Center for Coastal Environmental Health and Biomolecular Research

Marine Biotoxins Program - Manuscripts

Submitted Manuscripts

• Spliced Leader Trans-Splicing In A Dinoflagellate: Insight Into Gene Regulation In The Absence of Nucleosomes
  (CL# 1305, BMC Molecular Biology)… Kristy Lidie and Fran VanDolah
  Background: Spliced leader (SL) trans-splicing is a form of mRNA processing originally described in parasitic kinetoplastida. During this reaction, a short RNA sequence, the spliced leader (SL), is transferred from the 5'-end of a non-coding mRNA to a splice acceptor site on pre-mRNA molecules. As a result, diverse mRNAs acquire a common 5'-sequence. The biological significance of SL trans-splicing is unknown; however, this processing reaction has been implicated in mediation of mRNA stability or translatability and in the resolution of polycistronic pre-mRNAs and therefore plays an important role in genome organization and gene expression.
  
  Results: We have identified numerous mRNA sequences from a unicellular dinoflagellate Karenia brevis that contain an identical 22-bp leader sequence. Furthermore, we have isolated a gene from K. brevis genomic DNA encoding a putative SL RNA. The 666 bp DNA fragment encoding the SL RNA gene also encodes a 5S rRNA gene and binding sites for the Sm proteins involved in RNA splicing. These organizational features are characteristic of many previously identified SL genes. In addition we show that the SL gene has a conserved splice donor site, the dinucleotide GU, immediately following the SL sequence and has a predicted secondary structure typical of previously characterized SL RNAs.
  
  Conclusions: The data presented here support the hypothesis that a stereotypical SL RNA is present in the K. brevis genome and that maturation of a percentage of mRNAs in K. brevis occurs via a trans-splicing process in which a common SL sequence is added to the 5'-end of mature mRNAs. This is the first evidence for the occurrence of SL trans-splicing in a dinoflagellate and extends the known phylogentic range of this mRNA maturation process.

• The Influence Of Microbial Interactions On The Susceptibility Of Karenia Species To Algicidal Bacteria
  (CL# 1297, Aquatic Microbial Ecology)… Patricia B. Roth, Christina M. Mikulski, Gregory J. Doucette
  Abstract: A bacterial strain (D38BY) belonging to the Flavobacteriaceae and antagonistic towards an algicidal bacterium (strain S03; Flavobacteriaceae) was isolated from a culture of the red tide dinoflagellate Karenia brevis characterized previously as resistant to algicidal attack from strain S03. This antagonistic bacterium increased the survival time of otherwise susceptible, bacteria-free K. brevis cultures in a concentration-dependent manner during exposure to algicidal strain S03. Experimental evidence indicated that direct contact was required in order for strain D38BY to inhibit the killing activity of algicidal strain S03. While further work is needed to determine its precise mode of action, the antagonistic properties of strain D38BY provide further evidence that the resistance of certain algal taxa to algicidal attack can be more a function of the ambient microbial community than an intrinsic property of the alga.

• Description Of A New, Epipsammonic, Prorocentrum Species, Prorocentrum Gillespii Sp. Nov. (Dinophyceae), From Muri Lagoon, Rarotonga, Cook Islands
  (CL# 1247, Phycologia)… Laurinda L. Smith, Mark P. Skinner , Tutu Turua, Ron Johnstone, Glen Shaw, Richard J. Lewis and Steve L. Morton
  Abstract: A new, epipsammonic species of dinoflagellate, from the genus Prorocentrum, is described using scanning electron microscopy. Prorocentrum gillespii cells are up to 50 ?m in length and up to 41 µm in width and the surface morphology is rugose. Its valve pores, found within the surface depressions on both valves, exhibit a distinct ring and are found in two sizes, 0.1-0.14 µm and 0.2-0.21 µm. The valve centers are devoid of pores. P. gillespii has a horizontally striated intercalary band. The periflagellar region is an indentation located apically on the right valve and has flagellar and auxillary pores that are unequal in size. P. gillespii is compared to other species of benthic and epipsammonic Prorocentrum species. P. gillespii was found as a bloom on the seabed of a fringing, coral reef lagoon of a ciguatera endemic island.

• Design Of An Affinity Matrix For Solid-Phase Extraction Of The Algal Toxin Domoic Acid
  (CL# 1250, Analytica Chemica ACTA)… Elena V. Piletska, Fernando Navarro Villoslada, Iva Chianella, Alessandra Bossi, Kal Karim, Michael J. Whitcombe, Sergey A. Piletsky, and John S. Ramsdell
  Abstract: Two polymers, one imprinted and one non-imprinted, with high affinity for the neurotoxin domoic acid were designed and tested. A major challenge was the poor stability of domoic acid which precluded its use as a template in molecular imprinting. Domoic acid also contains double bonds which could lead to irreversible attachment of the template to the polymer matrix during polymerisation. For these reasons domoic acid was replaced in the polymerisation protocol with dihydrokainic acid (DHKA) a close structural analogue with greater stability due to the absence of double bonds. Computational screening of a virtual library of functional monomers was used to select monomers with affinity for both domoic acid and DHKA. The functional monomer 2-trifluoro(methyl) acrylic acid (TFMAA) was selected for polymer preparation. All synthesised polymers were tested using solid-phase extraction (SPE). It was found that both MIP and blank polymers demonstrated good recovery of domoic acid from aqueous solution with binding capacities of up to 0.5 mg/g. The synthesised materials had very low affinity for interfering compounds such as glutamic and aspartic acids.

• Morphology And Ecology Of A New Species Of Ostreopsis (Dinophyceae), O. Tholus Sp. Nov., Isolated From Hilo, Hawaii, USA
  (CL# 1215, Botanica Marina)… Steve L. Morton, Karen S. Sifleet, Laurinda L. Smith, And Michael L. Parsons
  Abstract: The morphology of a new species, Ostreopsis tholus sp. nov., is described from cultures isolated from Hilo, Hawaii using both light and scanning electron microscopy. Differences in the cell shape, size, and thecal morphology differentiates this new species from other described Ostreopsis. The thecal plate formula of this new species is the same as other species of the genus: Po, 3’, 7’’, 6C, 6S(?), Vp, Rp, 5’’’, 1p, 2’’’’. A morphological comparison of Ostreopsis tholus with the 9 known Ostreopsis is presented.

• Domoic Acid Contamination Within Eight Representative Species From The Benthic Food Web Of Monterey Bay, California
  (CL# 1223, MEPS)… Judah D. Goldberg, Rikk G. Kvitek, G. Jason Smith, Gregory J. Doucette, Mary W. Silver
  Abstract: Below the euphotic zone, the benthic food web ultimately relies on food sources from shallower waters, including phytoplankton in overlying waters. If the phytoplankton are harmful algal species like Pseudo-nitzschia australis, a diatom producing the neurotoxin domoic acid (DA), the benthic food web can become a depository for phycotoxins. We tested the hypothesis that DA contaminates benthic organisms during local blooms of P. australis, a widespread toxin producer along the US west coast. To test for trophic transfer and uptake of DA into the benthic food web we sampled eight benthic species comprising four feeding types: filter feeders (Emerita analoga and Urechis caupo); a predator (Citharichthys sordidus); scavengers (Nassarius fossatus and Pagurus samuelis); and deposit feeders (Neotrypaea californiensis, Dendraster excentricus, and Olivella biplicata). Sampling occurred before, during, and after blooms of P. australis, in Monterey Bay, CA, USA during 2000 and 2001. Domoic acid was detected in all eight species, with contamination persisting over variable time scales. Maximum DA levels in N. fossatus (673 ppm), E. analoga (278 ppm), C. sordidus (514 ppm), N. californiensis (144 ppm), P. samuelis (55 ppm), D. excentricus (13 ppm), and O. biplicata (2 ppm) coincided with P. australis blooms, while DA levels in U. caupo remained above 200 ppm (max = 751 ppm) throughout the study period. Domoic acid in six species exceeded levels thought to be safe for higher-level consumers (i.e., ? 20 ppm) and thus is likely to have deleterious effects on marine birds, sea lions, and the endangered California Sea Otter, known to prey upon these benthic species.

• Maternal-Fetal Transfer Of Domoic Acid In Rats At Two Gestational Time Points
  (CL# 1351, Environmental Health Perspectives) … Jen Maucher and John Ramsdell
  Abstract: Prenatal exposure to asymptomatic doses of domoic acid has been shown to cause learning and memory deficits later in life, as well as increased susceptibility to seizure inducing agents. We sought to further elucidate the transfer of DA from exposed pregnant rats to their embryos by measuring DA concentrations in maternal plasma, neonate brain and amniotic fluid. Pregnant rats were given a single IV dose of domoic acid (0.6 or 1.6 mg/kg) at either gestational day (GD) 13 or GD20, which correspond to the beginning of rat embryo neurogenesis and the last day of gestation, respectively. Using a direct ELISA, dose-dependent levels of DA were detected in the plasma of dams, amniotic fluid and prenate brain. An average of 6.6 and 20 ng DA/g brain tissue was found in GD13 and GD20 prenatal rats, respectively. Brain concentrations of DA in the GD13 prenates were identical to amniotic fluid levels consistent with no restriction for DA to enter the GD13 prenate brain. At GD20 the prenate brain contained half the concentration of the amnionic fluid. . This study shows that after one hour, fetal brain and amniotic fluid contain between 1 and 5 percent of the toxin found in the maternal circulation, with levels in brain tissue sufficient to activate ionotropic glutamate receptors for DA. The amniotic fluid levels of DA in this study were also within the same range measured in stranded California sea lions, which were DA intoxicated and showed reproductive failure Amniotic fluid levels may therefore make a useful biomarker of DA exposure in sea lions and improve treatment of stranded marine mammals by wildlife managers and rehabilitation facilities.

• The Florida Red Tide Dinoflagellate Karenia brevis: New Insights Into Cellular And Molecular Processes Underlying Bloom Dynamics
  (CL# 1353, Harmful Algae) … Frances M. Van Dolah, Kristy B. Lidie, Emily A. Monroe, Debashish Bhattacharya, Lisa Campbell, Gregory J. Doucette, Daniel Kamykowski
  Abstract: The dinoflagellate Karenia brevis is responsible for nearly annual red tides in the Gulf of Mexico that are responsible for extensive marine mortalities and human illness due to the production of brevetoxins. Although the mechanisms regulating its bloom dynamics and toxicity have received considerable attention, investigation into these processes at the cellular and molecular level has only begun in earnest during the past decade. This review provides an overview of the recent advances in our understanding of the cellular and molecular biology on K. brevis. Several molecular resources developed for K. brevis, including cDNA and genomic DNA libraries, DNA microarrays, metagenomic libraries, and probes for population genetics, have revolutionized our ability to investigate fundamental questions about K. brevis biology. Two cellular processes have received particular attention, the vegetative cell cycle and vertical migration behavior, which are of key importance due to their roles in the development of both surface populations that constitute blooms and subsurface cell aggregations that may serve to initiate them. High throughput sequencing of cDNA libraries has provided the first glimpse of the gene repertoire in K. brevis, with approximately 12,000 unique genes identified to date. Phylogenomic analysis of these genes has revealed a high rate of horizontal gene transfer in K. brevis, which has resulted in a chimeric chloroplast through the selective retention of genes of red, green, and haptophyte origin, whose adaptive significance is not yet clear. Gene expression studies using DNA microarrays have demonstrated a prevalence of post-transcriptional gene regulation in K. brevis that involves an unusual spliced leader trans-splicing mechanism. Among the trans-spliced gene transcripts are type I polyketide synthases (PKSs) implicated in brevetoxin biosynthesis, which are unique among type I PKSs in that each transcript encodes an individual catalytic domain, suggesting a novel gene structure in the dinoflagellate. Metagenomic libraries developed from bloom waters have unveiled the temporal and spatial complexity of the microbial soup that coexists with K. brevis and its active involvement in both bloom growth and termination processes. Finally, the development and application of population genetic markers has revealed a surprisingly high genetic diversity in K. brevis blooms, long assumed to consist of essentially clonal populations. With these foundations in place, our understanding of K. brevis bloom dynamics is likely to grow exponentially in the next few years.

• Brevetoxin (PbTx) Dynamics Following Lysis Of Karenia Brevis By Algicidal Bacteria: Toxin Size Fractions And Characterization Of Open A-Ring Pbtx Hydrolytics
  (CL# 1222, Patricia B. Roth, Michael J. Twiner, Zhihong Wang, Marie-Yasmine Bottein Dechraoui, and Gregory J. Doucette)
  Abstract: Flavobacterium sp. (strain S03) and Cytophaga sp. (strain 41-DBG2) are algicidal bacteria active against the brevetoxin (PbTx)-producing dinoflagellate, Karenia brevis. Little is known about the fate of PbTx associated with K. brevis cells following attack by such bacteria. The dynamics of PbTx in K. brevis cultures exposed to these algicidal strains were thus examined by receptor binding assay and LC/MS in three sized fractions (> 5µm, 0.22 – 5 µm, < 0.22 µm). In control cultures, concentrations of brevetoxin in the > 5 µm particulate size fraction correlated with cell density, whereas significant increases in the dissolved < 0.22 µm brevetoxin concentration were observed in the later stages of culture growth. Exposure of K. brevis to either algicidal bacterium strain caused cell lysis, coinciding with a rapid decline in the > 5 µm sized fractioned PbTx and simultaneous release of dissolved < 0.22 µm toxin. Upon cell lysis, dissolved brevetoxin accounted for ca. 60% of total brevetoxin and consisted of 51-82% open A-ring hydrolytic derivatives. Semi-synthesized PbTx-2 and PbTx-3 open A-ring hydrolytic derivatives bound with lower affinity (25- and 58-fold, respectively) to voltage-gated sodium channels and were less cytotoxic (893- and 147-fold, respectively) to N2A cells than the parent toxins. These novel findings contribute to our understanding of the fate and possible trophic transfer routes of PbTx during bloom events and will be important to consider when assessing the potential of HAB control and mitigation strategies.

• Embryonic Exposure To Domoic Acid Increases The Susceptibility Of Zebrafish Larvae To The Chemical Convulsant Pentylenetetrazole
  (CL# 1352) ... Jessica Tiedeken and John Ramsdell
  Abstract: Domoic Acid (DA) is a neurotoxin produced by diatoms of the genera Pseudo-nitzchia that targets the limbic system to induce tonic-clonic seizures and memory impairment. In utero exposure of mice to DA leads to a reduction in seizure threshold to subsequent DA exposures that is delayed until mid postnatal life. Similar exposure studies in rats with DA cause learning and memory deficits delayed until adolescence. We describe here application of the zebrafish (Danio rerio) and in ovo microinjection exposure as an alternative species model to characterize the adverse effects of embryonic DA exposure on susceptibility to seizure inducing agents later in life. After in ovo DA exposure, larval animals were characterized for sensitivity to the chemical convulsant pentylenetetrazole (PTZ), a well-defined agent for acute seizure models in laboratory rodents and more recently zebrafish. We find that DA exposure in ovo reduces the latency time till first PTZ seizure in larval fish. The severity of PTZ-induced seizures increased in the DA exposed cohorts as determined by seizure stage and movement parameters. In ovo DA exposure caused clear seizure behavior to non-symptomatic doses (1.0 and 1.25mM) of PTZ. Also doses of DA (0.13 and 0.22 ng/mg), which did not exhibit transient embryo symptomatology exhibited an increased seizure behavior in response to the PTZ. These studies demonstrate that in ovo exposure to DA reduces the threshold to chemically induced seizures in larval fish and increases the severity of seizure behavior in a manner that is consistent with in utero studies of laboratory rodents.

• Novel Structure Of Polyketide Synthase Gene Transcripts In The Toxic Dinoflagellates, Karenia Brevis
  (CL# 1321, Marine Biotechnology)... Emily Monroe and Fran VanDolah
  Abstract: Karenia brevis is the toxic dinoflagellate that forms near annual blooms off the Gulf coast of Florida causing the Florida red tide. K. brevis and red tide events cause massive fish kills, marine mammal mortalities, and human intoxications through the production of a suite of potent neurotoxins, the brevetoxins. Brevetoxins are polyketide compounds thought to be synthesized by a modified polyketide synthase complex, but the gene cluster has yet to be identified. We have identified eight PKS transcripts in K. brevis by high throughput screening of two cDNA libraries to K. brevis. Because of the lack of successful axenic K. brevis cultures, the origin of these transcripts remains controversial. Through phylogenetic analysis and PKS gene expression in a bacteria free environment, we have confirmed the eight PKS transcripts are encoded by K. brevis and not associated bacteria. Further, a splice leader sequence has been identified at the 5’end of the identified PKS transcripts indicating we have identified full PKS transcripts in K. brevis. Sequence analysis identifies single catalytic domains in each transcript with the exception of one transcript, an organization not expected for a type I modular PKS. This is the first study to identify full PKS transcripts in a dinoflagellate and shed light on the structure of these genes in a primitive eukaryote.

• Accumulation And Retention Of Brevetoxin In Two Species Of Planktivorous Fish After Exposure To Karenia brevis: Implications To Foodweb Transfer To Bottlenose Dolphins
  (CL# 1313, Marine Ecology Progress Series)... Michael Hinton and John S. Ramsdell
  Abstract: Brevetoxin uptake has been analyzed in two common planktivorous fish that are likely foodweb vectors for dolphin mortality events associated with brevetoxin producing red tides. Exposure of the fish were conducted for ten hours in the presence of Karenia brevis under conditions previously reported to reach optimal uptake of toxin in blood by oral exposure. Striped mullet were used to characterize a low dose uptake into specific organs and evaluate depuration over a two month period. Atlantic menhaden were used to characterize a higher dose uptake into whole body components and evaluate depuration over one month. Our findings indicate a substantially higher uptake of toxin by menhaden but comparable rates of elimination with a whole body half time of elimination of 25 days in menhaden. Elimination occurs rapidly from the intestine (t1/2 < 1 week) and muscle (t1/2 ~ 1 week) compartments and redistributes to liver which continues to accumulate body stores of toxin for four weeks. The accumulation and elimination characteristics of the vectoring capacity of these two fish species are discussed in relation to data from the Florida Panhandle dolphin mortality event of 2004.