RESEARCH ARTICLE
Variability of subseafloor viral abundance at the geographically
and geologically distinct continental margins
Katsunori Yanagawa1,2, Yuki Morono3, Yukari Yoshida-Takashima1, Masamitsu Eitoku1,
Michinari Sunamura2, Fumio Inagaki3, Hiroyuki Imachi1, Ken Takai1 & Takuro Nunoura1
1
Subsurface Geobiology and Advanced Research (SUGAR) Project, Japan Agency for Marine-Earth Science and Technology, Yokosuka, Japan;
Department of Earth and Planetary Science, Graduate School of Science, The University of Tokyo, Tokyo, Japan and 3Geomicrobiology Group,
Kochi Institute for Core Sample Research, Japan Agency for Marine-Earth Science and Technology, Nankoku, Kochi, Japan
2
Correspondence: Katsunori Yanagawa,
Subsurface Geobiology and Advanced
Research (SUGAR) Project, Extremobiosphere
Research Program, Institute of
Biogeosciences, Japan Agency for MarineEarth Science and Technology (JAMSTEC),
2–15 Natsushima-cho, Yokosuka 237-0061,
Japan. Tel.: +81 46 867 9717;
fax: +81 46 867 9715;
e-mail: [email protected]
MICROBIOLOGY ECOLOGY
Present address: Masamitsu Eitoku,
Department of Environmental Medicine,
Kochi Medical School, Kochi University,
Nankoku, Kochi, Japan
Received 21 April 2013; revised 26 October
2013; accepted 1 December 2013. Final
version published online 23 December 2013.
DOI: 10.1111/1574-6941.12269
Abstract
We studied the relationship between viral particle and microbial cell abundances in marine subsurface sediments from three geographically distinct locations in the continental margins (offshore of the Shimokita Peninsula of Japan,
the Cascadia Margin off Oregon, and the Gulf of Mexico) and found depth
variations in viral abundances among these sites. Viruses in sediments obtained
offshore of the Shimokita and in the Cascadia Margin generally decreased with
increasing depth, whereas those in sediments from the Gulf of Mexico were relatively constant throughout the investigated depths. In addition, the abundance
ratios of viruses to microbial cells notably varied among the sites, ranging
between 10 3 and 101. The subseafloor viral abundance offshore of the Shimokita showed a positive relationship with the microbial cell abundance and the
sediment porosity. In contrast, no statistically significant relationship was
observed in the Cascadia Margin and the Gulf of Mexico sites, presumably due
to the long-term preservation of viruses from enzymatic degradation within the
low-porosity sediments. Our observations indicate that viral abundance in the
marine subsurface sedimentary environment is regulated not only by in situ
production but also by the balance of preservation and decay, which is associated with the regional sedimentation processes in the geological settings.
Editor: Patricia Sobecky
Keywords
subsurface viruses; phage; viral decay;
deep-sea sedimentary environment.
Introduction
The abundance of viral particles in coastal surface sediments is generally more than 108 per 1 cm3 of sediment
and is usually greater than the microbial cell abundance
(Glud & Middelboe, 2004; Middelboe & Glud, 2006;
Middelboe et al., 2006; Danovaro et al., 2008; SiemJørgensen et al., 2008). Currently, it has been suggested
that viral infection is responsible for more than 80% of
microbial cell mortality in the deep-sea sediment–water
interface, and thus, the huge viral population may play
an important ecological role in shallow deep-sea sedimentary environments (Danovaro et al., 2008). However, our
knowledge of viral distribution and its ecological role in
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
the deep subseafloor environment remains poorly understood, despite the fact that the presence of a large number
of microbial cells has been observed (Whitman et al.,
1998; Parkes et al., 2000; Lipp et al., 2008; Kallmeyer
et al., 2012).
The first report of viral abundances in the marine subsurface environment studied the Saanich Inlet, British
Columbia, Canada, during the Ocean Drilling Program
(ODP) Leg 169S (Bird et al., 2001). The investigation of
a Holocene to late-Pleistocene sedimentary sequence with
a relatively high sedimentation rate reported that more
than 109 viruses were present in 1 g of dry sediment and
that the viral number generally decreased with increasing
sediment depth down to 118 m below the seafloor
FEMS Microbiol Ecol 88 (2014) 60–68
61
Viral distribution in deep subseafloor sediments
(mbsf). The virus to prokaryotic cell ratio (VPR) was
constant throughout the depths (i.e. 2.7–3.5). During the
Integrated Ocean Drilling Program (IODP) Expedition
307, Middelboe et al. (2011) investigated viral abundance
and productivity in subseafloor sediment from a coldwater coral mound in the Porcupine Seabight off Ireland.
The viruses were higher than the microbial cells from the
seafloor sediment to 96 mbsf, corresponding to a sediment age of c. 2.5 Ma. Incubation-based experiments for
the viral production rate suggested that most of the subseafloor viruses persisted over geological time scales and
that viral production was not significant (Middelboe
et al., 2011). Another study also showed high viral abundances (i.e. ranging from 1.6 9 106 to 5.7 9 108 viruses
cm 3) in selected subseafloor sediment samples (down to
the depth of 320 m) from the Peru Margin and eastern
equatorial Pacific sites during the ODP Leg 201 (Engelhardt et al., 2011). This study further tested prophage
induction from indigenous subseafloor bacterial culture
collections and suggested that lysogeny of host microbial
cells is one of the major sources of viral production in
marine subsurface sedimentary habitats. In this regard,
the latest study revealed whole genome information for a
putative temperate phage, rhizobiophage RR1-A, which is
associated with Rhizobium radiobacter. This organism is
the most frequently isolated organism from the deep subsurface (Engelhardt et al., 2013). These previous studies
of subseafloor viruses have consistently demonstrated
that, as observed in the prokaryotic populations, viruses
are spatially widely distributed in marine subsurface sediments. However, it remains controversial whether significant viral production indeed occurs in marine subsurface
environment.
In this study, to determine biotic and abiotic factors
controlling viral distribution in the subseafloor, we investigated the viral and microbial cell abundances in deep
marine subsurface sediments from three geographically
and geologically distinct locations in the continental margins: the Northwest Pacific (offshore of the Shimokita
Peninsula, northeastern Japan, sediment depth: c. 365
mbsf), the Northeast Pacific (the Cascadia Margin, offshore of Oregon, sediment depth: c. 420 mbsf), and the
Gulf of Mexico (offshore of the Mississippi river, sediment depth: c. 603 mbsf). The sediment samples from
offshore of Shimokita and the Cascadia Margin represent
organic-rich hemi-pelagic sediments, some of which harbor gas hydrates, in the northwestern and northeastern
Pacific margin, respectively, whereas the Gulf of Mexico
samples consist mainly of turbidites with relatively low
total organic carbon (TOC) content in the continental
slope basin. Microscopic observations also demonstrated
distinctive patterns of microbial cell abundance in each of
the locations, as previously shown elsewhere (Inagaki
FEMS Microbiol Ecol 88 (2014) 60–68
et al., 2006; Morono et al., 2009; Nunoura et al., 2009).
In this study, based on the vertical profiles of virus and
microbial cell abundances in sediments, we discuss the
spatial distribution of viruses and their potential ecological roles in the subseafloor microbial ecosystem. In addition, we examine the relation between the viral and
microbial cell abundances and the possible physical,
chemical, and geological characteristics, such as TOC,
porosity, depth, and sedimentation rate and age, that
could potentially affect the subseafloor microbial biomass,
community compositions, and functions.
Materials and methods
Sample collection
Subseafloor sediment samples used in this study were collected from three geographically and geologically distinct
locations: (1) Site C9001 Hole C at the northwestern
Pacific site off the Shimokita Peninsula of Japan, which
were explored during the Chikyu Shakedown Expedition
CK06-06 in 2006 (Aoike et al., 2010), (2) Site 1251 Hole
B at the northeastern Pacific site of the Cascadia Margin
off Oregon, which was explored during the ODP Leg 204
in 2002 (Trehu et al., 2003), and (3) Site U1320 Hole A
and Site U1324 Hole B at the Gulf of Mexico, which were
explored during the IODP Expedition 308 in 2005 (Flemings et al., 2006). The summary of sampling site locations
and sediment characteristics is listed in Table 1.
Site C9001 is located approximately 80 km from the
eastern coast of the Shimokita Peninsula (41°10.638′ N,
142°12.081′ E; 1180 m below sea level [mbsl]). The sediment is mainly composed of siliceous ooze and hemipelagic clay intercalated with volcanic tephras and sand/silt
layers, within some of which methane hydrates are formed
within the pore spaces. The sedimentation rate at Site
C9001 is c. 90 cm ky 1 (Aoike et al., 2010; Domitsu et al.,
2011). Samples used in this study consisted mainly of
hemipelagic diatom ooze, and the porous horizons associated with methane hydrate formation were not examined.
ODP Site 1251 is located at the foot of the South
Hydrate Ridge at the Cascadia continental margin offshore of Oregon (44°34.21′ N, 125°4.44′ W; 1210 mbsl).
The site is located on the slope basin, where wellstratified sediments are deposited with a sedimentation
rate of 3–160 cm ky 1 (Trehu et al., 2003). A strong
bottom-simulating reflector (BSR) that may represent the
base of gas hydrate stability zone is observed at 196 mbsf,
and relatively thin disseminated methane hydrate-bearing
layers are observed at approximately 200 mbsf (Trehu
et al., 2003).
IODP Sites U1320 Hole A (27°18.0809′ N, 94°23.2527′ W;
1480 mbsl) and U1324 Hole B (28°4.7845′ N, 89°8.3422′ W;
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
K. Yanagawa et al.
62
Table 1. Summary of sediment samples used in this study
Research Cruise
Latitude
Longitude
Water depth (mbsl)
Sediment depth (mbsf)
TOC (%)
Porosity (%)
Sedimentation
rate (cm ky 1)
Sediment age (Ma)
References
Offshore Shimokita
Peninnsula
Cascadia Margin offshore
of Oregon
Gulf of Mexico continental slope
Site C9001
Site 1251
Site U1320 Hole
A (BT Basin)
Site U1324 Hole B
(Ursa Basin)
CK06-06
41°10.638′ N
142°12.081′ E
1180
0.9–363
0.8–2.0
63–85
90
ODP Leg 204
44°34.21′ N
125°4.44′ W
1210
4.5–375
0.7–1.8
55–72
3–160
IODP Exp. 308
27°18.0809′ N
94°23.2527′ W
1480
3–285
0.3–0.7
41–71
181–260
IODP Exp. 308
28°4.7845′ N
89°8.3422′ W
1068
2.9–603
0.2–1.5
40–76
550–2500
0.0015–0.59
Aoike et al. (2010), Domitsu
et al. (2011) and Morono
et al. (2009)
0.008–1.8
Tr
ehu et al. (2003), Inagaki
et al. (2006) and Nunoura
et al. (2008)
0.0003–0.15
Flemings et al.
(2006) and Nunoura
et al. (2009)
0.001–0.064
Flemings et al. (2006)
and Nunoura et al. (2009)
1068 mbsl) are located on turbidite depositional basins of
the Gulf of Mexico continental slope, the Brazos-Trinity
Basin IV (BT Basin), and the Mars-Ursa salt-withdrawal
basin (Ursa Basin), respectively. The BT Basin is the
terminal basin of a series of bowl-shaped basins on the
upper–middle continental slope where the sedimentation
rate is 181–260 cm ky 1. The Ursa Basin is characterized
by the deposition of Pleistocene sediments (1.8 million–
10 000 years before present) from the Mississippi River at
an extremely high sedimentation rate ranging between
550 and 2500 cm ky 1 (Flemings et al., 2006).
The cores retrieved at Site C9001, Site 1251, Site
U1320, and Site U1324 were cut into 1.5 m-long whole
round sections on deck, and whole round cores (WRCs;
c. 5–10 cm in length) were subsampled from the short
sections of cores. The samples for virus and microbial cell
counts were taken from innermost parts of the WRCs
using sterilized spatula and immediately stored at 80 °C
as described previously (Inagaki et al., 2006; Morono
et al., 2009; Nunoura et al., 2009). The sampling depth
was presented in Table 1, and the details were listed in
Supporting Information, Table S1.
Enumeration of viruses and microbial cells
To estimate the number of viruses in marine subsurface
sediments, 3 cm3 of frozen sediments taken from the
innermost part of the WRCs was promptly suspended in
10 mL of 2% formaldehyde in modified SM buffer
[50 mM Tris-HCl pH 7.5, 0.1 M NaCl, 8 mM MgSO4, 3%
NaCl (wt/v)] in a 50 mL centrifuge tube. The slurry was
shaken with a ShakeMaster (BioMedical Science, Tokyo,
Japan) for 1 min at maximum speed (1100 r.p.m.) and
then sonicated at 15 W on ice for 1 min with an ultrasonic
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
homogenizer UH-50 (SMT Co. Ltd., Tokyo, Japan) to
detach the viruses from sediment matrices (Middelboe
et al., 2011). After centrifugation, a fraction of large particles including microbial cells was removed from the supernatants through a 0.2 lm cut-off syringe filter (Millipore,
Billerica, MA). Although some very large viruses could not
pass through the filter in this step, the typical-sized viral
particles were filtered onto 0.02-lm-pore-sized Anodisc
membrane filters (GE Healthcare, Piscataway, NJ). The
filters were rinsed thoroughly three times with 2 mL of
SM buffer. The viruses on the filter were then stained with
209 SYBR Gold (Molecular Probes, Eugene, OR; Sano
et al., 2004) and observed with a fluorescence microscope
(model BX51; Olympus, Tokyo, Japan) using the WIB
fluorescent filter settings (excitation wavelength: 460–
490 nm and emission wavelength: > 510 nm). The viruses
were manually counted from at least 40 microscopic fields
for each sample.
Microbial cell abundances in the sediment samples at
ODP Site 1251, IODP Sites U1320 and U1324 were evaluated using the fluorescent image-based cell enumeration
technique (Morono et al., 2009). In short, 1 cm3 of frozen sediment sample was suspended in 3% paraformaldehyde at 4 °C for 3 h, washed twice with PBS (pH 7.6),
and then stored in PBS/ethanol [1 : 1 (v/v)] at 20 °C.
The fixed slurry sample was treated with a 1% hydrogen
fluoride solution and mildly sonicated for 1 min. An
aliquot was then filtered on 0.2-lm-pore-size polycarbonate filters (Isopore; Millipore). The microbial cells on the
filter were stained with 2509 SYBR Green I at room temperature for 10 min. After rinsing with TE buffer, each
filter was mounted on a slide glass with mounting solution (1 : 2 mixture of VECTASHIELD mounting medium
H-1000 and TE buffer). Enumeration of SYBR Green
FEMS Microbiol Ecol 88 (2014) 60–68
63
Viral distribution in deep subseafloor sediments
In the deep subseafloor sediment samples from depths of
0.9–363 mbsf at Site C9001 off Shimokita (CK06-06), the
microbial cell abundances were relatively high, ranging
from 1.2 9 106 to 1.3 9 109 cells cm 3 sediment (Morono et al., 2009). The viruses were usually one to two
orders of magnitude lower than the microbial cell abundances, ranging from 1.6 9 104 to 7.3 9 106 viruses cm 3
(Fig. 1a). As a general trend, the viruses in the sediments
at Site C9001 decreased with increasing sediment depth,
which was consistent with the vertical profile of microbial
cell abundance. In the shallower sediments at depths
above approximately 154 mbsf, viral abundance decreased
gradually at an exponential rate of 0.031 m 1 (r = 0.91;
P < 0.001; n = 16). The viral abundances were nearly
constant below 154 mbsf, ranging from 1.6 9 104 to
5.5 9 104 viruses cm 3. The values of VPR were c. 0.01
in the shallower sediments (down to c. 100 mbsf) and further decreased down to 0.001 in the deeper sediments
below approximately 154 mbsf (Fig. 1b). The low VPR
value may indicate that lowered host prokaryotic activity
and/or high viral decay in the deep subsurface sediments.
5.4 9 107 cells cm 3, which gradually decreased with
increasing depth (P < 0.1; n = 14; Fig. 1c). The viral
counts above 180 mbsf had no significant relation with
depth (P > 0.1; n = 10; Fig. 1d). However, we found
unexpectedly low viral abundances in the sediments at
depths between 228 and 320 mbsf, which were two orders
of magnitude lower than those in the other sedimentary
horizons. Triplicate counts led to average values obtained
with a precision of 78%. Contrastingly, the microbial
abundances did not show such a trend (Fig. 1c). During
ODP Leg 204, low-temperature anomalies were observed
in the recovered cores at depths of approximately
200 mbsf, indicating the presence of methane hydrates at
these horizons (Trehu et al., 2003). This observation may
allow us to hypothesize that certain physical and/or
chemical conditions of the subseafloor sediments induced
by the present and past methane hydrate formations regulate the balance of in situ viral production and viral
decay (i.e. lowering the productivity of the prokaryotic
hosts and/or promoting viral decomposition). Another
conceivable explanation for the anomalously low viral
abundances may relate to the sedimentation regimes as
represented by the variation in the sedimentation rate at
Site 1251. The notably low viral numbers were found in
sediments at depths between 228 and 320 mbsf, where
the sedimentation rate was much lower than in the
shallower sediment layers (i.e. 60–160 cm ky 1 above
160 mbsf, 17 cm ky 1 between 160 and 285 mbsf,
3 cm ky 1 between 285 and 310 mbsf, and 33 cm ky 1
between 310 and 436 mbsf; Trehu et al., 2003). Furthermore, the lowest viral number was detected at a depth of
278 mbsf, beneath which a significantly low sedimentation rate was estimated from the sedimentation model.
As it has been reported that the sedimentation rate has
an impact on the subseafloor microbial cell abundances
at the Cascadia Margin IODP sites (Nunoura et al.,
2008), the sedimentation rate may also affect the subseafloor viral abundances. Rapid sedimentation would likely
supply more fresh organic compounds in the sediments,
whereas sluggish sedimentation would allow for microbial
oxidative degradation over a long period. This would
result in the excess consumption of microbially available
organic material at the seafloor followed by a decrease in
microbial metabolic activity that would lead to a decrease
in viral production from host microbial cells in the sedimentary habitats.
Viral abundances at Site 1251 in the Cascadia
Margin (NE Pacific)
Viral abundances at Sites U1320 and U1324 in
the Gulf of Mexico continental slope
Methane hydrate-bearing sediments from the South
Hydrate Ridge on the Cascadia Margin (ODP Site 1251)
harbored microbial cell abundances of 4.4 9 105 to
The abundances of viruses and microbial cells in the subseafloor sediments at the turbidite depositional basins
(IODP Sites U1320 and U1324) of the Gulf of Mexico
I-stained cells was performed using an automated slideloader system (Morono et al., 2009), and the acquired
images were processed by MetaMorph software (Molecular Devices, Downingtown, PA).
Sediment-free negative controls were used to check for
experimental contamination during the virus and microbial cell counting. All the count data of viruses and
microbial cells are shown in Supporting Information,
Table S1.
TOC content and porosity
The TOC and porosity data were available in the Janus
web database (ODP Legs and IODP Expeditions; http://
www-odp.tamu.edu/database/) and in the JAMSTEC Data
Site for Research Cruises (CK06-06; http://www.godac.
jamstec.go.jp/darwin/cruise/chikyu/902/e). Selected data
used in this study are shown in Supporting Information,
Table S1.
Results and discussion
Viral abundances at Site C9001 off Shimokita
(NW Pacific)
FEMS Microbiol Ecol 88 (2014) 60–68
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
K. Yanagawa et al.
64
Offshore Shimokita Peninnsula
(CK06-06)
Cascadia Margin
(ODP Leg 204)
Site 1251
Hole U1324B in the Ursa basin
0
100
100
100
100
200
200
200
200
300
400
300
400
500
400
(c)
700
103
106
109
1012
Microbial and viral abundance
–3
(numbers cm )
300
400
500
600
600
(a)
700
103
300
500
600
600
Depth (mbsf)
0
Depth (mbsf)
0
500
(e)
700
103
106
109
1012
Microbial and viral abundance
–3
(numbers cm )
(g)
106
109
1012
Microbial and viral abundance
–3
(numbers cm )
700
103
0
0
100
100
100
100
200
200
200
200
300
400
400
500
500
600
300
700
10–1
101
103
10–3
Virus-to-prokaryote abundance ratio
(VPR)
300
400
(d)
700
10–3
10–1
101
103
Virus-to-prokaryote abundance ratio
(VPR)
600
106
109
1012
Microbial and viral abundance
–3
(numbers cm )
300
400
500
500
600
(b)
Depth (mbsf)
0
Depth (mbsf)
0
Depth (mbsf)
Depth (mbsf)
Hole U1320A in the BT basin
0
Depth (mbsf)
Depth (mbsf)
Site C9001
Gulf of Mexico continental slope
(IODP Exp. 308)
600
(f)
700
10–1
101
103
10–3
Virus-to-prokaryote abundance ratio
(VPR)
(h)
700
10–1
101
103
10–3
Virus-to-prokaryote abundance ratio
(VPR)
Fig. 1. Depth profiles of microbial cell (open circles) and virus (closed circles) abundances and VPR (gray circles) in the subseafloor sediments at
Site C9001 offshore of the Shimokita Peninsula (a, b); at IODP Site 1251 in the Cascadia Margin (c, d); and at ODP Site U1320 Hole A (e, f) and
Site U1324 hole B (g, h) on Gulf of Mexico continental slope. The microbial cell abundance at the Shimokita Peninsula (CK06-06) offshore site
was originally reported by Morono et al. (2009).
were nearly constant throughout the sediment depths
(Fig. 1e and g). The maximum microbial cell number was
2.6 9 107 cells cm 3, which was notably higher than the
previously determined microbial cell abundances by conventional cell counting using acridine orange (AO) and
4′,6′-diamidino-2-phenylindole (DAPI) (Nunoura et al.,
2009). In this study, the microbial cell abundance was
re-estimated using SYBR Green I staining and an imagebased automatic cell counting system that can enumerate
microbial cells more accurately and sensitively than the
manual counting method (Morono et al., 2009). The viral
abundances were consistently higher than 6.2 9 104
viruses cm 3, and the VPR ranged widely from 0.05 to
3.1 and 0.005 to 1.8 at Sites U1320 and U1324, respectively (Fig. 1f and h). The microbial hydrogenase activities of the bulk sediment samples have been previously
measured at Sites U1320 and U1324 (Nunoura et al.,
2009). The hydrogenase activity reflects the contributions
of active microbial populations from both hydrogenogens,
such as fermenters, and hydrogenotrophs, such as
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Published by John Wiley & Sons Ltd. All rights reserved
methanogens, homoacetogens, sulfate reducers, and iron
reducers (Soffientino et al., 2009). The microbial hydrogenase activities of bulk sediment samples decreased with
increasing depth, whereas microbial cell numbers were
nearly constant, with no significant changes with depth
(U1320; P > 0.4, n = 10, U1324; P > 0.5, n = 18). Furthermore, no significant relationship between the
microbial 16S rRNA gene phylotype composition and
hydrogenase activity was identified (Nunoura et al.,
2009). These results strongly suggest that total microbial
productivity or metabolic activity in the subseafloor sediments decreases with increasing depth. The lower productivity of prokaryotic populations may be coupled with a
lower rate of viral production in the deep subseafloor
sediments. Nevertheless, the viral abundances were nearly
constant, with no significant changes with depth (U1320;
P > 0.4, n = 11, U1324; P > 0.6, n = 21). Virus productively would still affect viral abundance, but clearly, it is
not the only factor that has a noticeable effect on viral
abundance.
FEMS Microbiol Ecol 88 (2014) 60–68
65
Viral distribution in deep subseafloor sediments
Viral ecology in the deep subseafloor
sedimentary biosphere in the continental
margins
The correlation between the microbial cell and the virus
abundances does not represent any significant relationship,
except for the case of the deep subseafloor sedimentary
habitats offshore of Shimokita, which exhibited significant
correlation (r = 0.95; P < 0.001; n = 23; Fig. 2a). This
finding suggests that only the subseafloor viral abundances
offshore of Shimokita are influenced by the in situ viral
production by the host microbial cellular populations in
the sedimentary environments. In other words, our enumeration of microbial cells and viruses in the subseafloor
sediment samples from three distinct locations indicates
that the viral abundance is not necessarily always associated with the host microbial cell abundance. To pursue
key insights into understanding the viral abundance distribution in subseafloor sedimentary environments, we
examined the statistical relationship of viral counts with
TOC and sediment porosity that may affect microbial
metabolic activity (Fig. 2b and c).
Viral abundance vs. TOC
It seems likely that the higher TOC content is associated
with higher microbial metabolic activity in the subseafloor sedimentary environments, which subsequently leads
to higher indigenous viral production in microbial communities. Indeed, relatively high viral abundances have
been reported from the subseafloor sediments containing
high TOC content in the Saanich Inlet (c. 3%; Bird et al.,
2001) and Porcupine Seabight (1.4–2.6%; Middelboe
et al., 2011). However, our results of viral abundances
did not show any significant correlation with TOC content in the subseafloor sedimentary environment of each
site (Fig. 2b).
Viral abundance vs. sediment porosity
It has been considered that small pore spaces in the subseafloor sedimentary environment primarily influence
microbial activity due to the limitation of chemical transport and habitable space (Boivin-Jahns et al., 1996; Fredrickson et al., 1997; Zhang et al., 1998). The lack of pore
connectivity also restricts the movements of microbial
cells, creates spatial isolation, and eventually reduces the
genetic and physiological diversity of the subseafloor
microbial community (Boivin-Jahns et al., 1996; Zhou
et al., 2004). Furthermore, the sediment–cell interaction
could cause the puncture or tensile failure of the microbial cell membrane (Rebata-Landa & Santamarina, 2006).
Hence, pore and pore-throat sizes might be an important
factor for the microbial host cell ecology and, of course,
the viral production in the subseafloor sedimentary environments.
With regard to the relationship between the viral abundance and the porosity of sediment (Fig. 2c), a relatively
high correlation was observed in the sediment samples at
C9001 off Shimokita (r = 0.85; P < 0.01; n = 22). This
may suggest that viral production from host microbial
cell is constrained by the pore space. Correspondingly,
the proportional relationship between the microbial cell
and virus abundance at C9001 most likely represents a
Offshore the Shimokita Peninsula (CK06-06, Site C9001)
Cascadia Margin (ODP Leg 204, Site 1251)
Gulf of Mexico (IODP Exp. 308 Site U1320A)
Gulf of Mexico (IODP Exp. 308, Site U1324B)
108
108
108
107
107
n = 22, r = 0.85, P<0.01
106
105
104
Viral numbers cm–3
Viral numbers cm–3
Viral numbers cm–3
n = 23, r = 0.95, P<0.001
107
106
105
104
(a)
103
105
10
10
7
10
8
10
9
Microbial cell numbers cm–3
105
104
(b)
(c)
103
6
106
10
10
0
1
TOC (%)
2
103
40
50
60
70
80
90
Porosity (%)
Fig. 2. The relationships between microbial cell abundance and virus abundance (a), TOC and viral abundance (b), and porosity and viral
abundance (c) in the seafloor sediments. Red triangles, deep sediments at Site C9001 off Shimokita (CK06-06); black circles, ODP Site 1251 in
the Cascadia Margin; light-blue squares, IODP Site U1320A on the Gulf of Mexico continental slope; blue squares, IODP Site U1324B in the Gulf
of Mexico. The line was determined from linear regression analysis of each graph.
FEMS Microbiol Ecol 88 (2014) 60–68
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
K. Yanagawa et al.
66
Viral numbers vs. sediment age
Our study suggests the significance of viral preservation
and decay in the subseafloor viral abundance and its variation. Here, to estimate the net decay rates of the subseafloor viral population in the geological time scale (i.e.
longevity), we compared the viral abundance with
sediment age (Fig. 3 and Table S1). The sediment age of
each sample has previously been estimated based on
ª 2013 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Offshore the Shimokita Peninsula (CK06-06, Site C9001)
Cascadia Margin (ODP Leg 204, Site 1251)
Gulf of Mexico (IODP Exp. 308 Site U1320A)
Gulf of Mexico (IODP Exp. 308, Site U1324B)
108
107
Viral numbers cm–3
certain steady state in which the in situ viral production
of the host microbial communities dominates the in situ
viral decay (Fig. 2a). The relatively high-porosity sedimentary habitats off Shimokita (62–85%) would provide
habitable space for a larger microbial population size and
higher metabolic activity.
In contrast, no significant correlation between the viral
abundance and porosity was found for the subseafloor
sediments in the Cascadia Margin and the Gulf of Mexico
(Fig. 2c). The porosity values of sediments in the Cascadia Margin and the Gulf of Mexico were < 65%, most of
which were smaller than those of the C9001 site off Shimokita. In particular, fine sediment deposition with extremely high sedimentation rates in the Gulf of Mexico sites
likely accelerates the sediment compaction and the following decrease in porosity. These low-porosity sediments
may have an advantage for the preservation of viruses in
the subsurface because the viral abundances were relatively high and constant throughout the sediment depth.
The degree of preservation of viruses in the sediments is
considerably influenced by the in situ extracellular enzymatic degradation (Middelboe & Glud, 2006; Corinaldesi
et al., 2010). Particularly, the viruses were significantly
diminished by the extracellular hydrolytic enzymes such
as proteases and nucleases (England et al., 1998; Fischer
et al., 2004). Exoenzyme activity was indeed detected in
subseafloor environments in the eastern Mediterranean
Sea (Coolen et al., 2002) and also Site C9001 offshore of
the Shimokita Peninsula (Kobayashi et al., 2008). Nevertheless, the previous studies clarified that infectious
cyanophages retain their infectious capability in the sediments at depths down to 40 cm, corresponding to 100year-old sediment (Suttle, 2000). Furthermore, it was
revealed that the addition of DNases and proteases to the
subsurface sediment samples did not significantly increase
viral decay rates (Corinaldesi et al., 2010). These previous
studies, as well as the trends of viral abundances in the
Cascadia Margin and the Gulf of Mexico, may point to
the potential long-term preservation of viruses in the subseafloor sedimentary environments, particularly in the
low-porosity sediments. The preservation of viruses would
have a significantly greater impact on the viral abundance
than the in situ viral production.
106
105
104
103
10–4
10–3
10–2
10–1
1
10
Sediment age (Ma)
Fig. 3. Relationship between viral abundance and sediment depositional
age in the seafloor sediments. Red triangles, deep sediments at Site
C9001 off Shimokita (CK06-06); black circles, ODP Site 1251 in the
Cascadia Margin; light-blue squares, IODP Site U1320A on the Gulf of
Mexico continental slope; blue squares, IODP Site U1324B in the Gulf of
Mexico.
sedimentation rates and sedimentation models (Trehu
et al., 2003; Flemings et al., 2006; Aoike et al., 2010;
Domitsu et al., 2011). The result suggests that the viral
abundance shifts in sediments at 12 mbsf, corresponding
to 0.02 million years ago (Ma) offshore of Shimokita. As
described above, in situ viral production dominates in situ
viral decay in the Shimokita offshore sediments, whereas
in situ viral decay likely controls the viral abundance in
the other subseafloor sedimentary environments. The significant depletion of viral numbers down to one tenth of
the viruses in the surface sediments was also observed in
1.0–1.7 Ma sediment at approximately 30 mbsf in the
Porcupine Seabight (Middelboe et al., 2011). In these sedimentary environments, the viral abundance typically
decreases with increasing sediment depth. However, the
viral abundances were constant through all the depths in
the subseafloor sediments of the Gulf of Mexico, where
the rapid sedimentation prepared thick young sediments
(0.064 Ma even at 600 mbsf). These results indicate that
subseafloor viral populations can be maintained for
geological time scales of millions of years, as reported
previously (Corinaldesi et al., 2010; Middelboe et al.,
2011). As described above, viral degradation seems to be
associated with sediment porosity, which controls the
physical spaces in the sediments (i.e. sediment pore size
and pore-throat size). The decline in viral abundance in
the subseafloor sediments of the Cascadia Margin site was
more gradual over longer periods than in the sediments
of the offshore Shimokita Peninsula site (Fig 1). The
FEMS Microbiol Ecol 88 (2014) 60–68
Viral distribution in deep subseafloor sediments
overall porosity in the sediments of the Cascadia Margin
site is lower than that in the Shimokita offshore site. Less
porous sediments may lead to the better preservation of
viruses for longer periods in subseafloor sedimentary
environments.
Conclusion and future perspectives
In this study, we showed the depth variation in virus
and microbial cell abundances in the subseafloor sediments at three geographically and geologically distinct
locations. We found extremely low VPRs through all the
depths in the subseafloor sediments offshore of the
Shimokita Peninsula and constant viral abundances even
in the deep subseafloor sediments in the Gulf of Mexico
sites. This variation in virus and microbial cell abundances suggests a potential interaction between the in
situ viral production, decay, and preservation in subsurface sedimentary environments. Thus, the viral abundance in subseafloor sedimentary habitats could be
affected by the host microbial productivity and metabolic
activity coupled with the viral production, and the viral
decomposition function of the extracellular enzymes
from the microbial communities. Furthermore, the physical space in the sediment, including such factors as
porosity and pore-throat size, seems to be a significant
factor for maintaining viral populations in subseafloor
sedimentary environments over geologic time scales. The
viral ecology in subseafloor habitats (e.g. the infection
longevity of deeply buried viruses, viral genetic diversity,
in situ viral production/decay mechanisms, biogeochemical significance, and horizontal gene transfer) other than
the viral abundance and its biogeographical variation is
quite uncertain. Further investigation of the deep subseafloor viral ecology will shed light on these unclear questions in the future.
Acknowledgements
We sincerely thank R. Colwell, M. Delwiche, M. Holland,
the shipboard science party and crews of the ODP Leg
204 (D/V JOIDES Resolution), the IODP Expedition 308
(D/V JOIDES Resolution), and the Chikyu Shakedown
Expedition CK06-06 for sample collection. We thank
S. Tanaka and S. Fukunaga for technical support and
F. Rohwer for technical advice in our fluorescence
microscopic observations of viruses.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. The abundance of microbial cells and VLPs,
TOC, porosity, and sedimentation age from samples
collected at three distinct geological locations.
FEMS Microbiol Ecol 88 (2014) 60–68