Protist, Vol. 169, 727–743, xx 2018 http://www.elsevier.de/protis Published online date 18 June 2018 ORIGINAL PAPER Nutritional Intake by Ectoplasmic Nets of Schizochytrium aggregatum (Labyrinthulomycetes, Stramenopiles) a,b b,c,1 Izumi Iwata , and Daiske Honda a Graduate School of Natural Science, Konan University, 8-9-1, Okamoto, Higashinada, Kobe, Hyogo 658-8501, Japan b Institute for Integrative Neurobiology, Konan University, 8-9-1, Okamoto, Higashinada, Kobe, Hyogo 658-8501, Japan c Faculty of Science and Engineering, Konan University, 8-9-1, Okamoto, Higashinada, Kobe, Hyogo 658-8501, Japan Submitted December 30, 2017; Accepted June 9, 2018 Monitoring Editor: Michael Melkonian Thraustochytrid cells attach to their food via ectoplasmic nets (ENs). Here, we analyzed the cause and effect relationship between the various forms and functions of ENs of Schizochytrium aggregatum. The ENs spread out over a large area forming a fine network to efficiently search for the experimental food source. After recognizing the experimental food source, the ENs that attached to the food source became thicker, and net elements developed. The thick ENs on the surface at the attachment site were enveloped in dense materials (fibrous materials), which were visualized as fibrous layers under a transmission electron microscope. Experiments using fluorescein diacetate and the fluorescent glu- cose analog 2-NBDG showed that the production rate of hydrolytic enzymes and the absorption rate of glucose by ENs of S. aggregatum increased in the presence of an experimental food source. Our results reveal that ENs change their shape and function according to the presence/absence of a food source. © 2018 Elsevier GmbH. All rights reserved. Key words: Thraustochytrids; CMCase activity; ultrastructure; SEM-EDX; Alcian blue staining; extracellular polymeric substances. 1 Corresponding author; fax +81 78 435 2539 e-mail [email protected] (D. Honda). Abbreviations: ASW, artificial seawater; CMC, carboxymethylcellulose; EN, ectoplasmic net; EDX, energy dispersive X-ray spectrometry; EPSs, extracellular polymeric substances; FDA, fluorescein diacetate; 2- NBDG, 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose; SEM, scanning electron microscopy; TEM, transmission electron microscopy. https://doi.org/10.1016/j.protis.2018.06.002 1434-4610/© 2018 Elsevier GmbH. All rights reserved. 728 I. Iwata and D. Honda Introduction specific hydrolytic activity in live cells. Perkins (1973) showed that ENs penetrate the sporopol- Thraustochytrids are osmoheterotrophic unicellu- lenin layer of pine pollen. Thus, hydrolytic enzymes lar protists (Stramenopiles, Labyrinthulomycetes) are thought to be secreted by ENs to digest organic (Adl et al. 2012; Honda et al. 1999; Leipe et al. material (Coleman and Vestal 1987; Leano˜ and 1994; Patterson 1989; Tsui et al. 2009) that are Damare 2012; Moss 1986; Perkins 1973). found in marine and estuarine waters worldwide Although ENs are known to be closely related (e.g., Raghukumar 2002; Kimura et al. 1999; to nutrient intake, there is a lack of information Naganuma et al. 1998; Ueda et al. 2015). They about their function. The diameter of ENs changes associate with organic detritus (e.g., mangrove according to culture conditions (Raghukumar leaves and brown algae), macroalgae, diatoms, 2002), and ENs attached to a substrate are up to zooplankton fecal pellets, and marine vascular eight times thicker than free ENs (Gaertner 1981; plants (Bremer 1995; Frank et al. 1994; Moss Goldstein and Belsky 1964; Moss 1980; Ueda 1986; Raghukumar et al. 1995; Raghukumar and et al. 2015; Yokoyama et al. 2007; Yokoyama and Raghukumar 1999; Sathe-Pathak et al. 1993), Honda 2007). Thick ENs are presumed to be thin and produce extracellular hydrolytic enzymes, ENs twisted together (Gaertner 1981; Goldstein such as amylases, cellulases, lipases, proteases, and Belsky 1964; Moss 1980; Ueda et al. 2015). phosphatases, pectinases, and xylanases, which Perkins (1973) observed ENs by transmission elec- decompose organic substrates (Bongiorni et al. tron microscopy (TEM), and found that they consist 2005; Bremer 1995; Bremer and Talbot 1995; of a limited wall-less membrane that contains only Raghukumar et al. 1994; Taoka et al. 2009). Pre- vesicles or narrow internal membrane cisternae vious studies have suggested that thraustochytrids but no organelles. The internal membrane cister- play an important role in degradation and mineral- nae form a tubular or fenestrated-anastomosing ization processes in marine ecosystems. Cellulase array that appears to proliferate when the cells are production is particularly important for degrading grown on natural substrates (e.g., plant or animal plant detritus (e.g., leaves and algae). Bremer cells) rather than artificial substrates (e.g, Vish- (1995) and Bremer and Talbot (1995) demon- niac’s medium) (Perkins 1972, 1973). The plasma strated that the thraustochytrid Schizochytrium membrane of the thick ENs of Thraustochytrium ␤ aggregatum produces extracellular cellulases ( - motivum was found to be enveloped in fibrous mate- 1,4-glucanases) using a water-soluble cellulose rials that were different from the cell wall and formed ether, carboxymethylcellulose (CMC) as the sub- a layered structure adhering to the EN surface strate. Nagano et al. (2011) evaluated cellulolytic (Perkins 1972, 1973). ␤ ( -1,4-glucanase) activity in nine genera of thraus- Although there have been several ultrastruc- tochytrids using the same experimental protocols tural studies on ENs, few studies have focused on used by Bremer and Talbot (1995) and found that S. their function. The morphologies of ENs observed ␤ aggregatum had the highest -1,4-glucanase activ- in previous reports suggested possible functions ity. including selective enzyme secretion, nutrient Despite the importance of thraustochytrids in absorption, and food sensing, but these functions marine biology and biotechnology (especially lipid have not been validated experimentally. The pur- production), there is still a lack of basic biolog- pose of this study was to provide basic information ical knowledge about their nutrient intake. The on the mechanism of nutrient intake by ENs and ectoplasmic net (EN) is a branched network of to clarify the cause and effect relationship between plasma membrane extensions like pseudopodial the various forms and functions of ENs. We tested extensions (Moss 1985; Perkins 1972, 1973; Porter two hypotheses: (1) Cells can sense whether or 1969, 1972, 1989). The EN can often be dis- not a substrate is food via their ENs and release cerned as being separate from the cell body. In enzymes from the ENs to break down food; (2) Cells thraustochytrids, the EN emanates from the basal absorb nutrients through their ENs. We tested these side where it extends from a single bothrosome hypotheses in the following six experiments: (Moss 1980; Perkins 1972; Porter 1989). Vegetative Experiment 1: To test hypothesis (1), we ana- cells attach to substrates via the EN (Bower et al. lyzed the CMCase activity of S. aggregatum in 1989; Perkins 1973; Porter 1972). Coleman and various conditions. We tested different experimen- Vestal (1987) confirmed that ENs have associated tal food sources to determine which ones were enzymatic activity in an experiment with fluores- recognized as food (as reflected by increased cein diacetate (FDA), a cell-permeating esterase CMCase activity). The substrates were materials substrate that can be used to measure non- that could be found in the natural environment: Download English Version: https://daneshyari.com/en/article/8392700 Download Persian Version: https://daneshyari.com/article/8392700 Daneshyari.com.