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Previous Article | Next Article 
Applied and Environmental Microbiology, March 1999, p. 898-902, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Growth Characteristics of Heterosigma
akashiwo Virus and Its Possible Use as a Microbiological Agent for
Red Tide Control
Keizo
Nagasaki,*
Kenji
Tarutani, and
Mineo
Yamaguchi
Harmful Phytoplankton Section, Harmful Algal
Bloom Division, National Research Institute of Fisheries and
Environment of Inland Sea, 2-17-5 Maruishi, Ohno, Saeki, Hiroshima
739-0452, Japan
Received 2 November 1998/Accepted 2 December 1998
 |
ABSTRACT |
The growth characteristics of Heterosigma akashiwo
virus clone 01 (HaV01) were examined by performing a one-step growth
experiment. The virus had a latent period of 30 to 33 h and a
burst size of 7.7 × 102 lysis-causing units in an
infected cell. Transmission electron microscopy showed that the virus
particles formed on the peripheries of viroplasms, as observed in a
natural H. akashiwo cell. Inoculation of HaV01 into a mixed
algal culture containing four phytoplankton species, H. akashiwo H93616, Chattonella antiqua (a member of the
family Raphidophyceae), Heterocapsa triquetra (a member of
the family Dinophyceae), and Ditylum brightwellii (a member of the family Bacillariophyceae), resulted in selective growth inhibition of H. akashiwo. Inoculation of HaV01 and
H. akashiwo H93616 into a natural seawater sample produced
similar results. However, a natural H. akashiwo red tide
sample did not exhibit any conspicuous sensitivity to HaV01, presumably
because of the great diversity of the host species with respect to
virus infection. The growth characteristics of the lytic virus
infecting the noxious harmful algal bloom-causing alga were considered,
and the possibility of using this virus as a microbiological agent
against H. akashiwo red tides is discussed.
| |
INTRODUCTION |
Heterosigma akashiwo
virus (HaV) is a relatively large DNA virus that infects
Heterosigma akashiwo (a member of the family Raphidophyceae), which is one of the typical harmful algal bloom (HAB)-causing microalgae (11). The host specificity of HaV, the effects of physicochemical conditions on HaV algicidal activity, and storage techniques for HaV have been examined previously
(11-14).
H. akashiwo occurs in coastal waters of subarctic and
temperate areas of both the Northern Hemisphere and the Southern
Hemisphere. It often kills cultured fish, including salmon, yellowtail,
and sea bream, and the damage to aquaculture caused by H. akashiwo has been increasing (4, 5). Cage-reared
chinook salmon worth seventeen million New Zealand dollars were killed
in Big Glory Bay, New Zealand, in 1989 (2). In Japan,
cultured yellowtail, amberjack, striped jack, etc. worth 1,090 million
and 237 million yen were killed in Kagoshima Bay in 1995 and in Nomi
Bay in 1997, respectively (the amounts of damage are estimates made by
the Fisheries Agency of Japan). Therefore, practical countermeasures against blooms caused by this noxious microalga are urgently needed. In
order to develop practical countermeasures for eliminating HAB, the
algicidal activities of microorganisms have been examined, and
algicidal bacteria and viruses have recently been isolated and studied
(7).
Any microbiological agent used for elimination of HAB in the natural
environment must fulfill the following three requirements: it must be
practical in terms of scale, cost, and safety (10). Thus,
scale and the specificity of algicidal activity must be considered
prior to application of HaV. The objective of this study was to examine
the growth characteristics of HaV and the algicidal activity of HaV in
mixed algal cultures and in natural waters. Based on the results
obtained, the possibility of using HaV as a microbiological agent
against H. akashiwo is discussed.
| |
MATERIALS AND METHODS |
Host strains and virus clones.
Two strains of H. akashiwo were used in this study; H. akashiwo H93616
was isolated from the northern part of Hiroshima Bay (Hiroshima
Prefecture) in June 1993, and H. akashiwo NM96 was isolated
from Nomi Bay (Kochi Prefecture) in July 1996 (12). Neither
algal culture contained bacteria. The algal strains were grown in
modified SWM3 medium (3, 8) enriched with 2 nM Na2SeO3 at 20°C by using a cycle consisting
of 14 h of cool white fluorescent illumination (ca. 45 µmol of
photons m
2 s1) and 10 h of darkness
prior to each experiment.
HaV clone 01 (HaV01), which infects both H. akashiwo NM96
and H93616 and was isolated from Unoshima Fishing Port (Fukuoka Prefecture) in 1996, was used in this study (12). This virus clone was made axenic by filtering a preparation through a
0.2-µm-pore-size Nuclepore filter and treating it with antibiotics.
Just prior to each experiment, the HaV01 stock preparation was
inoculated into a fresh culture of H. akashiwo NM96 or
H93616 and incubated at 20°C for 3 days for multiplication, and the
resulting viral suspension was used as the inoculum. The virus titer
was estimated by the extinction dilution method (11), and
the most probable number was calculated by using the computer program
developed by Nishihara et al. (16).
One-step growth experiment.
In order to estimate the latent
period and burst size of HaV01, a one-step growth experiment was
designed. An H. akashiwo NM96 culture in the late log phase
was inoculated with HaV01. The initial densities of the host alga and
HaV01 were 1.27 × 105 cells/ml and 2.58 × 105 lysis-causing units (LCU)/ml, respectively; viz., the
multiplicity of infection (MOI) was 2.04. The algicidal effects were
monitored by determining the host cell density directly by optical
microscopy. When most of the host cells had disappeared, the HaV01
density was titrated by the extinction dilution method, and the burst size was calculated. The incubation conditions were as described above
and were determined based on the effects of physicochemical conditions
on the algicidal activity of HaV01 (13). Aliquots of the
algal culture were periodically examined by transmission electron
microscopy. The samples used for transmission electron microscopy were
prepared by using the method of Nagasaki et al. (15).
Algicidal effects of HaV01 in the mixed algal culture.
Exponentially growing H. akashiwo H93616, Chattonella
antiqua OC-B5 (a member of the family Raphidophyceae),
Heterocapsa triquetra H9104 (a member of the family
Dinophyceae), and Ditylum brightwellii (a member of the
family Bacillariophyceae) cultures were mixed; the initial densities of
the cultures used were 2.0 × 103, 5.2 × 102, 2.2 × 102, and 1.4 × 102 cells/ml, respectively. HaV01 was inoculated into 50-ml
portions of the mixed culture so that the initial HaV01 densities were 6.4 × 103 and 6.4 × 101 LCU/ml;
viz., the MOI with respect to H. akashiwo cells were 3.2 and
0.032, respectively. As a control, an HaV01 suspension was incubated at
100°C for 5 min, cooled, and inoculated into a portion of the mixed
culture at an initial density of 6.4 × 103 LCU/ml.
The incubation conditions were the same as those described above. The
density of each alga was monitored by direct counting with an optical microscope.
Algicidal activity of HaV01 in the natural seawater culture.
The algicidal effects of HaV01 in natural seawater were examined twice.
First, surface water was collected in Kure Port in northern Hiroshima
Bay on 8 April 1998; the density of H. akashiwo in this
water was 5 cells/ml. H. akashiwo H93616 and HaV01 were
inoculated into 100 ml of this natural seawater at initial densities of
6.3 × 103 cells/ml and 1.7 × 106
LCU/ml, respectively; viz., the MOI with respect to H. akashiwo cells was 260. For the control experiments, we used a
filtrate obtained by passing an H. akashiwo H93616 culture
through a type GF/F filter and a HaV01 suspension that had been
incubated at 100°C for 5 min. The effects of the algal culture
filtrate and the heat-treated virus suspension on the growth of
H. akashiwo H93616 were also examined. The incubation
conditions and methods used to monitor algal growth were the same as
those described above. For the second experiment, surface water was
collected in Kusatsu Fishing Port in northern Hiroshima Bay on 28 April 1998; diatoms were the dominant organisms in this water, and H. akashiwo was not detected. H. akashiwo H93616 and HaV01
were inoculated into 100 ml of this natural seawater at initial
densities of 3.3 × 103 cells/ml and 2.3 × 103, 2.3 × 102, and 2.3 × 101 LCU/ml, respectively; viz., the MOI with respect to
H. akashiwo cells (including both natural cells and strain
H93616 cells) were 0.70, 0.07, and 0.007, respectively. For the control
experiment, we used an H. akashiwo H93616 culture filtrate
and a heat-treated suspension of HaV01. The incubation conditions and
monitoring methods used were the same as those described above.
| |
RESULTS |
One-step growth experiment.
In the one-step growth experiment,
H. akashiwo cells became roundish within 8 h after
inoculation of HaV01 (Fig. 1). Almost all
of the cells lost motility within 24 h. The H. akashiwo
cell density decreased drastically and the culture became transparent 30 to 33 h after inoculation (Fig.
2). After lysis of most of the cells in
the host culture at 33 to 47 h after inoculation, a small number
of surviving cells were observed; these cells were roundish and had
lost motility. At 47 h after inoculation, the host cell density
had decreased to less than 101 cells/ml, while the density
of HaV01 had increased to 9.8 × 107 LCU/ml (Fig. 2).
Considering the high initial MOI (2.04) and the synchronous lysis of
the host cells, it is probable that most of the H. akashiwo
cells did not escape HaV01 infection; that is, this experiment
established that one-step growth of HaV01 occurred. The data show that
9.8 × 107 HaV01 particles originating from 1.3 × 105 virus-infected H. akashiwo cells were
released into the culture, indicating that ca. 7.7 × 102 infectious particles were produced by each H. akashiwo cell infected with HaV01. The latent period of HaV01 is
considered to be 30 to 33 h.
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FIG. 1.
Optical microphotographs of H. akashiwo NM96
cells before inoculation (A) and 4 h (B) and 8 h (C) after
inoculation of HaV01.
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FIG. 2.
Changes in density of H. akashiwo cells in
the one-step growth experiment in which the initial MOI of HaV01 was
2.04. The error bars indicate standard deviations.
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|
At 24 h after virus inoculation, both mature and immature daughter
virus particles were observed in most of the thin sections of H. akashiwo cells. A typical thin section of an infected cell contained ca. 170 sections of virus particles in a circular area with a
diameter of 4 µm (Fig. 3A). Assuming
that virus particles were distributed at the same concentration in a
virus-synthesizing globular zone, ca. 103 virus particles
could have been present in a hypothetical globe. This value is
comparable to the burst size (7.7 × 102 LCU/ml)
calculated by the extinction dilution method in the one-step growth
experiment. HaV01 particles were presumably formed on the periphery of
the viroplasm (Fig. 3B), as previously observed in H. akashiwo samples obtained from natural waters (15),
suggesting that the viruslike particles observed in the previous field
studies and the HaV01 particles are identical. HaV01 particles were
also observed in the subsurface area, which presumably had been
released from the viroplasm and had virus DNA inserted (Fig. 3A,
arrowhead).
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FIG. 3.
(A) Transmission electron micrograph of an H. akashiwo NM96 cell 24 h after inoculation of HaV01. (B)
Higher magnification of viroplasm (VP). Virus particles were found in
the subsurface area, as well as in the viroplasm area (arrowheads).
Note the difference between the mature virions (double arrowhead) and
the immature virions (white arrowhead). CH, chloroplast.
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|
Algicidal effects of HaV01 in the mixed algal culture.
Specific algicidal effects of HaV01 were observed even when C. antiqua, H. triquetra, and D. brightwellii
were also present (Fig. 4). Although the
rate of disappearance of H. akashiwo was affected by the
MOI, H. akashiwo was specifically eliminated even with the
lower MOI used in this experiment (0.03). In contrast, HaV01 had no
conspicuous effect on the growth of the other three species of
phytoplankton.
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FIG. 4.
Changes in densities of H. akashiwo (A),
C. antiqua (B), H. triquetra (C), and D. brightwellii (D) cells in the mixed algal culture inoculated with
HaV01 at MOI of 3.23 ( ), 0.03 ( ), and 0 ( ). The error bars
indicate standard deviations.
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|
Algicidal activity of HaV01 in the natural seawater culture.
The algicidal effects of HaV01 in natural seawater are shown in Fig.
5 and 6.
HaV01 specifically affected H. akashiwo H93616 in
unsterilized natural seawater cultures containing numerous natural
microorganisms (data not shown). In addition, HaV01 had no obvious
effect on the growth of diatoms even at an MOI of 260, which in a
natural environment would correspond to an extraordinarily high HaV
density (Fig. 5). H. akashiwo H93616 was specifically eliminated even when the MOI was as low as 0.007 (Fig. 6).
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FIG. 5.
Changes in densities of H. akashiwo () and
diatom ( ) cells in the natural seawater sample collected at Kure
Port on 8 April 1998. The natural seawater was inoculated with an
H. akashiwo culture (a+) and nontreated HaV01 (v+) (A), an
H. akashiwo culture and heat-treated HaV01 (v ) (B), an
H. akashiwo culture filtrate (a) and nontreated HaV01 (C),
and an H. akashiwo culture filtrate and heat-treated HaV01
(D). The error bars indicate standard deviations.
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FIG. 6.
Changes in densities of H. akashiwo () and
diatom () cells in the natural seawater collected at Kusatsu Fishing
Port on 28 April 1998. Natural seawater samples were inoculated with
H. akashiwo and HaV01 at MOI of 0.7 (A), 0.07 (B), 0.007 (C), and 0 (D). The error bars indicate standard deviations.
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| |
DISCUSSION |
HaV01 has a latent period of 30 to 33 h and a burst size of
7.7 × 102 LCU per infected cell. Compared with the
other microalgal viruses whose growth characteristics have been
studied, the burst size and the latent period of HaV01 are greater and
longer, respectively (Table 1). If these
data are used, the potential infectivity can be provisionally
calculated based on the growth characteristics of HaV01 estimated in
the one-step growth experiment. If an HaV01 particle whose latent
period and burst size are 33 h and 7.7 × 102
LCU/ml, respectively, is placed in 1 ml of seawater and constantly supplied with sensitive fresh host cells, ca. 6.0 × 105 infectious particles should be released into the 1 ml
of seawater after two cycles of infection, which should take ca.
66 h. Considering that the highest density of H. akashiwo cells in natural red tide water is ca. 4 × 105 cells/ml (6), this virus density is high
enough to potentially infect all of the cells. The highest yield of
HaV01 that we have obtained in our laboratory is ca. 108
infectious LCU/ml, which implies that 1 ha of shallow (mean depth, 10 m) coastal water should have an HaV density of ca. 1 particle/ml if 1 liter of the HaV suspension is used. In addition,
low-cost small-scale production of HaV (in 2-liter flasks) is possible in a laboratory. Although the calculations described above are merely
theoretical, use of HaV as a microbiological agent appears to be
promising from the viewpoint of scale and cost. Of course, additional
technical improvements would be required for large-scale production of
HaV prior to practical use of HaV as a microbiological agent for
elimination of red tide.
The present experiments also showed that HaV01 specifically eliminates
H. akashiwo H93616 when other phytoplankton species are
present and in natural seawater containing numerous natural microorganisms, even at a low MOI. Thus, HaV01 specifically infects H. akashiwo even when other microorganisms are present in
the ambient water. From the viewpoint of the safety of using HaV as a
microbiological agent for elimination of red tide, HaV specifically affects the target alga, H. akashiwo, and appears to have
little influence on other phytoplankton in the natural environment.
On the basis of the characteristics determined so far, HaV01 is a
promising tool for controlling H. akashiwo red tides because (i) it has a high growth rate and thus can be applied to natural environments (i.e., it meets the scale requirement), (ii) it can be
produced at a low cost, and (iii) it specifically attacks the target
HAB-causing alga and has little or no effect on other organisms, guaranteeing its safety. In addition, HaV01 originates from natural coastal water and has not been genetically manipulated or altered in
any way from its natural form (11).
A preliminary examination on the algicidal effects of HaV01 on a
natural population of H. akashiwo red tide was performed by
using water collected in northern Hiroshima Bay on 18 May 1998, in
which H. akashiwo (8.0 × 103 cells/ml) and
Eutreptiella spp. (8.2 × 103 cells/ml)
dominated and diatoms were scarce. HaV01 was inoculated into the
natural seawater sample at an MOI with respect to the natural H. akashiwo cells of 0.12. However, little specific growth inhibition
of H. akashiwo was detected (data not shown). Although it
has not been determined why the natural H. akashiwo
population was not eliminated, it should be noted that natural
populations of both sensitive and resistant cells occur in natural
H. akashiwo red tide seawater samples (12).
Therefore, the intraspecies specificity of HaV appears to be the most
difficult obstacle to the use of this virus as a microbiological agent
against H. akashiwo red tides. To solve this problem, what
determines the infection specificity of HaV against H. akashiwo must be clarified. Another practical solution would be to
prepare a cocktail of HaV clones, each with a different specificity of infection.
In conclusion, although HaV is a possible microbiological agent when
scale, cost, and safety are considered, the effects of various HaV
clones on natural populations of H. akashiwo must be
assessed in more detail before this virus can be used for elimination of H. akashiwo red tides.
| |
ACKNOWLEDGMENTS |
This work was supported by the Fisheries Agency of Japan.
We thank S. Norland (University of Bergen) for preparing the
most-probable-number computer program and S. Itakura (National Research
Institute of Fisheries and Environment of Inland Sea) for isolating
H. akashiwo NM96.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Harmful
Phytoplankton Section, Harmful Algal Bloom Division, National Research
Institute of Fisheries and Environment of Inland Sea, 2-17-5 Maruishi,
Ohno, Saeki, Hiroshima 739-0452, Japan. Phone: 81-829-55-0666. Fax: 81-829-54-1216. E-mail: nagasaki{at}nnf.affrc.go.jp.
| |
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Applied and Environmental Microbiology, March 1999, p. 898-902, Vol. 65, No. 3
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
