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The role of viruses in marine phytoplankton mortality
Baudoux, Anne-Claire
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Chapter 1
Introduction
Marine viruses, also referred to as virioplankton, are the most abundant and
diverse biological entities in the ocean. The ecological implication of marine viruses
goes beyond the mortality of their host. Viruses can substantially influence plankton
community structure. The cell lysis of the infected host may furthermore affect processes
of global significance, notably the cycling of nutrients. Viral pathogens are found to
infect all the major classes of phytoplankton. Algal viruses appear to be involved in the
disintegration of algal blooms, but only a limited number of studies has investigated
their role in population dynamics of non-bloom forming phytoplankton. Therefore, the
prevalence of virally mediated mortality in phytoplankton still remains elusive, and the
significance of viral lysis related to other phytoplankton losses is essentially unknown.
The purpose of this thesis is to address these issues in order to better understand the
ecological significance of algal viruses, specifically examining the impact of these
pathogens in marine environments with contrasting trophic status (eutrophic vs.
oligotrophic).
This introductory chapter covers different aspects of viral ecology, with special
emphasis on viruses infecting phytoplankton and the interaction with their host. The
chapters that follow describe the results from field and laboratory studies investigating
the ecological role of viral infection of phytoplankton as compared to other sources of
phytoplankton losses (e.g. microzooplankton grazing) in different marine systems.
1. Viruses in marine ecosystems
In essence viruses are simple. They are defined as small particles composed of
nucleic acids (either DNA or RNA) embedded in a protein shell (named caspid) that may
be surrounded by an envelope. Viruses do not respire, move, or grow. They do not have
inherent metabolism, therefore, they utilize the cellular machinery of a suitable host to
replicate. A given virus infects a restricted range of hosts. Most viruses described to date
are species-specific, i.e., they infect a single host species and sometimes even a single
9
Chapter 1
Introduction
strain within a species. As they do not move, viruses depend on passive diffusion to
contact a suitable host. Consequently the encounter rate between a virus and a host is
directly affected by their relative abundance.
1.1. Global abundance and diversity of marine viruses
In the years following the discovery of high virus abundance (typically 106 to
10 viruses mL-1), field studies have emphasized that marine viruses are also a dynamic
component of the planktonic community (Bergh et al. 1989). For example, virioplankton
abundance varies with depth (Hara et al. 1996, Culley & Welschmeyer 2002), along
trophic gradients (Weinbauer et al. 1993, Noble & Fuhrman 2000), and during the course
of phytoplankton blooming events (Bratbak et al. 1990, Castberg et al. 2001, Brussaard
et al. 2004b). It is now well accepted that viruses are present wherever life is found, and
current estimates of ≈ 1030 viruses in the world’s ocean indicate that they are the most
abundant marine biological entities (Suttle 2005).
The observation of natural virioplankton assemblages using transmission
electron microscopy (TEM) has revealed that, beyond their high abundance, viruses vary
in size and shape (Flint et al. 2000); most of them are polyhedral and range between 20
and 200 nm. The isolation and the characterization of viruses infecting specific microbial
host cultures, including prokaryotic and eukaryotic microbes, consistently showed that at
least one, and usually multiple, viruses can infect a single host species (see reviews by
Brussaard 2004, Weinbauer 2004). It is, therefore, sensible to assume that the richness of
viruses is at least as high as that of cellular life forms (Weinbauer 2004).
The development of culture-independent methodologies has considerably
advanced our understanding of global virus diversity. The use of pulsed field gel
electrophoresis (PGFE), that discriminates viruses based on their genome size, showed
that natural viral assemblage typically comprise 7 to 35 distinct viral genome size classes
ranging between 12 and 560 kilobases (Auguet et al. 2005 and reference therein). Using
this approach, pronounced variations in virus community structure were detected in
response to phytoplankton bloom formation (Castberg et al. 2001, Larsen et al. 2001),
water column stratification (Wommack et al. 1999), or salinity gradient (Sandaa et al.
2003). Overall, PFGE results confirm that viral populations are spatially and temporally
dynamic as previously predicted from changes in viral abundance.
The most striking evidence of high viral richness in the ocean was reported by
metagenomic analyses of uncultured marine viral communities (Breitbart et al. 2002,
Angly et al. 2006). The recent sequencing of shotgun libraries of 168 viral assemblages
collected from four major oceanic regions revealed that several hundred thousand
distinct viral species were dispersed in these waters (Angly et al. 2006). Most of these
viral genotypes were not similar to previously reported sequences, indicating that much
of the viral diversity is actually uncharacterized. An emerging view of viral diversity
contends that the vast majority of viral species is widespread and that the local
environmental conditions select for certain viral type through selective pressure.
8
10
Chapter 1
Introduction
1.2. Production vs. decay of marine viruses
In theory, all living marine organisms from ‘bacteria to whale’ are likely to be
infected by at least one virus. However, the host-virus encounter is an abundancedependent process; hence the majority of viruses probably infect the organism they most
frequently encounter, i.e., the bacteria and phytoplankton.
Viruses are produced by their host through four types of replication. The lytic
infection results in the release of virus progeny upon host lysis. The number of viruses
produced per infected cells is called the burst size. The magnitude of viral burst size has
important ecological implications as it directly influences the viral abundance, hence the
propagation of viral infection.
Viruses can also interact with their host through lysogeny (or latency) where the
viral genome is incorporated into the host cell genome (termed as prophage) and
propagates along with host replication until an inducing event triggers the lytic pathway.
The incidence of lytic or lysogenic replications may relate to ecological strategies.
Lysogenic replication may prevail over lytic infection when successful host-virus
encounter rate is too low to sustain lytic replication (Lenski 1988, Weinbauer 2004). The
importance and mechanisms underlying lytic vs. lysogenic replication are unclear and
therefore still need further investigation. So far lysogeny has only been reported for
prokaryotic microbes.
Although the lytic and lysogenic infections are the most investigated forms of
replication in marine environments, two other types of replication are described. These
include the chronic infection where viruses are released by budding or extrusion without
killing the host and the pseudolysogeny infection whereby the virus either enter a
dormant intracellular phase or proceed with lytic infection. This type of infection
resembles a true lysogenic infection except that the viral genome does not integrate into
host genome (as cited in Williamson et al. 2001).
The term ‘viral decay’ includes the reduction in viral abundance and infectivity.
Many different biotic and abiotic factors are involved in the loss of virus in the ocean.
Among these, solar radiations, particularly ultraviolet B radiations (UV-B), are
considered a major source of loss as they damage viral DNA (Suttle 2000, Wilhelm et al.
2002, Jacquet & Bratbak 2003). The effect of damaging sunlight can still be significant
at 10 m depth, as reported in the Gulf of Mexico offshore waters (Wilhelm et al. 2002).
Viruses may also be inactivated, at least temporarily, by adsorption to host cells, high
molecular weight dissolved organic matter, and transparent exopolymeric particles
(Noble & Fuhrman 1997, Brussaard et al. 2005b, Ruardij et al. 2005). Viral losses have
been observed due to grazing by nanoflagellates (Suttle & Chen 1992, Gonzalez & Suttle
1993) and adsorption of viruses to particles that sink out of the photic zone (Proctor &
Fuhrman 1991). The latter observation is consistent with the report of high virus
numbers in the sediments (Danovaro et al. 2001, Lawrence et al. 2001).
11
Chapter 1
Introduction
2. Ecological implications of marine viruses
2.1. The influence of viruses on the community composition
The majority of the marine viruses has a narrow host specificity, implying that
only a small subset of the community is infected by a given virus. The current consensus
is that virus will preferentially infect the most abundant host species (because of higher
encounter rate) and by “killing the winner” viruses maintain the coexistence of
competing species (Thingstad 2000). The incidence of changes in viral and microbial
community structure following lysis events strongly support the role of viruses as a
driving force for the interspecies competition and succession (Castberg et al. 2001,
Larsen et al. 2001, Brussaard et al. 2005b). Lately, several field studies demonstrated
that many marine viruses may even show intraspecies specificity, suggesting that viruses
may also influence the clonal composition of the host species (Tarutani et al. 2000,
Tomaru et al. 2004b, Mühling et al. 2005).
2.2. The effect of viruses on the biogeochemical cycles
Over the last two decades, it became evident that microbial processes largely
drive the cycle of matter and energy in the ocean. Whilst phytoplankton constitute the
base of the classical (grazing) food web, heterotrophic prokaryotes recycle dissolved
organic matter (DOM) to inorganic nutrients and bacterial biomass through the microbial
loop (Fig. 1). These microbes can be eaten by small predators, eventually leading back to
the classical (grazing) food web. In the original hypothesis of the ‘microbial loop’, the
primary source of DOM was assumed to derive from phytoplankton exudates and sloppy
feeding by zooplankton (Azam et al. 1983). Through the lysis of their host, viruses also
influence the cycling of DOM (Wilhelm & Suttle 1999). Prokaryotic and eukaryotic
viruses efficiently convert the particulate organic matter (i.e., living biomass) to DOM
that can be utilized by bacteria (Brussaard et al. 1996b, Middelboe et al. 2003). This
“viral shunt” results in diverting the flow of matter and nutrient away from the higher
trophic levels and, in turn, forces the food web towards a more regenerative system.
The incorporation of “viral module” in simple theoretical models systems
demonstrated that viral lysis enhanced bacterial respiration and production (Fuhrman
1999, Wilhelm & Suttle 1999) and reduced protist production (Fuhrman 1999). The first
mathematical ecosystem model based on empirical data confirmed that virally mediated
mortality of the bloom-forming algal species Phaeocystis globosa was an essential
regulating factor for the nutrient cycling (Ruardij et al. 2005). Experimental studies also
supported these models’ predictions. Viral mediated release of DOM can constitute a
significant supply of major nutrients (C, N, P) and trace nutrients (e.g. Fe) for other
12
Chapter 1
Introduction
photosynthetic and heterotrophic microorganisms (Middelboe et al. 1996, Göbler et al.
1997, Middelboe et al. 2003, Poorvin et al. 2004). Viral mediated nutrient cycling was
furthermore shown to influence bacteria and phytoplankton species composition and
succession (Göbler et al. 1997, Brussaard et al. 2005b).
Larger gazers
Bacteria
Debris,
exudates
Viruses
Viruses
Small grazers
(HNF, microzooplankton)
Inorganic
nutrients
DOM
Debris, exudates
Debris,
exudates,
sloppy
feeding
Viruses
Phytoplankton
Figure 1. Simplified diagram of the microbial loop. Through cell lysis of hosts, viruses divert
living biomass away from the higher trophic levels of the food web (microzooplankton,
heterotrophic nanoflagellates (HNF), larger grazers). Instead, living biomass is effectively
converted to dissolved organic matter (DOM), available for heterotrophic bacteria, hence forcing
the food web towards a more regenerative pathway. Black arrows indicate the flow of organic
matter and grey arrows represent the flow of inorganic nutrients.
Another possible effect of viruses on processes of global significance is to
accelerate the production of dimethylsulfide (DMS). The DMS is a biogas that
influences clouds formation, hence the global climate (Charlson et al. 1987). Many
phytoplankton species produce dimethylsulfoniopropionate (DMSP) that may be cleaved
into DMS and acrylic acid (AA) by the algal lyases and/or by the lyases of other
organisms. Laboratory studies have demonstrated that viral lysis of Micromonas pusilla,
Phaeocystis pouchetii, and Emiliania huxleyi was accompanied by a build up of DMSP
in the media (Hill et al. 1998, Malin et al. 1998, Wilson et al. 2002). Therefore, viral
lysis of phytoplankton may be an important source of DMSP in the environment.
13
Chapter 1
Introduction
3. Viruses and phytoplankton hosts
Soon after the discovery of high abundance of marine viruses, Suttle et al.
(1990, 1992) showed that adding virus concentrates to natural field samples could result
in decreased primary production. Complementing these studies, the observation of viral
infected algal cells suggested that viruses can account for significant phytoplankton
losses, particularly during blooming events (Bratbak et al. 1993, Nagasaki et al. 1994,
Brussaard et al. 1996b). With phytoplankton forming the base of the marine pelagic food
web and the awareness of the ecological and socio-economical consequences of algal
blooms (e.g., fisheries and tourisms activities), the ecology of algal viruses and their
contribution to phytoplankton mortality has gained considerable interest.
3.1 Taxonomy and phylogeny of algal viruses
Marine phytoplankton communities include a prokaryotic (cyanobacteria) and a
eukaryotic component. Currently, host specific viruses are reported for both groups of
phytoplankton. Viruses that infect cyanobacteria, referred to as cyanophages, were
reported in unicellular (Proctor & Fuhrman 1990) and colonial cyanobacteria (Ohki
1999, Hewson et al. 2004). The ecologically important marine Synechococcus sp. and
Prochlorococcus sp. are, by far, the most investigated cyanobacterial hosts (for reviews
see Suttle 2000, Mann 2003). All known cyanophages have tails, present double
stranded DNA, and belong to three families that also infect heterotrophic bacteria, the
Myoviridae, the Siphoviridae, and the Podoviridae. Besides morphological differences,
cyanophages belonging to these families also have variable “life styles”. For example,
the Myoviridae are typically lytic and have a broader host range than the other tailed
cyanophages. Conversely, the Podoviridae present the narrowest range of host. The
replication of Siphoviridae differs from other tailed phages as they can interact with their
host through lysogeny, and may thus propagate along with the host replication (see also
section 1.3).
Viruses have been isolated for the major existing classes of eukaryotic
phytoplankton. Unlike cyanophages, all known eukaryotic algal viruses propagate
through a lytic pathway. Until recently, most of these viruses were consistently assigned
to the family of large double stranded (ds)DNA viruses, the Phycodnaviridae (Wilson et
al. 2005b). Molecular based analysis using the highly conserved DNA polymerase (pol)
gene allowed distinguishing six genera within this family. With the increasing number of
viruses characterized, it became evident that viruses infecting phytoplankton are
extremely diverse with representatives in many more viral families than the
Phycodnaviridae. For example, picorna-like positive sense single stranded (ss)RNA
viruses were found to infect the diatom Rhizosolenia setigera (Nagasaki et al. 2004) and
the two toxic harmful algal bloom (HAB) species Heterocaspa circularisquama
14
Chapter 1
Introduction
(Tomaru et al. 2004a) and Heterosigma akashiwo (Tai et al. 2003). The recently
completed genomic sequence of the ssRNA H. akashiwo virus led to the creation of the
new, distinct viral family, the Marnaviridae (Lang et al. 2004). Previously unknown
nuclear inclusion viruses have also been reported to infect H. akashiwo (Lawrence et al.
2001) as well as the diatom Chaetoceros cf gracilis (Bettarel et al. 2005). Another
example of novel virus is the dsRNA virus that infects the cosmopolitan Micromonas
pusilla assigned to the Reoviridae family (Brussaard et al. 2004a, Attaoui et al. 2006).
These examples emphasize that many different viruses can infect the same algal species.
We are only starting to uncover the diversity of marine algal viruses. Many more viruses
need to be isolated and characterized in order to better evaluate algal virus richness.
3.2. Abundance and diversity of marine algal viruses
In the field, viral titer determination using the most probable number (MPN)
culture based method has been proven very useful in the study of algal virus ecology.
These studies indicated that infective algal viruses can be highly abundant (up to >105
mL-1), dynamic, and exhibit a high level of diversity (Waterbury & Valois 1993, Suttle
& Chan 1994, Cottrell & Suttle 1995a, Tomaru et al. 2004b).
The use of molecular tools allowed discriminating between different virus
isolates infecting the same species. For example, Cottrel and Suttle (1995b)
distinguished different Micromonas pusilla virus isolates using DNA hybridization. The
diversity of cyanophages (namely, myocyanophages) was estimated by the sequence
analysis of the gene encoding a structural protein g20 (Füller et al. 1998, Zhong et al.
2002, Mühling et al. 2005). Other genetic markers, such as the gene fragment of the
putative major caspid protein of Emiliania huxleyi viruses also revealed a high molecular
diversity among E. huxleyi viruses (Schroeder et al. 2002).
3.3 Algal viruses and phytoplankton mortality
The significance of algal viruses in terms of abundance, dynamics, and diversity
indicates a significant role of viral lysis in phytoplankton ecology. However, to fully
understand the role of viruses, it is essential to determine the extent of mortality they
impose on their host. The studies that have addressed this issue were mainly conducted
during conditions of high host cell abundance such as during phytoplankton blooms. In
theory, when the host cell abundance is high, the probability of collision between a host
and a virus increases, hence viruses may propagate rapidly through the host population.
This may result in bloom collapse if the viral lysis rates exceed the specific host growth
rates. This type of interactions is referred to as a control by “reduction” (Brussaard
2004). Several reports confirmed this theory and demonstrated that viruses are
profoundly involved in the disintegration of algal blooms. For example, high proportions
(10 - 50%) of algal cells were visibly (using TEM) infected at the end of a bloom of
15
Chapter 1
Introduction
Aureococcus anophagefferens (Sieburth et al. 1988, Gastrich et al. 2004), Heterosigma
akashiwo (Nagasaki et al. 1994), and Emiliania huxleyi (Bratbak et al. 1993, 1996,
Brussaard et al. 1996b). Other approaches determining cell lysis from the number of
putative algal viruses produced divided by an empirical viral burst size indicated that
viruses accounted for substantial mortality (7 - 100%) during the bloom of Phaeocystis
globosa (Brussaard et al. 2005a, Ruardij et al. 2005) and E. huxleyi (Jacquet et al. 2002).
While several studies have examined the role of viruses in controlling algal
bloom dynamics, fewer studies have investigated the potential role of viruses in
regulating non-blooming phytoplankton populations. Examinations of the picoeukaryote
Micromonas pusilla and its specific viruses indicated a turnover of host standing stock
between 2 and 25% d-1 in various marine systems (Cottrell & Suttle 1995a, Evans et al.
2003). Studies conducted on the cyanobacterium Synechococcus reported that virally
induced mortality daily removed <1 to 8% of the host population (Waterbury & Valois
1993, Suttle & Chan 1994, Garza & Suttle 1998). These results suggest a stable hostvirus coexistence, where the viruses seem to maintain host population size to nonblooming level rather than causing a rapid decline in host abundance. This type of
regulation is referred to as a “preventive” viral control (Brussaard 2004).
Overall, virally mediated mortality can occur at significant rates in
phytoplankton populations. However, our understanding of the global significance of
viral lysis is far from complete because non-blooming phytoplankton and more
generally, phytoplankton in oligotrophic environments have been inadequately sampled
as compared to bloom forming species, typically found in eutrophic (coastal) waters.
3.4. Potential factors regulating virally mediated mortality of
phytoplankton
The above referred field studies indicate that viral lysis can be responsible for
significant mortality in phytoplankton. Different factors can, however, regulate the
dynamics of virally mediated mortality. These regulatory parameters include
phytoplankton host abundance, morphology, physiology and their potential to develop
defense mechanisms.
As viral infection is a stochastic process, the rate of encounter depends on the
hosts and virus abundance and also on other morphological characteristics such as
particle size and motility (Murray & Jackson 1992). At a given virus concentration,
larger hosts will be more readily intercepted by a virus than the smaller counterparts.
Host cell motility enhances transport rates which, in turn, increase the probability of
collision with a given virus. Other host morphological characteristics can influence viral
infection rate. For example, field and laboratory evidence suggested that non-coccolithbearing Emiliania huxleyi are more readily infected than the lithed cells (Brussaard et al.
1996b, Jacquet et al. 2002). This is, however, not (yet) confirmed by controlled
experiments. Furthermore, a mesocosm study showed that Phaeocystis globosa cells
embedded in a colonial matrix tend to escape viral infection (Brussaard et al. 2005a,
16
Chapter 1
Introduction
Ruardij et al. 2005). Interestingly, this can be explained by the larger colonial size
(Ruardij et al. 2005).
The most efficient defense of phytoplankton against viral infection is to be
resistant. The incidence of resistant host strains has been reported for algal viruses in
culture (Thyrhaug et al. 2003) as well as in the field (Waterbury & Valois 1993). Theory
based on bacterial host-phages interactions suggests that resistance has a physiological
cost for the host cells, resistant cells may have a competitive disadvantage against
susceptible hosts (Levin et al. 1977). So far, the importance and the mechanisms
underlying resistance of phytoplankton against viruses remain largely unclear.
A potential phytoplankton chemical defense against viruses was recently
suggested by Evans et al. (2006). These authors related the negative effect of acrylic acid
(AA) and dimethylsulfide (DMS) on the titers of Emiliania huxleyi virus to the inability
to isolate viruses infecting E. huxleyi strains with high lyase activity (i.e., capable of
efficient conversion of dimethylsulfoniopropionate (DMSP) into the AA and DMS). It
was suggested that the cleavage of DMSP in DMS and AA during cell lysis of E. huxleyi
may reduce the titers of E. huxleyi viruses, and therefore decrease the probability of
infection of further cells. These observations led to argue that the DMSP system in
phytoplankton may operate as a chemical defense against viral infection. This study
supported the hypothesis that virucidal compounds can be produced alongside viruses
during viral infection, and, in turn, can reduce infection rates of other algal cells
(Thyrhaug et al. 2003). Another example of potential host defense strategy includes the
enhanced sinking rates of Heterosigma akashiwo cells when infected by a virus
(Lawrence & Suttle 2004). Viral infection may result in cells rapidly sink out of the
euphotic zone, which, in turn, may prevent viral infection of conspecifics.
As obligate parasites, viruses depend on the host cellular machinery to
propagate. Several studies have shown that the algal host growth stage may significantly
influence the lytic viral growth cycle. Reduction in viral burst size (Van Etten et al.
1991, Bratbak et al. 1998) and even prevention of viral infection were observed
(Nagasaki & Yamaguchi 1998) during the algal host stationary growth phase. The algal
host cell cycle stage may also influence the production of algal viruses (Thyrhaug et al.
2002). Viral infection of Pyramimonas orientalis at the onset of the light period led to a
higher viral production than when infected at the beginning of the dark period.
Conversely, Phaeocystis pouchetii infection was independent of the host cell cycle.
Different environmental variables known to influence phytoplankton growth
rates (i.e. light, nutrient, and temperature) may, furthermore, affect the viral growth
cycle. For instance, darkness could prevent viral infection of different prokaryotic and
eukaryotic algal hosts (MacKenzie & Haselkorn 1972, Allen & Hutchinson 1976,
Waters & Chan 1982). Temperature may alter the susceptibility of host species to virus
as shown for H. akashiwo (Nagasaki & Yamagushi 1998). Nutrient limitations were
found to have variable effects; phosphate depletion resulted in a reduction of the burst
size of P. pouchetii and Emiliania huxleyi viruses (Bratbak et al. 1993, 1998) or delayed
the cell lysis in Synechococcus (Wilson et al. 1996). Under nitrogen depletion, the
production of E. huxleyi viruses was delayed (Jacquet et al. 2002) and a reduction in the
17
Chapter 1
Introduction
viral burst size was observed for P. pouchetii (Bratbak et al. 1998).
In the ocean, phytoplankton cells experience strong fluctuations in natural
resources (e.g. light, nutrient, temperature). For instance, light and nutrient levels can
vary during phytoplankton bloom and across the water column of stratified systems (e.g.
open ocean). These variations may thus control the impact of viruses on the host
population. Furthermore, the contrasted nutrient conditions found in oligotrophic vs.
eutrophic environments may underlie differential virally mediated mortality of
phytoplankton in these respective environments.
4. Viral lysis and other sources of phytoplankton losses
4.1. Viral lysis and other sources of cell death by lysis
Algal cell lysis rates can be high and dynamic in marine environments
(Brussaard et al. 1995, 1996a, Agusti et al. 1998). Estimates up to 0.3 d-1 have been
reported not only during the termination of algal bloom (Brussaard et al. 1995, 1996a)
but also in oligotrophic marine environments (Agusti 1998). Algal cell lysis has
important implications on the trophic dynamics as the cell content released in
surrounding waters upon lysis provides DOM, potentially available for heterotrophic
bacteria. Several field studies indicated that DOM released upon algal cell lysis could be
sufficient to fulfill most of the bacterial carbon demand (Brussaard et al. 1996b, 2005b).
Viruses are considered important agents killing phytoplankton. Although
several studies have demonstrated that viruses can impose substantial mortality on their
host (see review Brussaard 2004 and section 3.3), the quantitative significance of viral
lysis in the ocean is not clear. One reason for this is that rates of virally mediated
mortality are assessed using different approaches; therefore results from these studies are
not necessarily comparable. More importantly, all known methodologies determining
virally mediated mortality rely on differing assumptions and conversion factors (Table
1). Very few studies have, thus far, determined the contribution of viral lysis to total
algal cell lysis. One recent mesocosm study has shown that viral lysis comprised 30 100% of the total lysis occurring during the bloom of Phaeocystis globosa (Brussaard et
al. 2005a).
In addition to viruses, several other mechanisms responsible for cell lysis are
currently described. For example, other pathogens (e.g., bacteria, fungi) are reported to
kill phytoplankton (Fukami et al. 1992, Mayali & Azam 2004). Another example
includes allelopathic interactions between phytoplankton species. In this type of
interaction, the production of a metabolite by a phytoplankton species has an inhibitory
effect on the growth or physiological function of another phytoplankton species that may
result in cell lysis (Vardi et al. 2002, Legrand et al. 2003).
18
19
- Rapid, inexpensive
- Direct observation of changes in
viral abundance/loss infectivity
- Rapid, inexpensive
- Direct changes in viral
abundance
- Rapid, inexpensive
- The only method excluding the
use of conversion factors
- Provides simultaneously viral
lysis and grazing rates
- Discriminates different algal
groups when combined with FCM
- All cells equally sensitive to viral infection
- All virus-host contact result in infection
- All cell lyse after viral infection
- Diffusion, adsorption rate, burst size, and
host cell size are constant
- Virus and host of interest can be
discriminated (Most Probable Number, MPN,
flow cytometry, FCM)
- Viral decay equals viral production
- Burst size is constant
- Virus of interest need be discriminated
(MPN, FCM)
- All virus produced from infected cells
- Virus of interest can be discriminated from
other virus (MPN, FCM)
- Burst size constant or within a stated range
- Algal growth rates unaffected by dilution
level and diluent
- Phytoplankton cell lysis starts after 12 h
- Losses are proportional to the dilution effect
of the loss agent (virus and grazers)
- No selective grazing on infected/
noninfected cells
Contact rates
Viral decay
Viral
production
Modified
dilution
method
19
Advantages
- No incubation required
Assumption
- All cells containing viruses will lyse
- The eclipse time (time from infection
to mature virus appearance) is constant
- Latent period equals host generation
- Host infection occurs continuously
Methodology
Frequency of
infected cells
using
transmission
electron
microscopy
(TEM)
- Only measures newly infected cells
- Initial phytoplankton concentration must be
high enough to allow up to 5-fold dilution
- A 24h incubation required
- Use of theoretical burst size
- MPN underestimates virus abundance
- FCM cluster may include other virus
- Viral loss not taken into account
- Use of theoretical burst size
- Relation between viral decay and
production is disputable
- Lytic and lysogenic virus not distinguished
- MPN underestimates virus abundance
- FCM cluster may include other virus
- Heavily dependent on theoretical
conversion factors (diffusion, adsorption
coefficient, cell size, burst size)
- MPN may underestimate virus abundance
- FCM cluster may include other viruses
- Cross infection is not taken into account
- Lytic and lysogenic virus not distinguished
Disadvantages
- Heavily dependent on theoretical
conversion factors (eclipse time, relationship
between latent period and host generation)
that are disputable
- Host of interest may be difficult to
discriminate in natural sample
- Selective losses of infected cells may occur
during sample preparation
- Time consuming
Evans et al. 2003
This thesis
Bratbak et al. 1993
Jacquet et al. 2002
Brussaard et al.
2005a
Suttle and Chan 1994
Cottrel and Suttle
1995a
Garza and Suttle
1998
Bongiorni et al. 2005
Suttle and Chan 1994
Garza and Suttle
1998
Source
Sieburth et al. 1988
Proctor et al. 1993
Nagasaki et al. 1994
Bratbak et al. 1993,
1996
Brussaard et al.
1996b
Binder 1999
Gastrich et al. 2004
Table 1. Summary of the assumptions, advantages, and disadvantages of the methods used to determine virally mediated mortality of
phytoplankton (adapted from Winget et al. 2005).
Chapter 1
Introduction
Non-pathogenic forms of algal cell lysis are also reported. For example, the
diatom Ditylum brightwellii was shown to experience a type of ‘intrinsic mortality’
under nitrogen controlled conditions using chemostat cultures (Brussaard et al. 1997).
Recently, another form of autocatalyzed cell death was shown to share similarities with
the programmed cell death (PCD) observed in higher plants and metazoans (Berges &
Falkowski 1998, Vardi et al. 1999, Berman-Frank et al. 2004). The PCD, unlike “natural
cell death” or “necrosis” refers to an active, genetically controlled degenerative process,
which involves series of apoptotic features such as morphological changes (e.g. cell
shrinkage, vacuolization) and complex biochemical events (e.g. activation of PCD
markers like caspases). The PCD ultimately leads to cell lysis. Laboratory studies
suggest that a wide range of phytoplankton is programmed to die in response to adverse
environmental conditions (see review by Franklin et al. 2006). The PCD pathway in
phytoplankton was found to operate under environmental stresses such as intense light
(Berman-Frank et al. 2004), darkness (Berges & Falkowski 1998), nutrient depletion
(Berman-Frank et al. 2004), CO2 limitation and oxidative stress (Vardi et al. 1999).
Some apoptotic features, possibly directing to PCD, have also been detected upon viral
infection (Berges and Brussaard unpubl. data, Lawrence et al. 2001). The complete
genome sequence of a virus infecting Emiliania huxleyi has recently revealed the
presence of genes encoding the biosynthesis of ceramide, which is known to suppress
cell growth and is an intracellular signal for apoptosis (Wilson et al. 2005a).
4.2. Viral lysis versus microzooplankton grazing
Prior to the recognition of algal cell lysis as an important loss factor,
phytoplankton cells were essentially treated as immortal unless they were preyed upon
by zooplankton or lost by sedimentation through the water column. Cell lysis,
sedimentation, and predation by zooplankton may thus, separately or in concert,
influence phytoplankton community structure.
Whether a phytoplankton cell sinks, is preyed upon, or undergoes lysis has
more implications than the structuring of phytoplankton community. As nicely
formulated by Kirchman (1999), “how phytoplankton die largely determines how other
marine organisms live”. The phytoplankton biomass that sinks is lost from the surface to
the benthic food web (Smetack 1985). In contrast, zooplankton grazing channels
phytoplankton biomass to the higher trophic levels whereas the release of cell
constituents mediated by lysis directly affects the standing stock of dissolved organic
carbon, forcing the food web towards a more regenerative pathway (Wilhelm & Suttle
1999, Brussaard et al. 2005b, Fig. 1). The quantification of cell lysis in relation to
sinking and grazing rates is, therefore, essential for an optimal understanding of the flow
of matter and energy in marine systems.
There are differential controls of phytoplankton in oligotrophic vs. eutrophic
environments. In oligotrophic waters (e.g. open ocean, surface coastal waters during
summer), the import rate of the controlling nutrient is low and the regeneration of this
limiting nutrient is critical to sustain high productivity. Small-sized picophytoplankton
20
Chapter 1
Introduction
dominate the autotrophic community due to their competitive growth characteristics.
Considering their micrometer size range, picophytoplankton cells are not prone to
sedimentation (Raven 1998). Instead, the rapidly growing small-sized predators
(heterotrophic nanoflagellates and microzooplankton) are thought to largely control this
phytoplankton biomass (Riegman et al. 1993, Kuipers & Witte 2000). As discussed
above (section 3.3), there is also evidence of significant viral lysis of smaller-sized
picophytoplankton. The relative importance of viral lysis as compared to grazing is,
however, largely unknown in these environments.
In eutrophic waters the import rate of the controlling nutrient is higher and
larger-sized phytoplankton can develop as they escape grazing by microzooplankton.
The larger-sized phytoplankton biomass may not be immediately controlled by larger
grazers as the development and the generation time of larger grazers is relatively long.
Size-selective escape of grazing or non-edible phytoplankton may form algal blooms.
Mass sedimentation can be involved in the disintegration of some of these blooms
(Smetack 1985, Brussaard et al. 1995). Cell lysis was also found to be responsible for
bloom termination (Brussaard et al. 1996a). As emphasized in this introductory chapter,
one possible agent causing the cell lysis is viral infection. Episodes of light and/or
nutrient limitations, typically occurring during algal blooms, may regulate the impact of
virus on host abundance. However, the extent to which viral lysis varies and the
underlying regulatory mechanisms remain poorly documented.
Summarizing the above, the understanding of the quantitative and qualitative
importance of phytoplankton viral lysis in the oligotrophic vs. eutrophic marine waters is
not clear. The present thesis aims to elucidate the role of algal viruses in these
contrasting environments. Therefore, virally induced mortality rates of different
phytoplankton groups were determined and related to microzooplankton grazing. In
order to compare results from different field studies, a single method assessing viral lysis
was used, namely an optimized version of the recently modified dilution method (Evans
et al. 2003). In its original form, the dilution method is routinely used to estimate grazing
on phytoplankton (Landry & Hassett 1982). The modified assay also includes virally
mediated losses of phytoplankton. Although this dilution method has some restrictions, it
has the benefits to exclude the use of conversion factors (i.e., it provides direct viral lysis
rates), to minimize the handling of sample, and it can be applied to the different
coexisting phytoplankton populations. In addition to these field studies, laboratory
experiment aimed to characterize specific virus-host model systems and to study the
virus-host interactions in relation to environmental relevant conditions.
21
Chapter 1
Introduction
5. This thesis
The aim of this thesis is to advance our knowledge on the ecological
significance of algal viruses for marine phytoplankton mortality. More specifically, this
research used a combination of field and laboratory approaches to explore three main
issues:
1.
2.
3.
The extent of virally induced lysis in phytoplankton mortality in marine
environments with contrasting trophic status (oligotrophic vs. eutrophic)
The comparison of viral lysis rates with other algal loss factors (mainly
microzooplankton grazing)
Possible factors regulating algal host-virus interactions.
Chapter 2 describes the significance of viruses during the course of an algal
bloom that developed in the eutrophic southern North Sea. The prymnesiophyte
Phaeocystis globosa is well known for its complex polymorphic life cycle (including
colonies and single cells) and its potential to develop dense blooms in temperate coastal
waters. The termination of P. globosa blooms is typically sudden. Earlier studies have
demonstrated that cell lysis can account for up to 75% of the bloom decline (Brussaard
et al. 1995). Recently, a mesocosm experiment showed that viruses could be a primary
cause of cell lysis for P. globosa (Brussaard et al. 2005a, Ruardij et al. 2005). However,
virally induced mortality of P. globosa has never been determined in the field. For
completeness and to allow the study of inter-annual variability, the significance of viral
lysis during 2 consecutive P. globosa blooms was investigated. To complement viral
lysis estimates of P. globosa, we monitored the total abundance of putative P. globosa
viruses (PgV) as well as the abundance of infective PgV.
Chapter 3 adds to the previous study by providing insight into the phenotypic
diversity of PgV. An earlier phylogenetic analysis of 24 PgV isolated from the Southern
North Sea revealed a close genetic relatedness among these isolates as they formed a
tight monophyletic group within the family of the Phycodnaviridae (Brussaard et al.
2004b). In order to address biodiversity issues, it was thus very challenging to explore
the phenotypic diversity among these isolates. Therefore, twelve of these isolates were
further characterized. This study includes a morphological (particle size and shape) and
molecular (genome size, major structural protein composition) characterization as well
as the investigation of ecologically relevant characteristics such as the length of the lytic
cycle, burst size, the range of host infected by these isolates, and their sensitivity to
temperature.
Chapters 4 and 5 investigate the role of viruses as mortality agents for
different picophytoplankton groups in oligotrophic waters. Chapter 4 describes a study
conducted in the northeastern subtropical Atlantic Ocean with a permanent oligotrophic
22
Chapter 1
Introduction
status whereas the study described in Chapter 5 was executed in the North Sea under
seasonal (summer) oligotrophic conditions. Sharp gradients of light, temperature, and
nutrient level are typically encountered across the water column in these environments.
At the bottom of the euphotic zone, an accumulation of phytoplankton, referred to as a
deep chlorophyll maximum (DCM), marks the transition between the nutrient-depleted
lit surface waters and the nutrient-enriched, light-depleted waters below the thermocline.
Different algal virus communities were observed in the surface and DCM waters (Zhong
et al. 2002). We investigated the role of algal viruses in the DCM (Chapter 4) and in the
surface waters (Chapter 5) and related rates of viral lysis to microzooplankton grazing
for 4 groups of picophytoplankton (including eukaryotes and prokaryotes).
Chapter 6 addresses the influence irradiance can have on virus-algal host
interactions. Given the changes in light conditions that a phytoplankton cell may
experience with depth or with time, investigating the effect of different irradiance on
host-virus interactions was timely. Chapter 6 describes a laboratory experiment testing
the effect of different light levels, including darkness, on two marine phytoplankton of
ecological relevance, namely the bloom former Phaeocystis globosa thriving in
eutrophic waters, and the picophytoplankter Micromonas pusilla ubiquitously distributed
including in oligotrophic environments.
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