Evidence for a Sexual Cycle in the Florida Red Tide Dinoflagellate, Ptychodiscus brevis (=Gymnodinium breve)

Abstract

Sexual stages (gametes, planozygotes) were observed in non-clonal strains of Ptychodiscus brevis stock cultures. Sexual stages were induced repeatedly in nonclonal isolates and crosses of six different isolates using nitrogen-deficient NH-15 medium, blue or green light, and/or lowered temperatures. Sexual stages also have been observed in field populations during Florida red tides. So far, hypnozygotic cysts have not been confirmed in laboratory cultures or field populations, although possible cysts have been observed during cold temperature experiments, initial experiments with multiple crosses, and during continuous 24-hour sampling during a red tide cruise in January 1980. Steidinger (1975a,b) proposed that blooms of the Florida toxic dinoflagellate, Ptychodiscus brevis (Davis) Steidinger, began 18-74 km offshore with a seed population and that the entire bloom cycle of initiation, support, and maintenance was mediated by hydrologic and meteorologic factors. Finucane (unpublished), Wilson (1967), and Steidinger & Ingle (1972) had postulated a benthic resting cyst stage for P. brevis and Steidinger suggested that cysts accumulated forming cyst beds. She further suggested that numerous cysts in these beds, when triggered by proper conditions, could excyst en masse to provide the seed population for a bloom. Oceanic fronts are thought to trigger excystment by changing the temperature over the sediment or lifting the cysts off the bottom into more suitable regions of light, temperature, and/or nutrients (Freeberg & Heyl, 1978; Hiaddad & Carder, 1979; Steidinger & Haddad, 1981). Although Wilson (1967) and Loper (unpublished) had observed possible gametic fusion, later sexual stages (planozygotes, hypnozygotes), necessary to confirm a sexual cycle, were not observed. This report describes gametes, planozygotes, and possible hypnozygotes (cysts) for P. brevis. MATERIALS AND METHODS Seven strains of Ptychodiscus brevis, isolated from waters off the west coast of Florida, were used in this study: a diploid (Loper et al., 1980) 1953 strain; haploid 1971, 1974, 1976, and 1977 strains; and haploid clonal 1978 P5 and P; strains. Cultures were maintained in stock TRIS-sulfide enriched NH-15 medium (based on Gates & Wilson, 1960, with four times as much TRIS and five times as much sulfide mix) of 30-32%o salinity at 24-26?C and ca. 6,200 /tW cm2/s (cool-white fluorescent bulbs) on a 10L:14D cycle. Culture transfers, inoculations, and medium additions were done with sterile Pasteur pipettes using a laminar flow hood. Sexual induction experiments TRANS. AM. MICROSC. Soc., 101(3): 287-293. 1982. ( Copyright, 1982, by the American Microscopical Society, Inc. This content downloaded from 157.55.39.243 on Wed, 05 Oct 2016 04:52:36 UTC All use subject to http://about.jstor.org/terms TRANS. AM. MICROSC. SOC. were conducted in the three-spot well/Petri dish isolation chambers described by Walker & Steidinger (1979). Crosses of the seven isolates were made by adding a small inoculum of each of two isolates to a spot-well. Cells were checked regularly for 3-8 weeks using a Zeiss Stereomicroscope IV-6 placed in the laminar flow hood. A Zeiss Photomicroscope III was used for more detailed observations, measurements, and photography. Sexual stages were looked for in the seven isolates and all possible crosses of the isolates grown in stock NH-15 medium. Additionally, isolates and crosses were subjected to inorganic nitrogen-deficient NH-15 medium, lowered salinity, lowered temperature, blue and green filtered light, and darkness in an attempt to induce sexual stages. Three crosses and the 1971 isolate also were subjected to seven modifications of stock NH-15 medium. Inorganic nitrogen-deficient medium was prepared by omitting KNO3, NH4Cl, and NH4VO3 from stock NH-15 medium. Five modified media were prepared by omitting: (1) vitamins; (2) vitamins and KNO3; (3) trace elements and chelator; (4) trace elements, chelator, and KNO3; or (5) vitamins, trace elements, and chelator. Two additional media were prepared by increasing: (1) vitamins by lOx and (2) trace elements and chelator by 10x. Initial experiments inoculating P. brevis directly into stock NH-15 or modified NH-15 media resulted in stress or death of the cells within 24-48 h. In all subsequent experiments, media were mixed either with equal volumes of inoculating medium or of filtered medium from a stationary 1974 P. brevis culture. The use of preconditioned medium eliminated this mortality. Double-distilled deionized water was used to lower the salinity of stock NH-15 medium to 26-28%o. Temperatures were lowered to 14-15?C, or 18?C, by placing the spot-well isolation chambers in water baths cooled by a chillercirculator. When testing the effect of cooler temperatures on sexual induction, stock cultures and crosses were subjected to lower temperatures immediately after inoculation. For cyst induction, crosses were made to obtain sexual stages before lowering the temperature to 18?C. Ziptone films were placed between the cultures and the fluorescent bulbs to produce blue and green light of approximately 488 and 532 nm, respectively.