David M Kulis

Publications of David M Kulis

• Transcriptome profiling of a toxic dinoflagellate reveals a gene-rich protist and a potential impact on gene expression due to bacterial presence.

  Authors: Ahmed Moustafa, Andrew N Evans, David M Kulis, Jeremiah D Hackett, Deana L Erdner, Donald M Anderson, Debashish Bhattacharya

  PloS one. 01/2010; 5(3):e9688.

  Dinoflagellates are unicellular, often photosynthetic protists that play a major role in the dynamics of the Earth's oceans and climate. Sequencing of dinoflagellate nuclear DNA is thwarted by theirDinoflagellates are unicellular, often photosynthetic protists that play a major role in the dynamics of the Earth's oceans and climate. Sequencing of dinoflagellate nuclear DNA is thwarted by their massive genome sizes that are often several times that in humans. However, modern transcriptomic methods offer promising approaches to tackle this challenging system. Here, we used massively parallel signature sequencing (MPSS) to understand global transcriptional regulation patterns in Alexandrium tamarense cultures that were grown under four different conditions. We generated more than 40,000 unique short expression signatures gathered from the four conditions. Of these, about 11,000 signatures did not display detectable differential expression patterns. At a p-value < 1E-10, 1,124 signatures were differentially expressed in the three treatments, xenic, nitrogen-limited, and phosphorus-limited, compared to the nutrient-replete control, with the presence of bacteria explaining the largest set of these differentially expressed signatures. Among microbial eukaryotes, dinoflagellates contain the largest number of genes in their nuclear genomes. These genes occur in complex families, many of which have evolved via recent gene duplication events. Our expression data suggest that about 73% of the Alexandrium transcriptome shows no significant change in gene expression under the experimental conditions used here and may comprise a "core" component for this species. We report a fundamental shift in expression patterns in response to the presence of bacteria, highlighting the impact of biotic interaction on gene expression in dinoflagellates.

• Fiber-optic microarray for simultaneous detection of multiple harmful algal bloom species.

  Authors: Soohyoun Ahn, David M Kulis, Deana L Erdner, Donald M Anderson, David R Walt

  Applied and environmental microbiology. 10/2006; 72(9):5742-9.

  Harmful algal blooms (HABs) are a serious threat to coastal resources, causing a variety of impacts on public health, regional economies, and ecosystems. Plankton analysis is a valuable component ofHarmful algal blooms (HABs) are a serious threat to coastal resources, causing a variety of impacts on public health, regional economies, and ecosystems. Plankton analysis is a valuable component of many HAB monitoring and research programs, but the diversity of plankton poses a problem in discriminating toxic from nontoxic species using conventional detection methods. Here we describe a sensitive and specific sandwich hybridization assay that combines fiber-optic microarrays with oligonucleotide probes to detect and enumerate the HAB species Alexandrium fundyense, Alexandrium ostenfeldii, and Pseudo-nitzschia australis. Microarrays were prepared by loading oligonucleotide probe-coupled microspheres (diameter, 3 mum) onto the distal ends of chemically etched imaging fiber bundles. Hybridization of target rRNA from HAB cells to immobilized probes on the microspheres was visualized using Cy3-labeled secondary probes in a sandwich-type assay format. We applied these microarrays to the detection and enumeration of HAB cells in both cultured and field samples. Our study demonstrated a detection limit of approximately 5 cells for all three target organisms within 45 min, without a separate amplification step, in both sample types. We also developed a multiplexed microarray to detect the three HAB species simultaneously, which successfully detected the target organisms, alone and in combination, without cross-reactivity. Our study suggests that fiber-optic microarrays can be used for rapid and sensitive detection and potential enumeration of HAB species in the environment.

• Cysts of Danish Gymnodinium nolleri Ellegaard et Moestrup sp. ined. (Dinophyceae): studies on encystment, excystment and toxicity

  Authors: Marianne Ellegaard, David M Kulis, Donald M Anderson

  Life cycle dynamics of Gymnodinium nolleri Ellegaard et Moestrup sp. ined. were studied under different temperature and nutrient conditions. Five culture strains originating from cysts found inLife cycle dynamics of Gymnodinium nolleri Ellegaard et Moestrup sp. ined. were studied under different temperature and nutrient conditions. Five culture strains originating from cysts found in Danish marine sediments were used for the experiments. Both encystment and excystment were found to vary with temperature. Maximal encystment occurred at 22–28°C, with no cysts formed below 13°C or above 33°C. Cyst production was slightly higher under phosphorus limitation than under combined nitrogen and phosphorus limitation. Maximal excystment occurred at 26.5°C with negligible excystment under 11°C and over 35°C. The resting period for cyst maturation was typically 3–4 weeks. Cysts produced under both phosphorus and nitrogen limitation were tested for paralytic shellfish poisoning (PSP) toxins by HPLC, and none were detected. It remains unclear why vegetative cells of this species have not yet been recorded in plankton samples from Scandinavia, despite the widespread distribution of the cysts.

• Outbreeding lethality between toxic Group I and nontoxic Group III Alexandrium tamarense spp. isolates: Predominance of heterotypic encystment and implications for mating interactions and biogeography

  Authors: Michael L. Brosnahan, David M. Kulis, Andrew R. Solow, Deana L. Erdner, Linda Percy, Jane Lewis, Donald M. Anderson

  Deep Sea Research Part II: Topical Studies in Oceanography.

  We report the zygotic encystment of geographically dispersed isolates in the dinoflagellate species complex Alexandrium tamarense, in particular, successful mating of toxic Group I and nontoxic GroupWe report the zygotic encystment of geographically dispersed isolates in the dinoflagellate species complex Alexandrium tamarense, in particular, successful mating of toxic Group I and nontoxic Group III isolates. However, hypnozygotes produced in Group I/III co-cultures complete no more than three divisions after germinating. Previous reports have suggested a mate recognition mechanism whereby hypnozygotes produced in co-cultures could arise from either homotypic (inbred) or heterotypic (outbred) gamete pairs. To determine the extent to which each occurs, a nested PCR assay was developed to determine parentage of individual hypnozygotes. The vast majority of hypnozygotes from pairwise Group I/III co-cultures were outbred, so that inviability was a result of hybridization, not inbreeding. These findings support the assertion that complete speciation underlies the phylogenetic structure of the Alexandrium tamarense species complex. Additionally, the ribosomal DNA (rDNA) copy numbers of both hybrid and single ribotype hypnozygotes were reduced substantially from those of haploid motile cells. The destruction of rDNA loci may be crucial for the successful mating of genetically distant conjugants and appears integral to the process of encystment.The inviability of Group I/III hybrids is important for public health because the presence of hybrid cysts may indicate ongoing displacement of a nontoxic population by a toxic one (or vice versa). Hybrid inviability also suggests a bloom control strategy whereby persistent, toxic Group I blooms could be mitigated by introduction of nontoxic Group III cells. The potential for hybridization in nature was investigated by applying the nested PCR assay to hypnozygotes from Belfast Lough, Northern Ireland, a region where Group I and III populations co-occur. Two hybrid cysts were identified in 14 successful assays, demonstrating that Group I and III populations do interbreed in that region. However, an analysis of mating data collected over an 18-year period indicated a leaky pre-mating barrier between ribosomal species (including Groups I and III). Whether the observed selectivity inhibits hybridization in nature is dependent on its mechanism. If the point of selectivity is the induction of gametogenesis, dissimilar ribotypes could interbreed freely, promoting displacement in cases where hybridization is lethal. If instead, selectivity occurs during the adhesion of gamete pairs, it could enable stable co-existence of A. tamarense species. In either case, hybrid inviability may impose a significant obstacle to range expansion. The nested PCR assay developed here is a valuable tool for investigation of interspecies hybridization and its consequences for the global biogeography of these important organisms.

• DSP toxin production de novo in cultures of Dinophysis acuminata (Dinophyceae) from North America

  Authors: Jeremiah D. Hackett, Mengmeng Tong, David M. Kulis, Elie Fux, Philipp Hess, Ronel Bire, Donald M. Anderson

  Harmful Algae. 8(6):873-879.

• DSP toxin production de novo in cultures of Dinophysis acuminata (Dinophyceae) from North America

  Authors: Jeremiah D. Hackett, Mengmeng Tong, David M. Kulis, Elie Fux, Philipp Hess, Ronel Bire, Donald M. Anderson

  Harmful Algae. 8(6):873-879.

• Identification and enumeration of Alexandrium spp. from the Gulf of Maine using molecular probes

  Authors: Donald M. Anderson, David M. Kulis, Bruce A. Keafer, Kristin E. Gribble, Roman Marin, Christopher A. Scholin

  Deep Sea Research Part II: Topical Studies in Oceanography.

  Three different molecular methods were used with traditional brightfield microscope techniques to enumerate the toxic dinoflagellate Alexandrium fundyense in samples collected in the Gulf of Maine inThree different molecular methods were used with traditional brightfield microscope techniques to enumerate the toxic dinoflagellate Alexandrium fundyense in samples collected in the Gulf of Maine in 1998, 2000, 2001, and 2003. Two molecular probes were used in fluorescent whole-cell (WC) microscopic assays: a large-subunit ribosomal RNA (LSU rRNA) oligonucleotide probe (NA1) and a monoclonal antibody probe thought to be specific for Alexandrium spp. within the tamarense/catenella/fundyense complex. Cell abundance estimates also were obtained using the NA1 oligonucleotide probe in a semi-quantitative sandwich hybridization assay (SHA) that quantified target rRNA in cell lysates. Here we compare and contrast the specificity and utility of these probe types and assay approaches.WC counts of the 1998 field samples demonstrated that A. fundyense cell densities estimated using the antibody approach were higher than those using either the NA1 oligonucleotide or brightfield microscopy due to the co-occurrence of A. ostenfeldii with A. fundyense, and the inability of the antibody to discriminate between these two species. An approach using cell size and the presence or absence of food vacuoles allowed more accurate immunofluorescent cell counts of both species, but small cells of A. ostenfeldii that did not contain food vacuoles were still mistakenly counted as A. fundyense. For 2001, a dual-labeling procedure using two oligonucleotide probes was used to separately enumerate A. ostenfeldii and A. fundyense in the WC format. In addition, the SHA was used in 2001 and 2003 to enumerate A. fundyense. Some agreement was observed between the two oligonucleotide methods, but there were differences as well. Not including samples with cell numbers below empirically determined detection limits of 25 cells l–1, good correlation was observed for surface samples and vertical profiles in May 2001 and June 2003 when the SHA estimates were, on average, equivalent, and 1.5× the WC counts, respectively. The worst correlations were for virtually all samples from the June 2001 cruise where the SHA both over- and under-estimated the WC counts. Some differences were expected, since the SHA and the WC assays measure different, but related parameters. The former quantifies intact cells and particulate material that might contain non-viable cells or fragments, whereas the latter measures only intact cells that survive sample processing and are visible in a sample matrix. A variety of factors can thus affect results, particularly with the WC method, including variable uptake of the oligonucleotide probe due to cell permeability changes, cell lysis during sampling, preservation and processing; variable rRNA content or accessibility due to nutritional or environmental factors; and the variable detection of intact cells or cell fragments in fecal pellets and detritus. The SHA offers dramatic increases in sample throughput, but introduces uncertainties, such as those due to sample matrix effects (non-specific labeling and cross-reactions), variable rRNA levels in intact cells or to the possible presence of target rRNA in cell fragments, fecal pellets, or detritus. Molecular probes are powerful tools for monitoring and research applications, but more work is needed to compare and refine these different cell enumeration methods on field samples, as well as to assess the general validity of brightfield or fluorescent WC approaches.

• Distribution and toxicity of Alexandrium ostenfeldii (Dinophyceae) in the Gulf of Maine, USA

  Authors: Kristin E. Gribble, Bruce A. Keafer, Michael A. Quilliam, Allan D. Cembella, David M. Kulis, Abigail Manahan, Donald M. Anderson

  Deep Sea Research Part II: Topical Studies in Oceanography.

  Alexandrium ostenfeldii is a thecate, mixotrophic dinoflagellate recently linked to a novel suite of toxins called spirolides. This study provides the first description of the regional distributionAlexandrium ostenfeldii is a thecate, mixotrophic dinoflagellate recently linked to a novel suite of toxins called spirolides. This study provides the first description of the regional distribution of A. ostenfeldii in the Gulf of Maine (GOM), and the first report and analysis of spirolide toxicity in A. ostenfeldii in waters south of Nova Scotia. Morphological examination of cells in field samples and of clonal cultures isolated from several stations in the GOM confirmed the presence of A. ostenfeldii. A genus-specific antibody probe, and an A. ostenfeldii species-specific oligonucleotide probe labeled these cells; a probe specific for the North American A. fundyense/tamarense/catenella species complex did not label A. ostenfeldii cells. Cell size ranged from 20 to nearly 90 μm, and most cells contained food vacuoles, with a total vacuole size from 1 to 48 μm.The hydrographic forcings controlling the distribution of A. ostenfeldii in the GOM are quite similar to those acting on the A. fundyense population at the same time of the year. The highest concentrations of A. ostenfeldii were observed nearshore, to the east of Penobscot Bay, at times with an offshore-turning branch of high cell concentration to the south of Penobscot Bay. Casco Bay appears to be an area of accumulation for A. ostenfeldii cells advected toward shore from the core of the population to the northeast. Concentrations of A. ostenfeldii were generally higher at the surface than deeper, except at locations where the pooling of lower-salinity water at the surface may have led to the subduction of the population flowing in from the east.PSP toxins were detected in field populations containing A. ostenfeldii and A. fundyense, but not in A. ostenfeldii cultures isolated from the GOM. Spirolide toxins were found in 36 of 60 field samples. More than 83% of samples containing A. ostenfeldii cells had one or more of spirolide congeners A, B, C2 and D2. The total concentration of spirolides per cell at each station where A. ostenfeldii was detected ranged from non-detectable to 282 fmol cell−1. A. ostenfeldii cultures originating from different locations showed a great diversity in spirolide content and composition. All cultures contained spirolides, ranging from 28 to 113 fmol cell−1. Spirolide congeners desMeC, C and D were present in some cultures, but were not detected in any of the field samples. Based on differences and similarities between their toxin profiles, five toxin phenotypes were identified. The highest per cell spirolide contents in the cultures were nearly two times lower than the highest levels observed in field samples. Currently there is no routine monitoring for spirolide toxins in shellfish in the region, but it may be necessary eventually to expand ongoing toxin monitoring in the GOM to include analysis for spirolides.

• An immunofluorescent survey of the brown tide chrysophyte Aureococcus anophagefferens along the northeast coast of the United States

  Authors: Donald M Anderson, Bruce A. Keafer, David M Kulis, Robert M. Waters, Robert Nuzzi

  Surveys were conducted along the northeast coast of the USA. between Portsmouth, NH, and the Chesapeake Bay in 1988 and 1990, to determine the population distribution of Aureococcus anophagefferens ,Surveys were conducted along the northeast coast of the USA. between Portsmouth, NH, and the Chesapeake Bay in 1988 and 1990, to determine the population distribution of Aureococcus anophagefferens , the chrysophyte responsible for massive and destructive ‘brown tides’ in Long Island and Narragansett Bay beginning in 1985. A species-specific immunofluorescent technique was used to screen water samples, with positive identification possible at cell concentrations as low as 10–20 cells ml−1. Both years. A.anophagefferens was detected at numerous stations in and around Long Island and Barnegat Bay, NJ, typically at high cell concentrations. To the north and south of thus ‘center’, nearly half of the remaining stations were positive for A.anophagefferens , but the cells were always at very low cell concentrations. Many of the positive identifications in areas distant from Long Island were in waters with no known history of harmful brown tides. The species was present in both open coastal and estuanne locations, in salinities between 18 and 32 practical salinity units (PSU). The observed population distributions apparently still reflect the massive 1985 outbreak when this species first bloomed, given the number of positive locations and high abundance of A.anophagefferens in the immediate vicinity of Long Island. However, the frequent occurrence of this species in waters far from this population ‘center’ is disturbing. Aureococcus anophagefferens is more widely distributed than was previously thought. Numerous areas thus have the potential for destructive brown tides such as those associated with the sudden appearance of the species in 1985.

• Probable origin and toxin profile of Alexandrium tamarense (Lebour) Balech from southern Brazil

  Authors: Graziela R. Persich, David M. Kulis, Emily L. Lilly, Donald M. Anderson, Virgínia M.T. Garcia

  Harmful Algae.

  The distribution of the toxic dinoflagellate Alexandrium tamarense Lebour has apparently expanded within the southern hemisphere during the last 2 decades. Toxic blooms of A. tamarense were recordedThe distribution of the toxic dinoflagellate Alexandrium tamarense Lebour has apparently expanded within the southern hemisphere during the last 2 decades. Toxic blooms of A. tamarense were recorded in Argentinean coastal waters since 1980; however, the first documented bloom in southern Brazil was in 1996. In this study, 13 strains of A. tamarense from southern Brazil were isolated and kept in culture. Phylogenetic analysis using RFLP and DNA sequences of the D1–D2 region of large subunit ribosomal DNA (rDNA) clearly indicates that Brazilian strains are most closely related to other South American strains. The strains from South America are placed firmly within a phylogenetic clade which contains strains from North America, northern Europe and northern Asia, previously called the North American clade. Possible dispersal hypotheses are discussed. The cultures were also analyzed for saxitoxin and its derivatives by high performance liquid chromatography (HPLC). The main saxitoxin groups found were the low toxicity N-sulfocarbamoyl group, C1, 2 (30–84%), followed by the high potency carbamate toxins, gonyautoxins 1, 4 (6.6–55%), gonyautoxins 2, 3 (0.3–29%), neosaxitoxin (1.4–24%) and saxitoxin (0–4.4%). The toxin composition is similar to that of other strains from South America, supporting a close relationship between A. tamarense from southern Brazil and other areas of South America. Toxicity values were variable (7.07–65.92 pg STX cell−1), with the higher range falling among the most toxic values recorded for cultures of A. tamarense, indicating the significant risk for shellfish contamination and human intoxication during blooms of this species along the southern Brazilian coast.