Original article Composition of small Thalassiosirales under highly diluted conditions in a eutrophic lake Masato CHUJO*, Luxi PANG*, Yoko FUJIMURA**, Yoshimasa AMANO**,***, and Motoi MACHIDA**,*** Abstract： Dominant algae have been changed from cyanobacteria to diatoms since 2000, as a consequence of the massive discharge of Tone River water, and the dominant diatom has been reported as Skeletonema potamos and small Thalassiosirales, although the species of the latter have not been identified. In this study, the dominant species of the small Thalassiosirales in the Lake Tega water from spring (April) to autumn (November) in 2017 were identified in detail by scanning electron microscopy (SEM). The results indicated that small Thalassiosirales were mainly composed of five genera, such as Cyclostephanos, Cyclotella, Discostella, Stephanodiscus, and Thalassiosira. The seasonal trend showed that the most abundant species in April, May, June, and November were Stephanodiscus binderanus, Cyclotella atomus, Discostella pseudostelligera, and C. atomus, respectively. Although Cyclotella meneghiniana occupied a small portion in April, it highly increased and became dominant from July to October. Key Words： diatoms, dilution, eutrophic lake, species identification, Thalassiosirales 20002). Then, the mean COD, TN, and TP INTRODUCTION concentrations were reduced to 9.7, 1.2, and 0.15 mg L-1, respectively, from 2011 to 20151). After Lake Tega (35º50'N, 140º03'E) is one of the 2000, cyanobacterial blooms have been remarkably eutrophic lakes in Japan. The water quality in this reduced, and diatoms have been dominant instead lake was significantly deteriorated in the 1970s, of M. aeruginosa (Iwayama and Ogura, 2015). due to the increase in population and urbanization According to the survey of Chiba Prefectural in the lake basin, and the mean values of chemical Government1), the dominant algae after the oxygen demand (COD), total nitrogen (TN), and dilution in Lake Tega have been observed as total phosphorus (TP) concentrations were as high Skeletonema potamos and small Thalassiosirales as 17.5, 3.5, and 0.29 mg L-1, respectively, in 1998 with light microscopy. Small Thalassiosirales have and 19991). In summer, cyanobacterial blooms been classified into some groups from the always appeared, and the main dominant algae, viewpoint of cell size, although species-level defined as the algal species with the largest cell identification has not been achieved yet. density among a certain taxonomic group, were Although a large-scale dilution has been carried observed as Microcystis aeruginosa. In order to out in several eutrophic lakes such as Green Lake improve the water quality and control (Oglesby, 1969), Moses Lake (Welch and Patmont, cyanobacterial blooms, a large-scale dilution 1980; Welch, 2009), and Lake Taihu (Hu et al., method has been applied to Lake Tega by the 2010), the successful result in the control of Ministry of Land, Infrastructure, Transport, and cyanobacterial blooms as observed in Lake Tega is Tourism (MLIT), the Japanese Government, from quite rare in the world. Thus, clarification of * Graduate School of Science and Engineering, Chiba University ** Graduate School of Engineering, Chiba University *** Safety and Health Organization, Chiba University Journal of Environmental Information Science Vol.2019, No.1 13 Ohori River Lake Te g a Tone River Te g a River Otsu River Somei-iriotoshi River Conveyance channel N Pump station Discharge point and flow direction Flow direction Sampling point 2 km Fig. 1 Inflow and outflow rivers and the North-Chiba Water Conveyance Channel in Lake Tega cyanobacterial blooms suppression mechanisms in are four rivers: the Ohori River, the Otsu River, Lake Tega could lead to the development of and the Somei-iriotoshi River flowing into Lake effective dilution methods for the control of Tega, and the Tega River outflowing the lake water. cyanobacterial blooms. As one of the clues of For the improvement of water quality in Lake elucidation of cyanobacterial blooms suppression Tega, the MLIT constructed the North-Chiba mechanisms, it is important to examine the Water Conveyance Channel (Fig. 1) and competitive interactions between bloom-forming discharged a large amount of the Tone River water species and the competitive species in Lake Tega to from the discharge points through the channel (10 find unfavorable conditions for the growth of m3 s-1 as the maximum) from 2000. As a result, the bloom-forming species. To perform the dilution rate, defined as the amount of inflow experimental investigations for the competitive water a day divided by the total water volume in interactions, exact information on the dominant Lake Tega, was varied approximately from 0.05 species of small Thalassiosirales would be day-1 (before 1999) to 0.15 day-1 (after 2000) required. (Amano et al., 2010; Mikawa et al., 2017). This study aimed to identify the dominant Water samples were taken at 5 cm below the species of small Thalassiosirales in Lake Tega with water surface on April 16, May 16, June 13, July a scanning electron microscope (SEM). SEM is a 15, August 14, September 14, October 12, and useful tool for the species-level identification of November 13, 2017, at the east part in Lake Tega small Thalassiosirales because it can distinguish (Fig. 1). Weather conditions and water the microstructures of frustules. Then, the temperatures on each sampling day were shown in seasonal trend of the composition of small Table 1. The samples were preserved in a cool dark Thalassiosirales was investigated. box and immediately transferred to the laboratory and stored at 4ºC in the dark until use. 1. METHODS The lake water samples were injected into a plankton counting vessel (MPC-200, Matsunami 1.1 Lake Tega and sampling points Glass Industry, Osaka, Japan), and the cell Lake Tega is located in the northwest part of number of small Thalassiosirales in water samples Chiba Prefecture, Japan, and is a small (water was directly counted by using a light microscope surface area of 6.5 km2), shallow (average water (ECLIPSE E100, Nikon, Tokyo, Japan) in depth of 0.86 m), and highly eutrophic lake. There triplicate, and the cell density was calculated. 14 Journal of Environmental Information Science Vol.2019, No.1 Table 1 Weather conditions and water temperatures on each sampling day Date Weather Water temperature (ºC) 2017/4/24 Sunny 17.7 2017/5/16 Sunny 20.2 2017/6/13 Cloudy 20.8 2017/7/18 Sunny 29.9 2017/8/14 Cloudy 26.5 2017/9/5 Cloudy 23.1 2017/10/12 Sunny 26.6 2017/11/13 Sunny 14.1 2.1 Cell densities of small Thalassiosirales Fig. 2 Cell densities of small Thalassiosirales in The number of small Thalassiosirales in the lake each month in 2017 water samples was depicted in Fig. 2. Approximately 1.3 × 104 cells mL-1 of small 1.2 Preparation for SEM observations Thalassiosirales were present in April and May. In The lake water samples were used for SEM June, the cell density of small Thalassiosirales was observations. Each sample was mixed with hot ca. 2.7 times greater than that in May, and the concentrated sulfuric acid and sonicated for 10 highest value (4.1 × 104 cells mL-1) was observed in min to clean diatom cells. Then, the sample was July. The cell density was then decreased, and the rinsed five times with distilled water, and the values in August and September were about 2.0 × harvested cells were suspended in distilled water. 104 and 2.7 × 104 cells mL-1, respectively. In A small portion of the cleaned sample was dripped October and November, the cell density was on an SEM stub and allowed to be dried in air recorded to be lower than that for other sampling overnight. The air-dried sample on the SEM stub periods, indicating ca. 7.3 × 103 and 3.8 × 103 cells was coated with platinum using a sputter coater mL-1, respectively. (JFC-1100, JEOL, Tokyo, Japan) and observed with SEM (JSM-6510A, JEOL, Tokyo, Japan). The 2.2 Species identification of small identification and counting were performed by Thalassiosirales by SEM observations scrolling the SEM screen (SEM stubs) linearly in The species identification of small the horizontal direction at 1000× magnification. Thalassiosirales was performed by SEM By referring to the references (Kobayashi et al., observations based on their morphological features. 2006; Tuji and Houki, 2001; Watanabe, 2005), the The dominant species of small Thalassiosirales species of small Thalassiosirales were identified were identified as Cyclotella atomus, Cyclotella based on the patterns of valve face, structures of meneghiniana, Discostella pseudostelligera, and fultoportulae or rimoportulae, and stria densities Stephanodiscus binderanus complex. S. (Fig. 5-7). Then, the composition of small binderanus and Stephanodiscus hantzschii were Thalassiosirales in the samples of April, May, June, referred to as S. binderanus complex because it July, August, September, October, and November was difficult to distinguish both species by was determined by identifying and counting 100, observing their inside valves. 101, 105, 106, 124, 110, 126, and 132 valves based Fig. 3 represents the monthly composition of on the SEM images, respectively. small Thalassiosirales except for some species that showed the relative cell abundance of less than 5%. 2. RESULTS The cell densities of dominant small Journal of Environmental Information Science Vol.2019, No.1 15 Fig. 3 Relative cell abundance of small Thalassiosirales in each month in 2017 Thalassiosirales were calculated from the cell complex was ca. 30% and it dominated over small densities and relative cell abundance of small Thalassiosirales communities in Lake Tega (Fig. 3). Thalassiosirales and were shown in Fig. 4. The relative cell abundance declined after April, Internal and external valve faces of each species and the percentage fluctuated around 10%. The listed in Fig. 3 are also shown in Figs. 5, 6, and 7. culture experiment for S.