliella is particularly advantageous as a feedstock because it is currently commercially mass cultured, thrives in salt water, and has no cell wall. Fourteen strains of Dunaliella have been investigated for growth rates and lipid production in mass culture and tested for enhanced lipid production under a range of environmental stressors including salinity, pH, nitrogen and phosphorus limitation, and light regime. The nuclear genome has been sequenced for four of these strains, with the objective of increasing carbon flux through genetic engineering. Electroflocculation followed by osmotic membrane rupturing may be a very energy and cost efficient means of harvesting the lipid bodies from Dunaliella. A technically feasible and scalable thermo‐catalytic process to convert the lipids into replacements for liquid transportation fuels has been developed. The lipids were converted into long‐chain alkanes through continuous thermal hydrolysis followed by fed‐batch thermo‐catalytic decarboxylation. These alkanes can be reformed into renewable diesel via conventional catalytic hydrocarbon isomerization reactions to improve cold flow properties, if desired. © 2013 American Institute of Chemical Engineers Environ Prog, 32: 916–925, 2013}, number={4}, journal={Environmental Progress & Sustainable Energy}, publisher={Wiley}, author={Wang, Wei-Cheng and Allen, Elle and Campos, Andrew A. and Cade, Rushyannah Killens and Dean, Lisa and Dvora, Mia and Immer, Jeremy G. and Mixson, Stephanie and Srirangan, Soundarya and Sauer, Marie-Laure and et al.}, year={2013}, month={Sep}, pages={916–925} }
 @article{flynn_stoecker_mitra_raven_glibert_hansen_graneli_burkholder_2013, title={Misuse of the phytoplanktonzooplankton dichotomy: the need to assign organisms as mixotrophs within plankton functional types}, volume={35}, ISSN={["1464-3774"]}, DOI={10.1093/plankt/fbs062}, abstractNote={The classic portrayal of plankton is dominated by phytoplanktonic primary producersand zooplanktonic secondary producers. In reality, many if not most planktontraditionally labelled as phytoplankto ...}, number={1}, journal={JOURNAL OF PLANKTON RESEARCH}, author={Flynn, Kevin J. and Stoecker, Diane K. and Mitra, Aditee and Raven, John A. and Glibert, Patricia M. and Hansen, Per Juel and Graneli, Edna and Burkholder, Joann M.}, year={2013}, pages={3–11} }
 @misc{glibert_burkholder_kana_2012, title={Recent insights about relationships between nutrient availability, forms, and stoichiometry, and the distribution, ecophysiology, and food web effects of pelagic and benthic Prorocentrum species}, volume={14}, ISSN={["1878-1470"]}, DOI={10.1016/j.hal.2011.10.023}, abstractNote={The genus Prorocentrum includes six planktonic species that form high-biomass blooms, and at least nine predominantly benthic toxigenic species. Four of the plankters, including P. minimum, the only plankter reported to be toxigenic, are among the most commonly recognized harmful algae that are increasing in frequency, duration, and magnitude globally. Culture studies suggest a species group that generally grows maximally at inorganic nutrient N:P ratios just below Redfield proportions. However, field studies indicate that planktonic Prorocentrum species bloom when nutrients are at high N:P ratios relative to Redfield proportions. In the benthic species P. lima complex, toxin production has been shown to be inversely related to nutrient limitation, increasing when nutrient ratios are above Redfield proportions. Mixotrophy and allelopathy can play an important role in the interactions among planktonic Prorocentrum species, diatoms and other dinoflagellates, but little information is available for benthic taxa. The available information suggests that there are allelopathic interactions among benthic species and other algae, and that benthic species also can adversely affect finfish and shellfish health. While high growth rates may allow these plankters to initiate blooms, adaptive physiology is hypothesized to allow blooms to be maintained at less than maximal growth rates and at non-optimal N:P ratios. Given the projection for land-based nutrient export to continue to increase, it is expected that there will be further expansion of planktonic harmful Prorocentrum spp. globally and more intensive or more toxic benthic occurrences in the future.}, journal={HARMFUL ALGAE}, author={Glibert, Patricia M. and Burkholder, JoAnn M. and Kana, Todd M.}, year={2012}, month={Feb}, pages={231–259} }
 @article{hathaway_moore_burkholder_hunt_2012, title={Temporal analysis of stormwater control measure effluent based on windows of harmful algal bloom (HAB) sensitivity: Are annual nutrient EMCs appropriate during HAB-sensitive seasons?}, volume={49}, ISSN={0925-8574}, url={http://dx.doi.org/10.1016/j.ecoleng.2012.08.014}, DOI={10.1016/j.ecoleng.2012.08.014}, abstractNote={Nutrient removal by stormwater control measures (SCMs) is typically reported in terms of an average annual percent removal or effluent concentration. However, when the performance of these systems is considered in light of downstream receiving aquatic ecosystems, which display seasonal sensitivities to nutrient loadings, the use of an annual-based metric seems arbitrary. To investigate the potential temporal mismatch between nutrient-sensitive periods in receiving water bodies and average annual reporting periods adopted for SCM performance metrics, a case study is presented for four SCM types (constructed stormwater wetlands, bioretention, vegetated filter strips, and swales) draining to the Neuse River Estuary (NRE) in North Carolina, USA. Outbreaks of harmful algal blooms (HABs) in the NRE have been related to different nutrient forms at different times of the year, resulting in a “window” of importance for a given nutrient. These windows were utilized herein to define seasons of interest for various pollutants, and thus how SCM effluent data should be grouped to evaluate seasonal differences in performance. Effluent SCM nutrient concentrations were analyzed on an annual and HAB-sensitive seasonal basis. Although the use of annual performance metrics was deemed either appropriate or conservative for total phosphorus and nitrate–nitrite, effluent concentrations of total ammonia–nitrogen were significantly higher during HAB-sensitive seasons from both bioretention areas and stormwater wetlands. In the case of the NRE, these data suggest SCMs such as bioretention and stormwater wetlands may perform less effectively during periods of HAB sensitivity and that the contribution of SCMs to HAB control in sensitive water bodies may be overstated using presently accepted annual evaluation metrics. Though the seasonal analysis presented is specific to HAB formation in the Neuse River Estuary, evaluation of SCM effluent nutrient concentrations on the basis of nutrient-sensitive periods in receiving water bodies has broad application to evaluation of SCMs in any nutrient-sensitive watershed. The present study suggests that evaluation of SCM performance with respect to nutrient-sensitive periods in receiving aquatic ecosystems warrants further study.}, journal={Ecological Engineering}, publisher={Elsevier BV}, author={Hathaway, Jon M. and Moore, Trisha L.C. and Burkholder, JoAnn M. and Hunt, William F.}, year={2012}, month={Dec}, pages={41–47} }
 @misc{burkholder_marshall_2012, title={Toxigenic Pfiesteria species-Updates on biology, ecology, toxins, and impacts}, volume={14}, ISSN={["1878-1470"]}, DOI={10.1016/j.hal.2011.10.022}, abstractNote={Abstract The genus Pfiesteria includes two toxigenic species, Pfiesteria piscicida and Pfiesteria shumwayae, that are thinly thecate dinoflagellates with apparently cosmopolitan distribution, especially in shallow, poorly flushed, eutrophic estuaries. They are heterotrophic prey generalists that typically feed via phagotrophy and prefer live fish or their fresh tissues as food. They can also engage in limited mixotrophy through temporary retention of kleptochloroplasts from algal prey. Toxicity is highly variable among strains, ranging from apparently nontoxic to highly toxic. Some strains produce a group of hydrophilic toxins with metal-mediated free radical production. Various metals can be involved in the toxin congeners, and the purified toxins are highly labile. These toxins can adversely affect mammalian cells as well as fish. Toxic strains are capable of killing fish by both toxins and physical attack from feeding upon epidermis and other tissues. Non-inducible strains do not produce sufficient toxin to kill fish, but some are capable of causing larval fish death by physical attack. From 1991 to 1998, Pfiesteria spp. were linked to major kills of juvenile Atlantic menhaden (Brevoortia tyrannus), mostly at densities of ≥4(3) × 102 to 103 (rarely, 104) flagellate cells mL−1. These kills mainly occurred in the second largest and largest estuaries on the U.S. mainland, especially two main tributaries of the Albemarle-Pamlico Estuarine System, following decades of hurricane-free conditions. Between kills, Pfiesteria abundance was low in surface waters (}, journal={HARMFUL ALGAE}, author={Burkholder, JoAnn M. and Marshall, Harold G.}, year={2012}, month={Feb}, pages={196–230} }
 @article{glibert_fullerton_burkholder_cornwell_kana_2011, title={Ecological Stoichiometry, Biogeochemical Cycling, Invasive Species, and Aquatic Food Webs: San Francisco Estuary and Comparative Systems}, volume={19}, ISSN={1064-1262 1547-6553}, url={http://dx.doi.org/10.1080/10641262.2011.611916}, DOI={10.1080/10641262.2011.611916}, abstractNote={Eutrophication has altered food webs across aquatic systems, but effects of nutrient stoichiometry (varying nutrient ratios) on ecosystem structure and function have received less attention. A prevailing assumption has been that nutrients are not ecologically relevant unless concentrations are limiting to phytoplankton. However, changes in nutrient stoichiometry fundamentally affect food quality at all levels of the food web. Here, 30-year records of nitrogen and phosphorus concentrations and ratios, phytoplankton, zooplankton, macroinvertebrates, and fish in the San Francisco Estuary (Bay Delta) were examined to collectively interpret ecosystem changes within the framework of ecological stoichiometry. Changes in nutrient concentrations and nutrient ratios over time fundamentally affect biogeochemical nutrient dynamics that can lead to conditions conducive to invasions of rooted macrophytes and bivalve molluscs, and the harmful cyanobacterium Microcystis. Several other aquatic ecosystems considered here have exhibited similar changes in food webs linked to stoichiometric changes. Nutrient stoichiometry is thus suggested to be a significant driver of food webs in the Bay Delta by altering food quality and biogeochemical dynamics. Since nitrogen-to-phosphorus ratios have increased over time, an overall implication is that remediation of fish populations in the San Francisco Estuary will require significant nitrogen reductions to restore the historic ecological stoichiometric balance and the food web.}, number={4}, journal={Reviews in Fisheries Science}, publisher={Informa UK Limited}, author={Glibert, Patricia M. and Fullerton, David and Burkholder, Joann M. and Cornwell, Jeffrey C. and Kana, Todd M.}, year={2011}, month={Oct}, pages={358–417} }
 @article{glibert_burkholder_2011, title={Harmful algal blooms and eutrophication: "strategies" for nutrient uptake and growth outside the Redfield comfort zone}, volume={29}, ISSN={["1993-5005"]}, DOI={10.1007/s00343-011-0502-z}, abstractNote={While many harmful algal blooms have been associated with increasing eutrophication, not all species respond similarly and the increasing challenge, especially for resource managers, is to determine which blooms are related to eutrophication and to understand why particular species proliferate under specific nutrient conditions. The overall goal of this brief review is to describe why nutrient loads are not changing in stoichiometric proportion to the “Redfield ratio”, and why this has important consequences for algal growth. Many types of harmful algae appear to be able to thrive, and/or increase their production of toxins, when nutrient loads are not in proportion classically identified as Redfield ratios. Here we also describe some of the physiological mechanisms of different species to take up nutrients and to thrive under conditions of nutrient imbalance.}, number={4}, journal={CHINESE JOURNAL OF OCEANOLOGY AND LIMNOLOGY}, author={Glibert, Patricia M. and Burkholder, JoAnn M.}, year={2011}, month={Jul}, pages={724–738} }
 @article{null_corbett_demaster_burkholder_thomas_reed_2011, title={Porewater advection of ammonium into the Neuse River Estuary, North Carolina, USA}, volume={95}, ISSN={["1096-0015"]}, DOI={10.1016/j.ecss.2011.09.016}, abstractNote={Radon-222 (222Rn) and ammonium (NH4+) were measured in interstitial water of the Neuse River Estuary (NRE), North Carolina, USA to determine the advective flux of NH4+ from sediments to the overlying water column. Porewater samples were collected over an annual cycle from multi-level piezometers installed in nearshore sites. NH4+ concentrations in sandy environments of the NRE were 10-fold higher than concentrations in the overlying water column. Shallow porewaters exhibited seasonal variations in NH4+ concentrations, which resulted in temporal changes in NH4+ flux from the sediment. Submarine groundwater discharge (SGD) was measured indirectly by using 222Rn as a tracer and directly via seepage meters. Discharge rates were variable depending upon the sampling location and season. The mean SGD was 9.1 ± 1.5 cm d−1 with a maximum SGD during spring at a rate of 13.6 cm d−1 based on 222Rn porewater distribution. High porewater NH4+ concentrations in sandy nearshore sediments contributed NH4+ to the overlying water via groundwater discharge as an advective process. The overall mean NH4+ flux was 11.2 ± 2.0 mmol NH4+ m−2 d−1. Seasonal trends in groundwater seepage rates and NH4+ concentration suggest that groundwater is an important mechanism advecting nutrients from porewaters to surface waters, which is comparable to riverine NH4+ discharge. SGD N:P ratios (NH4+ as N) were >16:1, indicating that SGD is an important contributor of inorganic N for phytoplankton growth and may influence the NRE toward a less N-limited system. The data from this study will advance current understanding about the role of NH4+ in the progressive eutrophication of shallow estuarine ecosystems.}, number={2-3}, journal={ESTUARINE COASTAL AND SHELF SCIENCE}, author={Null, Kimberly A. and Corbett, D. Reide and DeMaster, David J. and Burkholder, JoAnn M. and Thomas, Carrie J. and Reed, Robert E.}, year={2011}, month={Dec}, pages={314–325} }
 @article{may_burkholder_shumway_hegaret_wikfors_frank_2010, title={Effects of the toxic dinoflagellate Alexandrium monilatum on survival, grazing and behavioral response of three ecologically important bivalve molluscs}, volume={9}, ISSN={["1878-1470"]}, DOI={10.1016/j.hal.2009.11.005}, abstractNote={Little is known about interactions between shellfish and the toxic dinoflagellate Alexandrium monilatum. Toxic strains produce endotoxins with hemolytic and neurotoxic properties, and have been linked to fish and invertebrate kills. We experimentally assessed the survival, grazing and behavioral responses of three shellfish species to A. monilatum. Grazing studies were conducted with two size classes of Crassostrea virginica, Mercenaria mercenaria, and Perna viridis. These species inhabit areas where blooms of A. monilatum occur. Clearance rates of each species were depressed when exposed to toxic A. monilatum alone or with nontoxic Pavlova sp., in comparison to control animals fed only nontoxic algae. Exposure to toxic A. monilatum also caused shellfish to decrease shell valve gape. Intact cells of A. monilatum were found within shellfish feces, but the cells did not re-establish growing populations following gut passage. Survival of larval M. mercenaria and C. virginica was also tested when exposed to A. monilatum cells. Survival was significantly lower for larvae exposed to sonicated A. monilatum, in comparison to control larvae tested with nontoxic A. tamarense. Overall, the data indicate that blooms of A. monilatum can adversely affect some shellfish species by reducing valve gape and clearance rate, and by inducing larval mortality.}, number={3}, journal={HARMFUL ALGAE}, author={May, Susan P. and Burkholder, JoAnn M. and Shumway, Sandra E. and Hegaret, Helene and Wikfors, Gary H. and Frank, Dana}, year={2010}, month={Mar}, pages={281–293} }
 @article{skelton_burkholder_parrow_2009, title={Axenic Culture of the Heterotrophic Dinoflagellate Pfiesteria shumwayae in a Semi-Defined Medium}, volume={56}, ISSN={["1550-7408"]}, DOI={10.1111/j.1550-7408.2008.00368.x}, abstractNote={ABSTRACT.  A semi‐defined, biphasic culture medium was developed that supported the axenic growth of three strains of the heterotrophic dinoflagellate Pfiesteria shumwayae. Maximum cell yields and division rates in the semi‐defined medium ranged from 0.1 × 105 to 4.0 × 105 cells/ml and 0.5 to 1.7 divisions/day, respectively, and depended on the concentration of the major components in the medium as well as the P. shumwayae strain. The medium contained high concentrations of certain dissolved and particulate organic compounds, including amino acids and lipids. Pfiesteria shumwayae flagellated cells were attracted to insoluble lipids present in the medium and appeared to feed on the lipid particles, suggesting that phagocytosis may be required for growth in axenic culture. Development of a semi‐defined medium represents significant progress toward a completely defined axenic culture medium and subsequent determination of the biochemical requirements of P. shumwayae, needed to advance understanding of the nutritional ecology of this species. Further, this medium provides an economical, simplified method for generating high cell densities of P. shumwayae in axenic culture that will facilitate controlled investigations on the physiology and biochemistry of this heterotrophic dinoflagellate.}, number={1}, journal={JOURNAL OF EUKARYOTIC MICROBIOLOGY}, author={Skelton, Hayley M. and Burkholder, Joann M. and Parrow, Matthew W.}, year={2009}, pages={73–82} }
 @article{glibert_burkholder_kana_alexander_skelton_shilling_2009, title={Grazing by Karenia brevis on Synechococcus enhances its growth rate and may help to sustain blooms}, volume={55}, ISSN={["1616-1564"]}, DOI={10.3354/ame01279}, abstractNote={Grazing rates of Karenia brevis Clones CCMP2228 and CCMP2229 were determined in laboratory experiments using Synechococcus sp. Clone CCMP1768 as food. Growth (days to weeks) and uptake rates (hours to days) were assessed. In the growth experiments, K. brevis, previously depleted in nitrogen (N), was grown at 2 light intensities in the presence of varying concentrations of Synechococcus. Under high irradiance (300 µmol photons m -2 s -1 ; 14 h light:10 h dark cycle), expo- nential growth rates approximated those of phototrophic growth without Synechococcus (0.26 to 0.35 d -1 ). At this irradiance, K. brevis cells in all treatments grew for about 10 d. Under lower irradi- ance (43 µmol photons m -2 s -1 ), exponential growth rates of K. brevis cells varied with the enrichment level of Synechococcus, with rates under the highest Synechococcus enrichment level being nearly twice (0.58 d -1 ) those observed for the high irradiance treatments. Short-term uptake experiments (3 experiments, 2 clones) were done to examine the N-specific rates of grazing of 15 N-labeled Syne- chococcus. N-specific rates of grazing ranged from 9.28 × 10 -4 h -1 to 1.22 × 10 -2 h -1 and varied with the relative proportion of Synechococcus:K. brevis. These rates represent a range of 0.026 to 2.15 pmol-N K. brevis -1 d -1 , or 0.96 to 83.8 Synechococcus K. brevis -1 h -1 . Evidence of Synechococcus inside K. brevis was provided by confocal microscopy. Grazing by K. brevis thus enhances the range of nutritional substrates available to meet its growth requirements, and may play a substantial role in sustaining natural populations in inorganic N-poor waters.}, number={1}, journal={AQUATIC MICROBIAL ECOLOGY}, author={Glibert, Patricia M. and Burkholder, JoAnn M. and Kana, Todd M. and Alexander, Jeffrey and Skelton, Hayley and Shilling, Carol}, year={2009}, pages={17–30} }
 @article{rothenberger_burkholder_brownie_2009, title={Long-Term Effects of Changing Land Use Practices on Surface Water Quality in a Coastal River and Lagoonal Estuary}, volume={44}, ISSN={["1432-1009"]}, DOI={10.1007/s00267-009-9330-8}, abstractNote={The watershed of the Neuse River, a major tributary of the largest lagoonal estuary on the U.S. mainland, has sustained rapid growth of human and swine populations. This study integrated a decade of available land cover and water quality data to examine relationships between land use changes and surface water quality. Geographic Information Systems (GIS) analysis was used to characterize 26 subbasins throughout the watershed for changes in land use during 1992-2001, considering urban, agricultural (cropland, animal as pasture, and densities of confined animal feed operations [CAFOs]), forested, grassland, and wetland categories and numbers of wastewater treatment plants (WWTPs). GIS was also used together with longitudinal regression analysis to identify specific land use characteristics that influenced surface water quality. Total phosphorus concentrations were significantly higher during summer in subbasins with high densities of WWTPs and CAFOs. Nitrate was significantly higher during winter in subbasins with high numbers of WWTPs, and organic nitrogen was higher in subbasins with higher agricultural coverage, especially with high coverage of pastures fertilized with animal manure. Ammonium concentrations were elevated after high precipitation. Overall, wastewater discharges in the upper, increasingly urbanized Neuse basin and intensive swine agriculture in the lower basin have been the highest contributors of nitrogen and phosphorus to receiving surface waters. Although nonpoint sources have been emphasized in the eutrophication of rivers and estuaries such as the Neuse, point sources continue to be major nutrient contributors in watersheds sustaining increasing human population growth. The described correlation and regression analyses represent a rapid, reliable method to relate land use patterns to water quality, and they can be adapted to watersheds in any region.}, number={3}, journal={ENVIRONMENTAL MANAGEMENT}, author={Rothenberger, Meghan B. and Burkholder, JoAnn M. and Brownie, Cavell}, year={2009}, month={Sep}, pages={505–523} }
 @article{burkholder_glibert_2009, title={The importance of intraspecific variability in harmful algae-Preface to a collection of topical papers}, volume={8}, ISSN={["1568-9883"]}, DOI={10.1016/j.hal.2009.03.006}, abstractNote={Documenting the immense diversity of single-celled, eukaryotic organisms (protists) has been a formidable challenge for ecologists. These species were originally defined by morphological criteria, but shortcomings of the morphospecies concept, and a bewildering array of sizes and cellular attributes, has made constructing a taxonomy that is useful for ecologists nearly impossible. Consequently, physiological and genetic information has been integrated to address these shortcomings, and to develop the framework of a unifying taxonomy. DNA sequence information, in particular, has revolutionized studies of protistan diversity. However, the exponential increase in sequence-based protistan species richness published from field surveys in recent years raises the question of whether we have moved beyond characterizing species-level diversity and begun to reveal intraspecies diversity. The answer to that question appears to be ‘yes’, at least for some protistan lineages. The need to document such microdiversity may be justified, but it is important for protistologists to recognize and acknowledge that possibility, and its consequences.}, number={5}, journal={HARMFUL ALGAE}, author={Burkholder, JoAnn M. and Glibert, Patricia M.}, year={2009}, month={Jun}, pages={744–745} }
 @article{rothenberger_burkholder_wentworth_2009, title={Use of long-term data and multivariate ordination techniques to identify environmental factors governing estuarine phytoplankton species dynamics}, volume={54}, ISSN={["1939-5590"]}, DOI={10.4319/lo.2009.54.6.2107}, abstractNote={A continuous, 13‐yr record of environmental data and phytoplankton species and assemblage structure in the mesohaline Neuse River Estuary (biweekly, April‐October; monthly, November‐March) was used to evaluate phytoplankton assemblage responses to changing environmental conditions. Ordination techniques including nonmetric multidimensional scaling, indicator species analysis, and BIO‐ENV software were used to investigate potential environmental predictors of phytoplankton assemblage patterns under chronic eutrophication. Phytoplankton assemblages were strongly related to temperature and total nitrogen : total phosphorus ratios, with expected seasonal changes in species composition. Interannual changes in river discharge influenced whether phytoplankton assemblages were dominated by diatoms and phototrophic flagellates or by mixotrophic and heterotrophic dinoflagellates. Increasing ammonium concentrations also have been an important influence on phytoplankton assemblages. Raphidophytes (including the potentially toxic species Heterosigma akashiwo), haptophytes, chlorophytes, and the bloom‐forming dinoflagellate Heterocapsa rotundata have increased in more recent years (2000–2006), concomitant with increasing ammonium concentrations. Abundance of the potentially toxic dinoflagellates Prorocentrum minimum and the grouping Pfiesteria spp., “pfiesteria‐like” dinoflagellates, and Karlodinium veneficum remained stationary over time and rarely exceeded 103 cells mL−1. Abundance of P. minimum was positively related to dissolved organic nitrogen and suspended solids concentrations, whereas the highest abundance of the grouping Pfiesteria spp., “pfiesteria‐like” dinoflagellates, and K. veneficum occurred during summer and fall, related to high total phosphorus concentrations, temperature, and salinity. Overall, this study provides new species‐level insights to advance understanding about anthropogenic influences on phytoplankton assemblages. The data suggest an increasingly important role of ammonium in controlling phytoplankton assemblage structure, including increased abundance of some harmful species, in eutrophic estuaries.}, number={6}, journal={LIMNOLOGY AND OCEANOGRAPHY}, author={Rothenberger, Meghan B. and Burkholder, JoAnn M. and Wentworth, Thomas R.}, year={2009}, month={Nov}, pages={2107–2127} }
 @article{skelton_burkholder_parrow_2008, title={AXENIC CULTIVATION OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA SHUMWAYAE AND OBSERVATIONS ON FEEDING BEHAVIOR}, volume={44}, ISSN={["1529-8817"]}, DOI={10.1111/j.1529-8817.2008.00601.x}, abstractNote={ Pfiesteria shumwayae Glasgow et J. M. Burkh. [=Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester] is a heterotrophic dinoflagellate commonly found in temperate, estuarine waters. P. shumwayae can feed on other protists, fish, and invertebrates, but research on the biochemical requirements of this species has been restricted by the lack of axenic cultures. An undefined, biphasic culture medium was formulated that supported the axenic growth of two of three strains of P. shumwayae. The medium contained chicken egg yolk as a major component. Successful growth depended on the method used to sterilize the medium, and maximum cell yields (104 · mL−1) were similar to those attained in previous research when P. shumwayae was cultured with living fish or microalgae. Additionally, P. shumwayae flagellate cells ingested particles present in the biphasic medium, allowing detailed observations of feeding behavior. This research is an initial step toward a chemically defined axenic culture medium and determination of P. shumwayae metabolic requirements.}, number={6}, journal={JOURNAL OF PHYCOLOGY}, author={Skelton, Hayley M. and Burkholder, JoAnn M. and Parrow, Matthew W.}, year={2008}, month={Dec}, pages={1614–1624} }
 @article{glibert_burkholder_graneli_anderson_2008, title={Advances and insights in the complex relationships between eutrophication and HABs: Preface to the special issue}, volume={8}, ISSN={["1568-9883"]}, DOI={10.1016/j.hal.2008.08.020}, abstractNote={Dinoflagellate algal blooms (DABs), with Lingulodinium polyedrum as the dominant species, have increased over the past few years in coastal areas off Baja California, Mexico. Vertical and temporal variability of particulate cadmium (Cdp), dissolved Cd (Cdd), PO43 − and Cdd/PO43 − were investigated during two intense DABs of L. polyedrum that occurred during the fall of 2011 and 2012 in Todos Santos Bay. Results were then, compared with data gathered in the absence of algal blooms during the autumn of 2013. In both algal blooms, L. polyedrum tended to be concentrated near the surface throughout the duration; however, during DAB 2011 the number of cells was twice as abundant ([10.0 ± 8.0] × 105 cells L− 1) as in DAB 2012 ([5.0 ± 4.4] × 105 cells L− 1). During DAB 2011, Cdp increased significantly (up to 1.02 ± 0.99 nmol kg− 1) and was positively correlated with the cell abundance of L. polyedrum, suggesting that this dinoflagellate is able to assimilate and concentrate Cdd. Likewise, Cdd (up to 0.71 ± 0.17 nM) increased in the days of highest cell abundance, which could be attributed to uptake and subsequent regeneration of Cdd resulting from the remineralization of organic particulate matter produced during the bloom, as well as with the presence of organic ligands secreted by L. polyedrum that could keep Cdd in solution. During DAB 2011, dissolved Cdd/PO43 − ratios exhibited high vertical and temporal variability in the upper 5 m of the water column, but remained virtually constant near the bottom, suggesting a depth-dependent decoupling between these two dissolved components during the bloom development. Given the observed differences in the vertical and temporal variability of Cdd, Cdp, and PO43 − between these two intense DABs, we propose the existence of an abundance threshold of approximately 106 cells L− 1 of L. polyedrum above which Cd and PO43 − significantly increased due to remineralization in coastal waters during the bloom development.}, number={1}, journal={HARMFUL ALGAE}, author={Glibert, Patricia M. and Burkholder, JoAnn M. and Graneli, Edna and Anderson, Donald M.}, year={2008}, month={Dec}, pages={1–2} }
 @article{burkholder_touchette_allen_alexander_rublee_2008, title={Environmental conditions, cyanobacteria and microcystin concentrations in potable water supply reservoirs in North Carolina, USA}, volume={619}, journal={Cyanobacterial harmful algal blooms: state of the science and research needs}, author={Burkholder, J. M. and Touchette, B. W. and Allen, E. H. and Alexander, J. L. and Rublee, P. A.}, year={2008}, pages={293–294} }
 @article{heisler_glibert_burkholder_anderson_cochlan_dennison_dortch_gobler_heil_humphries_et al._2008, title={Eutrophication and harmful algal blooms: A scientific consensus}, volume={8}, ISSN={1568-9883}, url={http://dx.doi.org/10.1016/j.hal.2008.08.006}, DOI={10.1016/j.hal.2008.08.006}, abstractNote={In January 2003, the US Environmental Protection Agency sponsored a "roundtable discussion" to develop a consensus on the relationship between eutrophication and harmful algal blooms (HABs), specifically targeting those relationships for which management actions may be appropriate. Academic, federal, and state agency representatives were in attendance. The following seven statements were unanimously adopted by attendees based on review and analysis of current as well as pertinent previous data: 1) Degraded water quality from increased nutrient pollution promotes the development and persistence of many HABs and is one of the reasons for their expansion in the U.S. and the world; 2) The composition - not just the total quantity - of the nutrient pool impacts HABs; 3) High biomass blooms must have exogenous nutrients to be sustained; 4) Both chronic and episodic nutrient delivery promote HAB development; 5) Recently developed tools and techniques are already improving the detection of some HABs, and emerging technologies are rapidly advancing toward operational status for the prediction of HABs and their toxins; 6) Experimental studies are critical to further the understanding of the role of nutrients in HAB expression, and will strengthen prediction and mitigation of HABs; and 7) Management of nutrient inputs to the watershed can lead to significant reduction in HABs. Supporting evidence and pertinent examples for each consensus statement is provided herein.}, number={1}, journal={Harmful Algae}, publisher={Elsevier BV}, author={Heisler, J. and Glibert, P.M. and Burkholder, J.M. and Anderson, D.M. and Cochlan, W. and Dennison, W.C. and Dortch, Q. and Gobler, C.J. and Heil, C.A. and Humphries, E. and et al.}, year={2008}, month={Dec}, pages={3–13} }
 @misc{heisler_glibert_burkholder_anderson_cochlan_dennison_dortch_gobler_heil_humphries_et al._2008, title={Eutrophication and harmful algal blooms: A scientific consensus}, volume={8}, number={1}, journal={Harmful Algae}, author={Heisler, J. and Glibert, P. M. and Burkholder, J. M. and Anderson, D. M. and Cochlan, W. and Dennison, W. C. and Dortch, Q. and Gobler, C. J. and Heil, C. A. and Humphries, E. and et al.}, year={2008}, pages={3–13} }
 @misc{anderson_burkholder_cochlan_glibert_gobler_heil_kudela_parsons_rensel_townsend_et al._2008, title={Harmful algal blooms and eutrophication: Examining linkages from selected coastal regions of the United States}, volume={8}, ISSN={["1878-1470"]}, DOI={10.1016/j.hal.2008.08.017}, abstractNote={Coastal waters of the United States (U.S.) are subject to many of the major harmful algal bloom (HAB) poisoning syndromes and impacts. These include paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), amnesic shellfish poisoning (ASP), ciguatera fish poisoning (CFP) and various other HAB phenomena such as fish kills, loss of submerged vegetation, shellfish mortalities, and widespread marine mammal mortalities. Here, the occurrences of selected HABs in a selected set of regions are described in terms of their relationship to eutrophication, illustrating a range of responses. Evidence suggestive of changes in the frequency, extent or magnitude of HABs in these areas is explored in the context of the nutrient sources underlying those blooms, both natural and anthropogenic. In some regions of the U.S., the linkages between HABs and eutrophication are clear and well documented, whereas in others, information is limited, thereby highlighting important areas for further research.}, number={1}, journal={HARMFUL ALGAE}, author={Anderson, Donald M. and Burkholder, Joann M. and Cochlan, William P. and Glibert, Patricia M. and Gobler, Christopher J. and Heil, Cynthia A. and Kudela, Raphael M. and Parsons, Michael L. and Rensel, J. E. Jack and Townsend, David W. and et al.}, year={2008}, month={Dec}, pages={39–53} }
 @misc{burkholder_glibert_skelton_2008, title={Mixotrophy, a major mode of nutrition for harmful algal species in eutrophic waters}, volume={8}, ISSN={["1878-1470"]}, DOI={10.1016/j.hal.2008.08.010}, abstractNote={Historically most harmful algal species (HAS) have been thought to be strictly phototrophic. Mixotrophy, the use of phototrophy and heterotrophy in combination, has been emphasized as operative mainly in nutrient-poor habitats as a mechanism for augmenting nutrient supplies. Here we examine an alternate premise, that many harmful algae which thrive in eutrophic habitats are mixotrophs that respond both directly to nutrient inputs, and indirectly through high abundance of bacterial and algal prey that are stimulated by the elevated nutrients. From review and synthesis of the available data, mixotrophy occurs in all HAS examined thus far in the organic substrate- and prey-rich habitats of eutrophic estuarine and marine coastal waters. Where data are available comparing phototrophy versus mixotrophy, mixotrophy in eutrophic habitats generally is significant in nutrient acquisition and growth of HAS and, therefore, likely important in the development and maintenance of their blooms. In eutrophic habitats phagotrophic mixotrophs, in particular, have been shown to attain higher growth than when in phototrophic mode. Yet for many HAS, quantitative data about the role of mixotrophy in nutrition, growth, and blooms are lacking, especially relating laboratory information to natural field assemblages, so that the relative importance of photosynthesis, dissolved organic nutrients, and ingestion of prey largely remain unknown. Research is needed to assess simultaneously the roles of phototrophy, osmotrophy and phagotrophy in the nutritional ecology of HAS in eutrophic habitats, spanning bloom initiation, development and senescence. From these data, models that include the role of mixotrophy can be developed to gain more realistic insights about the nutritional factors that control harmful algae in eutrophic waters, and to strengthen predictive capability in predicting their blooms. An overall forecast that can be tested, as well, is that harmful mixotrophic algae will become more abundant as their food supplies increase in many estuaries and coastal waters that are sustaining chronic, increasing cultural eutrophication.}, number={1}, journal={HARMFUL ALGAE}, author={Burkholder, JoAann M. and Glibert, Patricia M. and Skelton, Hayley M.}, year={2008}, month={Dec}, pages={77–93} }
 @article{glibert_azanza_burford_furuya_abal_al-azri_al-yamani_andersen_anderson_beardall_et al._2008, title={Ocean urea fertilization for carbon credits poses high ecological risks}, volume={56}, ISSN={0025-326X}, url={http://dx.doi.org/10.1016/j.marpolbul.2008.03.010}, DOI={10.1016/j.marpolbul.2008.03.010}, abstractNote={The proposed plan for enrichment of the Sulu Sea, Philippines, a region of rich marine biodiversity, with thousands of tonnes of urea in order to stimulate algal blooms and sequester carbon is flawed for multiple reasons. Urea is preferentially used as a nitrogen source by some cyanobacteria and dinoflagellates, many of which are neutrally or positively buoyant. Biological pumps to the deep sea are classically leaky, and the inefficient burial of new biomass makes the estimation of a net loss of carbon from the atmosphere questionable at best. The potential for growth of toxic dinoflagellates is also high, as many grow well on urea and some even increase their toxicity when grown on urea. Many toxic dinoflagellates form cysts which can settle to the sediment and germinate in subsequent years, forming new blooms even without further fertilization. If large-scale blooms do occur, it is likely that they will contribute to hypoxia in the bottom waters upon decomposition. Lastly, urea production requires fossil fuel usage, further limiting the potential for net carbon sequestration. The environmental and economic impacts are potentially great and need to be rigorously assessed.}, number={6}, journal={Marine Pollution Bulletin}, publisher={Elsevier BV}, author={Glibert, Patricia M. and Azanza, Rhodora and Burford, Michele and Furuya, Ken and Abal, Eva and Al-Azri, Adnan and Al-Yamani, Faiza and Andersen, Per and Anderson, Donald M. and Beardall, John and et al.}, year={2008}, month={Jun}, pages={1049–1056} }
 @article{glibert_azanza_burford_furuya_2008, title={Ocean urea fertilization for carbon credits poses high ecological risks}, volume={56}, number={6}, journal={Marine Pollution Bulletin}, author={Glibert, P. M. and Azanza, R. and Burford, M. and Furuya, K.}, year={2008}, pages={1049–1056} }
 @article{hegaret_shumway_wikfors_pate_burkholder_2008, title={Potential transport of harmful algae via relocation of bivalve molluscs}, volume={361}, ISSN={["0171-8630"]}, DOI={10.3354/meps07375}, abstractNote={Aquaculture and restoration activities with bivalve molluscs often involve moving indi- viduals from one body of water to another. Our study tests the hypothesis that harmful algae ingested by source populations of shellfish can be introduced into new environments by means of these shell- fish relocations. Cultures of several harmful algal strains, including Prorocentrum minimum, Alexan- drium fundyense, Heterosigma akashiwo, Aureococcus anophagefferens, Karenia mikimotoi and Alexandrium monilatum, were fed to various species of bivalve molluscs, Crassostrea virginica, Argopecten irradians irradians, Mercenaria mercenaria, Mytilus edulis, Mya arenaria, Venerupis philippinarum and Perna viridis, to assess the ability of the algal cells to pass intact though the diges- tive tracts of the shellfish and subsequently multiply in number. Ten individuals of each shellfish species were exposed for 2 d to a simulated harmful algal bloom at a natural bloom concentration. The shellfish were removed after exposure, and maintained for 2 further days in ultra-filtered sea- water. Biodeposits (feces) were collected after 24 and 48 additional hours, and observed under light microscopy for the presence or absence of intact, potentially viable algal cells or temporary cysts. Subsamples of biodeposits were transferred into both algal culture medium and filtered seawater and monitored for algal growth. Intact cells of most harmful algal species tested were seen in biodeposits. Generally, harmful algae from the biodeposits collected in the first 24 h after transfer re-established growing populations, but algae could less often be recovered from the biodeposits collected after 48 h. These data provide evidence that transplanted bivalve molluscs may be vectors for the transport of harmful algae and that a short holding period in water without algae may mitigate this risk. Fur- ther, preliminary results indicate that emersion may also serve to mitigate the risk of transport.}, journal={MARINE ECOLOGY PROGRESS SERIES}, author={Hegaret, Helene and Shumway, Sandra E. and Wikfors, Gary H. and Pate, Susan and Burkholder, Joann M.}, year={2008}, pages={169–179} }
 @article{holm_stamper_brizzolara_barnes_deamer_burkholder_2008, title={Sonication of bacteria, phytoplankton and zooplankton: Application to treatment of ballast water}, volume={56}, ISSN={["1879-3363"]}, DOI={10.1016/j.marpolbul.2008.02.007}, abstractNote={We investigated the effect of high power ultrasound, at a frequency of 19 kHz, on the survival of bacteria, phytoplankton and zooplankton, in order to obtain estimates of effective exposure times and energy densities that could be applied to design of ultrasonic treatment systems for ballast water. Efficacy of ultrasonic treatment varied with the size of the test organism. Zooplankton required only 3–9 s of exposure time and 6–19 J/mL of ultrasonic energy to realize a 90% reduction in survival. In contrast, decimal reduction times for bacteria and phytoplankton ranged from 1 to 22 min, and decimal reduction energy densities from 31 to 1240 J/mL. Our results suggest that stand-alone ultrasonic treatment systems for ballast water, operating at 19–20 kHz, may be effective for planktonic organisms >100 μm in size, but smaller planktonic organisms such as phytoplankton and bacteria will require treatment by an additional or alternative system.}, number={6}, journal={MARINE POLLUTION BULLETIN}, author={Holm, Eric R. and Stamper, David M. and Brizzolara, Robert A. and Barnes, Laurie and Deamer, Nora and Burkholder, JoAnn M.}, year={2008}, month={Jun}, pages={1201–1208} }
 @article{reed_dickey_burkholder_kinder_brownie_2008, title={Water level variations in the Neuse and Pamlico Estuaries, North Carolina due to local and remote forcing}, volume={76}, ISSN={["1096-0015"]}, DOI={10.1016/j.ecss.2007.05.049}, abstractNote={Water level time series records from the Neuse and Pamlico River Estuaries were statistically compared to local and distant wind field data, water level records within the Pamlico Sound and also coastal ocean sites to determine the relative contribution of each time series to water levels in the Neuse and Pamlico Estuaries. The objectives of this study were to examine these time series data using various statistical methods (i.e. autoregressive, empirical orthogonal function analysis (EOF), exploratory data analysis (EDA)) to determine short- and long-time-scale variability, and to develop predictive statistical models that can be used to estimate past water level fluctuations in both the Neuse Estuary (NE) and Pamlico Estuary (PE). Short- and long-time-scale similarities were observed in all time series of estuarine, Pamlico Sound and subtidal coastal ocean water level and wind component data, due to events (nor'easters, fronts and tropical systems) and seasonality. Empirical orthogonal function analyses revealed a strong coastal ocean and wind field contribution to water level in the NE and PE. Approximately 95% of the variation was captured in the first two EOF components for water level data from the NE, sound and coastal ocean, and 70% for the PE, sound and coastal ocean. Spectral density plots revealed strong diurnal signals in both wind and water level data, and a strong cross correlation and coherency between the NE water level and the North/South wind component. There was good agreement between data and predictions using autoregressive statistical models for the NE (R2 = 0.92) and PE (R2 = 0.76). These methods also revealed significant autoregressive lags for the NE (days 1 and 3) and for the PE (days 1, 2 and 3). Significant departures from predictions are attributed to local meteorological and hydrological events. The autoregressive techniques showed significant predictive improvement over ordinary least squares methods. The results are considered within the context of providing long time-scale hindcast data for the two estuaries, and the importance of these data for multidisciplinary researchers and managers.}, number={2}, journal={ESTUARINE COASTAL AND SHELF SCIENCE}, author={Reed, Robert E. and Dickey, David A. and Burkholder, Joann M. and Kinder, Carol A. and Brownie, Cavell}, year={2008}, month={Jan}, pages={431–446} }
 @misc{touchette_burkholder_2007, title={Carbon and nitrogen metabolism in the seagrass, Zostera marina L.: Environmental control of enzymes involved in carbon allocation and nitrogen assimilation}, volume={350}, ISSN={["0022-0981"]}, DOI={10.1016/j.jembe.2007.05.034}, abstractNote={This study experimentally examined influences of environmental variables on the activities of key enzymes involved in carbon and nitrogen metabolism of the submersed marine angiosperm, Zostera marina L. Nitrate reductase activity in leaf tissue was correlated with both water-column nitrate concentrations and leaf sucrose levels. Under elevated nitrate, shoot nitrate reductase activity increased in both light and dark periods if carbohydrate reserves were available. When water-column nitrate was low, glutamine synthetase activity in leaf tissue increased with environmental ammonium. In contrast, glutamine synthetase activity in belowground tissues was statistically related to both nitrate and temperature. At the optimal growth temperature for this species (ca. 25 °C), increased water-column nitrate promoted an increase in glutamine synthetase activity of belowground tissues. As temperatures diverged from the optimum, this nitrate effect on glutamine synthetase was no longer evident. Activities of both sucrose synthase and sucrose-P synthase were directly correlated with temperature. Sucrose-P synthase activity also was correlated with salinity, and sucrose synthase activity was statistically related to tissue ammonium. Overall, the enzymatic responses that were observed indicate a tight coupling between carbon and nitrogen metabolism that is strongly influenced by prevailing environmental conditions, especially temperature, salinity, and environmental nutrient levels.}, number={1-2}, journal={JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY}, author={Touchette, Brant W. and Burkholder, JoAnn M.}, year={2007}, month={Nov}, pages={216–233} }
 @article{touchette_burkholder_2007, title={Effects of temperature and nitrate on phosphomonoesterase activities between carbon source and sink tissues in Zostera marina L.}, volume={342}, ISSN={["1879-1697"]}, DOI={10.1016/j.jembe.2006.11.005}, abstractNote={Inorganic phosphorus (Pi) is important in the regulation of many carbon and nitrogen metabolic processes of plants. In this study, we examined alterations of phosphomonoesterase activity (PA; both alkaline and acid) in a submersed marine angiosperm, Zostera marina, grown in Pi non-limiting conditions under elevated temperature and/or nitrate enrichment. Control plants (ambient water-column NO3− < 2.5 μM, with weekly mean water temperatures between 26.5–27.0 °C based on a 20-yr data set in a local embayment) were compared to treated plants that were exposed to increased water-column nitrate (8 μM NO3− above ambient, pulsed daily at 0900 h), and/or increased temperature (ca. 3 °C above weekly means) over eight weeks in late summer–fall. Under both nitrate regimes, increased temperature resulted in periodic increased leaf and root-rhizome tissue carbon content, and increased acid and alkaline PA activities (AcPAs and AlPAs, respectively). There was a positive correlation between AlPA and AcPA activities and sucrose synthase activities in belowground structures, and a negative correlation between AlPA activities and sucrose concentrations. There were also periodic changes in PA partitioning between carbon source and sink tissues. In high-temperature and high-nitrate treatments, AcPAs significantly increased in leaves relative to activities in root-rhizome tissues (up to 12-fold higher in aboveground than belowground tissues in as little as 3 weeks after initiation of treatments). These responses were not observed in control plants, which maintained comparable AcPA activities in above- and belowground tissues. In addition, AlPA activity was significantly higher in leaf than in root-rhizome tissues of plants in high-temperature (weeks 3 and 6) and high temperature combined with high nitrate treatments (week 8), relative to AlPA activities in control plants. The observed changes in PAs were not related to Pi growth limitation, and may allow Z. marina to alter its carbon metabolism during periods of increased carbon demand/mobilization. This response would make it possible for Z. marina to meet short-term P requirements to maximize carbon production/allocation. Such a mechanism could help to explain the variability in PA activities that has been observed for many plant species during periods when environmental Pi exceeds requirements for optimal growth.}, number={2}, journal={JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY}, author={Touchette, Brant W. and Burkholder, JoAnn M.}, year={2007}, month={Apr}, pages={313–324} }
 @article{touchette_burkholder_allen_alexander_kinder_brownie_james_britton_2007, title={Eutrophication and cyanobacteria blooms in run-of-river impoundments in North Carolina, USA}, volume={23}, ISSN={["1040-2381"]}, DOI={10.1080/07438140709353921}, abstractNote={Abstract We compared monthly data taken during the dry summer growing season of 2002 in 11 potable water supply reservoirs (19–85 years old based on year filled) within the North Carolina Piedmont, including measures of watershed land use, watershed area, reservoir morphometry (depth, surface area, volume), suspended solids (SS), nutrient concentrations (total nitrogen, TN; total Kjeldahl nitrogen, TKN; nitrate + nitrite, NO3− + NO2−; total phosphorus, TP; total organic carbon), phytoplankton chlorophyll a (chla) concentrations, cyanobacteria assemblages, and microcystin concentrations from monthly data taken during the dry summer 2002 growing season. The reservoirs were considered collectively or as two subgroups by age as “mod.” (moderate age, 19–40 years post-fill, n = 5) and “old” (74–85 yr post-fill, n = 6). The run-of-river impoundments were meso-/eutrophic and turbid (means 25–125 μg TP/L, 410–1,800 μg TN/L, 3–70 μg chla/L and 5.7–41.9 mg SS/L). Under drought conditions in these turbid systems, there was a positive relationship between chla and both TN and TP, supported by correlation analyses and hierarchical ANOVA models. The models also indicated significant positive relationships between TN and TP, and between SS and both TP and TN. Agricultural land use was positively correlated with TKN for the reservoirs considered collectively, and with TN, TKN, TP, and chla in mod. reservoirs. In models considering the reservoirs by age group, TN:TP ratios were significantly lower and NO3− + NO2− was significantly higher in old reservoirs, and these relationships were stronger when reservoir age was used as a linear predictor. Cyanobacteria assemblages in the two reservoir age groups generally were comparable in abundance and species composition, and comprised 60–95% (up to 1.9 × 106 cells/mL) of the total phytoplankton cell number. Potentially toxic taxa were dominated by Cylindrospermopsis raciborskii and C. philippinensis. Although known microcystin producers were low in abundance, microcystin (< 0.8 μg/L) was detected in most samples. TP and chla were significant predictors of total cyanobacterial abundance. The data suggest that at present these turbid, meso-/eutrophic reservoirs have moderate cyanobacteria abundance and low cyanotoxin (microcystin) levels over the summer growing season, even in low-precipitation seasons that favor cyanobacteria. Accelerated eutrophication from further watershed development is expected to promote increased cyanobacterial abundance and adversely affect the value of these reservoirs as potable water supplies.}, number={2}, journal={LAKE AND RESERVOIR MANAGEMENT}, author={Touchette, Brant W. and Burkholder, Joann M. and Allen, Elie H. and Alexander, Jessica L. and Kinder, Carol A. and Brownie, Cavell and James, Jennifer and Britton, Clay H.}, year={2007}, month={Jun}, pages={179–192} }
 @article{burkholder_libra_weyer_heathcote_kolpin_thorne_wichman_2007, title={Impacts of waste from concentrated animal feeding operations on water quality}, volume={115}, ISSN={["1552-9924"]}, DOI={10.1289/ehp.8839}, abstractNote={Waste from agricultural livestock operations has been a long-standing concern with respect to contamination of water resources, particularly in terms of nutrient pollution. However, the recent growth of concentrated animal feeding operations (CAFOs) presents a greater risk to water quality because of both the increased volume of waste and to contaminants that may be present (e.g., antibiotics and other veterinary drugs) that may have both environmental and public health importance. Based on available data, generally accepted livestock waste management practices do not adequately or effectively protect water resources from contamination with excessive nutrients, microbial pathogens, and pharmaceuticals present in the waste. Impacts on surface water sources and wildlife have been documented in many agricultural areas in the United States. Potential impacts on human and environmental health from long-term inadvertent exposure to water contaminated with pharmaceuticals and other compounds are a growing public concern. This work-group, which is part of the Conference on Environmental Health Impacts of Concentrated Animal Feeding Operations: Anticipating Hazards—Searching for Solutions, identified needs for rigorous ecosystem monitoring in the vicinity of CAFOs and for improved characterization of major toxicants affecting the environment and human health. Last, there is a need to promote and enforce best practices to minimize inputs of nutrients and toxicants from CAFOs into freshwater and marine ecosystems.}, number={2}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={Burkholder, JoAnn and Libra, Bob and Weyer, Peter and Heathcote, Susan and Kolpin, Dana and Thorne, Peter S. and Wichman, Michael}, year={2007}, month={Feb}, pages={308–312} }
 @article{burkholder_hallegraeff_melia_cohen_bowers_oldach_parrow_sullivan_zimba_allen_et al._2007, title={Phytoplankton and bacterial assemblages in ballast water of U.S. military ships as a function of port of origin, voyage time, and ocean exchange practices}, volume={6}, ISSN={1568-9883}, url={http://dx.doi.org/10.1016/j.hal.2006.11.006}, DOI={10.1016/j.hal.2006.11.006}, abstractNote={We characterized the physical/chemical conditions and the algal and bacterial assemblages in ballast water from 62 ballast tanks aboard 28 ships operated by the U.S. Military Sealift Command and the Maritime Administration, sampled at 9 ports on the U.S. West Coast and 4 ports on the U.S. East Coast. The ballast tank waters had been held for 2–176 days, and 90% of the tanks had undergone ballast exchange with open ocean waters. Phytoplankton abundance was highly variable (grand mean for all tanks, 3.21 × 104 viable cells m−3; median, 7.9 × 103 cells m−3) and was unrelated to physical/chemical parameters, except for a positive relationship between centric diatom abundance and nitrate concentration. A total of 100 phytoplankton species were identified from the ballast tanks, including 23 potentially harmful taxa (e.g. Chaetoceros concavicornis, Dinophysis acuminata, Gambierdiscus toxicus, Heterosigma akashiwo, Karlodinium veneficum, Prorocentrum minimum, Pseudo-nitzschia multiseries). Assemblages were dominated by chain-forming diatoms and dinoflagellates, and viable organisms comprised about half of the total cells. Species richness was higher in ballast tanks with coastal water, and in tanks containing Atlantic or Pacific Ocean source waters rather than Indian Ocean water. Total and viable phytoplankton numbers decreased with age of water in the tanks. Diversity also generally decreased with water age, and tanks with ballast water age >33 days did not produce culturable phytoplankton. Abundance was significantly higher in tanks with recently added coastal water than in tanks without coastal sources, but highly variable in waters held less than 30 days. Bacterial abundance was significantly lower in ballast tanks with Atlantic than Pacific Ocean source water, but otherwise was surprisingly consistent among ballast tanks (overall mean across all tanks, 3.13 ± 1.27 × 1011 cells m−3; median, 2.79 × 1011 cells m−3) and was unrelated to vessel type, exchange status, age of water, environmental conditions measured, or phytoplankton abundance. At least one of four pathogenic eubacteria (Listeria monocytogenes, Escherichia coli, Mycobacterium spp., Pseudomonas aeruginosa) was detected in 48% of the ballast tanks, but toxigenic strains of Vibrio cholerae were not detected. For ships with tanks of similar ballasting history, the largest source of variation in phytoplankton and bacteria abundance was among ships; for ships with tanks of differing ballasting histories, and for all ships/tanks considered collectively, the largest source of variation was within ships. Significant differences in phytoplankton abundance, but not bacterial abundance, sometimes occurred between paired tanks with similar ballasting history; hence, for regulatory purposes phytoplankton abundance cannot be estimated from single tanks only. Most tanks (94%) had adequate records to determine the source locations and age of the ballast water and, as mentioned, 90% had had ballast exchange with open-ocean waters. Although additional data are needed from sediments that can accumulate at the bottom of ballast tanks, the data from this water-column study indicate that in general, U.S. Department of Defense (DoD) ships are well managed to minimize the risk for introduction of harmful microbiota. Nevertheless, abundances of viable phytoplankton with maximum dimension >50 μm exceeded proposed International Maritime Organization standards in 47% of the ballast tanks sampled. The data suggest that further treatment technologies and/or alternative management strategies will be necessary to enable DoD vessels to comply with proposed standards.}, number={4}, journal={Harmful Algae}, publisher={Elsevier BV}, author={Burkholder, JoAnn M. and Hallegraeff, Gustaaf M. and Melia, Gregory and Cohen, Andrew and Bowers, Holly A. and Oldach, David W. and Parrow, Matthew W. and Sullivan, Michael J. and Zimba, Paul V. and Allen, Elle H. and et al.}, year={2007}, month={Aug}, pages={486–518} }
 @misc{burkholder_g. m._g._a._h. a._d. w._m. w._m. j._p. v._e. h._et al._2007, title={Phytoplankton and bacterial assemblages in ballast water of US military ships as a function of port of origin, voyage time, and ocean exchange practices}, volume={6}, number={4}, journal={Harmful Algae}, author={Burkholder, J. M. and G. M., Melia and G., Cohen and A., Bowers and H. A., Oldach and D. W., Parrow and M. W., Sullivan and M. J., Zimba and P. V., Allen and E. H., Kinder and et al.}, year={2007}, pages={486–518} }
 @misc{burkholder_tomasko_touchette_2007, title={Seagrasses and eutrophication}, volume={350}, ISSN={["1879-1697"]}, DOI={10.1016/j.jembe.2007.06.024}, abstractNote={This review summarizes the historic, correlative field evidence and experimental research that implicate cultural eutrophication as a major cause of seagrass disappearance. We summarize the underlying physiological responses of seagrass species, the potential utility of various parameters as indicators of nutrient enrichment in seagrasses, the relatively sparse available information about environmental conditions that exacerbate eutrophication effects, and the better known array of indirect stressors imposed by nutrient over-enrichment that influence seagrass growth and survival. Seagrass recovery following nutrient reductions is examined, as well as the status of modeling efforts to predict seagrass response to changing nutrient regimes. The most common mechanism invoked or demonstrated for seagrass decline under nutrient over-enrichment is light reduction through stimulation of high-biomass algal overgrowth as epiphytes and macroalgae in shallow coastal areas, and as phytoplankton in deeper coastal waters. Direct physiological responses such as ammonium toxicity and water-column nitrate inhibition through internal carbon limitation may also contribute. Seagrass decline under nutrient enrichment appears to involve indirect and feedback mechanisms, and is manifested as sudden shifts in seagrass abundance rather than continuous, gradual changes in parallel with rates of increased nutrient additions. Depending on the species, interactions of high salinity, high temperature, and low light have been shown to exacerbate the adverse effects of nutrient over-enrichment. An array of indirect effects of nutrient enrichment can accelerate seagrass disappearance, including sediment re-suspension from seagrass loss, increased system respiration and resulting oxygen stress, depressed advective water exchange from thick macroalgal growth, biogeochemical alterations such as sediment anoxia with increased hydrogen sulfide concentrations, and internal nutrient loading via enhanced nutrient fluxes from sediments to the overlying water. Indirect effects on trophic structure can also be critically important, for example, the loss of herbivores, through increased hypoxia/anoxia and other habitat shifts, that would have acted as “ecological engineers” in promoting seagrass survival by controlling algal overgrowth; and shifts favoring exotic grazers that out-compete seagrasses for space. Evidence suggests that natural seagrass population shifts are disrupted, slowed or indefinitely blocked by cultural eutrophication, and there are relatively few known examples of seagrass meadow recovery following nutrient reductions. Reliable biomarkers as early indicators of nutrient over-enriched seagrass meadows would benefit coastal resource managers in improving protective measures. Seagrasses can be considered as “long-term" integrators (days to weeks) of nutrient availability, especially through analyses of their tissue content, and of activities of enzymes such as nitrate reductase and alkaline phosphatase. The ratio of leaf nitrogen content to leaf mass has also shown promise as a “nutrient pollution indicator” for the seagrass Zostera marina, with potential application to other species. In modeling efforts, seagrass response to nutrient loading has proven difficult to quantify beyond localized areas because long-term data consistent in quality are generally lacking, and high inter-annual variability in abundance and productivity depending upon stochastic meteorological and hydrographic conditions. Efforts to protect remaining seagrass meadows from damage and loss under eutrophication, within countries and across regions, are generally lacking or weak and ineffective. Research needs to further understand about seagrasses and eutrophication should emphasize experimental studies to assess the response of a wider range of species to chronic, low-level as well as acute, pulsed nutrient enrichment. These experiments should be conducted in the field or in large-scale mesocosms following appropriate acclimation, and should emphasize factor interactions (N, P, C; turbidity; temperature; herbivory) to more closely simulate reality in seagrass ecosystems. They should scale up to address processes that occur over larger scales, including food-web dynamics that involve highly mobile predators and herbivores. Without any further research, however, one point is presently very clear: Concerted local and national actions, thus far mostly lacking, are needed worldwide to protect remaining seagrass meadows from accelerating cultural eutrophication in rapidly urbanizing coastal zones.}, number={1-2}, journal={JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY}, author={Burkholder, JoAnn M. and Tomasko, David A. and Touchette, Brant W.}, year={2007}, month={Nov}, pages={46–72} }
 @article{zimba_camus_allen_burkholder_2006, title={Co-occurrence of white shrimp, Litopenaeus vannamei, mortalities and microcystin toxin in a southeastern USA shrimp facility}, volume={261}, ISSN={["1873-5622"]}, DOI={10.1016/j.aquaculture.2006.08.037}, abstractNote={Various freshwater and marine algal toxins are known to affect plants, fishes, mammals, and invertebrates. During recent mortality events in Texas white shrimp aquaculture ponds, water and shrimp tissue samples were analyzed for cyanobacterial toxins and found to contain microcystin-LR. Cyanoprokaryota dominated the phytoplankton assemblage in water from the affected pond, particularly Microcystis aeruginosa and Anabaena sp. Water samples from the affected pond also contained high levels of microcystin-LR (45 μg/l), whereas adjacent ponds had a diatom-green algal assemblage and no measurable toxin. Unialgal isolates of M. aeruginosa from the affected pond produced microcystin-LR. Free microcystin-LR concentrations in dead shrimp hepatopancreas determined by HPLC were 55 μg/g total shrimp weight, whereas shrimp hepatopancreas from the adjacent pond without shrimp mortalities had no measurable toxin. Muscle toxin concentration was below 0.1 μg/g.}, number={3}, journal={AQUACULTURE}, author={Zimba, Paul V. and Camus, Al and Allen, Elle H. and Burkholder, JoAnn M.}, year={2006}, month={Dec}, pages={1048–1055} }
 @misc{burkholder_dickey_kinder_reed_mallin_mciver_cahoon_melia_brownie_smith_et al._2006, title={Comprehensive trend analysis of nutrients and related variables in a large eutrophic estuary: A decadal study of anthropogenic and climatic influences}, volume={51}, ISSN={["1939-5590"]}, DOI={10.4319/lo.2006.51.1_part_2.0463}, abstractNote={We used a decadal data set, with weekly to biweekly sampling in April—October and monthly sampling in November—March, to characterize climatic (hurricane‐level storms, a sustained 3‐yr drought) and anthropogenic influences on N and P concentrations and loadings to a large eutrophic, poorly flushed estuary, the Neuse Estuary of the Albemarle—Pamlico Estuarine System. Mass volume transport data were obtained with cross‐estuary transect flow measurements taken near the entrance to the estuary. Although trends were minimally influenced by hurricanes, analyses were significantly affected by the sustained drought near the end of the study. As examples, decreasing trends in total N (TN), total P (TP), and bottom‐water dissolved oxygen concentrations, and in TN loadings were significant considering all data, but these trends were not significant when the sustained drought was excluded from analysis. In addition, the trend in TN loading was especially sensitive to the initial sampling period. NH4+ concentrations dramatically increased (overall by ~500%) as a persistent trend regardless of attempts to control for climatic events. An increasing trend in NH4+ also was documented in an adjacent, rapidly flushed Coastal Plain estuary, the Cape Fear. The NH4+ data suggest a regional‐scale effect of high inputs from inadequately controlled, increasing nonpoint sources. The fragility of TN loading trends, the striking increase in NH4+ concentrations, and the lack of management emphasis on controlling nonpoint sources such as “new” industrialized swine production collectively do not support recent reports of achievement of a 30% reduction in TN loading to the Neuse. Nonpoint sources remain a critical target for reduction to alleviate the negative effects of cultural eutrophication in this system, as in many estuaries throughout the world.}, number={1}, journal={LIMNOLOGY AND OCEANOGRAPHY}, author={Burkholder, JoAnn M. and Dickey, David A. and Kinder, Carol A. and Reed, Robert E. and Mallin, Michael A. and McIver, Matthew R. and Cahoon, Lawrence B. and Melia, Greg and Brownie, Cavell and Smith, Joy and et al.}, year={2006}, month={Jan}, pages={463–487} }
 @article{glibert_burkholder_parrow_lewitus_gustafson_2006, title={Direct uptake of nitrogen by Pfiesteria piscicida and Pfiesteria shumwayae, and nitrogen nutritional preferences}, volume={5}, ISSN={["1878-1470"]}, DOI={10.1016/j.hal.2006.04.009}, abstractNote={The rates of uptake of a range of forms of nitrogenous nutrients were measured in cultures of Pfiesteria piscicida and Pfiesteria shumwayae maintained at varying physiological states. The measured rates of dissolved N uptake under some conditions approached the rates of N uptake that are achieved through phagotrophy. Rates of dissolved N uptake by P. piscicida contributed <10% of the cellular N of flagellated cells feeding on algae, but were equal to or greater than phagotrophic N acquisition in cells recently removed from fish cultures. Specific N uptake rates (V, h−1) were higher for cells that were maintained on algal prey for long periods (months) than those that were grown with live fish. However, rates of N uptake on a cellular basis for cells grown on or recently removed from fish were comparable to those maintained on algal prey, likely reflecting differences in the sizes of cells of different physiological condition. Preferences for form of N generally followed a decreasing trend of amino acids > urea > NH4+ > NO3−. Nitrate consistently was not a preferred form of N. Although Pfiesteria spp. are often found in eutrophic environments, the relationship between Pfiesteria spp. and nutrient availability is likely to be primarily indirect, mediated through the production of various prey on which Pfiesteria spp. feed. These findings also confirm, however, that when dissolved N concentrations are elevated, they can contribute to the supplemental nutrition of these cells, and thus may provide a significant source of N to Pfiesteria spp. in nature.}, number={4}, journal={HARMFUL ALGAE}, author={Glibert, Patricia M. and Burkholder, JoAnn M. and Parrow, Matthew W. and Lewitus, Alan J. and Gustafson, Daniel E.}, year={2006}, month={Sep}, pages={380–394} }
 @article{shumway_burkholder_springer_2006, title={Effects of the estuarine dinoflagellate Pfiesteria shumwayae (Dinophyceae) on survival and grazing activity of several shellfish species}, volume={5}, ISSN={["1878-1470"]}, DOI={10.1016/j.hal.2006.04.013}, abstractNote={A series of experiments was conducted to examine effects of four strains of the estuarine dinoflagellate, Pfiesteria shumwayae, on the behavior and survival of larval and adult shellfish (bay scallop, Argopecten irradians; eastern oyster, Crassostrea virginica; northern quahogs, Mercenaria mercenaria; green mussels, Perna viridis [adults only]). In separate trials with larvae of A. irradians, C. virginica, and M. mercenaria, an aggressive predatory response of three strains of algal- and fish-fed P. shumwayae was observed (exception, algal-fed strain 1024C). Larval mortality resulted primarily from damage inflicted by physical attack of the flagellated cells, and secondarily from Pfiesteria toxin, as demonstrated in larval C. virginica exposed to P. shumwayae with versus without direct physical contact. Survival of adult shellfish and grazing activity depended upon the species and the cell density, strain, and nutritional history of P. shumwayae. No mortality of the four shellfish species was noted after 24 h of exposure to algal- or fish-fed P. shumwayae (strains 1024C, 1048C, and CCMP2089) in separate trials at ≤5 × 103 cells ml−1, whereas higher densities of fish-fed, but not algal-fed, populations (>7–8 × 103 cells ml−1) induced low (≤15%) but significant mortality. Adults of all four shellfish species sustained >90% mortality when exposed to fish-fed strain 270A1 (8 × 103 cells ml−1). In contrast, adult M. mercenaria and P. viridis exposed to a similar density of fish-fed strain 2172C sustained <15% mortality, and there was no mortality of A. irradians and C. virginica exposed to that strain. In mouse bioassays with tissue homogenates (adductor muscle, mantle, and whole animals) of A. irradians and M. mercenaria that had been exposed to P. shumwayae (three strains, separate trials), mice experienced several minutes of disorientation followed by recovery. Mice injected with tissue extracts from control animals fed cryptomonads showed no response. Grazing rates of adult shellfish on P. shumwayae (mean cell length ±1 standard error [S.E.], 9 ± 1 μm) generally were significantly lower when fed fish-fed (toxic) populations than when fed populations that previously had been maintained on algal prey, and grazing rates were highest with the nontoxic cryptomonad, Storeatula major (cell length 7 ± 1 μm). Abundant cysts of P. shumwayae were found in fecal strands of all shellfish species tested, and ≤45% of the feces produced viable flagellated cells when placed into favorable culture conditions. These findings were supported by a field study wherein fecal strands collected from field-collected adult shellfish (C. virginica, M. mercenaria, and ribbed mussels, Geukensia demissa) were confirmed to contain cysts of P. shumwayae, and these cysts produced fish-killing flagellated populations in standardized fish bioassays. Thus, predatory feeding by flagellated cells of P. shumwayae can adversely affect survival of larval bivalve molluscs, and grazing can be depressed when adult shellfish are fed P. shumwayae. The data suggest that P. shumwayae could affect recruitment of larval shellfish in estuaries and aquaculture facilities; shellfish can be adversely affected via reduced filtration rates; and adult shellfish may be vectors of toxic P. shumwayae when shellfish are transported from one geographic location to another.}, number={4}, journal={HARMFUL ALGAE}, author={Shumway, Sandra E. and Burkholder, JoAnn M. and Springer, Jeffrey}, year={2006}, month={Sep}, pages={442–458} }
 @article{lewitus_wetz_willis_burkholder_parrow_glasgow_2006, title={Grazing activity of Pfiesteria piscicida (Dinophyceae) and susceptibility to ciliate predation vary with toxicity status}, volume={5}, ISSN={["1878-1470"]}, DOI={10.1016/j.hal.2006.04.012}, abstractNote={Variability has been reported in the toxicity potential of Pfiesteria piscicida that is partly a function of the history of exposure to live fish. Grazing properties of P. piscicida and its susceptibility to ciliate predation were compared in three functional types or toxicity states of this species: actively toxic cultures, cultures with temporary loss of demonstrable toxicity, and cultures with no demonstrable toxicity. Pronounced differences in predator–prey interactions were found between actively toxic cultures and cultures with reduced toxicity. When grown with Rhodomonas sp. (Cryptophyceae) prey, specific growth rates were relatively low in actively toxic cultures under both relatively high and low irradiances. In the cultures with reduced toxicity, prey chloroplast material was apparent in nearly 100% of dinoflagellate cells 3 h after feeding, while chloroplast inclusions were found in <40% of actively toxic cells for ≤16 h (high light) and ≤23 h (low light). These results suggest a relatively high reliance on phagotrophic carbon assimilation and more rapid response to algal prey availability in Pfiesteria cells with lower toxicity. Grazing by two euplotid benthic ciliates (Euplotes vannus and E. woodruffi) on P. piscicida also varied among functional types. Grazing on actively toxic P. piscicida cells did not occur, whereas net positive ingestion rates were calculated for the other prey cultures. These results support concurrent experimental findings that a natural assemblage of microzooplankton displayed lower grazing potential on actively toxic P. piscicida than on cultures with reduced toxicity. In summary, pronounced differences in trophic interactions were found between actively toxic cultures and those with reduced or undetectable toxicity, providing additional evidence of the importance of cellular toxicity in the trophic ecology of Pfiesteria.}, number={4}, journal={HARMFUL ALGAE}, author={Lewitus, Alan J. and Wetz, Michael S. and Willis, Bonnie M. and Burkholder, JoAnn M. and Parrow, Matthew W. and Glasgow, Howard B.}, year={2006}, month={Sep}, pages={427–434} }
 @article{burkholder_glibert_2006, title={Intraspecific variability: an important consideration in forming generalisations about toxigenic algal species}, volume={28}, ISSN={["1814-232X"]}, DOI={10.2989/18142320609504143}, abstractNote={Intraspecific variability (strain differences) in key characteristics such as life-history traits, behaviour, nutrition, genetics and toxicity, has been experimentally documented for many toxigenic microalgae, including species of cyanobacteria, dinoflagellates, haptophytes, raphidophytes and diatoms. This paper summarises findings from published studies on intraspecific variability in toxicity. The data show that different, often opposite, interpretations at the species level would have resulted from consideration of individual strains. A survey of recent literature on harmful algae revealed that intraspecific variation is still commonly overlooked in characterisations of these species and generalisations about their roles in foodwebs. The available data underscore the importance of including multiple strains in research to advance understanding about toxigenic algal species and the importance of tempering conclusions to consider the potential for differences beyond the strains studied.}, number={2}, journal={AFRICAN JOURNAL OF MARINE SCIENCE}, author={Burkholder, J. M. and Glibert, P. M.}, year={2006}, month={Sep}, pages={177–180} }
 @article{rublee_nuzzi_waters_schaefer_burkholder_2006, title={Pfiesteria piscicida and Pfiesteria shumwayae in coastal waters of Long Island, New York, USA}, volume={5}, ISSN={["1878-1470"]}, DOI={10.1016/j.hal.2006.03.007}, abstractNote={Water and sediment samples were collected during summer and early fall 1999–2004 from coastal waters of New York State, USA, to test for the presence of Pfiesteria piscicida and Pfiesteria shumwayae. Physical and chemical conditions were characterized, and real-time polymerase chain reaction assays were conducted. Both species were relatively common and found at most sites at least once, and the frequency of positive assays was higher in sediments than in the water column. In a subset of the data from Suffolk County, Long Island, the presence of Pfiesteria was related to high chlorophyll a and relatively high nutrient concentrations. Partial SSU rDNA sequences of four PCR amplicons generated using P. shumwayae primers indicated two sequences: three were identical to GenBank P. shumwayae entries, but one showed enough sequence difference (15 positions in a 454 bp amplicon) to suggest a possible new species. Three isolates were tested for toxicity, and one was found to kill fish in bioassays. Despite the widespread presence of both Pfiesteria species and demonstration of potential to harm fish, no blooms of these dinoflagellates have been observed, nor has there been evidence of Pfiesteria-related fish or human health problems in these waters, likely related to colder temperatures than optimal for Pfiesteria species.}, number={4}, journal={HARMFUL ALGAE}, author={Rublee, Parke A. and Nuzzi, Robert and Waters, Robert and Schaefer, Eric F. and Burkholder, JoAnn A.}, year={2006}, month={Sep}, pages={374–379} }
 @article{skelton_parrow_burkholder_2006, title={Phosphatase activity in the heterotrophic dinoflagellate Pfiesteria shumwayae}, volume={5}, ISSN={["1878-1470"]}, DOI={10.1016/j.hal.2006.04.010}, abstractNote={The ELF-97 phosphatase substrate was used to examine phosphatase activity in four strains of the estuarine heterotrophic dinoflagellate, Pfiesteria shumwayae. Acid and alkaline phosphatase activities also were evaluated at different pH values using bulk colorimetric methods. Intracellular phosphatase activity was demonstrated in P. shumwayae cells that were actively feeding on a fish cell line and in food limited cells that had not fed on fish cells for 3 days. All strains, whether actively feeding or food limited showed similar phosphatase activities. P. shumwayae cells feeding on fish cells showed ELF-97 activity near, or surrounding, the food vacuole. Relatively small, spherical ELF-97 deposits were also observed in the cytoplasm and sometimes near the plasma membrane. ELF-97 fluorescence was highly variable among cells, likely reflecting different stages in digestion and related metabolic processes. The location of enzyme activity and supporting colorimetric measurements suggest that, as in other heterotrophic protists, acid phosphatases predominate in P. shumwayae and have a general catabolic function.}, number={4}, journal={HARMFUL ALGAE}, author={Skelton, Hayley M. and Parrow, Matthew W. and Burkholder, JoAnn M.}, year={2006}, month={Sep}, pages={395–406} }
 @article{marshall_hargraves_burkholder_parrow_elbraechter_allen_knowlton_rublee_hynes_egerton_et al._2006, title={Taxonomy of Pfiesteria (Dinophyceae)}, volume={5}, ISSN={["1878-1470"]}, DOI={10.1016/j.hal.2006.05.001}, abstractNote={The dinoflagellate species originally described as Pfiesteria shumwayae Glasgow et Burkholder, recently transferred to a new genus, Pseudopfiesteria Litaker et al., is reclassified into the redefined genus Pfiesteria Steidinger et Burkholder, as Pfiesteria shumwayae within the order Peridiniales. This change is based upon consideration of a compilation of previous and new morphological analyses and molecular phylogenetic analyses. Morphological analysis with scanning and transmission electron microscopy supports previous findings except in the sulcal area. In the cells examined, the sulcus is partly concealed by the peduncle cover plate (p.c.), which originates at the right side of the sulcus along the left side of the 6c and 5‴ plates. The fine structure of the p.c. appears similar to that of other thecal plates. The 1″ plate can also extend slightly over the sulcus. Transmission electron microscopy revealed that Pfiesteria shumwayae can have at least six sulcal plates; the number remains uncertain and may vary. The sulcal plates of this small, delicately thecate species have not been clearly discerned by scanning electron microscopy of membrane-stripped and/or suture-swollen cells. The Kofoidian thecal plate formula for the genus Pfiesteria is Po, cp, X, 4′, la, 5–6″, 6c, p.c., ?s, 5‴, 0p, 2‴′. The monophyletic grouping of “pfiesteria-like” taxa within the order Peridiniales, as well as the grouping of Pfiesteria piscicida and Pfiesteria shumwayae within the same genus, is also supported by the preponderance of previous molecular evidence, and by the phylogenetic trees contributed in the present analysis. Pfiesteria appears to be closely related to as-yet informally described cryptoperidiniopsoids and calcareous dinoflagellates such as Thoracosphaera; thus, the family classification requires revision that is beyond the scope of this study.}, number={5}, journal={HARMFUL ALGAE}, author={Marshall, Harold G. and Hargraves, Paul E. and Burkholder, JoAnn M. and Parrow, Matthew W. and Elbraechter, Malte and Allen, Elle H. and Knowlton, Valerie M. and Rublee, Parke A. and Hynes, Wayne L. and Egerton, Todd A. and et al.}, year={2006}, month={Oct}, pages={481–496} }
 @article{parrow_elbraechter_krause_burkholder_deamer_htyte_allen_2006, title={The taxonomy and growth of a Crypthecodinium species (Dinophyceae) isolated from a brackish-water fish aquarium}, volume={28}, ISSN={["1814-232X"]}, DOI={10.2989/18142320609504145}, abstractNote={An unidentified heterotrophic dinoflagellate found growing in abundance in a brackish-water fish aquarium was isolated and serially cultivated using a fish cell line as the food source. Prominent characteristics of this dinoflagellate included a cingulum that did not fully encircle the motile cell, cell division in non-motile cysts, and a theca composed of thin but structured plates. Morphological analysis of flagellate cells by scanning electron microscopy revealed a Kofoid thecal plate tabulation of 4', 4a, 4", 'X', 5 or 6c, ?s, 5"', 1p, 1"", most consistent with the original description of Crypthecodinium setense Biecheler. This Crypthecodinium species exhibited a high maximum division rate (3.2 divisions day−1) and cell yield (>106 cells ml−1) when fed cultured fish cells. Small sub-unit rDNA phylogenetic analyses supported relatedness with a previously studied Crypthecodinium-like dinoflagellate, but a significant difference in aligned gene sequences was found. This study provides the first clear demonstration of the plate tabulation of a Crypthecodinium species since the original description over 60 years ago, allowing the original morphological conception of Crypthecodinium to be linked with molecular phylogenetic information.}, number={2}, journal={AFRICAN JOURNAL OF MARINE SCIENCE}, author={Parrow, M. W. and Elbraechter, M. and Krause, M. K. and Burkholder, J. M. and Deamer, N. J. and Htyte, N. and Allen, E. H.}, year={2006}, month={Sep}, pages={185–191} }
 @article{glibert_burkholder_2006, title={Toward an emerging consensus on the ecology of Pfiesteria}, volume={5}, ISSN={["1568-9883"]}, DOI={10.1016/j.hal.2006.05.002}, number={4}, journal={HARMFUL ALGAE}, author={Glibert, Patricia M. and Burkholder, JoAnn M.}, year={2006}, month={Sep}, pages={339–341} }
 @misc{glasgow_reed_toms_burkholder_2006, title={Variable depth automated dynamic water profiler}, volume={7,040,157}, publisher={Washington, DC: U.S. Patent and Trademark Office}, author={Glasgow, H., Jr. and Reed, R. E. and Toms, D. C. and Burkholder, J.}, year={2006} }
 @article{parrow_burkholder_deamer_ramsdell_2005, title={Contaminant-free cultivation of Pfiesteria shumwayae (Dinophyceae) on a fish cell line}, volume={39}, ISSN={["0948-3055"]}, DOI={10.3354/ame039097}, abstractNote={Geographically distinct strains of the heterotrophic dinoflagellate Pfiesteria shumwayae were cultivated on a fish cell line in the apparent absence of bacteria and other microbial conta- minants. Cultures were established with a high rate of success by inoculating single purified P. shumwayae cells into fish cell cultures containing a simple saltwater medium suitable for both cell types, and resulting isolates were serially cultivated on fish cells for months without visible signs of abnormality or reduced viability. P. shumwayae fed phagocytically on the fish cells and exhibited higher cell production than reported using other culturing methods. Compared to previous methods of studying the interaction between Pfiesteria spp. and fishes, this system enabled closer and more direct observation of the dinoflagellates and was also more economical and sustainable as a culturing method. The absence of bacteria and other contaminating microorganisms should facilitate important physiological and biochemical investigations. The methods used were inadequate for cultivating strains of P. piscicida, suggesting a possible difference in nutritional requirements between the 2 Pfiesteria species.}, number={1}, journal={AQUATIC MICROBIAL ECOLOGY}, author={Parrow, MW and Burkholder, JM and Deamer, NJ and Ramsdell, JS}, year={2005}, month={Apr}, pages={97–105} }
 @article{burkholder_gordon_moeller_law_coyne_lewitus_ramsdell_marshall_deamer_cary_et al._2005, title={Demonstration of toxicity to fish and to mammalian cells by Pfiesteria species: Comparison of assay methods and strains}, volume={102}, ISSN={0027-8424 1091-6490}, url={http://dx.doi.org/10.1073/pnas.0500168102}, DOI={10.1073/pnas.0500168102}, abstractNote={
            Toxicity and its detection in the dinoflagellate fish predators
            Pfiesteria piscicida
            and
            Pfiesteria shumwayae
            depend on the strain and the use of reliable assays. Two assays, standardized fish bioassays (SFBs) with juvenile fish and fish microassays (FMAs) with larval fish, were compared for their utility to detect toxic
            Pfiesteria
            . The comparison included strains with confirmed toxicity, negative controls (noninducible
            Pfiesteria
            strains and a related nontoxic cryptoperidiniopsoid dinoflagellate), and
            P. shumwayae
            strain CCMP2089, which previously had been reported as nontoxic. SFBs, standardized by using toxic
            Pfiesteria
            (coupled with tests confirming
            Pfiesteria
            toxin) and conditions conducive to toxicity expression, reliably detected actively toxic
            Pfiesteria
            , but FMAs did not.
            Pfiesteria
            toxin was found in fish- and algae-fed clonal
            Pfiesteria
            cultures, including CCMP2089, but not in controls. In contrast, noninducible
            Pfiesteria
            and cryptoperidiniopsoids caused no juvenile fish mortality in SFBs even at high densities, and low larval fish mortality by physical attack in FMAs. Filtrate from toxic strains of
            Pfiesteria
            spp. in bacteria-free media was cytotoxic. Toxicity was enhanced by bacteria and other prey, especially live fish. Purified
            Pfiesteria
            toxin extract adversely affected mammalian cells as well as fish, and it caused fish death at environmentally relevant cell densities. These data show the importance of testing multiple strains when assessing the potential for toxicity at the genus or species level, using appropriate culturing techniques and assays.
          }, number={9}, journal={Proceedings of the National Academy of Sciences}, publisher={Proceedings of the National Academy of Sciences}, author={Burkholder, J. M. and Gordon, A. S. and Moeller, P. D. and Law, J. M. and Coyne, K. J. and Lewitus, A. J. and Ramsdell, J. S. and Marshall, H. G. and Deamer, N. J. and Cary, S. C. and et al.}, year={2005}, month={Feb}, pages={3471–3476} }
 @article{springer_burkholder_glibert_reed_2005, title={Use of a real-time remote monitoring network (RTRM) and shipborne sampling to characterize a dinoflagellate bloom in the Neuse Estuary, North Carolina, USA}, volume={4}, ISSN={["1878-1470"]}, DOI={10.1016/j.hal.2004.08.017}, abstractNote={The spatial-temporal distribution of a dinoflagellate bloom dominated or co-dominated by Prorocentrum minimum was examined during autumn through early spring in a warm temperate, eutrophic estuary. The developing bloom was first detected from a web-based alert provided by a network of real-time remote monitoring (RTRM) platforms indicating elevated dissolved oxygen and pH levels in upper reaches of the estuary. RTRM data were used to augment shipboard sampling, allowing for an in-depth characterization of bloom initiation, development, movement, and dissipation. Prolonged drought conditions leading to elevated salinities, and relatively high nutrient concentrations from upstream inputs and other sources, likely pre-disposed the upper estuary for bloom development. Over a 7-month period (October 2001–April 2002), the bloom moved toward the northern shore of the mesohaline estuary, intensified under favorable conditions, and finally dissipated after a major storm. Bloom location and transport were influenced by prevailing wind structure and periods of elevated rainfall. Chlorophyll a within bloom areas averaged 106 ± 13 μg L−1 (mean ± 1 S.E.; maximum, 803 μg L−1), in comparison to 20 ± 1 μg L−1 outside the bloom. There were significant positive relationships between dinoflagellate abundance and TN and TP. Ammonium, NO3−, and SRP concentrations did not decrease within the main bloom, suggesting that upstream inputs and other sources provided nutrient-replete conditions. In addition, PAM fluorometric measurements (09:00–13:00 h) of maximal PSII quantum yield (Fv/Fm) were consistently 0.6–0.8 within the bloom until late March, providing little evidence of photo-physiological stress as would have been expected under nutrient-limiting conditions. Nitrogen uptake kinetics were estimated for P. minimum during the period when that species was dominant (October–December 2001), based on literature values for N uptake by an earlier P. minimum bloom (winter 1999) in the Neuse Estuary. The analysis suggests that NH4+ was the major N species that supported the bloom. Considering the chlorophyll a concentrations during October and December and the estimated N uptake rates, phytoplankton biomass was estimated to have doubled once per day. Bloom displacement (January–February) coincided with higher diversity of heterotrophic dinoflagellate species as P. minimum abundance decreased. This research shows the value of RTRM in bloom detection and tracking, and advances understanding of dinoflagellate bloom dynamics in eutrophic estuaries.}, number={3}, journal={HARMFUL ALGAE}, author={Springer, JJ and Burkholder, JM and Glibert, PM and Reed, RE}, year={2005}, month={Mar}, pages={533–551} }
 @article{burkholder_eggleston_glasgow_brownie_reed_janowitz_posey_melia_kinder_corbett_et al._2004, title={Comparative impacts of two major hurricane seasons on the Neuse River and western Pamlico Sound ecosystems}, volume={101}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.0306842101}, abstractNote={Ecosystem-level impacts of two hurricane seasons were compared several years after the storms in the largest lagoonal estuary in the U.S., the Albemarle–Pamlico Estuarine System. A segmented linear regression flow model was developed to compare mass-water transport and nutrient loadings to a major artery, the Neuse River Estuary (NRE), and to estimate mean annual versus storm-related volume delivery to the NRE and Pamlico Sound. Significantly less water volume was delivered by Hurricane Fran (1996), but massive fish kills occurred in association with severe dissolved oxygen deficits and high contaminant loadings (total nitrogen, total phosphorus, suspended solids, and fecal bacteria). The high water volume of the second hurricane season (Hurricanes Dennis, Floyd, and Irene in 1999) delivered generally comparable but more dilute contaminant loads, and no major fish kills were reported. There were no discernable long-term adverse impacts on water quality. Populations of undesirable organisms, such as toxic dinoflagellates, were displaced down-estuary to habitats less conducive for growth. The response of fisheries was species-dependent: there was no apparent impact of the hurricanes on commercial landings of bivalve molluscs or shrimp. In contrast, interacting effects of hurricane floodwaters in 1999 and intensive fishing pressure led to striking reductions in blue crabs. Overall, the data support the premise that, in shallow estuaries frequently disturbed by hurricanes, there can be relatively rapid recovery in water quality and biota, and benefit from the scouring activity of these storms.}, number={25}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Burkholder, J and Eggleston, D and Glasgow, H and Brownie, C and Reed, R and Janowitz, G and Posey, M and Melia, G and Kinder, C and Corbett, R and et al.}, year={2004}, month={Jun}, pages={9291–9296} }
 @article{coyne_burkholder_feldman_hutchins_cary_2004, title={Modified serial analysis of gene expression method for construction of gene expression profiles of microbial eukaryotic species}, volume={70}, ISSN={["1098-5336"]}, DOI={10.1128/AEM.70.9.5298-5304.2004}, abstractNote={ABSTRACT}, number={9}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Coyne, KJ and Burkholder, JM and Feldman, RA and Hutchins, DA and Cary, SC}, year={2004}, month={Sep}, pages={5298–5304} }
 @misc{burkholder_2004, title={Ongoing controversy over Pfiesteria}, volume={304}, DOI={10.1126/science.304.5667.46b}, abstractNote={READERS OF J. KAISER'S ARTICLE " THE Pfiesteria conundrum: More study, less certainty " (News of the Week, 2 Jan., p. 25) may have missed the fact that there is more certainty, not less, about the toxic microbe Pfiesteria. Some Virginia Institute of Marine Science (VIMS) scientists and their collaborators had earlier concluded that Pfiesteria as a whole is not toxigenic and only physically attacks fish (1, 2), based on one strain of P. shumwayae, CCMP2089. Yet Pfiesteria has nontoxic as well as toxic strains (3), and expression of toxicity by toxic strains depends on culture conditions (4). Other laboratories have now shown that when cultured and tested appropriately (5, 6), strain CCMP2089 is ichthyotoxic. Kaiser states that I declined to send these VIMS scientists toxic Pfiesteria culture. I had offered to provide them toxic culture and to show them how to grow it to express toxicity, if they would support the cost. Kaiser also describes me as not providing toxic Pfiesteria to the research community in general, although I have provided it to more than 40 scientists. Kaiser does not mention the two recent publications on Pfiesteria effects on fish and mammals: A highly toxic Pfiesteria strain killed shellfish larvae as a toxic effect, without physical contact (7), and Pfiesteria toxin caused hippocampal damage in rats (8). Kaiser mentions only an unpublished study that, logically, found no evidence of health impacts from Pfiesteria because there were no toxic Pfiesteria blooms during the study. Kaiser credits the VIMS scientists (1) for having found that Pfiesteria can kill larval finfish by physical attack, which colleagues and I had earlier published [(4), p. 200], and she asserts that I have attributed fish death from Pfiesteria only to toxin. I have described physical attack and toxin as important interactive factors [(9), p. 672]. Kaiser reports only negative findings about Pfiesteria amoebae. She mentions a study by Litaker et al. (10), who did not find amoebae in two P. piscicida strains (cultures 2 to 10 years old, of uncertain toxicity status) and, on that basis, concluded that the species does not form amoebae. She does not mention a peer-reviewed paper (11) with corrective information: Amoebae are minor to the toxicity issue; toxin is produced mostly by flagellated stages. Cultured toxic strains mostly have formed amoebae within the first few months after field isolation, and nontoxic strains rarely form amoebae. Kaiser describes a workshop presentation …}, number={5667}, journal={Science}, author={Burkholder, J. M.}, year={2004}, pages={46} }
 @article{glasgow_burkholder_reed_lewitus_kleinman_2004, title={Real-time remote monitoring of water quality: a review of current applications, and advancements in sensor, telemetry, and computing technologies}, volume={300}, ISSN={["1879-1697"]}, DOI={10.1016/j.jembe.2004.02.022}, abstractNote={Recent advances in communication and sensor technology have catalyzed progress in remote monitoring capabilities for water quality. As a result, the ability to characterize dynamic hydrologic properties at adequate temporal and spatial scales has greatly improved. These advances have led to improved statistical and mechanistic modeling in monitoring of water quality trends at local, watershed and regional scales for freshwater, estuarine and marine ecosystems. In addition, they have greatly enhanced rapid (e.g., real-time) detection of hydrologic variability, recognized as a critical need for early warning systems and rapid response to harmful algal bloom events. Here, we present some of the landmark developments and technological achievements that led to the advent of real-time remote monitors for hydrologic properties. We conclude that increased use and continuing advancements of real-time remote monitoring (RTRM) and sensing technologies will become a progressively more important tool for evaluating water quality. Recent engineering and deployment of RTRM technologies by federal and state regulatory agencies, industries, and academic laboratories is now permitting rapid detection of, and responses to, environmental threats imposed by increased nutrient loadings, development of hypoxic and anoxic areas, toxicants, and harmful algal bloom outbreaks leading to fish kill events and potential human health impacts.}, number={1-2}, journal={JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY}, author={Glasgow, HB and Burkholder, JM and Reed, RE and Lewitus, AJ and Kleinman, JE}, year={2004}, month={Mar}, pages={409–448} }
 @article{reed_glasgow_burkholder_brownie_2004, title={Seasonal physical–chemical structure and acoustic Doppler current profiler flow patterns over multiple years in a shallow, stratified estuary, with implications for lateral variability}, volume={60}, ISSN={0272-7714}, url={http://dx.doi.org/10.1016/j.ecss.2004.02.010}, DOI={10.1016/j.ecss.2004.02.010}, abstractNote={The overall goal of this study was to strengthen understanding of the hydrographic structure in shallow estuaries as influenced by seasonal and depth-dependent variability, and by variability from extreme meteorological events. The mesohaline Neuse Estuary, North Carolina, U.S.A., which was the focus, receives surface inputs from upriver and tributary freshwater sources and bottom inputs from downriver high-salinity sound water sources, resulting in varying degrees of stratification. To assess depth-dependent, estuary-wide changes in salinity, a multiple time series was created using data from four discrete depths (surface and 1, 2, and 3m±0.25m). The database was developed from weekly to biweekly sampling of the entire water column, and included side-channel as well as mid-channel data. We characterized seasonal differences in halocline depth affecting the hydrographic structure of the mesohaline estuary and site-specific variation in nutrient concentrations, based on a comprehensive eight-year physical/chemical database. The first two years of the record showed an expected seasonal signal and included events that impacted the surface layer from freshwater inputs. Remaining years had greater variability over seasons and depths, with freshening events that affected all depths. Halocline depth was compared at specific locations, and a “snapshot” view was provided of the relative depth of these water masses within the estuary by season. We also examined flow patterns at the same cross-estuary sites over a three-year period, using a boat-mounted acoustic Doppler current profiler (ADCP) with bottom-tracking capability. Composite visualizations constructed with single-transect ADCP data revealed a classical estuarine circulation pattern of outflow at the surface/southern shore and inflow at the bottom/northern shore. Although this pattern deviated under extreme climatological events and was sometimes variable, the estuary generally exhibited a high probability of direction of flow. Wind fields, hurricanes, and small-scale, high-precipitation events represented significant forcing variables.}, number={4}, journal={Estuarine, Coastal and Shelf Science}, publisher={Elsevier BV}, author={Reed, Robert E. and Glasgow, Howard B. and Burkholder, JoAnn M. and Brownie, Cavell}, year={2004}, month={Aug}, pages={549–566} }
 @article{parrow_burkholder_2004, title={The sexual life cycles of Pfiesteria piscicida and cryptoperidiniopsoids (Dinophyceae)}, volume={40}, ISSN={["1529-8817"]}, DOI={10.1111/j.1529-8817.2004.03202.x}, abstractNote={Sexual life cycle events in Pfiesteria piscicida and cryptoperidiniopsoid heterotrophic dinoflagellates were determined by following the development of isolated gamete pairs in single‐drop microcultures with cryptophyte prey. Under these conditions, the observed sequence of zygote formation, development, and postzygotic divisions was similar in these dinoflagellates. Fusion of motile gamete pairs each produced a rapidly swimming uninucleate planozygote with two longitudinal flagella. Planozygotes enlarged as they fed repeatedly on cryptophytes. In <12 h in most cases, each planozygote formed a transparent‐walled nonmotile cell (cyst) with a single nucleus. Zygotic cysts did not exhibit dormancy under these conditions. In each taxon, dramatic swirling chromosome movements (nuclear cyclosis) were found in zygote nuclei before division. In P. piscicida, nuclear cyclosis occurred in the zygotic cyst or apparently earlier in the planozygote. In the cryptoperidiniopsoids, nuclear cyclosis occurred inthe zygotic cyst. After nuclear cyclosis, a single cell division occurred in P. piscicida and cryptoperidiniopsoid zygotic cysts, producing two offspring that emerged as biflagellated cells. These two flagellated cells typically swam for hours and fed on cryptophytes before encysting. A single cell division in these cysts produced two biflagellated offspring that also fed before encysting for further reproduction. This sequence of zygote development and postzygotic divisions typically was completed within 24 h and was confirmed in examples from different isolates of each taxon. Some aspects of the P. piscicida sexual life cycle determined here differed from previous reports.}, number={4}, journal={JOURNAL OF PHYCOLOGY}, author={Parrow, MW and Burkholder, JM}, year={2004}, month={Aug}, pages={664–673} }
 @article{fan_glibert_burkholder_2003, title={Characterization of the affinity for nitrogen, uptake kinetics, and environmental relationships for Prorocentrum minimum in natural blooms and laboratory cultures}, volume={2}, ISSN={["1878-1470"]}, DOI={10.1016/S1568-9883(03)00047-7}, abstractNote={During the late spring and early summer of 1998, an extensive bloom of the dinoflagellate Prorocentrum minimum (>93% of phytoplankton cell density) developed in several tributaries of the Chesapeake Bay, USA. In January 1999, a bloom of mixed dinoflagellates (Heterocapsa rotundata, H. triquetra and P. minimum, with P. minimum forming 21% of total phytoplankton cells and 39% of the total biovolume) developed in the mesohaline Neuse Estuary, North Carolina, USA. During these blooms, experiments were carried out to characterize the nitrogen uptake kinetics of these assemblages with 15N isotopic techniques. Four nitrogenous substrates (NO3−, NH4+, urea, and a mixed amino acids substrate) were used to determine uptake rate and substrate preference. Rates of nitrogen uptake were also measured in P. minimum cultures grown on varying growth nitrogen substrates. The calculated kinetic parameters determined for the P. minimum-dominated field assemblages and the cultures indicated a preference for NH4+. NH4+ was also the primary nitrogen source supporting the blooms. In addition, a high affinity for urea was also found, and urea contributed significantly to the Neuse Estuary bloom. Furthermore, results showed that the regulation of uptake for each of the substrates was different: strong positive relationships between affinity and temperature were found for NH4+ and amino acids, while a negative response was found for NO3−, and very little response to temperature was noted for urea. These differences suggest that a diversity of nitrogen uptake mechanisms may aid the development and maintenance of P. minimum blooms.}, number={4}, journal={HARMFUL ALGAE}, author={Fan, CL and Glibert, PM and Burkholder, JM}, year={2003}, month={Oct}, pages={283–299} }
 @misc{parrow_burkholder_2003, title={Estuarine heterotrophic cryptoperidiniopsoids (Dinophyceae): Life cycle and culture studies}, volume={39}, ISSN={["1529-8817"]}, DOI={10.1046/j.1529-8817.2003.02146.x}, abstractNote={Cryptoperidiniopsoids are an unclassified group of delicately thecate heterotrophic dinoflagellates known to be common in eastern U.S. estuarine waters. Over the past 10 years cryptoperidiniopsoids were isolated from different geographical regions and cultured with cryptophyte algal prey. In the seven clonal isolates examined, reproduction was strongly linked to the availability of prey cells. The dinoflagellates phagocytized the contents of prey cells through a tube‐like peduncle, similarly as close relatives of Pfiesteria spp. and several other heterotrophic species. Cell division occurred while encysted, most commonly yielding two biflagellated offspring. Abundant fusing gametes, phagotrophic planozygotes, and cysts with a pronounced nuclear cyclosis characterized persistent sexuality. Cysts with nuclear cyclosis produced two flagellated offspring cells. The resistance of reproductive cysts to antimicrobial treatments was examined, and a simple high‐yield technique was developed for population synchronization while ridding the dinoflagellates of most contaminating vacuolar prey DNA and external contaminants. The DNA content and population DNA profiles of synchronously excysted cryptoperidiniopsoids from different isolates were measured using flow cytometry and were related to the life history of these and other dinoflagellates. Cryptophyte‐fed cultures with versus without extracellular bacteria were compared, and bacteria apparently promoted cryptoperidiniopsoid feeding and growth. Externally bacteria‐free dinoflagellates were cultured in media enriched with dissolved organic nutrients, and nutritional benefit may have occurred in some treatments. The potential for mixotrophic nutrition from maintenance of cryptophyte chloroplasts was examined using flow cytometrically sorted cells, but evidence of kleptoplastidy was not found in these isolates under the conditions imposed.}, number={4}, journal={JOURNAL OF PHYCOLOGY}, author={Parrow, MW and Burkholder, JM}, year={2003}, month={Aug}, pages={678–696} }
 @article{tengs_bowers_glasgow_burkholder_oldach_2003, title={Identical ribosomal DNA sequence data from Pfiesteria piscicida (Dinophyceae) isolates with different toxicity phenotypes}, volume={93}, ISSN={["1096-0953"]}, DOI={10.1016/s0013-9351(02)00087-7}, abstractNote={Complete small subunit ribosomal RNA, internal transcribed spacer 1 and 2, 5.8S, and partial large subunit ribosomal RNA gene sequences were generated from multiple isolates of Pfiesteria piscicida. Sequences were derived from isolates that have been shown to be ichthyotoxic as well as isolates that have no history of toxic behavior. All of the sequences generated were identical for the different cultures, and we therefore conclude that differences in toxicity seen between isolates of P. piscicida are linked to factors other than genetic strain variation detectable by ribosomal gene sequence analyses.}, number={1}, journal={ENVIRONMENTAL RESEARCH}, author={Tengs, T and Bowers, HA and Glasgow, HB and Burkholder, JM and Oldach, DW}, year={2003}, month={Sep}, pages={88–91} }
 @article{brownie_glasgow_burkholder_reed_tang_2003, title={Re-evaluation of the relationship between Pfiesteria and estuarine fish kills}, volume={6}, ISSN={["1435-0629"]}, DOI={10.1007/s10021-002-0194-5}, number={1}, journal={ECOSYSTEMS}, author={Brownie, C and Glasgow, HB and Burkholder, JM and Reed, R and Tang, YQ}, year={2003}, month={Jan}, pages={1–10} }
 @article{parrow_burkholder_2003, title={Reproduction and sexuality in Pfiesteria shumwayae (Dinophyceae)}, volume={39}, ISSN={["1529-8817"]}, DOI={10.1046/j.1529-8817.2003.03057.x}, abstractNote={ Pfiesteria shumwayae is a heterotrophic dinoflagellate with a widespread distribution in temperate‐subtropical estuarine waters. In this study, five clonal isolates from the eastern coast of North America, one from New Zealand, and a mixed composite of clones were cultured in aquaria and fed live fish. Division, sexuality, and phagotrophic feeding on fish were studied by LM, SEM, and flow cytometry. The development of reproductive cysts isolated from aquaria was followed. Synchronously excysted flagellate populations were examined for sexuality and then for feeding behavior and reproduction when given larval fish. Reproductive cysts varied in size and underwent protoplast division(s), most commonly producing two to eight biflagellated offspring. Fusing gametes, resulting planozygotes, and nuclear cyclosis were documented as evidence of sexuality. Gametes emerged from cysts, and fusions were approximately isogamous. Resulting planozygotes had two longitudinal flagella and one transverse flagellum and apparently fed before encysting. Distinct and lengthy chromosome movements (nuclear cyclosis) occurred in presumed zygotic cysts before nuclear division(s). These cysts did not exhibit dormancy in growing cultures and produced two or four biflagellated offspring. Flagellated cells fed on surficial fish tissues and then encysted for reproduction. Stages indicating a completed sexual cycle (fusion, planozygotes, and nuclear cyclosis) were uncommon or absent in clonal cultures but were relatively abundant in the mixed clone culture. Self‐sterility factors apparently influenced sexuality. Starved populations formed quiescent cysts that released swimming cells when food was provided. Pfiesteria shumwayae was similar in reproduction and sexuality to closely related species.}, number={4}, journal={JOURNAL OF PHYCOLOGY}, author={Parrow, MW and Burkholder, JM}, year={2003}, month={Aug}, pages={697–711} }
 @article{touchette_burkholder_glasgow_2003, title={Variations in eelgrass (Zostera marina L.) morphology and internal nutrient composition as influenced by increased temperature and water column nitrate}, volume={26}, ISSN={["0160-8347"]}, DOI={10.1007/BF02691701}, number={1}, journal={ESTUARIES}, author={Touchette, BW and Burkholder, JM and Glasgow, HB}, year={2003}, month={Feb}, pages={142–155} }
 @inbook{burkholder_2002, title={Chronic effects of toxic microalgae on finfish, shellfish, and human health}, ISBN={0195150937}, booktitle={Conservation medicine: Ecological health in practice}, publisher={Oxford; New York: Oxford University Press}, author={Burkholder, J. M.}, year={2002}, pages={229–249} }
 @article{jakobsen_tengs_vatne_bowers_oldach_burkholder_glasgow_rublee_klaveness_2002, title={Discovery of the toxic dinoflagellate Pfiesteria in northern European waters}, volume={269}, ISSN={["1471-2954"]}, DOI={10.1098/rspb.2001.1852}, abstractNote={Several dinoflagellate strains of the genus Pfiesteria were isolated by culturing techniques from sediment samples taken in the Oslofjord region of Norway. Pfiesteria piscicida, well known as a fish killer from the Atlantic coast of America, was identified by genetic methods and light microscopy. The related species Pfiesteria shumwayae was attracted from the sediment by the presence of fish, and has proved toxic. This present survey demonstrates the wide distribution of these potentially harmful species, but so far they have not been connected with fish kills in Europe.}, number={1487}, journal={PROCEEDINGS OF THE ROYAL SOCIETY B-BIOLOGICAL SCIENCES}, author={Jakobsen, KS and Tengs, T and Vatne, A and Bowers, HA and Oldach, DW and Burkholder, JM and Glasgow, HB and Rublee, PA and Klaveness, D}, year={2002}, month={Jan}, pages={211–214} }
 @article{parrow_burkholder_2002, title={Flow cytometric determination of zoospore DNA content and population DNA distribution in cultured Pfiesteria spp. (Pyrrhophyta)}, volume={267}, ISSN={["0022-0981"]}, DOI={10.1016/S0022-0981(01)00343-4}, abstractNote={The relative cellular DNA content from 23 different clonal cultures of Pfiesteria spp. zoospores was determined using a DNA fluorochrome and flow cytometry. Significant differences between Pfiesteria piscicida and P. shumwayae were detected, both in mean zoospore DNA content and population cell cycle DNA distribution. Intraspecific differences in DNA content were found between clonal zoospore cultures established from different geographical regions. Long-term cultures (years) of P. piscicida were available for testing, and a negative correlation was observed between zoospore DNA content and time in culture. Zoospore cell cycle-related DNA distributions were also markedly different between the two species in these clonal cultures. In most cultures tested, P. piscicida zoospores exhibited bimodal DNA flow histograms with G1-S-G2+M distributions, typical of eukaryotic asynchronously cycling cells. In contrast, cultures of P. shumwayae zoospores exhibited one DNA peak distribution, indicative of synchronized cells. The data are consistent with the hypothesis that P. shumwayae zoospores are interphasic cells, and mitosis in zoospore cultures of this species predominantly occurs as benthic or adherent non-motile division cysts. Light microscopy observations of the nuclear condition of electrostatically sorted zoospores of each Pfiesteria species also support this hypothesis. If highly conserved, this disparity in modes of vegetative reproduction would ramify the population dynamics of the two Pfiesteria species.}, number={1}, journal={JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY}, author={Parrow, MW and Burkholder, JA}, year={2002}, month={Jan}, pages={35–51} }
 @article{parrow_burkholder_2002, title={Flow cytometric determination of zoospore DNA content and population DNA distribution in cultured Pfiesteria spp. (Pyrrhophyta) (vol 267, pg 35, 2002)}, volume={268}, ISSN={["0022-0981"]}, DOI={10.1016/S0022-0981(02)00018-7}, number={2}, journal={JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY}, author={Parrow, MW and Burkholder, JM}, year={2002}, month={Feb}, pages={261–261} }
 @article{stoecker_parrow_burkholder_glasgow_2002, title={Grazing by microzooplankton on Pfiesteria piscicida cultures with different histories of toxicity}, volume={28}, ISSN={["1616-1564"]}, DOI={10.3354/ame028079}, abstractNote={Susceptibility of actively toxic (TOX-A) zoospores, temporarily non-toxic (TOX-B) zoospores, and zoospores non-inducible to toxicity (NON-IND) of Pfiesteria piscicida to microzoo- plankton grazing was compared in a laboratory experiment. Zoospores from all cultures were ingested by microzooplankton, but community grazing coefficients for TOX-A were < 20% of those for TOX-B or NON-IND zoospores in 6 h incubations. Tintinnids and strobilidiid ciliates that fed on P. piscicida declined in incubations containing TOX-A zoospores. There was no decline in a strom- bidiid ciliate or heterotrophic dinoflagellate populations that fed on TOX-A zoospores. These data suggest that, although microzooplankton grazing on non-toxic zoospores can be a significant source of mortality to planktonic populations of P. piscicida, grazing on toxic or very recently toxic zoospores is relatively low.}, number={1}, journal={AQUATIC MICROBIAL ECOLOGY}, author={Stoecker, DK and Parrow, MW and Burkholder, JM and Glasgow, HB}, year={2002}, month={May}, pages={79–85} }
 @misc{anderson_glibert_burkholder_2002, title={Harmful algal blooms and eutrophication: Nutrient sources, composition, and consequences}, volume={25}, ISSN={["0160-8347"]}, DOI={10.1007/BF02804901}, number={4B}, journal={ESTUARIES}, author={Anderson, DM and Glibert, PM and Burkholder, JM}, year={2002}, month={Aug}, pages={704–726} }
 @article{springer_shumway_burkholder_glasgow_2002, title={Interactions between the toxic estuarine dinoflagellate Pfiesteria piscicida and two species of bivalve molluscs}, volume={245}, ISSN={["1616-1599"]}, DOI={10.3354/meps245001}, abstractNote={Toxic strains of Pfiesteria spp. produce toxin(s) that can cause finfish death, but much less is known about impacts of Pfiesteria on shellfish. Here we conducted 4 experiments to examine interactions between shellfish and toxic (actively toxic or TOX-A from finfish-killing cultures and potentially toxic or TOX-B from cultures without finfish) and non-inducible (NON-IND, apparently incapable of killing fish via a toxic effect) strains of P. piscicida. First (Expt 1), we documented direct physical attack by P. piscicida TOX-A, TOX-B, and NON-IND zoospores on larvae of the bay scallop Argopecten irradians (Lamarck, 1819) and the eastern oyster Crassostrea virginica (Gmelin, 1791). Within 5 min zoospores swarmed around larvae that had discarded their vela, and attached with their peduncles. Within 15 min they had penetrated into the shellfish visceral cavity and had begun to feed aggressively; after 30 min all shellfish tissues except the adductor muscle had been consumed. Sec- ond, we tested the response of scallop larvae to P. piscicida (TOX-A or TOX-B) or cryptomonads (as controls) that were held in dialysis tubing (0.22 µm porosity) to prevent direct contact. After 60 min larval survival was 0% in the TOX-A treatment, 100% in the cryptomonad control, and intermediate in TOX-B and TOX-B + cryptomonad treatments. The data indicate a toxic effect of P. piscicida zoospores on the larvae, separate from the physical effect shown in Expt 1. Third, we compared grazing by juvenile and adult oysters on TOX-A, TOX-B, and NON-IND P. piscicida zoospores from the medium. After 60 min, grazing by juvenile oysters significantly differed as NON-IND >> TOX-B >> TOX-A. In contrast, adult oysters grazed significantly fewer TOX-A zoospores and main- tained comparable grazing on TOX-B and NON-IND zoospores. Thus juvenile oysters, but not adults, were sensitive to residual toxicity of TOX-B zoospores, and both life-history stages were sensitive to TOX-A zoospores. The adverse effects of toxic strains on larval survival and juvenile grazing indicate that P. piscicida could potentially affect shellfish recruitment. Fourth, we assessed zoospore survival after passage through the digestive tract of adult oysters. The feces contained many temporary cysts from zoospores, and within 24 h >75% of the cysts produced motile cells. The data indicate that adult oysters would be poor biocontrol agents of P. piscicida, given the high survival of ingested zoospores following gut passage and fecal elimination; and that oysters could act as vectors of toxic P. piscicida strains if transported from affected estuaries to other waters.}, journal={MARINE ECOLOGY PROGRESS SERIES}, author={Springer, JJ and Shumway, SE and Burkholder, JM and Glasgow, HB}, year={2002}, pages={1–10} }
 @article{lewitus_hayes_willis_burkholder_glasgow_holland_maier_rublee_magnien_2002, title={Low abundance of the dinoflagellates, Pfiesteria piscicida, P-shumwayae, and Cryptoperidiniopsis spp., in South Carolina tidal creeks and open estuaries}, volume={25}, ISSN={["0160-8347"]}, DOI={10.1007/BF02804892}, number={4A}, journal={ESTUARIES}, author={Lewitus, AJ and Hayes, KC and Willis, BM and Burkholder, JM and Glasgow, HB and Holland, AF and Maier, PP and Rublee, PA and Magnien, R}, year={2002}, month={Aug}, pages={586–597} }
 @article{rhodes_burkholder_glasgow_rublee_allen_adamson_2002, title={Pfiesteria shumwayae (Pfiesteriaceae) in New Zealand}, volume={36}, ISSN={["1175-8805"]}, DOI={10.1080/00288330.2002.9517117}, abstractNote={Abstract Pfiesteria shumwayae Steidinger et Burkholder is now known to be present in New Zealand and occurs in estuaries around the country. The presence of Pfiesteria was initially determined by a polymerase chain reaction (PCR)‐based detection assay, using oligonucleotide primers targeted at ribosomal DNA extracted from estuarine water and sediments. Presence was confirmed by isolation from fresh sediments in the presence offish (Oreochromis mossambicus), followed by identification by scanning electron microscopy. The New Zealand isolates of P. shumwayae were ichthyotoxic in bioassays, but there is no historic evidence offish kills in New Zealand associated with the dinoflagellate.}, number={3}, journal={NEW ZEALAND JOURNAL OF MARINE AND FRESHWATER RESEARCH}, author={Rhodes, LL and Burkholder, JM and Glasgow, HB and Rublee, PA and Allen, C and Adamson, JE}, year={2002}, month={Sep}, pages={621–630} }
 @article{touchette_burkholder_2002, title={Seasonal variations in carbon and nitrogen constituents in eelgrass (Zostera marina L.) as influenced by increased temperature and water-column nitrate}, volume={45}, ISSN={["0006-8055"]}, DOI={10.1515/BOT.2002.004}, abstractNote={Abstract Fluctuations in nitrogen and carbon compounds were examined over an autumn growing season in the submersed marine angiosperm Zostera marina L. (eelgrass). The experimental design included replicated controls (ambient NO3 −, typically < 2 μM), increased water-column nitrate (8 μMNO3 − above ambient, pulsed daily), increased environmental temperature (3 to 4 °C above 20-year weekly means), and combined increased water-column nitrate and temperature. Above- and belowground tissues were collected weekly to biweekly and assayed for total soluble carbohydrates, non-reducing carbohydrates, starch, α-cellulose, lipids, free amino acids, total protein, tissue nitrate, tissue nitrite, and tissue ammonium. Tissue nitrate declined, and amino acids, proteins, lipids, and cellulose increased as the growing season progressed in both control and treated plants. In addition, there were seasonal quadratic responses for tissue ammonium, soluble carbohydrates, and non-reducing sugars, with maxima during periods of optimal plant growth (mid- to late September). Increased temperature promoted periodic increases in amino acids and soluble carbohydrates, but decreased accumulation of α-cellulose by the end of the experiment. Moreover, increases in water-column nitrate led to periodic increases in tissue ammonium and amino acids, as well as decreases in non-reducing sugars. Toward the end of the experiment, increases in soluble carbohydrates for plants grown under higher temperatures may have been associated with an extension of the growing season. In contrast, decreased non-reducing sugars in nitrate-enriched plants may have resulted from an increased carbon demand during nitrate assimilation/reduction, as well as a reallocation of carbon to enhance amino acid synthesis.}, number={1}, journal={BOTANICA MARINA}, author={Touchette, BW and Burkholder, JM}, year={2002}, month={Jan}, pages={23–34} }
 @article{burkholder_glasgow_2002, title={The life cycle and toxicity of Pfiesteria piscicida revisited}, volume={38}, ISSN={["1529-8817"]}, DOI={10.1046/j.1529-8817.2002.02096.x}, abstractNote={Despite use of excellent molecular techniques, Litaker et al. (2002) cannot provide insights about the life history of toxic Pfiesteria piscicida because they showed no data in support of having used toxic strains; rather they presented evidence that they used non‐inducible strains. Litaker et al. did not find amoeboid stages or a chrysophyte‐like cyst stage in several cultures and unequivocally concluded that the stages do not exist in all P. piscicida strains. Thus, they did not consider the tenet that absence of evidence does not constitute proof of absence. Apparent discrepancies between the research by Litaker et al. and previous research on Pfiesteria can be resolved as follows: First, Litaker et al. did not use toxic strains. We have reported findings (similar to Litaker et al.) showing few amoeboid transformations in non‐inducible strains, which manifest some but not all of the forms that have been documented in some toxic strains. We, and others, have documented active toxicity to fish, transformations to amoebae, and chrysophyte‐like cysts in some clonal toxic strains. Second, the data from several recent publications, which were available but not mentioned by Litaker et al. or by Coats (2002) in accompanying commentary, have verified P. piscicida amoebae, chrysophyte‐like cysts, and other stages in some toxic strains through a combination of approaches including PCR data from clonal cultures.}, number={6}, journal={JOURNAL OF PHYCOLOGY}, author={Burkholder, JM and Glasgow, HB}, year={2002}, month={Dec}, pages={1261–1267} }
 @article{parrow_burkholder_deamer_zhang_2002, title={Vegetative and sexual reproduction in Pfiesteria spp. (Dinophyceae) cultured with algal prey, and inferences for their classification.}, volume={1}, DOI={10.1016/s1568-9883(02)00009-4}, abstractNote={Algal-fed clonal zoospore cultures of Pfiesteria piscicida and Pfiesteria shumwayae enabled description of certain conserved morphological and reproductive features. Common modes of reproduction (especially via division cysts) were documented in herbivorous P. piscicida and P. shumwayae using cultures fed algal prey, together with supporting photography and flow cytometric DNA measurements. Other cysts were characterized such as vacuolate cysts in starved P. piscicida cultures and temporary cysts in both species fed algal prey. This study also represents the first report of sexual reproduction in Pfiesteria spp. cultures fed algal prey rather than live fish; the first report of a technique for cell cycle synchronization for these heterotrophic dinoflagellates; and the first information on storage products of cells released from Pfiesteria reproductive cysts. Sexual reproduction in algal-fed P. piscicida clonal cultures was evidenced by fusing gametes, cells with two longitudinal flagella, and nuclear cyclosis. Both isogamous and anisogamous fusions were observed, and resulting cells with two trailing flagella (i.e., planozygotes and planomeiocytes) sometimes comprised ≥50% of the flagellated cells. These cells continued feeding activity and eventually (hours) lost their flagella and formed cysts. Nuclear cyclosis and a subsequent cell division were observed in thin-walled reproductive cysts prior to release of two flagellated cells. One gamete fusion event was also documented in 1 of 20 algal-fed clones of P. shumwayae, with an aplanozygote as the product. We obtained high cell synchrony (≥90% 1C) in the tested cultures using our preferential lysis technique and tracked the decline in lipid content of excysted zoospore populations over time. The data from this study were considered together with previous research to gain insights about relationships between Pfiesteria spp. and other heterotrophic dinoflagellates. Pfiesteria spp. should be regarded as free-living predators rather than parasites because they are prey generalists without demonstrated “host” specificity and their flagellated feeding stages are not morphologically distinct from swimming stages. Although they originally were placed within the Dinamoebales because amoebae can predominate, this study as well as other published research consistently has shown that the dominant stage varies depending on culture conditions, prey type/availability and strains. The peridinoid plate structure of each Pfiesteria species, which thus far has been conserved across culture conditions and strains, supports placement of Pfiesteria spp. within the Peridiniales. At the species level, plate structure (differing by one precingular plate) and molecular data (18S rDNA) indicate that the two Pfiesteria spp. are closely related in comparison to species grouped within other genera.}, number={1}, journal={Harmful Algae}, author={Parrow, M. and Burkholder, J. M. and Deamer, N. J. and Zhang, C.}, year={2002}, pages={5–33} }
 @article{glasgow_burkholder_morton_springer_2001, title={A second species of ichthyotoxic Pfiesteria (Dinamoebales, Dinophyceae)}, volume={40}, ISSN={["2330-2968"]}, DOI={10.2216/i0031-8884-40-3-234.1}, abstractNote={Abstract A second toxic species within the family Pfiesteriaceae, Pfiesteria shumwayae Glasgow & Burkholder sp. nov., is described from the New River Estuary and the Neuse Estuary of the Albemarle-Pamlico Estuarine Ecosystem, USA. The species is polymorphic and multiphasic, with flagellated, amoeboid and cyst stages. The flagellated zoospores (diameter 8–24 μm) have permanently condensed chromosomes (mesokaryotic nucleus); a chrysophyte-like cyst (diameter 6–25 μm)with organic scales and bracts; and thin thecal plates arranged in a Kofoidian series of Po, cp, X, 4′, la, 6″, 6c, 4s, 5″′, 2″″. The benthic filopodial (filose), lobopodia1 (lobose) and rhizopodial amoeboid stages (5–250 μm) have an outer covering that ranges from rough to smooth in texture, depending on the stage of origin and the prey source. Pfiesteria shumwayae amoebae have a normal eukaryote nucleus and cysts of multiple sizes (diameter 4–25 μm) with a reticulate outer covering. Toxic strains of the two Pfiesteria species have overlapping distributions in the mid-Atlantic and southeastern United States and Scandinavia, with toxic P. shumwayae also having been verified from New Zealand. Pfiesteria shumwayae is similar to P. piscicida in its complex life cycle, general nutrition, attraction to live fish prey, and ichthyotoxic activity that is stimulated by the presence of live fish or their fresh tissues and excreta. However, it can be distinguished from P. piscicida morphologically by having six precingular plates and a four-sided la plate, as well as genetically, on the basis of its ISS ribosomal DNA sequence.}, number={3}, journal={PHYCOLOGIA}, author={Glasgow, HB and Burkholder, JM and Morton, SL and Springer, J}, year={2001}, month={May}, pages={234–245} }
 @article{cancellieri_burkholder_deamer-melia_glasgow_2001, title={Chemosensory attraction of zoospores of the estuarine dinoflagellates, Pfiesteria piscicida and P-shumwayae, to finfish mucus and excreta}, volume={264}, ISSN={["1879-1697"]}, DOI={10.1016/S0022-0981(01)00299-4}, abstractNote={Toxic strains of the estuarine dinoflagellates, Pfiesteria piscicida and P. shumwayae, can cause fish death and disease, whereas other estuarine ‘lookalike’ species such as cryptoperidiniopsoids have not been ichthyotoxic under ecologically relevant conditions. Chemosensory attraction of three functional types of these Pfiesteria spp. were separately evaluated for their attraction to fresh fish mucus and excreta. Clonal cultures of actively toxic (TOX-A, engaged in killing fish) and temporarily nontoxic (tested as toxic but without access to live fish for >1 week to 5 months (in most experiments, ≤3 months) as ‘short-duration’ TOX-B; and without access to live fish for ≥1.5 years as ‘long-duration’ TOX-B) functional types of P. piscicida and P. shumwayae were derived from the same clones whereas the non-inducible cultures (NON-IND, tested as incapable of toxic activity in the presence of fish), of necessity, were from different clonal isolates. NON-IND cultures previously had been grown on algal prey for 3–8 months, and had repeatedly been tested as incapable of causing fish distress, disease or death via toxic activity. Attraction to fish materials was based on the number of zoospores that entered microcapillary tubes containing sterile-filtered 15-ppt water (controls), vs. entry into tubes with sterile-filtered mucus and excreta (collected in 15-ppt water) that had been collected from live tilapia, bluegill, hybrid striped bass, and Atlantic menhaden (tested separately within 3 h of removal from live fish). TOX-A zoospores of both Pfiesteria species exhibited the strongest attraction to the fish mucus and excreta, with comparable response to the materials from all four test fish species. Short-duration TOX-B zoospores showed an intermediate response that apparently depended on the duration of mucus separation from the live fish: the shorter the separation period, the stronger the zoospore attraction to the fish materials. In contrast to TOX-A and short-duration TOX-B zoospores, NON-IND and long-duration TOX-B zoospores generally showed little or no response to the fish materials. Zoospores of the cryptoperidiniopsoid demonstrated a moderate attraction that did not appear to depend on the time of isolation from fish. TOX-A zoospores were also tested for attraction to sterile-filtered vs. non-filtered fish mucus (time separated from the live animal, 3–96 h). These zoospores, which initially had been actively attracted, were no longer attracted to the unfiltered fish materials after 48 h, whereas attraction to the sterile-filtered fish mucus and excreta persisted throughout the duration of the experiment. Thus, the attractant signal in the materials was degraded or effectively blocked by the bacterial community within hours of isolation from live fish. This study indicates the importance of functional type or toxicity status, and the importance of the history of exposure to live fish, in the behavioral ecology of Pfiesteria spp. Initial attraction to fish materials strongly depended on the functional type, and on the history of toxic activity. Non-inducible and long-duration TOX-B cultures of Pfiesteria spp., unlike actively toxic and short-duration TOX-B (potentially toxic) strains, initially were virtually unresponsive to fish mucus.}, number={1}, journal={JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY}, author={Cancellieri, PJ and Burkholder, JM and Deamer-Melia, NJ and Glasgow, HB}, year={2001}, month={Sep}, pages={29–45} }
 @article{moeller_morton_mitchell_sivertsen_fairey_mikulski_glasgow_deamer-melia_burkholder_ramsdell_2001, title={Current progress in isolation and characterization of toxins isolated from Pfiesteria piscicida}, volume={109}, ISSN={["0091-6765"]}, DOI={10.2307/3454921}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={Moeller, PDR and Morton, SL and Mitchell, BA and Sivertsen, SK and Fairey, ER and Mikulski, TM and Glasgow, H and Deamer-Melia, NJ and Burkholder, JM and Ramsdell, JS}, year={2001}, month={Oct}, pages={739–743} }
 @article{glasgow_burkholder_mallin_deamer-melia_reed_2001, title={Field ecology of toxic Pfiesteria complex species and a conservative analysis of their role in estuarine fish kills}, volume={109}, ISSN={["1552-9924"]}, DOI={10.2307/3454919}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={Glasgow, HB and Burkholder, JM and Mallin, MA and Deamer-Melia, NJ and Reed, RE}, year={2001}, month={Oct}, pages={715–730} }
 @article{burkholder_glasgow_2001, title={History of toxic Pfiesteria in North Carolina estuaries from 1991 to the present}, volume={51}, ISSN={["1525-3244"]}, DOI={10.1641/0006-3568(2001)051[0827:HOTPIN]2.0.CO;2}, abstractNote={T fish kills for which Pfiesteria became well known began in North Carolina, when very little research had been conducted on this unusual dinoflagellate. North Carolina’s Albemarle-Pamlico Estuarine System, the epicenter of toxic Pfiesteria outbreaks, is the second largest estuary in area on the US mainland and the most important fish nursery ground on the US Atlantic Coast (Burkholder and Glasgow 1997, Mallin et al. 2000). As a conservative estimate, the state had sustained 48 toxic Pfiesteria outbreaks by 1997, involving more than a billion fish in an area more than 100 km2 (Burkholder et al. 2001a). These events had occurred nearly every summer beginning in 1991, when the organism was first recognized as an estuarine, fish-killing agent (Burkholder et al. 1992, 2001a, Burkholder and Glasgow 1997). In 1997, 50,000 fish died in a small area of the Chesapeake Bay from toxic Pfiesteria , and press coverage of the event was like an explosion. In the same summer, 1.2 million fish died during toxic Pfiesteria outbreaks in Albemarle-Pamlico estuaries, a 4-hour drive south of Washington, DC; those deaths went virtually unmentioned. North Carolina was the first state to encounter toxic Pfiesteria, and the knowledge gained there, especially about toxic Pfiesteria outbreaks and health impacts on laboratory workers (Burkholder et al. 1992, 1995, Burkholder and Glasgow 1995, 1997, Glasgow et al. 1995), benefited Maryland officials, who were challenged to act quickly and decisively. They evaluated and rapidly verified the role of toxic Pfiesteria in the Chesapeake Bay outbreak (MDNR 1998). Maryland was the first state from which people who reported neurocognitive, respiratory, and other symptoms from environmental exposure were clinically evaluated within a short period (1–3 weeks) after being exposed (Grattan et al. 1998). Maryland was also the first state to address the Pfiesteria problem by making significant advances in legislation for protection of water quality (State of Maryland 1998). Congressional attention following the toxic Pfiesteria outbreaks in Maryland led to the appropriation of many millions of dollars to federal agencies to research and manage toxic Pfiesteria outbreaks, resulting in excellent progress in some areas and setbacks in others. Here we examine the history of toxic Pfiesteria outbreaks before the four in Chesapeake Bay and a few aspects of the aftermath, from the perspective and experience of our research in the Albemarle-Pamlico Estuarine System with high toxic Pfiesteria activity. An important part of the Pfiesteria story is how North Carolina—with 98% of the Pfiesteria problem—subsequently moved to strengthen water resource protection, environmental education, and support of Pfiesteria research, actions that would not have been possible without the events that unfolded in Chesapeake Bay.}, number={10}, journal={BIOSCIENCE}, author={Burkholder, JM and Glasgow, HB}, year={2001}, month={Oct}, pages={827–841} }
 @article{kimm-brinson_moeller_barbier_glasgow_burkholder_ramsdell_2001, title={Identification of a P2X7 receptor in GH(4)C(1) rat pituitary cells: A potential target for a bioactive substance produced by Pfiesteria piscicida}, volume={109}, ISSN={["1552-9924"]}, DOI={10.2307/3454703}, abstractNote={We examined the pharmacologic activity of a putative toxin (pPfTx) produced by Pfiesteria piscicida by characterizing the signaling pathways that induce the c-fos luciferase construct in GH(4)C(1) rat pituitary cells. Adenosine-5'-triphosphate (ATP) was determined to increase and, at higher concentrations, decrease luciferase activity in GH(4)C(1) rat pituitary cells that stably express c-fos luciferase. The inhibition of luciferase results from cytotoxicity, characteristic of the putative P. piscicida toxin (pPfTx). The actions of both pPfTx and ATP to induce c-fos luciferase were inhibited by the purinogenic receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). Further characterization of a P2X receptor on the GH(4)C(1) cell was determined by the analog selectivity of P2X agonists. The P2X1/P2X3 agonist alpha,beta-methylene ATP (alpha,beta-MeATP) failed to increase or decrease c-fos luciferase. However, the P2X7 agonist 2',3'-(4-benzoyl)benzoyl ATP (BzATP), which had a predominant cytotoxic effect, was more potent than ATP. Immunoblot analysis of GH(4)C(1) cell membranes confirmed the presence of a 70-kDa protein that was immunoreactive to an antibody directed against the carboxy-terminal domain unique to the P2X7 receptor. The P2X7 irreversible antagonist oxidized-ATP (oxATP) inhibited the action of ATP, BzATP, and pPfTx. These findings indicate that GH(4)C(1) cells express purinogenic receptors with selectivity consistent with the P2X7 subtype and that this receptor pathway mediates the induction of the c-fos luciferase reporter gene by ATP and the putative Pfiesteria toxin}, number={5}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={Kimm-Brinson, KL and Moeller, PDR and Barbier, M and Glasgow, H and Burkholder, JM and Ramsdell, JS}, year={2001}, month={May}, pages={457–462} }
 @article{melo_moeller_glasgow_burkholder_ramsdell_2001, title={Microfluorimetric analysis of a purinergic receptor (P2X(7)) in GH(4)C(1) rat pituitary cells: Effects of a bioactive substance produced by Pfiesteria piscicida}, volume={109}, ISSN={["0091-6765"]}, DOI={10.2307/3454920}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={Melo, AC and Moeller, PDR and Glasgow, H and Burkholder, JM and Ramsdell, JS}, year={2001}, month={Oct}, pages={731–737} }
 @article{touchette_burkholder_2001, title={Nitrate reductase activity in a submersed marine angiosperm: Controlling influences of environmental and physiological factors}, volume={39}, ISSN={["0981-9428"]}, DOI={10.1016/S0981-9428(01)01278-5}, abstractNote={In plants, nitrate reductase (NR; EC 1.6.6.1) is considered to be a key enzyme in nitrate assimilation. Therefore, the activity of NR as influenced by major environmental factors may affect the survival of many aquatic nitrogen-limited plant species. In this study, the in vivo activity of NR following exposure to increased water-column nitrate was examined in a submersed marine angiosperm (eelgrass, Zostera marina L.). NR activity was primarily localized in new leaf tissue, and was related to light and/or soluble carbohydrate availability. Under extended periods of darkness (18 h), enzyme activity decreased by more than 60 %. Nevertheless, in vivo NR activity was induced during dark periods provided that water-column nitrate (≥ 8 μM NO3–) was available. Enzyme activities were lower in plants that were exposed to hypoxic conditions (< 3.5 mg O2·L–1 for 14 h), and/or elevated growth temperatures (3 to 4 °C above mean weekly temperatures). In contrast, exposure to the atmosphere for 90 min promoted a significant increase in in vivo NR activity. A temporal investigation (14 weeks in autumn) revealed that the intensity of in vivo NR response to water-column nitrate was directly correlated with the quantity of soluble carbohydrates within the leaf tissue. Many of the observed in vivo NR responses were likely related to carbohydrate availability. During periods where soluble carbohydrate availability was expected to be low, in vivo NR response to increased water-column nitrate was substantially compromised.}, number={7-8}, journal={PLANT PHYSIOLOGY AND BIOCHEMISTRY}, author={Touchette, BW and Burkholder, J}, year={2001}, pages={583–593} }
 @article{burkholder_glasgow_deamer-melia_2001, title={Overview and present status of the toxic Pfiesteria complex (Dinophyceae)}, volume={40}, ISSN={["2330-2968"]}, DOI={10.2216/i0031-8884-40-3-186.1}, abstractNote={Abstract This paper reviews the Pfiesteria issue and Pfiesteria science and presents new information on variation in toxicity among Pfiesteria strains, culture effects on their toxicity, the trophic interactions of Pfiesteria spp ., and impacts on fish and mammals. We also assess Pfiesteria spp. impacts on fish in comparison to certain other estuarine dinoflagellates of similar appearance. Species of the toxic Pfiesteria complex (TPC) thus far include P. piscicida and P. shumwayae. These species share morphological and genetic similarities, and both have toxic strains that (1) show strong attraction to live fish;(2) exhibit toxicity that is triggered by live fish or their fresh tissues and excreta; and (3) produce toxin(s) that cause fish stress, disease and death under ecologically relevant conditions (the standardized fish bioassay process involves testing live Pfiesteria cells at similar densities to those encountered during Pfiesteria-related fish kill/disease events). Both Pfiesteria species also have a complex life cycle with multiple amoeboid, flagellated and cyst stages, several of which are ichthyotoxic. TPC species are eurythermal and euryhaline, with prey spanning the estuarine food web, from bacteria to mammalian tissues. They can be stimulated directly or indirectly by nitrogen and phosphorus enrichment. Toxic strains can be either actively or potentially toxic (the TOX-A and TOX-B functional types, respectively); in addition, c.40% of randomly isolated clones have been found to be benign [the noninducible or NON-IND functional type, which apparently lacks the ability to produce bioactive substances (toxins) that cause fish disease or death]. These functional types differ significantly in response to algal prey, predators, nutrients and fish. Moreover, as an apparent artifact of culture conditions, toxic strains generally lose their ability to cause fish death and disease and become NON-IND within weeks to months. At low cell densities, toxic strains can be causative agents of acute and/or chronic diffuse and focal lesions and of other fish diseases, as demonstrated in fish bioassays. A partially purified, water-soluble Pjiesteria toxin disrupts calcium metabolism in rat pituitary cells and mimics an adenosine triphosphate neurotransmitter that targets P2X7 purinoreceptors found predominantly on immune cells. Respiratory, visual, and neurological impacts have been sustained by people exposed to aerosols from fish-killing Pfiesteria cultures or to water and aerosols during estuarine fish kills associated with toxic Pfiesteria. Neurocognitive impacts from exposure to toxic Pfiesteria have been replicated experimentally in small mammals. Toxic strains of Pfiesteria species have been confirmed from mid-Atlantic and Gulf Coast estuaries in the United States and from northern Europe and New Zealand, indicating that these toxic dinoflagellates are cosmopolitan in distribution.}, number={3}, journal={PHYCOLOGIA}, author={Burkholder, JM and Glasgow, HB and Deamer-Melia, N}, year={2001}, month={May}, pages={186–214} }
 @article{burkholder_glasgow_deamer-melia_springer_parrow_zhang_cancellieri_2001, title={Species of the toxic Pfiesteria complex, and the importance of functional type in data interpretation}, volume={109}, ISSN={["1552-9924"]}, DOI={10.2307/3454912}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={Burkholder, JM and Glasgow, HB and Deamer-Melia, NJ and Springer, J and Parrow, MW and Zhang, C and Cancellieri, PJ}, year={2001}, month={Oct}, pages={667–679} }
 @article{rezvani_bushnell_burkholder_glasgow_levin_2001, title={Specificity of cognitive impairment from Pfiesteria piscicida exposure in rats - Attention and visual function versus behavioral plasticity}, volume={23}, ISSN={["1872-9738"]}, DOI={10.1016/S0892-0362(01)00169-6}, abstractNote={Pfiesteria piscicida is a toxic dinoflagellate that has caused massive fish kills in estuaries along the East Coast of the United States, and exposure of humans to toxic Pfiesteria has been associated with cognitive impairment. A visual signal detection task was used to determine the possible importance of attentional and visual processes in Pfiesteria effects on cognitive function. Adult female rats were trained to perform the signal detection task. After training, the rats were injected subcutaneously with fish culture water containing toxic Pfiesteria (35,600 or 106,800 cells of Pfiesteria/kg of rat body weight) or with (control) fish culture water containing no Pfiesteria. Effects of toxic Pfiesteria on maintenance of signal detection behavior were assessed for 2 weeks after treatment. Then, the signal-response contingencies were reversed. After the discrimination was reestablished on the reversed levers, the rats received a second dose of toxic Pfiesteria. The rats were again tested for 2 weeks, after which a second reversal was imposed. Pfiesteria did not affect behavior in the signal detection task during 2 weeks of prereversal testing after either exposure. However, a significant Pfiesteria-induced deficit emerged when the signal-response contingencies were reversed. These findings suggest that Pfiesteria-induced deficits emerge during periods of behavioral transition and not during performance of previously learned tasks.}, number={6}, journal={NEUROTOXICOLOGY AND TERATOLOGY}, author={Rezvani, AH and Bushnell, PJ and Burkholder, JM and Glasgow, HB and Levin, ED}, year={2001}, pages={609–616} }
 @article{burkholder_marshall_glasgow_seaborn_deamer-melia_2001, title={The standardized fish bioassay procedure for detecting and culturing actively toxic Pfiesteria, used by two reference laboratories for Atlantic and Gulf Coast states}, volume={109}, ISSN={["1552-9924"]}, DOI={10.2307/3454922}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={Burkholder, JM and Marshall, HG and Glasgow, HB and Seaborn, DW and Deamer-Melia, NJ}, year={2001}, month={Oct}, pages={745–756} }
 @article{rublee_kempton_schaefer_allen_harris_oldach_bowers_tengs_burkholder_glasgow_2001, title={Use of molecular probes to assess geographic distribution of Pfiesteria species}, volume={109}, ISSN={["0091-6765"]}, DOI={10.2307/3454924}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={Rublee, PA and Kempton, JW and Schaefer, EF and Allen, C and Harris, J and Oldach, DW and Bowers, H and Tengs, T and Burkholder, JM and Glasgow, HB}, year={2001}, month={Oct}, pages={765–767} }
 @article{bowers_tengs_glasgow_burkholder_rublee_oldach_2000, title={Development of real-time PCR assays for rapid detection of Pfiesteria piscicida and related dinoflagellates}, volume={66}, ISSN={["0099-2240"]}, DOI={10.1128/AEM.66.11.4641-4648.2000}, abstractNote={ABSTRACT}, number={11}, journal={APPLIED AND ENVIRONMENTAL MICROBIOLOGY}, author={Bowers, HA and Tengs, T and Glasgow, HB and Burkholder, JM and Rublee, PA and Oldach, DW}, year={2000}, month={Nov}, pages={4641–4648} }
 @article{oldach_delwiche_jakobsen_tengs_brown_kempton_schaefer_bowers_glasgow_burkholder_et al._2000, title={Heteroduplex mobility assay-guided sequence discovery: Elucidation of the small subunit (18S) rDNA sequences of Pfiesteria piscicida and related dinoflagellates from complex algal culture and environmental sample DNA pools}, volume={97}, ISSN={["0027-8424"]}, DOI={10.1073/pnas.97.8.4303}, abstractNote={
            The newly described heterotrophic estuarine dinoflagellate
            Pfiesteria piscicida
            has been linked with fish kills in field and laboratory settings, and with a novel clinical syndrome of impaired cognition and memory disturbance among humans after presumptive toxin exposure. As a result, there is a pressing need to better characterize the organism and these associations. Advances in
            Pfiesteria
            research have been hampered, however, by the absence of genomic sequence data. We employed a sequencing strategy directed by heteroduplex mobility assay to detect
            Pfiesteria piscicida
            18S rDNA “signature” sequences in complex pools of DNA and used those data as the basis for determination of the complete
            P. piscicida
            18S rDNA sequence. Specific PCR assays for
            P. piscicida
            and other estuarine heterotrophic dinoflagellates were developed, permitting their detection in algal cultures and in estuarine water samples collected during fish kill and fish lesion events. These tools should enhance efforts to characterize these organisms and their ecological relationships. Heteroduplex mobility assay-directed sequence discovery is broadly applicable, and may be adapted for the detection of genomic sequence data of other novel or nonculturable organisms in complex assemblages.
          }, number={8}, journal={PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA}, author={Oldach, DW and Delwiche, CF and Jakobsen, KS and Tengs, T and Brown, EG and Kempton, JW and Schaefer, EF and Bowers, HA and Glasgow, HB and Burkholder, JM and et al.}, year={2000}, month={Apr}, pages={4303–4308} }
 @misc{mallin_burkholder_cahoon_posey_2000, title={North and South Carolina coasts}, volume={41}, ISSN={["1879-3363"]}, DOI={10.1016/S0025-326X(00)00102-8}, abstractNote={This coastal region of North and South Carolina is a gently sloping plain, containing large riverine estuaries, sounds, lagoons, and salt marshes. The most striking feature is the large, enclosed sound known as the Albemarle–Pamlico Estuarine System, covering approximately 7530 km2. The coast also has numerous tidal creek estuaries ranging from 1 to 10 km in length. This coast has a rapidly growing population and greatly increasing point and non-point sources of pollution. Agriculture is important to the region, swine rearing notably increasing fourfold during the 1990s. Estuarine phytoplankton communities in North Carolina are well studied; the most important taxonomic groups are diatoms, dinoflagellates, cryptomonads and cyanobacteria. Several major poorly flushed estuaries are eutrophic due to nutrient inputs, and toxic dinoflagellates (Pfiesteria spp) can reach high densities in nutrient-enriched areas. Fully marine waters are relatively oligotrophic. Southern species enter in subsurface intrusions, eddies, and occasional Gulf Stream rings, while cool water species enter with the flow of the Labrador Current to the Cape Hatteras region. The Carolinas have a low number of endemic macroalgae, but species diversity can be high in this transitional area, which represents the southernmost extension for some cold-adapted species and the northernmost extension of warm-adapted species. In North Carolina the dominant seagrass, Zostera marina, lies at its southernmost extension, while a second species, Halodule wrightii is at its northernmost extent. Widgeon-grass Ruppia maritima is common, growing in brackish water or low-salinity pools in salt marshes. Seagrass meadows are now much reduced, probably due to elevated nitrogen and increased sedimentation. In sounds, numerically dominant benthic taxa include bivalves, polychaetes and amphipods, many showing gradients in community type from mesohaline areas of the eastern shore to near marine salinities in western parts. The semi-enclosed sounds have extensive shellfisheries, especially of blue crab, northern quahogs, eastern oysters, and shrimp. Problems include contamination of some sediments with toxic substances, especially of metals and PCBs at sufficiently high levels to depress growth of some benthic macroinvertebrates. Numerous fish kills have been caused by toxic Pfiesteria outbreaks, and fish kills and habitat loss have been caused by episodic hypoxia and anoxia in rivers and estuaries. Oyster beds currently are in decline because of overharvesting, high siltation and suspended particulate loads, disease, hypoxia, and coastal development. Fisheries monitoring which began in the late 1970s shows greatest recorded landings in 1978–1982; since then, harvests have declined by about a half. Some management plans have been developed toward improving water quality and fisheries sustainability. Major challenges include; high coliform levels leading to closures of shellfish beds, a problem that has increased with urban development and increasing cover of watershed by impervious surfaces; high by-catch and heavy trawling activity; overfishing which has led to serious declines in many wild fish stocks; and eutrophication. Comprehensive plans limiting nutrient inputs are needed for all coastal rivers and estuaries, not only those that already exhibit problems. There is a critical need to improve management of non-point nutrient runoff through increased use of streamside vegetated buffers, preservation of remaining natural wetlands and construction of artificial wetlands. Improved treatment processes, based on strong incentive programmes, should also be mandated for present and future industrial-scale animal operations.}, number={1-6}, journal={MARINE POLLUTION BULLETIN}, author={Mallin, MA and Burkholder, JM and Cahoon, LB and Posey, MH}, year={2000}, pages={56–75} }
 @article{touchette_burkholder_2000, title={Overview of the physiological ecology of carbon metabolism in seagrasses}, volume={250}, DOI={10.1016/S0022-0981(00)00196-9}, abstractNote={The small but diverse group of angiosperms known as seagrasses form submersed meadow communities that are among the most productive on earth. Seagrasses are frequently light-limited and, despite access to carbon-rich seawaters, they may also sustain periodic internal carbon limitation. They have been regarded as C3 plants, but many species appear to be C3-C4 intermediates and/or have various carbon-concentrating mechanisms to aid the Rubisco enzyme in carbon acquisition. Photorespiration can occur as a C loss process that may protect photosynthetic electron transport during periods of low CO(2) availability and high light intensity. Seagrasses can also become photoinhibited in high light (generally>1000 µE m(-2) s(-1)) as a protective mechanism that allows excessive light energy to be dissipated as heat. Many photosynthesis-irradiance curves have been developed to assess light levels needed for seagrass growth. However, most available data (e.g. compensation irradiance I(c)) do not account for belowground tissue respiration and, thus, are of limited use in assessing the whole-plant carbon balance across light gradients. Caution is recommended in use of I(k) (saturating irradiance for photosynthesis), since seagrass photosynthesis commonly increases under higher light intensities than I(k); and in estimating seagrass productivity from H(sat) (duration of daily light period when light equals or exceeds I(k)) which varies considerably among species and sites, and which fails to account for light-limited photosynthesis at light levels less than I(k). The dominant storage carbohydrate in seagrasses is sucrose (primarily stored in rhizomes), which generally forms more than 90% of the total soluble carbohydrate pool. Seagrasses with high I(c) levels (suggesting lower efficiency in C acquisition) have relatively low levels of leaf carbohydrates. Sucrose-P synthase (SPS, involved in sucrose synthesis) activity increases with leaf age, consistent with leaf maturation from carbon sink to source. Unlike terrestrial plants, SPS apparently is not light-activated, and is positively influenced by increasing temperature and salinity. This response may indicate an osmotic adjustment in marine angiosperms, analogous to increased SPS activity as a cryoprotectant response in terrestrial non-halophytic plants. Sucrose synthase (SS, involved in sucrose metabolism and degradation in sink tissues) of both above- and belowground tissues decreases with tissue age. In belowground tissues, SS activity increases under low oxygen availability and with increasing temperatures, likely indicating increased metabolic carbohydrate demand. Respiration in seagrasses is primarily influenced by temperature and, in belowground tissues, by oxygen availability. Aboveground tissues (involved in C assimilation and other energy-costly processes) generally have higher respiration rates than belowground (mostly storage) tissues. Respiration rates increase with increasing temperature (in excess of 40 degrees C) and increasing water-column nitrate enrichment (Z. marina), which may help to supply the energy and carbon needed to assimilate and reduce nitrate. Seagrasses translocate oxygen from photosynthesizing leaves to belowground tissues for aerobic respiration. During darkness or extended periods of low light, belowground tissues can sustain extended anerobiosis. Documented alternate fermentation pathways have yielded high alanine, a metabolic 'strategy' that would depress production of the more toxic product ethanol, while conserving carbon skeletons and assimilated nitrogen. In comparison to the wealth of information available for terrestrial plants, little is known about the physiological ecology of seagrasses in carbon acquisition and metabolism. Many aspects of their carbon metabolism - controls by interactive environmental factors; and the role of carbon metabolism in salt tolerance, growth under resource-limited conditions, and survival through periods of dormancy - remain to be resolved as directions in future research. Such research will strengthen the understanding needed to improve management and protection of these environmentally important marine angiosperms.}, number={1-2}, journal={Journal of Experimental Marine Biology and Ecology}, author={Touchette, B. W. and Burkholder, J. M.}, year={2000}, pages={169–205} }
 @article{levin_rezvani_christopher_glasgow_deamer-melia_burkholder_moser_jensen_2000, title={Rapid neurobehavioral analysis of Pfiesteria piscicida effects in juvenile and adult rats}, volume={22}, ISSN={["0892-0362"]}, DOI={10.1016/S0892-0362(00)00080-5}, abstractNote={The estuarine dinoflagellate Pfiesteria piscicida is known to kill fish and has been associated with neurocognitive deficits in humans. We have developed a rat model to demonstrate that exposure to Pfiesteria causes significant learning impairments. This has been repeatedly seen as a choice accuracy impairment during radial-arm maze learning. Pfiesteria-induced effects were also seen in a locomotor activity test in the figure-8 apparatus. The current studies used the short-term radial-arm maze acquisition, the figure-8 activity test, and the functional observational battery (FOB) to assess Pfiesteria-induced neurobehavioral effects in adult and juvenile rats. In study 1, the neurobehavioral potency of three different Pfiesteria cultures (Pf 113, Pf 728, and Pf Vandermere) was assessed. Ninety-six (12 per group) adult female Sprague-Dawley rats were injected subcutaneously with a single dose of Pfiesteria taken from aquarium-cultured Pfiesteria (35,600 or 106,800 Pfiesteria cells per kilogram of rat body weight). One control group (N = 12) was injected with saline and one (N = 12) with aquarium water not containing Pfiesteria. All three of the Pfiesteria samples (p < 0.05) impaired choice accuracy over the first six sessions of training. At the time of the radial-arm maze choice accuracy impairment, no overt Pfiesteria-related effects were seen using an FOB, indicating that the Pfiesteria-induced choice accuracy deficit was not due to generalized debilitation. In the figure-8 apparatus, Pfiesteria treatment caused a significant decrease in mean locomotor activity. In study 2, the neurobehavioral effects of the Pf 728 sample type were assessed in juvenile rats. Twenty-four day-old male and female rats were injected with 35,600 or 106,800 Pf-728 Pfiesteria cells per kilogram of rat body weight. As with adult females, the juvenile rats showed a significant impairment in radial-arm maze choice accuracy. No changes in locomotor activity or the FOB were detected in the juvenile rats. Furthermore, there were no differences between male and female rats in the Pfiesteria-induced choice accuracy impairment. Pfiesteria effects on choice accuracy in the radial-arm maze in rats constitute a critical component of the model of Pfiesteria toxicity, because the hallmark of Pfiesteria toxicity in humans is cognitive dysfunction. Our finding that analysis of the first six sessions of radial-arm maze testing is sufficient for determining the effect means that this test will be useful as a rapid screen for identifying the critical neurotoxin(s) of Pfiesteria in future studies.}, number={4}, journal={NEUROTOXICOLOGY AND TERATOLOGY}, author={Levin, ED and Rezvani, AH and Christopher, NC and Glasgow, HB and Deamer-Melia, NJ and Burkholder, JM and Moser, VC and Jensen, K}, year={2000}, pages={533–540} }
 @misc{touchette_burkholder_2000, title={Review of nitrogen and phosphorus metabolism in seagrasses}, volume={250}, ISSN={["1879-1697"]}, DOI={10.1016/S0022-0981(00)00195-7}, abstractNote={Within the past few decades, major losses of seagrass habitats in coastal waters impacted by cultural eutrophication have been documented worldwide. In confronting a pressing need to improve the management and protection of seagrass meadows, surprisingly little is known about the basic nutritional physiology of these critical habitat species, or the physiological mechanisms that control their responses to N and P gradients. The limited available evidence to date already has revealed, for some seagrass species such as the north temperate dominant Zostera marina, unusual responses to nutrient enrichment in comparison to other vascular plants. Seagrasses derive N and P from sediment pore water (especially ammonium) and the water column (most nitrate). The importance of leaves versus roots in nutrient acquisition depends, in part, on the enrichment conditions. For example, a shift from reliance on sediment pore water to increased reliance on the overlying water for N and P supplies has been observed under progressive water-column nutrient enrichment. Seagrasses may be N-limited in nutrient-poor waters with sandy or (less so) organic sediments, and P-limited in carbonate sediments. On the basis of data from few species, seagrasses appear to have active uptake systems for NO3− and PO4−3, but NH4+ uptake may involve both low- and high-affinity systems. Pi uptake affinities reported thus far are much lower than values for active ammonium uptake, but comparable to values for nitrate uptake by leaf tissues. Beyond such basic information, seagrass species have shown considerable variation in nutritional response. Dominance of acropetal versus basipetal nutrient translocation appears to vary among species as an innate trait. While some species follow classic Michaelis–Menten kinetics for Ni uptake, others have exhibited sustained linear uptake with limited or negligible product feedback inhibition, perhaps in adaptation to oligotrophic environments. Zostera marina also is able to maintain nitrate reductase (NR) activity during dark periods if adequate carbohydrate reserves and substrate are available. Thus, this species can respond to nitrate pulses throughout a diel cycle, rather than being limited as most plants to nitrate uptake during the light period. Further adaptations may have occurred for seagrasses in extremely nitrate-depauperate conditions. For example, Halophila decipiens and H. stipulacea lack inducible NR and apparently have lost the ability to reduce nitrate; and a biphasic rather than hyperbolic Pi uptake curve, with ‘surge’ uptake, has been described for Zostera noltii. Many seagrasses respond favorably to low or moderate N and/or P enrichment. However, excessive Ni loading to the water column can inhibit seagrass growth and survival, not only as an indirect effect by stimulating algal overgrowth and associated light reduction, but—for some species—as a direct physiological effect. The latter direct impact has been most pronounced for plants growing in sandy (nutrient-poor) sediments, and is exacerbated by elevated temperatures and/or light reduction. Ammonia toxicity, known for many vascular plants, has been reported in seagrasses Ruppia drepanensis and Z. marina (125 μM water-column NH4+, 5 weeks). Z. marina has shown to be inhibited, as well, by pulsed water-column nitrate enrichment (as low as 3.5–7 μM NO3−, 3–5 weeks), which is actively taken up without apparent product feedback inhibition. Inhibition by elevated nitrate has also been reported, with description of the underlying physiological mechanisms, in certain macroalgae and microalgae. In Z. marina, this effect has been related to the high, sustained energy demands of nitrate uptake, and to inducement of internal carbon limitation by the concomitant ‘carbon drain’ into amino acid assimilation. In contrast, nitrate enrichment can stimulate growth of Z. marina when the sediment, rather than the water column, is the source. Because seagrass species have shown considerable variation in nutritional response, inferences about one well-studied species, from one geographic location, should not be applied a priori to that species in other regions or to seagrasses in general. Most of the available information has been obtained from study of a few species, and the basic nutritional physiology of many seagrasses remains to be examined and compared across geographic regions. Nonetheless, the relatively recent gains in general understanding about the physiological responses of some seagrass species to nutrient gradients already have proven valuable in both basic and applied research. For example, physiological variables such as tissue C:N:P content have begun to be developed as integrative indicators of nutrient conditions and anthropogenic nutrient enrichment. To strengthen insights for management strategies to optimize seagrass survival in coastal waters adjacent to exponential human population growth and associated nutrient inputs, additional emphasis is critically needed to assess the role of variable interactions—among inorganic as well as organic N, P and C, environmental factors such as temperature, light, and other community components—in controlling the physiology, growth and survival of these ecologically important marine angiosperms.}, number={1-2}, journal={JOURNAL OF EXPERIMENTAL MARINE BIOLOGY AND ECOLOGY}, author={Touchette, BW and Burkholder, JM}, year={2000}, month={Jul}, pages={133–167} }
 @misc{burkholder_pain_2000, title={Scary monsters super creeps}, number={2000 June 3}, journal={New Scientist}, author={Burkholder, J. and Pain, S.}, year={2000}, pages={42–45} }
 @article{glasgow_burkholder_2000, title={Water quality trends and management implications from a five- year study of a eutrophic estuary}, volume={10}, DOI={10.2307/2641015}, abstractNote={The Neuse River and Estuary, a major tributary of the second largest estuary on the United States mainland, historically has sustained excessive blooms of algae and toxic dinoflagellates, hypoxia, and fish kills. Previous attempts have been made to use short-term databases of 2–3 years, or data sets from infrequent (monthly) sampling, to assess whether nutrient inputs to the Neuse are increasing and supporting higher algal production. These previous efforts also have relied on single-point-determined flow velocity data, at upstream sites remote from the estuary, to estimate the volume of flow in quantifying nutrient loading to the estuary. We completed a five-year study of the Neuse, including a comparative inventory of nutrients to the watershed from point sources and from concentrated animal operations (CAOs) as recent nonpoint sources, as well as an intensive assessment of water quality over time in the mesohaline estuary. Estimates of nutrient loads were based on volume of flow data from shore-to-shore transect cross sections, taken with a boat-mounted acoustic Doppler current profiler at the westernmost edge of the estuary. A total of 441 point dischargers contributed at least 3.34 × 108 L effluent/d to the Neuse system, much of which came from municipal wastewater treatment plants (2.03 × 108 L effluent/d, excluding periods of plant malfunctions; total annual loadings of at least 9 × 105 kg P and 2.1 × 106 kg N, with a 17% increase in human population over the past decade). The Neuse basin also included 554 CAOs, with 76% in swine production (1.7 × 106 animals, from a 285% increase in the past decade) and 23% in poultry (5.5 × 105 animals). An estimated 5.9 × 109 kg manure produced by swine and poultry during 1998 contributed ∼4.1 × 107 kg N and 1.4 × 107 kg P to the Neuse watershed. About 20% of the area in the watershed now has enough manure from CAOs to exceed the P requirements of all nonlegume crops and forages. About two-thirds of the N- and P-rich feeds for these animals are imported (with 4.0 × 107 kg N and 1.6 × 107 kg P in 1998); thus, the watershed increasingly has become a nutrient sink. Over the five-year study in the Neuse Estuary study area, P loading significantly declined (by an estimated 14%), whereas TN (total nitrogen) loading significantly increased (by an average of 16%) and TNi (total inorganic nitrogen) increased by ∼38%. The increased inorganic N (Ni), partly related to severe storms with high precipitation in years 4–5, coincided with a decrease in phytoplankton biomass (as chlorophyll a) that likely reflected displacement/washout of algal populations and cysts. Thus, while both N and P supplies have increased in the watershed, there is evidence for a significant increase in Ni loading but, as yet, no apparent signal for increased P in the lower estuary. Weather patterns ultimately control when/whether the elevated Ni supply will support increased algal production, so that estuarine algal blooms, hypoxia, and fish kills will remain difficult, at best, to predict in modeling efforts. We recommend that decadal data sets, with sufficient sampling frequency to capture nutrient loadings from major storm events, be used to assess fluctuations in algal production of lower rivers and estuaries, and relationships with changing nutrient inputs. Given increased N and P supplies in the Neuse watershed from ongoing growth of both human and swine populations, a current management goal of 30% N reduction should be altered to include increased focus on Ni and strengthened comanagement of P. As for estuaries in other regions, nutrient reduction goals should be interpreted as “moving targets” that likely will have to be substantially adjusted upward, over time, to accomplish noticeable reductions in algal blooms, hypoxia, and fish kills in the lower Neuse River and Estuary.}, number={4}, journal={Ecological Applications}, author={Glasgow, H. B. and Burkholder, J. M.}, year={2000}, pages={1024–1046} }
 @article{burkholder_mallin_glasgow_1999, title={Fish kills, bottom-water hypoxia, and the toxic Pfiesteria complex in the Neuse River and Estuary}, volume={179}, ISSN={["0171-8630"]}, DOI={10.3354/meps179301}, abstractNote={A recent paper by Paerl et al. 'Ecosystem responses to internal and watershed organic matter loading: consequences for hypoxia in the eutrophying Neuse River Estuary, North Carolina, USA' (1998; Mar Ecol Prog Ser 166:17-25) makes the statement that increased nitrogen loading to the Neuse River Estuary has led to algal blooms which produced organic matter loads capable of causing extensive hypoxic and anoxic conditions that, in turn, have induced widespread mortality of resident finand shellfish (p l?) . In this Comment we demonstrate that Paerl et al.'s central conclusion about finfish kills is not supported either by their data or by any statistical analysis, despite invoking predictability ('Results and discussion', p 20-24, Paerl et al. 1998). The paper contains numerous misinterpretations and misuse of literature citations. Paerl et al. also made serious errors of omission, germane from the perspective of science ethics, in failing to cite peerreviewed, published information that attributed other causality to various fish kills that they described. When attempting to make the difficult step from correlation to implication of causality in a field setting, the available evidence for multiple causative factors should be considered-especially when dealing with a topic that has significant implications for policy makers, scientists, and the general public. Accordingly, formal correction of the Paerl et al. paper is necessary because of the authors' (1) lack of depth profiles of dissolved oxygen (DO) data to support any of their conclusions about kills of surface-schooling fish, (2) use of unrecorded or nonexistent fish kill data, as well as misconveyance of fish kills as reported in the State database upon which they relied, (3) apparent lack of understanding about the behavior of resident fish population~, (4) misuse of literature citations, (5) omission of a large body of peer-reviewed, published information on the same fish kills (1995-1996), and (6) lack of any supporting statistical analyses to demonstrate relationships among field dissolved oxygen, nutrient, and fish kill data. Lack of supporting dissolved oxygenlfish kill data and literature. The only DO data that Paerl et al. presented were from the bottom water. A much more complete database (laboratory of J.M.B. & H.B.G.; summary reports covering the period 1993 through 1996, available from the Division of Water Quality [DWQ] of the North Carolina Department of Environment & Natural Resources [NC DENR]) than that discussed by Paerl et al. contains information for physical, chemical, and biological factors on the mesohaline Neuse. This area (Fig. 1) includes the segments addressed in Paerl et al. and the segments where major fish kills historically have occurred in that estuary. Here we compare Paerl et al.'s contentions, based on biweekly data from 6 to 8 mainstem Neuse stations, with the data of our State-certified laboratory, including DO depth profiles. We report weekly data from 6 stations in the mainstem, mesohaline Neuse (Flanners Beach/Kennel Beach to MinnesottKherry Point), as well as data from freshwater segments following a major storm in late summer 1996. These data show that in summers of averageprecipitation years without hurricanes, hypoxic waters in the Neuse Estuary generally were constrained to the bottom third of the water column (Figs. 2 to 4 of this paper; hypoxia considered as in Paerl et al.). Paerl et al. stated that they used a fish kill database from NC DWQ (formerly the Division of Environmental Management [DEM] of the NC Department of Environment, Health & Natural Resources [NC DEHNR]) as}, journal={MARINE ECOLOGY PROGRESS SERIES}, author={Burkholder, JM and Mallin, MA and Glasgow, HB}, year={1999}, pages={301–310} }
 @article{lewitus_glasgow_burkholder_1999, title={Kleptoplastidy in the toxic dinoflagellate Pfiesteria piscicida (Dinophyceae)}, volume={35}, ISSN={["0022-3646"]}, DOI={10.1046/j.1529-8817.1999.3520303.x}, abstractNote={The ichthyotoxic dinoflagellate Pfiesteria piscicida Steidinger et Burkholder has a complex life cycle with several heterotrophic flagellated and amoeboid stages. A prevalent flagellated form, the nontoxic zoospore stage, has a proficient grazing ability, especially on cryptophyte prey. Although P. piscicida zoospores lack the genetic capability to synthesize chloroplasts, they can obtain functional chloroplasts from algal prey (i.e. kleptoplastidy), as demonstrated here with a cryptophyte prey. Zoospores grown with Rhodomonas sp. Karsten CCMP757 (Cryptophyceae) grazed the cryptophyte population to minimal densities. After placing the cultures in near darkness where cryptophyte recovery was restricted and further prey ingestion did not occur, the time‐course patterns in growth, prey chloroplast content·zoospore−1, and prey nucleus content·zoospore−1 were followed. Ingested chloroplasts were selectively retained in the dinoflagellate, as indicated by the decline and, ultimately, near absence of cryptophyte nuclei in plastid‐containing zoospores. Chloroplasts retained inside P. piscicida cells for at least a week were photosynthetically active, as indicated by starch accumulation and microscope‐autoradiographic measurements of bicarbonate uptake. Recognition that P. piscicida can function as a phototroph broadens our perspective of the physiological ecology of the dinoflagellate because it suggests that, at least during part of its life cycle, P. piscicida’s growth and survival might be affected by photoregulation and nutritional control of photosynthesis.}, number={2}, journal={JOURNAL OF PHYCOLOGY}, author={Lewitus, AJ and Glasgow, HB and Burkholder, JM}, year={1999}, month={Apr}, pages={303–312} }
 @article{lewitus_willis_hayes_burkholder_glasgow_glibert_burke_1999, title={Mixotrophy and nitrogen uptake by Pfiesteria piscicida (Dinophyceae)}, volume={35}, ISSN={["0022-3646"]}, DOI={10.1046/j.1529-8817.1999.3561430.x}, abstractNote={The nutritional versatility of dinoflagellates is a complicating factor in identifying potential links between nutrient enrichment and the proliferation of harmful algal blooms. For example, although dinoflagellates associated with harmful algal blooms (e.g. red tides) are generally considered to be phototrophic and use inorganic nutrients such as nitrate or phosphate, many of these species also have pronounced heterotrophic capabilities either as osmotrophs or phagotrophs. Recently, the widespread occurrence of the heterotrophic toxic dinoflagellate, Pfiesteria piscicida Steidinger et Burkholder, has been documented in turbid estuarine waters. Pfiesteria piscicida has a relatively proficient grazing ability, but also has an ability to function as a phototroph by acquiring chloroplasts from algal prey, a process termed kleptoplastidy. We tested the ability of kleptoplastidic P. piscicida to take up 15N‐labeled NH, NO, urea, or glutamate. The photosynthetic activity of these cultures was verified, in part, by use of the fluorochrome, primulin, which indicated a positive relationship between photosynthetic starch production and growth irradiance. All four N substrates were taken up by P. piscicida, and the highest uptake rates were in the range cited for phytoplankton and were similar to N uptake estimates for phagotrophic P. piscicida. The demonstration of direct nutrient acquisition by kleptoplastidic P. piscicida suggests that the response of the dinoflagellate to nutrient enrichment is complex, and that the specific pathway of nutrient stimulation (e.g. indirect stimulation through enhancement of phytoplankton prey abundance vs. direct stimulation by saprotrophic nutrient uptake) may depend on P. piscicida’s nutritional state (phagotrophy vs. phototrophy).}, number={6}, journal={JOURNAL OF PHYCOLOGY}, author={Lewitus, AJ and Willis, BM and Hayes, KC and Burkholder, JM and Glasgow, HB and Glibert, PM and Burke, MK}, year={1999}, month={Dec}, pages={1430–1437} }
 @article{levin_simon_schmechel_glasgow_deamer-melia_burkholder_moser_jensen_harry_1999, title={Pfiesteria toxin and learning performance}, volume={21}, ISSN={["0892-0362"]}, DOI={10.1016/S0892-0362(98)00041-5}, abstractNote={Pfiesteria piscicida is an estuarine dinoflagellate involved with fish kills along the east coast of the United States. We previously documented a radial-arm maze learning deficit in rats exposed to Pfiesteria that may be related to cognitive deficits seen in humans after accidental Pfiesteria exposure. The current study elucidated important behavioral parameters of this deficit. There were six dose groups. Forty (10/group) adult female Sprague–Dawley rats were injected (SC) with a single dose of Pfiesteria taken from aquarium-cultured Pfiesteria (35,600, 106,800, or 320,400 Pfiesteria cells/kg of rat body weight or a cell-free filtrate of the 106,800 cells/kg dose). One control group (N = 10) was injected with saline and one (N = 10) with aquarium water not containing Pfiesteria. Half of the rats in each group were tested on an 8-arm radial maze in a standard test room, and the other half were tested on the radial maze in a sound-attenuating chamber. In the standard maze room, there was a significant effect of Pfiesteria (p < 0.05) impairing choice accuracy improvement over the first six sessions of training among rats administered 106,800, 320,400, and the 106,800 cells/kg filtered sample. In contrast, there was no indication of an effect of Pfiesteria when the rats were tested on the same configuration radial maze in the sound-attenuating chamber. After 18 sessions of training in one room, the rats were switched for six sessions of testing in the other room and finally were switched back to their original room for three sessions. There was a significant Pfiesteria-induced deficit when the rats were tested in the standard test room but not when they were tested in the sound-attenuating chamber. When the Pfiesteria-exposed rats were initially switched from the sound-attenuating chamber to the standard test room they performed significantly worse than controls, whereas Pfiesteria-treated rats switched from the standard test room to the sound-attenuating chamber did not perform differently from controls. These results suggest that the Pfiesteria-induced learning impairment may result from the negative impact of distracting stimuli. At the time of the learning impairment, no overt Pfiesteria-related effects were seen using a functional observational battery and no overall response latency effects were seen, indicating that the Pfiesteria-induced choice accuracy deficit was not due to generalized debilitation. In the initial use of the figure-8 maze in this line of research, the rats in the same Pfiesteria treatment groups that showed significant deficits in the radial-arm maze showed greater declines in activity rates in a 1-h figure-8 locomotor activity test. Both the 106,800 and 320,400 Pfiesteria cells/kg groups showed significantly greater linear trends of activity decline relative to tank water-treated controls. This reflected an initial slight hyperactivity in the Pfiesteria-treated animals followed by a decrease to control levels. Pfiesteria effects in the figure-8 maze and in early radial-arm maze training may be useful in a rapid screen for identifying the critical toxin(s) of Pfiesteria in future studies. Published by Elsevier Science Inc.}, number={3}, journal={NEUROTOXICOLOGY AND TERATOLOGY}, author={Levin, ED and Simon, BB and Schmechel, DE and Glasgow, HB and Deamer-Melia, NJ and Burkholder, JM and Moser, VC and Jensen, K and Harry, GJ}, year={1999}, pages={215–221} }
 @article{fairey_edmunds_deamer-melia_glasgow_johnson_moeller_burkholder_ramsdell_1999, title={Reporter gene assay for fish-killing activity produced by Pfiesteria piscicida}, volume={107}, ISSN={["0091-6765"]}, DOI={10.2307/3434655}, abstractNote={Collaborative studies were performed to develop a functional assay for fish-killing activity produced by Pfiesteria piscicida. Eight cell lines were used to screen organic fractions and residual water fraction by using a 3-[4, 5-dimethylthiazol-(2-4)]-diphenyltetrazolium bromide cytotoxicity assay. Diethyl ether and a residual water fraction were cytotoxic to several cell lines including rat pituitary (GH(4)C(1)) cells. Residual water as well as preextracted culture water containing P. piscicida cells induced c-fos-luciferase expressed in GH(4)C(1) cells with a rapid time course of induction and sensitive detection. The reporter gene assay detected activity in toxic isolates of P. piscicida from several North Carolina estuaries in 1997 and 1998 and may also be suitable for detecting toxic activity in human and animal serum.}, number={9}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={Fairey, ER and Edmunds, JSG and Deamer-Melia, NJ and Glasgow, H and Johnson, FM and Moeller, PR and Burkholder, JM and Ramsdell, JS}, year={1999}, month={Sep}, pages={711–714} }
 @article{harvell_kim_burkholder_colwell_epstein_grimes_hofmann_lipp_osterhaus_overstreet_et al._1999, title={Review: Marine ecology - Emerging marine diseases - Climate links and anthropogenic factors}, volume={285}, ISSN={["1095-9203"]}, DOI={10.1126/science.285.5433.1505}, abstractNote={Mass mortalities due to disease outbreaks have recently affected major taxa in the oceans. For closely monitored groups like corals and marine mammals, reports of the frequency of epidemics and the number of new diseases have increased recently. A dramatic global increase in the severity of coral bleaching in 1997–98 is coincident with high El Niño temperatures. Such climate-mediated, physiological stresses may compromise host resistance and increase frequency of opportunistic diseases. Where documented, new diseases typically have emerged through host or range shifts of known pathogens. Both climate and human activities may have also accelerated global transport of species, bringing together pathogens and previously unexposed host populations.}, number={5433}, journal={SCIENCE}, author={Harvell, CD and Kim, K and Burkholder, JM and Colwell, RR and Epstein, PR and Grimes, DJ and Hofmann, EE and Lipp, EK and Osterhaus, ADME and Overstreet, RM and et al.}, year={1999}, month={Sep}, pages={1505–1510} }
 @article{burkholder_glasgow_1999, title={Science ethics and its role in early suppression of the Pfiesteria issue}, volume={58}, ISSN={["1938-3525"]}, DOI={10.17730/humo.58.4.976098q356672751}, number={4}, journal={HUMAN ORGANIZATION}, author={Burkholder, JM and Glasgow, HB}, year={1999}, pages={443–455} }
 @article{burkholder_1999, title={The lurking perils of Pfiesteria}, volume={281}, ISSN={["1946-7087"]}, DOI={10.1038/scientificamerican0899-42}, number={2}, journal={SCIENTIFIC AMERICAN}, author={Burkholder, JM}, year={1999}, month={Aug}, pages={42–49} }
 @inproceedings{glasgow_lewitus_burkholder_1998, title={Feeding behavior of the icthyotoxic estuarine dinoflagelate pfiesteria piscicida on amino acids, algal prey, and fish vs. mammalian erythrocytes}, booktitle={Harmful algae = Algas nocivas: Proceedings of the VIII International Conference on Harmful Algae, Vigo, Spain, 25-29 June 1997}, publisher={Vigo, Spain: Intergovernmental Oceanographic Commission  of Unesco}, author={Glasgow, H. B. and Lewitus, A. J. and Burkholder, J. M.}, year={1998}, pages={394–397} }
 @article{burkholder_1998, title={Implications of harmful microalgae and heterotrophic dinoflagellates in management of sustainable marine fisheries}, volume={8}, DOI={10.2307/2641362}, number={1 (suppl.)}, journal={Ecological Applications}, author={Burkholder, J. M.}, year={1998}, pages={S37–62} }
 @article{burkholder_larser_glasgow_mason_gama_parsons_1998, title={Influence of sediment and phosphorus loading on phytoplankton communities in an urban piedmont reservoir}, volume={14}, DOI={10.1080/07438149809354114}, abstractNote={ABSTRACT The response of phytoplankton communities to an imposed gradient of sediment loading under low versus high phosphate enrichment was assessed with enclosures (2-m-diameter polyethylene columns, open to the surface and the sediment) in a small piedmont reservoir within an urbanized watershed. The experimental design included replicated controls [CON, with ambient suspended solids (SS) and phosphorus (P)] and treatments low clay (LC), a natural hydrated sediment mix, sufficient clay initially added to effect an average daily concentration of 5 mg SS L−1 d−1), high clay (HC), 15 mg SS·L−1·d−1, phosphate enrichment (PHOS), ≥ 250 μg PO4 −3 P L−1); LC+P, and HC+P, imposed for ca. 10 weeks in the summer growing season. Clay additions without P led to decreased phytoplankton production (as chlorophyll a, chla) relative to controls, with colonial blue-greens and colonial greens increasing in LC, and HC dominated by filamentous blue-greens and mixotrophic flagellates. Significant increases in chla occurred ...}, number={1}, journal={Lake and Reservoir Management}, author={Burkholder, J. M. and Larser, L. M. and Glasgow, H. B. and Mason, K. M. and Gama, P. and Parsons, J. E.}, year={1998}, pages={110–121} }
 @book{burkholder_1998, title={Pfiesteria and pfiesteria-like dinoflagellates}, number={1998 Apr. 1}, journal={Report (Water Resources Research Institute of the University of North Carolina)}, institution={Raleigh, NC: University of North Carolina Water Resources Research Institute}, author={Burkholder, J. M.}, year={1998} }
 @inbook{burkholder_glasgow_lewitus_1998, title={Physiological ecology of Pfiesteria piscicida with general comments on 'ambush-oredator' dinoflagellates}, volume={41}, booktitle={Physiological ecology of harmful algal blooms}, publisher={New York: Springer}, author={Burkholder, J. M. and Glasgow, H. B. and Lewitus, A. J.}, editor={D. M. Anderson, A. D. Cembella and Hallegraeff, G. M.Editors}, year={1998}, pages={175–191} }
 @inproceedings{toffer_schaefer_glasgow_burkholder_rublee_1998, title={Ribosomal DNA from the toxic dinoflagellate pfiesteria piscicida}, booktitle={Harmful algae = Algas nocivas: Proceedings of the VIII International Conference on Harmful Algae, Vigo, Spain, 25-29 June 1997}, publisher={Vigo, Spain: Intergovernmental Oceanographic Commission  of Unesco}, author={Toffer, K. L. and Schaefer, E. F. and Glasgow, H. B. and Burkholder, J. M. and Rublee, P. A.}, year={1998}, pages={278–279} }
 @article{mallin_burkholder_mciver_shanks_glasgow_touchette_springer_1997, title={Comparative effects of poultry and swine waste lagoon spills on the quality of receiving streamwaters}, volume={26}, ISSN={["0047-2425"]}, DOI={10.2134/jeq1997.00472425002600060023x}, abstractNote={Abstract}, number={6}, journal={JOURNAL OF ENVIRONMENTAL QUALITY}, author={Mallin, MA and Burkholder, JM and McIver, MR and Shanks, GC and Glasgow, HB and Touchette, BW and Springer, J}, year={1997}, pages={1622–1631} }
 @article{burkholder_mallin_glasgow_larsen_mciver_shank_deamer-melia_briley_springer_touchette_et al._1997, title={Impacts to a coastal river and estuary from rupture of a large swine waste holding lagoon}, volume={26}, ISSN={["0047-2425"]}, DOI={10.2134/jeq1997.00472425002600060003x}, abstractNote={Abstract}, number={6}, journal={JOURNAL OF ENVIRONMENTAL QUALITY}, author={Burkholder, JM and Mallin, MA and Glasgow, HB and Larsen, LM and McIver, MR and Shank, GC and Deamer-Melia, N and Briley, DS and Springer, J and Touchette, BW and et al.}, year={1997}, pages={1451–1466} }
 @article{levin_schmechel_burkholder_glasgow_deamer-melia_moser_harry_1997, title={Persisting learning deficits in rats after exposure to Pfiesteria piscicida}, volume={105}, ISSN={["0091-6765"]}, DOI={10.2307/3433751}, abstractNote={Pfiesteria piscicida and other toxic Pfiesteria-like dinoflagellates have been implicated as a cause of fish kills in North Carolina estuaries and elsewhere. Accidental laboratory exposure of humans to P. piscicida has been reported to cause a complex syndrome including cognitive impairment. The current project was conducted to experimentally assess the possibility of cognitive effects of P. piscicida exposure in rats. Samples of water from aquaria in which P. piscicida zoospores were killing fish were frozen, a procedure that has been found to induce encystment. Thawed samples were injected into albino Sprague-Dawley rats. A significant learning impairment was documented in rats administered samples of P. piscicida that were recently frozen. Prolonged storage of Pfiesteria samples diminished the effect. No effect was seen in the recall of a previously learned task, but when the rats were called upon to learn a new task, the Pfiesteria-treated animals showed a significant learning deficit. This effect persisted up to at least 10 weeks after a single injection of Pfiesteria. The Pfiesteria-induced learning deficit did not seem to be associated with any obvious debilitation or health impairment of the exposed rats. Deficits in habituation of arousal and rearing behavior were detected using a functional observational battery. No Pfiesteria-induced effects on blood count and white cell differential or in a standard pathological screening of brain, liver, lung, kidney, and spleen tissue were seen at 2 months after exposure. These studies document a persistent learning impairment in rats after exposure to the dinoflagellate P.piscicida in otherwise physically well-appearing rats. This effect may partially model the symptoms of cognitive impairments that humans have shown after Pfiesteria exposure.}, number={12}, journal={ENVIRONMENTAL HEALTH PERSPECTIVES}, author={Levin, ED and Schmechel, DE and Burkholder, JM and Glasgow, HB and Deamer-Melia, NJ and Moser, VC and Harry, GJ}, year={1997}, month={Dec}, pages={1320–1325} }
 @article{burkholder_glasgow_1997, title={Pfiesteria piscicida and other Pfiesteria-like dinoflagellates: Behavior, impacts, and environmental controls}, volume={42}, ISSN={["0024-3590"]}, DOI={10.4319/lo.1997.42.5_part_2.1052}, abstractNote={Toxic Pfiesteria‐like dinoflagellates have been implicated as causative agents of major fish kills (affecting 103– 109 fish) in estuaries and coastal waters of the mid‐Atlantic and southeastern U.S. Transformations among an array of flagellated, amoeboid, and encysted stages in the complex life cycle of the representative species, Pfiesteria piscicida, are controlled by the availability of fresh secretions, blood, or other tissues of fish prey. P. piscicida also is a voracious predator on other estuarine microorganisms. Pfiesteria‐like dinoflagellates require an unidentified substance(s) commonly found in fresh fish excreta‐secreta to initiate toxin production. P. piscicida is lethal to fish at low cell densities (>250–300 cells ml−1), and at sublethal levels (∼100–250 cells m1−1) it has been shown to cause ulcerative fish diseases. P. piscicida also has been linked to serious human health impacts. This dinoflagellate is eurythermal and euryhaline, with optima for toxic activity by the most lethal stage (toxic zoospores, TZs) at ≥26°C and 15 psu, respectively. Thus far it has shown no light optimum and is capable of killing fish at any time during a 24‐h cycle. In warmer waters (≥ 15°C) flagellated stages predominate while fish are dying, whereas amoebae predominate in colder conditions and when fish are dead. Nutritional stimuli influencing P. piscicida are complex; inorganic phosphate apparently can directly stimulate TZs, whereas inorganic phosphate and nitrate indirectly promote increased production of nontoxic zoospores (NTZs, maintained in the absence of live fish, as potential precursors to lethal TZs) by stimulating their algal prey. Organic phosphate (Po) and nitrogen are taken up by P. piscicida osmotrophically, and Po is stimulatory to both TZs and NTZs. The available data point to a critical need to characterize the chronic and acute impacts of toxic Pfiesteria‐like dinoflagellates on fish and other targeted prey in estuarine and coastal waters that are adversely affected by cultural eutrophication.}, number={5}, journal={LIMNOLOGY AND OCEANOGRAPHY}, author={Burkholder, JM and Glasgow, HB}, year={1997}, month={Jul}, pages={1052–1075} }
 @article{burkholder_glasgow_1997, title={Trophic controls on stage transformations of a toxic ambush-predator dinoflagellate}, volume={44}, ISSN={["1066-5234"]}, DOI={10.1111/j.1550-7408.1997.tb05700.x}, abstractNote={ABSTRACT.  The toxic dinoflagellate, Pfiesteria piscicida, was recently implicated as the causative agent for about 50% of the major fish kills occurring over a three‐year period in the Albemarle‐Pamlico Estuarine System of the southeastern USA. Transformations between life‐history stages of this dinoflagellate are controlled by the availability of fresh fish secretions or fish tissues, and secondarily influenced by the availability of alternate prey including bacteria, algae, microfauna, and mammalian tissues. Toxic zoospores of P. piscicida subdue fish by excreting lethal neurotoxins that narcotize the prey, disrupt its osmoregulatory system, and attack its nervous system. While prey are dying, the zoospores feed upon bits of fish tissue and complete the sexual phase of the dinoflagellate life cycle. Other stages in the complex life cycle of P. piscidia include cryptic forms of filose, rhizopodial, and lobose amoebae that can form within minutes from toxic zoospores, gametes, or planozygotes. These cryptic amoebae feed upon fish carcasses and other prey and, thus far, have proven less vulnerable to microbial predators than flagellated life‐history stages. Lobose amoebae that develop from toxic zoospores and planozygotes during colder periods have also shown ambush behavior toward live fish. In the presence of abundant flagellated algal prey, amoeboid stages produce nontoxic zoospores that can become toxic and form gametes when they detect what is presumed to be a threshold level of a stimulatory substance(s) derived from live fish. The diverse amoeboid stages of this fish “ambush‐predator” and at least one other Pfiesteria‐like species are ubiquitous and abundant in brackish waters along the western Atlantic and Gulf Coasts, indicating a need to re‐evaluate the role of dinoflagellates in the microbial food webs of turbid nutrient‐enriched estuaries.}, number={3}, journal={JOURNAL OF EUKARYOTIC MICROBIOLOGY}, author={Burkholder, JM and Glasgow, HB}, year={1997}, pages={200–205} }
 @article{burkholder_noga_hobbs_glasgow_1992, title={NEW PHANTOM DINOFLAGELLATE IS THE CAUSATIVE AGENT OF MAJOR ESTUARINE FISH KILLS}, volume={358}, ISSN={["0028-0836"]}, DOI={10.1038/358407a0}, abstractNote={A worldwide increase in toxic phytoplankton blooms over the past 20 years has coincided with increasing reports of fish diseases and deaths of unknown cause. Among estuaries that have been repeatedly associated with unexplained fish kills on the western Atlantic Coast are the Pamlico and Neuse Estuaries of the southeastern United States. Here we describe a new toxic dinoflagellate with 'phantom-like' behaviour that has been identified as the causative agent of a significant portion of the fish kills in these estuaries, and which may also be active in other geographic regions. The alga requires live finfish or their fresh excreta for excystment and release of a potent toxin. Low cell densities cause neurotoxic signs and fish death, followed by rapid algal encystment and dormancy unless live fish are added. This dinoflagellate was abundant in the water during major fish kills in local estuaries, but only while fish were dying; within several hours of death where carcasses were still present, the flagellated vegetative algal population had encysted and settled back to the sediments. Isolates from each event were highly lethal to finfish and shellfish in laboratory bioassays. Given its broad temperature and salinity tolerance, and its stimulation by phosphate enrichment, this toxic phytoplankter may be a widespread but undetected source of fish mortality in nutrient-enriched estuaries.}, number={6385}, journal={NATURE}, author={BURKHOLDER, JM and NOGA, EJ and HOBBS, CH and GLASGOW, HB}, year={1992}, month={Jul}, pages={407–410} }