Influence of Temperature on Growth, Toxicity and Carbohydrate Production of a Japanese Ostreopsis Ovata Strain, a Toxic-Bloom-Forming Dinoflagellate
Total Page：16
File Type：pdf, Size：1020Kb
Vol. 65: 261–270, 2012 AQUATIC MICROBIAL ECOLOGY Published online March 2 doi: 10.3354/ame01555 Aquat Microb Ecol Influence of temperature on growth, toxicity and carbohydrate production of a Japanese Ostreopsis ovata strain, a toxic-bloom-forming dinoflagellate Nayani K. Vidyarathna*, Edna Granéli School of Natural Sciences, Marine Ecology Department, Linnaeus University, Kalmar 39182, Sweden ABSTRACT: Ostreopsis ovata is a benthic dinoflagellate that produces palytoxin and its ana- logues. Since the end of the 1990s, toxic blooms of O. ovata have been recorded in many tropical and temperate marine waters. These blooms often kill benthic invertebrates and cause health problems for humans. We hypothesize that increases in seawater temperature might induce these blooms. A strain of O. ovata isolated from the southern coast of Japan was selected for study. O. ovata cells were exposed to 7 different temperatures from 24 to 30°C for 30 d, and growth rates were noted. The specific growth rate was found to be highest at 25°C, next highest at 24°C and lower at 26, 28, 27, 30 and 29°C, in that order. The hypothesis that increased seawater temperature causes increases in growth rate was thus not supported. The cell toxicity and car bohydrate pro- duction of O. ovata were highest at the temperature range that is optimal for cell growth. Increas- esing sea surface temperature, as a result of global warming, is therefore not likely to have a substantial effect on the bloom formation and toxicity of this Japanese strain of O. ovata. KEY WORDS: Ostreopsis ovata · Benthic dinoflagellate · Palytoxin · Toxicity · Climate change · Carbohydrate production Resale or republication not permitted without written consent of the publisher INTRODUCTION ecosystems (IPCC 2007). These factors can have direct or indirect effects on phytoplankton growth. Over the past few decades, harmful algal bloom Climate change may therefore influence the forma- (HAB) incidents have increased in frequency, inten- tion and geographical expansion of HABs (Glibert et sity and geographical extent (Hallegraeff 2003, Glib- al. 2005, Dale et al. 2006). ert et al. 2005). Anthropogenic activities are consid- Elevated seawater temperatures may favour the ered to contribute to this increase in HABs (Glibert et dominance of warm-water harmful algae species in al. 2005). Nutrient enrichment, overfishing, agricul- the phytoplankton community and the extension of ture and aquaculture practices, the introduction of their range to higher latitudes (Tester et al. 2010). toxic species via ballast water dispersion or shellfish This is also evidenced by the expansion and the transplantation and climate changes increase HABs occurrence of the tropical benthic dinoflagellate (Hallegraeff 2003, Glibert et al. 2005). Ostreopsis ovata in temperate regions (Totti et al. The Intergovernmental Panel on Climate Change 2010, Pistocchi et al. 2011). (IPCC) predicts an increase in world average atmos- Ostreopsis ovata Fukuyo is found epiphytic on red pheric temperatures of 1.8 to 4°C over the 21st cen- and brown macroalgae and can also grow on other tury (IPCC 2007). Increases of sea surface tempera- benthic substrata, such as rocks, soft sediments and ture (SST), acidification, change in the stratification benthic invertebrates (Fukuyo 1981, Faust et al. of water masses, precipitation and the timing and 1996, Granéli et al. 2002, Totti et al. 2010). O. ovata volume of freshwater runoff are impacts on marine was found to be an important component in temper- *Email: [email protected] © Inter-Research 2012 · www.int-res.com 262 Aquat Microb Ecol 65: 261–270, 2012 ate marine waters (Vila et al. 2001, Adachi et al. 2008, medium was prepared with filtered and autoclaved Shears & Ross 2009). O. ovata produces palytoxin Skagerrack seawater (salinity 31 psu). Initial culture and its analogues, such as ovatoxin-a, b, c and d volumes of 225 ml were exposed to 24, 25, 26, 27, 28, (Ciminiello et al. 2010). Toxins produced by Ostreop- 29 and 30°C, which varied in the range of ±0.3°C sis spp. cause human fatalities — as the toxins accu- (Fig. 1). mulate higher up in marine food webs (fishes) — and Chlorophyll a was determined fluorometrically mortality of benthic invertebrates, such as sea (Turner Designs 10-AU) after filtration of sub-samples urchins (Granéli et al. 2002, Taniyama et al. 2003, (5 to 10 ml) through Whatman GF/C (25 mm) glass Durando et al. 2007, Shears & Ross 2009). microfibre filters and ethanol extraction of the filter Ostreopsis ovata produces copious amounts of for 2 h in the dark at room temperature (Jespersen & mucilage (Granéli et al. 2002, Guerrini et al. 2010, Christoffersen 1987). When the cultures reached sta- Totti et al. 2010). A mucilaginous matrix helps O. tionary phase, chlorophyll a levels were analysed ovata cells attach themselves to the substrate daily. The samples for cell counts were preserved (Granéli et al. 2002, Totti et al. 2010). Mucilaginous with acidic Lugol’s solution, settled in a Palmer-Mal- mats adhere loosely to the substrata so that the mat oney counting chamber (0.1 ml) and were manually can easily be resuspended in the water column by counted on an inverted microscope (Olympus CKX wave and mechanical action (Totti et al. 2010). 41) at 200× magnification (Palmer & Maloney 1954). Blooms of O. ovata are often associated with thick Samples for toxin analyses (15 to 25 ml) and for mucilaginous mats, which can have an ecological analyses of particulate and dissolved carbohydrate impact, thus their bloom expansion is of great con- concentrations (2 ml) were drawn from each replicate cern (Granéli et al. 2002 and 2011). The determina- on Day 10, 16, 24 or 26, corresponding to the early tion of the effects of temperature increases on the and mid-exponential and mid-stationary phases growth, toxicity and carbohydrate production of O. based on chlorophyll a growth curves. The cultures ovata is of major interest. were manually well mixed before each sampling to Temperature regulates the growth and toxicity of homogenize the samples. Ostreopsis ovata (Granéli et al. 2002, 2011, Pistocchi The experiment was terminated after the growth et al. 2011). Some studies show that Ostreopsis spp. curves entered the decaying phase with the final har- blooms coincide with increasing temperature, >25°C vest at Day 30. The specific growth rate (μ) of each (e.g. Pistocchi et al. 2011). Other studies show that treatment was calculated during the exponential seawater temperature is not a primary driver of growth phases using the following formula: Ostreopsis spp. proliferation (Totti et al. 2010, Man- μ = (ln N − ln N )/(t − t ) (1) gialajo et al. 2011). This suggests that the relation- 2 1 2 1 ship between temperature and the growth and/or where N1 and N2 are cell densities at time t1 and t2. − 3− toxicity of O. ovata might differ among strains. Inorganic nitrogen (NO3 ) and phosphorus (PO4 ) In this work, we investigated the effect of tempera- analyses were performed during the stationary and ture on the growth, toxicity and carbohydrate produc- tion of a Japanese strain of Ostreopsis ovata. MATERIALS AND METHODS Ostreopsis ovata strain s0662 was obtained from the Laboratory of Aquatic Environmental Science, Department of Aquaculture, Kochi University, Japan. Experimental set-up Prior to commencement of the experiment, Ostre- opsis ovata cells were grown in f/10 medium (Guil- lard & Ryther 1962) at 25°C, with a light irradiation of −2 −1 140 µmol photons m s by cool-white Osram tubes Fig. 1. Experimental set-up used to grow Ostreopsis ovata at and exposed to a 12:12 h light:dark cycle. The culture constant temperatures (24, 25, 26, 27, 28, 29 and 30°C) Vidyarathna & Granéli: Temperature and Ostreopsis ovata dynamics 263 decaying phases using the standard methods for sea- and centrifuged for 10 min at 10 000 × g (Eppendorf water analyses (Valderama 1995). 5417C). The supernatants were transferred to clean Eppendorf tubes without disturbing the cell pellets. Both the cell pellets and supernatants were stored at Haemolytic activity −20°C until analysis. The cell pellets, after adding 1.2 ml of 1M H2SO4, Sub-samples were filtered onto Whatman GF/C were incubated for 1 h in a boiling water bath and (25 mm) 1.2 µm mesh size glass microfibre filters and thereafter centrifuged for 5 min at 300 × g (Eppen- were stored at –20°C prior to analysis. The cells dorf 5417C). The total carbohydrate content in the retained on the filters were extracted in 1.5 ml of solution (particulate carbohydrates) was determined methanol for 30 min in the dark. The filtrates of these by the phenol-sulphuric method using D-(+)-glucose samples were tested for toxicity as well. as a standard (DuBois et al. 1956). The cell-free Measurement of the haemolytic activity of Ostre- medium, without further preparation, was tested for opsis ovata cells was based on the method of Igarashi dissolved carbohydrate (DuBois et al. 1956). et al. (1998) and Stolte et al. (2002), with some modi- Particulate and dissolved carbohydrate concentra- fications. Briefly, O. ovata cells methanolic extracts tions were expressed as ng of D (+) glucose equiva- and cell-free medium were mixed in different ratios lent per cell. The term ‘particulate carbohydrates’ in (4 to 50% of extracts or 8 to 100% of filtrates) with our experiment collectively include both intra- and isotonic phosphate buffer (IPB) in 96-well micro the extra-cellular carbohydrates that aggregate to plates (total volume 50 µl), after which 200 µl of 1% form the mucilaginous matrix. horse blood cell suspension in IPB was added and incubated for 4 h. Saponin (Sigma S-2149) in IPB (0 to 48%) was used to produce a standard haemolytic Statistical analyses curve as the reference. The haemolytic activity was measured as absorbance at 405 nm with a microplate Statistical analysis were performed with GraphPad reader (FLUOstar OPTIMA, BMG LABTECH).