Sei sulla pagina 1di 7

on April 9, 2016 by guesthttp://aem.asm.org/Downloaded

from

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Aug. 2005, p. 4516–4522

0099-2240/05/$08.00 0 doi:10.1128/AEM.71.8.4516–4522.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 71, No. 8

Isolation and Characterization of a Novel Single-Stranded RNA Virus Infectious to a Marine Fungoid Protist, Schizochytrium sp. (Thraustochytriaceae, Labyrinthulea)

Yoshitake Takao, 1 Keizo Nagasaki, 2 Kazuyuki Mise, 3 Tetsuro Okuno, 3 and Daiske Honda 1 *

Department of Biology, Faculty of Science and Engineering, Konan University, 8-9-1 Okamoto, Higashinada, Kobe 658-8501, Japan 1 ; National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency, 2-17-5 Maruishi, Ohno, Saeki, Hiroshima 739-0452, Japan 2 ; and Laboratory of Plant Pathology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan 3

Received 5 September 2004/Accepted 3 March 2005

Thraustochytrids are cosmopolitan osmoheterotrophic microorganisms that play important roles as decom- posers, producers of polyunsaturated fatty acids, and pathogens of mollusks, especially in coastal ecosystems. SssRNAV, a novel single-stranded RNA (ssRNA) virus infecting the marine fungoid protist Schizochytrium sp. (Labyrinthulea, Thraustochytriaceae) was isolated from the coastal water of Kobe Harbor, Japan, in July 2000, and its basic characteristics were examined. The virus particle is icosahedral, lacks a tail, and is ca. 25 nm in diameter. SssRNAV formed crystalline arrays and random assemblies within the cytoplasm of host cells, and it was also concentrated along the intracellular membrane structures. By means of one-step growth experi- ments, the lytic cycle and the burst size were estimated to be <8 h and 5.8 10 3 to 6.4 10 4 infectious units per host cell, respectively. SssRNAV had a single molecule of ssRNA that was approximately 10.2 kb long, three major proteins (37, 34, and 32 kDa), and two minor proteins (80 and 18 kDa). Although SssRNAV was considered to have some similarities with invertebrate viruses belonging to the family Dicistroviridae based on its partial nucleotide sequence, further genomic analysis is required to determine the detailed classification and nomenclature of SssRNAV. Our results indicate that viral infection is one of the significant factors controlling the dynamics of thraustochytrids and provide new insights into understanding the ecology of these organisms.

Thraustochytrids are marine fungoid protists classified in the class Labyrinthulea in the kingdom Chromista (8, 9). They are comprised of six genera (33, 47), Althornia (26), Aplanochy- trium (2), Japonochytrium (32), Schizochytrium (18), Thraus- tochytrium (59), and Ulkenia (13). However, it has been shown that the current classification of these genera based on mor- phology does not agree with the molecular phylogenetic rela- tionships based on the 18S rRNA gene sequences (21). Cur- rently, in order to resolve the confusion regarding the classification and nomenclature of the thraustochytrids, further comparative studies based on morphology, molecular phylog- eny, and chemotaxonomy are under way (R. Yokoyama, per- sonal communication). Hence, some of the thraustochytrid strains tested in the present study have not been fully identified yet (Table 1). Thraustochytrids are distributed in saline lakes and in ma- rine, estuarine, and deep-sea waters throughout the world (35, 47, 52), and they have been isolated from algal and plant material, as well as from sediments and water (14, 37, 55, 59). Recently, a rapid direct detection technique for thraus- tochytrids using the fluorogenic acriflavine dye was developed (53). Using this method, Naganuma et al. (39) estimated the abundance of thraustochytrids in the Seto Inland Sea of Japan

* Corresponding author. Mailing address: Department of Biology, Faculty of Science and Engineering, Konan University, 8-9-1 Oka- moto, Higashinada, Kobe 658-8501, Japan. Phone: 81 78-435-2515. Fax: 81 78-435-2539. E-mail: dhonda@konan-u.ac.jp.

4516

and demonstrated that the biovolume of thraustochytrids in coastal waters could reach 43% of the bacterial biovolume. The wide distribution and high abundance of these organisms indicate their ecological importance as decomposers (39, 52). In addition, thraustochytrids are known to produce large amounts of polyunsaturated fatty acids (PUFA), such as do- cosahexaenoic acid and docosapentaenoic acid (44), which are considered important food resources for higher organisms in marine systems (30, 34, 50). Furthermore, some species of thraustochytrids are known to be pathogens of mollusks, such as octopuses and bivalves (1, 46, 49). Because of these distinc- tive features, the ecological significance of thraustochytrids in the coastal ecosystems has been highlighted (50, 51). On the other hand, viruses are very abundant in marine environments (3, 48). Viruses and virus-like particles (VLPs) have been discovered in a variety of phytoplankton and bacte- ria (48, 54, 64) and have been recognized as important agents in controlling bacterial and algal biomass (4, 41, 48) and nu- trient cycling (17, 66) and in maintaining the biodiversity of bacteria and microalgae (5, 10, 12). So far, more than 13 viruses infecting marine microalgae have been isolated and characterized (5). The majority of these viruses are large (100- to 200-nm) double-stranded DNA viruses, and they are either included in the family Phycodnaviridae (5, 58, 65) or considered most likely to belong to this family based on some character- istics (7, 16, 23, 43, 57, 62). In contrast, there have been only a small number of reports describing RNA viruses infecting marine eukaryotic microor-

on April 9, 2016 by guesthttp://aem.asm.org/Downloaded

from

VOL. 71, 2005

ssRNA VIRUS INFECTING SCHIZOCHYTRIUM SP.

4517

TABLE 1. Infection specificities of SssRNAV with 19 strains of marine microorganisms

Taxon

Strain a

Group b

Isolation locality

Sensitivity

to

SssRNAV c

Kingdom Chromista Phylum Sagenista Class Labyrinthulea

Schizochytrium sp. strain NIBH N1-27 d

1

Nakaminato Harbor, Ibaragi, Japan

Schizochytrium sp. strain SEK 0209

1

Kobe Harbor, Hyogo, Japan

Schizochytrium limacinum IFO 32693 d

1

Colonia, Yap Island

Thraustochytrium aureum ATCC 34304 d

2

Woods Hole, Mass.

Thraustochytriaceae sp. strain MBIC 11066

1

Iriomote Island, Okinawa, Japan

Thraustochytriaceae sp. strain MBIC 11071

1

Iriomote Island, Okinawa, Japan

Thraustochytriaceae sp. strain MBIC 11072

1

Iriomote Island, Okinawa, Japan

Thraustochytriaceae sp. strain SEK 0210

2

Okinawa Island, Okinawa, Japan

Thraustochytriaceae sp. strain SEK 0211

2

Ishigaki Island, Okinawa, Japan

Thraustochytriaceae sp. strain SEK 0212

2

Ishigaki Island, Okinawa, Japan

Thraustochytriaceae sp. strain SEK 0213

2

Iriomote Island, Okinawa, Japan

Thraustochytriaceae sp. strain SEK 0214

2

Hiroshima Bay, Hiroshima, Japan

Class Bicoecea

Cafeteria sp. strain SEK 0124

Awaji Island, Hyogo, Japan

Phylum Ochrophyta Class Bacillariophyceae

Nitzschia sp. strain SEK 0215

Kobe Harbor, Hyogo, Japan

Skeletonema sp. strain SEK 0135

Kobe Harbor, Hyogo, Japan

Class Raphidophyceae

Heterosigma akashiwo SEK 0023

Kure Bay, Hiroshima, Japan

Class Chrysophyceae

Sulcochrysis biplastida MBIC 10502

Yokohama Harbor, Kanagawa, Japan

Kingdom Protozoa

Phylum Euglenozoa

Bodo sp. strain SEK 0126

Awaji Island, Hyogo, Japan

Phylum Dinozoa

Prorocentrum sp. strain SEK 0112

Takamatsu Harbor, Kagawa, Japan

a ATCC, American Type Culture Collection, (United States); MBIC, Marine Biotechnology Institute Culture Collection (Japan); IFO, Institute for Fermentation, Osaka (Japan); NIBH, National Institute of Bioscience and Human Technology (Japan); SEK, Laboratory of Systematics and Evolution, Konan University (Japan).

b Results of phylogenetic grouping for the strains tested (R. Yokoyama, personal communication).

c , lysed; , not lysed.

d Strain used for virus isolation.

ganisms. So far, three RNA viruses infecting marine eukaryotic microalgae have been isolated; two of them are single-stranded RNA (ssRNA) viruses (HaRNAV and HcRNAV), and one is

a double-stranded RNA (dsRNA) virus (MpRNAV).

HaRNAV is infectious to one of the most noxious bloom- forming phytoflagellates, Heterosigma akashiwo (Raphido-

phyceae), and has an ssRNA genome that is 9.1 kb long (61). HcRNAV is infectious to the bivalve-killing dinoflagellate Het- erocapsa circularisquama and has an approximately 4.4-kb ss- RNA genome (63). MpRNAV is infectious to the cosmopoli-

tan phytoplankter Micromonas pusilla and harbors 11 segments

of dsRNA as the viral genome, the total length of which is 25.5

kbp (6). In addition, the relationship between protists and their vi- ruses is poorly understood. Nagasaki et al. (40, 42) observed VLPs in marine apochlorotic flagellates and suggested that

viral infection might be one of the factors affecting their dy- namics. Garza and Suttle (15) isolated and characterized a large dsDNA virus infecting a marine heterotrophic nanoflagellate, Bodo sp., which also shared some characteris-

tics with viruses belonging to the family Phycodnaviridae. For

thraustochytrids, Kazama and Schornstein (28, 29) found her- pes-type VLPs in Thraustochytrium sp. (Thraustochytriaceae, Labyrinthulea) which were roundish, enveloped, 110 nm in diameter, and predicted to have a DNA genome. However, because the VLPs were not successfully brought into culture, further study could not be completed. In the present report, we describe the isolation and charac- terization of a novel ssRNA virus infecting Schizochytrium sp.

(Thraustochytriaceae, Labyrinthulea). To our knowledge, this is the first report describing the biological properties of an RNA virus infecting marine fungoid protists.

MATERIALS AND METHODS

Microorganism cultures. Strains of thraustochytrids and other microorgan- isms used in this study are listed in Table 1. All of these organisms are clonal, as established by the micropipetting method or an extinction dilution method. Thraustochytrids were grown at 20°C in 10 medium H (medium H is 0.2% glucose, 0.02% yeast extract, and 0.05% monosodium glutamate in seawater) (20). Other organisms were grown at 20°C in IMK medium (Wako Co., Ltd.) or f/2 medium (19). For cultivation of phytoplankton, the light conditions were 12 h of light (55 mol photons m 2 s 1 ; cool white fluorescent illumination) and 12 h of darkness. Isolation of lytic viruses. Surface water was collected in Kobe Harbor, Hyogo Prefecture, Japan, on 26 July 2000. It was filtered through a 0.2- m-pore-size filter (Nuclepore) to remove eukaryotic microorganisms and most bacteria. Ali- quots (100 l) of the filtrate were inoculated into exponentially growing cultures (150 l) of the three thraustochytrid strains shown in Table 1 and incubated at 20°C. Cultures inoculated with filtrates treated at 121°C for 15 min served as controls. Test cultures were checked by optical microscopy for 14 days to exam- ine whether cell lysis occurred. In the Schizochytrium sp. strain NIBH N1-27 culture inoculated with the filtrate, apparent growth inhibition was detected, although no lysis was observed in cultures of the other two strains and control cultures. Then a clonal lytic agent was obtained through two cycles of the extinction dilution procedure (43, 60). The lysate in the most diluted well of the second assay was inoculated into a 50-ml fresh culture of Schizochytrium sp. strain NIBH N1-27, and the resultant lysate was filtered through a 0.2- m-pore- size filter (Nuclepore); then 0.3 ml of the filtrate was mixed with 1 ml of 10% glycerol in 10 medium H and cryopreserved at 80°C as the original pathogen suspension. Serial transfers of a lysed culture to an exponentially growing culture of Schizochytrium sp. strain NIBH N1-27 were performed more than twice to verify the transferability. The concentration of the pathogenic agent was esti-

on April 9, 2016 by guesthttp://aem.asm.org/Downloaded

from

4518 TAKAO ET AL.

mated by the extinction dilution method (43, 60) using the computer program described by Nishihara et al. (45). Host range test. The host range of the pathogenic agent was examined by adding 100- l portions of the original pathogen suspensions to 1-ml cultures of the exponentially growing microorganisms listed in Table 1. The cultures were observed by optical microscopy. Cultures that were not lysed after 10 days were considered to be unsuitable hosts for the pathogen.

Growth experiment. In the one-step growth experiments, an exponentially growing culture of Schizochytrium sp. strain NIBH N1-27 was inoculated with the pathogen suspension at multiplicities of infection of 20.5, 21.2, and 30.1. Control cultures, to which an autoclaved (121°C, 15 min) filtrate was added, were also used for comparison. Aliquots of the cell suspension were removed every 8 h; then the cell density was estimated by optical microscopy, and the pathogen density was measured by the extinction dilution method (43, 60). Each experi- ment was performed in triplicate. TEM. For transmission electron microscopy (TEM) observations of the patho- gen, exponentially growing cultures of Schizochytrium sp. strain NIBH N1-27 were inoculated with the pathogen, and samples (2 ml) were removed at 0, 8, 16, and 24 h postinoculation. Each cell suspension was mixed with an equal volume

of a fixing cocktail (5% glutaraldehyde, 0.2 M sucrose, 0.2 M cacodylate buffer)

and kept on ice for 2 h. Cells were harvested by centrifugation at 640 g for 2 min; then the pellet was rinsed three times with 0.2 M cacodylate buffer and

postfixed with 1% buffered OsO 4 for 1.5 h on ice. Following three rinses with 0.2

M cacodylate buffer, the pellet was dehydrated in a graded ethanol series (30 to

100%) and embedded in Spurr’s resin (NISSHIN EM Co., Ltd). Thin sections were stained with 1% uranyl acetate and 3% lead citrate and observed at 80 kV using a JEOL JEM-1010 transmission electron microscope. Negatively stained pathogens were also observed by TEM. Briefly, a pathogen suspension was mounted on a grid (no. 780111630; JEOL DATUM Ltd.) for 30 s, and excess water was removed with filter paper (no. 1; TOYO Co., Ltd.). Then 4% uranyl acetate was added to the grid for 10 s, and the excess dye was removed with filter paper. After the grid was dried in a desiccator for 10 min, negatively stained pathogens were observed by TEM at an acceleration voltage of 80 kV. Particle diameters were estimated based on the negatively stained images. Analysis of nucleic acids and proteins. A Schizochytrium sp. strain NIBH

N1-27 culture (1.5 liters) was inoculated with 5 ml of a fresh pathogen suspension and lysed. Then the lysates were centrifuged at 14,000 g for 15 min to remove the cellular debris. The supernatant was added to polyethylene glycol 6000 (Wako Co., Ltd.) at a final concentration of 10% (wt/vol) and stored at 4°C overnight. After centrifugation at 3,600 g for 1.5 h, the pellet was suspended

in 10 mM phosphate buffer (10 mM Na 2 HPO 4 and 10 mM KH 2 PO 4 in distilled

water) and centrifuged at 100,000 g for 2 h. This purification process was

repeated twice. The pellet was resuspended in 750 l of distilled water; then the pathogen suspension was treated with proteinase K (final concentration, 1 mg

ml 1 ; Nippon Gene) at 55°C for 1.5 h. Nucleic acids were extracted from the

pellet by using TRIzol LS (Invitrogen), precipitated with ethanol, and then suspended in 50 l of distilled water. Aliquots (2 l) of the suspension were digested at 37°C for 1 h with RNase A (final concentration, 0.1 g l 1 ; Nippon

Gene) in 0.01 SSC or 2 SSC (1 SSC is 0.15 M NaCl plus 0.015 M trisodium citrate, pH 7.0) or with DNase I (final concentration, 10 U l 1 ; TAKARA Bio Inc.). Extract kept on ice with no enzymatic treatment served as a control. A

formaldehyde-agarose gel (1%; 15 by 20 cm; Seakem Gold Agarose; BMA Inc.) was loaded with 20 l of nucleic acid per lane and electrophoresed at 50 V for 14.5 h. Nucleic acids were visualized by SYBR Green II staining (Molecular Probes, Inc.). In addition, the pathogen suspension was mixed with one-third volume of 4 sample buffer (250 mM Tris-HCl, pH 6.8, 8% 2-mercaptoethanol, 8% sodium dodecyl sulfate [SDS], 40% glycerol, 0.04% bromophenol blue) and boiled for 5 min; then the proteins were separated by SDS-polyacrylamide gel electrophore-

sis (SDS-PAGE) using 15% polyacrylamide gels at 150 V for 20 min. Proteins

were visualized by Coomassie brilliant blue staining. SDS-PAGE standards (Bio-

Rad Laboratories, Inc.) with molecular masses ranging from 14.4 to 97.4 kDa were used for size calibration.

RESULTS AND DISCUSSION

Virus isolation and host range. A pathogenic agent causing lysis of Schizochytrium sp. strain NIBH N1-27 was isolated from the surface water collected in Kobe Harbor. This patho- gen was serially transferable to fresh host cultures, in which more than 95% of the host cells were lysed within 36 h after

APPL. ENVIRON. MICROBIOL.

were lysed within 36 h after A PPL . E NVIRON . M ICROBIOL . FIG.

FIG. 1. Optical microphotographs of Schizochytrium sp. strain NIBH N1-27. (A) Intact cells; (B) lysed cells 48 h after inoculation of SssRNAV.

virus inoculation (Fig. 1). In the exponential growth stage, Schizochytrium sp. strain NIBH N1-27 exhibits two distinct forms: vegetative growth and formation of zoospores. Based on the present observations, young zoospores appeared to be highly sensitive to viral infection because settlement to the bottom of vessels was immediately followed by cell lysis (data not shown). Further investigation is required to elucidate the relationship between the host’s life cycle and virus sensitivity. Based on the TEM observations, small VLPs were observed in the lysate of a Schizochytrium sp. strain NIBH N1-27 culture inoculated with the pathogen (Fig. 2A). Although healthy cells of Schizochytrium sp. strain NIBH N1-27 in the control cultures had cytoplasmic structures diagnostic of thraustochytrids and showed no symptoms of viral infection (Fig. 2B), VLPs whose sizes were similar were also observed in the cytoplasm of Schizochytrium sp. strain NIBH N1-27 cells inoculated with the pathogen (Fig. 2C, D, and E). Based on these results, it was demonstrated that (i) the pathogen was transferable to a fresh culture and caused cell lysis, (ii) the VLPs specifically appeared in lysed cultures, and (iii) the VLPs were not detected in healthy cultures, thus fulfilling Koch’s postulates. Therefore, we concluded that the VLP was a lytic virus infecting Schizochytrium sp. strain NIBH N1-27. We designated the virus SssRNAV (Schizochytrium single-stranded RNA virus). SssRNAV caused lysis of four thraustochytrid strains tested in the present experiments (NIBH N1-27, SEK 0209, MBIC 11066, and MBIC 11072) but had no effect on the other thraus- tochytrid strains and some unialgal strains (Table 1). As thraustochytrid strains from different localities showed sensi- tivity to SssRNAV, we predicted that this host-virus system is common at least along the central to western coast of Japan. Because the SssRNAV-sensitive Schizochytrium sp. strain SEK 0209 was isolated from the same sampling site as SssRNAV, it is likely that SssRNAV has some impact on the dynamics of thraustochytrids in natural environments. Because the classifications of thraustochytrids based on mor- phology and molecular phylogeny do not necessarily agree with each other (21), it is difficult to discuss whether SssRNAV is species specific or strain specific based only on the present results. However, it is notable that the four SssRNAV-sensitive strains (NIBH N1-27, SEK 0209, MBIC 11066, and MBIC 11072) belong to a particular group (Table 1) based on mo- lecular phylogeny (22; R. Yokoyama, personal. communica- tion).

VOL. 71, 2005

ssRNA VIRUS INFECTING SCHIZOCHYTRIUM SP.

4519

on April 9, 2016 by guesthttp://aem.asm.org/Downloaded from
on April 9, 2016 by guesthttp://aem.asm.org/Downloaded
from

FIG. 2. Transmission electron microphotographs of Schizochytrium sp. strain NIBH N1-27 infected by SssRNAV. (A) Negatively stained SssRNAV particles in the culture lysate; (B) thin section of a healthy cell of Schizochytrium sp. strain NIBH N1-27; (C) thin section of an SssRNAV-infected cell at 8 h postinoculation (the arrowheads indicate fibrils within vacuoles); (D) crystalline arrays of SssRNAV; (E) unordered aggregation of SssRNAV; (F) virus particles concentrated along the intracellular membranes. N, nucleus; G, Golgi body; Mt, mitochondrion.

on April 9, 2016 by guesthttp://aem.asm.org/Downloaded

from

4520 TAKAO ET AL.

htt p: // aem.asm.org/Downloaded from 4520 TAKAO ET AL. FIG. 3. Formaldehyde-agarose gel electrophoresis of viral

FIG. 3. Formaldehyde-agarose gel electrophoresis of viral nucleic acids. SssRNAV nucleic acids were not treated (lane 2), were treated with RNase A at 37°C under low-salt conditions (lane 3) or high-salt conditions (lane 4), or were treated with DNase I at 37°C (lane 5). Lane 1 contained an RNA molecular marker.

Morphology of SssRNAV. The particles of SssRNAV were 25 2 nm in diameter (average standard deviation) and angular and lacked a tail and an external membrane (Fig. 2A). SssRNAV often formed crystalline arrays (Fig. 2C and D) or random aggregations (Fig. 2E) in the host cell’s cytoplasmic area. Both crystalline array formation and unordered aggrega- tion within the cytoplasm are common features of several ssRNA viruses infectious to plants (36, 56), picornaviruses infectious to animals (11, 24), and marine algal ssRNA viruses (HaRNAV and HcRNAV) (61, 63). It was also notable that the virus particles were concentrated along the intracellular membrane structures (Fig. 2F). In SssRNAV-infected cells, vacuolation of the cytoplasm and the appearance of fibrils within small vacuoles were apparent (Fig. 2C). Vacuolation of the cytoplasm and the appearance of fibrils within vacuoles were also observed with HaRNAV (61). Genome of SssRNAV. Denaturing gel electrophoresis re- vealed that SssRNAV has a single molecule of nucleic acid that is approximately 10.2 kb long and is sensitive to RNase A treatment under both low- and high-salt conditions but is re- sistant to DNase I (Fig. 3). These data indicate that the SssRNAV genome is ssRNA. Based on these observations, SssRNAV is thought to be related to the picornavirus-like superfamily, the vertebrate virus families Picornaviridae (genomic RNA size, 7 to 8 kb) and Caliciviridae (7 to 8 kb), the plant virus family Sequiviridae (9 to 12 kb), and the inverte- brate virus family Dicistroviridae (9 to 12 kb), all of which have a poly(A) tail. Partial sequencing of the SssRNAV genome is now under way (data not shown), and the data reveal some similarity (E values, 1e-25 to 3e-22) with Triatoma virus, Dro- sophila C virus (DCV), Acute bee paralysis virus, and Taura syndrome virus belonging to the family Dicistroviridae. Further characterization of the viral genome, however, is necessary to determine the taxonomic position of SssRNAV. Considering that the hosts of the family Dicistroviridae are mainly crusta- ceans (insects and shrimps), the process of host range expan-

APPL. ENVIRON. MICROBIOL.

process of host range expan- A PPL . E NVIRON . M ICROBIOL . FIG. 4.

FIG. 4. SDS-PAGE of SssRNAV structural proteins. The gel was stained with Coomassie brilliant blue.

sion and evolution of SssRNAV and related viruses is of great interest. Proteins of SssRNAV. The protein analysis showed that SssRNAV has three major proteins (37, 34, and 32 kDa) and two minor proteins (80 and 18 kDa) (Fig. 4). The strength of the 16-kDa band was variable in the experiments (more than five experiments); thus, we could not verify if it originated from the host cells or SssRNAV particles. The band pattern of SssRNAV resembles that of DCV be- longing to the family Dicistroviridae, which has three major capsid proteins (31, 30, and 28 kDa) and one minor capsid protein (8.5 kDa) (25, 27). In addition, considering that capsid proteins of DCV are processed out of a precursor protein (100 kDa) (31, 38), the functions of the 80-kDa protein of SssRNAV are also of interest. Precise interpretation of the present results awaits future studies.

interpretation of the present results awaits future studies. FIG. 5. Changes in abundance of Schizochytrium sp.

FIG. 5. Changes in abundance of Schizochytrium sp. strain N1-27 cells with () or without ( ) viral inoculation and the viral titer (E). SssRNAV inoculation was performed in the exponential growth phase of host cultures (arrow). Results for only one of the triplicate experi- ments are shown. The error bars indicate the 95% confidence limits for the viral titer.

on April 9, 2016 by guesthttp://aem.asm.org/Downloaded

from

VOL. 71, 2005

Replication of SssRNAV. In the triplicate one-step growth experiments, a rapid decrease in host cell abundance within 8 h after virus inoculation was accompanied by a rapid increase in the viral titer (Fig. 5); thus, the latent period of SssRNAV was estimated to be 8 h. The burst sizes estimated from the experiments ranged from 5.8 10 3 to 6.4 10 4 infectious units cell 1 . When SssRNAV was compared with the other ssRNA viruses infecting microalgae, the latent period of SssRNAV was found to be much shorter than those of HaRNAV ( 12 days) (61) and HcRNAV (1 to 2 days) (63), and the burst size was found to be similar to that of HcRNAV (3.4 10 3 to 16 10 3 infectious units cell 1 ) (63). TEM observations revealed that 3.4 10 3 SssRNAV particles were scattered in a thin section of an infected cell 8 h after virus inoculation (Fig. 2C), which suggests that 6.0 10 5 virus particles can be present in a whole cell based on geomet- ric analysis. Thus, the possibility of underestimation of the burst sizes should be noted. Although the reasons for the underestimation have not been elucidated, the most likely ex- planations are that (i) a cluster of crystallized virus particles was counted as one infectious unit by the extinction dilution method and (ii) incomplete virus particles (lacking infectivity) accounted for a considerable proportion. In addition, it is also possible that PUFA excreted from burst cells interfered with the adsorption of viruses to host cells, because an increase in the viral titer of host lysates was often observed when the PUFA fraction was excluded by filtration through 0.2- m- pore-size filters (data not shown). Ecological implications. In the present study, we examined the characteristics of a novel ssRNA virus, SssRNAV, which infects the marine fungoid protist Schizochytrium sp. The char- acteristics of SssRNAV are quite different from those of the herpes-type VLPs found in Thraustochytrium sp. reported by Kazama and Schornstein (28, 29). As far as we know, this is the first report of an ssRNA virus infecting marine unicellular protists. Because of the recent progress in studies of marine viruses, the importance of viral impact on marine phytoplankton and bacteria has been highlighted. The present results emphasize the possibility that fungoid protists are also exposed to viral attack in marine systems. From the viewpoint of marine mi- crobial ecology, thraustochytrids are considered to have a role as decomposers; i.e., they presumably feed on dying and dead cells of microorganisms in marine environments. Actually, it has been reported that Schizochytrium sp. cells prey on a bloom-forming diatom (Thalassiosira sp.) (14), suggesting that this organism may have a role as a decomposer in the terminal stage of algal blooms. These considerations invite further em- pirical investigation of the dynamics of decomposers and their viruses, in addition to blooming algae and their viruses. Com- parisons of the dynamics of these microalgal components in bloom events are of great interest.

ACKNOWLEDGMENTS

This work was supported by the Kato Memorial Bioscience Foun- dation and the Asahi Glass Foundation. We are grateful to Toro Nakahara, Toshihiro Yokochi (National Institute of Advanced Industrial Science and Technology, Japan), and Rinka Yokoyama (Konan University, Japan), who kindly provided the thraustochytrid strains. We thank Hiroshi Kawai, Akio Murakami, Mitsunobu Kamiya (Kobe University, Japan), and Yuji Tomaru (Na-

ssRNA VIRUS INFECTING SCHIZOCHYTRIUM SP.

4521

tional Research Institute of Fisheries and Environment of Inland Sea, Japan) for their technical advice concerning transmission electron mi- croscopy. We also thank Tokushiro Takaso (University of the Ryukyus, Japan), who kindly provided seawater samples.

REFERENCES

1. Azevedo, C., and L. Corral. 1997. Some ultrastructural observations of a thraustochytrid (Protoctista, Labyrinthulomycota) from the clam Ruditapes descussatus (Mollusca, Bivalvia). Dis. Aquat. Org. 31: 73–78.

2. Bahnweg, G., and F. K. Sparrow. 1972. Aplanochytrium kerguelensis gen. nov. spec. nov., a new phycomycete from subantarctic marine waters. Arch. Mi- crobiol. 81:45–49.

3. Bergh, Ø., K. Y. Børsheim, G. Bratbak, and M. Heldal. 1989. High abun- dance of viruses found in aquatic environments. Nature 340:467–468.

4. Bratbak, G., J. K. Egge, and M. Heldal. 1993. Viral mortality of the marine alga Emiliania huxleyi (Haptophyceae) and termination of algal blooms. Mar. Ecol. Prog. Ser. 93: 39–48.

5. Brussaard, C. P. D. 2004. Viral control of phytoplankton populations—a review. J. Eukaryot. Microbiol. 51:125–138.

6. Brussaard, C. P. D., A. A. M. Noordeloos, R.-A. Sandaa, M. Heldal, and G. Bratbak. 2004. Discovery of a dsRNA virus infecting the marine photosyn- thetic protist Micromonas pusilla . Virology 319:280–291.

7. Brussaard, C. P. D., S. M. Short, C. M. Frederickson, and C. A. Suttle. 2004. Isolation and phylogenetic analysis of novel virus infecting the phytoplank- ton Phaeocystis globosa (Prymnesiophyceae). Appl. Environ. Microbiol. 70:

3700–3705.

8. Cavalier-Smith, T. 1997. Sagenista and Bigyra, two phyla of heterotrophic heterokont chromists. Arch. Protistenkd. 148: 253–267.

9. Cavalier-Smith, T., M. T. E. P. Allsopp, and E. E. Chao. 1994. Thraus- tochytrids are chromists, not fungi: 18S r RNA signature of Heterokonta. Phil. Trans. R. Soc. Lond. B Biol. Sci. 346: 387–397.

10. Cottrell, M. T., and C. A. Suttle. 1991. Wide-spread occurrence and clonal variation in viruses which cause lysis of a cosmopolitan, eukaryotic marine phytoplankter, Micromonas pusilla . Mar. Ecol. Prog. Ser. 78:1–9.

11. Dales, S., H. J. Eggers, I. Tamm, and G. E. Palade. 1965. Electron micro- scopic study of the formation of poliovirus. Virology 26:379–389.

1999. Marine viruses and their biogeochemical and ecolog-

12. Fuhrman, J. A

ical effects. Nature 399: 541–548.

13. Gaertner, A. 1977. Revision of the Thraustochytriaceae (lower marine fungi). I. Ulkenia nov. gen., with description of three new species. Vero¨ff. Inst. Meeresforsch. Bremerh. 16: 139–157.

1979. Some fungal parasites found in the diatom populations of

the Rusfjord area (South Norway) during March 1979. Veroeff. Inst. Meeres- forsch. Bremerhav. 18: 29–33.

15. Garza, D. R., and C. A. Suttle. 1995. Large double-stranded DNA viruses which cause the lysis of a marine heterotrophic nanoflagellate ( Bodo sp.) occur in natural marine viral communities. Aquat. Microb. Ecol. 9: 203–210.

16. Gastrich, M. D., O. R. Anderson, S. S. Benmayor, and E. M. Cosper. 1998. Ultrastructural analysis of viral infection in the brown-tide alga, Aureococcus anophagefferens (Pelagophyceae). Phycologia 37:300–306.

17. Gobler, C. J., D. A. Hutchins, N. S. Fisher, E. M. Cosper, and S. A. Sanudo- Wilhelmy. 1997. N release and bioavailability of C, P, Se, and Fe following viral lysis of marine chrysophyte. Limnol. Oceanogr. 42: 1492–1504.

18. Goldstein, S., and M. Belsky. 1964. Axenic culture studies of a new marine phycomycete possessing an usual type of asexual reproduction. Am. J. Bot. 51: 72–78.

19. Guillard, R. R. L., and J. H. Ryther. 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt, and Detonula confervacea (Cleve) Gran. Can. J. Microbiol. 8:229–239.

20. Honda, D., T. Yokochi, T. Nakahara, M. Erata, and T. Higashihara. 1998. Schizochytrium limasinum sp. nov., a new thraustochytrid from a mangrove area in the west Pacific Ocean. Mycol. Res. 102: 439–448.

21. Honda, D., T. Yokochi, T. Nakahara, S. Raghukumar, A. Nakagiri, K. Schaumann, and T. Higashihara. 1999. Molecular phylogeny of labyrinthu- lids and thraustochytrids based on the sequence of 18S ribosomal RNA gene. J. Eukaryot. Microbiol. 46: 637–647.

22. Huang, J., T. Aki, T. Yokochi, T. Nakahara, D. Honda, S. Kawamoto, S. Shigeta, K. Ono, and O. Suzuki. 2003. Grouping newly isolated docosa- hexaenoic acid-producing thraustochytrids based on their polyunsaturated fatty acid profiles and comparative analysis of 18S rRNA genes. Mar. Bio- technol. 5:450–457.

23. Jacobsen, A., G. Bratbak, and M. Heldal. 1996. Isolation and characteriza- tion of a virus infecting Phaeocystis pouchetii (Prymnesiophyceae). J. Phycol.

14. Gaertner, A

32:923–927.

24. Je´se´quel, A.-M., and J. M. Steiner. 1966. Some ultrastructural and histo- chemical aspects of coxackie virus-cell interactions. Lab. Investig. 15: 1055–

1083.

25. Johnson, K. N., and P. D. Christian. 1998. The novel genome organization of the insect picorna-like virus Drosophila C virus suggests this virus belongs to a previously undescribed virus family. J. Gen. Virol. 79: 191–203.

on April 9, 2016 by guesthttp://aem.asm.org/Downloaded

from

4522 TAKAO ET AL.

26. Jones, E. B. G., and D. J. Alderman. 1971. Althornia crouchii gen. et sp. nov., a marine biflagellate fungus. Nova Hedwigia 21: 381–400.

27. Jousset, F., M. Bergoin, and B. Revet. 1977. Characterization of the Dro- sophila C virus. J. Gen. Virol. 34: 269–285.

28. Kazama, F. Y., and K. L. Schornstein. 1972. Herpes-type virus particles associated with a fungus. Science 177: 696–697.

29. Kazama, F. Y., and K. L. Schornstein. 1973. Ultra-structure of a fungus herpes-type virus. Virology 52: 478–487.

30. Kimura, H., T. Fukuba, and T. Naganuma 1999. Biomass of thraustochytrid protoctists in coastal water. Mar. Ecol. Prog. Ser. 189: 27–33.

31. King, L. A., J. S. K. Pullin, and N. F. Moore. 1984. Characterization of a picornavirus isolated from a tumorous blood cell line of Drosophila melano- gaster . Microbios Lett. 26: 121–127.

32. Kobayashi, Y., and M. Ookubo. 1953. Studies on marine Phycomycetes [1]. Bull. Nat. Sci. Mus. (Tokyo) 33: 53–65.

33. Leander, C. A., and D. Porter. 2000. Redefining the genus Aplanochytrium (phylum Labyrinthulomycota). Mycotaxon 76: 439–444.

34. Lewis, T. E., P. D. Nichols, and T. A. McMeekin. 1999. The biotechnological potential of thraustochytrids. Mar. Biotechnol. 1: 580–587.

35. Lopez-Garcia, P., F. Rodriguez-Valera, C. Pedros-Alio, and D. Moreira. 2001. Unexpected diversity of small eukaryotes in deep-sea Antarctic plank- ton. Nature 409: 603–607.

36. Martelli, G. P., and M. Russo. 1972. Pelargonium leaf curl virus in host leaf tissues. J. Gen. Virol. 15: 193–203.

37. Miller, J. D., and E. B. G. Jones. 1983. Observation on the association of thraustochytrid marine fungi with decaying seaweed. Bot. Mar. 26: 345–351.

38. Moore, N. F., and J. S. K. Pullin. 1983. Heat shock used in combination with amino acid analogues and protease inhibitors to demonstrate the processing of proteins of an insect picorna virus ( Drosophila C virus) in Drosophila melanogaster cells. Ann. Virol. 134: 285–292.

39. Naganuma, T., H. Takasugi, and H. Kimura. 1998. Abundance of thraus- tochytrids in coastal plankton. Mar. Ecol. Prog. Ser. 162: 105–110.

40. Nagasaki, K., M. Ando, I. Imai, S. Itakura, and Y. Ishida. 1993. Virus-like particles in an apochlorotic flagellate in Hiroshima Bay, Japan. Mar. Ecol. Prog. Ser. 96: 307–310.

41. Nagasaki, K., M. Ando, S. Itakura, I. Imai, and Y. Ishida. 1994. Viral mortality in the final stage of Heterosigma akashiwo (Raphidophyceae) red tide. J. Plankton Res. 16: 1595–1599.

42. Nagasaki, K., M. Ando, I. Imai, S. Itakura, and Y. Ishida. 1995. Virus-like particles in unicellular apochlorotic microorganisms in the coastal water of Japan. Fish. Sci. (Tokyo) 61: 235–239.

43. Nagasaki, K., and M. Yamaguchi. 1997. Isolation of a virus infectious to the harmful bloom causing microalga Heterosigma akashiwo (Raphidophyceae). Aquat. Microb. Ecol. 13: 135–140.

44. Nakahara, T., T. Yokochi, T. Higashihara, S. Tanaka, T. Yaguchi, and D. Honda. 1996. Production of docosahexaenoic and docosapentaenoic acid by Schizochytrium sp. isolated from Yap Islands. JAOCS (J. Assoc. Oil Chem. Soc.) 73: 1421–1426.

45. Nishihara, T., N. Kurano, and S. Shinoda. 1986. Calculation of most prob- able number for enumeration of bacteria on a microcomputer. Eisei Kagaku 32: 226–228. (In Japanese with English abstract.)

46. Polglase, J. L. 1980. A preliminary report on the thraustochytrid(s) and labyrinthurid(s) associated with a pathological condition in lesser octopus Eledone cirrhosa . Bot. Mar. 23: 699–706.

47. Porter, D. 1989. Phylum Labyrinthulomycota, p. 388–398. In L. Margulis, J. O. Corliss, M. Melkonian, and D. Chapman (ed.), Handbook of protoc- tista. Jones and Bartlett Publishers, Boston, Mass.

APPL. ENVIRON. MICROBIOL.

48. Proctor, L. M., and J. A. Fuhrman. 1990. Viral mortality of marine bacteria and cyanobacteria. Nature 343: 60–62.

49. Ragan, M. A., G. S. MacCallum, C. A. Murphy, J. J. Cannone, R. R. Gutell, and S. E. McGladdery. 2000. Protistan parasite QPX of hard-shell clam Mercenaria mercenaria is a member of Labyrinthulomycota. Dis. Aquat. Org. 42: 185–190.

50. Raghukumar, S. 1996. Morphology, taxonomy, and ecology of thraus- tochytrids and labyrinthulids, the marine counterparts of zoosporic fungi, p. 35–60. In R. Dayal (ed.), Advances in zoosporic fungi. Publications Pvt. Ltd., New Delhi, India.

51. Raghukumar, S. 2002. Ecology of the marine protists, the Labyrinthulomy- cetes (thraustochytrids and labyrinthulids). Eur. J. Protistol. 38:127–145.

52. Raghukumar, S., N. Ramaiah, and C. Raghukumar. 2001. Dynamics of thraustochytrid protists in the water column of the Arabian Sea. Aquat. Microb. Ecol. 24: 175–186.

53. Raghukumar, S., and K. Schaumann. 1993. An epifluorescence microscopy method for direct detection and enumeration of the fungi-like marine pro- tists, the thraustochytrids. Limnol. Oceanogr. 38:182–187.

54. Reisser, W. 1993. Viruses and virus-like particles of freshwater and marine eukaryotic algae—a review. Arch. Protistenkd. 143:257–265.

55. Riemann, F., and M. Schrage. 1983. On the mass occurrence of a thraus- tochytroid protist (fungi or rhizopodan protozoa) in an Antarctic anaerobic sediment. Veroeff. Inst. Meeresforsch. Bremerhav. 19: 191–202.

56. Roberts, D. A., R. G. Christie, and M. C. Archer, Jr. 1970. Infection of apical initials in tobacco shoot meristems by tobacco ringspot virus. Virology 42:

217–220.

57. Sanddaa, R. A., M. Heldal, T. Castberg, R. Thyrhaug, and G. Bratbak. 2001. Isolation and characterization of two viruses with large genome size infecting Chrysochromulina ericina (Prymnesiophyceae) and Pyramimonas orientalis (Prasinophyceae). Virology 290: 272–280.

58. Schroeder, D. C., J. Oke, G. Malin, and W. H. Wilson. 2002. Coccolithovirus (Phycodnaviridae): characterization of a new large dsDNA algal virus that infects Emiliania huxleyi . Arch. Virol. 147:1685–1698.

59. Sparrow, F. K., Jr. 1936. Biological observations on the marine fungi of Woods Hole waters. Biol. Bull. 70:236–263.

60. Suttle, C. A. 1993. Enumeration and isolation of viruses, p. 121–137. In P. F. Kemp, B. Sherr, E. Sherr, and J. J. Cole (ed.), Handbook of methods in aquatic microbial ecology. Lewis Publishers, Boca Raton, Fla.

61. Tai, V., J. E. Lawrence, A. S. Lang, A. M. Chan, A. I. Culley, and C. A. Suttle. 2003. Characterization of HaRNAV, a single-stranded RNA virus causing lysis of Heterosigma akashiwo (Raphidophyceae). J. Phycol. 39: 343–352.

62. Tarutani, K., K. Nagasaki, S. Itakura, and M. Yamaguchi. 2001. Isolation of a virus infecting the novel shellfish-killing dinoflagellate Heterocapsa circu- larisquama . Aquat. Microb. Ecol. 23:103–111.

63. Tomaru, Y., N. Katanozaka, K. Nishida, Y. Shirai, K. Tarutani, M. Yamagu- chi, and K. Nagasaki. 2004. Isolation and characterization of two distinct types of HcRNAV, a single-stranded RNA virus infecting the bivalve-killing microalga Heterocapsa circularisquama . Aquat. Microb. Ecol. 34: 207–218.

64. Van Etten, J. L., L. C. Lane, and R. H. Meints. 1991. Viruses and virus-like particles of eukaryotic algae. Microbiol. Rev. 55:584–620.

65. Van Etten, J. L. 2000. Phycodnaviridae , p. 183–193. In M. H. V. Van Regen- mortel, C. M. Fauquet, D. H. L. Bishop, et al. (ed.), Virus taxonomy, classification and nomenclature of viruses, 7th report. Academic Press, San Diego, CA.

66. Wilhelm, S. W., and C. A. Suttle. 1999. Viruses and nutrient cycles in the sea. Virus play critical roles in the structure and function of aquatic food webs. BioScience 49:781–788.