Nutritional Intake by Ectoplasmic Nets of Schizochytrium Aggregatum (Labyrinthulomycetes, Stramenopiles)

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

Graduate School of Natural Science, Konan University, 8-9-1, Okamoto, Higashinada,

Kobe, Hyogo 658-8501, Japan

Institute for Integrative Neurobiology, Konan University, 8-9-1, Okamoto, Higashinada,

Kobe, Hyogo 658-8501, Japan

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.

Key words: Thraustochytrids; CMCase activity; ultrastructure; SEM-EDX; Alcian blue staining; extracellular

polymeric substances.

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

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

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