RESEARCH ARTICLE

Ecological Dynamics of Two Distinct Viruses

Infecting Marine Eukaryotic Decomposer
Thraustochytrids (Labyrinthulomycetes,
Stramenopiles)
Yoshitake Takao1*, Yuji Tomaru2, Keizo Nagasaki2¤, Daiske Honda3
1 Department of Marine Bioscience, Fukui Prefectural University, 1–1 Gakuencho, Obama, Fukui, 917–
0003, Japan, 2 National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research
Agency, 2-17-5 Maruishi, Hatsukaichi, Hiroshima, 739–0452, Japan, 3 Department of Biology, Faculty of
Science and Engineering, Konan University, 8-9-1 Okamoto, Higashinada, Kobe, 658–8501, Japan

¤ Current Address: Research Center for Fisheries and Environment in the Ariake and Yatsushiro Bay,
Fisheries Research Agency, 1551–8, Taira-machi, Nagasaki-shi, Nagasaki, 851–2213, Japan
* [email protected]

Abstract
OPEN ACCESS
Thraustochytrids are cosmopolitan osmotrophic or heterotrophic microorganisms that are
Citation: Takao Y, Tomaru Y, Nagasaki K, Honda D
(2015) Ecological Dynamics of Two Distinct Viruses
considered as important decomposers in coastal ecosystems. However, because of a lack
Infecting Marine Eukaryotic Decomposer of estimation method for each genus or systematic group of them, relatively little is known
Thraustochytrids (Labyrinthulomycetes, about their ecology in situ. Previously, we reported two distinct types of virus infecting
Stramenopiles). PLoS ONE 10(7): e0133395.
thraustochytrids (AuRNAV: reported as SssRNAV, and SmDNAV) suggesting they have
doi:10.1371/journal.pone.0133395
wide distributions in the host-virus systems of coastal environments. Here we conducted a
Editor: Arga Chandrashekar Anil, CSIR- National
field survey from 2004 through 2005 to show the fluctuation pattern of thraustochytrids and
Institute of Oceanography, INDIA
their viruses in Hiroshima Bay, Japan. During the field survey, we monitored the dynamics
Received: December 8, 2014
of the two types of thraustochytrid-infecting virus: small viruses causing lysis of Aurantio-
Accepted: June 25, 2015 chytrium sp. NIBH N1-27 (identified as AuRNAV) and the large viruses of Sicyoidochytrium
Published: July 23, 2015 minutum NBRC 102975 (similar to SmDNAV in physiology and morphology). Fluctuation
Copyright: © 2015 Takao et al. This is an open
patterns of the two distinct types of virus were different from each other. This may reflect the
access article distributed under the terms of the difference in the preference of organic substrates; i.e., it may be likely the host of AuRNAV
Creative Commons Attribution License, which permits (Aurantiochytrium sp.) increases utilizing algal dead bodies or feeble cells as the virus
unrestricted use, distribution, and reproduction in any
shows a large increase in abundance following raphidophyte blooms; whereas, the trophic
medium, provided the original author and source are
credited. nutrient supply for S. minutum may primarily depend on other constantly-supplied organic
compounds because it did not show any significant change in abundance throughout the
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files. survey. Further study concerning the population composition of thraustochytrids and their
viruses may demonstrate the microbial ecology (especially concerning the detrital food
Funding: This work was partially supported by the
Asahi Glass Foundation and Kato Memorial web) of marine environments.
Bioscience Foundation. The funder had no role in
study design, data collection and analysis, decision to
publish, or preparation of the manuscript.

Competing Interests: The authors have declared

that no competing interests exist.

PLOS ONE | DOI:10.1371/journal.pone.0133395 July 23, 2015 1 / 11

 Ecological Dynamics of Viruses Infecting Thraustochytrids

Introduction
Thraustochytrids are non-photosynthetic marine/estuarine stramenopile protists that are fre-
quently observed and/or isolated from marine and estuarine waters, sediments, algal and plant
materials both as saprotrophs and parasites [1]. Their bio-volume in coastal waters is estimated
at 43% of that of the bacterioplankton [2]. The ubiquitousness and ability to use a wide variety
of organic substrates (including bacterivory) argue for their ecological importance as decom-
posers [3, 4]. In addition, due to their high production of PUFAs (polyunsaturated fatty acids)
such as docosahexaenoic acid and docosapentaenoic acid [5], they are considered very impor-
tant as food resources for higher organisms in marine systems [6–8]. Because of these distinc-
tive features of the thraustochytrids, their ecological significance in the coastal ecosystems has
been studied [9, 10]. Kimura et al. [11] biogeographically demonstrated the abundance of
thraustochytrids was closely related with the density of POM (particulate organic matter). And
Bongiorni and Dini [12] show the abundance and composition of thraustochytrids change
with habitats and seasons in Mediterranean coastal areas. However, the effective techniques
that can separately estimate the abundance of each genus or systematic groups of thraustochy-
trids, still have not been established. In spite of their ecological significance, therefore, relatively
little is known about their ecological influence and impact in situ.
On the other hand, viruses are highly abundant in marine environments and are recognized
as important pathogens in controlling bacterial and algal biomass [13, 14], nutrient cycling
[15], and in maintaining the bio-diversity of bacteria and microalgae [14, 16]. To date, more
than thirty algal viruses have been isolated and characterized to different levels of resolution;
and particularly, the relationships between algal blooms and viruses have been intensively
investigated [17]. The viral infection is considered to affect the dynamics of algal blooms both
quantitatively (biomass) and qualitatively (clonal composition). Heterosigma akashiwo-HaV
(Heterosigma akashiwo virus) and Heterocapsa circularisquama-HcRNAV (Heterocapsa circu-
larisquama RNA virus) are well known host-virus systems [18, 19]. In both cases, the host and
their virus dynamics were tightly linked each other [20, 21]. Considering the fact that viruses
can’t reproduce without their specific host, fluctuations in abundance of certain virus may
reflect the host dynamics. Therefore, studies on viruses that infect thraustochytrids lead up to
novel information about their host.
Previously, we reported two distinct viruses infecting thraustochytrids: AuRNAV (Auran-
tiochytrium RNA virus: reported as SssRNAV) and SmDNAV (Sicyoidochytrium minutum
DNA virus) [22, 23]. AuRNAV is a single-strand RNA virus infecting Aurantiochytrium sp.
(formerly Schizochytrium sp., see Yokoyama and Honda, [24]); and SmDNAV is a double-
strand DNA virus infecting S. minutum. The two hosts are taxonomically distant within the
family Thraustochytriaceae. Here we describe the seasonal change in abundance of viruses
infecting the thraustochytrids in Hiroshima Bay, Japan and discuss the ecology of thraustochy-
trids from the viewpoint of the host-virus relationships.

Materials and Methods

Sample collection
Water samples from the surface layer (0 m) and 0.2 m above the sediment-water interface (B-
0.2 m) were collected in 2004 (May—Dec) and 2005 (Apr—Aug) at a semi-enclosed basin
(Itsukaichi Fishing Port; 34°21.400N, 132°21.864E, mean water depth = 5 m) located in Hiro-
shima Bay, Japan (Fig 1). No specific permits were required for the described field studies, as
the location is not privately-owned or protected in any way, and the field studies did not
involve endangered or protected species.

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 Ecological Dynamics of Viruses Infecting Thraustochytrids

Fig 1. Site map of the sampling location in Hiroshima Bay, Japan. This map was originally drawn by
using Adobe Illustrator (Adobe Systems Software Ireland Ltd.) based on the geological information.
doi:10.1371/journal.pone.0133395.g001

During the survey, the water temperature and salinity (psu) of the water column ranged at
16.4–28.1°C and 16.6–30.3, respectively.

Abundance of phytoplankton and thraustochytrids

Phytoplankton abundance was enumerated by direct count using light microscopy. During the
2005 survey, the abundance of thraustochytrids was estimated using a modified MPN (most
probable number) technique with pine pollen [25].

Virus titration
Virus titration of seawater samples (0 m and B-0.2 m) and sediment samples was conducted
using the extinction dilution method [26]. Six thraustochytrid strains were grown at 20°C in
10×medium-H [27] and were used as titration host strains for thraustochytrids-infecting
viruses in the water samples (Table 1). The cell culture plates were incubated at 20°C. The

Table 1. Host strains used to detect infecting viruses.

Taxon Strains* Isolation locality

Thraustochytriaceae Aurantiochytrium sp. NIBH N1-27 Nakaminato Harbor, Ibaragi, Japan
Aurantiochytrium limacinum NIBH SR-21 Colonia, Yap Island, Micronesia
Schizochytrium sp. SEK 0213 Iriomote Island, Okinawa, Japan
Sicyoidochytrium minutum NBRC 102975 Iriomote Island, Okinawa, Japan
Thraustochytrium aureum ATCC 34304 Woods Hole, Massachusetts, USA
Ulkenia amoeboidea SEK 0214 Hiroshima Bay, Hiroshima, Japan

*ATCC: American Type Culture Collection (USA), NBRC: National Institute of Technology and Evaluation (NITE) Biological Research Center (Japan),
NIBH: National Institute of Bioscience and Human-Technology (Japan), SEK: Laboratory of Systematics and Evolution, Konan University (Japan).

doi:10.1371/journal.pone.0133395.t001

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 Ecological Dynamics of Viruses Infecting Thraustochytrids

occurrence of cell lysis was monitored every other day for 14 days using optical microscopy;
and the most probable number (MPN) of viruses lytic to each host strain was calculated using
the computer program of Nishihara et al. [28]. Thus, viral abundance was estimated as the
MPN of infectious units that are lytic to each host strain. Cell lysates in the most diluted wells
were filtered through a 0.2-μm filter and then forwarded to the following isolation procedure.

Isolation of viruses
One clonal virus was isolated from each MPN assay. Clonal pathogens were obtained using
two cycles of the extinction dilution procedure [29] with the same thraustochytrid strain that
was initially used for titration. The resultant lysate at the final end-point dilution series from
the second extinction dilution procedure was regarded as a clonal virus suspension in which
the probability of two or more differing viruses occurring (i.e., failure in cloning) was estimated
at p  0.0106. The clonal viral suspensions were made free from bacterial contamination using
filtering through a 0.2-μm filter. The virus clones successfully isolated and free from bacterial
contamination were stored with 10% glycerol in 10×medium-H at -80°C.

TEM observation
TEM observation of the isolated viral clones was performed to show their morphology. Nega-
tively stained viruses were prepared according to the method of Takao et al. [22]; and observed
using TEM at an acceleration voltage of 80 kV using a JEOL JEM-1010 transmission electron
microscope. Particle sizes were estimated from negatively stained images.

Northern dot-blot analysis

Northern dot-blot analysis was performed to identify whether the viral isolate causing lysis of
Aurantiochytrium sp. NIBH N1-27 was AuRNAV (or a closely-ralated virus). An AuRNAV-
specific probe was synthesized using the AlkPhos Direct Labelling and Detection System with
CDP-Star (GE Healthcare) and the cDNA of AuRNAV including the non-coding and ORF2
region (nt 5231–5834; GenBank accession number AB193726) as the template [30]. Each virus
clone was inoculated into its suitable host strain (Aurantiochytrium sp. NIBH N1-27) and
incubated for 48 h. This is long enough for the tested-viruses to complete one replication cycle
as previously evaluated [22]. Two-μl of each viral suspension was dotted onto a positively-
charged nylon membrane, Hybond-N+ (Amersham Biosciences); then, hybridization was per-
formed according to the manufacturer's protocol using the above mentioned AuRNAV-specific
probe; and detected with chemical illumination using a LAS-3000 (Fuji Photo Film Co., Ltd.)

Results
Isolation and identification of viruses
We succeeded in isolating 118 viral isolates infecting thraustochytrids during the field surveys
conducted in 2004 and 2005. Among the clones, 52 and 66 were infectious to Aurantiochytrium
sp. NIBH N1-27 and S. minutum NBRC 102975, respectively. TEM observation revealed the
former were small icosahedral viruses similar to AuRNAV (ca. 25 nm in diameter, angular in
shape and lacking a tail) and the latter were large ellipse viruses resembling SmDNAV in mor-
phology (ca. 150 nm in diameter and lacking a tail) (S1 Fig). Northern dot-blot analysises
revealed all of the 52 AuRNAV-like viral isolates showed positive reaction to the specific
molecular probes, respectively (S2 Fig). Integrating the results of TEM observation with north-
ern dot-blot analysis, all of the isolated viral clones that caused lysis of Aurantiochytrium sp.

PLOS ONE | DOI:10.1371/journal.pone.0133395 July 23, 2015 4 / 11

 Ecological Dynamics of Viruses Infecting Thraustochytrids

NIBH N1-27 were identified as AuRNAV [22]; whereas, those lytic to S. minutum NBRC
102975 were considered to be (or closely related to) SmDNAV [23].

Field data collected in 2004

The temporal changes in the field data collected during 2004 are shown in Fig 2. Significant
decreases in practical salinity units (psu) were observed three times; in May, Jun, and Aug
(Fig 2). The diatoms in the genera Chaetoceros and Skeletonema dominated in the water col-
umn almost throughout the survey period except during a Heterosigma akashiwo bloom where
the highest cell density was 9.7 × 104 cells ml-1 on Jun 11 (Fig 2).
Six phylogenetically distant thraustochytrid clones were used to isolate and enumerate their
viruses throughout the survey; and consequently, only two types of virus respectively infecting
Aurantiochytrium sp. NIBH N1-27 (here regarded as AuRNAV) and Sicyoidochytrium minu-
tum NBRC 102975 (SmDNAV-like viruses) were detected and successfully isolated (Table 1
and Fig 2). AuRNAV showed a drastic increase in abundance (1.8 × 103 infectious units ml-1)
at ten days after the H. akashiwo bloom termination. Then, they rapidly decreased to below the
detection limit at 3.0 infectious units ml-1 after 23 Jul (Fig 2). Whereas, the viruses infecting
SmDNAV-like viruses did not show any changes in abundance during the survey period which
fluctuated at <2.5 × 101 infectious units ml-1 (Fig 2).
Considering the fact that viruses can’t reproduce without their specific host, fluctuations in
abundance of certain virus may reflect the host dynamics. In addition, our preliminary inocula-
tion experiments showed that Aurantiochytrium sp. NIBH N1-27 attached to H. akashiwo cells
and propagated more efficiently than other thraustochytrid strains tested (data not shown).
Therefore, we thought that the spike peak of thraustochytrids existed after the H. akashiwo
bloom but before the peak of AuRNAV. To confirm the hypothesis, we also estimated the
abundance of thraustochytrids during the field survey in 2005.

Field data collected in 2005

Temporal changes in field data collected in 2005 are shown in Fig 3. During 2005, there was lit-
tle precipitation during the rainy season (May to Jun) and a decrease in salinity was detected
only in Jul (Fig 3). Similar tendencies in algal dominance as in 2004 were also observed in
2005; i.e., diatoms such as genera Chaetoceros and Skeletonema were usually dominant in the
water column except during a H. akashiwo bloom and short period dominance of Thalassiosira
spp. (14–19 Jul) (Fig 3). H. akashiwo bloom (smaller but longer than 2004) was detected with a
maximum cell density of 8.8 × 103 cells ml-1 during Jun 23 to 27 (Fig 3).
In 2005, only the two similar types of virus were detected as in the case of 2004 (Fig 3); how-
ever, the fluctuation pattern differed from the preceding year. The abundance of AuRNAV
peaked (Jul 1; 2.2 × 102 infectious units ml-1) at the end of the H. akashiwo bloom, and one
month later (2 Aug; 3.0 × 102 infectious units ml-1) (Fig 3). SmDNAV-like viruses fluctuated at
less than 100 infectious units ml-1 from 26 May through 10 Jun, where the abundance was less
than the detection limit; and then, they moderately increased and reached a maximum
(3.0 × 102 infectious units ml-1) on 26 Jul (Fig 3).
Changes in abundance of thraustochytrids were estimated in 2005 (Fig 3). The abundance
ranged between 9.1 × 101 to 2.3 × 105 cells L-1. An increase accompanied with an increase of
AuRNAV was observed at the end of the H. akashiwo bloom (from 23 Jun through 5 Jul) and
was followed by a drastic decrease; then, they showed a moderate increase (Fig 3).

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 Ecological Dynamics of Viruses Infecting Thraustochytrids

Fig 2. Temporal changes in water temperature (A), salinity (B), and abundance of diatoms (C), Heterosigma akashiwo (D), viruses infecting
Aurantiochytrium sp. NIBH N1-27 (E), and viruses infecting Sicyoidochytrium minutum NBRC 102975 (F) during the field survey at Hiroshima Bay
in 2004. (■) and (□) indicate data at the surface and the B-0.2 m (0.2 m above the sediment-water interface) layer, respectively. Remarkable peaks and
valleys were highlighted by shadowing.
doi:10.1371/journal.pone.0133395.g002

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 Ecological Dynamics of Viruses Infecting Thraustochytrids

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 Ecological Dynamics of Viruses Infecting Thraustochytrids

Fig 3. Temporal changes in water temperature (A), salinity (B), and abundance of diatoms (C), Heterosigma akashiwo (D), thraustochytrids (E),
viruses infecting Aurantiochytrium sp. NIBH N1-27 (F), and viruses infecting Sicyoidochytrium minutum NBRC 102975 (G) during the field survey
at Hiroshima Bay in 2005. (■) and (□) indicate data at the surface and the B-0.2 m layer, respectively. Remarkable peaks and valleys were highlighted by
shadowing.
doi:10.1371/journal.pone.0133395.g003

Discussion
In the present field survey, we detected two distinct types of thraustochytrid virus; they were
AuRNAV and SmDNAV-like. Considering both virus types were isolated from a variety of
coastal environments in Japan, at least two distinct thraustochytrid-virus combinations may be
widely distributed and functioning in universal eukaryotic decomposing systems. However, no
viral agents causing lysis of the other four tested thraustochytrid clones were detected through-
out the present survey. Of course, this does not deny the possibility of the existence of other
thraustochytrid-infecting viruses. In this study, we used only six host strains to examine virus
abundance; as a result, only the viruses which caused lysis of tested hosts were isolated. It may
be possible to isolate a wider variety of viruses by using more thraustochytrid strains as hosts.
As well, it should be noted that host-virus combinations not accompanied with drastic cell lysis
may have been overlooked in this study.
The fluctuation patterns in abundance of the two virus types were different from each other.
AuRNAV showed a remarkable increase in abundance following the H. akashiwo bloom in
2004 (Fig 2). Since AuRNAV does not infect H. akashiwo [22], the increase is considered
reflecting the drastic increase and viral lysis of Aurantiochytrium sp. NIBH N1-27-type thraus-
tochytrids, which occurred following the H. akashiwo bloom. Actually, we succeeded in detect-
ing an increase in thraustochytrid abundance after the peak of the H. akashiwo bloom also in
2005, which was accompanied with the temporal increase of AuRNAV (Fig 3). Whereas, no
statistically significant relationship was found between the abundance of thraustochytrids and
AuRNAV from the Pearson's correlation coefficient analysis (data not shown).
A possible explanation for the ecological events which was observed in 2004 is reasonable;
i.e., H. akashiwo rapidly multiplied due to the enough amount of nutrient supply originated
from land water (Fig 2), and it caused drastic increase and dominance of Aurantiochytrium sp.
NIBH N1-27-type thraustochytrids (utilizing H. akashiwo cells). The spike peak of AuRNAV
detected in 2004 was considered as the result of virus infection to the dominant thraustochy-
trids. On the other hand, in 2005, land water supply was less than 2004 before the H. akashiwo
bloom. Then, the bloom scale was not as large as in 2004 (Fig 3). Although the similar events
should have occurred also in 2005, each event may have occurred at lower level, thus, the event
sequence was not so obvious in 2005 compared to 2004.
Another possibility is difference in the species composition of thraustochytrids. Untapped
organic matter remained in water column and/or released organic matter derived from virally
lysed NIBH N1-27-type thraustochytrid cells were considered to be substrates for multiplica-
tion of the other types of thraustochytrid. At the peak of thraustochytrids detected in 2005,
Aurantiochytrium sp. NIBH N1-27-type may not have been as dominant as in 2004. Although
we checked the total thuraustochytrids abundance to grasp the tendency of their dynamics, the
resolution quality was too low to verify the hypothesis. Techniques for separately estimating
the abundance of each genus or systematic groups of thraustochytrids is essential.
Peaks of AuRNAV and thraustochytrids were also detected from 19 Jul through 2 Aug
2005 (Fig 3) following the period when diatoms (genus Thalassiosira) dominated (14–19 Jul).
In this case, it is considered that Aurantiochytrium sp. NIBH N1-27-type thraustochytrids
increased by utilizing Thalassiosira sp. cells. Gaertner [31] reported that Schizochytrium sp.
cells parasitized on Thalassiosira sp. cells. In addition, our preliminary experiments showed

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 Ecological Dynamics of Viruses Infecting Thraustochytrids

that Aurantiochytrium sp. NIBH N1-27 attached and propagated on the surface of diatom
cells, as was observed in the case of H. akashiwo cells (data not shown). These results suggest
that Aurantiochytrium sp. NIBH N1-27-type thraustochytrids may have the ability to effec-
tively utilize the dead cells or feeble cells of phytoplankton (ex. Thalassiosira sp.).
On the other hand, SmDNAV-like viruses were detected more frequently than AuRNAV
throughout the field surveys. In 2004, they fluctuated at a relatively low level; and did not show
an increase either during or after the H. akashiwo bloom (Fig 2), suggesting that the dynamics
of S. minutum may depend on constantly supplied organic matter (ex. DOM/POM from land).
In 2005, the dynamics of SmDNAV-like viruses showed a moderate increase in abundance at
the surface layer after a drastic inflow of huge amount of river water (Fig 3), but the abundance
was not as high as AuRNAV. To this end, the fluctuation pattern of SmDNAV-like viruses was
different from that of AuRNAV. The difference is most likely due to the distinctive nutrient
acquisition strategies between S. minutum NBRC 102975-type and Aurantiochytrium sp.
NIBH N1-27-type thraustochytrids.
To our knowledge, this is the first report describing the seasonal changes in the abundance
of thraustochytrids and their viruses. Thraustochytrids is large taxa classified as a family, never-
theless only little is known about the differences and diversity of their ecological features. Our
results suggested that the community of thraustochytrids is comprised of plural groups that
have different ecological features and affected differently impacted by viruses. Further detailed
study focused on their internal population composition is necessary to reveal their ecological
impact in situ as a marine eukaryotic decomposer.

Supporting Information
S1 Fig. Transmission electron microscopy images of isolated viruses. AuRNAV (A),
SmDNAV (B), small viruses infecting Aurantiochytrium sp. NIBH N1-27 (C and E), and large
viruses infecting Sicyoidochytrium minutum NBRC 102975 (D and F).
(EPS)
S2 Fig. Northern dot-blot analysis of viruses infecting Aurantiochytrium sp. NIBH N1-27.
AuRNAV (A1), Rhizosolenia setigera RNA virus (A2), SmDNAV(A3), AuRNAV strains
(MD002, MD003, KM006, KM007, IS004) isolated from other part of Japan coast area (A4–8),
Viruses infecting Aurantiochytrium sp. NIBH N1-27 isolated in this study (A9—F10).
(EPS)

Acknowledgments
Thanks are due to T. Nakahara and T. Yokochi (National Institute of Advanced Industrial Sci-
ence and Technology, Japan) who kindly provided thraustochytrid strains. We would also like
to give our grateful acknowledgment to R. Yokoyama (Konan University, Japan) who kindly
provided thraustochytrid strains and their taxonomic information.

Author Contributions
Conceived and designed the experiments: Y. Takao Y. Tomaru KN DH. Performed the experi-
ments: Y. Takao Y. Tomaru. Analyzed the data: Y. Takao. Contributed reagents/materials/anal-
ysis tools: Y. Takao Y. Tomaru KN DH. Wrote the paper: Y. Takao Y. Tomaru KN DH.

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