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Molecular Typing of the Red-Tide Dinoflagellate Gonyaulax Polyedra in Phytoplankton Suspensions

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Molecular Typing of the Red-Tide Dinoflagellate Gonyaulax Polyedra in Phytoplankton Suspensions AQUATIC MICROBIAL ECOLOGY Vol. 9: 55-61, 1995 Published April 28 Aquat microb Ecol Molecular typing of the red-tide dinoflagellate Gonyaulax polyedra in phytoplankton suspensions Franco ~ollo'~~,Stefano Sassarolil, Laurita ~oni~,Isolina ~arota~ 'Dipartimento di Biologia Molecolare, Cellulare e Animale, Universita di Camerino. 1-62032 Camerino, Italy 2Scuola di Specializzazione in Biochimica e Chimica Clinica. Universita di Camerino, 1-62032 Camerino, Italy 3~ipartirnentodi Biologia Evoluzionistica e Sperimentale, Universita di Bologna, via Irnerio 42.1-40126 Bologna, Italy ABSTRACT: A group of closely related DNA sequences each approx. 240 base pairs long ('Gp240 family') was detected in the DNA extracted from cultured cells of the red-tide dinoflagellate Gonyaulax polyedra using the polyrnerase chain reaction. While members of the Gp240 family appeared to be pre- sent in the DNA of all the isolates of G. polyedra tested, no related sequence could be detected in the DNA of other dinoflagellates, diatoms or marine bacteria In a further set of experiments, the PCR sys- tem based on the Gp240 DNA was used to probe phytoplankton samples collected in the Northern Adriatic Sea during a red tide caused by G. polyedra. The results showed that the members of the Gp240 family contain a short simple-sequence stretch givlng nse to length polymorphism. The poten- tial of this molecular identification system to track G polyedra in its natural environment is discussed. Considerations are also made on the evolution of the simple-sequence loci in marine organisms. KEY WORDS: Gonyaulaxpolyedra . 'Gp240 family' - Simple-sequence loci . Dinoflagellate INTRODUCTION wild-type or genetically engineered microorganisms under a variety of environmental situations. These Dinoflagellate protists (Pyrrhophyta) represent a methods can offer significant advantages over tradi- highly diversified phylum, consisting of 2 classes, tional methods based on light microscope observations Dinophyceae, mainly including free-living sea-water and/or platings (Amici et al. 1991, Britschgi & Giovan- organisms, and Syndiniophyceae, intracellular para- noni 1991). As a first step towards the development of sites (Dodge 1984). The periodic blooms (red tides) of molecular methods to study the red-tide phenomena in the Dinophyceae are responsible for a number of eco- the Adriatic Sea, we have set up a PCR system aimed logical and hygienic problems all over the world. For at a repetitive tract 'Gp240 family' of the DNA of the this reason, the dynamics of the red-tide phenomenon dinoflagellate G. polyedra and we have tested it on and the biology of the causative organisms are the cultured dinoflagellate cells and on natural phyto- objects of growing interest (Loeblich 1976, Herzog & planktonic populations. Maroteaux 1986, Taylor 1987, Lenaers et al. 1989, 1991).Red tides are a recurrent phenomenon along the coastal waters of the Adriatic Sea and the earliest MATERIALS AND METHODS records of phytoplankton blooms date back to the last century (Forti 1906). The species mostly involved are In vitro cultivation of dinoflagellates and diatoms. Prorocentrum micans, Gonyaulax polyedra, Scripsiella Clones of the dinoflagellates Cachonina sp., Gonyau- trochoidea. Gymnodinium sp., Katodinium rotunda- lax polyedra Stein, Alexandrium tamarense Lebour, tum, and a few others (Boni 1983). Molecular detection Gymnodinium sp., G. catenatum Graham, G. corii Sch. and typing methods based on the use of labelled DNA and of the diatoms Nitzschia clostenum W., Sm. and probes or on that of the polymerase chain reaction Skeletonema costatum (Grev.) Cl. were isolated from (PCR) are finding increasing application in tracking phytoplanktonic associations in coastal (Emilia Rom- 0 Inter-Research 1995 Resale of full article not permitted Aquat microb Ecol9: 55-61, 1995 agna) waters of the Northern Adriatic Sea. The refer- conditions were as follows: 94°C for 20 S (denatura- ence cultures of G. polyedra were initiated from single tion), 55°C for 20 s (annealing) and 72°C for 30 S (elon- spores. All cultures were routinely maintained in f/10 gation) for a total of 40 cycles. To avoid DNA carryover, medium, 25%0salinity at 20°C under a 16:8 h 1ight:dark the reaction mixture (oligonucleotides, dNTPs and regime (ca 1600 pW cm-*, from cool white lamps). buffer) was pre-treated with 0.5 U DNase for 45 min at Antibiotics were added to the medium in the first room temperature. The design of the oligonucleotide phases of the cultivation to prevent bacterial growth primers (see below) is based on a clone of Gonyaulax (Guillard & Ryther 1963). Prior to harvesting, the cul- polyedra DNA in pUC13. The sequence of the insert tures were transferred in f/2 medium and grown to late was determined only in part. This clone was selected exponential phase. from a Sau3AI (partial digest) library of dinoflagellate DNA extraction and purification. About 300 m1 of DNA using a screening strategy aimed at the selection each culture were sedirnented by centrifugation at of clones carrying repeated sequences (Rollo et al. 2600 X g for 15 min. Cell pellets were mechanically 1987, Boni & Rollo 1992, Sassaroli 1992). disrupted in the presence of 2 volumes of a mixture Length polymorphism analysis of PCR amplified containing 50 mM Tris-HC1 pH 8.3, 50 mM Na2EDTA, DNA. To detect length polymorphisms in the members 6% (v/v) redistilled phenol and 2 % (w/v) sodium of the 'Gp240 family', 8 1.11 of the reaction mixture was dodecyl sulphate (SDS). An equal volume of redis- loaded on a 2.5% low-melting agarose minigel con- tilled phenol which had been previously saturated taining 0.5 pg ml-' ethidium bromide. After the run the with 50 mM Tris-HC1 pH 8.3 was added to the lysate 240 bp amplification band was visualized on a UV and the whole suspension vortexed for 1 min and cen- transilluminator and the gel cut. The agarose was trifuged for 3 min at maximum speed in a top-bench melted at 70°C for 10 min and 1 p1 of the agarose/DNA mini-centrifuge. The supernatant was transferred into suspension added to a PCR mixture containing 10 pCi a new tube, mixed with an equal volume of phenol: of a 32P-dATP in addition to the standard reagents. chloroform:isoamylic alcohol (24:24:1), vortexed for After 10 amplification cycles sodium acetate (200 mM 30 S and centrifuged as described above. Eventually final) and cold ethanol (2.5 vol) were added to the reac- the supernatant was washed 3 times with water-satu- tion mixture and the suspension stored overnight at rated ether, brought to 0.2 M (final) with sodium -25°C. The morning after, radioactive DNA was pre- acetate, mixed with 2.5 v01 of cold ethanol, and stored cipitated by centrifugation, and resuspended in 12 p1 overnight at -20°C. DNA was precipitated by 10 min distilled water plus 8 p1 of a standard formamide-dye centrifugation in a top-bench mini-centrifuge, desic- mixture. The sample was denatured at 100°C for 5 min, cated, resuspended in 30 p1 sterile distilled water, de- then loaded (4 p1) on a 50 X 21 X 0.025 cm 6% poly- salted using a 'Gene clean' kit (Bio 101, La Jolla, CA, acrylamide gel and the gel run for 6 h at 40 W constant USA) and stored at -20°C at a concentration of about power. After the run, the gel was oven desiccated and 100 ng p1-l. autoradiographed for 3 d. The same protocol was used to extract DNA from Molecular cloning and sequence analysis of PCR natural phytoplanktonic populations. In this case, how- amplified DNA. A fraction (8 p1) of the PCR mixture ever, the cells were lyophilized a few hours after their was directly loaded on a 2.5% agarose minigel con- recovery. DNA extraction was performed 2 wk later. taining 0 5 pg ml-' ethidium bromide or the whole Oligonucleotide synthesis. Oligonucleotide primers reaction volume (50 p1) was vacuum concentrated prior were prepared using a Beckman 200 A DNA synthe- to loading After the run the 240 bp amplification band sizer. After synthesis, the oligonucleotides were de- was visualized on a UV transilluminator, the gel cut, blocked in 14 M ammonium hydroxide for l? h at 55°C. and the DNA purified by electroelution followed by De-blocked oligonucleotides were purified by elution ethanol precipitation. Approxim.ately 100 to 300 ng on a 'DNA grade' G 50 sephadex resin (Pharmacia, purified DNA was phosphorylated using 3 units of Uppsala, Sweden). polynucleotide kinase, repaired using 2 units of T4 Amplification conditions. The PCR was done in a DNA polymerase and cloned into the Sma I site of the 50 1.11 volume and contained about 100 ng DNA, 50 mM polylinker of pUC18. Sequence analysis of the plasmid KC1, 10 mM Tris-HC1 (pH 8.4 at room temperature), inserts was performed using M13 primers and the 1.5 mM MgC12, 100 pg ml-l gelatin, 0.25 pM of each dideoxy chain termination method adapted to super- oligonucleotide primer, 200 pM of each deoxynu- coiled plasmid DNA sequencing (Chen & Seeburg cleotide triphosphate and 2.5 units of Taq polymerase. 1985). The reaction was performed in a DNA 'Thermal Analysis of sequence results. Nucleotide sequences Cycler' (Perkin-Elmer, Norwalk, CT, USA). Before were aligned using the Micro Genie (Beckman, Palo adding the polymerase to the reaction mixture, tem- Alto, CA) computer program. Pairwise comparisons plate DNA was denatured at 95°C for 7 min. Reaction were carried out manually. Rollo et al.: Typing of Gonyaulaxpolyedra Fig. 1. Gel electrophoresis (2.5%, agarose) of the amplifi- cation products (30 PCR cycles) of the DNA (1 ng) iso- latcd from cultured dinofla- gellates, diatoms and bacteria using the oligonucleotide primers described in the text. Molecular standard, '1 kb ladder', Bethesda Research Laboratories (lane l), Nitsz- chia closterium (lane 2),Skel- etonerna costatum (lane 3), G~~rnnodiniumcon; (lane 4), G.
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