Kimberly S. Reece

RECOGNIZING DINOFLAGELLATE SPECIES USING ITS rDNA SEQUENCES1

Dinoflagellate taxonomy is based primarily on morphology and morphometric data that can be difficult to obtain. In contrast, molecular data can be rapidly and cost‐effectively acquired, which has led to a rapid accumulation of sequence data in GenBank. Currently there are no systematic criteria for utilizing taxonomically unassigned sequence data to identify putative species that could in turn serve as a basis for testable hypotheses concerning the taxonomy, diversity, distribution, and toxicity of these organisms. The goal of this research was to evaluate whether simple, uncorrected genetic distances (p) calculated using ITS1/5.8S/ITS2 (ITS region) rDNA sequences could be used to develop criteria for recognizing putative species before formal morphological evaluation and classification. The current analysis used sequences from 81 dinoflagellate species belonging to 14 genera. For this diverse assemblage of dinoflagellate species, the within‐species genetic distances between ITS region copies (p=0.000–0.021 substitutions per site) were consistently less than those observed between species (p=0.042–0.580). Our results indicate that a between‐species uncorrected genetic distance of p≥0.04 could be used to delineate most free‐living dinoflagellate species. Recently evolved species, however, may have ITS p values <0.04 and would require more extensive morphological and genetic analyses to resolve. For most species, the sequence of the dominant ITS region allele has the potential to serve as a unique species‐specific “DNA barcode” that could be used for the rapid identification of dinoflagellates in field and laboratory studies.

New plasmids carrying antibiotic-resistance cassettes.

A series of new plasmid vectors is described that carry gene cassettes imparting resistance to the antibiotics chloramphenicol (CmR), kanamycin (KmR), tetracycline (TcR) and spectinomycin/streptomycin (Sp/SmR). The gene cassettes are symmetrically flanked by several restriction sites. In addition, several restriction sites that are normally found internal to the gene cassettes have been eliminated, thereby expanding the number of restriction enzymes available to excise an intact antibiotic-resistance gene. The gene cassettes are carried by high-copy-number plasmids that confer ampicillin resistance (ApR).

IDENTIFICATION OF PFIESTERIA PISCICIDA (DINOPHYCEAE) AND PFIESTERIA‐LIKE ORGANISMS USING INTERNAL TRANSCRIBED SPACER‐SPECIFIC PCR ASSAYS1

The putative harmful algal bloom dinoflagellate, Pfiesteria piscicida (Steidinger et Burkholder), frequently co‐occurs with other morphologically similar species collectively known as Pfiesteria‐like organisms (PLOs). This study specifically evaluated whether unique sequences in the internal transcribed spacer (ITS) regions, ITS1 and ITS2, could be used to develop PCR assays capable of detecting PLOs in natural assemblages. ITS regions were selected because they are more variable than the flanking small subunit or large subunit rRNA genes and more likely to contain species‐specific sequences. Sequencing of the ITS regions revealed unique oligonucleotide primer binding sites for Pfiesteria piscicida, Pfiesteria shumwayae (Glasgow et Burkholder), Florida “Lucy” species, two cryptoperidiniopsoid species, “H/V14” and “PLO21,” and the estuarine mixotroph, Karlodinium micrum (Leadbetter et Dodge). These PCR assays had a minimum sensitivity of 100 cells in a 100‐mL sample (1 cell·mL−1) and were successfully used to detect PLOs in the St. Johns River system in Florida, USA. DNA purification and aspects of PCR assay development, PCR optimization, PCR assay controls, and collection of field samples are discussed.

Phylogenetic analysis of Perkinsus based on actin gene sequences.

Perkinsus species presently are classified within the phylum Apicomplexa. This placement, however, is controversial. Based upon morphological observations and phylogenetic analyses of the small subunit ribosomal RNA gene, it has been suggested that Perkinsus may be more closely related to dinoflagellates. To reevaluate the phylogenetic position of Perkinsus, we obtained nucleotide sequence data for actin genes from Perkinsus marinus and 2 dinoflagellates, Prorocentrum minimum and Amphidinium carterae. Results indicated that there are 2 closely related actin genes in the genome of P. marinus. Phylogenetic comparisons of these actin gene fragments of P. marinus to available actin gene sequences for several ciliates and apicomplexans and to the 2 actin gene sequences from dinoflagellates obtained in this study supported a closer affinity of P. marinus to dinoflagellates than to apicomplexans.

Real-Time PCR for Detection and Quantification of the Protistan Parasite Perkinsus marinus in Environmental Waters

ABSTRACT The protistan parasite Perkinsus marinus is a severe pathogen of the oyster Crassostrea virginica along the east coast of the United States. Very few data have been collected, however, on the abundance of the parasite in environmental waters, limiting our understanding of P. marinus transmission dynamics. Real-time PCR assays with SybrGreen I as a label for detection were developed in this study for quantification of P. marinus in environmental waters with P. marinus species-specific primers and of Perkinsus spp. with Perkinsus genus-specific primers. Detection of DNA concentrations as low as the equivalent of 3.3 × 10−2 cell per 10-μl reaction mixture was obtained by targeting the multicopy internal transcribed spacer region of the genome. To obtain reliable target quantification from environmental water samples, removal of PCR inhibitors and efficient DNA recovery were two major concerns. A DNA extraction kit designed for tissues and another designed for stool samples were tested on environmental and artificial seawater (ASW) samples spiked with P. marinus cultured cells. The stool kit was significantly more efficient than the tissue kit at removing inhibitors from environmental water samples. With the stool kit, no significant difference in the quantified target concentrations was observed between the environmental and ASW samples. However, with the spiked ASW samples, the tissue kit demonstrated more efficient DNA recovery. Finally, by performing three elutions of DNA from the spin columns, which were combined prior to target quantification, variability of DNA recovery from different samples was minimized and more reliable real-time PCR quantification was accomplished.

THE CASE FOR SEQUENCING THE PACIFIC OYSTER GENOME

Abstract An international community of biologists presents the Pacific oyster Crassostrea gigas as a candidate for genome sequencing. This oyster has global distribution and for the past several years the highest annual production of any freshwater or marine organism (4.2 million metric tons, worth

In Vitro Propagation of Two Perkinsus spp. Parasites from Japanese Manila Clams Venerupis philippinarum and Description of Perkinsus honshuensis n. sp.

3.5 billion US). Economic and cultural importance of oysters motivates a great deal of biologic research, which provides a compelling rationale for sequencing an oyster genome. Strong rationales for sequencing the oyster genome also come from contrasts to other genomes: membership in the Lophotrochozoa, an understudied branch of the Eukaryotes and high fecundity, with concomitantly high DNA sequence polymorphism and a population biology that is more like plants than any of the model animals whose genomes have been sequenced to date. Finally, oysters play an important, sentinel role in the estuarine and coastal marine habitats, where most humans live, environmental degradation is substantial, and oysters suffer intense fishing pressures and natural mortalities from disease and stress. Consumption of contaminated oysters can pose risks to human health from infectious diseases. The genome of the Pacific oyster, at 1C = 0.89 pg or ~824 Mb, ranks in the bottom 12% of genome sizes for the Phylum Mollusca. The biologic and genomic resources available for the Pacific oyster are unparalleled by resources for any other bivalve mollusc or marine invertebrate. Inbred lines have been developed for experimental crosses and genetics research. Use of DNA from inbred lines is proposed as a strategy for reducing the high nucleotide polymorphism, which can interfere with shotgun sequencing approaches. We have moderately dense linkage maps and various genomic and expressed DNA libraries. The value of these existing resources for a broad range of evolutionary and environmental sciences will be greatly leveraged by having a draft genome sequence.

Pfiesteria shumwayae kills fish by micropredation not exotoxin secretion

ABSTRACT. Perkinsus species are destructive parasites of commercial Manila clams, Venerupis philippinarum, in Japan, Korea, and Spain. However, in vitro parasite cultures from this important host clam are not available. Tissues of Manila clams collected during April 2002 in Gokasho Bay, Japan harbored Perkinsus sp. parasites at a 97% prevalence (28/29) of moderate‐ and high‐intensity infections. Perkinsus sp. cells in tissue samples were enlarged in alternative Rays fluid thioglycollate medium, before propagation in DME:Hams F‐12 Perkinsus sp. culture medium. Enlarged parasite hypnospores zoosporulated at high frequencies to release motile zoospores, which gave rise to continuous schizogonic cell lines that also zoosporulated continuously at low frequencies. Four Perkinsus sp. in vitro isolates comprising two distinct morphotypes were cryopreserved, cloned, and archived for public distribution. For three isolates of one morphotype, nucleotide sequences of the ribosomal DNA internal transcribed spacer region, of the large subunit rRNA gene, and of actin genes, were consistent with those reported for P. olseni. Similar sequences from one morphologically unique isolate differed from those of all described Perkinsus species. These results show that at least two Perkinsus spp. infect Japanese Manila clams, and that one represents a new species, Perkinsus honshuensis n. sp.

Molecular, Morphological, and Experimental Evidence Support the Synonymy of Perkinsus chesapeaki and Perkinsus andrewsi

Pfiesteria piscicida and P. shumwayae reportedly secrete potent exotoxins thought to cause fish lesion events, acute fish kills and human disease in mid-Atlantic USA estuaries. However, Pfiesteria toxins have never been isolated or characterized. We investigated mechanisms by which P. shumwayae kills fish using three different approaches. Here we show that larval fish bioassays conducted in tissue culture plates fitted with polycarbonate membrane inserts exhibited mortality (100%) only in treatments where fish and dinospores were in physical contact. No mortalities occurred in treatments where the membrane prevented contact between dinospores and fish. Using differential centrifugation and filtration of water from a fish-killing culture, we produced ‘dinoflagellate’, ‘bacteria’ and ‘cell-free’ fractions. Larval fish bioassays of these fractions resulted in mortalities (60–100% in less than 24 h) only in fractions containing live dinospores (‘whole water’, ‘dinoflagellate’), with no mortalities in ‘cell-free’ or ‘bacteria’-enriched fractions. Videomicrography and electron microscopy show dinospores swarming toward and attaching to skin, actively feeding, and rapidly denuding fish of epidermis. We show here that our cultures of actively fish-killing P. shumwayae do not secrete potent exotoxins; rather, fish mortality results from micropredatory feeding.

Molecular phylogeny of the Haplosporidia based on two independent gene sequences.

Abstract. Diverse analytical and experimental results confirm that two protistan parasites, Perkinsus chesapeaki and Perkinsus andrewsi, described separately as parasites of Mya arenaria and Macoma balthica clams sympatric in Chesapeake Bay, USA, represent a single species. Ribosomal RNA (rRNA) internal transcribed spacer (ITS) regions, rRNA large subunit (LSU) gene, and actin gene sequences were obtained from clonal Perkinsus spp. cultured in vitro. Although multiple polymorphic sequences were found in DNA from clonal cultures at each locus, identical ITS region and actin gene sequences were found in the P. andrewsi holotype culture and in Perkinsus sp. clonal cultures from M. arenaria and Tagelus plebius. All sequences determined from cultures of P. chesapeaki and P. andrewsi at each locus grouped together in monophyletic clades with high support values in phylogenetic analyses. In vitro isolates of Perkinsus spp. from M. arenaria and M. balthica were reciprocally infective for each others cognate host. Lesions and histozoic parasite cell morphologies were consistent with those described for the original host/parasite interactions. In vitro isolate cell cycles and cell types of both parasites were indistinguishable. In accordance with the International Code of Zoological Nomenclature rules of priority, P. andrewsi is declared a junior synonym of P. chesapeaki.