Journal of Sustainability Science and Management eISSN: 2672-7226 Volume 14 Number 2, April 2019 : 71-81 © Penerbit UMT SUBSTRATE PREFERENCES AND EFFECTS OF MEDIUM WITH SOIL EXTRACT ON GROWTH OF GENUS Coolia (DINOPHYCEAE) NORMAWATY MOHAMMAD NOOR1*, AIMIMULIANI ADAM1, SHAHBUDIN SAAD1, FIKRI 1 2 AKMAL KHODZORI AND SITI RAEHANAH MUHD SHALEH 1Department of Marine Science, Kulliyyah of Science, International Islamic University Malaysia, Jalan Sultan Ahmad Shah Bandar Indera Mahkota, 25200 Kuantan, Pahang, Malaysia, 2Borneo Marine Research Institute, Universiti Malaysia Sabah, 88899 Kota Kinabalu, Sabah, Malaysia *Corresponding author email: [email protected] Abstract: Coolia is a benthic dinoflagellate that lives epiphytically with a substrate. It has been reported that several of the species produce biotoxins. Therefore, this study was done to identify the substrate preferred by Coolia and the effect of media with soil extraction on the growth of the species of C. tropicalis and C. malayensis. Several substrates; the seagrass (Enhalus), seaweed (Sargassum, Padina (Phaeophyta), Halimeda (Chlorophyta) and coral rubbles were collected and Coolia’s cell density was determined. C. tropicalis and C. malayensis were grown in ES-DK medium with and without soil extract at similar conditions. The specific growth rate, division per day and generation time were determined for both species. Results showed that Coolia flourished in the substrate with a greater amount of thalus from brown macroalgae (Phaeophyta). While the cell density of C. tropicalis was significantly higher in medium without soil extract, C. malayensis was significantly higher in the medium with soil extract. This indicates that both species have similar basic requirements, but differ in their specific needs. This information on the substrate and medium preferences increased our knowledge of the ecology of Coolia. KEYWORDS: Coolia tropicalis, Coolia malayensis, ES-DK medium, nutrient, benthic dinoflagellates Introduction the peritoneum and enlarged the spleen at a concentration of 900 mg/kg, while at the same Genus Coolia (Gonyaulacales, dose, toxin from C. tropicalis caused the mice Ostreopsidaceae) has a wide geographic to die (Rhodes et al., 2014). Both species tested distribution (Leaw et al., 2016). Seven species were isolated from Malaysian coastal waters, of Coolia have been reported, viz. C. monotis referred to Table 1 in Rhodes et al. (2014), C. Meunir, C. areolata L. Ten-Hage, J. Turquet, malayensis (KO92) and C. tropicalis (K1156). Quod & A. Couté, C. canariensis S. Fraga, C. malayensis Leaw, P. T. Lim & Usup, C. tropicalis Study of substrate preferences by benthic M. A. Faust, C. santacroce Karafas, Tomas & dinoflagellates have been reported elsewhere. York and C. palmyrensis Karafas, Tomas & Several reasons have been pointed out on York. So far, no Harmful Algal Bloom (HAB) substrate preference, mainly referring to the cases have been reported due to Coolia spp. morphology of the substrates (Accoroni et However, toxicity studies on these species have al., 2015). Substrates that have a wide surface revealed that the species produce biotoxins and area possess a higher cell density of benthic this raises the possibility that the species could dinoflagellates due to the greater number of cause problems in the future. A number of spaces for attachment. However, substrate toxins have been described from C. tropicalis, preferences can be species-specific as has been including cooliatoxin (in C. monotis) demonstrated for Gambierdiscus spp (Rains & Mohammad-Noor et al. (2013), a yessotoxin by Parsons, 2015). Study on substrate preference Holmes et al. (1995) and several more by Coolia is limited. This species occurrs yessotoxins were identified by Wakeman et al. naturally in the environment, particularly living (2015). Other studies reported the toxicological epiphytically with substrates such as seaweed effects of Coolia toxin in mice; C. malayensis and play an important role in the benthic caused low toxicity in mice with adhesion in ecosystem. 72 SUBSTRATE PREFERENCE AND EFFECTS OF MEDIUM WITH SOIL EXTRACT ON GROWTH OF GENUS Coolia (DINOPHYCEAE) In order to perform research on of marine diatoms and flagellates (Provasoli et phytoplankton, which includes Coolia, the al., 1957). Nevertheless, this medium is difficult establishment of viable cultures for the species to reproduce precisely because the soil used for will provide advantages. For Coolia, several making soil extract came from different sources media have been used to culture the species, (Provasoli et al., 1957). among them were L1 (Fraga et al., 2008), ES- DK (Leaw et al., 2010; Mohammad-Noor et al., In this study, substrate preferences for Coolia 2013), f/2 (Jeong et al., 2012), IMK (Tawong et were studied from two different locations, i.e., al., 2014), and modified K medium with f/2 coastal and lagoon areas. In addition, the effect trace metals (Karafas et al., 2015). Notably, of medium enriched with soil extract on the these studies focused on other aspects and not growth of C. tropicalis and C. malayensis using on the viability of the medium itself. Tawong et ES-DK medium was determined. Results of this al. (2014), on the other hand, compared the study can be used for the ecological study of suitability of a medium to enhance growth, Coolia and help to understand the suitability of reporting that f/2 medium was more suitable to medium with soil extract to culture C. tropicalis culture Ostreopsis, a benthic dinoflagellates in and C. malayensis for high biomass yields. comparison to PES medium. However, there is a limited study on the suitability of a medium to Materials and Methods enhance the growth rate and the use of Location of Field Study enrichment in the media. These are important aspects as unsuccessful culture establishment This study was carried out on Dinawan Island, will lead to a limited study of a particular Sabah. Samples were collected at two sampling species. For a detailed study to be performed on sites, i.e., coastal area and lagoon area (Figure a species, the establishment of healthy and high 1). The depth of the water during neap tide was biomass cultures is important, especially for about 2 to 3 m. The condition of the study sites species that have the potential to produce was explained in Mohammad-Noor et al. (2016). toxins and may harm human health such as Coolia. Sample Collection The soil is commonly used in microalgal Two replicates of substrate samples were cultures to enrich a culture by providing trace collected at both sampling sites. Substrates elements and vitamins needed for growth and samples collected were seaweed (Sargassum, reproduction of certain species or group of Padina, Halimeda), seagrass (Enhalus) and species while inhibiting the growth of other coral rubble. These substrates were kept species (Lee et al., 2013). Likewise, the soil in separate plastic bags containing filtered has been used in a biphasic culture to represent seawater. To dislodge benthic dinoflagellates a solid substrate. This culture technique has from the substrates, each of the substrates was been used to establish cultures of microalgae shaken vigorously before being filtered through especially benthic microlage (Brand et al., a 12 µm sieve. Then, samples were filtered using 2013). Herein, the soil enriched the medium a 20 µm sieve and samples collected on the filter with nutrients, ions and compounds that are were transferred into a urine bottle. Samples required in trace amounts. Erd-Schreiber were preserved with Lugol’s iodine andlabelled medium is one of the earliest mediums that with a location, date, and substrate type. used soil extract in producing unialgal cultures Journal of Sustainability Science and Management Volume 14 Number 2, April 2019 : 71-81 Mohammad Noor et al. 73 Figure 1: Sampling location in Dinawan Island, Sabah Physico-chemical parameters such as salinity, Algal Bloom Studies (UHABs) laboratory pH, temperature and dissolved oxygen were of Universiti Malaysia Sabah. Cultures were measured in situ using multiparameter Hydrolab. maintained using ES-DK media supplemented Water samples were collected and analysed for with soil extract at a temperature of 26-27 °C, nutrients as outlined in Mohammad-Noor et al. 12:12 light: dark cycle and light intensity of 100 (2016). Nutrients such as nitrate and phosphate µmol m-2 s-1. ES-DK was used to maintain both were determined according to Parson et al. Coolia species and for the growth experiments. (1984). The ES-DK medium used contained 32.55mg/L NaNO3, 4.65 mg/L Na-glycerophosphate, P2 Determination of Cell Abundance stock (Na2-EDTA, FeCl3.6H20, H3BO3, MnSO4. H2O, ZnSO4.7H2O and CoSO4.7H2O), Fe stock Cell abundance was determined by counting (Fe(NH4)2(SO4)2.6H20 and Na2-EDTA), vitamin 1mL of each sample twice under light B12, biotin and thiamine. The concentration of microscopy at a total magnification of 100 x. nitrate and phosphate in the ES-DK medium is Counting was done using the Sedgwick rafter presented in Table 1. Soil extract was added to counting procedure.` the medium. To prepare the soil extract, 300 g of garden soil was placed in a l L Erlenmeyer flask containing 600 mL of distilled water. Cultures and Medium Preparation The mixture then was autoclaved at 121 °C for 45 min and left to cool to room temperature. Cultures of C. tropicalis and C. malayensis were obtained from the Unit for Harmful Journal of Sustainability Science and Management Volume 14 Number 2, April 2019 : 71-81 74 SUBSTRATE PREFERENCE AND EFFECTS OF MEDIUM WITH SOIL EXTRACT ON GROWTH OF GENUS Coolia (DINOPHYCEAE) Next, the mixture was filtered through filter and perform the experiments were from the same paper (Whatmann no. 1). The liquid obtained batch. Nitrate and phosphate concentrations of was centrifuged at 4800 rpm for 15 min. The the soil extract were determined according to supernatant was collected and autoclaved at 121 the standard method by Parson et al. (1984). °C for 45 min.