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Molecular detection of the toxic marine diatom _pseudo-nitzschia multiseries_
h [electronic resource] /
by Jennifer Delaney.
[Tampa, Fla] :
b University of South Florida,
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Thesis (MS)--University of South Florida, 2010.
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ABSTRACT: The marine diatom genus Pseudo-nitzschia includes species that produce domoic acid, a neurotoxin responsible for illness and mortality in both humans and marine wildlife. Because of the expertise and time required for the microscopic discrimination of species, molecular methods that monitor environmental concentrations of Pseudo-nitzschia provide a rapid alternative for the early detection of blooms and prediction of toxin accumulation. We have developed a nucleic acid sequence-based amplification with internal control RNA (IC-NASBA) assay and a quantitative reverse transcription PCR (qRT-PCR) assay for the detection of the toxic species P. multiseries targeting the ribulose-1,5-biphosphate carboxylase/oxygenase small subunit (rbcS) gene. Both methods use RNA amplification and fluorescence-based real-time detection. Due to a limited rbcS sequence database, primers were designed and used to sequence this gene from 14 strains of Pseudo-nitzschia (including four P. multiseries) and 19 other marine diatoms. The IC-NASBA and qRT-PCR assays had a limit of detection of one cultured cell of P. multiseries and were linear over four and five orders of magnitude, respectively (r-squared ≥ 0.98). Neither of the assays detected closely related organisms outside the Pseudo-nitzschia genus, and the qRT-PCR assay was specific to P. multiseries. While cross-reactivity of primers with unknown species prevented reliable detection of P. multiseries in spiked environmental samples using IC-NASBA, the qRT-PCR assay had positive detection from 10,000,000 cells/L to 1,000 cells/L. Nearly a 1:1 relationship was observed between predicted and calculated cell concentrations using qRT-PCR. Based on a diel expression study, the rbcS transcript copy number per cell ranged from 21,600 to 53,500, with the highest expression during early to mid photoperiod. The rbcS qRT-PCR assay is useful for the detection and enumeration of low concentrations of P. multiseries in the environment.
Advisor: John Paul, Ph.D.
Harmful algae detection
x Marine Science
t USF Electronic Theses and Dissertations.
M olecular Detection of the Toxic Marine Diatom Pseudo nitzschia multiseries by Jennifer A. Delaney A thesis submitted in partial fulfillment of the requirements for the degree of Master of Science College of Marine Science University of South Florida Major Professor: John H. Paul, Ph.D. Mya Breitbart, Ph.D. Michael Parsons, Ph.D. Date of Approval: October 15, 2010 Keywords: Pseudo nitzschia NASBA, qRT PCR, harmful algae detection, rbcS Copyright 2010, Jennifer A. Delaney
ACKNOWLEDGEMENTS First, I would like to thank my major advisor, Dr. John Paul, for his support and guidance throughout this project. I would also like to acknowledge my committee members Dr. Mya Breitbart and Dr. Michael Parsons for their time and encouragement. I am very grateful to Dr. Stephen Bates and Claude LÂŽger of Fisheries and Oceans Canada, Dr. Sibel Bargu of Louisiana State University, and Dr. Bill Richardson and Julie Brame of the Florida Fish a nd Wildlife Research Institute for providing me with phytoplankton cultures Thank you to Sheila O'Dea and all members/former members of the Paul lab for their scientific advice and their friendship: Dave John, Lauren McDaniel, Jen Mobberley, Bob Ul rich, Beth Young, and Brian Zielinski. Last I want to thank all of my frien ds and family for their support. A s pecial thanks goes out to my parents and Anthony You have always believed in me, and t his thesis could not have been completed without you Th is work was funded by the Office of Naval Research (ONR) under grant no. N00014 07 1 0794 a STAR Fellowship Agreement no. FP91699801 awarded by the U.S. Environme ntal Protection Agency (EPA), and a Gulf Oceanographic Trust Fellowship award ed through the USF College of Marine Science.
! i TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ .. iii LIST OF FIGURES ................................ ................................ ............................... iv ABSTRACT ................................ ................................ ................................ ........ v i INTRODUCTION ................................ ................................ ................................ ... 1 Pseudo nitzschia : A cosmopolitan diatom and emerging HAB genus ................................ ................................ ................................ ..... 1 Global distribution ................................ ................................ ............ 2 Distribution in the United States and the Gulf of Mexico ................. 2 Domoic acid (DA) : A potent neu rotoxin to humans and wildlife .................. 3 Effects on humans and the marine food we b ................................ .. 5 Variability in toxin production and possible role(s) of DA ................. 7 Culture stud ies: Effect of growth phase and macro nutrients ................................ ................................ 7 Field studies: Effect of macronutrients and eutrophication ................................ ................................ .. 9 T race metals and DA production ................................ ........ 10 Bacteria and DA produ ction ................................ ................ 11 Possible role(s) of DA ................................ ......................... 11 Major toxic events in North A merica and around the world ...................... 12 Canada ................................ ................................ .......................... 12 The United States ................................ ................................ .......... 12 Mexico ................................ ................................ ............................ 15 Other global locations ................................ ................................ .... 16 Traditional monitoring strategies: T oxin detection and microscopy .......... 17 Molecular m onitoring strategies for Pseudo nitzschia .............................. 20 Commonly used genetic ta rget and methods ................................ 20 RuBisCO: A potenti al molecular detection target .......................... 24 Nucleic acid amplification based detect ion methods: qRT PCR and NASBA ................................ ................................ ...... 26 Research objectives ................................ ................................ ................. 29 MATERIALS AND METHODS ................................ ................................ ............. 30 Phytoplan kton cultures and cell counts ................................ .................... 30
! ii DNA extraction, rbcS primer design, and PCR ................................ ......... 30 Cloning, sequencin g, and bioinformatic analysis ................................ ...... 34 Syn thesis of in vitro transcript ................................ ................................ ... 36 IC NASBA assay d esign and reaction conditions ................................ ..... 36 TaqManÂ¨ qR T PCR assay d esign and reaction conditions ..................... 37 RNA extraction ................................ ................................ .......................... 39 Standard protocol ................................ ................................ .......... 39 Efficiency experiment ................................ ................................ ..... 39 Specificity testing ................................ ................................ ...................... 40 Spiked environmental sam ples ................................ ................................ 40 Determinatio n of IC NASBA assay inhibitor(s) ................................ ......... 42 Size fractionation experiment ................................ ........................ 42 K. brevis spike experiment ................................ ............................. 42 Sequence spe cific capture with DynabeadsÂ¨ ................................ .......... 42 Coupling of probe to beads ................................ ............................ 43 Hybridization of probe conjugated beads to target mRNA ............ 45 Expression patterns of the rbcS gene in P. multiseries ............................ 45 RESULTS ................................ ................................ ................................ ....... 47 Seque nce and phylogenetic analysis ................................ ....................... 47 Sensitivity and specificit y of IC NASBA and qRT PCR assays ................ 48 RNA ext raction efficiency experiment ................................ ....................... 53 Spiked environmental samples ................................ ................................ 54 Determination of the IC NASBA assay inhibitor ................................ ....... 56 Sequence spe cific capture with DynabeadsÂ¨ ................................ .......... 58 Probe coupling ................................ ................................ ............... 58 Target hybridization in cult ure and environmental samples ........... 59 Expression patterns of the rbcS gene ................................ ...................... 62 DISCUSSION ................................ ................................ ................................ ...... 63 REFERENCES ................................ ................................ ................................ .... 70 APPENDICES ................................ ................................ ................................ ..... 93 Appendix 1: IC NASBA assays for P. delicatissima and LA2 StC6C ....... 94 Appendix 2: P. m ultiseries CCMP2708 growth curve and toxin data ....... 97 Appendix 3: rbcS phylogeny of marine diat oms and environmental clones ................................ ................................ ................................ .. 99
! iii LIST OF TABLES Table 1 : Pseudo nitzschia species present in the Gulf of Mexico ......................... 4 Table 2: Phytoplankton culture growth conditions and GenBank accession numbers for the rbcS gene ................................ ................................ .... 31 Table 3: Sequences for primers, molecular beacons, TaqManÂ¨ probe and IC RNA oligonucleotides ................................ ................................ ....... 35 Table 4: Variability of rbcS nucleotide and amino acid sequences in Pseudo nitzschia and other marine diatoms ................................ ......... 47 Table 5: Specificit y of IC NASBA and qRT PCR assays ................................ .... 52 Table A1: P. delicatissima and LA2 StC6C beacon sequences .......................... 9 4 Table A2: Specificity of P. delicatissima and LA2 StC6C IC NASBA assays ................................ ................................ ................................ 95
! iv LIST OF FIGURES Figure 1: Chemical structure of domoic acid ................................ ......................... 5 Figure 2: Chains of P. multiseries CCMP2708 ................................ .................... 19 Figure 3 : Nucleic Acid Sequence Based Amplification (NASBA) wi th molecular beacon detection ................................ ................................ 27 Figure 4 : TaqManÂ¨ qR T PCR assay process ................................ ..................... 28 Figure 5 : General detection process incorporating b ead based target mRNA capture ................................ ................................ ...................... 43 Figure 6 : Activation and coupling of oligonucleotides to Dyna beadsÂ¨ MyOne Carboxylic Acid ................................ ................................ .... 44 Figure 7 : Evolutionary relationship of marine diatoms based on the rbcS gene ................................ ................................ ................................ ..... 49 Figure 8 : SYBR Â¨ Green dissociation curve ................................ ........................ 50 Figure 9 : Representative IC NASBA plots for P. multiseries ............................... 50 Figure 10 : Representative qRT PCR plot for P. multiseries ................................ 51 Figure 11 : Ty pical cell standard curves ................................ ............................... 51 Figure 12 : Comparison of three RNA extraction methods as determined by rbcS copy number ................................ ................................ .............. 54 Figure 13 : Inhibition of IC NASBA assay in spiked environmental samples ....... 55 Figure 14 : Calculated versus expected cells per liter for qRT PCR sp iked environmental experiment ................................ ................................ .. 56 Figure 15 : Results from size fractionation experiment ................................ ........ 57
! v Figure 16 : Comparison of P. multiseries and K. brevis IC NASBA assay performance ................................ ................................ ....................... 58 Figure 17 : Relative percentages of probe detected in various steps of DynabeadÂ¨ coupling procedure ................................ ......................... 59 Figure 18 : Comparison of IC NASBA plots for an environmental sample run without and with the Dynabe adÂ¨ target hybridization step .......... 60 Figure 19 : Detection of 10 6 cells/L using DynabeadsÂ¨ and IC N ASBA .............. 61 Figure 20 : Diel e xpression pattern of rbcS gene in exponential phase P. multiseries CCMP2708 culture ................................ ........................... 62 Figure A1: Transcript standard curves for P. delicatissima and LA2 StC6C IC NASBA assays ................................ ................................ ............. 9 4 Figure A2: Growth curve for P. multiseries CCMP2708 based o n cell counts an d fluorescence ................................ ................................ .... 9 7 Figure A3: Fluorescence growth curve and domoic acid measurement in stationary phase P. multiseries CCMP2708 cells .............................. 9 8 Figure A4: Evolutionary relationship of marine diatoms and environmental rbcS clones ................................ ................................ ........................ 9 9
! vi ABSTRACT The marine diatom genus Pseudo nitzschia includes species that produce domoic acid, a neurotoxin responsible for illness and mortality in both humans and marine wildlife. Because of the expertise and time required for the microscopic discriminati on of species, molecular methods that monitor environmental concentrations of Pseudo nitzschia provide a rapid alternative for the early detection of blooms and prediction of toxin accumulation. We have developed a nucleic acid sequence based amplification with internal control RNA ( IC NASBA) assay and a quantitative reverse transcription PCR (qRT PCR) assay for the detection of the toxic species P. multiseries targeting the ribulose 1,5 biphosphate carboxylase/oxygenase small subunit ( rbcS ) gene. Both meth ods use RNA amplification and fluorescence based real time detection. Due to a limited rbcS sequence database, primers were designed and used to sequence this gene from 14 strains of Pseudo nitzschia (including four P. multiseries ) and 19 other marine diat oms. The IC NASBA and qRT PCR assays had a limit of detection of one cultured cell of P. multiseries and were linear over four and five orders of magnitude, respectively (r 2 0.98). Neither of the assays detected closely related organisms outside the Pseu do nitzschia genus, and the qRT PCR assay was specific to P. multiseries While cross reactivity of primers
! vii with unknown species prevented reliable detection of P. multiseries in spiked environmental samples using IC NASBA, the qRT PCR assay had positive d etection from 10 7 cells/L to 10 3 cells/L. Nearly a 1:1 relationship was observed between predicted and calculated cell concentrations using qRT PCR. Based on a diel expression study, the rbcS transcript copy number per cell ranged from 2.16 x 10 4 to 5.35 x 10 4 with the highest expression during early to mid photoperiod. The rbcS qRT PCR assay is useful for the detection and enumeration of low concentrations of P. multiseries in the environment.
! 1 INTRODUCTION Pseudo nitzschia : A cosmopolitan diatom and emerging HAB genus Diatoms are a globally important group of phytoplankton, comprising approximately 40% of all primary production in the oceans (Mann, 1999; Sarthou et al., 2005) Because their frustules are composed of silica, they participate in the cycling of this critical nutrient, along with carbon, phosphorus, nitrogen, and iron (Sarthou et al., 2005) Diatoms require high nutrient concentrations for gr owth due to their relatively low surface to volume ratios, and they tend to dominate high nutrient regions in the ocean (Sarthou et al., 2005) They also respond rapidly to iron fertili zation experiments, becoming dominant members of the phytoplankton ass emblage (Coale et al., 1996; Marchetti et al., 2006) The sinking of diatom cells results in a n et downward flux of carbon, which has implications for carbon sequestration on a global scale (Bowler et al., 2010) Additionally, some diatoms of th e genus Pseudo nitzschia (Hasle, 1994) produce the neurotoxin domoic acid (DA). Members of this pennate diatom genus form colonies characterized by chains of overlapping cells (Skov et al., 1999) There are at least twelve toxic species of Pseudo nitzschia : P. australis, P. calliantha, P. cuspidata, P. delicatissima, P. fraudulenta, P. galaxiae, P.
! 2 multiseries, P. multistriata, P. pseudodelicatissima, P. pungens, P. seriata, and P. turgidula (Bates and Trainer, 2006; Lefebvre and Robertson, 2010) Global distribution. Pseudo nitzschia species have been observed in the waters of all seven continents, spanning tropical, temperate, and polar environments (Hasle, 2002, and references therein) In fact, one of the most studied species, P. multiseries has been found under the ice in Prince Edward Island, Canada at temperatures of 1.5 Â¡C (Bates et al. 1989) and during the summer in Galveston Bay, TX at temperatures up to 30 Â¡C (Dickey et al., 1992; Reap, 1991) DA production by this genus also occurs globally and has been most documented in waters of the United States, Canada, Ireland, Scotland, France, Italy, Japan, Denmark, Portugal, Chile, Australia, and New Zealand (Couture et al., 200 1; Pan et al., 2001; Trainer et al., 2001; Villac et al., 1993) The list of countries reporting toxin production by Pseudo nitzschia is continually growing, but the species responsible vary with location. Distribution in the United States and the Gulf of Mexico. The most severely toxic Pseudo nitzschia blooms in the United States occur along the west coast (Trainer et al., 2001) Ne vertheless, Pseudo nitzschia species (and DA production ) are prevalent in other areas of the country, including the northeast, southeast, and the Gulf of Mexico (Fire et al., 2009; Liefer et al., 2009; Thessen and Stoec ker, 2008; Verity, 2010; Villareal et al., 1994) In the Gulf of Mexico (GOM) Pseudo nitzschia can reach up to >10 7 cells/L (Dort ch et al., 1997) and comprise a large portion of the diatom
! 3 community (John et al., 2007) Their prevalence in the Gulf has increased during recent years, poss ibly due to an increase in eutro phication (Bates et al., 1998; Parsons et al., 2002) P. pseudodelicatissima found in Louisian a coastal waters has shown a high level of D A production (Pan et al., 2001; Parsons et al., 1999) and DA was found in P. multiseries isolated from the Texas coast (Dickey et al., 1992; Reap, 1 991) Pseudo nitzschia species have also been observed in Tampa Bay, Florida as a numerically dominant phytoplankton group (Badylak et al., 2007; Lundholm et al., 2003) Sixteen species of Pseudo nitzschia have been documented in the GOM nine of which fall on the list of potential domoic acid producers (Table 1). Domoic acid (DA) : A potent neurotoxin to humans and wildlife Domoic acid (DA) is a water soluble amino acid that affects neurotransmission (Figure 1). Based on isoto pic labeling studies, DA is derived from acetate via two separate precursor intermediates: 3 hydroxyglutamate, derived from the Krebs cycle, and an isoprenoid structure derived from gerany l pyrophosphate (Douglas et al., 1992; Ramsey et al., 1998; Smith et al., 2001) DA is structurally similar to kainic acid, which binds to a subgroup of the glutamate receptor family in the brain (reviewed by Jeffery et a l., 2004) Consequently, it has a high affinity for these receptors (known as the kainate receptors), which are heavily concentrated in the hippocampus. This interaction stimulates neuronal firing, leading to cell death and massive tissue degeneration (Jeffery et al., 2004)
! 4 Table 1. Pseudo nitzschia species present in the Gulf of Mexico. Species Sampling Location References P. americana Apalache Bay, FL (Del Rio et al., 2010) (Lundholm et al., 2002b) P. brasiliana Northeast GOM ; LA TX coast (Lundholm et al., 2002b) (Thessen et al., 2005) P. caciantha Near Tuxpam, MX (Lundholm et al., 2003) P. calliantha Tampa Bay and Apalache Bay, FL (Del Rio et al., 2010) (Lundholm et al., 2003) P. cuspidata Near Tuxpam, MX (Lundholm et al., 2003) P. decipiens Near Tuxpam, MX (Lundholm et al., 2006) P. delicatis sima LA TX coast; Near Tuxpam, MX (Lundholm et al., 2006) (Parsons et al., 1999) (Thessen et al., 2005) P. galaxiae Near Tuxpam, MX (Lundholm and Moestrup, 2002) P. linea Tampa Bay and Apalache Bay, FL (Lundholm et al., 2002b) P. multiseries Galveston Bay, TX; LA TX coast (Dickey et al., 1992) 2 (Fryxell et al., 1990) (Hasle, 1972) (Parsons et al., 1999) (Reap, 1991) (Thessen et al., 2005) P. m ultistriata LA TX coast (Thessen et al., 2005) P. pseudodelicatissima LA TX coast; North and east coasts of Yucatan Peninsula (Del Rio et al., 2010) (Ghinaglia et al., 2004) 1 (Pan et al., 2001) 1,2 (Parsons et al., 1999) 1,2 (Thessen et al., 2005) 1 P. pungens LA TX coast; Near Tuxpam, MX (Del Rio et al., 2010) (Fryxell et al., 1990) (Hasle, 1972) (Lundholm et al., 2006) (Parsons et al., 1999) (Thessen et al., 2005) P. seriata North, west, and east coasts of Yucatan Peninsula (Ghinaglia et al., 2004) P. subcurvata National Park Sistema Arreci fal Veracruzano (southern GOM ) (AkÂŽ Castillo and Okolodkov, 2009) P. subfraudulenta LA TX coast (Hasle, 1972) (Thessen et al., 2005) 1 Study found a member of the P. pseudodelicatissima/P. cuspidata complex, but the exact species is undetermined. 2 Study detected domoic acid production by this species.
! 5 Effects on humans and the marine food web When ingested by humans in hig h concentrations, DA can cause amnesic shellfish p oisoning, or ASP (Todd, 1993) The name of this condition stems from one of the most striking symptoms associated with DA poisoning, whi ch is permanent short term memory loss. Shortly after consumption of contaminated shellfish, both gastrointestinal (nausea, vomiting, diarrhea) and neurological (disorientation, memory loss) symptoms appear (CÂˆmpas et al., 2007) In severe cases, the individual may experience seizures, autonomic dysfun ction, coma, or even death. Figure 1. Chemical structure of domoic acid. Domoic acid bioaccumulates in organisms that feed on Pseudo nitzschia namely benthic and filter feeding invertebrates, and this toxin is then transferred to higher predators (Bejarano et al., 2008) DA is found primarily in the digestive gland of shellfish and does not appear to negatively impact these organisms, due
! 6 to their la ck of a complex central nervous system. Common invertebrate vectors include mussels ( Mytilus spp.; Bates et al., 1989; Horner et al., 1997) razor clams ( Siliqua patula ; Wekell et a l., 1994) various scallop species (Campbell et al., 2001; Couture et al., 2001) Dungeness crabs ( Cancer magister ; Horner and Postel, 1993 ) sand crabs ( Emerita analoga ; Ferdin et al., 2002; Powell et al., 2002) swimming crabs ( Polybius henslowii ; Costa et al., 2003) the common cuttlefish ( Sepia officinalis ; Costa et al., 2005) and Euphausiids (Bargu et al., 2002) In addition to invertebrates, planktivorous fish such as the northern anchovy ( Engraulis moordax ; Fritz et al., 1992; Lefebvre et al., 1999; Lefebvre et al., 2002b) and the Pacific sardine ( Sardinops sagax ; Lefebvre et al., 2002b; Tr ainer et al., 2001) as well as carnivorous fish such as mackerel ( Scomber japonicus ; Sierra BeltrÂ‡n et al., 1997) have been implicated in transfer of DA up the food web. Although few studies evaluating sub acute toxicity to fish exist, it appears that oral administration of DA to fish does not cause toxicological symptoms, indicating low gastrointestinal absorption of the toxin (Lefebvre et al., 2001) Nevertheless, DA has been detected in a wide variety of marine organisms, indi cating widespread cycling of this toxin in the marine environment (Lefebv re et al., 20 02a) Domoic acid poisoning of wildlife is apparent at the higher trophic levels, such as birds and marine mammals, where it results in 7 many of t he same neurological symptoms that humans e xperience including disorientation and seizures (Fritz et al., 1992; Goldstein et al., 2008; Gulland, 2000)
! 7 V ariability in toxin production and possible role(s) of DA. There has been significant variability observ ed in DA production, not only between different Pseudo nitzschia species, b ut also between different isolates of the same species (Bates et al., 1998) It is not completely understood why such variability in production of DA exists, nor does the toxin have any proven natural function in the physiology or life history of Pseudo nitzschia (Mos, 2001) However, many stud ies have established correlations between different environmental parameters/culture conditions and DA production, and there are also hypotheses as to the possible function of this toxin. Culture studies: Effect of growth phase and macro nutrients Most studies have shown that Pseudo nitzschia species produce the highest concentrations of DA in stationary phase (Bates, 1998; Bates et al., 1995b) A few exceptions, including P. pseudodelicatissima / cuspidata from the northern Gulf of Mexico (Pan et al., 2001) and isolates of P. australis and P. multiseries (Garrison et al., 1992; Pan et al., 1996b) have produced high DA concentrations in late exponential phase. An explanation for these results could be that some cells have stopped dividing and begin to produce DA, while others are still growing (Bates, 1998; Pan et al., 1 996b) Based on laboratory studies, nutrient conditions conducive to DA production include limitation by silicate or phosphate, and an excess of nitrogen (Bates et al., 1998) In P. multiseries cellular DA inversely correlates with both silicate and phosphate, in both batch and continuous cultures (Bates et al., 1991; Bates et al., 1996; Kudela et al., 2003; Pan et al., 1996a; Pan et al., 1996b; Pan
! 8 et al., 1996c) The same is true for P. seriata ( Fehling et al. 2004) This pattern could be the result of a competition for free energy between primary and secondary metabolism, in which free energy is diverted to toxin production when growth slows as a result of nutrient limitation (Pan et al., 1996a; Pan et al., 1996c) The obs ervation that DA accumulation occurs predominantly in stationary phase, when growth slows and nutrients become limiting, also supports this idea. DA is an amino acid, so nitrogen is required for its synthesis (Bates et al., 1991) Several studies have shown the ability of Pseudo nitzschia to grow (and produce DA) using a variety of nitrogen sources, including nitrate, urea, and ammonium (Bates et al., 1993b; Howard et al., 2007; Thessen et al., 2009) Bates et al. (1993b) found that high concentrations of ammonium, when compared to nitrate, resulted in a two to fourfold increase in DA produced in stationary phase. Another study using P. australis found an increase in particulate and dissolved DA production when cells were grown with urea as the sole nitrogen source, compared with both nitrate and ammonium (Howard et al., 2007) Recently, Thessen et al. (2009) examined the effect of different nitrogen sources (nitrate, ammonium, and urea) on growth and DA production for multiple strains of P. multiseries, P. fraudulenta, and P. calliantha The authors found that toxin productio n is possible when cells are provided with any of the three sources and that this production can vary with source. However, most of the variation observed was between strains, not nitrogen sources, indicating that other mechanisms are also involved in esta blishing conditions conducive to DA production.
! 9 Field studies: Effect of macronutrients and eut r ophication Field studies examining macronutrient concentrations and DA production have produced conflicting results, likely due to the complexity of the natural environment. Those conducted in southern California are consistent with laboratory studies, showing increa sed toxin production under phosphate and silicate limitation (Anderson et al., 2006; Schnetzer et al., 2007) Studies conducted in the Pacific Northwest have found no correlation between macronutrient concentrations and DA production (Marchetti et al., 2004; Trainer et al., 2009) Trainer et al. (200 9) concluded that there are no simple predictive relationships between concentration s of DA and macronutrients even in studies that establish correlations, it is not clear whether the nutrient concentrations play a direct role in toxin production or ar e simply a result of their drawdown during bloom formation. There has been speculation about the effect of eutrophication on development of toxic Pseudo nitzschia blooms. For example, in the northern Gulf of Mexico, the increase in nitrogen inputs relativ e to silicate have decreased the Si:N ratio by a factor of four over the last century (Turner and Rabalais, 1991) Simultaneously, an increase in Pseudo nitzschia has been observed since the 1950s (Dortch 1997). Previous studies have suggested the ability of Pseudo nitzschia to outcompete other phytoplankton at lo w Si:N ratios, thus suggesting that eutrophication may favor the formation of blooms (Sommer, 1994) I n an attempt to confirm the apparent correlation between nutrient input and Pseudo nitzschia abundance, Parsons et al. (2002) examined sediment cores from the
! 10 northern Gulf of Mexico. The authors found higher abundances of Pseudo nitzschia when nitrate concentrations were elevated and Si:N was low, thus providing evidence for the link between eutrophication and Pseudo nitzschia blooms. However, some studies have found toxic Pseudo nitschia blooms during silicate replete conditions, indicati ng that this ratio is not necessarily predictive of bloom formation (Traine r et al., 200 0) Although nutrient loading from rivers is hypothesized to be the cause of a number of toxic Pseudo nitzschia blooms, it is not responsible for every event (Trainer et al., 2000) The form of nitrogen present may also play a role in bloom toxicity. Coastal environments are especially rich in ammonium and organic forms of nitrogen (Bates et al., 1998) Based on laboratory studies utilizing different nitrogen sources, organic forms of nitrogen like urea may enhance DA production (see above) ; however more studies are needed to confirm this (Bates, 1998; Howard et al., 2007; Thessen et al., 2009) Trace metals and DA production Pseudo nitzschia cells also increase DA production under low iron and copper conditions (Maldonado et al., 2002; Rue and Bruland, 2001; Wells et al., 2005) Maldonado et al. (2002) found that iron uptake rates in P. multiseries were enhanced threefold by adding DA to the culture medium, and that DA helped to decrease the toxicity of copper. Pseudo nitzschia spp. have an inducible high affinity iron uptake capability, requiring copper and domoic acid, that enables them to outcompete other diatoms in low iron conditions (Wells et al., 200 5) In the Juan de Fuca eddy region, Trainer et al. (2009) found the highest cellular DA
! 11 concentrations and Pseudo nitz schia abundances in areas of iron limitation. Bacteria and DA production Several studies have examined the connection between bacteria associated with Pseudo nitzschia and the production of DA. Axenic cultures tend to accumulate less DA than non axenic c ultures (Douglas and Bates, 1992; Douglas et al., 1993; Kobayashi et al., 2009) and reintroducing the bacteria restores toxin production (Bat es et al., 19 95a) However, the bacteria themselves do not seem to be capable of DA production (Bat es et al., 200 4) It has been sugg ested that Pseudo nitzschia use a bacterially derived precursor to synthesize DA (Bates et al., 2004) Osada and Stewart (1997) fou nd that adding gluconic acid/glucolactone, a compound produced by the symbiont bacterium Altermonas sp., to axenic cultures increases DA production. The authors suggest that DA and gluconic acid/glucolactone are both nutrient scavengers, so Pseudo nitzschi a produces DA to counter the effects of the bacterially produced compound. Possible role(s) of DA The most widely accepted function of DA is to chelate extracellular iron and copper, copper being a necessary component of the high affinity iron transport ers present in Pseudo nitzschia (Bejarano et al., 2008; Wells et al., 2005) The presence of three carboxyl groups in the DA structure means it c ould chelate these trace metals (Bates et al 2001) Th is proposed function fits with observations of enhanced DA production in the presence of bacteria, due to a competition for micron utrients. DA may also simply serve as a way of dispensing
! 12 excess photosynthetic energy when cells can no longer grow optimally, since most of the toxin is excreted (Bates, 1998; Bates et al., 1991) Due to the low nitrogen content (4.5% molecular weight) of DA, it is unlikely to be used as an N storage compound (Bates et al., 1991) and the toxin lacks any apparent allelopathic effects against other phytoplankton (Lundholm et al 2005b) Major toxic events in North America and aroun d the world Canada. The first reported DA poisoning event occurred in 1987 on Prince Edward Island (PEI), Canada, during which 107 people became ill and three died from eating blue mussels contaminated with high levels of toxin (Bates et al., 1989; Perl et al., 1990; Todd, 1993; Wright et al., 1989) Pseudo nitzschia multiseries was subsequently implicated as the toxin producer, and shellfish were not cleared for harvesting until the following April (Bates et al., 1989) Shortly thereafter, in the summer/fall of 1988, another toxic bloom resulted in shellfish harvesting closures in Cardigan Bay PEI and the Bay of Fundy (Bates et al., 1998; Gilgan et al., 1990; Martin et al., 1990) Since then, high DA concentrations and harvesting closures have been common along the east and west coasts of Canada (Bates et al., 1998; Couture et al., 2001) The United States. In the fall of 1991, a major DA poisoning event affected the entire U.S. west coast, specifically California, Oregon, and Washington. In Santa Cruz, California, 95 Brandt's cormorants and 43 brown pelicans died as a result of consuming anchovies with high levels of DA (Fritz et al., 1992; Work et al., 1993)
! 13 Sick birds displayed central nervous system symptoms indicative of DA poisoning, and large numbers of P. australis frustules were found in the stomachs of birds and anchovies. Additionally, high levels of DA were found in Monterey Bay mussels (Wekell et al., 1994) In Oregon and Washington, razor clams and Dungeness crabs both contained high levels of DA, resulting in harvesting closures of almost one year in both of these states (Horner and Postel, 1993; Trainer, 2002; Villac et al., 1993) A study conducted later by the Washin gton Department of Health found that 21 people reported gastrointestinal symptoms after eating razor clams during the fall of 1991, and 13 of these exhibited mild neurological symptoms (Horner and Postel, 1993) The second significant DA poisoning event in the U.S. occurred in 1998 along the west coast. In May June in central California, there were over 400 sea lion mortalities due to the consumption of anchovies and sardines containing high levels of DA (Scholin et al. 2000) In addition, 70 sea lions and one northern fur seal stranded with neurological symptoms and were admitted to The Marine Mamma l Center; 48 of these sea lions later died (Gu lland, 2000) DA was detected in the serum, urine, and feces of several sea lions tested (Gulland, 2000; Lefebvre et al., 1999; Scholin et al., 2000) P. multiseries and P. australis were both discovered to be dominant Pseudo nitzschia species at the time of the strandings, and both were shown to produce DA (Scholin et al., 2000; Trainer et al., 2000) This event was the first to demonstrate trophic transfer of DA to marine mammals. In Oregon and Washington, DA rose to high levels in razor
! 14 clams by fall of 1998, resulting in harvesting closures of most beaches for over one year (Trainer, 2002; Trainer et al., 2001; Trainer and Suddleson, 2005) In California, marine mammals (namely sea lions) exhibiting symptoms of DA poisoning have stranded every year since 1998. Between 1998 2006, for example, there were a total of 715 strandings with neurological symptoms brought to The Marine Mammal Center in Sausalito, CA, and 298 of these either died or were euthanized (Goldstein et al., 2008) Some of the marine mammal mortality events attributed to DA in California have also included sea otters, dolphins, and northern fur seals (Gulland, 2006; Kreuder et al., 2003; Schnetzer et al., 2007) DA was detected in stranded Risso's dolphins, Cuvier's beaked whale, gray whales, and humpback whales in 2002, but the susceptibility of these mammals to DA is unknown (Torres de la Riva et al., 2009) Between 1998 and 2006, high concentrations of DA have often been found in shellfish on the U.S. west coast, resulting in commercial and recreati onal closures in Washington and Oregon (Holtermann et al., 2010; Trainer, 2002; Trainer and Suddleson, 2005; Tweddle et al., 2010) Such closures have resulted in significant economic losses for these states. For exa mple, an estimated $4.8 million was lost during the 2003 closures of razor clam and mussel beds in Oregon (Tweddle et al., 2010) Most of these closures have occurred in coastal waters. However in September 2003, shellfish harvesting was closed for the first time in Puget Sound due to high concentrations of Pseudo nitzschia ( Trainer et al., 2007) This occurred again in 2005 due to elevated concentrations of DA in oysters and clams in Sequim Bay (Trainer et al., 2007)
! 15 Elsewhere in the U.S., DA has been detected in shellfish and marine mammals but does not seem to accumulate in high concentrations and has not yet been definitively linked to DA poisoning in humans or animals. Interes tingly, DA was reported in shellfish in Nantucket as early as 1991, although at levels below the threshold for suspending harvesting (Nassif and Timperi, 1991) DA has been detected in stranded humpback whales in Maine (Gulland, 2006) as well as in stranded pygmy and dwarf sperm wh ales along the southeastern Atlantic coast from Virginia to Florida (Fire et al., 2009) In the Gulf of Mexico in 2004, a bottlenose dolphin mortality event occurred along the Florida panhandle; brevetoxin was determined to be the cause of death, but DA was also present in low amounts in some of these animals (NMFS, 2004) Mexico. In January 1996 in Cabo San Lucas, Baja California, over 150 pelicans died, and many more exhibited neurological symptoms that persisted for up to two months (Sierra BeltrÂ‡n et al., 1997) These birds ate mackerel containing high levels of DA, and about 50% of the colony was lost. The next year, another mortality event involving 766 common loons and 182 marine mam mals occurred in the Gulf of California (Sierra BeltrÂ‡n et al., 1998) P. australis frustules and DA were found in the stomachs of common dolphins and sardines. In January 2004, 112 dolphins, 195 sea lions 9 gray pelicans, and 20 tons of sardines died or were sickened in San Jorge Bay, Caborca, Sonora, presumably due to DA poisoning (Sierra BeltrÂ‡n et al., 2005) DA was detected in the blood of stranded dol phins for three out of four samples tested. Later in the year, pelicans were
! 16 also affected in Mazatlan Bay, Sinaloa. Although toxin analyses were not conducted on these animals, P. pseudodelicatissima was the dominant phytoplankton species, and the phytopl ankton toxin profile was similar to that of the dolphin blood in Carborca (Sierra BeltrÂ‡n et al., 2005) Other global locations. DA contamination of shellfish and planktivorous fish is also common in other areas of the world. A major DA event occurred in Scotland in 1999, when high concentrations were found in king scallops, queen scallops, and mussels (Gal lacher et al., 2001) Most scallop fisheries were closed from June 1999 to May 2000 and about Â£15 million were lost from this industry. DA has been detected frequently in Scottish shellfish since then (Campbell and Kelly, 2001; Campbell et al., 2003) In French waters, high DA concentrations in shellfish in the Bay of Seine and Western Brittany have resulted in harvesting closures (Amzil et al., 2001; Nezan et al ., 2006) During summer fall 1992, a bloom of Pseudo nitzschia and high DA concentrations in mussels forced the closure of harvesting along the east coast of the Jutland peninsula in Denmark (Lundholm and Skov, 1993) More recently, in 2005, mussel closures occurred in the same region (Lundholm et al., 2005a) High concentrations of DA have also been detected in shellfish, cephalopods, and sardines from Portugal (Costa and Garrido, 2004; Costa et al., 2003) Mediterranean mussels in Spain (Miguez et al. 1996) blue mussels in Greece (Kaniou Grigoriadou et al., 2005) shellfish and tunicates from Chile (LÂ—pez Rivera et al., 2009) and various shellfi sh
! 17 species from New Zealand (Rhodes et al., 1998a; Rhodes et al., 2001; Rhodes, 1998; Rhodes et al., 2003) Traditional monitoring strategies: Toxin detection and microscopy Following the ASP events of 1987 on Pri nce Edward Island, the Canadian government imposed a 20 g domoic acid per gram of mussel flesh (20 ppm) limit for harvesting (Jeffery et al., 2004) This limit is still enforced today and has been adopted by other countries, including those belonging to the European Union, the United S tates, New Zealand, and Australia (Jeffery et al., 2004) A slightly higher limit of 30 ppm for Dungeness crab viscera was established after the discovery that proper cleaning of crabs greatly reduces the risk of DA poisoning (Trainer, 2002) The U.S. Food and Drug Administration, through the National Shellfish Safety Program (NSSP), requires domestic commercial shellfish harvesting operations to monitor for biotoxins, as well as countries that export to the U.S. (Anderson et al., 2001) Several state agencies in the U.S. regularly monitor shellfish for DA, including those in Oregon, Washington, California, Alaska (Trainer, 2002) Some of the other states focus on other biotoxins but may test for DA if Pseudo nitzschia cell concentrations re ach a trigger level which varies with species and location Other countries that experience frequent DA contamination of shellfish have their own toxin monitoring programs, such as Canada, New Zealand, Scotland, Ireland, France, Norway, Denmark, Portugal, and Chile (Couture et al., 2001) There are two AOAC (Association of Official Analytical Chemists) ce rtified methods for detecting DA. These include HPLC coupled with UV detection
! 18 (AOAC, 2005a) and an Enzyme Linked ImmunoSorbent Assay (ELISA; AOAC, 20 05b) Although screening shellfish for DA is an important component of monitoring programs, the toxin is detected after it has already entered the food web, reducing warning time for regulatory agencies. There have also been questions about the reliabil ity of indicator species used, due to different toxin depuration rates and geographical distributions of these organisms (Ferdin et al., 2002) Most states within the U.S., as well as other countries, routinely monitor phytoplankton assemblages in order to de tect blooms of harmful algae such as Pseudo nitzschia before toxin accumulates. This method can serve as a type of "early warning" system, enabling better preventative strategies. There is no general consensus on the threshold cell concentration that shoul d be used, because toxin production/accumulation varies with Pseudo nitzschia species and geographic location (Anderson et al., 2001) In Washington State, research by the ORHAB partnership resulted in the establishment o f the following trigger cell concentrations for that region : P. multiseries / P. pungens : 1 x 10 5 cells/L P. heimii / P. fraudulenta / P. australis : 3 x 10 4 cells/L and P. delicatissima / P. pseudodelicatissima : 1 x 10 6 cells/L (Trainer and Suddleson, 2005) These concentrations initiate parti culate DA measurements of seawater to complement the shellfish monitoring and assist coastal managers with closure decisions. Around 5 x 10 4 cells/L of P. australis result in unsafe concentrations of DA in mussels and fish in California, which is similar t o the ORHAB data for this species (Busse et al., 2006) Some countries use phytoplankton monitoring as
! 19 part of a two tiered system, in whi ch the phytoplankton cell counts (first tier) trigger shellfish toxin testing (second tier). For example, New Zealand established 5 x 10 4 cells/L (when Pseudo nitzschia is >50% total phytoplankton) and 1 x 10 5 cells/L (when Pseudo nitzschia is <50% total p hytoplankton) as the concentration(s) to initiate DA testing of shellfish (Anderson et al., 2001) An inherent difficulty associated with traditional monitoring programs is the requirement for microscopy based techniques to identify species of Pseudo nitzschia. Some species can be grouped into general morphotypes using light microscopy (LM; Parsons et al., 1999; Trainer and Suddleson, 2005) The genus itself is easily distinguishable due to the formation of stepped colonies, or chains of overlapping cells (Figure 2; Hasle, 1994) However it is common to have multispecies blooms comprised of both toxic and non toxic species, some of which cannot be distinguished by LM. Figure 2 Chains of P. multiseries CCMP2708. ( A = phase contrast, B = dif ferential interference contrast )
! 20 For example, P. multiseries and P. pungens are nearly identical morphologically and often occur together, but the former is considerably more toxic (Bates et al., 1998; Miller and Scholin, 1996; Trainer et al., 1998) Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) can be used to examine the frustule patterns of these diatoms, which are unique for each species (e.g. Hasle and Lundholm, 2005; Lundholm et al., 2003; Lundholm et al., 2006) Because these techniques require a significant amount of time and expertise, as well as expensive equipment, they are not practical for most rout ine monitoring programs Several alternative identification techniques have been explored, including antibodies (Bates et al., 1993a) and lectins (Rhodes, 1998) However, most of the focus has been on the development of nucleic acid methods (utilizing DNA and RNA) for identification and enumeration of species within this harmful algal genus (i. e.: Diercks et al., 2008a; Miller and Scholin, 1998; Scholin et al., 1997) Molecular monitoring strategies for Pseudo nitzschia Commonly used genetic targets and methods Because the gene(s) involved in domoic acid production have not yet been elucid ated, molecular detection is typically based on the use of species specific sequences to detect potential toxin producers in the environment. Most previous studies utilizing molecular methods to detect Pseudo nitzschia have involved the use of ribosomal DNA or RNA (rDNA/rRNA; Anderson et al., 2001; Penna and Galluzzi, 2008) This includes the small subunit (18S), variable regions of the large subunit (28S), 5.8S, or the non coding internal and external
! 21 transcribed spacer regions (ITS an d ETS, respectively). Because it is relatively conserved, the 18S is typically used for discrimination above the species level, while the 28S and ITS regions are used for discrimination between species, the ITS bein g most variable (Penna and Galluzzi, 2008) Probes targeting variable regions of the 28S rRNA have been developed for a variety of Pseudo nitzschia species and used in two formats: whole cell hybridization and sandwich hybridization (Miller and Scholin, 1996, 1998; Scholin et al., 1996; Scholin et al., 1999; Scholin et al., 1997) Whole cell hybridization, also known as "Fluorescent in situ Hybridization" (FISH), involves fixing and permeabilizing cells, incubating them with a specific fluorescent oligonucleotide probe, and then viewing them under an epifluorescence microscope for identification/quantification of the target cell type (DeLo ng et al., 1989 ) Sandwich hybridization assays (SHAs) require homogenization of cells to liberate their nucleic acids, followed by two subsequent hybridizations: the first c aptures the target sequence to an anchored oligonucleotide, and the second uses a signal probe that binds near the capture site (Ness and Chen, 1991; Ness et al., 1991) An enzyme driven colourimetric reaction enables viewin g of the end products, and the amount of color is proportional to target abundance (Scholin et al., 1996) The SHA has been integrated into a remote field monitoring platform called the Environmental Sample Processor (ESP), an electro magnetic/fluidic system that concentrates water samples and uses various probe arrays to detect species of interest, as well as preserves samples to be analyzed later with whole cell hybridization (Greenfield et al., 20 08; Greenfield et al., 2006) Recently, a cELISA
! 22 was also added to the ESP for the detection of DA (Doucette et al., 2009; Scholin et al., 2009) Because the 28S probes were developed using Pseudo nitzschia strain s isolated from Monterey Bay, California, there have been mixed results when applying them to other geographical regions. In addition, the extensive cryptic diversity of this genus has caused difficulties for the discrimination of some species. Vrieling et al. (1996) and Bates et al. (1999) had success using the P. multiseries and P. pungens probes in Dutch and Canadian waters, respectively. The 28S probes also successfully detected six Pseudo nitzschia species in New Zealand (Rhodes et al., 2001) Other studies have shown the 28S probes to be variable in their discrimination of Pseudo nitzschia species (Lundholm et al., 2006; Orsini et al., 2002; Parsons et al., 1999; Rhodes et al., 1998b; Scholin et al., 1999; Turrell et al., 2008) For example, Lundholm et al. (2006) found that P. delica tissima like isolates from geographically diverse locations reacted with the probes fo r five different species When the 28S probes were applied to northern Gulf of Mexico waters, they agreed with the microscope on the presence or absence of four different Pseudo nitzschia species in 66% of the sample s analyzed (Parsons et al., 1999) However, a great deal of probe cross reactivity was observed with P. pseudodelicatissima and P. delicatissima indicating genetic variation in these species (Parsons et al., 1999) Another rR NA region that has been used for the development of Pseudo nitzschia detection a ssays is the 18S (Diercks et al., 2008a; Fitzpatrick et al., 2010) although it is generally more conserved than the 28S. Species specific
! 23 SHA probes were used to discriminate cultures of P. multiseries P. australis P. pungens and P. seriata and a genus probe detected all strains tested except for P. pseudodelicatiss ima (Diercks et al., 2008a) However, more extensive testing of these probes is needed. Eventually, these SHAs are to be integrated into a portable semi automated device developed in the EU project ALGADEC that performs SHAs with electrochemi cal detection (Dier cks et al., 200 8b) Fitzpatrick et al. (2010) used the 18S to design a SYBR Green qPCR assay for the Pseudo nitzschia genus, but species specific detection was not obtained, which the authors attributed to low sequence diversity for this gene. The ITS regions seem to be the most variable o f the rRNA, and examination of these regions has begun to unravel the cryptic diversity of the Pseudo nitzschia genus. Sequence analysis of the ITS2 has resulted in the description of new species, most notably within the P. pseudodelicatissima and P. delic atissima complexes (Amato and Montresor, 2008; Lundholm et al., 2003; Lundholm et al., 2006; Quijano Scheggia et al., 2009) This cryptic diversity may help to explain some of the variation observed with 28S probes. Because ITS sequence length can vary between species, the potential for species discrimination using automated ribosomal intergenic spacer analysis (ARISA) has also been examined (Hubbard et al., 2008) However, sequence variation has also been observed among isolates of the same species, and even within the same genome, or monocl onal culture (Casteleyn et al., 2008; D'Alelio et al., 2009; Orsini et al., 2004) For this reason Evans et al. (2007) concluded that the ITS regions alone were not suitable for barcoding diatoms. Recently, Moniz and
! 24 Kaczmarska (2010) proposed the use of the 5.8S + ITS2 region as a barcode, with the 5.8S gene acting as an "anchor" point and the ITS2 providing m ore resolution. Lastly, simple sequence repeats (SSRs), or microsatellites, and intersimple sequence repeats (ISSRs) have also been investigated as molecular tools for the discrimination of Pseudo nitzschia species (Bo rnet et al., 2004; Bornet et al., 2005; Evans et al., 2004; Evans and Hayes, 2004) SSRs are abundant polymorphic repeat motifs scattered throughout the genome, and ISSRs are the regions in between these motifs, which can be amplified with unique PCR pr imers (Bornet et al., 2004) These markers are highly variable, with the potential to discriminate discrete populations of the same species (Evans et al., 2004) RuBisCO: A potential molecular detection target. Ribu lose 1,5 biphosphate carboxylase/oxygenase (RuBisCO) catalyzes the first major step of carbon fixation: the carboxylation of ribulose 1,5 biphosphate (RuBP) using either carbon dioxide or molecular oxygen. In chromophytic and rhodophytic algae (non green a lgae), RuBisCO consists of eight large and eight small subunits encoded by the chloroplast (Hwang and Tabita, 1989) The genes encoding the large and small subunits ( rbcL and rbcS respectively) are cotranscribed and separated by a short, variable intergenic region (Hwang and Tabita, 1991) RuBisCO is one of the world's most abundant proteins; in fac t, it may comprise up to 50% of the total soluble proteins in a plant leaf or cell (Ellis, 1979)
! 25 Relatively few studies have focused on RuBisCO genes in Pseudo nitzschia Amato et al. (2007) examined the rbcL gene, along with the 28S and ITS rDNA, to reveal the presence of cryptic, reproductively isolated groups within P. pseudodelicatissima and P. delicatissima The rbcL and ITS regions were considerably more variable than the 28S g ene. The rbcL gene has been suggested as a potential barcode gene for diatoms (Evans et al., 2007) The authors initially chose this gene because it has straightforward alignment, sufficient variability, and because it is plastid encoded, amplifying contaminant DNA is unlikely. The rbcL and the cytoc hrome c oxidase ( cox 1) genes were concluded to be better barcode regions than either the 18S or ITS rDNA (Evans et al., 2007) McDonald et al. (2007) has also suggested the rbcL gene as a possible candidate gene for Pseudo nitzschia identification because of its high resolution compared to the 28S. Although the rbcS gene has been examined in a few marine diatoms no studies have focused on this gene in Pseudo nitzschia Hwang and Tabita (1991) sequenced the rbcL gene rbcL rbcS spacer region, and rbcS gene in Cylindrotheca sp. s train N1. The authors found that the rbcS gene encodes for 139 amino acids in this marine diatom (corresponding to 417 bp) and has a relativ ely high AT content (Hwang and Tabita, 1991) The same length was obse rved for the rbcS gene in a diatom endosymbiont of Peridinium foliaceum (Che snick et al., 199 6) Phylogenetic analyses revealed that the rbcS gene may be less conserved than the rbcL indi cating its potential useful ness in the resolution of closely related species (Chesnick et al., 1996)
! 26 Nucleic acid amplification based detection methods: qRT PCR and NASBA Real time nucleic acid amplification techniques have be en applied to the detection of several harmful algal species (reviewed by Humbert et al., 2010; Penna and Galluzzi, 2008) These methods enable higher sensitivity, which is necessary for the detection of pre bloom concentrations. Additionally, quantitative PCR (qPCR) allows for the semi quantificatio n or quantification of potentially toxic cells. Quantitative PCR assays have been designed for a variety of harmful phytoplankton, including dinoflagellates, raphidophytes, pelagophytes, diatoms, and cyanobacteria (Baxa et al., 2010; Bowers et al., 2000; Fitzpatrick et al., 2010; Galluzzi et al., 2004; Handy et al., 2005; Park et al., 2007; Popels et al., 2003) Although assay design is significantly more complex, multispecies detection is also possible with qPCR. Han dy et al. (2006) used multiplexing and multiprobing to detect several harmful raphidophyte species simultaneously. Furthermore, it has been demonstrated that morphotaxo nomy analyses and molecular methods such as qPCR and FISH all yield comparable cell concentrations (Touzet et al., 2009) Although most of these qPCR studies have targeted DNA, assay s using RNA (especially mRNA) amplification are advantageous because the rapid degradation of mRNA only permits the detection of viable cells, enabling a more relevant estimate of cell concentration. Additionally, the presence of high copy numbers of mRNA per cell combined with amplification provides the potential for a very low limit of detection in the environment. Two such RNA amplification methods are Nucleic Acid Sequence Based Amplification (NASBA; Figure 3;
! 27 Compton, 1991) and quantitative reverse transcription PCR (qRT PCR ; Figure 4 ). NASBA has been used for the detection of the RuBisCO large subunit ( rbcL ) gene in Karenia brevis and Karenia mikimotoi with a detection limit of one cell per reaction (Casper et al., 2004; Ulrich et al., 2010) A qRT PCR assay targeting the rbcL gene of K. brevis has also been deve loped (Gray et al., 2003) Coyn e and Cary (2005) used qRT PCR as a proxy for Pfiesteria piscicida cyst viability in estuarine sediments. Figure 3 Nucleic Acid Sequence Based Amplification (NASBA) with molecular beacon detection A) Reaction pathway. A 3' primer containing the T7 RNA polymerase promoter sequence (Primer 1) hybridizes to the single stranded sense RNA. Following reverse transcription, the RNA template is digested by RNAse H, which is specific for DNA/RNA h ybrids. Ne xt, Primer 2 anne als to the 5' end, and a sense DNA stran d is synthesized. Once a double stranded DNA molecule exists that contains the T7 promoter sequence, antisense RNA is generated by action of the RNA polymerase. The antisense RNA functions as a templ ate for re verse transcriptase, and the cycle repeats, with Primer 2 annealing first. B) Molecular beacon hairpin loop structure. Upon binding of the beacon to the target antisense RNA seque nce, fluorescence is emitted ( R = reporter dye ; Q = quencher).
! 28 Figure 4 TaqManÂ¨ qRT PCR assay process. Following reverse transcription PCR is conducted, with the TaqManÂ¨ probe binding to the antisense DNA strand during each cycle. The exonuclease activity of the DNA polymerase cleaves the probe, releasing the reporter dye into solution (R= reporter; Q= quencher) NASBA and qRT PCR can both incorporate fluorescent probe based detection of a specific genetic sequence, in the form of molecular beacons for NASBA (Leone et al., 1998) and TaqMan probes for qRT PCR (Livak et al.,
! 29 1995) NASBA quantification is accomplished by comparing the time to positivity (TTP) value of a sample, which is analogous to threshold cycle (Ct) in qRT PCR, to that of a standard curve. Addition of an internal control RNA enables higher precision in quantification (IC NASBA; Patterson et al., 2005; Ulrich et al., 2010) One major differe nce between these two methods is that NASBA is isothermal (41 Â¡C), while qRT PCR is conducted using traditional thermocycling conditions. Research objectives Molecular techniques are useful for the detection of Pseudo nitzschia due to the high morphologi cal similarity between species and the time required for microscopic identification. The ideal target gene should accurately delineate species, while the methodology employed should be sensitive and easily adaptable to high throughput screening and/or fiel d monitoring platforms. The major objectives of this research were to: 1. Evaluate the rbcS gene as a potential target for the detection of Pseudo nitzschia species by sequencing this gene from a variety of marine diatoms ; 2. Design an RNA amplification assay based on the rbcS gene for the detection of the domoic acid producer P. multiseries ; 3. Test the sensitivity and specificity of the assay(s) using cultures and RNA transcripts, as well as spiked environmental samples ; 4. Provide an estimate of rbcS trans cript copy number per cell and diel variability in expression
! 30 MATERIALS AND METHODS Phytoplankton cultures and cell counts Unialgal cultures were maintained in growth medias and conditions specified in Table 2. For cell enumeration, cultures were swirled gently to ensure a homogenous distribution of cells, and a subsample was fixed with Lugol's iodine at a final concentration of 2%. A 1 mL Sedgwick Rafter counting chamber (Wildlife Supply Company, Yulee, DL) with 1 L grids was used to count cells Counts were based on 50 fields of view, and at least 2 mL were enumerated per sample, which were then averaged to estimate cell concentration. DNA extraction, rbcS primer design, and PCR The DNA extraction protocol was adapted from Paul and Pichard (1995) as follows: A 50 mL aliquot of diatom culture underw ent centrifugation at 10,000 x g for 20 min or longer, depending on pellet stability. The super natant was decanted and the pellet was resuspended in 1 mL STE buffer. This volume was then transferred to a 1.5 mL microcentrifuge tube and the Paul and Pichard (1995) protocol was followed without further deviation. Total DNA w as quantified and purity was assessed with a NanoDrop spectrophotometer (ND 1000, NanoDrop Technologies, Wilmington, DE). Degenerate primers were designed by examining a ClustalW alignment (OMIGA 2.0, Accelrys, San Diego, CA) of the
! 31 Ta ble 2 Phytoplankton culture growth conditions and GenBank accession numbers for the rbcS gene. PSEUDO NITZSCHIA ISOLATES Species Strain designation Source 1 Media 2 Temperature Light regime ( mol photons m 2 s 1 ; light:dark) GenBank Accession Number for rbcS 3 P. cf. calliantha NWFSC 186 S. Bates, DFO L1 + Si 17 Â¡C 46 50; 13:11 HQ263183 P. cuspidata NWFSC 189 S. Bates, DFO L1 + Si 17 Â¡C 46 50; 13:11 HQ263184 P. delicatissima CL 252 S. Bates, DFO L1 + Si 17 Â¡C 46 50; 13:11 HQ263180 P. delicatissima CL 253 S. Bates, DFO L1 + Si 17 Â¡C 46 50; 13:11 HQ263181 P. delicatissima CL 260 S. Bates, DFO L1 + Si 17 Â¡C 46 50; 13:11 HQ263182 P. multiseries CCMP2708 CCMP L1 + Si 17 Â¡C 46 50; 13:11 HQ263174 P. multiseries CLNN 20 S. Bates, DFO L1 + Si 17 Â¡C 46 50; 13:11 HQ263175 P. multiseries CLNN 21 S. Bates, DFO L1 + Si 17 Â¡C 46 50; 13:11 HQ263176 P. multiseries PM O2 S. Bates, DFO L1 + Si 17 Â¡C 46 50; 13:11 HQ263177 P. pungens CL 249 S. Bates, DFO L1 + Si 17 Â¡C 46 50; 13:11 HQ263178 P. pungens CL 259 S. Bates, DFO L1 + Si 17 Â¡C 46 50; 13:11 HQ263179 P. sp. AL 2 5 AL 2 S. Bargu, LSU L1 + Si 22 Â¡C 40 45; 12:12 HQ263185 P. sp. LA1 St3 5 LA1 St3 S. Bargu, LSU L1 + Si 22 Â¡C 40 45; 12:12 HQ263186 P. sp. LA2 StC6C 6 LA2 StC6C S. Bargu, LSU L1 + Si 22 Â¡C 40 45; 12:12 HQ263187 OTHER PHYTOPLANKTON Species Strain designation Source 1 Media 2 Temperature Light regime ( mol photons m 2 s 1 ; light:dark) GenBank Accession Number for rbcS 3 DIATOMS Chaetoceros compressum CCMP168 CCMP f/2 17 Â¡C 35 40; 13:11 HQ263188 Cylindrotheca closterium CCMP1855 CCMP f/2 22 Â¡C 45 50; 12:12 HQ263189 Cylindrotheca fusiformis CCMP343 CCMP f/2 17 Â¡C 35 40; 13:11 HQ263190 Detonula confervacea CCMP353 CCMP f/2 4 Â¡C 8 12; 12:12 HQ263191
! 32 Table 2, Continued. DIATOMS Fragilaria pinnata CCMP395 CCMP f/2 17 Â¡C 35 40; 13:11 HQ263192 Leptocylindrus danicus CCMP1856 CCMP f/2 22 Â¡C 45 50; 12:12 HQ263194 Navicula incerta CCMP542 CCMP f/2 17 Â¡C 35 40; 13:11 HQ263195 Nitzschia curvilineata CCMP555 CCMP f/2 17 Â¡C 35 40; 13:11 HQ263196 Nitzschia frustulum CCMP558 CCMP f/2 17 Â¡C 35 40; 13:11 HQ263197 Nitzschia laevis CCMP559 CCMP f/2 17 Â¡C 35 40; 13:11 HQ263198 Nitzschia cf. ovalis CCMP1118 CCMP f/2 22 Â¡C 45 50; 12:12 HQ263199 Nitzschia cf. pusilla CCMP560 CCMP f/2 17 Â¡C 35 40; 13:11 HQ263200 Phaeodactylum tricornutum CCMP1327 CCMP f/2 17 Â¡C 35 40; 13:11 HQ263201 Rhizosolenia setigera CCMP1330 CCMP f/2 17 Â¡C 35 40; 13:11 HQ263202 Skeletonema costatum CCMP2092 CCMP L1 + Si 22 Â¡C 45 50; 12:12 HQ263203 Thalassiosira nordenskioeldii CCMP995 CCMP f/2 4 Â¡C 8 12; 12:12 HQ263204 Thalassiosira pseudonana CCMP1335 CCMP f/2 17 Â¡C 35 40; 13:11 HQ263205 Thalassiosira weissflogii CCMP1051 CCMP f/2 17 Â¡C 35 40; 13:11 HQ263206 Unknown Bacillariophyceae CCMP2297 CCMP L1 + Si 4 Â¡C 8 12; 12:12 HQ263193 PELAGOPHYTES Aureoumbra lagunensis CCMP1502 CCMP K 22 Â¡C 45 50; 12:12 N/A Aureococcus anophagefferens CCMP1794 CCMP L1 22 Â¡C 45 50; 12:12 N/A RAPHIDOPHYTES Chattonella subsalsa CCFWC75 FWRI N/A 4 N/A 4 N/A 4 N/A
! 33 Table 2, Continued. RAPHIDOPHYTES Heterosigma akashiwo CCMP452 CCMP L1 22 Â¡C 45 50; 12:12 N/A BOLIDOPHYTES Bolidomonas pacifica CCMP1866 CCMP Prov50 22 Â¡C 23 27; 12:12 N/A DINOFLAGELLATES Akashiwo sanguinea CCFWC31 FWRI N/A 4 N/A 4 N/A 4 N/A Alexandrium monilatum CCMP1305 CCMP L1 22 Â¡C 23 27; 12:12 N/A Gambierdiscus toxicus CCMP1649 CCMP K 22 Â¡C 23 27; 12:12 N/A Karenia brevis CCFWC261 FWRI N/A 4 N/A 4 N/A 4 N/A Karlodinium micrum CCFWC114 FWRI N/A 4 N/A 4 N/A 4 N/A Prorocentrum lima CCFWC381 FWRI N/A 4 N/A 4 N/A 4 N/A Pyrodinium bahamense CCFWC390 FWRI N/A 4 N/A 4 N/A 4 N/A CYANOBACTERIA Trichodesmium erythraeum CCMP1985 CCMP YBCII 22 Â¡C 23 27; 12:12 N/A 1 S. Bates, DFO: Stephen Bates and Claude LÂŽger of Fisheries and Oceans Canada; CCMP: Center for the Culture of Marine Phytoplankton; S. Bargu, LSU; Sibel Bargu of Louisiana State University; FWRI: Florida Fish and Wildlife Research Institute. 2 Except for YBCII (Chen et al., 1996) media was obtained from the CCMP (Guillard, 1960, 1975; Guillard and Hargraves 1993; Guillard and Ryther, 1962; Keller and Guillard, 1985; Keller and Selvin, 1987) 3 The rbcS gene was sequenced for all marine diatoms only. 4 Cultures were obtained from FWRI for specificity testing but not cultured in our lab. 5 Identified as P. cuspidata by electron microscopy (S. Bargu, personal communication). 6 ldentification pending.
! 34 rbcS gene for five marin e diatoms listed in GenBank (accession numbers M59080, AB018006, AB018007, EF067921, and AY819643) and were obtained from Eurofins MWG Operon (formerly Operon Biotechnologies, Huntsville, AL; Table 3). PCR amplification was conducted using GoTaqÂ¨ Green Mas ter Mix (Promega Corp., Madison, WI; final concentrations of 1.5 mM MgCl 2 and 0.2 mM each dNTP) and with final primer concentrations of 0.5 M. Thermal c ycling conditions were as follows: 10 min at 95 Â¡C; 40 cycles of 30 sec at 95 Â¡C, 30 sec at 54 Â¡C, and 45 sec at 72 Â¡C; and a final extension for 10 min at 72 Â¡C. Products were verified by gel electrophoresis and the amplicon was purified using the DNA Cle an & Concentrator 25 per manufacturer's instructions (Zymo Research Corp., Orange, CA). Cloning, sequencing and bioinformatic analysis PCR amplicons were TA TOPO cloned into either the pCRÂ¨2.1 TOPOÂ¨ or pCRÂ¨II TOPOÂ¨ Dual Promoter vectors according to the manufacturer's instructions (Invitrogen Life Technologies, Carlsbad, CA). White colonies were restreaked for purity at least twice and subsequ ently grown in Luria broth with 50 g/mL kanamycin. Plasmids were extracted using the Zyppy Plasmid Miniprep Ki t per manufacturer's instructions (Zymo Research Corp., Orange, CA). Bidirectional Sanger sequencing of the 420 bp insert was performed using M13 primers by the University of Florida DNA Sequencing Core. Sequences were manually examined, the vector and pri mers were trimmed, and orientation of the insert was verified using Sequencher 4.5 (Gene Codes Corp., Ann Arbor, MI). Sequences were aligned using the ClustalW algorithm (OMIGA 2.0, Accelrys,
! 35 San Diego, CA). Neighbor joining phylogenetic trees were constru cted using MEGA 4 ( Tamura et al., 2007) and the bootstrap consensus tree was inferred from 1,000 replicates. All other sequence and phylogenetic analyses were conducted using MEGA 4. Accession numbers for the rbcS gene sequences are given in Table 2. Table 3. Sequences for primers, molecular beacons, TaqManÂ¨ probe, and IC RNA oligonucleotides. Sequence name Sequence (5' to 3') Marine diatom rbcS gene primers Marine diatom rbcS forward primer GTGAGACTTACACAAGGTTG Marine diatom rbcS reverse primer 1 TTAGTAACGGCCACCTTCTGG Marine diatom rbcS reverse primer 2 TTAGTAACGGCTACCTTCAGG Marine diatom rbcS reverse primer 3 TTAGTAACGACCACCTTCTGG IC NASBA assay P. multiseries forward primer AGATTTAACTGATGAACAAA P. multiseries reverse primer 1 AATTCTAATACGACTCACTATAGGG AG AAGG GGTAAACCCCATAATTCCCA P. multiseries molecular beacon [6 FAM] CGTGCTCTATTAGCCGCGGTTTAGCA AGCACG [DABCYL] IC RNA molecular beacon [6 ROX] CATGCGTGGCTGCTTATGGTGACAAT CGCATG [DABCYL] P. multiseries IC RNA generation forward oligo 1 AATTCTAATACGACTCACTATAGGG AG AAAGATTTAACTGATGAACAAATTGAA AAGCAAATTGCTTACGTGGCTGCTTAT GGTGACAAT P. multiseries IC RNA generation reverse oligo GGTAAACCCCATAATTCCCAGTAACTA TTACGTGGGTGTGGATCATCTGTCCA TTCAACGTTCATATTGTCACCATAAGC AGCCA qRT PCR assay P. multiseries forward primer AGATTTAACTGATGAACAAA P. multiseries reverse primer GTAACTATTACGTGGGTGT P. multiseries TaqManÂ¨ probe [6 FAM] CTATTAGCCGCGGTTTA [TAMRA] 1 Text highlighted in grey indicates T7 RNA polymerase promoter site.
! 36 Synthesis of i n vitro transcript For each Pseudo nitzschia isolate, one sequenced rbcS clone was used for the synthesis of transcript. Plasmids were linearized by digestion with either HindIII or NotI restriction endonucleases, depending on the orientation of the insert Digested plasmids were purified using the DNA Clean & Concentrator 25 (Zymo Research Corp., Orange, CA). Run off transcripts were generated from either the T7 promoter or Sp6 promoter sites using the RiboprobeÂ¨ in vitro Transcription System (Promega Cor p., Madison, WI). Transcripts were purified using the RNEasyÂ¨ Mini Kit (Qiagen, Valencia, CA) per manufacturer's instructions and quantified with a NanoDrop spectrophotometer (ND 1000, NanoDrop Technologies, Wilmington, DE). Transcripts were mixed 1:1 in a n RNA storage buffer (8 M guanidinium isothiocyanate, 80 mM Tris HCl (pH 8.5), 24 mM MgCl 2 140 mM KCl) and frozen at 80 Â¡C until use. IC NASBA assay design and reaction conditions P rimers were designed to target a 126 bp region of the P. multiseries rbcS gene. The downstream primer contained the T7 promoter sequence at the 5' end followed by a purine rich spacer region. The molecular beacon was designed internal to the two primers and sequence parameters were confirmed using Mfold software ( http://bio web.pasteur.fr/seqanal/interfaces/mfold simple.html ) to ensure a calculated free energy of 3 0.5 kcal/mol. The P. multiseries beacon was labeled with 6 carboxyfluorescein (FAM ) at its 5' end and quencher DABCYL at its 3' end. Synthesis of IC RNA was co nducted as previously described by Ulrich et al. (2010) and transcripts were quantified and stored as previously
! 37 described The IC RNA beacon was labeled with 6 carboxy X rhodamine (ROX Â¨ ) at its 5' end and DABCYL at its 3' end. Primers and beacons were obtained from Eurofins MWG Operon (Huntsville, AL), while oligonucleotides for IC RNA synthesis were obtained from Integrated DNA Technologies, Inc. (Coralville, IA). Sequences for primers, beacons, and calibrator oligonucleotides are listed in Table 3. IC NASBA was performed using a NucliSENS Basic Kit and EasyQÂ¨ System according to manufacturer's instructions (bioMÂŽrieux Inc., Durham, NC). Final concentrations for primers and beacons were 400 and 100 nM, respectively, and the fin al concentration of KCl was 80 mM. The RNA template and reagent mix were incubated at 65 Â¡C for 2 min prior to the addition of the enzymes, with a final reaction volume of 10 L. Optimal IC RNA concentrations were determined empirically by titration to all ow for positive amplification of both the target and the internal control RNA ( IC RNA ) but ranged from 10 5 to 10 7 copies per reaction (data not shown). TTP ratios were calculated by dividing the TTP of the wild type (target) by the TTP of the IC RNA. The d etection threshold for all TTP calculations was 1.35 fluorescent units. A negative control (nuclease free wa ter) was included in each assay. TaqManÂ¨ qRT PCR assay design and reaction conditions Primers for a TaqManÂ¨ qRT PCR assay were designed to target a 106 bp region of the P. multiseries rbcS gene. The upstream IC NASBA primer was used as the forward primer for qRT PCR. Due to fewer thermal constraints on primer design compared to NASBA, the downstream primer for qRT PCR was
! 38 shifted upstream to exploi t additional nucleotide differences between Pseudo nitzschia and other marine diatoms (Table 3). The TaqManÂ¨ probe sequence was located in the same region as the IC NASBA molecular beacon but was shortened by three nucleotides on the 3' end (Table 3). The reverse primer was obtained from Integrated DNA Technologies, Inc. (Coralville, IA) and the TaqManÂ¨ probe from Eurofins MWG Operon (Huntsville, AL). SYBRÂ¨ Green dissociation ana lysis was conducted to aid in assay design RNA was extracted from P. multiseries cells as previously described, and cDNA was created using the reverse PCR primer and the SuperScriptÂ¨ III First Strand Synthesis System according to the manufacturer's instructions (Invitrogen Life Technologies, Carlsbad, CA). Reactions wer e performed in 50 L using SYBR Â¨ Green PCR Master Mix (Applied Biosystems, Carlsbad, CA) and a final primer concentration of 0.5 M Cycling conditions were as follows: 10 min at 95 Â¡ C; 35 cycles of 30 sec at 95 Â¡C, 3 0 sec at 51 Â¡C, and 45 sec at 60 Â¡C; an d a melting curve analysis (45 Â¡C to 95 Â¡C) TaqManÂ¨ a ssays were performed in 50 L reactions using the TaqManÂ¨ One Step RT PCR Master Mix Kit (Applied Biosystems, Carlsbad, CA). Final concentrations for pr imers and TaqManÂ¨ probe were 0.5 M and 0.25 M, respectively. Cycling conditions were as follows: 30 min at 45 Â¡C for reverse transcription; 10 min at 95 Â¡C; and 40 cycles of 15 sec at 90 Â¡C, 30 sec at 51 Â¡C, and 80 sec at 56 Â¡C. Fluorescence was measured after each 56 Â¡C incubation. Optimization of the extension temperature was also conducted. Reactions were
! 39 performed using a 7500 Real Time PCR System and analyzed with the software provided (Applied Biosystems, Carlsbad, CA). RNA extraction Standard protocol. P. multiseries cells (CCMP2708) were diluted in L1 + Si media (Guillard and Hargraves, 1993) based on t he desired number per IC NASBA or q RT PCR reaction. Cells were filtered onto a DuraporeÂ¨ HV polyvinylidene difluoride 0.45 m pore size filter (Millipore, Billerica, MA). The filter was placed in 500 L RLT lysis buffer ( Qiagen, Valencia, CA) with 0.143 M Â§ mercaptoethanol ( Â§ ME) and vortexed. Following a 10 min incubation, 350 L of 100% ethanol were added and the lysate was purified using RNEasyÂ¨ Mini Kit (Qiagen, Valencia, CA) according t o manufacturer's instructions. The on column DNAase d igestion step was included for q RT PCR samples. Final elution of RNA was carried out in 50 L of nuclease free water. Efficiency experiment. Two variations on the stan dard protocol were evaluated in parallel for the RNA extraction efficiency experiment, which was performed using the IC NASBA assay: 1) The same volume of culture was directly filtered onto an RNEasyÂ¨ column, incubated for 10 min with 500 L RLT/ Â§ ME + 350 L 100% ethanol, and then purified as normal; and 2) After filtration of culture, the filter was placed in a 2 mL tube containing zirconium beads + 500 L RLT/ Â§ ME and underwent bead beating for 2 min prior to purification using RNEasyÂ¨. Each of the three extraction methods was performed in triplicate to yield a theoretical concentration
! 40 of 100 cells per IC NASBA reaction. The TTP ratios for extracts were compared to a transcript standard curve to determine copy number per volume eluate. Percent recovery w as calculated as percent of the greatest recovery obtained. Specificity testing Specificity of the P. multiseries IC NASBA and qRT PCR assays was tested against the phytopl ankton strains listed in Table 2 Because rbcS transcripts were available for Pse udo nitzschia isolates, they were used at a concentration of 10 6 copies per reaction. For all non Pseudo nitzschia strains, several milliliters of culture were filtere d and extracted with the standard RNEasyÂ¨ prot ocol Total RNA was quantified with an Agil ent Bioanalyzer (Model 2100, Agilent Technologies, Santa Clara, CA) and extracts were diluted to 10 pg per reaction. Positive controls ( P. multiseries CCMP2708) were included in each assay and prepared in the same manner as the non targets. Spiked environ mental samples For the spiked seawater experiment using the P. multiseries TaqManÂ¨ qRT PCR assay, water was collected from Bayboro Harbor, an embayment in Tampa Bay (29 June 2010). Three replicate aliquots were examined by light microscopy for the presence of Pseudo nitzschia cells After determining that the natural presence of Pseudo nitzschia was below the detection limit, aliquots of untreated seawater were spiked with various concentrations of P. multiseries cells in a total volume of 50 mL. Due to the loss of strain CCMP2708 after extended time in culture, isolate CLNN20 was used for this experiment, which has an identical rbcS nucleotide sequence. Spiked concentrations spanned five
! 41 orders of magnitude, ranging from a theoretical 10 7 c ells/L to 10 3 cells/L. Each concentration and a non spiked sample were filtered in triplicate, and cultured cells were also filtered for the standard curve. Filters were stored in 500 L RLT/ Â§ ME at 80 Â¡C until processing, which occurred within one week. RNA was extracted using the standard RNEasyÂ¨ protocol incorporating the on column DNAse digestion step. One initial qRT PCR run was conducted to confirm the absence of P. multiseries in the unspiked samples as well as determine the proper dilution require d for minimization of chemical inhibition of the reaction. The optimal dilution factor for these samples was determined to be 1:5 (data not shown). Calculated versus expected cell concentrations were determined by comparing the Ct for each spiked concentra tion with the standard curve. The Ct value (cycle threshold) is the number of cycles required for fluorescence to reach a threshold value. Due to extensive and variable inhibition of the IC NASBA assay in environmental samples, a spiked seawater experiment similar to the one conducted for qRT PCR could not be performed. Water samples were collected from the Pinellas Point area in southern Tampa Bay on 14 August 2009. Seawater was spiked with P. multiseries cells at a theoretical concentration of 10 5 cells/L as described in the above qRT PCR experiment, only this was done in a total volume of 30 mL. RNA was extracted using the standard RNEasyÂ¨ protocol. Undiluted extracts and 1:10 dilutions were used in the IC NASBA reactions.
! 42 Determination of IC NASBA assay inhibitor (s) Size fractionation experiment. Seawater was collected from the Pinellas Point area on 6 August 2009, spiked with cultured P. multiseries at a concentration of 10 5 cells/L, and 30 mL was filtered through a 2.0 m pore size polycarbonate filt er (Whatman, Kent, UK). The filtrate was then spiked with P. multiseries at the same concentration of 10 5 cells/L and filtered through a DuraporeÂ¨ HV polyvinylidene difluoride 0.45 m pore size filter (Millipore, Billerica, MA). Both filter fractions were extracted using the standard RNEasyÂ¨ protocol An undiluted and a 1:10 dilution of each extract were used in IC NASBA Karenia brevis spike experiment Seawater was collected from the Pinellas Point area on 12 August 2009, spiked with cultured P. multiseries at a concentration of 10 5 cells/L, and 30 mL was filtered through a DuraporeÂ¨ HV polyvinylidene diflu oride 0.45 m pore size filter (Millipore, Billerica, MA). RNA was extracted as previously described. An undiluted and a 1:10 dilution of each extract were used in IC NASBA. In parallel, a subsample of the RNA extract was spiked with rbcL transcript from Karenia brevis and this was analyzed using the chemistry for a previously developed K. brevis IC NASBA assay (Casper et al., 2004) Sequence specific capture with Dynabeads Â¨ Dynabead s Â¨ MyOne Carboxylic Acid (Invitrogen Life Technologies, Carlsbad, CA ) were used for sequence specific capture of P. multiseries rbcS mRNA from RNEasy Â¨ elu ate prior to performing the IC NASBA assay (Figure 5 )
! 43 This step was included as an attempt to reduce inhibition of the IC NASBA assay. Figu re 5 General detection process incorporating bead based target mRNA capture Coupling of probe to beads. The capture probe contained the same sequence as the molecular beacon, with a primary amine group on its 5' end. Carbodiimide (N Ethyl N' (3 dimethylaminopropyl) carbodiimide hydrochloride ; EDC) activation was used for coupling of probe to the beads according to the manuf acturer's instructions
! 44 (Figure 6 ), with 5 nmol probe used per mg of beads. All solutions used were prepared RN Ase free. Figure 6 Activation and coupling of oligonuc leotides to DynabeadsÂ¨ MyOne Carboxylic Acid Probe coupling efficiency was measured using the Quant iT OliGreen Â¨ ssDNA Assay Kit (Invitrogen Life Tec hnologies, Carlsbad, CA). A low range oligonucleotide standard curve was prepared according to the manufacturer's instructions. Pure probe stock coupling buffer, and washes we re diluted with 1X TE as needed to fall within range of the standard curve S tandards and s amples were mixed wit h OliGreen Â¨ reagent and measured according to the manufacture r's instructions. The c oncentration of probe in pure stock, buffer, and washes was determined in ng/ L and total mass of probe exposed to beads and remaining in buffer/washes was calculated.
! 45 Hyb ridization of probe conjugated beads to target mRNA. The hybridization buffers and a modified protocol were derived from the Dynabeads Â¨ Oligo (dT) 25 manual (Invitrogen Life Technologies, Carlsbad, CA). Briefly, t he probe conjugated bead stock was mixed t horoughly and 25 L (0.1 mg) were transferred to a 2 mL tube. A magnetic particle concentrat or (MPC) was used to collect beads on the side of the tube, enabling removal of the storage buffer. Beads were subsequently washed twice in 200 L of 2X binding buf fer (20 mM Tris, 1 M LiCl, 2 mM EDTA; p H= 7.5; RNAse free). The RNEasy Â¨ eluate was brought to a volume of 1 00 L using nuclease free water, and 100 L of 2X bindin g buffer were added. T his volume (200 L) was transferred to the washed beads. The tube containing beads and target was heated for 5 min at 65 Â¡C and then immediately placed in a slow rotation device for 15 min at roo m temperature. After hybridization, the beads were separated using a MPC and washed twice in 200 L washing buffer (1 0 mM Tris, 0.15 mM LiCl, 1 mM EDTA; pH= 7.5; RNAse free). To elute the captured RNA, 50 L nuclease free water was added to the beads, and the tube was heated for 2 min at 80 Â¡C. The tube was then immediately placed on the MPC, and the supernatant was tran sferred to a 0.5 mL tube. Expression patterns of the rbcS gene in P. multiseries A diel expression study was conducted with P. multiseries CCMP2708 using cultures in exponential phase. Every four hours over a diel cycle, the culture was enumerated and 1 mL was filter ed in triplicate as previously described Filters were stored in 500 L RLT/Â§ME at 80 Â¡C until analysis with IC
! 46 NASBA, which was completed within two weeks for all samples. The extracted RNA from a known number of cells was compared to a n rbcS transcript standard curve to obtain copy number per cell.
! 47 RESULTS Sequence and phylogenetic analysis The rbcS primers successfully amplified the entire (420 bp) gene from Pseudo nitzschia and all marine diatoms tested. D ata presented are based on a 379 bp region of the rbcS gene af ter removal of the primer sites This sequence corresponds to 126 amino acids. The GC content of the rbcS gene ranged from 35.1% to 41.6%. For marine diatoms a lmost half of nucleotide site s were variable but only slightly more than 10% were variable within the Pseudo nitzschia genus specifically (Table 4) Table 4 Variability of rbcS nucleotide and amino acid sequences in Pseudo nitzschia and other marine diatoms. Alignment for Pseudo ni tzschia Variable sites Parsimony informative sites Percent similarity 1 Nucleotide 47/379 32/379 91 99% Amino acid 11/126 8/126 92 99% A lignment for a ll marine diatoms Variable sites Parsimony informative sites Percent similarity 1 Nucleotide 156/379 134/379 76 99% Amino acid 41/126 35/126 79 99% 1 Percent similarity calculated for different species only
! 48 All Pseudo nitzschia species shared at least 91% identity at the nucleotide level and 92% identity at the amino acid level (Table 4). The closely related species P. multiseries and P. pungens differed by only three nucleotides. Two Gulf of Mexico isolates, AL 2 and LA1 St3, ha d an identical rbcS sequence to P. cuspidata. The phylogenetic analysis (Figure 7 ) revealed Pseudo nitzschia as a well supported monophyletic group (bootstrap value = 99%). Within the genus, P. multiseries and P. pungens formed a well supported cl ade (boot strap value = 98%; Figure 7 ). P. cuspidata and P. delicatissima were sister groups in another clade, although bootstrap value were low. P. cf. calliantha and P seudo nitzschia sp. LA2 StC6C were located outside these two clades. Sensitivity and specifici ty of IC NASBA and qRT PCR assays Dissociation curve analysis revealed a single peak for the qRT PCR reaction, indicating that no primer dimers were present (Figure 8). The melting temperature of the amplicon was calculated to be 75.6 Â¡C and the TaqManÂ¨ assay had optimal results with a low extension temperature of 56 Â¡C. Both assays were sensitive to one cell per reaction in culture and demonstrated excellent linearity over four and five orders of magnitude for IC NASBA and qRT PCR, respectively (Figures 9 11). These ranges were also observed when using rbcS gene transcripts (data not shown). The IC RNA concentration optimal for detecting at least one cell (~10 4 copies of transcript) in IC NASBA was determined to be 10 6 copies per reaction (data not shown)
! 49 Fig ure 7 Evolutionary relationship of marine diatoms based on the rbcS gene. Bootstrap consensus tree was inferred from 1,000 replicates. Taxa include 14 Pseudo nitzschia isolates and 24 other marine diatoms. Sequences marked with a were used in the design of rbcS primers and have their GenBank accession numbers in parentheses. The remaining isolates were sequenced in this study.
! 50 Figure 8 SYBRÂ¨ Green dissociation curve. =1,000 cells; = 100 cells; # = 10 cells; = 1 cell. Figure 9 Representative IC NASBA plots for P. multiseries A) 100 cells; B) 1 cell The black line represents amplification of the target gene, while the grey line represents amplification of the IC RNA. The horizontal dashed line denotes the detection threshold value of 1.35. -0.04 0 0.04 0.08 0.12 0.16 0.2 45 55 65 75 85 95 Fluorescence Derivative Temperature (C) 0 1 2 3 0 50 Fluorescence value (A) Time (min) 0 1 2 3 0 50 Fluorescence value (B) Time (min)
! 51 Figure 10 Representative qRT PCR plot for P. multiseries =1,000 cells; = 100 cells; # = 10 cells; = 1 cell The h orizontal dashed line denotes Ct value. Figure 11 Typical c ell standard curves. A) IC NASBA; B ) qRT PCR. ! 0.00001 0.0001 0.001 0.01 0.1 1 10 0 10 20 30 40 Delta Rn Cycle number y = -0.125ln(x) + 1.26 R = 0.987 0.2 0.4 0.6 0.8 1 1.2 1.4 1 10 100 1000 TTP Ratio (A) Cells per Reaction y = -1.94ln(x) + 33.4 R = 0.995 10 15 20 25 30 35 1 10 100 1000 10000 Ct value (B) Cells per Reaction
! 52 Results of the specificity testing for both assays are presented in Table 5. The IC NASBA assay detected all four strains of P. multiseries as well as P. pungens, P. cuspidata, P. calliantha, and two of the Gulf of Mexico isolates, AL 2 and LA1 St3. The qRT PCR assay detected the four P. multiseries isolates but none of the other Pseudo nitzschia species. Neither assay detected any species outside the Pseudo nitzschia genus. Table 5 Specifici ty of IC NASBA and qRT PCR assays. Species Isolate IC NASBA result 1 qRT PCR result 1 P. multiseries CCMP2708 + + P. multiseries CLNN 20 + + P. multiseries CLNN 21 + + P. multiseries PM 02 + + Other Pseudo nitzschia species P. cf. calliantha NWFSC 186 + P. cuspidata NWFSC 189 + P. delicatissima CL 252 P. delicatissima CL 253 P. delicatissima CL 260 P. pungens CL 249 + P. pungens CL 259 + Pseudo nitzschia sp. AL2 + Pseudo nitzschia sp. LA1 St3 + Pseudo nitzschia sp. LA2 StC6C Other marine phytoplankton C. compressum CCMP168 C. closterium CCMP1855 C. fusiformis CCMP343 D. confervacea CCMP353 F. pinnata CCMP395 L. danicus CCMP1856 N. incerta CCMP542 N. alba CCMP2426 N. curvilineata CCMP555 N. frustulum CCMP558 N. laevis CCMP559 N. cf. ovalis CCMP1118 N. cf. pusilla CCMP560
! 53 Table 5, Continued. Species Isolate IC NASBA result 1 qRT PCR result 1 P. tricornutum CCMP1327 R. setigera CCMP1330 S. costatum CCMP2092 T. nordenskioeldii CCMP995 T. pseudonana CCMP1335 T. weissflogii CCMP1051 Unidentified Bacillariophyceae CCMP2297 A. lagunensis CCMP1502 A. anophagefferens CCMP1794 C. subsalsa CCFWC75 H. akashiwo CCMP452 B. pacifica CCMP1866 A. sanguinea CCFWC31 A. monilatum CCMP3105 G. toxicus CCMP1649 K. brevis CCFWC261 K. micrum CCFWC114 P. lima CCFWC381 P. bahamense CCFWC390 T. erythraeum CCMP1985 1 +, detected; not detected RNA extraction efficiency experiment Three RNA extraction methods, all based on the RNEasyÂ¨ protocol but with varying degrees of agitation for cell lysis, were compared to determine the method with the highest efficiency. The regular protocol, which had a 10 min incubation of the filter (plus cells) at room temperature with RLT/ Â§ ME, resulted in the highest rbcS copy n u mbers in RNEasy eluateÂ¨ (Figure 12 ). The bead beating and direct filtration methods yielded 36% and 32% as mu ch target in the elua te, respectively (Figure 12 ).
! 54 Figure 12 Comparison of three RNA extraction methods as determined by rbcS copy number. Error bars represent standard deviation of triplicate filters run using P. multiseries IC NASBA assay. Percent recovery is calculated as percent of the greatest recovery. Spiked environmental samples Examination of environmental samples spiked with P. multiseries cells revealed extensive inhibition of the IC NASBA assay, as is evident by the depressed target an d IC RNA signal compared to the po sitive control (Figure 13 A C ). This inhibition was not lessened with a 1:10 dilution of RNA extract (Figure 13 C). The qRT PCR assay successfully detected P. multiseries spiked into environmental samples ove r five orders of magnitude (Figure 14 ). Positive, linear detection was achieved from 10 7 cells/L to 10 3 cells/L, which corresponded to 5 x 10 4 and 5 cells/reaction, respectively. The calculated versus expected cells/L had a slope slightly above 1, with an r 2 of 0.986 ( Figure 14 ). 36% 100% 32% 0.E+00 5.E+06 1.E+07 2.E+07 2.E+07 3.E+07 3.E+07 4.E+07 4.E+07 Bead Beating with RLT/ ME 10 min incubation with RLT/ ME Direct filtration and incubation with RLT/ ME rbcS copy number in eluate Method of RNA extraction
! 55 Figure 13 Inhibition of IC NASBA assay in spiked environmental samples. Panel A shows amplification of the cell standard at 100 cells/reaction. Panels B and C show the spiked environmental sample (10 5 cells/L or ~150 cells/reaction) run as an undiluted extract and with a 1:10 dilution, respectively. Black curves represent amplification of target, grey curves represent amplification of IC RNA, and horizontal dashed lines denote detection threshold value 0 1 2 3 0 20 40 60 80 Fluorescence value (A) Time (min) 0 1 2 0 20 40 60 80 Fluorescence value (B) Time (min) 0 1 2 0 20 40 60 80 Fluorescence value (C) Time (min)
! 56 Figure 14 Calculated versus expected cells per liter for qRT PCR spiked environmental experiment. E quation and coefficient of determination (r 2 ) value were derived from log transformed data plotted on a linear scale. Error bars represent standard deviation of replicates. For each concentration, three filters were extracted, and each extract was run in duplicate in the qRT PCR reaction. Determination of IC NASBA assay inhibitor The size fractionation experiment revea led inhibition in the > 2.0 m size fraction while it appeared absent from the smaller fraction (Figure 15 ). Calculations from the smaller fraction produced an average number of c ells per reaction similar to the expected value (123 and 150 cells, respectively). The P. multiseries and K. brevis ass ays performed differently with the same seawater sample. While the P. multiseries assay demonstrated inhibition, the K. brevis assay had positive uninhibi ted detection (Figure 16 ). y = 1.03x + 0.0644 R = 0.986 1E+02 1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 1E+02 1E+03 1E+04 1E+05 1E+06 1E+07 1E+08 Calculated cells/L Expected cells/L
! 57 Figure 15 Results from size fractionation experiment. Panel A shows amplification of the cell standard at 100 cells/reaction. Panels B and C show the spiked environmental sample at 150 cells/reaction for the large and sma ll fractions, respectively. Black curves represent amplification of target, grey curves represent amplification of IC RNA, and horizontal dashed lines denote detection threshold value. 0 1 2 3 0 20 40 60 80 Fluorescence value (A) Time (min) 0 1 2 0 20 40 60 80 Fluorescence value (B) Time (min) 0 1 2 3 0 20 40 60 80 Fluorescence value (C) Time (min)
! 58 Figure 16 Comparison of P. multiseries and K. brevis IC NASBA assay performance Panel A shows inhibition in P. multiseries and Panel B shows detection by K. brevis assay. Black curves represent amplification of target, grey curves represent amplification of IC RNA, and horizontal dashed lines denote detection thresh old value. Sequence specific capture with DynabeadsÂ¨ Probe coupling. Probe coupling efficiency was approximately 40% (Figure 17 ) A large percentage of prob e exposed to the beads remained in the coupling buffer (~50%), while a lesser amount was lost in Wash 1 (~8%). Less than 1% of the total probe was detected in W ashes 2 and 3. 0 1 2 0 10 20 30 40 50 60 70 80 90 Fluorescence value (A) Time (min) 0 1 2 3 4 0 10 20 30 40 50 60 70 80 90 Fluorescence value (B) Time (min)
! 59 Figure 17 Relative p ercentage s of probe detected in various steps of the DynabeadÂ¨ coupling procedure. Ta rget hybridization in culture and environmental samples Afte r testing different bead amounts per hybridization reaction ( 0.4 mg, 0.2 mg, and 0.1 mg ), it was determined tha t 0.1 mg did not result i n significantly lower detection (data not shown). T hus, 0.1 mg was used for subsequent experiments in order to conserve bead stock. Using P. multiser i es RNA extracts, percent recovery by beads compared to a cell standard curve was variable ranging from approximately 10 20%. D etection of 10 cells per IC NASBA reaction was not consistently achieved, an d 1 cell per reaction was never detected. Hybridization and elution times were increased, but this did not significantly improve recovery (data not shown).
! 60 A replicate of a p reviously inhibited sample (collected 31 Ma rch 2009 from Mullet Key, Tierra Verde, FL ) was run using the IC NASBA assay and incorporating the bead hybridization step Although the target was below the limit of detection, successful amplification of IC RNA i ndicated removal of the i nhibitors (Figure 18 ). Figure 18 Comparison of IC NASBA plots for an environmental sample run without and with the DynabeadÂ¨ target hybridization step. Panel A shows inhibition of IC RNA amplification when bead step was not performed, and Panel B sh ows successful amplification of IC RNA after bead step. Black curves represent amplification of target, grey curves represent amplification of IC RNA, and horizontal dashed lines denote detection threshold value. 0 1 2 0 10 20 30 40 50 60 70 80 90 Fluorescence value (A) Time (min) 0 1 2 0 10 20 30 40 50 60 70 80 90 Fluorescence value (B) Time (min)
! 61 The bead hybridization step was also tested using environmental samples spiked with P. multiseries at concentrations of 10 4 cells/L and 10 6 cells/L. Detection of 10 4 cells/L (or 25 cells/reaction) was not consistently achieved, but the assay did detect 10 6 cells/L (2,500 cells/reaction; Figure 19). Percent recovery by beads compared to a cell standard curve usually ranged from 1.0 1.5%; however some experiments produced an inexplicably lower recovery (data not shown). Figure 19 Detection of 10 6 cells /L using DynabeadsÂ¨ and IC NASBA. Panel A is the cell standard curve, and Panel B shows successful amplification of target and IC RNA for the 10 6 cell/L spiked sample. Black curves represent amplification of target, grey curves represent amplification of I C RNA, and horizontal dashed lines denote detection threshold value. 0 1 2 3 0 10 20 30 40 50 60 70 80 90 Fluorescence value (B) Time (min) y = -0.169ln(x) + 1.64 R = 0.981 0.0 0.4 0.8 1.2 1.6 2.0 1 10 100 1000 TTP ratio (A) Cells per reaction
! 62 Expression patterns of the rbcS gene The overall average rbcS transcript copy number for cultured P. multiseries CCMP2708 was 3 x 10 4 copies/cell. There was some variability in expression over a diel cycle, with the highest occur r in g during early to mid photoperiod (Figure 20 ). The calculated copy number per cell was slightly higher for the second 8:00 a.m. time point compared to the sa me time on the previous day. Throughout the diel cycle, transcript abundance varied from 2.16 x 10 4 5.35 x 10 4 copies/cell. Figure 20 Diel expression pattern of rbcS gene in exponential phase P. multiseries CCMP2708 culture. Error bars represent standard deviation of replicates, and gray boxes denote dark phase of diel cycle.
! 63 DISCUSSION Although the marine diatom rbcS phylogeny placed all Pseudo nitzschia species into a well supported monophyletic group, this gene did not exhibit substantial variation between species within the Pseudo nitzschia genus Only 12% of nucleotide sites were variable for the species/strains examined. Nevertheless, the rbcS gene phylogeny in Pseudo nitzschia appears to follow the same general patt ern observed in ribosomal genes based on the small number of strains sequenced. Both the 28S and ITS regions of ribosomal RNA have shown P. multiseries and P. pungens to be closely related sister groups (Lundholm et al., 2002a; Lundholm et al., 2006) Within the rbcS gene the difference between these two species is only three base pairs. Similar to findings by Amato and Montresor (2008) and Lundholm et al. (2006) P. delicatissima and P. cuspidata are more closely related to each other than to P. calliantha based on the rbcS gene Additional sequencing of other Pseudo nitzschia species, especially those within groups containing significant cryptic or pseudo cryptic diversity, would show the extent to which the rbcS gene phylogeny parallels that of other genetic makers. Based on the rbcS gene sequence data, the identity of two of the Gulf of Mexico i solate s (AL 2 and LA1 St3) was predicted to be P. cuspidata T his was later confirmed by electron microscopy, however identification of LA2 StC6C is
! 64 still pending (Sibel Bargu, personal communication). It is interesting to note that P. multiseries isolates from locations as distant as eastern Canada (CCMP2708, CLNN 20, and CLNN 21) and Russia (PM O2) all contained 100% similarity in the rbcS gene. Using rbcS sequence data, IC NASBA and qRT PCR assays were developed for the detection of P. multiseries Both assay s were sensitive to one cultured cell per reaction and produced standard curves that spanned several orders of magnitude. Due to primer competition between target RNA and IC RNA, the dynamic range of the IC NASBA assay for a given concentration of IC RNA w as limited to four orders of magnitude. However, inclusion of IC RNA is advantageous because it increases assay precision and provides an indicator of inhibition in the sample, thus preventing false negatives (Patterson et al., 2005; Ulrich et al., 2010) Neither assay detected any species outside the Pseudo nitzschia genus. Moreover, w ithin the Pseudo nitzschia genus the qRT PCR assay was specific to P. multiseries wh ile IC NASBA exhibited some cross reactivity with other species. The IC NASBA assay detected sequences with one or two mismatches in the beacon site (e.g. P. pungens and P. calliantha respectively), but did not detect one of the Gulf of Mexico isolates (L A2 S t C6C, three mismatches) or any of the P. delicatissima strains (five mismatches). The molecular beacon exhibited greater specificity than the primers under NASBA reaction conditions, due to the stability of the stem loop structure as described by Tyag i and Kramer (1996) ; however single nucleotide disc rimination was not possible with this assay. The
! 65 qRT PCR assay discriminated a single mismatch, supporting the specificity of the TaqManÂ¨ probe to P. multiseries The most closely related species, P. pungens which had only one mismatch in the probe site, was not detected with as much as 10 9 copies of RNA transcript per reaction. Because of the low average GC content of the rbcS gene (38.5%), the primers had a low melting temperature, enabling the design of a low annealing temperature assay and a short prob e (17 bp). Short probes result in greater variability in melting temperature between matched and mismatched probes, enabling the discrimination of a single nucleotide as discussed by Kostina et al. (2007) Only the P. multiseries qRT PCR assay proved successful with field samples, effectively detecting cells spiked into natural seawater at concentrations ranging from 10 7 cells/L to 10 3 cells/L, which is the equivalent of 5 x 10 4 and 5 cells per reaction, respectively. The slope obtained from plotting calculated versus expected cells /L was very close to one (1.03 ), indicating good recovery of target cells and high specificity in complex natural samples This limit of detection cou ld be lowered further simply filtering larger volume s of seawater (> 50 mL) but is sufficient for early detection of most Pseudo nitzschia blooms. A study performed by Trainer and Suddleson (2005) defined the P. multiseries trigger concentration for initiation of particulate domoic acid measurements of seawater to be approximately 10 5 cells/L. A n IC NASBA assay suitable for the detection of the P. multiseries rbcS gene in environmental samples could not be achieved due to constraints on assay design and a lack of diversity i n the rbcS gene. Primer depletion caused by
! 66 cross reactivity with unknown species was determined to be the cause of inhibition in the IC NASBA assay based on the results of the following two experiments : 1.) Upon size fractionation of an "inhibited" environmental sample into particles larger and smaller than 2.0 m, cells spiked into the <2.0 um filtrate were readily detected, while the larger fraction inhibited detection of both target cells and IC RNA; and 2.) When cells of P. multiseries and cultured cells of Karenia brevis were spiked into the same inhibited environmental sample, the K. brevis assay, previously designed in our lab (Casper et al., 2004) had uninhibited positive detection. T ogether, these results indicate that the inhibitors were within the size fraction comprising single celled eukaryotes and that they were cross reactive to the P. multiseries assay. Sequence data for the rbcS gene in marine diatoms also supports this conc lusion. Several attempt s were made to reduce primer competition by non target organisms in environmental samples P rimer concentrations were increased to a maximum of 1.6 M and both primers were later redesigned to include a few additional mismatches be tween Pseudo nitzschia and other marine diatoms. However, neither of these approaches resulted in a reduction of inhibition (data not shown). A highly specific primer set could not be designed due to high conservation within the rbcS gene. Additionally, a magnetic bead based sequence specific capture method was incorporated into the RNA extraction protocol to select for P. multiseries transcripts. The capture probe contained the same sequence as the molecular beacon, which was highly specific for Pseudo nit zschia based on the results of non target testing While high concentrations
! 67 (10 6 cells/L) of P. multiseries were successfully detected assay sensitivity was lost due to low recovery by the beads. The variability in recovery observed in some bead hybridiz ation experiments also indicates the potential inability for reliable cell quantification using this method The increased occurrence of primer depletion in IC NASBA compared to qRT PCR may be attribut able to the relatively low isothermal temperature (41 Â¡C) at which NASBA is conducted. H ybridization conditions are less stringent than in PCR, which incorporates high temperature ( 90 Â¡C) melting cycles and variable annealing temperatures. Even though the molecular beacon can be very specific, the ideal IC NASBA target gene should exhibit a great deal of variation to avoid nonspecific primer binding in complex samples. Because quantification using these assays is based on rbcS copy number per cell at a given time, it is necessary to take variability of expression into account. We conducted an experiment to investigate the presence of any diel periodicity in rbcS gene expression by cultured cells of P. multiseries Ba sed on our results, expression wa s highest during early to mid photoperiod, with lowest being at night. This is in general agreement with studies measuring rbcL gene expression in diatom cultures, including Thalassiosira pseudonana (Granum et al., 2009) and Phaeodactylum tricornutum (Wawrik e t al., 2002) One study examining rbcL gene expression in natural populations of the Mississippi River plume found peak heterokont transcript abundance during morning hours; however this was not attributed solely to diatoms (John et al., 2007) Extraction of RNA for our experiments was typically done in th e morning, meaning that rbcS
! 68 gene expression was at its maximum. It is important to note that any slight under or over estimation of actual cell concentration using assays based on the rbcS gene could be due to natural fluctuations in expression. However the observed range in expression levels over a diel cycle was far less than an order of magnitude, so this would not translate to large differences in calculated cell concentration. Further studies using Pseudo nitzschia cultures, as well as natural pop ulations, should be conducted to provide a better understanding of fluctuations in rbcS gene expression These studies can include (but are not limited to) comparing rbcS copy number per cell with growth phase, cell size nutrient status, and light exposure. Additionally, we observed a difference in expression between the same time point s on consecutive days that was not attributable to measurement error (Figure 20 ) This discrepancy could have been caused by a change in growth conditions, or it could have resulted from changes in expression due to frequent sampling of the culture Future studies can also involve replicate culture flasks sampled over a longer period of time ( i.e. several days) t o balance out such dispariti es The molecular discrimination of Pseudo nitzschia species, as in other diatom genera, has proven to be an extremely difficult task and insight into the complexity of this genus continues to grow w ith the increased amount of genetic data available IC NASBA and qRT PCR are both sensitive and rapid methods for the detection of harmful algal species like P. multiseries The amplification of RNA transcripts enables a low limit of detection for viable cells within a natural
! 69 phytoplankton assemblage, providi ng early warning of a bloom before the potential accumulation of toxin. IC NASBA is advantageous, because its isothermal nature eliminates the need for complex thermocycling equipment and allows for easier integration into remote sensing platforms (i.e.: Casper et al., 2007) ; however PCR is more amenable to mod ification of reaction temperature Despite a lack of extensive diversity in the rbcS gene, it can be used as a genetic target in conjunction with molecular techniques that are able to discriminate regions of low diversi ty. The P. multiseries qRT PCR assay was specific and capable of detecting this species in spiked field samples over a wide range of concentrations, demonstrating its utility in the early detection of potentially toxic blooms.
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! 93 APPENDICES
! 94 Appendix 1: IC NASBA assays for P. delicatissima and LA2 StC6C Table A1. P. delicatissima and LA2 StC6C beacon sequences Sequence name Sequence (5' to 3') P. delicatissima beacon [5 HEX] CGCGATGATCAATCGTGGTTTAGCAA TCGCG [DABCYL] LA2 StC6C beacon [Cy5] CCGGATTATTAACCGTGGCTTAGCAAT CCGG [DABCYL] Figure A1 T ranscript standard curves for P. delicatissima and LA2 StC 6C IC NASBA assays A) P. delicatissima ; B) LA2 StC6C. y = -0.121ln(x) + 2.34 R = 0.984 0 0.5 1 1.5 2 2.5 1.E+04 1.E+05 1.E+06 1.E+07 TTP Ratio A rbcS Transcripts per Reaction y = -0.190ln(x) + 3.63 R = 0.954 0 0.5 1 1.5 2 2.5 1.E+04 1.E+05 1.E+06 1.E+07 TTP Ratio B rbcS Transcripts per Reaction
! 95 Appendix 1 Continued Table A2. Specificity of P. delicatissima and LA2 StC6C IC NASBA assays Species Isolate P.deli beacon 1 LA2 beacon 1 P. multiseries CCMP2708 P. multiseries CLNN 20 P. multiseries CLNN 21 P. multiseries PM 02 Other Pseudo nitzschia species P. cf. calliantha NWFSC 186 P. cuspidata NWFSC 189 P. delicatissima CL 252 + P. delicatissima CL 253 + P. delicatissima CL 260 + P. pungens CL 249 P. pungens CL 259 P. sp. AL2 P. sp. LA1 St3 P. sp. LA2 StC6C + Other marine phytoplankton C. compressum CCMP168 C. closterium CCMP1855 C. fusiformis CCMP343 D. confervacea CCMP353 F. pinnata CCMP395 L. danicus CCMP1856 N. incerta CCMP542 N. alba CCMP2426 N. curvilineata CCMP555 N. frustulum CCMP558 N. laevis CCMP559 N. cf. ovalis CCMP1118 N. cf. pusilla CCMP560 P. tricornutum CCMP1327 R. setigera CCMP1330 S. costatum CCMP2092 T. nordenskioeldii CCMP995 T. pseudonana CCMP1335 T. weissflogii CCMP1051 Unidentified Bacillariophyceae CCMP2297 A. lagunensis CCMP1502 A. anophagefferens CCMP1794
! 96 Appendix 1, Continued Table A2, Continued. C. subsalsa CCFWC75 H. akashiwo CCMP452 B. pacifica CCMP1866 A. sanguinea CCFWC31 A. monilatum CCMP3105 G. toxicus CCMP1649 K. brevis CCFWC261 K. micrum CCFWC114 P. lima CCFWC381 P. bahamense CCFWC390 T. erythraeum CCMP1985 1 +, detected; not detected
! 97 Appendix 2: P. multiseries CCMP2708 g rowth curve and toxin data Figure A2. Growth curve for P. multiseries CCMP2708 based on cell counts and fluorescence Counts were performed using a Sedgwick Rafter slide and chlorophyll a fluore scence was measured using a LS 5 fluorescence spectrophotometer (PerkinElmer, Waltham, MA ; excitation: 425 nm, emission: 680 nm). 0 5 10 15 20 25 30 35 40 45 50 0 20,000 40,000 60,000 80,000 100,000 120,000 140,000 0 5 10 15 20 Raw Fluorescence Cell counts (per mL) Day Cell counts Raw fluorescence
! 98 Appendix 2, Continued Figure A3. Fluorescence g rowth curve and domoic acid measurement of stationary phase P. multiseries C CMP2708 cells S tationary phase c ells were enumerated and 10 m L was filtered onto 25 mm GF/F filters and stored at 20 Â¡ C. For analysis of particulate domoic acid, filters were extracted in 5 mL of 20% methanol and vortexed for 2 min. After centrifugation for 10 min at 4,000 rpm, t he supernatant was removed diluted accordingly, and used in ASP ELISA (Biosense Laboratories, Bergen, Norway)
! 99 Appendix 3 : rbcS p hylogeny of marine diatoms and environmental clones
! 100 Appendix 3, Continued Figure A4. Evolutionary relationship of marine diatoms and environmental rbcS clones A n environmental clone library was created using diatom rbcS primers with cDNA from Pinellas Point in southern Tampa Bay ( collected 14 May 2009) The bo otstrap consensus tree was inferred from 1,000 replicates.